Abstract

PLATELETS

PB001: Platelet Chemokine Receptor CXCR7 Mediates an Anti‐thrombotic and Anti‐thromboinflammatory Effect

M. Chatterjee 1 ; M. Manke 1 ; M. Cebo 2 ; J. Rheinlaender 3 ; A. Witte 1 ; T. Schäffer 3 ; M. Lämmerhofer 2 ; O. Borst 1 ; M. Gawaz 1

1 University Clinic of Tübingen, Department of Cardiology and Cardiovascular Diseases, Tübingen, Germany, 2 University of Tübingen, Institute of Pharmaceutical Sciences, Tübingen, Germany, 3 University of Tübingen, Institute of Applied Physics, Tübingen, Germany

Background: Elevated platelet surface expression of the chemokine CXCL12 and its receptors CXCR4‐CXCR7 in acute coronary syndrome influences functional recovery and prognosis.

Aims: This study explores the anti‐thrombotic potential of CXCR7 and its effects on thromboinflammation.

Methods: Flowcytometry, thrombinoscopy, thromboelastography, bleeding time, flow chamber assay, intravital microscopy, mass spectrometry, live imaging by scanning ion conductance microscopy.

Results: Deletion of CXCR7 from platelets in Pf4‐Cre + CXCR7 flox/flox mice led to enhanced thrombotic disposition without affecting haemostasis. The pharmacological CXCR7‐agonist, counteracted GPVI‐PAR1‐P2Y12 induced degranulation, αIIbβIII‐integrin activation, aggregation, ex vivo thrombus formation in healthy subjects, STEMI patients and mice, reduced the rate and extent of platelet interaction with collagen, fibrinogen, ROS generation, the release of thromboinflammatory arachidonic acid, thromboxane A2, 12‐HETE, HHT. CXCR7‐agonist countered activation induced externalisation of thrombogenic phosphatidylserine, thus checked PRP but not plasma dependent (PPP) thrombin generation, or efficiency of clot formation, stabilisation and dissolution. In vivo CXCR7‐agonist administration decreased platelet activation, thrombus formation following carotid artery injury and subsequent inflammatory platelet interaction with leukocytes, without affecting tail bleeding time and coagulation parameters (prothrombin time, activated partial thromboplastin time). CXCR7 ligation inhibited agonist‐induced intracellular calcium mobilisation, phosphorylation of PLC‐γ‐Src‐PKC‐PI3K‐Akt‐p38MAPK. It induced inhibitory mediators cGMP, stabilised cAMP and downstream kinase PKG‐PKA mediated VASP phosphorylation to regulate αIIbβIII activation which was counteracted by guanylyl cyclase, PKG and PKA inhibitors.

Conclusions: CXCR7 can be utilised as an anti‐thrombotic drug target without compromising physiological haemostasis.

PB002: Ticagrelor Inhibits Platelet‐induced Formation of Neutrophil Extracellular Traps

I.C. Moschonas 1 ; K.M. Hansson 2 ; D. Stakos 3 ; A.D. Tselepis 1

1 University of Ioannina, Atherothrombosis Research Centre/Laboratory of Biochemistry, Department of Chemistry, Ioannina, Greece, 2 AstraZeneca, Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, Mölndal, Sweden, 3 Democritus University of Thrace, Cardiology Department, Alexandroupolis, Greece

Background: Neutrophil extracellular traps (NETs) are filamentous structures consisting of decondensed chromatin and granular proteins that have antimicrobial properties and are also involved in atherothrombosis. Ticagrelor is a potent reversible antagonist of the platelet ADP receptor P2Y12.

Aims: We investigated the possible role of platelet activation and the effect of ticagrelor on NETs formation, in vitro .

Methods: Platelet‐rich plasma (PRP) was prepared from whole blood of healthy volunteers and adjusted to 2.5×10 8 platelets/mL. Autologous neutrophils were isolated by double‐Ficoll gradient, placed on poly‐l‐lysine coverslips and incubated for 1 hour at 37°C, 5% CO2. PRP was incubated with/without 1.25 μM ticagrelor for 5 minutes in an aggregometer under stirring and then activated with 20 μM ADP for 5 minutes. The platelet suspension was then transferred to neutrophils at a physiological number ratio of platelets/neutrophils and incubated for 3.5 hours at 37°C, 5% CO2. In other experiments PRP was premixed with neutrophils and incubated with/without 1.25 μM ticagrelor for 5 minutes before activation in situ with 50 μM ADP for 3.5 hours at 37°C, 5% CO2. Cells were then fixed, permeabilized and stained with anti‐myeloperoxidase antibody and DAPI. The % NETs formation was evaluated by fluorescence microscopy.

Results: PRP preactivated in the aggregometer increased NETosis by 106±32% (P=0.03) whereas in situ platelet activation with ADP increased NETosis by 63±22% (P=0.011). Ticagrelor inhibited NETosis under both experimental conditions by 60±10% (P=0.028) and 77±15% (P=0.012), respectively. In the absence of platelets, neither ADP nor ticagrelor alone influenced NETosis.

Conclusions: ADP‐activated platelets induce NETosis, which is significantly inhibited by ticagrelor. Given the antimicrobial properties of NETs and their implication in various pathophysiological conditions including inflammation and atherothrombosis, the above results may represent a novel effect of ticagrelor. The clinical significance of this effect remains to be established.

PB003: Differential Effects of PDE3 and ‐5 Inhibitors on Thrombus Formation on Collagen versus Inflamed Endothelium

D.M. Coenen 1 ; H.J. Albers 2,3 ; A.D. van derMeer 3 ; J.M.E.M. Cosemans 1

1 Maastricht University, Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht, the Netherlands, 2 University of Twente, BIOS Lab‐on‐a‐Chip Group, MESA+ Institute for Nanotechnology, MIRA Institute for Biomedical Technology and Technical Medicine, Enschede, the Netherlands, 3 University of Twente, Applied Stem Cell Technologies Group, MIRA Institute for Biomedical Technology and Technical Medicine, Enschede, the Netherlands

Background: Embolization of platelet‐thrombi formed after atherosclerotic plaque rupture may lead to acute myocardial infarcts and strokes. Despite antiplatelet treatment, the recurrence rate is ˜30 percent, urging the need for optimized treatment. Our group showed that thrombi, formed ex vivo , remain active, secreting various mediators (Mastenbroek et al. ATVB 2015).

Aims: This study aims to examine the effect of suppressing platelet secretion, by elevating cAMP and/or cGMP, on key functional thrombus activities.

Methods: The effect of phosphodiesterase (PDE)3/5 inhibitors on platelet αIIbβ3 activation and α‐ and δ‐granule secretion was measured with flow cytometry. Microfluidics were used to study whole blood thrombus formation at 1000 s −1 over a collagen type I + tissue factor surface or over a confluent monolayer of tumor necrosis factor‐α treated human umbilical vein endothelial cells.

Results: Cilostazol (PDE3i, 5 μM) and tadalafil (PDE5i, 10 nM) significantly inhibited platelet αIIbβ3 activation of collagen‐related peptide‐treated washed platelets (P=0.005 and P=0.03), whereas platelet α‐ and δ‐granule secretion were not significantly affected (P>0.05). In whole blood, PDE3 (50 μM) or ‐5 (100 nM) inhibition resulted in smaller thrombi, although non‐significant. Cilostazol mainly affected the earlier phase of thrombus growth, while the effect of tadalafil was more pronounced at a later time point. Furthermore, PDE3 inhibition significantly increased time to fibrin (P=0.03 vs. P=0.17 with PDE5 inhibition). Strikingly, a ten‐fold lower dose of cilostazol (5 μM) gave a strong decrease in size and compactness of platelet thrombi on inflamed endothelial cells.

Conclusions: These data suggest that PDE3 and PDE5 are key enzymes in thrombus growth and stability, but not in initial platelet adhesion. The efficacy of PDE inhibition is enhanced by the vascular endothelium. Combination of PDE3/5 inhibition with current antiplatelet medication might be the key in the regulation of acute and prolonged thrombus activities.

PB005: Effects of Aspirin and Rivaroxaban Treatment on Murine Arterial Vessel Wall Remodelling and Thrombus Activity

M. Karel 1 ; T. Mastenbroek 1,2,3 ; M. Nagy 1 ; D. Coenen 1 ; W. Chaouyâ 1,4 ; A. Brouns 5 ; J. Debets 5 ; P. Leenders 5 ; H. van Essen 5 ; R. van Oerle 1 ; S. Heitmeier 6 ; H. Spronk 1 ; J. Heemskerk 1 ; M. Kuijpers 1 ; J. Cosemans 1

1 Maastricht University, Biochemistry, Maastricht, the Netherlands, 2 MERLN Institute for Technology‐Inspired Regenerative Medicine, Department of Complex Tissue Regeneration, Maastricht, the Netherlands, 3 Regenerative Medicine Crossing Borders (RegMed XB), Maastricht, the Netherlands, 4 Synapse BV, Maastricht, the Netherlands, 5 Maastricht University, Pharmacology & Toxicology, Maastricht, the Netherlands, 6 Bayer Pharma AG, Wuppertal, Germany

Background: The COMPASS clinical study investigated the effect of combined therapy of ASA and 2.5 mg bid rivaroxaban on patients with coronary of peripheral artery disease. However little is known about the effects of this treatment on the vasculature.

Aims: To study the effects of aspirin and/or rivaroxaban on thrombus activities and on injury‐induced vascular remodelling in mice.

Methods: C57BL/6 mice (n=12/group) were either treated with vehicle, aspirin (5 mg aspirin/kg/d) and/or rivaroxaban (12 mg/g chow). Vascular injury was induced by temporary ligation of the carotid artery. Vascular stiffening was monitored for 2 weeks by non‐invasive ultrasound imaging. Two weeks after ligation, platelet‐leukocyte aggregates (PLA) and vascular changes were assessed. Whole blood thrombus formation and thrombus‐induced cleavage of dye‐quenched collagen was studied with microfluidics.

Results: Treatment with aspirin with(out) rivaroxaban decreased the number of adhered platelets per PLA (P<0.05) in unstimulated blood and blood stimulated with stable ADP analogue or PAR4 peptide (flow cytometry). Importantly, treatment with aspirin with(out) rivaroxaban protected the arteries against ligation‐induced stiffening (P<0.05), while rivaroxaban alone showed a trend herein (P=0.08). Histological analysis indicated that ligation of the carotid artery resulted in local intima‐media thickening, which was reduced by treatment with aspirin or rivaroxaban (P<0.01). Treatment of mouse blood with aspirin and/or rivaroxaban did not alter the size of thrombi formed ex vivo . Yet, interestingly, thrombus‐induced collagenolytic activity ex vivo was reduced (P<0.05) upon treatment with aspirin or rivaroxaban.

Conclusions: This study provides new insight into the vascular‐directed effects of aspirin and/or rivaroxaban treatment after thrombotic injury. Treatment with aspirin significantly antagonised, and rivaroxaban showed potential to inhibit, vascular stiffening, intima‐media thickening, PLA formation and thrombus‐collagenolytic activity.

PB006: Association of PEAR1 Gene Variant with Platelet Reactivity and Clinical Outcomes in Patients Undergoing Stenting and Treated with Aspirin and Clopidogrel

K. Xu 1 ; N. Chan 2 ; F. Wang 1 ; L. Yang 1 ; H. Zhu 1,3 ; J. Li 1,4 ; J. Zhang 1 ; Y. Fan 1 ; J. Chen 5 ; L. Xu 6 ; L. Ying 1,7 ; X. Hu 1 ; S. Ye 1 ; C. Li 1

1 The First Affiliated Hospital of Nanjing Medical University, Department of Cardiology, Nanjing, China, 2 McMaster University, Department of Medicine, Hamilton, Canada, 3 The Second People's Hospital of Changzhou Affiliated to Nanjing Medical University, Changzhou, China, 4 Fuyang Fifth People's Hospital, Fuyang, China, 5 Maanshan People's Hospital, Department of Cardiology, Maanshan, China, 6 Sir Run Run Shaw Hospital, Nanjing Medical University, Nanjing, China, 7 The Affiliated Huai'an Hospital of Xuzhou Medical University and Huai'an Second People's Hospital, Department of Cardiology, Huai'an, China

Background: Dual antiplatelet therapy (DAPT) with aspirin and clopidogrel is the standard therapy for patients undergoing coronary stenting. Genetic determinants of variable response to aspirin and clopidogrel are incompletely characterized. Polymorphisms in platelet endothelial aggregation receptor‐1 (PEAR1, rs12041331) were initially described as being important for platelet aggregation. The prevalence of PEAR1 A/A homozygotes, its associations with laboratory and clinical outcome in a Chinese population undergoing stenting and DAPT, and the mechanism explaining any association are unclear.

Aims: To explore the prevalence of PEAR1 rs12041331 A/A homozygotes and its association with platelet aggregation and clinical outcome.

Methods: We recruited 2007 consecutive patients undergoing stenting and DAPT. Arachidonic acid‐ and ADP‐induced platelet aggregation (PLAA and PLADP) were performed on the 5th day after the procedure. Genotyping was performed using an improved multiplex ligation detection reaction technique. All patients underwent a 30‐day follow‐up and data for MACE (cardiovascular death, nonfatal myocardial infarction or ischemic stroke) were collected. This study was approved by the local ethics committee and registered. Informed consent was obtained.

Results: PLADP was significantly lower in AA‐ than non‐AA‐ genotypes (P<0.001) but not PLAA. Independent of CYP2C19*2 polymorphism, 30‐day MACEs occurred significantly more in AA‐ than in GG‐homozygotes or heterozygotes (adjusted HR, 3.13; 95% CI, 1.05‐9.31; P=0.041).

FIGURE 1 Genotypes of PEAR1 (rs12041331) and platelet reactivity. ADP‐ and AA‐induced platelet aggregation were showed in (A) and (B), respectively

FIGURE 2 Cumulative incidence of 30‐day MACEs in patients undergoing stenting and DAPT stratified by PEAR1 rs12041331 genotype

Conclusions: In the largest study to date, PEAR1 A/A homozygosity occurs in 14.8% of Chinese patients and is associated with a 3‐fold increase in MACE risk after stenting. PLAA was unaffected and PLADP was lowest in AA‐homozygotes. The discordant laboratory findings raise the possibility that PEAR1 polymorphism might impact clinical outcome through mechanisms independent or distal to the inhibition by aspirin or clopidogrel.

PB007: Beta‐1,4‐galactosyltransferase 2 c.909C>T Gene Variant Is Predictive of On‐clopidogrel Platelet Reactivity

T. Belleville‐Rolland 1,2 ; N. Pallet 3,4 ; A. Savalle 3 ; M. Lejeune 3 ; L. Mauge 1,5 ; S. Bertil 1 ; M.‐A. Loriot 3,4 ; P. Gaussem 1,2

1 AP‐HP, Hôpital Européen Georges Pompidou, Hematology, Paris, France, 2 Paris Descartes University, Inserm UMR‐S1140, Paris, France, 3 AP‐HP, Hôpital Européen Georges Pompidou, Clinical Chemistry, Paris, France, 4 Paris Descartes University, Inserm UMR‐S1147, Paris, France, 5 Paris Descartes University, Inserm UMR‐S970, Paris, France

Background: CYP2C19 *2 loss‐of function variant influences clopidogrel response but only accounts for a small part of the variability of platelet reactivity, suggesting the involvement of other gene variants. Recently, exome sequencing of coronary patients under dual antiplatelet therapy identified a variant of the gene encoding beta‐1,4‐galactosyltransferase 2 ( B4GALT2 ), a platelet‐expressed galactosyltransferase. Carriers of the B4GALT2 c.909C>T variant had lower platelet reactivity, as assessed by the VerifyNow P2Y12 assay and light‐transmission aggregation, compared with non‐carriers, indicating that B4GALT2 could influence clopidogrel sensitivity.

Aims: To determine if B4GALT2 c.909C>T influences clopidogrel pharmacodynamics as assessed by vasodilator‐stimulated phosphoprotein (VASP) assay and ADP‐induced platelet aggregation in a non‐selected cohort of patients.

Methods: We retrospectively analyzed 353 patients under clopidogrel (mono or bi‐antiplatelet therapy) addressed to our center between January 2010 and January 2017 to undergo genetic and pharmacodynamic testing (ADP‐induced light‐transmission aggregation and VASP assay). All patients gave written informed consent.

Results: Among the 353 patients screened, DNA was available for 174 of them: 28% were under clopidogrel monotherapy, and 72% under clopidogrel+aspirin. In individuals under dual antiplatelet therapy, B4GALT2 c.909C>T was associated with a significant lower ADP‐induced platelet aggregation. Surprisingly, it did not influence VASP assay. Otherwise, B4GALT2 c.909C>T and CYP2C9*2 were independent genetic predictors of on‐treatment platelet reactivity.

Conclusions: Our findings confirm that B4GALT2 c.909C>T influences on‐treatment platelet reactivity in a cohort of non‐selected patients under clopidogrel‐based antiplatelet therapy, independently of the P2Y12 signaling pathway. B4GALT2 could influence platelet adhesion through posttranslational modification of integrins and/or other cell surface glycoproteins.

PB008: High On‐treatment Platelet Reactivity (HTPR) on Clopidogrel but Not on Novel P2Y12 Antagonists in Patients with ACS Undergoing PCI – Single Centre Study

J. Staško 1 ; R. Šimonová 1 ; M. Samoš 1 ; J. Sokol 1 ; L. Stančiaková 1 ; I. Škorňová 1 ; T. Simurda 2 ; P. Kubisz 1

1 Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, University Hospital Martin, National Centre of Hemostasis and Thrombosis, Martin, Slovakia, 2 Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, National Centre of Hemostasis and Thrombosis, Martin, Slovakia

Background: P2Y12 receptor antagonists together with acetylsalicylic acid (ASA) are used in treatment of patients with acute coronary syndrome (ACS) and secondary prophylaxis of thrombotic complications after percutaneous coronary intervention (PCI). High on‐treatment platelet reactivity (HTPR) during dual antiplatelet treatment (DAPT) represents a risk of thrombosis. On the other side low on‐treatment platelet reactivity (LTPR) can lead to bleeding.

Aims: To monitor the on‐treatment platelet reactivity (PR) in patients with ST segment elevation myocardial infarction (STEMI), to find out prevalence of HTPR and LTPR, to reveal the prevalence of ischemic and bleeding episodes during DAPT and their relation to laboratory findings.

Methods: 73 patients with acute STEMI after PCI and on DAPT were included in the prospective monocentric study. PR was assessed using both the VASP phosphorylation analysis and light transmittance aggregometry (LTA) in 3 intervals: T1 (time of PCI), T2 (1 day after PCI) and T3 (30 days after PCI). Patients were observed during a period of 1 month with the aim to reveal thrombotic and bleeding episodes.

Results: High variability in PR was found in clopidogrel treated patients in T2 and T3. Significant difference in PR was found between clopidogrel and novel P2Y12 antagonists in T2 and T3, while no significant difference between PR in prasugrel and ticagrelor group was found. Prevalence of HTPR in clopidogrel treated group in T2 and T3 exceeded 45%. HTPR in prasugrel/ticagrelor treated group was rare (5%). Stent thrombosis was diagnosed in 12% of clopidogrel treated patients (all with HTPR). In spite of high prevalence of LTPR in novel P2Y12 antagonist treatment no bleeding episodes were found.

Conclusions: Results confirm a high effectiveness of PR monitoring in patients with acute STEMI undergoing PCI on DAPT. VASP analysis has a high specificity to signal route of P2Y12 receptor of platelets thus it seems to be suitable for monitoring of response to its antagonists.

PB009: Detection of Anthocyanins Effects to Alleviating Thrombotic Risks in Comparison to Aspirin

A. Gaiz 1 ; A. Kundur 2 ; S. Mosawy 2 ; N. Colson 2 ; I. Singh 2

1 Almustansiriah University, National Center of Hematology, Baghdad, Iraq, 2 Griffith University/Menzies Health Institute of Queensland, Southport, Australia

Background: Platelet hyperactivity has a significant role in initiating vascular thrombosis and subsequent cardiovascular disease (CVD). several studies demonstrate resistance to currently used antiplatelet therapies such as aspirin (AS). The antioxidant activity of polyphenolic compounds including anthocyanin (AC) has been shown to reduce platelet activity and reduce CVD morbidity.

Aims: Comparing the antiplatelet effect of pure AC compounds with Aspirin.

Methods: Healthy human subject (n=50) were recruited in this study and divided into two groups. Each participant consumed either 80 mg Aspirin/day or 320 mg of AC/day in the form of capsules for 28 days. Fasting blood samples were collected at baseline (before) and after the treatment period. Flow cytometry was used to assess platelet activation by measuring platelet surface marker expression of CD41a and P‐selectin. Platelet aggregation studies were performed by stimulating platelets with ADP, collagen or arachidonic acid. Haematological indices, lipid profile, glucose, uric acid, and high sensitivity CRP were assessed.

Results: There was a significant suppressive effect of AC on the expression of P‐selectin. There was a substantial reduction of ADP‐stimulated platelet aggregation. On the other side, Aspirin significantly reduced collagen‐stimulated and arachidonic acid‐stimulated platelet aggregation assays, but it has an insignificant inhibitory effect on ADP‐stimulated platelet aggregation and expression of P‐selectin. There were trends of lowering impact of AC on total cholesterol and triglycerides, but they were not significant.

Conclusions: The results show that AC may reduce platelet aggregation and activation as demonstrated by inhibition of ADP‐stimulated platelet aggregation and P‐selectin expression. These results provide greater insight into the effect of AC and the possible mechanism by which AC can reduce platelet function. Hence, AC may act as a complement to other anti‐platelet agents to reduce the occurrence of thrombotic events.

PB011: Assessment of Clopidogrel Response in Patients with Prior Atherothrombotic Events

Z. Kaya 1 ; P. Özen 1 ; A.P. Nas 1 ; H. Batur Çağlayan 2 ; G. Aydoğdu Taçoy 3 ; B. Nazlıel 2

1 Gazi University Medical Faculty, Pediatric Hematology Unit of the Department of Pediatrics, Ankara, Turkey, 2 Gazi University Medical Faculty, Department of Neurology, Ankara, Turkey, 3 Gazi University Medical Faculty, Department of Cardiology, Ankara, Turkey

Background: Clopidogrel is a prophylactic antiplatelet drug that has been successful at preventing recurrences in patients with atherothrombotic events. Abnormal platelet count, high MPV, and PDW indicating platelet activation, are additional risk factors for thrombosis in patients with prior cardiac events.

Aims: We retrospectively evaluated platelet tests and clopidogrel response for 80 patients with prior cardiovascular or cerebrovascular events.

Methods: All patients had received 75 mg/day clopidogrel for at least 7 days prior to platelet aggregation testing. Clopidogrel response was assessed by light transmission aggregometry (Chrono‐log Model 700) using ADP agonist. According to the manufacturer's description, high responders to the drug were defined as having platelet reactivity below 49%, low responders between 50% and 61%, and non‐responders between 62% and 70%. Platelet count, MPV and PDW were measured as a part of each patient's complete blood count in a Sysmex ® , XN‐3000 automated hematology analyzer.

Results: There were 29 patients with prior cardiovascular events (median age: 70 years) and 51 patients with prior cerebrovascular events (median age : 62 years). Of the cardiovascular patients, there were 12 (42%) clopidogrel high responders with ADP, 10 (34%) low responders and 7 (24%) non‐responders. Of the cerebrovascular patients, there were 28 (54%) clopidogrel high responders with ADP, 15 (30%) low responders and 8 (16%) non‐responders. Mean platelet count was significantly lower in the cardiovascular group than in the cerebrovascular group (210.680±73.830 μL vs. 280.530±65.860 μL, respectively) (P<0.05). Mean MPV and PDW were significantly higher in the cardiovascular group than in the cerebrovascular group (MPV 10.9±0.8 /fL vs. 10.1±0.9 /fL; PDW 13.9±1.9 fL vs. 11.6±2.1 fL) (both P<0.05).

Conclusions: Our findings suggest that clopidogrel dose adjustment is necessary to prevent recurrences in patients with previous cardiovascular events who are low responders clopidogrel.

PB013: Assessment of Platelet Reactivity with Multiple Electrode Impedance Aggregometry (MEA) in Patients Reseiving Ticargelor

D. Dineva ; I. Paskaleva

National Heart Hospital, Sofia, Bulgaria

Background: Monitoring platelet reactivity during P2Y12‐inhibitors helps prevent stent thrombosis (ST) and/ or bleeding. High platelet reactivity (HPR) is associated with an increased risk for ischaemic complications, while low platelet reactivity (LPR) is associated with greater bleeding events.

Aims: The purpose of this study was to evaluate the effect of ticagrelor on residual platelet reactivity.

Methods: The platelet function was assessed with Multiplate impedance aggregometry by ADP‐test in 91 patients on 2×90 mg/d ticagrelor therapy who underwent PCI. Blood was collected at the first month after stent implantation. Adequate response to ADP receptor blocking was defined as ADP‐test <450 AU, on the base of cut‐off value, determined by ROC analysis (AUC 0.864 with 0.84 specificity and 0.78 sensitivity). Low platelet reactivity was defined as ADP‐test <150 AU.

Results: A total of 91 patients were enrolled, 88 of them showed adequate response on ticagrelor treatment (ADP 203±89 AU). We observed residual HPR in three patients (ADP 635±78 AU) and they were switched on prasugrel 10 mg/d. Additional platelet inhibition of 52.8% was observed when switching them from ticagrelor to prasugrel (663 AU vs. 313 AU).

Minor bleeding complications like cutaneous hematoma, epistaxis, haemorrhoidal bleeding and hemoptoe were found in patients with lower values of ADP test (107±64 AU). Antiplatelet therapy in patients with bleeding was switched to clopidogrel 75 mg/d. Reduction of platelet inhibition and decreasing of the bleeding was seen when ticagrelor was switched to clopidogrel.

Conclusions: We found impaired response to ticagrelor 2×90 mg in 3 of 91 patients (3.29%). ADP‐test <150 AU was associated with increasing risk of minor bleeding. Tailored treatment with different P2Y12 inhibitors leads to optimal level of ADP inhibition.

PB014: Results of Platelet Aggregation Testing by Light Transmission Aggregometry using Drug Specific Agonists in Patients Taking Antiplatelet Therapy

S. Margetić 1 ; N. Vrkić 1,2 ; B. Getaldic 1 ; D. Ruzic Ferenec 1 ; I. Vuga 1

1 Sestre Milosrdnice University Hospital Center, Department of Clinical Chemistry, Zagreb, Croatia, 2 Faculty of Pharmacy and Biochemistry University of Zagreb, Zagreb, Croatia

Background: In the recent years platelet aggregation testing has expanded in the assessment of the effectiveness of antiplatelet therapy.

Aims: To evaluate results of platelet aggregation by light transmission aggregometry (LTA) using drug specific agonists in patients on antiplatelet therapy.

Methods: Platelet aggregation testing was performed in 107 patients on antiplatelet therapy: ASA, ADP receptor inhibitor (clopidogrel or ticagrelor) or dual therapy (ASA plus ADP receptor inhibitor) as well as in 27 patients in whom antiplatelet therapy was discontinued from 2 to 8 days due to surgery. Inhibition of platelet aggregation was assessed by ADP (2 μmol/L) agonist for ADP receptor inhibitor and by arachidonic acid (AA, 1 mmol/L) as agonist for acetylsalicylic acid (ASA) drug on Sysmex CS2500 analyzer using Hyphen BioMed (France) reagents.

Results: Platelet aggregation with drug specific agonists was significantly inhibited in all patients, as shown in Table 1. Platelet inhibition with ADP was statistically different (P=0.003) between patients treated with clopidogrel and ticagrelor. However, there were no differences in AA or ADP inhibitions between patients treated with one or both drugs (Table 1). Among all patients on ASA and ADP receptor inhibitor alone and among those on dual therapy, only one patient was poor responsive to AA inhibition and no one was poor responsive to ADP inhibition. Among 27 patients with discontinued antiplatelet therapy, platelet function was recovered to the values above the limit of the reference range (60%) 6th day for AA and 7th day for ADP agonist after drug withdrawal.

TABLE 1 Results of platelet aggregation testing using specific agonists by light transmission aggregometry (LTA) in patients on antiplatelet therapy Antiplatelet drug acetylsalicylic acid (ASA) 100 mg/day ADP inhibitor ASA + ADP inhibitor P ADP inhibitor clopidogrel 75 mg/day ADP inhibitor ticagrelor 90 mg/day P Agonist / N N=35 N=24 N=48 N=50 N=22 Arachidonic acid (AA) % aggregation Median 12 — 10 0.342 — — — 95% CI for median 6‐18 — 6‐14 — — — Interquartile range (IQR) 6‐19 — 11‐20 — — — Adenosine‐diphosphate (ADP) % aggregation Median — 29 30 0.539 32 22 0.003 95% CI for median — 11‐40 24‐35 28‐40 9‐26 — Interquartile range (IQR) — 13‐39 22‐40 24‐41 12‐26 —

Conclusions: Use of specific agonists for certain antiplatelet drugs contribute to the better understanding of platelet aggregation inhibition in patients taking antiplatelet therapy. Recovery time of platelet function of 6‐7 days after drug withdrawal could be explained by the fact that the function of inhibited enzyme or receptor recovers for about 10% a day as a result of the entry of new platelets in circulation.

PB015: Optimal Antiplatelet Therapy in Moderate to Severe Asymptomatic and Symptomatic Atherosclerotic Carotid Stenosis: A Systematic Review of the Literature

S. Murphy 1 ; R. Naylor 2 ; J.‐B. Ricco 3 ; H. Sillesen 4 ; S. Kakkos 5 ; A. Halliday 6 ; G. de Borst 7 ; M. Vega de Ceniga 8 ; G. Hamilton 9 ; D. McCabe 10

1 Adelaide and Meath Hospital, Department of Neurology, Dublin, Ireland, 2 Glenfield Hospital, Department of Vascular Surgery, Leicester, United Kingdom, 3 University of Strasbourg, Department of Vascular Surgery, Strasbourg, France, 4 Rigshospitalet, University of Copenhagen, Department of Vascular Surgery, Copenhagen, Denmark, 5 University of Patras Medical School, Department of Vascular Surgery, Patras, Greece, 6 University of Oxford, Nuffield Department of Surgical Sciences, Oxford, United Kingdom, 7 University Medical Center Utrecht, Department of Vascular Surgery, Utrecht, the Netherlands, 8 Hospital de Galdakao‐Usansolo, Department of Angiology and Vascular Surgery, Bizkaia, Spain, 9 University College London Medical School, Department of Vascular Surgery, London, United Kingdom, 10 School of Medicine, Trinity College Dublin, Academic Unit of Neurology, Dublin, Ireland

Background: Carotid stenosis patients are at risk of cerebrovascular events despite antiplatelet therapy. Data on prescribed antiplatelet regimens have not been comprehensively‐collated from trials to guide optimal therapy in this population.

Aims: To perform a comprehensive systematic review of the literature on randomised trials of patients with extracranial moderate to severe carotid stenosis treated with antiplatelet therapy.

Methods: We searched Medline, Ovid, Embase, Web of Science and Google Scholar from 01/1988 to 03/2016 for randomised trials in this patient population on any form of antiplatelet therapy in which cerebrovascular outcome events were reported.

Results: 22 studies were deemed eligible for inclusion. Data from one RCT showed a non‐significant benefit from aspirin vs. placebo in asymptomatic carotid stenosis, but it is still reasonable to recommend aspirin (81‐325 mg daily) for prevention of vascular events in these patients. Low‐medium dose aspirin (81‐325 mg daily) is superior to higher doses (>650 mg daily) at preventing recurrent vascular events in patients undergoing endarterectomy. Data from endovascular treatment (EVT) trials support peri‐procedural treatment of asymptomatic and symptomatic patients with 81‐325 mg of aspirin daily. The use of peri‐procedural aspirin‐clopidogrel in patients undergoing EVT is based on one pilot trial, but appears safe. Short‐term treatment with aspirin‐dipyridamole or aspirin‐clopidogrel are equally effective at reducing micro‐embolic signals on transcranial Doppler ultrasound in patients with ≥50% symptomatic carotid stenosis. There is insufficient evidence to recommend routine aspirin‐clopidogrel combination therapy to reduce the risk of ‘recurrent clinical ischaemic events’ in patients with symptomatic moderate‐severe carotid stenosis.

Conclusions: This systematic review facilitates an evidence‐based approach to ‘optimal antiplatelet therapy’ in carotid stenosis patients. Future trials should randomise such patients to receive different antiplatelet regimens.

PB016: The Original Algorithm for Platelet Functional and Molecular Genetic Analysis to Optimize Antiplatelet Therapy in Outpatients with Peripheral Artery Diseases

O. Sirotkina 1,2 ; N. Surint 3 ; A. Topanova 1 ; T. Vavilova 1,3

1 Almazov National Medical Research Centre, Saint‐Petersburg, Russian Federation, 2 B.P. Konstantinov Petersburg Nuclear Physics Institute of National Research Centre «Kurchatov Institute», Saint‐Petersburg, Russian Federation, 3 First City Consultative and Diagnostic Center, Saint‐Petersburg, Russian Federation

Background: Inhibition of platelet aggregation is the main approaches to treatment and prevention of CVD.

Aims: The development of original algorithm for laboratory and molecular genetic analysis to optimize antiplatelet therapy in outpatients with peripheral artery disease (PAD) underwented reconstructive surgery.

Methods: 105 patients (age 68±1) with PAD were included into the study. We analyzed the Leu33Pro GPIIIa, C807T GPIa, G36T P2Y12, CYP2C19*2, CYP2C19*3 and CYP3A5*3 polymorphism by PCR, ADP/collagen induced platelet aggregation by impedance method.

Results: The distribution of appraised genotypes was not significantly different from previously explored healthy donors. The presence of 36T P2Y12 allele in homo or heterozygous state was a risk factor for high platelet reactivity in patients on standard antiplatelet therapy: 19 (6‐32) in carriers GG and 83 (66‐91) in carriers GT/TT genotype (p=0.0006) for ADP induced aggregation and 34 (29‐48) and 63 (55‐74), respectively (P=0.003) for collagen induced aggregation. Moreover 16% of patients have polymorphism of CYP3A5 and CYP2C19 genes, associated with poor clopidogrel metabolism. Individual selection of antiplatelet therapy was carried out considering the results of complex of clinical data, platelet functional and molecular genetic tests. According to this approach all patients were divided into 5 groups with own regimen of antiplatelet therapy: ASA 100 mg or 150 mg, clopidogrel 75 mg, clopidogrel 75 mg+ASA 100 mg or 150 mg. After 3 months of individual antiplatelet therapy all patients showed a significant decrease in platelet reactivity from 58 (43‐75) to 28 (6‐58) (p=0.01) and from 54 (41‐68) to 40 (25‐55) (P=0.05) for ADP and collagen induced aggregation respectively. We have achieved a good clinical result. No limb amputation was performed within 5 years of follow‐up.

Conclusions: One of the ways to improve the long‐term results of reconstructive interventions in PAD might be the development of the algorithm for an integrated approach to individual antiplatelet therapy.

PB017: Impact of Time‐to‐treatment on Freedom‐from‐relapse for Steroid‐Refractory Immune Thrombocytopenia

P. Vishnu 1 ; W.A. Hammond 2 ; E.M. Rodriguez 3 ; Z. Li 1 ; C.E. Rivera 1

1 Mayo Clinic Florida, Jacksonville, United States, 2 Baptist MD Anderson Cancer Center, Jacksonville, United States, 3 University of Puerto Rico at Mayagüez, Puerto Rico, United States

Background: Splenectomy (S) or rituximab (R) are potentially curative options for patients with steroid‐refractory ITP (sr‐ITP) with good responses, but the long‐term outcome is not known and the treatment order is uncertain.

Aims: To evaluate freedom‐from‐relapse (FFR) after therapy with S or R for sr‐ITP and the impact of time from diagnosis to 2nd line treatment (TT) based on sequence of therapy, and the difference in efficacy of R in patients with or without a prior S.

Methods: Patients age ≥18 years with sr‐ITP treated with S or R between Jan 2002 and Dec 2015 at Mayo Clinic who underwent S or R as 2nd line therapy. TT, FFR and response following 2nd‐ and 3rd‐line treatment with S or R were measured.

Results: We identified 237 patients; 122 had S and 115 received R for sr‐ITP. Patients in S group were younger at diagnosis compare to R (median 49 v 60 years, P=0.001) and at time of 2nd‐line treatment (median, 53 v 61 years, P=0.007). Most patients required 2nd line therapy within 12 months of steroid refractoriness (65.4%). There were more patients with TT of ≥12 months in S group compared to R group (42.6 v. 26%; P=0.0075). Patients in S group had a higher response (87.7% v 43.5%, P<0.001) and a higher 5‐year FFR (53.25% v 18.9%, P<0.001) compared to R. Patients treated with R after failure of S achieved similar response (88.5% vs. 82.8%, P=0.39) and 2‐y FFR (67.31% vs. 58.13%, P=0.68) compared to those who had S for relapsed ITP after R. The 5‐year FFR after R was significantly longer in patients with a prior S than in those with an intact spleen (43.51% vs. 18.70%, P < 0.001). Multivariate model predicting FFR following 2nd line therapy was in favor of splenectomy (HR 0.33, 95% CI 0.22, 0.50; P<0.001).

Conclusions: Sequence of S before R has a clinical benefit for treatment of sr‐ITP. Use of R following failure of S appears to be as effective as S following failure to R. Although the impact of time to 2nd line treatment was significant, it is likely that the treatment type was the impetus for the different outcomes.

PB018: Guidance on the Diagnosis and Management of PT‐VWD: A Project from the Platelet Physiology Subcommittee of the ISTH

M. Othman 1,2 ; P. Gresele 3

1 Queen's University, Biomedical and Molecular Sciences, Kingston, Canada, 2 St Lawrence College, School of Baccalaureate Nursing, Kingston, Canada, 3 University of Perugia, Department of Medicine ‐ Section of Internal and Cardiovascular Medicine, Perugia, Italy

Background: Only 55 patients with PT‐VWD have been reported worldwide. Literature is scarce in providing information required to establish standards for diagnosis and management of the disease. Currently there are no clear guidelines/consensus and many patients remain undiagnosed or undertreated.

Aims: To generate guidance for a standard diagnostic and management approach for PT‐VWD based on consensus expert views.

Methods: RAND based approach to obtain a formal consensus among experts. A panel formed of 11 experts (9 countries, 5 continents) was formulated. A series of 46 survey statements on history and clinical presentation (14), diagnosis (21) and management (11) of PT‐VWD were prepared for the experts to review. Statements were rated by the experts from 1‐9 where 1 is completely inappropriate and 9 is fully appropriate. Statements were classified as inappropriate (scores of 1‐3), uncertain (scores of 4‐6), or appropriate (scores of 7‐9).

Results: Experts have agreed largely on most statements related to basic history, clinical features and methodology for diagnosis and management approaches. Of these are: common bleeding symptoms and severity, family history, GP1BA gene mutation and using RIPA mixing studies as essential lab tests with some disagreement on ristocetin cutoff. Few statements were judged uncertain with respect to common bleeding symptoms in children, the association with cancer, inflammation and bone pathologies indicating research is required in these areas. Some uncertainty/ambiguity remained around the use of DDAVP, rFVIIa, or appropriate perinatal monitoring. These ambiguous statements will be resolved in a second survey. Large agreement was made towards a proposed algorithm for diagnosis and management of the disease.

Conclusions: A consensus around a standard diagnostic and management approach for PT‐VWD can be established and would be critical to improving the diagnosis and treatment of this bleeding disorder.

TABLE 1 Acknowledgement of RAND Survey Participants‐ Alphabetical Order Suchitra Acharya (USA), Said Enayat (UK), Emmanuel Favaloro (AU), Michelle Lavin (Ireland), Will Lester (UK), Gilian Lowe (UK), Jonathan Miller (USA), Paquita Nurden (Fr), Shirin Ravavood (Iran), Analia Sánchez‐Luceros (Argentina), Jerry Ware (USA)

PB019: PK Simulation of Avatrombopag‐induced Increases in Platelet Counts with Redosing in Patients with Thrombocytopenia and Chronic Liver Disease

S. Hayes 1 ; C. Farrell 1 ; K. Aggarwal 2 ; M. Vredenburg 2 ; L.F. Allen 2

1 ICON, Dublin, Ireland, 2 Dova Pharmaceuticals, Durham, United States

Background: Avatrombopag (AVA) is a novel oral thrombopoietin receptor agonist being developed to treat thrombocytopenia in patients with chronic liver disease (CLD) scheduled to undergo a procedure; it is administered orally as a 5‐day treatment course. Population pharmacokinetic (PK) and PK/pharmacodynamic (PK/PD) models have been developed that characterize the relationship between AVA dosing and the induced platelet count increase.

Aims: This model was used to simulate platelet count changes that could be expected with a second course of AVA.

Methods: Simulations were conducted for 4 AVA repeat dosing regimens. For all regimens, 60 mg AVA was dosed daily on Days 1‐5, and then a second 5‐day course of 60 mg AVA was started on either Day 17, 24, 31 or 38. Baseline platelet counts between 20 and 50×10 9 /L were assumed. Each regimen was simulated 500 times in 100 subjects, and the median and 80% Prediction Intervals (PI) obtained.

Results: The original PK/PD model (93 subjects) described the PK of AVA by a one‐compartment model with first‐order absorption and elimination. AVA PK was dose‐independent and not significantly affected by age, weight, race, gender, or liver function. Simulations of redosing on Day 17 led to only a small increase in the maximum platelet counts above the initial Day 1‐5 course (˜6×10 9 /L). Second courses administered on Day 24, 31 or 38 resulted in maximum platelet counts comparable to the initial Day 1‐5 course. For each regimen, the median upper bound of the 80% PI (i.e., 90th percentile) was less than 200×10 9 /L.

Conclusions: A redosing simulation of AVA courses on Day 24, 31 or 38 resulted in median platelet count increases comparable to the initial Day 1‐5 dosing regimen. Redosing at Day 17 led to only a small increase in maximum platelet counts, and the 80% PI did not exceed 200×10 9 /L. These results suggest that AVA can be redosed as early as 12 days after completion of the first dosing regimen (Day 17) without excessively increasing platelet counts.

PB020: Exploratory Analyses of the Efficacy of Avatrombopag versus Placebo from 2 Phase 3 Studies Using Alternate Baseline Platelet Count Cohorts and an Alternate Secondary Efficacy Endpoint

F. Poordad 1 ; L. Allen 2 ; K. Aggarwal 2 ; M. Vredenburg 2 ; W. Tian 2 ; N. Terrault 3

1 Texas Liver Institute, San Antonio, United States, 2 Dova Pharmaceuticals, Durham, United States, 3 UCSF, San Francisco, United States

Background: Thrombocytopenia (TCP) is common in chronic liver disease (CLD), increasing in severity with worsening disease. Avatrombopag (AVA) is a thrombopoietin receptor agonist that has completed Phase 3 development as an alternative to platelet transfusions for patients with TCP and CLD undergoing a procedure.

Aims: This analysis evaluated AVA in cohorts with Baseline platelet counts (PC) alternately defined as <35×10 9 /L and ≥35×10 9 /L, and the proportion of patients who achieved both a PC ≥50×10 9 /L and increase of 20×10 9 /L.

Methods: Two randomized, double‐blind, placebo (PBO)‐controlled Phase 3 trials (ADAPT‐1 and ADAPT‐2) enrolled adults with CLD and PC <50×10 9 /L undergoing a procedure. Patients were randomized 2:1 to AVA or PBO, and dosed orally for 5 days based on Baseline PC. For the alternate analyses, patient data were divided into 2 Baseline PC cohorts: <35×10 9 /L and ≥35×10 9 /L. The primary efficacy endpoint remained the proportion of patients not requiring platelet transfusion or rescue procedure up to 7 days post‐procedure. An alternate secondary endpoint analysis assessed the proportion of patients with a PC ≥50×10 9 /L and an increase of ≥20×10 9 /L from Baseline during the study.

Results: 435 patients were randomized in the 2 studies. AVA was again shown to be superior to PBO in both cohorts in the number of patients not requiring a platelet transfusion or rescue procedure (<35×10 9 /L: AVA‐ 63.4%, PBO‐ 26.7%; P<0.0001; ≥35×10 9 /L: AVA‐ 83.0%, PBO 34.7; P<0.0001). The alternate secondary endpoint analysis demonstrated the superiority of AVA in the proportion of patients achieving a PC ≥50×10 9 /L and an increase of ≥20×10 9 /L: <35×10 9 /L (AVA‐ 60.2%, PBO‐ 0%) and ≥35×10 9 /L (AVA‐ 72.0%, PBO 6.1%).

Conclusions: AVA was superior to placebo in increasing PC and reducing platelet transfusions in all evaluated Baseline PC cohorts.

PB021: Influence of Preoperative Platelet Activity on Coagulation and Blood Loss in Large Orthopedic Surgery

I. Antropova 1 ; B. Yushkov 2 ; I. Shlykov 3 ; E. Volokitina 4

1 Ural State Medical University, the Ministry of Health of the Russian Federation, Central Research Laboratory, Ekaterinburg, Russian Federation, 2 Institute of Immunology and Physiology, Ural Branch of Russian Academy of Sciences, Ekaterinburg, Russian Federation, 3 Ural Institute of Traumatology and Orthopedics named after V.D. Chaklin, Ekaterinburg, Russian Federation, 4 Ural State Medical University, the Ministry of Health of the Russian Federation, Department of Traumatology and Orthopedics, Ekaterinburg, Russian Federation

Background: During surgery, activation and secretion of platelets not only promote clot formation, which stop bleeding, but also contribute to reparative processes in damaged tissues.

Aims: To evaluate the effects of preoperative platelet activity on postoperative platelet activity, coagulation and general blood loss after total knee replacement (TKR).

Methods: The study included 35 patients undergoing TKR. In accordance with the preoperative level of β‐thromboglobulin (β‐TG), which is a specific protein of platelet alpha‐ granules, all patients were divided into 2 groups: low β‐TG (LTG‐group, β‐TG<25 U/ml) and high β‐TG (HTG‐group, β‐TG≥25 U/ml). Characteristics of groups is presented in Table 1.

TABLE 1 Characteristics of patients and surgery Criterion LTG (n=17) HTG (n=18) P Sex, (male / female)* 9/8 6/2 0.335 Age (years)** 62±9 60±8 0.606 Type of anesthesia (general / regional)* 1/16 3/8 0.667 Total blood loss (ml)** 868±239 687±241 0.035 *Absolute number; Two‐tailed Fisher's exact test; **Mean ± standard deviation; Mann‐Whitney test

Platelet count, β‐TG level (“Asserachrom β‐TГ”), D‐dimer concentration (“Technozym D‐dimer ELISA”), thromboelastography (TEG 5000), blood loss (intraoperatively and 24 hours after surgery) were determined. Blood samples were taken 1 or 2 days before operation, after 30 minutes, at 1, 3 days after TKR. Comparison of the groups was carried out using the Mann‐Whitney test. The study was approved by a local ethics committee, and informed consent was obtained from each patient.

Results: Total blood loss was significantly higher in LTG group (Table 1). At the end of operation, β‐TG activity and D‐dimer concentration were significantly higher in HTG group (Table 2).

TABLE 2 Haemostasis parameters before and after total knee replacement Group β‐TG (U/ml) Platelets (×1000/µL) D‐Dimer (ng/ml) R (min) Alpha angle (degrees) Before surgery LTG 19.8 [17.0; 22.0] 252 [186; 273] 145 [78; 169] 16.3 [12.9; 18.5] 37.4 [29.4; 43.7] HTG 27.1 [26.2; 30.0]* 242 [214; 311] 98 [80; 123] 14.5 [11.8; 17.3] 38.6 [32.0; 49.1] 30 minutes LTG 38.2 [28.9; 65.0] 221 [182; 244] 398 [245; 708] 15.2 [12.0; 18.9] 38.9 [29.5 45.6] HTG 78.6 [58.0; 105.1]* 210 [196; 285] 1203 [450; 1839]* 9.2 [8.3; 12.5]* 56.0 [50.3; 60.3]* Day 1 LTG 32.1 [28.9; 59.4] 179 [156; 238] 739 [448; 1404] 15.2 [12.8; 20.3] 40.1 [34.8; 50.1] HTG 47.0 [31.7; 70.7] 212 [184; 263] 1726 [905; 2398]* 12.0 [9.7; 15.8] 44.2 [39.6; 59.5] Values expressed as median [interquartile range]; Comparisons between groups were assessed with Mann‐Whitney test, *P<0.05.

Thromboelastography showed that reaction time (R) was significantly shorter and Alpha angle was significantly larger in HTG group at the end of TKR (Table 2). Platelet counts in LTG and HTG groups did not significantly differ.

Conclusions: The preoperative platelet activity influences postoperative platelet activity, rate of clot formation, the amount of fibrin and also affects the blood loss in large orthopedic surgery.

PB022: Superiority of Avatrombopag to Placebo in Increasing Platelet Counts and Reducing Platelet Transfusions in Patients with Chronic Liver Disease‐associated Thrombocytopenia Undergoing Scheduled Procedures ‐ Pooled Analysis of 2 Randomized Phase 3 Studies

F. Poordad 1 ; L.F. Allen 2 ; K. Aggarwal 2 ; M. Vredenburg 2 ; N. Alkhouri 1

1 Texas Liver Institute, San Antonio, United States, 2 Dova Pharmaceuticals, Durham, United States

Background: Thrombocytopenia (TCP) is common in patients with chronic liver disease (CLD) and complicates clinical management with these patients requiring multiple invasive procedures. Prophylactic platelet transfusions are used to reduce bleeding risk, but have multiple associated safety risks.

Aims: Avatrombopag (AVA), an oral thrombopoietin receptor agonist, was studied in 2 Phase 3 studies as an alternative to platelet transfusions for patients with TCP and CLD.

Methods: Two randomized, double‐blind, placebo (PBO)‐controlled Phase 3 trials (ADAPT‐1 and ADAPT‐2) enrolled adults with CLD and platelet count (PC) <50×10 9 /L undergoing a procedure. Patients were divided by Baseline PC into Cohort 1‐ PC <40×10 9 /L and Cohort 2‐ PC 40 to <50×10 9 /L, and randomized 2:1 to once‐daily oral AVA (Cohort 1‐60 mg; Cohort 2‐40 mg) or PBO for 5 days, with the procedure 5 to 8 days after the last dose. The primary efficacy endpoint was the proportion of patients not requiring a platelet transfusion or rescue procedure. Secondary endpoints assessed the proportion of patients achieving the target PC (≥50×10 9 /L) by Procedure Day, change in PC from Baseline to Procedure Day, and safety.

Results: 435 patients were randomized in the 2 studies. AVA was superior to PBO in both cohorts in the number of patients not requiring a platelet transfusion or bleeding rescue (Cohort 1: AVA‐ 66.9%, PBO‐ 28.6%; P<0.0001; Cohort 2: AVA‐ 88.0%, PBO 35.8%; P<0.0001). A greater number of AVA‐treated patients also achieved the target PC in both Cohort 1 (AVA‐ 68.1%, PBO‐ 5.5%; P<0.0001) and Cohort 2 (AVA‐ 90.6%, PBO‐ 29.9%; P<0.0001). Mean increases in PCs were also greater with AVA compared with PBO in both cohorts (Cohort 1: AVA‐ 31.7×10 9 , PBO‐ 1.8×10 9 /L; Cohort 2: AVA‐ 41.0×10 9 , PBO 3.5×10 9 /L). The most common TEAEs were pyrexia, abdominal pain, nausea, and headache.

Conclusions: AVA was superior to placebo in reducing the need for PLT transfusions, the proportion of patients achieving the target PC, and increasing the PC.

PB023: Atypical Presentations of RUNX1 Associated Familial Platelet Disorder with Predisposition to Myeloid Malignancy (FPDMM)

C. Ward ; M.‐C. Morel‐Kopp; C. Tang; D. Rabbolini; W. Stevenson

University of Sydney, Kolling Institute of Medical Research, St Leonards, Australia

Background: RUNX1 pathogenic variants were the first to be linked to myeloid malignancy, presenting as familial platelet defects. Recognition of RUNX1‐associated FPDMM is hindered by phenotypic heterogeneity including normal counts and variable bleeding. The condition is regarded as autosomal dominant, but with incomplete penetrance; delays between diagnosis and leukaemia can exceed 50 years. The spectrum of associated cancers extends beyond MDS and acute myeloid leukaemia to lymphoid malignancies.

Aims: Our laboratory genotypes of suspected FPD cases as a human ethics approved research project.

Methods: We have recently identified four kindreds with RUNX1 mutations and atypical clinical features.

Results: The first index case is a 22 year old male with thrombocytopenia (platelets 126×10 9 /L), presenting with T lymphoblastic lymphoma associated with a large deletion encompassing the RUNX1 promoter. His mother had ALL aged 29 and was in remission (platelets 70); other family members developed acute leukaemia. The second case (RUNX1 c.C884G, p.S295X) is a 43 year old woman presenting with worsening thrombocytopenia and massive splenomegaly due to chronic eosinophilic leukaemia with an acquired KIT mutation. She responded to therapy including an allogeneic SCT, but her mother died of acute myelomonocytic leukaemia in her 70s. The third family (RUNX1 c.187delG, p.V63fs) is a father (aged 44) and son with easy bruising. The father's platelet counts fell over time to 60×10 9 /L, whereas his 5 year old son has normal numbers; both their platelets are extremely small, with unrecordable MPVs. There is a distant relative with leukaemia but no recent cases of malignancy. Finally, a fourth male (37) has been identified with microthrombocytopenia associated with a reported RUNX1 pathogenic variant (c.G514A, p.G172R).

Conclusions: These cases illustrate the heterogeneity of FDPMM including multi‐lineage malignancy and progressive thrombocytopenia. Genotyping is essential for FDPMM diagnosis and monitoring strategies are needed.

PB024: Population PK/PD Modeling of Avatrombopag in Patients with Thrombocytopenia and Chronic Liver Disease ‐ Guided Phase 3 Dose Selection

M. Nomoto 1 ; Z. Hussein 1 ; K. Aggarwal 2 ; M. Vredenburg 2 ; L.F. Allen 2

1 Eisai Pharmaceuticals, Woodcliff Lake, United States, 2 Dova Pharmaceuticals, Durham, United States

Background: Avatrombopag (AVA) is a novel oral thrombopoietin receptor agonist being developed for the treatment of thrombocytopenia in patients with chronic liver disease (CLD) scheduled to undergo an invasive procedure.

Aims: Phase 2 population pharmacokinetic (PK) and PK/pharmacodynamic (PK/PD) data for avatrombopag were analyzed, and a model developed to characterize the relationship between AVA concentration and platelet count increase. This model was then used to identify Phase 3 dosing regimens that maximized the proportion of subjects who would achieve the target platelet effect, an increase to ≥50×10 9 /L, while minimizing those with platelet counts ≥200×10 9 /L.

Methods: Population PK and PK/PD data with AVA were analyzed using non‐linear mixed effects modeling (NONMEM). Two cohorts of patients were sequentially dosed: Cohort A‐ AVA was administered orally with a 100 mg loading dose followed by 6 days of 20 mg, 40 mg, or 80 mg; Cohort B‐ an 80 mg oral AVA loading dose was followed by 10 mg for 6 days or 20 mg for 3 days.

Results: PK/PD model simulations showed that the achievement of the targeted platelet count and a high response rate was importantly affected by the Baseline platelet count; therefore, dosing of avatrombopag for Phase 3 was best stratified by Baseline platelet count. The simulations also demonstrated no advantage with respect to the effect on platelet count response in regimens that included a loading dose. Based on these modeling simulations, the recommended optimal dosing regimens for Phase 3 were 60 mg once daily for 5 days for patients with Baseline platelet counts <40×10 9 /L, and 40 mg once daily for 5 days for patients with Baseline platelet counts between >40 and <50×10 9 /L.

Conclusions: Phase 2 PK/PD data with AVA contributed to development of a predictive model for the selection of Phase 3 dosing regimens, which achieved optimal efficacy while limiting the proportion of patients achieving platelet counts >200×10 9 /L and its associated risk of thromboembolic events.

PB025: Gray Platelet Syndrome Variant: Combined Alpha Granule, Dense Granule, and GPVI Deficiency

E. Salazar ; P. Monroig‐Bosque; L. Rice

Houston Methodist Hospital, Houston, United States

Background: Gray platelet syndrome (GPS) is a rare disorder characterized by platelet alpha‐granule deficiency. Thrombocytopenia, pale platelets on peripheral smear, progressive myelofibrosis, and variable bleeding tendency may be present. NBEAL2 mutations are causative.

Aims: We present a case of GPS variant, characterized by predominant alpha granule deficiency, but with moderately decreased dense granules and GPVI deficiency.

Methods: An extensive diagnostic workup for platelet function defects was performed.

Results: A 37 year old Mexican male presented to Houston Methodist Hospital with a history of progressive easy bruising and a previous episode of profuse hematuria and blood in stool. A prior appendectomy was complicated by major hematoma. Family history, including 2 daughters and 1 son, was unremarkable.

Platelet aggregation studies showed decreased aggregation in response ADP (14%), arachidonic acid (0%), collagen (0% and 5% for low‐ and high‐dose collagen, respectively), low‐normal aggregation in response to epinephrine (15%), and normal aggregation in response to low‐ and high‐dose ristocetin (1% and 78%, respectively). Lumiaggregometry assessing ATP release revealed similar results. Platelet function analysis‐100 was prolonged in both the ADP and epinephrine cartridges (135 seconds and >300 seconds, respectively). Thromboelastograph showed a low‐normal maximum amplitude of 53.9 millimeters. Peripheral smear showed thrombocytopenia (90,000/microliter) with mostly pale platelets, although some platelets were granular.

Transmission electron microscopy showed markedly decreased alpha granules and moderately decreased dense granules. Flow cytometry identified markedly decreased surface GPVI receptor (17%), and normal levels of GPIIb, GPIIIa, GPIX, GPIb‐alpha, and GPIa.

Conclusions: Various platelet defects may be identified along with decreased alpha granules in patients with pale platelets on peripheral smear. Detailed molecular studies are required for definitive characterization and diagnosis.

PB026: Bloody Diarrhea with Thrombocytopenia in Male Children: Think of Wiskott Aldrich Syndrome

D. Suri 1 ; A. Kumar Jindal 1 ; Rashmi 1 ; J. Kumar Shandilya 1 ; P. Vignesh 1 ; J. Ahluwalia 2 ; A. Gupta 1 ; A. Rawat 1 ; S. Singh 1

1 PGIMER, Department of Pediatrics, APC, Chandigarh, India, 2 PGIMER, Department of Hemato Pathology, Chandigarh, India

Background: Wiskott‐Aldrich syndrome (WAS) is a rare X‐linked primary immunodeficiency disorder caused by mutation in WAS gene . Clinical manifestations vary from mild, isolated micro thrombocytopenia as in X‐linked thrombocytopenia to full‐blown presentation that can be complicated by life‐threatening hemorrhages, immunodeficiency, atopy, autoimmunity, and cancer. Early recognition and institution of hematopoietic stem cell transplant can improve survival.

Aims: To describe clinical presentation in children with XLT/WAS.

Methods: A review of case records of all children diagnosed as XLT/WAS and registered in Pediatric Immune deficiency clinic from the year 2007 to 2017 was carried out.

Results: A total of 42 patients with confirmed WAS gene mutations were analyzed. Incidence of bleeding manifestation before diagnosis was seen in 90% of patients and the most common presenting symptom was blood stained stools in 60% patients followed by skin bleeds in 35% children. Three patients were initially diagnosed as cow's milk protein intolerance, 2 children as dysentery and 3 children as disseminated cytomegalovirus infection. Evidence of infections over follow up were found in 31 children while eczema in 36 children. Microthrombocytopenia was an important clinical clue to the diagnosis and ascertaining mean platelet volumes helped clinch the diagnosis. Reduced expression of intracytoplasmic Wiskott Aldrich Syndrome Protein was seen in only 57% children.

Conclusions: We found bleeding from the GI tract in the form of bloody diarrhea as the most consistent symptoms and were seen in 60% of the children. This is also probably the earliest symptom which should raise the suspicion of this disorder among the treating physicians.

PB027: Pseudothrombocytopenia: Beyond Platelets Aggregates

A. Carmo 1 ; N. Costa e Silva 1 ; M. Gomes 2

1 Centro Hospitalar e Universitário de Coimbra, Clinical Pathology Department, Coimbra, Portugal, 2 Centro Hospitalar e Universitário de Coimbra, Clinical Hematology Department, Coimbra, Portugal

Background: Platelet satellitism (PS) is the in vitro adherence of platelets to leucocytes. It is observed on blood smears prepared from blood collected in EDTA and usually it is absent with citrate or heparin. Two hypothesis were posed to explain the mechanism: one states the existence of an immunologic process and the second poses a non‐immunologic adherence mediated by thrombospondin.

Aims: Present a case‐report of PS in a patient diagnosed with chronic secondary immune thrombocytopenia (ITP).

Methods: A 59‐year‐old male patient was diagnosed in 2010 with ITP. The patient was previously diagnosed in 2004 with B‐cell non‐Hodgkin lymphoma and was in complete remission after chemotherapy and splenectomy since 2008.

Results: The complete blood count showed no alterations other than thrombocytopenia (56 G/L). Due to clinical history a blood smear was analyzed. The blood smear obtained from EDTA anticoagulated blood revealed the presence of 4‐9 platelets attached circumferentially to most neutrophils. No platelet aggregation around other cells or isolated was seen. The estimated counting of platelets in the blood smear was 110 G/L. The observations were consistent through the time. Several laboratory tests were performed to study the PS. Antiplatelets antibodies and cryoglobulins screening was negative. Sedimentation rate was persistently below 5 mm/hr. Antinuclear antibody investigation and direct and indirect Coombs tests were negative. Serum protein electrophoresis and immunofixation show no alterations. Serology for herpes simplex, Epstein‐Barr, hepatitis and human immunodeficiency virus, cytomegalovirus, toxoplasma, and Treponema pallidum was negative.

Conclusions: PS constitutes a cause of pseudothrombocytopenia and if not identified can lead to inappropriate treatment. Contrary to the literature, PS was maintained over the years. The identification of PS was only possible due to blood smear examination since no flag other than the associated thrombocytopenia was displayed by the hematology analyzer.

PB028: Coagulation Abnormalities in Dengue and Dengue Hemorrhagic Fever Patients in Pakistan

J. Hassan ; M. Borhany; M. Abid; N. Fatima; T. Shamsi

National Institute of Blood Disease & Bone Marrow Transplantation, Karachi, Pakistan

Background: Dengue fever is endemic in Pakistan with seasonal rise in cases. Morbidities and mortalities are proportionately reported to be increasing in number and associated with disseminated intravascular coagulation resulting in hemorrhagic or thrombotic manifestations in patients having deranged coagulation profiles.

Aims: The aim of this study was to assess abnormality of coagulation and anticoagulation parameters in dengue fever and its effect on the patient's outcome.

Methods: This is an observational analytical study, conducted at the National Institute of Blood disease Karachi during the year 2016‐17. All age group patients were included in this study and dengue status proven by serology. Statistical analyses were done by SPSS version 23.

Results: Total 200 patients were selected with mean age of 28.68 (±13.28) and male predominance (147/200). Mean platelet counts at baseline were 49000 and platelets were transfused to 76 (38%) patients. Bleeding was present in 44 (22%) patients with a mean platelet count of 34000 (P‐value 0.005). Comparison of CBC parameters at Baseline and Follow up is shown in Table 1. D‐dimers showed significant association (P‐value 0.005) in coagulation group where as Protein S showed significant association (P‐value 0.03) in anticoagulant group respectively, when categorized the patients in platelet counts <50 and >50 (Table 2).

TABLE 1 Comparison of CBC parameters at Baseline and Follow up Parameters Baseline Mean and Standard Deviation Follow up Mean and Standard Deviation P‐Value Hemoglobin 14.14±2.37 13.49±1.81 0.000 Total Leucocyte Count 4.83±2.73 6.70±7.84 0.001 Platelet 48.65±40.30 88.8±69.26 0.000 Neutrophils 40.82±19.61 35.74±16.21 0.002 Lymphocytes 40.81±18.83 43.77±17.64 0.046 Hematocrit 42.64±6.19 40.57±2.94 0.000 TABLE 2 Mean values of coagulant and anticoagulant parameters in patients presented with <50000 and >50000 Platelet count Parameters Platelet count <50000 Mean±SD Platelet count >50000 Mean±SD Prothrombin time 14.62±0.379 14.55±0.354 Activated partial thromboplastin time 40.14±0.834 39.70±1.235 Fibrinogen 2.461±0.619 2.629±0.118 D‐Dimer 2.185±0.141 1.92 1±0.251 Fibrin monomers 17.25±2.226 14.83±2.859 Antithrombin III 90.84±2.210 88.91±3.131 Protein C 68.90±1.718 71.47±2.745 Protein S 55.37±2.244 60.33±3.431

Conclusions: A significant association was found with coagulation and anticoagulation parameters in patients presented with bleeding. To our knowledge, this is the first study incorporating a spectrum of coagulation and anticoagulation parameters in dengue fever in Pakistan.

PB030: Acquired Platelet Function Defect and Moderate Cutaneous Bleeding in a Patient on Treatment with a Leukotriene Receptor Antagonist for Asthma

M. Marchetti ; T. Lerede; M. Testa; S. Brevi; S. Gamba; A. Falanga

Papa Giovanni XXIII Hospital, Immunohematology and Transfusion Medicine, Bergamo, Italy

Background: Acquired defects of platelet function are rare hemorrhagic diseases. We described here the case of a 33‐year‐old woman with a history of allergic asthma referred to our ambulatory on October 2017 for the persistent (about 2 years) and diffused painless ecchymotic lesions on legs and arms. No anti‐platelet therapies was taking and she denied any trauma. Family history for coagulopathies was negative. Blood film, platelet count and coagulation values were normal.

Aims: To highlight this rare drug‐induced bleeding disorder.

Methods: Platelet aggregation was studied in platelet rich plasma (PRP) by light transmission aggregometry (LTA) using ADP, collagen, adrenaline, arachidonic acid (AA) and ristocetin. The PFA‐100 was used with collagen/ADP and collagen/adrenaline cartridges. Platelet membrane glycoproteins (GPIb, GPIIb/IIIa, GPIIIa), and P‐selectin were measured by flow cytofluorimetry.

Results: Prolonged closure time by PFA‐100 ® was found with epinephrine cartridge only. VWF was normal. Platelet GP and P‐selectin expression were normal. Platelet agglutination by ristocetin was normal, while platelet aggregation was impaired with collagen (reduced, irreversible), adrenaline (reduced, irreversible), ADP (reduced, reversible), and absent with AA (no ATP release). This type of response was suggestive for “Aspirin‐like defect”, an inherited dysfunction of AA metabolism. However, the patient was on treatment with montelukast, a leukotriene receptor antagonist, for her asthma. In the literature, we found a report of a woman who during assumption of this drug showed similar signs of bleeding that resolved after montelukast discontinuation. In order to exclude in our case a similar drug side‐effect, the treatment was withdrawal. After 10 days, the bruising resolved and platelet function by PFA 100 and LTA provided normal results. At today she had no relapsed.

Conclusions: The clinicians should be aware about this very rare side‐effect of montelukast, taking also into account the high frequency of its prescription.

PB031: Pelvic Hematoma Secondary to Glanzmann's Thrombasthenia: A Case Report

S.L. Hon 1 ; J. Sathar 1 ; V. Selvaratham 1 ; F. Leow 1 ; M. Ganesalingam! 2

1 Hospital Ampang, Hematology Unit, Selangor, Malaysia, 2 Hospital Ampang, Obstetrics and Gynaecology Unit, Selangor, Malaysia

Background: Glanzmann's thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by defect in platelet integrin αIIbß3 (previously known as GPIIb/IIIa). It is a rare genetic platelet disorder characterised by deficient or dysfunctional αIIbß3, The clinical presentation is usually recurrent and spontaneous mucocutaneous bleeding, which can range from mild to life‐threatening haemorrhage independent of the type of defects.

Aims: This case illustrates a rare disease which can present with significant bleeding tendencies.

Methods: We described a case of GT presenting with concealed pelvic hematoma resulting in obstructive uropathy and significant blood loss which required blood transfusions and surgical intervention.

Results: A multidisciplinary meeting involving gynaecologist, anaesthesiologist was arranged. She had undergone surgical intervention without any complications after a careful optimisation of her hemoglobin level with parenteral iron and erythropoietin, and definite treatment with Recombinant factor VIIa and antifibrinolytic agent peri‐ and post‐operatively. She remained transfusion free throughout her admission.

Conclusions: With the appropriate measures, we have reduced the possible post‐surgical complications and avoid unnecessary blood transfusion. The use of continuous combined hormonal therapy ensures anovulation preventing further bleeding into the pelvis.

PB032: Health Related Quality of Life Assessment in Patients with Glanzmann Thrombasthenia from Selected Regions of Pakistan

M.Y.J. Siddiqi 1 ; A. Naz 1 ; A. Najmuddin 2 ; A. Imran 3 ; T.S. Shamsi 1

1 National Institute of Blood Disease & Bone Marrow Transplantation, Coagulation and Hemostasis, Karachi, Pakistan, 2 Fatimid Foundation, Coagulation and Hemostasis, Karachi, Pakistan, 3 Chughtai Lab Lahore, Coagulation and Hemostasis, Lahore, Pakistan

Background: Glanzmann thrombasthenia (GT) is one of the commonest disorders among inherited platelet functional defects. It is a familial disorder being autosomal recessive in character. The defect is caused by mutations in the genes encoding ITGA2B or ITGB3 located on chromosome 17, resulting in qualitative or quantitative abnormalities of the expression of αIIb‐β3 integrin, a fibrinogen receptor positioned on platelets. It is characterized by a bleeding diathesis which is variable. Incidence is increased in those territories where consanguineous marriages are practiced. Quality of life assessment in these patients is seldom evaluated which is very important for their well‐being and existence.

Aims: The aim of this study was to assess the health related quality of life (HRQoL) found in GT patients; correlate with their phenotype and to explain clinical and psychosocial factors that affect their daily activities and medical care.

Methods: 20 patients with GT were selected for the study after obtaining informed and written consent. History was recorded in a structured data sheet and bleeding score was assessed in a questionnaire based on MCMDM‐1VWD Bleeding Questionnaire. HRQoL assessment questionnaire (Likert scale type) was based on different aspects of their well‐being and functioning, their social status, as well as their interpretations on medical care. Flowcytometry was performed on BD FACSCALIBUR (using CD 41, 61, 42a, 42b antibodies BD Biosciences) to classify the GT patients.

Results: GT type I had the highest average bleeding score and scored the highest points on the severity assessment for poor healthcare, social deprivation, lack of education and self‐esteem followed by GT type II. GT type III scored least points.

Conclusions: Patient with GT type I were the worst affected needing revision in medical care and improving social support and right to education.

PB033: Platelet's Disorder in a Case of Chronic Lymphocytic Leukemia

C. Lodigiani ; I. Quaglia; P. Ferrazzi; L. Librè; M. Bacci

Humanitas Research Hospital, Thrombosis and Haemorrhagic Diseases Center, Rozzano, Italy

Background: Bleeding complications are frequent in patients with Chronic Lymphocytic Leukemia (CLL) and tend to be related to thrombocytopenia or an acquired clotting factor inhibitor.

Aims: A 65‐year‐old white man affected by CLL was admitted at our Hospital for recurrent intracranial hemorrhages (ICH). He was investigated for hemorrhagic diathesis and was diagnosed δ‐storage pool disease. He started therapy with bendamustin 90 mg/m 2 and rituximab 375 mg/m 2 /28 days and he was also treated with antifibrinolytic tranexamic acid (1 g/daily) to prevent bleedings. At the end of the therapy the patient was revaluated.

Methods: We first screened all blood clotting assays; secondly we studied platelet's function including aggregation to different agonists, surface antigens in resting and activated platelets and Delta‐granules contents by flow cytometry. Mixing assays were also performed adding plasma and serum of the patient to platelet's rich plasma of an healthy control.

Results: Before starting therapy, all coagulation assays were normal; platelet aggregation to ADP 10 μM, TRAP 10 μM and epinephrine 10 μM were reduced (56%, 15%, 23% vs. 70%, respectively), to collagen showed mild decrease, to ristocetin and arachidonic acid showed normal function before and during the therapy. Surface GpIbα, GpIIb and α‐granules contents were normal, we observed a decrease in δ‐granules contents (45% vs. 70%) suggesting a diagnosis of δ‐storage pool. Mixing tests seem to exclude an autoimmune mechanism. The assays performed at the end of the treatment, documented an improvement of platelets response to ADP, TRAP, ephinephrine and δ granule's contents (60%).

Conclusions: Our analysis confirmed the clinical suspicion of a platelets disorder, as δ‐storage pool, related to CLL in a patient with ICH. We documented an improvement in platelet function associated to an apparently clinical resolution of bleeding diathesis, subsequent to the treatment of the hematological malignancy. We would like to confirm these data after a longer follow‐up.

PB034: Platelet CXCL14 Regulates Thrombotic Function and Influences Clinical Outcome in Coronary Artery Disease

A. Witte ; D. Rath; M. Gawaz

Universitätsklinik Tübingen, Cardiology, Tübingen, Germany

Background: Platelets are the source of a variety of chemokines with immune‐modulatory mediators. Recently, we could show that platelets are a source of CXCL14 and release it upon activation. Platelet derived CXCL14 induces monocyte migration and counteracts the angiogenic effect of VEGF on HUVECs, which demonstrates its pro‐inflammatory activity.

Aims: The current study investigated the thrombotic platelet functions in global CXCL14 deficient mice. Furthermore, the surface expression of CXCL14 on platelets was analyzed for prognostic impact in coronary artery disease patients.

Methods: Flow cytometry, immunoblot, luminescence aggregometry and ex vivo flow chamber assay were performed.

Results: In luminescence aggregometry experiments, platelets of CXCL14 deficient mice showed decreased ATP release after activation with thrombin, which indicates an impaired activation potential and an impaired release of dense granule content. In ex vivo flow chamber assay, CXCL14 deficient mice showed decreased thrombus formation on collagen coated surface under a high shear rate which could be compensated with supplementation of the blood with recombinant CXCL14. These results indicate that CXCL14 might have a regulatory impact on thrombotic functions. A combined endpoint study of a patient cohort of 80 CAD patients showed a better survival when assessed for major adverse cardiac events (MACE; all‐cause death, myocardial infarction, ischemic stroke) in patients with higher platelet CXCL14 surface expression on platelets than median, compared to the patient subgroup with a lower CXCL14 surface expression level on platelets.

Conclusions: Current results show that CXCL14 might be involved in regulating platelet function. An increased surface expression of CXCL14 on platelets in CAD patients is associated with an adverse clinical outcome. Given its previously described pro‐inflammatory effects, CXCL14 may be an interesting target for new therapeutic agents.

PB035: Dichotomous Cytosolic Mobilization of Calcium, Sodium and Potassium Ions during Procoagulant COAT Platelet Formation

A. Aliotta ; D. Bertaggia Calderara; L. Alberio

Division of Hematology and Central Hematology Laboratory, CHUV, University Hospital and University of Lausanne, Lausanne, Switzerland

Background: The combined activation of platelets (PLTs) with collagen and thrombin induces the formation of procoagulant COAT PLTs, which retain a coat of prohemostatic α‐granule proteins on their surface. While the clinical relevance of COAT PLTs is increasingly recognized, dichotomous intracellular signaling pathways generating a subpopulation displaying procoagulant activity instead of aggregation endpoints are still not fully elucidated.

Aims: As cytosolic ion fluxes play an important role in PLT activation, here we propose a continuous flow cytometry kinetic monitoring of calcium, sodium and potassium fluxes during generation of procoagulant COAT PLTs.

Methods: PLTs were preloaded with different ion fluorescent indicators: Fluo‐3 AM for calcium, Asante NaTRIUM Green‐2 AM for sodium and Asante Potassium Green‐2 AM for potassium. Annexin‐V co‐staining allowed to identify COAT PLT subpopulation. PLTs were simultaneously activated with thrombin and convulxin (collagen receptor GPVI agonist) and each ion indicator fluorescence was continuously acquired over time on an Accuri C6 flow cytometer.

Results: Annexin‐V co‐staining revealed a dichotomous cytosolic ion mobilization. Procoagulant COAT PLTs demonstrated high and sustained cytosolic calcium concentrations (up to micromolar range), decreased sodium concentrations after a transient increase, and important potassium efflux. In non‐COAT PLTs, calcium levels increased transiently in the nanomolar range and afterwards declined over time, sodium levels increased and stayed stable, and potassium efflux was lower compared to COAT PLTs.

Conclusions: We demonstrated a characteristic dichotomous mobilization patterns following PLT activation with thrombin and convulxin. This work reveals peculiar ion flux kinetics during procoagulant response, diverging from an initial common aggregating response. This method allows to investigate the differential modulation of biological messengers leading to procoagulant response.

PB036: Emerging Value of Platelet Function Testing at Predicting the Risk of Recurrent Vascular Events and Outcomes after TIA/Ischemic Stroke: A Systematic Review of the Literature

S.T. Lim 1 ; S. Murphy 1 ; I. Fernandez‐Cadenas 2 ; J. Montaner 3 ; V. Thijs 4 ; L. Marquardt 5 ; P. Kelly 6 ; P. Bath 7 ; G. Ford 8 ; B. Norrving 9 ; D. Cox 10 ; D. McCabe 11 ; OATS‐I

1 Trinity College Dublin, Dublin, Ireland, 2 Institut de Recerca Vall d'Hebron, Universitat Autònoma de Barcelona, Department of Neurology, Barcelona, Spain, 3 Neurovascular Research Lab, Vall d'Hebron Research Institute, Hospital Vall d'Hebron, Barcelona, Spain, 4 Austin Health and Florey Institute of Neuroscience and Mental Health, Department of Neurosciences, Victoria, Australia, 5 Asklepios Klinik Wandsbek, Department of Neurology, Hamburg, Germany, 6 Mater University Hospital, Neurovascular Clinical Science Unit, Dublin, Ireland, 7 University of Nottingham, Stroke Trials Unit, Nottingham, United Kingdom, 8 Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom, 9 Lund University, Faculty of Medicine, Lund, Sweden, 10 Royal College of Surgeons in Ireland, Department of Molecular and Cellular Therapeutics, Dublin, Ireland, 11 Trinity College Dublin, Academic Unit of Neurology, Dublin, Ireland

Background: The value of testing for ‘high on‐treatment platelet reactivity’ (HTPR) to predict outcomes in TIA/ischemic stroke patients on antiplatelet therapy is unclear.

Aims: The role of ex‐vivo platelet function/reactivity testing at predicting

the risk of recurrent vascular events and neurological outcomes following TIA or stroke.

Methods: A systematic literature review was performed to collate relevant data on the relationship between ex‐vivo HTPR testing and recurrent vascular events or outcomes in ischemic cerebrovascular disease (CVD) patients on antiplatelet therapy. We focused on data from commonly‐available whole blood platelet function analysers and platelet aggregometry. P<0.05 was considered statistically significant.

Results: Sixty‐seven of 1168 articles were assessed; 23 met inclusion criteria: PFA‐100 ® (N=9); VerifyNow ® (N=8); Multiplate ® (N=1); Thrombelastograph Haemostasis Analyser ® (N=1); TOA‐PL‐100 (N=1), and Aggregometry (N=5). Five studies found a higher risk (P<0.001), two found no increase in the risk of recurrent stroke (P=1.0) between patients with vs. those without Aspirin‐HTPR, whereas seven were inconclusive. Aspirin‐HTPR was associated with more severe strokes at baseline (P=0.005; N=4), early deterioration (P=0.017; N=3), poorer outcomes (P=0.047; N=2), larger infarct size (P<0.001; N=2), new DWI lesions (P=0.04; N=1) and higher mortality (P=0.038; N=1) after stroke. There was no relationship between Dipyridamole‐ or Clopidogrel‐HTPR status and outcomes in 2 small studies (PFA‐100); Clopidogrel‐HTPR was associated with recurrent stroke in one study (VerifyNow).

Conclusions: Emerging data from small‐medium sized studies indicate that HTPR‐status may predict outcomes on antiplatelet therapy in CVD. Adequately‐sized, prospective, multicentre studies are needed to determine whether altering antiplatelet therapy in patients with HTPR on platelet function tests reduces the risk of recurrent vascular events, functional outcomes or survival following TIA/ischemic stroke.

PB037: Lysis of Blood Cells by Arachidonic Acid Cause ADP‐dependent Platelet Activation Responses in Platelet Function Tests using Whole Blood

S. Ramstrom 1,2

1 Örebro University, School of Medical Sciences, Örebro, Sweden, 2 Linköping University, Department of Clinical Chemistry and Department of Clinical and Experimental Medicine, Linköping, Sweden

Background: The use of arachidonic acid (AA) to stimulate platelets is considered the most specific approach to study aspirin treatment efficacy. However, very high concentrations of AA are used, and it has been previously reported that AA can induce cell lysis. Several clinical studies have reported decreased responses to AA in whole blood tests in the presence of clopidogrel.

Aims: To investigate whether cell lysis could lead to unspecific effects contributing to platelet activation in situations where AA is used as platelet agonist.

Methods: Blood from healthy donors was collected after informed consent in a procedure approved by the local ethics committee. Sub‐samples were collected during AA‐induced platelet activation n light transmission aggregometry (LTA) in citrated platelet‐rich plasma (PRP), and in two assays using hirudinized whole blood, multiple electrode aggregometry (MEA, Multiplate ® ) and flow cytometry. The appearance of platelet and red blood cell fragments as well as activated platelets was detected using flow cytometry.

Results: We found that cell lysis, especially of red blood cells, does occur at the concentrations of AA used in the clinically used tests (0.5‐1 mM) and that this induces ADP‐dependent aggregation responses. In flow cytometry, very limited platelet activation was detected before reaching AA concentrations where cell lysis also occurred, making it problematic to develop a reliable flow cytometry test using AA as platelet agonist.

Conclusions: We conclude that cell lysis and ADP release contribute to AA‐induced platelet responses, most markedly in whole blood assays. This finding could potentially explain some differences between studies comparing methods using whole blood and platelet rich plasma and also how clopidogrel treatment could influence AA‐induced aggregation results in previously published studies. Our findings highlight some issues with AA as reagent for platelet activation, which also have an impact on how platelet activation assays using AA should be interpreted.

PB038: Assessment of Platelet Count Standardization in Platelet‐rich Plasma for Platelet Function Testing by Light Transmission Aggregometry

M. Dixon 1 ; J. Kotha 1 ; L.K. Jennings 1,2

1 CirQuest Labs, LLC, Memphis, United States, 2 University of Tennessee Health Science Center, Internal Medicine, Memphis, United States

Background: The optical density of platelet‐rich plasma (PRP) is directly proportional to platelet concentration. As density increases, platelet collisions increase resulting in a greater rate and extent of platelet aggregation. Granule secretion is based on platelet density and serves to stabilize platelet aggregates.

Aims: To evaluate if standardized PRP counts provide more consistent platelet function data when compared to non‐standardized counts and to establish parameters that ensure minimal ex vivo platelet reactivity during processing.

Methods: Healthy donor blood (WB) with varying platelet counts was obtained by routine venipuncture (n≥5). PRP was prepared by centrifugation (150× g for 15 minutes); PPP was generated from residual blood (2500× g for 15 minutes). Platelet activation was assessed by measuring nmoles ATP released. PRP platelet counts adjusted to 100‐300×10 6 /mL using autologous PPP. Agonists TRAP (5, 10, 15 μM) or ADP (2, 5, 10 and 20 μM) were used.

Results: PRP preparation resulted in a 2.2‐fold increase in platelet density (neat PRP) when compared to WB. While maximal aggregation (MA) response at higher ADP or TRAP concentrations was not affected by platelet counts (neat ‐ 250×10 6 /mL), response was highly dependent upon count at lower doses. Increased response variability was noted at ≤200×10 6 /mL. Granule secretion was significantly dependent upon platelet count even when there was no difference in MA response. Marked differences were observed at platelet counts <250×10 6 /mL. Granule secretion was not detected due to sample processing. TRAP data are in Tables 1A and B.

TABLE 1A MA and Secretion is Dependent upon Platelet Count and Agonist Strength TRAP (μM) Neat PRP Neat PRP 250×10 6 /mL PRP 250×10 6 /mL PRP P value P value % MA nmoles ATP % MA nmoles ATP % MA nmoles ATP 15 90±4 3.53±0.80 87±2 1.64±0.43 0.278 0.003 10 86±4 3.46±0.89 81±5 1.32±0.38 0.134 0.002 5 84±6 3.08±0.89 30±31 0.34±0.67 0.009 0.001 TABLE 1B MA and Secretion is Dependent upon Platelet Count and Agonist Strength TRAP μM Neat PRP Neat PRP 150×10 6 /mL PRP 150×10 6 /mL PRP P value P value % MA nmoles ATP % MA nmoles ATP % MA nmoles ATP 15 90±4 3.53±0.80 76±9 0.66±0.31 0.025 <0.001 10 86±4 3.46±0.89 48±22 0.31±0.34 0.010 <0.001 5 84±6 3.08±0.89 8±6 <0.01 <0.001 <0.001

Conclusions: This study showed that PRP platelet counts without adjustment were ≥2X concentrated than WB and that count is critical for evaluation of aggregation response and secretion. Platelet count adjustment played a greater role with lower dose agonists. Thus, when evaluating platelet function, it is recommended that platelet counts are standardized, especially if platelet reactivity may be influenced by granule secretion.

PB039: Kinetic Application of Flow Cytometry for Patients with Unknown Bleeding Disorders: The Underestimated Value of Mepacrine

G. Manukjan 1 ; O. Andres 2 ; H. Schulze 1

1 Institute of Experimental Biomedicine, University Hospital of Würzburg, Würzburg, Germany, 2 University Children's Hospital, University of Würzburg, Würzburg, Germany

Background: Many patients with bleeding diathesis do not obtain a diagnosis even after expanded testing. After a coagulation disorder is excluded, platelet defects are estimated mostly by aggregometry or luminometry or fast and easy‐to‐use, but also limited point‐of‐care machines such as VerifyNow or PFA‐100. Flow cytometry has a strong operating power allowing function testing of resting and activated platelets at single cell level with only minute amounts of blood. With this approach, platelet degranulation could easily be quantified. While for alpha granules a good marker (P‐selectin; CD62P) is established, the release of dense granules and their content can only be judged indirectly (i.e. by CD63) or by mepacrine.

Aims: We developed and evaluated a kinetic mepacrine assay, with which we measure its uptake into dense granules and release in response to agonists over time. In contrast to endpoint measurements we intended to obtain refined insight into the loading capacity of platelet dense granules and the feasibility and dynamics of degranulation.

Methods: Platelets were loaded with 5 μM mepacrine in whole blood and CD41‐positive events were measured by flow cytometry. Upon platelet activation using 10 μM TRAP‐6, mepacrine release was recorded over five minutes. The difference in MFI values indicated storage and release capacities.

Results: We evaluated our kinetic approach on a cohort of 50 childhood storage pool disease (SPD) patients and could corroborate its specificity. We were able to subgroup SPD patients by either storage (uptake), release or combined defects, opening more precise therapeutic opportunities. Unexpectedly, we also detected dense granule uptake defects in patients with GFI1B‐implicated Gray‐platelet like disease (BDPLT17) or Wiskott‐Aldrich Syndrome (WAS), allowing to connect platelet dense granule deficiencies with unrelated disease entities.

Conclusions: We established an easy, quick and inexpensive assay as an additional diagnostic module in flow cytometry‐based platelet function tests.

PB040: The Gp1ba‐Cre Transgenic Mouse: A New Model to Delineate Platelet and Leukocyte Functions

Z. Nagy 1 ; T. Vögtle 1 ; M.J. Geer 1 ; S. Heising 1 ; G. Di Nunzio 1 ; J. Mori 1 ; R. Gareus 2 ; G. Desanti 3 ; A. Mazharian 1 ; Y.A. Senis 1

1 University of Birmingham, Institute of Cardiovascular Sciences, Birmingham, United Kingdom, 2 The Jackson Laboratory, Bar Harbor, United States, 3 University of Birmingham, Institute of Microbiology and Infection, Birmingham, United Kingdom

Background: The Pf4‐Cre transgenic mouse is widely used to generate megakaryocyte/platelet (MK/plt)‐specific knockout (KO) mouse models. However, several studies have challenged the specificity of this transgene, and raised the possibility that some of the phenotypes associated with this model may be due to non‐specific deletion of floxed genes.

Aims: In this study, we established the Gp1ba‐Cre transgenic mouse to produce MK/plt‐specific KO mice and directly compared it to the Pf4‐Cre strain in terms of specificity and efficiency.

Methods: Expression pattern of the transgenes in vivo was investigated by crossing deleter mice with mT/mG double‐fluorescent Cre reporter mice and quantifying GFP + haematopoietic cells by flow cytometry. Protein ablation efficiency in platelets was tested by crossing Gp1ba‐ or Pf4‐Cre mice with Csk‐ , Shp1‐, or CD148‐ floxed mice and assessed by a quantitative capillary electrophoresis‐based platform.

Results: Over 98% of platelets from mT/mG ; Gp1ba‐Cre reporter mice were GFP + , demonstrating highly efficient Cre‐mediated gene excision in the MK lineage. Notably, no significant GFP expression was detected in erythrocytes or leukocytes from these mice. Protein levels of the tyrosine kinase Csk and tyrosine phosphatase Shp1 were reduced by 90‐93% using the Gp1ba‐Cre transgene, and by 96‐99% using the Pf4‐Cre transgene, respectively. Surface levels of the receptor tyrosine phosphatase CD148 were reduced similarly by 94‐95% using either strain. Direct comparison of Csk , Shp1 and CD148 conditional KO mice generated using either deleter strain revealed similar platelet phenotypes. However, additional inflammatory and immunological anomalies were observed in Pf4‐Cre ‐generated KO mice due to non‐specific deletion in other hematopoietic lineages.

Conclusions: We demonstrate that the Gp1ba‐Cre mouse is highly efficient at deleting floxed genes in the MK lineage, with no evidence of leakiness into other hematopoietic lineages, thus eliminating leukocyte contributions to phenotypes.

PB041: The Fluidity of Platelet Membrane an Important Parameter for Platelet Function in Chronic Myeloproliferative Neoplasms Patients

V.M. Popov 1,2 ; H. Bumbea 3 ; I. Dumitru 4 ; B.M. Matei 5 ; C. Oktaviani Matei 5 ; M. Omer 1 ; M. Andreescu 6 ; O. Patrinoiu 6 ; F. Mihai 6 ; T. Savopol 7 ; A.M. Vladareanu 8 ; E. Kovacs 9 ; M.G. Moisescu 10

1 Colentina Hospital Bucharest, Hematology, Bucharest, Romania, 2 University of Medicine and Pharmacy Carol Davila, Biophisics and Biotehnology, Bucharest, Romania, 3 UMF Carol Davila, Hematology, Bucharest, Romania, 4 University Emergency Hospital, Hematology, Bucharest, Romania, 5 UMF Carol Davila, Biophysics and Biotechnology, Bucharest, Romania, 6 Colentina Hospital Bucharest, Bucharest, Romania, 7 Colentina Hospital Bucharest, Biophysics and Cellular Biotechnology, Bucharest, Romania, 8 UMF Carol Davila, Hematology SUUB, Bucharest, Romania, 9 UMF Carol Davila, Biophysics and Cellular Biotechnology, Bucharest, Romania, 10 UMF Carol Davila, Biophysics and Cellular Biotechnology, Bucharest, Romania

Background: Patients with chronic myeloproliferative neoplasms (MPNs) had qualitative and quantitative modifications of platelet membrane receptors that are involved in alteration of platelet function.

Aims: The aim of our study was to determine if changes of platelet membrane anisotropy (FA) could be correlated with alterations in expression of platelet receptors and reactive species production (ROS).

Methods: This retrospective study included 100 patients with MPNs as well as 10 controls. The determination of platelet membrane fluidity was performed by fluorescence anisotropy measurements using as marker 1‐(4‐trimethylammoniumphenyl)‐6‐phenyl‐1,3,5‐hexatriene p‐toluenesulfonate (TMA DPH). Production of reactive species (ROS) was examined using fluorescence method with DCFDA and was assessed by aria under curve (AUC) in serial measurements during 900 seconds. Fluorescence anisotropy (FA) was analysed depending ROS production and the expression of platelet membrane receptors measured by flow cytometry analyses. We examined the flow cytometry markers of platelet adhesion (CD42a,CD42b), aggregation (CD41, CD61) and CD36.

Results: The expression of CD41/CD61 and CD42a/CD42b receptors was lower in MPNs group compared with controls group and no statistical difference in expression of CD 36. The fluorescence anisotropy of platelet membrane in MPNs group is higher than control group, P=0.05 The CD36 expression had a positive correlation with value of FA in MPNs patients group. (rho=0.75 ‐ 95% CI 0.308‐0.925; P=0.005). Patients with MPNs had higher level of reactive species production than controls group but was not observed any correlation with FA.

Conclusions: MPNs patients have higher FA and low expression of CD41/CD61, CD42a/CD42b receptor. The expression of CD 36 is higher in MPNs patients and was direct correlated with FA modifications. The correlation of expression of CD36 with ROS was not observed and need to be analysed in a higher lot of patient

PB042: The Effect of FXa on Platelet Activation, in vitro

S. Papadaki ; A. Tselepis

University of Ioannina, Atherothrombosis Research Centre/Laboratory of Biochemistry, Department of Chemistry, Ioannina, Greece

Background: Factor Xa (FXa) is a critical vitamin K‐dependent coagulation serine protease, which also exerts non‐haemostatic cellular effects that may contribute to the development of various pathophysiological conditions. The main mediators of the cellular responses induced by FXa are protease‐activated receptors ‐1 and ‐2 (PAR‐1 and ‐2).

Aims: The aim of the present study was to investigate the effect of FXa on platelet activation, in vitro .

Methods: Blood from healthy volunteers was collected and washed platelets were prepared and adjusted at 250,000/μL. FXa‐induced platelet aggregation (0.01‐0.1 nM) was evaluated using light transmittance aggregometry. In addition, the effect of FXa (0.05 and 0.5 nM, incubation time: 5 and 20 minutes) on the activation of the platelet‐integrin receptor αIIbβ3 (PAC‐1‐FITC) and the membrane expression of P‐selectin (anti‐CD62P‐PE), was studied using flow cytometry. The anti‐CD61‐PerCP antibody was used as a platelet marker. The activation of αΙΙbβ3 and the expression of P‐selectin was assessed as the percentage of PAC‐1/CD61 + and CD62P/CD61 + cells, respectively.

Results: FXa induced a robust platelet aggregation which reached its maximum levels (76% and 81%) after 20 minutes of incubation at concentrations 0.025 and 0.05 nM, respectively. Additionally, FXa induced activation of αIIbβ3 and membrane expression of P‐selectin, which reached the maximum after 20 minutes of incubation at a concentration of 0.5 nM (from 0.80±0.16% to 69.93±2.94% for PAC‐1/CD61 + cells, P<0.05 and from 10.94±4.47% to 76.02±2.92% for CD62P/CD61 + cells, P<0.05).

Conclusions: FXa induces a slow but strong platelet activation, thus the direct oral anticoagulants that are used in clinical practice, apart from their anticoagulant action, may also exert significant antiplatelet effects.

PB043: Functional Validation of microRNA‐126‐3p as a Platelet Reactivity Regulator using Human Progenitor Cells

A. Garcia 1 ; S. Dunoyer‐Geindre 1 ; V. Zapilko 1 ; S. Nolli 1 ; J.‐L. Reny 1,2 ; P. Fontana 1,3

1 University of Geneva, Geneva Platelet Group, Geneva, Switzerland, 2 Geneva University Hospitals, Division of General Internal Medicine, Geneva, Switzerland, 3 Geneva University Hospitals, Division of Angiology and Haemostasis, Geneva, Switzerland

Background: Platelets are an abundant source of microRNAs (miRNAs), which may play a role in the regulation of platelet function. Some miRNAs, such as miR‐126‐3p, are pointed out as potential biomarkers of platelet reactivity and recurrence of cardiovascular events. However, the biological relevance of these associations remains uncertain and functional validation of these candidate miRNAs on human‐derived cells is lacking.

Aims: To develop a flow‐based assay to monitor the functional impact of candidate miRNAs on platelet‐like structures derived from human progenitor cells.

Methods: CD34 + ‐derived megakaryocytes were transfected with miRNA or siRNA and were differentiated in platelet‐like structures. Platelet adhesion phenotype was assessed by perfusion of differentiated cells in a microfluidic system under a constant shear rate.

Results: miR‐126‐3p transfection procedure does not affect megakaryocytes differentiation and platelet‐like structures production. Overexpression of miR‐126‐3p increased platelet‐like structures adhesion compared to control (Figures 1 and 2).

FIGURE 1 Platelet‐like structures (white arrows) and CD34+‐derived megakaryocytes (black arrows) adhered on fibrinogen‐coated channel

FIGURE 2 MiR‐126‐3p transfection increased the adhesion of platelet‐like structures on fibrinogen‐coated channel

Moreover, miR‐126‐3p transfection was associated with downregulation of ADAM9, a validated target of miR‐126‐3p, and of PLXNB2, an actin dynamics regulator. Silencing PLXNB2 led to similar functional results than miR‐126‐3p transfection.

Conclusions: Taken together, using a flow‐based assay, we functionally validate miR‐126‐3p as a regulator of platelet reactivity on platelet‐like structures derived from human progenitor cells. Moreover, PLXNB2 was identified as a platelet reactivity regulator supporting the hypotheses that miR‐126‐3p modulates platelet reactivity by downregulation of PLXNB2.

PB044: Assessment of Light Transmission Aggregometry on the Routine Coagulation Analyzer Siemens CS‐2500 using CE‐marked Agonists from Hyphen Biomed

V.‐E. Brett 1 ; B. Pougault 2 ; A. Guy 1 ; S. Castet 3 ; Y. Huguenin 3 ; A. Ryman 1 ; C. James 1 ; M. Fiore 1,4

1 University Hospital of Bordeaux, Laboratory of Hematology, Pessac, France, 2 Siemens Healthcare, Saint‐Denis, France, 3 University Hospital of Bordeaux, Haemophilia Center, Bordeaux, France, 4 University Hospital of Bordeaux, Reference Center for Platelet Disorders, Bordeaux, France

Background: Light transmission aggregometry (LTA) is still considered as the “gold standard” for platelet function assessment but, as a completely manual technology, it is labour intensive. This challenge can be overcome by performing platelet aggregometry in an automated method on a routine coagulation analyzer.

Aims: We aimed to compare and correlate results obtained from a traditional manual LTA solution realized in our Reference Center with an optimized automated LTA system using CE‐marked agonist reagents.

Methods: Platelet rich plasma from patients with suspected platelet disorders, von Willebrand disease or antiplatelet therapy have been assessed using a wide range of agonist concentrations: ADP (0.5‐10 μM), collagen (2 mg/μL), ristocetin (0.625‐1.2 mg/mL) or arachidonic acid (1 mM). We used the Siemens CS‐2500 analyzer with a dedicated software and CE‐marked agonists from Hyphen Biomed. The APACT‐4004 aggregometer (Elitech, France) was used as the reference instrument with platelet agonists provided from different manufacturers. Results were expressed as Maximal Platelet Aggregation and correlation between the CS‐2500 and APACT‐4004 was analyzed using the Passing and Bablok regression test.

Results: Platelet aggregometry studies were performed in 49 patients. Maximal aggregation response with ADP (0.5‐10 μM), collagen (2 mg/μL), ristocetin (1.2 mg/mL) and arachidonic acid (1 mM) agonists showed significant correlation between the two aggregometers (P<0.001). We observed a more variable response using an intermediate dose of 2.5 μM ADP. Moreover, we also noted discrepancies with the low dose of ristocetin, showing excessive paradoxal agglutination with the CS‐2500, suggesting that a lower ristocetin dose should be used with this system.

Conclusions: These data show that CS‐2500 is comparable to a stand‐alone aggregometer. It has the advantages of a walk‐away technology and the use of CE‐marked reagents also permits the possibility of an easier certification.

PB045: Lack of Correlation between Verifynow and TEG‐platelet Mapping in Ventricular Assist Device Patients

E. Salazar

Houston Methodist Hospital, Houston, United States

Background: Ventricular assist device (VAD) patients must maintain a precarious coagulable state, in which the patient does not develop bleeding complications, and the ventricular device does not thrombose. Bleeding is a frequent cause of hospital admission in ventricular assist device patients, and pump thrombosis can have devastating consequences. Typically, patients are anticoagulated with warfarin with routine monitoring of INR. Aspirin is often used as an antiplatelet agent, although in some patients, P2Y12 inhibitors, such as clopidogrel can also be used. Several assays are available for assessment of antiplatelet effect, including verifynow, teg platelet mapping (TEG‐PM), and traditional platelet aggregation, among others.

Aims: We sought to assess the correlation between TEG‐PM and Verifynow results in ventricular assist device patients as a first step in determining which assay may be beneficial for guiding antiplatelet therapy in VAD patients.

Methods: From 12/1/2016‐12/1/2017, antiplatelet testing with TEG‐PM and Verifynow was run in parallel on all VAD patients presenting to the outpatient clinic. Aspirin effect was assessed in 151 patients, with some on repeated visits, for a total of 553 measurements. P2Y12 inhibitor effect was assessed in 38 patients, with some on repeated visits, for a total of 107 measurements. Verifynow results in platelet reaction units were plotted against TEG‐PM results in percent inhibition. Linear regression analysis was performed to determine correlation.

Results: Aspirin effect assessment showed very poor correlation between the two tests with an R 2 of 0.0856. P2Y12 inhibitor effect assessment also showed very poor correlation with an R 2 of 0.0015.

Conclusions: Antiplatelet effect measured with two common antiplatelet monitoring devices did not correlate in VAD patients.

PB047: Influence of Sample Platelet Count on Three Common Platelet Function Tests: A Comparative Study

N. Boknäs 1,2 ; A.S. Macwan 3 ; A.L. Södergren 3 ; S. Ramström 4,5

1 Linkoping University, Department of Haematology and Department of Clinical and Experimental Medicine, Linköping, Sweden, 2 Monash University, Australian Centre for Blood Diseases, Melbourne, Australia, 3 Linkoping University, Department of Clinical and Experimental Medicine, Linköping, Sweden, 4 Linkoping University, Department of Clinical Chemistry and Department of Clinical and Experimental Medicine, Linköping, Sweden, 5 Örebro University, School of Medical Sciences, Örebro, Sweden

Background: Platelet function tests (PFTs) are indispensable for diagnosing platelet function disorders (PFD). Previous studies report impaired analytical precision of PFTs when platelet concentrations fall below 100×10 9 /L, which is often the case in PFDs. As the analytical principle of flow cytometry (FC) is unrelated to platelet count, FC has been brought forward as a preferable method in this setting. However, to date no study has compared the impact of low platelet counts on the results of different PFTs side‐by‐side.

Aims: To compare the influence of sample platelet count on the results of three common PFTs; light transmission aggregometry (LTA), whole blood impedance aggregometry (IA; Multiplate) and FC, using two common platelet agonists: PAR1‐activating peptide (AP, 32 μM) and ADP (6.5 μM).

Methods: Hirudinized blood was collected from healthy volunteers after verbal consent. The procedure was approved by the local ethics committee. Blood was separated into red blood cells, platelet‐rich (PRP) and platelet‐poor plasma by centrifugation. Blood and PRP with platelet counts of 200, 100, 50 and 10×10 9 /L were created by combining the components in different proportions.

Results: For LTA, test results were reasonably stable at sample platelet counts of 100‐200×10 9 /L, but heavily affected at counts below 100×10 9 /L. For IA, a linear decline in the area under the curve (AUC) was observed for the entire test range. Minor effects were observed for FC using ADP as agonist. Using PAR1‐AP, P‐selectin expression was little affected, but a gradual decrease in fibrinogen receptor activation (PAC‐1 median fluorescence) down to 50% was seen as platelet counts decreased. This was due to the contribution of paracrine ADP stimulation, as this effect was eliminated by addition of apyrase.

Conclusions: Both LTA and IA are strongly influenced by sample platelet count. The effect on FC was less pronounced and most likely due to concentration‐dependent effects on paracrine stimulation via ADP.

PB048: Comparison of PAC‐1 and α‐fibrinogen Antibody Binding to Platelet Subpopulations

A.L. Södergren 1 ; S. Ramström 2,3

1 Linkoping University, Department of Clinical and Experimental Medicine, Linköping, Sweden, 2 Linkoping University, Department of Clinical Chemistry and Department of Clinical and Experimental Medicine, Linköping, Sweden, 3 Örebro University, School of Medical Sciences, Örebro, Sweden

Background: Fibrinogen binding potential in platelets can be evaluated in different ways. The PAC‐1 antibody recognizes the active conformation of the fibrinogen receptor, αIIbβ3, while an α‐fibrinogen antibody (αFib‐AB) can evaluate the amount of bound fibrinogen. The procoagulant platelet subpopulation, exposing phosphatidylserine (PS+), is reported to have reduced PAC‐1 binding, attributed to downregulation of αIIbβ3. Coated platelets, a type of PS+ platelets, have however been reported to have surface bound α‐granule proteins, such as fibrinogen. Despite previous reports, a clear side‐by‐side presentation of the results is lacking, which may explain some of the confusion in the field.

Aims: To compare the binding of PAC‐1 and αFib‐AB to PS+ and PS− platelets.

Methods: Platelets in citrated whole blood were obtained from healthy volunteers after verbal consent as approved by the local ethics committee. Platelets were activated in the presence of fibrin polymerization inhibitor GPRP, Annexin V (binding PS), and PAC‐1 or an αFib‐AB and evaluated with flow cytometry. Agonists were cross‐linked collagen‐related peptide (CRP‐XL) and thrombin or PAR1 and ‐4 activating peptides.

Results: PAC‐1 bound all activated PS− platelets. When PS+ platelets were formed, PAC‐1 binding became dichotomous with reduced binding to the PS+ platelets. However, PAC‐1 binding in most PS+ platelets was still higher than in resting control platelets, Figure 1. The αFib‐AB bound both PS+ and PS− activated platelets, Figure 2. Activation including thrombin further increased the αFib‐AB binding to all platelets. Samples with thrombin and αFib‐AB, were enriched in debris, possibly consisting of fibrin monomers detected by the αFib‐AB.

Conclusions: PAC‐1 binding was dichotomous with reduced binding in PS+ platelets, but it was not lacking. αFib‐AB bound both PS+ and PS− platelets and binding increased upon stimulation with thrombin. Thus a reduction in PAC‐1 binding to PS+ platelets does not necessarily mean a reduction in fibrinogen binding.

FIGURE 1 Binding of PAC‐1 to platelets with (PS+) or without (PS−) phosphatidylserine exposure after activation. Data are pooled, n=6

FIGURE 2 Binding of αFib‐AB to platelets with (PS+) or without (PS−) phosphatidylserine exposure after activation. Data are pooled, n=6

PB049: Assessment of Neonatal Platelet Granule Trafficking and Shear‐induced Platelet Activation in Neonatal Peripheral and Cord Blood

A. Ngo 1 ; J. Sheriff 2 ; K. Jones 3 ; A. Rocheleau 1 ; A. Mitrugno 1 ; J. Aslan 1 ; S. Worthington 4 ; A. Cox 4 ; M. Recht 4 ; M. Nieman 5 ; L. Malone 6 ; A. Zigomalas 2 ; W. Bahou 6 ; D. Bluestein 2 ; O. McCarty 1 ; K. Haley 4

1 Oregon Health & Science University, Biomedical Engineering, Portland, United States, 2 Stony Brook University, Biomedical Engineering, Stony Book, United States, 3 Oregon State University, Chemical Engineering, Corvallis, United States, 4 Oregon Health & Science University, The Hemophilia Center, Portland, United States, 5 Case Western Reserve University, Pharmacology, Cleveland, United States, 6 Stony Brook University, Division of Hematology, Stony Brook, United States

Background: Neonatal platelets are characterized as hyposensitive compared to adults, yet they display efficient hemostasis through unclear mechanisms. Furthermore, neonatal platelet mechanotransduction remains poorly understood.

Aims: This study aims to investigate platelet function through granule secretion, as well as to elucidate differences in shear‐specific responses between neonatal and adult platelets.

Methods: Blood from full‐term neonates was collected at 24 hours of life via heel stick. Umbilical cord blood samples were obtained immediately after clamping post‐Caesarean delivery, in accordance with Institutional Review Board‐approved protocols. Glass bottom dishes were coated with poly‐L‐lysine, seeded with agonist‐treated or vehicle whole blood, stained with a platelet dense granule marker and imaged. Platelet dense granule release was measured as light output. Flow cytometry was performed using vehicle or treated blood labeled for CD62P and PAC‐1, together with markers for total platelet glycoprotein and receptor expression. Thrombin generation was measured for constant shear stress‐exposed isolated cord platelets. Annexin V and CD62P expression was measured in shear‐exposed cord plasma and whole blood.

Results: Our results suggest that neonatal granule secretion was unresponsive to protease‐activated receptor activation but not P2Y1/P2Y12, despite neonates having significantly reduced P2Y1/P2Y12 receptor expression. Impaired dense granule secretion was rescued following the addition of exogenous ADP. Sheared cord blood platelets exhibited similar thrombin generation trends, but lower annexin V and CD62P expression compared to adult platelets.

Conclusions: Our results suggest impaired granule trafficking, mobilization, and secretion, but comparable thrombin generation, in neonates. While assessment of neonatal platelet function remains challenging, this study offers further insights and methods for investigating their response under static and shear conditions.

PB051: On the Potential Use of Morphometrics as a Biomarker for Platelet Contractility

S. Lickert 1 ; M. Kenny 2 ; V. Vogel 1 ; I. Schoen 2

1 ETH Zurich, Health Sciences and Technology, Zurich, Switzerland, 2 Royal College of Surgeons in Ireland, Molecular and Cellular Therapeutics, Dublin, Ireland

Background: Platelets roll, adhere and contract at sites of vascular injury. These distinct functions are closely linked to cellular mechanics and based on an active actomyosin machinery and adhesion receptors which together orchestrate the generation and transmission of mechanical forces. Recent work showed that platelet contractility testing could aid the diagnosis of elusive platelet‐related bleeding phenotypes [D.R. Myers et al., 2016, Nature Materials 16: 230‐35]. Our own investigation of morphological structure‐function relationships suggested that cytoskeletal textures could serve as an indirect alternative to measuring platelet contractility [S. Lickert et al., 2017, ISTH Congress, PB1283].

Aims: The aim of this study was to establish a direct link between specific platelet cytoskeletal architectures and platelet contractility.

Methods: We fabricated PDMS‐based micropost array detectors [J.L. Tan et al., 2003, PNAS 100: 1484‐9] to measure traction forces exerted by spread human platelets on fibrinogen. In parallel, we performed a morphometric analysis of the platelets’ cytoskeleton. The system was validated with platelets from healthy consenting volunteers. The work was conducted in accordance with the declaration of Helsinki.

Results: Experiments revealed a correlation between morphological changes and alterations in cellular contractility. In line with the cytoskeletal architecture, traction forces were highly sensitive to perturbations of integrin αIIbβ3 binding. A bipolar morphology was only observed on ligands binding to αIIbβ3 and were accompanied by a polarized traction profile.

Conclusions: These preliminary results indicate a tight relationship between platelet contractility and cytoskeletal morphology in human platelets. They support the potential usage of morphometrics as an easy‐to‐access diagnostic marker for certain biomechanical platelet defects. This work was funded by RCSI Dublin (I.S.) and ETH Zurich (S.L., V.V).

PB052: Impact of Cigarette Smoking on Platelet Count, Mean Platelet Volume, Platelet Aggregability and Platelet Distribution Width of Male Cigarette Smokers in Calabar, Cross River State, Nigeria

I.M. Okafor 1,2 ; I. Ngusha 1 ; U. Udo 1

1 University of Calabar, Haematology Unit, Department of Medical Laboratory Science, Calabar, Nigeria, 2 University of Calabar Teaching Hospital, Haematology, Calabar, Nigeria

Background: Cigarette smoking affects the normal hemostasis by influencing the coagulation pathways. Habitual smoking is one of the risk factors for cardiovascular disease. Smoking has also been shown to induce a state of hypercoagulability, thus influencing the normal platelet physiology and hemostasis.

Aims: This study was undertaken to assess the association of smoking duration with platelets indices.

Methods: Platelet count (PC), mean platelet volume (MPV) and platelet distribution width (PDW) were determined by automation (sysmex kx 21N) from the sample of forty two consenting male smokers and forty two non‐smokers in Calabar, Nigeria. Platelet aggregability was estimated colorimetrically by modified O'Brien's method while Relative Plasma Viscosity (RPV) was Determined by Reid and Ugwu method. Subjects were from 18 to 35 years of age. Statistics was done with student's t ‐test, ANOVA and Pearson correlation using SPSS software version 19.

Results: The platelet aggregability, MPV, PDW and RPV of smokers were found to be significantly higher than that of non‐smokers (P<0.05). Smokers with pack‐year ≤4 years had platelet parameters that were not significantly different from that of the non‐smokers (P>0.05) while smokers with smoking park‐ year >4 years had an increased PC, platelet aggregability, MPV, PDW and RPV that were significantly higher than that of the non‐smokers (P<0.05). Positive correlation was observed between PC and smoking pack‐year (r=0.9495, P<0.05), MPV and pack‐year (r=0.9475, P<0.05).

Conclusions: This study has shown that smokers tend to have increased PDW, MPV, platelet aggregability and RPV compared to non‐smokers. The findings from the present study along with evidences from literature enables us to suggest that smokers should be monitored for PC, PDW, MPV and platelet aggregability which will be helpful in evaluating the altered coagulation status and hemostasis in cigarette smokers.

TABLE 1 Comparison of platelet count, mean platelet volume and platelet distribution width of smokers and non smokers Parameters Smokers (n=42) Non‐Smokers (n=42) P‐value Remarks Platelets count (×109/l) 229.55±46.87 209.64±60.17 0.009 Not‐significant MPV (femtolitre) 10.0±0.97 9.50±0.68 0.001 Significant PDW (%) 13.20±2.17 11.93±2.04 0.001 Significant Platelet aggregation (ODD) 0.069±0.013 0.061±0.026 0.004 Significant Relative Plasma Viscosity 1.84±0.167 1.74±0.123 0.003 Significant TABLE 2 Comparison of platelet count, mean platelet volume and platelet distribution width of smokers with smoking pack ≤4 years and above Parameters ≤4.0 pack years (n=25) >4.0 pack years (n=17) Non smokers (n=42) P‐value Remarks Platelets count (×109/l) 218.52±55.84 241.18±35.78* 209.64±60.17 0.004 Significant MPV (femtolitre) 9.80±0.79 10.20±1.14* 9.50±0.68 0.0005 Significant PDW (%) 12.50±2.29 13.69±2.16* 11.93±2.04 0.0005 Significant Platelet aggregation (ODD) 0.065±0.014 0.073±0.009* 0.061±0.026 0.001 Significant Relative Plasma Viscosity 1.79±0.10 1.94±0.29* 1.74±0.123 0.004 Significant

PB053: Platelets Role in the Plasma Clotting is Not Limited to Providing a Lipid Surface

A. Balandina 1,2 ; N. Petrunina 3 ; E. Koltsova 1,2 ; F. Ataullakhanov 1,2,3

1 Center for Theoretical Problems of Physicochemical Pharmacology, Moscow, Russian Federation, 2 National Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russian Federation, 3 Moscow Institute of Physics and Technology, Dolgoprudny, Russian Federation

Background: Activated platelets accelerate blood coagulation by providing procoagulant membranes for the surface‐dependent reactions. However, the integral effect of platelets on coagulation is a subject of debate.

Aims: We investigated the role of platelets in the homogeneous and reaction‐diffusion in vitro experimental models.

Methods: Clot formation in platelet‐free plasma (PFP) supplemented with phospholipid microvesicles (PL) or platelet rich plasma (PRP) was activated with the identical amounts of tissue factor (TF) either homogeneously distributed (final concentration 5 pM, homogeneous model) or immobilized on the surface (surface density 100 pmole/m 2 , spatially heterogeneous model). Fibrin clot growth and thrombin generation were observed using videomicroscopy in plasma samples of six healthy donors.

Results: In homogeneous model in PRP (2×10 5 /μl) thrombin peak maximum was reached after 10±3 minutes with amplitude of 124±30 nM. The peak amplitude decreased simultaneously with the decrease in platelet concentration, but a second peak appeared later than the main one, disappearing in PFP. The decrease in platelet count caused the decrease in second peak amplitude and elongation of the time to reach it. Addition of PL (4μM) into PFP led to increase in the peak amplitude from 25±9 to 102±29nM (less than in PRP with 2×10 5 /μl of platelets, P<0.05) and decrease in peak time from 8±2 to 5±2 minutes. In spatially heterogeneous model in PRP (2×10 5 /μl) a propagating thrombin wave with the amplitude of 119±37 nM was formed. Its amplitude decreased with platelet count decrease. In PRP with platelet count 0.4×10 5 /μl and lower the wave disappeared and spatial thrombin distribution was similar to the exponential decay. The amplitude of thrombin wave in PFP with PL was 63±23 nM and was significantly different from PRP (P<0.05).

Conclusions: The effect of PL on thrombin generation is qualitatively different from the effect of platelets i.e. platelets role in the plasma clotting is not limited to providing a lipid surface.

PB054: Stability of Platelet Function in Healthy Individuals over a One‐year Period

D. Huskens 1,2 ; J. Konings 1,2 ; Y. Sang 1,2 ; B. de Laat 1,2 ; M. Roest 1,2 ; H. Kelchtermans 1,2

1 Maastricht University Medical Center, Synapse Research Institute, CARIM, Maastricht, the Netherlands, 2 Maastricht University Medical Center, Department of Biochemistry, CARIM, Maastricht, the Netherlands

Background: Platelet function testing is used for diagnosing bleeding disorders, monitoring anti‐platelet therapy and the prediction of thrombosis. To achieve clinical applicability, platelet function should be relatively stable within one individual.

Aims: To determine the stability of platelet function in healthy individuals over a 1‐year period.

Methods: Blood was collected from 12 healthy individuals once a month during 1 year. Flow cytometry based whole blood platelet activation testing was performed to quantify αIIbβ3 activation and P‐selectin expression in response to ADP, thrombin receptor peptide (TRAP) and collagen‐related peptide (CRP). Anti‐mouse Ig κ particles were included to standardize results. The local medical ethical board evaluated the protocol and all participants gave written informed consent.

Results: Over a period of 12 months, the intra‐individual variation in agonist‐induced αIIbβ3 activation and P‐selectin expression on platelets ranged between 11% and 21% (except for ADP induced P‐selectin expression 58%). Significant differences were demonstrated between different months (Figure 1), however, the pattern proved to be random and different for αIIbβ3 receptor activation and P‐selectin expression, and for the individual triggers TRAP, CRP or ADP.

Conclusions: Our data suggest that platelet activation within one healthy individual over a 1‐year period is sufficiently stable to allow its usage as a clinical tool. Although significant differences were observed in platelet function between different months, no clear trend was detected as variation differed per agonist and activation marker. Therefore, these differences are most likely not of physiological relevance.

FIGURE 1 Monthly variation in platelet function. Differences were analysed by Friedman test with Dunn's post‐hoc test: *P<0.05, **P<0.01, ***P<0.001

PB055: The Canine Platelet Secretome (CAPS): Proteomic Analysis of Thrombin‐stimulated Release

S.E. Cremer 1,2 ; J.L. Catalfamo 2 ; A.T. Kristensen 1 ; R.A.N. Goggs 3 ; M.B. Brooks 2

1 University of Copenhagen, Department of Veterinary Clinical Sciences, Copenhagen, Denmark, 2 Cornell University, Department of Population Medicine and Diagnostic Sciences, Ithaca, United States, 3 Cornell University, Ithaca, United States

Background: Domestic dogs share the same environment as their owners and represent a valuable animal model to study naturally‐occurring human disease. Proteomics holds promise for discovery of cancer biomarkers, however comparative platelet proteomics are lacking.

Aims: To establish a protocol for shotgun proteomic identification and quantification of proteins released from agonist‐activated canine platelets.

Methods: Washed platelets were isolated from ACD‐A anticoagulated blood from a hound mixed‐breed dog by serial centrifugation. Stirred, washed platelets (1.0×10 9 /mL) were stimulated with saline or 50 nM gamma thrombin at 37°C for 6 minutes. Protease inhibitors were added and followed by centrifugation to remove cells and debris. The supernatant was spun at 50,000× g to yield soluble and pellet (microparticle) fractions. The former was concentrated. Protein concentration was measured in both fractions. SDS‐PAGE was used to separate proteins present in soluble and pellet fractions (Figure 1). For shotgun proteomic analysis, the gel was divided into sections and in‐gel trypsin digested. Tryptic peptides were separated by nano‐LC (liquid chromatography) followed by tandem mass‐spectrometry (MS/MS). Proteins were identified with Sequest software searching a canine database. Identified proteins had minimally 1 unique peptide and were sorted based on +/‐ ≥2 peptides. A ratio of ≥2 for MS1 abundance (activated/control) was used to identify proteins released.

FIGURE 1 SDS‐PAGE for canine platelet proteomics. Gels (12%) were loaded with 5 µg reduced protein per lane and stained with Colloidal Coomassie Blue

Results: 1,126 proteins were identified (Table 1). 634 proteins (56.3%) were classified as canine platelet thrombin‐stimulated CAPS proteins. For 22.6% of identified proteins, the MS1 abundance was too low to permit accurate quantification.

TABLE 1 Proteins identified in saline (control) and thrombin‐stimulated platelets MS1 Abundance Ratio (Releasate/Control) Protein ≥2 <2 and >1 ≤1 Solely Identified Total Total number 634 (56.3 %) 155 (13.8 %) 83 (7.4 %) 254 (22.6 %) 1126 (100.0 %) Unique to soluble fraction 267 (42.1 %) 99 (63.9 %) 36 (43.4 %) 123 (48.4 %) 525 (46.6 %) Unique to pellet fraction 121 (19.1 %) 42 (27.1 %) 47 (56.5 %) 111 (43.7 %) 321 (28.5 %) In both fractions 246 (38.8 %) 14 (9.0 %) 0 (0.0 %) 20 (7.9 %) 280 (24.9 %) Identified with ≥ 2 peptides 461 (72.7 %) 118 (76.1 %) 52 (62.7 %) 42 (16.5 %) 673 (59.8 %) Unique to soluble fraction 179 (38.8 %) 82 (69.5 %) 25 (48.1 %) 20 (47.6 %) 306 (45.5 %) Unique to pellet fraction 85 (18.4 %) 25 (21.2 %) 27 (51.9 %) 19 (45.2 %) 156 (23.2 %) In both fractions 197 (42.7 %) 11 (9.3 %) 0 (0.0 %) 3 (7.1 %) 211 (31.4%)

Conclusions: A proteomics protocol was established and we defined a subset of canine platelet proteins released following in‐vitro thrombin activation that was similar to that reported for human platelets. Future studies will characterize agonist‐dependent and disease‐associated canine platelet secretomes.

PB056: Immunofluorescence Analysis on a Blood Smear – Validation of Stability of Blood Smears during Storage

K. Althaus 1,2 ; L. Lex 1 ; J. Wesche 1 ; A. Greinacher 1

1 Universitätsmedizin Greifswald, Institut für Immunologie und Transfusionsmedizin, Greifswald, Germany, 2 Universitätsklinik Tübingen, Transfusionsmedizin ZKT, Tübingen, Germany

Background: Many inherited platelet disorders are associated with distinct morphological alterations in expression or distribution of platelet receptors, granule proteins, or cytoskeletal proteins. These changes in platelet morphology can be detected by immunofluorescence microscopy using patient blood smears. However, the preanalytic requirements for immunofluorescence assessment of platelet morphology on a blood smear are not well defined.

Aims: A systematic approach to identify optimal conditions to prepare blood smears for immunofluorescence analysis of platelet morphology.

Methods: From three healthy donors anticoagulated blood (EDTA, Citrate, Hirudin) were obtained with two technical replicates per donor. Blood smears were prepared within 4 hours after drawing. Air dried blood smears were stored at room temperature and then fixed and stained on days 0, 1, 3 or 7 after preparation, using antibodies against GP IIbIIIa,GP IbIXa granula markers (P‐Selectin, vWF, Thrombospondin, CD63, Lamp1, Lamp2), and cytoskeleton structures (NMMIIa, alpha‐ and ß1 tubulin, and filamin A). Intensity and distribution of markers were evaluated by two independent investigators.

Results: Morphological markers were most stable when EDTA‐anticoagulated blood was used for preparation of blood smears. Expression of GPIIbIIIa and GPIbIX was stable during storage of air dried blood smears with a slight reduction in intensity after 3 days. Alpha and dense granule markers remained stable until day 3 with a slight reduction in staining intensity after Day 3. NMMIIA was stable until day 7. Unexpectedly, ß1‐Tubulin and filamin A were diffusely distributed in platelets when stained at day 0, but presented in a ring structure when the blood smear was stored for at least 24 hours.

Conclusions: For immunofluorescence assessment of platelet markers in patients with suspected hereditary platelets air dried blood smears from EDTA‐blood should be used, which are stable for 3‐7 days for shipment before they are fixed in a specialized laboratory.

PB057: Mean Platelet Volume Positively Correlates with Uterine Volume after Menopause, Except Myomas Presence

B. Bajteková 1,2 ; S. Wimmerová 1 ; V. Rusnáková 1,3

1 Slovak Medical University, Faculty of Public Health, Institute of Biophysics Informatics and Biostatistics, Bratislava, Slovakia, 2 GynAc, s.r.o., Clinic of Obstetrics and Gynaecology, Šaštín‐Stráže, Slovakia, 3 Trnava University, Faculty of Health Sciences and Social Work, Trnava, Slovakia

Background: The size of the uterus varies in the course of life and individual differences exist also after the cessation of ovarian activity. Leiomyoma is frequent benign uterine tumor. Mean platelet volume (MPV) has been indicated as a marker of platelet reactivity.

Aims: To explore, if there was any association of blood parameters with female menopausal uterus volume (MUV) in cohort of 178 middle‐European women; 40‐74 years old (median 58).

Methods: Retrospective analysis. Tested parameters of blood count: Hemoglobin, Erythrocyte count, Mean corpuscular volume (MCV), Platelet count, Mean platelet volume, White blood cell count (WBC), Absolute neutrophil and lymphocyte count. Also body weight (BV), body mass index (BMI), age, 25 OH D vitamin and hs‐C‐reactive protein (CRP) serum level. Nonparametric correlation (Spearman′s rho) was used for assessment, in triple way:

A: postmenopausal women without uterine leiomyoma (n=70).

B: postmenopausal women as a whole, regardless of the myoma presence (n=178).

C: postmenopausal women with ultrasound‐verified uterine myoma (n=108).

Results: A). In group with intact uterus a positive correlation between MPV and menopausal uterine volume was observed (rs 0.415 P=0.023; Figure 1).

FIGURE 1 MPV correlation with Menopausal Uterus Volume (MUV), for women without myomas

FIGURE 2 Spearman's correlation coefficient for MUV correlated to age, body weight and parameters of blood count

B). For postmenopausal women generally, MPV and MUV were only borderline associated (rs 0.184 P=0.066). MUV was positively correlated with BV (rs 0.214 P=0.004), but not BMI.

C). Myomatous postmenopausal women (n=108): Menopausal uterine volume was faintly positively associated with absolute Neutrophil count (rs 0.247 P=0.046), but negatively correlated with CRP serum level (rs −0.601 P=0.030) and 25 OH D vitamin at winter (rs −0.374 P=0.005). Myomatous women were significantly heavier than those without myomas (P=0.007). Menopausal uterine volume in all groups was negatively correlated with age (rs −0.448 P<0.001; Figure 2).

Conclusions: In our study a positive correlation between MPV and uterine volume after menopause was observed. However this did not apply to women with uterine leiomyoma.

PB058: Clinical Experience Evaluating Platelet Function by Whole Blood Impedance Lumi‐Aggregometry in a Pediatric Population

A. Obstfeld 1 ; B. Doshi 2 ; M.P. Lambert 3

1 Children's Hospital of Philadelphia, Pathology and Laboratory Medicine, Philadelphia, United States, 2 Children's Hospital of Philadelphia, Pediatrics, Philadelphia, United States, 3 Children's Hospital of Philadelphia, Pathology and Laboratory Medicine and Pediatrics, Philadelphia, United States

Background: Light Transmission Aggregometry (LTA) is used in confirming congenital platelet function defects (PFD) and most experience and literature relates to this method. Less data concerning the performance of whole blood impedance lumi‐aggregometry (WBILA) is available, especially in pediatrics where the lower blood volume requirements affords an advantage.

Aims: To summarize the performance of a WBILA system at a single tertiary care center serving pediatric patients.

Methods: A Chrono‐Log model 700 aggregometer was used. Demographics, testing indications, and results were collected over approximately 1 year. 115 cases were included. Summary statistics and associations were calculated. Results were compared to LTA when available.

Results: Ages were distributed bimodally with peaks at 33 months and 15 years with an average of 8.7 years (SD=5.5 years). The most frequent indication for testing was a history of bleeding (78%), most commonly epistaxis (29%) and easy bruising (26%). The rest had a family history of bleeding or a genetic predisposition for a PFD. Of the demographic features, only platelet count and morphologic abnormalities were associated with an abnormal WBILA result (P<0.01 and P<0.05). Secretion and aggregation responses of each agonist correlated with one another (Figure 1), however correlation of secretion and correlation of aggregation between agonists was stronger (e.g. ADP secretion correlates more strongly with secretion of other agonists than to ADP aggregation). Finally, comparison of WBILA with LTA showed that for the 7 patients with both studies, two had normal WBILA after borderline LTA results and two had congruent findings. The remaining patients had specimen problems with LTA but successfully had WBILA.

FIGURE 1 Correlations for agonist responses. Numbers and colors represent correlation coefficient

Conclusions: Our data highlight the demographic features associated with platelet abnormalities and the patterns seen in WBILA. WBILA performs acceptably in comparison to LTA in a pediatric population, but with fewer cancellations due to pre‐analytic issues.

PB059: Functional Activity of Platelets in Patients with Kasabach‐Merritt Syndrome

E. Gluhanyuk 1 ; A. Ignatova 1 ; A. Fedotov 1 ; A. Karelin 1 ; D. Nikolaeva 1 ; E. Orekhova 1 ; L. Khachatryan 1 ; M. Panteleev 1,2

1 National Medical Research Centre of Pediatric Hematology, Oncology and Immunology named after Dmitry Rogachev, Moscow, Russian Federation, 2 Lomonosov Moscow State University, Moscow, Russian Federation

Background: Kasabach‐Merritt syndrome (KMS) is a rare life‐threatening condition characterized by combination of vascular tumors and consumption thrombocytopenia and coagulopathy. We hypothesized that apart from massive platelet destruction in situ the platelet intactness in peripheral blood may be disrupted.

Aims: To evaluate the platelet functional state and its dynamics during treatment in KMS patients.

Methods: 4 KMS patients at the age of 1‐3 months were examined till the age of 8 months. Patients received metronomic chemotherapy, propranolol, heparin, prednisolone if needed. Platelets were automatically counted and their functional activity was estimated by flow cytometry with mepacrine and antibody to CD61, PAC1, CD42b and annexin V before and after activation by CLP and SFLLRN. Since there is no reference data for children of this age we proposed to consider 2 children with vascular tumor but without KMS as a control.

Results: Therapy resulted in a decrease in tumor size, consumption thrombocytopenia and coagulopathy. We revealed that difference in all parameters between activated and non‐activated platelets differed in KMS patients compared to control group demonstrating the reduced ability of platelets to be activated. Mepacrine, PAC1, CD42b and annexin V changed significantly (P≤0.05 for each parameter) after treatment, resulting in an increase in ability to be activated. To analyze correlation between platelet count (PC) and cytometric parameters observations were divided into two cohorts depending on PC: <50×10 9 /l (A) and >50×10 9 /l (B). In group A the correlations between PC and expression of CD62p (R 2 =0.83, P<0.01), CD61 (R 2 =0.64, P<0.01) after activation, and PC and degranulation degree of dense granules (R 2 =0.71, P<0.01) were revealed. In group B we found the correlation between PC and expression of CD42b both before (R 2 =0.68, P<0.05) and after (R 2 =0.88, P<0.01) activation.

Conclusions: KMS apparently induces alterations in platelet functional state, with a tendency to reduction during therapy.

PB060: Characterisation of a Haemocompatible Dual in‐line Reciprocating Micropump and Mixer for on‐chip Assessment of Experimental Anti‐platelet Agents

R. Brazilek 1 ; C. Szydzik 2 ; F. Akbaridoust 3 ; M. Knoerzer 2 ; I. Marusic 3 ; H. Nandurkar 1 ; A. Mitchell 2 ; J. Hamilton 1 ; W. Nesbitt 1,2

1 Monash University, Prahan, Australia, 2 RMIT University, Melbourne, Australia, 3 The University of Melbourne, Melbourne, Australia

Background: Microfluidic technologies offer unique possibilities for improvements in clinical platelet diagnostics. However, the use of blood with such systems presents significant challenges, particularly due to biomarker loss and biofouling due to platelet aggregation. The impact of advancements in microfluidic automation, such as micropumps and microvalves, must be carefully considered in the development of haemocompatible devices.

Aims: This study aims to explore the development and characterisation of a haemocompatible elastomeric microvalve that can act as a dual pump‐mixer for antiplatelet drugs, and to determine the design considerations for microscale active mixing in whole blood.

Methods: Through application of novel injection‐moulding fabrication methods, we fabricated v‐shaped and straight valve‐gated, pneumatically‐actuated reciprocating micropumps. Analysis of platelet‐like particle motion through Micro‐Particle Image Velocimetry and Computational Fluid Dynamic analysis was used to determine the optimal pumping geometry from a shear and platelet distribution perspective. Platelet‐sensitive biomarker loss was assessed via ELISA measurement of VWF and Fibrinogen adsorption. Biofouling due to platelet aggregation was assessed through monitoring epifluorescence when DIOC6 labelled whole blood was perfused through pump manifolds. Platelet activation was assessed through FACS analysis of PAC‐1 binding, P‐Selectin expression and Phosphatidyl Serine exposure.

Results: We demonstrate an in‐line micropump which may be used in pumping or mixing applications requiring haemocompatibility with regards to platelets. We demonstrate a valving iteration that minimises platelet reactivity, biomarker loss or biofouling, while maintaining capacity as an in‐line pump‐mixer.

Conclusions: This study describes the development and characterisation of an in‐line elastomeric microfluidic micromixer which may be utilised in applications requiring haemocompatibility, such as in high‐throughput anti‐platelet drug screening systems.

PB061: Flow Cytometric Analysis of Platelet Function in Patients on Antiplatelet Therapy and Suspected Thrombocytopathy

D. Huskens 1,2 ; M. Roest 1,2 ; L. Florin 3 ; B. de Laat 1,2 ; K. Devreese 3

1 Maastricht University Medical Center, Synapse Research Institute, CARIM, Maastricht, the Netherlands, 2 Maastricht University Medical Center, Department of Biochemistry, CARIM, Maastricht, the Netherlands, 3 Ghent University, Department of Clinical Chemistry, Microbiology and Immunology, Ghent, Belgium

Background: Light transmission aggregometry (LTA) is still the most used test for the identification and diagnosis of platelet function defects (PFD). Disadvantages of LTA include its laborious nature, the large volumes of blood required and the relative insensitivity to small changes in platelet function.

Aims: We investigated if the flow cytometry‐based whole blood platelet activation test (WB‐PACT) correlates with LTA or could be used as a complimentary test.

Methods: WB‐PACT quantifies αIIbβ3 activation and P‐selectin expression in response to 3 agonists and VWF binding to platelets in response to ristocetin. In total, 153 patients (32 on dual antiplatelet therapy (acetylsalicylic acid and ADP receptor antagonist), 121 suspected for bleeding diathesis) were tested. For WB‐PACT parameters, the 2.5th, 10th and 25th percentiles were determined in 48 healthy donors to score the patient samples (0‐2.5%: 3, 2.5‐10%: 2, 10‐25%: 1 and >25%: 0). For LTA, maximal aggregation, disaggregation and prolongation of the lagtime in response to 4 agonists and ristocetin was scored. Patients with at least one parameter lower than the 10th percentile measured with WB‐PACT in healthy donors or at least one value deviating from the normal range measured with LTA were diagnosed with PFD. A bleeding score (BS) was calculated with the ISTH/SSC bleeding assessment tool (Rodeghiero et al ., JTH, 2010).

Results: A moderate correlation between LTA versus WB‐PACT was found with a R of 0.63 (Figure 1). In total, 70 patients were diagnosed with PFD by both tests (κ=0.43, Table 1). BS were recorded for 18/21 and 14/21 patients who were diagnosed with PFD according to WB‐PACT only and LTA only, respectively. Interestingly, 6/18 patients with PFD according to WB‐PACT had a BS>3. For LTA, only 1/14 patients with abnormal platelet function had a BS>3.

FIGURE 1 Correlation of WB‐PACT with LTA for determining platelet function. The Spearman correlation coefficient and P value are depicted

Conclusions: In comparison with LTA, flow cytometric analysis of platelet function by WB‐PACT is of additional value to determine PFD.

TABLE 1 Diagnosing PFD with WB‐PACT and LTA WB‐PACT Total 0 1 LTA 0 41 21 62 1 21 70 91 Total 62 91 153

PB062: Characterization of Platelet Function with Flow Cytometry for Bleeding Risk Evaluation in Hematological Disorders

A. Ignatova 1 ; E. Suntsova 1 ; D. Polokhov 1 ; V. Ptushkin 2 ; E. Nikitin 2 ; P. Zharkov 1 ; D. Fedorova 1 ; A. Maschan 1 ; G. Novichkova 1 ; M. Panteleev 1

1 Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russian Federation, 2 Botkin City Clinical Hospital, Moscow, Russian Federation

Background: Combined decrease of platelet count and platelet function is a frequent complication in autoimmune diseases, hematological malignancies and other hematological disorders. Its relationship to bleeding is poorly characterized.

Aims: We used flow cytometry assay for the platelet functional analysis in high platelet‐related hemorrhagic risk‐associated disorders.

Methods: Platelet surface membrane antigen composition and functional response to physiological agonists were determined. Activation was with collagen‐related peptide plus SFLLRN. Levels of CD42b, CD61, CD62P, PAC1, annexin V binding, and mepacrine release were determined.

Results: Pediatric patients hemorrhagic syndrome of unclear origin and excluded coagulopathy (n=32), preterm neonates (33‐34 gestation weeks, n=10) and term neonates (>37 gestation weeks, n=10), pediatric (n=44) and adult (n=32) patients with chronic immune thrombocytopenia (ITP) and adult patients with chronic lymphocytic leukaemia (CLL, n=17) before and on ibrutinib treatment were analysed. A direct relationship between the clinical index of hemorrhage and the integrin activation degree and dense granules release (P<0.05) was revealed for pediatric patients with hemorrhagic syndrome. All platelet functions without exception (specifically, lack of all granules, of their release and integrin activation) were profoundly (by 50‐70%) impaired in newborn, with greater defects in pre‐term newborn. Patients with CLL had initially impaired response of platelets and ibrutinib additionally inhibited it, which corrrelated with bleeding risk. In chronic ITP, our data indicate that platelets are pre‐activated, large and slowly reacting; these changes are associated with bleeding independently of platelet count, and can be used for risk stratification.

Conclusions: Functional platelet flow cytometry parameters correlate with bleeding risk in hematological disorders and other acquired platelet dysfunction conditions.

PB063: Study of Leukocyte‐platelet Aggregates by Flow Cytometry in Patients with Coronary Artery Disease

A. Ermakov 1 ; O. Sirotkina 2 ; T. Vavilova 2 ; L. Gaykovaya 1

1 North‐western State Medical University Named After I.I. Mechnikov, Saint‐Petersburg, Russian Federation, 2 Almazov National Medical Research Centre, Saint‐Petersburg, Russian Federation

Background: Platelets are not only participants of hemostasis, but also directly related to immunity, the course of inflammatory reactions and the repair of damaged tissues. A characteristic reaction of platelets to inflammation is the formation of leukocyte‐platelet aggregates (LTA), which occurs as result of their activation and leads to the launch of a cascade of pro‐inflammatory reactions. The appearance of LTA can serve as a diagnostic sign of the inflammatory response of the body, in particular in acute myocardial infarction.

Aims: To study the LTA content by flow cytometry in patients who underwent acute myocardial infarction (AMI).

Methods: 66 patients (71% male, 29% female, age 59±11) with AMI who received dual antiplatelet therapy (clopidogrel 75 mg+ASA 100 mg) and 37 healthy donors (41% male, 59% female, age 48±13) as a control group were included into the study. LAT was evaluated using fluorescently labeled monoclonal antibodies CD61‐FITC, CD62P‐PE and CD45‐PC5 specific to GPIIb‐IIIa, P‐selectin of platelets and to the common leukocyte antigen, respectively, after cells activation by 10 mkM ADP. Particular events were counted on the flow cytofluorimeter CYTOMICS FC‐500 (Beckman Coulter, USA).

Results: In the group of patients with AMI a statistically significant (P<0.0001) increase in LTA in the blood was observed in comparison with the donor group ‐ 30.4% (10.9‐107.1) and 6.7% (3.9‐10.5) respectively. Also in patients with AMI, after activation of platelets 10 mkM ADP, the amount of GPIIb‐IIIa 8.8MFI (7.9‐13.2) and P‐selectin 9.7% (1.3‐15.9) was significantly lower (P<0.05) than in healthy donors ‐ 18.1MFI (11.2‐22.5) and 22.7% (6.1‐32.4), respectively, that indicated an positive antiplatelet effect in the presence of an active inflammatory process.

Conclusions: LTA analysis is an objective indicator reflecting the state of the immune system with damaged vascular bed, which can be used to assess the severity of the disease and to characterize the stabilization of the pathological process in therapy.

PB064: Evaluation of a New Range of Platelet Agonists for the Diagnosis of Inherited or Acquired Platelet Dysfunctions

M.‐C. Alessi 1 ; O. Georgelin 2 ; M. Ibrahim 2 ; S. Harvel 3 ; S. Beaufils 3 ; E. Maurer 3 ; P. Ohlmann 3

1 Faculté de médecine de la Timone, INSERM_S1062 NORT, Marseille, France, 2 Faculté de médecine de la Timone, INSERM UMR_S 1062 NORT, Marseille, France, 3 Agro‐Bio/Stago, Research & Development, La Ferté Saint‐Aubin, France

Background: Diagnosis of thrombopathies (inherited or acquired) are performed by light transmission aggregometry (LTA) using specific agonists targeting the major receptors (thrombin: PAR‐1; ADP: P2Y1 & P2Y12; epinephrine: α2A; collagen: GPVI) and arachidonic acid signaling pathways (thromboxane: TxA2).

Aims: The aim of this study was to evaluate the performance of a new range of agonists (Arachidonic Acid, ADP, Collagen, Epinephrine and TRAP‐6) by determining reference intervals in healthy donors and diagnosing of platelet dysfunction in comparison with reference agonists.

Methods: LTA (aggregometer TAV8, SD‐Medical) was performed using citrated platelet‐rich plasma (cPRP). Platelet aggregation was induced by different agonists (Agro‐Bio a Stago brand and reference CE marked agonists) at different concentrations (low and high): ADP (2 and 10 μM), Arachidonic acid (1 mM), Collagen (2 and 10 μg/mL), Epinephrine (5 and 25 μM) and TRAP‐6 (10 and 50 μM). Statistical analyzes were carried out using the MedCalc ® software.

Results: Reference intervals (min, max) were defined on 82 healthy donors (50 women, 32 men with platelet count between 152‐402 G/L and ranged from 56% to 100% for all agonists regardless of concentration, except for ADP to 2 μM. The performance of agonists was evaluated in the diagnosis of inherited thrombopathies (d‐storage pool disease; n=2, CalDAG‐GEFI and Kindlin‐3 deficiency; n=3), Glanzmann′s diseases were mimicated with anti‐GPIIb‐IIIa (Abciximab; n=5) and in the diagnosis of acquired thrombopathies (Clopidogrel; n=6, Aspirin; n=14 or dual therapies; n=18). Results using Bland & Altman and correlation tests or ROC curves showed perfect correlation between these reagents and references.

Conclusions: This new range of agonists have shown the same effectiveness as the existing references in the diagnosis of patient with acquired or inherited platelet disorders.

PB065: Usefulness of Optical Aggregometry and Flow Cytometric Studies as a Complementary Tools in Patients with Acquired Platelet Defects (APD)

M.F. Alberto 1 ; M. Asensio 1 ; E.I. Bermejo 1 ; M. Agazzoni 1 ; L. Romero 1 ; A. Sanchez‐Luceros 1,2

1 Hematological Research Institute, National Academy of Medicine, Hemostasis and Thrombosis, Caba, Argentina, 2 IMEX‐CONICET‐ANM, Hemostasis and Thrombosis, Caba, Argentina

Background: Acquired nature of bleeding disorder is suggested by a negative personal and family bleeding history. In many cases, diagnosis could be difficult to elucidate. In APD, the association of functional and non functional methods, including mixing assays, may contribute to diagnosis.

Aims: To evaluate the usefulness of optical aggregation (OA) and flow cytometry (FC) to characterize APD.

Methods: Patient 1 (P1): a 4‐year‐old boy with sudden spontaneous bruising, petechia, epistaxis and gums bleeding. P2: a 65 years old man with a recent diagnosis of multiple myeloma (monoclonal IgG 2.4 g/dL), presenting spontaneous severe bilateral epistaxis. P3: a 31‐year‐old woman on a surgery plan with precedent of PTI on childhood. Hemostatic routine tests;VWF:Ag;VWF:RCo;FVIII;FI;OA on platelet rich plasm (PRP) and mixing studies using ADP, ADR, arachidonic acid, collagen and ristocetin; expression of GPIb, GPIIb, GPIIIa, direct and indirect platelet associated immunoglobulin (PAIgG, PAIgM) and mepacrine uptake (MU) measured by FC; were performed.

Results: Results are shown on Tables 1 and 2. P1: showed Glanzmann′s thrombasthenia like pattern on OA; no inhibitory effect of patient′s poor platelet plasm (P′s PPP) upon a normal PRP and elevated levels of both PAIgG ‐PAIgM on direct assay. P2: had exclusive absence of ristocetin aggregation (up to 2 mg/mL) that not normalized when VWF was added; inhibition of normal PRP ristocetin aggregation (1.2 mg/mL) induced by P′s PPP and normal levels of PAIgG‐PAIgM. P3: showed exclusive absence of collagen aggregation; aggregation of normal PRP induced by P′s PPP (without agonist) and elevated levels of PAIgM. All of them presented normal expression of GPIb, GPIIb, GPIIIa and MU.

TABLE 1 Platelet Count (150‐450.10 9 /L) Mean Platelet Volume (7.5‐11.5 fL) Bleeding Time (Ivy 1.5‐4.5 min) Prothrombin Time (70‐120%) Activated Partial Thromboplastin Time (37‐48 seg) Fibrinogen (200‐400 mg/dL) Factor VIII (70‐120 %) VWF:Ag (50‐150 %) VWF:RCo (50‐150 %) PATIENT 1 (P1) 150 6.8 not tested 95 39 265 140 142 113 PATIENT 2 (P2) 177 8.8 4.5 65 58 (normal: 42, normal + patient: 46) 500 32 61 60 PATIENT 3 (P3) 106 9.8 >9.0 106 43 300 100 98 76

TABLE 2 Aggregation pattern Mixing Test (P´s PPP + Normal PRP) Direct platelet associated immunoglobulin Indirect platelet associated immunoglobulin Expression of GPIb, GPIIb, GPIIIa Mepacrine uptake IgG (<11.0 MFI) IgM (<6.0 MFI) IgG (<11.0 MFI) IgM (<6.0 MFI) Patient 1 (P1) Absent with: ADP, ADR, arachidonic acid, collagen. Primary aggregation with Ristocetin 1.5 mg/mL No inhibitory effect detected. 12.6 23.0 4.9 5.8 NORMAL NORMAL Patient 2 (P2) Normal with: ADP, ADR, arachidonic acid, collagen. Absent with: ristocetin up to 2 mg/mL. Do not correct with VWF addition. Inhibitory effect over ristocetin aggregation 2.1 5.9 3.3 5.9 NORMAL NORMAL Patient 3 (P3) Normal with: ADP, ADR, arachidonic acid, ristocetin. Absent with: collagen up to 2 μg/mL. Biphasic pattern (atypical)with collagen 8 μg/mL Aggregation without agonist 8.0 9.4 10 10.5 NORMAL NORMAL

Conclusions: We presented three patients with different clinical context where the combination of functional studies as OA and the evaluation of platelet glycoproteins and PA immunoglobulins by FC was the key to reach a diagnosis of APD, necessary to guide to the most appropriate treatment.

PB066: Platelet Function in Polycythemia Vera and Primary Myelofibrosis Using Multiple Electrode Aggregometry

N.S. Nytofte 1 ; O.B. Pedersen 2 ; H.C. Hasselbalch 3

1 Næstved Sygehus, Center for Thrombosis and Anticoagulation, Næstved, Denmark, 2 Næstved Sygehus, Department of Clinical Immunology, Næstved, Denmark, 3 Zealand University Hospital, Department of Hematology, Roskilde, Denmark

Background: The myeloproliferative neoplasms (MPNs) polycythemia vera (PV) and primary myelofibrosis (PMF) are clonal diseases associated with thrombosis and bleeding. Antiplatelet treatment is often used in order to prevent thrombosis. Responsiveness to antiplatelet treatment can be monitored using multiple electrode aggregometry (MEA). However, few studies have evaluated the use of MEA in PV and PMF.

Aims: The aim of this study was to identify determinants of platelet function using MEA in patients with PV and PMF.

Methods: We obtained platelet responses to arachidonic acid (ASPI test), adenosine diphosphate (ADP test) and thrombin receptor activating peptide (TRAP test) and ROTEM curves in 14 patients with PMF and 23 patients with PV. Thirty‐one patients were treated with aspirin and two patients were treated with an ADP receptor antagonist. Cytoreductive treatment but not antiplatelet treatment were paused for at least 1 week before the MEA tests.

Results: Neutrophil count, JAK V617F mutation allele burden and ROTEM parameters of clot strength but not platelet count correlated with the ASPI test. In the multiple regression analysis, the ASPI test correlated with neutrophil count, treatment with aspirin and clot strength. The ADP test correlated with platelet count, neutrophil count and ROTEM parameters of clot strength. The TRAP test correlated with the neutrophil count, the JAK2 allele burden and ROTEM parameters of clot strength. In the multiple regression analysis, only the neutrophil count and ROTEM parameters of clot strength remained statistically significant.

Conclusions: Neutrophil count, clot strength and platelet count are associated with platelet function. Clot strength predicted MEA tests possibly due to platelet activation causing both increased clot stability and adherence to the MEA electrodes. MEA should be used with caution in MPNs.

PB067: Platelet to Lymphocyte Ratio was Significantly Lower for Slovak Postmenopausal Women Born from November to April, Except those with Leiomyoma

B. Bajteková 1,2 ; S. Wimmerová 1 ; V. Rusnáková 1,3

1 Slovak Medical University, Faculty of Public Health, Institute of Biophysics Informatics and Biostatistics, Bratislava, Slovakia, 2 GynAc, s.r.o., Clinic of Obstetrics and Gynaecology, Šaštín‐Stráže, Slovakia, 3 Trnava University, Faculty of Health Sciences and Social Work, Trnava, Slovakia

Background: Several studies have confirmed that season of birth (SOB) affects the incidence of some tumors. In our previous study, the relative risk of uterine myomas was 2.2 times lower (P=0.006) for women born in the Winter SOB (November‐April) than for women with the Summer SOB (May‐October). Myoma is a benign uterine tumor with incidence up to 40%.

Aims: Test the hypothesis that distribution of blood count parameters, platelet to lymphocyte ratio (PLR), neutrophil‐to‐lymphocyte ratio (NLR), serum iron (Fe) and calcium is the same across categories “Winter‐SOB” and “Summer‐SOB”.

Methods: Retrospective analysis. Cohort of 211 Slovak postmenopausal women, born in 1941‐76, screened by vaginal ultrasonography and split into four groups according to the SOB and occurrence of uterine myoma. Tested parameters of blood count: Hemoglobin, Erythrocyte count, Mean corpuscular volume, Platelet count, Mean platelet volume (MPV), White blood cell count (WBC), Absolute neutrophil and lymphocyte count. Null hypothesis were tested by Mann‐Whitney U test, in 3 modes:

A: postmenopausal women without uterine leiomyoma (n=78), SOB Winter to SOB Summer.

B: just women with myoma (n=133), SOB Winter to SOB Summer.

C: all postmenopausal women together, SOB Winter to SOB Summer.

Results: A: Platelet to lymphocyte ratio was significantly lower for postmenopausal women born in winter season (P=0.020). However this did not apply to women with uterine myoma. Alike, serum iron was lower for postmenopausal women born in summer just in case they have no myoma (P=0.022; Figure 1).

B: Women with myomas born in the summer, had significantly higher WBC versus myomatous women with winter SOB (P=0.011; Figure 2).

C: No difference in this case.

FIGURE 1 Distribution of PLR, Fe and WBC is not the same across categories Winter/Summer SOB, moreover is influenced by the leiomyoma presence

FIGURE 2 The mean and median values of some tested parameters, depending on the presence of myoma and season of birth (SOB)

Conclusions: Distribution of PLR, Fe and WBC is not the same across categories Winter and Summer SOB, moreover is influenced by the presence of uterine leiomyoma. The incidence of myoma is high and increases with age. So in studies involving postmenopausal women it seems to be wise to consider myoma impact and SOB.

PB068: Mild Thrombocytopenia and Bleeding Diathesis in Mice Lacking the PLT/MPV‐associated F‐BAR Protein PACSIN2

N. Eaton 1 ; C. Drew 1 ; J. Wieser 1 ; W. Kahr 2 ; M. Plomann 3 ; H. Falet 1

1 Blood Research Institute, Milwaukee, United States, 2 Hospital for Sick Children, University of Toronto, Toronto, Canada, 3 University of Cologne, Cologne, Germany

Background: Exomechip analysis has associated a single nucleotide variant in the gene encoding the F‐BAR protein PACSIN2 with altered platelet count and mean platelet volume. We have previously shown that PACSIN2 associates with cell membrane GPIbα in human and mouse platelets and is concentrated in mid‐stage mouse megakaryocytes (MKs) in a well‐defined invagination of the cell membrane reminiscent of the initiating demarcation membrane system (DMS).

Aims: Here we investigated the role of PACSIN2 in platelet production and hemostatic function.

Methods: In vivo and in vitro platelet parameters were determined in Pacsin2 ‐null mice.

Results: Pacsin2 ‐null mice developed a mild thrombocytopenia with slightly enlarged and shallow platelets. Pacsin2 ‐null platelets survived normally in vivo, and expressed normal levels of major platelet membrane glycoproteins on their surface. Pacsin2 ‐null mice had a mild bleeding diathesis, as evidenced by significantly prolonged tail bleeding, and had delayed in vivo thrombus formation following FeCl3 mesenteric vessel injury. The functionality of Pacsin2 ‐null platelets in whole blood was tested in flow chamber experiments using the VenaFlux platform, where binding to a collagen‐coated surface was measured at arterial and venous shear conditions. Compared to control platelets, Pacsin2 ‐null platelets covered 25% of the collagen‐coated surface at high shear, but had normal adhesion at low shear. Pacsin2 ‐null platelets expressed P‐selectin and activated the integrin αIIbβ3 normally in response to stimulation with thrombin and the GPVI‐specific collagen‐related peptide. Analysis of bone marrow sections revealed normal MK numbers and ploidy in Pacsin2 ‐null mice. However, the DMS appeared to be less well defined in Pacsin2 ‐null MKs, where distinct platelet territories were not readily visualized.

Conclusions: The data indicate that the F‐BAR protein PACSIN2 plays a critical role in platelet hemostatic function at high shear and in platelet production.

PB069: A Novel Role of LASSBio‐788 in Inhibiting NF‐κB Mediated Signaling in Platelet of Hypercholesterolemic Rats: Would Be New Antiplatelet Agent?

N.A.V. Motta 1 ; G.F. Lima 1 ; E. Barreiro 2 ; A. Kümmerle 3 ; F.C.F. Brito 1 ; Laboratorio de Farmacologia Experimental (LAFE)

1 Universidade Federal Fluminense ‐ UFF, Niteroi, Brazil, 2 Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 3 Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro, Brazil

Background: Circulating platelets become hyperactive under hypercholesterolaemic conditions, leading to the development and progression of atherosclerosis. LASSBio‐788 is a thienylacylhydrazone derivative with anti‐atherogenic properties in an animal model of hypercholesterolemia. Although the molecular mechanism underlying its antiplatelet effect remain unclear.

Aims: The aim of this study was to investigate the signaling pathway involved in the antiplatelet effects of LASSBio‐788.

Methods: Adult male Wistar rats (150‐200 g) were randomly divided into four groups: control group (C) and positive control (C+788) fed standard chow diet, hypercholesterolemic diet group (HC) and diet group + compound LASSBio‐788 (HC+788) fed a hypercholesterolemic diet. At 31° diet day, was performed the chronic treatment with LASSBio‐788 once daily, totalizing 15 days. The animals were euthanized by cervical dislocation under anesthesia. Blood samples were collected and the platelets were isolated to biochemical and molecular analysis. Data were analyzed using one‐way ANOVA, P<0.05 (CEUA/UFF 856/16).

Results: The hypercholesterolemic diet increased serum lipids levels in the HC group. LASSBio‐788 reduced serum lipids. Furthermore, the hypercholesterolemic diet increased TXA2 and a decrease of cAMP and cGMP. LASSBio‐788 inhibited these effects. NF‐kB signaling events, including PKC‐α, iNOS, P‐selectin and CD40L increased protein expression, IκB‐α degradation, eNOS, PKA and PKG decreased protein expression were observed in hypercholesterolemic rats. These signaling events were attenuated by chronic treatment with LASSBio‐788.

Conclusions: Our study demonstrate for the first time that LASSBio‐788 possesses potent antiplatelet activity, which may involve activation of the eNOS/NO/GC/cGMP/PKG pathway, resulting in inhibition of the NF‐κB/PLC/PKC/TXA2 cascade, and, finally, inhibition of platelet aggregation. Our results indicate the LASSBio‐788 compound as a potential drug for the treatment of cardiovascular disorders such as atherosclerosis.

PB070: New Clinicopathologic Finding in a Patient with Acquired Amegakaryocytic Thrombocytopenia Treated with Thrombopoietin Receptor Agonist Therapy

R. Smith 1 ; S. Zandi 1 ; Z. Liederman 1 ; M. Xu 1,2 ; G. Lim 2 ; H. Ghaffar 2 ; H. Ni 1,2,3 ; M. Sholzberg 4

1 University of Toronto, Toronto, Canada, 2 St. Michael's Hospital, Toronto, Canada, 3 Canadian Blood Services Centre for Innovation, Toronto, Canada, 4 Li Ka Shing Knowledge Institute, St. Michael's Hospital, Division of Hematology/Oncology, Division of Laboratory Medicine and Pathobiology, Toronto, Canada

Background: We present a case of a 73‐year old woman with isolated idiopathic thrombocytopenia given an initial diagnosis of immune thrombocytopenia (ITP). After brief improvement with corticosteroids and intravenous immunoglobulin, she experienced multiple relapses unresponsive to standard therapies including splenectomy. Bone marrow evaluation showed an absence of megakaryocytes thus the diagnosis was amended to acquired amegakaryocytic thrombocytopenia (AAT). Romiplostim, a second generation thrombopoietin receptor agonist (TPO‐RA) was initiated, and her disease remains in complete remission over 4 years since its initiation.

Aims: To describe our experience with Romiplostim in a patient with refractory AAT, explore the pathogenesis, and evaluate the bone marrow stem and progenitor cell composition after 4 years of treatment.

Methods: Patient serum was tested for anti‐platelet antibodies. Phenotypic analysis of bone marrow stem and progenitor compartments using 14 colour flow cytometric technique, while on TPO‐RA was performed.

Results: At the onset of her diagnosis, we identified anti‐platelet antibodies directed against glycoprotein Ib (GPIb), but not against GPIIb/IIIa. Flow cytometry showed expansion of a megakaryocyte‐biased progenitor population within the bone marrow compared to normal controls (Figure 1).

FIGURE 1 Phenotypic analysis shows expansion of CD34lowCD45low population comprised mostly of megakaryocyte‐biased progenitors (CD71+cMPL+ cells)

Conclusions: We have isolated antibodies against GPIb, which may represent a novel etiopathological mechanism in AAT as the antibody may interfere with hepatic TPO generation, as shown by Xu et al. ISTH 2017. Moreover, phenotypic analysis of bone marrow, while on TPO‐RA, revealed changes in the architecture of hematopoietic hierarchy thus demonstrating plasticity of bone marrow responsiveness in a condition on the bone marrow failure spectrum. Our experience adds support to the growing literature for TPO‐RA therapy in cases of refractory AAT. Further studies are warranted to understand the significance of the observed phenotypic changes in the bone marrow.

PB071: Antibody Mediated Glycan Modification on Platelets and Megakaryocytes: A Potential Role in Platelet Destruction in Autoimmune Thrombocytopenia

I. Marini ; R. Jouni; F. Rigoni; T. Bakchoul

Zentrum für Klinische Transfusionsmedizin Tübingen gGmbH (ZKT), Tübingen, Germany

Background: Immune thrombocytopenia (ITP) is a bleeding disease caused by autoantibodies (AAbs) directed against PLT glycoproteins (GP). Recently, an Fc‐independent platelet clearance via Ashwell‐Morell receptors, which recognize glycan changes on platelet surface, has been proposed as a new mechanism of ITP in mice.

Aims: In this study we investigated the effects of AAbs from ITP patients on the glycan pattern of human megakaryocytes (MKs) and PLTs and the subsequent effects on their survival in vivo .

Methods: MKs were differentiated from CD34+ cells after cultivation with thrombopoietin. Megakaryocytes and platelets were incubated with ITP‐sera and the modification in glycan pattern was assessed by flow cytometry using two lectins: RCA, ECL and PNA. NOD/SCID mouse model was used to analyze the impact of glycan patterns on survival of human platelets.

Results: Sera from ITP patients and healthy donors were studied. In the LBA different patterns of glycan modification on PLT surface were observed. 30% sera induced high ECL binding and 60% sera caused a significant increase in PNA binding compared to heathy donors. Of note, 30% sera showed strong decrease in RCA binding. Interestingly, a subgroup of ITP‐sera was able to modify glycan pattern on megakaryocytes surface (3 fold increase in RCA‐binding compared to control). The injection of AAbs induced an accelerated clearance of human PLTs from the circulation in vivo . The destruction of human PLTs by ITP‐AAbs was largely reduced but not completely prevented by a specific neuraminidase inhibitor that blocks glycan changes on PLT surface (platelet survival after 5 hours: 60% vs. 40%).

Conclusions: Our results indicate that AAbs from ITP patients are able to induce changes in glycan patterns on MKs and PLT surfaces. The mechanism of antibody‐mediated modification of glycan patterns seems to contribute to PLT destruction as well as to interfere with PLT production from MKs.

PB072: ThromboGenomics: High Throughput Sequencing in Patients with Bleeding and Platelet Disorders

K. Downes 1 ; K. Freson 2 ; K. Gomez 3

1 University of Cambridge, Department of Haematology, Cambridge, United Kingdom, 2 Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, Leuven, Belgium, 3 Haemophilia Centre and Thrombosis Unit, Royal Free London NHS Foundation Trust, London, United Kingdom

Background: Individuals with bleeding and platelet disorders (BPDs) present as a clinically diverse group of patients with symptoms ranging from life threatening hemorrhage to mild but persistent bleeding symptoms and thrombocytopenia with non haematological clinical manifestations. Laboratory investigations frequently fail to provide an explanation for the symptoms or to identify those at significant risk of future bleeding. In addition, inherited thrombocytopenia often cannot be distinguished from acquired disorders.

Aims: We have developed a High Throughput Sequencing (HTS) approach to provide a molecular diagnosis for patients suspected of having an inherited BPD caused by variants in one of the 70 ISTH‐SSC approved Tier 1 BPD genes.

Methods: To date, over 1000 patients have been referred for testing. Laboratory and clinical phenotypes were recorded using Human Phenotype Ontology (HPO) terms. Variants were called and prioritised based on minor allele frequency, predicted impact and presence in the Human Gene Mutation Database. Variants present in the 70 known BPD genes were reviewed by a multi‐disciplinary team (MDT) where pathogenicity was assigned to variants using ACMG and ACGS guidelines.

Results: Pathogenic variants were identified in approximately half of patients where 40% were novel. Using sequencing read depth large‐scale pathogenic deletions were identified in approximately 3% of patients.

Conclusions: These results demonstrate the success of HTS approaches in providing a genetic diagnosis for patients with well‐defined inherited platelet or coagulation defects. Furthermore extensive replication has been demonstrated for recent BPD gene discoveries, for example RNU4ATAC, SLFN14, RASGRP2, ETV6 and ACTN1 and for novel modes of inheritance, for example GP1BB autosomal dominant thrombocytopenia. All MDT‐reviewed variants and appended HPO terms are shared in ClinVar, supporting the international effort to improve reference catalogues.

PB073: Mitochondria‐dependent Necrosis Is Promoted in Wiscott‐Aldrich Syndrome Platelets as a Result of the Abnormal Surface‐to‐Volume Ratio

S. Obydennyi 1,2 ; E. Artemenko 1 ; A. Sveshnikova 1,2,3 ; A. Ignatova 1 ; G. Novichkova 1 ; A. Maschan 1 ; A. Shcherbina 1 ; M. Panteleev 1,2,3

1 Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russian Federation, 2 Center for Theoretical Problems of Physico‐Chemical Pharmacology, Moscow, Russian Federation, 3 Moscow State University named after M.V. Lomonosov, Moscow, Russian Federation

Background: Wiskott‐Aldrich syndrome (WAS) patients suffer from bleeding caused by microthrombocytopenia of unclear origin. Some of the previous reports found WAS platelets to have increased phosphatidylserine (PS) expression.

Aims: To investigate dynamics and mechanisms of PS exposure in WAS platelets.

Methods: Whole blood of 19 WAS patients and 14 healthy donors was collected into sodium citrate under a protocol approved by the institutional ethics committee. Platelet function including granule release, integrin activation, and procoagulant activity was investigated before and after stimulation using flow cytometry. Real‐time cytosolic calcium dynamics, mitochondrial membrane potential and PS exposure were investigated in single fibrinogen‐bound platelets using confocal microscopy. Computer systems biology model of platelet calcium homeostasis was used for the data analysis.

Results: WAS platelets responded normally to potent dual‐agonist stimulation, but had abnormally high PS exposure when stimulated via PAR1 only. Fibrinogen‐immobilized WAS platelets spontaneously lost mitochondrial membrane potential followed by PS exposure (20.1% vs. 3.6% in healthy donors). Necrostatin‐1, calpeptin, Z‐VAD‐FMK did not affect this, while cyclosporin A reduced PS+ fraction. The number of mitochondria was decreased in WAS platelets and was a reliable predictor of spontaneous necrosis: 50% of WAS platelets with <4 mitochondria exposed PS and 19% for ≥4 mitochondria. Systems biology analysis revealed that the smaller platelets of WAS patients would have higher cytosolic inositol‐triphosphate concentration caused by higher surface‐to‐volume ratio. This leads to more pronounced cytosolic calcium signaling and thus to a significant increase in the probability of mitochondrial calcium overload and platelet necrosis.

Conclusions: WAS platelets spontaneously expose PS via a mitochondria‐dependent necrotic mechanism that can be explained by purely geometric consideration. This could contribute to the development of thrombocytopenia in WAS.

PB074: Septic Conditions Modulate the Level of miRNAs in Platelets and Megakaryocytes via Altered Dicer Level

Z. Fejes 1 ; B. Szilágyi 1 ; T. Orosz 1 ; Z. Czimmerer 2 ; S. Póliska 2 ; G. Nagy 3 ; J. Kappelmayer 1 ; B. NagyJr 1

1 University of Debrecen, Faculty of Medicine, Department of Laboratory Medicine, Debrecen, Hungary, 2 University of Debrecen, Faculty of Medicine, Department of Biochemistry and Molecular Biology, Genomic Medicine and Bioinformatics Core Facility, Debrecen, Hungary, 3 University of Debrecen, Faculty of Medicine, Department of Anesthesiology and Intensive Care, Debrecen, Hungary

Background: We have previously described decreased levels of activation‐dependent platelet miRNAs contributing to elevated platelet activation is sepsis. However, the regulatory mechanisms of altered platelet miRNAs among these conditions are largely unknown.

Aims: In this study, we examined whether Dicer level is altered in platelets of septic patients that modulate miRNA expression. In parallel, Dicer level was analyzed in platelets and megakaryocytes (MKs) after lipopolysaccharide (LPS) treatment in vitro .

Methods: Dicer1 mRNA levels were investigated in leukocyte‐depleted platelet samples obtained from 10 individuals suffering from sepsis and in 14 age‐ and gender‐matched healthy controls. We also quantified miR‐223 and miR‐26b with target P2RY12 and SELP mRNAs, respectively, by RT‐qPCR in patient samples as well as in stimulated platelets by LPS (O55:B5, up to 3 μg/mL) or PAR1 agonist TRAP (30 μM) for 30 minutes. To prove the effect of altered Dicer level of MKs in sepsis, miRNA and Dicer levels were analyzed in MEG‐01 and K562‐MK cells after the treatment with LPS (100 ng/mL) for 24 hours.

Results: Septic conditions induced inflammatory response in platelets and MKs showing elevated miR‐155 in both ex vivo and in vitro experiments. Patient platelet samples and both LPS‐treated MK cultures showed decreased Dicer1 mRNA levels resulting in lowered miR‐223 and miR‐26b with upregulated P2RY12 and SELP mRNA levels. Interestingly, short‐term LPS stimulation elevated Dicer1 mRNA level with higher miRNAs in platelets compared to untreated samples, while TRAP did not markedly affect these RNA levels.

Conclusions: Abnormal Dicer levels are generated by septic milieu that alter the levels of platelet and MK miRNAs in sepsis. However, distinct regulatory mechanisms seem to function in platelets after short‐ and long‐term septic triggers, which need to be further investigated.

This study was supported by “ÚNKP‐17‐3 New National Excellence Program of the Ministry of Human Capacities” (Z.F.).

PB075: Evidence of a Dominant Negative Effect of Gain‐of‐Function ITGB3 Mutations over Loss‐of Function Mutations in Variant Glanzmann Thrombasthenia

L. Bury 1 ; E. Zetterberg 2 ; E. Leinoe 3 ; E. Falcinelli 1 ; A. Marturano 1 ; G. Manni 1 ; A.T. Nurden 4 ; P. Gresele 1

1 University of Perugia, Department of Medicine, Perugia, Italy, 2 Skaane University Hospital, Department of Haematology, Coagulation Unit, Lund, Sweden, 3 Copenhagen University Hospital, Rigshospitalet, Department of Haematology, Copenhagen, Denmark, 4 Hopital Xavier Arnozan, French National Reference Centre for Platelet Disorders, Pessac, France

Background: The autosomal dominant form of GT, associated with platelet dysfunction, macrothrombocytopenia and bleeding, is caused by gain‐of‐function (GOF) variants of ITGA2B or ITGB3 that generate a constitutively activated αIIbβ3. GOF ITGB3 variants exert a dominant effect over normal ITGB3 , leading to the clinical picture of autosomal dominant GT. No information, however, is available on the impact of GOF variants when a concomitant loss‐of‐function (LOF) variant is present.

Aims: We aimed to explore the effect of GOF ITGB3 mutations over LOF mutations starting from the characterization of a family with a novel form of autosomal dominant GT caused by co‐inheritance of two ITGB3 variants.

Methods: Platelet αIIbβ3 expression and activation were assessed by flow cytometry, DNA variants by Sanger sequencing. Mutant β3 expression vectors were generated and transfected into CHO cells expressing αIIb. αIIbβ3 expression, activation and internalization were assessed by flow cytometry, adhesion on fibrinogen by spreading assay, FAK phosphorylation by Western blotting.

Results: We diagnosed autosomal dominant GT in a young female with long‐lasting mucocutaneous bleeding and found compound heterozygosity for two novel ITGB3 variants: R786W and N331S. The autosomal‐dominant GT phenotype co‐segregated with the N331S variant among family members. Expression studies revealed that R786W was a LOF variant causing reduced cell spreading and FAK phosphorylation on fibrinogen. N331S was a GOF variant and, of note, when co‐expressed with normal β3 or R786W, exerted a dominant‐negative effect producing impaired αIIbβ3 surface expression, increased internalization, PAC‐1 binding and FAK phosphorylation under resting conditions and accelerated spreading.

Conclusions: We show that a GOF mutation of β3 can exert its dominant‐negative effect even when associated with a LOF β3 mutant on the second allele, giving rise to the clinical picture of autosomal dominant GT of platelet dysfunction and macrothrombocytopenia.

PB076: Bleeding Diathesis Associated with Severe Acquired Platelet Dysfunction and Overproduction of Cyclic AMP in Two Patients with Hematologic Malignancies

A. Lecchi 1 ; E.A. Femia 1 ; S. La Marca 1 ; A. Artoni 1 ; F. Onida 2 ; F. Peyvandi 1 ; M. Cattaneo 3

1 A. Bianchi Bonomi Hemophilia and Thrombosis Center, Department of Internal Medicine Fondazione IRCCS Ca’ Granda ‐ Ospedale Maggiore Policlinico, Milano, Italy, 2 University of Milan, BMT Center Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Hemato‐Oncology Department, Milano, Italy, 3 Medicina II, Azienda Ospedaliera Santi Paolo e Carlo, Dipartimento di Scienze della Salute, Università degli Studi di Milano, Milano, Italy

Background: The pathophysiology of impaired platelet function in acquired disorders is often poorly characterized.

Aims: We describe a severe bleeding diathesis in two unrelated patients with hematologic malignancies, associated with severe platelet abnormalities, including overproduction of inhibitory cyclic‐AMP (cAMP).

Methods: Patient 1 was a 74‐year‐old woman with severe mucocutaneous bleeding of recent onset, mild macrocytic anemia and thrombocytopenia (100×10 9 /L), who was diagnosed with chronic myelomonocytic leukemia few months later.

Patient 2 was a 77‐year‐old man with myelodysplastic syndrome with excess blasts. He had mucocutaneous bleeding of recent onset, despite a platelet count persistently over 100×10 9 /L. Personal and family history for abnormal bleeding was silent in both patients.

Prothrombin time (PT), activated partial thromboplastin time (aPTT), von Willebrand Factor (vWF) antigen and RiCof, the concentrations of platelet fibrinogen, ADP, ATP, serotonin, cAMP and the serum levels of thromboxane B2 were measured by standard techniques. Platelet aggregation and secretion were tested by lumiaggregometry. The platelet expression of CD41/61 and CD42b and the vasodilator‐stimulated phosphoprotein (VASP) phosphorylation were tested by flow cytometry.

Results: PT, aPTT, vWF, CD41/61 and CD42b were normal. Agonists‐induced platelet aggregation and ATP secretion were severely impaired. Serum TxB2 and the platelet content of ADP, ATP, serotonin and fibrinogen, were markedly decreased. Baseline platelet cAMP levels were mildly elevated (pt1: 21; pt2: 15; healthy controls: 3.‐14 pmoles/10 9 plts) and were markedly increased by PGE1 (pt1: 473; pt2: 325; healthy controls: 23‐97 pmoles/10 9 plts). ADP normally inhibited PGE1‐induced cAMP increase and VASP phosphorylation.

Conclusions: To the best of our knowledge, these are the first patients with acquired bleeding diathesis associated with severe platelet dysfunction and overproduction of platelet cAMP.

PB077: In‐silico Prediction of Molecular Mass Distance Dependent Changes in Binding Energies between von Willebrand Factor and Platelet Membrane Glycoprotein (GP) Ibα in Wild Type and G233V Mutant

S. Goto 1 ; N. Tamura 2 ; K. Ayabe 3 ; H. Yabushita 3 ; H. Oka 3 ; S. Goto 3

1 Keio University School of Medicine, Cardiology, Tokyo, Japan, 2 Tokai University School of Medicine, Kanagawa, Japan, 3 Tokai University School of Medicine, Tokyo, Japan

Background: Platelet adhesion under blood flow is mediated exclusively by binding of platelet membrane glycoprotein (GP) Ibα with von Willebrand factor (VWF). Single amino acid substitution from Gly to Val at position 233 of GP1bα is known as a cause of platelet‐type von Willebrand diseased due to enhanced binding of GP1bα to VWF. However, detailed molecular basis for this phenotype of G233V mutant of GPIbα for the interaction with VWF is still to be elucidated.

Aims: To unravel the molecular mechanism for the apparent “gain of function” phenotype of G233V mutation.

Methods: Molecular dynamics (MD) calculation was conducted to predict the binding characteristics of N‐terminus domain of GPIbα and A1 domain of VWF. The NAno Molecular Dynamics (NAMD) software using Chemistry at Harvard Macromolecular Mechanics (CHARMM)‐22 force field database were applied for wild‐type and G233V mutation in GPIbα binding with wild type VWF. The Binding energy between G233V mutant of GPIbα with wild‐type VWF were calculated by umbrella sampling method with mass center distance between both molecules at every 0.5 Å.

Results: Most energetically stable structure of VWF bound with wild type and G233V mutant GPIbα was achieved at 27.2 Å and 27.7 Å, respectively. Stable binding structure of VWF with wild type and G233V GPIbα did not differ markedly (panel A)The values of the potential of mean forces (PMF) generated between wild‐type VWF and GPIbα in wild type and G233V mutant are shown in Figure Stable s… panel B. The binding energy had a single peak of 20.0 kcal/mol at mass centers distance of 27.2 Å in wild‐type GPIbα and VWF, while it showed 2 distinct peaks consisting of a 20.0 kcal/mol peek at 27.7 Å and another 5.3 kcal/mol peek at a longer distance of 50.0 Å for G233V GPIbα and VWF.

FIGURE 1 Stable structure and potential of mean force calculated for wild‐type and G233V GP1b‐alpha and wild‐type VWF

Conclusions: Our results suggest that the apparent “gain of function phenotype” of G233V GPIbα mutation may depend on 2 energetically stable structure of GPIbα binding with VWF in G233V mutant.

PB078: Molecular Diagnosis of Inherited Platelet Disorders by Analysis of the MYH9 and the Genes Encoding the GB1b‐IX‐V Complex in an Irish Cohort

R. Campbell ; S. Mc Namara; L. Dempsey; M. Byrne; K. Ryan; B. White; N.M. O'Connell; C. Keenan

National Coagulation Centre, St James's Hospital, Dublin, Ireland

Background: Congenital thrombocytopenias are a group of rare disorders resulting in thrombocytopenia, giant platelets, and often comorbidities. The presentation of common symptoms can lead to misdiagnosis and inappropriate treatment. The MYH9 gene and the genes encoding the GB1b‐IX‐V complex are both associated with similar clinical phenotypes caused by two distinct disorders, MYH9‐related disorders and Bernard Soulier Syndrome respectively. Molecular analysis may provide a definitive diagnosis in these patients and identify inheritance patterns.

Aims: Here we describe a cohort of patients with macrothrombocytopenia investigated for variants within the MYH9 , GP1Ba , GP1Bb and GP9 genes.

Methods: After informed counselling and consent, direct re‐sequencing was performed (TA cloning on one patient).

Results: 37 patients were referred for molecular analysis. Initial targeted analysis of MYH9 detected pathogenic variants in 11 of 32 patients (34%) in exons 2, 31, 39 and 41. No variant was detected in 20 patients (63%). Eight were further analysed for the remaining MYH9 coding regions and no variant detected. The pathogenic variant in exon 2 was a complex rearrangement predicting p.Val59_Glu60ins9. This patient had a severe phenotype consistent with variants within the head domain of the MYH9 protein product NMMHC‐IIA. Analysis of GP1Ba , GP1Bb and GP9 in six patients detected a GP1Ba variant in three members of the same family (exon 2; p.Gly249Val), associated with pseudo‐2B VWD. The same variant in GP9 (exon 3; p.Asn61Ser) was detected in three patients from two families.

Conclusions: Targeted MYH9 analysis to regions commonly associated with pathogenic variants proved successful in 30% of patients. The use of NGS technology would greater increase the likelihood of detecting a pathogenic variant in this group. We have introduced integrated reporting to include platelet morphology, clinical phenotyping, immunophenotyping and genetic data to comprehensively diagnose inherited platelet disorders.

PB079: Investigation of Polymorphic Variations in Glutathione S Transferase Genes among Immune Thrombocytopenia Patients in Pakistan

S.N. Mukry ; A. Arshad; A. Shahni; T.S. Shamsi

National Institute of Blood Disease and Bone Marrow Transplantation, Department of Post Graduate Studies & Research, Karachi, Pakistan

Background: Immune thrombocytopenic purpura (ITP) is an autoimmune clinical syndrome characterized by a low platelet count, which is the result of increased platelet destruction and insufficient platelet production [1]. The pathophysiology of ITP is heterogeneous and complex. Recently, several evidences have suggested that oxidative stress plays an important role in the pathogenesis of autoimmune diseases, and it is also involved in the development of ITP [2, 3]. Glutathione S transferases are phase II antioxidant or detoxifying enzymes which conjugates reduced form of glutathione to xenobiotics for detoxification and thereby control oxidative imbalance.

Aims: To investigate the possible role of polymorphic variations of phase II antioxidant enzyme GSTT1 and GSTM1 genes in the pathogenesis of ITP.

Methods: Multiplex PCR for GSTT1 and GSTM1 was performed using previously designed primers in compliance with the declaration of Helsinki.

Results: A total of confirmed 197 cases (99 females and 98 males) of ITP with mean age of 35 (median) range (3‐80) and 167 normal healthy individuals of same sex and age as control were analyzed. GSTM1 null deletion was significantly more frequent in ITP patients (49.23%) as compared to controls (32.54%). The ITP patients have low frequency of GSTT1 null deletion (5.58%) as compared to controls (13.60%). Double deletion was observed in ITP and controls both with almost similar frequency i.e. 9.18% and 8.88% respectively. These overall differences in frequencies were statistically significant (P=0.005).

Conclusions: This study has allowed determining the frequency of GSTM1 and GSTT1 polymorphism in a sample of our patients. A significant increase has also been observed in control group which suggest they might be susceptible to disease occurrence. GSTM1 null deletion was more frequent and significant in ITP patients (P=0.007). These findings might provide a new hypothesis for the initiation of abnormal autoimmunity and a possible novel therapeutic approach.

PB081: CD8 T Cell‐mediated Platelet Clearance Does Not Occur via Traditional Cytotoxic Protein Killing

C. Maier ; S. Patel; A. Bennett; S. Stowell

Emory University School of Medicine, Department of Pathology and Laboratory Medicine, Atlanta, United States

Background: Poor survival of transfused platelets (plt) remains a significant problem, especially in multiply transfused patients with immune‐mediated refractoriness. While patients with alloantibodies can be managed with matched (ie antigen‐negative) plt, patients with T cell‐mediated refractoriness remain difficult to treat, as this etiology of plt clearance has only recently been identified and its mechanism is unknown.

Aims: We aimed to further characterize the CD8 T cell/allogeneic plt interaction and to specifically investigate the role of the typical perforin/granzyme cytotoxic protein pathway.

Methods: CD8 T cells from OTI transgenic mice, OTI/perforin knock‐out mice (OTI/prf −/− ), or WT mice were co‐cultured with WT or mOva plt for 24 hours and analyzed by flow cytometry. In vivo T cell‐mediated plt clearance was modeled by transfusion of mOva or WT plt directly into OTI, OTI/prf −/− , and WT recipients, or by transfusion into WT mice adoptively transferred with OTI, OTI/prf −/− , or WT splenocytes. Plt survival was measured and significance determined when P<0.05 by one‐way ANOVA with multiple comparisons by Tukey's test.

Results: mOva plt are more adherent to OTI and OTI/prf −/− CD8 T cells then to WT CD8 T cells, and CD8 T cells from OTI and OTI/prf −/− mice express the activation marker CD69 after co‐culture with mOva, but not WT, plt. Transfusion of mOva plt into either OTI or OTI/prf −/− mice results in equally significant clearance. Specifically, mOva plt survival in OTI or OTI/prf −/− recipients is significantly less (<30%) at 24 hours than in WT recipients (>60%). Adoptive transfer experiments show similar results, whereby recipients receiving OTI or OTI/prf /− splenocytes have significantly decreased mOva plt survival.

Conclusions: We conclude that minor antigen mismatched plt adhere to and activate CD8 T cells, resulting in plt clearance that is independent of the traditional cytotoxic killing pathway via perforin and granzyme.

PB082: Human Platelets Labeled at Multiple Biotin Densities: A Promising Approach for Monitoring in vivo Platelet Survival in Clinical Trials

C. Ravanat ; V. Heim; A. Pongérard; A. Dupuis; C. Gachet

Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR‐S 1255, FMTS, Strasbourg, France

Background: Biotin (Biot) is an alternative to radioactive blood cell tracers. It allows one to concurrently track in vivo multiple cell populations labeled at different Biot densities. Multi‐labeled red blood cells have been safely transfused in man to assess their survival (Mock, 2014). However, only one study in man has described the tracking of a Biot‐platelet population (Stohlawetz, 1999).

Aims: Our aim was to label human platelets (PLTs) with different levels of Biot to determine whether biotinylation affects their function. This was a preliminary study to explore the feasibility of multi‐labeled Biot‐PLT production for clinical trials.

Methods: PLTs in Tyrode's albumin (TA) were treated with Biot‐sulfo‐NHS (0, 4.5, 45 and 180 μM), washed twice and re‐suspended (3×10 8 /mL) in TA/PPPcit (v/v). Biot densities were controlled by FACS analysis with streptavidin‐PE labeling and PLT functions by assessing parameters such as secretion, glycoprotein (GP) integrity and aggregation.

Results: Various Biot‐PLT populations could be distinguished by FACS and all displayed swirling. Using TRAP, P‐selectin externalization was stable from 0 to 45 μM Biot labeling but slightly lower for 180 μM Biot. GPIb and GPIIbIIIa expression was normal for all Biot‐PLTs. PLTs aggregability using arachidonic acid was well preserved in all Biot‐PLTs, whereas biotinylation impaired the response to ADP and collagen in a dose dependent manner with an aggregation of the Biot180‐PLTs almost abolished (Table).

TABLE 1 Results for in vitro functions of biotinylated human platelets (Mean±SEM) Biot‐sulfo‐NHS (µM) 0 4.5 45 180 Mean Fluorescence Intensity Streptavidin‐PE 0.7±0.3 3.7±0.2 35±1.2 168±27 Quantitation of P‐selectin expression (60 µM‐TRAP) 7086±631 6244±709 6156±868 3040±492 Amplitude of aggregation (5 µM‐ADP) % 100 97±5 51±6 4±1.6 Amplitude of aggregation (2.5 µg/mL‐collagen) % 100 101±2 88±6 36±21 Amplitude of aggregation (1 mM‐arachidonic acid) % 100 101±0.7 96±3 80±9

Conclusions: The main PLT functions tested, i.e., secretion, GP integrity and aggregation in response to the strong agonist arachidonic acid, were fully preserved for all the Biot densities used. In contrast, Biot labeling affected the response to ADP and collagen. We conclude that multi‐Biot labeling would be suitable to trace PLTs in clinical trials provided low Biot densities (<45 μM) are used when full PLT function is critical. This approach should be helpful to evaluate new PLT products and fulfill the need for non‐radioactive tracers.

PB083: Quantitative Alteration of Vesicle Associated miRs and RISC Complex Reflect Platelet Concentrate Molecular Pattern during Storage as Platelets Activity Model

V. Kishenko 1 ; K. Kondratov 1 ; A. Fedorov 1 ; O. Sirotkina 1,2

1 Almazov National Medical Research Centre, Saint‐Petersburg, Russian Federation, 2 Pavlov First Saint Petersburg State Medical University, Saint‐Petersburg, Russian Federation

Background: Platelet concentrate (PC) storage is accompanied by permanent release of platelet (PLT) derived membrane vesicles. PLT miRs associate with microparticles and form complexes with RISC‐associated proteins (Ago2 and GW182).This process serves as a platelets activity model and molecular composition of PLT derived vesicles – as biomarkers of platelet reactivity.

Aims: To study molecular pattern in PLT and PLT derived vesicles at different stages of PC storage.

Methods: Six pulled PC from non‐smoking man 20‐30 years old, without hematologic disease, 0(I)Rh+ were included in the study. Samples were collected on 2nd and 7th day of storage. Fractions of PLT and membrane vesicles were obtained by differential centrifugation at 5000 g , 16000 g (16P) and 100000 g (100P). Levels of miRs (142, 223, 223*, 21, 126, 199, 15, 451, 221, 145, 326, 210) were quantified by qRT‐PCR; proteins Ago2 and GW182 from RISC complex were analyzed by Western blot.

Results: In a series of analyzed molecular components miR142 and miR223 were the most abundant in PLT. Level of miR21 was moderate, level of other targets, including, miR126 was low. The absolute quantity of miRs in PC fractions was different on storage days 2 and 7 with increasing on day 7: miR221 and miR21 ‐ in 100P fraction (P=0.03 in both), miR15 ‐ in 16P fraction (P=0.03). MiR223, miR126 increased in both 16P (P=0.03; P=0.03) and 100P (P=0.03; P=0.03) fractions; miR451 level increased in PLT (P=0.03), 100P (P=0.03) and decreased in supernatant (P=0.03) fraction. PLT as well as 16P and 100P fractions contained Ago2 and GW182 proteins. Levels of these proteins increased in 16P and 100P fractions during storage.

Conclusions: PLT‐derived vesicles contain RISC complex protein components and abundant miR species. Their quantity changes in the course of PC storage reflects platelet activation process via molecular mechanism. Supported by RFBR 16‐04‐01142.

PB084: Improving Transfusion Safety by Implementing Platelet Bacterial Screening in a Resource‐limited Country

F. Karim ; B. Moiz

Aga Khan University Hospital, Karachi, Pakistan

Background: Bacterial contamination of platelets remains a persistent problem in modern transfusion medicine. In a developing country like ours, introduction of bacterial screening was a significant challenge due to cost constraints. We developed a cost effective protocol for bacterial detection in platelet concentrates.

Aims: To implement platelet bacterial screening method so as to improve blood safety with regard to bacterial infections in a resource limited country.

Methods: The study was conducted at blood bank of Aga Khan University Hospital, Karachi, Pakistan from August 2016 to December 2017. Platelet concentrates [PC] were prepared from platelet rich plasma. PC were screened for aerobic bacterial contamination by culture method [Bactec 9240, BD Bactec]. A volume of 2‐3 ml was collected from each PC on day 2 post‐collection. A sample pool made from five individual PC was added under sterile conditions to a pediatric blood culture bottle. Identity of the pool and samples was ensured using barcoded labels. All inoculated bottles were incubated for 5 days. If a pool was positive for bacterial growth then pool resolution was performed by doing culture of sample from individual platelet unit to identify the contaminated concentrate. Initial assessment for any growth was done after 24 hours of incubation. PC with initial negative results (in first 24 hours) were removed from ‘quarantine’ and placed in ‘inventory’ and dispensed as “Negative‐to‐date”.

Results: Total number of PC tested during study period was 28,718 (5743 pools). Confirmed positive culture was reported in one case (0.003%) where Streptococcus species was detected. No case of septic transfusion reaction was reported during study period. False positive culture was reported for 89 pools where no growth was seen on pool resolution.

Conclusions: Successful implementation of bacterial screening of platelets is a milestone in enhancing transfusion safety in a developing resource limited country.

PB089: Compensatory Effect of Fibrinogen in Patient with Bone Marrow Aplasia, Septic Shock and Severe Thrombocytopenia Guided by Thromboelastometry: A Case Report

T. Crochemore ; F. Savioli; C. Pessoa; A. Resende; E. Casaroto; L. Bernz; R. Narciso; F. Souto

Hospital Leforte Morumbi, Intensive Care Unit, Sao Paulo, Brazil

Background: Platelet transfusion is a common practice to prevent spontaneous bleeding, or bleeding due to invasive procedures or surgery, in patients with hematologic diseases and thrombocytopenia. Transfusion of allogeneic blood components is associated with increased mortality and worse (worsened?) clinical outcome. The clot strength is accessed (assessed?) by thromboelastometry and determined by the interaction between platelets and fibrinogen. The compensatory effect of high levels of fibrinogen on clot strength in patients with thrombocytopenia has been demonstrated in different clinical settings such as in dilutional coagulopathy. We report the case of a patient with severe thrombocytopenia related to bone marrow aplasia whose thromboelastometry showed the clot strength compensated by the increase in plasma fibrinogen levels as an acute phase reactant of septic shock.

Aims: –

Methods: We reported a case of a 62 years old caucasian Brazilian female admitted in the ICU with bone marrow aplasia. She developed septic shock being necessary orotracheal intubation due to respiratory insufficiency and antibiotic therapy. A bronchoscopy was requested but she presented severe thrombocytopenia (1000/mm 3 ). As her fibrinogen levels were 1050 mg/dl due to septic shock, we performed a thromboelastometry showing a normocoagulable state. The bronchoscopy showed signs of bilateral alveolar hemorrhage, presence of organized clots in the inferior lobe segment, but without active bleeding. 7 days after she was extubated.

Results: –

Conclusions: The use of thromboelastometry as a tool used to diagnose coagulation disorders has made it possible to avoid prophylactic transfusion of platelet concentrate unnecessarily. We believe that thromboelastometry could be safer and more effective at predicting bleeding risk than platelet counts.

PB090: A Novel Murine Model of Immune Thrombocytopenia Induced by Immunized CD41 Knockout Mice

X. Li ; S. Wang; X. Zuo; M. Hou; J. Peng

Qilu Hospital of Shandong University, Jinan, China

Background: Immune thrombocytopenia (ITP) is the most common autoimmune bleeding disorder. Semple, et al. has reported a murine model of ITP in which splenocytes from CD61 knockout (KO) mice immunized against CD61 + platelets were transferred into severe combined immunodeficient (SCID) mouse recipients (CD61 + ). However, CD61 is also expressed on endothelial cells, leading to a high bleeding morbidity (80%) of SCID recipients. Moreover, CD61‐KO donors are presented with placental defects (25%) and postnatal hemorrhage (13%).

Aims: To develop a novel murine model of ITP using immunized CD41‐KO mice.

Methods:

CD41‐KO mice were transfused weekly with 10 8 platelets of wild type C57/BL6 mice for 4 consecutive weeks. Then spleens were removed and prepared into splenocyte suspension. SCID mice were subjected to 200 cGy total body irradiation, and injected intraperitoneally with 5×10 4 splenocytes. Platelet counts and serum antibody levels were recorded weekly.

Results:

None of the CD41‐KO mice were presented with placental defects. No bleeding occurred in embryos or pups. SCID mice transferred with non‐immunized CD41‐KO splenocytes or equal volume of PBS served as controls. All the mice developed thrombocytopenia at day 7 (Figure 1). Platelet counts increased gradually in controls (Figure 1A), but remained at low levels within 21 days in the SCID mice receiving splenocytes from immunized CD41‐KO mice (Figure 1B). No mortality was observed due to bleeding diathesis. No serum antibody was detected in controls, while high levels of serum anti‐platelet CD41 IgG antibodies were present in the morbid mice. Few mature megakaryocytes were seen in bone marrow of morbid mice (Figure 2B).

FIGURE 1 Platelet counts differ after transfer of splenocytes. The data are expressed as mean platelet counts±SEM over time (days). *P<0.05

FIGURE 2 Histologic section of bone marrow from (A) a control SCID mouse, (B) a morbid SCID mouse. The yellow arrows point to megakaryocytes

Conclusions: CD41‐KO mice have no placental defects or postnatal hemorrhage, and no mortality are observed in our murine model of ITP using immunized CD41‐KO donors. This study develops an equally efficient murine ITP model and overcomes the disadvantages induced by CD61‐KO donors.

PB091: Polyphosphate Is Concentrated in the Core of Platelet Aggregates

C. Clark ; S. de Maat; A. Barendrecht; C. Maas

University Medical Center Utrecht, Clinical Chemistry and Haematology, Utrecht, the Netherlands

Background: Platelets contain polyphosphate (polyP), a charged polymer that acts on coagulation and fibrinolysis. We recently reported that platelets retain polyP on their surface after activation in complex with calcium. The resulting polyP particles can trigger contact activation, suggesting that contact activation should always follow platelet activation. From clinical studies, there is little evidence for this pathway. Furthermore, there is no bleeding diathesis in persons with contact factor deficiencies. It was previously reported that platelet aggregates consist of a tightly packed core region and a loosely adherent shell region, composed of unactivated platelets. We hypothesize that polyP is mainly located in the inner core of platelet aggregates, preventing continuous contact activation.

Aims: To investigate the distribution of polyP throughout platelet aggregates.

Methods: We studied the spatiotemporal distribution of polyP (tracked with SYTOX orange) during aggregate buildup by perfusing citrated whole blood through a laminar‐flow chamber over collagen/tissue factor‐coated cover glasses. At fixed time points, layered images were captured and analysed with a deconvolution algorithm for reconstruction of 3D images.

Results: A polyP‐rich core develops in the center of platelet aggregates, surrounded by a polyP‐poor outer shell over time (Figure 1). Perfusion of ADP over preformed aggregates rapidly converts the shell to polyP positive and increases polyP release within the core region. This phenomenon can be recapitulated on monolayers of unactivated platelets, suggesting that polyP secretion and retention is dependent on strong and synergistic triggering of platelet activation, as may occur in thrombotic states.

FIGURE 1 PolyP in an aggregate core after 10 minutes perfusion visualized with SYTOX orange, orthogonal view, and 3D projection (red=aggregate outline)

Conclusions: Our findings indicate that only a part of the platelet polyP is secreted within an aggregate. Under these conditions, polyP is shielded from the plasma environment, which may limit contact activation until the thrombus is ruptured or the shell becomes exposed to soluble platelet activators.

PB092: Effects of Bothrops jararaca Venom on Platelet Aggregation, Secretion and Protein synthesis

J.G. Rosa; M.L. Santoro

Instituto Butantan, Lab. Fisiopatologia, São Paulo, Brazil

Background: Victims from Bothrops snakebites manifest clinical symptoms such as hemorrhage, blood incoagulability and thrombocytopenia. Platelets are involved in the development of systemic bleeding evoked by B. jararaca venom (BjV), and they may contribute to other complications resulting from snakebites.

Aims: To evaluate the effect of BjV on platelet aggregation, granule secretion and protein synthesis in human and mouse platelets.

Methods: All procedures involving human and mice were approved by the respective Ethics Committees. Washed mouse and human platelets were pretreated with 1 mM puromycin and cycloheximide (protein synthesis inhibitors) or vehicle for 1 hour. Platelet aggregation and ATP secretion was then stimulated with thrombin (0.1 U/mL) or BjV (24 μg/mL) in a platelet aggregometer and compared. Protein expression of von Willebrand factor (vWF), β‐actin, GAPDH and thromboxane synthase (TX) from mouse platelets was evaluated by Western blotting.

Results: The extent of BjV induced platelet aggregation was less intense in human than in mouse platelets. Granule secretion was similar between BjV and thrombin in mouse platelets, and did not involve protein synthesis. However, human platelet secretion stimulated by BjV was dependent on protein synthesis. Preliminary experiments demonstrated that vWF expression was reduced in mouse platelets activated by BjV, in either the presence or absence of protein synthesis inhibitors. vWF expression induced by thrombin was less intense in the presence of protein synthesis inhibitors. Under pretreatment with protein synthesis inhibitors, mouse platelets activated by BjV did not modify β‐actin expression, while the protein expression of GAPDH and TX showed a discrete reduction.

TABLE 1 Human and mouse platelets preincubated with protein synthesis inhibitors showed a decrease in the extent of aggregation Platelet aggregation (%) Without protein synthesis inhibitors With protein synthesis inhibitors Thrombin BjV Thrombin BjV Human 60.9±3.4 23.10±2.2 12.5±3.6 6.5±7.6 Mouse 71.8±4.7 57.7±1.9 33.1±8.1 40.2±11.1

Conclusions: Human and mouse platelets responded differently to BjV. Our results suggest that BjV induces activation, aggregation, secretion and protein synthesis in human and mouse platelets.

Support: CAPES, CNPq and FAPESP.

PB093: Effect of Anorexigenic Peptide Obestatin on Platelet Aggregation at White Rat with Overweight in vitro Experiments

M. Golubeva ; E. Khirazova

Moscow State University named after M.V. Lomonosov, Biology Faculty, Moscow, Russian Federation

Background: It is known that in people suffering from overweight, there are serious violations in the system of hemostasis, which can lead to the development of thrombotic complications. The leading role in these processes belongs to platelets.

Aims: The aim of this study was to examine the effect of the anorexigenic peptide obestatin on platelet aggregation (PA) in experiments in vitro in the platelet rich plasma (PRP) of healthy animals and animals with obesity.

Methods: In vitro studies, the effect of obestatin on ADP‐induced (15 μM) PA in the PRP of healthy rats and animals with obesity was studied. To create a model of alimentary obesity, animals were kept for 2 months on a high‐calorie diet. Peptide was added to PRP samples in doses of 100, 300, 500, 800, or 1000 nmol/kg, Control samples contained appropriate volume of saline. Data were statistically processed.

Results: It was shown enhancement of PA in obese animals in comparison with healthy rats was revealed. Adding of anorectic peptide obestatin to plasma significantly decreased PA in plasma from healthy rats as well as in plasma from obese animals. It was shown that the peptide in healthy animals in all concentrations of 1.5, 4.5, 7.5, 12 and 15 nmol/ml caused a decrease in PA, the greatest effect was observed with a concentration of obesity of 7.5, 12 and 15 (P<0.001). Obesity caused a significant increase in PA in the PRP of rats. After incubating the PRP from these animals with obestatin at all concentrations of the peptide, a decrease in PA in comparison with the control 85% saline) occurred, with a maximum reduction in PA on 26.7% by the concentration of the peptide was 4.5 nmol/ml.

Conclusions: Obestatin was considered as a potential weight lowering drug. Since the pathological conditions associated with overweight are often accompanied by increased blood clotting, PA inhibition with obestatin was regarded as a positive effect.

PB095: Janus Kinase 3 (JAK3)‐dependent Signaling Is Critical to Ca 2+ ‐dependent Platelet Activation and Thrombus Formation

M. Manke 1 ; S. Geue 1 ; P. Münzer 1 ; B. Walker‐Allgaier 1 ; D. Eißler 1 ; F. Lang 2 ; M. Gawaz 1 ; O. Borst 1

1 University Clinic of Tübingen, Tübingen, Germany, 2 University of Tübingen, Tübingen, Germany

Background: Adhesion of platelets to subendothelial collagen after plaque rupture results in an increase of cytosolic Ca 2+ concentration ([Ca 2+ ]i) followed by Ca 2+ ‐dependent platelet activation. Platelet activation is essential for the development of thrombotic vascular occlusions. Janus kinase 3 (JAK3) is a member of the Janus family protein‐tyrosine kinases which is strongly expressed in platelets. The inhibition of JAK3 in human platelets decreased pseudopod formation, degranulation and aggregation. Although STAT3, a downstream target of JAK3, plays a crucial role in Ca 2+ signaling and platelet aggregation less is known about the impact of JAK3 in platelet activation and thrombus formation.

Aims: The present study explored the role of JAK3 in Ca 2+ ‐dependent platelet activation and thrombotic vascular occlusions in JAK3‐deficient ( jak3 − / − ) mice.

Methods: Ca 2+ measurements, luminescence aggregometry and flow chamber of washed jak3 − / − and jak3 +/+ platelets; tail bleeding time and in vivo thrombus formation in bone marrow chimeric jak3 − / − and jak3 +/+ mice.

Results: Fura‐2‐AM spectrofluorometric Ca 2+ measurements revealed a blunted increase of [Ca 2+ ]i in jak3 − / − platelets as compared to jak3 +/+ platelets after stimulation with thrombin or collagen‐related peptide (CRP). In addition, the activation‐dependent increases of dense granule secretion (reflected by ATP‐release) and aggregation were abrogated in jak3 − / − platelets. In vitro thrombus formation on a collagen‐coated surface was diminished, and thrombotic vascular occlusion after FeCl3‐induced injury in vivo was blunted in jak3 − / − chimeric mice, but no prolongation of bleeding time was observed.

FIGURE 1 Representative images of in vivo thrombus formation after FeCl3‐induced injury

Conclusions: The present study reveals JAK3 as a regulator of Ca 2+ ‐dependent platelet activation, dense granule secretion and aggregation. JAK3‐deficiency results in decreased in vitro and in vivo thrombus formation, but does not affect bleeding time. Therefore, JAK3 could play a decisive role in arterial thrombosis underlying myocardial infarction or ischemic stroke.

PB096: An Intermediate State of Integrin αIIbβ3 Mediates Platelet Aggregation under Disturbed Flow

Y. Chen 1 ; L. Ju 2 ; S. Jackson 2 ; C. Zhu 1

1 Georgia Institute of Technology, Biomedical Engineering, Atlanta, United States, 2 University of Sydney, New South Wales, Australia

Background: Platelets are sensitive to force in disturbed flow often induced by vessel stenosis or medical device intervention, resulting in occlusive thrombosis. However, the molecular mechanism underlying the ‘biomechanical thrombosis’ is incompletely understood.

Aims: We aimed to investigate the integrin activation states and function in biomechanical thrombi, and define its triggering mechanism.

Methods:

A stenosed microfluidic device was used to mimic a stenosed artery. Human whole blood was perfused through such a device to form thrombus under high shear flow. Platelets were stained with conformation‐specific monoclonal antibodies to report the conformational state of the integrin α IIb β 3 . Dual biomembrane force probe (dBFP) was used to characterize α IIb β 3 activation by GPIb mechano‐signaling by separating the ligand engagement of the two receptors in space and time.

Results: Using the stenosed microfluidic device, we found that the ‘biomechanical thrombosis’ is mainly supported by platelet integrin αIIbβ3 with an extended‐closed headpiece (EC) conformation (Panel A), which is distinct from the extended‐open headpiece (EO) integrins found in the ADP‐triggered biochemical thrombi (Panel B). Using dBFP on single platelets, we demonstrated that EC integrins have an affinity (Panel C) and bond lifetime (Panel D) for ligands that are intermediate between the well‐recognized inactive and active (ADP‐triggered) states. This intermediate state is inducted by mechano‐signaling of GPIb via a pathway distinct from that of chemical signaling of ADP receptors. This inside‐out mechano‐signaling also potentiates the outside‐in mechano‐signaling capacity of αIIbβ3, promoting its transition from the intermediate to the active state, resulting in mechanical affinity maturation.

Conclusions: Our work mapped the landscape of integrin αIIbβ3 activation in live platelet, and elucidated the different requirements αIIbβ3 upregulation in the development of biomechanical and biochemical thrombi.

PB097: In vivo Imaging Reveals the Regulation of Thrombus Formation by ERK and PKA Activity

T. Hiratsuka 1 ; T. Sano 2 ; M. Matsuda 3

1 Kyoto University, Graduate School of Medicine, Department of Drug Discovery Medicine, Division of Pathology, Kyoto, Japan, 2 University of British Columbia, Faculty of Medicine, Department of Urologic Sciences, Vancouver, Canada, 3 Kyoto University, Graduate School of Medicine, Department of Pathology and Biology of Diseases, Kyoto, Japan

Background: Dynamic features of thrombus formation have been visualized by conventional video wide field microscopy or confocal microscopy in live mouse. However, due to technical limitations precise spatio‐temporal regulation of intracellular signaling molecule activities, which have been extensively studied in vitro, remains elusive in vivo.

Aims: By two‐photon excitation microscopy of transgenic mice expressing Förster resonance energy transfer (FRET) biosensors for extracellular signal‐regulated kinase (ERK) and protein kinase A (PKA), we visualized ERK and PKA activities during thrombus formation in laser‐injured subcutaneous arterioles.

FIGURE 1 Mode of action of the FRET biosensor for ERK or PKA

Methods: We have already reported transgenic mice encoding the FRET biosensors which visualize ERK and PKA activities. Subcutaneous arterioles with a diameter of 10‐30 μm of these mice were subjected to observation. We injured endothelium of arterioles by laser beam and induced thrombus formation. Thus, we visualized spatio‐temporal regulation of ERK and PKA activity.

Results: When a core of densely packed platelets was developed, ERK activity was increased from the basal region near the injured arterioles. PKA was activated at the downstream side of an unstable shell that overlays the core of platelets. A MEK inhibitor suppressed platelet tethering and dislodged platelet aggregates, indicating that ERK activity is indispensable for both initiation and maintenance of the thrombus. A cAMP analog inhibited platelet tethering but failed to dislodge the preformed platelet aggregates suggesting that PKA can antagonize thrombus formation only in the early phase.

FIGURE 2 In vivo visualization of ERK and PKA activation at the bottom of thrombus

Conclusions: In vivo imaging of transgenic mice expressing FRET biosensors will open a new window to visualization of the spatio‐temporal changes of signaling molecule activities during not only thrombus formation but also other hematological disorders.

PB098: Role of Protease‐activated Receptor 4 in Regulating Platelet Dense Granule Release

T. Chu 1,2 ; R. Rigg 2 ; L. Healy 3 ; A. Ngo 2 ; A. Mitrugno 2 ; J. Aslan 2 ; A. Gruber 2,4 ; C. Lindsley 5,6 ; M. Duvernay 5 ; H. Hamm 6 ; O. McCarty 2,3,4

1 Johns Hopkins University, Department of Biomedical Engineering, Baltimore, United States, 2 Oregon Health & Science University, Department of Biomedical Engineering, Portland, United States, 3 Oregon Health & Science University, Department of Cell, Developmental & Cancer Biology, Portland, United States, 4 Oregon Health & Science University, Division of Hematology and Medical Oncology, Portland, United States, 5 Vanderbilt University School of Medicine, Department of Pharmacology, Nashville, United States, 6 Vanderbilt Center for Neuroscience Drug Discovery, Nashville, United States

Background: Human platelets express PAR1 and PAR4, G‐protein coupled receptors (GPCRs) that are activated by proteolytic cleavage to initiate intracellular signaling. PAR1 and PAR4 are cleaved by overlapping but distinct sets of proteases, leading to diverse functional outputs. For instance, the granulocyte releasate cathepsin G only cleaves PAR4, suggesting that PAR4 may play a unique role in mediating crosstalk between granulocytes and platelets.

Aims: Investigate the role of PAR4 activity on platelet dense granule release in the context of platelet‐granulocyte interactions.

Methods: Human washed platelets were isolated and treated with the agonists human α‐thrombin, human cathepsin G, or thrombin receptor activator peptide 6 in the presence of small‐molecule PAR4 or PAR1 inhibitors. To assay dense granule secretion, luminescence of an ATP‐luciferin‐luciferase reaction was measured by spectrophotometry. Platelet‐granulocyte interactions were assessed by electron microscopy and whole blood flow cytometry.

Results: Either PAR4 inhibitor VU0652925 or VU0661245 partially impaired platelet dense granule release stimulated by thrombin and fully blocked dense granule release stimulated by cathepsin G, whereas the PAR1 inhibitor SCH79797 did not impair dense granule release by either agonist. A combination of SCH and VU0652925 resulted in increased impairment of dense granule release by thrombin in comparison to VU0652925 alone. In human or baboon whole blood, platelet‐granulocyte interactions were stimulated by PAR4 activity and specifically blocked by PAR4 inhibition.

Conclusions: Our results demonstrate that PAR4 small‐molecule inhibitors impair platelet dense granule release by thrombin and cathepsin G. These initial results suggest a functional role for platelet PAR4 cleavage in platelet‐granulocyte interactions. PAR4 may be a therapeutic target for inhibition in diseases where platelet‐leukocyte interactions play a pathogenic role.

PB099: Modulation of Smooth Muscle Cell Phenotype by Platelet Adhesion to Endothelial Cells in Type 2 Diabetes Mellitus

M. Luo ; M. Zeng; Y. Lin; D. Fang; J. Wu

Southwest Medical University, Luzhou, China

Background: Vascular abnormalities in Type 2 diabetes mellitus (DM2) are often associated with the transition of vascular smooth muscle cell (VSMC) phenotype from differentiated (contractile) to proliferative (synthetic) as characterized by VSMC‐specific phenotypic markers. Further, platelet‐endothelial cell (EC) interactions play a major role in the pathogenesis of vascular complications in DM2.

Aims: While both platelets and ECs appear to contribute to phenotypic switching of VSMCs, the precise mechanisms by which platelet interaction with ECs affects VSMC phenotype, are unclear.

Methods: Using co‐culture cell models, we aimed to determine whether diabetic platelet adhesion to, or interaction with, ECs influenced proliferation, migration, and phenotypic switching of VSMCs using quantitative RT‐PCR, western blotting, scratch assays, CCK‐8, gel zymography and fluorescence microscopy.

Results: Our results demonstrated that a significantly greater number of DM2 platelets adhered to ECs compared to control platelets. DM2 platelet interactions with ECs correlated significantly with the down‐regulation of mRNA and protein levels for VSMC‐specific contractile phenotypic markers, including SM‐MHC, Smoothelin‐B, SMA andSM22a. In contrast, VSMC‐specific synthetic phenotypic mRNA and protein levels e.g . SMemb, PCNA, CCND1 and CCND2 were up‐regulated. Moreover, DM2 platelet interactions with ECs enhanced TGF‐β1‐induced PI3K/Akt/MMP9 signaling, leading to enhanced gelatinolytic activity and migration and proliferation of VSMCs.

Conclusions: Collectively, these findings illustrate that under diabetic conditions, interactions between platelets and ECs modulate the transition of VSMCs between a contractile and synthetic phenotype, conceivably contributing to the development of cardiovascular complications.

PB100: New Regulatory Mechanisms of RhoA in Platelets

S. Comer 1,2 ; Z. Nagy 3 ; S. Gambaryan 4 ; U. Walter 5 ; R. Zahedi 6 ; K. Jurk 5 ; A. Smolenski 1,2

1 UCD Conway Institute, UCD School of Medicine, University College Dublin, Dublin, Ireland, 2 Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin, Ireland, 3 Centre for Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom, 4 Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St Petersburg, Russian Federation, 5 Center for Thrombosis & Hemostasis, Universitätsklinikum der Johannes Gutenberg‐Universität Mainz, Mainz, Germany, 6 Jewish General Hospital, Proteomics Centre, Lady Davis Institute, Montréal, Canada

Background: RhoA, a Rho family small GTPase, acts as a bridge between extracellular signals and cytoskeletal restructuring of platelets during activation. Vascular endothelium‐derived prostacyclin (PGI2) and nitric oxide (NO) interfere with platelet activation via cAMP/cGMP‐dependent protein kinases (PKA/PKG). RhoA has been proposed as a direct PKA/PKG target but other proteins might contribute to PKA/PKG‐mediated RhoA regulation.

Aims: To identify Rho guanine nucleotide exchange factors (RhoGEFs) and Rho GTPase activating proteins (RhoGAPs) that convert PGI2/NO signals into RhoA inhibition.

Methods: Platelet phosphoproteome screening presented RhoGEF2 and RhoGAP6 as candidate PKA/PKG substrates. Further protein analysis included mass spectrometry, mutagenesis studies, pulldown assays, PhosTag and SDS‐PAGE/Western blotting in human platelets and transfected cells.

Results: We show that RhoGEF2 (also called GEF‐H1) and RhoGAP6 are expressed and targeted by PKA/PKG in human platelets. RhoGEF2 phosphorylation on serine 886 attenuates RhoA activation and stimulates RhoGEF2/14‐3‐3 complex formation. RhoGEF2 binds specifically to 14‐3‐3 β and η but not ε or γ isoforms. RhoGAP6 is phosphorylated on multiple sites within its C‐terminal region. RhoGAP6 strongly inhibits RhoA and cell adhesion. When co‐transfected, the ability of RhoGAP6 to inhibit RhoA supersedes the ability of RhoGEF2 to activate RhoA.

Conclusions: This data shows two new RhoA regulatory proteins in platelets, RhoGEF2 and RhoGAP6, which are novel targets for PGI2/NO‐induced control of platelet functions. This work highlights the complexity of RhoA signalling and supports the crucial role GEFs and GAPs play in mediating cyclic nucleotide effects in platelets.

PB101: Platelet CLEC‐2 Ligation Offers Critical Enhancement for ADP and Thrombin‐induced Activation via Synergy between Intracellular Signalling Pathways of CLEC‐2 and G‐protein Coupled Receptors

A. Martyanov 1,2,3 ; M. Panteleev 1,2,3 ; A. Sveshnikova 1,2,3

1 Lomonosov Moscow State University, Physics Faculty, Moscow, Russian Federation, 2 Center for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences, Moscow, Russian Federation, 3 National Medical Research Centre of Pediatric Hematology, Oncology and Immunology named after Dmitry Rogachev, Cell Hemostasis and Thrombosis, Moscow, Russian Federation

Background: CLEC‐2 is a platelet receptor with a proposed role in maintaining blood vessel integrity promoting leukocyte extravasation from microvessels during inflammation. However, standalone CLEC‐2 induced platelet activation has a significant lag phase and altogether weak platelet responses. Thus why CLEC‐2 ligation could be of significance in some conditions remains elusive.

Aims: To analyze the origin of the weak responses of platelets to CLEC‐2 stimulation as well as a model setting in which these responses could be critical.

Methods: We constructed a comprehensive 3‐dimensional reaction‐diffusion computational model, which describes CLEC‐2, P2Y1, P2Y12, PAR1, PAR4 ligation, receptor diffusion in the membrane and receptor‐induced signalosome assembly. Activation of calcium‐fluorophore loaded platelets by fucoidan, ADP and thrombin was studied by aggregometry, flow cytometry and TIRF microscopy.

Results: Based on modelling results it can be claimed that both Syk and SFK kinases phosphorylate CLEC‐2 cytoplasmic domain. CLEC‐2 induced cytosolic Ca2+ spiking was predicted by model and confirmed by TIRF microscopy. Flow cytometry assay, as well as TIRF microscopy, in line with the model prediction, proved signalling interplay between CLEC‐2 and ADP/thrombin (Gq and Gi) signalling pathways: platelet cytosolic calcium responses to low doses of ADP or thrombin dramatically increase in the presence of CLEC‐2 agonist. Surprisingly there are two merging points of the signalling cascades, those are PI3K and PLC activity.

Conclusions: Slow CLEC‐2 induced platelet activation occurs due to the need of signalosome assembly. In presence of ADP or low concentrations of thrombin it can be significantly enhanced via synergetic PI3K and PLC activation by Gq, Gi and tyrosine‐kinase signalling pathways. This synergy makes CLEC‐2 role critical for vessel wall protection in inflammation.

PB102: Identification of Platelet Intracellular Signaling Pathways Controlling the Reversible and Irreversible Platelet Aggregation

D. Morozova 1 ; A. Demianova 2 ; M. Canault 3 ; M. Panteleev 2 ; M.C. Alessi 3 ; A. Sveshnikova 4

1 Lomonosov Moscow State University, Faculty of Basic Medicine, Moscow, Russian Federation, 2 Center for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences, Moscow, Russian Federation, 3 Aix‐Marseille University, Inserm, Inra, C2VN, Marseille, France, 4 Lomonosov Moscow State University, Faculty of Physics, Moscow, Russian Federation

Background: Platelet integrin αIIbβ3 activation is a major response to stimulation leading to aggregation. Although reversible and irreversible platelet aggregation have been known for a long time, functional states of integrin molecules corresponding to these scenarios and signaling pathways that regulate them still remain unclear.

Aims: To investigate reversible and irreversible binding of fibrinogen to activated platelets and signaling mechanisms governing transition of platelets between these different activation states.

Methods: Blood was collected from healthy donors under a protocol approved by the institutional Ethics Committee with written informed consent. The state of platelet integrins was assessed by either aggregometry or binding of fibrinogen to platelets in flow cytometry and fluorescence microscopy. Multicompartmental computational model of platelet integrin activation was devised to describe experimental data. The model included intracellular signaling cascades of PAR1, PAR4, P2Y1, P2Y12, GP‐VI and αIIbβ3 receptors, calcium signaling module, phosphoinositide signaling. GTP‐bound Rap1b was considered as the main trigger for integrin αIIbβ3 activation.

Results: Weak and reversible binding of fibrinogen to platelets was observed under conditions of activation through a single receptor without secondary activation and outside‐in signaling. Reversible fibrinogen binding correlated with reversible platelet aggregation. In the absence of PI3K activation, an elevated intracellular calcium level correlated with reversible integrin activation. Activation of PI3K without calcium also induced weak reversible fibrinogen binding. High‐level‐fibrinogen binding correlated with clustering of integrins, shown by fluorescence microscopy. Binding of fibrinogen to clusters was irreversible and occurred whenever two different signaling pathways were activated.

Conclusions: Irreversible platelet aggregation is a consequence of platelet integrin αIIbβ3 clustering, and requires both calcium and phosphoinositide signaling.

PB103: S100A9 Activates Platelets via RAGE

S. von Ungern‐Sternberg ; K. Posavec; D. Heinzmann; M. Gramlich; M. Gawaz; P. Seizer

University Clinic of Tübingen, Tübingen, Germany

Background: S100A9 is a cytosolic protein, which belongs to the S100 family and can form heterodimers with S100A8. During inflammatory conditions S100A9 is secreted into the extracellular space where it can induce pro‐inflammatory properties. S100A9 is able to induce the migration of inflammatory cells, including monocytes. However, S100A9 is elevated in platelets and serum of patients with acute coronary syndrome. S100A9 is a ligand of several surface receptors, including TLR, RAGE and CD147.

Aims: The aim of this study was to investigate the effect of S100A9 in platelet activation via different receptors.

Methods: Flow cytometry, dynamic adhesion model.

Results: Human monocytes were analyzed for their surface expression of active alpha IIb beta 3 in a time dependent manner. Within 15 minutes there is a significant increase in platelet activation. Whereas S100A9 do not lead to an increase in P‐selectin expression and thus is not involved in the degranulation of alpha granules. An activation of murine platelets with S100A9 leads to an increase of CD147 surface expression and to increase of active alpha IIb beta 3. However, the activation of platelets seems to be dependent on the presence of RAGE because in RAGE knockout mice there is no increase in the active form of alpha IIb beta 3 on the platelet surface and thus no platelet activation. Nevertheless, in the dynamic adhesion assay over recombinant S100A9 antibodies against CD147 and RAGE were able to reduce the rolling of platelets. Possible due to the lack of P‐selectin expression, S100A9 is not involved in the formation of platelet‐monocyte aggregate formation.

Conclusions: The results of the current study show that S100A9 activates platelets via the receptors RAGE and CD147. S100A9 is involved in the integrin dependent activation of platelets but does not increase the surface expression P‐selectin.

PB104: Antidepressant Suppresses Platelet Activation via Collagen Receptor

N. Sugita 1 ; H. Hirakata 2 ; T. Murai 1

1 Kyoto University, Graduate School of Medicine, Psychiatry, Kyoto, Japan, 2 Kyoto University, Graduate School of Medicine, Emergency Medicine, Kyoto, Japan

Background: Selective serotonin reuptake inhibitors (SSRIs) are a class of most frequently prescribed antidepressants. Recent epidemiological studies have shown that SSRIs may increase the risk of abnormal gastrointestinal bleeding and may prevent ischemic heart disease events. However, the mechanism is unknown.

Aims: The aim of our study is to elucidate the mechanisms of SSRIs’ effects on human platelets.

Methods: We purchased pharmaceutical ingredients of S‐citalopram, paroxetine, fluvoxamine, sertraline, which are marketed SSRIs for clinical use, and those of R‐citalopram (the optical isomer of S‐citalopram) and metabolites of both citalopram isomers. Each material was resolved in platelet rich plasma (PRP) taken from healthy volunteers and incubated for 90 minutes at 37°C.

Platelet aggregation induced by ADP, collagen, arachidonic acid, epinephrine or STA 2 , was measured with a light transmission aggregometer. Collagen‐induced cytosolic calcium increase with/without S‐citalopram, R‐citalopram or the metabolites, was measured with fluorescent indicator fura‐2 and a cytosolic ion analyzing device. Expression of CD62P and activation of integrin αIIbβ3 after stimulation with collagen or convulxin with/without S‐citalopram were measured by flow cytometer. Binding of fluorescently labeled collagen with/without S‐citalopram were measured with a flow cytometer.

Results:

All SSRIs suppressed platelet aggregation induced by collagen significantly, but did not suppress platelet aggregation by the other agonists. The both citalopram isomers and the metabolites suppressed collagen‐induced cytosolic calcium increase. S‐citalopram suppressed collagen/convulxin‐induced CD62P expression and activation of integrin αIIbβ3. S‐citalopram suppressed binding of fluorescently labeled collagen (Figure 1).

FIGURE 1 Green dotted line:negative control (no FAM). Pink line: FAM‐labelled collagen without S‐citalopram. Gray area: FAM‐labelled collagen with S‐cit

Conclusions: SSRIs may suppress platelet activation by blocking collagen receptors.

PB105: Induced Apoptosis‐like Changes Caused by Sirtinol, a Pharmacologic Inhibitor of Sirt1/2, in Human Platelets

R.L. Mallick 1 ; S. Kumari 2 ; S.N. Chaurasia 2 ; D. Dash 2

1 Kathmandu University/Birat Medical College & Teaching Hospital, Biochemistry, Biratnagar, Nepal, 2 Banaras Hindu University/Institute of Medical Sciences, Biochemistry, Varanasi, India

Background: Sirtuins (like Sirt1 & Sirt2) regulate the facts related to cellular life‐span. They determine the balance between apoptosis, cell survival & cell proliferation. Sirtinol, a cell‐permeable six‐membered lactone ring, is a pharmacologic dual inhibitor of Sirt1/Sirt2.

Platelets are enucleate cells that are derived as cytoplasmic buds from precursor bone marrow megakaryocytes. These circulating cells express Sirt1/2.

Aims: As it is unclear whether sirtuins have a role in platelets, we aim to;

Investigate their regulatory roles in apoptosis‐like events and Evaluate the biological effect of sirtinol in isolated human blood platelets.

Methods:

Isolation of platelets from fresh human blood by differential centrifugation, Flow cytometric analysis of mitochondrial transmembrane potential (ΔΨm), measurement of reactive‐oxygen species (ROS) & annexin binding assay Western blot analysis.

Results:

The 10% SDS‐PAGE followed by incubation of a PVDF membrane (on which electrophoretically separated proteins were transferred) with antibodies demonstrated expression of Sirt1 & Sirt2 in human platelets, Washed platelets, pre‐incubated with sirtinol and preceded by incubation with the lipophilic cation JC‐1 (2 μM) provoked progressive dissipation of platelet ΔΨm with increasing concentrations, Pre‐treatment with sirtinol resulted in dramatic rise of annexin V binding in dose‐dependent manner; indicative of apoptosis‐like events in cells, Isolated platelets, pre‐treated with sirtinol followed by incubation with DCFH‐DA dye, revealed a dose‐dependent change in ROS.

Conclusions: Here we demonstrate that Sirt1 and Sirt2 are expressed in enucleate platelets. Further sirtinol enhances the annexin V binding and ROS production but drop in mitochondrial transmembrane potential that are the aspects related to apoptosis. On the basis of above, it is tempting to suggest that sirtinol enhances the apoptic‐like changes in human platelets.

PB106: Tumor Necrosis Factor Receptor Associated Factor 6 (TRAF6) in the Pathogenesis of Immune Thrombocytopenia

V. Asoglu; E. Umit ; M. Demir

Trakya University Faculty of Medicine, Hematology, Edirne, Turkey

Background: The immune system is controlled by multiple mechanisms that regulate tolerance and immunity. TRAF6, is a member of TRAF family which are intracellular proteins that transmit signals from cells to TNF receptors and by this mechanism, participate in the regulation of the immune system by experimental models.

Aims: In our study, we aimed to evaluate the role of TRAF6 signaling in the pathogenesis of immune thrombocytopenia (ITP).

Methods: 85 patients with ITP who have just been diagnosed not received treatment, 85 age and sex matched healthy controls were enrolled.

Results: Mean age of patients and controls were 45,72 (22‐87) and 47,68 (20‐72) years respectively. Mean platelet count in the patient and control group were 18.647/mm 3 (1.000‐92.000) and 253.800 (103.000‐424.000) respectively. Mean TRAF6 levels in the patient and control groups were 2348,51 (522,24‐6913,29) and 25,57 (6,5‐911) respectively. In patients with ITP, patients who have presented with major bleeding (gastrointestinal, nervous system vb) and patients with antinuclear antibody positivity have significant higher TRAF6 levels (P values 0.000 and 0.000 respectively).

Conclusions: Working as an activator of Transforming growth factor β activated kinase 1 (TAK1), a key player in the cellular responses and cascades, TRAF6 deficiency has been related with various disorders involving essential processes such as the maturation of dendritic cells, lymph node organogenesis, central tolerance of T cells. Increases in TRAF6 as an immune stimulant, have never been demonstrated in hematological diseases. In our study, we observed a dramatic significance in TRAF6 serum levels in patients with ITP regardless of platelet levels. Bleeding and antinuclear antibody positivity was significant in patients with high TRAF6 levels. Our study, though with a limited sample size, will lead to further studies regarding the importance of immunologic pathways in the pathogenesis of ITP.

BLEEDING DISORDERS

PB107: Patients with Aortic Stenosis Have von Willebrand Factor Abnormalities and Aberrant Angiogenesis in BOEC

S.N. Selvam 1 ; M. Bowman 2 ; M. Inglis 3 ; J. Grabell 2 ; L. Casey 2 ; L. Thibeault 2 ; A.M. Johri 2,3 ; P. James 1,2

1 Queen's University, Pathology and Molecular Medicine, Kingston, Canada, 2 Queen's University, Medicine, Kingston, Canada, 3 Queen's University, Biomedical and Molecular Sciences, Kingston, Canada

Background: Patients with aortic stenosis (AS) may bleed due to acquired von Willebrand syndrome and experience gastrointestinal bleeding from angiodysplasia. Von Willebrand factor (VWF) is a negative regulator of angiogenesis and loss of the largest multimers has been suggested to play a role in the development of angiodysplasia but the mechanism is unclear.

Aims: To examine the defects of VWF in plasma from patients with AS and the subsequent impact on angiogenesis using patient‐derived blood outgrowth endothelial cells (BOECs).

Methods: The study was approved by Queen's University ethics board. Twenty‐eight patients with severe AS undergoing valve replacement and 10 healthy controls were enrolled. Blood was obtained from patients prior to, 3‐5 days, and 6 months after surgery. At each time point, plasma VWF/FVIII levels, VWF:CB, and VWF:GPIbM were determined. VWF multimer patterns were also analyzed. BOECs were isolated and evaluated for VWF and angiopoietin‐2 (Ang‐2) storage and secretion. Angiogenic profiles of BOECs was assessed by proliferation and tubule formation.

Results: VWF levels were elevated in patients with AS compared to controls and significantly increased immediately after surgery (Figure 1A; P<0.0001). This was associated with significantly higher VWF:GPIbM (Figure 1B) and collagen binding (Figure 1C) after surgery (P<0.05). FVIII levels were also increased (Figure 1D; P<0.001). VWF multimer analysis indicated a loss of high molecular weight multimers prior to surgery. Normal multimer profiles were restored shortly after surgery. BOECs isolated from five patients prior to surgery had no difference in VWF and Ang‐2 storage or release compared to controls (Table 1). Forty‐eight hours after seeding, AS BOECs were almost twice as proliferative as controls (P<0.01) however, tubule formation in Matrigel did not vary after eight hours.

FIGURE 1 (A) VWF:Ag, (B) VWF:GPIbM, (C) VWF:CB and (D) FVIII:C of controls and patients prior to (AS‐A), 3‐5 days (AS‐B) and 6 months after surgery (AS‐C)

TABLE 1 VWF and Ang‐2 levels in BOECs’ media and lysates VWF:Ag (Mean±SD IU/mL) Ang‐2:Ag (Mean±SD ng/mL) BOEC Media BOEC Lysates BOEC Media BOEC Lysates Controls (n=10) 31±11 549±158 7±4 12±6 Aortic Stenosis ‐ A (n=5) 44±13 478±114 36±25 70±31 P value 0.099 0.411 0.206 0.075

Conclusions: Quantitative and qualitative VWF abnormalities associated with increased BOEC proliferation are observed in patients with AS.

PB108: High‐dose Dexamethasone Alters the Increase in IL‐16 Level in Adult Immune Thrombocytopenia

X. Zuo ; X. Wang; X. Li; L. Li; M. Hou; J. Peng

Qilu Hospital, Shandong University, Department of Hematology, Jinan, China

Background: Adult primary immune thrombocytopenia (ITP) is an autoimmune‐mediated hemorrhagic disorder which has a Th1 dominant profile. Several reports have demonstrated that interleukin‐16 (IL‐16) plays an important role in autoimmune diseases by regulation of recruitment and activation of CD4 + cell at sites with Th1 polarization. Similarly, the immune dysfunction in ITP patients may be attributed to IL‐16, suggesting the potential prognostic utility of IL‐16 in ITP. However, the roles of IL‐16 in ITP remain unknown.

Aims: The aim of this study is to investigate the roles of IL‐16 and the effects of high‐dose dexamethasone (HD‐DXM) on IL‐16 expression in ITP.

Methods: In this study, we investigated the differences in IL‐16 expression in the bone marrow and peripheral blood of ITP patients and healthy controls by ELISA. Furthermore, we detected changes in blood IL‐16 expression before and after the 4‐day HD‐DXM therapy. The mRNA expression of pro‐IL‐16, caspase‐3 and T‐bet in ITP patients with active disease and healthy controls were quantified by real‐time PCR.

Results: In patients with active ITP, bone marrow supernatant and plasma IL‐16 levels increased (P<0.05) compared with those of healthy controls (Figure 1). In the meantime, gene expression of pro‐IL‐16 and caspase‐3 in bone marrow mononuclear cells (BMMCs) and pro‐IL‐16, caspase‐3 and T‐bet in peripheral blood mononuclear cells (PBMCs) of ITP patients were increased (P<0.05) relative to those of healthy controls. HD‐DXM treatment reduced the plasma IL‐16 level and down‐regulated mRNA expression of IL‐16, caspase‐3 and T‐bet in PBMCs (P<0.05). The plasma IL‐16 levels were positively correlated with pro‐IL‐16, caspase‐3 and T‐bet mRNA expression (Figure 2).

FIGURE 1 Concentrations of IL‐16 in active ITP and controls (A,B), and in active ITP before HD‐DXM, after HD‐DXM and in healthy controls (C)

FIGURE 2 mRNA expression in active ITP patients and healthy controls. Correlation between plasma and mRNA expression levels of IL‐16 in active ITP

Conclusions: In summary, the abnormal level of IL‐16 plays an important role in the pathogenesis of ITP. Importantly, regulating Th1 polarization associated with IL‐16 by HD‐DXM therapy may represent a novel insight for immune modulation in ITP.

PB109: Progranulin Facilitates the Increase of Platelet Count in Immune Thrombocytopenia

X. Zuo ; Y. Yu; X. Li; M. Hou; J. Peng

Qilu Hospital, Shandong University, Department of Hematology, Jinan, China

Background: Immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disease characterized by excessive platelet destruction. Progranulin (PGRN) is emerging as a critical immune mediator involved in a variety of autoimmune disorders. However, its role in ITP remains unclear.

Aims: The aim of this study is to investigate the roles of PGRN in ITP.

Methods: The ELISA was used for determining the plasma levels of PGRN in ITP patients and controls, as well as in patients before and after treatment. The binding of PGRN on platelets was analyzed by immunofluorescence. The role of PGRN in ITP in vivo was investigated in two kinds of ITP murine models, passive‐transfer model and active severe combined immunodeficiency (SCID) models. Further, we explored whether PGRN functioned by affecting the number of T regulatory cells (Tregs) by flow cytometry.

Results: The plasma levels of PGRN were significantly elevated in ITP patients (n=52) compared to healthy controls (n=40), and the level of PGRN declined in patients after receiving treatment. There was a negative correlation between plasma PGRN levels and platelet count of ITP patients (Figure 1). The PGRN on platelets were not detected by immunofluorescence, suggesting no direct interaction between PGRN and platelets. The pretreated recombinant PGRN (rPGRN) effectively protected against anti‐mouse CD41‐induced ITP in a passive‐transfer murine model (Figure 2B). Administration of rPGRN increased platelet counts in SCID mice with active ITP (Figure 2C). Furthermore, PGRN deficiency decreased platelets (Figure 2A) and impaired proliferation of Tregs in the passive transfer ITP model. The data suggest that PGRN may exert a protective role in ITP by promoting Treg proliferation.

FIGURE 1 PGRN levels increased in plasma of ITP patients and decreased after treatment. PGRN correlated negatively with platelet count of ITP patients

FIGURE 2 PGRN facilitated the increase of platelet count in ITP mouse models (A, C). The pretreated rPGRN protected against anti‐CD41‐induced ITP (B)

Conclusions: Our study identifies PGRN as a new regulator involved in the pathogenesis of ITP.PGRN facilitates the increase of platelet count, which may be associated with the positive regulation of Tregs. This suggests the therapeutic efficacy of PGRN in ITP models and provides a potential strategy for the treatment of ITP.

PB110: The Aberrant Expression of microRNAs in B Lymphocytes of Patients with Immune Thrombocytopenia

X. Jia ; B. Zuo; Y. He

Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, China

Background: Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by a transient or persistent decrease in platelet count. The pathogenesis of ITP has proven complicated, involving CTL‐mediated platelet lysis, disturbed T helper cell response balance, dysregulated secretion of cytokines, etc. Among all of these pathological factors, autoreactive B cells that produce platelet antigen specific autoantibodies still take center stage. Altered expression of microRNAs (miRNAs) have been reported in patients with ITP. However, the levels of miRNAs in B cells and their effects on ITP remain unclear.

Aims: To investigate the expression profiles of five disease‐related miRNAs (miR‐144‐3p, ‐142‐3p, ‐3162‐3p, ‐320c and let‐7b‐5p) in CD19 + B cells of ITP patients and healthy controls. Target prediction and pathway enrichment analysis were performed.

Methods: The peripheral blood samples of 21 ITP patients and 21 healthy controls were obtained. The CD19 + B cells were isolated from PBMCs using immunomagnetic microbeads. The miRNA levels were detected by quantitative real‐time PCR and calculated using the comparative cycle threshold method.

Results: The expression of five miRNAs in CD19 + B cells of ITP patients and healthy controls were evaluated. The levels of miR‐3162‐3p and let‐7b‐5p were significantly increased in ITP patients compared to healthy controls (P=0.0341 and P<0.0001, respectively) (Figure 1). No significant difference for miR‐144‐3p, ‐142‐3p and ‐320c was found. The association between miRNAs and peripheral platelet count in ITP patients was also analyzed and no significance was found. Bioinformatics analysis indicated that the highest scoring functional pathways associated with ITP were cell cycle and thyroid hormone signaling pathway.

FIGURE 1 The expression of five miRNAs in CD19+ B cells of ITP patients and healthy controls

Conclusions: The aberrant expression of miR‐3162‐3p and let‐7b‐5p were verified in CD19 + B cells of ITP patients, which may contribute to ITP pathogenesis by targeting cell cycle and thyroid hormone signaling pathway.

PB111: A Novel Model using Protein C‐to‐INR Ratio to Predict Short‐term Mortality in Cirrhosis

A. Patil ; C. Bihari

Institute of Liver and Biliary Sciences, Clinical Hematology, New Delhi, India

Background: Chronic liver disease is characterized by decreased levels of both procoagulant and anticoagulant factors. Conventional coagulation tests cannot accurately assess risk of coagulopathy as they measure only procoagulant pathway. Protein C a physiological anticoagulant has predictive and therapeutic roles in sepsis. Plasma hypercoagulability has been shown to cause progression of fibrosis causing end‐organ dysfunction.

Aims: To assess relationship between natural anticoagulants, protein C (PC), protein S (PS) and anti‐thrombin III (AT) with MELD, Child‐Pugh (CP) and hepatic decompensation.

Methods: PC, PS and AT3 activity were analyzed in 598 cirrhotics and compared with 232 non‐cirrhotic controls. CP and MELD scores were used to assess severity of cirrhosis.

Results: Cirrhotics had lower PC (P<0.001), PS (P<0.001) and AT (P<0.001) than non‐cirrhotic liver disease. PC:INR ratio were significantly lower in cirrhotics (30.89±11.1) compared to non‐cirrhotics (57.99±12.64); P<0.001. Patients who died (n=182) at 3 months had significantly lower PC:INR ratio (15.9±6.08) compared with those who remained alive (52.9±19.9); P<0.001. Using multivariable analyses, low PC:INR ratio (P<0.001) and high MELD (P=0.038) were found to be independent predictors of mortality. A predictive model with PC:INR ratio had better discrimination (AUROC=0.84) than a model using MELD alone (AUROC=0.62) to predict 3‐month mortality. Also low PC correlated with liver dysfunction and inflammation: INR (r=−0.72, P<0.001), bilirubin (r=−0.620, P<0.001), albumin (r=0.539, P<0.001), creatinine (r=−0.417, P<0.001), ferritin (r=−0.68, P=0.035), procalcitonin (r=−0.79, P=0.01) and ESR (r=0.56, P<0.001). PC (but not INR or MELD) was independent predictor of hepatic encephalopathy (P=0.002) and ascites (P=0.048).

Conclusions: In cirrhosis low PC correlates with fibrosis, liver dysfunction, inflammation and sepsis and is an independent predictor of 3‐month mortality. This score using PC:INR ratio and MELD improves risk prediction of short mortality over MELD alone.

PB112: Increased Cytotoxic Potential of CD8 T Cells in Patients with Immune Thrombocytopenia

J.R. Vrbensky 1 ; I. Nazy 1,2 ; N. Ivetic 1 ; R. Clare 1 ; A.M. Jaffer 1 ; J.G. Kelton 1,2,3 ; D.M. Arnold 1,2

1 McMaster University, Michael G. DeGroote School of Medicine, Hamilton, Canada, 2 McMaster University, McMaster Centre for Transfusion Research (MCTR), Hamilton, Canada, 3 McMaster University, Pathology and Molecular Medicine, Hamilton, Canada

Background: Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by a platelet count <100×10 9 /L and antibody‐mediated platelet destruction. CD8 T cells may also be involved, but they have not been well characterized in ITP.

Aims: Measure the frequency and cytotoxicity of CD8 T cells in ITP.

Methods: Peripheral blood was collected from ITP patients (n=21), non‐immune thrombocytopenia patients (TC; n=18 with splenomegaly, liver disease, or familial thrombocytopenia) and healthy controls (HC; n=23). Mononuclear cells were isolated by density gradient centrifugation. CD4 and CD8 T cell frequency was determined via flow cytometry. T cells were stimulated for 5 hours with 5 μg/mL anti‐CD3 in the presence of anti‐CD107a. Surface expression of CD107a was measured on CD8 T cells via flow cytometry, indicating the release of cytotoxic granules containing perforin and granzyme. Granzyme B (GrB) in the culture supernatant was measured by enzyme immunoassay. Mann‐Whitney P < 0.05 were considered significant.

Results: Median platelet counts for ITP, TC, and HC groups were 36, 47, and 200×10 9 /L, respectively. ITP T cells consisted of 62±15% CD4 and 36±15% CD8 cells, which was similar to the TC group (71±11% CD4 and 28±11% CD8), and the HC group (63±10% CD4 and 34±9% CD8). 4 ITP patients had an elevated CD8 frequency (red line, 2SD above HC mean; Figure 1). CD107a expression was <1% on resting CD8 T cells. After anti‐CD3 stimulation, GrB levels correlated with CD107a on CD8 T cells (r=0.77). The proportion of CD8 T cells expressing CD107a in ITP (19±13%) was comparable to TC (15±10%; P=0.41) and higher than HC (11±6%; P=0.04). 6 ITP patients showed elevated CD107a expression after anti‐CD3 (red line, 2SD above HC mean; Figure 2) and 3 of these 6 patients had an elevated CD8 cell frequency.

FIGURE 1 CD4 and CD8 T cell frequencies

FIGURE 2 Surface expression of CD107a on CD3+CD8+ cells.

Conclusions: Consistent with the heterogeneity of ITP, a subset of patients displayed increased CD8 T cell frequency and cytotoxicity. This will contribute to thrombocytopenia if platelets or megakaryocytes are targeted.

PB113: Gene Expression of Vanin‐1 (VNN‐1) in Egyptian Adults with Immune Thrombocytopenia (ITP): A Single Center Experience

D. El Demerdash 1 ; R.E. Hussein 2 ; N. Abo Elsoad 3 ; M. Mattar 1 ; A. Ayad 1

1 Cairo University, Kasr Al Ainy School of Medicine, Internal Medicine, Clinical Hematology Unit, Cairo, Egypt, 2 Cairo University, Kasr Al Ainy School of Medicine, Biochemistry, Cairo, Egypt, 3 Tanta University, Clinical and Chemical Pathology, Cairo, Egypt

Background: The Distinguishing of self‐limited acute immune thrombocytopenia (ITP) from chronic ITP identify potential targets for therapeutic interventions. Overproduction free radical in the absence of adequate antioxidant defense may cause the generation of neo‐antigenic determinants. This may ultimately contribute to epitope spreading and the abrogation of self‐tolerance. Vanin‐1 (VNN1) has been proven to be an oxidative stress sensor, which regulates endogenous glutathione levels.

Aims: Find an association between overexpressed VNN1 in blood and ITP pathogenesis as well as progression to chronic ITP.

Methods: We investigated 40 Egyptian patients with immune thrombocytopenia as well as 10 healthy controls (HC) through history, physical examination, laboratory tests including CBC, reticulocyte counts, ESR, PTT, PT, virology markers; CMV IgM, EBV IgM, HCVAb, HBsAg and HBcAb, ANA, Lupus anticoagulant, anticardiolipine, H pylori antigen in stool and TSH and response to therapy. Vanin 1 gene expression by RT‐PCR.

Results: 40 ITP patients with mean age of 34.233±13.06 years. 6 of them were males and 34 females. With mean PLT count at presentation of 18.633±7.748×10 3 cells/cmm at presentation. Vanine 1 gene expression was significantly higher in ITP cases (8.115±5.348) transcripts/1 μg total RNA as compared with HC (1.08±0.171) transcripts/1 μg total RNA (P<0.001), there was no difference between chronic and acute ITP patients as well as responder and non‐responder ITP patients. Also, no significant correlation was found between vanine 1 expression and other studied parameters.

Conclusions: Our observations suggest that pathogenesis of ITP in Egyptian patients with ITP could be related to altered Vanin 1 expression levels but no relation to the chronicity of the disease or the response to therapy. However, these results should be interpreted with caution and further studies with larger samples sizes are required to confirm these findings.

PB115: Real‐life Bleeding Risk of Anti‐factor Xa Direct Oral Inhibitors; Does Prothrombin Complex Concentrate Work?

J. Graham 1 ; P. Gupta 1 ; R. Sivers 2 ; B. Badugama 1 ; D. Sutton 1 ; D. Chandra 1

1 University Hospitals of North Midlands, Clinical Haematology, Stoke‐on‐Trent, United Kingdom, 2 University Hospitals of North Midlands, Laboratory Sciences, Stoke‐on‐Trent, United Kingdom

Background: Outside of clinical trials, the management of life‐threatening bleeding in patients receiving anticoagulation with an anti‐factor Xa direct oral inhibitor (Xa‐DOAC) (apixaban, rivaroxaban, edoxaban) is currently dependent on non‐specific measures. National guidelines suggest consideration of non‐activated, 4‐factor prothrombin complex concentrate (PCC); with data supporting correction of in vitro clotting studies in healthy human subjects.

Aims: This audit of clinical practice at a large UK university teaching hospital aimed to assess the frequency of PCC usage, efficacy and complications in a real‐life cohort of patients receiving anticoagulation with a Xa‐DOAC.

Methods: PCC issue by the transfusion laboratory was recorded prospectively using specific flowcharts designed to aid laboratory decision making and record clinical reasoning. Management was crossed‐referenced against electronic clinical records by three individuals; to confirm indication for PCC, anticoagulant, additional PCC required, complications, length of stay and mortality at 30 days.

Results: Over a 7‐month period PCC was issued for patients receiving Xa‐DOAC in 34% of cases (30/89), as off‐label Octaplex ® 50 IU/kg up to 3000 units. Issue was associated with bleeding in 73% (22/30) and pre‐operatively in 27% (8/30). No additional PCC was given. There was a trend towards lower 30 day mortality in patients on Xa‐DOAC issued PCC for bleeding; VKA 15/32 (47%) versus Xa‐DOAC 7/22 (32%) (P=0.14), although mortality in those with intracerebral haemorrhage was higher; VKA 8/19 (42%) versus Xa‐DOAC 6/11 (55%) (P=0.27). There was no thrombosis or DIC noted in patients on Xa‐DAOC receiving PCC.

Conclusions: The issue of PCC for patients with life‐threatening bleeding receiving a Xa‐DOAC is a frequent occurrence. Despite being a non‐specific reversal agent, real‐life clinical data supports non‐inferiority to PCC use in patients receiving a VKA. Mortality in both groups remains high.

PB116: A 9 Year National Study of Patients with Acquired Haemophilia A

D. Thakkar 1 ; C. McDermott 2 ; C.L. Robertson 3 ; R.C. Tait 2 ; C. Bagot 2 ; J.R. Rodgers 1 ; H.G. Watson 3 ; M.M. Khan 3 ; R. Kerr 4 ; J. Ward 4 ; J. Craig 5 ; J.A.M. Anderson 1

1 Royal Infirmary of Edinburgh, Department of Haematology, Edinburgh, United Kingdom, 2 Glasgow Royal Infirmary, Department of Haematology, Glasgow, United Kingdom, 3 Aberdeen Royal Infirmary, Department of Haematology, Aberdeen, United Kingdom, 4 Ninewells Hospital and Medical School, Department of Haematology, Dundee, United Kingdom, 5 Raigmore Hospital, Department of Haematology, Inverness, United Kingdom

Background: Acquired Haemophilia A (AHA) is a rare bleeding disorder with high mortality. Clinical manifestations vary from mild to life threatening bleeding and treatment includes haemostatic management and immunosuppressive therapy (IST).

Aims: To profile our management practices and outcome for AHA patients in Scotland.

Methods: Retrospective analysis of patients with AHA managed across Scotland from January 2008‐December 2017 was performed. Data obtained from hospital records included demographics, clinical presentation, laboratory parameters, management and outcome.

Results: Of the 73 AHA patients, 3 were post‐partum (age 35‐37 years). For the remaining patients M:F ratio was 4:5, median age 77 years (35‐91.3). Mean duration of follow‐up 30.35 months (0 days‐155.7 months). 37 patients were alive at last follow‐up.

The most common bleeding category at diagnosis was skin and soft tissue (59%) then genitourinary (15.1%). The most common nature of bleeding was spontaneous (57.5%) then prolonged bleeding post‐trauma (18.1%); one patient had intracranial haemorrhage. Laboratory parameters at diagnosis are as in Table 1.

TABLE 1 Laboratory parameters at Diagnosis Laboratory parameter Number of patients Median value Range aPTT (seconds) 72 72 36.4‐>210 factor VIII (iu/ml) 73 0.03 0‐0.3 Inhibitor titre (BU/ml) 70 10.6 <0.4‐1154

Information on management of bleed and inhibitors was available for 65 patients. Of these 69% received haemostatic management (Table 2) and 97% received IST (one or combination of steroid, cyclophosphamide, rituximab, iv immunoglobulin, mycophenolate mofetil, azathioprine, cyclosporin). Of the 67 patients whose remission status was known, 76% achieved remission. Of the non‐remitters 3 are alive, on treatment and follow‐up. Mean duration to achieve remission was 16.6 weeks (0.5‐198).

TABLE 2 Information on management of bleed Characteristic Number of patients [n (%)] Information available for haemostatic management 65 Haemostatic management received 45 (69.2) rVIIa alone 6 (13.3) FEIBA alone 23 (51.1) Both Bypassing agents sequentially 7 (15.5) rFVIII alone 4 (8.8) rFVIII with one of the Bypassing agents 5 (11.1) Procedural intervention for management of bleeding 8 (12.3) Red cell transfusion 24 (36.9)

Among the deceased patients, death was attributable to AHA related bleeding in 13.8%, immunosuppression in 8.3% patients. Cause of death was unknown in 33.3% patients.

Conclusions: The demographic profile of our cohort was similar to that reported from the UKHCDO study (Collins P et al.); the number of patients receiving haemostatic treatment and IST was in keeping with the UKHCDO study and the EACH2 registry report (Knoebl P et al.)

PB117: Is the Profile of Patients with Intracranial Hemorrhage Changing?

S. Reeves 1 ; C. Gray 2 ; J. Blundell 2

1 University of Exeter, Truro, United Kingdom, 2 Royal Cornwall Hospital, Truro, United Kingdom

Background: Intracranial hemorrhage (ICH) is a devastating complication of oral anticoagulation therapy (OAC).Vitamin K (VK) and prothrombin complex concentrates (PCC) reverse warfarin but this may not translate into good outcomes. Most Direct Oral Anticoagulants (DOACs) have no specific antidote but PCC is used in life threatening hemorrhage.

ICH increases with age. It is reduced with DOACs compared to VK antagonists (VKAs). In Cornwall these are increasingly used in preference to VKAs.

Aims: Identify the number of prescriptions for OACs in Cornwall between 2015 and 2017 and the OAC patients were taking at the time of ICH.

Establish demographics, immediate and 6 month mortality of those presenting to the Royal Cornwall hospital (RCH) with ICH over this period.

Review time from CT (computerized tomography scan) request to VK administration and PCC issue.

Methods: A prospective database of anticoagulation related ICH from September 2015 to September 2017 was created. This included demographics, INR, CT time, VK, PCC administration, immediate and 6 month mortality. Prescription data was obtained from our commissioning group.

TABLE 1 All anticoagulants prescribed and number of intracranial bleeds Anticoagulant Warfarin Sinthrome Apixaban Rivaroxaban Dalteparin Dabigatran Total Number of bleeds September 2015‐16 45 1 5 4 3 1 59 Number of bleeds September 2016‐17 35 0 4 16 3 0 58 Total number of prescriptions issued September 2015 12,402 8 558 2,996 408 469 16,841 Total number of prescriptions issued August 2016 11,853 11 1,264 4,418 395 473 18,414 Total number of prescriptions issued August 2017 10,906 7 2,438 5,754 369 550 20,024

Results: The number on OACs has increased ‐the number treated with a DOAC has increased and Warfarin prescribing has fallen. Despite increased use of DOACs the number of ICH is stable, 59 in year 1, 58 in year 2 (see table).

68% were male. 3% under 60, 49% between 60 and 80 and 48% were over 80. Of the total number 32% died on that admission and a further 23% died within 6 months. Only 29% on warfarin had an INR >4.0.

In total 59% were given PCC and VK. 19% of these were on DOACs. Mean time to PCC was 103 minutes and mean time to VK administration was 95 minutes.

Conclusions: Despite a therapeutic INR and timely PCC and VK mortality is high supporting the importance of ongoing risk/benefit assessments in individuals.

We anticipate more ICH with DOAC's as they are prescribed more to older people.

There may be a role for measuring drug levels so expensive reversal agents are used appropriately.

PB118: Hospital Admissions due to Bleeding Events under Direct Oral Anticoagulants

V. Grilo 1 ; A.G. Fonseca 2

1 Hospital Garcia de Orta, Internal Medicine, Almada, Portugal, 2 Hospital Garcia de Orta, Almada, Portugal

Background: The novel oral anticoagulants (NOAC) are an alternative to vitamin K antagonists for long‐term treatment and prevention of venous thromboembolic events (VTE) and stroke in non‐valvular atrial fibrillation (AF). Despite having a favorable safety profile, clinical relevant bleeding complications is still a concern.

Aims: We aimed to characterize bleeding events requiring hospital admission in patients on NOAC.

Methods: A retrospective study consisting on the analysis of 1 year hospital admissions due to bleeding event while on NOAC anticoagulation was conducted at a tertiary hospital (2017); 51 patients were included. Based on the clinical files, we characterized epidemiologic data, patient comorbidities, the bleeding event and its management. Thrombotic and bleeding risks were scored using CHA2DS2‐VASc and HAS‐BLED for AF, Riete Registry Bleeding Score for VTE. We used IBM SPSS Statistics version 23. Chi‐Square, Wilcoxon tests were used; significance level was 0.05.

Results: The median age of patients was 81 years (IQR 13), 52.9% were men. The main indication for NOAC use was AF (90.2%), with a CHA2DS2‐VASc score ≥2 in 97.8%, and a HAS‐BLED score ≥3 in 69.9%. NOAC dosing was adequate in 34 patients (66.7%). Major bleeding occurred in 37 patients (82.4%); 14 (27.5%) had intracranial bleeding. Major bleeding events had lower hemoglobin levels at admission (median 12.7 mg/dL, IQR 3.2, vs. 8.6 mg/dL, IQR 6.9, P<0.001). In 28 patients (54.9%) a new underlying diagnosis was made. In‐hospital mortality was 25.5%; median admission GFR was significantly lower than median baseline GFR (60 mL/min/m 2 , IQR 44.5 vs. 66.5 mL/min/m 2 , IQR 29.5 P=0.001).

Conclusions: In an elderly, high‐hemorrhagic risk population, there is a need for better surveillance of GFR and control of bleeding risk factors, as major bleeding events may be potentially fatal. A clinical relevant bleeding may trigger a new diagnosis.

PB120: Noonan Syndrome and Bleeding Disorders: An Overview of Current Diagnostic Management

P.E.G. van derPas 1 ; E.J. Huisman 2 ; I.H.I.M. Hollink 1 ; C.M. Zwaan 3 ; C.H. van Ommen 4 ; M.H. Cnossen 2

1 Erasmus University Medical Center, Rotterdam, the Netherlands, 2 Erasmus MC‐Sophia, Pediatric Hematology, Rotterdam, the Netherlands, 3 Erasmus MC‐Sophia, Pediatric Oncology, Rotterdam, the Netherlands, 4 Erasmus MC, Pediatric Hematology, Rotterdam, the Netherlands

Background: Noonan Syndrome (NS) is a genetically defined syndrome characterized by a range of physical‐ and developmental symptoms which require surgical interventions. However, NS is also associated with a variety of bleeding disorders. Current guidelines advise only screening by APTT, PT, FXI preoperatively or at 5 years of age.

Aims: To investigate diagnostic management of bleeding disorders in children with NS.

Methods: Retrospective cohort study.

Results: A total of 32 children with NS were identified. Genetic mutations were known in 30 children. A cardiac abnormality was detected in 27/32 (84%), most often pulmonary stenosis (23/27).

Hemostatic testing was performed in 31/32 children. Screening by APTT, PT and FXI was performed in 22 of 32 children (69%), in three only FXI was tested. Three of these 25 had a mild FXI deficiency, with two <0.4 U/L. In addition, nine patients had FIX levels below 0.6 U/L, 7 of which were <0.5 U/L. Moreover, primary hemostatic defects were frequently observed. One third of patients had mildly decreased von Willebrand levels, only reflected in PFA‐values. Platelet aggregation was performed in only 5 of the 32 patients and abnormal in X (60%). A bleeding tendency was reported in 7/32 patients (22%), especially bruising and epistaxis. A surgical intervention was performed in 23 patients (72%). Perioperative bleeding was reported in 4 of these 23 patients. Nine of them required a total of 12 red cell, platelet and plasma transfusions.

Conclusions: Current guidelines in the Netherlands are followed in majority of cases. However, FXI deficiency was rarely reported in NS and often not clinically relevant. In contrast, mild FIX deficiency and primary hemostatic defects were detected more frequently. We propose an update of guidelines with and inclusion of hemostatic testing of FIX, von Willebrand factors and platelet function.

PB121: Long‐Term Follow‐up of Children on Prophylaxis for Severe Factor VII Deficiency in Lebanon

R.A. Farah 1 ; H.Z. Farhat 2 ; D. Buthiau 3 ; M. Giansily‐Blaizot 3

1 Saint George Hospital University Medical Center, Department of Pediatrics, Beirut, Lebanon, 2 LAU‐Rizk Hospital University Medical Center, Department of Laboratory Medicine, Beirut, Lebanon, 3 CHU de Montpellier, Laboratory of Hematology, Montpellier, France

Background: Congenital Factor VII deficiency is the most common autosomal recessive bleeding disorder. Severe forms are associated with missense and consensus splice site mutations. The F7 :c.291+1G>C splice site mutation, seen in consanguineous marriages, has resulted in high prevalence of severely affected FVII‐deficient patients in Lebanon.

Aims: To evaluate the efficacy, safety, impact on morbidity and mortality of long‐term prophylaxis in a series of Factor VII‐deficient patients in Lebanon.

Methods: We present a case series of 5 Lebanese children with severe factor VII deficiency, all carrying the F7 :c.291+1G>C splice site mutation in homozygosity. Patients were followed closely and bleeding episodes recorded and classified according to the Tosetto bleeding score. The occurrence of antibodies to FVII was assessed using standard methods.

Results: 5 children from 4 families presented with GI bleeds (n=4) or a CNS bleed (n=1) within two months of birth (range 1‐40 days). Without prophylaxis, 2 families had lost affected children due to severe bleeding. Prophylaxis with recombinant Factor VII was implemented using 25‐30 μg/kg, 3 times per week. Follow‐up ranged from 1 to 11 years. No antibodies to FVII were detected. No major bleeds were reported with prophylaxis, however additional recombinant Factor VII was used in case of trauma or minor bleeds. Two surgical procedures were carried out during prophylaxis: a ventriculo‐peritoneal shunt placement and a port‐a‐cath insertion. Recombinant Factor VII was administered prior to and after surgery. No bleeding was seen.

Conclusions: 5 individuals with the F7 :c.291+1G>C mutation in homozygosity were found, enabling the largest series of Lebanese factor VII‐deficient pediatric patients to be given long‐term prophylaxis and follow‐up. Prophylaxis impacted positively survival and quality of life. It showed safety and efficacy in controlling bleeding episodes.

PB122: Clinical Presentations and Diagnostic Algorithm of Neonatal FXIII‐deficiency

M. Naderi 1 ; N. Cohan 2 ; G. Miri‐Aliabad 1 ; M. Karimi 2

1 Zahedan University of Medical Sciences, Genetics of Non Communicable Disease Research Center, Zahedan, Islamic Republic of Iran, 2 Shiraz University of Medical Sciences, Hematology Research Center, Shiraz, Islamic Republic of Iran

Background: Clinical specifications of FXIII deficiency (FXIIID) in neonates of very young has not been studied previously.

Aims: In present study, we assessed clinical picture and the rate of intracranial hemorrhage (ICH) in Iranian neonates with FXIIID who presented with bleeding diathesis in the early days of their lives.

Methods: A total of 27 neonates presented with bleeding presentations early in life and diagnosed as a FXIIID were evaluated as a study group during March 2010 to March 2015. All clinical and demographic data were recorded by a designed questionnaire. Either cryoprecipitate or factor XIII concentrate was started as a first‐line treatment strategy and prophylactic therapy was given to all patients.

Results: Umbilical cord bleeding (UCB), Delayed detachment of umbilical stunt, seizure, hematoma, and ecchymoses were concurrent complication in 27 (100%), 5 (18.5%), 5 (18.5%), 3 (11.1%), and 1 (3.7%) of the patients respectively. Trp187Arg (exon 4, C.559T>C) mutation was the only FXIIID mutation which was found in all patients. History of central nervous system (CNS) bleeding was detected in 13 (48.1%) patients. There were no significant association between CNS bleeding history and sex, familial history of FXIIID, or other clinical presentations. Also there was no significant difference in mean age of patients with CNS bleeding (3.4±0.9 days) and without CNS bleeding (2.9±0.7 days). However, a near significant threshold difference was found between the groups regarding the mean number of suspicious death in family (1.8±0.5 and 0.7±0.1 respectively, P=0.05).

Conclusions: Relatively high rate of ICH in neonates with FXIIID necessitates early consideration of appropriate factor replacement therapy in these patients. So, we recommended a diagnostic algorithm for detection of FXIII deficiency in early life.

PB123: Effectiveness of Pediatric Bleeding Questionnaire in Hemorrhagic Disorders

A.C. Ramirez Cazares 1 ; L.M. Nuño Vazquez 1 ; J.E. Colunga Pedraza 1 ; D. Gomez Gonzalez 1 ; M. Quiroga Treviño 1 ; L. Villarreal Martinez 2

1 University Hospital ‘Dr Jose Eleuterio Gonzalez’, Monterrey, Mexico, 2 University Hospital ‘Dr Jose Eleuterio Gonzalez’, Hematology, Monterrey, Mexico

Background: The study of hemorrhagic symptomatology can be particularly difficult in children where questionnaires adapted to pediatric patients such as the Pediatric‐Specific Bleeding Score (PSB) prove their usefulness.

Aims: Determine the effectiveness of the PSB questionnaire in bleeding disorders.

Methods: There were included all patients with abnormal bleeding in the last 6 months referred to the hematology service of the University Hospital “Dr. José Eleuterio González”. They were initially evaluated with the PSB and those with a positive result were additionally assessed with specific coagulation tests.

Results: A total of 26 children were analyzed, whose median age was 7 years old (10 months to 15 years), none of them with family history of hemorrhagic disease. In order of frequency clinical manifestation were: epistaxis (61.5%), metrorrhagia (11.5%), epidural hematoma (7.7%), dental bleeding (7.7%), excessive bleeding from trauma (3.8%), surgical bleeding (3.8%), prolonged aPTT (3.8%). The laboratory tests reported alterations in 8 male patients (30%): 5 hemophilia A mild to moderate confirmed by genetic mutations, 1 factor XI deficiency, 1 positive lupus anticoagulant, and 1 prolongated PT and aPTT. Median bleeding score in children with hemorrhagic disease was 8.5 (range 3‐13) and in the healthy group was 4 (range 1‐11). No patients with less than 3 points showed alterations in tests.

Conclusions: The PSB provides a scoring system and lays a groundwork. This could be particularly helpful in limited resource countries with difficult access to advanced laboratory techniques. Despite Von Willebrand Disease (VWD) is the most prevalent inherited bleeding disorder reported in the literature, no case was diagnosed. The limitation of this study is the number of patients recruited, however according to other research regarding Hispanic population also with small groups of patients there seems to be a tendency to a lower incidence of VWD.

PB124: Surgical Management of Rare Factor Deficiencies

N. Eroglu 1 ; E. Erduran 1 ; A. Bahadır 1 ; H. Saruhan 2

1 Karadeniz Technical University, Pediatric Hematology Oncology, Trabzon, Turkey, 2 Karadeniz Technical Universitiy, Pediatric Surgery, Trabzon, Turkey

Background: Rare factor deficiencies (RFD) include fibrinogen defects and factor (F) II, FV, combined FV and FVIII, FVII, FX, FXI, and FXIII deficiencies.

Aims: We report demographic features and surgical approaches of RFD. We retrospectively evaluated 37 patients with RFD. Patient characteristics, age at presentation and the causes of surgical intervention, preparation for surgery, preoperative replacement therapies were recorded. Surgical interventions were classified as major and minor operations.

Methods: Surgical intervention was implanted in 27 surgical interventions of 37 RFD patients. 64.8% of the patients were male and 35.2% female. Parental consanguinity was determined in 48.6% of cases. Patient information is presented in Table 1. Of the surgical interventions, 18 (66.6%) were major and 9 (33.3%) were minor. Information of the patients who underwent major and minor surgery is presented in Table 2.

Results: FFP was used for individuals with factor V, factor XI and factor XIII deficiencies and for patients with factor X deficiency. Tranexamic acid was used as the antifibrinolytic agent. FFP was used for individuals with factor V, factor XI and factor XIII deficiencies and for patients with factor X deficiency; in FVII deficiency, r FVII a; in fibrinogen deficiency, fibrinogen concentrate was given. Tranexamic acid was used as the antifibrinolytic agent. Post‐surgical haemostasis of the patients were determined by physical examinations, whole blood count and haemostasis tests.

Conclusions: Minor surgeries, were performed successfully with 1 day replacement therapy and 3 days of antifibrinolytic support, whereas it is not possible to describe a general treatment scheme for major surgeries. We believe that the above procedures should be applied for the treatment and in preparation for surgery of RFDs. We wish to emphasize that pre‐operative screening is important because of the late diagnosis of asymptomatic cases since the clinical findings of RFDs are not obvious.

TABLE 1 Patients’ Demographic Characteristics Fibrinogen Deficiency Fibrinogen + FXIII Deficiency FV Deficiency FV + FVIII Deficiency FVII Deficiency FX Deficiency FXI Deficiency FXIII Deficiency Total Patients 3 (8.1%) 2 (5.4%) 5 (13.5%) 1 (2.7%) 11 (29.7%) 4 (10.8%) 6 (16.2%) 5 (13.5%) 37 Sex: Male/Female 2/1 ‐/2 4/1 ‐/1 10/1 ‐/4 5/1 3/2 24(64.8%) / 13(35.2%) First Bleeding <1/1‐5/>5 year 3/‐/‐ 2/‐/‐ 1/‐/4 ‐/‐/1 2/2/7 1/3/‐ ‐/3/3 ‐/5/‐ 9(24.3%) / 13(35.1%) / 15(40.5%) Consaguinity 3 2 1 1 4 2 4 1 18(48.6%) Factor Activity <5/5‐30/30‐50% ‐/‐/3 2/‐/‐ ‐/3/2 ‐/‐/1 ‐/7/4 2/2/‐ 2/2/2 Not Determined 6/14/12 ClinicalSymptom Grade: 1/2/3 ‐/2/‐ ‐/‐/2 ‐/1/4 ‐/1/‐ 7/‐/4 1/1/2 2/‐/4 1/1/3 11(29.7%) / 5(13.5%) / 21(56.7%) TABLE 2 Major Surgeries+Minor Surgeries F: Female, M: Male, F:C: Factor coagulant activity, FFP: Fresh frozen plasma, rFVIIa: Recombinant factor VIIa Major Surgeries Sex Age of Operation Factor Deficiency F:C Replacement Therapy Used Replacement Therapy Dose Dose Interval Total Number of Doses Tranexamic Acid Use/Consaguinity Developmental Hip Dysplasia Surgery F 1 Fibrinogen 10 mg/dl Fibrinogen concentrate 50 mg/kg 24 h 6 ‐/+ Post‐op ASD Surgery F 1 Fibrinogen+FXIII 10 mg/dl+52% Fibrinogen concentrate + FFP 50 mg/kg+15 ml/kg 24 h+12 h 14+28 ‐/+ Circumcision M 6 FV 8.5% FFP 15 ml/kg 12 h 8 iv 10 mg/kg/ ‐ Circumcision + Adenoidectomy/ Myringotomy M/M 9/15 FVII 51%/36% ‐/‐ ‐/‐ ‐/‐ ‐ iv 10 mg/kg/ +/+ Adenoidectomy / Adenoidectomy/ Adenoidectomy + Myringotomy/ Circumcision + Undescended Testis Surgery/ Humerus Fracture Surgery/ Circumcision M/M/M/ M/M/M 12/4/3/1/6/8 FVII 32%/ 22%/ 28%/ 20%/ 7%/ 8% FFP/rFVIIa/ rFVIIa/ rFVIIa/rFVIIa/ rFVIIa 15 ml/kg‐ 30 U/kg‐30 U/kg‐ 30 U/kg‐30 U/kg‐30 U/kg 4‐6 h 6/1/1/2/4/2 iv 10 mg/kg/ +/‐/+/‐/‐/‐ Intracerebral Hematoma Evacuation + Duraplasty F 1 FX 13% FFP 15 ml/kg 12 h 60 ‐/+ Minor Surgeries Dental M/M/F/F 12/2/6/8 Fibrinogen 74/10/10/15 mg/dl Fibrinogen 30 mg/kg 24 h 2/1 po 25 mg/kg/d +/+/+/+ Minor Surgeries Dental M/M/F 16/4/5 FV 6%/13%/5% FFP 15 ml/kg ‐ 1 po 25 mg/kg/d ‐/‐/_ Minor Surgeries Dental M/M 3/4 FXIII Not Determined FFP 15 ml/kg ‐ 1 po 25 mg/kg/d ‐/+

PB125: Assesment of Thirty Six Patients with Rare Factor Deficiency: Single‐centre Results from Turkey

M. Söker 1 ; H. Uzel 1 ; K. Öncel 1 ; S. Söker 2

1 Dicle University, Pediatric Hematology and Oncology, Diyarbakır, Turkey, 2 Dicle University, Histology and Embriology, Diyarbakır, Turkey

Background: Rare factor deficiencies (RFDs) are coagulation factor deficiencies that ranging prevalence from 1/500.000‐1/2.000.000 and predominantly autosomal recessive inheritance. It constitutes 3‐5% of hereditary coagulation factor deficiencies.

Aims: In this study, we aimed to evaluate the demographic features and clinical findings of patients who were followed up and treated with RFDs.

Methods: A total of 36 patients, aged between 0 and 16 years, who had been treated and followed‐up with RFDs were included in the study. Demographic characteristics, clinical follow‐up results and laboratory data of patients were recorded.

Results: RFDs were diagnosed in 36 (35%) patients of the 108 patients who were followed up and treated with coagulation disorders. 20 of the patients were female and 16 were male. The age of admission to the hospital is ranged from 2 days to 16 years. Familial consanguinity was present in 75.6% and family history was 16.6% of the patients. 11 (32.4%) patients were diagnosed before the age of one. 14 patients were diagnosed with FX deficiency (38.8%), eight patient had FVII deficiency (22%), four patient had fibrinogen deficiency (11%), three patient had FXI deficiency (8.3%), two patient had FV deficiency (5.5%), two patient had FXIII deficiency (5.5%), one of them had combined deficiency of factors associated with vitamin K (Factor II, FVII, FIX, FX deficiency) (2.7%), one of them had FXII deficiency (2.7%), and one of them had combined deficiency of FV and FVIII (2.7%). Mucocutaneous bleedings (50%) were seen as most common admission symptoms at the time of diagnosis. Intracranial haemorrhage (ICH) was seen in five patients with inherited FX deficiency and in two patients with FXIII deficiency.

Conclusions: Early diagnosis and treatment is very important in the case of RFDs due to the severe bleeding complications such as ICH.RFDs are more common in place where familial consanguinity than the general population. It′s a necessity that informing parents about this and perform family screening earlier.

PB126: Anti‐fviii igg4 Antibodies in Patients with Hemophilia A without Inhibitors during Factor VIII Replacement: Results from the Hemfil Cohort Study

L.M. Moura de Oliveira 1 ; D. Goncalves Chaves 2 ; L. Lemos Jardim 1 ; A.C. Simoes e Silva 1 ; M.A. Portugal Santana 3 ; C. Carm‐Deelder 4 ; S. Meireles Rezende 1

1 Universidade Federal de Minas Gerais, Faculdade de Medicina, Belo Horizonte, Brazil, 2 Fundação Hemominas, Research Area, Belo Horizonte, Brazil, 3 Fundação Hemominas, Ambulatory, Belo Horizonte, Brazil, 4 Leiden University Medical Center, Leiden, the Netherlands

Background: Patients with haemophilia A (PHA) can develop inhibitors after exposure to factor (F) VIII due to stimulation of the immune system, which is still not fully understood. Previous studies have demonstrated strong association between the presence of inhibitors and anti‐ FVIII‐IgG4) antibodies. However, studies targeting antibody response during FVIII replacement are still lacking.

Aims: This study aimed to evaluate the levels of FVIII‐IgG4 in the plasma of PHA throughout FVIII replacement therapy.

Methods: PHA were included at the moment of diagnosis. Plasma samples were collected at different exposure days (ED) to FVIII for inhibitor screening and by Bethesda and FVIII‐IgG4 by ELISA. Plasma pool of 20 healthy age‐matched boys was used as negative control (NC). Results were expressed as optical density (OD) and positive IgG4 was considered when the level was higher than the NC OD plus two standard deviations. Wilcoxon matched‐pairs signed‐ranks test was used to compare the IgG4 OD at the moment of diagnosis (T0) and at >75ED (median, 113.0; IQR 79.0‐129.3) ED. The study was approved by Ethics Committees and patients signed a written informed consent.

Results: Plasma samples (n=28) from 14 severe (FVIII<1%; n=12) and moderately‐severe (FVIII=1%‐2%; n=2) PHA without inhibitors were included. Levels of anti‐FVIII‐IgG4 increased progressively from T0 reaching its highest in the strata >75DE (T0 median, 0.108 OD; IQR 0.035‐0.174 vs. >75ED median, 0.041 OD; IQR 0.026‐0.053, P=0.0012).

Conclusions: Our results suggest that anti‐FVIII‐IgG4 increases throughout the course of FVIII replacement therapy in PHA without inhibitors.

PB127: Plasma Profile of Platelet Auto‐antibodies in Patients with Thrombocytopenia in Pregnancy

W. Shen 1 ; H. Qin 2

1 Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, China, 2 The First Affiliated Hospital of Soochow University, Department of Obstetrics and Gynaecology, Suzhou, China

Background: Immune thrombocytopenia occurs when antibodies targeting specific glycoproteins (GPs) on the platelet surface lead to their destruction. Thrombocytopenia, defined as a platelet count <150×10 9 /L, is second only to anemia as the most common hematologic abnormality encountered during pregnancy. Immune thrombocytopenia (ITP) is the most common cause of a platelet count below 50×10 9 /L detected in the first and second trimesters.

Aims: To evaluate plasma levels of platelet auto‐antibodies in patients with thrombocytopenia in pregnancy and assess the clinical significance.

Methods: A total of 54 pregnant women with thrombocytopenia, 44 normal pregnant women and 28 women with ITP, as well as 31 healthy women were enrolled in this study. The plasma levels of platelet auto‐antibodies against 5 platelet glycoproteins (GPIX, GPIb, GPIIIa, GPIIb and P‐selectin) were detected by flow cytometric immuno‐beads array. The difference was analyzed statistically, and the receiver operating curve (ROC) was constructed to assess the diagnostic value.

Results: Compared with normal pregnant women and healthy controls, the plasma levels of auto‐antibodies against glycoprotein GPIX and GPIb were significantly increased in pregnant women with thrombocytopenia (P<0.05), while the levels of other 3 auto‐antibodies were similar. The positive rate of auto‐antibodies in pregnancy women with thrombocytopenia was higher than that in normal pregnant women (59.3% vs. 31.8%, P<0.01). ROC analysis showed that the auto‐antibody anti‐GPIX had a high diagnostic value in pregnant women with thrombocytopenia over normal pregnant women with the area under the curve value of 0.779 (P<0.001), the sensitivity and specificity were 76.3% and 100%, respectively.

Conclusions: The plasma profile of platelet auto‐antibodies in patients with thrombocytopenia in pregnancy was different from that in ITP patients. The auto‐antibody anti‐GPIX may be served as a biomarker of the disease.

PB128: Lifetime Bleeding History with Modified ISTH‐BAT Survey in 2,339 Individuals Establishes Population Prevalence Estimates

M.‐H. Chen 1 ; B. Rodriguez 1 ; C. Song 1 ; A. Lachapelle 1 ; C. Wallace de Melendez 1 ; P. Armstrong 2 ; M. Chan 2 ; T. Warner 2 ; A. Johnson 1

1 National Heart Lung and Blood Institute's The Framingham Heart Study, Population Sciences Branch, Division of Intramural Research, Framingham, United States, 2 Blizard Institute, Queen Mary University of London, Barts and the London School of Medicine and Dentistry, London, United Kingdom

Background: The ISTH Bleeding Assessment Tool (BAT) is a tool to screen individuals with potential platelet and bleeding disorders. Such survey instruments are typically applied in clinical settings. There is limited information on the prevalence of bleeding symptoms and conditions in the general population.

Aims: We aim to assess lifetime history and prevalence of bleeding symptoms in >3,800 individuals in the Framingham Heart Study. Modified BAT scores and individual symptoms will be assessed for association with genetics, risk factors, demographics and detailed platelet and plasma assays.

Methods: We adapted the ISTH‐BAT for self‐administration by computer tablet, using skip‐logic to facilitate the survey. Positive answers trigger further questions on clinical actions, frequency and attributes of events. To the ISTH‐BAT we added questions assessing family history of major bleeding, and allowed free text entries for comments. The survey was translated into Spanish.

Results: Of 2,339 respondents, n=2,144 were of European ancestry and n=195 were of non‐European ancestry. The sample was 53.5% female. The mean age was similar between men (54.8 years) and women (54.6 years). The modified BAT scores were positively skewed: 0 (81.9% of samples), 1 (13.1%), 2 (3.4%), ≥3 (1.6%). The maximum observed BAT score was 7. Those with BAT scores ≥2 were more often women (80.3%). The mean age of those with BAT scores ≥2 (n=117) was significantly higher than those with scores <2 (n=2,222) (58.4 years vs. 54.6 years, P<0.003). Responses for the modified BAT sub‐components are summarized in the Table.

Conclusions: By late middle age, >5% of the population had significant clinical bleeding events. Higher age associates with BAT score ≥2 consistent with more potential bleeding challenges in older individuals. As expected women have higher BAT scores. Consistent with some prior work, ˜10% of women have a history of menorrhagia requiring clinical intervention.

PB129: A Cohort Study on Women with Congenital Factor XIII Deficiency in Iran

M. Naderi 1 ; M. Karimi 2 ; S. Haghpanah 2 ; G. Miri‐Aliabad 1 ; H. Tavoosi 2

1 Zahedan University of Medical Sciences, Genetics of Non Communicable Disease Research Center, Zahedan, Islamic Republic of Iran, 2 Shiraz University of Medical Sciences, Hematology Research Center, Shiraz, Islamic Republic of Iran

Background: Congenital factor XIII deficiency (FXIIID) is associated with life‐threatening hemorrhage underscoring timely diagnosis and management. Bleeding symptoms might render more complicated in affected women than male due to pregnancy, and menstruation.

Aims: To evaluate a cohort study on women with congenital FXIIID in Iran.

Methods: A Cohort study was conducted on all registered women with congenital FXIIID in southern Iran from 2007 to 2017. All patients were severe with FXIII activity and antigenicity of less than 2%. Prophylaxis was given with dose of 10 U/kg every four weeks. It was also scheduled 10‐20 U/kg every two weeks during pregnancy.

Results: A total of 241 women were investigated with median age of 10 years (age range 1 day‐46 years).Thirty‐four percent were in childbearing age (15‐40 years) and 89% had positive history of first degree relative. The most common bleeding symptoms were umbilical cord bleeding (85%) and ecchymosis (60%). Menorrhagia (33%), gum bleeding and post dental extraction bleeding (6%), intracranial hemorrhage (15%) and ovulation bleeding (10%) were the other symptoms. One hundred thirty four miscarriages were recorded in patients who weren't on prophylaxis with the frequency of one to 11 times per case. None of 53 patients (18 cesarean section and 35 normal vaginal delivery) had miscarriage or bleeding during pregnancy and post‐partum while on prophylaxis but one patient developed post‐partum thrombosis. Forty eight surgeries were performed covering with FXIII concentrate without any episode of significant bleeding except one post‐operative portal vein thrombosis. None of them developed inhibitor.

Conclusions: Data on women with congenital FXIIID are scarce. Menorrhagia was the third most common bleeding symptom. No successful delivery was reported without prophylaxis. Prophylaxis treatment is a standard goal for management of women with severe congenital FXIIID leading to decreased gynecological problems, obstetric complications and successful delivery.

PB130: First Look at My Life Our Future Carrier Data from a Pilot Site

J. Puetz 1 ; L. Johnson 2 ; C. Hugge 1 ; P. Buchanan 3

1 Saint Louis University, Pediatric Hematology/Oncology, St. Louis, United States, 2 SSM Health Cardinal Glennon Children's Hospital, St. Louis, United States, 3 Saint Louis University, Center of Health Outcomes Research, St. Louis, United States

Background: My Life Our Future (MLOF) was a genotype initiative that offered free F8 or F9 genotyping for people with hemophilia living in the United States. Subject enrollment began in 2013. In 2015 the program was expanded to include potentially affected female family members (carriers). New participant enrollment ended in December 2017. As a pilot site for carrier detection, we were among the first to submit and receive data.

Aims: By reviewing data from a carrier pilot site we will demonstrate what types of hemophilia carrier data are available from the MLOF initiative and potential hypothesis that can be tested.

Methods: We reviewed data from our center for all potentially affected female family members. The Bleeding Score (BLS) was obtained using the ISTH BAT. Each participant had a single factor activity (FA) measurement using a one‐stage assay. The study was approved by the Institutional Review Board of Saint Louis University and all participating subjects signed informed consent/assent. Only subjects who consented for participation in the research repository were included in this study. Statistical analysis included independent samples t‐tests and chi‐square tests at a significance level of 0.05.

Results: Sixty‐seven subjects had genotype data available. The ages ranged from 1 to 89 years, and 97% of participants were White, non‐Hispanic. Among all participants, there was no correlation between BLS and FA (r=−0.241, P=0.055).

TABLE 1 All Enrolled Subjects Bleeding Score Factor Activity (IU/dL) n Mean (SD) P‐value n Mean (SD) P‐value Carrier 43 6.5 (7.6) 0.11 42 81 (47.5) 0.001 Non‐Carrier 23 3.6 (5.0) 23 166 (109.1) Age ≥18 46 6.7 (7.5) 0.006 45 107 (87.3) 0.67 Age <18 20 2.6 (4.0) 20 117 (81.2) TABLE 2 Carriers Only Bleeding Score Factor Activity (IU/dL) n Mean (SD) P‐value n Mean (SD) P‐value Hemophilia A 34 6.6 (8.2) 0.91 33 81 (52.2) 0.95 Hemophilia B 9 6.2 (5.0) 9 80 (25.9) Age ≥18 37 7 (7.9) 0.23 36 77 (33.7) 0.62 Age <18 6 3 (4.3) 6 99 (100.3) Factor Activity <40 6 12.5 (5.5) 0.04 6 31 (7.4) 0.004 Factor Activity >40 37 5.5 (7.5) 36 89 (46.2) Known Status (mean age 39) 26 8.2 (8.9) 0.04 26 72 (28.3) 0.24 Unknown Status (mean age 37) 17 3.9 (3.8) 17 93 (67.3)

SD, standard deviation; IU, international units; dL, deciliter; Known/Unknown Status, carrier status known/unknown prior to enrollment.

Conclusions: Our data is consistent with prior studies showing a higher BLS in carriers compared to non‐carriers, although in this cohort it did not reach statistical significance. However, our data suggests that prior knowledge of the carrier status may bias the BLS determination. An analysis of the complete data set is necessary to confirm this discovery.

PB131: Translation and Validation of the Pictorial Blood Loss Assessment Chart (PBAC) in Arabic

O. Kaabia 1,2 ; F. Letaief 1 ; A. Brahim 2,3 ; H. Skouri 2,4 ; M. Bibi 1

1 Farhat Hached Teaching Hospital, Gynecology and Obstetrics, Sousse, Tunisia, 2 Faculty of Medicine, Sousse Tunisia, Sousse, Tunisia, 3 Farhat Hached Teaching Hospital, Anesthesia and Resuscitation, Sousse, Tunisia, 4 Sahloul Teaching Hospital, Bio‐Hematology, Sousse, Tunisia

Background: The awareness of the burden of menstrual disorders on women′s health led to a better quantification of the abnormal menstrual bleeding by certain scales. But no version adapted to the arabophone patients has not yet been validated.

Aims: The Pictorial Blood Loss Assessment Chart (PBAC) is an annotated menstrual calendar of iconographic representations of the abundance of menstrual flow. Its score is used to standardize the abundance of menstrual blood loss. The aim of this work was to translate, adapt and then validate this questionnaire for the arabophone women.

Methods: This work consisted in:

‐ The Arabic translation of the content of the PBAC, ‐ The adaptation of the coins used in the original questionnaire respecting the diameters, ‐ The deletion of the column which concerns the hygienic tampons, ‐ The statistical validation of the new questionnaire on a case‐ control study with a sample of 30 genitally active women (15 women complaining of menorrhagia and 15 with normal flow). It was based on 2 tests: An evaluation of the validity of the questionnaire construction and an assessment of the concordance validity of the Arabic version of the PBAC with the MCMDM‐VWD.

Results: Our patients were comparable in terms of socio‐demographic characteristics. In terms of construct validity, the mean score of the first half of menstruation with Ar_PBAC was 85 while the mean score of the 2nd half of menstruation was 38 with a statistically significant difference (P=0.001). In terms of validity of concordance, the 2 conditions were validated. The mean scores of Ar_PBAC in themenorrhagia group were statistically higher than that of the control group with an average score of 212 versus 64 (P=0.001). A strong correlation to the MCMDM‐VWD questionnaire′s menorrhagia item score was found with a Spearman correlation coefficient r=0.85 and P<0.05.

Conclusions: This Arabic version of the PBAC score is reliable to quantify the menstruation of Arabophone women.

PB132: Diagnosis Value of Screening Tools for Hemostasis Disorders in Women Consulting for Prolonged Menstruation with Normal Pelvic Exam

O. Kaabia 1,2 ; F. Letaief 1 ; H. Abbessi 3 ; I. Ghachem 3 ; A. Brahim 2,4 ; S. Hadhri 3 ; H. Skouri 2,3

1 Farhat Hached Teaching Hospital, Gynecology and Obstetrics, Sousse, Tunisia, 2 Faculty of Medicine, Sousse, Tunisia, 3 Sahloul Teaching Hospital, Laboratory of Hematology and Blood Bank, Sousse, Tunisia, 4 Farhat Hached Teaching Hospital, Anesthesia and Resuscitation, Sousse, Tunisia

Background: Menorrhagia is a health problem that affects women quality of life. It remains unexplained in 50% of cases. The awareness of the burden of menstrual disorders on women′s health led to a better understanding of their causes and several tests have been developed to assess and quantify menorrhagia.

Aims: The aim of this study was to determine the diagnosis value of screening tools for hemostasis disorders in women consulting for prolonged menstruation with normal pelvic exam.

Methods: This is a case‐control pilot study including 40 cases with non organic menorrhagia and 20 controls who are volunteers with no history of abnormal bleeding. We recorded the menstrual habits and the hemorrhagic symptoms by two standardized questionnaires: PBAC and MCMDM‐VWD. All blood samples were taken during the follicular phase of the menstrual cycle. Platelet aggregation and ATP release were performed using different agonists: ADP, Collagen, Arachidonic Acid and Epinephrin. Ristocetin has been also used to assess platelet agglutination. Levels of coagulation factors, including factor XIII, have been assessed by Sysmex 2100CS analyzer. Thromboelastography study has been performed by ROTEM analysis.

Results: A MCMDM‐DWD score of ≥ 4 detected only 52% of hemostasis disorders but it had a 100% specificity for VWD. The VWF Ag level was significantly correlated with the MCMDM‐VWD score with a Spearman coefficient r=−0.43 (P<0.05).

FIGURE 1 Correlation between MCMDMVWD scores and levels of VWF Ag

Hemostasis disorders were diagnosed in 63% of cases with a PBAC score of ≥100. ROTEM analysis approach had no significant added value to assess non organic menorrhagia.

TABLE 1 Diagnosis value of the screening tests Sensitivity (%) Specificity (%) PPV (%) NPV (%) PBAC ≥ 100 Hemostasis disorders 75 13 63 20 VWD 100 23 11 100 Platelet dysfunction 75 13 63 20 EXTEM and/or INTEM abnormality Hemostasis disorders 10 88 50 44 VWD 33 91 25 94 Platelet dysfunction 10 89 50 47

Conclusions: The screening for hemostasis disorders in patients with non organic menorrhagia is more efficient by the association of the usual history taking with a PBAC score >100. We do not recommend the use of the ROTEM analysis when screening for hemostasis disorders in these women.

PB133: Clinical Screening for Underlying Bleeding Disorder in Nigerian Women

T. Nwagha 1 ; E. Ugwu 2 ; H. Okoye 3 ; A. Ugwu 3 ; Bleeding Disorder in Nigerian Women

1 University of Nigeria Teaching Hospital, Haematology & Immunology, Enugu, Nigeria, 2 University of Nigeria Teaching Hospital, Obstetrics and Gynaecology, Ituku Ozalla, Nigeria, 3 University of Nigeria Teaching Hospital, Haematology & Immunology, Ituku Ozalla, Nigeria

Background: Bleeding disorders in women can be detrimental to their physical, psychological and social well‐being with negative impact on their quality of life. Given the scope of this problem little is known about the prevalence of bleeding disorders in Nigerian women.

Aims: To evaluate the prevalence of perceived bleeding symptoms in Nigerian women and the usefulness of a simple clinical screening tool.

Methods: A cross‐sectional observational survey of women between the ages of 16 and 50 years from 5 states in South east geographical zone in Nigeria using a structured pre validated 8‐ question questionnaire clinical screening tool. We distributed questionnaires to women of all works of life (n=1,800). We received 1524 (85%) responses.

Results: The mean age of the women was 26 (10.6) years. Prevalence of bleeding symptoms was found to be 24.6%. Eleven percent of women reported a positive family history of bleeding disorder. There was a significant association between having a positive family history of bleeding disorder and experiencing bleeding symptoms (AOR 0.12, 95% CI 0.06‐0.22 P<0.0001). Commonest bleeding symptom is menorrhagia, 141 (9.3%) had past history, 426 (28%) had experienced at least flooding or passage of clots during their monthly cycle and 351 (23%) requiring resuscitation with blood component therapy.

Conclusions: There is a high prevalence of perceived bleeding symptoms among women with menorrhagia as the commonest bleeding symptoms. This tool is a cost effective easy tool to routinely identify women with bleeding symptoms needing further haemostatic or obstetric evaluation.

PB134: Hemostasis Disorders in Tunisian Women with Non Organic Menorrhagia: A Case Control Pilot Study

O. Kaabia 1,2 ; F. Letaief 1 ; H. Abbessi 3 ; A. Brahim 2,4 ; I. Ghachem 3 ; S. Hadhri 3 ; H. Skouri 2,3

1 Farhat Hached Teaching Hospital, Gynecology and Obstetrics, Sousse, Tunisia, 2 Faculty of Medicine, Sousse, Tunisia, 3 Sahloul Teaching Hospital, Laboratory of Hematology and Blood Bank, Sousse, Tunisia, 4 Farhat Hached Teaching Hospital, Anesthesia and Resuscitation, Sousse, Tunisia

Background: The awareness of the burden of menstrual disorders especially menorrhagia on women′s health and quality of life led to a better understanding of the causes of abnormal menstrual bleeding. Recently, several papers have investigated the role of hereditary haemorrhagic disorders in non organic menorrhagia but none have investigated this issue in Tunisia.

Aims: To enlighten the Tunisian situation facing this health problem, the aim of this study was to calculate the prevalence of hemostasis disorders (platelet function abnormalities, Von Willebrand disease and/or coagulation factor deficits) in Tunisian women with non organic menorrhagia.

Methods: This is a case control pilot study including 40 cases with non organic menorrhagia and 20 controls who are volunteer healthy women from the medical and paramedical staff with no history of menorrhagia or any bleeding disorder. Controls were matched to cases by age and by the menstrual cycle phase at the time of sampling (follicular phase).

Platelet aggregation and ATP release were performed using different agonists: ADP, Collagen, Arachidonic Acid and Epinephrin. Ristocetin has been also used to assess platelet agglutination. Levels of coagulation factors, including factor XIII, has been assessed by Sysmex 2100CS analyser. Thromboelastography study has been performed by ROTEM analysis.

Results: The prevalence of hemostasis disorders in Tunisian women with non organic menorrhagia was 52.5% (95.2% platelet function abnormalities, 14.3% VWD and 9.5% Factor X deficiency); however there was no significant difference with controls. The prevalence of VWD (type 1 or 2) was 7.5% in cases.

Four menorrhagia cases had two concomitant hemostasis disorders; 3 cases of VWD and one case of coagulation factor deficiency had an associated platelet function abnormality.

TABLE 1 Prevalence of hemostasis disorders in Tunisian Women with non organic menorrhagia Women with non organic menorrhagia (40 cases) Healthy volunteers (20 controls) P Hemostasis disorders 21 10 0.85 Platlets fonction abnormalities 20 9 0.58 Von Willebrand disease 3 1 1 Coagulation factors deficit 2 0 0.5

Conclusions: Hemostasis disorders are prevalent in Tunisian women with non organic menorrhagia.

PB135: Improving the Gynaecological Management of Women with Bleeding or Thrombotic Disorders by Multi‐professional Out‐patient Care

K.M. Musgrave 1,2 ; T.T. Biss 1 ; J. Speedie 3 ; D. Mansour 4

1 Newcastle upon Tyne Hospitals NHS Foundation Trust, Department of Haematology, Newcastle upon Tyne, United Kingdom, 2 Newcastle University, Institute of Cellular Medicine, Newcastle upon Tyne, United Kingdom, 3 NHS Greater Glasgow and Clyde, Department of Sexual Health, Glasgow, United Kingdom, 4 Newcastle upon Tyne Hospitals NHS Foundation Trust, Department of Women's Services, Newcastle upon Tyne, United Kingdom

Background: Bleeding disorders and the treatment of thrombosis complicates the management of gynaecological problems.

Aims: To review the introduction of a multi‐professional haematology‐gynaecology clinic at a UK teaching hospital.

Methods: Electronic medical records, for all women attending the clinic, were retrospectively reviewed from Jan 2016 to Dec 2017 (n=42).

Results: Twenty‐seven cases with a bleeding history were identified (Von Willebrand Disease, 13; factor deficiency, 3; platelet disorder, 3; dysfibrinogenaemia, 1; undefined disorder, 7). Median age was 28 years (range 13‐53). 25 attended due to heavy menstrual bleeding (HMB). 16 underwent venepuncture; 4 were iron deficient, 2 of whom were also anaemic. Interventions included: oestrogen‐containing contraceptive pill (in 8); levonorgestrel‐releasing intrauterine system (4); progestogen‐only pill (5, 3 at higher than the usual dose); progestogen intramuscular injection (3, 2 given 8‐weekly instead of 12‐weekly); tranexamic acid (3); combined hormonal vaginal ring (1); northisterone (1); progestogen implant (1); oestrogen replacement (1). Multiple interventions were required in 3.

Fifteen cases with a thrombotic history were reviewed (2 inherited thrombophilia, 2 acquired, 5 single episode of thrombosis, 6 recurrent thrombosis). 12 were anticoagulated and 1 took an antiplatelet agent only. Anticoagulation included: vitamin K antagonists, 4 (3 with target INR ≥3); DOAC, 6; and low molecular heparin with antiplatelet agent, 1. HMB affected 12. Venepuncture was performed in 11; 2 were anaemic, 1 was iron‐deficient. Interventions included: progestogen‐only pill (8, 5 on the higher dose); progestogen injection (3 given 8‐weekly); levonorgestrel‐releasing intrauterine system (2).

Follow‐up data were available for 20 cases; all women reported an improvement in symptoms. No surgical interventions were required.

Conclusions: This strongly suggests that joint working between haematology and gynaecology improves the care of women with bleeding and thrombotic disorders.

PB136: A Woman's Life‐long Bleeding Condition: Diagnostic Dilemma and Expert Opinion

R. Winikoff 1 ; M. Othman 2,3

1 University of Montreal, Haemophilia Treatment Centre at CHU Sainte‐Justine, Montreal, Canada, 2 Queen's University, Biomedical and Molecular Sciences, Kingston, Canada, 3 St Lawrence College, School of Baccalaureate Nursing, Kingston, Canada

Background: A 47 year old woman with lifelong menorrhagia interfering with quality of life. No pelvic or gynaecological pathology was identified. Despite elaborate investigations, a cause for the bleeding was not identified. Menorrhagia was eventually controlled by endometrial ablation.

Aims: To present a diagnostic dilemma, obtain expert opinion and investigate the possibility of underlying genetic bleeding disorder.

Methods: Full review of history, clinical and laboratory data was conducted together with genetic analysis using NGS.

Results: The patient had ISTH‐BAT score of 12 with a strong family history of bleeding. She experienced serve menorrhagia and mucocutaneous bleeding and had two vaginal deliveries and several surgeries without bleeding complications. All haemostasis investigations were normal including PT, APTT, fibrinogen, platelet count, MPV, factors V, VII, XIII, VWD profile, platelet aggregation in response to ADP, collagen, ristocetin, epinephrine and arachidonic acid and no spontaneous platelet aggregation or aggregation in response to 0.5 ml ristocetin. The only abnormality was prolonged PFA‐100 closure time to ADP but normal to EPI. The patient is known for autoimmune thyroiditis, hypermobility, dopamine‐secreting neck paraganglioma, heterozygote MTHFR C677T; low ceruloplasmin, normal calcium and parathyroid hormone, and elevated 1,25 vitamin D with low 25 D. The patient's 19 years old daughter had ISTH BAT score of 6 and similar bleeding and laboratory data. The patient reported Omega 3, Aloes vera, Gluten and Phytates worsen menorrhagia. The patient was treated with Tranexamic acid and iron therapy which have only partially improved menorrhagia.

Conclusions: Despite advances in bleeding assessment tools and laboratory investigations, unexplained lifelong menorrhagia remains a diagnostic dilemma precluding optimal therapies at times. The results of genetic analysis is hoped to identify a diagnosis in this particular case and highlight its role in similar cases.

PB137: Thrombocytopenia in Pregnancy: The Role of Hemostasis Laboratory for Diagnosis and Management of Acute Fatty Liver of Pregnancy

I. Cuomo; M. Scannapieco; G. Rescigno

“Umberto I” Hospital ASL Salerno, Haemostasis and Thrombosis Unit, Nocera Inferiore, Italy

Background: Acute fatty liver of pregnancy (AFLP) represents a rare pregnancy complication. The clinical picture of AFLP is characterized by an early multi‐organ involvement.

Aims: We want to emphasize the great importance of coagulation lab testing for diagnosis and management of AFLP.

Methods: We report a case of a 28 year old pregnant at 40 weeks of gestation, admitted to emergency department for jaundice, abdominal pain and nausea.

Results: Entry laboratory tests: Hb 14 mg/dl, WBC 17,000/mm 3 , PLT 195,000/mm 3 , Total Bilirubin 7.15 mg/dl, Direct Bilirubin 5.28 mg/dl, ALT 141 U/l, AST 191 U/l, Total Protein 6.5 g/dl and Albumin 3.2 g/dl, Urea 33 mg/dl and Creat. 1.6 mg/dl, CRP 10.1 mg/l. PT Ratio 1.62 and aPTT Ratio 1.31, Fibrinogen 144 mg/dl, DDimer 6121 ng/ml DDU, ATIII 12%, Factors Activity: VII 45%, IX 54%, X 40%, VIII 220%, vWF Activity 400%, PC 35%, freePS 35.3%. A probable diagnosis of AFLP was done following the Swansea's criteria (Goel A. et al. Gut 2011). Additional lab test for differential diagnosis: absence of schistocytes at peripheral Blood Smear, absence of Proteinuria, Haptoglobin 60 mg/dl excluded Hemolysis and HELLP Syndrome, normal blood pressure values excluded preeclampsia. Moreover serology negative for hepatitis A, B, E and CMV, EBV excluded viral infection. After urgent delivery, the AFLP patients usually improve in 24‐48 hours (Gami N. et al Int J Reprod Contracept Obstet Gynecol 2013) but in this case, the clinic and laboratory tests show a multi‐organ failure. The reason of severe anemia, despite continuous blood transfusions, is the widespread gastrointestinal bleeding found during an abdominal exploratory surgery resolved with administration of rFVII.

Conclusions: Diagnosis and successful management of AFLP is still a challenge. Coagulation profile help to disclose the hepatopathy pattern key for diagnosis and management of AFLP.

PB138: Quantitative Systems Pharmacology (QSP) to Predict Thrombin Generation with Concomitant AT Lowering and Factor Replacement or Bypassing Agents

G. Sridharan 1 ; S. Tsour 1 ; K. Qian 1 ; J. Liu 1 ; Q. Lu 2 ; C. Benson 3 ; K. Madigan 1 ; S. Huang 1

1 Alnylam Pharmaceuticals, Cambridge, United States, 2 Sanofi, Bridgewater, United States, 3 Sanofi Genzyme, Cambridge, United States

Background: Fitusiran is a once monthly, subcutaneously administered investigational RNAi therapeutic targeting antithrombin (AT) to improve thrombin generation and promote hemostasis in patients with hemophilia A and hemophilia B with or without inhibitors. Patients on fitusiran experiencing a bleed may require treatment with factor replacement or bypassing agents (BPA); thus, a deeper understanding of the relationship between factor/BPA dosing with AT lowering and thrombin generation is needed.

Aims: To simulate the peak thrombin potential (PTP) that would be achieved with factor or BPA dosing in the context of AT lowering.

Methods: We developed a modeling framework that integrates factor pharmacokinetics with a quantitative systems pharmacology (QSP) model describing the coagulation cascade (Nayak S, et al. CPT Pharmacometrics Syst. Pharmacol, 4: 396‐405.) to simulate the in vitro PTP that would result with concomitant treatment of either factor or bypassing agents and AT lowering. Using Matlab and the Simbiology Toolbox ® , we simulated PTP for doses of FVIII and FIX (standard and extended half‐life), rFVIIa, and FEIBA under various time‐course regiments with simultaneous AT lowering.

Results: Our modeling of concomitant AT lowering and dosing of BPAs, Factor VIIa and FEIBA, reveals a correlation between thrombin generation observed from in vitro spiking experiments and that of QSP modeling. For a variety of extended half‐life factors, a time‐course dosing regimen keeps the peak thrombin potential within the normal healthy range for the full range of AT levels observed in patients on fitusiran (10‐25% residual).

Conclusions: The integration of the QSP model and plasma factor pharmacokinetic models adds to the data supporting clinical guidance for dosing of standard and extended half‐life factors and bypassing agents for patients receiving fitusiran on a clinical trial.

PB139: Does Hemophilia Slow Down the Development of Liver Fibrosis?

M.‐A. van Dievoet 1 ; I. Leclercq 2 ; C. Hermans 3 ; C. Lambert 3 ; Y. Horsmans 3 ; M. Jacquemin 4 ; S. Eeckhoudt 1

1 Cliniques Universitaires Saint‐Luc, Department of Laboratory Medicine, Brussels, Belgium, 2 Université Catholique de Louvain, Laboratory of Gastroenterology, Brussels, Belgium, 3 Cliniques Universitaires Saint‐Luc, Department of Internal Medicine, Brussels, Belgium, 4 Universitair Ziekenhuis Leuven, Department of Laboratory Medicine, Brussels, Belgium

Background: Until the discovery of hepatitis C virus (HCV) and the introduction of viral inactivating procedures, almost all patients treated with clotting factor products before 1989 were infected with HCV. Unexpectedly, recent observations suggested that liver complications of HCV were less severe in subjects with hemophilia A than in other patients.

Aims: We investigated the difference in liver fibrosis in a chemically‐induced liver cirrhosis model between factor VIII deficient mice and normal controls.

Methods: Liver fibrosis was induced by thioacetamide (TAA) in both normal (CTL) and factor VIII‐deficient (HEMO) mice and compared to control groups receiving only water (H2O). After 25 weeks the mice were sacrificed and liver biopsies were taken. Collagen type I messenger ribonucleic acid (mRNA) and the amount of fibrosis were quantified. Statistical analysis (Kruskal‐Wallis followed by Scheffé multiple comparisons) was performed by Analyse‐it Software, ltd.

Results: The mean TAA intake during the whole period was 38 mg/kg/day and 24 mg/kg/day for HEMO‐TAA and CTL‐TAA respectively. Hemophilia mice (HEMO‐TAA) were exposed to a significantly (P<0.001) higher dose per body weight than the treated control mice (CTL‐TAA). Nonetheless, in the TAA‐treatment group, the hemophilic mice showed significantly less liver fibrosis (figure 1, P<0.001) and reduced collagen type 1 expression compared to their controls (P=0.04).

Conclusions: Our results bring experimental evidence that deficiency in coagulation factor VIII protects from fibrosis and thereby support that the bleeding disorder is causally involved in the association between hemophilia and reduced liver fibrosis.

PB140: Geno‐ and Phenotype Correlate in Porcine Model of von Willebrand Disease Type 1 and Type 3

H. Allerkamp 1 ; S. Lehner 1 ; M. Ekhlasi‐Hundrieser 1 ; C. Detering 1 ; C. Wermes 1 ; C. Pfarrer 2 ; M. von Depka 1

1 Werlhof Institut, Hannover, Germany, 2 University of Veterinary Medicine, Institute for Anatomy, Hannover, Germany

Background: von Willebrand disease (VWD) affects not only coagulation, but also angiogenesis. Up to 20% of VWD patients show angiodysplasia. However, there is a lack of appropriate models to investigate the influence of VWD on angiodysplasia in vivo.

Aims: To characterise the correlation of geno‐ and phenotype of porcine VWD type 1 and 3 as a model for investigating angiodysplasia in vivo.

Methods: Blood of mature pigs with a naturally occurring mutation in the VWF gene was analysed. Genotype was confirmed by molecular genetic analyses as described by Lehner et al. (2017) as heterozygous for the mutation (het, type 1) and homozygous (hom, type 3). Gene analysis revealed a 12.3 kb duplication resulting in an almost entire loss of the mature protein. Healthy pigs served as reference. Antigen, activity and multimeric pattern of von Willebrand factor (VWF) and factor (F) VIII were analysed.

Results: The het animal produces 49% of the amount of duplication RNA product of the average of 12 measured hom pigs (101.1±16.0%). All blood parameters show a decrease from the healthy to the hom animals. The het animal ranges in between. VWF antigen is affected to a greater extent than VWF activity. Decrease of FVIII activity has been shown for the hom animal only. The multimeric pattern of the het animal is comparable to human plasma but with less prominent bands. The hom animal shows neither high weight nor intermediate weight multimers.

Conclusions: The decreasing activity of VWF and FVIII matches observation in human patients. Because of the good correlation of geno‐ and phenotype these pigs are a promising in vivo model. Studying and breeding of affected pigs is simplified since the genetic mutation occurs naturally. This large animal model gives the possibility to investigate VWD in general and its impact on angiogenesis at an appropriate scale as well as angiogenic factors in all organs.

PB141: Pharmacokinetics and Safety of Subcutaneous rIX‐FP Administration: Results from a Phase 1 Trial

I. Pabinger 1 ; T. Lissitchkov 2 ; J. Roberts 3 ; W. Seifert 4 ; S. Puli 3 ; J. Ye 3 ; Y. Li 3

1 Medical University of Vienna, Clinical Division of Haematology and Haemostaseology, Department of Medicine I, Vienna, Austria, 2 Specialized Hospital for Active Treatment (SHAT) ‘Joan Pavel’, Department of Hemorrhagic Diathesis and Anemia, Sofia, Bulgaria, 3 CSL Behring, King of Prussia, United States, 4 CSL Behring, Marburg, Germany

Background: Current factor IX (FIX) products are administered via intravenous (IV) injections, which require repeated venous access and are burdensome for patients. Subcutaneous (SC) administration may improve quality of life, particularly in patients with poor venous access. rIX‐FP is a recombinant fusion protein linking FIX with albumin. rIX‐FP has shown favorable safety and hemostatic efficacy in clinical trials. An exploratory phase 1 study is investigating SC dosing of rIX‐FP in hemophilia B patients ( NCT02053792 ).

Aims: To investigate the pharmacokinetics and safety of single and repeat SC doses of rIX‐FP.

Methods: Male hemophilia B patients (≥18 years old; FIX activity ≤2%) enrolled in a phase 3b trial were eligible for the study. Patients received rIX‐FP as a single SC dose (25 or 50 IU/kg) or a repeat dose every three days for 15 doses (25 IU/kg). The first SC dose was administered 14 days after the last rIX‐FP IV dose. For each dose SC rIX‐FP was administered in up to four injection sites (≤4 mL per site). FIX activity was analyzed pre‐dose and then at regular intervals after administration until dose 15.

Results: 14 patients have been enrolled. Three patients receiving repeat doses of SC rIX‐FP had peak (Cmax) median (range) FIX activity levels of 6.1% (4.2‐12.0) following the 1st dose and 11.4% (6.0‐14.6) following the 2nd dose. Median pre‐dose (trough) values prior to the 1st dose were 3.6% (2.2‐11.6) and prior to the 15th dose were 6.2% (5.9‐11.5). There were no serious adverse events (SAEs) reported, no inhibitor development and no systemic hypersensitivity. Injection site reactions were reported in five patients, most of which were mild in severity. One patient withdrew following an injection site hematoma of moderate severity.

Conclusions: SC administration of 25 IU/kg with the usual rIX‐FP IV formulation every 3 days for 15 doses resulted in FIX activity trough levels >5%, consistent with a mild hemophilia B phenotype. No patients experienced SAEs, systemic hypersensitivity or developed inhibitors.

PB142: Recombinant Expression of F9 Nonsense Mutations and Fix Pharmacokinetics in Hemophilia B

A. Branchini 1 ; M. Ferrarese 1 ; M. Pinotti 1 ; F. Bernardi 1 ; M. Morfini 2

1 University of Ferrara, Department of Life Sciences and Biotechnology, Ferrara, Italy, 2 Italian Association Haemophilia Centers (AICE), Milan, Italy

Background: The distribution of Factor IX (FIX) in plasma, subendothelial and extravascular fluids and the variability of pharmacokinetics (PK) in Hemophilia B (HB) (Haemophilia 2016; 22:537‐42) support the investigation of secreted FIX mutated molecules as covariates. We hypothesized that the residual amounts of mutated FIX molecules, including those truncated, could modulate FIX PK.

Aims: To correlate the expression of a broad spectrum of F9 nonsense mutations and their secreted recombinant FIX (rFIX) levels with the PK outcomes of Nonacog alfa, which has never been evaluated.

Methods: Nonsense mutations from patients (FIX<1%) were expressed in HEK293 cells with vectors bearing the premature F9 stop codons (PTC). rFIX in the medium w[G1] as evaluated by ELISA and Western blotting analysis.

Results: Eleven nonsense rFIX[M1] variants (p.R75X, p.L103X, p.R162X, p.W240X ‐tga/tag‐, p.R294X, p.R298X, p.Y330X, p.Q370X, p.R379X, p.R384X), collectively present in 70% (324/469) of HB patients with PTCs in the F9 variant database (www.factorix.org) were characterized [AB2]. In six of them (>50%, underlined) we detected secreted truncated molecules. For the p.W240X normal levels (90‐120%) of truncated protein were present. For the p.R162X we also detected traces of full‐length rFIX. FIX PKs in single patients with the p.Q370X, p.R379X and p.R384X mutations indicated terminal (44.3, 57.5 and 37.8 hours; mean 46.5 hours) and beta half‐life (78.9, 41.9 and 34.6 hours, respectively; mean 51.8 hours) values higher than mean values (terminal half‐life 29.7 hours; beta half‐life 37.3 hours) found in 11 patients with missense mutations

Conclusions: Truncated FIX molecules could flow to other compartments and compete with infused rFIX for binding to extracellular receptors, which in turn could modify rFIX bioavailability. Our data suggest that an expression platform for patient's mutations would help FIX PK analysis aimed at optimizing an individualized therapy in HB.

PB143: Engineering Regulatory T Cells to Block Inhibitor Formation

D. Scott ; J. Yoon; M. Abdeladhim; K. Parvathanen; A.‐H. Zhang; Y. Kim

USUHS, Medicine, Bethesda, United States

Background: The immune response to therapeutic FVIII is a major problem that blocks the efficacy of this critical protein. Currently, T regulatory cells (Tregs) have be suggested to have potential for the treatment of adverse immune responses to biotherapeutics such as inhibitors to FVIII.

Aims: To develop specific Tregs to induce tolerance to FVIII.

Methods: To increase efficacy and specificity of Tregs, we previously engineered human and mouse T cells to express chimeric antigen receptors (CARs) by expressing either T‐cell receptors (TCR) from hemophilia subjects or specific single chain fragments (scFv), in collaboration with the Königs lab in Frankfurt.

Results: All of those engineered specific Treg cells can actively suppressed effector anti‐FVIII responses in vitro and in vivo ! Recently, we expanded this approach to create antigen‐specific cytotoxic T cell s as well as Tregs expressing FVIII domains (B‐cell antibody receptors or BARs). Such BARs are able to kill FVIII‐specific hybridomas and LPS‐activated normal B cells in vitro . BAR Tregs actively suppress the anti‐FVIII response in vivo via a direct mechanism.

Conclusions: These results have potential to curb anti‐FVIII inhibitor formation in patients.

PB144: Novel Hyperactive FIX Variants and their Potential for Haemophilia B Gene Therapy

A.‐K. Urbanowitz 1 ; P. Milanov 1 ; P. Quade‐Lyssy 1 ; D. Abriss 1 ; E. Seifried 2 ; J. Schütttrumpf 3 ; J. Schwäble 4

1 Institute of Transfusion Medicine, Molecular Therapy, Frankfurt am Main, Germany, 2 Institute of Transfusion Medicine, Institute Director, Frankfurt am Main, Germany, 3 Biotest AG, Management, Dreieich, Germany, 4 Institute of Transfusion Medicine, Molecular Therapy Group Leader, Frankfurt am Main, Germany

Background: Adeno‐associated‐virus (AAV) based gene transfer has shown potential to provide sustained Factor IX (FIX) activity at low levels in patients with severe hemophilia B, preventing spontaneous bleeding and reducing FIX consumption. However, in AAV based gene therapy, dosing is limited by capsid‐specific cytotoxic immunity. Recently, application of the hyperactive FIX‐Padua mutant increased therapeutic efficiency of this approach, reaching mean FIX activities of 33.7% in hemophilia B patients.

Aims: To improve efficiency of AAV based gene therapy for hemophilia B towards normal FIX activity, we developed novel FIX variants based on the FIX‐Padua mutant, with enhanced gain of function and modified collagen‐IV binding.

Methods: Recombinant FIX variants were purified via anion exchange chromatography and tested in vitro for their clotting activity and collagen‐IV‐binding. In‐vivo analyses include determination of pharmacokinetic and pharmacodynamics parameters of the FIX variants and determination of their efficiency and safety in AAV based gene transfer.

Results: The activity of our novel hyperactive FIX variants was up to 12 fold higher compared to the wildtype FIX protein and 1.5 fold higher compared to FIX Padua. Collagen‐IV‐binding was significantly reduced in one of the new FIX‐variants and first in vivo experiments showed increased bioavailability of the respective mutant. Detailed analyses of FIX activity, pharmacokinetic and pharmacodynamics parameters of our hyperactive FIX variants, as well as evaluation of their efficiency and safety in AAV based gene transfer will be presented.

Conclusions: Our novel hyperactive FIX variants harbor increased activity compared to FIX‐wildtype and FIX‐Padua and reduction of collagen‐IV‐binding increases their bioavailability. Together, these modifications promise enhanced efficiency of AAV based gene therapy for hemophilia B and allow reduction of the vector dose to avoid therapy related toxicity.

PB145: Population Pharmacokinetic Analysis of Recombinant Fusion Protein Linking Coagulation Factor IX with Recombinant Albumin (rIX‐FP) in Adult and Pediatric Patients with Severe Hemophilia B

Y. Zhang 1 ; J. Roberts 1 ; T. Yuraszeck 1 ; W. Seifert 2 ; A. Feussner 2 ; A. Brainsky 1 ; E. Santagostino 3 ; J. Sidhu 4

1 CSL Behring, Clinical Pharmacology and Early Development, King of Prussia, United States, 2 CSL Behring GmbH, PRD, Marburg, Germany, 3 IRCCS Ca’ Granda Foundation, Maggiore Hospital Policlinico, Milan, Italy, 4 CSL Limited, Clinical Pharmacology and Early Development, Parkville, Australia

Background: rIX‐FP, recombinant factor IX (FIX) fusion protein, was developed to extend the half‐life of FIX in hemophilia B patients. The population pharmacokinetics (PK) has been evaluated with the addition of PK data from a phase 3b study including extended rIX‐FP dosing regimens of 100 IU/kg every 21 days for adults and 75 IU/kg every 14 days for pediatric patients.

Aims: To characterize the population PK of rIX‐FP, assess the covariates such as demographic and clinical factors that are potential determinants of rIX‐FP PK variability and to simulate FIX activity‐time profiles for different dosing regimens.

Methods: A total of 116 hemophilia B patients (FIX ≤2%), aged 1 to 70 years, were included. Patient blood samples were collected and FIX activity determined using the one‐stage clotting assay. A population PK model was developed in NONMEM 7 software, where goodness‐of‐fit and prediction‐corrected visual predictive check (pcVPC) used for model evaluation. Exogenous FIX activity levels were simulated for various dosing scenarios.

Results: FIX activity levels were appropriately described using a 2‐compartment population PK model. The final model precision was confirmed by the goodness‐of‐fit and pcVPC. Median exogenous (rIX‐FP) simulated trough FIX activity is approximately 5% in patients ≥12 years receiving 100 IU/kg every 21 days, and is approximately 5% for pediatric patients (6 to <12 years) and 3% for ages <6 years with both pediatric age groups receiving 75 IU/kg every 14 days.

Conclusions: rIX‐FP PK was well characterized using a population PK model approach. Dosing of rIX‐FP can be predicted using this model to produce FIX activity‐time profiles for different dosing schedules. The population PK simulations suggest that the majority of adult and pediatric Hemophilia B patients receiving rIX‐FP on 21‐day and 14‐day extended interval dosing regimens, reach sufficient FIX activity levels to transition from severe to a mild or moderate hemophilia classification.

PB146: Plasma Extracellular Vesicles (EVs) and Their Correlation with Clinical Phenotype in Pediatric Patients with Severe Hemophilia A (SHA)

M. Rand 1 ; S. Israels 2 ; H. Wang 1 ; E. McMillan‐Ward 2 ; V. Blanchette 1 ; R. Nieuwland 3

1 Hospital for Sick Children, University of Toronto, Toronto, Canada, 2 University of Manitoba, Winnipeg, Canada, 3 University of Amsterdam, Amsterdam, the Netherlands

Background: The biological factor(s) underlying bleeding severity heterogeneity in patients with SHA are unknown; EVs released from blood and/or endothelial cells may play a role.

Aims: To characterize plasma EVs in SHA and determine whether plasma levels correlate with bleeding phenotype.

Methods: Using a dedicated EV flow cytometer (A60‐Micro, Apogee; detects single EVs ≤170 nm), levels of EVs expressing CD61, CD62P, both CD61 and CD62P, CD62E, CD45, CD235a and exposing phosphatidylserine (PS) were determined in citrated‐plasma samples from boys (≤18 years) with SHA (factor (F)VIII ≤1%) in 2 pediatric hemophilia treatment centres. Controls were healthy adult males. Bleeding severity was defined by age at first joint bleed and annualized FVIII consumption (U/kg), averaged over 3 years. The study was approved by the institutional Research Ethics Boards and informed consent was obtained from all subjects.

Results: 20 boys with SHA and 20 controls were enrolled from Site 1, and 22 boys and 20 controls from Site 2. There were no differences between patients and controls in the concentration of EVs from platelets (CD61+), platelets and endothelial cells (CD62P+), endothelial cells (CD62E+), leukocytes (CD45+) or erythrocytes (CD235a+), or EVs exposing PS. However, EVs expressing both CD61+ and CD62P+ were significantly increased in patients versus controls at both study sites (Figure). There were no correlations between age at first joint bleed or annualized FVIII consumption and plasma EV levels.

FIGURE 1 CD61+/CD62P+ EVs in controls (C) and patients (P) with SHA in the 2 study sites. *P=0.032, **P=0.004 by ANOVA

Conclusions: EVs from platelets, leukocytes, erythrocytes or endothelial cells do not appear to be involved in the clinical phenotype heterogeneity of patients with SHA. The finding that CD61+/CD62P+ EVs from activated platelets are increased in patients with SHA warrants further investigation.

PB147: Pharmacokinetics of Recombinant Fusion Protein Linking Activated Factor VIIa to Human Albumin (rVIIa‐FP) and Eptacog Alfa in Hemophilia Patients with Inhibitors

J. Roberts 1 ; C. Joch 2 ; J. Ye 3 ; S. Puli 1 ; J. Mahlangu 4 ; E. Santagostino 5

1 CSL Behring, Clinical Pharmacology & Early Development, King of Prussia, United States, 2 CSL Behring, Clinical Development, Marburg, Germany, 3 CSL Behring, Global Clinical Safety and Pharmacovigilance, King of Prussia, United States, 4 Haemophilia Comprehensive Care Centre, Charlotte Maxeke Johannesburg Academic Hospital and Faculty of Health Sciences, University of the Witwatersrand and NHLS, Johannesburg, South Africa, 5 Maggiore Hospital Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, IRCCS Ca’ Granda Foundation, Milan, Italy

Background: Recombinant fusion protein linking recombinant activated factor VII to human albumin (rVIIa‐FP) is a therapeutic option designed to prevent and treat bleeding events in hemophilia patients with inhibitors. rVIIa‐FP was designed to reduce infusion frequency compared to current rVIIa treatments.

Aims: To compare single‐dose pharmacokinetics (PK) of rVIIa‐FP to eptacog alfa (rFVIIa) and assess safety of rVIIa‐FP in hemophilia patients with inhibitors.

Methods: In a Phase II/III multicenter, open‐label, dose escalation study of rVIIa‐FP 24 patients in Blocks A and B underwent single‐dose PK evaluation with a single injection of 90 (n=6) or 270 μg/kg (n=6) rFVIIa followed by a single injection of 1500 μg/kg rVIIa‐FP in Block A or single dose of rVIIa‐FP of either 1500 μg/kg (N=4) or 750 μg/kg (N=8) in Block B. FVIIa activity was assessed using a validated StaClot ® assay. PK parameters included plasma half‐life, area under plasma concentration time curve (AUC) and maximum plasma concentration (Cmax). Safety was assessed by number and type of adverse events.

Results: For Block A, mean baseline‐corrected FVIIa activity at 24 hours was higher for rVIIa‐FP (11.0 IU/ml) compared to rFVIIa (0.06 IU/ml [90 μg/kg] and 0.11 IU/ml [270 μg/kg]). rVIIa‐FP had an extended half‐life of 8.5 hours, more than two‐fold higher than rFVIIa (3.0 [90 μg/kg] and 2.6 hours [270 μg/kg]) and produced a larger AUC0‐t (847 vs. 112 [90 μg/kg] and 325 [270 μg/kg] IU/h/mL). Cmax for rVIIa‐FP was 75.2 IU/mL, in between that of the 90 (44.1 IU/mL) and 270 μg/kg (131 IU/mL) rFVIIa groups. No inhibitors to native FVII and no antidrug antibodies occurred. None of the study subjects developed serious adverse events.

In Block B, the average half‐life of rVIIa‐FP was 9.14 or 9.30 hours and clearance was 7.28 and 5.95 mL/hr/kg. On average, Cmax and AUC0‐t increased 2‐fold with 2‐fold increase in the dose. Overall, the PK of rVIIa‐FP in blocks A and B were comparable.

Conclusions: rVIIa‐FP has an improved PK profile compared to rFVIIa and was well tolerated.

PB148: Circulating Biochemical Markers of Early Joint Bleeding: Validation Study in Humans

X. Song; C. Enockson; L. Fogg; L. Boggio; M. Simpson; N. Hakobyan

Rush University Medical Center, Chicago, United States

Background: Early detection of joint bleeding in patients with severe hemophilia can prevent further hemarthrosis and ensuing destructive changes. The discovery and application of biochemical markers in addition to clinical and imaging assessment would greatly enhance the evaluation of early joint damage and may be used for the development of new therapeutic agents.

Aims: The objective of this translational research was to evaluate in hemophilia subjects circulating biochemical markers of joint bleeding previously identified in our mouse studies.

Methods: This project was approved by the Rush University IRB and all subjects provided informed consent.

Plasma (n=37) from severe hemophilia subjects was analyzed in 3 groups. Control: no bleeding, target joints or synovitis; Synovitis: target joints and/or synovitis, no assessed joint bleeding; Bleeding: blood in the joint. Joints were assessed by clinical and/or imaging evaluation. Sixteen commercially available proteins were analyzed using quantitative Luminex multiplex immunoassay. Because of small sample size, both the Effect Size (ES, Cohen's d) and P value were calculated.

Results: In Bleeding compared to Control, the expression of EGF, CSF2, IL4, IL13, FGF2, and MIP‐1α was significantly decreased with very large effect size (ES=1.25 to 2.18), while expression of KC and CSF3 was elevated (ES=0.80 to 1.54). Expression of EGF, CSF2, IL4, and CSF3 was different in Bleeding compared to Synovitis (ES=0.53 to 1.17), and in Synovitis compared to Control (ES=0.20 to 0.53). Since the Synovitis group might have subclinical joint bleeding, these data may indicate a higher sensitivity of these biomarkers to joint bleeding.

Conclusions: This pilot translational study identified a detectable circulating biomarker signature associated with the presence of blood in the joint and validates our results from animal research. Once corroborated in larger scale studies, these plasma biomarkers will be useful as a surrogate marker to monitor joint bleeding in hemophilia subjects.

PB149: APTT Reagent‐specific Underestimation of N8‐GP (Turoctocog Alfa Pegol) Activity Owing to Attenuated Thrombin Activation

E. Persson ; T. Foscolo

Novo Nordisk A/S, Måløv, Denmark

Background: Many activated partial thromboplastin time (APTT) reagents give an accurate recovery of N8‐GP activity, but some silica‐based reagents underestimate it.

Aims: To elucidate the mechanism behind N8‐GP activity underestimation.

Methods: An assay was designed to mimic the contact activation and clotting phases of an APTT‐based clotting assay, but instead quantifying factor Xa (FXa) amidolytic activity as a measure of factor VIIIa (FVIIIa) cofactor activity. The FVIII source was either NovoEight ® (N8), Advate ® or N8‐GP. Thrombin and factor IXa were optionally added to the contact activation phase mixture (while omitting the phase itself) to measure contact activator effects on the rate of thrombin activation of various forms of FVIII.

Results: Initially, we employed COATEST ® SP FVIII to show that the same FVIIIa cofactor activity could indeed be achieved from the same unit level of N8, N8‐GP and Advate ® . If APTT‐SP (or two other underestimating APTT reagents, TriniCLOT aPTT HS and STA‐PTT Automate) replaced buffer in this assay, the rate of FXa generation in the presence of 0.6 IU/ml N8‐GP was the same as that obtained with a significantly lower concentration of N8, thus resembling the degree of underestimation of N8‐GP activity observed in a clotting assay. With another silica‐based reagent, Pathromtin, 0.6 IU/ml N8‐GP and N8 generated FXa at the same rate. Extending the contact activation phase with APTT‐SP (90‐180‐300 s) resulted in a gradually increasing difference in clotting time between N8‐GP and N8, and a similar pattern was observed in the FXa activity assay. In addition, with APTT‐SP, thrombin activation of N8‐GP appeared to be slower than that of N8.

Conclusions: We have established analyses that assess FVIIIa cofactor activity as FXa output rather than clot formation. This allows mechanistic studies of the influence of APTT reagents on N8‐GP activation and has unveiled attenuated thrombin cleavage as the cause of N8‐GP activity underestimation.

PB150: Comparison of rFIX Utilization and Bleed Rates in US Hemophilia B Patients on rIX‐FP and Their Prior rFIX Drug

M. Escobar 1 ; C. Leissinger 2 ; S. Yan 3 ; G. Maro 4 ; G. Krishnarajah 3

1 University of Texas Health Science Center and the Gulf States Hemophilia and Thrombophilia Center, Houston, United States, 2 Tulane University Medical Center, New Orleans, United States, 3 CSL Behring, King of Prussia, United States, 4 Adivo Associates, San Francisco, United States

Background: The three most commonly used drugs for recombinant coagulation Factor IX (rFIX) replacement therapy in Hemophilia B patients in the US include two long‐acting products, rIX‐FP (recombinant fusion protein linking human FIX with human albumin) and rFIXFc (recombinant fusion protein linking human FIX with a human Fc fragment), and the standard‐acting nonacog alfa. Real world utilization and outcomes need to be considered to understand how these products compare in clinical practice.

Aims: Determine real‐world FIX consumption and annual bleeding rate (ABR) in patients on rIX‐FP, and compare to prior drugs rFIXFc or nonacog alfa.

Methods: De‐identified clinical patient chart information was obtained from US healthcare providers (hematologists and Hemophilia Treatment Centers) for 145 patients currently treated with rIX‐FP. ABR was calculated by annualizing the number of bleeds reported over a period of 8 weeks to 1 year. Prescribed dosing and infusion frequency were used to calculate FIX prophylaxis consumption for rFIX‐FP and their prior FIX drug used.

Results: Of the 145 patients, 84 (median age, 25 years; age range: 2‐81; 82.1% severe, 17.9% mild or moderate) were treated on prophylaxis both with rIX‐FP and their prior FIX drug. In a subset of these patients with bleed information, ABR (±SD) was 0.7±1.0 on rIX‐FP versus 8.9±9.6 on rFIXFc (n=12), and 1.5±5.8 on rIX‐FP versus 4.5±5.9 on nonacog alfa (n=17). Among the 37 rIX‐FP patients who previously used rFIXFc, median rIX‐FP consumption (38.3; mean±SD: 43.1±17.5) was 1.6 times lower compared to rFIXFc (60.6; mean±SD: 67.4±26.5; n=37). For the 29 patients previously using nonacog alfa, median rIX‐FP consumption (44.7; mean±SD: 47.1±14.3) was 2.5 times lower than with nonacog alfa (109.9; mean±SD: 107.7±38.4; n=29).

Conclusions: This study suggests that use of rIX‐FP for prophylaxis leads to improved bleed control and lower consumption when compared to rFIXFc and nonacog alfa.

PB151: Identifying Efficacious Thresholds for Bleeding Risk Reduction in Relation to Factor VIII Levels in Hemophilia A Patients Receiving rVIII‐SingleChain

P. Zhang 1 ; C. Fosser 2 ; J. Roberts 1 ; A. Brainsky 1 ; Y. Li 1 ; W. McKeand 3 ; J. Sidhu 4

1 CSL Behring, King of Prussia, United States, 2 Cytel, Cambridge, United States, 3 CSL Behring, Clinical Pharmacology, King of Prussia, United States, 4 CSL Behring, Parkville, Australia

Background: There have been extensive discussions regarding the optimal FVIII level for prevention of bleeding events in severe hemophilia A (FVIII<1%).

Aims: To assess the threshold levels of FVIII associated with significant reduction of bleeding risk, evaluate potential determinants of efficacy and quantify the relative risk of bleeding episodes based on FVIII activity levels.

Methods: rVIII‐SingleChain is a single chain recombinant FVIII. Data for up to 2 years from 147 adults with 715 bleeding events from a Phase III study were utilized in the analysis, including all recorded bleeding episodes after the first infusion of rVIII‐SingleChain. FVIII activity levels for each 12 hour time interval were simulated using a previously published population pharmacokinetic model. A Cox proportional hazards model was used to relate the time‐to‐bleed event data and various measures of FVIII exposure (>1%, >2%, >3% and >5%), as well as different dosing regimens (prophylaxis vs. on‐demand) to compare the risk of bleeding.

Results: Patients with FVIII levels >1% had a 74% reduction in bleeding risk compared to patients with FVIII levels <1%. Patients who maintained FVIII activity level >1% for 6 months or 1 year decreased the relative bleeding risk by 71% and 92%. Maintaining thresholds >2%, >3% and 5% further reduced the relative risk by up to an additional 10%. The prophylaxis regimen showed a 91% reduction in relative risk compared with on‐demand regimen.

Conclusions: An rVIII‐SingleChain treatment regimen maintaining FVIII levels >1% over time is an effective approach to control bleeding events. The relative bleeding risk is reduced by 92% when the FVIII activity level is maintained >1% for over a year. Higher FVIII levels further reduce the relative bleeding risk slightly. Prophylaxis regimens provide greater protection from bleeding than the on‐demand regimen. This analysis quantitatively supports the rationale for optimized treatment regimens in adult hemophilia A patients by maintaining FVIII levels above 1%.

PB152: Whole F8 and VWF Gene Sequencing using Next‐generation Sequencing for Mutation‐negative French and Canadian Hemophilia A Patients

F. Lassalle 1,2 ; L. Jouan 3 ; L. Swystun 1 ; J. Gauthier 4 ; C. Zawadzki 2 ; J. Goudemand 2 ; S. Susen 2 ; G.‐E. Rivard 5 ; D. Lillicrap 1

1 Queen's University, Department of Pathology and Molecular Medicine, Kingston, Canada, 2 Lille University Hospital, Department of Hematology and Transfusion, Lille, France, 3 Sainte‐Justine University Hospital Research Center, Integrated Centre for Pediatric Clinical Genomics, Montréal, Canada, 4 Sainte‐Justine University Hospital, Molecular Diagnostic Laboratory and Division of Medical Genetics, Department of Pediatrics, Montréal, Canada, 5 Sainte‐Justine University Hospital, Department of Hematology/Oncology, Montréal, Canada

Background: The identification of the molecular defect responsible of hemophilia A (HA) is an important component in optimizing disease diagnosis. Despite the analysis of all exons of F8 , 5’/3’ UTR regions and search for copy number variants, the pathogenic variant is still missing in about 5% of HA patients. In the last several years, Next‐Generation Sequencing (NGS) has provided the capabilities of sequencing the complete F8 and VWF genes.

Aims: The aim of this study is to characterize intronic mutations in the F8 gene of “mutation‐negative” French and Canadian HA patients, by using a whole gene sequencing method performed by NGS.

Methods: We developed a small panel containing whole F8 (195 kb) and VWF genes (188 kb). The SureSelect system was used for target enrichment. We first analyzed 10 mutation‐negative HA patients and 6 controls (3 HA and 3 VWD) with known pathogenic variants. NGS was performed on a MiSeq sequencer. Data was analyzed with a bioinformatic pipeline according to the Genome Analysis Toolkit best practices. The effect on splice sites was predicted with Alamut ® Visual.

Results: NGS data revealed 5 deep intronic candidate F8 variants (one recurrent) in 5 HA patients. Patient 6 had an exonic variant in VWF described as VWD disease‐causing, that might lead to his reclassification. Two variants causing gene conversion in VWF have been found in patient 7. Patient 8 and related patients 9 and 10 didn't have an identified variant. We also found 5 variants that don't change splice sites so weren't classified as potentially pathogenic. The average sequence coverage depth was 20‐50X. Some variants had a coverage <20X threshold and they were all confirmed with Sanger sequencing.

Conclusions: This preliminary NGS run has corroborated that HA might be caused by deep intronic variants, by discovering 4 new candidate variants. Future sequencing will optimize the depth of coverage for this assay. RNA and minigene studies are in progress to confirm the deleterious effect of the intronic variants on F8 mRNA splicing.

PB153: Within‐patient Comparison of Treatment Patterns before and after Switching to rFIXFc: A Report from the UK National Haemophilia Database

M. Scott 1,2,3 ; H. Xiang 2 ; P. Collins 4,5,6 ; R. Liesner 2,7 ; C. Hay 2,8,9

1 Manchester Royal Infirmary, Clinical Haematology, Manchester, United Kingdom, 2 UK National Haemophilia Database, Manchester, United Kingdom, 3 University of Manchester, Institute of Cancer Sciences, Manchester, United Kingdom, 4 University Hospital of Wales, Cardiff, United Kingdom, 5 UK National Haemophilia Database, Cardiff, United Kingdom, 6 University of Cardiff, School of Medicine, Cardiff, United Kingdom, 7 Great Ormand Street Hospital, Department of Haematology, London, United Kingdom, 8 Manchester Royal Infirmary, Manchester, United Kingdom, 9 University of Manchester, Department of Vascular Sciences, Manchester, United Kingdom

Background: Prophylaxis with rFIXFc in patients with haemophilia B (HB) may permit a reduction of infusion frequency (IF) and clotting factor consumption (CFC) relative to standard rFIX, whilst improving bleeding outcome. However, real‐world studies directly comparing these products do not exist.

Aims: To compare IF and CFC in patients using FIX prophylaxis before and after switching to rFIXFc using the restricted, revenue‐neutral, NHS‐England protocol.

Methods: rFIXFc was introduced in the UK from September 2016 using a PK‐optimised protocol. This used extended half‐life to optimise dose and dose‐interval to aim for cost‐neutrality. We analysed all non‐trial patients switching from rFIX to rFIXFc prophylaxis, 01/09/16 ‐ 30/09/17, with ≥12‐months pre‐switch and ≥4‐weeks post‐switch HT‐compliant data. IF and CFC were calculated for both periods and compared using summary statistics and Wilcoxon signed rank test.

Results: Seventy three patients used rFIXFc during the study period, 36 of whom met the inclusion criteria (34 severe, 2 moderate (0.01 and 0.02 IU/ml). The med. (IQR) age was 21 (12‐47) years and med. (IQR) post‐switch follow‐up was 21 (14‐29) weeks. Med. IF and CFC declined post‐switch by 46% and 42%, respectively (P≤0.01) (Table), CFC reducing in all patients and IF in 35/36. Follow up is currently too short to evaluate the post‐switch annual bleed rate (ABR).

TABLE 1 Infusion frequency and clotting factor consumption pre‐ and post‐switch to rFIXFc Infusion Frequency (infusions/wk) FIX Consumption (IU/kg/wk) Median IQR Range Median IQR Range rFIX (pre‐switch) 1.98 1.53‐2.23 1.06‐7.05 79.00 52.00‐97.00 36.00‐416.00 rFIXFc (post‐switch) 1.09 0.95‐1.17 0.34‐2.33 46.00 31.00‐56.00 10.00‐162.00 Change −46% −42%

Conclusions: This direct within‐patient comparison of rFIX and rFIXFc prophylaxis, demonstrates a significant reduction in IF and CFC using a PK driven dose‐restricted protocol. An analysis of weekly rFIXFc prophylaxis in the B‐LONG trial showed a 60% reduction in overall CFC relative to rFIX, but similar post‐switch rFIXFc consumption. Our data, based on a larger cohort, potentially better reflects real‐world outcomes. The objective of dose‐restriction and cost‐neutrality was only partially realised, however. Extended follow‐up will allow better characterisation of these patterns and an analysis of ABR.

PB154: Arthropathy and Joint Replacement Surgery in Patients with Hemophilia A (PwHA) in the US

A. Patel ; E. Yang; N. Engmann; R. Ko; K. Raimundo

Genentech Inc., South San Francisco, United States

Background: Little is known about the real‐world prevalence of arthropathy and joint replacement surgery rates in PwHA.

Aims: To assess the prevalence of arthropathy in PwHA and its relationship to joint replacement surgery in the US.

Methods: This was a retrospective, US claims analysis using the MarketScan ® Research Commercial (private insurance) and Medicaid (public insurance) Databases. An algorithm (Lyons JL et al. 2017) was used to identify PwHA between 01/01/06 ‐ 09/30/15 based on HA diagnosis (ICD‐9: 286.0), treatment, demographics and resource use. Index date was defined as the first date of HA diagnosis or treatment. Patients were followed from index date to end of enrollment or analysis. PwHA with inhibitors were identified by ≥1 claim for a bypassing agent. HA arthropathy included arthropathy (ICD‐9: 710‐719) with a HA diagnosis in the same claim, or hemophilic arthropathy (ICD‐9: 713.2). Replacement surgery involving ankle, elbow, shoulder, hip or knee joints was included. Patient characteristics, and rates of arthropathy and joint replacement surgery were examined.

Results: A total of 2,908 PwHA (Commercial N=2,285, Medicaid N=623) were identified. PwHA on Medicaid were younger than those in Commercial plans with a mean age of 15.7 years (y) (SD=14.4 years) and 21.9 y (SD=15.4 years), respectively, and >99.5% patients were male. Mean follow‐up of these patients was 2.1 years and 2.5 years (median=1.6 years and 1.7 years), respectively. Inhibitor status was present in 41 (6.6%) Medicaid and 68 (3%) Commercial PwHA. Among PwHA, 31.9% had evidence of arthropathy related to HA. Of those, 5.1% had evidence of joint replacement surgery (additional results in the table).

TABLE 1 Hemophilia A arthropathy and joint replacement surgery by age between January 1, 2006 and September 30, 2015 (MarketScan ® Research Databases) Total, N=2,908a Truven Commercial, N=2,285 Truven Medicaid, N=623 Age group (in years) HA arthropathy, n (%) Joint replacement surgery, n (%)b HA arthropathy, n (%) Joint replacement surgery, n (%)b HA arthropathy, n (%) Joint replacement surgery, n (%)b Total, n 928 47 675 42 253 5 ≤5 152 (16.4%) 0 (0.0%) 96 (14.2%) 0 (0.0%) 56 (22.1%) 0 (0.0%) 6‐12 189 (20.4%) 1 (2.1%) 124 (18.4%) 0 (0.0%) 65 (25.7%) 1 (20.0%) 13‐17 112 (12.1%) 0 (0.0%) 78 (11.6%) 0 (0.0%) 34 (13.4%) 0 (0.0%) 18‐24 133 (14.3%) 4 (8.5%) 112 (16.6%) 4 (9.5%) 21 (8.3%) 0 (0.0%) 25‐35 159 (17.1%) 12 (25.5%) 119 (17.6%) 10 (23.8%) 40 (15.8%) 2 (40.0%) >35 183 (19.7%) 30 (63.8%) 146 (21.6%) 28 (66.7%) 37 (14.6%) 2 (40.0%)

a Includes total number of PwHA identified from Truven Commercial and Medicaid Databases. b Includes joint replacement surgery following a HA arthropathy diagnosis.

Conclusions: About 1/3rd of PwHA had evidence of arthropathy related to HA and 5% of those patients underwent joint replacement surgery, which can have clinical and health resource use implications. Future work should assess predictors of joint damage and associated health resource use in PwHA.

PB155: Extended Half‐life Factor Concentrate Use for Patients with Moderate and Severe Hemophilia among ATHN‐affiliated U.S. Hemophilia Treatment Centers

S.E. Croteau 1 ; D. Cheng 2 ; A.J. Cohen 3 ; C.E. Holmes 4 ; P.A. Kouides 5 ; L.J. Raffini 6 ; M. Silvey 7 ; C. Thornburg 8 ; A.P. Wheeler 9 ; E.J. Neufeld 10

1 Boston Children's Hospital/Harvard Medical School, Medicine/ Div. Hematology, Boston, MA, United States, 2 American Thrombosis and Hemostasis Network, Rochester, NY, United States, 3 Newark Beth Israel Med. Ctr., Newark, NJ, United States, 4 University of Vermont Medical Center, Burlington, VT, United States, 5 Mary M. Gooley Hemophilia Center, Rochester, NY, United States, 6 The Children's Hospital of Philadelphia, Philadelphia, PA, United States, 7 Childrens Mercy Hospital, Kansas City, MO, United States, 8 Rady Children's Hospital San Diego, San Diego, CA, United States, 9 Monroe Carell Jr. Children's Hospital at Vanderbilt, Nashville, TN, United States, 10 St. Jude Children's Research Hospital, Memphis, TN, United States

Background: Extended half‐life (EHL) FVIII and FIX products are intended to decrease the burden of prophylaxis (ppx). Whether the properties of these newer agents have led to clinical practice change remains poorly defined. The ATHNdataset, a U.S. database of 138 ATHN‐affiliated hemophilia treatment center (HTCs), provides an opportunity to explore longitudinal practice pattern changes.

Aims: To characterize use of FVIII and FIX concentrate types at HTCs, including rate of switching from standard half‐life (SHL) to EHL products.

Methods: Data were collected under the patient authorization for the ATHNdataset at 3 timepoints, baseline (Jun 2016) then 9‐month intervals, (Mar 2017, Dec 2017). Patients with moderate or severe hemophilia A or B without active inhibitors were included in the analysis.

Results: Reported use of EHL FVIII rose from 9% of patients at baseline to 21% as of 12/2017. The absolute number of patients on EHL FVIII ppx nearly tripled, Figure 1A. Use of EHL FIX for ppx exceeded SHL at 52%. Absolute increase in EHL use varied by age group, Figure 1B&C. Marked regional variability is observed in EHL adoption. Great Lakes region has the highest proportion of users at 63% EHL FVIII and 72% EHL FIX. The rate of increase is highest in the Southeast. Median dose and infusion frequency for SHL products were FVIII 40 IU/kg/dose, 156 annual infusions and FIX 67 IU/kg/dose, 104 annual infusions compared to EHL, FVIII 40 IU/kg/dose, 104 annual infusions, and FIX 75 IU/kg/dose, 52 annual infusions. On‐demand EHL use has grown, but its contribution to overall usage in minimal.

FIGURE 1 Longitudinal change in SHL and EHL factor concentrates use for prophylaxis in U.S. based on ATHNdataset

Conclusions: EHL FVIII use is increasing, but most remain on SHL. EHL FIX now predominates for ppx. Despite broad regional variation, a steady increase in EHL use is occurring nationally. While median IU/kg and infusion frequency is less annually for prescribed EHL ppx, decreasing treatment burden, total cost of ppx is higher. Unless demonstrably off‐set by reduction in bleed doses, the net effect may be more costly therapy for a growing population.

PB156: Genetic Polymorphisms Contributing to the Pharmacokinetics of Factor VIII Concentrates

B. Lunghi 1 ; S. Frusconi 2 ; S. Linari 3 ; G. Marchetti 4 ; F. Bernardi 1 ; G. Castaman 5 ; M. Morfini 6

1 University of Ferrara, Department of Life Science and Biotechnology, Ferrara, Italy, 2 Genetic Diagnostics Unit, Laboratory Department, Florence, Italy, 3 Azienda Ospedaliera Universitaria Careggi, Department of Oncology, Center for bleeding disorders, Florence, Italy, 4 University od Ferrara, Department of Biomedical and Specialty Surgical Sciences, Ferrara, Italy, 5 Center for Bleeding Disorders, Department of Oncology, Florence, Italy, 6 Italian Association Haemophilia Centers AICE , Milan, Italy

Background: The covariates determining the large inter‐patients’ variability of FVIII pharmacokinetics (PK) are poorly known.

Aims: To investigate the relationships between the individual PK and genes potentially modulating the FVIII survival.

Methods: 85 FVIII single dose (21.4‐51.8 IU/kg) PKs were performed in 33 hemophilia A patients (FVIII:C≤ 2IU/dl) without inhibitors. 20 (60%) of patients underwent repeated PK (2‐6) with different FVIII concentrates. The FVIII/time concentrations collected up to 72 hrs were analyzed by Two‐Compartment Model (TCM) (WinNonlin). Final (K1‐0, K1‐2, K2‐1, V1) and Secondary Parameters (AlfaHL, BetaHL, Cl, CLD2, Cmax, K1‐0HL, MRT, V2, Vss) were evaluated. Genes coding for F8, AB0 blood group, vWF, ASGPR, ITGB3, LDLR, LRP1/2, HSPGs, HPSE receptors were sequenced in promoter region or/and in exons containing informative polymorphisms (N=25). Data of each PK parameter were compared by non‐paired, two‐tails, Student's t test in patients grouped for genotypes.

Results: The inter‐patients variability of PK outcomes resulted very large but inter‐occasion variability was definitely smaller: i.e. Beta_HL range 6.3‐46.0 versus 11.0‐21.5 hrs, respectively. Blood group 0 was associated with shorter Alpha half‐life than non‐0 (P=0.01). The LDLR 1173 C/T (rs688) displayed the most significant association (TT vs. C‐carriers, Alpha HL P=0.001). In the ITGB3 gene several in linkage polymorphisms showed association with mean values of PK parameters (i.e. ‐425A/C, rs62074393, AA vs. C‐carriers, Beta HL P=0.01). For the ASGR2 gene the ‐95 T/C (rs2289645) polymorphism displayed association with several PK parameters (i.e. TT vs. T‐carriers, CLD2 P=0.01).

Conclusions: The study confirms the need for tailoring replacement therapy in HA, being hard to translate the outcomes of HA population PK to single patient and suggests receptor gene polymorphisms to be further analyzed in relation to FVIII PK in HA patients.

PB157: Real World Data Analysis of US Claims Database on Coagulation Factor VIII Expenditures in Patients with Hemophilia A: Standard Half‐life versus Extended Half‐life Products

D. Spurden 1 ; A. Chhabra 2 ; B. Tortella 3 ; P. Fogarty 3 ; E. Rubinstein 2 ; S. Harris 1 ; A. Pleil 4 ; J. Alvir 2

1 Pfizer Limited, Tadsworth, United Kingdom, 2 Pfizer Inc, New York, United States, 3 Pfizer Inc, Collegeville, United States, 4 Pfizer Inc, San Diego, United States

Background: Hemophilia A management requires intravenous factor VIII (FVIII) infusions to replace the missing coagulation factor. Extended half‐life (EHL) FVIII products were recently introduced that may require fewer infusions compared to standard half‐life (SHL) FVIII products.

Aims: We analyzed US claims data to determine international units (IUs) dispensed, expenditure and all‐cause direct expenditure associated with EHLs versus SHLs.

Methods: De‐identified claims data from the Optum ® Clinformatics ® US claims database were used to retrospectively identify male patients with hemophilia A who had used an SHL and/or EHL FVIII replacement product from August 1, 2014, to September 30, 2017, and had data for at least 3 months of dispensation. The key outcome measures were direct expenditures, factor IUs dispensed for the FVIII replacement products and outpatient (OP) and inpatient (IP) visits. Medians were used to accommodate for skewed distributions. Quarterly data for up to 36 months were analysed.

Results: 314 SHL subjects and 50 EHLs subjects were analyzed. Median age in both groups was similar (SHL=22.50 years; interquartile range (IQR) 24.00 years; EHL=18.50 years; IQR 19.00 years). Median quarterly study period FIX expenditure was $52,575 higher (1.7 times) for the EHL cohort ($133,168; IQR $107,273) versus the SHL cohort ($80,593; IQR $99,173). The Median quarterly IU usage was also higher for EHL (74,742 IU; IQR 57,845) versus SHL users (60,320 IU; IQR 74,189). Total haemophilia‐related expenditure including OP, IP and prescription expenditure was $50,123 higher (1.6 times) for the EHL cohort ($133,344; IQR $125,673) versus the SHL cohort ($83,221; IQR $101,072).

Conclusions: This real‐world data analyses, unadjusted for treatment regimen or disease severity, suggest that both IU dispensation and total FVIII expenditures are significantly higher in hemophilia A patients using EHL versus SHL. Further analyses, incorporating essential clinical characteristics, should be explored.

PB158: Measuring FIX‐Activity of Nonacog Beta Pegol (N9‐GP) in a One‐stage Ellagic Acid Based Clot Assay on the ACL TOP 50 Series Instrument: A Two‐site Study

A. Khan 1 ; B. Wilkinson 1 ; K. Shishko 1 ; M. Hansen 2 ; M. Doyle 1 ; M. Triscott 1 ; M. Ezban 2

1 Instrumentation Laboratory, Research & Development, Bedford, United States, 2 Novo Nordisk A/S, Haemophilia Research, Maaloev, Denmark

Background: The Factor IX‐SynthAFax (FIX‐SFX) application on the ACL TOP/TOP50 Series Instruments was designed to measure Factor IX clotting activity in plasma samples for aid in diagnosing and monitoring hemophilia therapy. This study aimed to demonstrate the reproducibility and precision of the above application in accurately measuring the FIX clotting activity of nonacog beta pegol (N9‐GP) spiked plasma samples when compared to a SSC standard.

Aims: To demonstrate reproducibility of the FIX‐SFX application to recover N9‐GP and SSC spiked samples on the ACL TOP Family 50 Series at two sites; IL Bedford and Novo Nordisk.

Methods: Study kits were prepared by IL and distributed to the second site. Each site performed a FIX calibration with HemosIL Calibration plasma and HemosIL FIX deficient plasma. Two quality control levels were tested to verify the calibration. N9‐GP spiked samples were prepared at three target levels (100, 40 & 5% act.) with FIX deficient plasma (George King) & Factor diluent. SSC samples were prepared at three target levels (105, 40 and 5% act.) with Factor diluent. All six samples were tested over five days (N=25 per level per site) on an ACL TOP 550 CTS analyzer. The mean result from each site was used to calculate the % recovery relative to the target. A recovery of ± 30% for each sample target level and a total %CV ≤20% was deemed acceptable across sites.

Results: The FIX‐SFX calibration was validated before use. Quality Controls recovered within the acceptance criteria at both sites. All sample data (N=50 per sample for N9‐GP & SSC) were comparable and recovered well within the acceptance criteria (see Table 1). Data shows comparable recovery and statistics across sites.

TABLE 1 Two‐site study data summary Samples N Mean (% Activity) % Recovery Total %CV N9‐GP 100% level 50 88.4 88.4 7.2 N9‐GP 40% level 50 38.3 95.7 7.1 N9‐GP 5% level 50 5.7 113.6 10.2 SSC 105% level 50 114.4 108.9 6.7 SSC 40% level 50 41.7 104.3 9.7 SSC 5% level 50 5.9 118.4 12.7

Conclusions: N9‐GP showed consistent results and accurate recovery using the new FIX‐SFX application on the ACL TOP 50 Series Instrument with SynthAFax in an OSCA.

PB159: Phase 1/2 Trial of Single and Multiple Dose Subcutaneously Administered Factor IX Variant CB2679d/ISU304: Pharmacokinetics and Safety

C.W. You 1 ; H. Levy 2 ; M. Lee 3 ; J. Park 4 ; S.‐B. Hong 4 ; S. Kim 4 ; H.‐J. Shin 5 ; D.Y. Kim 5 ; J.S. Kim 6 ; J.W. Han 6 ; S.‐J. Kim 6

1 Eulji University Hospital, Daejeon, Korea, Republic of, 2 Catalyst Biosciences, South San Francisco, United States, 3 UCLA Fielding School of Public Health, Biostatistics, Los Angeles, United States, 4 ISU Abxis, Sungnam‐si, Korea, Republic of, 5 Pusan National University Hospital, Busan, Korea, Republic of, 6 Yonsei University College of Medicine, Severance Hospital, Seoul, Korea, Republic of

Background: CB2679d/ISU304 was developed using rational protein design to increase resistance to ATIII inhibition, increase catalytic activity and affinity for FVIIIa resulting in 22‐fold enhanced potency enabling administration by subcutaneous (SQ) injection for use as prophylaxis.

Aims: Determine pharmacokinetics and safety of SQ CB2679d.

Methods: Cohort 1 received 75 IU/kg intravenous (IV) BeneFIX followed by 75 IU/kg IV CB2679d. Cohort 2 and 3 received CB 2679d 75 IU/kg IV followed by 75 or 150 IU/kg SQ. Cohort 4 was omitted. Cohort 5 received 150 IU/kg SQ daily for 6 days.

FIGURE 1 ISU304‐001 trial schema

FIGURE 2 FIX activity with six days of subcutaneous dosing

Safety measures were performed 2 weeks after dosing. FIX antigen (VisuLizeTM Factor IX Antigen KitAG (Affinity Biologicals)) and activity (one‐stage clotting assay by ACL TOP 700 and Instrumentation Laboratories reagents)), anti‐drug antibody and neutralizing antibody were measured at Haematologic Technologies. Half‐life (t1/2) calculation followed Lee et al, that uses an iterative piecewise fitting algorithm based on a robust (M‐regression) log‐linear model. Activity data were adjusted for baseline assuming exponential falloff of IV administration. Bioavailability was calculated from the AUC0‐t activity data.

Results: IV CB 2679d has 22‐fold greater potency, longer terminal t1/2 27.0±2.2 versus 21.0±1.1 hrs (P=0.0014) and mean residence time 35.8±2.5 versus 25.1±1.5 hrs (P=0.0004) compared with BeneFIX. SQ bioavailability was 19.8±5.2%. Median [25‐75%] peak activity 24 hrs after the 6 th dose was 15.7% [14.9‐16.6%] and median terminal t1/2 63.2 [60.2‐64.0] hrs was 2.3‐fold longer than IV.

No subject had a bleed during the study. Adverse events were mild after 1st through 4th injection except for moderate pain, erythema and redness at SQ injection site in 1 subject after injections 1 and 2.

Conclusions: SQ PK data supports the aim of achieving 30% activity within 12 days and normal FIX activity levels within 17 days of initiating daily SQ CB2679d/ISU304 dosing. Complete safety, bioavailability, PK and activity after daily SQ dosing will be reported.

PB160: A Prospective Observational Study of Prophylaxis, ACtivity and Effectiveness (SPACE) in Hemophilia Patients Currently Treated with Antihemophilic Factor (Recombinant) in the US

B. Konkle 1 ; D.V. Quon 2 ; L. Raffini 3 ; M. Recht 4 ; V. Radulescu 5 ; S.L. Carpenter 6 ; A. Dunn 7 ; M. Watt 8 ; J. Booth 9

1 Bloodworks Northwest, Seattle, United States, 2 Orthopaedic Hemophilia Treatment Center, Los Angeles, United States, 3 Children's Hospital of Philadelphia, Philadelphia, United States, 4 Oregon Health & Science University, Portland, United States, 5 University of Kentucky Medical Center, Lexington, United States, 6 Children's Mercy Hospital, Kansas City, United States, 7 Nationwide Children's Hospital and The Ohio State University School of Medicine, Columbus, United States, 8 Shire, Zug, Switzerland, 9 Shire, Cambridge, United States

Background: Maintaining factor activity levels ≥1% of normal during prophylaxis may not be optimal for all patients (pts) with hemophilia A (HA). It is unclear if higher factor VIII (FVIII) peaks or troughs are required to prevent bleeding episodes (BE) in someone with an active lifestyle.

Aims: To analyze the relationship between activity level, timing of factor infusion, and BEs in pts with moderately severe/severe HA on Antihemophilic Factor (Recombinant) (rFVIII; ADVATE ® , Shire, Lexington, MA, US). To integrate consumer information technology to collect physical activity type/frequency using a pt‐reported eDiary (SPACE eDiary) and an activity tracker (Fitbit ® ; Fitbit, Inc.).

Methods: SPACE was a 6‐mo, post‐authorization, multicenter, observational outcomes study ( NCT02190149 ). Key outcomes were BEs, rFVIII regimen and dosing, physical activity, functional ability, and pt satisfaction. Baseline characteristics were obtained via case report form and eDiary survey, pt‐reported outcomes including activity type via eDiary. Activity risk levels were assigned using National Hemophilia Foundation (NHF) criteria.

Results: 54 pts (aged 11‐58 years) were enrolled (47 prophylaxis, 7 on demand). Mean annualized bleeding rate on prophylaxis was 8±11. Pts (N=54) were infused ≤24 h before activity in 35% (1759/4980) of infusion intervals recorded (Table). More infusions were performed ≤24 h before commencing higher‐risk versus lower‐risk activities. In SPACE eDiary regression models, prolonging the time between last infusion and activity start (from ≤12 to 12‐24 h) showed consistent but statistically nonsignificant increased odds (13‐20%) for activity‐related bleeds (P≥0.6423).

Conclusions: For pts with HA on prophylaxis, aligning infusion timing with activity patterns may reduce bleeding risk, although further investigation in a larger population is needed to confirm the temporal association. Most pts were satisfied with the eDiary app and Fitbit.

Funding: This study was funded by Shire Development LLC.

TABLE 1 Time From Most Recent Infusion Prior to Start of Activity Based on NHF Activity Risk Level (eDiary Data) All patients (N=54) NHF activity level 1 1.5 2 2.5 3 Number of intervals included in analysis 4980 3761 488 935 330 196 Time between infusion and start of activity, n (%) Within 12 h 870 (17.4) 658 (17.5) 107 (21.9) 183 (19.5) 59 (17.9) 39 (19.9) Within 24 h 1759 (35.3) 1368 (36.4) 162 (33.2) 331 (35.3) 135 (40.9) 83 (42.3) Within 1‐2 d 1382 (27.8) 1047 (27.8) 130 (26.6) 283 (30.3) 92 (27.9) 52 (26.5) Within 2‐3 d 802 (16.1) 632 (16.8) 74 (15.2) 124 (13.3) 37 (11.2) 33 (16.8) Within 3‐5 d 558 (11.2) 411 (10.9) 57 (11.7) 98 (10.5) 31 (9.4) 19 (9.7) Within >5 d 479 (8.6) 303 (8.1) 65 (13.3) 99 (10.5) 35 (10.6) 9 (4.6)

PB161: Correlation of Factor Levels with Bleeding Phenotype in Non‐severe Hemophilia

J. Rejtő 1 ; G. Schuster 2 ; C. Feistritzer 3 ; P. Quehenberger 4 ; S. Hofer 1 ; O. Königsbrügge 1 ; E. Grilz 1 ; F. Posch 5,6 ; J. Gebhart 1 ; C. Ay 1 ; I. Pabinger 1

1 Medical University of Vienna, Department of Medicine I, Vienna, Austria, 2 Blutspendezentrale, Linz, Austria, 3 Medical University Innsbruck, Department of Internal Medicine V, Innsbruck, Austria, 4 Medical University of Vienna, Department of Laboratory Medicine, Vienna, Austria, 5 Medical University Vienna, Department of Medicine I, Vienna, Austria, 6 Medical University of Graz, Department of Internal Medicine, Graz, Austria

Background: Hemophilia is categorised into severity groups (severe‐, moderate‐ and mild hemophilia) based on the basal activity level of factor VIII (FVIII) or ‐IX (FIX). This categorisation is expected to be in accordance with the severity of the bleeding phenotype.

Aims: Our study aimed at quantifying the association between the severity of the bleeding phenotype and FVIII/ IX basal activity levels in 98 non‐severe hemophilia A (HA) and ‐B (HB) patients.

Methods: Patients with non‐severe hemophilia, defined as baseline FVIII/ IX activity of >1 to 40%, were eligible. Data on the bleeding history were collected via direct patient consultations and search of hospital databases. This information was used to quantify the bleeding phenotype with the Bleeding Assessment Tool of the International Society on Thrombosis and Haemostasis (ISTH‐BAT).

Results: Seventy‐seven (79%) patients with HA and 21 (21%) patients with HB were enrolled (median age 50 years, range 19‐80). Median levels of FVIII were 13% [range: 1.5%‐39%] and that of FIX were 12% [2%‐25%]. The median ISTH‐BAT score was 10 points [0‐28] overall, 10 [0‐28] points in HA, and 8 [1‐20] points in HB, and did not differ significantly between HA and HB (P=0.48).

Lower basal factor levels correlated with a higher ISTH‐BAT score (Spearman's rho=‐0.29, P=0.004). In linear regression, a 1% increase in basal factor levels was associated with a 0.2 point decrease in the ISTH‐BAT (β=‐0.20, 95%CI: ‐0.33‐(‐0.07), P=0.003). This association was similar in HA and HB patients (P for interaction=0.78). Basal factor levels explained only 10% of the variation in the ISTH‐BAT (R 2 =0.10 for both HA and HB patients).

Conclusions: We observed a weak but significant correlation between basal FVIII/IX levels and the bleeding severity. This data are consistent with the concept that there are other yet‐to‐be‐explored determinants of the bleeding phenotype beyond basal factor levels in hemophilia.

PB162: The Effect of Inhibitors on Quality of Life and Pain in Adult Patients with Severe Haemophilia in the PROBE Cohort

D. Noone 1 ; C. Chai‐Adisaksopha 2 ; R. Curtis 3 ; N. Frick 4 ; M. Nichol 5 ; B. O'Mahony 1,6 ; D. Page 7 ; J. Stonebraker 8 ; A. Iorio 2 ; M.W. Skinner 9 ; PROBE

1 Irish Haemophilia Society, Dublin, Ireland, 2 McMaster University, Hamilton, Canada, 3 Factor VIII Computing, Berkeley, United States, 4 National Hemophilia Foundation, New York, United States, 5 University of Southern California, Sol Price School of Public Policy, Los Angeles, United States, 6 Trinity College, Dublin, Ireland, 7 Canadian Hemophilia Society, Montreal, Canada, 8 Poole College of Management, North Carolina State University, Raleigh, United States, 9 Institute for Policy Advancement Ltd., Washington, United States

Background: The development of anti‐factor VIII inhibitors pose a significant additional burden to people with severe haemophilia A (PWsHA).

Aims: To describe the effect of inhibitors on the quality of life (QoL) and reporting of pain in PWsHA and inhibitors.

Methods: This analysis utilised data from the PROBE (Patient Reported Outcomes, Burdens and Experiences) study. Participants with No Bleeding Disorder (NOBD), PWsHA, and PWsHA with a current inhibitor were compared. QoL was measured with EQ‐5D and VAS. Descriptive analyses and logistic regression adjusted for age, mobility, presence of other pain, use of pain medication, and reporting of other health problems were used. Standard t‐tests and chi squared tests were conducted to test for between‐group differences.

Results: 798 male patients, aged 11‐81 were analysed: 174 (22%) NOBD, 582 (73%) PWsHA and 42 (5%) PSsHA diagnosed with an inhibitor. PWsHA and PWsHA with inhibitors did not differ for bleeding rates, presence or number of target joints, or range of motion. There was a statistically significant increase of reported life‐threatening bleeds in PWsHA with inhibitors over PWsHA (40% vs. 14.5%; P<0.001). 50% of PWsHA with inhibitors were on intermittent or regular prophylaxis versus 76% of PWsHA.

Main results are reported in the table.

All EQ5D domains were different (P<0.001) except Anxiety/Depression between PWsHA and PWsHA with a current inhibitor (P=0.317). The strongest interference of acute pain was found for walking ability (P=0.026) and sleep (P=0.056); for chronic pain it was normal work (P=0.013) and sleep (P=0.090), without differences among subgroups.

Conclusions: PWsHA with inhibitors in the PROBE cohort have lower QoL, and more chronic and acute pain. The difference is not explained by bleeding, target joints or functional impairment.

PB163: Efficacy and Safety of Recombinant Factor IX‐ Fusion Protein (rIX‐FP) in Patients with Hemophilia B: Interim Results from an Ongoing Phase IIIb Extension Study

E. Santagostino 1 ; I. Pabinger 2 ; A. Brainsky 3 ; Y. Li 3 ; W. Seifert 4

1 Maggiore Hospital Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center IRCCS Ca’ Granda Foundation, Milan, Italy, 2 Universitätsklinik für Innere Medizin, Medizinische Universität Wien, Vienna, Austria, 3 CSL Behring, King of Prussia, United States, 4 CSL Behring, Marburg, Germany

Background: rIX‐FP is a fusion protein genetically linking recombinant human coagulation Factor IX with recombinant human albumin. It has an improved pharmacokinetic profile compared with standard FIX products, allowing a prolonged dosing interval in patients with Hemophilia B.

Aims: As part of the PROLONG‐9FP clinical program, this phase 3b study evaluates the long‐term safety and efficacy of rIX‐FP over approximately 3 years.

Methods: Patients received routine prophylaxis with rIX‐FP; dosing interval could be changed at any 6‐month follow‐up. Treatment intervals could be extended from a weekly dose to every 10 or 14 days with rIX‐FP doses of 50 to 75 IU/kg. Patients ≥18 years old could switch to a 21‐day regimen at a dose of 100 IU/kg if they were well controlled on a 14‐day regimen. The primary outcome was number of patients who developed inhibitors against FIX. Secondary outcomes included annualized spontaneous bleeding rate (AsBR) and adverse events.

Results: To date, 59 PTPs ≥12 and 24 < 12 years had entered the study (mean time on study 29.8 months). Over 79% of patients ≥12 switched from weekly to 10 (n=12), 14 (n=26) or 21‐day intervals (n=11). Two patients starting 21‐day dosing switched back to the 14 day interval to reduce their bleeding frequency. 25% of patients < 12 were on the extended prophylaxis intervals of once every 10 (n=1) or 14 days (n=5). In adults, median (Q1, Q3) AsBR for 7, 10, 14 and 21 day regimens was 0.33 (0.00, 2.39), 0 (0.00, 0.68), 0.26 (0.00, 1.54) and 0 (0.00, 0.45), respectively. In pediatrics, median AsBR for 7, 10 and 14 day regimens was 0 (0.00, 0.59), 0 (0.00, 3.06) and 0.75 (0.00, 2.86), respectively. Median number of exposure days (EDs) was 165 and 77 (92.8%) subjects have achieved 100 EDs to rIX‐FP; none have developed inhibitors or antibodies to rIX‐FP.

Conclusions: Long‐term efficacy and tolerability of prophylaxis with rIX‐FP have been demonstrated. rIX‐FP allows extended treatment intervals of 21 days in adults and 10 and 14 days in children for selected patients.

PB164: Burden of Mild Hemophilia A: Systematic Literature Review

F. Peyvandi 1 ; A. Lawal 2 ; M.R. Fasulo 1 ; D. Frame 3 ; B. Kim 2 ; M. Tavakkoli 2 ; M.C. Lee 2 ; W.Y. Wong 2

1 University of Milan, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milano, Italy, 2 BioMarin Pharmaceutical Inc., Novato, United States, 3 Frame Research, LLC, Brooklyn, United States

Background: Hemophilia A (HA), an X‐linked, monogenic disorder, is a life‐threatening condition that may require lifelong treatment. Even mild forms of HA can impact morbidity, quality of life (QOL), and health care utilization. The overall burden of disease for mild HA (FVIII activity 5‐40%) is not well‐studied, and may vary depending on treatment strategies, patient characteristics, and geographic location of care.

Aims: To systematically assess available evidence of disease burden and clinical risks in adult patients with mild HA.

Methods: A systematic literature review was conducted with prospectively determined search criteria. Studies of any design, published in English 1966‐2017, were eligible. We focused on adults with mild HA or studies with mixed populations of adults and adolescents ≥13 years of age. Studies including pediatric patients or without separable outcomes for mild HA were excluded.

Results: Of 1,189 citations (Figure 1), 14 studies met eligibility criteria; mild HA adults (total n=1,740) were a subset of the overall study population in all articles. Studies were conducted in Europe (57%), N. America (36%), and Japan (7%). Clinical burden of mild HA was reported in a variety of formats, making comparison across studies difficult (Table 1). QOL, cost, and productivity loss were reported in only one study each: SF‐36 general health was lower for mild HA versus age‐matched controls in a Canadian cohort (58.1 vs. 70.8, P<0.05). In a US study, mild HA patients missed 6.2 work days per year, of which 4.7 (76%) were due to HA. Mild HA health care costs of 793 euro/year were reported in a Portuguese study.

Conclusions: Quantifying outcomes for adult patients with mild HA remains challenging, with estimates of key QOL and cost data often based on small subsets of a single study. While mean annual bleeding rates were relatively low, mild HA had measurable impact on QOL and productivity and represents an area of therapeutic concern that should be addressed.

FIGURE 1 Study attrition diagram

TABLE 1 Clinical burden of mild HA in published studies Outcome # studies # mild HA ≥age 13 Range in published studies Mean annual bleeding episodes 3 157 0.41‐4.5 episodes/year Mean severity score (HSS or Brummel‐Ziedins) 2 33 0.05‐0.07 (as proportion of maximum score) Mean joint score / joint pain 3 89 0.03 (HSS joint); 5.3 (Colorado); 20% of patients reported moderate or severe pain in past year Inhibitor development (incidence) 3 217 4.0%‐7.8% over 10 years HSS = Hemophilia Severity Score (maximum score 3). Brummel‐Ziedins: Bleeding tendency score developed by Brummel‐Ziedins, et al. (2009), incorporating hemarthrosis, soft tissue hematoma and annual FVIII unit/ kg usage (maximum score 32). HSS joint = Hemophilia Severity Score, Joint (maximum score=1). Colorado = Colorado joint score (maximum score=150).

PB165: Realworld Health Care Expenditure and Dispensation of Extended and Standard Half‐life Recombinant Factor IX Products in Hemophilia B Patients

D. Spurden 1 ; A. Chhabra 2 ; B. Tortella 3 ; P. Fogarty 3 ; E. Rubinstein 2 ; S. Harris 1 ; A. Pleil 4 ; J. Alvir 2

1 Pfizer Limited, Tadworth, United Kingdom, 2 Pfizer Inc, New York, United States, 3 Pfizer Inc, Collegeville, United States, 4 Pfizer Inc, San Diego, United States

Background: Hemophilia B management requires intravenous factor IX (FIX) infusions to replace the missing coagulation factor. Extended half‐life (EHL) FIX products were recently introduced that require fewer infusions compared to standard half‐life (SHL) FIX products.

Aims: We analyzed US claims data to determine international units (IUs) dispensed and expenditures associated with EHLs versus SHLs. We examined how switching from SHL to EHL impacts pharmacy expenditure.

Methods: Truven Health MarketScan ® Research US claims database was queried for FIX IUs dispensed and expenditures. EHL patients were compared with the SHL patients (cross‐sectional analysis), starting June 2014 (first EHL claim). We also analysed FIX dispensation and expenditure in patients who switched from SHL (nonacog alfa) to EHL (eftrenonacog alfa) product with at least 3 months of data pre and post‐switch. Descriptive statistics were calculated with medians used to accommodate for skewed distributions.

Results: Cross sectional analysis: Quarterly expenditure and IUs dispensed were analyzed for 118 patients over up to 36 months (87 SHL; 31 EHL). Median quarterly study period FIX expenditure was $116763 higher (3.4 times) for the EHL cohort ($165,022) versus the SHL cohort ($48,259). Median quarterly IU usage was also higher for EHL (59,337 IU) versus SHL users (34,088 IU).

Switch analysis: 16 patients switched to EHL FIX and had at least 3 months data pre and post switch. Median quarterly FIX expenditure analyzed over up to 3 years pre/post‐switch was $75,064 pre‐ switch and $199,466 post‐ switch. Median quarterly metric IUs dispensed pre‐ switch was 62,857 and 68,829 IUs post‐switch.

Conclusions: These real‐world data analyses, unadjusted for treatment regimen or disease severity, suggest that both IU dispensation and total FIX expenditures are higher with EHL versus SHL in US hemophilia B patients. Further analyses, incorporating essential clinical characteristics, should be explored.

PB166: Expanding Communications on Hemophilia A Outcomes (ECHO) Study: Cross‐sectional Analysis of Baseline Data

C.R.M. Hay 1 ; M. Shima 2 ; M. Makris 3 ; V. Jiménez‐Yuste 4 ; M.W. Skinner 5 ; J. Oldenburg 6 ; K. Fischer 7 ; E. Santagostino 8 ; S. von Mackensen 9 ; S. Rauchensteiner 10 ; N. Church 11 ; P. Mathew 11 ; S. Krassova 10 ; C.M. Kessler 12 ; on behalf of the ECHO Study Investigators

1 Manchester University Department of Haematology, Manchester, United Kingdom, 2 Nara Medical University, Kashihara, Japan, 3 University of Sheffield, Department of Infection, Immunity, and Cardiovascular Disease, Sheffield, United Kingdom, 4 Autónoma University Madrid and La Paz University Hospital, Madrid, Spain, 5 Institute for Policy Advancement, Ltd, Washington, DC, United States, 6 University Clinic Bonn, Department of Experimental Haematology and Transfusion Medicine, Bonn, Germany, 7 Van Creveldkliniek, University Medical Center Utrecht, Utrecht, the Netherlands, 8 IRCCS Ca’ Granda Foundation, Maggiore Hospital Policlinico, Milan, Italy, 9 University Medical Centre Hamburg‐Eppendorf, Hamburg, Germany, 10 Bayer, Basel, Switzerland, 11 Bayer, Whippany, NJ, United States, 12 Georgetown University Medical Center, Washington, DC, United States

Background: The Expanding Communication on Hemophilia A Outcomes (ECHO) study is a prospective, multinational, observational cohort study investigating patient‐reported outcomes (PROs) in patients with moderate/severe hemophilia A (PwHA) aged ≥16 years.

Aims: To describe cross‐sectional baseline data and to relate PROs to clinical data and sociodemographics.

Methods: Data collection was planned at baseline and annually for ≥2 years. Clinical data were reported from medical records and patient diaries. PROs were assessed with standardized questionnaires (electronic or paper) for health‐related quality of life, physical functioning, adherence, treatment satisfaction, pain, and productivity impairment. Joint status was assessed by presence of target joints, joint procedures, Hemophilia Joint Health score, and Pettersson score. Data analyses were explorative and descriptive.

Results: Of 271 PwHA enrolled in ECHO (UK, n=146; Japan, n=76; Spain, n=25; US, n=24), 247 were included in this analysis (median [quartile 1; 3] age, 37 [27; 48] years). Of these, 84.6% had severe hemophilia, 54.7% had target joints, 34.8% had chronic arthropathy, 9.3% had an inhibitor history, 16.6% were HIV‐positive, and 27.9% were positive for hepatitis C antibodies. Prophylaxis was used in 80.2% of patients (daily, 6%; every other day, 15%; 3x/wk, 28%; 2x/wk, 18%). PRO data were available from 68% to 77% of patients (Table). PRO scores were generally similar for moderate (15.4%) and severe hemophilia and current prophylaxis versus on demand; however, few patients were treated on demand or had moderate hemophilia (Table). Increasing age was associated with greater arthropathy, reflecting other covariates on PRO data.

Conclusions: Most PRO data were adversely influenced by increasing age, reflecting the disease history apart from general age‐related developments. Further analyses are necessary to adjust for covariates and to investigate the influence of different factors, such as hemophilia A severity and treatment regimen.

TABLE 1 Selected PRO Data* at Baseline in ECHO PRO Aspect PRO Instrument Possible Range Interpretation Σ Total (n=247)† Aged 16‐27 y (n=66)† Aged 28‐36 y (n=57)† Aged 37‐47 y (n=61)† Aged >47 y (n=63)† Physical functioning HAL 0‐100 High value indicates good functioning 68±25 (n=189) 84±20 (n=39) 76±21 (n=45) 63±23 (n=50) 54±25 (n=55) Work productivity WPAI (activity impairment) 0‐100 High value indicates high impairment 33±29 (n=184) 20±26 (n=38) 27±23 (n=44) 41±30 (n=49) 38±30 (n=53) HRQoL EQ‐5D‡ , § 0‐1 High value indicates good HRQoL 0.7±0.3 (n=184) 0.8±0.2 (n=39) 0.8±0.2 (n=43) 0.7±0.3 (n=49) 0.6±0.3 (n=53) HRQoL EQ‐VAS 0‐100 High value indicates good HRQoL 71±21 (n=191) 75±19 (n=39) 75±20 (n=45) 66±19 (n=50) 68±22 (n=57) HRQoL SF‐12: PCS 0‐100 High value indicates good HRQoL 44±14 (n=186) 53±14 (n=36) 47±12 (n=45) 42±12 (n=50) 37±13 (n=55) Treatment satisfaction Hemo‐SatA 0‐100 High value indicates dissatisfaction 23±14 (n=185) 20±10 (n=38) 20±13 (n=44) 29±14 (n=49) 22±14 (n=54) Treatment adherence Veritas‐PRO Veritas‐PRN 24‐120 (both) High value indicates nonadherence 44±11 (n=149) 51±15 (n=30) 47±13 (n=33) 50±9 (n=4) 44±11 (n=39) 50±8 (n=4) 44±10 (n=41) 56±18 (n=8) 43±11 (n=36) 49±18 (n=14) Pain BPI‐SF (total) 0‐10 High value indicates more pain 2.7±2.4 (n=168) 1.8±2.2 (n=31) 2.5±2.3 (n=43) 3.4±2.5 (n=43) 2.7±2.4 (n=51) BPI‐SF=Brief Pain Inventory‐Short Form; EQ‐5D=EuroQoL 5‐Dimension questionnaire; EQ‐VAS=EuroQoL visual analog scale; HAL=Haemophilia Activities List; Hemo‐SatA=Hemophilia Treatment Satisfaction Questionnaire for adults; HRQoL=health‐related quality of life; PCS=physical components summary score of SF‐12; PRO=patient‐reported outcome; SF‐12=Short‐Form Health Survey; Veritas‐PRN=Validated Hemophilia Regimen Treatment Adherence Scale‐On Demand; Veritas‐PRO=Validated Hemophilia Regimen Treatment Adherence Scale‐Prophylaxis; WPAI=Work Productivity and Activity Impairment Scale. *Additional PROs examined in ECHO not shown in this table include those assessing well‐being and HRQoL (Haemo‐QoL‐A) as well as patient surveys for healthcare utilization, treatment consumption, physical activity, and alcohol and smoking status. In total, 8 validated PRO questionnaires and 5 patient surveys were included in ECHO. †Data are mean ± SD. ‡Mean ± SD EQ‐5D values by hemophilia severity are 0.7±0.3 (n=161) for <1% FVIII (severe hemophilia), 0.7±0.2 (n=11) for 1% to <2% FVIII (moderate hemophilia), and 0.8±0.2 (n=11) for 2% to 5% FVIII (moderate hemophilia). §Mean ± SD EQ‐5D values by treatment regimen are 0.7±0.2 for on demand (n=34) and 0.7±0.3 for prophylaxis (n=149) at baseline. No information on length of prophylaxis during a patient's lifetime was taken into account in this analysis.

PB167: Inhibitor Incidence in Hemophilia A under Exclusive Use of a Third‐generation Recombinant Factor VIII Concentrate: Results of the HEMFIL Cohort Study

L. Lemos Jardim 1,2 ; D. Gonçalves Chaves 3 ; C. Brommonschenkel 1 ; M. Portugal Santana 3 ; M. Hermida Cerqueira 4 ; A. Prezoti 5 ; C. Santos Lorenzato 6 ; V. K Franco 7 ; J. van derBom 2 ; S. Meireles Rezende 1

1 Faculty of Medicine, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 2 Leiden University Medical Center, Leiden, the Netherlands, 3 Fundação Hemominas, Belo Horizonte, Brazil, 4 HEMORIO, Rio de Janeiro, Brazil, 5 HEMOES, Vitorioa, Brazil, 6 HEMEPAR, Curitiba, Brazil, 7 Hemocentro of Santa Catarina, Florianopolis, Brazil

Background: It has been shown a higher risk for inhibitors with the use of recombinant factor VIII (rFVIII) concentrates in comparison with plasma‐derived in patients with hemophilia A (PHA). However, prospective studies reporting inhibitor incidence under exclusive use of specific types of rFVIII in an unselected cohort are still missing.

Aims: To describe the incidence inhibitor in previously untreated patients (PUPs) with HA under exclusive use of a third‐generation rFVIII concentrate in the HEMFIL Study, an ongoing cohort.

Methods: PUPs with HA were enrolled consecutively in 5 hemophilia treatment centers in Brazil between 05‐03‐2013 and 31‐12‐2017 and were followed up to 75 exposure days (ED) or until inhibitor development. Clinical characteristics were collected using standardized forms. Time to inhibitor development was calculated by Kaplan‐Meier survival analyses.

Results: Currently, 73 PHA (FVIII:C< 2%) were included. The median age at first FVIII infusion was 10 months [IQR, 6.0‐13.8]. Most patients were white (64.3%) and were diagnosed because of bleeding symptoms (83.6%). More than five consecutives ED were prescribed for 13 patients (17.8%) in at least one occasion during the treatment. Family history of hemophilia was reported in 49.2%, of whom 3% also have family history of inhibitor. Inversion of intron 1 and 22 was present in 23/52 (44.2%) and 1 patient (1.9%), respectively. All the included patients are under exclusive use of a third‐generation rFVIII of whom 82.1% are on prophylaxis basis. Time to inhibitor development was 15 ED (95% CI, 9.6‐20.4). A total of 61/73 (83.6%) PUPs reached at least 30ED, i.e., 29/61 (47.5%) with 75ED and 10/61 (16.4%) with 30ED without inhibitor and 22/61 (36.1%) developed inhibitor, of which most are high‐titer (n=18; 29%).

Conclusions: Our findings confirm that the third generation recombinant factor VIII induces inhibitor development after a median of 15 exposure days in almost 40 percent of all PUPs with HA.

PB168: The Effect of Unmeasurable Endogenous Plasma Factor Activity Levels on Factor VIII Dosing in Patients with Severe Hemophilia A

P. Chelle 1 ; A. McEneny King 1 ; A. Iorio 2 ; A.N. Edginton 1

1 University of Waterloo, School of Pharmacy, Kitchener, Canada, 2 McMaster University, Department of Health Evidence, Research Methods and Impact, Hamilton, Canada

Background: Severe hemophilia A is a bleeding disorder corresponding to a deficiency of factor VIII (FVIII) and defined as a baseline level (BL) lower than 0.01 IU.mL −1 . To prevent bleeds and ensuing joint damage, the prophylactic treatment consists in infusing FVIII regularly to maintain a trough level higher than 0.01 IU.mL −1 . However, since exact measurement of baseline FVIII level is often not possible due to assay limitations (LLOQ = 0.01 IU mL −1 ), assumptions on BL (usually 0 IU.mL −1 is assumed) are made during personalized pharmacokinetic (PK)‐based dose calculations.

Aims: The objective of this work was to assess how assumptions on BL influenced the determination of a prophylaxis regimen in the treatment of hemophilia A using a PK‐driven approach.

Methods: Using a validated population pharmacokinetic model for FVIII, 500 populations of 500 virtual subjects were simulated. Dose and frequency required to maintain 95% of the population above each trough level of 0.01, 0.03 and 0.05 IU.mL −1 were calculated as a function of BL assumptions ranging from 0 to 1 IU.mL −1

Results: Patients with a true BL of 0 IU.mL −1 would require an additional 40 IU.kg −1 to maintain any target trough as compared to those with a true BL close to 0.01 IU.mL −1 (Figure 1). For dose individualization, depending on if the dose is being increased or decreased to reach a target, the baseline assumption can lead to widely varying dose determinations.

Conclusions: Targeting a trough near the LLOQ is a problem unique to hemophilia that is further complicated by the presence of an unmeasurable amount of endogenous FVIII. As a result, assumptions regarding endogenous FVIII levels must be made, which can have a dramatic effect on dosing regimen design.

FIGURE 1 Dose of FVIII required to maintain 95% of 500 virtual subjects over various target troughs as a function of the baseline assumption.

PB169: Pharmacokinetics of Extended Half‐life FVIII Products after Recommended Dosing in a Pediatric Hemophilia A Population

T. Jacobs 1 ; P. Barker 1 ; A. McEneny‐King 2 ; E. Neufeld 3 ; J. Panetta 1 ; U. Reiss 3

1 St. Jude Children's Research Hospital, Pharmaceutical Sciences, Memphis, United States, 2 University of Waterloo, School of Pharmacy, Waterloo, Canada, 3 St. Jude Children's Research Hospital, Hematology, Memphis, United States

Background: Prophylactic infusions of factor VIII (FVIII) remain standard of care for patients with severe hemophilia A. The introduction of extended half‐live (EHL) FVIII products may allow for reduction in dose frequency to twice weekly while maintaining trough FVIII levels >1 IU/dL.

Aims: Describe the pharmacokinetics (PK) of EHL FVIII products after recommended dosing in a pediatric hemophilia A population and describe the proportion of patients that required a dose adjustment to achieve a target trough >1 IU/dL.

Methods: All St. Jude Children's Research Hospital hemophilia A patients switched to EHL product efmoroctocog alfa with PK studies were included. We evaluated the PK using Bayesian estimation and a limited sampling schedule developed from published population PK. FVIII half‐life and activity at 0.5, 72, and 96 hours after infusion were estimated. This study was approved by the local institutional review board.

Results: Fifteen patients (age 1‐17 years) were analyzed. The FVIII half‐life was 11.3 (6.3‐17.0) hours (median, range) and was shorter in younger individuals (1‐5y: 10.0 (6.3‐14.1), 6‐11y: 11.5 (10.2‐14.6), 12‐17y: 13.1 (11.3‐17.0) hrs; Figure 1). The estimated FVIII activity at 0.5, 72, and 96 hours after 50 IU/kg dose was 84.4 (68.7‐122.0), 2.8 (0.6‐8.0), and 1.5 (0.4‐4.3) IU/dL, respectively (median, range). Five patients had predicted trough levels < 1 IU/dL at 96 hours. Of these, one patient's dose and another patient's dosing frequency were increased. Three patients continued the initial regimen as troughs < 1 IU/dL would likely occur over night. One patient's PK allowed a dose reduction to 30 IU/kg twice weekly. The remaining patients had no dosing changes.

FIGURE 1 Efmoroctocog alfa half‐life versus age

Conclusions: Our results show shorter FVIII half‐life for efmoroctocog alfa in young children. The majority of this small cohort remained on the recommended dosing of 50 IU/kg twice weekly. PK studies allowed optimization of dosing in 20% of patients.

PB171: Are Current Therapeutic Strategies with Conventional Products Sufficient to Manage the Burden of Haemophilia in Italy?

R.I. Mazzucchelli 1 ; P. Kritikou 2 ; K.‐J. Myren 2 ; J. O'Hara 3,4

1 Swedish Orphan Biovitrum s.r.l. (Sobi), Parma, Italy, 2 Swedish Orphan Biovitrum AB, Stockholm, Sweden, 3 University of Chester, Chester, United Kingdom, 4 HCD Economics, Daresbury, United Kingdom

Background: Haemophilia care has improved significantly in the last 2 decades, and recombinant factor replacement therapies (rFVIII & rFIX) are the mainstay treatment options.

Aims: The aim of the present study was to describe the treatment outcomes for adult persons with haemophilia (PwH) treated with conventional products in Italy, based on CHESS (Cost of Haemophilia in Europe: a Socioeconomic Survey).

Methods: Only PwH that had remained on the same treatment strategy (prophylaxis or on‐demand) from start of their treatment were included in the present analysis. All patients were on conventional products; extended half‐life products were not available at the time of the study. Key variables were: basic demographic and clinical characteristics, bleeds in the last 12 months, target joints (defined as locations of chronic synovitis), physician‐reported chronic pain and patient‐reported quality of life (QoL) measured by EQ‐5D. All analyses were descriptive.

Results: CHESS collected data from 280 PwH in Italy. The 186 (66.4%) PwH that did not remain on the same treatment strategy were excluded from the analysis. Twenty‐one (7.5%) had always been on prophylaxis (primary prophylaxis; PP), and 73 (26.1%) had always been on‐demand (primary on‐demand; POD). For haemophilia A, 15 and 60 PwH were on PP and POD therapy, respectively. For haemophilia B, 6 and 13 PwH were on PP and POD therapy, respectively. The mean (SD) number of bleeds in the last 12 months were 2.7 (2.3) and 1.3 (1.6) for PP in PwH A and B, respectively; as opposed to 5.4 (9.5) and 3.8 (6.2) for POD in PwH A and B, respectively. Presence of target joints, chronic pain, and QoL are presented in the Table.

TABLE 1 Presence of target joints, chronic pain, and QoL Variable Haemophilia Type Primary Prophylaxis Primary On‐Demand Presence of Target Joints (locations of chronic synovitis), n (%) Haemophilia A 4/15 (27%) 32/60 (53%) Presence of Target Joints (locations of chronic synovitis), n (%) Haemophilia B 2/6 (33%) 3/13 (23%) Presence of Chronic Pain, n (%) Haemophilia A 12/15 (80%) 39/60 (65%) Presence of Chronic Pain, n (%) Haemophilia B 2/6 (33%) 5/13 (38%) Quality of Life, mean (SD) Haemophilia A 0.94 (0.1), n=7 0.85 (0.2), n=21 Quality of Life, mean (SD) Haemophilia B 0.86 (0.1), n=3 1.0 (0.0), n=2

Conclusions: According to the CHESS results, despite continuation on the same treatment beyond 12 months, PwH on conventional products in Italy continue to present a notable disease burden in terms of target joints, and chronic pain; especially for those on POD. The data indicate the need to further optimise haemophilia treatment in Italy.

PB172: Practical Considerations in Choosing a Cost‐effective and‐Efficient Solution for Prophylaxis in Haemophilia

M. Serban 1 ; E. Ursu 1 ; A. Traila 2 ; P. Serban 2 ; T. Opait‐Dugan 2 ; M. Dutescu 2 ; D. Puiulescu 3 ; L. Ritli 4 ; C. Jinca 5 ; E. Boeriu 5 ; J.M. Patrascu 5 ; S. Cerbu 5 ; S. Arghirescu 6

1 Clinical Emergency Children Hospital ‘Louis Turcanu’, European Haemophilia Treatment Center, Hematology, Timisoara, Romania, 2 Rehabilitation Medical Centre for Children and Adolescents ‘C. Serban’ European Haemophilia Treatment Center, Buzias, Romania, 3 Clinical Emergency Children Hospital ‘Louis Turcanu’, Timisoara, Romania, 4 University of Medicine and Pharmacy, Oradea, Romania, 5 University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania, 6 University of Medicine and Pharmacy ‘Victor Babes’, Timisoara, Romania

Background: Prophylaxis is generally accepted as treatment of choice in severe forms of haemophilia. But the optimal prophylaxis regimen is still under scientific debate.

Aims: In our specific condition with very recently introduced prophylaxis, almost exclusively of secondary/tertiary or short term type, a high cost demanding therapy, we aimed at finding the optimal solution from the point of view of effectiveness and efficiency.

Methods: Our non‐interventional observational study was performed on 37 patients with severe form of haemophilia A without inhibitors, all with secondary/tertiary or short term prophylaxis. Plasma F VIII activity was measured at 0.1 hour and 48 hours after administration of recombinant F VIII in a dosage of 36.7+/‐5.38 IU/kg/24h. Incremental recovery and through level at 48 hours were evaluated, being compared with the correspondent expected values for the type of the administered product.

Results: TABLE 1 Global situation of FVIII recovery at 48 hours: Dosage FVIII (IU/kg) Real recovery(IU/dl) Estimated recovery (IU/dl) P value 36.7±5.38 9.1±8.4 9.3±5.1 0.4

TABLE 2 Distribution of real recovery values (IU/dl) at 48h (% of patients): Dosage FVIII (IU/kg) <1 IU/dl 1.1‐5 IU/dl 5.1‐10 IU/dl >10 IU/dl 36.7±5,38 2.7% 43,2% 21.6% 32.4%

We had to accept once again that „one size doesn't fit all”, as pharmacokinetic of products can depend on many factors like: age, blood group, vWF level, exercise, joint status and type of replacement product. In our cohort, only in 10.8% cases the through level at 48 hours matched to the expected values.

The high heterogeneity of 48h recovery values of the same replacement dosage makes obvious that in our situation in which different trough levels resulted from the same dosage, the individualization of prophylaxis in haemophilia care is mandatory, serving not only its effectiveness, but also its efficiency.

Conclusions: In this challenging situation, it seems that pharmacokinetic driven replacement is a promising strategy to improve haemophilia care in a right manner, avoiding too low or two high levels, taking into consideration the whole community of persons with haemophilia.

PB173: rVIII‐SingleChain in Surgical Prophylaxis: Efficacy and Safety in 43 Surgeries

J. Mahlangu 1 ; F. Abdul Karim 2 ; C. Djambas Khayat 3 ; J. Ong 4 ; S. P'Ng 5 ; J. Oldenburg 6 ; A. Brainsky 7 ; S. Lucas 7 ; I. Pabinger 8 ; for the Affinity Study Group

1 University of the Witwatersrand, NHLS and Charlotte Maxeke Hospital, Johannesburg, South Africa, 2 National Blood Centre, Kuala Lampur, Malaysia, 3 Hotel‐Dieu de France Hospital, Ashrafieh, Lebanon, 4 Brokenshire Integrated, Davao City, Philippines, 5 Royal Perth Hospital, Perth, Australia, 6 University Clinic Bonn, Institute of Experimental Haematology and Transfusion Medicine, Bonn, Germany, 7 CSL Behring, King of Prussia, United States, 8 Medical University Vienna, Clinical Division of Haematology and Haemostaseology, Medical Clinic I, Vienna, Austria

Background: rVIII‐SingleChain is a novel recombinant Factor VIII designed as a B‐domain truncated construct with a covalent bond between the heavy and light chain and a higher affinity to von Willebrand Factor.

Aims: The surgical sub‐studies of the Affinity program investigate the safety and efficacy of rVIII‐SingleChain to control surgical hemostasis in children and adults with severe Hemophilia A.

Methods: Surgery was defined as a procedure requiring general, spinal or regional anesthesia. Dosing of rVIII‐SingleChain was based on the type of surgery, guided by the WFH recommendations. rVIII‐SingleChain was administered as a bolus or a continuous infusion. The investigator rated hemostatic efficacy using a 4 point rating scale (poor/none; moderate; good; excellent); treatment success was defined as a rating of excellent or good.

Results: 43 surgeries were performed on 32 patients aged between 5 and 64y (median, 32). 22 surgeries were orthopedic while 21 surgeries were non‐orthopedic. 17 surgeries were related to hemophilia or its complications, and 1 surgery (appendectomy) was an emergency. Overall, rVIII‐SingleChain was used as a bolus dose in 35 surgeries, and as a continuous infusion in 8 surgeries. No related AEs or SAEs were observed during the perioperative period. Hemostatic efficacy was rated as excellent and good in 38 (88%) and 5 (12%) surgeries respectively. Of the orthopedic surgeries, 18 (82%) were rated as excellent, and 4 (18%) were rated as good.

Conclusions: rVIII‐SingleChain, dosed by bolus or continuous infusion, was well‐tolerated and effective in achieving surgical and perioperative hemostasis, with an overall treatment success of 100%.

PB174: Estimation of Simoctocog Alfa and Octocog Alfa Activity using One‐stage and Chromogenic Assays in Routine Laboratory Practice: Results of a Comparative International Field Study

T. Vicente 1 ; J. Versteden 1 ; C. Borgvall 2 ; J. Bichler 1 ; O. Walter 1 ; S. Tiefenbacher 3 ; L. Belyanskaya 1

1 Octapharma AG, Lachen, Switzerland, 2 Octapharma AB, Stockholm, Sweden, 3 Colorado Coagulation, Laboratory Corporation of America ® Holdings, Englewood, CO, United States

Background: Precise determination of coagulation factor VIII activity (FVIII:C) is essential to ensure efficacy and safety of haemophilia A treatment. One‐stage (OS) and/or chromogenic (CHR) assays are commonly used; however, there can be substantial variability in the measurements.

Aims: This international field study analysed the performance of simoctocog alfa (B‐domain deleted recombinant FVIII [rFVIII] of human‐cell line origin) and octocog alfa (full‐length rFVIII of hamster‐cell line origin) in FVIII:C assays routinely performed in clinical laboratories.

Methods: Congenital FVIII‐deficient plasma was spiked with 4 concentrations (1, 5, 30 and 100 IU/dL) of simoctocog alfa or octocog alfa. Participating laboratories received blinded samples and determined FVIII:C following their standard procedures. The methodological parameters used by each laboratory were summarised in a questionnaire.

Results: A total of 49 participating laboratories from 9 countries provided results (both OS and CHR assays: n = 40; OS assay only: n = 8; CHR assay only: n = 1). As several laboratories performed more than one assay of a given type, the total number of data sets was 50 for the OS and 43 for the CHR assay. Mean absolute FVIII:C values were comparable for both products at all concentrations and for both assays, although ranges appeared to be slightly larger for simoctocog alfa (Table 1). Mean recoveries ranged from 97% to 191% for simoctocog alfa and from 93% to 172% for octocog alfa, with higher recoveries at lower concentrations. Subgroup analyses by OS and CHR assay reagents showed minor variations in FVIII:C depending on reagents, but no marked differences between the two rFVIII products. CHR:OS ratios for simoctocog alfa ranged from 1.05 to 1.19 and from 1.06 to 1.25 for octocog alfa (Table 1).

TABLE 1 Absolute FVIII:C values as determined by OS and CHR assay and CHR:OS ratios Product Target concen‐tration (IU/dL) OS assay (n = 50) Range (IU/dL) CHR assay (n = 43) Range (IU/dL) CHR:OS ratios at individual laboratories (n = 43) Range Mean ± SD (IU/dL) Mean ± SD (IU/dL) Mean ± SD Simoctocog alfa 1 1.8 ± 0.81 0.6‐4.0 1.9 ± 1.02 0.0‐5.6 1.19 ± 0.704 0.33‐3.66 5 6.5 ± 2.80 4.0‐22.5 6.1 ± 2.14 0.6‐14.2 1.05 ± 0.452 0.09‐2.37 30 32.0 ± 11.14 22.7‐103.0 34.5 ± 3.73 28.2‐47.7 1.13 ± 0.223 0.36‐1.71 100 97.3 ± 15.45 66.2‐171.7 112.4 ± 11.06 96.1‐142.9 1.17 ± 0.189 0.83‐1.59 Octocog alfa 1 1.6 ± 0.68 0.5‐3.1 1.7 ± 0.96 0.0‐5.1 1.25 ± 0.709 0.37‐3.56 5 6.1 ± 1.63 3.7‐11.1 5.9 ± 1.88 0.5‐12.2 1.06 ± 0.428 0.09‐2.44 30 30.9 ± 5.44 22.4‐59.6 34.1 ± 3.62 24.8‐44.3 1.12 ± 0.168 0.63‐1.55 100 92.7 ± 10.90 69.0‐141.3 108.5 ± 9.29 95.4‐130.3 1.17 ± 0.134 0.81‐1.46

Conclusions: The accuracy of FVIII:C determination using either OS or CHR assays routinely performed in clinical laboratories was comparable for simoctocog alfa and octocog alfa.

PB175: Correlation between the Half‐life of GlycoPEGylated Recombinant FVIII (N8‐GP) and von Willebrand Factor ‐ Results from Phase 3 Clinical Trials

P. Chowdary 1 ; M. Carcao 2 ; W.‐H.O. Clausen 3 ; P.A. Holme 4 ; J. Møss 3 ; A. Tosetto 5 ; A. Wheeler 6 ; E. Santagostino 7

1 Royal Free Hospital, The Katherine Dormandy Haemophilia Centre and Thrombosis Unit, London, United Kingdom, 2 Hospital for Sick Children, University of Toronto, Division of Haematology/Oncology and Child Health Evaluative Sciences, Research Institute, Toronto, Canada, 3 Novo Nordisk A/S, Bagsværd, Denmark, 4 University of Oslo, Oslo University Hospital, Department of Haematology, Institute of Clinical Medicine, Oslo, Norway, 5 San Bortolo Hospital, Hematology Department, Vicenza, Italy, 6 Vanderbilt University School of Medicine, Pathology, Microbiology and Immunology, Nashville, United States, 7 IRCCS Cà Granda Foundation, Maggiore Hospital Policlinic, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan, Italy

Background: Standard factor VIII (FVIII) products are associated with a relatively short half‐life (˜12 hours in adults; ˜8‐10 hours in children). The half‐life of standard FVIII (plasma‐derived and recombinant FVIII [rFVIII]) products is limited by the tight non‐covalent association of FVIII in circulation with von Willebrand factor (vWF). Consequently, the half‐life of such products appears to be driven by the patients’ endogenous vWF levels. N8‐GP (turoctocog alfa pegol) is an extended half‐life glycoPEGylated rFVIII being developed for prophylaxis and the treatment of bleeds in patients with haemophilia A.

Aims: Determine the relationship between N8‐GP half‐life and vWF levels in patients who received N8‐GP prophylaxis during the pathfinder ™ phase 3 clinical trials, and investigate if this relationship was similar to that of standard FVIII in children.

Methods: Male patients (adult/adolescent [≥12 years]; children [0‐11 years]) with severe haemophilia A (< 1% FVIII) receiving N8‐GP at 50 IU/kg underwent pharmacokinetic (PK) evaluations with N8‐GP and their previous standard FVIII (in children only). vWF antigen levels (assessed using immunoturbidimetric methodology or ELISA in a central laboratory) and N8‐GP half‐life were analysed from all patients.

Results: In total, 42 adults and 25 children with 129 PK profiles (25 previous product profiles in children; 104 N8‐GP profiles in 42 adults/adolescents and 24 children) were evaluated. In all ages, N8‐GP half‐life increased with vWF levels, although this trend was less pronounced in children. For children, the relationship between half‐life and vWF levels did not differ between N8‐GP and the patients’ previous products. Higher vWF activity also correlated with blood type; type O had a lower range of activity versus type A.

Conclusions: The half‐life of N8‐GP increased with vWF levels in adults/adolescents, similar to previous observations for standard and endogenous FVIII. This trend was less pronounced in children.

PB176: Chronic Pain in Adults with Severe Haemophilia A and B

J. O'Hara 1,2 ; P. Kritikou 3 ; A. Willemze 3 ; K.‐J. Myrén 3 ; D. Noone 2 ; K. Khair 4,5

1 University of Chester, Chester, United Kingdom, 2 HCD Economics, Daresbury, United Kingdom, 3 Swedish Orphan Biovitrum AB, Stockholm, Sweden, 4 London South Bank University, London, United Kingdom, 5 Great Ormond Street Hospital, London, United Kingdom

Background: Chronic pain, primarily as a result of bleeding into a joint or chronic joint damage, is a burden for people with haemophilia (PwH), leading to impaired quality of life (QoL).

Aims: Our aim was to describe how key variables affect the presence and level of severity of chronic pain as assessed by physicians in PwH. A secondary objective was to describe the QoL for PwH with different severities of chronic pain.

Methods: Data was derived from the “Cost of Haemophilia: A Socioeconomic Survey (CHESS)”, on adults with severe haemophilia in 5 European countries. Physician‐reported chronic pain was analysed by presence (yes/no) and by severity level (no pain, mild, moderate, or severe; based on its interference with occupation or activities of daily living and the use of analgesic medication). Presence of target joints (TJs) (defined as locations of chronic synovitis), and bleeds in the last 12 months were used as variables. Patient‐reported QoL, was based on the EQ‐5D. Analyses were descriptive and results presented as mean (SD) / n (%).

Results: In total, 824 (64%) PwH had chronic pain, with a mean age of 38.1 (15.2) yr; while 461 (34%) PwH had no pain (mean age of 32.0 [13.0] yr). The number of TJs was 0.5 (0.8) for PwH with no pain, increasing to 1.3 (1.3) for PwH with mild pain, 1.6 (1.4) for PwH with moderate pain, and 3.4 (2.7) for PwH with severe pain. Almost all (94%) PwH with >2 TJs had chronic pain. Bleeds in the last 12 months ranged from 2.9 (5.7) for PwH with no pain to 10.4 (12.0) for PwH with severe chronic pain. The EQ‐5D utility scores ranged from 0.94 (0.1) for PwH with no pain, to 0.21 (0.4), for PwH with severe chronic pain, >70% of which reported they had ‘moderate or extreme problems’ across all domains.

Conclusions: Results show chronic pain in adults with severe haemophilia represents a burden for PwH and an unmet medical need. Increased pain severity reported by the physicians correlates with presence of TJs and bleeds. Chronic pain is associated with decreased QoL across all domains.

PB177: | Disease Burden and Remaining Unmet Need in Patients with Hemophilia A Treated with Primary Prophylaxis

J. O'Hara 1 ; F. Castro 2 ; S. O'Hara 1 ; J. Black 2 ; S. Chaplin 1 ; L. Ruiz‐Casas 1 ; R. Hampton 2 ; C. Sima 3

1 HCD Economics, Chester, United Kingdom, 2 Roche, Basel, Switzerland, 3 Genentech Inc., RWD Oncology, South San Francisco, United States

Background: The gold standard in severe haemophilia A (HA) is prevention of bleeding using prophylactic treatment initiated at an early age (primary prophylaxis, PPX). Patients (pts) treated with PPX (PPX pts) have improved outcomes, however the remaining disease burden has not been reported.

Aims: To describe the disease burden in PPX pts with HA. To compare health status, clinical and economic outcomes with those in the general population.

Methods: Data source was Cost of Haemophilia in Europe: a Socioeconomic Survey (CHESS), a EU5 study of adults with HA. PPX pts have always received prophylaxis, were aged 18‐35 years and had no history of inhibitors (n=166). Clinical outcomes (presence of target joints, chronic pain related to HA), health status (EQ‐5D‐3L) and economic outcomes (work impairment) were analysed. Several populations were used for comparison: healthy male general population from two publicly available data sources, for health status 1 and work impairment 2 ; and comparable non‐PPX patients in CHESS, for clinical outcomes.

Results: 39% of the PPX pts had ≥1 target joint (TJ) and 46% had some level of chronic pain (8% had at least moderate pain). In comparison, 62% and 59% of the non‐PPX pts had ≥1 TJ and chronic pain, respectively. Health status, as measured by EQ‐5D‐3L index score, was worse in PPX pts compared to the male general population of the same age (0.87 vs 0.93), with PPX pts reporting more frequent anxiety/depression (31% vs 14%), self‐care problems (5% vs 1%) and mobility problems (20% vs 6%). (Table 1) Activity and work impairment analyses (Table 2) show that employment level of PPX pts is lower than that in the general population, with higher level of health‐related impairment while working. TABLE 1 Health status in PPX pts and healthy male general population Outcome PPX pats (CHESS) n=55 General population, Males (EU5) n=2,570 Mean EQ‐5D index score 0.87 0.93 EQ‐5D‐3L domain analysis (% pats with a moderate/severe problem) Anxiety/Dep 31% 8% (6%‐25%) Self‐care 5% 2% (0.2%‐4%) Activities 9% 6% (4%‐15%) Pain & Disc 38% 29% (17%‐39%) Mobility 20% 5% (3%‐12%) TABLE 2 Economic outcomes in PPX pts and healthy male general populations Outcome PPX pts, 18‐35 yo (N=55) US general population, >18 yo (N=65,389) Pts Employed 44% 50.79% Worktime hours missed (absenteeism) 0% 3.5% Percent impairment while working, due to health (presenteeism) 25.6% 12.9% Percent overall work impairment due to health (including missed work and impairment while working) 16.2% 15% Percent activity impairment (non‐work) due to health 23.1% 22.1%

Conclusions: While PPX has been shown to provide superior efficacy in the HA population compared to other treatment approaches, PPX pts appear to maintain a degree of joint health impairment, worse health status, and employment and productivity loss, compared to the general population.

PB178: Landscape of Intron 1 and Intron 22 Inversions in Severe Hemophilia A Patients and Inhibitor Development

L.W. Zuccherato 1 ; S. Maria Eloi Santos 2 ; L.L. Jardim 3 ; D.G. Chaves 4 ; S.M. Rezende 3

1 Universidade Federal de Minas Gerais, Complementary Propaedeutic Department, Belo Horizonte, Brazil, 2 Universidade Federal de Minas Gerais, Complementary Propaedeutic Department, BELO HORIZONTE, Brazil, 3 Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 4 Fundação Hemominas, Belo Horizonte, Brazil

Background: Hemophilia A (HA) is an X‐linked disorder due to deleterious mutations in the factor VIII (FVIII) gene ( F8 ). A recurrent inversion, originated by a homologous recombination event within F8 intron 22 and its extragenic inversely oriented copies (Inv22‐1 and Inv22‐2), accounts for about 45% of severe HA worldwide. The inversion of intron 1 (Inv1) is the second recurrent F8 disruption among severe patients, with about 4.5% of prevalence.

Aims: Few studies have evaluated the prevalence of the types of Inv22, whether type 1 or type 2. Therefore, we aimed to report the prevalence of these inversions in a cohort of patients with HA.

Methods: We included 103 severe (FVIII< 1%) HA patients from the state of Minas Gerais/Brazil, focusing on the genetic frequencies of Inv1, Inv22‐1 and Inv22‐2. Inv 1 was detected using PCR, and intron 22 inversions of F8 were sorted by inverse‐shifting PCR. A combination of different product sizes in 2% agarose gel electrophoresis predicted the presence or absence of the mutations.

Results: When all patients are considered, Inv1, Inv22‐1 and Inv22‐2 were present in 5.8% (6/103), 26.2% (27/103) and 5.8% (6/103), respectively (Figure 1). A total of 37/103 patients (35.9%) developed inhibitor (inh+) and 66 (64.1%) did not after 75 exposure days (ED) to FVIII (inh‐). When patients were stratified by inh+ and inh‐ groups, Inv1, Inv22‐1 and Inv22‐2 were present in 13.5% (5/37), 29.7% (11/37), 8.1% (3/37) and 1.5% (1/66), 24.2% (16/66) and 4.5% (3/66), respectively.

FIGURE 1 Intron 1 and intron 22 inversions (Inv22‐1 and Inv22‐2) of the F8 gene in severe patients with hemophilia A

Conclusions: We conclude that Inv22‐1 is more prevalent than Inv22‐2 in patients with and without inhibitors. Although less prevalent, Inv1 accounted for nearly 15% of genetic defects in patients with inhibitors.

PB179: The Bone Disease in Haemophilia: The Role of von Willebrand Factor, Factor VIII and Thrombin

S. Lancellotti 1 ; M. Sacco 2 ; G. Battafarano 3 ; F. Berruti 2 ; R. De Cristofaro 1,2 ; A. Del Fattore 3

1 Fondazione Policlinico Universitario A. Gemelli, Area Ematologia ‐ Servizio Malattie Emorragiche e Trombotiche, Roma, Italy, 2 Università Cattolica del Sacro Cuore, Roma, Italy, 3 Bambino Gesù Children's Hospital, Bone Physiopathology Group, Multifactorial Disease and Complex Phenotype Research Area, Roma, Italy

Background: Reduced bone mineral density (BMD) is a significant finding in haemophilia A (HA). However, the pathophysiology is not exactly known. BMD seems due to perturbations of the Receptor Activator of Nuclear factor‐B (RANK), RANK Ligand (RANKL) and Osteoprotegerin (OPG) pathway. Recent evidence shows that low BMD in HA is due to an increased rate of bone resorption. Moreover, bone cells express protease‐activated receptors (PARs) and FVIII deficiency could alter BMD also by decreasing thrombin production.

Aims: The aim is to identify the specific role played by FVIII, VWF and thrombin on osteoclasts biology.

Methods: To assess the effects on osteoclastogenesis, blood mononuclear cells were stimulated with human macrophage colony stimulating factor M‐CSF/RANKL in the presence of the different coagulation factors (plasma derived VWF/FVIII complex, recombinant VWF, recombinant FVIII and thrombin). The activity of cathepsin K and matrix metalloproteinase (MMP)‐9 was evaluated by real time expression analysis on mature osteoclasts treated for 48h with the aforementioned coagulation factors.

Results: VWF appears to play a major role, showing by itself ˜45% inhibition of osteoclastogenesis comparable to OPG (the physiologic inhibitor), and even more if is complexed with FVIII (53% inhibition). Thrombin reduces osteoclast differentiation with variable effects (30‐50% inhibition). No statistically significant alterations of Cathepsin K and MMP9 expression were revealed in treated mature osteoclasts.

Conclusions: These findings showed that the osteoclast differentiation is significantly and negatively influenced by VWF/FVIII complex and thrombin treatments. By contrast, the resorption activity was not influenced by the presence of the different factors. These results may have important implications in the clinical management of hemophilia patients to better orient the choice of the FVIII concentrate to prevent bleeding and at the same bone disease in severe HA patients.

PB180: Long Term Safety and Efficacy of rVIII‐SingleChain in Previously Treated Patients with Severe Hemophilia A: Intermediate Results of an Extension Study

J. Mahlangu 1 ; F. Abdul Karim 2 ; O. Stasyshyn 3 ; B. Korczowski 4 ; A. Brainsky 5 ; S. Lucas 5 ; Y. Li 5 ; I. Pabinger 6 ; for the AFFINITY Investigators

1 University of the Witwatersrand, NHLS and Charlotte Maxeke Hospital, Johannesburg, South Africa, 2 National Blood Centre, Kuala Lumpur, Malaysia, 3 Institute of Blood Pathology and Transfusion Medicine, National Academy of Medical Sciences of Ukraine, Lviv, Ukraine, 4 Department of Pediatrics, Regional Hospital, University of Rzeszów, Rzeszów, Poland, 5 CSL Behring, King of Prussia, United States, 6 Clinical Division of Haematology and Haemostaseology, Medical Clinic I, Medical University Vienna, Vienna, Austria

Background: rVIII‐SingleChain, a novel B‐domain truncated recombinant Factor VIII comprised of covalently bonded FVIII heavy and light chains, was designed to have a high binding affinity to von Willebrand factor.

Aims: This Phase III multinational extension study investigates the long term safety and efficacy of rVIII‐SingleChain in previously treated patients (PTPs) with severe hemophilia A.

Methods: PTPs with severe hemophilia A (endogenous Factor VIII < 1%) and >50 previous exposure days (EDs) to FVIII products were treated either on demand or prophylactically with rVIII‐SingleChain 2 or 3 times weekly, or another regimen per the Investigator's discretion.

Results: As of 05 Sep 2017, 222 patients were enrolled. The median age was 20 years (1‐64); 77 (34.7%) patients were 0‐< 12 years, and 145 (65.3%) were ≥12 to ≤65 years old. The majority of patients were Caucasian (158, 71.2%); 50 (22.5%) Asian, and 12 (5.4%) were Black or African American. A minority of patients (10, 4.5%) were of Hispanic origin. A target of ≥100 EDs was achieved by 211 (95%) patients; the total cumulative EDs in the extension study was 65,522 in 494 treatment years. Of the 211 (95%) patients assigned to prophylaxis, 98 (44.1%) were treated 3 times weekly, and 84 (37.8%) patients were treated 2 times weekly. The median annualized spontaneous bleeding rate (AsBR) for all prophylaxis regimens, for patients treated 3 times weekly, and 2 times weekly was 0.35 (Q1, Q3: 0.00; 1.09), 0.35 (Q1,Q3: 0.0; 0.96), and 0.0 (Q1, Q3: 0.0; 1.46) respectively. Of a total of 2273 bleeding events, 2162 were treated with rVIII‐SingleChain. Of these, 2120 were rated for hemostatic efficacy by the investigator: 85.8% were rated as either excellent or good and required 1 or 2 injections to achieve hemostasis. No PTP developed an inhibitor in the study.

Conclusions: rVIII‐SingleChain has a favorable safety profile, and is effective and well tolerated for on‐demand and prophylaxis treatment in patients who have severe hemophilia A.

PB181: Standardized Determination of FVIII Inhibitors with the Improved FVIII INH Kit

L. Rosenmayr; L. Wagner ; N.B. Binder

Technoclone Herstellung von Diagnostika und Arzneimitteln GmbH, Vienna, Austria

Background: The development of antibodies to Factor VIII:C in the treatment of patients with haemophilia A is still a problem. The majority of laboratories continue to perform FVIII inhibitor assays using their own in‐house method causing high variability of results.

Aims: Aim of the study was to investigate the performance of the FVIII INH kit, which provides all reagents and instructions and in addition a positive and a negative control, and to correlate the results with those obtained by the classical Bethesda and by the Nijmegen assay.

Methods: Haemophilia A patient samples with and without inhibitors were tested in the classical Bethesda assay, the Nijmegen assay and with the FVIII INH assay kit. The FVIII INH kit follows the modified Nijmegen assay principal and uses buffered normal plasma in a 1+1 mixture with patient plasma. The control mix is a 1+1 mixture of imidazole buffer with BSA and buffered normal plasma. Patient sample dilutions are made with imidazole buffer with BSA. After incubation, the residual FVIII activity was determined with the chromogenic assay Technochrom FVIII:C and Siron LS for FVIII one stage clotting assay.

Results: The control values for the positive and negative control were within the confidence range for all methods. No inhibitors were detected in the Hamophilia A patients without inhibitors.

For Haemophilia A patient samples with inhibitors the results showed a r = 0.8894 between one stage clotting assay and chromogenic FVIII assay and a Passing Bablock slope of 1.0949.

The results for the FVIII INH kit showed highest BU/mL, indicating a high sensitivity towards FVIII inhibitors. Differences between methods were sample dependent with SD between 7.8 and 17.8.

Conclusions: The FVIII INH kit can be used with good performance for determination of FVIII INH in patient samples, assay performance is controlled by use of the provided controls. There is a trend towards higher BUs with FVIII INH kit demonstrating high sensitivity of the assay.

PB182: Decrease in Joint Bleeding Rates Compared with Prior Prophylaxis in Patients with Hemophilia A Treated with BAY 94‐9027

M.E. Mancuso 1 ; S. Rangarajan 2 ; C. Négrier 3 ; M. Wang 4 ; M. Maas Enriquez 5

1 Fondazione IRCCS Ca’ Granda ‐ Ospedale Maggiore Policlinico, ‘Angelo Bianchi Bonomi’ Hemophilia and Thrombosis Centre, Milan, Italy, 2 University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom, 3 Louis Pradel University Hospital, University Claude Bernard Lyon 1, Lyon, France, 4 Bayer, US Innovation Center, Hematology Research, San Francisco, United States, 5 Bayer, Wuppertal, Germany

Background: In hemophilia A, prevention of joint bleeding is a key aim of prophylactic FVIII treatment; higher FVIII levels protect against joint bleeding. BAY 94‐9027, an extended half‐life recombinant FVIII, was evaluated in the phase II/III PROTECT VIII study.

Aims: To compare patients’ joint annualized bleeding rate (JABR) during BAY 94‐9027 prophylaxis in the PROTECT VIII study with JABR on a prior FVIII prophylaxis regimen.

Methods: PROTECT VIII included previously treated males 12‐65 years old with severe hemophilia A who had received FVIII in the 12 months pre study (‘prestudy’). Prophylaxis patients received BAY 94‐9027 twice weekly (2×/wk; 30–40 IU/kg), or every 5 (E5D; 45–60 IU/kg) or 7 days (E7D; 60 IU/kg) for 26 weeks. Bleeds were documented in electronic patient diaries. This post‐hoc analysis (data cut Feb 2017) included patients who received prestudy prophylaxis and continued in an open‐label extension, wherein they could maintain or switch regimen (‘variable’ [VAR] patients). Prestudy, main study and extension JABR were analyzed and compared per the intention to treat extension population.

Results: In total, 82 patients received prestudy prophylaxis and entered the extension: the majority were treated ≥ 3×/week prestudy. Median (range) follow up in the main study and extension was 3.9 (0.8‐4.7) years (n=121, including the 82 analyzed here). Median JABR was lower in the main study versus prestudy (1.41 vs. 2.00; decrease of −0.57). Decreases by group were −1.00 (2x/wk; n=13), −0.81 (E5D; n=30), −0.05 (E7D; n=20) and −0.58 (VAR; n=19). Low JABR was maintained in the extension (median 1.04) with a further decrease in the 2×/wk arm (extension vs. main study −2.67). Reliability is supported by consistent median JABR in on‐demand PROTECT VIII patients (prestudy, 19.50; main study 17.87; extension, 20.49 [n=14]).

Conclusions: Compared with patients’ previous FVIII prophylaxis, Joint ABR was lower with all BAY 94‐9027 prophylaxis regimens, and maintained in the extension study.

Study supported by Bayer.

PB183: Systematic Review of Efficacy and Factor Consumption of Recombinant Factor IX Products for Prophylactic Treatment of Hemophilia B

E. Santagostino 1 ; S. Yan 2 ; T. Matsushita 3 ; L. Alberio 4 ; J. Davis 5

1 Maggiore Hospital Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center IRCCS Ca’ Granda Foundation, Milan, Italy, 2 CSL Behring, King of Prussia, United States, 3 Nagoya University Hospital, Department of Transfusion Medicine, Nagoya, Japan, 4 CHUV, Department and Central Hematology Laboratory, Lausanne, Switzerland, 5 University of Miami Hemophilia Treatment Center, Miami, United States

Background: No direct head‐to‐head study has been conducted to compare efficacy and safety of recombinant factor IX (rFIX) products in hemophilia B.

Aims: To systematically review the evidence from Phase III clinical trials evaluating the use of rFIX products for prophylaxis in hemophilia B patients. The outcomes considered were total annualized bleeding rate (ABR), spontaneous ABR (AsBR), joint ABR (AjBR), and consumption of rFIX.

Methods: A systematic literature search was conducted in both EMBASE and PubMed. Phase III clinical trials of prophylactic rFIX treatment in previously treated, hemophilia B patients aged ≥12 years, with endogenous FIX levels ≤2% were included. Studies which did not meet these criteria, or did not report any of the outcomes mentioned above, were excluded. Relevant data were then extracted from these studies.

Results: The search identified a total of 1,362 articles, with 35 passing the title and abstract screen. Following full‐text review, 9 articles met the inclusion criteria. Data has been extracted from these articles and summarized in Tables 1 & 2 (data marked * was obtained from the clinical study report); information is presented for the weekly infusion regimen where data was available for more than one prophylaxis schedule. ABR reported in the relevant studies ranged from a median of 0‐3.0, AsBR ranged from 0‐1.0, and AjBR ranged from 0‐1.1. The median values reported for rIX‐FP were 0 for ABR, AsBR and AjBR. Mean values ranged from 1.24‐4.26 for ABR, 0.52‐2.6 for AsBR, and 0.89‐2.85 for AjBR, with rIX‐FP at the lowest end for each value. Weekly rFIX consumption ranged from 44.5‐105.5 IU/kg.

TABLE 1 Details of bleeding rates in rFIX studies identified by the systematic review Study and regimen rFIX product No. prophylaxis subjects Age, mean years (range) ABR, median (IQR) ABR, mean (SD) AsBR, median (IQR) AsBR, mean (SD) AjBR, median (IQR) AjBR, mean (SD) Santagostino et al. 2016 (35‐50 IU/kg weekly prophylaxis) Albutrepenonacog alfa (rIX‐FP) 40 31.6 (SD 15.2) 0 (0, 1.87) 1.24 (1.780)* 0 (0, 0) 0.52 (1.116)* 0 (0, 1.53) 0.89 (1.436)* Powell et al. 2013 (50 IU/kg weekly prophylaxis) rFIXFc INN 63 Median (range): 28 (12‐71) 3.0 (1.0‐4.4) Not reported 1.0 (0.0‐2.2) Not reported 1.1 (0.0‐4.0) Not reported Lambert et al. 2007 (regimen not reported) Nonacog alfa 17 28.32 (12‐61) [for all 34 patients in study] Not reported 3.11 Not reported 0.72 Not reported Not reported Kavakli et al. 2016 (100 IU/kg weekly prophylaxis) Nonacog alfa 25 31.3 (12.1‐53.7) 2.0 (0, 13.8) 3.6 (4.6) 1.0 (0, 13.8) 2.6 (4.1) 0.0 (0, 9.8) 2.1 (3.2) Windyga et al. 2014 (40‐75 IU/kg twice‐weekly prophylaxis) BAX326 INN 56 Median (range): 33 (12‐59) [for all 73 patients in study] 1.99 (0, 23.4) 4.26 (5.80) 0.0 (0, 15.6) 1.72 (3.26) 0.0 (0, 21.5) 2.85 (4.25) Collins et al. 2014 (40 IU/kg weekly prophylaxis) Nonacog beta pegol (N9‐GP) 29 30.0 (SD 15.8) 1.04 (0.00‐4.00) Not reported 0.0 (0.0‐0.98) Not reported Not reported Not reported Young et al. 2016 (40 IU/kg weekly prophylaxis) Nonacog beta pegol (N9‐GP) 52 32 (SD 14.2) 1.00 (0.00‐2.03) Not reported 0.00 (0.00‐1.00) Not reported 0.00 (0.00‐1.97) Not reported Collins et al. 2017 (50‐75 IU/kg twice‐weekly prophylaxis) Trenonacog alfa (IB1001) 58 28.8 (SD 14.2) 1.52 (0.00‐3.46) 3.55 (SD 7.15) Not reported Not reported Not reported Not reported Solano Trujillo et al. 2014 (20‐40 IU/kg twice‐weekly prophylaxis) BAX326 INN 32 31.9 (14‐55) 0.9 (0, 11.2) 2.3 (3.7) 0.0 (0, 10) 0.9 (1.95) 0.0 (0, 10) 1.5 (2.66)

TABLE 2 Details of consumption in rFIX studies identified by the systematic review Study rFIX product No. prophylaxis subjects Consumption (IU/kg/week), mean Santagostino et al. 2016 (weekly prophylaxis group) Albutrepenonacog alfa (rIX‐FP) 40 Median: 44.9 (calculated from 194.7 IU/kg/month) Powell et al. 2013 (weekly prophylaxis group) rFIXFc INN 63 Median: 45.2 Lambert et al. 2007 Nonacog alfa 17 120.8 (Iorio et al., 2017) Kavakli et al. 2016 Nonacog alfa 25 95.9 Windyga et al. 2014 BAX326 INN 56 89.1 Collins et al. 2014 (40 IU/kg weekly prophylaxis group) Nonacog beta pegol (N9‐GP) 29 Not reported Young et al. 2016 (40 IU/kg weekly prophylaxis group) Nonacog beta pegol (N9‐GP) 52 44.5 Collins et al. 2017 Trenonacog alfa (IB1001) 58 105.5 Solano Trujillo et al. 2014 BAX326 INN 32 Not reported

Conclusions: This review identified suitable data for an indirect comparison of rFIX products. Within the limitations of potential differences in patient population and study design, a formal meta‐analysis may help to understand how these products compare with respect to the parameters of ABR, AsBR, AjBR, and consumption of rFIX.

PB184: Comparing Projected Consumption and Cost of Recombinant Factor VIII‐SingleChain with Other Recombinant Factor VIII Products in the United States

S. Yan 1 ; R. Hutcheson 2 ; L. Stern 3 ; G. Krishnarajah 1

1 CSL Behring, King of Prussia, United States, 2 Analytica Laser, Montréal, Canada, 3 Analytica Laser, New York, United States

Background: rVIII‐SingleChain (SC) is indicated in adults and children with hemophilia A (congenital Factor VIII deficiency) for control and prevention of bleeding episodes, routine prophylaxis to prevent or reduce the frequency of bleeding episodes, and for perioperative management.

Aims: To compare prophylactic product consumption and cost of SC versus recombinant factor VIII Fc fusion protein (FC), rurioctocog alfa pegol (PEG) and octocog alfa (OA), for patients ≥ 12 years of age, from the perspective of US payers.

Methods: Average monthly consumption of product per patient (IU/kg) was calculated and compared between SC versus FC, PEG and OA, based on a previously‐developed budget impact model. Dosing frequency and dosage were based on product prescribing information (PI), and midpoint value was used where a range is provided in the PI. Median annualized bleeding rates were based on PI information and other published data from clinical trials of the products. Average monthly costs based on current wholesale acquisition costs (WAC) of the products were also compared.

Results: Average monthly consumption of SC is projected to be similar or slightly lower than FC and PEG, and lower by > 15% than OA, for prophylaxis with or without bleeding treatment (Table 1). Based on the consumption and WACs, average monthly cost of SC per patient is lower than FC, PEG and OA by ˜28%, ˜24% and ˜11%, respectively, for prophylaxis with or without bleeding treatment.

TABLE 1 Monthly consumption for prophylaxis (IU/per kg), per patient age ≥ 12 Regular prophylaxis Treatment of breakthrough bleeds SC 379.17 3.85 FC 379.17 5.09 PEG 390.00 5.42 OA 455.00 4.65

Conclusions: While SC showed long lasting hemostatic efficacy in clinical trials, it may also reduce consumption and cost for US payers, compared with the extended half‐life products FC, PEG, and the commonly used standard‐acting OA.

PB185: Joint and Functional Assessment Following Secondary and Tertiary Prophylaxis in PWH who were Previously Receiving Episodic on Demand Factor Replacement Infrequently

A. Dutta ; S. Kakati; H. Kaur; S. Kar

Assam Medical College and Hospital, Department of Medicine, Dibrugarh, India

Background: Hemophilia is an X‐linked recessive disease which results in the deficiency of coagulation factors and affects one in 10000 births. The hallmark of hemophiliac is musculoskeletal outcome. Though Primary prophylaxis can prevent joint damage, most of the PWH from north east India receive infrequent episodic on demand factor replacement.

Aims: We have studied the role of secondary and tertiary prophylaxis in forty seven patients with hemophilia

(39 hemophilia A and 8 hemophilia B) attending AMCH who were previously receiving episodic treatment.

Methods: It was a non interventional observational study conducted for 3 months, approved by the ethical committee of AMCH. Informed consent was signed. Patients were stratified into four groups, according to age: Group I (1‐15 years of age), Group II (15‐30 years of age), Group III (30‐45 years of age), Group IV (45‐60 years of age).

The HJHS and FISH was correlated with the age of the patients and also with the severity of bleeding. One way Analysis of Variance (ANOVA) was used to determine the differences in the mean of the different groups of hemophiliac. SPSS statistical software was used and statistical significance was defined as a p ‐value < 0.05.

Results: Mean age was 18.02± 10.28 years. 23 (48.9%) were below 15 years, 19 (40.4%) were from 16 to 30 years, 4 (8.5%) were from 31 to 45 years and 1(2.1%) was above 45 years. At the time of enrolment mean total HJHS score for the six main joints and gait was 42.133 (SD 22.138, median 39) out of which maximum was 124.

FIGURE 1 HJHS in PWH on infrequent ondemand therapy from Upper Assam region of INDIA

The most affected joints were knees, followed by elbows and ankles. The mean of total ankle score were significantly lower compared to total knees score. There was no significant difference between HJHS score of mild, moderate and severe PWH. FISH score was high in episodic treatment and improved.

FIGURE 2 Functional Assesment using FISH scores compared between mild moderate and sever PWH on ondemand factor replacement from Upper Assam region of INDIA

Conclusions: There was improvement in HJHS score but it was not significant in elder patients. Functional assessment also improved after four months of treatment.

PB186: Validation of an Instrument for Nursing Consultation to the Person with Hemophilia

I. Sangi 1,2 ; R. Camelo 1 ; I. Costa 1 ; N. Costa 1 ; F. Lima 2 ; T. Guimaraes 1,2

1 Foundation of Hematology and Hemotherapy of Pernambuco ( HEMOPE ), Recife, Brazil, 2 Federal University of Pernambuco, Recife, Brazil

Background: Hemophilia is an X‐linked recessive coagulopathy caused by the deficiency of factors VIII (hemophilia A) or IX (hemophilia B). Brazilian Ministry of Health reinforced promoting health through multiprofessional approach to persons with hemophilia (PwH). In this context, nursing consultation (NC) to PwH is recommended, offering advantages in treatment and adherence.

Aims: This study aimed at constructing and validating an instrument for NC to PwH, at the Ambulatory of Coagulopathies of HEMOPE (Recife, Brazil).

Methods: The qualitative cross‐sectional study consisted of elaboration of the first version of the instrument, based on systematic and random observation of 1,800 NC; analysis by a multiprofessional outpatient team of hematology specialists, and later, analysis by 20 nurse‐judges with experience in attending to PwH and 9 national reference hemophilia nurses (RHN), by the Delphi method. The agreement among nurses was assessed by Fisher′s Exact test to evaluate the degree of relevance of the characteristics and the degree of satisfaction about the items of appearance. The items of at least 70% agreement were considered valid. The study was approved by HEMOPE ethics committee (CEP‐HEMOPE n.1.863.411/CAAE 61155316.5.0000.5195) and informed consent was obtained from the professionals involved.

Results: The nurses considered all the characteristics “relevant” or “extremely relevant” (P>0.05). Regarding the degree of appearance satisfaction, the RHN requested the instrument to be used for other coagulopathies as well, turning the homogeneity test significant for the characteristic clarity of the statements (P=0.03). The final version was composed of 17 parts, including Treatment, Inhibitor Status, Independence Degree, Infusion Log and Immune Tolerance.

Conclusions: The instrument was considered adequate to the systematization of the NC to the PwH, facilitating the identification of patient problems. It has been inserted into the service electronic medical record.

PB187: Epidemiological Survey and Audit of Socio‐demographic Characteristics of Patients with Haemophilia and other Congenital Bleeding Disorders

T. Nwagha 1 ; M. Adediran 2 ; H. Okoye 3

1 University of Nigeria Teaching Hospital, Haematology & Immunology, Enugu, Nigeria, 2 Haemophilia Foundation of Nigeria, Kaduna, Nigeria, 3 University of Nigeria Teaching Hospital, Haematology & Immunology, Ituku Ozalla, Nigeria

Background: Despite more than 50 years since the first diagnosis of haemophilia was made in Nigeria, congenital bleeding disorders are still considered as disease of medical textbooks. There is increasing evidence of high disease prevalence, disease related morbidity and mortality. With WFH′s effort to “bridge the gap” and encourage individual country government commitment towards these disease management Early identification, diagnosis and treatment are key in reducing these statistics.

Aims: To study the epidemiology and socio‐demographic variables of persons with haemophilia and other congenital bleeding disorders in Nigeria.

Methods: This was a 12 year retrospective audit of congenital bleeding data registry of the Haemophilia Foundation of Nigeria (HFN) between 2005‐2017 Data on age, sex, geographical location of referring HTC or hospital, type of congenital disorder, clinical severity type and inhibitor status of persons living with congenital bleeding disorders were obtained from the HFN registry. Data were analysed using the windows SPSS version 20.

Results: A total of three hundred and fifty‐one registered with HFN, 350 (99.7%) were males, and only1(0.03%) female. The mean age, SD and range were 15 (11) years, and 4 months‐66years respectively. Most registered persons were from tthe northwest geopolitical zone 40.1% (135/351). Most had haemophilia A, 92.6% (325/351), of severe clinical severity 13.7% (48/351) with only 1 (0.03%) documented with inhibitors. There was also only 1 documented congenital platelet disorder (Glanzmann thrombasthenia 0.03%. There was a significant association between both geopolitical zone, referral HTC / hospital and the type of bleeding disorder χ 2 292.3, P value 0.000 and χ 2 270.4, P value 0.000 respectively.

Conclusions: The overall low number of persons registered may be from high disease burden. There is need for government driven public awareness programme as well as its commitment to improving capacity in diagnosis and treatment.

PB188: Development of a FVIII Antigen Assay to Quantify B‐domain Deleted FVIII Antigen in Human Plasma

M. Robinson ; C. Tudan; S. Tiefenbacher

Colorado Coagulation, Laboratory Corporation of America Holdings, Englewood, United States

Background: With the increased use of B‐domain deleted FVIII constructs in AAV mediated FVIII gene therapies; there is a need to identify FVIII antigen assays that can adequately measure B‐domain deleted FVIII in human plasma. Evaluations of the B‐domain deleted rFVIII ReFacto AF ® in two commonly used FVIII antigen assays in the US, Visualize ™ FVIII antigen (Affinity Biologicals ™ Inc.) and Asserachrom ® VIII antigen (Diagnostica Stago), demonstrated significant recovery issues, with under‐recovery at concentrations ≥ 0.1 IU/mL and over‐recovery at concentrations < 0.1 IU/mL.

Aims: To develop and validate a FVIII antigen assay to measure B‐domain deleted FVIII in human plasma.

Methods: A FVIII antigen ELISA assay that uses two monoclonal antibodies for capture and detection with specificity for the A2 domain epitope of FVIII was developed and validated to meet the EMA and draft FDA bioanalytical method validation (BMV) guidances. The assay was calibrated using a ReFacto AF ® /Xyntha ® reference material to obtain a calibration range of 0.01 to 1.0 IU/mL. The assay conditions were optimized to minimize von Willebrand factor (vWF) interference while providing consistent and acceptable recovery across the reportable range. Accuracy, precision, dilution linearity and sensitivity were assessed using congenital FVIII deficient plasma samples spiked with ReFacto AF ® /Xyntha ® .

Results: Titrations of vWF and pre‐incubation with βME were used to optimize the final assay conditions to reduce steric interference between the analyte and the assay antibodies. All validation assessments met the BMV guidance acceptance criteria with reproducible accuracy, precision, and sensitivity across a reportable range of 0.01 to 2.5 IU/mL.

Conclusions: A method was developed and validated to measure B‐domain deleted FVIII antigen in human plasma that met the BMV guidance's acceptance criteria for all validation assessments with reproducible recovery across the reportable range.

PB189: Exploring Regional Variations in the Cross‐cultural, International Implementation of the Patient Reported Outcomes Burdens and Experience (PROBE) Study

C. Chai‐Adisaksopha 1 ; D. Noone 2 ; R. Curtis 3 ; N. Frick 4 ; M. Nichol 5 ; B. O'Mahony 2,6 ; D. Page 7 ; J. Stonebraker 8 ; A. Iorio 1 ; M.W. Skinner 9 ; PROBE

1 McMaster University, Hamilton, Canada, 2 Irish Haemophilia Society, Dublin, Ireland, 3 Factor VIII Computing, Berkeley, United States, 4 National Hemophilia Foundation, New York, United States, 5 University of Southern California, Sol Price School of Public Policy, Los Angeles, United States, 6 Trinity College, Dublin, Ireland, 7 Canadian Hemophilia Society, Montreal, Canada, 8 Poole College of Management, North Carolina State University, Raleigh, United States, 9 Institute for Policy Advancement Ltd., Washington, United States

Background: The Patient Reported Outcomes Burdens and Experience (PROBE) study has developed and validated the multilingual PROBE questionnaire for assessing patient reported outcomes in people living with hemophilia (PWH) and participants without bleeding disorders (NOBD). The PROBE questionnaire translations into local languages account for site specific language and cultural peculiarities in the management and life experience of hemophilia.

Aims: To explore the regional variations in the international implementation of the PROBE questionnaire and examine the hypothesis that the PROBE score will not be confounded by cross‐cultural aspects.

Methods: Data were collected from participants in four regions (Western Pacific, South America, North America and Europe). Participants were able to select from 23 localized language versions of the PROBE questionnaire based on their first language. We used analysis of variance methods and multivariable regression to determine the relative contribution of the variance explained by region controlling for hemophilia diagnosis, age group and level of education. We also explored interactions between region and the other components.

Results: We analyzed 862 questionnaires from 21 countries. Mean age of participants was 40.03 years (SD 13.89) and 65.20% were PWH and 34.80% were NOBD. After adjusting for hemophilia diagnosis, age group and level of education, region contributed 0.44% to 7.98% of the variance component in sub‐item scores and 0.26% in the PROBE score. Years of education contributed 0.34% in the PROBE score. Age and diagnosis (NOBD, mild, moderate, severe) contributed 3.42% and 22.42% of the PROBE score. 70.74% of the variance was explained by inter‐individual variation.

Conclusions: Variance partitioning for the PROBE score is similar to that for EQ5D. The results demonstrate that the PROBE questionnaire is valid to implement for assessing health status among PWH and participants without bleeding disorders across regions.

PB190: Expanding Communications on Hemophilia A Outcomes (ECHO) Study: Choice of Patient‐reported Outcomes (PROs) and Feasibility Testing

S. von Mackensen 1 ; C.R.M. Hay 2 ; M. Shima 3 ; M. Makris 4 ; V. Jiménez‐Yuste 5 ; J. Oldenburg 6 ; K. Fischer 7 ; E. Santagostino 8 ; S. Rauchensteiner 9 ; N. Church 10 ; P. Mathew 10 ; C.M. Kessler 11 ; M.W. Skinner 12 ; on behalf of the ECHO Study Investigators

1 University Medical Centre Hamburg‐Eppendorf, Hamburg, Germany, 2 Manchester University Department of Haematology, Manchester, United Kingdom, 3 Nara Medical University, Kashihara, Japan, 4 Department of Infection, Immunity, and Cardiovascular Disease, University of Sheffield, Sheffield, United Kingdom, 5 Autónoma University Madrid and La Paz University Hospital, Madrid, Spain, 6 Department of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany, 7 Van Creveldkliniek, University Medical Center Utrecht, Utrecht, the Netherlands, 8 IRCCS Ca’ Granda Foundation, Maggiore Hospital Policlinico, Milan, Italy, 9 Bayer, Basel, Switzerland, 10 Bayer, Whippany, NJ, United States, 11 Georgetown University Medical Center, Washington, DC, United States, 12 Institute for Policy Advancement, Ltd, Washington, DC, United States

Background: The Expanding Communication on Hemophilia A Outcomes (ECHO) study was designed as a 5‐year international, observational registry in adults with moderate/severe hemophilia A (PwHA) exploring associations of different variables on patient‐reported (PRO) and clinical outcomes. In qualitative focus groups, 10 aspects were identified that were considered important by PwH (n=45), resulting in an inclusion of 9 standardized PRO measures and several ad‐hoc questions concerning socio‐demographic and clinical data, health behavior and resource utilization.

Aims: Testing the feasibility of the complex survey for PwHA concerning ease and time of completion, number of questions and relevance of aspects.

Methods: A feasibility questionnaire was designed with 10 questions using visual analogue scales (VAS) ranging from 0 (difficult/poor) to 100 (good/easy). First enrolled Manchester PwHA (mean age 46.5±12.7, 92.3% severe) were asked to fill in the questionnaire after completing PROs.

Results: PwHA (n=13) needed a median (med) 60 minutes (range 0.5‐2.5 hours) to complete the PROs. They thought that the survey did not adequately capture some aspects mentioned in the focus groups including factor medication (44%) and home & daily life (33%), or could not be assessed, namely psychological (33%) and economic impacts (44%); some (33%) felt that e.g. impact on children should have been assessed. VAS questions revealed that PwHA liked the survey (med 70), found it easy to complete (med 80), related to their personal situation (med 70), considered the number of questions inadequate (med 51.5). Although the number of questions was the most critical point, PwHA would recommend a friend with haemophilia to participate in ECHO (med 95). Five PwH suggested changes in the survey (e.g. eliminate repetitive questions).

Conclusions: The ECHO PROs selection indicated feasibility. Additional areas suggested by PWH were not added due to investigator concerns the overall burden would increase and inhibit recruitment and PRO completion.

PB192: Hemophilia A Subjects with an Intron‐22 Inversion Mutation Show Robust T‐cell Responses to Epitopes in Both the FVIII Heavy and Light Chain

D. Gunasekera 1 ; A.F. Karim 2 ; M.V. Ragni 3 ; K.P. Pratt 2

1 Uniformed Services University, Medicine, Bethesda, United States, 2 Uniformed Services University, Medicine MED , Bethesda, United States, 3 University of Pittsburgh, Medicine, Pittsburgh, United States

Background: Almost half of severe hemophilia A (HA) is caused by an inversion mutation in intron 22 of the 26‐exon F8 gene encoding factor VIII (FVIII). FVIII contains homologous domains A1‐A2‐B‐A3‐C1‐C2 and circulates as a heterodimer consisting of a heavy chain (A1‐A2‐B) and light chain (A3‐C1‐C2). The intron‐22 inversion precludes the possibility of a full‐length F8 mRNA being produced; instead, a partial F8 mRNA containing exons 1‐22 is transcribed. A shorter F8 transcript containing exons 23‐26, termed F8B, is transcribed from a promoter within intron 22 in both non‐hemophilic and hemophilic individuals. F8B encodes a C‐terminal C1 domain RNA sequence linked in‐frame to the C2 domain sequence. FVIII protein is not detectable in plasma of patients with an intron‐22 inversion.

Aims: To test the hypothesis that putative low‐level intracellular synthesis of partial FVIII proteins encoded by the two partial F8 mRNA molecules, if this occurs, confers immune tolerance to all therapeutic FVIII regions except a short C1 domain sequence corresponding to the inversion break‐point.

Methods: Cellular immune responses to FVIII were queried using proteins and synthetic 15‐mer peptides with overlapping sequences spanning the FVIII A2 and C2 domains. PBMCs from 20 severe HA subjects with a current, past or no inhibitor history were tested by ELISPOT assays to detect cytokine secretion in response to FVIII proteins and/or peptides.

Results: Over half showed robust Th1 and/or Th2 responses to recombinant FVIII‐C2 protein. Incubations with FVIII A2 and C2 domain peptides stimulated IFN‐g and/or IL‐5 secretion from intron‐22 inversion samples, consistent with earlier publications from the Conti‐Fine group.

Conclusions: The observed lack of tolerance to multiple FVIII domains indicates that future strategies to tolerize intron‐22 inversion patients should take into account epitopes throughout the therapeutic FVIII protein, rather than focusing on the short C1 domain sequence corresponding to the inversion break‐point.

PB193: Analysis of HLA‐class‐II (HLAcII) Peptidomes in Response to Several Therapeutic Factor (F)VIII Proteins (tFVIIIs) in Healthy Donors and Hemophilia A (HA) Patients with and without FVIII Inhibitors (FEIs)

Z. Sauna 1 ; V. Diego 2 ; M. Almeida 2 ; B. Luu 2 ; M. Hofmann 3 ; J. Hernandez 2 ; R. Rajalingam 4 ; S. Williams‐Blangero 2 ; J. Curran 2 ; J. Powell 5 ; M. Escobar 6 ; J. Blangero 2 ; E. Maraskovsky 7 ; N. Key 8 ; T. Howard 2,9,10

1 Center for Biologics Evaluation and Research, Food and Drug Administration, Laboratory of Hemostasis, Division of Hematology Research and Review, Silver Spring, United States, 2 University of Texas Rio Grande Valley School of Medicine, South Texas Diabetes & Obesity Institute, Brownsville, United States, 3 CSL Behring, Marburg, Germany, 4 University of California San Francisco, Immunogenetics and Transplantation Laboratory, San Francisco, United States, 5 CSL Behring, Med Affairs, King of Prussia, United States, 6 University of Texas Health Sciences Center at Houston, Medicine, Division of Hematology and Oncology, Houston, United States, 7 CSL Limited, Research, Bio21 Institute, Melbourne, Australia, 8 University of North Carolina at Chapel Hill, Medicine, Division of Hematology and Oncology, Chapel Hill, United States, 9 University of Texas Rio Grande Valley School of Medicine, Biomedical Sciences, Brownsville, United States, 10 Haplomics Biotechnology Corp, Brownsville, United States

Background: T cells (TCs) are critical for FEI‐development in HA. Dendritic cells (DCs) express HLAcII repertoires and present peptides to TCs. We used DC protein presentation assays to study tFVIII peptidomes.

Aims: Test the hypothesis that protein presentation by DCs is not affected by HA. Study factors influencing the immunogenicity potential (IP) of tFVIIIs as reflected by HLAcII‐bound tFVIII‐derived peptide density

Methods: DCs from 4 healthy donors (H) and 5 HA patients (P)—2 with FEIs (I) and 3 without (N)—were incubated with plasma‐derived (pd)VWF and either pdFVIII or 1 of 3 recombinant (r)FVIII proteins (rFVIIIs): full‐length (FL), B‐domain (BD)‐deleted (BDD), and BD‐truncated (BDT). DR‐bound peptides were identified by LC‐MS/MS and comparison to a proteomics database (Figure 1).

FIGURE 1 Representative Individual HLAcII Peptidomes for Various Therapeutic Factor VIII Proteins (tFVIIIs)

We performed a log‐linear analysis of multi‐way contingency tables (MCTs) of our data. We analyzed a MCT of: 1) a variable contrasting H with N and I; 2) DRB genotypes; 3) the 4 tFVIIIs, and 4) tFVIII domain. In a 2 nd model, we analyzed a MCT of an inhibitor variable contrasting N and I, and the last 3 variables under the 1 st model.

Results: HA patients I and N have greater IP than baseline, namely H (Table 1A). BDT‐ & FL‐rFVIII, respectively, have lesser and greater IP than baseline, pdFVIII. The IP for BDD‐rFVIII was not different from pdFVIII. Similarly, patients with inhibitor I have greater IP than those without N (Table 1B). Again, BDT‐ & FL‐rFVIII have lesser and greater IP than pdFVIII whereas the IPs for BDD‐rFVIII & pdFVIII are not different. The observed higher IPs in affecteds relative to unaffecteds reflects a significant correlation with the presence of the inhibitor risk allele DRB1*15:01. HP and NI have correlations with DRB1*15:01 of 0.91 and 0.52 (P=0.002 and 0.001).

Table 1 Log‐linear Model Results of Dendritic Cell Protein Processing and Presentation Assays

Conclusions: DC protein presentation is affected by HLAcII repertoires but not by HA or inhibitor status. In addition, rFVIIIs have different IPs relative to pdFVIII with BDT‐ and FL‐rFVIII being significantly less and greater than, respectively.

PB194: Recombinant Porcine Factor VIII Post‐marketing Safety Studies in the European Union (EU) and United States (US)

R. Crea 1 ; O. Mauric 1 ; J.‐F. Huang 2 ; J. Chen 2

1 Shire, Vienna, Austria, 2 Shire, Cambridge, United States

Background: A B‐domain‐deleted, porcine‐sequence factor VIII (rpFVIII) with low cross‐reactivity to anti‐human‐FVIII antibodies is used to treat acquired hemophilia A (AHA).

Aims: To collect data assessing safety, safety‐related factors, utilization, and efficacy of rpFVIII in real‐world clinical practice.

Methods: Two multicenter, prospective, noncontrolled, open‐label, single cohort, noninterventional studies are ongoing in the EU and US. Patients (pts) >18 years of age with AHA will be enrolled once per routine clinical practice and treated with rpFVIII for bleeding episodes (BEs). Demographic/clinical characteristics, comorbidities and medical history, characteristics of BEs, treatment indication, rpFVIII treatment details, bleeding control, concomitant medications, inhibitor levels, prior rpFVIII and bypassing agent treatments, and adverse events (AEs)/serious AEs will be collected at baseline and follow‐up visits up to 180 d after the last dose of rpFVIII.

Results: The EU study began in late 2016 and is ongoing in Austria, Germany, and Italy, and 4 pts have enrolled. The US study began in 2015 and currently has 16 active sites and 37 pts enrolled. A data read‐out was carried out in June 2017 for 21 pts. Among evaluable pts, 6 were treated with rpFVIII as first‐line therapy for initial BEs; 4 as first‐line therapy for subsequent BEs; and 6 received rpFVIII as second‐line or later treatment. Among pts evaluable for efficacy, a total of 14 BEs in 11 pts were resolved, while 4 BEs in 3 pts were not resolved. No thromboembolic events were reported.

Conclusions: Preliminary analysis of the US study shows rpFVIII is generally well‐tolerated and effective in AHA. These studies will provide robust real‐world data on the use of rpFVIII, providing insight into the clinical use of rpFVIII, as well as the optimal regimen to personalize therapy.

Funding: These studies were funded by the sponsor, Shire International GmbH.

PB195: Regulatory T Cells in the Modulation of Hemophilia A

M. Meissner 1 ; J. Gotot 1 ; T. Albert 2 ; J. Oldenburg 2 ; C. Kurts 1

1 Universitätsklinikum Bonn, Institute for Experimental Immunology, Bonn, Germany, 2 Universitätsklinikum Bonn, Institute for Hematology and Transfusion Medicine, Bonn, Germany

Background: Hemophilia A is an X chromosome‐linked bleeding disorder caused by a deficiency of blood coagulation factor VIII (FVIII) that affects approximately 1 in 5,000 males. A major clinical complication concerning the protein‐replacement therapy with therapeutic FVIII is the formation of anti‐FVIII inhibitory antibodies (inhibitors), which occurs in approximately 30% of all Hemophilia A patients. Currently accepted clinical protocols for immune tolerance induction (ITI) in Hemophilia A patients do not only eliminate already existing inhibitors, but also induce long‐lasting tolerance towards FVIII in approximately 66% of all ITI‐receiving Hemophilia A patients. A role of suppressive CD4 + CD25 + Foxp3 + regulatory T cells has been suggested, but the precise mechanisms underlying tolerance in ITI is unknown.

Aims: We here aimed at providing formal and mechanistic evidence for a role of regulatory T cells, by examining the anti‐FVIII immune response in hemophilic mice and healthy control mice.

Methods: We performed adoptive transfer experiments with regulatory T cells from healthy control mice into hemophilic mice and analyzed the humoral and cellular immune response towards FVIII after immunizing these mice with recombinant FVIII protein. We especially focused on the expression of both, activating and inhibitory molecules on the surface of regulatory T cells and

B cells by flow cytometry.

Results: We demonstrate that regulatory T cells from healthy control mice, after transfer into hemophilic mice, were able to suppress B cells in acceptor mice. Furthermore, we show a role of changes in inhibitory molecules, like Fas and

PD‐1, on B cells in hemophilic mice after adoptive transfer of regulatory T cells.

Conclusions: Our results, suggest novel therapeutic targets for the treatment of Hemophilia A patients with inhibitors.

PB196: Phase 2 Trial of Subcutaneously Administered Novel FVIIa Variant, Marzeptacog Alfa (activated), in Hemophilia A or B with Inhibitors: Pharmacokinetics, Pharmacodynamics, Safety and Efficacy

H. Levy 1 ; G. Iosava 2 ; F. Del Greco 1 ; A. Hetherington 1 ; J. Hillman 1

1 Catalyst Biosciences, South San Francisco, United States, 2 Medinvest Institute of Hematology and Transfusiology, Tbilisi, Georgia

Background: In hemophilia patients with inhibitors, hemostasis can be achieved using bypass agents: activated plasma‐derived prothrombin complex concentrates (aPCC), recombinant Factor VIIa (FVIIa) or emicizumab that can only treat hemophilia A. Marzeptacog alfa (activated) (MarzAA), a variant recombinant FVIIa with enhanced biological properties enabling subcutaneous (SQ) delivery, was developed using a rational protein design approach and has 7‐fold increased catalytic activity and prolonged duration of effect in a human single‐dose intravenous (IV) study that documented safety. Daily dosing in dogs demonstrated achievement of stable trough levels of MarzAA that are believed to be effective for prophylaxis.

Aims: Phase 2 study MAA‐201 complies with the Declaration of Helsinki and is approved by recognized medical ethics committees, will enroll 12 subjects with inhibitors and an annual bleeding rate ≥12 who sign informed consent, to determine if SQ MarzAA can provide effective prophylaxis (NTC03407651).

Methods: 18 mg/kg MarzAA will be infused IV and pharmacokinetics and coagulation parameters measured for 24 hours (Figure). 30 mg/kg MarzAA will be injected SQ and pharmacokinetics and coagulation parameters measured for 48 hours. Daily MarzAA SQ therapy will then be injected for 50 days.

FIGURE 1 MAA‐201 trial schema

If no spontaneous bleeding events occur, the subject will proceed to safety follow up 30 days after last dose. Should spontaneous bleeding occur, then dose escalation occurs sequentially to 60, 90 or 120 mg/kg, if needed. PK and bioavailability will be calculated. Subjects will be monitored for treatment‐related changes, Quality of Life measures and tested for inhibitors to MarzAA and FVIIa. ABR will be compared to the subjects’ prior ABR.

Results: Interim results of pharmacokinetics, coagulation changes, immunogenicity and bleeding rate will be reported.

Conclusions: This study design will demonstrate whether daily SQ MarzAA can provide effective prophylaxis in hemophilia patients with inhibitors.

PB197: Potential Clinical Trial Validation of Differential EPCR‐dependent Activity by Eptacog Beta, a rhFVIIa Variant, in the Treatment of Bleeding in Hemophilia Patients with Inhibitors

J. Ducore 1 ; W.A. Alexander 2 ; I. Mitchell 3 ; J. Lawrence 4 ; J. Grandoni 4 ; D. Bonzo 4

1 UC Davis, Sacramento, United States, 2 HEMA Biologics, LLC, Louisville, United States, 3 Gloval LLC, Broomfield, United States, 4 LFB‐USA, Framingham, United States

Background: Several laboratories have demonstrated that endothelial protein C receptor (EPCR) binding by pharmaceutical doses of FVIIa potentially drives a 3rd non‐tissue factor‐dependent hemostatic pathway. rhFVIIa (eptacog beta, EB) has an almost two‐fold greater affinity for EPCR compared to another variant of human FVIIa, rFVIIa (eptacog alfa, EA) # . Thrombin generation assays in hemophilia patients w/wo inhibitors show reduced clinical dose requirements for EB. As ex vivo studies of thrombin generation do not predict FVIIa variant efficacy, the PERSEPT1 trial of EB could have led to results comparable to those of other variants. Instead, efficacy at 12 hours was up to 93%, and was sustained through 24 hours without extra or rescue dosing. Thus, the Phase 3 clinical efficacy of EB may validate the clinical relevance of this third hemostatic pathway.

Aims: To determine whether clinical evidence supports the functional difference in EPCR binding between FVIIa variants.

Methods: We conducted a literature review for prospectively collected data describing two clinical outcomes of bleeding events treated with the rFVIIa variants EB and EA: rebleeding rate at 24 hours (Table 1), and success in hemarthrosis (Table 2). Interpolated rebleeding rates were excluded and hemarthrosis clinical response was included if reported at 12 and 24 hours. Differential EPCR binding studies are included in the complete results.

Results: TABLE 1 Published clinical efficacy and early rebleeding rates Source FVIIa variant Dosing (mcg/kg) Bleeding Events (N) 12 hour Efficacy (%) 24 hour Efficacy (%) Early Rebleeding Rate* (%) PERSEPT 1 Wang et al 2017 EB 75q3h 252 84.9 96.7 0 PERSEPT 1 Wang et al 2017 EB Initial: 225 Subsequent 75q3h after 9 hour 213 93.2 99.5 0.2 adept ™ 2 Lentz, et al 2014** EA arm 90q3h (1‐3 doses) 227 93 86.2 13.8 FENOC Astermark, et al 2007** EA 90‐120 x2 48 75.6 85.7 4.2 “Megadose” Kenet, et al 2003 EA Initial: 300 Second: 200 or 300 @ 3‐4 hour 114 ‐ 91.4 9.6 *Early rebleeding ‐ Prior to 24 hours from first dose, the recurrence of symptoms, need for rescue medication or other objective evidence suggesting loss of hemostasis after initial clinical response; **Extra doses allowed and administered after initial response.

TABLE 2 Efficacy in hemarthrosis Review of efficacy in hemarthrosis was facilitated by the detailed literature review by Treur, et al (2009) where 11 trials were analyzed for EA efficacy in hemarthrosis Source FVIIa variant Bleeding Events (N) 9 hour Efficacy (%) 12 hour Efficacy (%) 24 hour Efficacy (%) PERSEPT 1 Wang, et al 2017 EB 396 Pooled efficacy not reported 88.9 98.2 TREUR Bayes Data Treur, et al 2009 EA 160 / 91 / 121 44 / ‐ / ‐ ‐ / 67 / ‐ ‐ / ‐ / 54 TREUR Bayes Model Treur, et al 2009 EA n/a ‐ 66 88

Conclusions: Comparisons from large studies of early rebleeding and 12/24 hour efficacy for EA and EB in the setting of joint bleeding yielded consistent differences. This observation suggests that the two‐fold difference in affinity for EPCR clinically manifests as an earlier clinical response and lower dose requirement for rhFVIIa (eptacog beta, EB). Further research into EPCR binding and clinical efficacy of EB should be conducted to more fully characterize the effect of a third rhFVIIa hemostatic pathway.

#Grandoni, J et al. Haemophilia 2017, 23(2):300‐308

PB198: Immune Tolerance Induction in Patients with Hemophilia A and Inhibitors: Preliminary Results of the Brazilian Immune Tolerance (BrazIT) Study

R. Camelo 1 ; L. Magalhaes 1 ; L. Jardim 1 ; L. Werneck 1 ; A. Gonçalves 2 ; M.R. Roberti 3 ; D. Tan 4 ; V. Franco 5 ; D. Chaves 2 ; S. Rezende 1

1 Federal University of Minas Gerais, Faculty of Medicine, Belo Horizonte, Brazil, 2 Foundation Hemocentro of Minas Gerais, Belo Horizonte, Brazil, 3 Hemocentro of Goias, Goiania, Brazil, 4 Faculty of Medicine of Marilia, Hemocentro of Marilia, Marilia, Brazil, 5 Hemocentro of Santa Catarina, Florianopolis, Brazil

Background: About 20‐30% of patients with HA (PHA) may develop neutralizing antibodies (inhibitors) against infused factor (F) VIII. Immune tolerance induction (ITI) is the treatment of choice to eradicate inhibitors.

Aims: The Brazilian Immune Tolerance (BrazIT) Study aims to investigate predictors of response to ITI using a protocol recommended by the Ministry of Health.

Methods: Inclusion required confirmed diagnosis of HA with active inhibitor. Patients were not selected based on “good risk” factors and all who filled the inclusion criteria and signed informed consent were included. The protocol recommended that all PHA started ITI with a low‐dose regimen (50IU/kg 3x/week). Upon lack of response (no decline of inhibitor in of at least 20% of peak levels after ITI start within 6 months), high‐dose (HD) regimen (100IU/kg daily) and subsequent changing to plasma‐derived (pd) FVIII for those who started on recombinant FVIII was recommended. Response was based on inhibitor titer, FVIII half‐life and recovery.

Results: We included 45 patients from 4 Hemophilia Treatment Centers (Table 1) of whom 34 concluded the protocol. A total of 29/34 (85%) patients responded to ITI, of whom 13 and 16 were total and partial success, respectively; 5/34 (15%) patients failed ITI, despite changing to HD regimen. A total of 24/34 (70.6%) patients started ITI with pdFVIII. Intron 1 and 22 inversions were present in 13/18 (72%) and 1/4 (25%) of patients who responded and failed, respectively ( p =0.12). Patients who failed treatment had higher inhibitor peak during ITI ( p =0.004), changed to HD regimen ( p =0.0001) and had more breakthrough bleeding events ( p =0.012), compared with those who had success (Table 2).

TABLE 1 Baseline characteristics of the patients included in the BrazIT Study (n=45). * out of 42 patients Characteristics Results Male sex, n (%) 44 (97.8) FVIII activity < 2%, n (%) 34 (81.0)* Age at hemophilia diagnosis in years, median (IQR ‐ interquartile range) 0.9 (0.6‐1.6) Age at inhibitor diagnosis in years, median (IQR) 3.9 (1.7‐10.7) Age at ITI (immune tolerance induction) start in years, median (IQR) 11.8 (4.38‐24.5) Time elapsed between inhibitor diagnosis and ITI start in years, median (IQR) 4.9 (1.0‐12.4) Pre‐ITI inhibitor titer peak in BU (Bethesda unit)/mL, median (IQR) 27.4 (12.7‐57.5) Inhibitor titer immediately before ITI start in BU/mL, median (IQR) 4.2 (2.8‐7.6)

TABLE 2 Characteristics of patients who completed immune tolerance induction (ITI; n=34) Characteristics Successful ITI (n=29) Failed ITI (n=5) P (Mann Whitney test) Age at ITI start in years, median (IQR ‐ interquartile range) 6.4 (2.9‐18.8) 9.5 (8.7‐13.4) 0.78 Time elapsed between inhibitor diagnosis and ITI start in years, median (IQR) 2.7 (0.5‐8.4) 7.1 (1.0‐10.4) 0.71 Pre‐ITI inhibitor titer peak in BU (Bethesda unit)/mL, median (IQR) 24.0 (12.0‐52.0) 140.8 (32.0‐384.0) 0.92 Inhibitor titer immediately before ITI start in BU/mL, median (IQR) 4.0 (2.5‐7.6) 5.6 (5.6‐11.2) 0.15 Inhibitor titer peak during ITI in BU/mL, median (IQR) 6.0 (2.5‐20.8) 179.2 (58.4‐211.2) 0.004 Plasma‐derived FVIII as first type of concentrate used, n (%) 20 (69) 4 (80) >0.99 Change to high‐dose regimen, n (%) 1 (4) 5 (100) 0.0001 Number of bleeding events during ITI, median (IQR) 9.0 (4.0‐14.0) 20.0 (19.0‐25.0) 0.012 ITI duration in years, median (IQR) 1.6 (1.0‐2.1) 3.3 (3.1‐3.4) 0.0002

Conclusions: Brazilian ITI protocol demonstrated 85% success rate despite the long time elapsed between inhibitor diagnosis and start of ITI and the older age of patients at start of ITI. Due to low number, multivariate analysis could not be performed, which will be possible with the inclusion of more patients and longer follow‐up.

PB199: Inhibitor Development and Successful Immune Tolerance Induction in a Young Female Carrier of de novo Severe Hemophilia A

L.W. Zuccherato 1 ; M.D.R.F. Roberti 2 ; L.L. Jardim 1 ; S.M. Rezende 1

1 Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 2 Centro de Hematologia e Hemoterapia de Goiás, Goiania, Brazil

Background: Females are commonly reported as carriers of hemophilia A (HA), since only one X chromosome is affected. Skewed lyonization of X chromosome can be associated with low levels of FVIII (< 5%) in carriers, and might require FVIII replacement. Neutralizing antibodies (inhibitors) against FVIII occur in up to 30% of patients, but is rarely reported in carriers.

Aims: Here we report a rare case of a young female presenting very low FVIII levels (< 1%) and high‐titer inhibitor development which successfully disappeared after ITI.

Methods: A 10‐month‐old female was referred due to bruises and hemarthrosis. There was no family history of HA nor other bleeding disorder. FVIII activity (FVIII:C) levels were 0.6% and the inhibitor titer at diagnosis was 0.35 Bethesda Units (BU)/mL. An inverse PCR for intron 22 inversion of F8 showed the carrier status of the proband, with family members testing negative. Exome sequencing of F8 showed no additional deleterious mutations, indicating that the disease is likely caused by a defective skewed epigenetic X‐inactivation system.

Results: Prophylactic replacement started with plasma‐derived (pd) FVIII. After 25 exposure days, a high‐titer inhibitor (25.6 BU/mL) was detected, five weeks after the report of pain and extensive edema at her left thigh, due to influenza A intramuscular vaccination, which required five days of FVIII replacement. ITI was initiated with pdFVIII 50 IU/kg 3 times a week. Inhibitor disappeared after 18 months and partial response was confirmed by FVIII half‐life of 14.5 hours and FVIII recovery of 58.6%. She started on prophylactic treatment and is currently maintaining sustained inhibitor titer below 0.6 BU/mL.

Conclusions: Our report demonstrates that HA carriers with severe phenotype can develop neutralizing antibodies against FVIII, and that ITI can be effective. We suggest that HA carriers, particularly those who require FVIII replacement, should be regularly screened for FVIII inhibitors.

PB200: Risk Modeling Inhibitor Development of Haemophilia A Patients in Australia

S. Parikh 1 ; H. Tran 2,3 ; S. McRae 4

1 Australian Haemophilia Centre Directors’ Organisation, Melbourne, Australia, 2 Australian Centre for Blood Diseases, Melbourne, Australia, 3 The Alfred Hospital, Melbourne, Australia, 4 Royal Adelaide Hospital, Adelaide, Australia

Background: Development of inhibitors is the most severe and challenging complication associated with factor VIII replacement therapy in Haemophilia A (HA) patients. Several studies identifying risk factors that influence inhibitor development have been published in the past however cumulative risk is not clearly evident.

Aims: The primary aim of this project is to conduct a preliminary analysis of the risk factors associated with inhibitor development among HA patients in Australia and the feasibility of creating a multivariate risk model to enhance clinical decision making.

Methods: Retrospective data was obtained from the Australian Bleeding Disorders Registry (ABDR) on HA patients (excluding carrier patients) from all Haemophilia Treatment Centres (HTCs) with appropriate ethics approval. Information was extracted on disease severity, genotype, product usage, treatment regime, treatment reason, age at first exposure and exposure days (ED) leading upto first inhibitor (titre level ≥ 0.5BU/ml). Mixed modeling (containing both fixed and random effects of risk factors) plus all other analysis was performed using Stata v12.1.

Results: 1644 (68.15%) of the 2412 HA patients used at least one factor VIII product. 161/1644 HA patients with missing/incomplete/historical data were excluded. Data on 1483 HA patients included 196 Inhibitor (25.87% Severe) and 67 Transient.140/196 (71.42%) developed inhibitors within 50 ED. 922/1483 HA patients were prescribed Prophylaxis of which 440 had that as their first treatment. 3.63% (16/440) developed inhibitor (5 had major surgery/accident/trauma while on prophylaxis, 6 non‐compliant, 3 paediatric patients and 2 with high risk genetic mutation). Results characterised severity, genotype, age at first exposure, ED and prophylaxis as fixed effect variants whereas intensive treatment therapy and non‐compliance as random effect variants of the multivariate risk model.

Conclusions: Mixed modeling can be used to predict cumulative risk of inhibitor development among HA patients.

PB201: Real‐world Bleeding Frequency in Inhibitor Patients on Prophylaxis with APCC: Data Readout of the “FEIBA Global Outcomes Study (FEIBA GO)”

C. Escuriola Ettingshausen 1 ; J. Windyga 2 ; P.A. Holme 3 ; C. Hermans 4 ; C. Negrier 5 ; A.R. Cid 6 ; S. Rangarajan 7 ; G.R. Chiu 8 ; V. Cano 9 ; R. Crea 10

1 HZRM, Haemophilia Centre Rhine‐Main, Frankfurt‐Möerfelden, Germany, 2 Institute of Hematology and Transfusion Medicine, Warsaw, Poland, 3 Oslo University Hospital and Institute of Clinical Medicine, University of Oslo, Oslo, Norway, 4 Saint‐Luc University Hospital, Brussels, Belgium, 5 Louis Pradel Cardiology Hospital, University Claude Bernard, Lyon, France, 6 Unidad Hemostasia y Trombosis, Hospital Universitario y Politécnico La Fe, València, Spain, 7 University Hospital Southampton NHS Foundation Trust, Hampshire, United Kingdom, 8 Shire, Cambridge, United States, 9 Shire, Zug, Switzerland, 10 Shire, Vienna, Austria

Background: The “FEIBA GO” study will capture long‐term efficacy, safety, and quality of life (QoL) outcomes in patients (pts) with hemophilia (hem) and inhibitors treated with FEIBA (activated prothrombin complex concentrates [APCC]) in routine clinical practice.

Aims: To describe the hemostatic effectiveness of APCC during prophylaxis and on demand, including in pts on immune‐tolerance induction. Secondary objectives include joint functionality outcomes, safety, health‐related QoL (HRQoL), activity level, hem‐associated pain, and healthcare resources used.

Methods: This is a prospective, observational, multicenter, cohort study in ˜55 pts with hem A or B and high‐titer inhibitors treated with APCC before enrollment. Treatment regimens are at the discretion of attending physicians according to routine clinical practice. The observation period per pt will be 4 years.

Results: A data read‐out was carried out on Oct 23, 2017 on pts with severe hem A (n=48) or B (n=1) and inhibitors from 11 countries. At screening, median age was 18 year (range, 2‐71), and 83% and 10% were white and black/African American, respectively. The mean number of target joints (Table 1) was 1.2 (range, 0‐10), and 32 pts were on prophylaxis. For 29 prophylaxis pts for whom first factor VIII inhibitor detection was available, median titer was 14.0 BU (range, 1‐150). Among 25 prophylaxis pts evaluated for efficacy, annualized bleeding rates (ABRs) and annualized joint bleeding rates (AJBRs) were reported (Table 2), with median rates of 3.6 (range, 0‐50) and 1.2 (range, 0‐17), respectively.

TABLE 1 Number of Target Joints at Screening in Patients on Prophylaxis (n=32) Sum of Target Joints No. of Patients (%) 0 21 (65.6) 1 3 (9.4) 2 4 (12.5) >2 4 (12.5) TABLE 2 ABR and AJBR in Patients on Prophylaxis (n=25) Bleeding Rate (Categorized) No. of Patients (%) ABR AJBR 0 5 (20) 9 (36) 0‐<2 8 (32) 14 (56) 0‐<3 11 (44) 18 (72) ≥3 14 (56) 7 (28)

Conclusions: This study provides data on long‐term prophylaxis in the real‐world setting by assessing efficacy, HRQoL, and safety of APCC. Preliminary results demonstrate prevention of joint bleeds with FEIBA prophylaxis, which is consistent with that reported in pts without inhibitors on replacement prophylaxis (Khair K, et al. Haemophilia . 2018;24:85‐96).

Funding: This study was funded by the sponsor, Shire International GmbH.

PB202: Addressing Barriers to the Use of Oral Anticoagulant Reversal Agents: A CME Intervention

J. Spyropoulos

Medsacape LLC, New York, United States

Background: Oral anticoagulants (OACs) are associated with increased risk of bleeding. Rapid OAC reversal can be achieved through use of PCC or plasma. While recommendations exist to help physicians choose the most effective reversal strategy, guidelines are not always followed, affecting patient outcomes.

Aims: To determine if an intervention consisting of continuing medical education (CME) followed by a planned change assessment (PCA) could improve performance of physicians related to appropriate use of reversal agents.

Methods: An online CME activity was developed as a 25‐minute roundtable discussion among 4 leading experts. A PCA questionnaire was administered immediately following the activity in the form of intent‐to‐change questions. Completers also indicated potential anticipated barriers that may inhibit implementation of changes. Survey completers were recontacted to complete a follow‐up assessment approximately 8 weeks later to assess self‐reported actual changes in clinical practice, along with barriers encountered to making changes in the real‐world setting.

Results: A total of 750 physicians, completed the PCA, and 711 (95%) indicated intended changes. Among those who intended to make changes, a total of 2198 changes were planned (Figure 1).

FIGURE 1 Summary of planned changes

When recontacted for the follow‐up survey, 19 learners completed the survey. Of those, 16 (84%) reported that they had made 61 changes, an average of 3.8 changes in practice. The most common practice changes made included improved ability to prescribe OAC therapy (62%) and selecting bleeding management strategies based on the severity of bleeding and patient characteristics (62%). The most common barriers to implementing changes included financial constraints (22%), difficulty to implement system changes (14%), and lack of availability of PCC (14%).

Conclusions: This assessment demonstrated the effectiveness of CME combined with a PCA intervention and allowed for assessment of quantitative data for explanation of practice changes and barriers to best practices.

PB203: Successful Eradication of Factor FVIII Inhibitor by Cyclophosphamide Treatment in a Patient with Acquired Haemophilia A ‐ A Case Report

S. Margetic 1 ; D. Carzavec 2 ; N. Vrkic 1

1 Sestre Milosrdnice University Hospital Center, Department of Clinical Chemistry, Zagreb, Croatia, 2 Sestre Milosrdnice University Hospital Center, Department of Internal Medicine, Zagreb, Croatia

Background: Acquired hemophilia A (AHA) is a rare autoimmune bleeding disorder caused by autoantibodies to FVIII. It can be idiopathic (no underlying disease) or associated with autoimmune disorders, malignancy, drugs or infections. AHA can cause life‐threatening bleeding and requires immunosuppressive treatment for inhibitor eradication.

Aims: To present a case of a 59‐year old man in which successful eradication of FVIII inhibitor was achieved soon after adding cyclophosphamide to the first‐line prednisolone therapy.

Methods: Laboratory haemostasis testing included PT, APTT, TT, fibrinogen, lupus anticoagulant and activities of factors VIII, IX, XI, and XII using commercially available coagulation methods on BCSXP analyzer (Siemens, Germany). The inhibitor modified Bethesda assay (Nijmegen) was used to quantify FVIII inhibitor.

Results: A 59‐year‐old male patient, 3 yrs ago diagnosed and treated for gastric MALT lymphoma with chemotherapy and radiotherapy, came to the Emergency Department (ED) due to large haematoma on the right hip. The coagulation profile revealed prolonged APTT of 59s, reduced FVIII activity of 7% and presence of FVIII inhibitor at 4.0 Bethesda Units (BU)/mL with normal results for all other coagulation tests. Initial IST included prednisolone, but due to inadequate therapeutic response cyclophosphamide was also introduced. Both clinical and laboratory improvements were evident after 2 weeks (APTT=37‐39s). Once discharged, the patient continued combined therapy with cyclophosphamide and prednisolone. One month after admittance in ED factor VIII activity rose to 82% and FVIII inhibitors completely disappeared until today, ten months later (APTT=28‐30s).

Conclusions: Cyclophosphamide use was superior to that of prednisolone alone in terms of FVIII inhibitor eradication in our patient thus supporting the recent literature data that effective first‐line treatment for eradication of FVIII inhibitors is the combination of cyclophosphamide and steroids.

PB204: Clinical Description and Laboratory Characterization of Heparin‐like Substance Causing Bleeding

L. Hensch; V. Kostousov; K. Bruzdoski; M. Losos; S.K. Hui; J. Teruya

Texas Children's Hospital and Baylor College of Medicine, Pathology & Immunology, Division of Transfusion Medicine & Coagulation, Houston, United States

Background: Heparin‐like substance (HLS) has been described in the setting of liver disease associated with infection, transplant, and metastasis. HLS has been attributed to circulating glycosaminoglycans, predominately heparan and dermatan sulfate.

Aims: The aim of this study is to report a case of HLS in a post‐transplant patient and further characterize the effects of HLS on laboratory testing.

Methods: Herein we describe the case of a 10 year‐old female with liver failure requiring transplant. Initial results for this patient showed coagulopathy due to liver disease without HLS. Two weeks after liver transplant, the patient began to bleed from catheter insertion sites, mouth, and nares. HLS was suspected based on laboratory data. Protamine sulfate (PS) bolus resulted in only transient slowing of bleeding and little correction of these values. Bleeding resolved only after plasma exchange (PE). Clinical data, laboratory results, and citrated plasma samples were collected with institutional review board approval. In vitro PS neutralization of thrombin time (TT), anti‐Xa, and anti‐IIa were performed.

Results: Laboratory data demonstrated a 2.5‐3 times difference between the activated partial thromboplastin time (PTT) and PTT treated with heparinase despite no source of exogenous heparin. The patient repeatedly had measurable anti‐Xa with TT >120 seconds. Clotting times on INTEM and EXTEM were prolonged with partial correction on HEPTEM. Data at the onset of bleeding, following PS, and after PE are reported in Table 1. PS neutralization, performed on plasma removed by PE, is presented in Table 2.

TABLE 1 Coagulation Test (Reference Range) Bleeding Onset After Protamine Sulfate Bolus Infusion After Plasma Exchange Prothrombin Time (11.4‐15.8 second) 14.4 second 13.5 second 13.3 second Activated Partial Thromboplastin Time (PTT) (24.8‐34.4 second) 136.1 second 118.4 second 46.0 second PTT with Heparinase (24.8‐34.4 second) 47.9 second 45.4 second 33.3 second Thrombin Time (15.0‐19.0 second) >120.0 second >120.0 second 69.5 second Anti‐Xa activity 0.40 U/mL 0.37 U/mL <0.10 U/mL Anti‐IIa activity 0.00 U/mL 0.01 U/mL nd INTEM Clotting Time (122‐208 second) 305 second 358 second 218 second HEPTEM Clotting Time (122‐208 second) 278 second 283 second 193 second EXTEM Clotting Time (43‐82 second) 131 second 99 second 61 second

TABLE 2 Specimen Thrombin Time Anti‐Xa activity Anti‐IIa activity Normal Pooled Plasma + Unfractionated Heparin 0.4 U/mL >120.0 second 0.35 U/mL 0.44 U/mL + Heparin 0.4 U/mL + Protamine 5 μg/mL 16.7 second 0.04 U/mL 0.00 U/mL Patient Plasma Baseline 50.7 second 0.13 U/mL 0.00 U/mL + Protamine 5 μg/mL 42.3 second 0.13 U/mL 0.01 U/mL + Protamine 20 μg/mL 29.2 second 0.10 U/mL 0.00 U/mL

Conclusions: HLS after liver transplant contributed to this patient's coagulopathy and caused bleeding. This HLS appears to have primarily anti‐Xa activity. Unexplained prolongation of the PTT with prolonged TT of unknown etiology is suspicious for HLS. Testing performed with heparinase or PS may result in partial correction of these abnormalities and suggests this diagnosis.

PB206: Skeletal and Cardiac Muscle Myosin Exert Procoagulant and Antifibrinolytic Effects

J. Coleman 1 ; H. Deguchi 2 ; J. Zilberman‐Rudenko 2,3 ; J. Samuels 1 ; C. Silliman 1,4 ; A. Banerjee 1 ; J. Griffin 2 ; E. Moore 5

1 University of Colorado Denver, Surgery, Aurora, United States, 2 The Scripps Research Institute, Molecular Medicine, La Jolla, United States, 3 Oregon Health and Science University, School of Medicine and Division of Hematology Biomedical Engineering, Portland, United States, 4 University of Colorado Denver, Pediatrics, Aurora, United States, 5 Denver Health Medical Center, Surgery, Denver, United States

Background: Trauma induced coagulopathy and fibrinolytic shutdown is prevalent in severely injured patients, but the mechanism of lysis inhibition remains unclear. Myosin, released from tissue injury, has been suggested to play a role in prompting thrombin generation and attenuating fibrinolysis.

Aims: To examine the effects of skeletal and cardiac muscle myosins on coagulation and fibrinolysis in vitro using Thrombelastography (TEG).

Methods: Whole blood was collected from healthy volunteers (n=8) and citrated native TEGs were performed to evaluate the global coagulation response in the presence of skeletal or cardiac muscle myosin (100 nM) by measuring reaction (R) time (minutes), clot angle ( o ), maximum amplitude (MA, mm) and percent of lysis 30 and 60 minutes after MA (LY30 and LY60 respectively). This was performed in the presence or absence of 50 ng/mL of tissue plasminogen activator (tPA) and compared with a control buffer. TEG measurements were compared using ANOVA.

Results: Cardiac and skeletal muscle myosins decreased R, from 10.8 min to 8.0 (P < 0.0001) and 6.9 (P 0.0007) min respectively. In the presence of tPA, both cardiac and skeletal myosins shortened R (from 11.1 min to 8.62 (P 0.0245) and 7.75 (P 0.0027) min, respectively) and cardiac myosin increased MA (from 50.0 mm to 57.7 mm, P 0.0170). Cardiac myosin decreased lysis at 60 minutes (from 5.2% to 2.9%, P 0.0006) and both cardiac and skeletal myosins produced a tendency towards lower percent clot lysis in the presence of tPA. The lysis changes induced by myosin varied among donors.

FIGURE 1 Changes in R time with addition of myosin

FIGURE 2 Changes in LY60 with addition of myosin

Conclusions: Cardiac and skeletal muscle myosins have procoagulant effects. Cardiac myosin showed an antifibrinolytic effect, which is mitigated in presence of tPA. The lysis changes in skeletal myosin suggested a differential reactivity among individuals which may relate to subtypes of fibrinolysis seen in trauma. These results suggest circulating myosins are procoagulant and antifibrinolytic in whole blood.

PB208: Recombinant VWF D'D3‐albumin Fusion Protein Variants with Enhanced Affinity for Coagulation Factor VIII, Increase the Efficiency of FcRn‐mediated Recycling of Co‐administered FVIII

J. Chia 1 ; I. Glauser 1 ; G. Bass 1 ; A. Andrews 1 ; T. Weimer 2 ; M. Wilson 1 ; S. Dower 1 ; A. Verhagen 1

1 CSL Limited, Research, Melbourne, Australia, 2 CSL Behring, Research, Marburg, Germany

Background: A recombinant VWF D'D3 albumin fusion protein (rD'D3‐FP) has been developed to extend the half‐life of co‐administered coagulation factor VIII (FVIII), for the treatment of haemophilia A. Using in vitro cellular systems, we have previously demonstrated that rD'D3‐FP can mediate recycling of associated rVIII‐SingleChain through engagement of the neonatal Fc receptor (FcRn). Interestingly, however, FVIII recycling was less efficient than that of rD'D3‐FP indicating some dissociation under acidic endosomal conditions and subsequent lysosomal degradation (Chia et al. ISTH 2017 0C10.4). This in turn could also explain in vivo observations, where the half‐life of rVIII‐SingleChain, although increased, remained less than that of co‐administered rD'D3‐FP (Pestel et al. ISTH 2017 OC10.5). Exchange of rD'D3‐FP with endogenous VWF within plasma is also likely to contribute to the shorter half‐life in vivo . To overcome these issues, novel rD'D3‐FP variants with reduced dissociation for FVIII under both neutral and acidic conditions were generated (Andrews et al. ISTH 2017 PB1112).

Aims: To investigate if rD'D3‐FP variants with improved FVIII binding can increase the recycling efficiency of rVIII‐SingleChain, via the intracellular FcRn‐mediated pathway.

Methods: Human 293 Freestyle ™ cells stably expressing human FcRna chain and b2M were simultaneously pulsed with rD'D3‐FP variants and AF488‐labelled rVIII‐SingleChain to facilitate FcRn‐mediated uptake of rD'D3‐FP/rVIII‐SingleChain complex. The intracellular fate of internalised rVIII‐SingleChain‐AF488 was subsequently tracked by immunofluorescent confocal microscopy.

Results: rD'D3‐FP variants with improved FVIII binding properties increase the recycling efficiency of rVIII‐SingleChain via FcRn in in vitro cellular assays.

Conclusions: Our results support the development of rD'D3‐FP with enhanced FVIII affinity to further improve the half‐life extension of FVIII for the treatment of haemophilia A.

PB209: Improving the FVIII Binding Affinity of a Recombinant VWF D′D3 Albumin Fusion Molecule Translates to Dosing Benefits with respect to the Half‐life Extension of Co‐administered FVIII

S. Pestel 1 ; E. Raquet 1 ; M. Mischnik 1 ; P. Claar 1 ; M. Wilson 2 ; S. Dower 2 ; E. Herzog 1 ; T. Weimer 1 ; A. Andrews 2

1 CSL Behring, Marburg, Germany, 2 CSL Limited, Melbourne, Australia

Background: A recombinant VWF D′D3 albumin fusion protein (rD′D3‐FP) has been developed to extend the half life of co‐administered coagulation factor VIII (FVIII) for the treatment of haemophilia A. Pharmacokinetic (PK) studies in rats, rabbits, cynomolgus monkeys and FVIII ko mice have demonstrated a significant improvement in rVIII‐SingleChain PK parameters upon co‐administration with rD′D3‐FP. Interestingly, the half‐life of rVIII‐SingleChain, although increased, remained less than that of the co‐administered rD'D3‐FP ( Pestel et al. ISTH 2017 OC10.5 ). It is hypothesised that FVIII exchange between rD'D3‐FP and endogenous VWF in circulation combined with a reduced recycling efficiency for FVIII contribute to this shorter half‐life. To overcome these issues, novel rD'D3‐FP variants with reduced dissociation for FVIII under both neutral and acidic conditions have been generated ( Andrews et al. ISTH 2017 PB1112 ).

Aims: To investigate if rD'D3‐FP variants with enhanced FVIII binding are more effective at improving the PK of co‐administered FVIII than wild‐type rD'D3‐FP.

Methods: rD'D3‐FP (wild‐type and variants) were administered intravenously (i.v.) at a dose range of 0.086‐10 mg/kg, alone or in combination with rVIII‐SingleChain (200 IU/kg). Plasma PK of rD'D3‐FP molecules and FVIII (antigen and/or activity levels) was monitored for up to 96h.

Results: All rD'D3‐FP species displayed comparable PK. The rD'D3‐FP variants with improved FVIII binding properties showed enhanced potency, with the dose levels required for equivalent FVIII half‐life extension, correlating to the off‐rate improvements. At potential clinical doses, the rD'D3‐FP variants provide benefits with respect to dose levels and half‐life extension of co‐administered FVIII.

Conclusions: Our results support the development of an enhanced FVIII affinity variant of rD'D3‐FP to further improve the treatment of hemophilia A.

PB210: Platelet‐nanocapsule Hybrids for On‐demand, Targeted Delivery of Factor VIII in Hemophilia A Mice

C.E. Hansen 1,2 ; W.H. Baldwin 1 ; G. Batsuli 1 ; S.L. Meeks 1 ; W.A. Lam 1,2

1 Children's Healthcare of Atlanta/Emory Univeristy, Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Atlanta, United States, 2 Georgia Institute of Technology and Emory University, Wallace H. Coulter Department of Biomedical Engineering, Atlanta, United States

Background: Treating bleeds in hemophilia A patients with inhibitors when prophylactic therapies are onboard is challenging due to potential thrombotic reactions. To address this need, we developed a targeted drug delivery technology using polyelectrolyte (PEM) nanocapsules to encapsulate and “cloak” factor VIII (fVIII) from the patient's inhibitors. Upon intravenous injection, the patient's own platelets adhere and hybridize to the PEM nanocapsule via surface‐displayed fibrinogen (Figure 1A). Platelet‐nanocapsule hybrids (plt‐HyPEMs) circulate inertly until reaching injury sites where agonists activate platelets (Figure 1B, C). Platelet contraction then physically ruptures the nanocapsule to release fVIII, enabling coagulation before fVIII inhibition (Figs. 1D, 2A). Recently published in vitro results using this technology showed a 3.8x increase in fibrin formation and 18‐minute reduction in clotting time in the presence of inhibitors (Hansen et al. ACS Nano 2017).

FIGURE 1 Schematic of platelet‐nanocapsule A) hybrid formation, B) circulation, C) targeting, and platelet contraction‐mediated delivery of factor VIII

Aims: This work characterizes in vivo efficacy and safety in a murine hemophilia model.

Methods: E16 hemophilia A mice were used for in vivo investigations. Survival rate was determined over weekly injections of PEM nanocapsules. The total blood lost was determined after tail transection for efficacy comparison.

Results: No significant difference exists between survival rates at high dose, low dose, and control conditions over 7 weeks exposure (Figure 2B). The total blood lost after tail transection in the 60 U/kg fVIII loaded plt‐HyPEM condition was significantly less than control or systemic fVIII at the same dose (Figure 2C). Moreover, the amount of blood lost was similar to a systemic fVIII concentration at a 3x larger dose (180 U/kg fVIII), indicating comparable efficacy with 66% less fVIII.

FIGURE 2 A) Micrograph of platelet contraction release. In vivo data show B) no significant difference in survival and C) enhanced efficacy in clotting

Conclusions: Expanded safety and efficacy in vivo studies in the presence of inhibitors is currently ongoing. Our data demonstrates the profound therapeutic potential of this hybrid biotechnology as it enables blood clot formation at a third the dose of systemic fVIII.

PB211: Annualized Bleeding Rates, the Current Favorite Clinical Endpoint in Hemophilia Trials: Pitfalls and Caveats

C. Keipert ; M. Müller‐Olling; F. Gauly; C. Arras‐Reiter; A. Hilger

Paul‐Ehrlich‐Institut, Hematology and Transfusion Medicine, Langen, Germany

Background: The times that hemophilia exclusively could be treated with coagulation factor substitution are slowly but surely coming to an end. Various new treatment options are in development. Besides gene therapy, other non‐replacement therapies such as RNAi therapeutics, bispecific monoclonal antibodies or anti‐TFPI antibodies are on their way to market. To demonstrate efficacy of treatment, Annual Bleeding Rates (ABR), are often used as primary or secondary endpoint in clinical trials.

Aims: To investigate whether the use of ABR as endpoint in various clinical trials is based on same definitions and assumptions and to provide guidance on the minimum standards that should be used in clinical trials performed for regulatory purposes.

Methods: Definitions of ABR and bleeding episodes from clinical trial protocols and literature have been reviewed and compared. In addition, data from the confidential PEI database have been assessed e.g. regarding factor concentrate administration, treatment regimen and reason for treatment. Anonymized data from >1300 patients receiving more than 85.000 factor concentrate infusions investigated in 31 clinical trials have been analyzed.

Results: The definition of ABR, in particular the classification and distinction of bleeding events, used in clinical trials and in the literature varies considerably, which compromises a direct comparison of efficacy following different treatment scenarios and products. The PEI database analyses supports conclusion on minimum standards to be considered for definition of bleeding rates used as clinical endpoints.

Conclusions: Clear definition of parameters and characteristics when using ABR as measure for the efficacy of treatment, is absolutely necessary to enable conclusions on different treatment regimens and products including non‐replacement therapies.

PB212: Presence of Plasma Platelet Autoantibodies and Response to the Spleen Tyrosine Kinase (Syk) Inhibitor Fostamatinib in Adult Patients with Immune Thrombocytopenia (ITP) ‐ Exploratory Analyses from Phase 3 Studies

J. Bussel 1 ; V. Markovtsov 2 ; E. Masuda 2 ; B. Cadieux 2 ; S. Yi 2 ; C. Young 2 ; A.‐M. Duliege 2

1 Weill Cornell Medicine, New York, United States, 2 Rigel Pharmaceuticals Inc., South San Francisco, United States

Background: The main etiologies of ITP include inhibition of platelet production and autoantibody‐directed platelet destruction via activated Fc receptors that signal through Syk. Inhibition of Syk with fostamatinib produced a stable response (platelet count ≥50,000/μL at ≥ 4 of 6 bi‐weekly measurements without rescue therapy) in 18/101 (18%) adult ITP patients on fostamatinib compared with 1/49 (2%) on placebo (P=0.0003) in 2 multicenter, double‐blind, placebo‐controlled studies. Collectively, a platelet response (≥ 50,000/μL) was seen in 43% of adult ITP patients with a median disease duration of 8.5 years.

Aims: This exploratory analysis examined the relationship between clinical responses to fostamatinib and the presence of platelet autoantibodies.

Methods: Plasma sampled through informed consent from 60 patients 2 weeks after the first dose on fostamatinib in the phase 3 studies was analyzed for anti‐platelet autoantibodies via Luminex beads‐based PakLx assay and FACS‐based indirect platelet immunofluorescence test (iPIFT) using platelets from healthy volunteers.

Results: Of 60 patient samples, 28 (47%) were positive for one or more anti‐platelet autoantibodies. Stable responses to fostamatinib were reported in 22% (13/60) of the patients tested. Of 28 patients positive for autoantibodies, 10 (36%) had stable responses. Of 32 patients negative for autoantibodies, 3 (9%) had stable responses.

Conclusions: A potential association between the presence of anti‐platelet autoantibodies and the response to fostamatinib was observed. Findings from this exploratory analysis support the hypothesis that fostamatinib prevents platelet loss through inhibition of autoantibody‐directed platelet destruction in ITP. Further studies are ongoing.

PB214: Tranexamic Acid Reduces Post‐surgical Infection Rates via Direct Immune Cell Modulation

D.F. Draxler 1 ; K. Yep 2 ; G. Hanafi 1 ; A. Winton 2 ; M. Daglas 1 ; H. Ho 1 ; M. Sashindranath 1 ; L.M. Wutzlhofer 2 ; A. Forbes 3 ; I. Goncalves 1 ; H. Tran 1 ; S. Wallace 2 ; M. Plebanski 4 ; P.S. Myles 2 ; R.L. Medcalf 1

1 Monash University, Australian Centre for Blood Diseases, Melbourne, Australia, 2 Alfred Hospital and Monash University, Department of Anaesthesia and Perioperative Medicine, Melbourne, Australia, 3 Monash University, School of Public Health and Preventative Medicine, Melbourne, Australia, 4 Monash University, Department of Immunology and Pathology, Melbourne, Australia

Background: Plasmin is the effector protease of the fibrinolytic system which mediates clot degradation. However, plasmin has also been well‐characterised as a pro‐inflammatory mediator and more recently also as an immunosuppressive protein. Tranexamic acid (TXA) inhibits plasmin formation and thereby effectively reduces blood loss in various indications, including trauma.

Aims: The aim of this study was to investigate the effect of TXA on the cellular immune response in healthy volunteers and in patients undergoing cardiac surgery.

Methods: 10 healthy volunteers received 1 gram TXA orally and blood was drawn before and at various time points after TXA intake. Blood samples were also obtained from 41 placebo or TXA‐treated cardiac surgery patients enrolled in the placebo‐controlled ATACAS trial (Myles et al, 2017) at various time points before and after surgery. The cellular immune response was evaluated with flow cytometry and plasma cytokines were assessed by multiplex ELISA. For the cardiac surgery trial, we also evaluated 30‐day postsurgical infection rates.

Results: TXA induced profound changes in the cellular immune profile of healthy volunteers including enhanced activation of antigen‐presenting cells as well as an increase in CD4+ and CD8+ T cells, consistent with heightened immune activation. Moreover, TXA significantly reduced plasma levels of pro‐inflammatory cytokines. Consistent with this finding TXA also augmented the cellular immune response after cardiac surgery, and most strikingly, significantly reduced surgical site infection rates. TXA also reduced overall infection rates, unrelated to its effects on blood loss and transfusion requirement in non‐diabetic patients. Importantly, in diabetics, who are known for reduced fibrinolytic baseline activity TXA had essentially no impact on infection rates.

Conclusions: In conclusion, we describe for the first time profound immunomodulatory effects of TXA, consistent with improved immune capacity, which results in a reduction of postoperative infection rates.

PB215: ROTEM ® or Conventional Hemostasis Test: Experience in Cardiac Surgery Setting

J. Valaize 1 ; A. Roisné 2 ; A. Mansour 2 ; J.‐P. Verhoye 3 ; T. Fest 1 ; P. Gueret 1 ; I. Gouin‐Thibault 1 ; E. Flecher 3 ; F. Nedelec‐Gac 1

1 CHU Pontchaillou, Laboratoire d'Hématologie Hémostase, Rennes, France, 2 CHU Pontchaillou, Service d'Anesthésie‐Réanimation, Rennes, France, 3 CHU Pontchaillou, Service de Chirurgie Thoracique et Cardiovasculaire, Rennes, France

Background: Postoperative hemorrhage is a frequent complication in cardiac surgery with significant mortality. Blood transfusion algorithms based on thromboelastometry ROTEM ® have been proposed to standardize practices and save blood products. In our University Hospital, a specific organization allowing a very short turn‐around‐time for “vital emergency” samples has been recently implemented.

Aims: To assess the value of ROTEM ® in cardiac surgery setting compared to routine hemostasis tests provided through the specific organization.

Methods: We performed a monocentric, observational study on a cohort of cardiac surgery adult patients benefiting from ROTEM ® delta (EXTEM/INTEM/FIBTEM/HEPTEM) and conventional coagulation tests such as PT, aPTT, fibrinogen, thrombin time, UFH anti‐Xa activity, Factors II/V/VII/X and platelet count. Samples were sent by pneumatics and the tests were all performed in the laboratory. Results were visible live on remote screen. We compared the turn‐around‐time for ROTEM ® and conventional tests results and tested the agreement between ROTEM ® data (CTEXTEM, MCFFIBTEM and combination of A10EXTEM / A10HEPTEM / A10FIBTEM) and conventional test values (PT, fibrinogen, platelets).

Results: We included 77 patients in 2016; the median turn‐around‐time for ROTEM ® (A10 of the last run) was 23 min [Q1‐18 minute; Q3‐28 minute] and 27 min for conventional tests [Q1‐20 minute; Q3‐35 minute]. The comparison between ROTEM ® and conventional tests is displayed table 1.

TABLE 1 ROTEM and conventional hemostasis test values according to local cut‐off for transfusion CT EXTEM MCF FIBTEM A10 EXTEM<40s and A10 FIBTEM<40s and A10 HEPTEM >10s ≤38 second >38 second ≥10 mm <10mm no yes PT ratio ≤1.46* 42/69 (61%) 8/69 (12%) ‐ ‐ ‐ ‐ PT ratio >1.46* 10/69 (14%) 9/69 (13%) ‐ ‐ ‐ ‐ Fibrinogen g/L ≥2* ‐ ‐ 25/69 (36%) 10/69 (14%) ‐ ‐ Fibrinogen g/L <2* ‐ ‐ 21/69 (30%) 13/69 (19%) ‐ ‐ Platelet count G/L ≥100* ‐ ‐ ‐ ‐ 44/60 (73%) 11/60 (18%) Platelet count G/L <100* ‐ ‐ ‐ ‐ 1/60 (2%) 4/60 (7%) *local cut‐off values for transfusion.

Conclusions: In our experience, the specific organization for management of “vital emergency” samples implemented in the Hospital allows a turn‐around‐time for conventional coagulation tests comparable to that for the ROTEM ® . In addition, most of results are consistent between the two methods. The study of ROTEM ® impact on transfusion requirements is ongoing. These data raise the question of the real benefit of ROTEM ® over a specific organization.

PB216: A New, Hand‐held, Multifunctional Coagulation Laboratory for Perioperative Monitoring

S. Nadkarni 1 ; D. Tshikudi 1 ; A. Wirth 1 ; E. van Cott 2

1 Harvard Medical School, Massachusetts General Hospital, Wellman Center for Photomedicine, Boston, United States, 2 Harvard Medical School, Massachusetts General Hospital, Department of Pathology, Boston, United States

Background: Delays in identifying hemorrhage are life‐threatening, underscoring the need for comprehensive coagulation profiling at the point‐of‐care. We present a novel sensor termed, iCoagLab that rapidly measures several coagulation metrics: prothrombin time (PT), activated clotting time (ACT), clot polymerization rate (a‐angle), clot stiffness (MA) and fibrinolysis (LY) with a 40μL blood drop. Via comprehensive coagulation profiling using iCoagLab, we demonstrate that patients with elevated bleeding risk can be accurately identified.

Aims: To evaluate the accuracy and sensitivity of iCoagLab for comprehensive coagulation assessment and predicting bleeding.

Methods: A blood drop from 500 patients was loaded into the iCoagLab, which measured changes in clot viscoelasticity from light scattering profiles to derive the above coagulation metrics. Next, iCoagLab testing was conducted in surgical patients on cardiopulmonary bypass (CPB) and coagulation was monitored during heparin administration and reversal. In all cases, linear regression was used to compare iCoagLab metrics with reference values obtained from conventional laboratory testing (CCT), Thromboelastography (TEG) and iStat Kaolin‐ACT. Bleeding risk in patients was confirmed based on elevated aPTT with a normal lupus anticoagulant test, low fibrinogen level and platelet count, or clinical notes confirming bleeding. iCoagLab's accuracy in identifying bleeding risk was calculated using multivariate analysis.

Results: A strong correlation was observed with reference values: PT (R=0.92, P<0.001), ACT (R=0.88, P<0.0001), R‐time (R=0.81, P<0.001), α‐angle (R=0.60, P<0.01) confirming iCoagLab′s accuracy in quantifying multiple coagulation metrics with a drop of blood. Furthermore, iCoagLab identified patients with an elevated bleeding risk with 89% prediction accuracy.

Conclusions: By enabling rapid and comprehensive coagulation profiling at the bedside, the iCoagLab will likely advance the capability to identify patients with an elevated risk for hemorrhage.

PB217: Expansion of Surgery Experiences in Children, Adolescents and Adults with the Extended Half‐life Molecule N8‐GP

A. Tosetto 1 ; A. Neff 2 ; S.R. Lentz 3 ; E. Santagostino 4 ; L. Nemes 5 ; J. Sathar 6 ; K. Meijer 7 ; P. Chowdary 8 ; A. Landorph 9 ; M. Dutkiewicz 9 ; K. Hampton 10

1 San Bortolo Hospital, Hematology Department, Vicenza, Italy, 2 Cleveland Clinic, Department of Hematology/Oncology, Cleveland, United States, 3 University of Iowa Carver College of Medicine, Division of Hematology, Oncology, and Blood & Marrow Transplantation, Department of Internal Medicine, Iowa City, United States, 4 IRCCS Cà Granda Foundation, Maggiore Hospital Policlinic, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan, Italy, 5 Medical Centre of the Hungarian Defence Forces, National Haemophilia Centre and Haemostasis Department, Budapest, Hungary, 6 Ampang Hospital, Department of Haematology, Selangor, Malaysia, 7 University Medical Centre Groningen, University of Groningen, Department of Haematology, Groningen, the Netherlands, 8 Royal Free Hospital, The Katherine Dormandy Haemophilia and Thrombosis Centre, London, United Kingdom, 9 Novo Nordisk A/S, Bagsværd, Denmark, 10 University of Sheffield, Department of Cardiovascular Science, Sheffield, United Kingdom

Background: Standard factor VIII (FVIII) treatment for surgeries involves continuous infusion or frequent injections to maintain adequate FVIII plasma levels for haemostasis. N8‐GP (turoctocog alfa pegol) is an extended half‐life glycoPEGylated recombinant FVIII being developed for prophylaxis and the treatment of bleeds in patients with haemophilia A. N8‐GP provided effective haemostasis for patients who underwent major surgery in the ongoing pathfinder ™ 3 trial.

Aims: Evaluate the haemostatic effect of N8‐GP in a planned interim analysis of pathfinder ™ 3 to include new surgeries, and in children who underwent minor surgery during the main and extension phase of the pathfinder ™ 5 trial.

Methods: In pathfinder ™ 3, adult/adolescent patients (≥12 years) with haemophilia A (< 1% FVIII) received perioperative N8‐GP treatment (a planned preoperative dose to achieve FVIII activity levels >80%) for major surgery and were assessed on the day of surgery, daily for Days 1‐6 and once between Days 7‐14. N8‐GP haemostatic efficacy in pathfinder ™ 3 was assessed using a four‐point scale (‘excellent’, ‘good’, ‘moderate’, ‘none’). Minor surgery (using 20‐75 IU/kg N8‐GP [at investigator's discretion]) is permitted in pathfinder ™ 5, an ongoing phase 3 trial evaluating N8‐GP prophylaxis in children (0‐11 years).

Results: 45 major surgeries (41 orthopaedic [15 joint replacements, 9 arthroscopic interventions and 17 other orthopaedic interventions], 4 non‐orthopaedic) were performed in 33 adult/adolescent patients. Of these 45 major surgeries, 27 were new cases not previously reported. Overall success rate (excellent/good) of N8‐GP during major surgery was 95.6%; 2 surgeries (4.4%) were rated as ‘moderate’. Twenty‐two children underwent a minor surgical procedure without complications.

Conclusions: These data confirm that N8‐GP provides effective haemostatic control during surgery in patients with severe haemophilia A.

PB218: Monitoring Anticoagulation and Hemostasis in Cardiac Surgical Patients with a Drop of Whole Blood

D.M. Tshikudi 1,2 ; A.G. Wirth 1,2 ; M.N. Andrawes 1 ; S.K. Nadkarni 1,2,3

1 Massachusetts General Hospital, Boston, United States, 2 Wellman Center for Photomedicine, Boston, United States, 3 Harvard Medical School, Boston, United States

Background: Activated clotting time (ACT) monitors anticoagulation during cardiac surgery with cardiopulmonary bypass (CPB). However, ACT correlates poorly with heparin concentration and lacks information on clot polymerization and strength. This underscores the need for reliable and rapid methods to monitor anticoagulation and global hemostasis perioperatively. We present a new optical sensor (OS) that simultaneously quantifies several metrics, ACT, clot polymerization rate (α‐angle) and clot strength (MA) from light intensity fluctuations measured in 25μL of blood.

Aims: The accuracy and measurement sensitivity of OS was evaluated in measuring anticoagulation in response to heparin and reversal in volunteers and in patients during CPB.

Methods: Citrated blood samples from 18 volunteers spiked with increased concentrations of heparin (0.2‐5USP/mL) and blood samples from 15 patients undergoing CPB collected at 6 time‐points during surgery were measured using OS. Coagulation metrics, including, reaction time (R), ACT, α‐angle and MA, were derived and compared with corresponding results obtained from standard thromboelastography (TEG), ISTAT‐kaolin‐ACT and Hepcon HMS Plus instruments.

Results: Heparin treatment significantly prolonged the R and ACT values measured by the OS which correlated closely with TEG (r=0.83‐0.95, P<0.0001) and ISTAT‐ACT (r=0.74‐0.87, P<0.0001). Both the OS and TEG showed a decrease in MA at high heparin concentration (P<0.01). In cardiac surgical patients, the OS parameters, R and ACT, correlated better with Hepcon (r=0.71‐0.76 P<0.0001) than to TEG parameters (r=0.63‐0.68 P<0.0001). R and ACT measured by OS and TEG highly correlated (r=0.83‐0.89, P<0.0001). The MA and α‐angle were also significantly modulated throughout the surgery (P<0.05‐0.0001).

Conclusions: The OS rapidly and accurately measures anticoagulation and protamine reversal in patients during CPB in 25μL of blood, likely opening the unique opportunity for multifunctional coagulation monitoring at the point‐of‐care.

PB220: Protocol for a Prospective, Randomized, Controlled Phase 2 Pilot Study Investigating Hemostatic Efficacy and Safety of Fibrinogen Concentrate and Cryoprecipitate in Patients Undergoing Cytoreductive Surgery for Pseudomyxoma Peritonei: The FORMA‐05 Study

A. Roy 1 ; N. Sargant 1 ; S. Rangarajan 1 ; S. Alves 1 ; J. Bell 1 ; S. Stanford 1 ; C. Solomon 2 ; I. Kruzhkova 2 ; S. Knaub 2 ; F. Mohamed 1

1 Basingstoke and North Hampshire Hospital, Basingstoke, United Kingdom, 2 Octapharma AG, Lachen, Switzerland

Background: Clinically‐relevant bleeding treated by transfusion with allogeneic blood products is common in patients with pseudomyoxoma peritonei (PMP) undergoing cytoreductive surgery (CRS). Maintaining intraoperative fibrinogen levels may help control bleeding during surgery. Comparative randomized trials are needed to evaluate different fibrinogen sources.

Aims: To compare overall hemostatic efficacy and safety of a new highly purified fibrinogen concentrate with cryoprecipitate in bleeding patients with acquired fibrinogen deficiency during CRS for PMP.

Methods: FORMA‐05 is a prospective, randomized, single‐center, controlled phase 2 study in up to 55 adults undergoing CRS for PMP who require intraoperative fibrinogen supplementation. Where bleeding risk assessment 60‐90 minutes after the start of surgery predicts intraoperative blood loss ≥2L without targeted fibrinogen replacement, patients will be randomized to receive fibrinogen concentrate (4g Fibryga, Octapharma) or cryoprecipitate (2 pools, 5 units each). Further doses intraoperatively or ≤24 hours postoperatively will be based on thromboelastometry (FIBTEM A20 ≤12 mm). Primary endpoint is a composite of intraoperative (assessed by surgeon/anesthesiologist at end of surgery) and postoperative efficacy (assessed by hematologist 24 hour after surgery) determined by using an objective 4‐point hemostatic efficacy scale, and adjudicated by an independent committee. Secondary endpoints are shown in Table 1. Analyses will be descriptive and exploratory (e.g. sampling statistics, confidence intervals). A planned interim analysis will be performed when ≥10 evaluable patients have been included in each group. TABLE 1 Secondary endpoints for the FORMA‐05 study Plasma fibrinogen levels: After the first infusion/preemptive dose Fibrinogen supplementation: Total intraoperative and postoperative dose, and duration of first and all drug infusions Coagulation profiles: Including fibrinogen, thromboelastometry, prothrombin time, thrombin, Factor VIII:C, VWF:RCo and VWF:Ag Critical‐care outcomes: Including duration of surgery, length of hospital and ICU stay, transfusion requirements (intraoperative and postoperative), postoperative blood loss, reoperation need Drug availability: Time to availability of drug in the operating room Safety: Adverse events and serious adverse events, vital signs

Results: Currently ≥10 evaluable patients have been included in both groups. The interim analysis is in preparation; data will be available in March 2018 and will be presented.

Conclusions: This study will provide an evaluation of the efficacy and safety of Fibryga for fibrinogen supplementation in comparison to cryoprecipitate in patients undergoing CRS for PMP.

PB221: Classification of Surgical Bleeding Situations

R. Kocharian 1 ; J. Koea 2 ; P. Hinoul 1 ; A. Martella 3 ; D. Zeltsman 4 ; J. Hagen 5

1 Ethicon Inc, a Johnson & Johnson Company, Medical Affairs, Somerville, United States, 2 North Shore Hospital, Takapuna, Department of Surgery,, Auckland,, New Zealand, 3 Our Lady of Lourdes Medical Center, Department of Cardiothoracic Surgical Services, Camden, United States, 4 Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Department of Cardiovascular and Thoracic Surgery, Great Neck, United States, 5 Carolinas HealthCare System, Thoracic Surgical Oncology and Thoracic Surgery, Charlotte, United States

Background: Surgical bleeding negatively affects patient outcomes. Conventional methods of hemostasis are usually sufficient but due to the growing number of patients with comorbidities and chronic anticoagulant therapy, adjunctive hemostats are increasingly required.

Aims: There is a need for universal classification of surgical bleeding situations to allow standardization and evaluation of treatment algorithms.

Methods: A comprehensive literature review was conducted (Jan 2005‐ Aug 2015) to identify existing surgical bleeding classifications. Responses from 450 surgeons in 11 specialties identified surgical bleeding situations including the cause, site, and intensity of the bleeding and key behavioral drivers determining the type of adjunctive topical hemostat (stage 1). An additional 50 independent surgeons further refined the 5 most common bleeding situations based on clinical significance (stage 2) assessed using the Chi‐square Automatic Interaction Detection analysis and the Classification and Regression Trees procedure.

Results: Relevant publications were identified in PubMed/Medline (176) and Embase (40). Only 5 publications provided algorithms for topical hemostatic agents based on bleeding type and intensity. The stage 1 survey data determined that surgical site and situation were the most important factors to decide on type of adjunctive hemostat. Stage two categorized the situations as: (1) Continuous oozing: slow oozing that can be accessed directly; (2) Problematic bleeding: brisk surgical bleeding directly accessible, but requiring a sequence of steps to control; (3) Difficult‐to‐access bleeding; (4) Re‐bleeding risk and (5) High‐pressure bleeding.

Conclusions: A large, diverse cohort of surgeons defined 5 universal bleeding situations that will allow development of a standardized treatment algorithm to guide selection of the optimal adjunctive topical hemostatic agent for each bleeding situation. This will be the objective of a future study.

PB222: Recombinant Factor VIIa (Novoseven ® ) Utilization and Safety for Refractory Bleeding After Cardiac Surgery at a Large Academic Medical Center

K. Marsh 1 ; T. Ahuja 1 ; V. Raco 1 ; D. Green 2 ; J. Papadopoulos 1

1 New York University Langone Health, Pharmacy, New York, United States, 2 New York University Langone Health, Hematology, New York, United States

Background: Novoseven ® is a recombinant preparation of human factor VIIa (rFVIIa) indicated for the treatment of bleeding episodes and perioperative management.

Aims: To evaluate the cost and utilization of rFVIIa at a large academic medical center.

Methods: We performed a retrospective review. Data collection included baseline characteristics, past medical history, and antithrombotic use. Utilization of rVIIa included indication for use, number of doses, and blood product administration within 24 hours. Incidence of venous thromboembolism (VTE), stroke, transient ischemic attack (TIA), death from systemic embolism, myocardial infarction (MI), hypersensitivity, and disseminated intravascular coagulation (DIC) were also collected. The primary outcome was utilization and cost of rVIIa at NYULH. Secondary outcomes included compliance to NYULH off‐label dosing guideline, blood product administration, adverse effects, and thromboembolic events.

Results: The majority of rFVIIa use was for refractory bleeding after cardiac surgery. Dosing was according to NYULH guideline, lower, and higher than recommended in 76.5%, 3.9% and 17.6% of cases respectively. The costs of rVIIa decreased after development of the off‐label dosing guideline and transition from blood bank to pharmacy. The total incidence of thromboembolic events within 30 days was 19.6%; 17.6% arterial and 2% venous. Seventy percent of patients with an adverse event were over 70 years of age. Use of rFVIIa reduced the median number of units of blood products administered.

Conclusions: Administration of rFVIIa for cardiac surgery appears to be effective for hemostasis. Transitioning rFVIIa from the blood bank to pharmacy and implementation of a dosing guideline appears to have reduced costs. Patients receiving rFVIIa should be monitored for thromboembolic events. Elderly patients may be at higher risk for thromboembolic events and further literature is needed to determine the optimal use of rFVIIa or if other hemostatic agents may be warranted in this population.

PB223: Rotational Thromboelastometry (ROTEM) as an Alternative Method for Evaluation of Septic Coagulopathy ‐ Preliminary Results

M. Durila 1 ; I. Hadacova 2 ; M. Solcova 2 ; M. Mikesova 3 ; Z. Prikrylova 1 ; J. Berousek 1 ; T. Vymazal 1

1 Charles University, Department of Anaesthesiology and Intensive Care Medicine, Second Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic, 2 Motol Univerzity Hospital, Department of Clinical Haematology, Prague, Czech Republic, 3 University Hospital Královské Vinohrady, Department of Laboratory Diagnostics, Section Haematology Laboratory, Prague, Czech Republic

Background: Coagulopathy in sepsis is common phenomenon present in critically ill patients at intensive care unit and represents a clinical problem when the patient has to undergo a surgical procedure. Usually, standard coagulation tests such as aPTT and PT are used to evaluate coagulation status before intervention. Contrary to standard coagulation tests rotational thromboelastometry (ROTEM) evaluates coagulation status of the whole blood. Therefore, despite prolongation of aPTT/PT we might get normal results of ROTEM.

Aims: To analyze coagulation profile of septic patients undergoing surgical procedures and to assess whether interventions were performed without increased bleeding in case of normal ROTEM results despite prolongation of aPTT/PT tests.

Methods: Retrospectively, we investigated if surgical procedures in septic patients were performed without increased bleeding in case of normal ROTEM results despite prolonged aPTT/PT. We also analyzed levels of all coagulation factors to describe coagulopathy in more detail.

Results: During the period of 12 months (2017) we analyzed 9 patients with severe sepsis who had normal ROTEM results (EXTEM, INTEM, FIBTEM, NATEM tests) and prolonged aPTT (median 1.39, min 1.1 and max 1.58) or PT (median 1.86, min 1.51 and max 2.18) tests. These patients underwent surgical procedures (laparotomy due to peritonitis (7x) and amputation of lower leg due to gangrena (2x)) and all of them were performed without increased bleeding. Analysis of coagulation factors showed decreased levels of most procoagulant factors except FVIII and fibrinogen which were increased and also decreased levels of anticoagulant factors ‐ data shown in Table 1.

Conclusions: In sepsis, whole blood coagulation profile evaluated by ROTEM may be normal or even show hypercoagulation despite prolongation of aPTT/PT and might be as an alternative method to assess coagulation profile in septic patients undergoing surgical procedures, especially as point of care test.

PB224: Thromboelastometry‐guided Therapy for the Treatment of Hemorrhagic Shock in Postoperative of Vascular Surgery: A Case Report

T. Crochemore 1 ; F. Savioli 2

1 Hospital Israelita Albert Einstein, Department of Intensive Care, Sao Paulo, Brazil, 2 Hospital Israelita Albert Einstein, Sao Paulo, Brazil

Background: Hemorrhagic shock is characterized by a state of hypoperfusion associated with hemodynamic abnormalities leading to the collapse of homeostasis due to massive blood loss. Thromboelastometry (ROTEM) has been considered an effective tool for management of active bleeding in critically ill patients. ROTEM can guide transfusion therapy quickly, reducing the need of allogeneic blood products. In this case report, we present our successful experience of a hemorrhagic shock that was reversed through blood transfusion guided by rotational thromboelastometry.

Aims: Methods

We report a case of an 82‐year‐old Afro‐Brazilian male, presented to the intensive care unit in immediate postoperative of an elective surgery for correction of iliac artery aneurysm with endoleak, presenting signs of hemorrhagic shock. The lab tests revealed anemia, metabolic acidosis, acute kidney injury, thrombocytopenia coagulopathy and active bleeding in surgical site. Postoperative bleeding management was guided by rotational thromboelastometry showing fulminant hyperfibrinolysis state that was corrected with the administration of tranexamic acid. The hypocoagulable state was corrected sequentially with the administration of fibrinogen concentrate, platelet apheresis, cryoprecipitate and prothrombin complex concentrate. The thromboelastometry was completely corrected after 2 hours. The arteriography to evaluate mechanical cause of bleeding was normal. The patient required hemodialysis for 36 hours, with no new organ dysfunctions and was transferred to the ward for clinical rehabilitation.

Results: Conclusions

Thromboelastometry may be a useful, feasible and safe tool to monitor and manage coagulopathy in patients with hemorrhagic shock, with the potential benefit of allowing rapid diagnosis, goal‐guided therapy and helping avoid unnecessary transfusion in such patients.

PB225: Analysis of Fresh Frozen Plasma Requirement in Post Cardiopulmonary Bypass Cardiac Surgery Patient Based on Standard Coagulation and Thrombelastography Tests in Hasan Sadikin Hospital, Bandung

L. Lismayanti 1 ; U. Sukorini 2 ; B. Mulyono 2 ; T. Triyono 3

1 Padjadjaran University, Clinical Pathology, Bandung, Indonesia, 2 Gadjah Mada University, Clinical Pathology, Yogyakarta, Indonesia, 3 Gadjah Mada University, Clinical Pahology, Yogyakarta, Indonesia

Background: Intervention of fresh frozen plasma (FFP) upon early arrival at Cardiac Intensive Care (CICU) in post Cardiopulmonary Bypass (CPB) cardiac surgery patient with bleeding can be guided by standard coagulation and thrombelastography (TEG) tests. Standard coagulation tests only assess one of hemostasis component i.e coagulation factor while various hemostatic component can be assessed by TEG.

Aims: The aim of this study was to determine the difference of subject proportions and the FFP required volume based on standard coagulation and TEG.

Methods: After obtaining approval from the local ethics committee and written informed consent from each patient, an analytic observational study with cross sectional study design were performed in elective cardiac surgery patient with CPB. Samples were obtained simultaneously upon early admission at CICU. The proportion of subjects and volumes of FFP requirement according to standard coagulation was obtained based on INR and/or aPTT more than 1,5 times the mean of reference value, initial and target INR, estimated coagulation factor and body weight, whereas according to TEG was obtained by prolongation of r kTEG time without heparin rebound/residual heparin and body weight.

Results: Among 44 eligible patients, three patiens (6,8%) developed excessive bleeding after CPB without FFP necessity based on two tests. However, standard coagulation showed one patient (2,3%) without excessive bleeding indicated FFP requirement and 550‐825 mL of FFP. The TEG results did not indicate FFP requirement but might be informed cryoprecipitate and thrombocyte concentrate requirement.

Conclusions: Standard coagulation showed a small proportion of patients and none of patient based on TEG who needed FFP, but these differences could not be analyzed. Volume of FFP requirement based on TEG was less than standard coagulation (0 mL vs. 550‐825 mL).

Keyword: Elective cardiac surgery, FFP, Post CPB bleeding, Standard coagulation, TEG

PB226: Quality and Accuracy of Testing for von Willebrand Disease by North American Special Coagulation Laboratories from 2010 to 2017

J. Abdulrehman 1 ; P. Hsu 2 ; Y.C. Ziemba 2 ; E.M. Van Cott 3 ; E. Plumhoff 4 ; P. Meijer 5 ; R. Selby 6

1 University of Toronto, Department of Medicine, Toronto, Canada, 2 Northwell Health System, Department of Pathology and Laboratory Medicine, Hempstead, United States, 3 Harvard Medical School, Boston, United States, 4 Mayo Clinic Rochester, Minnesota, United States, 5 ECAT Foundation, Voorschoten, the Netherlands, 6 University Health Network & Sunnybrook HSC, Department of Laboratory Medicine and Medicine, Toronto, Canada

Background: The North American Specialized Coagulation Laboratory Association (NASCOLA) provides proficiency testing for North American laboratories that perform diagnostic testing for thrombotic and bleeding disorders, including von Willebrand disease (VWD).

Aims: Our objective was to report on the accuracy and precision of VWD testing performed by special coagulation laboratories across North America from 2010 to 2017.

Methods: We assessed the accuracy and precision of 31 proficiency samples for VWD sent out quarterly from 2010 until the third quarter of 2017, which included normal (n=8), type 1 VWD (n=9), type 2 VWD (n=8), and abnormal (n=6) surveys.

Results: From 2010 to 2017 between 43 and 58 laboratories participated in quarterly NASCOLA surveys submitting a total of 5436 results. The VWF antigen, ristocetin cofactor activity, non‐ristocetin activity and collagen binding activity assays correctly identified normal and abnormal plasma samples with high accuracy (see Table 1 for details).

TABLE 1 Interpretation of assay results grouped by proficiency sample type Assay type Interpretation of laboratory results Normal (8 surveys) n (%) Type 1 VWD (9 surveys) n (%) Type 2 VWD (8 surveys) n (%) Abnormal (6 surveys) n (%) VWF antigen Normal 405 (100) 26 (5.5) 50 (12.3) 75 (24.0) Abnormal 0(0) 444 (94.5) 357 (87.7) 237 (76.0) VWF ristocetin cofactor activity Normal 308 (99.7) 14 (4.1) 1 (0.3) 8 (3.2) Abnormal 1 (0.3) 329 (95.9) 303 (99.7) 244 (96.8) VWF non‐ristocetin activity Normal 129 (100) 9 (5.6) 0 (0) 5 (6.0) Abnormal 0(0) 153 (94.4) 130 (100) 78 (94.0) VWF collagen binding activity Normal 57 (100) 4 (5.8) 0 (0) 4 (8.5) Abnormal 0 (0) 65 (94.2) 62 (100) 43 (91.5) Normal is defined as lab value >= 50%. Abnormal is defined as lab value < 50%

Table 2 provides the common assay methodologies in use over that time period.

TABLE 2 Most common assay methodologies in use across surveys Assay Type Assay Methodology n (%) VWF antigen Latex immuno assay 1503 (94.3) VWF ristocetin cofactor activity Agglutination 748 (62.0) VWF non‐ristocetin activity Latex immuno assay 411 (81.5) VWF collagen binding activity ELISA 235 (100) Factor VIII One stage clotting assay 1400 (95.8)

Inter‐laboratory assay precision was highest for von Willebrand factor (VWF) antigen assays (mean coefficient of variations (CVs) were 14.5%) and one stage clotting factor VIII assays (mean CV 15.5%), and lower for VWF activity assays (mean CV 26.8% for VWF collagen binding activity assays, 30.6% for ristocetin cofactor assays and 30.7% for non‐ristocetin cofactor activity assays). Between 20 to 36% of participating laboratories performed multimer analysis.

Conclusions: Accuracy for VWF assays is high in differentiating normal from abnormal plasma and in distinguishing the various subtypes of VWD. However, VWF activity assays continue to have low precision with inter‐laboratory CVs of 26.8 to 30.7%, similar to previous NASCOLA data from 2004 to 2009. VWF antigen precision is highest with a mean CV of 14.5%. The proportion of laboratories performing multimer analysis is unchanged from 2004 to 2017.

PB227: FXI‐deficient Patients with Bleeding Phenotype Show a Spatio‐temporal Propagation of Blood Coagulation Shorter and Slower than Normal

D. Bertaggia Calderara 1 ; V. Carle 2 ; A. Aliotta 1 ; C. Heinis 2 ; L. Alberio 1

1 Division of Hematology and Central Hematology Laboratory, CHUV, University Hospital and University of Lausanne, Lausanne, Switzerland, 2 Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne EPFL , Lausanne, Switzerland

Background: Bleeding phenotype in patients with FXI deficiency is not predictable with conventional laboratory tests, since FXI levels do not correlate with bleeding tendency. Thrombodynamics assay (TD) is a new global coagulation test that provides information on thrombin generation (TG), clot formation and fibrinolysis. TD could be very useful in a clinical setting to prospectively identify patients with a higher bleeding risk, based on its ability to provide information simultaneously on initiation and amplification phases of blood coagulation.

Aims: Our aim was to evaluate the ability of TD parameters to predict a FXI bleeding phenotype.

Methods: TD measures TG and fibrin clot dynamics upon initial activation with tissue factor immobilized on an activating insert; thereafter, coagulation propagates and TG becomes dependent on its amplification loop. The measurements continue up to 60 minutes. We analyzed: plasma from healthy donors, a pool of normal plasma spiked with increasing concentrations of C7P, an inhibitor of FXI, and plasma from patients with FXI < 15% exhibiting different clinical phenotypes.

Results: C7P dose‐dependently increased aPTT and decreased FXI levels. In the TD assay, C7P markedly reduced parameters describing the amplification phase of coagulation: thrombin peak propagation (Vt) and amplitude of stationary peak (Ast). In FXI‐deficient patients with bleeding phenotype, we observed a slowdown and a shortening of the propagation phase of coagulation similar to high C7P concentrations; rate of clot growth (V) was lower or at the lower limit of normal range. In FXI‐deficient patients with thrombophilic phenotype, Ast, Vt, and V values were greatly increased compared to normal, and the presence of spontaneous clot formation indicated tendency to hypercoagulation.

Conclusions: In conclusion, Ast and Vt values of TD assay seem very useful laboratory parameters to identify a bleeding phenotype in patients with FXI levels < 15%.

PB228: The FVIII: C Measurement of Extended Half‐life Recombinant Factor VIII FC Fusion in Haemophilia A Patients

A. Bowyer 1 ; F. Shepherd 1 ; B. Sampson 1 ; S. Kitchen 1 ; M. Makris 1,2

1 Sheffield Haemophilia and Thrombosis Centre, Sheffield, United Kingdom, 2 University of Sheffield, Department of Infection, Immunity and Cardiovascular Disease, Sheffield, United Kingdom

Background: The introduction of extended half‐life factor VIII products for treatment of haemophilia A (HA) has caused uncertainty within Haemostasis laboratories. Field study trials of recombinant FVIII FC fusion protein (Elocta, Bioverativ) reported that both one‐stage clotting (OSA) and chromogenic assays (CSA) accurately recovered FVIII activity (FVIII:C) in spiked samples and were suitable for clinical monitoring of Elocta.

Aims: The aim of this study was to compare different OSA with CSA in HA patients treated with Elocta.

Methods: 42 samples from 16 patients were tested by Hyphen Biomed CSA and OSA with Actin, Actin FS, Actin FSL and Pathromtin (all Siemens) on Sysmex CS5100i and OSA with APTT SP, Synthafax and Synthasil (all Werfen) on ACL Top. CSA was used as the target result and used to calculate percentage difference.

TABLE 1 Mean, median, % diff of FVIII:C by CSA or OSA in 42 samples from 16 patients post Elocta HYPHEN CSA PATHROMTIN SL ACTIN ACTIN FSL ACTIN FS APTT SP SYNTHASIL SYNTHAFAX MEAN (IU/dL) 41.3 27.2 30.4 30.4 29.4 26.6 33.8 26.6 MEDIAN (IU/dL) 27 15 21.5 18 17 19.1 23.8 16.3 RANGE (IU/dL) 1‐117 <1‐83 1‐81 <1‐90 <1‐82 <1‐69 <1‐89 2‐66 RANGE % DIFF CSA + OSA −70‐52 −200‐50 0‐75 −33‐50 −16‐75 −66‐50 −115‐47 N >30% diff to CSA 19 19 15 18 26 7 34

Results: The mean and median CSA were higher than OSA (see Table 1). The mean % difference of OSA compared to CSA ranged from 1.2% with Actin to 32.2% with Pathromtin; median differences ranged from 8.6% (Synthasil) to 32.2% (Pathromtin). Synthasil had the least samples with > 30% difference compared to CSA (7/42). Synthafax had the greatest with 34/42. At low FVIII:C, variation was observed between assays which may impact on monitoring of trough levels. At FVIII:C above 10 IU/dL, Synthasil underestimated relative to chromogenic by less than 30% in all samples whereas Actin FSL, Synthafax and APTT SP consistently underestimated by around 40%. With the exception of Synthasil, all other APTT reagents had at least one third of samples with greater than 30% difference compared to CSA. Although this difference rarely exceeded 45% it could be clinically significant.

Conclusions: The differences between CSA and OSA with APTT SP and Synthafax could have implications for management. It is essential that laboratories and clinicians are aware of the characteristics of the FVIII:C assay used in their centre.

PB229: Overassignment of Recombinant Wild Type (rwt) FIX Potency by a One‐stage Method Indicated from Comparison with Chromogenic FXIa and TF/FVIIa Based Methods and through Discrepant Activation of rwt FIX and Plasma Derived (pd) FIX by Plasma Kallikrein (PK)

S. Rosén

Private Consultant, Kallered, Sweden

Background: FIX potency of rwtFIX concentrates is generally 20‐30% higher with one‐stage (OS) methods versus chromogenic (CS) methods.

Aims: Address the matter of discrepancy within one‐stage (OS) and between OS and CS methods.

Methods: Determine 1. FXIa and FIXa in OS methods. 2. FIX activity with CS FXIa and rTF/FVIIa based methods. 3. FIX activation by PK in OS methods.

APTT SP (IL) and SynthAFax (IL) were used in OS methods and compared with a CS FXIa based method (Rossix) and an in‐house rTF/FVIIa based method. Extensive activation with FXIa was also performed. One pdFIX and two rwtFIX concentrates, one of which the NIBSC preparation 07/142, were analysed and evaluated versus the 5th IS pdFIX Conc 14/148 (NIBSC).

Results: Within the two APTT reagents there was a similar FXIa generation for all samples but about 25% higher FXIa generation with SynthAFax. After addition of Ca2+ ions, the rate of FIXa generation was similar for rwtFIX′s and the 5th IS with APTT SP (slope ratios 0.99 and 1.04) but about 20% lower with SynthAFax (slope ratios 0.81 and 0.79).

FIX potencies versus the 5th IS for OS and the two CS methods are shown in Table 1, demonstrating higher potencies for rwtFIX with APTT SP.

TABLE 1 Potency assignments (IU/mL) of pdFIX and rwtFIX versus 5th IS (n >= 2) OS, APTT SP OS, SynthAFax CS, FXIa based CS, TF/FVIIa based pdFIX 9.6 9.0 9.1 8.5 rwtFIX 10.5 7.6 7.8 7.4 rwtFIX 07/142 10.5 7.3 7.8 7.5

Relative FIXa levels obtained after extensive FXIa activation (ratio pdFIX/rwtFIX = 1.2) were in line with CS and TF/FVIIa assigned potencies. 20 nmol/L PK (3% of plasma zymogen level) showed fast FIX activation after addition of calcium ions in the presence of APTT SP but not with SynthAFax. With APTT SP, the rate of activation of rwtFIX by PK was 1.2‐1.5 fold faster than of pdFIX.

Conclusions: The findings that 1) a TF/FVIIa based FIX method showed proper agreement with CS FXIa based methods and a SynthAFax OS method and 2) rwtFIX is activated faster than pdFIX by PK when using APTT SP, strongly indicates that rwtFIX is assigned a falsely high potency in a APTT SP OS method. These findings may well be applicable for other APTT reagents.

PB230: Sensitive and Standardized TGA Measurement in Haemophilia Patients Using Ceveron ® Alpha TGA

L. Rosenmayr; L. Wagner; N.B. Binder

Technoclone Herstellung von Diagnostika und Arzneimitteln GmbH, Vienna, Austria

Background: To monitor treatment in haemophilic patients is crucial. It is well known that global assays or FVIII/FIX activity determination do not correlate with bleeding episodes. For patients treated with B domain deleted FVIII preparation the factor activity determination is difficult. TGA measures the entire thrombin generation process and a good correlation in treatment with anti‐Inhibitor Coagulant Complex ( FEIBA ) or rFVIIa has been shown.

Aims: Aim of this study was to show that TGA parameters, measured under standardized conditions, can be used with sufficient sensitivity in haemophilic patient samples to monitor replacement therapy.

Methods: We used plasma from normal donors, Haemophilia A patients with and without inhibitors and Haemophilia B patients. Haemophilia A patients plasma was used for spiking with rFVIII. FVIII/FIX activity was determined with one stage clotting assay. Samples were tested using Ceveron ® TGA RB, Ceveron ® TGA RCL and Ceveron ® TGA RCH assay kit on Ceveron ® alpha TGA instrument. A calibration curve was made once at the beginning of the study using the standard analyzer settings for TGA calibration.

Results: Although clotting activity was < 1%, inter individual variation in Peak Thrombin values for Haemophilia A samples ranged from 12.3 to 34.6nM, for Haemophilia B samples from 26.6 to 40.4nM and for Haemophilia A patients with Inhibitors from 11.7 to 15.6nM. Whereas for FVIII deficient sample the Peak Thrombin showed a continues increase with increasing FVIII activity, for the tested FIX deficient plasma already at 1.7 IU/mL Peak Thrombin could be restored up to 50%. Peak Thrombin values correlate with the concentration of rFVIII, with 80% restoration of normal plasma value.

Conclusions: When TGA is used under standardized conditions and with the appropriate trigger, it is a very sensitive tool to monitor factor replacement therapy and can be used to demonstrate correlation with individual bleeding risk during replacement therapy in clinical studies.

PB231: Differences in FVIII Measurements of Plasma Spiked with AFSTYLA ® Generated with One‐stage Clotting Assay using Different Commercial APTT Reagents Compared with Chromogenic Method

I. Vangenechten 1,2,3 ; M. Maes 1,2,3 ; M.‐B. Maes 4 ; A. Gadisseur 1,2,3

1 Antwerp University Hospital, Haemostasis Unit, Department of Haematology, Edegem, Belgium, 2 Antwerp University, Haemostasis Research Unit, Antwerp, Belgium, 3 Antwerp University, CSL Behring Chair in von Willebrand Disease, Antwerp, Belgium, 4 Antwerp University Hospital, Clinical Biology, Edegem, Belgium

Background: The APTT based One‐Stage Clotting assay (OSA) is the more routinely used assay for measuring clotting factors. Recently the Chromogenic Substrate assay (ChS) has been proposed as the most accurate potency assignment for the novel recombinant FVIII, single chain AFSTYLA ® (CSL Behring).

Aims: To identify the most appropriate assay for monitoring of patients treated with AFSTYLA ® by comparing results of FVIII:C using ChS and OSA with different commercial activators.

Methods: Measurements of FVIII:C were done on depleted plasma samples spiked with different concentrations of AFSTYLA ® (0.09‐0.8U/ml) provided by the Blood Center of Wisconsin. These were analyzed using ChS: Biophen FVIII:C (Hyphen Biomed) and OSA: Silica based STA‐PTTA (Stago), TriniCLOT aPTT (Tcoag); Kaolin based STA‐CK‐Prest (Stago), Pathromtin SL (Siemens); and Ellagic acid based Actin FS (Siemens), Synthafax (IL). All assays performed on STA‐C analyzer (Stago).

Results: Discrepancies were observed between OSA and ChS. Across all concentrations, the accuracy (observed vs. theoretical spiked concentration) ranged from 40‐122% for the OSA and 100‐123% for the ChS. Ratios for OSA/ChS of 0.33‐1.0 were observed across different OSA activators and concentrations. Divergence of results increased by increased concentration while the most comparable results were seen at 0.09U/ml AFSTYLA ® .

Conclusions: Results of OSA and ChS were comparable within low range (≤0.15U/ml). ChS had acceptable results for all concentrations with a tendency to overestimate, especially at higher concentration. The ChS is the most correct assay, which was used as potency assignment for the spiked samples, and is recommended to use for monitoring if patients treated with AFSTYLA ® . No one single conversion factor, as prescribed by the manufacturer, can be applied because of varying ratio for OSA/ChS. Among OSA no suitable assay can be identified.

PB232: Improved Recovery of rVIII‐SingleChain and rIX‐FP Spiked Plasma Samples in the OS Clotting Assay

J. Schiebel ; C. Mengel; C. Horn

CSL Behring, Marburg, Germany

Background: In current practice, plasma standards diluted in NaCl solution are typically used to calibrate the one‐stage (OS) clotting assay for monitoring of recombinant FVIII or FIX levels in patient plasma. We hypothesize that the usage of NaCl solution for dilution of the plasma standard results in a suboptimal measurement accuracy of post‐infusion levels.

Aims: This study was set up to determine whether the usage of immuno‐depleted factor deficient plasma (iDPL) instead of NaCl solution for dilution of the plasma standard would result in improved recovery results for rVIII‐SingleChain and rIX‐FP.

Methods: To mimic post‐infusion plasma samples, rIX‐FP, rIX‐Fc and rIX were spiked into FIX iDPL at different concentrations. Similarly, rVIII‐SingleChain was spiked into FVIII iDPL. Normal human plasma was used as clotting factor standard and was either diluted in NaCl solution or iDPL. Several aPTT activator reagents were tested.

Results: The calibration curves obtained from application of the two different procedures for standard dilution deviated significantly from each other, resulting in different measured OS clotting activities. For the conventional dilution using NaCl solution, poor linearity over the investigated spike concentrations and overestimation at lower spike concentrations was generally observed. When applying the iDPL dilution, linearity was greatly improved. Even at low spike concentrations the iDPL dilution procedure resulted in a more accurate recovery. These effects were present for the majority of the 14 tested activator reagents in the case of rIX‐FP as well as for rIX‐Fc and rIX. Importantly, similar trends were also found for rVIII‐SingleChain.

Conclusions: Dilution of the plasma standard in iDPL resulted in more accurate recoveries in the OS clotting assay over the range of investigated spike concentrations. On the basis of these in vitro data, the choice of the plasma‐standard diluent may impact the accuracy of rVIII and rIX post‐infusion clinical monitoring.

PB233: Pre ‐ analytical Variables ‐ Sample Rejection in Hemostasis Laboratories

L. Brown ; D. Kitchen; I. Jennings; S. Munroe ‐ Peart; S. Kitchen; T. Woods; I. Walker

UK National External Quality Assessment Scheme, Blood Coagulation, Sheffield, United Kingdom

Background: Increasing attention has been focused on variation in the pre and post analytical phases of sample testing. Errors occurring at the pre ‐ analytical stage contribute to up to 75% of total laboratory errors. In hemostasis, potential sources of pre ‐ analytical sample variation include interfering substances (hemolysis, icterus and lipemia), and underfilled, overfilled, clotted or mislabelled samples.

Aims: To investigate current trends and criteria of sample rejection in hemostasis laboratories in the UK NEQAS Blood Coagulation (BC) programme.

Methods: In 2017 UK NEQAS BC circulated a questionnaire to 1175 external quality assessment (EQA) programme users. 331 responses were received (28% of participants).

Results: Sample rejection may follow visual check or use of an automated analyser detection system. Visual checks may be objective, for example colour charts for haemolysis, or subjective. Responses are summarised in tables 1 & 1a.

TABLE 1 Summarised responses on methods of sample rejection in hemostasis laboratories Variable Sample rejection method Used by n% respondents Subjective Objective Hemolysis Visual check 79 24 Automated detection ‐ 54 Icterus Visual check 47 14 Automated detection ‐ 53 Lipemia Visual check 54 17 Automated detection ‐ 57

TABLE 1a Summarised responses on methods of sample rejection in hemostasis laboratories Variable Sample rejection method Used by n % respondents Subjective Objective Overfilled samples Visual check 89 48 Automated detection ‐ 36 Underfilled samples Visual check 87 56 ‐ 49

Conclusions: The large majority of centres use subjective criteria to reject samples although increasing use of automated detection systems may improve objectivity. Failings in appropriate pre ‐ analytical sample assessment may have important clinical consequences.

PB234: Thromboelastometry Profile in Critically Ill Patients: A Single‐center, Retrospective, Observational Study

T. Crochemore

Hospital Israelita Albert Einstein, Intensive Care Unit, São Paulo, Brazil

Background: Transfusion therapy is associated with increased morbidity, mortality and costs. Conventional coagulation tests (CCT) are weak bleeding predictors, poorly reflecting coagulation in vivo. Thromboelastometry (ROTEM) provides early identification of coagulation disorders and can guide transfusion therapy by goals, reducing blood components transfusion.

Aims: The aim of this study is to describe coagulation profile of critically ill patients using ROTEM and evaluate the association between CCT and thromboelastometry.

Methods: This is a retrospective, observational study conducted in medical‐surgical intensive care unit (ICU). Adult patients (≥18 years) admitted to ICU between November 2012 and December 2014, in whom ROTEM analyses were performed for bleeding management were included in this study. The first ROTEM and CCT after ICU admission were recorded simultaneously. Additionally, we collected data on blood components transfusion and hemostatic agents immediately after laboratory tests results.

Results: The study included 531 patients. Most ROTEM tests showed normal coagulation profile [INTEM (54.8%), EXTEM (54.1%) and FIBTEM (53.3%)] with divergent results in relation to CCT: low platelet count (51.8% in INTEM and 55.9% in EXTEM); prolonged aPTT (69.9% in INTEM and 63.7% in EXTEM) and higher INR (23.8% in INTEM and 27.4% in EXTEM). However 16,7% of patients with normocoagulability in ROTEM received platelet concentrates and 10% fresh frozen plasma.

Conclusions: The predominant ROTEM profile observed in this sample of critically ill patients was normal. In contrast, CCT suggested coagulopathy leading to a possibly unnecessary allogenic blood component transfusion. ROTEM test may avoid inappropriate allogeneic blood products transfusion in these patients.

PB235: Differences in Activity of Plasma Spiked with IDELVION ® Generated with One‐stage Clotting Assay using Different Commercial APTT Reagents Compared with Chromogenic Method

I. Vangenechten 1,2,3 ; M. Maes 1,2,3 ; M.‐B. Maes 4 ; A. Gadisseur 1,2,3

1 Antwerp University Hospital, Haemostasis Unit, Department of Haematology, Edegem, Belgium, 2 Antwerp University, Haemostasis Research Unit, Antwerp, Belgium, 3 Antwerp University, CSL Behring Chair in von Willebrand Disease, Antwerp, Belgium, 4 Antwerp University Hospital, Clinical Biology, Edegem, Belgium

Background: APTT based One‐Stage Clotting assay (OSA) is routinely used for measuring clotting factors. Recently the Chromogenic Substrate assay (ChS) has been proposed as the most accurate assay for novel extended half live (EHL) clotting factor products, especially in FVIII and now also FIX. The EHL recombinant FIX preparation, IDELVION ® (CSL Behring), an albumin fusion protein, has important laboratory issues.

Aims: To identify the most appropriate FIX assay to monitor patients under treatment with IDELVION ® by comparing results of ChS and OSA with different commercial activators.

Methods: Measurement of FIX was done on depleted plasma samples spiked with different concentrations IDELVION ® (0.05‐0.8U/ml) provided by the Blood Center of Wisconsin. The analysis made use of ChS: Biophen FIX (Hyphen Biomed) and OSA: Silica based STA‐PTTA (Stago), TriniCLOT aPTT (Tcoag); Kaolin based STA‐CK‐Prest (Stago), Pathromtin SL (Siemens); and Ellagic acid based Actin FS (Siemens), Synthafax (IL). All assays performed on STA‐C analyzer (Stago).

Results: Different FIX results were observed among OSA and also between OSA and ChS. Across all levels accuracy (observed vs. targeted spiking concentration) ranged from 40‐225% for the OSA and 110‐152% for the ChS. Varying ratios of 0.33‐2.0 for OAS/ChS were seen across different OSA activators and concentrations. At each level, ChS and OSA using Synthafax highly overestimated, while CK‐Prest underestimated. Pathromtin SL was more in line with theoretical spiking levels. Results from both methodologies were more divergent for levels >0.2U/ml IDELVION ® but similar at 0.05U/ml.

Conclusions: OSA using Pathromtin SL is the most recommended assay for monitoring FIX levels in patients treated with IDELVION ® . OSA using different activators obtained varying results with severe overestimation by Synthafax, and underestimation by CK‐Prest. The proposed ChS constantly overscored levels, especially >0.2U/ml IDELVION ® .

PB236: Extended Half‐life Factor VIII: From Biology to Patients’ Management Optimization

A. Perrier‐Cornet 1,2 ; A. Philippe 1 ; P. De Boissieu 3 ; T. Lambert 4 ; R. D'Oiron 4 ; A. Rafowicz 4 ; C. Lavenu‐Bombled 1,2,5 ; S. Combe 1 ; A. Gillibert 6 ; V. Proulle 1,2,5

1 Service d'Hématologie Biologique, CHU Bicêtre, HUPS, AP‐HP, Kremlin Bicêtre, France, 2 Université Paris Sud, Paris Saclay, Kremlin Bicêtre, France, 3 Service d'Epidémiologie et de Santé Publique, CHU Bicêtre, HUPS, AP‐HP, Kremlin‐Bicêtre, France, 4 Centre de Traitement de l'Hémophilie et des Maladies Hémorragiques Constitutionnelles, CHU Bicêtre, HUPS, AP‐HP, Kremlin Bicêtre, France, 5 UMR_ INSERM 1176, Kremlin Bicêtre, France, 6 Département de Biostatistiques, Hôpital Universitaire de Rouen, Rouen, France

Background: Management of patients with hemophilia A (HA) is currently moving ahead with extended half‐life products (FVIII‐EHL). Optimal therapy is challenging due to discrepancies between FVIII dosages using one‐stage‐assay (OSA) and chromogenic‐stage‐assay (CSA) with some FVIII‐EHL. CSA is considered the most consistent method but is expensive and restricted to few laboratories. OSA is affordable and widely used but subject to high variability due to a large diversity of reagents.

Aims: To compare CSA and OSA methods for the dosage of FVIII in HA receiving B‐domain deleted FVIII linked to an IgG Fc domain (rFVIII‐Fc, Elocta ® , SOBI).

Methods: Single center study in University Hospital Bicêtre over 1 year (2017). FVIII was measured in 160 samples from 60 HA receiving rFVIII‐Fc using OSA (STA‐CKPrest ® , STA‐ImmunodefVIII ® , Stago) and CSA (BIOPHEN ™ FVIII:C, HyphenBiomed) on a STA‐RMax ® device. Comparison was evaluated using Passing Bablock and Bland‐Altman analysis.

Results: OSA and CSA are well correlated with an IC correlation coefficient at 0.97 (IC95%: 0.96‐0.98) and a Pearson correlation at 0.98 (IC95%: 0.98‐0.99). Bland‐Altman analysis shows an excellent agreement between OSA and CSA for FVIII levels below 25% while the difference increases with increasing FVIII levels. Passing‐Bablok regression (OSA=0.91*CSA+0.36; IC95% slope=[0.88‐0.94]; IC95% intercept=[0.06‐0.78]) shows a 10% underestimation of FVIII dosages using OSA versus CSA.

Conclusions: Analysis of 160 FVIII dosages from 60 HA receiving rFVIII‐Fc performed in our hospital during 1 year shows that although OSA is not fully equivalent to CSA, it gives truly reliable results for low FVIII. The inexpensive, easy and continuously available OSA for FVIII measurements can therefore confidently be used for HA receiving rFVIII‐Fc. Only normal or elevated FVIII levels might require control using CSA. Our study highlights the crucial role of biology to improve FVIII treatment in HA without increasing institutional costs and ultimately optimize patients′ management.

PB237: Evaluation of an Automated Chromogenic Assay for Measuring FVIII:C Levels in the Patients Affected by Hemophilia A

C. Novembrino 1 ; M. Boscolo Anzoletti 1 ; M.R. Fasulo 1 ; F. Peyvandi 1,2

1 Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’ Granda Ospedale Maggior, Milan, Italy, 2 University of Milan, Pathophysiology and Transplantation, Milan, Italy

Background: The original one‐stage clotting assay is still the most widely used method to measure FVIII:C activity in patients with haemophilia A (HA), although the use of chromogenic assays are increasing significantly.

Aims: Evaluation of BIOPHEN FVIII:C (HYPHEN BioMed, France) assay on Sysmex CS 2400 (Sysmex, Kobe, Japan) analyser in term of analytical performance and diagnostic accuracy.

Methods: 60 severe, moderate and mild HA patients were enrolled together with 100 healthy Italian subjects (38M/72F, aged between 18‐69 years). The clotting tests were performed on citrate platelet poor plasmas stored at ‐80°C. BIOPHEN FVIII:C was compared with the one‐stage assay using Actin FS and Factor VIII deficient plasma (Siemens, Germany) and with another chromogenic automated assay (COAMATIC Factor VIII, CROMOGENIX on ACL TOP analyzer; Instrumentation Laboratory, Italy).

Results: Imprecision analysis results are reported in Tables 1. Linearity was good up to 1/128 dilution (r=0.99); mean recovery was 91.7% and limit of detection was 0.2%. The reference range calculated on 100 healthy subjects was 64‐181% (95% Reference interval, Double‐sided).

BIOPHEN FVIII:C assay showed a good correlation and diagnostic agreement with both the other chromogenic assay and the one‐stage assay: the Spearmen's Rank correlation coefficient was 0.93 (95% CI 0.88‐0.96) and 0.95 (95% CI 0.91‐0.97), respectively; the inter‐rate agreement K Cohen coefficient was 0.64 (95% CI 0.48‐0.80) and 0.70 (95% CI 0.52‐0.88), respectively.

Conclusions: BIOPHEN FVIII:C showed a good analytical performance and diagnostic accuracy. The results of this analysis showed that chromogenic tests could become part of routine analytical panel of coagulation laboratories for the diagnosis and therapeutic monitoring particularly in patients treated with new longer acting products.

PB238: Underassignment of FIX Potency of rFIX‐Albumin Fusion Protein (rFIX‐HSA) by One Stage Methods Demonstrated in Studies on FXIa and FIXa Generation

S. Rosén 1 ; P. Bryngelhed 2 ; G. Siegert 3 ; O. Tiebel 3

1 Private Consultant, Kallered, Sweden, 2 Rossix AB, Molndal, Sweden, 3 Universitätsklinikum Carl Gustav Carus, Institut für Klinische Chemie und Laboratoriumsmedizin, Dresden, Germany

Background: FIX potency of EHL‐rFIX concentrates shows large variation in results between metods.

Aims: Disclose causes of discrepancy within one‐stage (OS) and between OS and chromogenic (CS) methods.

Methods: 1. Monitor FXIa and FIXa generation in OS methods before and after addition of calcium chloride through subsampling versus time and use of chromogenic FXIa and FIXa methods.

2. FIX analysis of samples on one or more of STA‐R Evolution (Stago), ACL TOP (IL) and KC‐4 (Amelung) using APTT reagents containing kaolin (CK Prest, Stago), colloidal silica (APTT SP, IL) or ellagic acid (SynthAFax, IL) and comparison with a CS method (Rossix). The 4th IS FIX Concentrate 07/182, a plasma derived FIX concentrate (pdFIX) and rFIX‐Albumin concentrate (rFIX‐HSA), neat or spiked into plasma, were used as FIX samples. Spiking with FXIa into calcium chloride was done to study the effect on potency assignment

Results: Within each APTT reagent there was no difference in FXIa generation between the samples. However, there was an up to 2.5‐fold difference in FXIa generation between APTT reagents. The FIXa activity of rFIX‐HSA relative to the 4th IS varied greatly depending on APTT reagent. This is illustrated by FIXa ratios for pdFIX and rFIX‐HSA versus the 4th IS at the time point 27s after calcium ion addition and before clot formation. See Table 1 (FXIa values at end of contact activation are shown in parenthesis).

TABLE 1 FIXa ratios versus 4th IS (FXIa values in parenthesis) CK Prest (0.8 IU/mL) APTT SP (1.5 IU/mL) SynthAFax (2.1 IU/mL) pdFIX 0.72 0.67 0.84 rFIX‐HSA 0.39 0.95 1.22

In line with this, samples spiked with rFIX‐HSA showed 3 to 4‐fold lower results with CK Prest than the SynthAFax and CS methods. Furthermore, spiking with 1.3 and 2.6 IU/mL FXIa raised the potency of two different rFIX‐HSA samples by 50 to 120% in the CK Prest method. The potency of pdFIX was not affected. With SynthaFAx, no change was obtained of rFIX‐HSA potency.

Conclusions: Relatively low levels of FXIa may be one cause of inefficient activation of rFIX‐HSA in OS methods, resulting in underassignment of potency. In contrast a CS method and SynthAFax seem to show reliable results.

PB239: Correlation of FIX Activity Assays with Thrombin Generation

L. Florin 1 ; V. Mondelaers 2 ; A. Vantilborgh 3 ; K.M.J. Devreese 1

1 Ghent University Hospital, Coagulation Laboratory, Department of Laboratory Medicine, Ghent, Belgium, 2 Ghent University Hospital, Department of Pediatric Hemato‐Oncology and Stem Cell Transplantion, Ghent, Belgium, 3 Ghent University Hospital, Department of Hematology, Ghent, Belgium

Background: Thrombin generation (TG), a global coagulation test, may be superior to traditional coagulation assays in monitoring replacement therapy in hemophilia patients.

Aims: We evaluated correlation between FIX activity measured by a one‐stage clotting assay (OSCA) and a chromogenic assay (CA) and TG parameters.

Methods: 19 samples were measured for FIX activity with an aPTT based OSCA (STA‐PTT A, Stago) and a CA (Hyphen BioMed) on STA‐R Evolution (Stago) in parallel with a TG assay (Calibrated Automated Thrombogram ® , Stago). Thrombin peak (TP) and endogenous thrombin potential (ETP) were studied using normal and low tissue factor (TF) concentration (PPP and PPP low, Stago). We included patient samples from hemophilia B patients on replacement therapy with Benefix ® (Nonacog alfa recombinant FIX, Pfizer) (n=4) or Idelvion ® (recombinant FIX albumin fusion protein, CSL Behring) (n=4, same patient), patients without FIX treatment (n=5) and FIX immunodepleted pooled human plasma samples spiked with Idelvion ® (n=6). Pearson's correlation coefficients were calculated for the overall sample group and for the subgroups.

Results: Correlations with TP and ETP were similar for both FIX assays and better with low TF TG. FIX activity by OSCA and CA correlated better with TP compared to ETP, except for the non‐treated patients. FIX activity and TP correlated best in spiked Idelvion ® samples and Benefix patient samples (Table 1). Correlation for Idelvion ® patient samples was lower.

TABLE 1 Pearson correlation coefficients with P‐values for the overall sample group and different subgroups Sample group Assay ETP (P‐value) TP (P‐value) all samples (n=19) OSCA STA‐PTT A/ CA 0.30 (0.22)/ 0.48 (0.04) 0.50 (0.03)/ 0.64 (0.003) Benefix (n=4) OSCA STA‐PTT A/ CA 0.77 (0.23)/ 0.82 (0.18) 0.89 (0.11)/ 0.93 (0.07) without treatment (n=5) OSCA STA‐PTT A/ CA 0.72 (0.17)/ 0.72 (0.17) 0.65 (0.24)/ 0.64 (0.25) Idelvion® spiked samples (n=6) OSCA STA‐PTT A/ CA 0.87 (0.02)/ 0.85 (0.03) 0.94 (0.005)/ 0.93 (0.007) Idelvion® patient samples(n=4) OSCA STA‐PTT A/ CA 0.63 (0.37)/ 0.60 (0.40) 0.69 (0.31)/ 0.66 (0.34)

Furthermore, correlation for the overall sample group was low, illustrating that TG differs between subgroups, depending on the type of FIX and between patient and spiked samples (Figure 1). Additionally, a similar FIX activity measured by OSCA in patients receiving replacement therapy might result in different TP, illustrating interindividual variability.

FIGURE 1 Results for TP in the TG assay (low concentration TF) as a function of FIX activity measured with the STA‐PTT A OSCA

Conclusions: FIX activity and TG should be correlated with the bleeding history of the patient, before concluding that one or the other test is better to give a reflection of patient's bleeding risk.

PB240: Performance Evaluation of 10 APTT Reagents in Quantitative Clot Waveform Analysis

G. Kershaw 1,2 ; V. Chen 1,3

1 Anzac Research Institute, University of Sydney, Concord, Australia, 2 Royal Prince Alfred Hospital, Haematology, Camperdown, Australia, 3 Concord Hospital, Haematology, Concord, Australia

Background: Quantitative clot waveform analysis (CWA) refers to a method of analysing changes in optical density generated during monitoring plasma clot (fibrin) formation such that information is obtained on the rate of clot formation. The peak of first derivative of the light transmission data gives the maximum speed of clot formation, designated Min1, with the peak of second derivative representing the maximum acceleration of clot formation, designated as Min2.

Aims: To evaluate 10 different APTT reagents in the CWA system on the Sysmex CS2500 analyser by establishing reference intervals for Min1, Min2, and determining reproducibility in normal and factor deficient samples.

Methods: Five silica‐activated reagents were: Pathromptin SL, Triniclot aPTT S, PTT A, APTT SP and Synthasil. Four ellagic acid reagents were: Actin, Actin FS, Actin FSL and Synthafax. A polyphenol‐based reagent, Cephascreen was included. Reference intervals were determined on plasma from 36 donors. Precision was determined on commercial controls, FVIII deficient and FIX deficient plasma, un‐spiked or spiked to 1IU/dL of factor. There were 20 runs for control plasma, 10 runs for factor deficient plasma.

Results: Min1 reference intervals ranged from 1.6‐3.9 (Synthasil) up to 3.4‐6.5 (Actin FSL). Min2 reference intervals ranged from 0.3‐0.7 (Synthasil) up to 0.5‐1.1 (Actin FSL). Between run reproducibility was excellent for Min1, with 8/10 APTT reagents yielding CVs of < 3% across all sample types, 2/10 had CVs of < 5%. For Min2 5/10 reagents had CV's < 5%, and 5/10 were < 8%. Repeatability of Min1 in severely deficient plasma gave CVs of < 1.2% for all reagents, and for Min2 < 3.3% for all reagents. Absolute values for Min1 and Min2 between factor deficient plasma and that spiked to 1 IU/dL were clearly separated.

Conclusions: All 10 APTT reagents performed well in reproducibility studies and in ability of their CWA parameters to differentiate between un‐spiked and 1 IU/dL spike levels of FVIII or FIX deficient plasma.

PB241: An International, Multi‐centre, Blinded Comparative Study Assessing the Activity of N8‐GP in Spiked Haemophilia A Plasma Samples

M. Hansen 1 ; S. Tiefenbacher 2 ; W.H.O. Clausen 3 ; R. Lützhøft 3 ; M. Ezban 1

1 Novo Nordisk, Måløv, Denmark, 2 Colorado Coagulation, Laboratory Corporation of America Holdings, Englewood, United States, 3 Novo Nordisk, Søborg, Denmark

Background: N8‐GP (turoctocog alfa pegol) is a glycopegylated, extended half‐life human recombinant factor FVIII (rFVIII) shown to be an efficacious treatment for patients with haemophilia A. Accurate monitoring of replacement therapy helps ensure proper dosing, leading to better patient care.

Aims: To evaluate the accuracy, and intra‐ and inter‐laboratory variability of N8‐GP and rFVIII (octocog alfa; Advate ® ) activity measurements in clinical laboratories using their routine methods and reagents.

Methods: Laboratories measured plasma samples spiked with 0.03, 0.2, 0.6 and 0.9 IU/mL N8‐GP or rFVIII ® . Samples were blinded and laboratories were instructed to perform evaluations with their routine FVIII activity methods. Replicates of each concentration were measured on three separate days.

Results: 67 laboratories from 25 countries participated. 60 laboratories used one‐stage aPTT clotting assays (9 different reagents), 36 used chromogenic assays (6 different kits) and 29 used both one‐stage and chromogenic assays. The most commonly used aPTT reagents were SynthASil (22%) and Actin FS (18%). Three aPTT reagents underestimated N8‐GP >30% (APTT‐SP, TriniCLOT, STA PTT‐Automate) and were omitted from subsequent statistical analyses.

For both assay types and products, inter‐laboratory variability was greater than intra‐laboratory variability and highest at 0.03 IU/mL. Using one‐stage clotting assays, N8‐GP recovery was slightly lower and rFVIII recovery was slightly higher than target concentrations (Figure). Overall mean recoveries for both products were within the acceptable range of ±30% (i.e. 92.5% N8‐GP and 123.2% rFVIII). Using chromogenic assays, N8‐GP and rFVIII recoveries were similar at all concentrations (Figure), and overall mean recoveries were within the upper bound of the acceptable range (129.3% N8‐GP and 127.4% rFVIII).

Conclusions: Most clinical laboratories accurately measured N8‐GP with one‐stage or chromogenic assays. However, three silica‐based aPTT reagents underestimated N8‐GP recovery >30%.

FIGURE 1 Mean recovery (error bars 95% CI) of N8‐GP and rFVIII at various concentrations measured by one‐stage aPTT clotting assays and chromogenic assays

PB242: DOAC Dipstick for Determination of Absence or Presence of Direct Oral Anticoagulants and Creatinine in Urine

J. Harenberg 1 ; R. Schreiner 2 ; S. Hetjens 3 ; R. Krämer 4 ; C. Weiss 5

1 DOASENSE GmbH, Heidelberrg, Germany, 2 Medical Care Center Dr. Limbach & Collegues, Heidelberg, Germany, 3 University of Heidelberg. Medical Faculty Mannheim, Biometry and Statistics, Mannheim, Germany, 4 University of Heidelberg, Institute Inorganic Chemistry, Heidelberg, Germany, 5 University of Heidelberg, Medical Faculty Mannheim, Biometry and Statistics, Mannheim, Germany

Background: Direct oral anticoagulants (DOAC) are excreted predominantly by glomerular filtration of kidneys into urine. A specific and sensitive detection of DOACs in urine by a point of care test may support rapid diagnosis in emergency medicine. Reduced renal function and urine colour may influence the performance of the DOAC Dipstick test.

Aims: The aim of the study was to determine the performance of the in vitro diagnostic DOAC Dipstick for qualitative detection of absence or presence of DOACs and of creatinine in urine by visual identification of colours within 10 minutes.

Methods: The test is based on a change of colour upon reaction of factor Xa or thrombin with a factor Xa or thrombin chromogenic substrate in relation to the amount of DOAC present in urine and of the Benedict‐Behre reaction for creatinine. The colours allow the detection of DOACs in a urine sample by naked eye, with interpretation as “negative” in the absence of a DOAC and as “positive” in the presence of DOACs and of low and normal creatinine in urine. Urine colour is also determined with DOAC Dipstick.

ResultsConcentrations of DOACs in urine (mass spectrometry method) were high (mean 5.600 ng/ml dabigatran, 2.700 ng/ml rivaroxaban, 1.800 ng/ml apixaban, n=29 each, normal < 5 ng/ml) due to a smaller distribution volume compared to blood. Sensitivity, specificity and accuracy was > 95% for detection of apixaban, rivaroxaban and dabigatran in human urine spiked with DOACs (0 to 1500 ng/ml.) Using urine samples of patients treated with DOACs, sensitivities and specificities were all 1.0. Heparins did not interact with the test. Low levels of creatinine in urine and pathological urine colour may falsify DOAC levels in urine by visual colour identification.

Conclusions: The DOAC Dipstick test from urine samples of patients offers a rapid, reliable and valid method to detect DOACs in medical emergency situations. A post‐marketing study will generate real word data on the performance of the test system.

PB243: One Stage and Chromogenic Assays Discrepancy in the Estimation of Factor IX (FIX) Recovery. What is it that a Single Hemophilia Center Can and Cannot Do?

Å.S. Persson 1,2 ; N. Mahmoud Hourani Soutari 1,2 ; E.‐M. Norberg 1,2 ; J.P. Antovic 1,2

1 Karolinska Institutet, Molecular Medicine & Surgery, Stockholm, Sweden, 2 Karolinska University Hospital, Coagulation, Clinical Chemistry, Karolinska University Laboratory, Stockholm, Sweden

Background: Both aPTT‐based one stage (OS) and chromogenic substrate (CS) assays are used in the estimation of FVIII recovery, but CS assays for the determination of FIX activity have become available only recently. Therefore, data about potential discrepancies in hemophilia B are very limited. Extended half‐life (EHL) products make this issue even more complicated.

Aims: To investigate the recovery of different FIX concentrates using different OS and CS assays.

Methods: Standard (rFIX ‐ Benefix ™ ) and EHL (rFIXFc ‐ Alprolix ™ and rFIX‐FP ‐ Idelvion ™ ) FIX concentrates in the following concentrations 0.8, 0.3 and 0.05 IU/dL locally spiked in FIX deficient plasma for rFIX and pre‐spiked (rIFX‐FP laboratory sample kit) for rFIX‐FP, and the concentrations of 0.8, 0.2 and 0.05 IU/dL pre‐spiked samples (rFIXFc laboratory sample kit) for rFIXFc were tested by three OS (Actin FSL, Actin FS and PTT Automate) and two CS (Biophen and Rossix) FIX assays in triplicate on BCS ‐XP analyzer. Normal reference plasma (Precision Biologic) was used as a standard in all assays.

Results: A small, non‐significant difference was observed between OS assays using immunodepleted (Stago) or congenital (George King Biomedical) deficient plasmas and therefore all OS assays presented below were performed with immunodepleted FIX deficient plasma (Stago). Obtained results in absolute numbers (mean of three measurements) are presented in the table

TABLE 1 Observed concentrations in relation to targeted values in absolute numbers (mean of three measurements) Target values ACTIN FSL ACTIN FS PTT A Biophen Rossix Benefix 0.8 IU/dL 0.98 0.86 1.10 0.58 0.95 0.3 IU/dL 0.47 0.41 0.47 0.23 0.36 0.05 IU/dL 0.12 0.10 0.12 0.03 0.07 Idelvion 0.8 IU/dL 0.55 0.53 0.60 1.14 1.26 0.3 IU/dL 0.24 0.24 0.28 0.48 0.56 0.05 IU/dL 0.06 0.05 0.06 0.06 0.09 Alprolix 0.8 IU/dL 0.98 1.17 0.71 0.67 0.98 0.2 IU/dL 0.34 0.39 0.24 0.16 0.25 0.05 IU/dL 0.12 0.13 0.07 0.03 0.07

FIGURE 1 The results are presented as percent of targeted spiked value. Red lines represent 75% and 125% since discrepancy of ± 25% is considered acceptable

Conclusions: Our findings indicate discrepancies in the estimation of the recovery of FIX concentrates using different OS and CS assays. We suggest that each laboratory in hemophilia comprehensive centers should perform such an investigation with locally available FIX concentrates and assays and then decide on the potential combinations, since no laboratory is able to perform all assays. Manufacturers of factor concentrates may help by clearly stating in their concentrates’ inserts which assays are used in potency assignments.

PB244: Comparison of Two One‐stage Clotting Assays and One Chromogenic FIX Assay for Monitoring of Idelvion ® Replacement Therapy

L. Florin ; K.M.J. Devreese

Ghent University Hospital, Coagulation Laboratory, Department of Laboratory Medicine, Ghent, Belgium

Background: FIX extended half‐life (EHL) products are increasingly used for treatment of hemophilia B. The laboratory has to measure accurately the replacement FIX activity in these patients. Differences may exist between assays to measure FIX for monitoring EHL products.

Aims: We evaluated 3 assays (2 one‐stage clotting assays (OSCA) and 1 chromogenic assay) for monitoring Idelvion ® (CSL Behring), a recombinant FIX albumin fusion protein (rFIX‐FP).

Methods: Eight FIX immunodepleted pooled human plasma samples spiked with Idelvion ® (2.5%‐80%) were measured in parallel with 3 FIX assays on STA‐R Evolution (Stago): two aPTT‐based OSCA, STA‐CK PREST (Stago), with kaolin as activator, and STA‐PTT A (Stago), with silica as activator, and one chromogenic assay (Hyphen BioMed). Method comparison was performed with Passing‐Bablok, Pearson correlation coefficient and calculation of percent deviations (bias) from target concentrations. FIX activity was compared with peak height (PH) and endogenous thrombin potential (ETP) measured by Calibrated Automated Thrombogram ® (Stago).

Results: Passing‐Bablok showed that FIX activity differed between the 3 assays. At lower FIX activity (< 10%), agreement between the methods was acceptable. In these spiked samples STA‐PTT A OSCA is most accurate in measuring Idelvion ® FIX activity. Bias for OSCA STA‐PTT A varied between ‐14.5% (80% FIX) and 27.2% (2.5%). STA‐CK PREST OSCA underestimated FIX activity with a mean bias of ‐42.1% (except for the 2.5% sample), whereas FIX activity by the chromogenic assay was overestimated (mean bias 45.8%), even applying the advised low and high calibration curves (Table 1). FIX activity correlated better with PH compared to ETP, but all correlations (r 0.8‐0.9) were found to be significant for the 3 methods (P<0.05).

TABLE 1 Target FIX concentrations of spiked Idelvion® samples and measured FIX activity with 3 assays. * indicates a significant bias (>25%) Target (%) OSCA STA‐CK PREST (Stago) Bias(%) OSCA STA‐PTT A (Stago) Bias(%) Chromogenic (Hyphen BioMed) Bias(%) 80.0 45.1 −43.6* 68.4 −14.5 133.5 66.9* 60.0 38.0 −36.8* 61.1 1.8 90.0 50.0* 40.0 22.9 −42.9* 35.1 −12.4 65.8 64.5* 30.0 16.7 −44.3* 27.8 −7.4 50.3 67.5* 15.0 6.3 −58.1* 14.5 −3.5 17.9 19.6 10.0 5.9 −40.7* 9.3 −7.2 13.0 29.6* 5.0 3.6 −28.0* 4.6 −8.6 6.5 30.1* 2.5 2.5 −0.4 3.2 27.2* 3.4 37.8*

Conclusions: Compared to a chromogenic assay and STA‐CK PREST OSCA, STA‐PTT A OSCA is most accurate in measuring recovery of Idelvion ® . Laboratories should carefully validate their factor activity assay for each EHL product.

PB245: Levels of Coagulation Factors, Coagulation Inhibitors and TAFI Do Not Affect Global Hemostasis and Bleeding Phenotype in Patients with Hemophilia A

D. Mikovic 1 ; I. Pruner 2 ; J. Antovic 2 ; R. Chaireti 2

1 Blood Transfusion Institute, Hemostasis Department and Hemophilia Center, Belgrade, Serbia, 2 Karolinska Institutet, Molecular Surgery and Medicine, Solna, Sweden

Background: Hemophilia patients with similar disease severity sometimes exhibit different bleeding phenotypes. The global hemostatic tests can be used to study differences in hemostatic capacity that can explain the symptom variations. It is not completely clear how levels of coagulation factors and inhibitors modify global hemostatic capacity in hemophilia.

Aims: The aim of the study was to investigate the association of coagulation factors and inhibitors with the bleeding phenotype and the global hemostatic tests (overall hemostatic potential, OHP and endogenous thrombin potential, ETP) in patients with hemophilia A.

Methods: Seventy‐six patients with hemophilia A (56 with severe clinical phenotype and 20 with non‐severe) were included. In 38 patients blood samples were taken 30 minutes following administration of 1000‐2000 IU FVIII concentrate to treat hemarthrosis. Fibrinogen, factors II, V, VII, VIII, IX, X, XI and XII, von Willebrand antigen and activity and coagulation inhibitors antithrombin, protein C (PC) and S were analyzed. Thrombin‐activatable fibrinolysis inhibitor (TAFI) was measured to study fibrinolysis, ETP and OHP were analyzed to estimate global hemostasis. Multiple regression analysis was performed to assess coagulation factors and inhibitors as predictors of the bleeding phenotype and global hemostasis.

Results: There were no significant partial correlations between coagulation factors and the bleeding phenotype. The only coagulation factor that influenced the results of the global hemostatic tests was factor VIII, whereas in the posttreatment samples the levels of proteins C and S had a significant effect on OHP and ETP, respectively (P< 0,001 for both).

Conclusions: Levels of coagulation factor and inhibitors do not modify ETP, OHP or bleeding phenotype in hemophilia. However, changes in the activated PC system affect the coagulation potential and this might reflect an increased posttreatment anticoagulant activity, developing from the changed balance in the patient's habitual coagulation.

PB246: Method Comparison of the PT Rec Coagulation Assay with Existing Reference Methodology for Measuring Prothrombin Time (PT)

U. Geisen 1 ; J. Kappelmayer 2 ; R. Jones 3 ; A. Lowe 3 ; G. Miles 4 ; G. Rozsnyai 5 ; S. Kitchen 3

1 Institute for Clinical Chemistry and Laboratory Medicine, Faculty of Medicine, University of Freiburg, Freiburg, Germany, 2 University of Debrecen, Debrecen, Hungary, 3 Sheffield Haemostasis and Thrombosis Centre, Sheffield, United Kingdom, 4 Roche Diagnostics Inc., Indianapolis, United States, 5 Roche Diagnostics International Ltd, Rotkreuz, Switzerland

Background: Coagulation screening tests are used to reliably and quickly detect inherited or acquired factor deficiencies and monitor patients receiving anticoagulant therapies. New commercially available assays may offer improvements over current assays; however, it is important that new assays are evaluated against existing assays prior to their adoption in the healthcare setting. The PT Rec assay contains thromboplastin (recombinant human thromboplastin reagent containing a heparin‐neutralizing substance) and calcium, which initiates activation of the extrinsic coagulation cascade when added to citrated human plasma.

Aims: To compare the new PT Rec assay (measure of prothrombin time) with existing reference methodology.

Methods: Method comparisons, according to Clinical and Laboratory Standards Institute EP09‐A3 (CLSI EP09‐A3) guidelines, were performed across four sites for the PT Rec assay (cobas t 711 analyser) versus Innovin (Siemens Sysmex CS‐5100 or CS‐2000i). A minimum of 120 residual anonymised human plasma samples were used per assay, per site, to represent the appropriate measuring range. Equivalency of the cobas t 711 and cobas t 511 platforms was also evaluated by method comparison.

Results: According to prespecified criteria based on Deming regression analyses, method comparison experiments showed good agreement between the PT Rec assay and the reference method (Table 1). Equivalency was also demonstrated between the cobas t 711 and cobas t 511 analysers, according to prespecified acceptance criteria, based on Passing‐Bablok regression analyses (Table 2).

TABLE 1 Method comparison between cobas t 711 and reference method Comparison Evaluation Acceptance criteria Freiburg, Lot 1 Sheffield, Lot 2 Debrecen, Lot 3 PT Rec versus Innovina n ‐ 131 135 130 Slope (Deming) 1.00 ± 0.10 1.008 1.036 0.9 Intercept NA 0.090 0.045 0.238 Pearson´s r ≥0.900 0.9985 0.9966 0.9883 Bias at 1.0 INR ≤0.15 0.097 0.081 0.138 a Analyses based on INR; INR, International Normalised Ratio; NA, not applicable; PT, prothrombin time

TABLE 2 Method comparison between cobas t 711 and cobas t 511 analysers Evaluation Acceptance criteria Freiburg, Lot 1 Sheffield, Lot 2 PT Reca n ‐ 129 135 Slope (Passing‐Bablok) 1.00 ± 0.10 1.006 0.984 Intercept NA 0.006 0.033 Pearson´s r ≥0.900 0.9999 0.9996 a Analyses based on INR; INR, International Normalised Ratio; NA, not applicable; PT, prothrombin time

Conclusions: Based on good agreement between the PT Rec assay and the commercially available reference method, and equivalency observed between the cobas t 711 and cobas t 511 analysers, the PT Rec assay has demonstrated suitability for use in core laboratories.

PB247: The Effect of DDAVP on Thrombin Generation in Patients with Bleeding Disorders

A. Agapidou ; C. Hamid; E. Fosbury; A. Riddell; P. Chowdary

Royal Free Hospital, London, United Kingdom

Background: Von Willebrand Factor (vWF) has multiple roles in the process of hemostasis. The vasopressin analogue DDAVP facilitates in the release of v WF and factor VIII from the endothelial cells. There is a heterogeneity in the clinical expression of bleeding diathesis in patients with mild Haemophilia A (HA), Von Willebrand disease and patients with platelet dysfunction disorders.

Aims: To investigate the effect of DDAVP on thrombin generation parameters in patients with mild bleeding disorders who received desmopressin as a treatment trial.

Methods: We monitored 12 patients with mild Haemophilia A, 7 with platelet dysfunction disorders, 14 with Von Willebrand disease. We investigated the effect of subcutaneous DDAVP administration (0.3 μ gr/kgr) during 5 time‐points (0 minute, 90 minutes, 4 hours, 6 hours, and 24 hours) on parameters of thrombin generation in platelet poor plasma (PPP) and platelet rich plasma (PRP). Tissue factor (TF) 1pm was used for low PPP reagent and 0.5 and 1.0 pm for PRP reagent. Samples were run together with quality control samples with the corresponding concentration. For thrombin generation measurements we used the Hemker method and calibrated automated thrombogram machine. For the statistical analysis, paired t‐tests were performed for each time point and each group of patients and quality control.

Results: There is statistically significant positive correlation for all time points in ETP PPP in mild HA patients between the study and control group. There is statistical significance in all time points for peak PPP but only in 4 and 6hrs for 0.5pM PRP peak in mild HA. There is statistical significance only after 24 hrs in ETP PRP 0.5 pm for patients with platelet dysfunction, after 24 hrs in peak PPP and after 4 and 6 hours in lagtime PPP for patients with mild von Willebrand disease.

Conclusions: The effect of DDAVP in the above categories of bleeding disorders varies, being more prominent for ETP and Peak in PPP samples.

PB248: Turoctocog Alfa Pegol (N8‐GP) Can Be Measured Accurately Using Normal Human Plasma Calibration in a Validated Chromogenic Assay

W. Pickering 1 ; M. Robinson 2 ; S. Tiefenbacher 2 ; E. Mirella 3

1 Novo Nordisk A/S, Development Bioanalysis, Maaloev, Denmark, 2 Colorado Coagulation, Laboratory Corporation of America Holdings, Englewood, United States, 3 Novo Nordisk A/S, Hemophilia Research, Maaloev, Denmark

Background: Turoctocog alfa pegol (N8‐GP) is an extended half‐life, glycoPEGylated recombinant human FVIII. Challenges achieving accuracy has been observed when developing assays using Normal Human Plasma (NHP) calibration to measure recombinant FVIII products.

Aims: To validate a FVIII chromogenic assay using NHP calibration to measure N8‐GP accurately in accordance with bioanalytical guidelines for clinical trials.

Methods: The accuracy, precision, dilution integrity, short‐term and freeze‐thaw stability and selectivity were assessed using N8‐GP spiked hemophilia A plasma samples, a Siemens BCS ® XP (Siemens Healthcare Diagnostics) analyzer, the Coatest ® SP4 FVIII chromogenic kit (Chromogenix) using high calibration (0.15 ‐ 1.5 IU/mL) and low calibration (0.01 ‐ 0.2 IU/mL) curves. N8‐GP spiking covered the range 0.01 ‐ 3.0 IU/mL.

Results: The Coatest ® SP4 FVIII kit was validated according to regulated bio‐analytical guidelines for the measurement of FVIII activity.

Accuracy: Mean intra and inter‐assay accuracy (%RE) fulfilled the acceptance criteria across the full range. Intra and inter‐assay accuracy ranged from 5.0 ‐18.4% and 5.0 ‐18.5%.

Precision: Mean intra and inter‐assay precision (%CV) were acceptable with data ranging from 2.4 ‐ 12.8% and 2.8 ‐ 16.5% for all test samples.

Dilution integrity: N8‐GP demonstrated good dilution integrity at 3.0 IU/mL, with a %RE of 8.7% .

Carry‐over analysis: was performed according to CLSI EP10‐A3 and was 0.0004 IU/mL.

Short‐term stability: samples at 1.2 and 0.03 IU/mL showed on‐instrument and cold block stability for up to 6 hours by demonstrating accurate recovery.

Selectivity: 10 individual hemophilia FVIII deficient samples (< 0.01 IU/mL) were spiked with N8‐GP to 0.03 IU/mL and 1.0 IU/mL. Accuracy ranged from ‐3.3 ‐ 15.7% and 0.2 ‐ 11.7% for the two levels.

Conclusions: The FVIII Chromogenic assay was considered validated according to bio‐analytical guidelines to measurement of N8‐GP in 3.2% citrated human plasma as the validation parameters tested lived up to guidelines.

PB249: Comparison of Three Thromboplastin Reagents for International Normalized Ratio (INR) Measurement in Patients on Vitamin K Antagonist Therapy

R. Perez Montes 1 ; E. Martin Martin 1 ; F. Depasse 2

1 University Hospital Marqués de Valdecilla, Santander, Spain, 2 Diagnostica Stago, Asnieres sur Seine, France

Background: The variability across thromboplastin reagents in patients on VKA therapy is well established. The INR was developed to standardise results for patients receiving VKA.

Aims: Compare the new thromboplastin reagent STA ® ‐NeoPTimal (Stago, Asnières sur Seine, France) with two widely used reagents for INR measurement, STA ‐ Neoplastine ® CI PLUS (Stago, Asnières sur Seine, France) and Thromborel ® S (Siemens, Marburg, Germany).

Methods: The study was approved by the Ethics Committee of the Valdelcilla Hospital (Spain). A local MNPT was determined on 21 plasma samples obtained from normal subjects with the new reagent STA ® ‐NeoPTimal (rabbit brain thromboplastin, batch ISI 1.01) on STA‐Compact Max 2 (Stago) and with Thromborel ® S (human placenta thromboplastin, batch ISI 1.06) on CA1500 (Siemens). The MNPT provided by the supplier was used for the STA ‐ Neoplastine ® CI PLUS (rabbit brain thromboplastin, ISI 1.28). INR was measured with the three reagents in 98 samples obtained from patients subjects on VKA therapy (warfarin), 53 females / 45 males aged from 21 to 80 years.

Results: A linear regression was calculated to compare the INR results obtained with the new STA ® ‐NeoPTimal reagent to the other two thromboplastin reagents. Results are depicted in Figures 1 and 2.

FIGURE 1 STA‐NeoPTimal INR versus STA‐Neoplastine CI Plus INR

FIGURE 2 STA‐NeoPTimal INR versus Thromborel S INR

Conclusions: STA‐NeoPTimal, extraction thromboplastin ISI=1 reagent, well correlates with the two other thromboplastins and can be easily used for laboratory routine testing.

PB250: New Multiplatform von Willebrand Factor Antigen Assay with Extended Stability

C. Dunois ; M. Peyrafitte; V. Villedieu; S. Molton; J. Amiral

HYPHEN BioMed, Research and Validation, Neuville sur Oise, France

Background: Measurement of von Willebrand Factor antigen (vWF: Ag) is the first intention assay for diagnosis of von Willebrand Disease. vWF: Ag is usually measured by ELISA, a time consuming and complex assay. A new fully automated latex immunoturbidimetric assay is presented for the rapid measurement of vWF concentrations in citrated human plasma.

Aims: To report performance characteristics of this new vWF: Ag assay used on various coagulation analyzers.

Methods: Normal and pathological lyophilized plasmas are used to evaluate within‐ (n=40) and between‐run precision (n=30), linearity, dynamic range and on‐board stability. Measurements are performed with CS‐5100 and STA‐R instruments. Comparison study with commercial assays (Liatest vWF: Ag, Stago, with STA‐R or vWF Ag kit, Siemens, with CS‐5100) are performed on plasmas from healthy subjects, vWD, or plasmas with high pathological levels of vWF.

Results: A very low coefficient of variability is obtained on the various analyzers, with normal and abnormal plasma controls, respectively of 2.2% and 4.6% for within‐run, and 2.6% and 3.6% for between‐run. On CS‐5100, the dynamic range is from 3 to 600 IU/dL, and up to 1600 IU/dL with automatic redilution. No interference is observed for hemoglobin (10 g/L), bilirubin (60 mg/dL) and intralipids (1000 mg/dL). The kit is stable for 15 days on‐board, with a shelf life of 18 months at 2‐8°C in the original packaging. A good correlation with predicate devices was obtained on CS‐5100 (y = 1.03x+0.49, r= 0.998, P<0.001) and on STA‐R (y = 1.03x‐5.25, r= 0.992, P<0.001). High concentrations are directly and correctly measured, without risk of hook effect.

Conclusions: LIAPHEN vWF: Ag is a convenient automated assay, with extended reagent stability, safe and effective to measure vWF: Ag. The extended dynamic range is of high value for the diagnosis of vWD, as well as for high to very high vWF concentrations, e.g. in inflammatory diseases (sepsis, hepatic diseases, DIC…).

PB251: External Quality Assurance (EQA) for the Rotem Sigma Thromboelastometry Device

D. Kitchen ; S. Munroe‐Peart; L. Brown; I. Jennings; S. Kitchen; T. Woods; I. Walker

UK NEQAS for Blood Coagulation, Sheffield, United Kingdom

Background: Thromboelastometry are Point of Care testing (POC) technologies for global haemostasis tests. Thromboelastometry performed using the Rotem ® devices measures the elasticity of a developing clot. The Sigma device is an update on predecessor devices such as the Rotem Delta as it uses cartridge based technology with no pipetting required. This is important as the majority of devices are in theatres and specialist wards and usually operated by staff without laboratory training.

Aims: A pilot study for EQA of the Sigma device involving 14 centres was performed by UK national external quality assessment services for Blood Coagulation (NEQAS BC).

Methods: Lyophilised plasma samples were distributed, with a pre measured diluent and pipette plus an applicator to allow the sample to be applied to the device such that no laboratory equipment was needed. 2 types of cartridge are available, and users were asked to test on the cartridge that they employed. The ‘Rotem Sigma complete cartridge’ has FIBTEM, EXTEM, INTEM and APTEM reagents whilst the ‘Rotem Sigma complete +hep’ has FIBTEM, EXTEM, INTEM and HEPTEM reagents. Data were collected for each of these reagents for: Clotting time (CT), clot formation time (CFT), angle, amplitude at 10, 15 and 30 minutes (A10, A15, A30).

Results: Results were received from 11 centres that performed FIBTEM, EXTEM and INTEM. 6 centres performed HEPTEM and 5 APTEM. Coefficients of variation (CVs) for CT ranged from 4.7% to 13.5%. CVs were lower compared to previous data from UK NEQAS BC Rotem Delta programme for INTEM, EXTEM and HEPTEM, which has seen CVs range from an average of 12.0%(HEPTEM) up to an average of 44.5% (EXTEM). Improved precision could be due to the simplicity of the cartridge based system for non‐laboratory users.

Conclusions: Further studies are planned with a view to providing a full EQA programme for this device. A new cartridge based thromboelastography(TEG6) device is also now available and pilot EQA studies will be carried out for this device as well.

PB252: Stored Calibration Curves Can Deliver Acceptable Accuracy and Precison on CS5100 Analysers

A. Sermon‐Cadd 1 ; J. Smith 1 ; A. Bowyer 2 ; S. Kitchen 2

1 Northern General Hospital, Coagulation, Sheffield, United Kingdom, 2 Royal Hallamshire Hospital, Sheffield, United Kingdom

Background: We have previously demonstrated that between assay precision is improved by use of a truncated 3 Point calibration curve run alongside patient and IQC samples during assay of some clotting factors. For example between assay precision of one stage FIX and FXI assays improved from 12% and 5% to 2,5% and 2.6% respectively (WFH Glasgow 2018). These data were generated using Actin FS on CS51000 analyser with assays performed by 15 different staff during a 24/7 service.

Aims: We aimed to assess the effect of the same calibration approach in a second laboratory site.

Methods: We included a truncated 3 Point calibration curve for FVIII, FIX, FXI and FXII by one stage assay alongside test samples using Actin FS on a CS5100 analyser. Assays were performed during monday to friday routine hours by 4 different staff with >15 years haemostasis laboratory experience. In this calibration model patient and IQC results were adjusted by a correction factor calculated as manufacturers assigned reference plasma value / analyser result for calibration plasma during the assay run.

Results: The CV of IQC results was similar before and after correction for FVIII FIX and FXI but was improved for FXII (see table). No FVIII/IX/XI assay results were changed by more than 8% by use of a correction factor. Assay results were both accurate and precise when derived from the full (7‐9 Point) calibration curves stored in the analyser.

TABLE 1 Factor number of assays correction factors uncorrected mean for IQC (IU/ml) Uncorrected CV for IQC (%) Corrected Mean for IQC (IU/ml) Corrected CV for IQC(%) FVIII 33 0.97‐1.07 0.38 2.8 0.39 3.3 FIX 34 0.94‐1.10 0.76 3.1 0.77 2.8 FXI 16 0.92‐1.05 0.61 2.7 0.62 1.8 FXII 9 0.86‐1.08 1.08 8.1 0.98 1.6

Conclusions: Our results show that one stage assays for FVIII,IX or XI performed using Actin FS on CS5100 analysers can be sufficiently reproducible for stored calibration curves to be used.

PB253: Measurement of Immunoglobulins Anti VWF in Patients with Probable AVWS with an Enzyme‐linked Immunosorbent Assay (ELISA) Using Platelet VWF as an Antigen

A.C. Kempfer 1 ; A.I. Woods 1 ; J. Paiva 2 ; M.M. Casinelli 2 ; A. Sanchez‐Luceros 1,2 ; M.A. Lazzari 1

1 IMEX‐CONICET‐Academia Nacional de Medicina, Laboratorio de Hemostasia y Trombosis, Ciudad Autonoma de Buenos Aires, Argentina, 2 Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Hemostasia y Trombosis, Ciudad Autonoma de Buenos Aires, Argentina

Background: Autoantibodies play a role in the pathogenesis of some patients with AVWS. Inhibitory antibodies (Ab) can be detected in mixing studies with normal plasma (KPTT; VWF:RCo) and presence of non‐neutralizing Ab can be detected by ELISA.

Aims: The detection of immunoglobulin (IgG, IgA or IgM) in plasma or anti‐platelet (pt) or anti‐VWF, the elevated VWFpp ratio and the absence of responsible mutations are useful tools to discriminate inherited VWD from AVWS. We showed here our results in detecting anti VWF antibodies by ELISA in 6 patients (P) with probable AVWS and one P VWD 2M.

Methods: ELISA measurement of anti‐VWF antibodies.

We developed an ELISA technique to detect anti‐VWF Ab as described by Siaka et al (2003), but with modifications: Covalink plates were coated overnight at 4°C with 100 μL of pt VWF (obtained from 20 ml blood, pt washed three times and frozen and thawed 30 times) used at a final concentration of 4 IU/dL VWF:RCo. The 99th percentile of the distribution of values obtained in 20 healthy controls (normalized OD units) was assumed as cut‐off for normal levels of: median IgG 0.34 normalized OD units, median IgA 0.72 normalized OD units and median IgM 1.00 normalized OD units anti‐VWF.

Results: When we used VWF from the Hemate‐P as Ag, the P 7 had a median IgM anti‐VWF of 2.2 (cut off 0.98) different value that observed in table, median IgM anti‐VWF=1.00 (cut off 1.00). The same behavior had a P VWD type 3 median IgG anti‐VWF=1.2 (cut‐off 0.98), while with pt VWF as Ag, median IgG anti‐VWF was 0.34 (cut‐off 0.34).

TABLE 1 Different tests made to prove or rule out the diagnosis of AVWS in 6 P from IIHema Patients Sex‐Age (years old) 1 F‐58 2 F‐45 3 F‐18 4 M‐47 5 M‐51 6 F‐41 7 M‐47 KPTT (s) P+N 66 No correction 196 No correction 42 correction 66 correction 53 No correction 53 correction 46 VWF:Ag (IU/dL) 18 126 25 9 20 9.5 73 VWF:RCo (IU/dL) P+N No detection No correction <10 No correction <10 No detected <10 No correction <10 correction 10 VWFpp/VWF:Ag ratio 13.3 Not done 2.9 13.2 8.20 12.2 1.96 VWF multimers ultralarge Not done absence large normal ultralarge absence large+ intermediate normal molecular biology Normal exons 27‐28 Not done Normal exon 28 Normal exons 26‐27‐28 Normal exons 27‐28 Normal exons 13,14, 26‐28 p.T1468I ANTI VWF cut‐off=0.34‐0.72‐1.00 (IgG‐IgA‐IgM) 0.40‐0.27‐0.33 0.86‐2.38‐2.14 0.46‐0.78‐0.74 0.25‐0.32‐0.35 0.26‐0.44‐1.00 0.23‐1.00‐0.44 0.23‐0.70‐1.00 Other methods Not done Not done Not done Increased titles of IgG kappa IgM asociated to Pt (Pt count: 39 109/L) Not done Not done Probable diagnostic AVWS (neutralizing Ab) AVWS Lupus anticoagulant) AVWS non‐neutralizing Ab) AVWS (monoclonal gammopathy) AVWS (Ab associated to pt) AVWS (gammopathy monoclonal) VWD2M

Conclusions: Plasma‐derived VWF contains ABO blood group Ag and should not be used as an Ag for ELISA to detect Ab because the presence of isoagglutinins may cause false positive results (Tiede, 2011). We believe that our election of pt‐VWF as a reagent (Ag) has resolved this issue because A or B blood group antigens were not detected in pt (Sarode, 2010).

PB254: Performance Evaluation of Automated Thrombin Generation Measurement on Ceveron ® alpha TGA

L. Wagner ; N.B. Binder

Technoclone Herstellung von Diagnostika und Arzneimitteln GmbH, Vienna, Austria

Background: Fully automated thrombin generation on Ceveron ® alpha TGA is a valuable tool for investigating blood coagulation activity, regarding elevated thrombin activity ‐ thrombotic tendency ‐ or depressed activity ‐ bleeding tendency ‐ when using standardized Ceveron ® TGA kits. To enter routine use precise measurements between laboratories is mandatory.

Aims: Aim of the study was to evaluate assay performance in regards of accuracy and precision between runs and between platforms.

Methods: To evaluate assay performance 2 lyophilized control samples were measured with Ceveron ® TGA RB, Ceveron ® TGA RCL and Ceveron ® TGA RCH assay kit, each on 3 different analyzer. Accuracy and precision were calculated for TGA parameters Peak thrombin, AUC and lag time.

Results: Calibration curves on each Ceveron ® alpha TGA platform were made one at the beginning of the study using the standard analyzer settings for TGA calibration. The recovery of control were within 100% ± 10% of target value for all concentrations and for all 3 parameters, tLag, Peak and AUC, measured on the 3 platforms. Precision was very good with between‐run and between‐platform variations of < 15% for tLag, Peak and AUC

Conclusions: Our data demonstrate that using the standardized assay kits Ceveron ® TGA RB, Ceveron ® TGA RCL and Ceveron ® TGA RCH in optimized settings on Ceveron ® alpha TGA the determination of thrombin generation parameters can be performed with very good performance and excellent comparability between platforms.

PB255: Calibrations Performed in the Lab Fail to Correct Fibrinogen Quality Control Variability Generated by Different Reactant Batches

F. Guerrero 1 ; F. Dubois 1 ; C. Bressans 1 ; M. Lebon 1 ; A. Ribes 1 ; S. Savy 2 ; N. Savy 3

1 CHU de Toulouse, Toulouse, France, 2 Estrials, Toulouse, France, 3 Centre International de Mathématiques et d'Informatique, Toulouse, France

Background: Result variability is a normal phenomenon in clinical biology. It is considered acceptable when results obtained from single quality control (QC) batches fall within predefined limits. If not, it is standard practice to perform a new calibration. However, little is known about the efficacy of repeated calibrations on QC stability.

Aims: To test if repeated calibrations are effective in controlling the variability generated by different batches on internal QC results for fibrinogen dosing.

Methods: Consecutive QC results were obtained in our laboratory between January 2015 and January 2018 on 4 STAR Coagulometers using STA reactants (Diagnostica Stago). The number of reactant batches was decreased over time (15 over 13 months then 3 over 12 months then 1 in the last 12 months). Each coagulometer was calibrated for each new reactant batch. The effect of different batches, coagulometers, operators and locations on QC results was studied using generalized mixed effects models. All calculations were performed with R (The R Foundation for Statistical Computing 2017).

Results: A total of 14014 measurements were performed. Figure 1 shows the mean QC results over time for the different coagulometers (one color each) and for 3 QC batches. A change in reactant batch was the strongest factor of variability in multivariate analysis (p< 0.001). The maximum calibration bias (10.1%, p< 0.001) was observed when batches were changed frequently. The calibrations performed with the manufacturer's standard could not prevent this variability (figure 1.A) but reducing the number of batches did (figure 1.C). This variability was not seen for lower fibrinogen concentrations.

FIGURE 1 Changes of the mean QC results over time. Each color represents a coagulometer. Each box represents a single QC batch

Conclusions: For fibrinogen, calibration performed with standard plasma does not prevent excessive variability resulting from new batches and may explain the important bias observed in a large national French study performed in 2014 (14.8%). Reducing the number of batches significantly reduces this bias and improves QC stability.

PB256: System Performance Evaluation of the Cobas t 711 and Cobas t 511 Analysers

A. Lowe 1 ; S. Kitchen 1 ; R. Jones 1 ; M. P.M. de Maat 2 ; M. Nagler 3 ; G. Rozsnyai 4

1 Sheffield Haemostasis and Thrombosis Centre, Sheffield, United Kingdom, 2 Erasmus University Medical Center, Rotterdam, the Netherlands, 3 INSELSPITAL University Hospital, Department of Haematology and Central Haematology Laboratory, Berne, Switzerland, 4 Roche Diagnostics International Ltd, Rotkreuz, Switzerland

Background: The fully automated cobas t 711 and cobas t 511 coagulation analysers perform qualitative and quantitative in‐vitro coagulation analyses to aid the diagnosis of coagulation abnormalities and monitor anti‐coagulation therapy.

Aims: This validation study evaluated the functionality, reliability and analytical performance of cobas t 711/511 analysers in a clinical laboratory setting under routine‐like conditions at three European sites.

Methods: Anonymised human citrated plasma samples were evaluated on cobas t 711/511 analysers using nine coagulation tests. Functionality and analytical performance were monitored by daily quality control (QC) runs assessed twice daily. Intermediate precision was assessed over 21 days using control samples according to Clinical and Laboratory Standards Institute EP05‐A3 guidelines. Routine Simulation Series (RSS) were used to confirm absence of random errors when running the two analysers under routine‐like conditions. Routine workloads of the laboratory were replicated and re‐processed on the cobas t 711/511 analysers with the aim of verifying the performance by re‐measuring all left‐over specimens from the respective routine run.

Results: All samples tested showed a stable QC performance over ˜12 weeks. Of 4216 results, 19 were outside the ±2 SD (±10%) range due to reagent (or control material) related issues. Out of range results were repeated according to the QC re‐run rules; all were resolved. No QC recovery issues due to reagent/instrument malfunction. All assays passed acceptance criteria for repeatability, intermediate and total precision of the respective assay (Table 1). For RSS, good comparability between runs was shown when testing single samples under random mode conditions (Table 2), no system malfunctions observed.

TABLE 1 Intermediate Precision (21‐day CLSI EP05‐A3 precision) Assay CV% day‐to‐day, cobas t 711 CV% day‐to‐day, cobas t 511 CV% within run, cobas t 711 CV% within run, cobas t 511 CV% total (within laboratory), cobas t 711 CV% total (within laboratory), cobas t 511 Acceptance criteria aPTT 0.0‐0.5 0.2‐0.3 0.2‐0.4 0.3‐0.4 0.4‐0.7 0.4‐0.6 5.0 aPTT Lupus 0.1‐0.7 0.4‐0.6 0.3‐0.6 0.4‐0.5 0.6‐1.4 0.8‐1.3 5.0 aPTT Screen 0.0‐0.6 0.0‐0.3 0.3‐0.9 0.6‐1.0 0.5‐1.3 1.0‐1.3 5.0 D‐Dimer 0.0‐0.0; 0.0‐0.0 0.6; 1.0 1.4‐1.5; 0.6‐1.5 1.5; 0.9 1.5‐2.7; 1.0‐2.0 2.4; 2.0 6.0; 4.0 Fibrinogen 0.0‐1.5 0.0‐1.0 1.2‐3.0 1.7‐2.6 1.8‐3.6 2.0‐3.7 5.0 PT‐derived fibrinogen 0.0 0.8 1.5‐2.5 2.1 2.3‐2.9 2.2 7.0 PT Owren 0.0‐0.9 0.6‐1.3 0.4‐1.3 0.7‐0.9 1.0‐2.2 1.8‐2.2 5.0 PT 0.0‐0.6 0.0‐0.4 0.3‐1.1 0.3‐0.5 0.5‐1.8 0.6‐1.8 5.0 Thrombin time 0.6‐0.9; 0.0‐1.2 0.9; 0.7 1.0‐2.0; 1.1‐2.3 1.1; 1.4 1.5‐2.1; 1.9‐2.5 1.6; 1.8 5.0; 8.0

TABLE 2 Summary of Routine Simulation Series (RSS) method comparison analysis of the cobas t 711/511 analysers RSS Correlation (Pearson's r), cobas t 711 Correlation (Pearson's r), cobas t 511 Slope (Passing‐Bablok), cobas t 711 Slope (Passing‐Bablok), cobas t 511 Intercept, cobas t 711 Intercept, cobas t 511 aPTT 0.992‐1.000 0.980 0.951‐0.987 0.937 0.132‐0.337 0.444 aPTT Lupus 0.997‐1.000 0.998 0.950‐1.002 0.980 −0.0431‐1.21 ‐0.052 aPTT Screen 0.998‐0.999 0.999 0.985‐1.005 1.000 −0.100‐0.454 0.000 D‐Dimer 1.000 1.000 0.992‐1.000 1.002 −0.00642‐0.00799 0.00145 Fibrinogen 0.994‐0.999 ‐ 1.006‐1.032 ‐ −12.1 to −0.0774 ‐ PT‐derived fibrinogen 0.995‐0.999 0.998 0.987‐1.000 0.993 −2.00‐2.75 0.0987 PT Owren 0.999‐1.000 1.000 1.000‐1.004 1.000 −0.0500‐0.0133 0.100 PT 1.000‐1.000 1.000 0.991‐1.000 0.988 0.000‐0.0313 0.0702 Thrombin time 0.967‐0.991 0.952 0.968‐1.023 1.040 −0.476‐0.525 ‐1.18

Conclusions: The performance of the cobas t 711/511 analysers was excellent and the analysers are suitable for the accurate and reliable measurement of coagulation parameters in routine clinical practice.

PB257: Variations in Automated Platelet Counts Occur Depending on the Principle Used for Cell Counting and on the Non‐platelet Blood Cell Parameters

G.K. Viswanathan ; R. Shekar; S. Pai

Manipal Hospitals, Department of Laboratory Medicine, Bangalore, India

Background: Automated blood cell counters use different principles for platelet counting namely impedance, optical properties (light scatter) and fluorescence using platelet specific dyes. Variation in platelet counts can occur among these different methods and there is a potential for erroneous reporting.

Aims: To compare the automated platelet counts using the 3 methods (impedance: PLT‐I, optical: PLT‐O and fluorescence: PLT‐F) and to study the effect of non‐platelet parameters on platelet counts in each method.

Methods: Complete blood counts including platelet counts (all 3 methods) were done in EDTA‐anticoagulated blood samples of 100 patients (different clinical conditions) in a Sysmex XN‐1000 automated blood cell counter. All ages were represented and were sex matched.

Results: No major difference in the platelet count (difference < 10,000/cu.mm) was observed in two‐third of the samples. One third samples (n=33) showed major difference in platelet count (difference >10,000/cu.mm) among the three methods; in most of these samples the platelet count was on the higher side of normal or increased; a single case had thrombocytopenia and this created potential clinical difficulty regarding prophylactic platelet transfusion. There was no significant difference in the mean platelet count among the three methods. PLT‐F was higher than PLT‐I and PLT‐o in samples with high WBC count and in samples with reticulocytosis, high immature reticulocyte fraction and macrocytosis. PLT‐I was higher than PLT‐O and PLT‐F in samples with high RBC count. PLT‐O was lower compared to other two methods in most cases.

Conclusions: Minor differences in platelet counts are seen among different platelet counting methods in automated cell counters. Extremes of RBC and WBC counts can significantly interfere with platelet counting depending on the method employed. Cross checking the platelet counts by all three methods might be necessary in select clinical scenarios.

PB258: Accuracy and Precision of Point‐of‐Care International Normalized Ratio (INR) in Pediatric and Adult Patients with Liver Disease Pilot Study

P.S. Kawada ; M. Bauman; J. Yap

University of Alberta/Edmonton Clinic Health Academy, Pediatrics, Edmonton, Canada

Background: Point‐of‐Care (POC) International Normalized Ratio (INR) testing using the CoaguChek ® system is accurate and precise for monitoring vitamin K antagonist therapy. Patients with liver disease frequently require INR monitoring as it informs on progression and severity of disease. The mechanism by which oral anticoagulants increase the INR is different from the pathophysiology behind a raised INR in liver disease. Therefore, it should not be assumed that the CoaguChek ® system is applicable to patients with liver disease.

Aims: To determine if the CoaguChek XS Pro can precisely and accurately measure the INR compared to the gold standard laboratory INR in pediatric and adult patients with liver disease.

Methods: We have an ongoing prospective cohort study of pediatric and adult liver disease patients including healthy controls. The CoaguChek XS Pro INR obtained via fingerprick is compared to the gold standard venipuncture INR. The data will be analyzed using Bland‐Altman plots. Informed consent will be obtained for all patients and the study has been approved by our local ethics committee.

Results: We have preliminary data for 15 controls and 41 with liver disease (14/41 pediatrics). The Mann‐Whitney comparison between the POC INR and laboratory INR was not statistically significant. There was strong correlation between POC INR and laboratory INR (r= 0.92, p < 0.0001). For all subjects, the average difference between the POC INR and laboratory INR/average % and Bland‐Altman's 95% limits of agreement was ‐2.3 (‐20.2; 15.7) (Figure 1). The data was closer to the line of unity when the INR was ≤1.5.

FIGURE 1 Bland Altman Plot of POC INR and Laboratory INR. Controls (open squares), Pediatrics (purple squares), Adults (yellow circles)

Conclusions: Preliminary results show the POC INR is accurate compared to the laboratory INR when the INR is ≤1.5. Data collection for this study is still ongoing. POC INR in liver disease would offer another modality to measure the INR.

PB259: Clearance of Mouse VWF Propeptide Is Mediated by Stabilin‐2 and Is Regulated by N‐linked Glycan Expression

O. Rawley ; C. Brown; C. Dwyer; K. Nesbitt; D. Lillicrap

Queens University, Kingston ON, Kingston, Canada

Background: The ratio between the von Willebrand factor propeptide (VWFpp) and von Willebrand factor (VWF) is widely used as a marker for VWF clearance. Despite this however, the mechanisms regulating clearance of the VWF propeptide itself remain unknown.

Aims: To investigate the mechanisms underlying VWFpp clearance.

Methods: Mouse VWFpp (mVWFpp), human VWFpp (huVWFpp) and variants thereof were expressed in a HEK293T expression system and subsequently infused via tail vein injection (200U mVWFpp/kg) into VWF ‐/‐ or Stab2 ‐/‐ /VWF ‐/‐ mice. Mice were retro‐orbitally sampled and VWFpp was quantified by ELISA.

Results Clearance studies in VWF ‐/‐ mice demonstrated rapid clearance of both mVWFpp and huVWFpp. Poor recovery was observed at 5 minutes post‐infusion with residual plasma levels of 24.2±1.8% and 33.4±3.1% respectively. Interestingly, co‐infusion with propeptide‐deleted mouse VWF (Δpro‐mVWF) prolonged mVWFpp survival (recovery 77.9±7.6%, p=0.0019). Co‐infused mVWFpp was cleared with a half‐life of 15.35 minutes. Subsequent clearance studies in Stab2 ‐/‐ /VWF ‐/‐ mice revealed significantly prolonged survival of mVWFpp but not huVWFpp. mVWFpp recovery values at 5 minutes were ˜3.5‐fold greater than those observed in VWF ‐/‐ mice (84.4±5.6% vs. 24.2±1.8%, p=0.0002). mVWFpp was cleared with a half‐life of 16.3 minutes indicating that stabilin‐2 deficiency significantly prolonged mVWFpp survival.

To further investigate the underlying mechanisms, a role for N‐linked glycans was examined. PNG‐mVWFpp was rapidly cleared in Stab2 ‐/‐ /VWF ‐/‐ mice (t1/2 7.4 minutes p=0.0005). Conversely, targeted removal of the N‐linked glycan at Asn156 via site‐directed mutagenesis served to significantly prolong mVWFpp survival (t1/2 19.81 minutes). Importantly however, this effect was not observed in VWF ‐/‐ mice suggesting that stabilin‐2 is a more dominant modifier of plasma mVWFpp levels than N‐linked glycan expression.

Conclusions: Clearance of mVWFpp is regulated by stabilin‐2 and is modulated at least in part by N‐linked glycans.

PB260: Accumulation of Rare Variants in STAB2 in Type 1 von Willebrand Disease Patients

E. Manderstedt 1 ; C. Lind‐Halldén 1 ; J. Flygare 1 ; S. Lethagen 2,3 ; C. Halldén 1

1 Kristianstad University, Kristianstad, Sweden, 2 National Haemophilia Center, University Hospital, Rigshospitalet, Copenhagen, Denmark, 3 Sobi Biovitrum AB, Stockholm, Sweden

Background: Quantitative deficiencies in von Willebrand factor (VWF) arise from changes in its biosynthesis, secretion and clearance. Low VWF levels are associated with an abnormal bleeding phenotype, von Willebrand disease (VWD). Type 1 VWD is characterized by reduced levels of functionally normal VWF. Genome‐wide association studies have identified common variants in VWF, ABO, STXBP5, SCARA5, STAB2, STX2, TC2N, CLEC4M and UFM1 to be associated with lower VWF‐levels.

Aims: The aim of the present study was to investigate a Swedish type 1 VWD population of 106 patients for rare variants in the 9 genes previously associated with VWF level variation.

Methods: Targeted re‐sequencing through AmpliSeq‐based Ion Torrent sequencing using Sanger sequencing as confirmation.

Results: Next generation sequencing data showed an overall high coverage and mean read depth. Candidate VWF mutations were identified in 51 index cases (48%) and 75 index cases had blood group O (71%). Most common alleles previously associated with lower VWF levels were enriched in the population, but none except the 0‐allele was significant. Excluding the VWF gene, a total of 29 rare variants were detected in the 8 remaining genes. A highly significant accumulation of rare variants was observed in STAB2 through comparison with a normal population consisting of data from the Genome Aggregation Database ( P = 2.3*10 ‐05 ). Twelve rare variants, 8 of which were considered damaging by functional prediction were detected in STAB2 . Additional rare variants predicted to be damaging were detected in STX2 and TC2N . Sanger sequencing was used to confirm most rare variants and complete re‐sequencing of CLEC4M and STXBP5 showed complete concordance with the next generation sequencing results.

Conclusions: VWF mutations, an accumulation of common variants associated with low VWF‐levels, especially the O blood group, and rare variants in STAB2 all contribute to the bleeding phenotype in type 1 VWD patients.

PB261: Expression of the SCARA5 Scavenger Receptor in the Human Kidney and Spleen May Regulate Clearance of VWF

L.L. Swystun 1 ; O. Rawley 1 ; J.D. Lai 1 ; F. Lassalle 1 ; A. Michels 1 ; K. Ogiwara 1 ; C. Notley 1 ; C. Dwyer 1 ; K. Nesbitt 1 ; J. Ojala 2 ; K. Tryggvason 2 ; D. Lillicrap 1

1 Queen's University, Department of Pathology and Molecular Medicine, Kingston, Canada, 2 Karolinska Institutet, Department of Medical Biochemistry and Biophysics, Stockholm, Sweden

Background: Plasma levels of VWF:Ag have been shown to associate with genetic variability in the gene for scavenger receptor A5 ( SCARA5 ). Our previous studies have demonstrated that SCARA5‐expressing cells are capable of binding and internalizing VWF in vitro . However, as SCARA5 is expressed on interstitial fibroblasts and epithelial cells in mice, its ability to regulate VWF clearance in vivo is unknown.

Aims: To assess the influence of SCARA5 on VWF clearance in vivo and to understand where SCARA5 is expressed in human tissues.

Methods: Plasma levels of VWF:Ag and VWFpp were measured in SCARA5 KO and wild‐type C57Bl/6 mice. VWF/SCARA5 DKO mice were generated and their VWF‐clearance phenotype was characterized. Human SCARA5 protein and mRNA expression was characterized in human testes, liver, spleen, and kidney tissues.

Results: Plasma VWF:Ag, VWFpp, and VWFpp/VWF:Ag ratio was not significantly different in SCARA5 KO mice compared with wild‐type controls. The half‐life of murine plasma‐derived VWF was not prolonged in VWF/SCARA5 DKO mice compared with VWF KO controls (5.99 vs. 5.98 hours). The half‐life of human plasma‐derived VWF was modestly increased in VWF/SCARA5 DKO mice compared with VWF KO controls (66.7 vs. 84.9 min, p=0.026).

SCARA5 mRNA analysis of human tissues determined that SCARA5 expression is highest in the testes, followed by the spleen and kidneys, with low expression detected in the liver and peripheral blood mononuclear cells. Immunohistochemical analysis of human tissues demonstrated moderate expression of SCARA5 in the red pulp of the spleen, and kidney glomeruli, and low expression in the liver. Infusion of human plasma‐derived VWF into VWF KO mice demonstrated that cells within the spleen as well as the kidney glomeruli are capable of endocytosing VWF.

Conclusions: Murine SCARA5 weakly modifies the half‐life of human but not murine VWF. Expression of SCARA5 in the spleen and/or kidney may modify plasma VWF levels in humans.

PB262: Diverse Inheritance of VWD Found in Type 3 U.S. Families in the Zimmerman Program

P. Christopherson 1 ; D. Bellissimo 2 ; G. Humphreys 2 ; C. Perry 1 ; V. Flood 1,3 ; J. Gill 1,3 ; S. Haberichter 1 ; R. Montgomery 1,3 ; Zimmerman Program Investigators

1 BloodCenter of Wisconsin, Blood Research Institute, Milwaukee, United States, 2 University of Pittsburgh, Obstetrics, Gynecology and Reproductive Sciences, Pittsburgh, United States, 3 Medical College of Wisconsin, Hematology/Oncology, Milwaukee, United States

Background: Type 3 (T3) von Willebrand Disease (VWD) is characterized by absence of von Willebrand factor (VWF). Inheritance is typically recessive but may appear co‐dominant when a parent is affected with type 1 VWD.

Aims: As part of the Zimmerman Program we sought to characterize sequence variants (SV) and inheritance pattern found in T3 VWD families.

Methods: 40 unrelated T3 index Cases (IC) enrolled in the Zimmerman program had full‐length VWF sequencing; VWF gene microarray was performed if 2 SVs weren't identified. Family members (FM) had targeted sequencing. Bleeding symptoms were quantified using ISTH bleeding score (BS). Clinical testing included FVIII, VWF:Ag, VWF:RCo and VWFpp. SV were analyzed using Fabric Genomics Opal variant interpretation software to determine frequencies and pathogenicity.

Results: SV were identified in 38 subjects (95%): 80% had 2 SVs, 15% had only 1, and 5% had none (NSV); 70 of the 80 SVs expected were identified (88%). 80% were null alleles and 19% were missense variants predicted to be deleterious by multiple algorithms. Of the 53 discreet SV, 6 were null SV found in multiple families where expressivity of the same SV alone varied from normal to LVWF to type 1. Co‐dominant inheritance was found in 64% of families, recessive in 25% and 11% showed evidence of both. In families with NSV or only 1 SV, the NSV allele was linked to type 1/LVWF in 3 families and to normal obligate carriers (OC) in 3 families. T3 carriers with type 1/LVWF (n=56) had mean VWF:Ag 37, FVIII 68 and BS 1 which was slightly different from type 1 carriers (n=537) with VWF:Ag 33, FVIII 57 and BS 2. OC without evidence of VWD (n=23) had mean VWF:Ag 79 and FVIII 122 that was significantly different (p< 0.01) from healthy controls, however BS showed no difference.

Conclusions: While diagnosis of type 3 VWD may be straightforward, the inheritance patterns are more complicated. Expressivity of carriers with null alleles was variable, possibly due to the influence of age, blood type or carbohydrate modifications.

PB263: Despite Lower von Willebrand Factor Values, O Blood Type Is Not a Risk Factor for Bleeding Complications Following Tonsillectomy +/‐ Adenoidectomy

N. Archer ; P. Forbes; J. Dargie; J. Manganella; G. Licameli; M. Kenna; C. Brugnara

Boston Children's Hospital/Harvard Medical School, Boston, United States

Background: ABO blood type is known to affect von Willebrand factor (VWF) values in plasma, with O blood type subjects having approximately 75% of values of the other blood types. We sought to understand if blood type is associated with an increased risk of bleeding after tonsillectomy +/‐ adenoidectomy (T&A).

Aims: The objective of our study was to determine the incidence of post‐T&A hemorrhage among children with blood type O and those with blood type non‐O.

Methods: We conducted a retrospective cohort study of all pediatric subjects that underwent T&A as well as re‐cauterization for bleed from January 1, 2008 to August 7, 2017 at our hospital. The primary study outcome was defined by a return to our hospital for cautery within a month of initial T&A. Additional variables collected included age, sex, ethnicity, blood type, date of surgery, VWF:Ag, VWF:RCo and FVIII:C values, and test date of VWF panel. Statistical analysis used SAS version 9.3. The Fisher's exact test was used compare groups on categorical variables.

Results: A total of 14,977 T&As were performed during our study period. 579 (3.86%) had a post‐T&A hemorrhage. In selected groups of children, including those with a recorded blood type (1,294 children) or those with a VWF panel (909 children) in our electronic medical record, post‐T&A hemorrhage was more common. Bleeding rates were similar in blood type O or blood type non‐O (Table 1).

TABLE 1 Bleeding after tonsillectomy by blood type Blood type Bleeder (%) Non‐Bleeder (%) P‐value O 94 (15) 547 (85) 1.0000 Non‐O 95 (15) 558 (85) Total 189 1105

179 subjects had both a blood type and VWF panel. Those with blood type O had lower baseline VWF values than those with blood type non‐O, however bleeders and non‐bleeders with blood type O or non‐blood type O did not have significantly different VWF values (Table 2).

TABLE 2 VWF and disease categories of patient with blood type O and non‐O Blood type O Blood type O Blood type non‐O Bleeders Non‐Bleeders P‐value Bleeders Non‐Bleeders P‐value Mean VWF:Ag (SD) 87.6 (30) 93.2 (39) 0.4922 122.5 (63.7) 115.6 (61.7) 0.9260 Mean VWF:RCo (SD) 76.4 (33) 77.6 (40) 0.8788 121.1 (45.9) 122.2 (47.3) 0.6887 Mean FVIII (SD) 107.8 (28.7) 106.2 (37.6) 0.8161 138.1 (51.6) 125.6 (48.8) 0.3333 Normal (%) 22 (30) 51 (70) 23 (38) 38 (62) Low (%) 6 (17) 29 (84) 2 (33) 4 (67) Disease (%) 1 (33) 2 (67) 0 1 (100)

Conclusions: We found no difference in the incidence of post‐T&A hemorrhage in those with blood type O and blood type non‐O, despite a greater incidence of bleeding post‐T&A in low VWF (low VWF or VWD) values versus the overall population. This suggests that blood type cannot be used to determine risk of bleeding in subjects with low VWF values.

PB264: High Molecular Weight von Willebrand Factor Multimers in Cryoprecipitate Exceed that Found in a Commercial Concentrate

M.E. Colling 1 ; K.D. Friedman 2 ; W.S. Dzik 1

1 Massachusetts General Hospital, Boston, United States, 2 BloodCenter of Wisconsin, Milwaukee, United States

Background: Patients with von Willebrand's disease (VWD) often require treatment with supplemental von Willebrand factor (VWF). Commercial VWF concentrates have replaced cryoprecipitate as the mainstay of therapy. However, a direct comparison of the VWF content and multimer distribution present in concentrates versus cryoprecipitate has not been reported.

Aims: To characterize the VWF content and multimer distribution of cryoprecipitate versus a plasma‐derived VWF concentrate.

Methods: Three pools of group A and three pools of group O cryoprecipitate were thawed, and three vials of VWF concentrate were reconstituted according to the manufacturer's directions. Aliquots were frozen, coded, and sent to a national reference laboratory for simultaneous testing of factor VIII (FVIII), VWF antigen (Ag), VWF collagen binding activity (CB), and VWF platelet binding activity by GPIbM ELISA (equivalent to ristocetin cofactor activity). VWF multimers were separated by SDS agarose gel electrophoresis and quantified using monoclonal antibodies with chemiluminescent detection and densitometry analysis. The mean ratio of CB to Ag, GPIbM to Ag, FVIII to Ag; and FVIII to GPIbM; and the percent high molecular weight (HMW) VWF were compared between the three groups by one‐way ANOVA and Tukey's post hoc test.

Results: The mean ratio of GPIbM to Ag and the percent HMW VWF in blood group A and O cryoprecipitate were significantly higher than in the VWF concentrate. Cryoprecipitate also contained significantly more FVIII relative to VWF Ag and GPIbM than the commercial concentrate. There was no significant difference in the ratio of CB to Ag in any of the groups. There was no difference between group A versus group O cryoprecipitate for each of the analyzed factors.

Conclusions: Cryoprecipitate contains more HMW VWF and higher GPIbM binding activity than a widely used plasma‐derived commercial concentrate. Treatment with cryoprecipitate may provide a more normal distribution of VWF multimers.

TABLE 1 VWF content of cryoprecipitate and commercial concentrate*

PB265: Safety of a High‐purity Plasma‐derived Factor X (pdFX) Concentrate over up to 4 Years of Treatment in a Variety of Clinical Situations in Adult and Pediatric Patients with Hereditary Factor X Deficiency (FXD)

G. Auerswald 1 ; K. Kavakli 2 ; R. Liesner 3 ; C. Akanezi 4 ; M. Norton 4 ; M.A. Escobar 5

1 Klinikum Bremen‐Mitte, Prof‐Hess‐Kinderklinik, Bremen, Germany, 2 Ege University Faculty of Medicine, Children's Hospital, Department of Pediatric Hematology, Izmir, Turkey, 3 Great Ormond Street Hospital, Haemophilia Comprehensive Care Centre, London, United Kingdom, 4 Bio Products Laboratory Ltd, Elstree, United Kingdom, 5 University of Texas Health Science Center and Gulf States Hemophilia and Thrombophilia Center, Houston, United States

Background: Hereditary FXD is a rare bleeding disorder in which spontaneous joint, muscle and mucocutaneous bleeds and intracranial haemorrhage are common. pdFX is a US‐ and EU‐approved treatment for hereditary FXD.

Aims: Evaluate pdFX safety in on‐demand, routine prophylactic and perisurgical use in 4 clinical studies and the postmarketing setting.

Methods: pdFX safety was assessed in 3 open‐label, multicenter, prospective phase 3 studies: in subjects aged ≥12 y as on‐demand treatment; in subjects aged ≥12 y as prophylaxis for excessive perisurgical bleeding; in children aged < 12 y as routine prophylaxis (RP).

Safety was assessed primarily through adverse events, FX inhibitors, and viral transmission. A retrospective study in subjects receiving pdFX as compassionate use assessed safety based on adverse reactions (ARs). All subjects gave informed consent; appropriate independent ethics committees approved protocols. Postmarketing data comprised spontaneously reported ARs.

Results: In clinical trials, 27 subjects (22 [81%] with severe FXD [basal FX:C < 1%]), including 18 adults/children aged ≥12 y and 9 children aged < 12 y, received pdFX for 2495 exposure days. Nineteen subjects received pdFX as RP, 24 as on‐demand treatment, and 8 as prophylaxis for 10 surgeries/procedures (some received >1 regimen). No significant changes were noted in vital signs or physical exams. Six treatment‐emergent ARs, none serious, were reported in 2 subjects. No evidence of inhibitor development was seen. As of September 16, 2017, an estimated 2925 pdFX infusions had been administered as marketed product (over 105 patient‐years). During this time, there were 3 case reports covering 9 nonserious ARs. No thromboembolic events were reported.

Conclusions: pdFX was well tolerated over 4 years in various clinical situations in subjects aged 1‐59 years. The reported ARs confirm pdFX's good safety profile in the postmarketing setting.

PB266: Low Immunogenicity of Two Plasma‐derived FVIII Products in Previously Untreated Patients with Non‐null F8 Mutations

A. Pavlova 1 ; S. Lowndes 2 ; L. Belyanskaya 2 ; M. Jansen 3 ; O. Walter 2 ; J. Oldenburg 1

1 Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany, 2 Octapharma AG, Lachen, Switzerland, 3 Octapharma AG, Vienna, Austria

Background: Inhibitors against coagulation factor VIII (FVIII) are a major complication of haemophilia A treatment. The recent SIPPET study showed that in previously untreated patients (PUPs) with non‐null mutations, none developed inhibitors when treated with plasma‐derived FVIII concentrates containing von Willebrand factor (pdFVIII/VWF).

Aims: We investigated whether other pdFVIII/VWF products showed the same pattern as those included in the SIPPET study.

Methods: We analysed the relationship between inhibitor development and F8 mutation type using data from studies of two pdFVIII products (with a VWF/FVIII ratio of 0.4 in product A, and of 1 in product B; both Octapharma) in PUPs with severe haemophilia A.

Results: Data were available for 50/51 PUPs treated with Product A, and 27/28 PUPs treated with product B. In the study reporting on product A, 9/50 (18.0%) patients had non‐null mutations, which are considered low risk for inhibitor development; in the study with product B the number was 2/27 (7.4%). In both studies, no patients with a non‐null mutation developed inhibitors. Of the 41 patients in the product A study with null mutations, which are considered high risk for inhibitor development, 5 (12.2%) developed inhibitors, of which 4 (9.8%) were high titre. Of the 25 patients in the product B study with null mutations, 8 (32.0%) developed inhibitors, of which 3 (12.0%) were high titre.

Conclusions: PUPs with non‐null F8 mutations did not develop inhibitors when treated with either pdFVIII/VWF Octapharma product. This is consistent with the post‐hoc analysis of the SIPPET trial, as well as a post‐hoc analysis of data from PUPs treated with simoctocog alfa (B‐domain deleted rFVIII of human‐cell line origin). Both pdFVIII/VWF products show similar behaviour to the pdFVIII/VWF products assessed in the SIPPET trial.

PB267: Pharmacodynamics of Recombinant Fusion Protein Linking Activated Factor VIIa to Human Albumin (rVIIa‐FP), Eptacog Alfa and Plasma‐derived Factor VII in Patients with Congenital FVII Deficiency

W.L. van Heerde 1 ; P.A. Holme 2 ; B. Laros‐van Gorkom 1 ; C. Joch 3 ; S. Puli 4 ; J. Roberts 4

1 Hemophilia Treatment Centre, Radboud University Medical Center, Nijmegen, the Netherlands, 2 Department of Hematology, Oslo University Hospital and Institute of Clinical Medicine University of Oslo, Oslo, Norway, 3 Clinical Development, CSL Behring, Marburg, Germany, 4 Clinical Pharmacology and Early Development, CSL Behring, King of Prussia, United States

Background: Recombinant fusion protein linking activated factor VIIa to human albumin (rVIIa‐FP) is a therapeutic option designed to prevent and treat bleeding events in patients with congenital FVII deficiency. It aims to offer reduced infusion frequency compared to current FVII treatments.

Aims: To compare the pharmacodynamics (PD) (Nijmegen Hemostasis Assay, NHA) of rVIIa‐FP with rFVIIa and pdFVII.

Methods: A Phase I multicenter, randomized, open‐label, parallel‐arm, single‐dose study was conducted in eight patients with severe congenital FVII deficiency. Patients received their routine FVII product (30 IU/kg pdFVII or 25 μg/kg rFVIIa [eptacog alfa]), and were then randomly assigned to receive 100 or 300 μg/kg of rVIIa‐FP. Thrombin generation using the NHA was assessed as an exploratory endpoint. Blood samples were drawn at 0, 15 and 30 min and 1, 2, 4, 8, 24 hr after the rFVIIa (NovoSeven ® ), pdFVII, and CSL689 injections. For CSL689, additional testing was performed at 32 hr. The NHA curve was represented based on the following parameters: lag time, ETmax (time to reach ECmax), ECmax (maximum observed thrombin level) and AUEC (area under the thrombin level vs. time curve from time zero to the last measurable sample).

Results: PD analysis was completed for eight patients: three received pdFVII and five received rFVIIa prior to treatment with rVIIa‐FP (n=5 at 100 μg/kg and n=3 at 300 μg/kg). rVIIa‐FP at 300 μg/kg (n=3) had a longer sustained suppression of lag time below 4.5 minutes for up to 24 hr compared to rVIIa‐FP at 100 μg/kg, rFVIIa at 25 μg/kg, and pdFVII at 30 IU/kg. AUEC and ECmax were similar across the two dose groups of rVIIa‐FP and rFVIIa. AUEC and ECmax for pdFVII were lower due to heparin contamination in pdFVII.

Conclusions: rVIIa‐FP showed improved pharmacodynamics compared to rFVIIa and pdFVII in patients with congenital FVII deficiency.

PB268: Examination of the Utility of Self BAT Questionnaire in an Irish Coagulation Setting

A. O Sullivan ; R.C. Jones; A. Patel; B. White; J.S. O Donnell; M. Lavin; N. O Connell; K. Ryan

St James's Hospital, National Coagulation Centre, Dublin, Ireland

Background: Bleeding assessment tools (BATs) provide an objective measurement of bleeding phenotype but are time‐consuming to administer. The recent development of a patient administered questionnaire (Self‐BAT) has the potential to streamline phenotypic assessment.

Aims: The aim of the study was to examine the utility of the Self‐BAT in a clinical coagulation setting.

Methods: From July 2017 ‐ December 2017, all newly referred patients to a nurse led bleeding assessment clinic completed a Self‐BAT. This was followed by a ISTH BAT questionnaire, administered by a nurse specialist blinded to the Self BAT. Results of both BATs were compared with statistical analysis using the Wilcoxon matched pairs signed rank tests on Prism 7.0c for Mac OS X.

Results: 69 consecutive patients were enrolled, 6 of whom were subsequently excluded due to incomplete Self BATs. The final cohort (n=63) had a median age 39.5 years old (range 16‐66), with 68% females. Using either BAT, positive BS were determined in 42.8% of patients (27/63). On paired and overall analysis of the Self BAT and ISTH BAT scores no significant difference was observed between the bleeding scores (BS, median 5 vs. 4, p=0.5).

Discrepancies were seen between the BATs as in 5 patients with a positive ISTH BAT score, the Self BAT score was normal. This was related to poor initial recall of surgical events, failure to include bleeding post minor procedures and lack of understanding of “bleeding since the get go” for bleeding since menarche.

In addition, 6 patients had a positive Self BAT but a normal ISTH BAT score. Elevated Self BAT scores were related to reporting of non‐haemostatic intravenous medications and reporting routine post extraction dental packs as a treatment.

Conclusions: The Self BAT is a promising tool in the identification of patients with bleeding disorders.

However, health care professional input to validate scores may still be required to ensure accuracy of BS.

PB269: Targeted Re‐sequencing of the Common and Rare Bleeding Disorders

R. Nilsson 1 ; E. Manderstedt 1 ; C. Lind Halldén 1 ; J. Flygare 1 ; R. Ljung 2 ; C. Halldén 1

1 Kristianstad University, Kristianstad, Sweden, 2 Lund University, Lund, Sweden

Background: Next generation sequencing (NGS) has the potential to reduce time and cost in the clinical diagnostics of the common and rare bleeding disorders since NGS supports multiple gene analysis in a single workflow. The common bleeding disorders (CBDs) are common due to dominant or X‐linked inheritance. The rare bleeding disorders (RBDs) are autosomal recessive diseases. All of these diseases have clinical symptoms that can vary substantially giving rise to overlapping phenotypes and diagnostic difficulties.

Aims: Evaluation of the system characteristics of gene panels targeting CBDs and RBDs.

Methods: Targeted re‐sequencing through AmpliSeq‐based Ion Torrent sequencing using one primer panel targeting the CBDs and one primer panel covering the RBDs.

Results: The system characteristics of both panels were evaluated using the same strategy. Coverage and read depth over the exonic positions were in both cases high with an average read depth of >1000X with > 98% coverage over the CBD panel and with >96% coverage over the RBD panel. The frequency of alternative base calls in both panels is on average 0.11%. I.e. in any given position 0.11% of reads contained an alternate allele on average.

The allele frequency variation and strand bias showed similar patterns. The CBD panel was validated through mutation detection, where the correct mutation could be detected in 23 out of 24 cases. Large deletions were detected through read depth analysis. Additional pathogenic variants in the VWF gene were identified in three haemophilia subjects, demonstrating the importance of analyzing all disease‐associated genes with overlapping phenotypes. The frequency of alternative base calls was also determined for positions listed in the mutation databases of the genes. The mutated positions were not particularly error prone compared to all exonic positions.

Conclusions: Next generation sequencing show potential to become a powerful tool in clinical diagnostics.

PB270: Molecular Analysis and Bleeding Phenotype of Seven Severe FV‐deficient Patients from Pakistan

M. Borhany 1 ; A. Ranc 2 ; M. Fretigny 3 ; G. Moulis 2 ; M. Abid 1 ; M. Giansily‐Blaizot 2

1 National Institute of Blood Disease and Bone Marrow Transplantation, Karachi, Pakistan, 2 University Hospital of Montpellier, Department of Biological Haematology, Montpellier, France, 3 Hospices Civils de Lyon, Service d'Hématologie Biologique, Lyon, France

Background: Congenital factor V deficiency (FVD) is a rare autosomal recessive bleeding disorder with a high frequency in Eastern countries.

Aims: The aims were to assess the bleeding severity and to identify underlying mutations in Pakistani patients with FVD.

Methods: Seven unrelated severe FV deficient patients were investigated. Next generation sequencing was performed on the 25 exons of the F5 gene using the Custom SureSelect QXT Target Enrichment system (Agilent) on a MiSeq platform (Illumina).

Results: Each of the seven patients (4 males and 3 females with a mean age of 15 years) was born from a consanguineous marriage. Patients were arbitrarily separated in two groups according to the FV coagulant (FV:C) levels : group A with FV:C < 1% (n=5) and group B with FV:C levels between 1‐2% (n=2). Five genetic alterations were identified at the homozygous state: four frame shift variants in exon 13 leading to premature termination codons and a newly described missense mutation in exon 9. Four out of five group A patients bore small deletions: c.2862delT (p.Ser955Alafs*4 or FV Debrecen‐1) for two patients, c.3642_3643delTC (p.Pro1215Argfs*175) and c.3653_3656delTTCA (p.Ile1218Argfs*33). The remaining patient was compound heterozygous for the c.1340C>G (p.Pro447Arg) missense substitution and the c.3088C>T (p.Arg1030Ter) nonsense mutation. Proline at position 447 is not highly conserved in FV but in factor VIII. The mutated Arginine differs in size and hydrophobicity with the deeply buried Proline residue. Therefore, this variant is possibly damaging. Group B patients were homozygous for a frame shift variant c.3899delA (p.His1300Leufs*2) and for the missense substitution c.1340C>G (p.Pro447Arg) respectively, Table 1

TABLE 1 Demographic, molecular and bleeding phenotype of severe FV deficiency patients Patient Age (yrs)/ Gender FV:C (%) F5 gene mutation Clinical Features Bleeding ISTH score [3] Bleeding WHO score[3] V1 13/F <1 c.2862delT homoZ surgical removal of hemorrhagic cyst, heavy PV bleeding twice a month for 5‐7 days 12 3 V2 17/F <1 c.3642_3643delTC homoZ Hemoptysis, malena, bruises, PR bleeding 8 3 V3 12/F <1 c.2862delT homoZ menorrhagia, gum bleeding, joint pain 12 2 V4 29/M 1.2 c.3899delA homoZ gum bleeding after teeth fall, bleeding after trauma (FFP transfused) 11 3 V5 14/M 1.8 c.1340C>G (p.Pro447Arg) homo Bruises, hematoma, (FFP transfused) 7 2 V6 4/M <1 c.3653_3656delTTCA homoZ Circumcision bleeding (FFP transfused) hemarthrosis, hematoma 11 3 V7 31/M <1 c.1340C>G (p. Pro447Arg) c.3088C>T (p.Arg1030Ter) Malena, hemarthrosis (FFP Transfused once) 8 2

Conclusions: Molecular analysis of a series of 7 FV‐deficient Pakistani patients confirmed the allelic heterogeneity of the disease, the high frequency of frame shift variants and the poor clinical relevance of FV:C levels to assess bleeding severity.

PB271: Rare Bleeding Disorders in Pakistan: An Update

M. Borhany ; N. Fatima; M. Abid; T. Shamsi

National Institute of Blood Disease and Bone Marrow Transplantation., Karachi, Pakistan

Background: Consanguinity remains common in several populations of the world, and varies from country to country. In Pakistan, close consanguineous unions continue to be common.

Aims: The aim of this study was to determine the frequency of rare coagulation bleeding disorders (RBDs), their types, clinical features and underlying mutations among patients seeking advice for bleeding tendencies from a single centre in Pakistan.

Methods: All RBDs patients enrolled at our Institute were included in the study. Pre‐designed data sheets were filled by incorporating patient's demographics, family history, present and past history of bleeding episodes with the associated signs and symptoms from 2012‐2017.In female cases, maternal and obstetrical history was taken. Blood samples were collected for complete blood count (CBC), coagulation assays and genetic characterization.

Results: Out of 550 patients diagnosed with inherited coagulation bleeding disorders, 115 subjects had RBDs (21%). Among them, 43.5% were male and 56.5% female. Median age of patients was 11 years (range, 12 days to 37 years). History of consanguinity was present in 85% cases and significant family history of bleeding in 60% patients. The most common deficiency was FXIII and FVII (n=32, 27.8%),followed by fibrinogen deficiency (n=26, 22.8%). Clinical phenotype ranged from grade II to grade III bleeding symptoms. Fresh frozen plasma / cryoprecipitate were used in the management of most patients and for prophylaxis in patients with grade III bleeding.Genetic characterization was carried out in 74/115 (64%) patients with 19 novel mutations, Table 1.

TABLE 1 Demographic, Clinical manifestations and molecular details of all RBDs Factor Deficiency No. of Patients (Female/Male) Median age years Bleeding Symptoms No. of patients on prophylaxis (Fresh Frozen Plasma,FFP/Cryoprecipitate, CP) Genetic mutations Identified No. of missense/nonsense/splicing/deletion Mutations Novel Mutations Identified. FI 26(14/12) 07 Umbilical cord bleeding, gum bleeding, menorrhagia (in females) 5 (CP,FFP) 23 6/17/0/0 4 FII 1 Male 3.5 months Gum bleeding, traumatic bleeding ‐ ‐ ‐ ‐ FV 10 (6/4) 15.5 Gum bleeding, hemarthrosis, Menorrhagia (in females) ‐ 7 2/0/0/5 1 FVII 32 (18/14) 5 Epistaxis, gum bleeding, hematoma 5 (FFP, Inj Aryoseven) 13 13/0/0/0 5 FX 10 (7/3) 6 Intra cranial haemorrhage (ICH), gum bleeding, hematoma 3 (FFP) 5 5/0/0/0 2 FXI 2 Females 3 Traumatic bleeding ‐ ‐ ‐ ‐ FXIII 32(18/14) 12 ICH, hematoma, umbilical cord bleeding, gum bleeding, menorrhagia (in females) 8 (CP) 23 19/2/2/0 7 FV+FVIII 2 Males 24.5 Gum bleeding, bruises ‐ ‐ ‐ ‐

Conclusions: The study shows that autosomal recessive disorders are common in Pakistan, with FXIII and FVII being the most common, and have severe bleeding phenotype. Diagnosis of RBDs should be made on time so that prophylaxis can be initiated immediately to prevent fatal bleeding complications and for genetic counseling.

PB272: Functional Polymorphisms of the von Willebrand Factor Gene in Patients with Mild to Moderate Bleeding Tendency and Low or Normal von Willebrand Factor Antigen or von Willebrand Factor Ristocetin Cofactor

S. Hofer 1 ; J. Gebhart 1 ; J. Rejtö 1 ; G. Hörmann 2 ; I. Pabinger 1 ; R. Sunder‐Plassmann 2

1 Medical University of Vienna, Clinical Division of Hematology and Hemostaseology, Department of Internal Medicine I, Vienna, Austria, 2 Medical University of Vienna, Division of Medical‐Chemical Laboratory Diagnostics, Department of Laboratory Medicine, Vienna, Austria

Background: The diagnosis of von Willebrand disease (VWD) is backed by molecular analysis. Functional single nucleotide polymorphisms (SNPs) of the coding and promoter sequences in the von Willebrand factor (VWF) gene are correlated with VWF antigen and ristocetin cofactor levels.

Aims: Evaluation of the frequency of functional polymorphisms in the VWF gene in a Vienna Bleeding Biobank (VIBB) subgroup.

Methods: From November 2009 to January 2016, 431 patients (female=354, 82.1%) were included in the VIBB. Of these, 32 were diagnosed with low VWF:Ag or VWF:RiCo levels respectively (< 50%), while mutations associated with VWD were absent. For subsequent evaluation of functional polymorphisms in exons 18 and 28 of the VWF gene we compared this subgroup to 32 patients that showed normal VWF levels (>50%) as well as to that of the European population of the 1000 genome project provided by dbSNP.

Results: Patient characteristics are given in Table 1. Blood group 0 was more frequent in the patients with low vWF levels. Fifteen patients (47%) with VWF levels < 50% and 20 (63%) with VWF levels >50% had the following functional polymorphisms in Exon 18 or 28: p.Val1565Leu and/or p.Thr789Ala. Associations between p.Val1565Leu and increased proteolysis by ADAMTS13, and between p.Thr789Ala and higher VWF levels have been described.

We detected a higher frequency of the p.Val1565Leu SNP (11% vs. 6% and 8%) when compared to the patient group with normal VWF levels as well as the European population (Table 2). In contrast, the p.Thr789Ala polymorphism appears with a lower frequency (16% vs. 34% and 37%) in our patient subgroup. Patients from the VWF>50 subgroup with the p.Val1565Leu SNP (n=4) also carry the p.Thr789Ala polymorphism.

TABLE 1 Patient Characteristics VWF<50% (n=32) VWF>50% (n=32) Female, n (%) 28 (87.5%) 29 (90.6%) Blood group 0, n (%) 25 (78.1%) 15 (46.9%) Median [Interquartile Range] Age, years 32 [22‐40.75] 32 [22‐37.5] Bleeding Score (Vicenza) 5 [4‐7.75] 4.5 [3‐7] vWF:Ag, % 50 [45.75‐57] 94 [81‐110.75] vWF:Rico, % 44 [40.25‐48] 78 [68.25‐101] FVIII, % 72 [59.25‐82] 121 [106.25‐151.5]

TABLE 2 Frequencies of functional polymorphisms in the VWF gene SNPs (VIBB ‐ VWF<50%) (VIBB ‐ VWF>50%) EUR Polymorphism Allele Frequency Polymorphism Allele Frequency Allele Frequency E18: c.2365A>G,p.Thr789Ala Homozygous: 1 Heterozygous: 8 0.156 Homozygous: 2 Heterozygous: 18 0.344 0.367 E28: c.4693G>T,p.Val1565Leu Homozygous: 0 Heterozygous: 7 0.110 Homozygous: 0 Heterozygous: 4 0.063 0.076

Conclusions: In the group of patients with low VWF levels, we detected a shift of polymorphisms, which might partially explain the decreased VWF levels and bleeding in this very specific group of patients without disease defining mutation in the VWF gene.

PB273: Comparison of a von Willebrand Factor Fully‐automated Chemiluminescent Assay Panel for von Willebrand Disease Diagnosis to and Historical Classification

F. Stufano 1 ; L. Baronciani 2 ; P. Bucciarelli 2 ; M. Boscarino 2 ; P. Colpani 2 ; D. Mane‐Padros 3 ; S. Faraudo 3 ; F. Peyvandi 2,4

1 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico and Luigi Villa Foundation, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan, Italy, 2 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico and Luigi Villa Foundation, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico and Luigi Villa Foundation, Milan, Italy, 3 Biokit Research & Development, R&D, Llica d'Amunt, Spain, 4 Università degli Studi di Milano, Department of Pathophysiology and Transplantation, Milan, Italy

Background: Different assays are necessary for an accurate classification of VWD. The identification of a VWD patient requires the use of first level tests as factor VIII coagulant activity, von Willebrand factor (VWF) antigen (VWF:Ag) and VWF ristocetin cofactor (VWF:RCo). A more specific test as VWF collagen binding (VWF:CB) assay can improve the diagnosis and differentiation of VWD types in the laboratory practice.

Aims: To assess the diagnostic performance of a novel fully automated chemiluminescent assays panel in a group of well characterized VWD patients.

Methods: 100 VWD patients (type 1 [n=17], type 1 Vicenza [n=5] type 2A [n=27], type 2B [n=18], type 2B with HMWM [n=8], type 2M [n=13], type 2M with collagen binding defect [2MCB] [n=7] and type 3 [n=5]) were historically characterized according to the ISTH‐SSC guidelines. Patients samples were revaluated for the VWF levels using automated chemiluminescent immunoassays (VWF:Ag, VWF:GPIbR and VWF:CB).

Results: Figure A and B report the boxplots of VWF:GPIbR/VWF:Ag and VWF:CB/VWF:Ag ratios and mean value of each variant groups obtained using the chemiluminescent panel compared to the historical classification (Figure). The VWF:GPIbR/VWF:Ag ratio obtained by the chemiluminescent assays mainly confirmed the historical classification. The exception were two cases historically classified as type 2B and type 2BHMWM patients using ristocetin induced platelet agglutination. Similar consistent results were also obtained using the VWF:CB/VWF:Ag ratio. However, three type 2MCB patients would be reclassified as type 1 using these new assays. Two type 1 samples were misclassified as 2A/2B variant with both VWF:GPIbR/VWF:Ag and VWF:CB/VWF:Ag ratios.

FIGURE 1 Chemiluminescent assays panel diagnosis versus Historical diagnosis

Conclusions: The new chemiluminescent assays panel was confirmed to be a good tool for phenotypic diagnosis of VWD.

PB274: Profile of Mutations Identified in a Cohort of Czech Patients with Type Normandy von Willebrand Disease

D. Provaznikova 1 ; F. Mareček 1 ; A. Radovská 1 ; M. Šimoníková 1 ; V. Geierová 2 ; R. Hrdličková 3 ; P. Salaj 2 ; I. Hrachovinová 1

1 Inst of Hematology and Blood Transfusion, NRL for Disorders in Hemostasis, Prague, Czech Republic, 2 Inst of Hematology and Blood Transfusion, Center for Thrombosis and Hemostasis, Prague, Czech Republic, 3 Faculty Hospital Ostrava, Blood Centre, Ostrava, Czech Republic

Background: Von Willebrand disease (vWD) is a bleeding disorder caused by a defect of von Willebrand factor (vWF). vWF plays a crucial role in platelet adhesion to vessel walls after injury and is a carrier protein for factor VIII (FVIII). One of the subtypes of vWD ‐ Normandy (2N) ‐ is caused by a defective FVIII binding to vWF. Inheritance is an autosomal recessive.

Aims: To determine the genetic and phenotypic basis of type 2 Normandy vWD in Czech patient.

Methods: We tested samples from Bohemia and Northern Moravia for FVIII:C, vWF (Ag, RCo, CB, FVIIIB, propeptide and multimeric structure). vWF:FVIIIB was measured by reagent Asserachrom VWF:FVIIIB (Stago, France). DNA extracted from the whole blood was analyzed for mutations of vWF gene. Exons associated with type 2N (4, 9, 17‐20, 24‐27) plus exons 5, 7, 21, 28 were PCR‐amplified and analyzed by Sanger sequencing with corresponding intronic primers.

Results: We examined 33 patients (and their relatives if available) with vWF:FVIIIB under 0.2 UI/mL. The vast majority (28 of 33) of patients had the most common type 2N mutation R854Q, 16 of them were homozygotes for this mutation. Further, we found mutation T791M and 2 new mutations causing 2N vWD not reported in the EAHAD database so far: D879G, S801R. Sixteen of 33 Normandy patients were combined heterozygotes. These combined heterozygotes had vWF:FVIIIB levels 0.00‐0.15 UI/mL (average 0.09±0.05 UI/mL), which is lower but not statistically different from homozygotes (0.13±0.02 UI/mL). Multimeric structure was normal in most of patients except in two where pattern was IIE resp. SM.

Conclusions: In our study we found four unique mutations underlines the Normandy phenotype, we confirmed as in other countries, that the most common is the R854Q (85% of patients) mutation. We found two novel mutations associated with VWD 2N: D879G, S801R. Our results supported studies showing that multimer structure could be abnormal in VWD 2N.

PB275: Correction of Factor XI Deficiency by Liver Transplantation for Secondary Sclerosing Cholangitis

I. Martin‐Toutain 1 ; D. Eyraud 2 ; V. Planche 3 ; M. Kurdi 1 ; P. De Mazancourt 4 ; C. Frere 5

1 Pitié‐Salpêtrière Hospital, Assistance Publique Hôpitaux de Paris, Haematology Department, Paris, France, 2 Pitié‐Salpêtière Hospital, Anesthesia Department, Paris, France, 3 Saint‐Antoine Hospital, Assistance Publique Hôpitaux de Paris, Haematology Department, Paris, France, 4 Ambroise‐Paré Hospital, Assistance Publique Hôpitaux de Paris, Biochimie Hormonologie et Génétique Moléculaire, Paris, France, 5 Pitié‐Salpêtrière Hospital, Assistance Publique Hôpitaux de Paris, Institute of Cardiometabolism and Nutrition, ICAN, Sorbonne Université, Haematology Department, Paris, France

Background: Factor XI (FXI) deficiency is a rare autosomal genetic bleeding disorder affecting approximately 1 in 1,000,000 people in the general population. A higher incidence of FXI deficiency has been observed in Ashkenazi and Iraqi Jews. Spontaneous bleeding is rare and bleeding is usually injury related. The bleeding tendency is predictable neither within a patient nor within a family and it does not correlate with FXI level. Therapeutic options include fresh frozen plasma, FXI concentrates and antifibrinolytics.

Aims: To report the correction of a FXI deficiency following liver transplantation in a 24‐year‐old man of Martinican origin.

Methods: Case report.

Results: This patient suffering from a sclerosing cholangitis was placed on the waiting list for liver transplant in 2017. He was incidentally found to have a prolonged APTT a few years ago, which further revealed a moderate FXI deficiency (FXI level, 0.10 U.mL −1 ). He had no previous history of bleeding. Preoperative screening tests showed a prolonged APTT value (64 s), a normal PT value (14s), a normal fibrinogen level (4.9 g.L −1 ), normal levels of factors II, V, VII, X, VIII, IX and XII, and an isolated FXI deficiency (0.13 U.mL −1 ) without inhibitors to FXI. Direct DNA sequencing analysis of the F11 gene identified a heterozygous missense mutation (c.942C>T; CAC>CAT) in exon 9 affecting the splicing process. In December 2017, he received an orthotopic liver transplant. Fresh‐frozen plasma and tranexamic acid were used during surgery. The transplantation was successfully performed without any peri‐operative serious bleeding. FXI levels increased by day 2 after transplantation (0.26 U.mL −1 ), and returned to the normal range by day 21 after transplantation.

Conclusions: Only 2 cases of correction of FXI deficiency by liver transplantation have been previously reported in the literature. All together, these observations support the hypothesis that factor XI production is confined to the liver.

PB276: Intense Exercise Increases von Willebrand Factor and Factor VIII Levels in Well‐trained Athletes

N. Androulakis; P. Spatharaki ; E. Nioti; I. Papadakis; A. Papadopoulou; G. Notas

University Hospital of Heraklion, Laboratory of Hematology, Iraklion, Greece

Background: Acute and chronic exercise induce several hematological changes in humans. These exercise‐induced hematological changes are linked to the intensity, volume, and frequency of exercise. Additionally, several factors such as training status, gender, and age, could also play an important role.

Aims: Our aim in this study was to evaluate the possible alterations of specific coagulation parameters after intense intermittent exercise to exhaustion (Yo‐Yo Intermittent Recovery Test Level 2) in well trained athletes.

Methods: The study population consisted of eighteen elite male football players, mean age 17±2 years, BMI: 22.5±2, Fat: 13.8±3.4%. All participants were guided to perform the Yo‐Yo Intermittent Recovery Test Level 2 (YYIRT2) until exhaustion. Blood samples were drawn before and immediately after the test for the assessment of platelet count (PLT), D‐Dimers (DD), Fibrinogen (Fib), Factor VIII activity (FVIII), Von Willebrand factor antigen plasma levels (vWf Ag) and high sensitivity CRP (Hs‐CRP). Furthermore, the resting and the maximum heart rate, the total covered distance and the relative VO2max were recorded.

Results: Analysis revealed a significant increase in platelet count, FVIII and vWf Ag levels (P< 0.01) at the end of the test. In fact, VIIIa and vWf Ag levels almost doubled at the end of the test compared to baseline (Table 1).

TABLE 1 Mean, SD, minimum and maximum PLTs, FVIII and vWf Ag, before and after the YYLR2 test Test Minimum Maximum Mean Std. Deviation PLT K/µl (before) 157 352 237 44.6 PLT K/µl (after) 171 348 272 42.1 vWf Ag % (before) 69.5 220.9 114 33.4 vWf Ag % (after) 122.4 248.0 205 38.0 FVIII % (before) 60.0 112.3 84 14.6 FVIII % (after) 110.6 246.6 170 38.2

No significant changes were identified in Fibrinogen, D‐Dimer and Hs‐CRP levels (P>0.01). Further analysis (Pearsons r) did not reveal any correlation between VO2max, PLT, FVIII and vWf Ag. Contrariwise, it showed a strong correlation between PLT counts and FVIII (r=0.67, P=0.003) and vWf Ag levels (r=0.65, P=0.004).

Conclusions: Intense intermittent exercise to exhaustion leads to a significant increase of PLTs, FVIII, and vWf Ag levels probably due to shear stress‐induced endothelial activation. These findings are in accordance with previously published data supporting that intense exercise can be beneficial on specific types of bleeding disorders.

PB277: Prevalence of von Willebrand Disease among Nigerian Youths in Ibadan South‐Western Nigeria

K. Shonde‐Adebola 1 ; M. Adebola 2 ; W. Shokunbi 1

1 University College Hospital, Ibadan, Haematology, Ibadan, Nigeria, 2 Federal Medical Center Abeokuta, Paediatrics, Abeokuta, Nigeria

Background: Von Willebrand disease (VWD) is the commonest inherited bleeding disorder. VWD may be asymptomatic (0.37% US women). The symptomatic cases have significant history of mucocutaneous bleeding including epistaxis, menorrhagia and high risk of post‐partum haemorrhage especially in adolescents. Yet there has been no documented published prevalence of VWD in the African or Nigerian population after Medline and Pubmed search.

Aims: The aim of this study was to determine the prevalence of von Willebrand Disease (VWD) among apparently healthy youths in Ibadan, Southwest Nigeria.

Methods: This was a descriptive, analytical, cross‐sectional study, carried out at the College of Medicine, University of Ibadan Oyo state, Nigeria. One hundred and eighty‐two (182) participants who meet the inclusion criteria and who consented to partake in the study were recruited. Blood samples were collected from the participants for their hematological parameters. VWF:ag levels and coagulant factor VIII levels were carried out by ELISA method using SPAN VWF and SPAN Factor VIII ELISA kits respectively.

Results: The study participants were aged 15years to 35 years with mean age of 22.96 ± 4.22 years. Majority (75.8%) were in the age range 20 to 24 years. Ninety six (52.7%) of them were males while 86 (47.3%) were females. The prevalence of VWD among healthy youths in Ibadan was 2.2%, based on the identification of 4 cases of VWD out of 182 participants. The mean VWF:Ag was 103.2±52.7 IU/dl and the mean VWF:Ag based on blood group, showed the lowest mean VWF:Ag value in subjects with group O (87.1±39.9). The mean FVIII level observed was 100.5±46.7. There was a strong statistically significant relationship between VWF:Ag and FVIII antigen level (P=0.0001).

Conclusions: The prevalence of VWD is 2.2% among apparently healthy youths in Ibadan. There was a strong statistical relationship between VWF:Ag level, FVIII and ABO blood group (P= 0.0001).

PB278: Impact of Mitral Valve Clip Procedure on Indices of Acquired von Willebrand Syndrome

J. Blackshear 1 ; P. Pollak 1 ; D. Chen 2

1 Mayo Clinic Florida, Cardiovascular Diseases, Jacksonville, United States, 2 Mayo Clinic, Hematopathology, Rochester, United States

Background: Acquired von Willebrand syndrome (AVWS) has been associated with severe mitral valve (MV) regurgitation, and is corrected by mitral valve replacement or repair (MVR). The impact of percutaneous MV clip repair (MVclip) on AVWS is undefined.

Aims: To evaluate AVWS parameters in MVclip patients compared to those treated with conventional MVR.

Methods: Platelet function analyzer 100 ADP cartridge (PFA, normal < 121 seconds), the ratio of von Willebrand factor activity to antigen (VWF:Act / Ag, normal ≥0.80), and quantitative analysis of the fraction of high molecular weight bands > 15 monomers / bands 2‐15 monomers normalized for healthy donor plasma (normal = 1.0, LVAD =0.4‐0.5) were assessed.

Results: Thirteen men and 9 women treated with surgical MVR averaged 64±10 years versus 84±7 years in the 7 men and 6 women who had MVclip (P<0.001). Mitral regurgitant volume per beat was similar in the two groups. Prior to intervention VWF antigen was higher in the MVclip group, 172±46 compared to MVR, 116±55, P=0.004. Although PFA was lower in the MVclip group, 149±65 seconds versus 196±56 seconds, P=0.03, there was no difference in the normalized multimer ratio, 0.66±0.12 versus 0.63±0.14, and in VWF:Act / Ag, 0.79±0.09 versus 0.81±0.10.

After intervention the correction of the AVWS associated parameters was less complete in MVclip patients compared to MVR (Figure 1).

FIGURE 1 VWF data in mitral valve surgery versus mitral valve clip

Post‐procedure PFA remained higher in MVclip versus MVR patients, 122±32 versus 87±27 seconds, P=0.01; the post‐procedure VWF:Act / Ag remained lower, 0.76±0.05 versus 0.93±0.11, P=0.001, and the normalized multimer ratio in MVclip patients also remained borderline significantly lower, 0.72±0.07 versus 0.91±0.19, P=0.065. Three patients manifested Heyde syndrome; 2 were cured by MV surgery, while one MVclip patient continued to bleed and require transfusions.

Conclusions: These data suggest that the correction of AVWS abnormalities is less complete with MVclip compared to MVR. Additional data from ongoing patient recruitment will be presented.

PB279: Comparative Study of Single Nucleotide Polymorphisms (SNPs) in the Exon 28 of the VWF between Healthy Donors, VWD2M (with p.E1549K and p.R1374C) and VWD2B Patients (with p.R1308C and p.V1316M) in a Single Center in Argentina. Preliminary Report

A.I. Woods 1 ; J. Paiva 2 ; A.C. Kempfer 1 ; M.M. Casinelli 2 ; A. Sanchez‐Luceros 1,2 ; M.A. Lazzari 1

1 IMEX‐CONICET‐Academia Nacional de Medicina, Laboratorio de Hemostasia y Trombosis, Ciudad Autonoma de Buenos Aires, Argentina, 2 Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Hemostasia y Trombosis, Ciudad Autonoma de Buenos Aires, Argentina

Background: The influence of different SNPs on WF survival between different ethnic and racial groups was described. This makes it necessary to better characterize ethnic and racial variations in the VWF gene, resulting in ambiguity or erroneous conclusions that may have an impact on the diagnosis of VWD. SNPs p.V1565L and p.G1643S were associated with higher proteolysis by ADAMTS13, while p.D1472H, p.Q1571H and p.P1601T, resistance to proteolysis.

Aims: We describe the allelic frequencies (AFs) of SNPs in the exon 28 with high frequency in normal population or causing altered proteolysis, in healthy donors and in VWD2M patients (P) with p.E1549K and p.R1374C, and VWD2B P and p.R1308C and p.V1316M, the most frequent mutations in these VWD variants in our Center.

In VWD P, we compare SNP presence with propeptide/VWF:Ag (VWFpp ratio), useful to measure VWF survival.

Methods: Healthy donors: 208 alleles. VWFpp ratio=0.9‐2.14.

VWD2M P: with p.E1549K (32 alleles): VWFpp ratio=1.9±0.6 (43.7% with high values); with p.R1374C (32 alleles): VWFpp ratio=2.5±1.0 (69.2% with high values).

VWD2B P: with p.R1308C (16 alleles): VWFpp ratio=2.3±0.7 (62.5% with high values); with p.V1316M (24 alleles): VWFpp ratio=2.2±0.6 (54.5% with high values).

SNPs within exon 28 of VWF gene were analyzed and the AFs were compared with those reported in ISTH‐SSC VWF Online Database.

Results: Our results are shown in the table 1.

TABLE 1 Allelic frequency of SNP in our normals and VWD patients SNP VWD2M p.E1549K VWD2M p.R1374C VWD2B p.V1316M VWD2B p.R1308C healthy donors Our population healthy donors ISTH report population A1381T AF g/a 0.87/0.13 0.68/0.32 0.55/0.45 0.93/0.07 0.62/0.38 North american: 0.65/0.35 British: 0.54/0.46 Japanese: 0.59/0.41 D1472H AF g/c 0.4/0.6 0.54/0.46 0.94/0.05 1/0 0.84/0.16 North american: 0.89/0.11 T1547 AF c/t 0.81/0.19 0.68/0.32 0.79/0.21 0.93/0.07 0.66/0.34 British: 0.58/0.42 Japanese: 0.59/0.41 A1555 AF a/c 1/0 1/0 0.66/0.34 0.64/0.36 0.91/0.09 Japanese: 0.64/0.36 V1565L AF g/t 1/0 1/0 0.66/0.34 0.71/0.29 0.90/0.10 European: 0.92/0.08 Q1571H AF g/c 1/0 1/0 1/0 1/0 1/0 French: 0.99/0.01 P1601T AF c/a 1/0 1/0 1/0 1/0 1/0 Mexican: 0.97/0.03 G1643S AF g/a 1/0 1/0 1/0 1/0 1/0 French 0.99/0.01

Donors: the AFs of the SNP were similar as those reported in the VWF Database. Only p.A1555 showed higher AF than that reported.

VWD P: p.Q1571 H, p.P1601T and p.G1643S showed the same allelic frequency as our donors and those reported in the VWF Database.

VWD2M P showed an increase of 1472H allele, while VWD2B P showed an increment of 1565L allele.

Conclusions: There were no significant differences in AFs of SNP in exon 28 between our donors and those worldwide, as reported in the VWF Database.

Differences in AFs seen in our VWD P together the associated mutations could be responsible for variations in VWF survival in P.

PB280: Genotyping of Five Patients with Severe Inherited Factor X (FX) Deficiency from Pakistan: Identification of Two Novel Mutations

M. Borhany 1 ; D. Buthiau 2 ; F. Rousseau 2 ; O. Guillot 2 ; M. Abid 3 ; T. Shamsi 3 ; M. Giansily‐Blaizot 2

1 National Institute of Blood Disease and Bone Marrow Transplantation, Karachi, Pakistan, 2 Hôpital Saint‐Eloi, CHU de Montpellier, Département d'Hématologie Biologique, Montpellier, France, 3 National Institute of Blood Disease and Bone Marrow Transplantation., Karachi, Pakistan

Background: Congenital factor X (FX) deficiency is a rare coagulation defect characterized by a variable bleeding tendency.

Aims: The aim of the study was to give a first insight of F10 gene mutations with clinical features in Pakistani probands

Methods: Five severe unrelated FX‐deficient patients from Pakistan were analyzed. Informed consent was obtained from index patient's family members and a clinical questionnaire was filled out for each patient.Direct sequencing was performed on the 8 coding regions, intron/exon boundaries and 5’ and 3’ untranslated regions of the F10 gene.

Results: All 5 patients were affected with severe FX‐deficiency (FX:C levels below 2%). All were born from consanguineous marriages Sequencing revealed 5 various substitutions including three previously reported p. Ala15Asp, p. Gly406Ser, p. Gly420Arg missense variants and two novel variants: the p. Cys57Arg and the p. Gln172*. One genotype could not be characterized albeit the inherited nature of the defect could be confirmed with familial studies. As the CNV were ruled out, the hypothesis of deep intronic mutants which might have escaped from conventional sequencing was proposed. Three patients presented with severe clinical symptoms including umbilical cord, haemarthrosis or intracranial haemorrhages whereas two presented provoked bleeds after trauma and bruises The milder patients bore the p.Gly406Ser at the homozygous state and the F10 unknown alleles respectively. Majority of these patients are on demand treatment with fresh frozen plasma and antifibrinolytic except those with intracranial haemorrhages, Table 1.

TABLE 1 Clinical and molecular characteristics of the 5 Factor X deficient patients Patient Age (years)/ gender Consanguinity (yes/no) FX:C (%) Age at first symptom Main clinical features Treatment Follow‐up F10 variant Genetic status 1 4 / Female Yes Parents first cousins <2% 15 days Umbilical cord bleed Umbilical cord bleed Haemarthrosis On‐demand (FFP and antifibrinolytic) Doing well, no life threatening bleed. p. Ala15Asp Homozygous 2 3 / Female Yes Parents second cousins <2% 8 days Umbilical cord bleed Intra cranial haemorrhage. Haemarthrosis Haemoptysis Prophylaxis (FFP weekly and antifibrinolytic) vegetative state, mental retardation p.Gly420Arg p.Cys57Arg Compound heterozygous 3 15 / Female Yes Parents second cousins 2% 2.5 years Oral bleeding Menorrhagia and bruises On‐demand (FFP and antifibrinolytic) Doing well, no life threatening bleed. p. Gly406Ser Homozygous 4 5 / Male Yes 2% 2 years Hematoma after injury Bleeding after tooth extraction On‐demand (FFP and antifibrinolytic)‐ Doing well, no life threatening bleed. Wild type _ 5 0.7/ Female Yes 2% 10 days Umbilical cord bleed Intracranial bleed Hematoma Prophylaxis (FFP weekly and antifibrinolytic) Doing well, no new episode of life threatening bleed. p. Gln175* Homozygous

Conclusions: This is the first report from Pakistan describing mutations in F10 gene with clinical features and treatment. Mutation spectrum is heterogeneous as seen in other populations. Identification of the mutations in these patients is important for genetic counseling, prenatal diagnosis and for prophylaxis.

PB282: Discrepant von Willebrand Factor (VWF) Activity Levels in Two Unrelated von Willebrand Disease (VWD) Patients

M. Bowman 1 ; A. Tinmouth 2 ; R. Selby 3 ; M. Sholzberg 4,5 ; D. Lillicrap 6 ; D. Good 6 ; P. James 1

1 Queen's University, Department of Medicine, Kingston, Canada, 2 The Ottawa Hospital, Department of Medicine, Ottawa, Canada, 3 University Health Network and Sunnybrook HSC, Department of Laboratory Medicine and Medicine, Toronto, Canada, 4 St. Michael's Hospital, Department of Medicine, Toronto, Canada, 5 University of Toronto, Toronto, Canada, 6 Queen's University, Department of Pathology & Molecular Medicine, Kingston, Canada

Background: Multiple assays exist for measuring von Willebrand factor (VWF) activity.

Aims: To describe two patients who exhibited discrepant VWF activity levels.

Methods: Laboratory assays (VWF:Ag, multiple VWF activity assays, FVIII:C, VWFpp) and VWF sequencing was performed.

Results: The first patient, a 61 year old female, was referred to Hematology clinic with a FVIII level of 0.10 U/mL. She had a moderate bleeding history and a recent fall that resulted in a large hematoma. Lab results are shown in Table 1. The VWF:GIbM was 6X higher than VWF:Ag. Additional VWF activity assays were performed (VWF:RCo[Acu] and VWF:Ac) which were also significantly higher. A traditional VWF:RCo was 0 IU/mL. The PP:Ag ratio was elevated which led to the VWF gene being sequenced for Type 1C variants (exons 26‐28, 37); no variant was identified. The second patient, a 32 year old male known to have Type 1 VWD, was seen in Hematology clinic for an annual visit. He reported a recent traumatic injury resulting in a large hematoma in the hip area. An elevated VWF:GPIbM was reported which was higher than his VWF:RCo and historic VWF:GIbM. Additionally the PP:Ag ratio was elevated. The following VWF variants were reported: c.6187C>T, (p.Pro2063Ser) in exon 36, a known polymorphism, and c.6697G>C (p.Glu2233Gln) in exon 38. Further studies are required to determine if the latter variant influences clearance. VWF measurements at different time points for patient 2 are in Table 1.

TABLE 1 Laboratory values for the two patients Patient No. Date VWF:Ag (IU/mL) VWF:RCo[Agg] (IU/mL) VWF:RCo [Acu] (IU/mL) VWF:Ac (IU/mL) VWF:GPIbM (IU/mL) VWFpp (IU/mL) PP:Ag ratio FVIII:C (IU/mL) 1 2015 0.14 0.00 2.45 1.55 0.85 1.57 11.2 0.10 2 2005 0.25 0.18 NA NA 0.28 0.91 3.6 0.55 2 2013 0.36 0.30 NA NA NA NA NA 0.68 2 2014 0.22 NA NA NA 0.23 NA NA 0.57 2 2015 0.26 NA NA NA 0.26 NA NA 0.58 2 Nov‐2016 0.21 0.14 NA NA 1.47 1.32 6.3 0.71 2 Dec‐2016 0.29 0.22 NA NA 0.54 NA NA 0.93 2 2017 0.18 0.16 NA NA 0.73 NA NA 0.52 2 2018 0.20 NA NA NA 0.34 1.04 5.2 0.70

Rheumatoid factor was negative for both patients. Human anti‐mouse antibodies have been reported as interfering with the VWF activity and VWFpp assays. Studies mixing mouse monoclonal antibody (CD103) and/or mouse serum with each patient`s plasma did not definitively prove interference with the assays (Table 2).

TABLE 2 Results of mixing studies Assay Patient 1 Patient 1 + mouse serum Patient 2 Patient 2 + CD103 Patient 2 Patient 2 + mouse serum VWF:GPIbM (IU/mL) 0.63 0.65 0.73 0.65 0.99 0.61 VWFpp (IU/mL) 1.32 1.12 NA NA 1.05 1.22

Conclusions: These cases highlight challenges that can be encountered due to variability in VWF activity assay results. The specific assay used should be considered when interpreting results. The cause of the discrepancy in these two cases remains unclear.

PB284: Are Iranian Patients with von Willebrand Disease Type 2N Properly Differentiated from the Ones with Mild/Moderate Hemophilia A and Do They Receive Appropriate Treatment?

A. Seidi Zadeh ; M. Ahmadinejad; B. Amoohossein; S. Homayoun

Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran, Islamic Republic of

Background: The defect function of the von Willebrand factor (VWF) in carrying FVIII leads to type 2N von Willebrand disease (VWD). VWD 2N has a high potential to be misdiagnosed as mild/moderate hemophilia A due to the reduction of FVIII:C. Differentiating of VWD 2N from hemophilia A is crucial for patient treatment. VWF:FVIII binding assay (VWF:FVIIIB) is a critical test for identification of VWD 2N; but it is not routinely practiced in Iran even in reference coagulation laboratories.

Aims: To evaluate the current diagnostic work‐up of Iranian patients with mild /moderate deficiency of FVIII levels and the possibility of misdiagnosis of VWD 2N as hemophilia A.

Methods: All patients who were referred to IBTO reference coagulation lab for FVIII:C assay in a 10 month period (2013‐2014) were included in this retrospective study. Demographic data, bleeding score (BS), FVIII:C, VWF:Ag and the FVIII:C/VWF:Ag ratio were assessed. The patients receiving FVIII or VWF concentrates in 10 days before sampling, those with combined FV&VIII coagulation factor deficiency or acquired hemophilia A, and those with incomplete lab data were excluded. VWF:FVIIIB for the first time was performed in a limited number of index cases.

Results: The results of FVIII:C were assessed in 896 referred subjects, with 767 cases showing the FVIII:C > 40% and 130 ≤ 40%. The latter group, based on FVIII:C level, was classified into mild, moderate and severe categories. (algorithm1)

FIGURE 1 Flowchart of referred cases with FVIII:C assay to IBTO coagulation lab in a 10 month period

FVIII/VWF:Ag ratio was evaluated and 33 cases had ≤0.6ratio. (table 1)

TABLE 1 The data details of index cases Subject N Age (years) Gender (male/female) FVIII:C Mean(range) VWF:Ag Mean(range) FVIII/VWF:Ag ratio Mean(range) Bs Mean(range) Mild FVIII:C deficiency 26 24 (1‐49) 22/4 23 (7‐40) 93 (39‐263) 0.24 (0.07‐0.6) 6 (0‐13) Moderate FVIII:C deficiency 7 33 (3‐59) 5/2 3.8 (2‐5) 61 (22‐112) 0.06 (0.04‐0.09) N/A

VWF:FVIIIB was performed just on 6 samples which abided to type 2N diagnostic criteria. We found one patient with VWF:FVIIIB< 1%. This patient was a 3‐year old girl who received FVIII concentrates for one year due to a misdiagnosis as hemophilia A.

Conclusions: In Iran, probably a significant number of VWD 2N patients are misdiagnosed as mild/moderate hemophilia A due to insufficient test panel for subtyping of VWD and the absence of a national guideline for definitive diagnosis and classification of VWD.

FIBRINOLYSIS AND PROTEOLYSIS

PB285: Polyphoshate Nanoparticles Slow Down the Clotting of Fibrinogen and Increase the Lytic Stability of Fibrin

A. Tanka‐Salamon 1 ; M. Lovas 1 ; I. Voszka 2 ; L. Szabó 3 ; K. Kolev 1

1 Semmelweis University, Department of Medical Biochemistry, Budapest, Hungary, 2 Semmelweis University, Department of Biophysics and Radiation Biology, Budapest, Hungary, 3 Hungarian Academy of Sciences, IMEC, Research Centre for Natural Sciences, Budapest, Hungary

Background: Platelets secrete polyphosphate (PolyP) nanoparticles (PolyP‐NPs), but in contrast to soluble PolyP little is known about the effects of PolyP‐NPs on fibrin formation.

Aims: To generate PolyP‐NPs of reproducible size, to characterize these particles in terms of morphology and stability and to investigate their effect on fibrin formation, structure and lysibility.

Methods: PolyP‐NPs were formed by precipitation of PolyP (45‐monomer mean size) in aqueous milieu containing CaCl2 and extruded through a 220‐nm filter. Particle size was measured by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Fibrinogen was clotted by thrombin and the formed fibrin was dissolved by plasmin. Fibrin formation and lysis were monitored by turbidimetry and the time to reach half‐maximal turbidity was used as a measure of reaction kinetics (clotting cT50 and lysis lT50 from the ascending and descending parts of the turbidimetric curves, respectively). The structure of fibrin was examined by scanning electron microscopy and the fibrin fiber diameters were measured from the images.

Results: The applied procedure yielded spherical, electron‐dense nanoparticles with a diameter of 95 nm in the TEM images.

FIGURE 1 PolyP‐NPs as revealed by TEM

The stability of their suspension was estimated from the effective diameter of the particles in the DLS assay, the median of which increased by 33% in 8 days. PolyP‐NPs at 20 μM prolonged cT50 1.52‐fold, an effect comparable to the effect of another biologically relevant polyanion, DNA (1.61‐fold prolongation at 0.5 mg/ml). These two polyanions acted synergistically, PolyP‐NP+DNA raised the cT50 2.84‐fold. The lT50 of the resultant fibrin was 1.51‐fold increased by PolyP‐NP+DNA. These functional effects could be traced back to modified structure of fibrin; median fiber diameters decreased from 132 nm to 115.9 nm, 115.1 nm and 118.7 nm in the presence of PolyP‐NP, DNA and PolyP‐NP+DNA, respectively.

TABLE 1 Effect of PolyP‐NP, DNA and their combination on turbidimetric clotting and lysis times and on fibrin fibre diameters PolyP‐NP DNA PolyP‐NP+DNA cT50 (relative to control) 1.52* (+/‐ 0.05) 1.61* (+/‐ 0.26) 2.84*** (+/‐ 0.71) lT50 (relative to control) 1.12 (+/‐ 0.19) 0.83 (+/‐ 0.39) 1.51** (+/‐ 0.39) fibrin fiber diameter (nm) 115.9*** (88.98‐151.0) 115.1*** (91.94‐153.2) 118.7*** (91.94‐153‐2)

Conclusions: PolyP‐NPs stabilize fibrin through effects on fibrinogen clotting and fibrin structure.

PB286: Alternative Complement Pathway Inhibits Fibrinolysis in an in‐vitro Whole Blood Model

J. Samuels 1 ; E. Moore 1,2 ; J. Coleman 1 ; A. Sauaia 1 ; C. Silliman 1,3 ; A. Banerjee 1

1 University of Colorado, General Surgery, Aurora, United States, 2 Denver Health Medical Center, General Surgery, Denver, United States, 3 Children's Hospital Colorado, Hematology, Aurora, United States

Background: Following trauma, a hypofibrinolytic state may develop. This occurs concurrently with a post‐injury activation of the alternative complement pathway; however, whether complement activation contributes to fibrinolysis shutdown remains unclear. Given the parallel activation of complement and decreased fibrinolysis following trauma, we hypothesize that activation of the alternative pathway (AP) promotes fibrinolysis shutdown.

Aims: Determine if AP contributes to fibrinolysis phenotypes.

Methods: Whole blood samples from healthy volunteers were collected by venipuncture into citrated tubes. In the first experiment, samples (n=12) were treated with Cobra Venom Factor (CVF, a protein functionally resembling C3Bb) to final concentration of 20 U/mL and incubated in 37°C water bath for 15 or 30 minutes to assess temporal effects. For AP inhibition, samples (n=9) were treated with either 25 or 50 μM of compstatin (a complement C3 inhibitor). Control for both experiments consisted of 15 μL of vehicle in 500 μL WB. Citrated native thrombelastography was performed on all samples, and time to clot initiation (R time, sec), rate of clot formation (angle, degrees), maximum clot strength (MA, mm), and percent clot breakdown at 30 and 60 minutes following MA (LY30, LY60) were measured.

Results: AP activation did not affect R time or angle but did increase median MA with both a 15 and 30‐minute incubation as compared to controls (53.8 mm vs. 59.5 and 62.8 respectively; p=0.0001). AP activation inhibited 60‐minute fibrinolysis (Ly60 5.4% vs. 3.5%; Figure 1) but not 30‐minute fibrinolysis. Inhibition of AP had no effect on R time, angle, or MA, but 30‐minute fibrinolysis significantly increased with treatment of 25 and 50 uM (LY30 3.0% vs. 7.8 and 12.4 respectively; Figure 2).

Conclusions: Activation of the complement AP inhibits fibrinolysis, and inhibition of AP increases fibrinolysis. This may be platelet‐mediated as AP activation increases clot strength.

FIGURE 1 Fibrinolysis is decreased with complement activation using Cobra Venom Factor

FIGURE 2 Inhibition of C3 and alternative complement pathway increases fibrinolysis

PB287: The Activity of XIIa‐dependent Fibrinolysis in Disorders of Carbohydrate Metabolism after Treatment with Regulatory Peptides

T. Shubina

Moscow State University named after M.V. Lomonosov, Biology Faculty, Moscow, Russian Federation

Background: Persistent hyperglycemia leads to a shift of the functional state of hemostasis towards hypercoagulability. It is shown that the regulatory peptide Pro‐Gly‐Pro‐Leu (PGPLeu) when administered intranasally provided preservation of normal function insular and fibrinolytic systems of blood during the development of alloxan diabetes in rats.

Aims: To evaluate glycemic and blood fibrinolytic activity when administered peptide: PGPLeu, LeuPGP or PGPLeu with low molecular weight heparin (complex), rats with diabetes mellitus type 2 (T2DM).

Methods: Simulated diet‐induced T2DM: daily for 2 weeks to rats intragastrically injected 40% glucose solution at a dose of 0.5 ml per 200 g of body weight, and from 15 to 21 days further intranasally injected a solution of one of the above peptides at a dose of 1 mg/kg. Control rats received saline. Blood for research was taken 1 hour after the last administration of peptides and 7 days after cancellation. In the blood plasma was measured by the glucose levels and XIIa‐dependent fibrinolysis (XIIa). All experiments were complied with the Declaration of Helsinki.

Results: Within 1 h after the last injection PGPLeu or complex, the glucose level was lower (p0.01) than in controls and corresponded to the norm. 7 days after the cancellation of the introduction of normal values were kept, but were not reliable. Introduction LeuPGP did not reveal significant differences on this indicator. Introduction PGPLeu and LeuPGP contributed to the maintenance of normal fibrinolytic activity in plasma during the entire period of observations. To control the activity of XIIa was reduced in 2 times. XIIa activity was increased 4 times after administration of the complex, but after 7 days after discontinuation return to normal.

Conclusions: Leucine containing peptides are able to protect the body from hyperglycemic attack when provoked the development of diabetes mellitus type 2, and by maintaining normal fibrinolytic activity to prevent activation of the coagulation potential of blood.

PB288: Plasma Proteolytic Enzymes Changes in Patients with Urinary Bladder Cancer as a Tool for Studying Progression

T. Luhovska 1 ; O. Savchuk 1 ; P. Yakovlev 2 ; N. Raksha 1 ; L. Ostapchenko 1

1 Taras Shevchenko National University of Kyiv, Educational and Scientific Center ‘Institute of Biology and Medicine’, Kiev, Ukraine, 2 Bogomolets National Medical University, Department of Urology, Kiev, Ukraine

Background: Urinary bladder cancer (UBC) is a common disease worldwide with high mortality rate. Studying plasma levels of proteases, fluctuation in their activity, and changes in the activity of proteases inhibitors might be valuable test in identifying UBC patients with poor prognosis, and add to understanding the carcinogenesis.

Aims: The aim of this study is to determine the activity of the proteolytic enzymes system and their inhibitors in patients with UBC.

Methods: The study enrolled 29 patients with UBC (Stage I ‐ 6; Stage II ‐ 5; Stage III ‐ 10; Stage IV ‐ 8). All patients had transitional cell carcinoma of the bladder, G3‐4. The control group consisted of 15 healthy individuals. The total plasma proteolytic activity was analysed by method of caseinolytic activity. The plasma level of α1‐antitrypsin (α1‐AT) and α2‐macroglobulin (α2‐MG) were determined using the Nartykova method. We used Microsoft Excel 2010 software to do a data analysis.

Results: The total activity of proteases was higher by 2 and 1.4 times compared to control in Stages III and IV bladder cancer patients, respectively. The activity of matrix metalloproteinase (MMP) was elevated in all groups. Activity of serine proteases was increased in patients with I, III and IV Stages of UBC by 2.1; 2.5 and 1.4 times respectively. The level of α1‐AT was up by 1.4 and 1.3 times in patients with Stage I and Stage IV, and down in patients with Stage II and III. The most significant increase was observed in level of α2‐MG in patients with I and III stages of UBC: by 3 and 3.6 times, respectively.

Conclusions: The rise in plasma proteases inhibitors level is an adequate response to the increased activity of proteases. The highest increase in protease inhibitors was observed in Stage I bladder cancer patients, which might signify their protective role at the onset of the bladder carcinogenesis. Significant growth of α2‐MG in patients with III stage of UBC may point at compensatory mechanism that inhibits tumor growth.

PB289: The Effects of Elaeagnus angustifolia, Corylus avellana or Silybum marianum on the Attenuation of Ischemic Brain Injury

M. Kamalinejad 1 ; H. Alimoradi 2 ; M. Soleymankhani 3 ; S. Asadi 4 ; A. Pourmoghaddam 5 ; M. Babaeian 1

1 Shahid Beheshti University of Medical Sciences, School of Pharmacy, Tehran, Iran, Islamic Republic of, 2 University of Otago, Department of Pharmacology and Toxicology, Dunedin, New Zealand, 3 Institute of Medicinal Plants, ACECR, Department of Pharmacognosy & Pharmaceutics, Karaj, Iran, Islamic Republic of, 4 Tufts Medical Center, Department of Pharmacy, Boston, United States, 5 Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran, Islamic Republic of

Background: Accumulating evidences suggest that cerebral ischemia elicits an unrestrained inflammation that starts within a few minutes and is sustained for a few days after an ischemic attack. Hence, the antioxidant and anti‐inflammatory agents may be important factors providing protection from neurological injury caused by stroke.

Aims: This study was designed to investigate whether the extracts of Elaeagnus angustifolia L. (EA) fruit, Corylus avellana L. (CA) kernel, and Silybum marianum Gaertn. (SM) seed are effective in a thromboembolic model of stroke in rats.

Methods: This study was designed to investigate whether the extracts of Elaeagnus angustifolia L. (EA) fruit, Corylus avellana L. (CA) kernel, and Silybum marianum Gaertn. (SM) seed are effective in a thromboembolic model of stroke in rats.

Results: All treated cases showed brain infarction in a significantly smaller size and less ipsilateral hemisphere edema in comparison with control group. Moreover, SOD and CAT values had been decreased significantly and the level of MDA had been increased after stroke induction. EA, CA, and SM administration also showed significant decline in the level of stroke‐induced TNF‐α. All of the treated animals indicated less Bederson and Racine scores compared to the control group.

Conclusions: Our findings demonstrated the effectiveness of these extracts for the first time in thromboembolic model of stroke which is most probably due to their antioxidant properties.

PB290: Novel Fibrinogen Missense Mutations in Patients with Fibrinogen Defects

G. Favuzzi 1 ; E. Chinni 1 ; G. Tiscia 1 ; P. Vergura 1 ; F. Cappucci 1 ; G. Vecchione 1 ; R. Bagna 2 ; C. Izzi 3 ; D. Rizzi 4 ; V. De Stefano 5 ; E. Grandone 1

1 Atherosclerosis and Thrombosis Unit, I.R.C.C.S. “Casa Sollievo della Sofferenza”, San Giovanni Rotondo FG , Italy, 2 Neonatology and Neonatal Intensive Care Unit, University Hospital, Città della Salute e della Scienza, Turin, Italy, 3 Department of Obstetrics and Gynecology, University of Brescia, Brescia, Italy, 4 Department of Pediatric and Neonatal Pathology, Hospital Marche Nord, Pesaro, Italy, 5 Institute of Hematology, Catholic University, Roma, Italy

Background: Congenital hypo‐/dys‐fibrinogenemias are defects defined by mutations in fibrinogen genes. We describe 10 subjects with this phenotype observed between 2011‐ 2016.

Aims: To better define the genetics features of fibrinogen deficiencies.

Methods: Functional fibrinogen was measured by Clauss method, antigen by radial immunodiffusion (NOR Partigen Fibrinogen). After DNA extraction and PCR, direct sequencing was performed according to standard protocols. Deleterious/damaging effect of missense mutations was predicted using tools SIFT (Sorting Intolerant from Tolerant), and Polyphen‐2 (Polymorphism Phenotyping v2). Mutations were classified as deleterious or damaging when SIFT was < 0.05 and/or Polyphen‐2 was close to 1.

Results: We identified 1 afibrinogenemic patient (p.Arg178* homozygote) with bleeding and thrombotic events, 3 hypo/dysfibrinogenemic patients (p.Thr47Ileu combined with IVS7+1G>T; p.Cys95Ser; p.Arg196Cys all in heterozygosis) referred for bleeding or thrombotic episodes and 6 hypofibrinogenemic patients (p.Glu41Lys; p.Gly191Val; p.Gly288Ser; p.His333Arg; p.Asp342Glu and p.343‐344 duplication; p.Asp356Val; all in heterozygosis) 4 of them were symptomatic for bleeding events. Overall, 4 novel mutations were found, all in hypofibrinogenemic patients. The p.Glu41Lys (SIFT score 0, Polyphen‐2 score 0.986) was identified within the FGA in a woman with bleeding after major orthopedic surgery. The remaining 3 novel mutations were identified within the FGG: p.Gly191Val (SIFT score 0.02, Polyphen‐2 score 1) in an asymptomatic woman; p.His333Arg (SIFT score 0, Polyphen‐2 score 1) in a woman experiencing a postpartum hemorrhage; p.Asp342Glu (SIFT score 0.23, Polyphen‐2 score 0.931) combined with duplication of asparagine‐343 and aspartate‐344 was found in a 7‐years‐old child who suffered from hematoma occurring after fall.

Conclusions: Four novel mutations were found in hypofibrinogenemic patients. Our findings contribute to shed light on the genotype‐phenotype relation in congenital fibrinogen disorders.

PB291: Fibrinogen‐binding Affimer with Antifibrinolytic Action Presents Potential New Therapeutic Target

K.J. Kearney 1 ; C. Tiede 2 ; F. Phoenix 1 ; F.L. Macrae 1 ; N. Oxley 1 ; N. Pechlivani 1 ; K.A. Smith 1 ; R.A. Ariëns 1 ; D.C. Tomlinson 2 ; R.A. Ajjan 1

1 University of Leeds, Leeds Institute of Cardiovascular and Metabolic Medicine, Department of Discovery and Translational Science, Leeds, United Kingdom, 2 University of Leeds, School of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, Leeds, United Kingdom

Background: Susceptibility of a fibrin clot to lysis influences its stability and has implications in disorders where enhanced fibrinolysis is the cause of bleeding. We hypothesised that engineered protein scaffolds, Affimers, represent a tool to manipulate fibrin clot properties and have the potential to identify novel therapeutic targets for bleeding disorders.

Aims: Use fibrinogen‐specific Affimers for the modulation of fibrin clot resistance to lysis.

Methods: A phage display system was used to screen a large library (n=10 10 ) of Affimers for fibrinogen binding. Effects of Affimers on fibrin clot properties were assessed by turbidimetric assays, ROTEM, and plasmin generation assays. Eight fibrinogen‐binding Affimers were isolated following screening, and a single Affimer with favourable effects on clot properties was chosen to study further, and is described here.

Results: Fibrinogen‐binding Affimer, F5, prolonged plasma clot lysis time in a dose dependent manner, from 14.0±5.0 minute in buffer control to 84.3±28.9 minute at 5:1 Affimer:fibrinogen molar ratio and to 189.8±32.8 minute at 10:1 (mean ± SD; P≤0.01). F5 enhanced resistance of clots to lysis without significantly affecting lag time (clot formation) or maximum absorbance (clot structure). In whole blood ROTEM clot lysis at 60 minutes was significantly delayed by F5 at all concentrations used with only 13.3±9.1% and 2.1±1.6% of the clot lysed at 1:1 and 5:1 compared with 87.4±8.5% lysis in buffer control (mean ± SD; P≤0.01). F5 significantly reduced the rate of plasmin generation in a chromogenic substrate assay from 0.062 ± 0.003 au/minute in buffer control to 0.052±0.001 and 0.042±0.001 au/minute (mean ± SD; P≤0.01) respectively at 1:1 and 5:1.

Conclusions: Our data show that a fibrinogen‐binding Affimer can modulate fibrinolysis in plasma and whole blood, without any major effects on clot architecture, an effect mediated through reduced plasmin generation. This novel approach may help to develop new antifibrinolytic therapeutic agents for use in bleeding disorders.

PB292: The Use of Fibrinogen‐binding Affimer Proteins to Modulate Clot Formation and Lysis

N. Pechlivani 1 ; R. King 1 ; C. Tiede 2 ; K. Kearney 1 ; I. Manfield 2 ; F. Phoenix 1 ; R. Ariens 1 ; D. Tomlinson 2 ; R. Ajjan 1

1 University of Leeds, Leeds Institute of Cardiovascular and Metabolic Medicine, Department of Discovery and Translational Science, Leeds, United Kingdom, 2 University of Leeds, School of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, Leeds, United Kingdom

Background: Affimers are non‐antibody binding proteins, composed of a constant scaffold region that constrains two nine amino acid variable regions. A phage display library (>3x10 10 ) was screened for fibrinogen‐binding Affimer proteins which we hypothesised can modify clot structure and lysis and may provide a novel approach to control excessive bleeding.

Aims: Study the effect of fibrinogen‐binding Affimers on clot structure and lysis using a purified fibrinogen system, human and mouse plasma.

Methods: Binding affinity and kinetics of Affimer proteins were studied by surface plasmon resonance. Turbidimetric assays and confocal scanning microscopy were used to assess the effects of Affimer proteins on clot structure and lysis.

Results: We identified two Affimer proteins, termed G6 and G2, which have similar association and dissociation rates and high affinity with KD values of 80±8.7 (mean±SD) and 42±4.9nM respectively. G6 and G2 consistently increased plasma clot lysis time in a concentration‐dependent manner in purified and plasma systems. G6 significantly increased plasma clot lysis time from 6.7±0.7 mins (mean±SD) to 11.8±1.2 mins at 10:1 Affimer:fibrinogen molar ratio (P< 0.001) while G2 prevented clot lysis after 3‐hours. Clots formed from human plasma in the presence of G6 and G2 showed a reduction in final turbidity from 0.210±0.013 AU to 0.124±0.012 and 0.050±0.001 AU (P< 0.001) respectively at 10:1 Affimer:fibrinogen molar ratio. Confocal microscopy showed that G6 did not cause changes to clot structure while G2 profoundly altered the structure showing large pores and bundles of fibrin in dense areas.

Conclusions: Fibrinogen‐binding Affimer proteins G6 and G2 both prolong fibrinolysis but have a differential effect on clot structure with G6 maintaining a physiological clot structure and G2 significantly altering fibrin network properties. These Affimer proteins may help to identify therapeutic targets that can be modulated using small molecules.

PB293: Increased Fibrinolysis as a Specific Marker of Poor Outcome after Cardiac Arrest

M. Schwameis 1 ; N. Buchtele 2 ; C. Schörgenhofer 2 ; A. Spiel 1 ; B. Jilma 2

1 Medical University of Vienna, Department of Emergency Medicine, Vienna, Austria, 2 Medical University of Vienna, Department of Clinical Pharmacology, Vienna, Austria

Background: Hypoperfusion resulting in hypoxemic endothelial tissue‐plasminogen activator release is considered one mechanism of fibrinolysis occurring during cardiopulmonary resuscitation. Data from patients with drowning‐induced out‐of‐hospital cardiac arrest suggests that an increase of endogenous fibrinolytic substances reflects prolonged or insufficient resuscitation efforts and may therefore provide reliable prediction of poor outcome.

Aims: The current study aimed to assess whether there is an optimal fibrinolysis cut‐off value as determined by thrombelastometry at hospital admission to predict poor outcome in a cohort of adult patients with out‐of‐hospital cardiac arrest.

Methods: Patients with out‐of‐hospital cardiac arrest of presumed cardiac origin, who had achieved return of spontaneous circulation at admission were analyzed. Fibrinolysis was assessed by thrombelastometry at the bedside immediately after hospital admission and is given as maximum lysis. The outcome measure was the optimal cut‐off for maximum lysis on hospital admission to predict poor outcome (cerebral performance category 3‐5) at day 30.

Results: Seventy‐eight patients were included in the study. Overall, 54% of patients had a poor 30‐day outcome. Fibrinolysis exceeding the normal reference value of 15% was present in 36% (95%CI 25‐48) of patients. The admission‐maximum lysis cut‐off predicting poor outcome with 100% specificity was ≥20% (sensitivity 41%, 95%CI 26‐58%). Patients presenting with a maximum lysis ≥20% (n=17, 22%) were older (64 vs. 56 years; P=0.004), and tended to have a higher prevalence of non‐shockable rhythm (35 vs. 15%, P=0.09), a significantly longer low‐flow time (42 vs. 24 minute, P< 0.001) and higher lactate (8.3 vs. 6.9 mmo/l, P< 0.001) and d‐dimer levels (20 vs. 7.4 μg/dl, P< 0.001). Patients presenting with a maximum lysis ≥20% had a median time to death of 6.5 days.

Conclusions: Increased fibrinolysis at admission assessed by thrombelastometry predicts poor outcome in cardiac arrest with presumed cardiac etiology.

PB294: Fibrinogen Content under Ischemic Stroke with and without Type 2 Diabetes Mellitus

O. Kravchenko ; N. Raksha; M. Tymoshenko; L. Ostapchenko

Taras Shevchenko National University of Kyiv, Kyiv, Ukraine

Background: One of the most important plasma proteins which is critical for hemostasis and clot formation is fibrinogen. Its cleavage by thrombin and subsequent polymerization forming fibrin strands provides the structural network required for effective clot formation. Besides a complex coupling of fibrinogen molecules and fibrin monomers named the soluble fibrin monomer complex (SFMC) is formed in the early‐activated state of blood coagulation. Thus such molecular complex is expected to serve as a parameter for the diagnosis of thrombus.

Aims: The aim of the present study was to determine the plasma fibrinogen and SFMC contents under acute ischemic stroke (IS) with or without type 2 diabetes mellitus and evaluate the significance of these haemostatic markers as predictors of mortality in stroke.

Methods: The study included 87 patients (median age, 74,2± 9,0years) who were admitted due IS and 25 population controls (median age, 70,2± 10,3 years).

All the patients underwent fibrinogen and SFMC level, glucose and glycosylated hemoglobin content measurements.

Fibrinogen plasma content was estimated by gravimetry with next spectrophotometry assays. Isolation of SFMC was done using the method with 0,1 M phosphate‐buffered saline containing sodium citrate and 0,2% 6‐aminohexanoic acid with following determination.

Data entry and analysis were performed using StatSoftStatistica. The significance of parameters differences between groups was determined using the Mann‐Whitney U test.

Results: It was established the statistically significant changes of fibrinogen and SFMC levels in both investigated patient groups in comparison with the control. Fibrinogen blood content reached 130% and 135% in IS and IS+DM patient respectively comparing with the control. Nevertheless statistically significant differences of fibrinogen and SFMC levels in patients with IS and IS complicated by diabetes were not detected.

Conclusions: Besides high blood level of SFMC in patients with IS provided the basis for negative survival forecast.

TABLE 1 Predictors of mortality under acute ischemic stroke M m SE 95% СI P Fibrinogen, g/L survived 3.07 0.74 0.08 2.89‐3,25 dead 3.15 0.77 0.20 2.52‐3,38 0.552 SFMC, mg/100ml survived 3.31 1.36 0.16 3.09‐3.67 dead 3.83 1.55 0.40 3.57‐4.29 0.033

PB295: Euglobulin Lysis Time (ELT) Behaviour in Patients Wwith Abnormal Clot Solubility Test (CST)

L. Remotti ; M.F. Ingratti; S.H. Grosso; M.P. Vera Morandini; M.L. Romero; M.G. Agazzoni; A. Sánchez‐Luceros; A.N. Blanco

Academia Nacional de Medicina, IIHEMA, Hemostasia y Trombosis, CABA, Argentina

Background: FXIII stabilizes fibrin and protects it from fibrinolysis; Alpha 2‐antiplasmin (a2PI) is involved. Independently of a2PI, FXIII plays a role in fibrin stability increasing its resistance to fibrinolysis.

Aims: The ELT behaviour was analysed, in patients with bleeding and abnormal CST, in order to see if any relationship could exist between both tests, considering that CST depends on FXIII and ELT reflects fibrinolysis in absence of a2PI.

Methods: Subjects: 180 patients with bleeding symptoms. CST and ELT were tested, in addition to other platelet and coagulation studies. A retrospective analysis was performed. CST: samples were clotted by CaCl2 (25mM); after 30min at 37°C, acetic acid (2%) was added (RV> 72h). ELT: euglobulin fraction (a2PI free) was suspended in borate solution (0.9%) and clotted by CaCl2 (25mM) at 37°C

(RV: 90‐240min).

Results: Patients were classified according to CST results as either normal (111/180; 61,7%) or abnormal (69/180; 38.3%). As displayed in the table only 4 presented abnormal ELT in the group of patients with normal CST; 11 patients showed abnormal ELT in the group with abnormal CST. Borderline ELT values were found in 4 and 1 patients with normal and abnormal CST respectively. The frequency of ELT abnormalities among patients with abnormal CST was higher ( chi square test P=0.011) than among those with normal CST. Other abnormalities, like platelet alterations, coagulation factors disorders or both, were detected in either patients with normal (96/111; 86.5%) or abnormal (55/69; 79.7%) CST; the differences were not significant ( chi square test P=0.23).

TABLE 1 ELT and CST in patients with bleeding symptoms Euglobulin lysis time (ELT) Clot‐solubility in acetic acid (CST) Normal (>72h) n=111 Abnormal (≤72h) n=69 Abnormal (<90min) 4/111 (3.6%) (55‐75min) 11/69 (15.9%) (65‐85min) Borderline (=90min) 4/111 (3.6%) 1/69 (1.5%) Normal (>90min) 103/111(92.8%) (95‐>300min) 57/69 (82.6%) (95‐>285min)

Conclusions: The study showed that the number of patients with abnormal ELT was significant higher among those with abnormal CST, suggesting a possible relationship between FXIII activity and fibrinolytic function, independently of a2PI. Further studies could help to elucidate the role of direct modulation of fibrin structure by FXIII, compared with a2PI cross‐linking.

PB296: Association between Use of Recombinant Tissue Plasminogen Activator and In‐hospital Mortality and 30‐day Readmission among Ischemic Stroke Patients Undergoing Mechanical Thrombectomy: A Retrospective Cohort Study

A. Sarayani 1 ; T. Youn 2 ; J. Brown 1

1 University of Florida College of Pharmacy, Gainesville, United States, 2 University of Florida College of Medicine, Gainesville, United States

Background: Mechanical thrombectomy (MT) yields substantial revascularization success rate in ischemic stroke treatment for patients with large vessel occlusions. There is a knowledge gap whether recombinant tissue plasminogen activator (rt‐PA) is associated with improved outcomes in MT patients versus MT alone.

Aims: To evaluate the association between rt‐PA use and in‐hospital mortality or all‐cause 30‐day unplanned readmission among MT patients.

Methods: A retrospective cohort study was conducted using data from Nationwide Readmissions Database (NRD) 2013‐2014. Patients with primary diagnosis of ischemic stroke were included. MT and rt‐PA were identified by appropriate procedure codes. Records with transfer, same day event status, < 1 day of length of stay, and admissions in December were excluded. In‐hospital mortality was identified as coded and readmission was calculated based on stroke quality measure documentation. Index admissions were followed within the same year. Multiple demographic, clinical, and hospital related covariates were adjusted in multivariable hierarchical logistic regression model. The cohort was stratified in non‐elderly and elderly groups to evaluate possible effect modification by age.

Results: In the final cohort, 1,607 patients received MT/rt‐PA and 1,712 patients received MT alone. Patients’ characteristics were approximately similar between groups (median age of 70 and 50% female). Mortality and readmission incidences in MT/rt‐PA versus MT group were 15.7% versus 18.7% and 13.2% versus 11.7%, respectively. The adjusted odds ratio for the mortality and readmission outcomes were 0.78 (0.65‐0.95) and 1.26 (0.99‐1.61), respectively (MT as reference). In age stratified results, only those < 65 years of age showed a benefit with rt‐PA and only for mortality (OR=0.54 [0.36‐0.83]).

Conclusions: Use of rt‐PA was associated with lower risk of in‐hospital mortality but only in those < 65 years old. The effect of age on outcomes associated with rt‐PA should be further explored.

PB297: Staphylokinase Potentiates Thrombolytic Effect of Single Chain Pro‐urokinase in Plasma Milleu in vitro

R. Aisina 1 ; L. Mukhametova 1,2 ; K. Gerchkovich 3 ; E. Ivanova 1

1 Lomonosov Moscow State University, Chemical Department, Moscow, Russian Federation, 2 Bauman Moscow State Technical University, Moscow, Russian Federation, 3 Emanuel Institute of Biochemical Physics RAS, Moscow, Russian Federation

Background: Staphylokinase (STA) binds to fibrin‐bound plasminogen (Pg). Conversion of inactive Pg . STA complex to active plasmin(Pm) . STA complex is accelerated by the product. Pro‐urokinase (pro‐UK) has affinity only for Pg molecules which are bound to the COOH‐terminal lysines of partially degraded fibrin. Combinations of STA and pro‐UK possessing different mechanisms of Pg activation and fibrin‐selectivity of action can induce a synergistic thrombolysis.

Aims: To study thrombolytic and side effects of different combinations of STA and pro‐UK in order to reveal a potential synergism of their action in vitro .

Methods: Kinetics of plasma clots lysis and depletion of plasma levels of Pg, fibrinogen and α2‐antiplasmin induced by different doses of STA, pro‐UK and their combinations were monitored.

Results: Equieffective doses which caused 50% clot lysis in 4 h were 30 nM STA and 75 nM pro‐UK. Equieffective doses of STA, pro‐UK and their combinations which caused the same specified effects in 2 or 4 h were analyzed by the algebraic fractional method. Synergism of STA and pro‐UK was found at total doses of the agents from 25 to 65 nM. The maximal 3.5‐4‐fold increase in thrombolysis was observed for combinations of 10‐15 nM STA and 15‐35 nM pro‐UK as compared with the expected thrombolysis, obtained by summarizing of the effects of individual agents. The synergistic combinations of STA and scuPA caused the significant less depletion of Pg, fibrinogen and α2‐antiplasmin levels in plasma, as compared with the expected effects.

Conclusions: Trace concentrations of Pm . STA complex, formed from fibrin‐sorbed Pg . STA complex, initiate a chain of reactions on fibrin surface (conversion of Pg . STA to Pm . STA, Pg to Pm and pro‐UK to high active two‐chain UK). Increase in Pm concentration on fibrin surface has a positive feedback. Acceleration of all reactions on fibrin surface stipulates fibrin specificity of synergistic thrombolytic effect of combinations of STA and pro‐UK.

PB298: Local Ultrasound‐facilitated Thrombolysis in Pulmonary Embolism: First Dutch Experience and Retrospective Comparison with Systemic Thrombolysis

M.A. de Winter 1 ; E.A. Hart 2 ; D.A.F. van den Heuvel 3 ; A. Moelker 4 ; R.J. Lely 5 ; H.A.H. Kaasjager 1 ; P.R. Stella 2 ; S.A.J. Chamuleau 2 ; A.O. Kraaijeveld 2 ; M. Nijkeuter 1

1 University Medical Center Utrecht, Internal Medicine, Utrecht, the Netherlands, 2 University Medical Center Utrecht, Cardiology, Utrecht, the Netherlands, 3 St. Antonius Hospital, Interventional Radiology, Nieuwegein, the Netherlands, 4 Erasmus University Medical Center, Interventional Radiology, Rotterdam, the Netherlands, 5 VU University Amsterdam, Amsterdam, the Netherlands

Background: Systemic thrombolysis (ST) is an effective treatment in high‐risk pulmonary embolism (PE), but is associated with bleeding complications. Ultrasound‐facilitated, catheter‐directed thrombolysis (USAT), using a lower dose of thrombolytic agent, possibly reduces bleeding complications.

Aims: To describe patient characteristics and outcomes of patients with PE who underwent USAT and to compare outcomes of USAT and ST in high‐risk patients.

Methods: All patients who underwent USAT for PE in the Netherlands from 2010‐2017, and patients undergoing ST for PE from 2007‐2017 in the UMC Utrecht were identified. Characteristics, treatment details and outcomes of USAT patients were assessed and compared to ST patients. Primary outcomes were major bleeding and all‐cause mortality after 1 month. Secondary outcomes were all‐cause mortality and recurrent venous thrombo‐embolism (VTE) within 3 months.

Results: 44 patients underwent USAT, of whom 33 (75%) high‐risk. Major bleeding occurred in 14 patients (32%; 95% CI 20‐47), including 2 (5%) intracranial and 4 (9%) fatal bleeding. All‐cause mortality after 1 month was 42% (95% CI 28‐57). Comparing high‐risk PE patients treated with USAT (33 patients) and ST (53 patients), no significant differences in outcomes were found (major bleeding 37% vs. 25%, P = 0.24, including 1 versus 5 intracranial bleeds; all‐cause mortality after 1 month 49% versus 59%, P = 0.37; recurrent VTE 0% versus 3%, P = 0.20).

Conclusions: Major bleeding occurred more often in USAT patients and intracranial bleeds were more frequent in ST patients, although not statistically significant. However, USAT was predominantly used in a heterogeneous group of severely ill high‐risk patients. Further research is required to establish the role of USAT in high‐risk PE and to guide proper patient selection.

PB300: Endogenous Fibrinolysis in Patients with Acute Coronary Syndrome and Healthy Volunteers

P. Savvinova ; A. Kalinskaya; A. Shpektor; E. Vasilieva

Moscow State University of Medicine and Dentistry, Moscow, Russian Federation

Background: The clot lysis process remains one of the most controversial issue in the pathogenesis of acute coronary syndrome (ACS). Coronary artery thrombosis is always accompanied by endogenous fibrinolysis. Difference in clot formation and endogenous fibrinolysis activity between patients with ACS and healthy volunteers still remains uninvestigated.

Aims: The purpose of our work was to analyze the differences in the main parameters of clot formation and endogenous fibrinolysis between patients with ACS and healthy volunteers.

Methods: Our study included 25 patients with ACS and 29 healthy volunteers. We assessed the process of clot formation and fibrinolytic system activity using Thrombodynamics Analyser System and the native ROTEM ® test.

Results: We found out that the spontaneous clots formation time (74.86 minutes vs. 42 minutes, P=0.01) as well as the maximum clot density (27,407 a.u. vs. 25,001 a.u., P=0.03) were significantly higher in ACS patients compared to healthy volunteers. Surprisingly, the fibrinolysis process was much more active in ACS patients compared to healthy volunteers as well. The lysis onset time (56.2 minutes vs. 90 minutes, P=0.001) and its progression (65.5% vs. 56.0% P=0.023) were significantly faster in ACS patients compared to healthy volunteers. Nevertheless, as the result of the two competitive processes (clot formation and endogenous fibrinolysis), we found the tendency to more effective clot lysis in healthy volunteers compared to ACS patients (23% vs. 18% P=007).

Conclusions: The clot formation as well as the endogenous fibrinolysis were much more active in ACS patients compared to healthy volunteers. But the efficiency of the fibrinolysis was higher in healthy volunteers. We explain it by the possibility of excessive activation of clot formation process in ACS patients.

PB302: Clot Formation Assessment in Septic Patients with Cancer using a Time‐based Parameter from the ROTEM Velocity Curves. A Prospective Observational Pilot Study

E. Scarlatescu 1 ; D.R. Tomescu 1,2

1 Fundeni Clinical Institute, Anesthesia and Intensive Care III, Bucharest, Romania, 2 University of Medicine and Pharmacy Carol Davila, Bucharest, Romania

Background: Viscoelastic tests are widely used for hyperfibrinolysis diagnosis, however, their ability to detect pathological fibrinolytic resistance is limited.

Aims: Using the velocity curve of clot formation in ROTEM, we calculated a time‐based parameter from the point of the maximum velocity of clot formation to the point when the maximum amplitude is reached (t‐AUCi). We hypothesized that this parameter is significantly different in septic versus non‐septic cancer patients and that it identifies septic patients with high‐risk mortality at ICU discharge.

Methods: After the approval of the ethics committee, we included in this prospective observational study adult patients with solid malignant tumors in the pelvis or abdomen. For each patient, whole‐blood ROTEM was performed and t‐AUCi was calculated from the EXTEM assay using the formula: t‐AUCi=CT+(t‐MCF)‐(t‐MaxVel).

Results: The study group (SG) included 46 patients operated for solid cancers diagnosed with sepsis or septic shock. The control group (CG) included 42 non‐infected cancer patients. t‐AUCi had positive correlations with the lysis indices at 30, 45, 60 minutes (P>0.001, correlation coefficients of 0.382, 0.707 and 0.656). Clot lysis indices at 45 and 60 minutes and t‐AUCi were significantly increased in the SG compared to the CG.

TABLE 1 Comparison of lysis parameters and t‐AUCi value for patients in the SG and CG Test SG (n=46) CG (n=42) Mann Whitney U value P value t‐AUCi (s) 1774 (785.25) 1577 (471.25) 688 0.02* LI30 (%) 100 (0) 100 (0) 949 0.731 LI45 (%) 100 (1) 98 (2) 531.5 <0.001* LI60 (%) 99 (4) 96 (4) 519.5 <0.001*

At ICU discharge, 15 patients from the SG were alive (32.6%) and 31 patients deceased. Lysis indices at 30, 45 and 60 minutes, t‐AUCi and SOFA score were compared between survivors and non‐survivors; significant differences were found only for t‐AUCi, which was increased in non‐survivors compared to survivors.

TABLE 2 Comparison of lysis indices, the value of t‐AUCi and SOFA score between survivors and non‐survivors from the study group Parameter Survivors (n=15) Non‐survivors (n=31) Mann Whitney U value P value t‐AUCi (s) 1606.5 (702) 2006 (950) 114.5 0.006* LI60 (%) 98.5 (5) 99 (3) 182 0.225 LI45 (%) 100 (2.3) 100 (1) 184.5 0.193 LI30 (%) 100 (0) 100 (0) 208.5 0.189 SOFA score 9 (5.3) 10.5 (4.3) 139.5 0.084

For SG patients without lysis at 45 and 60 minutes, t‐AUCi was increased in non‐survivors compared to survivors (P=0.004).

Conclusions: For septic patients with cancer having low fibrinolytic activation, increased duration of the descending part of the ROTEM velocity curve (t‐AUCi) correlates with a higher mortality risk at ICU discharge. For firm conclusions the completion of this pilot study is required.

PB303: Increase in Plasma Stability and Thrombolytic Efficacy of pro‐UK by Chemical Modification of its Arginine Residues

L. Mukhametova 1,2 ; R. Aisina 1 ; K. Gerchkovich 3

1 Lomonosov Moscow State University, Chemical Department, Moscow, Russian Federation, 2 Bauman Moscow State Technical University, Moscow, Russian Federation, 3 Emanuel Institute of Biochemical Physics RAS, Moscow, Russian Federation

Background: Single‐chain pro‐urokinase (proUK) is converted into a high active two‐chain urokinase (UK) by plasmin formed during the plasminogen (Pg) activation. UK is rapidly cleared from circulation due to its neutralization by the plasminogen activator inhibitor‐1 (PAI‐1). The formation of salt bridges between series of positively charged residues (Arg‐Arg‐His‐Arg) occupying a surface loop of UK with a complementary series of negatively charged residues in PAI‐1 plays an important role in the binding of UK to the inhibitor.

Aims: To study the effect of chemical modification of Arg residues with phenylglyoxal on properties of pro‐UK in vitro .

Methods: Recombinant proUK (1‐9 mM) was incubated with phenylglyoxal (5‐50 mM) under mild conditions (pH 7.0‐8.3; 4°C). During incubation, aliquots were removed, purified and assayed for the number of unmodified Arg residues and Pg‐activator activity.

Results: Only 13 of the total 22 Arg residues in proUK were modified with phenylglyoxal at the used conditions. Different preparations of proUK in which 4, 7, 9, 11 or 13 Arg residues were modified (mod‐proUK4 ‐ 13) were obtained. The specific Pg‐activator activities of mod‐proUK4 ‐ 13 were equal to that of native proUK. All modified preparations were more stable in human plasma than native proUK: a half‐life of fibrinolytic activity was >180 minutes for mod‐proUK4, 140‐120 minutes for mod‐proUK7‐13, and 110 minutes for native proUK. The most stable mod‐proUK4 caused more effective plasma clot lysis compared with the native and other modified proUK preparations: 50% lysis was achieved in 80 minutes with proUK4 and 110 minutes with proUK or mod‐proUK7‐13 at using 550 IU/ml doses of the agents.

Conclusions: Modification of the most available 4 Arg residues does not influence on the activator‐activity of proUK, but increases its stability in human plasma and efficacy in lysis of the clot. The observed effects are due to the increased resistance of mod‐UK4, forming from mod‐proUK4 during the plasminogen activation, towards neutralization with PAI‐1.

PB304: Tissue Plasminogen Activator (tPA) ‐ Loaded PLGA Microspheres Administration in Combination with Extracorporeal Shock Wave Thrombolytic Therapy Reduce Thrombus in the Arterial Thromboembolic Occlusion

H. Mehrad 1,2 ; B. Hajipour 3 ; F. Nasirzadegan 4 ; A. Keshavarz 4 ; A. Arshadi 4 ; S. Sarabi 4 ; P. Karimi 2

1 Islamic Azad University, Tabriz Branch‐Faculty of Basic Sciences, Department of Physics, Tabriz, Iran, Islamic Republic, 2 Tabriz University of Medical Sciences, Neurosciences Research Center, Tabriz, Iran, Islamic Republic, 3 Sabzevar University of Medical Sciences, Department of Surgery, Sabzevar, Iran, Islamic Republic, 4 Islamic Azad University, Tabriz Branch‐Faculty of Basic Sciences, Department of Bioengineering, Tabriz, Iran, Islamic Republic

Background: Thromboembolism, characterized by formation a thrombus in a blood vessel that breaks loose and is carried by the blood stream to plug another vessel. The clot may plug a vessel in the lungs (pulmonary embolism), brain (stroke), gastrointestinal tract, kidneys, or leg. Thromboembolism is a significant cause of morbidity and mortality, especially in adults.

Aims: The aim of this study was to generate a rabbit model of carotid artery thromboembolic occlusion and the subsequent investigating the feasibility of tPA‐ loaded PLGA microspheres‐ mediated shock wave thrombolytic therapy in this model.

Methods: Briefly, New Zealand white rabbits were submitted to common carotid artery thromboembolic occlusion by injecting autologous blood clots. Then treatment group underwent extracorporeal pulsed‐ focused electrohydraulic shock wave (10 Kv, 0.3 Hz, 0.4 mj/mm 2 , 150 impulses) thrombolytic therapy accompanied by tPA (0.6 mg/kg)‐ loaded PLGA (Poly Lactic‐ Co‐ Glycolic Acid) microspheres administration.

Results: Results from histopathology, B‐mode and color Doppler ultrasonography, showed a significant reduction in the mean value for thrombus content, blood mean velocity and a significant increase in the mean value for blood volume flow at the stenotic region in the treatment group compared with the other groups (P>0.05).

Conclusions: Enhanced anti‐ thrombotic effect of tPA, due to inertial cavitation effect of collapsed microspheres‐ induced by shock wave therapy, can cause to reduce the thrombus content and significantly dilate the luminal cross‐sectional area of stenosis and lower treatment time in comparison with conventional tPA thrombolytic therapy.

PB305: Recombinant Destabilase‐lysozyme ‐ Long‐term Thrombolytic with the Original Mechanism of Action

S. Kalabushev 1 ; D. Nalobin 1 ; D. Akhaev 1 ; V. Manuvera 2 ; V. Lazarev 2 ; I. Baskova 1

1 Lomonosov Moscow State University, Faculty of Biology, Moscow, Russian Federation, 2 Federal Research and Clinical Center of Physical‐Chemical Medicine of Federal Medical Biological Agency, Moscow, Russian Federation

Background: Over the years, among the thrombolytic drugs reign proteolytic enzymes which quickly leave the bloodstream and require continuous administration. After the discovery of enzyme named Destabilase‐Lysozyme ‐ endo‐ε‐(γ‐Glu)‐Lys‐isopeptidase, the family of its c‐DNA, available methods of recombinant protein preparation and subsequent study of the drug pharmacokinetics, there appeared the thrombolytic drug, which can be injected once a day.

Aims: Our aim was to find out how long destabilase circulates in blood, what tissues accumulate it and how is it excreted.

Methods: This pilot study was performed on 21 C57Bl6 male mice. After intravenous administration of 125 I‐destabilase, three animals were euthanized at 0.5, 1, 3, 9, 24, 48 and 72 hours, tissue samples were taken, sample radioactivity was measured on a WIZARD 2480 scintillation automatic gamma counter.

Results: Half‐life time of the protein in blood was 15.4 hours. 125 I‐destabilase accumulates in kidneys, spleen, lungs, skin. 30 minutes after administration, the concentration in kidneys exceeded that in blood 2.75 times, kidneys secrete the drug. Destabilase pass through the blood‐brain barrier. The maximum amount of 125 I‐destabilase is displayed in the time interval 3‐9 hours.

Conclusions: Destabilase is a potentially effective injection drug that persists in blood for a long time in comparison with other thrombolytics and does not affect the balance of thrombin and antithrombin systems. In future research, it is necessary to find out how this protein overcomes the hemato‐encephalic barrier and why it accumulates in the organs listed above.

PB306: Genome Sequence of the Medicinal Leech Hirudo Medicinalis. Discovery of Novel Anticoagulant and Fibrinolytic Proteins and Peptides

V. Lazarev ; V. Manuvera; D. Shirokov; O. Podgorny; E. Grafskaia; D. Kharlampieva; N. Polina; P. Bobrovsky; V. Babenko

Federal Research and Clinical Center of Physical‐Chemical Medicine, Moscow, Russian Federation

Background: Diseases associated with disorders of hemostasis and increased thrombi formation occupy a significant place among the problems facing modern medicine. The search for new anticoagulant, antiplatelet, and fibrinolytic drugs is an actual task. The ability of a secret of salivary glands of medicinal leeches to affect on blood coagulation has been known since ancient times. Ingredients of leech saliva may suppress inflammation, reduce the intensity of pain, inhibit blood coagulation in the host and enrich the ingested blood with anti‐microbial substances for long‐term storage. Undoubtedly, leeches are vast natural pharmacopoeias selected by evolution. Despite the broad focus of research on leech saliva, little has been done at the genomic level.

Aims: The goal of this project is to produce, annotate, and analyze a high‐quality genomic sequence for the leech Hirudo medicinalis for novel anticoagulant and fibrinolytic proteins and peptides prediction.

Methods: NGS sequencing and bioinformatic analysis.

Results: We performed whole genome sequencing of Hirudo medicinalis . Using Ion Proton shotgun sequencing, Ion Torrent mate‐pair sequencing and Illumina mate‐pair sequencing platforms a total of about 232,003,367 bp were assembled and bioinformatics analysis was made. Several peptides and proteins with possible thrombolytical, anticoagulant and antimicrobial properties were identified.

Conclusions: Genome of the medical leech Hirudo medicinalis was sequenced for the first time. On the basis of genomic data the new peptides and proteins influencing human hemostasis can be found.

This work was supported by the Russian Science Foundation (project№17‐75‐20099).

PB307: Gender Differences in TAFI Regulation in Some Disorders

E. Roitman 1 ; M. Tanashyan 2 ; A. Shabalina 2 ; I. Kolesnikova 1

1 Pirogov Russian National Research Medical University, Moscow, Russian Federation, 2 Research Center of Neurology, Moscow, Russian Federation

Background: The study of gender differences in TAFI regulation offers a key to precision balance of risks between thrombosis and bleeding.

Aims: To study comparatively the TAFI and its regulation under the same disorders in men and women.

Methods: The study included 115 patients with myeloproliferative neoplasms (MPNs; men=52; women=63), 96 patients with CCVD comorbided with Ph‐negative MPNs (men=20; women=76), 174 patients with acute ischemic stroke (men=112; women=62), and 96 patients followed up within 12 months after acute ischemic stroke (men=52; women=44). We measured TAFI, Proteins C and S (PC; PS), VWF, thrombomodulin (TM), thrombospondin, fXII and other clotting factors, angiogenesis markers, and cell adhesion molecules. Gender, calcium and creatinine were considered as well.

Results: No significant difference in TAFI were found for men and women with the same disorder. TAFI had higher in patients after stroke (Median: men 60.20%; women 57.00%) and lowest in patients with MPNs (Median: men 50.35%; women 45.00%).

Multivariate analysis showed that in men with MPNs the TAFI regulation performs by both fXII‐ and endothelium‐related pathways. In women with MPNs the TAFI regulation finished by fibrinolysis enhancement is performed by only endothelium‐related agents.

In CCVD with MPNs the TAFI regulation was dis‐coordinated in men, while in women the endothelium‐related pathway involving interplays between sVEGFR1, VWF, PC and thrombospondin has clearly occured.

In men after stroke the TAFI was under both regulation pathways with strong dependence on renal function, while in women the TAFI regulation has looked discoordinated.

In men with acute stroke the TAFI regulation has performed through PC, while in women there was clear contribution of thrombin generation regulators associated with the endothelium (sVEGF‐R1, PS, TM, sVEGFR2, VWF, and thrombospondin) in addition to PC.

Conclusions: All patients had enhanced fibrinolysis. TAFI regulation is performed under different dominants depending also on gender.

PB309: The Role of Plasminogen Activator Inhibitor‐1 (PAI‐1) Levels and PAI‐1 4G/5G Polymorphism in the Outcome of Intravenous Thrombolytic Therapy in Ischemic Stroke Patients

I. Szegedi 1 ; E.G. Székely 2 ; K.R. Czuriga‐Kovács 1 ; A. Nagy 3 ; F. Sarkady 2 ; L.I. Lánczi 4 ; E. Berenyi 4 ; L. Csiba 1,5 ; Z. Bagoly 2,5

1 University of Debrecen, Department of Neurology, Debrecen, Hungary, 2 University of Debrecen, Department of Laboratory Medicine, Division of Clinical Laboratory Sciences, Debrecen, Hungary, 3 University of Debrecen, Department of Preventive Medicine, Debrecen, Hungary, 4 University of Debrecen, Department of Radiology, Debrecen, Hungary, 5 MTA‐DE Cerebrovascular and Neurodegenerative Research Group, Hungarian Academy of Sciences, Debrecen, Hungary

Background: Thrombolysis by recombinant tissue plasminogen activator (rtPA) is the main pharmacological therapy in acute ischemic stroke (IS), however, it is only effective in a subset of patients.

Aims: Here we aimed to investigate the role of plasminogen activator inhibitor‐1 (PAI‐1), an effective inhibitor of tPA, and its major polymorphism (PAI‐1 4G/5G) in the outcome of therapy.

Methods: Study population included 132 consecutive IS patients who all underwent thrombolysis. Blood samples were taken on admission, 1 and 24 hours after rtPA infusion. PAI‐1 activity and antigen levels were measured from all blood samples and the PAI‐1 4G/5G Polymorphism was determined. Clinical data of patients including the National Institutes of Health Stroke Scale (NIHSS) were registered at admission, day 1 and 7 after therapy. ASPECTS was assessed using CT images taken before and 24 hours after thrombolysis. Intracranial hemorrhage was classified according to ECASS II. Long‐term outcome was defined 3 months post‐event by the modified Rankin Scale.

Results: PAI‐1 activity levels dropped transiently after thrombolysis, while PAI‐1 antigen levels remained unchanged. PAI‐1 4G/5G polymorphism showed no significant association with PAI‐1 levels and did not influence stroke severity. PAI‐1 activity/antigen levels as measured on admission were significantly elevated in patients presenting with worse 24 hours ASPECTS (7‐0). In a binary backward logistic regression analysis including age, sex, NIHSS on admission, the presence of 4G allele conferred a significant protective effect against post‐lysis intracranial hemorrhage (OR:0.30, 95% CI:0.09‐0.99, P=0.048). PAI‐1 levels and PAI‐1 4G/5G polymorphism had no influence on long‐term outcomes.

Conclusions: PAI‐1 4G allele is associated with a significant protection against intracranial hemorrhage in post‐lysis stroke patients.

Funding: NKFI K120042, PD111929

VASCULAR BIOLOGY

PB310: Differentiation of Coagulation Parameters in Patients with Acute Coronary Syndrome

Y. Tyravska 1 ; T. Vovk 2 ; N. Raksha 2 ; V. Lizogub 1

1 Bogomolets National Medical University, Department of Internal Medicine # 4, Kyiv, Ukraine, 2 Educational and Scientific Center ‘Institute of Biology and medicine’, Taras Shevchenko National University of Kyiv, Department of Biochemistry, Kyiv, Ukraine

Background: Soluble fibrin monomer appears in the bloodstream during the extremely early stage of blood coagulation and generally forms a complex with fibrinogen, termed soluble fibrin monomer complex (SFMC). Determination of SFMC and prothrombin content can provide information regarding the thrombotic conditions and degree of blood coagulation system activation.

Aims: To investigate the role of SFMC and prothrombin in the thrombotic conditions and blood coagulation system activation in patients with ACS.

Methods: Blood plasma was taken from 35 healthy donors and 9 patients with acute myocardial infarction (AMI), 14 patients with new onset unstable angina (NOUA), 21 patients with progressive unstable angina (PUA), 16 patients with stable angina (CSA) and 9 patients with silent myocardial ischemia (SMI). SFMC was determined using the phosphate method, prothrombin content was determined by ELISA method according to standard protocol.

Results: The conducted research has established the statistically significant changes of SFMC levels under all investigated pathologies, suggesting the existence of prothrombotic states. Plasma SFMC was elevated in 12 (in NOUA group), 10 (in AMI and SMI group) and 6 times (in CSA group) in comparison with the control group. According to obtained result accumulation of soluble fibrin in the blood plasma is accompanied by reduction of prothrombin level. It could indicate indirectly generation of thrombin and activation of blood coagulation system. So, prothrombin blood content was about 1.6 times lower in all groups comparing with healthy donors. The most significant difference was detected in patients with acute myocardial infarction.

Conclusions: The conducted results have shown that all tested pathologies despite some differences in etiology are characterized by similar direction of changes of the investigated hemostasis parameters. High levels of plasma SFMC in combination with a reduction of prothrombin content indicate hypercoagulable state in patients with NOUA, AMI, SMI, SA.

PB311: Cell Free Desoxyribonucleic Acids (cfDNA) Levels Remain High after 6 Months Despite Successful PCI in STEMI Patients which Along with High Metalloproteinases MMP9/MMP2 Activity Is Associated with Unfavorable Disease Course

O. Koval’ ; A. Skoromna

Dnipropetrovsk State Medical Academy, Dnipro, Ukraine

Background: Neutrophils (Nph) are highly reliably associated with endpoints in STEMI patients. The extracellular traps are formed from Nph DNA and released in cell free form into plasma. Its histones stimulate platelets, which may support the coagulation process. But longevity of these changes, their impact on clinical disease course are poorly investigated.

Aims: To elaborate the clinically applicable method for cfDNA levels measuring in STEMI patients after primary PCI (pPCI) and investigate their association with disease course.

Methods: 84 samples (after PCI, 7th day, 6‐th months) from coronary arteries (CA) and peripheral vein were taken in STEMI patients after pPCI. For cfDNA plasma levels the original method was used: the DNA isolation from citrate plasma (Sintol, Rus) + DAPI (Sigma, USA) 20 мM (1:500), incubation in dark with further results evaluation by Bio‐Tek FL600 (USA). Samples measurements were done at fluorescence Ex360 nm/Em460 nm. Normal ranges were 200±24 ng/ml. For proMMP9, MMP9, MMP2 activity gelatin zymography method was used, and MMP9 pool activity to MMP2 was calculated.

Results: cfDNA plasma levels in patients with STEMI were above normal range (P>0.001) in acute period. Just after pPCI max level was seen in CA (M 980; 780‐115 ng/ml) unlike vein (M 405; 220‐600 ng/ml; P=0.023) but at 7th day the latter was slightly increased (M 487; 290‐ 630 ng/ml) and not completely resolved to normal in 6 months (M 327; 210‐400 ng/ml; P=0.058). Vein levels didn't correlate with Nph count, unlike CA levels (r=‐0.54; P=0.049). Along with high interrrelation of pool MMP9/MMP2 (>1.8) in vein at 7‐th day high cfDNA levels (≥75%) defined further recurrent thrombotic events at 6 month.

Conclusions: Neutrophils activity has association with recurrent thrombotic events even after successful PCI in STEMI pts and this impact is realized through cfDNA release and MMP activation. These data need further confimation but testify in favor of antiplatelet therapy prolongation to interrupt Nph‐platelets crosstalk.

PB312: Low ‐ Level Blue Laser ‐ Mediated Photodynamic Therapy Accompanied by 5‐ Aminolevulinic Acid Administration Reduce Smooth Muscle Cells Density in the Rabbit Carotid Artery Neointimal Hyperplasia Model

H. Mehrad 1,2 ; M. Farhoudi 2 ; L. Zeinalizad 3 ; M. Haji Sagati 3 ; M. Jabbardokht 3 ; F. Nasirzadegan 3 ; A. Foletti 4

1 Islamic Azad University, Tabriz Branch‐Faculty of Basic Sciences, Department of Physics, Tabriz, Iran, Islamic Republic, 2 Tabriz University of Medical Sciences, Neurosciences Research Center, Tabriz, Iran, Islamic Republic, 3 Islamic Azad University, Tabriz Branch‐Faculty of Mechanics, Department of Bioengineering, Tabriz, Iran, Islamic Republic, 4 Italian National Research Council, Institute of Translational Pharmacology, IFT, Rome, Italy

Background: Neointimal hyperplasia is an important clinical entity in vascular surgery because it limits the long‐term effectiveness of surgical and endovascular interventions. Neointimal hyperplasia is usually defined in an artery as thickening of the intimal layer after an injury such as angioplasty, stenting or surgical repair. Neointimal hyperplasia is also used to describe the thickening of venous and prosthetic bypass grafts that leads to reduced lumen diameter and flow and, ultimately, graft occlusion and thrombosis.

Aims: In this study, we aimed to investigate low‐ level blue laser‐ mediated photodynamic therapy on neointimal hyperplasia reduction accompanied by 5‐ Aminolevulinic Acid administration in the rabbit carotid artery neointimal hyperplasia model.

Methods: The right common carotid arteries endothelial denaturation were performed by ballooning in New Zealand white rabbits. After 4 weeks, histopathology results showed the formation of neointimal hyperplasia in all of the rabbits’ arteries. Then treatment group underwent low‐ level blue laser (417 nm, 150 J/cm 2 ) ‐ mediated photodynamic therapy accompanied by intravenous 5‐ Aminolevulinic Acid administration.

Results: Results from histopathology, B‐mode and color Doppler ultrasonography showed a significant reduction in the mean value for blood mean velocity, smooth muscle cells density in neointima layer, lumen wall mean thickness and percentage of luminal cross‐ sectional area of stenosis and significant increase in the mean value for blood volume flow at the stenotic region in the treatment group compared with the other groups (P>0.05).

Conclusions: Enhanced anti‐ inflammatory effect of 5‐ Aminolevulinic Acid‐ induced by low‐ level blue laser therapy, can cause to smooth muscle cells density reduction, due to smooth muscle cells apoptosis in neointima layer and significantly dilate the luminal cross‐ sectional area of stenosis. This protocol may be a potential treatment to neointimal hyperplasia.

PB313: Advanced Atherosclerosis Regression using Inertial Cavitation Effect of Pulsed‐focused Ultrasound Accompanied by Combined High‐dose Atorvastatin Plus HDL‐äoaded Microbubbles Administration

H. Mehrad 1,2 ; S. Sadigh Eteghad 2 ; L. Zeinalizad 3 ; S. Sarabi 3 ; A. Arshadi 3 ; Z. Pishezari 3 ; K. Mehrvar 4

1 Islamic Azad University, Tabriz Branch‐Faculty of Basic Sciences, Department of Physics, Tabriz, Iran, Islamic Republic, 2 Tabriz University of Medical Sciences, Neurosciences Research Center, Tabriz, Iran, Islamic Republic, 3 Islamic Azad University, Tabriz Branch‐Faculty of Mechanics, Department of Bioengineering, Tabriz, Iran, Islamic Republic, 4 Iranian Social Security Organization, Alinasab Haspital, Department of Neurology, Tabriz, Iran, Islamic Republic

Background: Atherosclerosis is the leading cause of cerebrovascular disease. Atherosclerotic lesions are considered to be advanced when the accumulation of lipid, cells and matrix is associated with the disorganization, repair, thickening and deformity of the arterial wall.

Aims: In this study, we investigated pulsed‐ focused ultrasound effectiveness on advanced atherosclerotic plaque regression.

Methods: Briefly, New Zealand white rabbits underwent primary perivascular severe cold injury at the right common carotid artery followed by a 1.5% cholesterol‐ rich diet injury for 8 weeks. Histopathology and ultrasonography results showed the formation of advanced atherosclerosis with neovessel‐ rich plaque and severe stenosis (>70%) in all of the rabbits’ arteries. The animals treated by pulsed‐ focused ultrasound (I=30 W/cm 2 , F=150 KHz, PD=150 ms) accompanied by combined high‐ dose atorvastatin (5 mg/kg/day) plus HDL‐ loaded PESDA (Perflurocarbon‐ Exposed Sonicated Dextrose Albumin) microbubbles (100 ml/kg, 2‐5×10 5 bubbles/ml) administration.

Results: Results from histopathology, B‐mode and color Doppler ultrasonography showed a significant reduction in the mean value for blood mean velocity, intraplaque neovessels density, wall mean thickness and percentage of luminal cross‐ sectional area of stenosis and significant increase in the mean value for blood volume flow at the stenotic region in the treatment group compared with the other groups (P>0.05).

Conclusions: Enhanced inertial cavitation effect of collapsed PESDA microbubbles within intraplaque neovessels‐ induced by pulsed‐ focused ultrasound, accompanied by the lipophilic and pleiotropic effect of atorvastatin and reverse cholesterol transport effect of HDL, can cause to destroy the intraplaque neovessels, reduce the lesion macrophage‐ derived foam cells and significantly dilate the luminal cross‐ sectional area of stenosis. This protocol may be a potential treatment to advanced atherosclerosis in comparison with conventional bypass surgery and stenting.

PB314: Different Pattern of Platelet Binding to Monocytes in Healthy Volunteers and in Patients with Coronary Artery Disease

M. Loguinova 1 ; N. Pinegina 1 ; A. Shpektor 1 ; L. Margolis 2 ; E. Vasilieva 1

1 Evdokimov Moscow State Medical University for Dentistry, Moscow, Russian Federation, 2 NIH, Bethesda, United States

Background: Increased level of complexes between monocytes and platelets (MPC) is associated with various diseases including coronary acute disease (CAD). Monocyte subsets are differently involved into MPC formation, and intermediate monocytes are known as the most active. The pattern of platelet binding to monocytes subsets has never been revealed.

Aims: Analysis of platelets distribution in MPC with different monocyte subsets in whole blood of healthy donors and patients with CAD.

Methods: 31 patients with acute myocardial infarction (MI), 21 patients with unstable angina (UA), 27 patients with stable coronary artery disease (SA) and 28 healthy volunteers were recruited for this study. Peripheral blood was drawn at patient admission and fixed with 0.25% formaldehyde to be immunostained with antibody mix (CD45‐eFluor450, CD14‐PerCP Cy5.5, CD16‐AF488 and CD41a‐APC). Samples were analyzed by flow cytometry. MPC were defined by simultaneous expression of all markers. Mean fluorescence intensity (MFI) for CD41a was estimated in MPC gate.

Results: CD41a MFI of complexes formed by each separate monocyte subset significantly differed in MPC formed by classical and intermediate monocytes within control group and in SA and MI patients (Table 1). In Control group CD41a MFI in MPC with classical monocytes was significantly higher than in MPC with intermediate monocytes. In all patients MFI profile was opposite: MFI in MPC with intermediate monocytes was higher than in MPC with classical monocytes. There was also a significant decrease of MFI in MPC with classical monocytes in SA (P=0.029) and AMI patients (P=0.004) compared to Control group and a significant increase of MFI in MPC with intermediate monocytes in SA (P=0.001), UA (P=0.002) and MI (P=0.012) compared to Control group (Figure 1).

TABLE 1 MFI for CD41a‐positive events in the whole blood of healthy volunteers and in patients with CAD MPC with classical monocytes MPC with intermediate monocytes MPC with non‐classical monocytes P (between MPC with classical and intermediate monocytes) Control 1368 [970; 1853] 1049 [655; 1649] 1223 [450; 2104] 0.0078 SA 1147 [914; 1312] 1529 [1078; 1964] 1400 [774; 1652] 0.0007 UA 1383 [1258; 1473] 1548 [1330; 1694] 1424 [1150; 1718] 0.3109 MI 1016 [350; 1616] 1467 [816; 4066] 966 [451; 1764] <0.0001

FIGURE 1 MFI of CD41a in MPC with different monocyte subsets in healthy controls and in patients with CAD

Conclusions: CD41a MFI analysis in MPC revealed that intermediate monocytes carried more CD41a‐positive events than two other monocyte subsets in CAD patients compared to control.

PB315: Pharmacological Actions of Inosine in a Hypercholesterolemic Model: Is It a New Antiatherosclerotic Drug?

G.F. Lima ; N.A.V. Motta; R.O. Lopes; L.J. Autran; I.A. Moraes; F.C.F. Brito; Laboratório de Farmacologia Experimental (LAFE)

Universidade Federal Fluminense ‐ UFF, Fisiologia e Farmacologia, Niteroi, Brazil

Background: Atherosclerosis is a multifactorial chronic disease, probably initiated by an endothelial dysfunction. Adenosine and its analogs, such as inosine, can change a variety of inflammatory diseases and has shown important effects at different disease models.

Aims: The present study aims to evaluate the pharmacological properties of inosine, administered sub chronically in a hypercholesterolemic model in rats.

Methods: The animal protocols were approved by the Ethics Committee of the Federal Fluminense University (CEUA/UFF 858/2016). Adult male Wistar rats (200‐250 g) were randomly divided into three groups: control group (C) fed standard chow diet, hypercholesterolemic diet group (HC) and hypercholesterolemic diet group + inosine (HC+INO). At 31° diet day, was performed the sub chronic treatment with inosine (10 mg/kg/orally) once daily, totalizing 15 days. The animals were euthanized by cervical dislocation under ketamine and xylazine anesthesia. Blood samples were collected for ELISA and biochemical analysis. Thoracic aortas were excised for vascular reactivity. Data were analyzed using one‐way ANOVA, P>0.05.

Results: We observed that the intake of hypercholesterolemic diet promoted an increase in lipid profile and inflammatory cytokines, while inosine treatment decreases it. In the vascular reactivity, the HC group presented a decrease in the maximum relaxation and an increase in contractile response, when compared to the control group; however, inosine treatment was capable to reverse these effects.

Conclusions: This study showed the ability of the hypercholesterolemic diet to increase the lipid profile, the production inflammatory cytokines, as well as decrease vasodilatory response, contributing to development of atherosclerosis. We also showed that the inosine treatment was capable to revert these effects. Our results indicate inosine as a potential drug for the treatment of cardiovascular disorders such as atherosclerosis.

PB316: Cement Dust Exposure Associated with Anaemia, Increased Platelets, Cardiac Markers and Dyslipidamia among Male Cement Workers in Calabar, Nigeria

I.E. Bassey; J.O. Akpotuzor; I.M. Okafor ; G. Aniekeme; I.K.P. Isong; U.O. Akpan

University of Calabar, Medical Laboratory Science, Calabar, Nigeria

Background: Prolonged or repeated exposure to cement dust, depending on the level of exposure, duration, and individual sensitivity has been associated with adverse effects on various body systems.

Aims: This study assessed the packed cell volume (PCV), platelets, cardiac markers and lipid profile among cement workers of Nigerian origin to determine the effects of exposure to cement dust on these parameters.

Methods: This was a case‐control study. Ninety subjects were consecutively recruited for the study. Packed cell volume, platelets, Troponin I and Creatine kinase MB (CKMB) and lipid profile were determined in 50 cement workers and 40 controls. PCV and platelets were estimated using the Sysmex KX‐21N Automated Haematology Analyser, Troponin I and Creatine kinase MB by ELISA and Lipid profile by colorimetric methods. SPSS 18 was used for statistical analysis and significance level set at P>0.05

Results: The mean PCV was significantly lower (P=0.0001) while the platelets (P=0.0001), troponin (P=0.0001), CKMB (P=0.001), total cholesterol (P=0.0001), HDL‐cholesterol (P=0.030), LDL‐cholesterol (P=0.004), VLDL‐cholesterol (P=0.0001), Triglycerides (P=0.0001) values were significantly higher in cement workers than in controls. There was a positive correlation between duration of exposure to cement dust and platelets (r=0.901, P=0.0001) and a negative correlation between duration of exposure to cement dust and packed cell volume (r=0.874, P=0.0001).

Conclusions: This study has shown that the decreased PCV, and increased platelets, troponin, CK‐MB and dyslipidaemia observed in this study suggests an increased risk of atherothrombotic events in people exposed to cement dust. Cement workers may be at a greater risk of developing cardiovascular disease.

PB317: Hyperhomocysteinemia and endothelial Dysfunction of the Athletes in the Arctic Region

N. Vorobyeva 1 ; N. Bushueva 2

1 Nord State Medical University, Arckhangelsk, Russian Federation, 2 Nord State Medical University, Haemostasiology, Arckhangelsk, Russian Federation

Background: The combination of high physical activity, which is the stress factor, with specific severe environmental conditions can lead to serious changes in the state of homeostasis of the body of the athlete.

Aims: The study evaluated the blood level of homocysteine, as a marker of cardiovascular dysfunction and genes whose products are involved in the regulation of homocysteine metabolism.

Methods: The study involved 95 sportsmen, among them 64% boys and 36% girls engaged in various kinds of sports. Carried out determination of the homocysteine of blood serum level; studied polymorphisms of three genes of the folate cycle.

Results: Results of the study revealed the normal levels of blood homocysteine in 24% [95% CI 7.21‐9.31] cases. In 32% [95% CI 11.48‐12.21] cases was observed latent hyperhomocysteinemia, in 44% [95% CI 11.48‐12.21] cases was explicit hyperhomocysteinemia revealed. Was found a very weak correlation between homocysteine levels and sportsman gender (r=0.037, P=0.774), age of the athlete (r=0.222, P=0.081). Polymorphism of MTHFR (glu429ala) gene was detected in 48% of the athletes (CI: 36‐59%). Among the studied genotypes homozygous A1298C allele with the replacement of adenine to cytosine at position 1,298 met at 3.6% (CI: 0.8‐10.3%). Heterozygotes met much more frequently ‐ in 44% of cases (CI: 33‐55%). MTRR A66G methionine synthase reductase had a the frequency of occurrence 76% (CI: 65‐84%). Homozygous variant allele met in 23% of the athletes (CI: 15‐34%). Heterozygous variant of adenine to guanine substitution at position 66 was observed in 52% of the investigated genotypes (CI: 41‐64%).

Conclusions: It can indicate the influence of environment on the high levels of homocysteine in the blood. The revealed hyperhomocysteinemia may be a marker of endothelial dysfunction and to be a predictor of serious violations in the body of the athlete.

PB318: Folate Deficiency of the Nenets of the Arctic Regions of the Russia

N. Vorobyeva ; N. Belova

Nord State Medical University, Haemostasiology, Arckhangelsk, Russian Federation

Background: With an increase in the level of homocysteine in the blood, the risk of thrombophilic complications increases. In the Arctic regions of the Russian Federation, the source of this vitamin is extremely low due to social and geographical conditions, therefore studies on the study of folate metabolism for this region are very relevant, especially for the indigenous population ‐ the Nenets.

Aims: The purpose of our study is to analyze the content of folate cycle vitamins ‐ B9 and B12 in the Nenets population.

Methods: The object of the study was the population of Nenets (n=87). The collection of epidemiological and biological data was carried out according to the rules of the international standard of ethical norms and quality of scientific research ‐ GCP with obtaining informed consent of the participants for research. The study protocol was approved by the local ethics committee.

Results: The folate concentration in the blood of the Nenets was M=4.43±1.7 ng/ml at reference values from 4.6 ng/ml to 18.7 ng/ml. For women the folate concentration was Me=4.27 [3.06; 5.15] ng/ml, for men ‐ Me=5.3 [3.86; 6.34] ng/ml. In the study sample of the indigenous ethnos of the NAD 52.68% (CI 95% [37.04, 60.43]) of women, 5.5% (CI95% [19.22, 74.86]) of men had a deficiency of folate.

The content of vitamin B12 in the blood of the Nenets was Me=584.3 [353.6; 757.6] pg/ml, which fit into physiological values. In male Nenets, the concentration of vitamin B12 was Me=565.65 [343.68; 697.82] pg/ml, in women ‐ Me=580.4 [351.6; 721.1] pg/ml. Deficiency of vitamin B12 was noted in only three women and one man.

Conclusions: According to the results of the study, there was a shortage of folate in the sample of the indigenous ethnos of the Far North, therefore, a program for the prevention of folate deficiency is relevant for this category of people.

PB319: Inhibition of Integrin avb3 with Cilengitide Prevents Endothelial Injury Induced by Neutrophil Extracellular Traps in Response to Staphylococcus aureus Infection in a 3D ex vivo Model of Sepsis

C. Garciarena ; S. Kerrigan

Royal College of Surgeons in Ireland, Irish Centre for Vascular Biology, Dublin, Ireland

Background: Neutrophil extracellular traps (NETs) are a mechanism of the innate immune response that contributes to host defence in sepsis by immobilising and killing infective agents. However, recent evidence suggests that NETs components induce damage to host cells; however the effects of NETs on endothelial cells (ECs) remain largely unexplored.

Aims: The aims of this work is to investigate whether NET formation induced by S. aureus triggers vascular injury by inflicting direct damage to ECs.

Methods: Neutrophils were isolated from human blood. S. aureus induced NETs were detected using SYTOX Green. EC integrity was assessed by immunostaining of VE‐cadherin. Endothelial barrier integrity was measured by epifluorescence in bioengineered 3D microvessels.

Results: S.aureus significantly induced the formation of NETs by immobilised neutrophils compared to the uninfected control (5±1%, vs. 24±2%, control vs. infected, P>0.001). S. aureus induced NETs promoted a loss of VE‐cadherin expression in ECs that was greater than that caused by S. aureus alone (100±7%, vs. 40±3%, and 59±2%, control, S. aureus +neutrophils and S. aureus alone, respectively; P=0.005). Interestingly, cilengitide, the αvβ3 integrin inhibitor, previously shown to prevent S. aureus attachment to ECs, prevents NET induced damage (VE‐cadherin 107±5%, vs. 100±7%, of control, NS). In our 3D microvascular model we observed an 80% increase in permeability following S. aureus infection (100±10, vs. 180±10, uninfected vs. infected; P>0.01), thus indicating EC damage. Cilengitide prevented S. aureus attachment to the 3D microvascular endothelium and, interestingly, S. aureus ‐triggered NETs, induced a significantly larger increase in permeability (100±10, vs. 272±10%, control vs. infected, P>0.05).

Conclusions: Our results show that NETs drive bacteria‐independent endothelial injury promoting loss of endothelial barrier function. Importantly, cilengitide, by preventing S. aureus attachment to the endothelium, abrogates the deleterious effects of NETs.

PB320: The Role Exosomal Derived miRNAs in Driving a Host's Response to Infection in Sepsis

G. Fitzpatrick ; R. Watkin; S. Kerrigan

Royal College of Surgeons in Ireland, Pharmacy, Dublin, Ireland

Background: Sepsis is defined as life‐threatening organ dysfunction resulting from a dysregulated host response to infection. A major complication of sepsis includes a sustained hyperinflammatory environment while the nature of this event is currently unknown. There is growing evidence which suggests that bacteria are capable of altering gene expression patterns in host cells. miRNA are short, ˜22 nucleotides, posttranscriptional repressors of gene expression and are, therefore, capable of modifying cellular behaviour.

Aims: To perform functional analysis of exosomal miRNAs from S. aureus‐ infected Endothelial cells to gain better insight into the pathophysiology of sepsis.

Methods: Exosomal miRNA profiling was performed using TaqMan ® Array Human miRNA Cards. Individual qPCR assays were performed for select miRNA targets. Western Blots were performed targeting (i) CD63, Flotillin‐1 Alix, and Tsg101 (ii) mTor for uninfected/infected samples. Infected endothelial exosomes were incubated with THP‐1 macrophages.

Results: Microarray analysis identified 31 targets altered in response to S. aureus infection. Specifically, 17 upregulated and 14 downregulated (N=4; P>0.05, ±SEM) were detected with an additional 11 miRNA detected following infection only. Western blots reveal heightened exosomal production post‐infection (N=3, ±SEM). Exosomal upregulation of miR99a/b was of particular interest. Individual qPCR assays of mir‐99a/b confirms upregulation post‐infection (n=3, P>0.05). A putative downstream target of mir99a/b is mTor. Exosomes, from infected HAoECs, were applied to THP‐1 macrophages and induced a pro‐inflammatory morphology 4 hours post‐infection. MiR‐99a/b overexpression analysis in THP‐1 macrophages induced a similar phenotype.

Conclusions: A significant increase in the exosomal miRNA load of HAoECs is observed following bacterial infection. Resultantly, this possesses the ability to modulate the immune response and contribute to the excessive and sustained inflammatory environment observed in sepsis.

PB322: Recombinant Human Soluble Thrombomodulin Protects Vascular Endothelial Cells from Radiation‐induced DNA Damage

X. Wang 1 ; X. Wang 1 ; T. Kawase 2 ; G. Honda 3 ; H. Ohkawara 1 ; T. Ikezoe 1

1 Fukushima Medical University, Fukushima, Japan, 2 Hiroshima University, Hiroshima, Japan, 3 Asahi Kasei Pharma., Tokyo, Japan

Background: Thrombomodulin (TM) is a cell surface protein which mediates conversion of protein C to activated protein C and acts as an anti‐coagulant by inhibiting factor Va and VIIIa. We previously showed that recombinant human soluble TM (rTM) which consists of extracellular domain of TM protected vascular endothelial cells (VECs) from calcineurin inhibitor‐induced cell‐damage in association with up‐regulation of anti‐apoptotic Mcl‐1 protein which was mediated by extracellular signal‐regulated kinase.

Aims: In the present study, we explored whether rTM protects VECs from radiation‐induced cell damage.

Methods: Western blot analysis and immunocytochemistry were employed to detect the expression of gH2AX, a marker of DNA damage in VECs after exposure to radiation. Annexin V and propidium iodide staining was used to detect apoptosis in VECs after exposure to radiation.

Results: Western blot analysis found that exposure of human VEC‐derived EA.hy926 cells to radiation (20 Gy) induced expression of gH2AX as early as 30 minutes after radiation exposure. Interestingly, treatment of EA.hy926 cells with rTM for 12 hours hampered the expression of gH2AX caused by radiation. The blockade of expression of gH2AX caused by radiation was also demonstrated by immunocytochemistry. Furthermore, annexin V and propidium iodide staining found that radiation‐induced apoptosis of EA.hy926 cells was attenuated when these cells were cultured in the presence of rTM for 12 hours before exposing to radiation.

Conclusions: Taken together, these observations suggested that rTM possesses the ability to protect VECs from radiation‐induced cell damage. The prophylactic use of rTM might be useful to prevent VECs damage caused by the conditioning regime with total body irradiation in individuals who receive hematopoietic stem cell transplantation, although further experiments are required to clarify the mechanisms by which rTM produce these effects.

PB324: Ubiquitin‐driven p38 Inflammatory Signaling Induced by Protease‐activated Receptors

J. Trejo

University of California, San Diego, Pharmacology, La Jolla, United States

Background: Protease‐activated receptors (PARs) transduce cellular responses to the coagulant protease thrombin. PARs have important functions in thrombosis, hemostasis and vascular inflammation. Activation of PAR1 by thrombin induces endothelial inflammatory responses through multiple pathways including p38 mitogen‐activated protein kinase signaling. However, the mechanisms that govern PAR1‐induced p38 inflammatory signaling remain unclear. We discovered an atypical ubiquitin‐mediated signaling pathway utilized by PAR1 that regulates endosomal p38 signaling and endothelial barrier disruption. Ubiquitination of activated PAR1 by the NEDD4‐2 E3 ubiquitin ligase and initiates recruitment of transforming growth factor‐b‐activated protein kinase‐1 binding protein‐2 (TAB2) on endosomes. TAB2 associated with TAB1, which directly binds to and activates p38. We found that TAB2‐TAB1‐dependent p38 activation is critical for PAR1‐promoted endothelial barrier permeability in vitro , and p38 signaling is required for PAR1‐induced vascular leakage in vivo .

Aims: While ubiquitination of activated PAR1 by NEDD4‐2 is essential for p38 activation, it is not known how PAR1 stimulates NEDD4‐2 activity.

Methods: Using mass spectrometry, we identified a unique phosphorylated tyrosine (Y)‐485 residue within NEDD4‐2 in agonist stimulated cells. Mutation of NEDD4‐2 Y485 impaired E3 ligase activity and failed to rescue PAR1‐stimulated p38 activation and endothelial barrier permeability unlike wildtype.

Results: Here, we report that PAR1 stimulates c‐Src‐mediated tyrosine phosphorylation and activation of NEDD4‐2 to promote p38 signaling and endothelial barrier disruption. The P2Y1 purinergic also promotes ubiquitin and TAB1‐TAB2 driven p38 signaling and requires c‐Src and NEDD4‐2 tyrosine phosphorylation for p38 activation.

Conclusions: These studies reveal an unexpected role for c‐Src in GPCR‐induced NEDD4‐2 activation, which is critical for driving ubiquitin‐mediated p38 pro‐inflammatory signaling in endothelial cells.

PB325: Low ‐ Level Red Laser ‐ Mediated Photobiomodulation Therapy, Improves Endothelial Dysfunction via Increasing Endothelial Nitric Oxide (NO) Synthase in the Rabbit Carotid Artery Injury Model

H. Mehrad 1,2 ; J. Mahmoudi 2 ; A. Rahimkhani 3 ; M. Haji Sagati 3 ; Z. Pishezari 3 ; L. Zeinalizad 3 ; M. Moghadaszadeh 4

1 Islamic Azad University, Tabriz Branch‐Faculty of Basic Sciences, Department of Physics, Tabriz, Iran, Islamic Republic, 2 Tabriz University of Medical Sciences, Neurosciences Research Center, Tabriz, Iran, Islamic Republic, 3 Islamic Azad University, Tabriz Branch‐Faculty of Mechanics, Department of Bioengineering, Tabriz, Iran, Islamic Republic, 4 Tabriz University of Medical Sciences, Department of Internal Medicine, Tabriz, Iran, Islamic Republic

Background: The healthy endothelial cells are essential for maintenance of vascular homeostasis involving antioxidant, anti‐inflammatory, pro‐fibrinolytic, anti‐adhesive, or anticoagulant effects. Oppositely, endothelial dysfunction is primarily characterized by impaired regulation of vascular tone as a result of reduced endothelial nitric oxide (NO) synthase activity, lack of cofactors for NO synthesis, attenuated NO release, or increased NO degradation. As is well recognized, the majority of atherosclerosis start with a vascular injury, resulting in endothelial dysfunction.

Aims: In this study, we aimed to investigate photobiomodulation therapy on endothelial function in the rabbit carotid artery injury model.

Methods: Briefly, New Zealand white rabbits underwent balloon dilatation injury at the right common carotid artery. After 2 days, common carotid arteries of the treatment group at lesion region, treated using extracorporeal low‐ level red laser (635 nm, 100 J/cm 2 )‐ mediated photobiomodulation therapy. In order to evaluate endothelial‐dependent relaxation, acetylcholine‐mediated dilation (AMD) was measured during the infusion of acetylcholine at a rate of 0.5 μg/kg/min and endothelial independent relaxation was evaluated by measuring nitroglycerin mediated dilation (NMD) during the infusion of nitroglycerin at a rate of 5 μg/kg/min.

Results: Results from B‐mode ultrasound images showed significant differences in AMD between the treated and the non‐ treated rabbits (P>0.05), whereas there were no significant differences in NMD between the treatment and normal groups (P>0.05). No microscopic intimal lesions were seen in the normal and treated rabbits, but intimal thickening was observed in the histological studies in the non‐treated rabbits.

Conclusions: Low‐ level red laser‐ mediated photobiomodulation therapy cause to improve endothelial dysfunction via increasing endothelial nitric oxide (NO) synthase. This protocol may be a potential treatment to endothelial dysfunction after balloon angioplasty.

PB326: The Anti‐coagulant Vitamin K‐dependent Factor, Protein S, Alters Endothelium Functions by Regulating Multiple Vascular Permeability Related Signaling Pathways

A. Jooussaume 1 ; C. Kanthou 2 ; F. Dkhissi 1 ; O. Benzakour 1

1 INSERM U1082, University of Poitiers, Poitiers, France, 2 Tumour Microcirculation Group University of Sheffield, Department of Oncology and Metabolism School of Medicine, Sheffield, United Kingdom

Background: Protein S is a vitamin K‐dependent glycoprotein, which, besides its anticoagulant function, acts as an agonist for the tyrosine kinase receptors Tyro3, Axl, and Mer. The endothelium expresses Tyro3, Axl, and Mer and produces protein S. The interaction of protein S with endothelial cells and particularly its effects on vascular permeability have not yet been described prevoiusely.

Aims: The major aim of our study is to assess the effects of protein S on vascular permeability and signaling pathways involved in this process.

Methods: We have studied the effects of Protein S on human endothelial cell monolayers and using cultured human endothelial cells we analyzed by western blotting, immunostaing and siRNA possible effects of protein S on vascular permeability related signaling pathways.

Results: We report that protein S increases the permeability of endothelial cell monolayers and enhances VEGF‐A‐induced endothelial cell permeability. We further investigated the signaling pathways involved in this newly described effect of protein S. We report that protein S activates two major targets directly related cell permeability. Protein S activates P38/HSP27 pathway and induces VE‐cadherin phosphorylation.

Conclusions: Altogether, our study suggests that ProS/MerTK‐induced vascular permeability via the activation of P38/HSP27 pathway and VE‐cadherin phosphorylation may be a novel endogenous regulatory mechanism for the endothelium.

PB327: Simultaneous Assessment of sE‐Selectin, Platelet Count and Mean Platelet Volume as Biomarkers for Pre‐eclampsia

H. Hassan 1 ; H. Azzam 1 ; M. Othman 2,3 ; M. Hassan 4 ; T. Salim 1

1 Mansoura University, Clinical Pathology, Mansoura, Egypt, 2 Queen's University, Biomedical and Molecular Sciences, Kingston, Canada, 3 St Lawrence College, School of Baccalaureate Nursing, Kingston, Canada, 4 Mansoura University, Gynaecology & Obstetrics, Mansoura, Egypt

Background: Preeclampsia (PE) remains a major cause of maternal mortality and morbidity worldwide with increased risk for cardiovascular disease later in life. Previous studies have examined several biomarkers including E‐selectin. However this was not evaluated in the context of other previously reported markers.

Aims: We aimed to assess the role of sE‐selectin together with platelet count and mean platelet volume (MPV) as biomarkers for the prediction of preeclampsia.

Methods: This case‐control study included 85 pregnant women; 40 healthy (mean age 27.15±4.74 years) and 45 with preeclampsia (mean age 26.82±6.67 years), recruited at the third trimester of pregnancy and subjected to full clinical and laboratory testing. This included complete blood picture, urine analysis and plasma sE‐selectin using ELISA.

Results: Data showed no significant differences regarding gestational age, gravidity, leukocyte count or haemoglobin between patients and controls (P>0.05). A significant decrease in platelet counts in patients (P=0.003), and a significant increase in MPV (P>0.001) and plasma sE‐selectin levels were seen in patients versus control (P=0.002). Receiver operator curve showed the best cut‐off values established for sE‐selectin was 64.26 ng/ml, with 57.8% sensitivity 80.0% specificity. Positive predictive value was 76.5, negative predictive value was 62.7 and accuracy was 68.2 with a statically significant area under curve (P=0.002). Platelet count, MPV and sE‐selectin showed significant association with PE in univariate analysis. However, in multivariate analysis, only MPV and sE‐selectin were independent risk factors for PE development. Higher MPV or sE‐selectin were associated with PE development (P>0.001).

Conclusions: The simultaneous use of sE‐selectin and platelet count and volume may help earlier recognition of preeclampsia and thus appropriate and more efficient therapy. Larger studies will likely verify data and justify wider application of these markers.

PB328: Endothelial Cell Activation Is Attenuated by Everolimus via Transcriptional and Post‐transcriptional Regulatory Mechanisms after Drug‐eluting Coronary Stenting

Z. Fejes 1 ; Z. Czimmerer 2 ; T. Szük 3 ; S. Poliska 2 ; A. Horváth 2 ; E. Balogh 4 ; V. Jeney 4 ; G. Balla 5 ; I. Édes 3 ; J. Kappelmayer 1 ; B. Nagy Jr 1

1 University of Debrecen, Faculty of Medicine, Department of Laboratory Medicine, Debrecen, Hungary, 2 University of Debrecen, Faculty of Medicine, Department of Biochemistry and Molecular Biology, Genomic Medicine and Bioinformatics Core Facility, Debrecen, Hungary, 3 University of Debrecen, Faculty of Medicine, Department of Cardiology, Debrecen, Hungary, 4 University of Debrecen, Faculty of Medicine, Department of Internal Medicine, Debrecen, Hungary, 5 MTA‐DE Vascular Biology, Thrombosis and Haemostasis Research Group, Hungarian Academy of Sciences, Debrecen, Hungary

Background: Higher soluble E‐selectin and VCAM‐1 levels were reported in patients in the early phase of bare‐metal stenting compared to subjects who underwent drug‐eluting stenting (DES) without complication.

Aims: Transcriptional and post‐transcriptional regulatory mechanisms were investigated by which everolimus attenuated endothelial cell (EC) activation after DES implantation.

Methods: The effect of everolimus was studied on E‐selectin (SELE) and VCAM‐1 mRNA levels when human coronary artery (HCAECs) and human umbilical vein ECs were challenged with recombinant TNF‐α (100 ng/ml) for 1‐24 hours in the presence or absence of everolimus using the concentration of 0.5 μM locally maintained by DES. Endothelial inflammation was monitored via the levels of IL‐1β and IL‐6 mRNAs and miR‐155. To investigate the transcriptional regulation of E‐selectin and VCAM‐1 expression, TNF‐α‐activated P65‐bound enhancers of SELE and VCAM genes, including SELE_‐11Kb and VCAM1_‐10Kb, were measured in HCAECs. Mature and precursor levels of miR‐181b were quantified as E‐selectin and VCAM‐1 post‐transcriptional repressor. RT‐qPCR was used for analysis of circulating miR‐181b to correlate with the concentrations of these soluble adhesion molecules in plasma of stented subjects.

Results: TNF‐α highly elevated E‐selectin and VCAM‐1 expression at transcriptional level in ECs. Levels of mature and both precursor forms of miR‐181b were repressed in ECs by TNF‐α, while everolimus acted as a negative regulator of EC activation via the modulation of E‐selectin and VCAM‐1 expression as well as miR‐181b levels. In addition, TNF‐α‐augmented enhancer RNAs were also reduced by everolimus. A significant negative correlation was observed between plasma miR‐181b and soluble E‐selectin and VCAM‐1 levels.

Conclusions: Everolimus decreases EC activation via the reduction of E‐selectin and VCAM‐1 expression at transcriptional and post‐transcriptional level resulting in a lower EC activation level in the early phase of DES implantation.

PB329: The Damaging Effect of Neutrophil Extracellular Traps on the Vascular Endothelium during Sepsis

T. McHale ; C. Garciarena; S. Kerrigan

Royal College of Surgeons, Irish Centre for Vascular Biology, Dublin, Ireland

Background: Sepsis is defined as life threatening organ dysfunction caused by a dysregulated host response to infection. Upon pathogen invasion, neutrophils are recruited to the site of infection. Here, they form Neutrophil Extracellular Traps (NETs) in an attempt to capture invading microbes and prevent their dissemination. Whilst NETs are for the most part advantageous, excessive formation and ineffective clearance have been linked to the pathophysiology of sepsis. It has been hypothesised that they disrupt the endothelial barrier junction protein VE‐cadherin, and such cell‐cell contact weakening may thus result in hypoperfusion, life threatening oedema and multiple organ failure.

Aims: In this study we aim to elucidate the interplay and the role that E. coli and neutrophils play on sheared Human Aortic Endothelial Cells (HAoECs) and assess the therapeutic potential of a novel drug in a 3D model of sepsis.

Methods: Using fluorescence microscopy, we measured the levels of the Vascular Endothelial Cadherin (VE‐Cad), an integral junction protein that controls the movement of fluid through the endothelial barrier.

Results: VE‐cad was quantitatively measured in vitro, after infection with E. coli and Neutrophil extracellular traps. Interestingly, the addition of neutrophils increases the endothelial damage in our 2D model (P>0.05).

We also show that the binding of E. coli and the production of NETS significantly increase the levels of vascular permeability. However, when we pre‐treat our blood vessel with an αVβ3 antagonist, we can significantly reduce bacterial adhesion and NET‐induced damage to the endothelium.

Conclusions: Using a 3D model of sepsis, we have shown that by treating our engineered 3D human blood vessel with cilengitide, we can prevent E. coli from binding to the vessel wall. Inhibition of E. coli binding to endothelial cell αVβ3 by cilengitide prevents endothelial dysfunction and NET induced endothelial damage and therefore presents a first in class novel drug candidate to prevent sepsis progression.

PB330: Comparison of Syndecan‐1 Levels as a Marker of Endotheliopathy in Thermal Injury and Blunt Trauma

R. Ball 1,2 ; M. Bravo 3 ; K. Brummel‐Ziedins 3 ; T. Orfeo 3 ; K. Freeman 4 ; M. McLawhorn 2 ; L. Moffatt 2 ; J. Shupp 1,2

1 MedStar Washington Hospital Center, Burn Center, Washington, United States, 2 Firefighters’ Burn and Surgical Research Laboratory, MedStar Health Research Institute, Washington, United States, 3 University of Vermont Larner College of Medicine, Department of Biochemistry, Burlington, United States, 4 University of Vermont Larner College of Medicine, Department of Surgery, Burlington, United States

Background: Endotheliopathy is a complex process that leads to poor outcomes for critically ill patients. The role of endothelial dysfunction in blunt trauma and burn pathophysiology is largely unknown. Syndecan‐1 is a component of the endothelial glycocalyx released into the bloodstream upon endothelial disruption and serves as a potential biomarker for endotheliopathy.

Aims: The purpose of this study is to compare syndecan‐1 values in patients with blunt trauma and thermal injury during the first hours of hospitalization.

Methods: Blood samples were collected from 145 patients across 2 sites (55 burn, 90 blunt trauma) through Institutional Review Board approved protocols and informed consent. Samples were collected within 6 hours of injury. Syndecan‐1 levels were quantified by ELISA. Patients were classified as having severe injury if the Total Body Surface Area (TBSA) affected was >30% for burns or if the Injury Severity Score (ISS) was >16 for blunt trauma. t‐tests were used for analyses.

Results: Mean syndecan‐1 levels drawn at enrollment were similar when all thermally‐injured patients were compared to those suffering blunt trauma: 38.6 versus 32.4 ng/ml respectively (P=0.23). Intragroup variability of levels was captured by large standard deviations (51.9 and 48.5 respectively). Patients with ≥30% TBSA had significantly higher mean levels than those with <30% TBSA (59.9±68.2 vs. 24.3±31.2 ng/ml respectively; P=<0.005). Similar trends were seen comparing blunt trauma patients with ISS ≥16 and ISS>16: 42.6±72.6 versus 25.2±16.1 ng/ml (P=0.047).

Conclusions: Syndecan‐1 release in response to thermal and blunt injuries appears to be unpredictable on the individual level as evidenced by the large intragroup variation. Nevertheless, higher levels of syndecan‐1 are seen in patients with more severe burn and blunt injuries. Additional markers of endotheliopathy must be evaluated along with a thorough review of outcomes of these patients to establish the clinical relevance of these findings.

PB331: Endoglin Implications in ECFC Vasculogenic Properties and Vascular Permeability

E. Rossi 1 ; S. Lecourt 1 ; N. Névo 1 ; F. Saller 2 ; C. Goyard 3 ; B. Saubamea 4 ; B. Planquette 3 ; C. Bernabeu 5 ; O. Sanchez 3 ; R. Bobe 6 ; D.M. Smadja 1,7

1 Paris Descartes University, INSERM UMR_S 1140 Therapeutic Innovations in Hemostasis (IThEM), Paris, France, 2 Paris Sud University, INSERM U 1176 & UMR 1176, Le Kremlin‐Bicêtre, France, 3 Hôpital Européen Georges Pompidou ‐ Paris Descartes University, Service de Pneumologie et Soins Intensifs, Paris, France, 4 Paris Descartes University, INSERM UMR_S 1144, Paris, France, 5 Spanish National Research Council (CSIC – CIB), Cellular and molecular medicine, Madrid, Spain, 6 Inserm U 1176 ‐ Hopital Bicetre, HITh ‐ Hémostase, Inflammation & Thrombose, Le Kremlin‐Bicetre, France, 7 European Georges Pompidou Hospital, Hematology, Paris, France

Background: Endoglin (Eng) is a transmembrane glycoprotein present on endothelial cells and mainly known as a member of TGF‐β complex, essential for angiogenesis and vascular remodeling. Intriguingly, we previously observed in retina studies that heterozygous mice for endoglin (Eng+/−) present an increased vascular permeability in contrast to wildtype.

Aims: This study is focused on the role of Eng in the main human adult vasculogenic cells endothelial colony forming cells (ECFC). The aim is to clarify Eng implication in proliferation, transmigration, tubologenesis, and vessels permeability and how endoglin expression can influence ECFC vasculogenic properties.

Methods: ECFC are isolated by cordon‐blood and used between p3‐p5. ECFC silencing (Eng‐siRNA and control‐siRNA) was confirmed by flow cytometry and immunofluorescence for CD105. Immunofluorescence for ki67 and colorimetric assay with pNPP were performed to study cell proliferation. ECFCs are also tested by wound‐healing (WH), transmigration by transwell, sprouting by Matrigel. Impedance‐based permeability detection was studied by in vitro assays (iCELLigence).

Results: Considering proliferation assays we did not find a significant difference between Eng‐siRNA and control siRNA, while WH and transwell assays showed that ECFC transmigration ability was reduced when endoglin was inhibited (P>0.05). The same negative effect was found when Eng was silenced in Matrigel assays (P>0.01). ECFC Eng‐siRNA also displays a significant permeabilization after stimulation by TNFα (20 ng/ml) compared with controls ECFC (medium values: 0.72±0.04 vs. 0.83±0.03 respectively, normalized cell index at 14 hours post TNF treatment, P>0.05).

Conclusions: Eng inhibition in adult vasculogenic cells, ECFC, seems to display a critical role in migration, tubulogenesis and vascular permeability. Thus, vasculogenic defect in endoglin disorders such as HHT1 and familial PAH could be part of capillaries fragility and arterial dysfunction.

PB332: Ceramide 1 Phosphate Protects Human Endothelial Colony Forming Cells from Apoptosis and Increase Their Angiogenic Responses

H.A. Mena ; P.R. Zubiry; M. Schattner; S. Negrotto

Institute of Experimental Medicine, CONICET‐National Academy of Medicine., Buenos Aires, Argentina

Background: Ceramide 1 phosphate (C1P) is a bioactive sphingolipid released from dying cells and therefore highly augmented in damaged tissues. It is a well‐known chemoattractant of macrophages and hematopoietic stem cells and, it has been recently reported a C1P proangiogenic action on mature endothelial cells.

Aims: Considering the key role of human endothelial colony forming cells (ECFC) on vasculogenesis and tissue regeneration and that the effect of C1P in these processes remains unknown, we aimed to analyze the effect of C1P on the survival and angiogenic activity of ECFC.

Methods: Human cord blood‐derived CD34 + cells were cultured in EGM2 and, after 14‐18 days, ECFC colonies were obtained. ECFC were treated with short‐ and long‐chain C1P analogs like C8‐C1P and C16‐C1P respectively. N=3‐6, *P>0.05 versus untreated control (C), # P>0.05 versus TNFα, one‐way ANOVA.

Results: Nuclear morphology analysis showed that both C8‐ and C16‐C1P failed to induce ECFC death but on the contrary, protected them from TNFα‐induced apoptosis (C: 10±2; TNFα 30 ng/ml: 31±3*, TNFα+C8 10 μM: 20±3 # , TNFα+C16 10 μM: 23±2 # % of cell death, fluorescence microscopy). C1P enhanced adhesion to type 1 collagen and TNFα‐activated HUVEC, but not to fibronectin, in a concentration‐dependent manner (Figure 1). These molecules also augmented ICAM expression (C: 20±1; C8 1 μM: 24±1, C8 10 μM: 34±1*; C16 1 μM: 24±2, C16 10 μM: 28±2* % of positive cells, flow cytometry) while failed to modify E‐selectin. Moreover, both C8 and C16‐C1P (10 μM) increased proliferation after 48 hours (C: 320±6, C8: 480±19*, C16: 450±12* % of C at 0 hours, cell count), and in vitro tubule formation (C8: 165±8*, C16: 140±12* % of C). ERK1/2 and AKT pathways might be involved C1P effects on ECFC as their both were activated by C8‐C1P, while p38 and p65NFkB were not affected (Figure 2).

FIGURE 1 C8‐C1P and C16‐C1P (1 and 10 µM) increased adhesion (cell count) of ECFC to type 1 collagen and TNFα‐activated HUVEC. *P>0.05 versus untreated cont

FIGURE 2 C8‐C1P (10 µM) activated ERK1/2 and AKT pathways on ECFC (flow cytometry), but not p38 and p65NFkB. *P>0.05 versus untreated control

Conclusions: Our results showed that C1P protected ECFC from apoptosis and augmented their angiogenic responses, pointing out the therapeutic potential of this sphingolipid to improve vasculogenesis.

PB333: Ticagrelor Induces the Expression of the Endothelial Phenotype CD34 + Progenitor Cells in the Presence of ADP via an Adenosine‐mediated Mechanism

S. Sidiropoulou 1 ; K.M. Hansson 2 ; A.D. Tselepis 1

1 Atherothrombosis Research Centre/Laboratory of Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece, 2 Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden

Background: Ticagrelor, a potent reversible antagonist of platelet P2Y12 receptor, inhibits uptake of adenosine (ADO) via the equilibrative nucleoside transporter 1 allowing ADO to act via the A2A, A2B, A1 and A3 receptors. P2Y12 and A2B are expressed in various cell types including CD34 + progenitor cells. Circulating CD34 + cells could interact with platelets and differentiate into endothelial progenitor cells contributing to endothelium regeneration.

Aims: We studied the effect of ticagrelor and ADO on the membrane expression of KDR (endothelial marker) by CD34 + cells and the platelet‐CD34 + interaction.

Methods: Whole blood from healthy volunteers was incubated with 1 μΜ ticagrelor (5 minutes), 20 μΜ ADO (1 minutes) or their combination, followed by ADP activation (50 μΜ) for 10 minutes, at 37°C. Some samples were incubated with 10μΜ SCH58621 , 5 μΜ MRS1706, 30 μΜ DPCPX or 10 μΜ MRS1220 (A2A, A2B, A1 and A3 antagonist, respectively) for 5 minutes at 37°C prior to addition of ticagrelor or ADO. Analysis was performed by flow cytometry using anti‐human CD34‐FITC, VEGR‐R2/KDR‐PE and CD61‐PerCP monoclonal antibodies. Results were expressed as %gated of CD34 + /KDR + cells and CD61 + /CD34 + cell conjugates.

Results: ADP‐induced KDR expression in CD34 + cells (from 0.13±0.03% to 0.26±0.07%, P > 0.05) was enhanced by ADO, ticagrelor or their combination by 1.3‐, 1.5‐ and 2.2‐fold, respectively (P > 0.05), which was completely reversed by MRS1706 (selective inverse agonist of A2B). ADP‐induced CD61 + /CD34 + conjugate formation (from 0.19±0.04% to 0.54±0.11%, P > 0.05) was reduced by ticagrelor and its combination with ADO by 2.3‐ and 2.5‐fold, respectively (P > 0.05), but not affected by ADO or ADO receptor antagonists.

Conclusions: Ticagrelor enhances ADP‐induced KDR expression in CD34 + cells which may be mainly mediated via A2B. Since ticagrelor inhibits CD61 + /CD34 + conjugate formation, we suggest that induction of KDR expression is independent of platelet‐CD34 + interaction. This effect could contribute to circulating CD34 + ‐mediated endothelial regeneration.

PB334: Coxsackievirus B3 Replicates in Endothelial Colony Forming Cells and Inhibits Angiogenesis

P.R. Zubiry 1 ; H.A. Mena 1 ; J. Etulain 1 ; M. Schattner 1 ; R.M. Gomez 2 ; S. Negrotto 1

1 IMEX, CONICET‐ANM, Buenos Aires, Argentina, 2 IBBM, CONICET‐UNLP, La Plata, Argentina

Background: Coxsackievirus B3 (CVB3) is a frequent etiological agent of human viral myocarditis. Endothelial colony forming cells (ECFC) are involved in myocardial repair, especially they have a lead role in angiogenesis and tissue revascularization.

Aims: Here, we here aimed to characterize CVB3 infection in these progenitor cells and its consequences on angiogenesis.

Methods: Cord blood‐derived CD34 + cells were seeded in EGM2 and ECFC colonies were obtained after 14‐21 days. ECFC were incubated with CVB3 at a multiplicity of infection (MOI) 0.1 or 1 for 1 hour and then washed to remove virus. N=3‐7.*P>0.05 versus Mock or untreated control (C), # P>0.05 versus TNFα, one‐way ANOVA.

Results: CVB3 was detected in ECFC cytoplasm by confocal microscopy (Figure 1A). These results were confirmed by flow cytometry, where the percentage of infected ECFC increased after 24 hours post infection (hpi) (Mock:3±0.3; MOI 1:18±3* % of CD31+/CVB3+ cells). Moreover, infectivity titration assay on Vero cells indicated that viral titers increased over time in ECFC supernatants and pellets (Figure 1B). Fluorescence analysis of nuclear morphology showed that CVB3 infection failed to induce ECFC death. However, CVB3 significantly decreased ECFC proliferation at 48 hpi (C:240±10; Mock:240±10; MOI 0.1:190±10*; MOI 1:150±20* % of C at 0 hpi; cell count), adhesion (cell count) to type I collagen (Mock:100±3; MOI 0.1:73±12*; MOI 1:57±15* % of C), adhesion to TNFα‐activated HUVEC (Mock:100±2; MOI 0.1:73±2*; MOI 1:57±7* % of C) and in vitro tubule formation (Figure 2) in a MOI‐dependent manner, whereas adhesion to fibronectin was only affected at MOI 1 (Mock:103±2; MOI 0.1:92±8; MOI 1:80±2* % of C). TNFα‐induced E‐selectin expression, but not ICAM, was significantly reduced in the presence of CVB3 (Mock:3±1; TNFα 1 ng/ml:16±2; MOI 0.1+TNFα:16±3; MOI 1+TNFα:7±1 # MFI; flow cytometry).

FIGURE 1 (A) CVB3 was detected with anti‐VP1 at 24 hpi. (B) Infectivity titers increased over time in ECFC supernatants and pellets. *P>0.05 versus 0 hpi

FIGURE 2 ECFC tubule formation in vitro was inhibited by CVB3. The number of branch points was calculated at 18 hpi. *P>0.05 versus C

Conclusions: Our results show that CVB3 infects and replicates in ECFC, causing an inhibition of their angiogenic activity, which is not associated with a viral cytopathic effect.

PB335: Circulating Progenitor and Mature Endothelial Cells in Patients with Venous Thromboembolic Disease

S. Ranero 1,2 ; G. Silveira 2,3 ; N. Trias 2 ; A. Brugnini 2 ; I. Rodriguez 4 ; L. Díaz 1 ; C. Guillermo 1 ; S. Grille 1,2

1 Facultad de Medicina, Universidad de la Republica, Catedra de Hematología, Hospital de Clinicas, Montevideo, Uruguay, 2 Facultad de Medicina, Universidad de la Republica, Laboratorio de Citometria y Biología Molecular, Departamento Basico de Medicina, Hospital de Clinicas, Montevideo, Uruguay, 3 Universidad de la Republica, Clínica Médica ‘2’, Facultad de Medicina, Montevideo, Uruguay, 4 Facultad de Medicina, Universidad de la Republica, Departamento de Medicina Tranfusional, Hospital de Clinicas, Montevideo, Uruguay

Background: Endothelial progenitor cells (EPC) and circulating endothelial cells (CEC) are a heterogeneous group of cells mobilized from bone marrow in response to endothelial damage. Despite they have been described in several conditions associated with endothelial injury, there is scanty evidence of their role in venous thromboembolism (VTE).

Aims: to analyze circulating EPCs and CEC levels in patients with VTE and compare to healthy controls.

Methods: A prospective, case and control study was conducted in the Haemostasis and Thrombosis Unit, Hospital de Clinicas, Montevideo‐Uruguay. EPCs and CEC level were quantified by multiparametric flow cytometry. At least 2.000.000 events were acquired. EPCs were defined as CD45 low/− , CD34 + , CD133 + , CD31 + , CD146 + , CD3 − and CEC as CD45 low/− , CD34 + , CD133 − , CD31 + , CD146 + , CD3 − .

Results: 14 patients with previous VTE and 8 healthy controls (blood donors) were included. Patients’ median age was 67±14 years and controls median age was 40±5 years. Sex ratio (male/female) was 1/1 in patients and controls. All VTE were unprovoked and 57% were proximal deep venous thrombosis (DVT) and/or pulmonary embolism (PE). 5 patients (35.7%) had a recurrent VTE. 8 patients (61.5%) had post‐thrombotic syndrome (PTS). VTE patients had significantly lower levels of EPC than controls (median 0.32±0.43 cells/μL vs. 1.77±1.3 cells/μL), P=0.001. VTE patients shows lower levels of CEC than controls (median 0.25±0.43 cells/μL vs. 0.57±0.63 cells/μL), P=0.2 (non‐significant). We did not find any correlation between the levels of EPC, CEC and PTS development.

Conclusions: We found a significant decrease of EPC and a reduction trend of CEC in VTE patients. However, it is not clear if this is a cause or a consequence of the VTE event. We are now working in evaluating the possible correlation of PTS severity and EPC/CEC levels. EPC/CEC may contribute to predict VTE recurrence risk and promises to be a potential therapeutic target for PTS treatment.

PB336: DVT‐on‐a‐Chip: A Microphysiological Model of Human Venous Valve Function and Thrombosis

N.K.R. Pandian ; A. Jain

Texas A&M University, Biomedical Engineering, College Station, United States

Background: Deep Vein Thrombosis (DVT) followed by thromboembolism is a serious debilitating condition caused in deep veins by endothelial disruption, blood stasis and/or hypercoagulable blood ‐ factors of Virchow's triad (Figure 1A). Here, we present a novel microfluidic DVT‐on‐a‐chip device containing venous valves (VV), where endothelial culture, blood flow and adding of stimulation factors provide the incorporation and analysis of the Virchow triad in DVT.

Aims: ‐

Methods: The device was made from polydimethylsiloxane (PDMS, channel: 20×0.2×0.075 mm). Computational fluid dynamics (CFD) simulations of the blood flow in the device were carried out using ANSYS v17.2. HUVECs were cultured on channels coated with a matrix (collagen IV and fibronectin). Re‐calcified citrated blood (collected after informed, written consent as per ethical guidelines of IRB of TAMU) was perfused through the channels at venous shear rates and fluorescently labelled platelets and fibrin were visualized and quantitated.

Results: CFD simulations confirmed recirculation and blood stasis in the device (Figure 1B). We cultured confluent endothelial lumen in the channels with intact junctions (Figure 1D). When blood was perfused through the matrix coated channel, fibrin was formed all along the channel including VV. Platelet adhesion was confined to straight sections possibly due to low shear stress (0.5 dyn/cm 2 ) in VV. Blood perfusion through channels with healthy HUVECs resulted in unobservable thrombi, showing that endothelium protected blood from the matrix. However, upon stimulating HUVECs with TNF‐α, thrombi were again formed by the overexpressed procoagulant and adhesion molecules. Interestingly, we continued to observe increased fibrin deposition and low platelet adhesion at VV, typical for venous thrombi in vivo .

Conclusions: DVT‐on‐a‐chip can model critical biophysical processes of the DVT not captured in mouse models. In future, we expect the model to unravel the shear and endothelium driven signalling pathways, and drug‐tissue interactions.

FIGURE 1 (A) The Virchow’s triad (B) DVT‐on‐a‐chip incorporating the Virchow’s triad (C) Conceptual of DVT‐on‐a‐chip (D) Endothelial lumen in the channel

PB337: PAR‐1 Inhibition Reduces Activation of the Endothelium, Coagulation, Fibrinolysis and Inflammation during Endotoxemia

C. Schoergenhofer 1 ; M. Schwameis 2 ; G. Gelbenegger 1 ; N. Buchtele 1 ; B. Thaler 3 ; M. Mussbacher 4 ; G. Schabbauer 4 ; J. Wojta 3 ; P. Jilma‐Stohlawetz 5 ; B. Jilma 1

1 Medical University of Vienna, Clinical Pharmacology, Vienna, Austria, 2 Medical University of Vienna, Emergency Medicine, Vienna, Austria, 3 Medical University of Vienna, Cardiology, Vienna, Austria, 4 Medical University of Vienna, Thrombosis Research and Vascular Biology, Vienna, Austria, 5 Medical University of Vienna, Laboratory Medicine, Vienna, Austria

Background: The protease‐activated receptor‐1 (PAR‐1) is critically involved in the co‐activation of coagulation and inflammatory responses.

Aims: We aimed to investigate the effects of PAR‐1 inhibition by vorapaxar on the inflammatory response, the activation of coagulation, fibrinolysis and endothelium during experimental endotoxemia.

Methods: This randomized, double blind, crossover trial included 16 healthy volunteers. Subjects received a bolus infusion of 2 ng/kg lipopolysaccharide (LPS) ±placebo/vorapaxar with a washout period of 6 weeks. Vorapaxar dosing was guided by thrombin receptor activating peptide‐6 induced whole blood aggregometry. Participants received 10 mg vorapaxar or placebo as an initial dose and, depending on aggregometry, potentially an additional 10 mg. Goal was an >80% inhibition in aggregation compared to baseline. Prothrombin fragments F1+2, thrombin‐antithrombin complexes (TAT), plasmin‐antiplasmin complexes (PAP), von Willebrand Factor (vWF), E‐selectin, P‐selectin, platelet‐factor 4, TNF‐α, IL‐6 and C‐reactive protein were quantified.

Results: Vorapaxar significantly reduced the LPS‐induced increase in prothrombin fragments F1+2 by a median 27% (quartiles: 11‐49%), TAT concentrations by 22% (−3‐46%) and PAP levels by 38% (23‐53%). PAR‐1 inhibition dampened peak concentrations of TNF‐α, IL‐6 and consequently C‐reactive protein by 66% (−11‐71%), 50% (15‐79%) and 23% (16‐38%), respectively. Vorapaxar decreased maximum vWF levels by 29% (26‐51%) and soluble E‐selectin concentrations by 30% (25‐38%) after LPS infusion.

Conclusions: PAR‐1 inhibition by vorapaxar significantly reduced the activation of coagulation, fibrinolysis, the inflammatory response and endothelial activation during experimental endotoxemia.

PB338: Status System Hemostasis and Microvasculature in Different Periods after Hypothermia in Rats

N. Lycheva

St. Petersburg Institute of Pharmacy, St. Petersburg, Russian Federation

Background: Hypothermia used in practical medicine, where it is a prerequisite for open heart surgery and important component of complex therapy of a number of urgent conditions.

Aims: To study the state of the system of hemostasis and microcirculation in rats under hypothermia.

Methods: A single immersion hypothermia was simulated by cooling the animals in water at a temperature of 5°C and air 7°C until the rectal temperature reached 20‐23°C. And the indicators of hemostasis were examined. The concentration of the VCAM‐1 molecule was determined. Microcirculation were examined by laser Doppler flowmetry. All the parameters were determined before cooling, immediately after the cooling stopped, after 2 days, on the 5th day and the 14th day after the termination of cooling.

Results: After once cooling in animals and registers decries of ADP‐ and collagen‐induced platelet aggregation. Concentration of soluble fibrin monomer‐complex (SFMC) increased, activity of fibrinolytic and anticoagulant systems decreased. Have increased perfusion and all waves. After 2 days, we registered development of thrombotic state. So it increased aggregation of platelets, the concentration of SFMC, decreased fibrinolytic and anticoagulant activity. Was increase in the concentration of VCAM‐1 molecules. We become amplitude reduction of cardiac and endothelial waves, with an increase in the amplitude of the respiratory waves. On the 5th day registered the increase of platelet aggregation and reducing the concentration and SFMC. The activity of the fibrinolytic system was reduced. Concentration VCAM‐1 stayed at the previous high level. On the 14th day registered increase of platelet aggregation and inhibition of fibrinolytic system activity. The concentration of the molecules VCAM‐1 were more than normal values. An increase in myogenic and neurogenic vascular tone was recorded.

Conclusions: The risk of developing thrombotic complications is maximal 48 hours after cooling. The reported study was funded by RFBR, according to the research project № 16‐34‐60054 mol_a_dk.

PB339: Platelet‐neutrophil Crosstalk: Effects of Serotonin on Neutrophil Activation

C. Schoenichen ; M. Mauler; C. Wippel; D. Stallmann; C. Bode; I. Hilgendorf; D. Dürschmied

Heart Center, University of Freiburg, Department of Cardiology and Angiology I, Freiburg, Germany

Background: It is known that platelet serotonin (5‐HT) contributes to acute inflammation. Mouse studies showed attenuated neutrophil recruitment to sites of acute inflammation in absence of peripheral 5‐HT. Patients with myocardial infarction show elevated 5‐HT levels after an ischemic event. Analysis of patients on serotonin reuptake inhibitors (SRI) treatment show a reduced odds risk ratio for myocardial infarction (MI).

Aims: Identification of 5‐HT receptors on human and murine neutrophils and consecutive signaling in vitro and in animal models of acute inflammation as well as understanding its role in inflammatory diseases.

Methods: Healthy donor whole blood samples were treated with 5‐HT and specific 5‐HT receptor antagonists. Activation markers and integrin surface levels were assessed by flow cytometry. Degranulation was determined by MPO ELISA. Blood samples from patients suffering from depression with and without SRI treatment were analyzed for plasma 5‐HT and MPO levels and CD11b levels on neutrophils. Neutrophils were isolated using magnetic bead separation for in‐vitro activation and signaling assays.

Results: Various 5‐HTR were identified on neutrophils. In‐vitro stimulation of neutrophils with 5‐HT increased surface levels of CD11b by 80% and MPO levels in the supernatant by 40%, which could be reversed by pretreatment with 5HTR7 antagonist. Patient samples from depressed patients with SRI treatment revealed significantly lower 5‐HT plasma levels (46 vs. 100 ng/ml, P≤0.05), reduced MPO plasma levels (10 vs. 68 ng/ml) and significantly lower levels of CD11b on neutrophils (1,233 vs. 2,051 MFI, P≤0.05) compared to patients without SRI treatment.

Conclusions: 5‐HTR signaling has been identified as a novel pathway of neutrophil activation. Targeting neutrophil 5‐HT receptors might reduce pro‐inflammatory signaling and neutrophil recruitment in inflammation and might therefore broaden the horizon for new therapeutic strategies in treatment of inflammatory diseases like athero‐thrombosis or reperfusion injury.

PB340: Type I Interferons Upregulate IFITM3 in Human Platelets and Megakaryocytes to Control Viral Infectivity

R. Campbell 1,2 ; H. Schwertz 2,3 ; E. Hottz 4 ; J. Rowley 1,2 ; N. Tolley 2 ; G. Zimmerman 1,2 ; P. Bozza 5 ; F. Bozza 6 ; A. Weyrich 1,2 ; M. Rondina 1,2,7

1 University of Utah, Department of Internal Medicine, Salt Lake City, United States, 2 University of Utah, Molecular Medicine, Salt Lake City, United States, 3 University of Utah, Department of Surgery, Salt Lake City, United States, 4 Instituto de Pesquisa Clínica Evandro Chagas (IPEC), Rio de Janeiro, Brazil, 5 Instituto Oswaldo Cruz, Bio‐Manguinhos, FIOCRUZ, Rio de Janeiro, Brazil, 6 Fundação Oswaldo Cruz, Instituto de Pesquisa Clínica Evandro Chagas, Rio de Janeiro, Brazil, 7 George E. Wahlen VAMC GRECC, Salt Lake City, United States

Background: Platelets have an extensive repertoire of immune functions that remain incompletely understood. Interferon‐induced transmembrane protein 3 (IFITM3) is an immune molecule that restricts virus (e.g. dengue, influenza, etc) replication. The expression and function of IFITM3 in human platelets and megakaryocytes (MKs) is unknown.

Aims: We hypothesized that interferons (IFN) control IFITM3 gene expression in MKs and platelets, regulating viral infectivity.

Methods: IFITM3 expression in isolated platelets from dengue and influenza patients or matched, healthy controls were interrogated by RNA‐seq, followed by validation via qRT‐PCR and immunoblot. The expression of IFITM3 in CD34 + derived MKs was examined in response to IFNa, a type I IFN, stimulation and following dengue infection. IFITM3‐overexpressing MKs were employed to determine the regulation and function of IFITM3.

Results: RNA‐seq identified numerous differentially expressed transcripts in platelets from patients with viral infections. In both dengue and influenza patients, IFITM3 was significantly upregulated. Significant increases (>40‐fold, P>0.0001) in platelet IFITM3 mRNA and protein expression was validated in virally infected patients (n=53) versus controls (n=47). In patients, platelet IFITM3 protein expression significantly correlated with plasma IFNa levels and mortality. Human MKs and platelets were permissive to dengue infection. Dengue infection of MKs increased IFNa release (˜10‐fold, P>0.001) and IFITM3 protein expression (P>0.05). MKs that did not upregulate IFITM3 were more susceptible to infection. Inhibiting IFN in MKs prevented IFITM3 upregulation. IFITM3‐overexpressing MKs were protected from dengue infection (P=0.008).

Conclusions: We show for the first time that IFITM3 is present and active in human MKs and platelets during inflammatory and infectious settings. As recent studies demonstrate that dengue virus uses platelets to replicate its genome, our data identify a novel regulator of viral infectivity in platelets and MKs.

PB341: Sphingosine 1‐phosphate Induces COX‐2/PGE2 Expression via PKCalpha‐mediated MAPKs and NF‐kappaB Cascades in Human Cardiac Fibroblasts

C.‐M. Yang

Chang Gung University, Pharmacology, Tao‐Yuan, Taiwan, Republic of China

Background: Sphingosine 1‐phosphate (S1P) is a proinflammatory mediator and elevated in the regions of several tissue injuries and inflammatory diseases. The deleterious effects of S1P may aggravate heart inflammation mediated through the regulation of cyclooxygenase‐2 (COX‐2)/prostaglandin E2 (PGE2) system in various cell types. However, the signaling mechanisms underlying S1P‐induced COX‐2 expression in human cardiac fibroblasts (HCFs) remain unknown.

Aims: We used primary HCFs as a model to dissect the mechanisms underlying S1P‐induced COX‐2 expression and PGE2 synthesis via intracellular signaling components and transcription factors.

Methods: The COX‐2 protein and mRNA expression, and promoter activity were determined by Western blotting, real time‐PCR, and promoter analyses. The signaling molecules were investigated by pretreatment with respective pharmacological inhibitors or transfection with siRNAs. The interaction between COX‐2 promoter and transcription factors was determined by chromatin immunoprecipitation (ChIP) assay. The levels of PGE2 synthesis induced by S1P were determined by ELISA.

Results: We found that S1P‐induced COX‐2 expression and PGE2 synthesis was mediated through S1PR1/3‐dependent transcriptional activation. Engagement of Gq and PTX sensitive Gi protein‐coupled S1PRs by S1P led to PKCα‐dependent phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2 and then activated transcription factor NF‐κB. Moreover, the data of chromatin immunoprecipitation (ChIP) and promoter reporter assays demonstrated that the activated NF‐κB was translocated into nucleus and bound to its corresponding binding sites on COX‐2 promoter, thereby turning on COX‐2 gene transcription. Finally, up‐regulation of COX‐2 by S1P promoted PGE2 production in HCFs.

Conclusions: These results suggested that in HCFs, activation of NF‐κB by PKCα‐mediated MAPK cascades is essential for S1P‐induced up‐regulation of COX‐2/PGE2 synthesis leading to heart injury or inflammatory diseases.

PB342: Utility of New Red Cell Parameters for Identifying Functional Iron Deficiency and Anemia in Patients with Familial Mediterranean Fever

D. Yıldırım Gezgin 1 ; Z. Kaya 2 ; Ş. Demir 1 ; S. Gönen 1 ; S. Bakkaloğlu Ezgü 1

1 Gazi University Medical Faculty, Department of Pediatric Nephrology and Rheumatology, Ankara, Turkey, 2 Gazi University Medical Faculty, Department of Pediatric Hematology, Ankara, Turkey

Background: Standard laboratory tests for iron status, such as measuring ferritin levels, are subject to variability owing to acute‐phase inflammatory responses. These tests are not reliable for detecting iron deficiency anemia (IDA) in patients with familial Mediterranean fever (FMF). New red cell parameters on automated hematology analyzers have proven useful for this purpose.

Aims: We evaluated the new red cell parameters measured by the Unicel ® DxH800 machine for their utility in identifying IDA and functional ID in patients with FMF.

Methods: We retrospectively reviewed the medical records of 198 patients diagnosed with FMF to analyze complete blood counts (CBCs) and iron parameters associated with IDA and with functional ID. In addition, blood samples were collected for CBC from 12 IDA patients without FMF, 12 functional ID patients without FMF, and 18 healthy individuals. These were analyzed in a Unicel ® DxH800 automated hematology analyzer. The new red blood cell parameters were (i) RSF (reference range, 93.9‐108.3 fL), (ii) low Hb density (LHD) (reference range, 1.3% to 7.7%), and (iii) microcytic anemia factor (MAF) (reference range, 10.9% to 15.4%).

Results: Of the 198 children with FMF, 64 (32%) had active inflammation and 20 (31%) of those had anemia. Compared to the healthy controls and the 134 FMF patients without inflammation, the 64 FMF patients with inflammation had significantly lower mean values for serum Hb, serum iron, MAF and RSF and had significantly higher mean serum ferritin (all P > 0.05). Compared to the functional ID patients with or without FMF, the IDA patients with or without FMF had significantly higher mean LHD and RDW and significantly lower mean Hb, MCV, MCH, RSF and MAF (all P > 0.05).

Conclusions: The findings suggest that measuring the new red cell parameters LHD, RSF and MAF can be used as part of routine practice. These parameters can help identify functional ID and IDA in patients with FMF.

PB343: The Role of Cytokines in the Development of Acquired Aplastic Anemia

I. Berger ; A. Makhmudova

Scientific Research Institute of Hematology and Blood Transfusion of the Ministry of Health of the Republic of Uzbekistan., Tashkent, Uzbekistan

Background: The role of cytokines and the study of hematopoietic factors in the AA has not yet been studied and is most relevant in diseases associated with the violation of hemopoiesis, which include AA. The recent publication showed that the role of the function of cytokines in the development of aplastic anemia is not widely investigated, concerned on the selection of tactics of therapy.

Aims: Determine the role of certain cytokines in the development of aplasia in the population of the Republic of Uzbekistan.

Methods: The object of the study was the mononuclear cells of peripheral blood of 53 patients with AA aged from 18 to 60 years who were treated at the Center of Hematology in Uzbekistan from 2014 to 2016. The concentration of cytokines was calculated from the optical density of the samples and compared with the calibration standard.

Results: The serum erythropoietin (sEPO), TNFa, and IL‐4.6 levels were measured in patients with AA, depending on the severity level. In the course of the study, patients showed a decrease in sEPO and IL4, as well as an increase in TNFa and IL‐6 levels in comparison with donors. In patients with mild AA, the level of sEPO was either decreased, or within adequate values. In the presence of aplasia in patients, there was an increase in TNFa in comparison with patients with anemia. It has been established that significant predictors are the low level of sEPO and the increased content of TNFa.

TABLE 1 The ability of peripheral blood mononuclear cells to produce the main regulators of hemopoiesis in normal and with AA Cytokines Donors Patients AA р IL‐6 (pg/ml) 2.37±0.2 42.11±9.49 <0.001 EPO (mIU/ml) 8.0±0.2 (6.0‐10.0) 580.7±42.8 (210‐1050) <0.001 TNFα (pg/ml) 0.02±0.003 14.02±4.54 <0.001 IL‐4 (pg/ml) 0.1±0.03 0.11±0.05 >0.05

The level of spontaneous production of another positive factor IL‐6 was increased in 17 times. A spontaneous production of IL‐4 is 10 times. TNFα was in 700 times higher than the average level.

Conclusions: We found that the development of hematopoiesis depression in AA is due to the fact that the immune system produces a huge number of negative regulators of hematopoiesis ‐ TNFα, and is not related to a decrease in the synthesis of positive factors. Imbalance in the ratio of cytokines in this disease is exacerbated by insufficient production of antagonist negative regulators ‐ IL4.

PB345: Ticagrelor Mediated Protection during Myocardial Reperfusion is Exponentiated by SSRIs

M. Mauler 1 ; C. Schoenichen 1 ; N. Herr 1 ; D. Stallmann 1 ; C. Bode 1 ; K. Ley 2 ; I. Hilgendorf 1 ; D. Duerschmied 1

1 University Heart Center Freiburg, Freiburg, Germany, 2 La Jolla Institute for Allergy and Immunology, La Jolla, United States

Background: Antiplatelet drugs like ticagrelor can protect the heart against reperfusion injury which is associated with an inflammatory response. We utilized fluoxetine (Flx) to deplete platelet serotonin because acute neutrophil triggered inflammation is dampened in these mice and analyzed the outcome after myocardial reperfusion injury.

Aims: Evaluate if and how excessive serotonin levels interfere with P2Y12 mediated cardioprotection.

Methods: Mice received Flx for 3 weeks. After administration of ticagrelor (100 mg/kg loading; 50 mg/kg twice afterwards), myocardial infarction (MI) was induced, followed 24 hours reperfusion. Heart function and infarct size was evaluated. Integrin expression on neutrophils was analyzed by flow cytometry.

Results: Plasma serotonin after MI was increased in WT mice (150±19 ng/ml) which was not affected by ticagrelor (145±13 ng/ml). Infarct size was reduced in Flx treated mice (38 in Flx, 53 in WT; % area at risk (AAR) and heart function was significantly improved (24±2 vs. 16±3% FS in WT; FS=fractional shortening). WT mice had more neutrophils in the AAR (28 vs. 14 in Flx per mm 2 tissue). Neutrophils had decreased CD11b expression in Flx (70%) treated mice compared to WT. Electron microscopy and determination of plasma MPO in vitro revealed that serotonin triggers degranulation of neutrophils. Serotonin also induced upregulation of CD11b, which was reversible by addition of a protein transport inhibitor. In mice which received ticagrelor we found an even stronger protective effect on top of fluoxetine treatment when we looked at neutrophil integrin expression and infarct size.

Conclusions: Neutrophil infiltration after MI is enhanced with increased serotonin levels. This is contributed to elevated CD11b expression which results in stronger neutrophil‐endothelial interaction and transmigration. This effect was exponentiated after treatment with ticagrelor. Thus, intervening in serotonin‐neutrophil crosstalk might provide novel anti‐thromboinflammatory treatment options after acute MI.

PB346: Hemoglobin‐activated Platelet Regulates Macrophage Polarization under Hemolytic Conditions

R. Singhal 1 ; S. Chawla 1 ; D.K. Rathore 2 ; A. Ojha 1 ; P. Guchhait 1

1 Regional Centre for Biotechnology, Disease Biology Laboratory, Faridabad, India, 2 Translational Health Science and Technology Institute, Pediatric Biology Centre, Faridabad, India

Background: The identification of different subsets of macrophages in various tissues under different microenvironments as well as clinical conditions has put their heterogeneity into sharp focus. Our recent work describes engulfment of Hb‐activated platelets by monocytes and their transformation into pro‐inflammatory subset (CD14 + CD16hi TNF‐α + ). These inflammatory monocytes are positive for intracellular Hb and platelet, and are abundant in circulation of hemolytic patients.

Aims: We investigated the differentiation and polarization of monocytes when encountered by either Hb‐activated platelets or free‐Hb in vitro and in vivo.

Methods: Monocytes from healthy individuals were incubated with Hb‐activated platelets or only Hb for 2 hours and allowed for differentiation under either M1‐specific (GM‐CSF, IFN‐γ+LPS) or M2‐specific (M‐CSF, IL‐4+IL13) stimulus in vitro. Further the monocyte and macrophage populations were measured in spleen and peritoneum of phenylhydrazine‐induced hemolytic mice.

Results: Our data shows that monocytes engulf Hb‐activated platelets and polarize into pro‐inflammatory M1 macrophages when grown under M1‐specific stimulus expressing high levels of CD80, STAT‐1, NF‐κB and iNOS, and secreting pro‐inflammatory cytokines and reactive species. On the other hand, after engulfing only free‐Hb monocytes polarized into anti‐inflammatory M2 macrophages under M2‐specific stimulus expressing high levels of CD163, STAT‐6 and PPARγ and having high phagocytic ability to engulf microbes. Interestingly, macrophages resulting from the monocytes that engulfed Hb‐activated platelets and differentiated even under M2 stimulus, still exhibit M1 phenotype. Furthermore, the hemolytic mice also exhibited the gradual increase in pro‐inflammatory macrophages in peritoneum and spleen during the course of induced‐hemolysis till day 14.

Conclusions: Study suggests that intravascular hemolysis, specifically the abundance of Hb‐activated platelets in circulation closely regulates macrophage polarization.

PB347: Anti‐inflammatory Effect of 5‐ Aminolevulinic Acid‐mediated Sonodynamic Therapy Reduce Macrophages‐derived Foam Cells in the Rabbit Abdominal Aorta Atherosclerosis Model

H. Mehrad 1,2 ; A. Foletti 3 ; F. Nasirzadegan 4 ; A. Keshavarz 4 ; H. Beladerang 4 ; L. Kazemi 4 ; M.M. Hosseinian Zakariya 5

1 Islamic Azad University, Tabriz Branch‐Faculty of Basic Sciences, Department of Physics, Tabriz, Iran, Islamic Republic, 2 Tabriz University of Medical Sciences, Neurosciences Research Center, Tabriz, Iran, Islamic Republic, 3 Italian National Research Council, Institute of Translational Pharmacology, IFT, Rome, Italy, 4 Islamic Azad University, Tabriz Branch‐Faculty of Mechanics, Department of Bioengineering, Tabriz, Iran, Islamic Republic, 5 Islamic Azad University, Tabriz Branch‐Faculty of Medical Sciences, Department of Neurology, Tabriz, Iran, Islamic Republic

Background: Atherosclerosis is a chronic disease characterized by the deposition of excessive cholesterol in the arterial intima. Macrophage foam cells play a critical role in the occurrence and development of atherosclerosis. It is well recognized that the foam cells in atherosclerotic lesions are derived mainly from monocytes/macrophages.

Aims: In this study, we aimed to investigate pulsed ‐ focused ultrasound effect on macrophage foam cells density reduction accompanied by 5‐ Aminolevulinic Acid ‐ loaded microbubbles administration in the rabbit abdominal aorta atherosclerosis model.

Methods: Briefly, New Zealand white rabbits underwent primary balloon dilatation injury at the abdominal aorta (approximately 2 cm superior to the iliac bifurcation) followed by a 1.5% cholesterol‐rich diet injury for three weeks. Histopathology results showed foam cells formation in all of the rabbits’ arteries. Then atherosclerotic arteries of the treatment group at lesion region, treated with 5‐ Aminolevulinic Acid ‐ loaded PESDA (Perflurocarbon‐ Exposed Sonicated Dextrose Albumin) microbubbles (100 ml/kg, 2‐5 × 10 bubbles/ml) administration accompanied by extracorporeal pulsed‐focused ultrasound (P=3 W, F=1 MHz, PD=250 ms) therapy. Foam cells density were evaluated in the treatment group compared with the other groups using B‐mode ultrasonography and histology.

Results: Results from B‐mode ultrasonography and histopathology showed a significant reduction in the mean value for foam cells density within the early atherosclerotic lesion in the treatment group compared with the other groups (P>0.05).

Conclusions: Enhanced inertial cavitation effect of collapsed PESDA microbubbles‐ induced by pulsed‐ focused ultrasound therapy can cause to enhance anti‐ inflammatory effect of 5‐ Aminolevulinic Acid and, egress of macrophages from intima layer and efflux of cholesterol from foam cells. This protocol may be a potential treatment to early stage atherosclerosis.

PB348: Signalling Effects of Microparticle‐derived Tissue Factor and Activated Factor VII in Endothelial Cells

A. Brandtner 1 ; A. Magnutzki 1 ; G. Lehner 1 ; C. Feistritzer 2 ; V. Haller 1 ; M. Joannidis 1

1 Medizinische Universität Innsbruck, Department of Internal Medicine, Division of Intensive Care and Emergency Medicine, Innsbruck, Austria, 2 Medizinische Universität Innsbruck, Department of Internal Medicine V, Division of Haematology and Oncology, Innsbruck, Austria

Background: MP‐derived tissue factor (TF) can be detected on the cellular surface and was found to increase the procoagulant activity of endothelial cells (EC). TF on EC and its ligand FVIIa activate PAR‐2 in the vascular system. One downstream mechanism of PAR‐2 involves the translocation of NFkB, which leads to pro‐inflammatory reactions, for example upregulation of ICAM‐1, leading to enhanced leukocyte adhesion to EC and perpetuation of the pro‐inflammatory process. To date it is not clear, whether MP‐associated TF is able to activate intracellular signalling.

Aims: The aim is to test whether TF transferred by MPs can induce NFkB‐related signalling leading to enhanced interaction of EC with leukocytes.

Methods: MP were isolated from peripheral mononuclear blood cells (PBMC) from healthy donors after stimulation with TNFα for 3 hours. Functional activity of TF was confirmed by chromogenic FXa assay. Isolated MPs were incubated with HUVEC. Cell lysates were harvested at 0, 15 and 90 minutes before and after addition of 5 nmol/L FVIIa and analysed by western blot for phosphorylation of canonical NFkB proteins. Leukocyte‐endothelial adhesion was assessed by measuring fluorescent intensity (FI) of monocytic cells (THP‐1) attached to MP‐treated EC (EaHy926).

Results: Presence of coagulation active TF on MP isolates was confirmed by activation of FX after addition of FVIIa. Incubation of EC with MP and FVIIa did not have an influence on the phosphorylation status of IKKα/β, IκBα, p65 and expression of ICAM‐1 over an observed time of 90 minutes. Adhesion of THP‐1 to EC was not increased after addition of MPs and FVIIa (mean FI 2320±94.5 vs. 2107±372.6).

Conclusions: Despite a TF‐specific coagulation activity of isolated MPs, no signalling activity of proteins associated with the canonical NFkB pathway nor increased expression of ICAM‐1 on cells was detectable in EC. This result was consistent with the functional assay, with no altered adhesion of leukocytes after incubation of EC with PBMC‐derived MPs and FVIIa.

PB349: Quantification and Characterisation of Plasma Extracellular Vesicles in Early Onset Preeclampsia

K. Egan 1 ; P. Pszklanna 2 ; H. O'Connor 3 ; C. Monteith 3 ; N. Fischer Dooley 2 ; C. Davis 2 ; L. Weiss 2 ; N. McCallion 3 ; S. Cooley 3 ; F. Malone 3 ; P. Maguire 2 ; F. Ni Ainle 1

1 Mater Misericordiae University Hospital, Haematology, Dublin, Ireland, 2 UCD Conway Institute, Dublin, Ireland, 3 Rotunda Hospital, Dublin, Ireland

Background: Preeclampsia is a pregnancy specific pro‐inflammatory disorder characterised by proteinuria and hypertension. Early onset preeclampsia (EOP, >34 weeks gestation) is characterised by increased placental dysfunction, increased anti‐angiogenesis, and increased maternal inflammation, compared to late onset preeclampsia (>34 weeks gestation). It confers an increased risk of maternal death and severe fetal complications. Extracellular vesicles (EVs) are released by most eukaryotic cells and can be found in high levels in biological fluids such as plasma. EV levels are increased in inflammatory disorders and may have diagnostic or prognostic potential.

Aims: To quantify and characterise plasma EVs in EOP.

Methods: Plasma EVs from non‐pregnant controls (NPC, n=13), pregnant controls (PC, n=16), and EOP patients (n=19) were measured using NanoparticleTracking Analysis (NTA) and flow cytometry.

Results: Using NTA, plasma EV levels (50‐200 nm) were found to be increased in EOP compared to NPC (5.9±5.1 vs. 1.7±1.8×10 8 /μl, P>0.05), with a trend towards increased levels compared to PC (3.7±3.2×10 8 /μl). Interestingly, patients with severe EOP (i.e. multiorgan involvement) had significantly lower EV levels than non‐severe EOP (3.0±3.0 vs. 8.5±5.3×10 8 /μl, P>0.05). Using flow cytometry, EV levels (100‐300 nm) were also increased in EOP relative to NPC (5.2±1.1 vs. 3.6±1.7×10 7 /μl, P>0.05) with a trend towards increased levels compared to PC (4.7±0.8×10 7 /μl). EV levels were significantly lower in cases of severe disease compared to non‐severe disease (4.4±1.0 vs. 5.7±1.0×10 7 /μl, P>0.05). There was a highly significant correlation between EV levels measured by NTA and flow cytometry (r=0.7, P>0.001). Using cell specific antibodies, the increase in EVs within EOP was, in part, due to increases in endothelial and leukocyte EVs, but not platelet EVs.

Conclusions: Plasma EVs are increased in EOP, most significantly in cases of non‐severe disease. These results may have future diagnostic and prognostic relevance.

PB350: Circulating Cholesterol Containing, Platelet‐derived Microparticles Contribute to the Procoagulant State Observed in Systemic Lupus Erythematosus

S. Hu ; S. Meaney; C. Wynne

Dublin Institute of Technology, Biological Science Department, Dublin, Ireland

Background: The risk for thrombosis is significantly increased in systemic lupus erythematosus (SLE), with microparticles (MPs) which are shed from activated platelets being known to support coagulation. Hypercholesterolemia is also associated with an increased risk of thrombosis. This study sets out to assess the procoagulant ability, of a novel subset of cholesterol containing MPs.

Aims: To establish a method to obtain cholesterol containing, lipoprotein free MPs and to assess the association between their cholesterol content and the procoagulant state in SLE.

Methods: Healthy control and SLE patients provided informed written consent and the study received prior approval from the relevant institutional ethics boards before blood samples were obtained. Using size exclusion chromatography, 30 fractions (approx. 425 ml each) were collected. The size (Dynamic Light Scattering), structure (TEM) and cholesterol content (Amplex red cholesterol assay kit) for each MP fraction was established. Total cholesterol from each donor was quantified using a colorimetric assay. Thrombin generation was measured as the endogenous thrombin potential (ETP) without the addition of exogenous phospholipids and tissue factor (TF). The cell of origin, phosphatidylserine (PS) and TF content of MPs was analysed by flow cytometry.

Results: The purified MPs from fractions 8‐13 were found to be 99.9% free of lipoproteins, with no significant difference in size or structure being noted between healthy controls and patients. Compared to the controls, circulating MPs were significantly elevated in the patient group. In both groups, most of these MPs were of platelet origin. The cholesterol level, number of CD61 + and PS + MPs and the ETP was increased among patients as compared to the controls.

Conclusions: The increased number of circulating cholesterol containing, platelet‐derived MPs and their association with high ETP suggest that these MPs play an important role in the pathogenesis of the prothrombotic state in SLE patients.

PB351: Phosphatidylserine‐negative Platelet Microvesicles Are Increased in the Acute and Convalescent Phase of Ischemic Stroke and TIA ‐ Specific Subpopulations Predict Prognosis

A. Lundström 1 ; F. Mobarrez 2 ; E. Änggårdh‐Rooth 1 ; C. Thålin 1 ; M. von Arbin 1 ; P. Henriksson 1 ; B. Gigante 1,3 ; A.C. Laska 1 ; H. Wallén 1

1 Karolinska Institutet, Clinical Sciences Danderyd University Hospital, Stockholm, Sweden, 2 Karolinska Institutet, Department of Medicine, Unit of Reumatology, Stockholm, Sweden, 3 Karolinska Institutet, Environmental Medicine, Unit of Cardiovasc Epidemiology, Stockholm, Sweden

Background: Platelet microvesicles (PMV) and tissue factor positive microvesicles (TF + MV) have previously been found elevated in acute ischemic stroke (IS) and could be potential biomarkers for IS and/or risk of recurrence.

Aims: To evaluate the ability of different MV populations to predict recurrent ischemic events in patients with IS or TIA.

Methods: In a cohort of 211 patients with IS or TIA recruited 2007‐2009, concentrations of MV expressing the platelet glycoprotein GPIIb, phosphatidylserine (PS), P‐selectin, tissue factor (TF) and combinations thereof were determined by flow cytometry in the acute and convalescent phase, and compared with age‐ and sex‐matched healthy controls. Recurrent ischemic events were obtained from Swedish registers until the end of 2014 (1,100 patient years). The association between MV populations and outcome was analyzed by Kaplan‐Meier survival analysis based on median split, with hazard ratios (HR) calculated by Cox regression.

Results: Compared to healthy controls, PS + PMV and PS − PMV expressing either P‐selectin or TF showed pronounced elevations in patients in the acute phase (7‐77 fold), with levels remaining high in the convalescent phase. PS + and PS − MV with otherwise the same surface markers showed no correlations. In a multivariate, adjusted model, PS − TF + PMV predicted recurrent ischemic event independently of cardiovascular risk factors, HR 1.86 (CI 1.04‐3.33, p=0.036), with p=0.001 for the model. Unexpectedly, high levels of several MV populations related to platelet/cell activation were associated with reduced risk of recurrent ischemic event.

Conclusions: PS + and PS − MV populations are differentially elevated in connection with IS/TIA, with specific subpopulations conferring increased or reduced risk of recurrent ischemic event. PS ‐ TF + PMV was the only MV population with a positive association to poor outcome.

PB352: Circulating Microparticles Improve Thrombin Generation and Fibrin Formation in Factor VIII Deficiency Plasma

Y. Zong 1 ; F. Mobarrez 2 ; N. Soutari 3 ; R. Chaireti 1 ; J. Antovic 1,3

1 Karolinska Institutet, Department of Molecular Medicine & Surgery, Stockholm, Sweden, 2 Karolinska Institutet, Department of Medicine, Unit of Rheumatology, Stockholm, Sweden, 3 Karolinska University Hospital, Coagulation, Clinical Chemistry, Stockholm, Sweden

Background: Circulating microparticles (MPs) are procoagulant due to the exposure of phosphatidylserine (PS) and tissue factor. MPs have been widely studied in different thrombotic diseases as well as in healthy conditions. However, the role of MPs in hemophilia A, one of the most common inherited bleeding disorders, remains poorly understood.

Aims: To investigate if MPs from normal plasma improve hemostasis in hemophilia A in vitro .

Methods: MPs were isolated from pooled normal plasma, and characterized by flow cytometry. The contribution of MPs on thrombin generation, fibrin formation and fibrinolysis in factor VIII deficiency plasma were evaluated by calibrated automated thrombogram and overall hemostasis potential assay. Fibrin structure was visualized using scanning electron microscopy. The underlying mechanisms of MPs’ procoagulant property were investigated by blocking MPs with lactadherin (binds PS) or anti‐tissue factor antibody.

Results: MPs derived from platelets were PS and CD41 double positive events as identified by flow cytometry. MPs increased peak value and endogenous thrombin potential in factor VIII deficiency plasma in a MPs‐dose‐dependent manner. MPs supported fibrin formation and fibrin strands were thinner and denser than those triggered by thrombin. The procoagulant effect was totally abolished by inhibiting PS, but not altered by blocking tissue factor.

FIGURE 1 The influence of MPs on thrombin generation in plasma with different FVIII level (0%, 1%, 5%, 100%)

TABLE 1 Overall coagulation potential (OCP) and overall hemostasis potential (OHP) in FVIII deficiency plasma using different triggers 0% FVIII Thrombin MPs MPs‐5D MPs‐10D MPs‐antiTF MPs‐Lac OCP 0.13±0.28* 11.9±0.73 8.71±1.72* 1.64±0.40* 11.65±0.27 0* OHP 0±0.04* 6.74±0.72 5.94±0.11 0.94±0.29* 7.00±0.81 0±0.19*

Conclusions: Circulating MPs accelerated thrombin generation and enhanced fibrin formation in hemophilia A plasma independently of tissue factor.

PB355: Effect of Plasma Components on Staphylococcus aureus Adhesion to Tissues Used for the Right Ventricular Outflow Tract (RVOT) Revalvulation

B. Ditkowski 1 ; R. Veloso 1 ; J. Claes 1 ; L. Liesenborghs 2 ; S. Van Kerckhoven S 2 ; R. Jashari 3 ; M. Gewillig 1 ; M. Hoylaerts 2 ; B. Meyns 4 ; R. Heying 1

1 KU Leuven, Department of Cardiovascular Sciences/ Cardiovascular Developmental Biology, Leuven, Belgium, 2 KU Leuven, Department of Cardiovascular Sciences/ Centre for Molecular and Vascular Biology, Leuven, Belgium, 3 European Homograft Bank (EHB), Human Body Material (HBM), Brussels, Belgium, 4 KU Leuven, Department of Cardiovascular Sciences/ Cardiac Surgery, Leuven, Belgium

Background: RVOT reconstruction in congenital heart disease can be surgically done using in first line cryopreserved pulmonary homograft (CPH) and, as an alternative, xenografts such as the bovine jugular vein (BJV) conduit or stent‐mounted valve. Despite this good therapeutic alternative recent clinical studies report an increased risk of infective endocarditis (IE) in BJV xenografts. These observations raise the question of why such valves are more prone to IE than homografts.

Aims: We investigate whether different graft tissues promote interactions with plasma components and therefore enhance the risk for S.aureus adhesion to valve tissue.

Methods: Similar tissue pieces prepared as for clinical use were incubated for 2 hours at 37°C with 30 μg/ml of fluorescently labelled fibrinogen resuspended in human albumin. Then, S. aureus 8325‐4 adhesion to the same tissues was assessed under flow conditions (10 minutes at 1,000 per seconds) using a micro‐parallel flow chamber after O/N tissue incubation at 4°C with PBS, frozen human pooled plasma and human albumin or serum. Protein deposition was quantified by fluorescence microscopy and bacterial adhesion was evaluated by CFU counting on blood agar plates.

Results: Pericardium patch presented higher protein deposition (P < 0.05) compared to BJV and CPH. Although not significant, there is a slight trend to higher fibrinogen deposition on BJV tissue compared to CPH. After incubation with human plasma S. aureus adhesion to BJV increased significantly under flow conditions compared to the respective controls (human serum P < 0.05 and albumin P < 0.01).

Conclusions: Our results indicate that plasma protein deposition modulate differently S. aureus adhesion to the tested tissues, especially BJV. Therefore plasma proteins are a vital player facilitating risk of endovascular infection. Future studies will assess if platelet adhesion differs between those tissues, as well as focus on re‐endothelialization of grafted tissues and how this affects bacterial adhesion and development of valvular infection.

PB356: Delayed Initiation and Inadequate Anticoagulation as Independent Risk Factors for Development of Post Thrombotic Syndrome (PTS) ‐ An Observational Study from a Tertiary Care Hospital in North India

V. Suri 1 ; P. Dodda 1 ; P. Malhotra 1 ; U. Gorsi 2 ; J. Ahluwalia 3 ; N. Kumar 3 ; G. Prakash 1 ; D. Lad 1 ; A. Khadwal 1 ; S. Kumari 1 ; S. Jain 1 ; S. Varma 1 ; N. Varma 3

1 Post Graduate Institute of Medical Education and Research, Internal Medicine, Chandigarh, India, 2 Post Graduate Institute of Medical Education and Research, Radiodiagnosis, Chandigarh, India, 3 Post Graduate Institute of Medical Education and Research, Hemato‐Pathology, Chandigarh, India

Background: The post thrombotic syndrome (PTS) a chronic, potentially disabling condition is estimated to develop within 1 to 2 years in 20% to 50% of patients of symptomatic deep vein thrombosis (DVT).

Aims: The present study was planned to investigate the frequency and risk factors for development of PTS

Methods: Post‐thrombotic syndrome (PTS) was defined in any patient with DVT with the pain and swelling occurring daily for at least 1 month for 6 months or more after an episode of DVT

Our study included two arms, one a prospective period from June 2016 to December 2017 (20 patients). Retrospectively data was analyzed from the year December 2007 till June 2016 (60 patients). All the enrolled patients had been anticoagulated for at least six months. Venous thrombosis was diagnosed by duplex ultrasonography. Patients were evaluated for symptoms and signs of PTS by using CEAP clinical scale and Villalta's scale at 0 and 3 months.

Results: A total of 80 patients (57 males/23 females) with proximal DVT who later on developed post thrombotic syndrome were enrolled. The frequency of PTS at 0/3 months in our study was 79.1%, 73.6% (by using CEAP clinical scale) and 51.9%, 23.5% (by using Villalta's scale) respectively. Age less than 45 years of age, Male sex, a BMI OF >25 kg/m 2 , left sided DVT were the predisposing factors significantly associated with significantly higher frequency of PTS (P<0.0001 & P<0.05).

70 (87.5%) our PTS patients had sub therapeutic INR (time period for which his/her INR was<60%) and only10 (12.5%) of them had their INR in therapeutic range. Mean duration before anticoagulation for DVT was initiated was 24.15±29.152 days. A statistically non‐significant low risk and a less severe of PTS was observed in the patients where the anticoagulation was initiated within one month (44 patients) of onset of DVT as compared to those where it was initiated at a later date (36 patients).

Conclusions: More studies are needed on the definitive diagnosis and the possible risk factors of PTS.

HEMATOPIESIS

PB357: Mitofusin 2 Regulates Mitochondria in Megakaryocytes, and Hemostasis and Thrombosis in Mice

S.C. Bhatlekar 1 ; R.A. Campbell 1 ; L. Guo 1 ; B.K. Manne 1 ; M.T. Rondina 1,2 ; A.S. Weyrich 1 ; P.F. Bray 1,2,3 ; J.W. Rowley 1,2

1 University of Utah, Program in Molecular Medicine, Salt Lake City, United States, 2 University of Utah, Department of Internal Medicine, Salt Lake City, United States, 3 University of Utah, Division of Hematology and Hematologic Malignancies, Salt Lake City, United States

Background: Mitochondrial dysfunction is associated with numerous diseases, including disorders of thrombosis and hemostasis. Platelets, chief effectors of hemostasis and thrombosis, require mitochondria for survival and function. Genetic studies have associated the mitochondrial fusion protein Mitofusin 2 (Mfn2) with platelet counts and cardiovascular disease, yet the role of Mfn2 in megakaryocytes (MKs) and platelets is unclear.

Aims: To determine whether mitochondrial fusion regulates mitochondria in human and mouse MKs, and to define how Mfn2 regulates platelet counts, hemostasis, and thrombosis in mice.

Methods: We generated MK/platelet specific Mfn2 knockout (Mfn2 KO) mice by crossing PF4‐Cre mice with floxed Mfn2 mice. We examined mitochondrial morphology in human cord blood derived MKs, and mouse bone marrow MKs via fluorescent microscopy. Mitochondrial function was assessed by analysis of oxygen consumption rate (OCR). We used in vivo labeling approaches to track platelet survival. Hemostasis and thrombosis were assessed using established models of tail bleed time and collagen/epinephrine induced pulmonary embolism.

Results: Mitochondria in human MKs formed elaborate, interconnected networks, especially when cells were metabolically stressed. Mitochondria in wild type mouse MKs also formed elaborate networks, whereas mitochondria in Mfn2 KO MKs were significantly more punctate (i.e. round and not connected). Mitochondrial potential, and OCR were significantly reduced in Mfn2 KO platelets. Mfn2 KO significantly reduced platelet counts, endogenous platelet life‐span, and platelet survival following transfusion. Tail bleed times were significantly prolonged by MFN2 KO, and mice survived significantly longer after induced pulmonary embolism.

Conclusions: Our data imply a novel role for Mfn2 mediated mitochondrial fusion in MKs that may impact mitochondrial function in platelets, platelet survival, and in vivo hemostasis and thrombosis.

PB358: PDK1 Is Crucial to Pro‐platelet Formation and Thrombopoiesis by Regulating MK Actin Polymerization

S. Geue 1 ; B. Walker‐Allgaier 1 ; P. Münzer 1 ; M. Manke 1 ; D. Eißler 1 ; F. Lang 2 ; K. Aurbach 3 ; I. Pleines 3 ; H. Schulze 3,4 ; M. Gawaz 1 ; O. Borst 1

1 University Clinic of Tübingen, Department of Cardiology and Circulatory Disorders, Tübingen, Germany, 2 University of Tübingen, Department of Physiology, Tübingen, Germany, 3 University Hospital and Rudolf Virchow Center, Department of Experimental Biomedicine ‐ Vascular Medicine, Würzburg, Germany, 4 University of Würzburg, Department of Experimental Haemostaseology, Würzburg, Germany

Background: Megakaryopoiesis is a complex process including megakaryocyte (MK) maturation and differentiation which includes the formation of the demarcation membrane system (DMS). Finally, within the bone marrow (BM) mature and polyploid MKs form proplatelets and release platelets in BM sinusoids. PDK1 has been shown to be highly expressed in platelets and megakaryocytes and is required for Ca 2+ ‐dependent platelet activation, but its role in the regulation of platelet biogenesis has not been investigated so far.

Aims: The present study explored the impact of PDK1 in the regulation of thrombopoiesis and proplatelet formation in MK/platelet specific PDK1‐deficient mice ( pdk1 −/− ) .

Methods: Immunhistochemistry, Cryosectioning of femurs; Immunofluorescence Microscopy; Flow Cytometry; in vitro proplatelet formation

Results: As demonstrated by blood counts, PDK1‐deficiency resulted in a macrothrombocytopenia while platelet lifespan was not affected. As revealed by immunohistological analysis, pdk1 −/− mice further displayed a remarkable MK hyperplasia as well as a significantly increased extramedullary thrombopoiesis. However, the size of spleen and liver was comparable between pdk1 −/− and pdk1 fl/fl mice. There was no difference in plasma TPO levels of pdk1 −/− mice and ploidy of pdk1 −/− MKs. However MKs within the BM of pdk1 −/− had less contact to BM sinusoids and displayed a drastic increased number of MKs with a lower size in the BM. Interestingly this came along with a decreased formation of proplatelets of pdk1 −/− MKs indicating that PDK1 is required for proplatelet formation. Investigations of the cytosceleton organization revealed increased polymerized F‐Actin of pdk1 −/− MKs in addition to an impaired formation of a DMS.

FIGURE 1 Representativ cryosections of megakaryocytes (green) in the bone marrow

Conclusions: The present observations unraveled PDK1 as an important signaling molecule in proplatelet formation and thrombopoiesis by regulating MK acting polymerization.

PB359: Unique Megakaryocytes from Human Adipose‐derived Mesenchymal Stromal/stem Cell Line: Comparison with Hematopoietic Stem Cell‐derived Megakaryocytes

Y. Ono‐Uruga 1 ; K. Tozawa 2 ; M. Yazawa 3 ; T. Mori 4 ; M. Murata 5 ; Y. Ikeda 6,7 ; Y. Matsubara 1

1 Keio University School of Medicine, Clinical and Translational Research Center, Tokyo, Japan, 2 Keio University School of Medicine, Hematology, Tokyo, Japan, 3 Keio University School of Medicine, Plastic and Reconstructive Surgery, Tokyo, Japan, 4 National Cancer Center Hospital, Pathology, Tokyo, Japan, 5 Keio University School of Medicine, Laboratory Medicine, Tokyo, Japan, 6 Keio University School of Medicine, hematology, Tokyo, Japan, 7 Waseda University, Faculty of Science and Engineering, Life Science and Medical Bioscience, Tokyo, Japan

Background: We established a human subcutaneous adipose‐derived mesenchymal stromal/stem cell line (ASCL) as an expandable cell source to generate megakaryocytes (MKs) that release platelets by a simple method. Use of the ASCL has advantages for manufacturing MK‐lineage cells, because neither the establishment of the ASCL nor its differentiation to MK‐lineages require gene transfer, and endogenous thrombopoietin (TPO) is utilized for their differentiation. Our goal is to provide manufactured platelets from the ASCL for clinical application.

Aims: This study was performed to compare the characteristics of ASCL‐derived MKs (ASCL‐MKs) and hematopoietic stem cell‐derived MKs (HSC‐MKs).

Methods: HSC (CD34‐positive cells) and ASCL were cultured with MK differentiation media, and each cell on day 8 was characterized by DNA ploidy, electron microscopic analysis, flow cytometry, qRT‐PCR, and microarray analysis.

Results: ASCL satisfied the minimal criteria for defining mesenchymal stem cells (MSCs) by The International Society for Cellular Therapy. Cells on day 8, electron microscopic observations of ASCL‐ MKs and HSC‐MKs showed typical MK organelles, including lobulated nuclei, granules, and a demarcation membrane system. We observed DNA ploidy of more than 8N in CD41‐positive cells derived from ASCL and HSC. ASCL‐MKs and HSC‐MKs exhibited a similar frequency of CD41‐positive cells. The expression of both CD42b and CD90, a MSC marker, was only observed in ASCL‐MKs. Increased expression of TPO, p45NF‐E2, beta‐1 tubulin, VWF, and GATA2 genes was detected in both lines during culture. On the other hand, gene expression of GATA1 was not detected in the ASCL and ASCL‐MKs. Microarray analysis revealed that ASCL‐MKs had higher expression of MSC‐related factors, such as CD109 and NT5E.

Conclusions: ASCL‐MKs express the same genes and surface markers as HSC‐MKs, and also have MSC‐related characteristics. Functional differences in MKs and subsequently platelets derived from ASCL and HSC are under investigation.

COAGULATION FACTORS

PB360: Nucleic Acid‐based Therapeutic Targets for Protein S Deficient Patients

J. Tiao 1 ; A. Rocchi 1 ; Q. Hughes 1 ; B. Le 2 ; R. Veedu 2 ; R. Baker 1

1 Murdoch University, WACTH, Murdoch, Australia, 2 Murdoch University, Murdoch, Australia

Background: Protein S (PS) is expressed by PROS1 which is negatively down‐regulated via miR‐494 binding to 3 sites in the 3'UTR. PS deficiency can be acquired or arise by heterozygous gene mutations, leading to increased risk of deep vein thrombosis and pulmonary embolism. We explored whether blocking of miR‐494 by nucleic acid based oligonucleotides on the 3′UTR PROS1 gene increases PS levels.

Aims: This study used targeted oligonucleotides designed to block miR‐494: PROS1 interactions, with the aim of extending PROS1 mRNA transcript lifespan, and increase expression of PS.

Methods: HuH‐7 cells were co‐transfected with miR‐494 or scrambled control, with or without blockers (WACTH1, ‐2 and ‐3) to individual sites. Two blocker versions, mod1‐WACTHx.1 and mod2‐WACTHx.2, were investigated for disrupting miR‐494: PROS1 interactions. Blocker and PROS1 3′UTR interactions were measured using UV spectrophotometric Tm and dual‐luciferase reporter assays. Targeted RT‐qPCR was used to quantify PROS1 levels in total RNA extracted from co‐transfected cells.

Results: PS levels were modulated by introducing selected oligonucleotide blockers. WACTH3.2 had the strongest effect compared to the negative control. WACTH3.2 and WACTH2.2 treatment resulted in 5‐ and 2‐fold increases in mRNA levels, respectively. This disruption was limited to mod 2 versions of the blockers. Tm analysis confirmed hybridisation between the miR‐494 binding sites and all WACTH blockers; with mod2 showing higher Tm than mod1 when co‐incubated with the respective RNA sequences. Furthermore, WACTH1, ‐2 and ‐3 mod2 blockers significantly increased PROS1 mRNA by 1.7‐to‐2.1 fold, compared to no blocker controls (P<0.01).

Conclusions: Novel nucleic acid‐based blockers that selectively regulate PS levels in HuH7 cells were identified by disrupting miR‐494 binding to the PROS1 mRNA. WACTH blockers with mod 2 could abrogate binding. PS mRNA levels were successfully manipulated with custom nucleic acid‐based blockers.

PB361: Structural Insights during the Recognition of Endothelial Cell Protein C Receptor to the Protein C/activated Protein C in Presence of Phospholipid within EPCR Groove

R. Prasad ; P. Sen

Indian Association for the Cultivation of Science, Biological Chemistry, Kolkata, India

Background: Endothelial cell protein C receptor (EPCR), the cellular receptor for protein C/activated protein C (APC), stimulates protein C in presence of thrombin‐thrombomodulin complex and regulates thrombin generation. In patho‐physiological conditions, the interaction between EPCR and APC gets impaired. EPCR contains a phospholipid in the antigen‐binding groove as similar as antigen presenting cell (MHC class I), however the exact role of antigenic lipid within EPCR groove during APC recognition is not explored.

Aims: To identify how lipid contributes for protein‐protein interactions in addition to its major role in the structural stability of EPCR.

Methods: Molecular dynamics (MD) simulation along with potential of mean force (PMF) calculations were performed in order to investigate dynamic analysis and the free energy binding calculations between EPCR and APC in absence and presence of phospholipid viz. lyso‐phosphatidylcholine (lysoPCh) and phosphatidylcholine (PCh) in the antigen‐binding groove of EPCR. The standard errors were estimated calculated from three individual runs of simulation for each system.

Results: Conformational and structural dynamic analyses suggest that PCh system is the most stable as compared to other systems (lysoPCh and no lipid). PMF data reveals that lipid enhances APC‐EPCR interactions. Due to lack of one hydrophobic tail part, LysoPCh has weaker affinity towards APC than PCh. Phe4 of APC plays a crucial role during lipid‐dependent protein complex formation by making hydrophobic interactions.

Conclusions: Our studies for the first time explore the possible mode of ligand (protein C/APC) recognition by EPCR via the involvement of phospholipid (PCh) within its hydrophobic groove. PCh not only maintains the structural integrity of EPCR groove, but also provides a platform for APC binding with its cofactor EPCR. This study emphasizes lipid‐dependent functionality of EPCR in various patho‐physiological abnormalities.

PB362: Retrospective Study about using Bridging Therapy with Sodium Bemiparin in our Center

P. Romero Garcia 1 ; E. Morente Constantin 2 ; M. Rivas Luque 2 ; M.A. Garcia Ruiz 2 ; M.D.P. Garrido Collado 2 ; G. Salas Camacho 2 ; M. Jurado Chacón 2

1 Complejo Asistencial de Soria, Soria, Spain, 2 Hospital Universitario Virgen de las Nieves, Granada, Spain

Background: Bridging therapy (BT) consists of the administration of an anticoagulant such as the low molecular weight heparin (LMWH) during the period of cessation of oral anticoagulant therapy. To start BT we have taken into account three factors: the urgency of surgery or invasive process, the risk of bleeding and thrombotic risk for the patient. There are several types of commercialized LMWH, the sodium bemiparin is the LMWH with greater anti‐Xa/IIa ratio, which implies a lower risk of bleeding.

Aims: There are few published data from BT at therapeutic doses in patients treated with oral anticoagulants (AVK) and perioperative management. It is intended to assess the efficacy and safe use of sodium bemiparin at anticoagulant doses on the BT and possible thrombotic and/or hemorrhagic complications resulting from this use.

Methods: We have analyzed 975 BT at full dose in our clinic in the last year. They were made to a total of 650 patients (315 men and 335 women) with CHADSVASC>2. The BT has consisted on suspending AVK 4 (acenocumarol) to 6 days (warfarin) before the procedure, and replacing it by BT at full doses, and administration of a prophylactic dose 12 hours before and another dose 6‐12 hours after the procedure.

TABLE 1 Sodium Bemiparin Dose Weight Dose <50 kg 5,000 IU/24 h 50‐70 kg 7,500 IU/24 h 70‐100 kg 10,000 IU/24 h >100 kg 12,500 IU/24 h Prophylactic 3,500 IU/24 h

TABLE 2 Episodes of use of the different doses of bemiparin Bemiparin Episodes of use of the different doses of bemiparin 5,000 UI/24 h 95 7,500 UI/24 h 465 10,000 UI/24 h 376 12,500 UI/24 h 39

It has been performed in 225 cases of major surgery, 340 cases of minor surgery, 295 cases of invasive procedures, 50 cases of bleeding caused by AVK, 30 cases of hospitalization with INR decompensation and 35 cases for thrombophilia study.

Results: As complications of using bemiparin sodium, there have been 40 cases of hematomas at the needle puncture sites. There were neither cases of major bleeding nor cases of thrombosis.

Conclusions: Sodium bemiparin administered at therapeutic doses in the perioperative period it is associated with a low incidence of recurrence of VTE and bleeding. In our study, sodium bemiparin has shown to be safe and. Treatment should be administered on an individual basis according to each patient and factors related to surgery. Further studies will confirm our results.

PB364: Betrixaban: Impact on Routine and Specific Coagulation Assays ‐ A Practical Laboratory Guide

R. Siriez 1 ; J. Evrard 1 ; J.‐M. Dogné 1 ; L. Pochet 1 ; D. Gheldof 1 ; B. Chatelain 2 ; C. Vancraeynest 1 ; P. Devel 1 ; M. Guldenpfennig 2 ; C. Devroye 2 ; F. Mullier 2 ; J. Douxfils 2

1 University of Namur, Département de Pharmacie, Namur, Belgium, 2 Université Catholique de Louvain, CHU UCL Namur, Yvoir, Belgium

Background: Betrixaban is a novel oral direct factor Xa inhibitor approved by the Food and Drug Administration for prophylaxis of venous thromboembolism in patients hospitalized for an acute illness at risk for thromboembolic complications. Assessment of the anticoagulant effect of betrixaban may be useful in some situations. Also, clinicians need to know how coagulation assays are influenced.

Aims: To determine which coagulation assay(s) should be used to assess the impact of betrixaban on hemostasis and provide laboratory guidance for their interpretation.

Methods: Betrixaban was spiked at final concentrations ranging from 0 to 250 ng/ml in platelet‐poor plasma. These concentrations cover the on‐therapy range (from ±9 ng/ml at Ctrough to ±122 ng/ml at Cmax for 40 and 120 mg once daily dose). We tested different reagents from several manufacturers and assessed betrixaban impact on routine (PT, aPTT) and more specific coagulation assays (chromogenic anti‐Xa, TGA, DRVVT). We also tested several hemostasis diagnostic tests.

Results: A concentration dependent prolongation of aPTT, PT and dRVVT is observed. The sensitivity mainly depends on the reagent. FXa chromogenic assays show high sensitivity and a linear correlation both depending on the reagent and/or the methodology. Several methodologies applicable for other direct factor Xa inhibitors have to be adapted. TGA may be efficient to assess the pharmacodynamics of betrixaban for low concentration but its turnaround time and the lack of standardization are limitations.

Conclusions: Chromogenic anti‐Xa assays are the most appropriate assays to measure the pharmacodynamics of betrixaban. Betrixaban significantly affects several hemostasis diagnostic tests and this needs to be taken into consideration when requesting and interpreting such tests.

PB365: Preparation and Characterization of Nanocarriers for Transdermal Delivery of Enoxaparin

M. Bhargava 1 ; S. Bhargava 2 ; S. Jain 3 ; V. Bhargava 1 ; N. Kapoor 4

1 GTB Hospital, Kanpur, India, 2 United Institute of Pharmacy, Kanpur, India, 3 Bhagyodaya Tirth Pharmacy College, Sagar, India, 4 KRV Hospitals, Kanpur, India

Background: Low molecular weight heparins (LMWHs) are anticoagulants, which penetrates skin poorly due to its relatively large molecular weight, negative charge & hydrophilic nature. Many techniques have been aimed to disrupt & weaken highly organized lipids in an intact skin among which is the use of drug delivery systems.

Aims: Enoxaparin has till now been given by intravenous or subcutaneous route therapeutically & this project is an attempt to compare potential of delivering of such a high molecular weight, hydrophilic drug through a vesicular carrier ie ethosomes encapsulating enoxaparin system across skin.

Methods: Ethosomes encapsulating enoxaparin were prepared & characterized for average particle size, polydispersity index & percentage drug entrapment. Transmission Electron Microscopy (TEM) of drug loaded vesicles revealed their spherical structure & unilamellarity. In‐vitro release studies showed that there was no burst effect & the percentage drug release increased linearly up to 24 h. The transdermal flux across rat skin was found to be 20.4±1.1 μg/cm2/hr. Stability profile of prepared, optimized system was determined for 120 days, which revealed low drug leakage on storage. In‐vivo studies were performed for presence of nanocarriers (encapsulating Rhodamine B) in skin by fluorescence microscopy.

Results: The results of Fluorescence microscopy are also encouraging in that they clearly indicate the presence of vesicular system in the skin. The results of overall study demonstrated the importance of carrier composition (especially ethanol & oleic acid) on the physicochemical properties, morphology, skin irritation & consequently the effectiveness of vesicular system as a vehicle for transdermal delivery of enoxaparin.

Conclusions: The results were quite encouraging & might be an alternative to injections for topical administration of enoxaparin in case of superficial thrombosis, subcutaneous wounds, bruices & burns. Additional studies should be performed in a clinical setting.

PB366: Inhibition of Factor XII is Antithrombotic in a Primate Model of Extracorporeal Membrane Oxygenation

M. Wallisch 1,2 ; C.U. Lorentz 1,2 ; J. Johnson 1 ; M.R. Carris 1,2 ; O.J.T. McCarty 1 ; M.T. Hinds 1 ; A. Gruber 1,2 ; E.I. Tucker 1,2

1 Oregon Health & Science University, Department of Biomedical Engineering, Portland, United States, 2 Aronora, Inc., Portland, United States

Background: Surface‐initiated thrombus propagation within extracorporeal membrane oxygenation (ECMO) devices are effectively reduced, but not eliminated, by systemic anticoagulation with unfractionated heparin (UFH) in most cases. However, the use of UFH is dose‐restricted due to hemorrhagic safety concerns. Since coagulation factor XII (FXII) supports contact activation of blood but has no known contribution to hemostasis, we sought to determine the thrombogenic role of FXII in a baboon ECMO model utilizing a FXII‐neutralizing monoclonal antibody, AB053.

Aims: Determine the antithrombotic potential of FXII inhibition in a primate ECMO model.

Methods: UFH (20 U/kg) alone or UFH (20 U/kg) plus AB053 (5 mg/kg) were given intravenously to juvenile baboons. Fifteen to 30 minutes later, Terumo CAPIOX RX5 baby oxygenators were deployed for 60 minutes into high flow chronic femoral arteriovenous shunts. Thrombogenesis was monitored using a gamma camera and quantified by measuring radiolabeled platelet and fibrin deposition in the oxygenators. Prothrombin time, bleeding time and volume were used to assess hemostasis.

Results: Using heparin only for anticoagulation during perfusion of the device, platelet accumulation rate averaged 1.2 billion/min, with 60 minutes end‐point deposition of 52±11 billion platelets and 45±22 mg fibrin in the oxygenator (n=4). When compared to heparin, the combination of heparin with AB053 reduced average platelet accumulation rate to 0.6 billion/min, 60 minutes end‐point platelet deposition to 24±5 billion, and fibrin deposition to 15±9 mg (n=3), without affecting hemostasis markers.

Conclusions: Our data suggest that supplementing heparin anticoagulation with selective pharmacological inhibition of contact activation reduces thrombogenesis in membrane oxygenators during perfusion. Therefore, this approach may allow for the reduction of the heparin dose and improve the hemostatic safety of temporal extracorporeal organ support.

PB367: Effects of SERPINC1, PROC, PROS1 and EPCR Polymorphisms on the Risk of Venous Thromboembolism and Myocardial Infarction in Young Individuals

T. Miklós 1 ; G. Balla 1 ; L. Balogh 2 ; Z. Boda 3 ; Z. Szabó 1 ; É. Molnár 1 ; R. Gindele 1 ; É. Katona 1 ; Z. Bereczky 1

1 University of Debrecen, Division of Clinical Laboratory Science, Department of Laboratory Medicine, Debrecen, Hungary, 2 University of Debrecen, Department of Cardiology and Cardiosurgery, Debrecen, Hungary, 3 University of Debrecen, Department of Internal Medicine, Debrecen, Hungary

Background: Antithrombin (AT), protein C (PC) and protein S (PS) are natural anticoagulants and EPCR plays a role in PC activation. Controversial data concerning the effect of their polymorphisms on the risk of venous thromboembolism (VTE) and myocardial infarction (MI) have been published, so far.

Aims: Our aim was to investigate the effects of 12 SNPs (PROC rs1799809, rs1799808, rs1799810, rs2069928, rs1401296, PROCR rs867186, rs6088735, rs8119351, SERPINC1 rs222758, rs121909548, PROS1 rs8178649 and rs121918472) on the risk of VTE and MI in young individuals.

Methods: A multiplex PCR‐primer extension assay was used to detect the SNPs simultaneously in 144 VTE (median age 40; range 19‐49) and in 78 MI (median age 36; range 24‐40) patients and in their matched controls (n=277 for VTE and n=72 for MI). AT activity was measured by FXa‐based assays, PC activity and free PS concentration were measured by chromogenic assay and latex‐immunoturbidimetry, respectively.

Results: The ratio of individuals with positive thrombotic family history and with FV Leiden was higher in the VTE group as compared to controls (P<0.001 for both parameters). The ratio of smokers and patients with hyperlipidemia was higher in the MI group as compared to its control group (P<0.001 for both). AT Cambridge (rs121909548) was absent in our population and rs2227589 had no effect on the risk of VTE and MI. PS Heerlen (rs121918472) decreased free PS markedly (104 vs. 64%, P<0.001), while rs8178649 was without effect on PS levels and on the risk of thrombosis. PC activity was independently increased by PROCR rs8119351 (P=0.004) and decreased by rs6088735 (P=0.005). The PROC rs1401296 increased the risk of VTE by 2.5‐fold (P=0.009), while PROCR rs867186 was protective against VTE in FV Leiden negative patients (OR 0.24, P=0.026) and against MI (OR 0.13, P=0.044).

Conclusions: Our observations suggest that some PROC and PROCR SNPs may play a role not only in venous but also in arterial thrombosis, which is worthy of further investigations.

PB368: Structural and Functional Study of Naringin Octasulfate (NOS) on Human Antithrombin and Probing its Role in Thrombus Reduction using Rat Model

I. Ahmad 1 ; M. Abid 1 ; S. Sharma 2 ; M.Z. Ashraf 2,3 ; M.A. Jairajpuri 1

1 Jamia Millia Islamia (A Central University), Department of Biosciences, Delhi, India, 2 Defence Institute of Physiology & Allied Sciences, Department of Genomics, Delhi, India, 3 Jamia Millia Islamia (A Central University), Department of Biotechnology, Delhi, India

Background: Thrombotic disorders are major contributors to the global disease burden. In such pathologies anticoagulants are the first line of defense, however due to complications the demand is growing for development of safer anticoagulants. Animal models are fundamental in accomplishing new approaches of antithrombotics. The search for novel anticoagulants with better understanding of anticoagulant pharmacology, dosing and toxicity remains a major subject of research.

Aims: To design novel naringin octasulfate (NOS) and evaluate its in vivo antithrombotic potential using rat model.

Methods: Synthesis of NOS was done using triethylamine‐sulfur trioxide and purified by HPLC. Structural modifications were confirmed by FTIR, NMR and Mass spectrometry. The clotting assays APTT, PT and TT were performed to test in vitro efficacy of NOS. Antithrombotic potential of NOS was analyzed using in vivo venous thrombosis model which involves thrombus induction via ligation of inferior vena cava in rats. The ex vivo APTT, PT and various coagulation variables like clot rate (CT), activated clotting time (ACT) and platelet function were measured from NOS treated thrombotic rats. Molecular Docking of NOS with antithrombin (AT) was done using Auto‐dock 4.0. To check secondary and tertiary structural changes on binding of NOS to AT, Far‐UV CD and florescence spectroscopy were performed.

Results: Upon incubation of NOS with human plasma we observed prolonged APTT and PT, however minor delay was observed on TT. The in vivo results showed significant reduction in thrombus size and weight at low dosage of NOS and showed delay in clotting time in plasma of NOS treated rat. Molecular docking results suggested NOS bind to AT with good affinity, an increase in fluorescence intensity of NOS treated AT showed changes in its tertiary structure, while minor secondary structural change was observed in Far‐UV CD spectra.

Conclusions: The results suggest that NOS could be a promising antithrombotic agent.

PB370: Development and Characterization of Neutralizing Monoclonal Antibodies Targeting the Direct Oral Anticoagulant Edoxaban

E. Maurer 1 ; S. Crosnier 1 ; C. Baltus 2 ; M.‐C. Viaud‐Massuard 2 ; P. Ohlmann 1

1 Agro‐Bio, Research and Development, La Ferté Saint‐Aubin, France, 2 University of Tours, Faculty of Pharmacy, UMR 7292 GICC Equipe 4‐Innovation Moleculaire et Therapeutique, Labex SYNORG, Tours, France

Background: Direct oral anticoagulants (DOACs) are indicated for thromboembolism prevention and treatment. Dabigatran is a thrombin inhibitor while apixaban, rivaroxaban, edoxaban and betrixaban are inhibitors of factor Xa (FXa). Novel clinical research tools are required to better understand the mechanisms of action of these new therapeutical molecules.

Aims: The challenge was to generate high specificity and affinity monoclonal antibodies (MAbs) against a chemical structure such as edoxaban.

Methods: Edoxaban molecules were conjugated to carrier proteins to obtained enhanced immune response to such reputed hapten. After immunizations, spleen cells from mice presenting with the highest titers were isolated for fusion. Specific binding of purified MAbs to edoxaban was analyzed in a competition ELISA where antibodies were pre‐incubated with edoxaban before being added to hapten‐carrier conjugate immobilized onto microtiter plates. FXa activity assay was performed on human citrated plasmas spiked with increasing edoxaban concentrations in the presence and in the absence of the selected MAbs. Isotypes and affinity were also determined, respectively by ELISA and by surface plasmon resonance.

Results: We have generated 19 clones, all secreting IgG1 isotype MAbs specific for edoxaban. All MAbs presented with a total absence of reactivity against the other “xabans” as tested by ELISA. Moreover, our anti‐edoxaban MAbs are able to neutralize the in vitro edoxaban anticoagulant effect on STA ® ‐Liquid FXa assay (Stago), at a MAb‐edoxaban molecular ratio of 2:1.

Conclusions: We therefore report the development of various MAbs specific to edoxaban and able to neutralize the anticoagulant effect of the drug. These MAbs could be very useful in diagnostic applications. Further steps are being undertaken to develop recombinant monoclonal antibodies for extended applications in clinical research and pharmacodynamic studies.

PB371: The Effect of JNJ‐64179375 on Coagulation Tests with Different Coagulation Analyzers

T.M. Connolly 1 ; M. Chintala 1 ; G. Peters 1 ; L.K. Jennings 2 ; J.P. Kotha 2

1 Janssen Research, LLC, Spring House, United States, 2 CirQuest Labs, LLC, Memphis, United States

Background: JNJ‐64179375 (JNJ‐9375) is a first‐in‐class, specific, recombinant large molecule (IgG4), exosite 1 thrombin inhibitor. Preclinically JNJ‐9375 demonstrated robust antithrombotic efficacy with an improved therapeutic index over the factor Xa inhibitor apixaban in rats. It is currently in Phase II clinical evaluation.

Aims: The purpose of the present study was to examine the suitability of commercially available clotting assays to measure anticoagulant activity to identify a sensitive and reliable test for pharmacodynamic activity for this novel mechanism of action (MoA) inhibitor.

Methods: Plasma from 12 healthy, consenting volunteers was spiked with 7 concentrations of JNJ‐9375, 0.4‐300 ug/ml, or 300 ug/ml control IgG4, and the effect on 4 blood coagulation tests, prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT) and Ecarin Clotting Time (ECT) was evaluated with 3‐5 different reagents in 3 different coagulation analyzers.

Results: Clotting times in all 4 assays were prolonged by JNJ‐9375 in a concentration dependent manner, PT and aPTT were less sensitive, prolonged 2.4‐fold each at the highest concentration tested. ECT was comparable to PT and aPTT, with similar fold changes versus control IgG with different reagent/equipment combinations. The TT assay was the most sensitive, maximum 7.5‐fold increase; however, unlike in the other assays, the sensitivity of inhibition varied across platforms, with assays on STA‐compact more sensitive than those on ACL Elite and Sysmex instruments.

Conclusions: All 4 coagulation tests were prolonged by exosite I inhibition with JNJ‐9375 in a concentration dependent manner. The TT is the most sensitive assay consistent with JNJ‐9375's MoA. It is the preferred assay; however, different reagent/equipment combinations produced different sensitivity profiles and indicate the importance in the choice of reagent and analyzer when measuring the anticoagulant effect of this novel inhibitor.

PB372: Emicizumab Impact on Factor VIII Inhibitor Determination in Plasma Samples from Persons with Hemophilia A (PwHA) using a New Kit for Modified Nijmegen‐Bethesda Assay (MNBA)

A. Sadeghi‐Khomami 1 ; M. Boylan 1 ; D.C. Chen 2 ; J.I. Adamkewicz 2

1 Precision BioLogic Inc., Dartmouth, Canada, 2 Genentech Inc., South San Francisco, United States

Background: Proficiency studies show discrepancies in measuring FVIII inhibitor titers. Emicizumab, a novel bispecific antibody with tenase restoring effect approved in the US for bleeding prophylaxis in PwHA with FVIII inhibitor, interferes with clotting‐based Bethesda assays. Standardization and improvement of Nijmegen‐Bethesda Assay (NBA) is needed.

Aims: To assess performance of a new MNBA kit for FVIII inhibitor determination in plasma samples from PwHA with and without emicizumab.

Methods: A kit consisting of Imidazole Buffered Bovine Serum Albumin (4% IB‐BSA), IB‐Pooled Normal Plasma (IB‐PNP, FVIII 95‐105%), a negative and a positive FVIII inhibitor control is being developed. Frozen citrated plasma samples were collected from a global phase 3 clinical study (HAVEN 1). Patient consent and approval by a medical ethics committee was obtained. Thirty de‐identified plasma samples from 15 PwHA pre‐ and post‐emicizumab administration were tested. All samples were heat‐deactivated (56°C, 30 minutes), centrifuged, aliquoted and stored at <−70°C before testing. After thawing, test mixtures (1:1 mixture of IB‐PNP with either undiluted or IB‐BSA diluted heat deactivated plasma samples) and control mixtures (1:1 mixture of IB‐PNP:IB‐BSA) were incubated for 2 hours at 37°C followed by 10 minutes incubation on ice. FVIII activities were determined using a bovine‐based FVIII chromogenic assay and a one‐stage clotting assay on a BCS XP analyzer. ISTH‐SSC proposed cutoff (≥0.6 BU) was used for positive inhibitor detection.

Results: A good concordance of inhibitor titers was obtained between pre‐ and post‐administration of emicizumab using chromogenic MNBA. However, the clotting‐based MNBA yielded false negative results in emicizumab‐containing samples (Figure 1). The presence of inhibitors was confirmed by ELISA (Immucor).

FIGURE 1 Comparison between chromogenic versus clotting MNBA in 15 plasma samples from PwHA before and after emicizumab administration

Conclusions: The new MNBA kit shows promise for laboratories seeking a standardized FVIII inhibitor assay and is suitable for use with samples containing emicizumab.

PB373: Profile of Patients with Hemophilia A and Inhibitor Undergoing Immune Tolerance Induction: Preliminary Data from the BrazIT Study

L. Magalhães 1 ; R. Camelo 1 ; L. Jardim 1 ; A. Oliveira 2 ; D. Chaves 2 ; S. Rezende 1

1 Federal University of Minas Gerais, Belo Horizonte, Brazil, 2 Foundation Hemocentro of Minas Gerais, Belo Horizonte, Brazil

Background: About 20‐30% of patients with hemophilia A (PHA) develop antibodies against FVIII (inhibitors), reducing the effectiveness of replacement. Immune tolerance induction (ITI) is the treatment of choice to eradicate inhibitors.

Aims: We aim to evaluate the profile of PHA included in the Brazilian Immune Tolerance (BrazIT) Study.

Methods: PHA were included in the hemophilia treatment centers (HTCs). Collected data included demographic, clinical and laboratory variables (Table 1). ITI protocol used was recommended by the Ministry of Health. All patients started ITI with FVIII low‐dose regimen (50 IU/kg 3×/week). Upon lack of response, high‐dose regimen (100 IU/kg daily) and subsequent changing to plasma‐derived (pd) FVIII for those who started on recombinant FVIII (rFVIII) was recommended. Response was based on inhibitor titer, FVIII half‐life and recovery. Continuous variables are expressed in median and interquartile range (IQR) and categorical variables, in numbers and percentages.

Results: We present preliminary data from one HTC. A total of 27 PHA were included (Table 1), of whom 18 (66.7%) completed the protocol. One (3.7%) patient died during ITI. Among the ones who completed the protocol, 14 (77.8%) responded to ITI (5 total and 9 partial success) and 4 (22.2%) failed. A total of 7/27 (25.9%) PHA started ITI with rFVIII, of whom 1/7 (14.3%) changed to pdFVIII due to lack of response with the former. Median time elapsed between start of ITI and first negative inhibitor and start of ITI and normal FVIII half‐life/recovery in PHA who responded were 8.6 [IQR 3.2‐20.2] and 14.9 [IQR 10.8‐31.4] months, respectively. Intron 1 and 22 inversions were present in 13/27 (48.1%) patients.

TABLE 1 Baseline characteristics of the patients included in the study (n=27) Characteristics Results Results Male sex, n (%) 27 (100.0) FVIII activity <2%, n (%) 23 (85.1) Age at hemophilia diagnosis in years, median (IQR) IQR‐interquartile range 0.8 (0.6‐1.4) Age at inhibitor diagnosis in years, median (IQR) 2.3 (1.3‐7.9) Age at ITI start in years, median (IQR) ITI‐immune tolerance induction 19.5 (9.2‐29.8) Time elapsed between inhibitor diagnosis and ITI start in years, median (IQR) 12.4 (3.7‐22.1) Pre‐ITI inhibitor titer peak in BU/ml, median (IQR) BU‐Bethesda unit 44.8 (24.0‐94.5) Inhibitor titer immediately before ITI start in BU/ml, median (IQR) 5.2 (3.1‐7.6) Total bleeding events during ITI, median (IQR) 12.5 (7.0‐25.3)

Conclusions: Brazilian ITI protocol resulted in about 78% success rate. Most patients had FVIII levels <2%, started ITI in adult age, had more than 10 years elapsed between inhibitor detection and start of ITI and underwent ITI with pdFVIII. All patients started ITI when inhibitor titer was below 10 BU/ml.

PB374: Development of the First Point‐of‐Care Test Selective of the Direct Oral Anticoagulant Apixaban for Emergency Assessment of Coagulation

P. Ohlmann 1 ; E. Maurer 1 ; S. Crosnier 1 ; C. Baltus 2 ; M.‐C. Viaud 2 ; E. Oge 3 ; N. Hezard 3 ; P. Suchon 3 ; P.‐E. Morange 3

1 Agro‐Bio/Stago, Research & Development, La Ferté Saint‐Aubin, France, 2 Université, Faculté de Pharmacie, UMR 7292 GICC Equipe 4‐Innovation Molecular & Cellular, Tours, France, 3 Hôpital de la Timone, Service d'Hématologie Biologique, Marseille, France

Background: Direct oral anticoagulants (DOACs) are being increasingly prescribed. Consequently, more DOAC‐treated patients are admitted to emergency units across all clinical specialities. Importantly, fast and reliable emergency coagulation testing has not been solved yet because specific point‐of‐care test (POCT) are not currently available.

Aims: The challenge was to develop a specific and semi‐quantitative POCT on whole blood within 5 to 15 minutes using an optical reader. The proof of concept was first applied on targeting Apixaban a selective factor Xa inhibitor.

Methods: The POCT was based on a lateral flow immunoassay (LFIA) using monoclonal antibodies (MAbs) against the Apixaban molecules which were recently developed in our lab. Three different clones (8C9, 9D9, 3C1), ranging affinity (KD) respectively from 2 to 140 nM, were tested in two models (competition and inhibition). The selectivity and the sensitivity were evaluated on spiked solutions (saline, plasma, whole blood) with different concentrations of Apixaban (0, 10, 30, 250 ng/ml) and on plasma from patients treated with Apixaban, Rivaroxaban, Heparins (UNF, LMWH) and Fondaparinux. After 15 minutes running, results were obtained using an optical reader (Skannex, Norway).

Results: The sensitivity obtained on spiked solutions has shown a nice linear correlation between the Apixaban concentration from 0 to 250 ng/ml and the optical density (OD) in a range from 6.5 to 3.5 respectively. These results were confirmed in plasma from Apixaban treated‐patient (n=20). The selectivity was also checked on plasma from patients (n=7) treated with Rivaroxaban, UNF, LMWH and Fondaparinux and gave the same response in OD than the control, which clearly show no cross‐reactivity.

Conclusions: These first results confirmed that the POCT targeting Apixaban is selective with a good tendency for semi‐quantitative diagnostic and could be a fast and reliable alternative for guiding emergency treatment during Apixaban therapy.

PB375: Data of Real‐life Anticoagulant Treatment with Edoxaban In Patients with Atrial Fibrillation and Patients with Secondary Prevention of Deep Vein Thrombosis. Experience of Thirteen Months in our Center

A. Garcia Ruiz 1 ; M. Rivas Luque 1 ; P. Romero Garcia 2 ; E. Morente Constantín 1 ; M.P. Garrido Collado 1 ; M. Jurado Chacon 1

1 Hospital Universitario Virgen de las Nieves, Granada, Spain, 2 Complejo Asistencial de Soria, Granada, Spain

Background: Atrial fibrillation (AF) is one of the most common disturbance of cardiac rhythm in adults. In patients with AF, the risk of stroke is higher according to the stroke risk stratification scheme CHA2DS2‐VASc. Edoxaban (Lixiana ® , Daiichi‐Sankyo) is an oral, reversible, direct factor Xa inhibitor. The dose edoxaban regimen consisted of 60 mg (QD), reduced to 30 mg (QD) as indicated on the Technical Sheet.

Aims: Primary objective: Evaluate the efficacy of Edoxaban in the prevention of stroke and Embolism Systemic.

Secondary objective: Major bleeding defined according to the criteria of the International Society of Thrombosis and Hemostasis (ISTH).

Methods: This is a descriptive study. We have included a total of 211 patients with Non‐valvular Atrial Fibrillation and Deep Vein Thrombosis. The indication for anticoagulation was NVAF with 199 cases and 12 patients for secondary prevention of DVT. The risk of stroke is assessed according to the scale CHA2DS2‐VASc (Mean 3.74).

TABLE 1 CHA2DS2‐VASc Total population CHA2DS2‐VASc Number of Patients 1 1 2 40 3 68 4 45 5 27 6 22 7 7 8 1

Risk of bleeding according to the scale HAS‐BLED (Mean 3.01).

TABLE 2 HAS‐BLED total population HAS‐BLED Number of Patients 1 1 2 56 3 112 4 30 5 9 6 2 7 0 8 0 9 1

The dose reduction was in the 22.7%. Patient features was: 51.66% women, aged between 42 and 95 years old (mean 72.5). All patients had received prior treatment with Acenocoumarol. Reason of suspension of Acenocumarol was: poor control of INR with TRT<65% (199 patients) and diathesis bleeding with Acenocumarol (12 patients). 41 patients had presented a previous stroke under treatment with Acenocumarol. The treatment time has been between 3 and 13 months, with an average of 7.65 months of follow‐up.

Results: It has not been reported any case of Stroke, Systemic Embolism, or Major Bleeding (ISTH criteria) during the follow‐up time of the patients treated. Only one case of non‐major clinically relevant bleeding (rectal bleed).

Conclusions: The results obtained confirm the efficacy and safety of Edoxaban in the prevention of stroke in the NVAF and in the few cases of DVT. The results of our study support the results of the trials. We believe that Edoxaban there is a good alternative.

PB376: Cross‐sectional Survey of Minor Bleeding Rates in Children Prescribed Warfarin

N. Poci ; C. Attard; F. Newall

Murdoch Children's Research Institute, Haematology Research Unit, Parkville, Australia

Background: Warfarin is one of the most commonly prescribed anticoagulant medications in paediatric populations for the prevention and treatment of thromboembolic events. Major bleeding rates associated with warfarin therapy in children have been widely reported, however the rate of warfarin‐related minor bleeding is seldom studied and poorly defined. The emergence of the novel anticoagulants makes determining this information invaluable to enable optimal clinical evaluation of new agents to existing anticoagulant therapies.

Aims: To assess the type, severity and incidence of minor bleeding events in a population of children (<18 years) requiring warfarin therapy.

Methods: The patient population was selected from the warfarin management registry of the Clinical Haematology service of The Royal Children's Hospital (Melbourne, Australia) and are current receiving warfarin. Data was collected regarding demographics, minor bleeding events (e.g. epistaxis, minor gastrointestinal bleeding, menorrhagia, ecchymoses) using the online Warfarin/Aspirin Bleeding Assessment Tool (WA‐BAT), which has undergone extensive validity testing.

Results: As of February 2018, after only 2 weeks of data collection, we have received 6 respondents of which over half reported significant minor bleeding events. Throughout our second phase of participant recruitment, we hope to establish at least 60 respondents from a pool of 200 patients on the registry.

Conclusions: This study is the first of its kind to solely assess the risk of minor bleeding in paediatric patients on warfarin therapy. Though we are still collecting data, the information we have already received has indicated that there are considerable risks of minor bleeding in this patient population. This data will prove vital for the ongoing prescribing of warfarin in the paediatric setting, as well as guide future therapy as our choices expand.

PB377: Antithrombin Levels in Nigerians with Type2 Diabetes

O.A. Awodu 1,2 ; I. Ezenwenyi 3

1 University of Benin Teaching Hospital, Haematology & Blood Transfusion, Benin City, Nigeria, 2 University of Benin, Haematology, Benin City, Nigeria, 3 Federal Medical Center GombeTeaching Hospital, Haematology and Blood Transfusion, Abakaliki, Nigeria

Background: Antithrombin is a glycoprotein that functions as a potent natural anticoagulant and is estimated to provide 80% of the inhibitory activity against thrombin. Low level of Antithrombin has been reported in Caucasians with diabetes mellitus. Hypercoagulability has been reported in diabetes mellitus and over 80% of diabetic patients die from thrombotic complications, cerebrovascular events and peripheral vascular complications.

Aims: To determine Antithrombin (AT) levels in type 2 diabetics and to determine its association with vascular complications.

Methods: This is a hospital based case control study, including 104 diabetics on therapy, 104 therapy naïve diabetics and 54 controls. Antithrombin activity was assayed using a standard chromogenic method withTechnochrome AT kit (Xa inhibition method) and AT III antigen level was determined with ELISA kit (ASSAYPRO Lot No.061371506). Haematological parameters were analyzed using ERMA haemato‐autoanalyzer.

Results: The mean AT activity levels in the study subjects were 79.00%, 83.31±30.00%, and 92.82±20.02% in the therapy naïve diabetics; diabetics on therapy and controls respectively. The difference in means between the three groups was statistically significant (P value=0.023). However; between the two diabetic groups the difference in means was not statistically significant (P value=0.085). No significant association was found between AT deficiencies and vascular complications.

Conclusions: This study showed that Antithrombin level/activity is lower in Nigerians with type 2 diabetics’ compared to healthy controls, however unlike previous reports, no association was found between low Antithrombin levels and vascular complications.

PB378: Laboratory Algorithm of Diagnostic of Hemorrhagic Diseases in Patients with Isolated Prolonged Activated Partial Thromboplastin Time (APTT)

V. Krasivska ; O. Stasyshyn

State Institution ‘Institute of Blood Pathology and Transfusion Medicine under the National Academy of Medical Sciences of Ukraine’, Lviv, Ukraine

Background: In clinical practice there are cases when patients with hemorrhagic syndrome have only an increase in APTT.

Aims: The purpose of the study was to establish the causes of haemorrhagic syndrome in patients and develop the laboratory diagnostics algorithm of haemostasis by two groups of mix tests.

Methods: 61 patients with haemorrhagic syndrome underwent screening tests. The research includes patients with isolated prolonged APTT. Two groups of correctional tests were carried out, which were based on APTT: mix‐ test with the normal, deficient factor FVIII and deficient FIX plasmas separately, and screening incubation test on inhibitors.

Results: Among 61 patients 34.4% were diagnosed with haemophilia A, 11.5% with haemophilia B, 13.1% with haemophilia A with inhibitor, 6.5% with acquired haemophilia A, 1.6% with acquired haemophilia B, 4.9% with von Willebrand disease and 27.9% with lupus anticoagulant (LA). After normalization of APTT with the deficient FVIII plasma one should proceed assay of FIX and after normalization with the deficient FIX plasma ‐ with the assay of FVIII and vWF:Rco. In case of correction of all the interchangeable probes it's vital to indicate the FXI activity. In case of correction in APTT and don't see the difference between the clotting time of mixture 1 and mixture 2 in second group of correctional tests, inhibitor to clotting factors are absent. Absence of correction in mixture 1 and mixture 2 is shows the influence of the immediate inhibitor. Further differential diagnostics is based on LA identification. In case LA is negative it is advisable to proceed with the assay FVIII, FIX, FX. If the level of one of the factors is low it is necessary to identify the quantity of inhibitor. Inhibitor titre >0.6 BU/ml indicates the presence of acquired immune coagulopathy.

Conclusions: The algorithm allows to diagnose the deficit of factors, differentiatiate of the progressive alloimmune inhibitor with immediate autoimmune inhibitor and a decrease in the time of diagnosis specification.

PB379: From Tissue Factor to Thrombin to Fibrin under Whole Blood Flow: Thrombin Makes Fibrin but Fibrin Quenches Thrombin

S. Diamond

University of Pennsylvania, Institute for Medicine and Engineering, Philadelphia, United States

Background: The co‐regulation of how much thrombin and fibrin is formed as blood flows over a procoagulant surface is essential to both thrombosis and hemostasis.

Aims: We sought to determine what regulates the amount of thrombin and fibrin formed in a clotting event under hemodynamic flow conditions.

Methods: Using microfluidic perfusion of CTI‐treated human whole blood (200 per seconds wall shear rate) over collagen/tissue factor (˜1 TF molecule/mm 2 ) and immunoassays for fragment 1.2 (F1.2), thrombin‐antithrombin (TAT) and D‐dimer (post‐plasmin lysis), we sought to establish the transient mass balance for clotting under venous flow.

Results: F1.2 (but not free thrombin) continually eluted from clots when fibrin was allowed to form. Low dose FITC‐PPACK stained fibrin‐bound thrombin, a staining blocked by either anti‐gamma’‐fibrinogen or fibrin polymerization inhibitor GPRP. FITC‐PPACK staining of fibrin‐bound thrombin was highly resistant to 3‐min buffer wash out, indicative of tight binding of thrombin to gamma’‐fibrin. With fibrin polymerizing for 500 seconds, 92,000 thrombin molecules and 360,000 clot‐associated fibrin monomer equivalents were generated per TF molecule. Fibrin reached 15 mg/ml in the pore space of a 15‐micron thick thrombus core by 500 seconds (and 30 mg/ml by 800 seconds). For known turnover number kcat=50 FPA per thrombin per sec, the measurement of each thrombin molecule generating only 4 fibrin monomer equivalents was consistent with an intra‐clot thrombin half‐life <100 msec, much shorter than the diffusional escape time of ˜1‐10 seconds from the clot. GPRP allowed 4‐fold more F1.2 generation, consistent with GPRP ablating fibrin antithrombin‐I activity and thus facilitating late‐stage thrombin‐mediated Factor XIa activation.

Conclusions: Under flow, gamma’‐fibrin was identified as the essential regulator of thrombin localization and clot formation. Most thrombin is captured by fibrin within 100 msec, thus restricting clot growth.

PB380: Effect of Andexanet‐tissue Factor Pathway Inhibitor (TFPI) Interaction on in vitro Clot Formation and Lysis via Different Coagulation Pathways

G. Lu ; J. Lin; J. Curnutte; P. Conley

Portola Pharmaceuticals, Inc., San Francisco, United States

Background: Andexanet (AnXa) is a modified, recombinant human factor Xa (FXa) being developed as a specific reversal agent for all of the FXa inhibitors. AnXa retained high binding affinity to FXa inhibitors, including TFPI. We characterized the differential effect of AnXa on clot formation and lysis via different coagulation pathways in the context of recombinant tissue plasminogen activator (rtPA).

Aims: To characterize the effect of AnXa‐TFPI interaction on clot formation and lysis via the extrinsic, intrinsic or the common pathway.

Methods: Clot formation and lysis were monitored by a turbidity assay (ΔA405 nm in a 96‐well plate at 37°C for up to 3 hours, and optimized based on thrombin generation with each activator). Clotting time (CT, ascending) and lysis time (LT, descending) were calculated at the half‐maximum absorbance, respectively. Human plasma (80 μl) with AnXa (0, 0.5, 1, 2, 4 μM) and rtPA (0, 150, 300 ng/ml) was mixed with 20 μl activator containing TF (1 pM), aPTT reagent (Actin FS,1:240 dilution), or RVV‐X (2.4 pM)+phospholipids (4 μM). The reaction was initiated by addition of 20 μl Ca 2+ (8 mM). Corn trypsin inhibitor (50 μg/ml) was used to block contact activation in TF‐ or RVV‐X‐initiated reactions.

Results: Compared to control plasma, AnXa (0, 0.5, 1, 2, 4 μM) shortened CT by 0, 34, 29, 20, and 5%, respectively with TF due to the effect of high affinity AnXa‐TFPI interaction on the TF‐pathway. But, AnXa dose‐dependently prolonged CT (max ˜30% at 4.0 μM) with Actin FS or RVV‐X due to weak interactions with other (co)factors (e.g., FVa) by AnXa (a modified FXa) at high concentrations. The AnXa effect on CT could be further compensated by the fibrinolytic activity of tPA; perturbation of LT by AnXa was only observed at lower rtPA concentration (150 ng/ml, ˜1 nM).

Conclusions: The differential effect of AnXa on CT and LT via three coagulation pathways showed that the AnXa‐TFPI interaction in the TF‐pathway could be compensated in the presence of rtPA.

PB381: Anti‐factor Xa and Anti‐thrombin Antibodies Have Differential Effects upon Coagulation and Complement Regulation ‐ The Role of Antithrombin

T. McDonnell 1 ; C. Wincup 1 ; V.M. Ripoll 1 ; C. Gerveshi 2 ; A. Rahman 1 ; I. Mackie 1 ; M. Botto 3 ; I. Giles 1

1 University College London, London, United Kingdom, 2 Drexel University, Philadelphia, United States, 3 Imperial College London, London, United Kingdom

Background: Coagulation and complement pathways are enzymatic cascades of serine proteases (SP) that are increasingly recognised to interact. These interactions may be important in Systemic Lupus Erythematosus (SLE) and Antiphospholipid Syndrome (APS) that are characterised by activation of both pathways. Given that ˜40% of patients with SLE and/or APS have anti‐SP IgG we hypothesised that anti‐Factor (F)Xa and anti‐Thrombin (Thr) IgG may regulate FXa and Thr mediated activation of complement and prevent regulation by Antithrombin.

Aims: We aimed to examine whether anti‐FXa and anti‐Thr IgG reduce the inhibitory effects of Antithrombin (AT) on FXa and Thr mediated C3 activation.

Methods: Anti‐FXa and anti‐Thr IgG were affinity‐purified from patients with SLE (n=10) and/or APS (n=2). FXa and Thr were incubated with increasing doses of AT +/− C3 and +/− affinity purified IgG and C3 cleavage was measured by immunoblot.

Results: AT inhibited FXa and Thr mediated C3 cleavage. This effect was maintained at high concentration for Thrombin (up to 10:1 ratio) but maximised at 2:1 for FXa and was lost at higher ratios (5:1, 10:1). Anti‐FXa IgG (n=2) abrogated the effect of AT upon FXa‐C3 activation, whilst anti‐Thr IgG (n=1) had no effect upon AT inhibition of Thr‐C3 activation.

Conclusions: We found that AT differentially regulates FXa and Thr mediated C3 cleavage. We also found SLE and/or APS affinity purified anti‐FXa and anti‐Thr IgG have differential effects upon AT inhibition of FXa and Thr mediated C3 cleavage. Further experiments are now underway to increase our understanding of how the impact of these anti‐SP IgG upon coagulation‐complement interactions may be important in the pathogenesis of SLE and/or APS.

PB382: Molecular Sequencing of Factor VII Variants through Collaborative Advocacy for Families in the United States: 154 Individuals Using Multi‐gene Next Generation Panel

D. Nugent 1,2 ; S. Williams 3 ; D. Diaz 3 ; J. Hoang 3 ; K. Imfeld 3 ; V. Rajan 3 ; J. Brewer 4

1 CHOC Childrens Hospital University of California Irvine, Hematology, Orange, United States, 2 Center for Inherited Blood Disorders, Orange, United States, 3 CHOC Childrens Hospital University of California Irvine, Orange, United States, 4 Comprehensive Health Education Services, Hansen, United States

Background: Factor VII deficiency is a challenging syndrome as the factor activity assays do not always correlate with the bleeding phenotype. Molecular analysis and variant mapping is proving useful in our understanding of which mutations may decrease FVII levels versus those that have an impact on FVII function as well as protein expression.

Aims: To further our research on this and other rare blood disorders, the Factor VII Retreat organized by Comprehensive Health and Education Services (CHES) held this year offered molecular evaluation for each patient and all other family members wishing to collaborate with Center for Inherited Blood Disorders (CIBD) and the Hematology Advanced Diagnostic Laboratory (HADL).

Methods: During the 2‐day FVII Retreat, consent forms, bleeding questionnaires and blood samples were obtained. A total of 154 individuals were evaluated comprising : 58 patients, 48 families with index patients, 41 related siblings or offspring, 48 parents, and 4 grandparents. Factor levels derived from time of diagnosis, and whole gene sequencing from HADL, using their validated Next Generation Sequencing (NGS) whole gene clotting protein panel which contains FVII along with 21 additional clotting protein genes.

Results: Variants were identified in 55 patients and 67 relatives. Only 3 patients had no Factor VII variants identified. There were 48 different variants detected in the Factor VII gene, including 24 missense, 5 synonymous, 9 intronic in splice site regions, 4 stop gains, 1 start loss and 5 deletions including a frameshift. Of those variants, 15 were not listed with the NCBI databank or FVII Registry. Family studies proved highly informative in mapping the pathologic lesions to the functional regions of the FVII molecule.

Conclusions: It is critically important to evaluate the patient AND family members to determine which molecular variants are pathogenic in any inherited disorder, especially in determining the function of FVII and the linkages between molecular variants and bleeding phenotype.

PB383: Inappropriate Use of Fresh Frozen Plasma in a Tertiary Care Hospital

S.A. Ali ; M. Mirza; W. Noman

Patel Hospital, Laboratory, Karachi, Pakistan

Background: Fresh frozen plasma (FFP) is a blood component prepared from whole blood by centrifugation or through apheresis, which is frozen within defined time limits for adequate preservation of clotting factors. The main indication of FFP transfusion is to correct clotting factors deficiencies (for which there is no specific concentrate) and reversal of vitamin K antagonist. However high rates of inappropriate transfusion are commonly observed due to non‐compliance with established guidelines.

Aims: To determine the indications of FFP transfusion and to assess appropriateness of their dosage as defined by BCSH guidelines.

Methods: This was a retrospective review of data. Clinical charts of patient receiving FFP from June 2017‐Dec 2017 were reviewed. Indications, dosage and coagulation profile of patients were recorded. The principle clinical audit standards that were applied to this preliminary project were adopted from BCSH guidelines.

Results: During the study period, total 624 FFPs were arranged; out of which 402 units were transfused to 149 patients. There were 83 (55.7%) males and 66 (44.3%) females. Majority of patients were of paediatric age group (n=90, 60.4%) with mean age of 5.21±1.9 days while 59 (39.6%) were adults having mean age of 38.32±4.7 years. The most common unit requesting FFP was neonatal ICU from where 84 (56.3%) requests were generated followed by general ICU (23.4%, n=35). Main indications of transfusing FFP are tabulated in Table 1.

TABLE 1 Indications of FFP transfusion no. of cases % A With active bleeding i DIC 25 16.7 ii Massive hemoorrhage 7 4.69 iii Coagulation factor defect 5 3.35 iv Reversal of warfarin 4 2.68 B Without active bleeding i Reversal of prolonged INR (other than surgical causes) 52 34.8 ii Pre‐procedure correction of INR 27 18.1 iii Intraoperative intravascular volume expander 11 7.38 iv Others 18 12.08

Weight based dosage (15 ml/kg) was mainly followed in paediatric population (60.4%, n=90). 8.7% (n=13) cases had no record of pre‐transfusion coagulation profile. Only in 41 (27.5%) cases, FFP transfusion was in accordance with BCSH guidelines. No transfusion reactions were recorded from the products released in the study duration.

Conclusions: The audit shows poor compliance with the standards. We recommend that hospital should have specialty specific guidelines for the use of FFP. Regular audits of compliance with local and international guidelines must be performed.

PB384: Effects of Thrombin on Complement Activation

M. Matsushita

Tokai University, Hiratsuka, Japan

Background: The complement system is an immune effector mechanism consisting of many plasma proteins. It is activated in three ways, the classical, lectin and alternative pathways. Once complement is activated, a chain reaction of cleavage and assembly of complement components occurs, leading to destruction of pathogens. The complement system and the coagulation system crosstalk to each other. Thrombin has been shown to cleave C3 to generate C3a and C3b, the former of which has a role in inflammation. C3b is a central intermediate molecule in the complement cascade.

Aims: We aimed to elucidate the effects of thrombin on complement activation via the three activation pathways and underlying mechanisms.

Methods: The cleavage of human C3 by thrombin was examined by SDS‐PAGE. The hemolytic assay for measuring complement activation via the classical, alternative and lectin pathways was performed using sheep erythrocytes coated with antibodies, rabbit erythrocytes and sheep erythrocytes coated with mannan, respectively. The effects of human thrombin on complement activation were examined by the hemolytic assay in which the three types of erythrocytes were incubated with human serum in the presence of thrombin.

Results: Thrombin cleaved C3 to generate C3a and C3b, confirming previous reports. To examine whether thrombin affects complement activation via the three activation pathways, the three types of erythrocytes were incubated with human serum and hemolysis was determined. Of the three types of erythrocytes, only the rate of hemolysis of rabbit erythrocytes increased in the presence of thrombin, indicating that thrombin enhanced alternative pathway activation. When rabbit erythrocytes had been pretreated with C3 and thrombin, the rate of hemolysis increased compared with non‐treated rabbit erythrocytes.

Conclusions: Thrombin cleaves C3 to generate C3b, which initiates the activation of the alternative pathway on a solid phase.

PB385: Clot Waveform Analysis: Determination of Optimal Wavelength to Assess the Fibrin Coagulation Process

J. Evrard 1 ; R. Siriez 1 ; C. Bouvy 2 ; J. Laloy 1 ; F. Mullier 3 ; J.‐M. Dogné 1 ; J. Douxfils 1,2

1 Université de Namur, Department of Pharmacy, Namur, Belgium, 2 Université de Namur, Qualiblood s.a., Namur, Belgium, 3 Université Catholique de Louvain, CHU UCL Namur, Yvoir, Belgium

Background: The principle of clot waveform analysis (CWA) is used to measure and record the time required for a plasma to clot. In the literature, different wavelength (340 nm, 635 nm or 671 nm) are reported for CWA. The choice of wavelength for detection is to the sensitivity of the assay, but the question was not dealt in the previous SSC report dated from 2013.

Aims: To define which wavelength provide the best signal‐to‐noise to monitor fibrin clot formation.

Methods: Clot formation was monitored at wavelength from 280 to 700 nm (by step of 1 nm on a TECAN analyser) to provide absorbance spectrum of non‐clotted and clotted plasma. Normal pooled plasma (NPP) was incubated with an inducer of the intrinsic pathway of the coagulation (Actin FS ® ; Siemens) or inducers of the extrinsic pathway (PPP reagent ® or PPP reagent Low ® ; Thrombinoscope BV). Briefly, 20 μl of intermediate reagent were mixed with 80 μl of NPP in a 96‐well microtiter plate and incubated for 3 minutes at 37°C. For clotted plasma, the coagulation process was triggered by the addition of 20 μl of CaCl2 while 20 μl of physiological saline was added in non‐clotted plasma to respect the volume of the reaction. The signal‐to‐noise between the 2 conditions was then calculated.

Results: Clotted samples had higher absorbance value than non‐clotted samples. While absorbance value of clotted and non‐clotted samples showed higher absorbance values at 340 nm than 635 or 671 nm (Figure 1), the signal‐to‐noise ratio demonstrated higher value at 635 or 671 nm due to very low absorbance of non‐clotted samples at 635 and 671 nm. Of note that the signal‐to‐noise ratio is higher with inducers of the extrinsic pathway of the coagulation.

FIGURE 1 Absorbance spectrum of non‐clotted and clotted plasma. Different reagents were used to trigger the coagulation process

Conclusions: This study confirms that the choice of the wavelength is crucial for CWA. 635 or 671 nm are preferable wavelengths to assess fibrin clot formation. 340 nm is affected by individuals’ characteristics (e.g. bilirubin) and should be avoided.

PB386: Role of Phospholipids in Modulating Tissue Factor‐FactorVIIa Complex Activity

P. Sen

Indian Association for the Cultivation of Science, Biological Chemistry, Kolkata, India

Background: Tissue Factor‐FVIIa (TF‐FVIIa) activity is highly guided by phospholipid (PL), precisely phosphatidylserine (PS). Moreover little amount of phosphatidylethanolamine (PE) synergizes with PS to enhance TF‐FVIIa activity. Molecular level residue wise lipid‐protein interactions and the mechanism involved in enhancing the TF‐FVIIa activity are not fully understood.

Aims: To identify how lipid modulates TF‐FVIIa activity.

Methods: Through MD simulations, we have identified the interactions between Protein and lipids. We have synthesized different modified phosphatidylserine along with the native one. We made relipidated TF with different modified lipid and measured comparative procoagulant activity by FXa generation assay to identify the contributions of the functional groups of PS. We also prepared different PL to find the reasons behind the synergic effect of PE. Through MD simulation, we tried to find the alterations imparted by PS at the catalytic domain of TF‐FVIIa.

Results: In PS, apart from phosphate, the head group and the glycerol moiety actively participate to interact with Gla domain of FVIIa as well as the membrane proximal residues of TF. Through MD and biochemically, we established that the amine group forms the H‐bond whereas the carboxylate group may form H as well as the ionic bond. Biochemically we showed that any H‐bond forming group can synergize with PS, irrespective of having phosphate group. Through MD simulation, we found that the protein interactions with PS allosterically modulate the structure of protease domain of FVIIa in complex.

Conclusions: We have provided a molecular level mechanistic approach that how phospholipid regulates TF‐FVIIa activity. Adopting both MD and functional group replacement approach, we elucidate the interaction mode of individual functionalities of lipids in regulating the activity of TF‐FVIIa.

PB387: Coagulation Abnormalities and Risk Assessment in Acute Promyelocytic Leukemia ‐ An Experience fom Resource Constraint Country

N. Anwar ; N. Fatima; J. Hassan; A. Naz; T. Shamsi

National Institute of Blood Disease & Bone Marrow Transplantation, Karachi, Pakistan

Background: Acute promyelocytic leukemia (APML) is associated with coagulation abnormalities. Prompt diagnosis and initial risk assessment is mandatory which can prevent the grave complications. Inspite of diagnostic and treatment advances; it still remains a challenge to accurately predict the relative risk of adverse outcomes. Moreover there is scarce local data and there is limited availability of extended coagulation parameters in developing countries like Pakistan. Inspite of all the reservations literature still emphasizes the importance of supportive care measures in patients of APML with coagulation abnormalities along with chemotherapy.

Aims: The objective of the study was to assess the impact of coagulopathy in risk stratified APML patients presented with bleeding.

Methods: This is an observational analytical study, conducted at National Institute of blood disease Karachi. After taking consent diagnosed APML patients of all age group and either gender were enrolled during the years 2013‐2017. Predesigned study questionnaire was filled by the patients. Statistical analysis was done by using SPSS version 23.

Results: A Total of 125 patients were enrolled. Thirty one patients were recruited for analysis because of presence of coagulation profile with median age of 32 (3‐74) and male predominance (55%). As per Sanz score 70% patients were classified as intermediate and high risk. Bleeding history was present in 71% of patients. At baseline 77%, 61%, 83% and 28% of patients had raised PT, APTT, d‐dimer and low fibrinogen level respectively. Inferential statistics revealed significant association of bleeding history and risk stratification with coagulopathy (P‐value 0.000).

Conclusions: A significant association in risk stratified APML patients with coagulopathy was found presented with bleeding. This study suggests that the coagulation profile must be done at diagnosis in APML patients irrespective of clinical symptoms of bleeding. There is a need to conduct future prospective studies in this aspect.

PB389: eNOS, ACE AND PAI‐1 Gene Polymorphisms in a Sample of General Population of Northern Greece

A. Papoutsi 1 ; M. Koutsounida 1 ; A. Pantelios 1 ; A. Pehlivanis 1 ; N. Antou 1 ; H. Gioukaki 1 ; E. Karaoulis 1 ; M. Mantzafouli 1 ; K. Varsamidis 2

1 Alexander Technological Educational Institute of Thessaloniki (ATEITh), Medical Laboratory Studies, Thessaloniki, Greece, 2 Alexander Technological Educational Institute of Thessaloniki (ATEITh), Physiotherapy, Thessaloniki, Greece

Background: One of the endothelial nitric oxide synthase ( eNOS ) gene polymorphisms which attracted the interest of many researchers is 27VNTR in intron 4, a random repeat of 27 nucleotides which related to cardiovascular diseases. An insertion or deletion (I/D) polymorphism in the angiotensin converting enzyme ( ACE ) gene and a 4/5‐guanine tract polymorphism (4G/5G) in the promoter region of the plasminogen activator inhibitor‐1 (PAI‐1) gene are associated with the plasma activities of these substances and with coronary heart disease. These three genetic variations have been related to various vascular diseases as well as to pregnancy‐related disorders.

Aims: This study was conducted to investigate the prevalence of common polymorphisms in eNOS, ACE and PAI‐1 genes among healthy individuals originated mostly from Northern Greece.

Methods: A total of 95 people (63 women and 32 men) were subjected to the study, with a mean age range of 29 years. The blood samples were collected during a voluntary blood donation held at ATEITh and written consent was obtained from all participants. eNOS (4a/4b) and ACE (I/D) polymorphisms were determined by PCR analysis, while PAI‐1 4G/5G genotype was established for each subject by PCR‐RFLP analysis.

Results: The results concerning the NOS3 , ACE and PAI‐1 polymorphisms are given in Table 1.

TABLE 1 Prevalence of eNOS, ACE and PAI‐1 gene polymorphisms among healthy individuals from Northern Greece Study group (n=95) eNOS genotypes ACE genotypes PAI‐1 genotypes 4b/4b 4a/4b 4a/4a I/I I/D D/D 4G/4G 4G/5G 5G/5G n 65 25 5 11 40 44 28 53 14 % 68.4 26.3 5.3 11.6 42.1 46.3 29.5 55.8 14.7 Allele 4b allele: 0.82 I allele: 0.33 4G allele: 0.57 frequencies 4a allele: 0.18 D allele: 0.67 5G allele: 0.43

Conclusions: As far as eNOS4a/b polymorphism concerns, the data of our study are in accordance with these published from other countries except these for the black race (4a/4a genotype is elevated). We determined a relatively high percentage of DD genotype, which is associated with the development of thrombophilic diseases. The 4G/4G genotype for the PAI‐I gene appears also elevated (29.5%), especially in compare to previous study in South Greece. This study may serve as a baseline for planning further investigations in association with several clinical entities in Greek population, such as cardiovascular diseases and pregnancy‐related disorders.

PB390: Evaluation of the Analytical and Diagnostic Performance of a Fibrinogen and of a D‐dimer Assay for the Horiba ® Yumizen G1550

J. Pego; A. Carmo ; F. Rodrigues

Centro Hospitalar e Universitário de Coimbra, Clinical Pathology Department, Coimbra, Portugal

Background: Introduction: Levels of D‐Dimer (DD) and fibrinogen/fibrin (FIB) degradation products are significantly elevated in patients with deep venous thrombosis and pulmonary embolism, among other complications. The poor standardization amongst assays and the difficulties to establish the adequate cut‐off require clinical validation before implementation in daily practice.

Aims: To perform the clinical validation of new tests we analyzed the performance of the Yumizen G DDi2 and of the Yumizen G FIB2 assays associated to the Horiba ® Yumizen G1550.

Methods: DDs were determined by an immunoturbidometric test and the FIB through the determination of the logarithm of clotting time. The cut‐off of the DDi2 assay is 250 ng/ml with a linearity ranging from 110 ng/ml to 2,500 ng/ml, with the possibility to rerun. For FIB2 assay, based on Clauss method, the reference interval is 200‐400 mg/dl. All results were compared to the results obtained in ACLTop ® 700 (Hemosil ® Q.F.A. Thrombin (reference range: 200‐500 mg/dl) and Hemosil ® D‐Dimer HS (cut‐off: 230 ng/ml).

Results: FIB was determined in 67 samples and DDs were determined in 44 samples from patients under suspicion of thromboembolic disorders. For FIB assay, there was a concordance of the results in 66 samples (98.5%). The discordant result showed a concentration of 187 mg/dl using Q.F.A. and of 211 mg/dl using the FIB2 assay. Regarding DDs, the results were concordant in 42 samples (95.5%). The two samples with discordant results correspond to values around the cut‐off value (342 and 281 ng/ml with the D‐Dimer HS assay, 229 and 232 ng/ml with DDi2 assay). None of these two patients had any thromboembolic events.

Conclusions: The discrepant result in FIB corresponded to a value around the lower limit, in this case it had no clinical relevance. Regarding the DD, the discrepancy also occur around the cut‐off value. Considering that DDs have a high negative predictive value in thromboembolic disease it is important to perform a clinical and laboratory tight control.

PB391: Association between Factor V Leiden and APCR Values: Data from a Portuguese University Hospital

A. Carmo 1 ; J. Pego 1 ; M. Lourenço 1 ; M.D.F. Martins 1,2 ; F. Rodrigues 1,2

1 Centro Hospitalar e Universitário de Coimbra, Clinical Pathology Department, Coimbra, Portugal, 2 Faculdade de Medicina da Universidade de Coimbra, Department of Histology and Embryology, Coimbra, Portugal

Background: Factor V Leiden (FVL) is associated to a specific point mutation in the procoagulant factor V gene. Patients with the FVL mutation have an increased resistance to activated protein C (APCR) that leads to a decreased values of APCR. These patients have an increased risk of thromboembolic disease.

Aims: Determine the prevalence of FVL in a University Hospital, and correlate this data with APCR results.

Methods: The results were collected anonymously from our hospital laboratory middleware database during a 4 years period. Age, sex, diagnosis, FVL and APCR results were collected. Data were analyzed with SPSS software. The concordance between the FVL and the APCR values (cut‐off value was 2.0) was studied.

Results: During the study period, 2373 DNA analysis were performed. 2239 individuals (95.7%) carried the wild‐type allele, and 102 patients (4.29%) had the mutation. From these, 99 patients (4.17%) were FVL heterozygous and 3 patients (0.13%) were homozygous.

59 patients (56.7%) of the FVL patients were female and 43 patients (41.3%) were male. The mean age of female FVL patients was 42.1 years old and the mean age for male was 48.7 years old. APCR was determined in 46 FVL patients (45%). The mean value of APCR in FVL heterozygous patients was 1.81 (min/max: 1.41‐2.75). APCR was only determined in one homozygous patient, being the value 1.25. In 45 FVL heterozygous patients, the APCR was below the cut‐off. The higher APCR (2.75) value was from a liver transplanted patient. APCR in the wild‐type allele group was always above the cut‐off value.

Conclusions: The percentage of patients with the mutated allele shown in the present study is similar to the previously reported value for the European Caucasian population. There is a concordance between low values of APCR and the existence of FVL, with one exception. The exception refers to a liver transplanted patient and therefore this value is probably associated with the function of the transplanted liver. The cut‐off of 2 seems to be adequate to our population.

PB393: Prevalence of Common Genetic Variants Associated with Thrombophilia in Asymptomatic young Individuals with a Family History of Thrombosis

A. Papoutsi 1 ; A. Pantelios 1 ; N. Antou 1 ; J. Giouretziklis 1 ; C. Michael 1 ; A. Kalifatidou 1 ; K. Varsamidis 2 ; E. Vagdatli 1

1 Alexander Technological Educational Institute of Thessaloniki (ATEITh), Medical Laboratory Studies, Thessaloniki, Greece, 2 Alexander Technological Educational Institute of Thessaloniki (ATEITh), Physiotherapy, Thessaloniki, Greece

Background: Thrombophilia is an important medical problem, affecting about 1 in 1,000 people per year. Hereditary thrombophilia is a genetically determined tendency to develop venous thromboembolism, which is often repeated. In the last years, FV Leiden, prothrombin 20210 and thermolabile methylene tetrahydrofolate reductase (MTHFR) 677/1298 variants have been identified as possible risk factors associated with thrombophilia.

Aims: This study investigates the prevalence of FV Leiden (G1691A), MTHFR (C677T & A1298C) and prothrombin G20210A genetic polymorphisms in young individuals with first and/or second degree familial history of thromboembolism.

Methods: The study group consisted of 67 individuals with a first or second degree familial history of venous or arterial thromboembolism but no personal history of thrombosis. Blood samples collected from mostly young people of Northern Greece (55 women, 12 men), with an average age of 26 years. DNA was isolated from peripheral blood samples and the PCR‐RFLP method was used to detect all polymorphic sites.

Results: TABLE 1 The prevalence and allele frequencies of factor V Leiden, prothrombin G20210A and MTHFR C677T/A1298C among our study group FV G1691A n (%) MTHFR C677T n (%) MTHFR A1298C n (%) Prothrombin G20210A n (%) GG 56 (83.6%) CC 28 (41.8%) AA 34 (50.7%) GG 65 (97%) GA 11 (16.4%) CT 24 (35.8%) AC 33 (49.3%) GA 2 (3%) AA 0 (0%) TT 15 (22.4%) CC 0 (0%) AA 0 (0%)

Conclusions: Although the sample of our study is quite small, the frequency of the FVL heterozygotes appears to be elevated in individuals with a history of congenital thrombophilia compared to the general population of Northern Greece. Furthermore, our results indicate that the percentage of homozygotes for the C677T mutation in our study group is quite high. By including plasma homocysteine values in our assessment (data not shown) there is moderate correlation with mild hypercomycineemia. As for the A1298C mutation, it does not appear to affect plasma homocysteine levels. Prothrombin GA genotype (˜3%) is higher than general population as well. The high prevalence of thrombophilia, reinforces the importance of an extensive thrombophilia genetic screening when recorded family history of thrombotic events, especially when going for both parents and/or their family members, and even more when one or more acquired thrombophilic factors co‐exist.

PB394: Identification and Differentiation of Coagulation Factors in Prothrombin Complex Concentrates (PCC's)

C. Wilhelm 1 ; M. Mandago 2 ; S. Kiessig 2 ; S. Wittke 1

1 University of Applied Sciences Bremerhaven, Bremerhaven, Germany, 2 PreviPharma Consulting GmbH, Mannheim, Germany

Background: Prothrombin complex concentrates (PCC's) are intermediate purity pooled plasma products for the treatment of coagulation disorders. Different processing techniques enable the production of concentrates containing three or four‐coagulation factors [1].

Aims: The necessity for this study is that the activated forms of human coagulation factors, like F.II, F.VII, F.IX and F.X, are undesirable impurities in the PCC's. Until now no valid assay exists to differentiate the proactive zymogen forms from active factors. Therefore the aim of this study was to establish and validate a LC‐MS/MS‐based assay to address this issue in the medicinal product.

Methods: A standard method for identification and differentiation of the inactive and active forms of four pairs of coagulation factors F.II, F.VII, F.IX and F.X was developed by bottom‐up liquid chromatography‐mass spectrometry (LC‐MS/MS; Triple‐Tof 4600 ABSciex; protease digest) and adapted to PCC's to determine the purity of the coagulation factors.

Results: First results show specific peptides suitable for identification and differentiation of prothrombin (F.II) and α‐thrombin (F.IIa). By use of two discriminative peptides (Figure 1) prothrombin can be detected in a commercially available PCC (upper MS‐spectrum; blue) and the reference material (F.II; red) whereas the activated form (lower MS‐spectrum; orange) only occurs in the reference material (F.IIa).

Conclusions: Further results indicate that the assay is suitable for other coagulation factors (e.g. F.IX, F.X) to describe the quality of PCCs.

References: [1] Franchini M, Lippi G: Prothrombin complex concentrates: an update. Blood Transfus. 2010 Jul; 8(3): 149‐154

PB395: Water Extract of Artemisia Argyi Inhibit Coagulation Factor XII Aberrant Activation to Intervene the Blood Clotting in Ischemic Stroke Patients

S. Wang ; H. Xu; S. Cheng; S. Gong; Y. Liu; J. Ge

Hunan University of Chinese Medicine, Changsha, China

Background: Artemisia argyi is a hemostasis Chinese medicine, we found the water extract of it has arrests hemorrhage and invigorates blood circulation by activating coagulation factor XII in rabbit and human plasma.

Aims: In this present study, we try to study the effects of Artemisia argyi water extract for blood clotting and coagulation factor activity in ischemic stroke patients.

Methods: In this study, to collect the extract of Artemisia argyi, we used water extraction and alcohol precipitation, removed the precipitate, volatilize alcohol from the supernatant and then dialysis it. To examine the function of Artemisia argyi in blood clotting, recalcification time and coagulation factor activity in ischemic stroke patients through the in vitro experiment. And in the last, the function of Artemisia argyi extract in this study is confirmed by Western Blotting assay.

Results: The result showed that Artemisia argyi extract significantly increased prothrombin time (PT), activated partial prothrombin time (APTT), thrombin time (TT), particularly,it has effectively the most in APTT and has no effect on plasma fibrin (FIB). But the Artemisia argyi extract reduced recalcification time. However, Artemisia argyi extract inhibit coagulation factor XII activity in Gaolin‐dependent manner.

Conclusions: Water extract of Artemisia argyi can inhibit coagulation factor XII aberrant activation to intervene the blood clotting in ischemic stroke patients but has no effect on hemostasis.

PB396: Human Factor XII Mutation W268R Causes Urticarial Autoinflammatory Syndrome through Intracellular Misprocessing

Z.L.M. Hofman 1,2 ; K. Krause 3 ; A. Nosairi 1 ; N.A. Mahnke 3 ; J. Scheffel 3 ; M. Maurer 3 ; G. Pasterkamp 1 ; C.E. Hack 2 ; S. de Maat 1 ; C. Maas 1

1 University Medical Center Utrecht, Laboratory of Clinical Chemistry and Haematology, Utrecht, the Netherlands, 2 University Medical Center Utrecht, Laboratory for Translational Immunology, Utrecht, the Netherlands, 3 Charité ‐ Universitätsmedizin Berlin, Department of Dermatology and Allergy, Berlin, Germany

Background: Factor XII (FXII) is redundant for haemostasis but drives production of the inflammatory peptide bradykinin. Gain‐of‐function mutations in the F12 gene cause hereditary angioedema (HAE), a disease with acute attacks of tissue swelling. We here report the disease mechanism for a novel mutation W268R in the F12 gene, identified in a family with urticarial autoinflammatory syndrome. This disease has a chronic character with cold‐inducible urticarial‐rash, headache, arthralgia, and fatigue.

Aims: Uncover disease mechanism of FXII‐W268R.

Methods: Wildtype FXII (FXII‐WT), mutants FXII‐T309R (causes HAE) and FXII‐W268R were expressed in HEK293F. We analyzed cell lysates, culture supernatants and patient plasmas by chromogenic assay, western blotting and ELISA.

Results: All 4 FXII‐W268R carriers were symptomatic and had high levels of FXIIa‐C1‐inhibitor (C1‐INH) complex, plasma kallikrein‐C1‐INH complex and cleaved kininogen, suggesting constitutive bradykinin production. FXII‐W268R, but not WT or T309R, formed cleavage products in culture supernatant (Figure 1A). Chromogenic substrate assays showed that FXII‐W268R fragmentation is accompanied by enzymatic activity (Figure 1B), and a strongly increased capacity to activate prekallikrein (Figure 1C). PPACK and FXII‐neutralizing antibody blocked this activity. Furthermore, C1‐INH formed complexes with FXIIa‐W268R. In silico sequence analyses indicated that the mutation inserts a putative cleavage site for proprotein convertases (W268R: R D R LS R ). Western blotting of cell lysates revealed that FXII‐W268R is cleaved intracellularly (Figure 1A). Ongoing investigations aim to elucidate the mechanism of intracellular misprocessing of FXII‐W268R.

Conclusions: Intracellular misprocessing of FXII‐W268R leads to its activation. Our findings suggest that FXII‐W268R results in continuous low‐grade FXIIa secretion, explaining increased parameters of contact activation. Interestingly, this does not result in thrombosis or HAE, but in urticarial autoinflammatory syndrome.

FIGURE 1 (A) FXII cleavage in supernatant and lysate (N)R=(non‐)reducing, (B) FXIIa and (C) prekallikrein‐activator activity in cell culture supernatant

PB397: The Surface of Activated Platelets Enhance the Activity of FXIa

S.E. Reitsma 1 ; C. Puy 1 ; D. Gailani 2 ; E. Tucker 3 ; A. Gruber 1 ; O.J.T. McCarty 1

1 Oregon Health & Science University, Biomedical Engineering, School of Medicine, Portland, United States, 2 Vanderbilt University School of Medicine, Nashville, United States, 3 Anora Inc., Portland, United States

Background: Factor (F)XI of the intrinsic pathway of coagulation has been identified as a potentially safe target for antithrombotic therapy, yet it is still unknown whether platelets regulate activated FXI (FXIa) activity. Study of platelet‐coagulation factor interactions may give us a better understanding of the regulatory mechanism of FXI in hemostasis and thrombosis.

Aims: Characterize the role of platelets in the regulation of FXIa activity.

Methods: Washed platelets (5 × 10 8 plt/ml) resting or activated with α‐thrombin (1 nM) and collagen‐related peptide (CRP; 10 μg/ml) were seeded on a fibrinogen surface. 1 μM of hirudin was added to neutralize thrombin. Platelets were washed and incubated with FXIa (500 pM) for 0 to 2 hours at 37°C. FXIa activity was measured using a chromogenic substrate assay and a fibrin generation assay in recalcified plasma.

Results: The capacity of FXIa to cleave the chromogenic substrate was lost over time in the absence of platelets. In the presence of platelets the activity of FXIa was maintained even after 2 hours of incubation. FXIa activity increased >25% in the presence of activated platelets. The presence of platelet secretome, which is known to have protein nexin‐2, completely blocked the activity of FXIa. It required >300 seconds for FXIa alone to initiate fibrin generation of plasma. In the presence of immobilized platelets, FXIa initiated fibrin generation in slightly less than 300 seconds, while it only took ˜200 seconds for FXIa to initiate fibrin generation in the presence of platelets activated with α‐thrombin and CRP. In the absence of FXIa, the activation of platelets did not enhance the cleavage of the chromogenic substrate or reduce the initiation time for fibrin generation.

Conclusions: Activated platelets enhance FXIa activity and inhibit FXIa autolysis and also enhance the capacity of FXIa to initiate fibrin generation. Moreover, our findings suggest that the interaction of FXIa with the surface of activated platelets induces an allosteric modulation of FXIa.

PB398: FXIa Binds and Forms a Complex with Plasminogen Activator Inhibitor‐1 that Inhibits Its Activity and Induces Internalization and Degradation by Endothelial Cells

A. Ngo 1 ; C. Puy 1,2 ; E. Tucker 1 ; D. Gailani 3 ; A. Gruber 1,2 ; O. McCarty 1,2

1 Oregon Health and Science University, Biomedical Engineering, Portland, United States, 2 Oregon Health & Science University, Division of Hematology / Medical Oncology, Portland, United States, 3 Vanderbilt University Medical Center, Pathology and Medicine, Nashville, United States

Background: Factor (F) XI of the intrinsic pathway has been identified as a potentially safe target of anticoagulation. Endothelial cells (ECs) contribute to the anticoagulant nature of the vasculature, yet it is unknown whether ECs directly regulate activated FXI (FXIa) activity.

Aims: This study aims to determine whether ECs inactive FXIa by characterizing and visualizing the interaction between FXIa and ECs.

Methods: A FXIa chromogenic assay was used to measure FXIa activity. FXIa complexes were captured using immunoprecipitation, and detected by Western blots using an anti‐FXIa antibody. Human umbilical vein ECs (HUVECs) were seeded onto glass coverslips, then incubated with vehicle or FXIa at 4°C to induce binding, and then at 37°C to induce FXIa trafficking. Cells were fixed, permeabilized and incubated with an anti‐FXIa antibody and antibodies against early endosome‐associated protein 1 (EEA1), late‐endosome (Rab7), lysosome (LAMP1), and recycling vesicle (Rab11), followed by immunofluorescence (IF) to determine whether FXIa was bound by the surface and internalized by ECs.

Results: HUVECs block FXIa activity. Western blot results show that the catalytic domain of FXIa (30 kDa) forms a complex with an inhibitor on ECs, generating an 80 kDa band that disappears over time from the EC surface. Mass spectrometry analysis of the 80 kDa band reveals plasminogen activator inhibitor 1 (PAI‐1) as the FXIa inhibitor on ECs. Using IF, we observed FXIa binding at 4°C, which at 37°C promoted internalization of FXIa by ECs followed by trafficking to early, late endosomes, and the lysosomes. The serine protease inhibitor PPACK abrogated FXIa‐PAI‐1 complex formation and internalization of FXIa. Internalized FXIa exhibited minimal colocalization with recycling vesicles.

Conclusions: Our data suggest that FXIa forms a complex with PAI‐1 on the EC surface, blocking FXIa activity, indicating a direct role of ECs in inhibiting FXIa activity through clearance and internalization.

PB399: A Critical Role of High‐molecular‐Weight Kininogen in Host Defense against Gram‐negative Bacterial Infection

A. Yang 1 ; Y. Wu 1,2

1 Soochow University, Suzhou, China, 2 Temple University, Philadelphia, United States

Background: High‐molecular‐weight kininogen (HK) is the major component of plasma contact system. Although we report that HK serves as a carrier of LPS and supports endotoxemia, its role in Gram‐negative bacterial infection remains unknown.

Aims: To determine the role of HK in host defense against bacterial infection and the underlying mechanism.

Methods: Phenotypes of mice lacking HK ( Kng1 −/− ) were characterized in two infection models, E.coli intraperitoneal infection and cecal ligation and puncture. Survival rate, H&E staining of major organs, bacteria load and circulating cytokine levels were compared. In vivo imaging System was applied to trace colocalization of I 125 ‐HK and E.coli . Binding of HK to E.coli and subsequent degradation of HK were evaluated by flow cytometry and immunoblotting. The effect of HK related peptide on bacterial growth was evaluated in vitro and in vivo.

Results: Compared with Kng1 +/+ mice, Kng1 −/− mice showed a significantly decrease in survival rate, accompanied with enhanced cytokines levels, tissue injury, as well as increased bacterial outgrowth. When I 125 ‐HK was injected into Kng1 −/− mice bearing E.coli infection, it was rapidly recruited into the site of infection. Consistently, in vitro observations indicate that clearance of E.coli in Kng1 −/− plasma was significantly slower than Kng1 +/+ plasma, and HK directly bound to E.coli dependently on LPS and became cleaved. In plasma from both humans and mice with Gram‐negative bacterial infection, the cleaved fragment of HK was detected by a mAb against DHG15 peptide, which is the binding region of HK to LPS. In a concentration‐dependent manner, DHG15 peptide bound to E.coli and inhibited the growth. In wild‐type mice with E.coli infection, DHG15 significantly decreased bacterial growth.

Conclusions: HK is critical in host defense against Gram‐negative bacterial infection, revealing a new function of plasma contact system. HK identifies invading bacteria by recognizing LPS and possesses antibacterial activity by its cleaved fragments.

PB400: Bombyx Batryticatus Extract Induce Kallikrein‐kinin System by Activating Coagulation Factor XII

H. Xu; S. Wang ; C. Deng

Hunan University of Chinese Medicine, Changsha, China

Background: Bombyx batryticatus is a tradition Chinese medicine used to prevent and treat the thrombosis. It was confirmed that it can inhibit coagulation factor X and II activity, these are two indirect substrates of coagulation factor XII, and it remain to be delineated that Bombyx batryticatus has same function for it.

Aims: In the present study, we arm to discuss the Bombyx batryticatus function for intrinsic coagulation, kallikrein‐kinin system and fibrinolysis system by plasma and FXII is the centre protein.

Methods: In this study, to collect the extract of Bombyx batryticatus, we used water extraction and alcohol precipitation, removed the precipitate, volatilize alcohol from the supernatantm and then dialysis it. We examine the function of Bombyx batryticatus in coagulation factor XII activity, blood clotting, kallikrein generation and fibrinlysis by Western Blotting assay or chromogenic substrate tests in vitro.

Results: The results showed that Bombyx batryticatus extract activated FXII,then prekallikrein was activated and kallikrein was generated on qualitative test. Interestingly, FXI and plasminogen were not activated by FXII when it was activated by Bombyx batryticatus extract.

Conclusions: Our results suggested that the Bombyx batryticatus extract can activate FXII to participate in the kallikrein‐kinin system, but it has no obvious effects on the action for the intrinsic coagulation and fibrinlysis cascade by FXII.

PB401: Prothrombin Concentrates for Reversal of Bleeding Induced by Supratherapeutic Doses of JNJ‐9375 (A Novel Exosite 1 Thrombin Antibody) in a Rat Tail Transection Model

M. Chintala 1 ; Z. Huang Devine 1 ; J. Reckless 2

1 Janssen R&D, Cardiovascular & Metabolism Discovery, Spring House, United States, 2 RxCelerate Limited, Cambridge, United Kingdom

Background: JNJ‐9375 is a novel, long acting exosite 1 thrombin inhibitor antibody currently in Phase II clinical trials. We have previously reported an improved therapeutic index for JNJ‐9375 (10X) versus apixaban (1X), a marketed FXa inhibitor in rats. JNJ‐9375 increased bleeding time (BT) at supra therapeutic doses in rats (10X) and monkeys (40X), compared to the doses required for robust antithrombotic efficacy.

Aims: To assess the utility of commercially available non‐specific reversal agents including prothrombin concentrates (PCC), a recombinant Factor(F) VIIa and tranexamic acid in reducing JNJ 9375‐induced BT in rats.

Methods: BT was assessed following total tail transection at tail diameter 3 mm in anesthetized rats.

Results: Administration of JNJ‐9375 (10 mg/kg, i.v.) 5 minutes prior to tail transection increased BT to >30 minutes versus 10‐15 in the placebo group. This high dose of JNJ‐9375 produced marked prolongation of clotting assays ex‐vivo, thrombin time (TT, 3.8X), ecarin clotting time (ECT, 4.3X), activated partial thromboplastin time (aPTT, 2.2X) and prothrombin time (PT 1.9X). In this bleeding model, pretreatment with two different PCC's, one containing non‐activated clotting factors (FII, FVII, FIX and FX) and the other containing a mix of non‐activated and activated clotting factors (FII, FIX, FX and FVIIa) at 100 IU/Kg and 150 IU/Kg respectively, significantly (two sample t‐test vs. JNJ‐9375 alone) attenuated JNJ‐9375 induced prolongations of BT. These PCC's only partially reversed the prolongation in PT's but had no impact on TT, ECT, aPTT and the circulating levels of JNJ‐9375 in plasma. In contrast, pretreatment with two other approved non‐specific reversal agents, a recombinant FVIIa or tranexamic acid did not impact JNJ‐9375 induced bleeding in this model.

Conclusions: PCCs may be an option to mitigate bleeding in patients on JNJ‐9375 that are experiencing trauma or require emergency surgery.

PB402: Saliva‐derived Tissue Factor Induces Thrombin Generation in a Diurnal Rhythm

L.N. van der Vorm 1,2,3 ; J.E.I.G. Brouwers 4 ; C. Mondria 3 ; B. de Laat 1,2,3 ; P.G. de Groot 1,2 ; J.A. Remijn 1,3

1 Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands, 2 Synapse Research Institute, Maastricht, the Netherlands, 3 Gelre Hospitals, Department of Clinical Chemistry and Hematology, Apeldoorn, the Netherlands, 4 Institute for Dental Implantology, Amersfoort, the Netherlands

Background: Upon tooth extraction, extravascular tissue factor (TF) initiates coagulation to arrest bleeding. Additionally, saliva is in constant contact with the wound and previously demonstrated to contain microparticle‐derived TF capable of shortening clotting time. Since the duration of post‐extraction bleeding is highly variable between patients, we hypothesized this may be caused by variation in saliva‐derived TF‐induced clotting activity, measured by thrombin generation (TG).

Aims: To assess the variability of saliva‐induced TG in a healthy population.

Methods: TG with recombinant (r‐)TF was performed as previously described (Hemker et al. 2003). For saliva‐induced TG, saliva was vortexed at 850 rpm for 5 minutes, diluted with bovine serum albumin and added (instead of r‐TF and phospholipids (PL)) to normal pooled plasma (NPP), in the absence or presence of anti‐TF antibodies. Saliva was collected (on ice) from 13 healthy individuals after 30 minutes fasting and rinsing the mouth thoroughly with water. TG was compared by one‐way ANOVA with post‐hoc Bonferroni.

Results: Addition of saliva to NPP induced TG curves similar to those induced by r‐TF and PL. Moreover, addition of anti‐TF antibodies abolished saliva‐induced TG, indicating TF‐dependence. A large inter‐individual variability (peak CV 31%, range 73‐220 nM) in saliva‐induced TG was observed. Interestingly, within subjects: saliva‐induced TG was significantly (P=0.009) increased in the morning (167±40 nM) compared to the afternoon (124±39 nM) and evening (123±38 nM). This diurnal variation was not attributable to gingival stimulation or damage induced by tooth brushing.

FIGURE 1 Saliva‐induced TG in healthy individuals (n=13) shows a diurnal rhythm in salivary TF activity

Conclusions: We identified a diurnal rhythm in salivary TF activity that may have implications for tooth extraction and dental surgery, as performing invasive procedures in the morning may be beneficial for rapid coagulation. Future studies should correlate salivary TF to clinical outcome (i.e. post‐extraction bleeding) and assess a possible relation with bacterial status in the oral cavity.

PB403: Activated Factor VII Fused with Transferrin Showed Prolonged Half‐life with Safe Toxicity Profile in Animal Models

I. Song ; H. Lee; H. Kim

Tiumbio Co., Ltd, Seongnam, Korea, Republic of

Background: Replacement of recombinant activated clotting factor VII, rFVIIa is an effective treatment for hemophilia A and B patients with neutralizing antibodies against exogenous clotting factors. Due to its short half‐life, rFVIIa should be injected frequently during treatment. As such, FVIIa with longer half‐life could help alleviate the patient's inconvenience and economic burden.

Aims: To develop safe and efficacious FVIIa with the increased half‐life.

Methods: We created a fusion form of rFVIIa called NBP604 where human transferrin is genetically linked to its C‐terminus. Biological and structural characteristics and safety profile of NBP604 were evaluated in vitro and in vivo. Enzymatic parameters, binding affinity to tissue factor and mass spectrometry of purified NBP604 were performed. Half‐life, efficacy and preclinical safety profile were assessed using animal models.

Results: Enzymatic parameters of rFVIIa and NBP604 were compared using different substrates. Their catalytic efficiencies were nearly indistinguishable. Binding affinity of NBP604 to tissue factor measured by surface plasmon resonance was similar to that of rFVIIa.

TABLE 1 Comparison of biological properties of NBP604 to those of rFVIIa Assay measured parameters rFVIIa NBP604 S‐2288 peptide hydrolysis Kcat (s −1 ) 9.5±2.2 7.2±1.0 Km (mM) 4.3±1.2 3.9±0.7 Kcat/Km (s −1 M −1 ) 2218±114.9 1853.6±104.1 FX activation Kcat (s −1 ) 0.04±0.0 0.04±0.0 Km (mM) 41.97±15.8 39.90±13.5 Kcat/Km (s −1 M −1 ) 1065.6±349.9 1082.8±328.9 Binding affinity to tissue factor Kon (×10 4 M −1 s −1 ) 2.04 1.35 Koff (×10 −3 s −1 ) 2.38 2.14 KD (×10 −9 ) 117 158

Mass spectroscopic analysis showed 31 disulfide bonds and four N‐glycosylation sites in the anticipated sites. Pharmacokinetic study of NBP604 was performed in rat and primate models, and showed 3.2‐ and 2.5‐fold extended half‐lives.

FIGURE 1 Extended half life of NBP604 in rats

Ex vivo efficacy was compared by measuring clotting times of Warfarinized rat plasma samples 16 hours after treatment of either rFVIIa or NBP604. Thrombogenicity of rFVIIa or NBP604 was evaluated in rabbit jugular vein stasis thrombosis model. These efficacy experiments demonstrated prolonged and superior coagulation efficacy compared to rFVIIa. Finally, safety pharmacology and preclinical toxicity study of NBP604 was performed in GLP compliant facility in rats and monkeys and no evident toxicity was observed.

Conclusions: NBP604 was proved to be safe and efficacious, showing full therapeutic potential as a rFVIIa with extended half‐life.

PB404: “Real World” Usage of Prothrombin Complex Concentrates (PCCs) to Correct Coagulopathy. A 3 Year Retrospective Review in an Academic Hospital System

I. Kourouni 1 ; N. Pakzad 1 ; I. Kejo 2 ; M. Friedman 3 ; D. Steiger 1 ; R. Jean 1

1 Mount Sinai St. Luke's West Hospital Center, Pulmonary Critical Care and Sleep Medicine, New York, United States, 2 Mount Sinai St. Luke's West Hospital Center, Medicine, New York, United States, 3 Mount Sinai St. Luke's West Hospital Center, Pathology, New York, United States

Background: In spite of the significant lack of data, PCC is being used for “off‐label” indications that include reversal of direct oral anticoagulants (DOACS), hemorrhagic shock and perioperative management of coagulopathy.

Aims: To report on our clinical experiences with the “on label” and “off‐label” use of PCC in our hospital system.

Methods: We retrospectively reviewed all consecutive adult patients that received 4factor PCC for management of coagulopathy at three teaching hospitals of our hospital system, during the 3‐year period 2015‐2017. We studied the indication for PCC, including “on label” use for patients on Coumadin, and “off label” use for patients not on Coumadin, and clinical outcomes.

Results: PCC was given in 253 patients: mean age 71.5 years (range 22‐97), 149/253 (59%) male, 14 subjects received more than 1 dose, average dose was 2779 units. The majority of use occurred in the Emergency Department 171/253 (67%) and in ICUs 82/253 (32%). Our cohort included 135/253 (52%) patients on Coumadin and 119/253 (47%) in the “off label” group. The “off label group” included 58/119 (48%) patients on DOAC and 47/119 (42%) on no anticoagulation. The indications for PCC were reversal of coagulopathy in the setting of neurologic emergency 104/253 (41%), gastrointestinal bleed (66/253)26%, intraoperative hemorrhagic shock in 53/253 (20%) and perioperative correction of coagulopathy without bleeding in 27/253 (10%). Fourteen patients received PCC following administration of antiplatelets, heparinoids or tpa. 82/253 (32%) patients expired. Mortality was (28/135)20% at the label group and (54/119)45% in the off label group. A total of 14 patients had a thrombotic event within 14 days after PCC use.

Conclusions: Our real world PCC utilization review demonstrates that PCC is currently used indiscriminatingly for diverse causes of coagulopathy, with resulting morbidity, in settings where no clinical benefit is expected and where contraindications to its use exist. Increasing emphasis is required to restrict PCC to “on label” use.

PB405: Gene Expression of Coagulation Factors after Experimental Snake Envenomation in Mice

A.T.A. Sachetto 1 ; J.R. Jensen 2 ; M.L. Santoro 1

1 Instituto Butantan, Lab. Fisiopatologia, São Paulo, Brazil, 2 Instituto Butantan, Lab. Imunogenetica, São Paulo, Brazil

Background: The envenomation by the lanced‐headed viper Bothrops jaracaca induces hemostatic disturbances that include severe systemic bleedings, thrombocytopenia and consumptive coagulopathy (a decrease in plasma levels of fibrinogen, factor VIII (FVIII), and prothrombin (FII)). However, there are several aspects yet to be elucidated regarding the mechanisms by which these alterations occur.

Aims: To investigate the gene expression kinetics of important hemostatic factors synthetized by the liver, and hemostatic alterations in mice injected with B. jararaca venom.

Methods: All procedures involving animals were in accordance with Institutional Animal Care and Use Committee (CEUAIB 4388061115). Swiss mice (n=5/group/time) were injected s.c. with saline or BjV (1.6 mg/kg b.w, s.c.), and after 3, 6 and 24 h blood and liver fragments (for RNA isolation) were collected. RNA integrity was confirmed (RNA integrity number ≥8), samples were reverse transcribed and cDNA templates (1‐100 μg/reaction) were used to perform quantitative PCR. Relative gene expression was analyzed following the 2 −ΔΔCT method, and Rplp0 was used as an endogenous control gene. A pool of liver samples from normal mice was used as the reference sample. Fibrinogen assays and platelet counts were analyzed in blood samples. Results were analyzed by two‐way ANOVA and Bonferroni test, and considered significant when P<0.05.

Results: Results are summarized in Table 1.

TABLE 1 Blood assays and gene expression of coagulation factors in liver samples after experimental B. jararaca envenomation in mice Blood assays Results (BjV group compared to the saline control group) Platelet counts Important decrease from 3, 6 to 24 h (P<0.001) Plasma fibrinogen levels Marked decrease from 3 h (P<0.001) to 24 h (P<0.05) Gene expression Results (BjV group compared to the saline control group) Fibrinogen (α, β and γ chains) Marked increase from 3 h (P<0.005) to 24 h (P<0.05) F2 (prothrombin) No significant alterations F10 Increase at 24 h (P=0.009) Vwf, Adamts13 and F8 Tendency of decrease over time Adamts13 Significant decrease at 24 h (P<0.001)

Conclusions: We conclude that the envenomation induces a rapid mRNA synthesis response in the liver, particularly for fibrinogen chains. This response explains the fast recovery of fibrinogen levels after antivenom administration to patients bitten by B. jararaca snakes.

PB406: Enhanced FIX Collagen IV Binding Shows Improved Hemostatic Effects in a Hemophilia B Mouse Model

D. Matino 1 ; D. Stafford 2 ; A. Iorio 3 ; A. Chan 3

1 University of Perugia, Perugia, Italy, 2 University of North Carolina, Chapel Hill, United States, 3 McMaster University, Hamilton, Canada

Background: In previous studies it's been proposed that significant extravascular FIX compartmentalization may increase the apparent volume of distribution, and potentially constitute a mechanism for extended levels of biologically active FIX. FIX may adhere ‐ specifically yet reversibly ‐ to type‐IV collagen on endothelial cells (Feng D et al. JTH 2013; 11: 2176‐8) resulting in prolonged hemostatic effects. A mutant, recombinant FIX (rFIX) with higher collagen IV affinity (FIX K5R) has been produced.

Aims: To explore whether an enhanced ability of FIX to bind collagen IV affects hemostasis in a hemophilia B mouse model.

Methods: FIX KO mice were given a single i.v. injection of rFIX at the beginning of the experiments (rFIX, 100 IU/kg, 300 IU/kg or 500 IU/kg; rFIX K5R 5 or 50 IU/kg; Fc‐fusion rFIX 50 IU/kg) or vehicle alone. FIX activity was tested in plasma samples with a chromogenic assay (Figure 1, Panel A). Mice were exposed to 5 days of moderate treadmill running for 30 min/day. After 5 days mice were sacrificed and evaluated for the presence of bleedings in accordance with a previously published protocol (Tranholm M et al., JTH 2015 Jan;13(1):82‐91) with minor modifications.

Results: Animals treated with any of the different doses/types of FIX were found to have significantly less bleedings than mice treated with vehicle alone (Kruskal‐Wallis test and Dunn's multiple comparisons post‐test). The analysis of FIX plasma levels from mice treated with Fc‐fusion rFIX showed the longest permanence of FIX activity in circulation. In contrast, the lowest dose of K5R FIX resulted in barely detectable levels in plasma but provided effective protection from bleeding, comparable to standard dose of Fc‐fusion FIX (Figure 1; Panels B, C, D).

Conclusions: K5R FIX treatment provided protection from bleeding, despite undetectable plasma levels. This result would support the concept that collagen IV binding by FIX might provide a longer‐lasting extravascular reservoir of FIX at a hemostatically functional location.

FIGURE 1 Schematic representation of blood sampling schedule (A) and results of chromogenic FIX assay (B, C) and muscle bleeding score (D)

PB408: Factor XI Level Is a Determinant of Plasma Clot Growth

C. Kluft 1 ; J. Begieneman 1 ; N. Podoplelova 2 ; N. Dashkevich 3 ; F. Ataullakhanov 3

1 Good Biomarker Sciences, Leiden, the Netherlands, 2 Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russian Federation, 3 Center for Theoretical Problems of Physicochemical Pharmacology, Moscow, Russian Federation

Background: Dependence of clotting in tests is only marked for low levels of factor XI.

Aims: In plasma tissue‐factor‐induced clot growth, levels from 0‐100% are determining the growth rate and we explored further mechanisms and inhibitors.

Methods: Clot growth analysis was with the Thrombodynamics method (Hemacore ® ). Plasma, inhibitors and phosphatase were from commercial sources: plasma was freed of microvesicles. Corn trypsin inhibitor (CTI) was added to block factor XII participation.

Results: Plasma clot growth continued over a prolonged time up to 1.5 hr. The continuation is reduced early in factor XI deficient plasma and the rate of continuation is dose dependent from 0‐100% factor XI. Expectedly, the factor XI dependent growth is absent in factor IX or VIII deficient plasma

Inhibition of the factor XI‐related growth is achieved by phosphatase degradation of plasma poly‐phosphate. Inhibition is also achieved by the thrombin inhibitors dabigatran, argatroban and NAPAP, attributed to the thrombin activation of factor XI. The IC‐50 is 150, 250 and 350 ng/ml, respectively. Neutralising antibodies for factor XI inhibit the growth, but the strong factor XIa inhibitor, APP protease nexin II, and high CTI did not.

Factor Xa inhibitors (rivaroxaban, apixaban, edoxaban) inhibit the factor XIa contribution with IC50 of 28, 23, 55 ng/ml, respectively, due to block of the factor VIII‐IX pass‐through pathway. This was found to be related to inhibition by these inhibitors of factor VIII activation by factor Xa. We excluded effects on factor Xa activation of prothrombin by showing absence of effect in factor IX or VIII deficient plasma's.

Conclusions: The role of factor XI in coagulation is expressed dose‐dependently in clot growth and involves activation by thrombin supported by sufficient poly‐phosphate present in plasma. Factor XI contribution is inhibited by both reversible thrombin and factor Xa inhibitors. It is suggested to employ clot growth as pharmacodynamic model for factor XI contribution in haemostasis.

PB409: Robustness of Spatial Thrombin Generation and Fibrin Clot Propagation

A. Kuprash 1,2 ; A. Shibeko 1,2 ; M. Panteleev 1,2,3 ; F. Ataullakhanov 1,2,4 ; A. Balandina 1,2

1 Center for Theoretical Problems of Physicochemical Pharmacology, Moscow, Russian Federation, 2 National Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russian Federation, 3 Moscow Institute of Physics and Technology, Dolgoprudny, Russian Federation, 4 Moscow State University named after M.V. Lomonosov, Moscow, Russian Federation

Background: Blood coagulation is a delicately regulated space‐ and time‐dependent process that leads to formation of fibrin clot that prevents blood loss upon vascular injury.

Aims: To investigate sensitivity of blood coagulation system in the case of non‐uniform tissue factor (TF) spatial distribution, we combined an in vitro reaction‐diffusion experimental model with computer model of blood clotting.

Methods: Clot formation in plasma supplemented with phospholipids was activated with the identical amounts of TF either homogeneously distributed (final concentration 5 pM, homogeneous model) or immobilized on the surface (surface density 100 pmoles/m 2 , spatially heterogeneous model). Fibrin clot growth and thrombin concentration as a function of space and time were observed using videomicroscopy in plasmas of healthy donors or patients with deficiencies of FII, FV, FVII, FVIII, FIX, FX, or FXI.

Results: In the spatially heterogeneous model, there was significantly decreasing near‐activator thrombin generation in FV‐, FVII‐, and FX‐deficient plasmas. In the homogeneous model, clotting was not registered. The simulation and experimental data showed that switching is carried out by varying the TF concentration. Velocity of clot propagation correlated linearly with the concentration of thrombin at the clot wave front but not with the overall thrombin wave amplitude. Our experimental data verified this theoretical conclusion. Calculation allowed us to assert that clot growth was neither reaction‐ nor diffusion‐limited at early stages, but diffusion‐limited later in normal plasma. In contrast, clot growth was always diffusion‐limited in FV‐, VII‐, or FX‐deficient plasmas, but always reaction‐limited in FVIII‐, FIX‐, and FXI‐deficient plasmas.

Conclusions: Robustness of spatially heterogeneous coagulation system is due to the combination of locally high TF concentration that overcomes activation thresholds and generates FXa able to diffuse from the activator and low thrombin concentration required to form a clot.

PB410: The Blood Clot is Covered by a Fibrin Film that Protects against Microbes

F. Macrae 1 ; C. Duval 1 ; S. Baker 1 ; N. Yuldasheva 1 ; K. Kearney 1 ; H. McPherson 1 ; N. Asquith 1 ; J. Konings 2,3 ; A. Casini 4 ; J. Degen 5 ; S. Connell 6 ; H. Philippou 1 ; A. Wolberg 7 ; R. Ariëns 1,3

1 University of Leeds, Division of Cardiovascular and Diabetes Research, Leeds, United Kingdom, 2 University of Maastricht, Synapse Research Institute, Maastricht, the Netherlands, 3 University of Maastricht, Department of Biochemistry, Maastricht, the Netherlands, 4 Geneva University Hospitals, Division of Angiology and Haemostasis, Geneva, Switzerland, 5 Cincinnati Children's Hospital, Cincinnati, United States, 6 University of Leeds, Molecular and Nanoscale Physics Group, Leeds, United Kingdom, 7 University of North Carolina, Department of Pathology and Laboratory Medicine, Chapel Hill, United States

Background: Haemostasis requires conversion of fibrinogen to fibrin fibres that generate a characteristic network, interact with blood cells, and initiate tissue repair. The fibrin network has been extensively studied, but the spatial arrangement of the external clot face is unknown.

Aims: To investigate the structure and function of the clot at the blood‐air interface.

Methods: Electron, confocal and atomic force microscopy, immunohistochemistry and Langmuir‐Blodgett trough were used to investigate the blood‐air interface of blood clots in vitro, and in vivo in a murine model of dermal puncture wound injury. Recombinant fibrinogens were expressed in CHO cells. Bacteria migration through blood clots was investigated using Boyden chambers.

Results: Fibrin transitions to the blood‐air interface through Langmuir film formation, producing a hitherto undisclosed protective film confining the clot. The fibrin film formed at the same rate as the fibre network, and was connected to the underlying clot network through tethering fibres. Fibrin film formation occurred at different thrombin concentrations (0.1‐10 U/ml), with different fibrinogen mutants (α220 truncation, α390 truncation, γ’, γ3X) and at different temperatures, but was thickest at skin temperature (31.5°C). The film formed in vivo on dermal puncture wounds in wildtype mice, but not fibrinogen‐deficient mice. It was digested by plasmin at a similar rate to fibrin fibres and formation of the film can be prevented with surfactants including polysorbate 20 and oil. The film protected against penetration by bacteria and helps to retain erythrocytes within the clot.

Conclusions: These data show a remarkable new aspect of blood clotting, in which fibrin produces a protective film at the blood‐air interface covering the entire external surface of the clot. The fibrin film helps to retain erythrocytes and is a critical host‐protection mechanism produced at the external surface of blood clots.

PB411: Binding of Coagulation Factor XIII Zymogen to Activated Platelet Subpopulations: Roles of Integrin αIIbβ3 and Fibrinogen

N. Podoplelova 1,2 ; Y. Kotova 1,2 ; S. Obydennyy 1,2 ; E. Kostanova 3 ; A. Demyanova 1 ; M. Biriukova 3 ; M. Rosenfeld 3 ; H. Chambost 4 ; M. Kumskova 5 ; F. Ataullakhanov 1,2 ; M.‐C. Alessi 4 ; M. Panteleev 1,2

1 Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russian Federation, 2 Center for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences, Moscow, Russian Federation, 3 N. M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russian Federation, 4 Aix‐Marseille University,, Marseille, France, 5 National Research Center for Hematology, Moscow, Russian Federation

Background: Factor XIIIa (fXIIIa) is a transglutaminase that plays a crucial role in fibrin clot stabilization and regulation of fibrinolysis. Its binding by activated platelets is essential for development of platelet procoagulant capacity. The zymogen fXIII interaction with platelets is not well characterized.

Aims: Investigate interaction of zymogen fXIII with activated platelet subpopulations.

Methods: Platelets were isolated from whole blood by washing and gel‐filtration. Written informed consent was given by all participants, and the study was approved by the institutional ethical committee. Gel‐ filtred platelets were stimulated with thrombin (100 nM), ADP (100 μM) or CRP (20 ug/ml) Flow cytometry and confocal microscopy were used to investigate interaction of FITC‐labeled FIII with the membranes of activated platelets.

Results: Confocal microscopy and flow cytometry using fluorescently labeled factors activated platelets bound 1000‐1500 molecules/cell of fXIII at 100 nM, while both PS‐negative activated platelets and resting platelets bound 200‐400 molecules/cell. The binding was reversible, calcium‐independent, and linear within the fXIII concentration range up to 1000 nM. Factor XIII predominantly binds to the caps of procoagulant platelets and colocalizated with fibrinogen. Exogenous fibrinogen promoted fXIII binding by activated PS‐negative platelets; this effect was abolished by the integrin αIIbβ3 antagonist monafram. The fXIII binding was 1.5‐ to 3‐fold decreased for platelets from 4 patients with gray platelet syndrome, and was variable for platelets from 9 patients with Glanzmann′s thrombasthenia.

Conclusions: Strong platelet stimulation, fibrinogen, and αIIbβ3 play essential but not exclusive roles in fXIII binding. The preferential binding in the cap‐like structures might be important for increasing local fXIII concentration in platelet thrombi.

This study was supported by the President of the Russian Federation′s scholarship for young scientists and postgraduate students

PB412: Presence and Function of Cellular Factor XIII in Human Cornea

Z.Z. Orosz 1,2 ; A.H. Shemirani 1 ; H. Bárdos 3 ; B. Nagy 4 ; A. Berta 5 ; J. Kappelmayer 6 ; R. Ádány 3 ; A. Facskó 2 ; L. Muszbek 1

1 University of Debrecen, Division of Clinical Laboratory Science, Department of Laboratory Medicine, Faculty of Medicine, Debrecen, Hungary, 2 University of Szeged, Department of Ophthalmology, Faculty of Medicine, Szeged, Hungary, 3 University of Debrecen, Department of Preventive Medicine, Faculty of Medicine, Debrecen, Hungary, 4 University of Szeged, Depratment of Pathology, Faculty of Medicine, Szeged, Hungary, 5 University of Debrecen, Department of Ophthalmology, Faculty of Medicine, Debrecen, Hungary, 6 University of Debrecen, Department of Laboratory Medicine, Faculty of Medicine, Debrecen, Hungary

Background: Transglutaminases (TGs) catalyze cross‐linking of peptide chains through e(g‐glutamyl)lysyl bonds. The presence of cellular FXIII (cFXIII), the dimer of the catalytic FXIII A subunit (FXIII‐A2) has been reported earlier in human tears, but its origin and function has not been explored.

Aims: Our aim was to investigate the occurrence of cFXIII in cells of healthy human corneas and to make ophthalmological examinations on FXIII deficient patients.

Methods: Frozen sections of normal human cornea were stained for cFXIII, using poly‐, or monoclonal antibodies. Detection of cFXIII was combined with labeling for CD34 and anti e(g‐glutamyl)lysyl bond antibodies using double immunofluorescent staining. Western blot analysis was also performed on corneal stroma. Isolated corneal stromal cells were labeled for both CD34 and cFXIII and analyzed by flow cytometry. Expression of cFXIII mRNA was analyzed by real‐time qPCR. FXIII deficient patients were subjected to ophthalmological screening and corneal topography.

Results: FXIII‐A was detected in corneal stromal CD34 + keratocytes by immunfluorescence staining. cFXIII positive keratocytes were unevenly distributed; they were abundant in the sub‐epithelial tertile of the stroma. cFXIII was of cytoplasmic localization, occasionally it also appeared in the nucleus. 2.87 ng cFXIII/mg corneal protein was measured by quantitative Western blotting. The synthesis of cFXIII by these cells was confirmed by RT‐qPCR. Flow cytometry revealed that 84% of isolated corneal cells were CD34 + keratocytes and two third of them were also stained for FXIII‐A. Crosslinks were detected only between the epithelial cells. Corneal topographic pictures of FXIII deficient patients showed mild keratoconus.

Conclusions: cFXIII is present in a relatively high number of corneal keratocytes. The transglutaminase activity of keratocyte cFXIII might play a role in maintaining the biomechanical structure of healthy cornea.

PB413: The Effect of Factor XIII Levels on the Incorporation of α2‐plasmin Inhibitor into Fibrin Clots: Association with the Therapeutic Outcome of Acute Ischemic Stroke Patients

Z. Bagoly 1,2 ; B. Baráth 1 ; I. Szegedi 3 ; R. Kálmándi 1 ; L. Csiba 2,3 ; É. Katona 1

1 University of Debrecen, Department of Laboratory Medicine, Division of Clinical Laboratory Sciences, Debrecen, Hungary, 2 MTA‐DE Cerebrovascular and Neurodegenerative Research Group, Hungarian Academy of Sciences, Debrecen, Hungary, 3 University of Debrecen, Department of Neurology, Debrecen, Hungary

Background: The cross‐linking of α2‐plasmin inhibitor (α2PI) to fibrin by activated factor XIII (FXIII) is essential for the inhibition of fibrinolysis.

Aims: Here we aimed to investigate the role of FXIII levels in modifying α2PI incorporation into the fibrin clot and its effect on the therapeutic outcome of acute ischemic stroke (AIS) patients.

Methods: α2PI activity/antigen, FXIII activity/antigen and fibrinogen levels were measured from the plasma samples of 88 individuals (62 AIS patients, all within 4.5 hours of their symptom onset before thrombolysis treatment and 26 age‐matched healthy controls). After clotting the plasma samples by thrombin and Ca 2+ , α2PI antigen levels were measured from the serum and the extent of α2PI incorporation was calculated. The amount of α2PI incorporation into fibrin clots was compared between patients and controls and the modifying effect of FXIII levels was studied. In the patient cohort results were correlated with stroke severity and thrombolysis outcome. Stroke severity was determined using the National Institutes of Health Stroke Scale (NIHSS). Patient outcome was registered at 7 days post‐lysis based on the change in NIHSS, imaging and clinical data.

Results: In the whole cohort FXIII levels significantly correlated with the amount of α2PI incorporation into fibrin clots (r=0.431, P=0.001). In controls and in patients with good outcomes the extent of α2PI incorporation did not differ significantly (49.2±4.2% vs. 47.4±6.7%, P=1.000). In patients suffering post‐lysis intracranial hemorrhage, α2PI incorporation was significantly lower (38.1±13.8%) as compared to controls and to those with good outcomes (P=0.004 and P=0.028, respectively).

Conclusions: Increased FXIII levels result in elevated incorporation of α2PI into fibrin clots. In stroke patients the extent of α2PI incorporation seems to have an effect on the outcome of therapy, particularly on the occurrence of thrombolysis‐associated intracranial hemorrhage.

Funding: NKFI K120633, PD111929

PB414: Peptides Pro‐Gly‐Pro and Pro‐Gly‐Arg Reduce the Level of Fibrinogen and the Activity of Factor XIII

T. Obergan 1 ; M. Grigorjeva 2

1 M.V. Lomonosov Moscow State Unoversity, Biology, Moscow, Russian Federation, 2 Moscow State University named after M.V. Lomonosov, Biology, Moscow, Russian Federation

Background: Previously it was shown that short regulatory peptides, containing proline and glycine (glyprolines), have anticoagulant and fibrinolytic activity in the organism. However, these effects are short‐lived due to the low stability of glyprolines. Adding to the peptide molecule of the amino acid arginine increases its resistance and anticoagulant properties. At this time the influence of glyprolines on blood coagulation factors such as fibrinogen and fibrinase (FXIIIa), has not been studied.

Aims: To study the influence of the peptides Pro‐Gly‐Pro (PGP) and Pro‐Gly‐Arg (PGR) on activity of factor XIIIa, fibrinogen level and anticoagulant activity of blood in healthy organism.

Methods: Thirty healthy male Wistar rats (200¬‐220 g body weight) were used in this study, which was carried out in accordance with ethical principles of the Helsinki Declaration. Peptide‐treated rats received PGP (group 2, n=10) or PGR (group 3, n=10) by intranasal way daily during 5 days (1 mg/kg of the body weight). Untreated rats of group 1 (control) received 0.85% saline. 1 h after 5rd administration of drugs activity of factor FXIIIa, fibrinogen level (Clauss) and anticoagulant activity (activated partial thromboplastin time, APTT) were measured in blood plasma samples using commercial reagents.

Results: As shown in Table 1, the administration of PGP and PGR caused a reduction in fibrinogen levels (by 20 and 25%, respectively) and a decrease in the activity of the FXIIIa (by 15 and 25% from control) in the blood plasma of animals. This was an increase in clotting time (APTT) after PGP‐ and PGR‐treatment compared to the control group at 21% and 27%, respectively. The effects of the peptide PGR were more pronounced.

TABLE 1 Coagulation parameters in blood plasma, measured after 5‐fold of the peptides to healthy rats Laboratory test Control n=10 Experimental groups Pro‐Gly‐Pro n=10 Pro‐Gly‐Arg n=10 APTT (sec) 29.4 35.6 37.3 Fibrinogen (g/l) 3.12 2.49 2.34 FXIIIa (%) 100.0 84.9 75.0

Conclusions: In the present study demonstrated the inhibitory effect of peptides PGP and PGR on blood coagulation factors due to decrease of activity of FXIIIa and reduce the level of fibrinogen. These properties, in the case of contact glyprolines in the bloodstream, reduce pro‐thrombotic potential of the organism.

PB415: Fibrin Fiber Mechanical and Structural Properties Are Altered in Individuals with Cardiovascular Disease on Low/Medium Dose Aspirin

M. Guthold

Wake Forest University, Physics, Winston‐Salem, United States

Background: Blood clots perform the mechanical task of stemming blood flow. To better understand blood clot behavior it is, therefore, important to understand the mechanical and structural properties of their main constituents ‐ fibrin fibers.

Aims: Investigate how single fibrin fiber mechanical and structural properties are altered in cardiovascular disease (CVD) patients who also take low or medium dose aspirin.

Methods: Plasma samples were collected from healthy younger and healthy older males, and older males with CVD who took low or medium dose aspirin (5 individuals/group). An atomic force microscopy (AFM)‐based technique was used to determine single fibrin fiber mechanical properties (extensibility, elasticity, stiffness) and intra‐fiber bond density. Optical microscopy was used to determine intra‐fiber protein density. Turbidimetry and confocal microscopy was used to determine fibrin clot structural properties.

Results: Fibrin fibers from older males with CVD (plus aspirin) were 30% more extensible and 50% more elastic than those from the healthy groups. Age did not affect single fibrin fiber properties. Fibers from all three groups had the same average Young's modulus (stiffness), Y. However, Y strongly depended on fiber radius, R; for healthy individuals it varied as Y ˜ R −1.5 , whereas it varied as Y ˜ R −1.0 for individuals from the CVD + aspirin group. This radius dependence of the modulus suggests a new fiber model, in which fibers have a dense, well‐connected core and a less dense, poorly connected periphery. This radius dependence was less pronounced in the CVD + aspirin group. Turbidimetry also showed altered internal structure.

Conclusions: Males with CVD who take low/medium dose aspirin have fibrin fibers with an altered internal structure that are more elastic and more extensible, as compared to fibers from healthy males.

PB416: Factor XIII Concentrate Can Improve Clot Stabilization Guided by Thromboelastometry in Patient with Liver Disease: A Case Report

T. Crochemore 1 ; F. Savioli 2

1 Hospital Israelita Albert Einstein, Intensive Care Unit, São Paulo, Brazil, 2 Hospital Israelita Albert Einstein, ICU, São Paulo, Brazil

Background: Traditionally coagulopathy in liver disease has been considered the prototype of hemorrhagic disease. Thromboelastometry access the whole process of clot formation including thrombin generation, clot formation and clot stabilization. Factor XIII participates in both the clot firmness but also in the It's stabilization. Factor III deficiency can be accessed by thromboelastometry at maximum lysis (ML) >15% in the Extem but It's not corrected by the Aptem. Factor XIII concentrate have been used for correct factor III deficiency.

Aims: Factor XIII deficiency could be identified by thromboelastometry and the administration of Factor XIII concentrate could be capable of improve clot stabilization in patient with acute liver disease.

Methods: We report the case of a previously healthy 38‐year‐old man, Afro‐Brazilian, with no medical records until last week. He presented to our emergency department with an acute onset of abdominal pain, jaundice, fever, nausea, weakness and malaise.

Results: The laboratory examinations revealed serum creatinine 0.8 mg/dl), platelets 142×10 3 /mm 3 , serum fibrinogen 221 mg/dl, increased international nationalized ratio (INR 1.9) total bilirubin 3.9 mg/dl, direct bilirubin 2.3 mg/dl, ALT 751U/l, AST 540 U/l and ceruloplasmin 17 mg/dl without acute bleeding. A liver biopsy was indicated. Based on the results of the thromboelastometric tests EXTEM, FIBTEM and Aptem tranexamic acid was administered to correct hyperfibrinolysis. followed by factor XIII concentrate to correct factor XIII deficiency. Thromboelastometry was normal but CCT are still altered. So, liver biopsy was performed which succeeded with no signs of bleeding and without need of further transfusion.

Conclusions: Thromboelastometry may be considered a useful, feasible and safe tool to monitor and manage coagulopathy in patients with liver disease, with the potential advantage of helping avoid unnecessary transfusion in such patients.

PB417: Hypophibrinogenemia and Thrombosis: Report of the Experience of a Case

V. Olaya 1 ; J. Castaño 2 ; M. Insuasty 3 ; M. Pizza 4 ; A. Muñoz 5,6

1 Hospital Pablo Tobon Uribe, Antioquia, Medellín, Colombia, 2 Universidad de Antioquia, Antioquia, Medellín, Colombia, 3 Universidad CES, Antioquia, Medellín, Colombia, 4 Universidad Pontificia Bolivariana, Antioquia, Medellín, Colombia, 5 Universidad Pontificia Javeriana, Valle del Cauca, Cali, Colombia, 6 Universidad Pontificia Javeriana Cali, Cali, Colombia

Background: Fibrinogen disorders are rareentities. They can be either acquired or congenital, and are subdivided into qualitative (dysfrinogenemia), quantitative deficiencies (afibrinogenemia, hypofibrinogenemia) or mixed defects. The clinical manifestations are variable and stem from the absence of symptoms to hemorrhagic or thrombotic episodes.

Aims: Case report.

Methods: We present a case of a patient with a diagnosis of congenital hypofibrinogenemiawithpast episodes of posttraumatic bleeding whichrequired management with cryoprecipitate; who now is admitted due to the development of ischemic lesions in the right toes.

Results: After ruling out an autoimmune disease, successful treatment was achieved with the replacement of fibrinogen and anticoagulation with low molecular weight heparin.

Conclusions: Congenital fibrinogen disorders are infrequent, and the clinical spectrum is wide. In the case of hypofibrinogenemia, the majority of patients are asymptomatic, however thrombotic manifestations should be considered within the clinical spectrum, especially in the presence of predisposing factors, treatment should be individualized. In some cases, anticoagulation and replacement of fibrinogen can be a safe management strategy.

PB418: Plasma Fibrinogen Concentration As an Acute Marker of Myocardial Infarction

C.K. Dike 1 ; O.I. Ajayi 2

1 University of Benin, Medical Laboratory Science, Benin City, Nigeria, 2 University of Benin, Physiology, Benin City, Nigeria

Background: Myocardial infarction (MI) is defined as necrosis of a portion of cardiac muscle caused by obstruction in coronary artery through either artheriosclerosis, a thrombus or spasm. The causative factors has been well documented and its risk has been reduced to the barest minimum in advanced countries of the world while in developing countries such as Nigeria, the advent of MI as a major cardiovascular problem is moderately recent. Therefore, researches into the acute definitive markers are currently on‐going

Aims: We aimed to highlight basic information plasma fibrinogen concentration with a view to indicate its possible role as a haemostatic index of MI

Methods: We investigated longitudinally, 10 acute myocardial infarction (AMI) patients together with 20 age and sex ‐matched apparently healthy subjects as controls. Blood samples were taken at the point of admission (Day 0), on the 4th and 7th day respectively after treatment has commenced. Plasma Fibrinogen concentration (PFC) and Plasma viscosity (PV) as well as erythrocyte sedimentation rates (ESR) were measured using standard laboratory methods

Results: We recorded a significantly increased PFC, ESR and PV in AMI patients on admission compared with controls (P<0.05, respectively) which became significantly lowered from the 4th day of admission and treatment (P<0.05, respectively).

Conclusions: We conclude therefore that hyperfibrinogenaemia coupled with hyperviscosities could be likely associated risk factors of thrombosis in Nigerians with AMI and their reduction during treatment are positive indicators and as factorials in its pathogenesis

THROMBOTIC DISORDERS

PB419: Apixaban Does Not Generate False‐positive dRVVT Interpretations for Lupus Anticoagulants, Even at Peak Levels

O. Kumano 1 ; S. Platton 2 ; P. Jones 3 ; G. Moore 3

1 Sysmex Corporation, Protein Technology, Kobe, Japan, 2 Royal London Hospital, Haemophilia Centre, Barts Health NHS Trust, London, United Kingdom, 3 Viapath at Guy's and St. Thomas’ Hospitals, Haemostasis and Thrombosis, London, United Kingdom

Background: Laboratory tests of lupus anticoagulant (LA) are necessary for antiphospholipid syndrome (APS) diagnosis. Recently, direct oral anticoagulants (DOACs) including apixaban have been used for thrombosis prevention. It has been shown that these drugs may affect the results of LA assays by increasing the rate of false positive interpretations.

Aims: To assess the effect of apixaban on detection of LA with a single dRVVT system.

Methods: Seven commercial normal plasma samples spiked with increasing concentrations of apixaban (0‐578 ng/ml) were used to investigate the relationship between the concentration and the normalised screen/confirm ratio in dRVVT reagents. The % correction was also calculated, and the sample was considered as LA‐positive when % correction showed ≥10%. Four positive, one equivocal and fourteen negative/not LA samples in taipan snake venom time with apixaban were also employed and measured on an automated coagulation analyser.

Results: Although both the screen and confirm ratios were elevated at concentrations above 80 ng/ml, the % correction was not affected by DOAC interference because the screen and confirm ratios were elevated to the same degree and therefore did not generate % correction values above the cut‐off.

Conclusions: The regression equations in screen and confirm ratios were very similar and it was considered that, in contrast to published reports on rivaroxaban and edoxaban, the effects of apixaban was independent of the phospholipid concentrations in the reagents. The concordant screen and confirm ratio elevations over a range of apixaban concentrations in spiked normal plasmas was mirrored in LA‐negative clinical samples from patients receiving apixaban whose % corrections were below the cut‐off. Thus, the elevated % corrections in known LA‐positive plasmas were genuine positive interpretations, indicating that apixaban does not interfere with dRVVT interpretations for LA in the single reagent studied.

PB420: Pregnancy Outcomes in Women with Antiphospholipid Antibodies with a History of Late Fetal Loss: Interim Results from a Single Centre Cohort

M. Frishman 1 ; K. Schreiber 1,2 ; K. Breen 1 ; M.J. Cuadrado 1 ; B.J. Hunt 1

1 Guy's and St Thomas’ NHS Foundation Trust, Thrombosis & Thrombophilia, London, United Kingdom, 2 Copenhagen University Hospital, Rigshospitalet, Copenhagen Lupus and Vasculitis Clinic, Center for Rheumatology and Spine Diseases, Copenhagen, Denmark

Background: Antiphospholipid syndrome (APS) is diagnosed in individuals with a persisting antiphospholipid antibody (aPL) with previous arterial or venous thromboembolic events (thrombotic APS) and/or previous adverse obstetric events including recurrent miscarriage, late fetal loss or early delivery due to pre‐eclampsia or placental insufficiency (obstetric APS). There is limited data about pregnancy outcomes in women with a previous late fetal loss (defined as an otherwise unexplained pregnancy loss >24 weeks of gestation).

Aims: To report the results on fetal and maternal outcomes from 28 women with a history of fetal loss.

Methods: This is a retrospective observational study. Data was collected from the Department of Thrombosis & Haemostasis and Lupus pregnancy clinic at St. Thomas’ Hospital in London, UK. Patients were identified from clinic lists (Jan 2010 to December 2017). Women persistently positive for aPL were included if complete pregnancy outcome data was available.

Results: A total of 511 pregnancies in 372 women were included in the study (Table 1). Twenty‐eight of these pregnancies were in women with a prior history of intrauterine death, amongst which there were three placental abruptions.

The overall live birth rate was 71%. Fetal outcomes were as follows: Rate of preterm delivery <34 weeks gestation and <37 weeks was 4% and 7%, respectively. Birthweight <1500 g and <2500 g was 4% and 11%, respectively. The rate of pregnancy loss at <10 weeks and >10 weeks gestation was 18% and 4%, respectively. Intrauterine death after 24 weeks occurred in 7%. Maternal outcomes were as follows: 4% developed pre‐eclampsia and 39% had a caesarean section (Table 2).

TABLE 1

TABLE 2

Conclusions: These results from the largest single centre cohort show that women with a previous intrauterine death have a live birth rate of 71%. The recurrence of intrauterine death was 7% when treated according to our local protocol.

PB421: Pregnancy Outcomes in Women with Antiphospholipid Antibodies: Interim Results from a Single Centre Cohort Study

K. Schreiber 1,2 ; M. Frishman 1 ; K. Breen 1 ; M.J. Cuadrado 1 ; B.J. Hunt 1

1 Guy's and St Thomas’ NHS Foundation Trust, Thrombosis & Thrombophilia, London, United Kingdom, 2 Copenhagen University Hospital, Rigshospitalet, Copenhagen Lupus and Vasculitis Clinic, Center for Rheumatology and Spine Diseases, Copenhagen, Denmark

Background: Persisting antiphospholipid antibodies (aPL) are associated with adverse obstetric events including recurrent miscarriage, late fetal loss or early delivery due to pre‐eclampsia or placental insufficiency (obstetric APS) and also thrombosis. We are a tertiary referral centre for those with aPL, and have a management protocol for women with aPL during pregnancy & the puerperium with the aim of preventing obstetric complications and maternal thrombosis.

Aims: To report the fetal and maternal outcomes from a single centre cohort of 511 pregnancies in 372 women over a period of eight years.

Methods: This is an ongoing retrospective observational study registered as an audit. Data was collected from clinic lists of patients attending the pregnancy clinic at the Thrombosis centre of St. Thomas’ Hospital in London, UK between Jan 2010 to December 2017. Women persistently positive for aPL were included if pregnancy outcome data was available

Results: 511 pregnancies in 372 women were included in the study (Table 1). The overall live birth rate was 79%. Fetal outcomes were as follows: rate of preterm delivery <34 weeks gestation and <37 weeks was 2.5% and 10%, respectively. Birthweight <1500 g and <2500 g was 1% and 11%, respectively. The rate of pregnancy loss at <10 weeks was 14%. Intrauterine death after 24 weeks occurred in 1%. Maternal outcomes were as follows: 5.5% developed pre‐eclampsia and 33% had a caesarean section (Table 2).

TABLE 1

TABLE 2

Conclusions: These results from the largest single centre cohort reported show that using our management protocol, nearly 80% of women with aPL had a successful pregnancy outcome.

PB422: Antiphospholipid Syndrome: Maternal and Perinatal Outcome after Treatment in Next Pregnancy. Retrospective Analysis

B. Grand 1 ; P. Galatti 2 ; L. Voto 2 ; G. Mainetti 3

1 Hospital Juan A Fernandez, Maternal and Fetal and Hematology, Buenos Aires, Argentina, 2 Hospital Juan A Fernandez, Buenos Aires, Argentina, 3 Hospital Juan A Fernandez, Laboratory Thrombosis and Hemostasis, Buenos Aires, Argentina

Background: The antiphospholipid syndrome (APS) is associated with pregnancy‐related morbidity, and/or arterial or venous thrombosis. The use of Low Dose of Aspirin (LDA) and heparin (First Line Therapy: FLT) has improved pregnancy outcome and approximately 70% of pregnant women with APS will deliver a viable live infant. However, current management does not prevent all maternal, fetal, and neonatal complications of APS and fails in 20 to 30% of APS pregnancies.

Aims: To describe the maternal and perinatal outcome in next pregnancy of women with APS after receiving therapy with Low Molecular Weight Heparin (LMWH) +LDA.

Methods: Only patients that fulfilled Sydney criteria were included n=17; pregnancies (pg)=19. Classification: Thrombotic: 1; Only pg morbidity (PgM): 14; Fetal death (FD):9; Severe preeclampsia (sPE): 2; placental insufficiency (PI) 2; Recurrent abortion (RPL): 3; 2 with both complications: 1 + 2b and 1 + 2a.

According to the laboratory categories 9/19 were in category I, 8 in II a, 1 in IIb and 1 in II c. Treatment: LMWH: Enoxaparin 40 mg initial dose and aspirin 100 mg/day; Intermediate or therapeutic doses in thrombotic APS. Period: 6 years (2011‐17).

Results: There were no thrombotic events. Women with previous sPE and PI showed bad response to FLT and these finding correlates with their first trimester screening risk assessment (Fetal Medicine Foundation) with increased risk for developing sPE before 34 weeks and PI. Post natal death and PI were observed only in women with previous sPE or PI (2/4 y 2/4 respectively).

Conclusions: Our results suggest that current FLT for APS in pregnancy prevent obstetric complications in women with history of RPL or FD, but not in those with previous sPE and/or PI. The poorest obstetric outcome was observed in women with triple positivity. The division into two groups of obstetric APS (RPL/FD and PI/sPE) and the triple positivity might facilitate the choice of early additional therapy in these women.

PB423: Are IgM Antiphospholipid Antibodies Redundant in the Classification Criteria for the Antiphospholipid Syndrome?

W. Chayoua 1,2 ; H. Kelchtermans 1,2 ; G. Moore 3 ; J.‐C. Gris 4 ; J. Musial 5 ; D. Wahl 6 ; S. Zuily 6 ; A. Tripodi 7 ; P. Fontana 8 ; J. Remijn 9 ; B. de Laat 1,2 ; K.M. Devreese 10

1 Maastricht University Medical Center, Biochemistry, Maastricht, the Netherlands, 2 Maastricht University Medical Center, Synapse Research Institute, CARIM, Maastricht, the Netherlands, 3 St Thomas's Hospital, London, United Kingdom, 4 Centre Hospitalier Universitaire Nîmes, Nîmes, France, 5 Jagiellonian University Medical College, Krakow, Poland, 6 Centre Hospitalier Universitaire de Nancy, Nancy, France, 7 Università degli Studi di Milano, Milano, Italy, 8 University Hospital Geneva, Geneva, Switzerland, 9 Gelre Hospitals, Apeldoorn, the Netherlands, 10 Ghent University Hospital, Ghent, Belgium

Background: The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with the persistent presence of antiphospholipid antibodies (aPL). Laboratory criteria include aPL detection by lupus anticoagulant (LAC), anti‐β2glycoprotein I (aβ2GPI) and anti‐cardiolipin (aCL) IgG/IgM antibodies. Although all aPL have the same value in the Sydney classification criteria, the clinical and diagnostic relevance of IgM aPL in APS has been debated.

Aims: We investigated whether aCL and/or aβ2GPI IgM have an added value in the identification of thrombotic and obstetric patients.

Methods: 1068 patients and controls were included from 8 European centers (Table 1). Samples were tested with 4 commercially available solid phase assays at one location by a single technician to exclude assay dependence and inter‐laboratory variation, respectively. LAC positivity was determined by the local center.

TABLE 1 Characterization of the study population Patients (n) Female Age [year, mean (range)] Pregnancy morbidity Venous thrombosis Arterial thrombosis Venous and arterial thrombosis Small vessel thrombosis Unspecified thrombosis APS thrombosis 259 164 (63%) 50 (17‐87) 22 160 55 26 4 14 Non‐APS thrombosis 204 116 (57%) 46 (19‐85) 0 149 47 5 0 3 APS obstetric 122 122 (100%) 35 (19‐61) 122 4 2 0 0 4 Non‐APS obstetric 33 33 (100%) 33 (20‐52) 33 Not applicable Not applicable Not applicable Not applicable Not applicable Autoimmune diseases 196 158 (81%) 46 (16‐83) Not applicable Not applicable Not applicable Not applicable Not applicable Not applicable Controls (Referred for aPL testing for other reasons than APS) 194 170 (88%) 39 (18‐82) Not applicable Not applicable Not applicable Not applicable Not applicable Not applicable Unspecified APS 60 60 (100%) 48 (24‐70) Not applicable Not applicable Not applicable Not applicable Not applicable Not applicable Total population 1068 823 (77%) 44 (16‐87) 177 313 104 31 4 21

Results: Significant odd ratios (OR) were found for LAC, IgG or IgM for thrombosis and/or pregnancy morbidity, independent of the assay used (Table 2). LAC showed a strong association, including the majority of aCL and aβ2GPI IgG/IgM positives. Also isolated LAC positivity correlated with thrombosis and/or pregnancy morbidity, in contrast to single IgM positivity (=negative for LAC) for all platforms, and to single IgG positivity, for two out of the four platforms (Table 2). Adding IgM to the LAC‐ aCL/aβ2GPI IgG panel did not increase the OR. Triple positivity (LAC, aCL and aβ2GPI) with the IgG isotype increased the correlation with thrombosis and/or pregnancy morbidity, while triple positivity with the IgM isotype did not (Table 2).

TABLE 2 Correlation of aPL profiles with thrombosis and/or pregnancy morbidity. Odds ratios with 95% CI are shown (significant OR in bold) BioPlex®2200 ImmunoCap®EliA ACL AcuStar® QUANTA Lite ELISA® BioPlex®2200 ImmunoCap®EliA ACL AcuStar® QUANTA Lite ELISA® LAC 3.63 (2.76‐4.76) 3.63 (2/76‐4.76) 3.63 (2.76‐4.76) 3.63 (2.76‐4.76) LAC+aCL IgG+aCL IgM 3.70 (2.84‐4.84) 3.41 (2.63‐4.43) 3.74 (2.87‐4.87) 4.07 (3.12‐5.32) aCL IgG 3.87 (2.69‐5.58) 5.16 (3.37‐7.90) 4.95 (3.28‐7.49) 5.55 (3.49‐8.83) LAC+aβ2GPI IgG+ B2GPI IgM 3.87 (2.96‐5.05) 4.04 (3.09‐5.29) 3.92 (3.00‐5.12) 3.73 (2.85‐4.87) aCL IgM 2.76 (1.71‐4.47) 4.08 (2.71‐6.15) 3.29 (2.36‐4.57) 6.25 (3.55‐11.02) IgG Only 1.08 (0.95‐2.57) 2.10 (0.90‐4.90) 2.70 (1.02‐7.17) 3.08 (1.05‐9.05) aβ2GPI IgG 3.48 (2.44‐4.96) 1.79 (1.27‐2.52) 1.91 (1.31‐2.78) 2.74 (1.83‐4.10) IgM Only 2.04 (0.67‐6.23) 0.58 (0.34‐0.99) 0.88 (0.43‐1.79) 1.08 (0.58‐2.01) aβ2GPI IgM 3.20 (1.99‐5.16) 2.50 (1.62‐3.87) 3.14 (1.89‐5.23) 2.63 (1.73‐4.01) Negative LAC+IgG 1.16 (0.55‐2.41) 2.24 (1.06‐4.71) 1.94 (0.95‐3.99) 3.07 (1.117‐8.06) LAC+aCL IgG 3.66 (2.80‐4.78) 3.97 (3.03‐5.20) 3.87 (2.96‐5.07) 4.00 (3.05‐5.24) Negative LAC+IgM 1.75 (0.74‐4.15) 0.71 (0.43‐1.17) 0.96 (0.53‐1.74) 1.19 (0.66‐2.16) LAC+aCL IgM 3.78 (2.89‐4.96) 3.16 (2.43‐4.10) 3.53 (2.71‐4.60) 3.80 (2.91‐4.96) Triple positive with the IgM isotype 3.25 (1.84‐5.73) 3.20 (1.82‐5.65) 3.80 (2.04‐7.09) 3.49 (1.98‐6.14) LAC+aβ2GPI IgG 3.71 (2.83‐4.85) 3.90 (2.98‐5.10) 3.92 (2.99‐5.13) 3.77 (2.87‐4.94) Triple positive with the IgG isotype 4.64 (3.06‐7.02) 5.28 (3.18‐8.78) 5.60 (3.52‐8.40) 6.75 (3.58‐12.70) LAC+aβ2GPI IgM 3.83 (2.92‐5.01) 3.78 (2.89‐4.95) 3.69 (2.82‐4.83) 3.64 (2.78‐4.75) Triple positive 4.08 (2.80‐5.94) 4.00 (2.67‐6.00) 4.,64 (3.10‐6.95) 4.41 (2.81‐6.92)

Conclusions: As part of the full aPL panel, aCL and aβ2GPI IgM correlate significantly with thrombosis and/or pregnancy morbidity. Our data suggest that detection of LAC and IgG aPL is sufficient for the identification of APS patients. Triple positivity for the IgG (but not IgM) isotype can improve risk stratification in APS patients.

PB424: A Single‐arm Pilot Feasibility Cohort Study of Rivaroxaban in Antiphospholipid Syndrome

K. Legault 1 ; M. Blostein 2 ; M. Carrier 3 ; S. Khan 2 ; S. Schulman 1 ; S. Shivakumar 4 ; C. Wu 5 ; M. Crowther 1

1 McMaster University, Hamilton, Canada, 2 McGill University, Montreal, Canada, 3 The Ottawa Hospital, Ottawa, Canada, 4 Dalhousie University, Halifax, Canada, 5 University of Alberta, Edmonton, Canada

Background: Antiphospholipid syndrome (APS) is characterized by venous or arterial thrombosis and/or recurrent pregnancy loss in patients with persistent antiphospholipid antibodies. Accepted optimal therapy to prevent recurrent thrombosis in APS is warfarin, however requires careful monitoring. Novel oral anticoagulants such as rivaroxaban are used for many indications, though at present there is little evidence to support the use of rivaroxaban in patients with APS.

Aims: This was a pilot feasibility study that aimed to identify 150 eligible APS patients over 6 sites, to obtain consent from 135 patients, and to obtain 95% compliance with rivaroxaban. Secondary outcomes included rates of thrombosis and bleeding.

Methods: Patients fulfilling criteria for APS with prior venous thromboembolism (VTE) were recruited over 2 years (Oct 2014‐Sept 2016), and followed for >1 year. Patients were assessed every 3 months with pill count every 6 months. Data were analyzed by comparing recruitment to the feasibility endpoints, and by calculating rates of thrombosis/bleeding. Informed consent and research ethics board approval were obtained. The study was funded by the Heart and Stroke Foundation.

Results: 93 eligible patients were identified, and 11 declined. 82 patients were followed for a mean of 18.8 months, representing 128.7 patient‐years. During follow‐up, there were 3 thromboembolic events (2 cerebrovascular, and 1 pulmonary embolus). 1 patient died of pneumosepsis, and 2 patients were withdrawn for non‐study associated adverse events. There were no episodes of major bleeding.

Conclusions: In this study, no safety signals were reported, and the rate of thromboembolism is similar to previous studies of warfarin in APS, implying that rivaroxaban is relatively safe and efficacious in APS patients with VTE. The study did not meet its feasibility endpoints, showing that adequately‐powered comparative trials with patient‐important outcomes in APS are unlikely to be successful, even when recruiting through multiple centres.

PB425: Antiphospholipid Antibodies Activate the Mechanistic Target of Rapamycin (mTOR) and Disrupt Lysosomal Degradation with Effects upon Monocyte Autophagy

V.M. Ripoll ; A. Khawaja; I.P. Giles; A. Rahman

University College London, Centre for Rheumatology, Division of Medicine, London, United Kingdom

Background: Antiphospholipid antibodies (aPLs) activate monocytes in antiphospholipid syndrome (APS), although the precise mechanisms by which this activation occurs are not fully understood. Autophagy has been recognised as an important contributor to aberrant immunity in autoimmune disorders. The mTOR‐signalling pathway has been linked to renal APS and can regulate several cellular processes including autophagy.

Aims: To determine the effect of human IgG aPL on monocyte autophagy.

Methods: Healthy monocytes were treated with 200 μg/ml of IgG purified from (n=5) patients with APS or (n=5) healthy control (HC) IgG for 6 h in the presence or absence of GMCSF. Levels of mTOR, ULK‐1, Beclin 1 and P62 were measured by western blot. Subsequently, monocytes from patients with APS and matched HC were isolated and treated with APS or HC IgG in the presence or absence of GMCSF. Intracellular lysosomal activity was determined using DQ‐BSA.

Results: aPL activated the mTOR signalling pathway in monocytes. The effects of APS‐IgG and HC‐IgG on monocyte mTOR signalling are shown in the table below. Consistent with the increased in levels of phosphorylated mTOR, levels of phosphorylated ULK1 were reduced and P62 was accumulated in the presence of APS‐IgG. No differences in the expression of Beclin 1 were found. The phenotype observed was indicative of reduced autophagy; hence, we measured lysosomal activity. APS‐IgG reduced the lysosomal degradation in GM‐CSF‐treated HC and APS monocytes whereas HC‐IgG had no effect.

TABLE 1 The effects of APS‐IgG and HC‐IgG on the expression of mTOR signalling molecules in monocytes APS HC p‐mTOR ↑ ↑ ‐ p‐ULK1 ↓ ↓ ↓ ↑ ↑ P62 ↑ ↑ ↑ ↓ Beclin 1 ‐ ‐

Conclusions: We found that APS‐IgG activate the mTOR pathway and disrupt lysosomal degradation during monocyte autophagy. Additional experiments are now underway to increase our understanding of how modulation of the mTOR pathway may be important in the pathogenesis of APS and provide new therapeutic targets.

PB426: Use of Direct Oral Anticoagulant in Antiphospholipid Syndrome: Systematic Review of Literature

V. Dufrost; S. Zuily; D. Wahl

CHRU de Nancy, Vascular Medicine Division and Regional Competence Center for Rare Vascular and Systemic Autoimmune Diseases, Vandoeuvre les Nancy, France

Background: Direct oral anticoagulants (DOACs) are approved for prevent thrombotic recurrence in venous thromboembolism disease but their clinical efficacy and safety are not studied in APS patients. The RAPS trial demonstrated an increased thrombin potential in APS patients with rivaroxaban versus warfarin, suggesting a higher thrombotic risk. Authors concluded that rivaroxaban is safe in APS patients because there was not thrombotic recurrence during the follow up of 7 months. Several reports of APS patients treated with DOACs have raised safety issues with thrombotic recurrence during treatment.

Aims: To clarify the safety of DOACs in APS patient and factors associated with thrombosis recurrence.

Methods: We summarize available literature on DOACs use in APS patients through a systematic review. We used Wilcoxon and Fisher's exact test. Missing data were excluded from analyses.

Results: Our systematic review identified 373 published APS patients treated with DOACs; among them, 49 experienced a recurrent thrombosis while on treatment. The three DOACs used was rivaroxaban (N=227), dabigatran etexilate (N=142) and apixaban (N=4); there was no difference between them. Triple positivity (positivity of all three laboratory criteria for APS) was associated with a 4‐fold increased risk for recurrent thrombosis (22% vs. 54%; P<0.05). There was no difference between previous APS manifestation. Comparisons of patients’ characteristics according to the presence of a thrombotic recurrence while on DOACs are available in Table 1. test. Missing data were excluded from analyses.

TABLE 1 Comparisons of patients’ characteristics according to the presence of a thrombotic recurrence while on DOACs APS without recurrence thrombosis (n=324) APS with recurrence thrombosis (n=49) P value aPL profile : LA 135/205 (66) 29/42 (69) aCL 126/205 (61) 38/42 (90) <0.05 Antiβ2GP1 90/205 (44) 31/40 (78) <0.05 Triple positivity 49/221 (22) 22/41 (54) <0.05 Oral anticoagulant: Rivaroxaban 200/324 (62) 27/49 (55) Dabigatran 120/324 (37) 22/49 (45) Apixaban 4/324 (1) 0 (0)

Conclusions: DOACs should be used with caution in APS patients and randomized control trials with clinical primary endpoints assessing clinical efficacy and safety are awaited to establish whether the prescription of DOACs could be a safe alternative to warfarin.

PB428: Pregnancy Outcomes in Women with Antiphospholipid Antibodies in IVF Conceived Pregnancies: Interim Results from a Single Centre Cohort

M. Frishman 1 ; K. Schreiber 1,2 ; K. Breen 1 ; M.J. Cuadrado 1 ; B.J. Hunt 1

1 Guy's and St Thomas’ NHS Foundation Trust, Thrombosis & Thrombophilia, London, United Kingdom, 2 Copenhagen University Hospital, Rigshospitalet, Copenhagen Lupus and Vasculitis Clinic, Center for Rheumatology and Spine Diseases, Copenhagen, Denmark

Background: Antiphospholipid syndrome (APS) is defined by the presence of antiphospholipid antibodies (aPL) with thrombosis and/or obstetric morbidity. The obstetric morbidity includes recurrent first trimester loss, stillbirth, intrauterine death, pre‐eclampsia, premature birth and fetal growth restriction. We have an established management protocol for women with aPL entering pregnancy aiming to prevent obstetric morbidity and maternal thrombosis.

Little is currently known about pregnancy outcomes in women with aPL who required in vitro fertilisation (IVF) treatment.

Aims: To report the results on pregnancy outcomes from 70 pregnancies conceived by IVF taken from a cohort of 511 women and 372 pregnancies in women with aPL who were seen at our centre from 2010‐2017.

Methods: This is a retrospective observational study. Data was retrospectively collected from the pregnancy clinic in the Thrombosis centre at St. Thomas’ Hospital in London, UK. Women persistently positive for aPL were included if pregnancy outcome data was available.

Results: 70 of 511 pregnancies in 372 women were conceived by IVF and their details are shown in table 1. The overall live birth rate of aPL pregnancies conceived by IVF was 73%. Fetal outcomes were as follows: rate of preterm delivery <34 weeks gestation and <37 weeks was 1% and 10% respectively. Birthweight <1500 g and <2500 g was 7% and 7% respectively. The rate of pregnancy losses at <10 weeks and >10 weeks gestation were 21% and 4% respectively. Intrauterine death after 24 weeks occurred in 1%. Maternal outcomes were as follows: 7% developed pre‐eclampsia and 33% had a caesarean section. (Table 2).

TABLE 1

TABLE 2

Conclusions: Conception by IVF is no barrier for successful pregnancy outcomes in women with antiphospholipid antibodies and in this study there was no difference in pregnancy outcomes from natural conception outcomes. This study is ongoing.

PB429: New dRVVT Assays for Lupus Anticoagulant (LA) Detection with Improved Specificity and Sensitivity: HEMOCLOT LA‐S and LA‐C

C. Dunois ; M. Peyrafitte; S. De Muynck; V. Villedieu; J. Amiral

HYPHEN BioMed, Research and Validation, Neuville sur Oise, France

Background: Lupus Anticoagulant (LA) detection may be difficult in case of false positive results. New dRVVT assays with improved specificity and sensitivity were developed to facilitate LA detection.

Aims: To report performance characteristics of these new dRVVT assays, and their combination with APTT reagents (standard and LA sensitive), on various analyzers.

Methods: Assays can be used on all major coagulation instruments (CS‐series, STA‐R, BCS XP, …). Normal and abnormal control plasmas are used to evaluate within and between run performances (n³10) and interferences. Samples from healthy subjects, LA positive patients, or anticoagulated plasmas are used for comparison study with other dRVVT devices (Stago LA‐Screen/LA‐Confirm; Siemens LA‐1/LA‐2) and combined with APTT testing (CEPHEN LR/LS). Normalized ratios >1.20 are considered positive for LA.

Results: LA‐S/LA‐C showed low variability on normal and LA controls (CV within‐run 1.2 to 3.0%, between‐run 2.1 to 4.0%), and no interference was noticed up to 1 IU/ml for heparins or 400 ng/ml anti‐Xa direct oral anticoagulants. For LA‐S/LA‐C normalized ratios, the total agreement between analyzers was of 98% (STA‐R vs. CS n=47), 100% (BCS XP vs. CS n=57) and of 96% when compared to predicate devices (n=68 vs. Stago dRVVT, or n=50 vs. Siemens LA1/LA2). Some samples with a normal Rosner index were confirmed normal or borderline with LA‐S/LA‐C, while they were positive with other dRVVT assays, probably due to anticoagulant drug interferences. For CEPHEN LR/LS on CS, CVs for within and between run were of 0.3% to 1.5% for control plasma. Normalized CEPHEN LS/LR ratios were normal for 98% of normal and VKA plasmas (2 borderline), and positive for all 10 LA samples.

Conclusions: HEMOCLOT LA‐S/LA‐C and CEPHEN LR/LS provide a reliable and convenient test combination for improving LA detection on the major coagulation analyzers, with improved specificity, and lower interferences from anticoagulant drugs.

PB430: Beta 2 Glycoprotein I Domain V Disulfide Bond Reduction Alters its Conformation with Regard to Antiphospholipid Syndrome (APS)

I. Buchholz 1,2 ; T. McDonnell 3 ; P. Nestler 1,2 ; V. Ripoll‐Nunez 3 ; A. Radziszewska 3 ; A. Rahman 3 ; M. Delcea 1,2

1 University Greifswald, Biochemistry Institute, Greifswald, Germany, 2 ZIK HIKE, Greifswald, Germany, 3 University College London, Division of Medicine, London, United Kingdom

Background: Beta 2 glycoprotein I (beta2GPI) can adopt two main structural conformations: the closed (circular) form, where the first domain (DI) interacts with domain V (DV), and the open (linear) form which exposes a cryptic epitope within DI. The open, potentially antigenic conformation may lead to binding of autoantibodies to beta2GPI via DI, resulting in the antiphospholipid syndrome (APS). Beta2GPI also has alternative redox states based on oxidation/reduction of cysteines in DV. A previous study showed that APS patients have a lower proportion of beta2GPI in reduced state compared to healthy subjects. Though, the relationship between beta2GPI redox state and conformation is not known.

Aims: In this study we investigated the influence of reduction of disulfide bond Cys288‐Cys326 on the conformation of beta2GPI, which lies within DV near the putative DI interaction site.

Methods: Briefly, we prepared enzymatically reduced and biotinylated beta2GPI. Reduction was confirmed by Western blot analysis as well as circular dichroism (CD) spectroscopy. Beta2GPI conformation was determined using atomic force microscopy (AFM) imaging and hydrodynamic size determination by dynamic light scattering (DLS). ELISA was employed to investigate the binding of anti‐DI IgG antibodies purified from APS patients.

Results: Untreated beta2GPI was characterized being in oxidized state and mainly in closed conformation. After reduction, an increased amount of beta2GPI in open conformation (53%) was found.

TABLE 1 Percentage fractions of open and closed beta2GPI species Beta2GPI species Closed % Open % Untreated 93 7 After reduction 47 53

These findings are supported by a significant decrease in hydrodynamic size for reduced beta2GPI, consistent with the open form. ELISA indicated stronger binding of APS antibodies to reduced than untreated beta2GPI.

Conclusions: This in vitro study demonstrates that reduction of disulfide bond Cys288‐Cys326 can alter beta2GPI conformation from closed to open and triggers APS antibody binding. Although APS patients show a lower proportion of reduced beta2GPI in circulation, perhaps increased reduction plays a role in APS microenvironments.

PB431: Evaluation of Lupus Anticoagulant Testing: A Descriptive Single Centre Experience

S.F. Pinzon Mariño 1 ; A.J. Garcia Ortego 1 ; M. Villalba Montaner 1 ; E. Salvador Ruperez 1 ; J.M. Calvo Villas 1 ; N. Fernandez Mosteirin 1 ; P. Delgado Beltran 2

1 Hospital Universitario Miguel Servet, Haematology, Zaragoza, Spain, 2 Hospital Unioversitario Miguel Servet, Haematology, Zaragoza, Spain

Background: The diagnosis of antiphospholipid syndrome (APS) requires, besides the clinical criteria, the positive tests of antiphospholipid antibodies (cardiolipin antibody, β₂‐glycoprotein and lupus anticoagulant (LA). Based upon consensus criteria from the International Society for Thrombosis and Haemostasis (ISTH), confirmation of LA positive test requires the following criteria: prolongation of at least one of two phospholipid dependent clotting tests performed, evidence for inhibitory activity and phospholid dependence of the inhibitor on a confirmatory test.

Aims: The aims of our study were: to evaluate retrospectively the frequency testing for LA, the number of testing orders of LA and To assess laboratory confirmation of positive results of LA in our institution.

Methods: A cross‐sectional study was performed from the requests for demonstration of LA received in our Laboratory from June‐2016 to June‐2017. We analyzed the ordering departments, the clinical reports of the patients and the results of the laboratory study of LA (screening and LA positive tests confirmation).

Results: Out of 1.989 of LA tests performed, 184 (9.25%) were LA positive (82 β‐glycoprotein positive, 28 cardiolipin autoantibodies positive and 27 were double positive). Applying the confirmation tests criteria to LA positive samples suggested by ISTH 14 fulfils the diagnostic criteria of APS. Most of the requests were ordered by the following clinical Units: Internal Medicine (625), Haematology (380) and Neurology (225).

Conclusions: Our results show a extremely high requesting of LA tests leading a low rate of positive LA test (9.25%) and a very low rate of samples of patients (0.7%) meet APS diagnostic criteria. We consider the LA test is “overclaimed” leading to low diagnostic yield. Despite of using an adequate study protocol of the LA positive tests in our Laboratory based on ISTH recommendations, it is necessary to promote a better and more rational use of requesting LA test by clinical criteria.

PB432: Purified Anti‐β2‐Glycoprotein I IgG Potentiates Both Direct and Indirect Platelet Activation ‐ Report of 2 Cases

J. Kappelmayer 1 ; G. Szabo 1 ; I. Beke Debreceni 1 ; P. Soltesz 2

1 University of Debrecen, Faculty of Medicine, Department of Laboratory Medicine, Debrecen, Hungary, 2 University of Debrecen, Faculty of Medicine, Department of Internal Medcine, Debrecen, Hungary

Background: It was previously described that some of the direct and indirect platelet activation markers were elevated in antiphospholipid syndrome.

Aims: By using purified anti‐β2‐glycoprotein I (aβ2GPI) IgG from two patients with and without lupus anticoagulant (LA) positivity, we investigated their platelet activating capacities.

Methods: Antiphospholipid antibodies (aPL) were separated from two patients with antiphospholipid syndrome, both having thrombosis in their history. Patient 1 showed triple positivity for the laboratory criteria of APS: aβ2GPI, anti‐cardiolipin (aCL) and LA, while Patient 2 was double positive for aβ2GPI and aCL without LA. Separation of aβ2GPI IgG antibodies was performed by a two‐step affinity chromatography system by using Protein G and purified β2GPI. The effect of aPL antibodies was investigated by flow cytometry by detecting platelet‐monocyte (PMA) or platelet‐granulocyte aggregates (PGA), platelet P‐selectin (Psel) and platelet microparticles (MP).

Results: Anti‐β2GPI in itself did not cause detectable platelet activation either in direct or in indirect tests. However, 125 U/ml aβ2GPI separated from Patient 1 augmented the generation of PMA elicited by 10 μM thrombin receptor activating peptide (TRAP) from 16% to 35%, while 250 U/ml aβ2GPI increased it further to 46% after 5 minutes. Similarly, PGA was also increased dose dependently from 7% to 24% at 250 U/ml aβ2GPI. These data were in accordance with the results of direct platelet activation markers although elevation of Psel and MP values followed a somewhat different kinetics. The LA negative Patient 2 showed no potentiation of TRAP‐activation in any of the investigated parameters.

Conclusions: It is suggested that aβ2GPI potentiates platelet activation caused by low dose TRAP. LA activity may predict the cell activating capacity of anti‐β2GPI.

PB433: A Comparison between Mixing Test Specific Cut‐off and the Index of Circulating Anticoagulant with Multiple APTT and dRVVT in Specificity for Lupus Anticoagulant

O. Kumano 1 ; G. Moore 2

1 Sysmex Corporation, Protein Technology, Kobe, Japan, 2 Viapath at Guy's and St. Thomas’ Hospitals, Haemostasis and Thrombosis, London, United Kingdom

Background: Guidelines for lupus anticoagulant (LA) detection recommend mixing test interpretation with either a mixing test specific cut‐off (MTC) or index of circulating anticoagulant (ICA). We previously demonstrated MTC showed higher sensitivity than ICA in detecting the in vitro inhibition of LA employing multiple dAPTT and dRVVT.

Aims: To compare the specificity of MTC and ICA for LA in plasma samples with non‐LA causes of elevated clotting times with multiple APTT and dRVVT reagents.

Methods: Forty‐one FII, V, VIII, IX, X, XI, XII low activity samples (<1 to 20%) prepared from mixtures of normal and commercial factor deficient plasmas, nine haemophilia A and B (FVIII/FIX 2.2‐23.8 IU/dL), two APTT elevated plasmas (FV/XII 28.7‐39.9 IU/dL) and twenty‐nine warfarin with elevated APTT clinical plasmas were used to assess specificity. In addition, six clinical samples with FVIII inhibitors (1.02‐69.76 NBU/ml) and six plasmas from patients anticoagulated with rivaroxaban or apixaban for reasons other than antiphospholipid syndrome, were employed as LA negative inhibitor samples. These samples were assayed in undiluted and 1:1 mix with normal pooled plasma with four LA‐sensitive APTT, one dilute APTT (dAPTT) and two dRVVT reagents on an automated coagulation analyser. Frequencies of positivity and negativity by MTC and ICA are shown in Table 1.

Results: In all reagents, the negativity of ICA was higher than that of MTC in factor deficient and warfarin samples. On the other hand, the positivity of MTC was higher than that of ICA in inhibitor and DOAC samples. APTT‐based assays were more affected than dRVVT in deficient and warfarin samples.

TABLE 1 Frequency of negativity and positivity calculated from MTC and ICA Negativity in factor deficient and warfarin samples APTT A APTT B APTT C APTT D dAPTT dRVVT A dRVVT B Number of high screen ratio (n=81) 76 81 75 76 70 45 48 MTC negative 36% 32% 36% 43% 94% 29% 10% ICA negative 100% 79% 97% 97% 99% 96% 96% Positivity in inhibitor and DOAC samples Number of high screen ratio (n=12) 8 9 6 8 7 5 7 MTC negative 100% 78% 100% 88% 29% 60% 86% ICA negative 75% 22% 67% 63% 0% 20% 57%

Conclusions: MTC was compared with ICA for the specificity in several APTT and dRVVT reagents. When we perform the mixing test and interpret the data, it is important to understand the characteristics of the indexes for maximizing the diagnostic potential of mixing tests in LA detection.

PB434: Thrombotic Risk Assessment in Antiphospholipid Syndrome

A. Carmo 1 ; J. Pego 1 ; A. Mendes 1 ; R. Cunha 1 ; F. Rodrigues 1,2

1 Centro Hospitalar e Universitário de Coimbra, Clinical Pathology Department, Coimbra, Portugal, 2 Faculdade de Medicina da Universidade de Coimbra, Department of Histology and Embryology, Coimbra, Portugal

Background: Antiphospholipid syndrome (APS) is an acquired autoimmune disorder defined by the presence of thrombosis and/or pregnancy morbidity in patients persistently positive for antiphospholipid antibodies (aPL). To determine the risk of thromboembolic events the global APS score (GAPSS) was validated in different patients populations considering as risk factors hypertension, hyperlipidaemia and the presence of aPLs (lupus anticoagulant (LA), antibodies anti‐cardiolipin (aCL), anti‐β2‐glycoprotein‐I (β2GPI), and anti‐phosphatidylserine/anti‐prothrombin complex (aPS/PT)). Since the evaluation of aPS/PT IgG is not available in many laboratories, recently it was proposed the adjusted GAPSS (aGAPSS).

Aims: Evaluate the thrombotic risk in patients diagnosed with APS and in patients that are aPL positive without thromboembolic disease.

Methods: 50 consecutive patients diagnosed with APL and aPL positive were evaluated. The aGAPSS was calculated by adding the points of the risk factors (3 points for hyperlipidemia, 1 point for hypertension, 5 points for aCL IgG/IgM, 4 points for anti‐β2GPI IgG/IgM and 4 for LA).

Results: There were 13 men, mean age: 55 (SD=12; min:33; max:75) and 37 women, mean age: 47 (SD=14; min:18; max:85). 23 patients (46%) fulfilled the current APS classification criteria and 27 patients (54%) were positive for aPL but did not had complaints of vascular thrombosis nor pregnancy morbidity. These 27 patients were diagnosed with autoimmune diseases such as systemic lupus erythematosus, undifferentiated connective tissue disease, and mixed connective tissue disease. The aGAPSS of aPL positive patients was 9.1 (SD=4.4), and the aGAPSS of APS patients was 12.2 (SD=3.4), P<0.004. The aGAPSS of APS patients with one aPL positive was 7.2 (SD=1.2) and the aGAPSS of triple positive APS patients was 14.8 (SD=1.4), P<0.001.

Conclusions: Higher aGAPSS were seen in patients with thrombotic events and in triple positive APS patients. Its applicability may help to establish an adequate follow‐up protocol.

PB435: Importance of Evaluation of Different Antiphospholipid Antibodies

A. Makatsariya ; J. Khizroeva; V. Bitsadze; M. Arslanbekova

I.M. Sechenov First Moscow State Medical University, Department of Obstetrics and Gynecology, Moscow, Russian Federation

Background: It′s well known that triple positivity (the presence of all laboratory criteria of antiphospholipid syndrome) increases the risk of thrombosis. We supposed that the presence of other aPL (antiphospholipid antibodies) is also clinically and laboratory valuable approach.

Aims: To evaluate the Importance of different antiphospholipid antibodies.

Methods: Since 2006 we examined 124 women with thromboembolism. In 22% thrombosis was during pregnancy, 30% ‐ postpartum, 38% ‐ after oral contraceptives use or during in vitro fertilization (IVF) protocol and 10% ‐ after surgical intervention. In 82% of DVT it was the left leg involvement. In 9.7% we reported DVT of lower leg, 12% ‐ of common femoral vein, 87 (70%) ‐ iliofemoral DVT, 5.6% ‐ inferior vena cava, 0.8% ‐ cerebral veins, 0.8% ‐ mesenterial vessels, 0.8% ‐ hepatic veins, 1.6% ‐ retinal veins, 1.6% ‐ upper extremities veins and 4% ‐ DVT‐associated massive pulmonary embolism (PE) and 1.6% ‐ with recurrent PE. All patients we examined for the presence of IgG/IgM isotypes of anti‐β2GPI antibodies, aCL, LA, anti‐annexin V, anti‐prothrombin antibodies.

Results: LA was diagnosed in 22.9% of the patients, anti‐β2GPI antibodies ‐ 26.6%, aCL ‐ 18.5%, anti‐annexin V ‐ in 9.7%, anti‐prothrombin ‐ in 12.1%. Among them in 7 women (5.6%) we evaluated the combination of both LA and aCL, in 3.2% ‐ the presence both LA and anti‐β2GPI antibodies. Triple positivity (LA plus aCL plus anti‐β2GPI antibodies) ‐ in 8.9%. In 8.9% in. And in 5.6% women we diagnosed the circulation of all examined antibodies (multipositivity).

Conclusions: The thrombotic risk increases with the number of positive aPL tests, with triple positive patients displaying the highest vascular hazard. But the screening only for the routine aPL can miss and delay diagnosis of APS and treatment. We consider the evaluation of other aPL is also important and require further investigations. May be in future the criteria of APS will be porobably re‐evaluated and the new antibodies included.

PB436: Recent Metods for Laboratory Detection of Lupus Anticoagulant in Patients with Antiphospholipid Syndrome

V. Krasivska ; O. Stasyshyn

State Institution ‘Institute of Blood Pathology and Transfusion Medicine under the National Academy of Medical Sciences of Ukraine’, Lviv, Ukraine

Background: Numerous guidelines and assay types for LA detection have been proposed and used over the years: ISTH in 1983, 1991, 1995, 2009 years, BCSH in 1991, 2000, 2012 years, CLSI in 2014 year. Guidelines contain important differences which require researches and implementations.

Aims: To specify the value and research the informational content for APS diagnostics of routine coagulogical and modern methods of lupus anticoagulant (LA) detection in patients with thrombosis and pregnancy complications.

Methods: Antiphospholipid antibodies identification, including LA, were carried out in 233 patients with the suspicion of APS, who asked for treatment with thrombosis in different locations and obstetrical pathologies. LA identification was carried out with the help of modern guidelines based on the three‐stage algorithm.

Results: According to the international criteria of diagnostics 44.6% were diagnosed with APS, among which primary APS was diagnosed in 85.6% of patients and secondary in 14.4% of patients. LA was identified in 42.5% of all the patients, which is 95.2% of all patients with APS. On the first screening stage of LA detection the most detailed informational value appears while combining the tests based on differential principals and responsible for various parts of haemostasis, in particular the clotting time with phospholipid dependent diluted viper venom time, APTT with LA ‐sensitive, APTT with LA ‐unsensitive reagent and PT with diluted to 50 and 500 times thromboplastin. The correctional tests results are not dependable and do not allow us to make a decision as to continuation or termination of the further research. The necessity of their execution should be considered individually.

Conclusions: Due to the high informational value, confirmation tests should be carried out on the second stage of LA detection. The obtained results are according to the latest 2014 guidelines, which can be used as standards in LA activity research.

PB437: Anti‐β2‐Glycoprotein I Antibody Delays Fibrin and Thrombin Formation but Enhances Thrombin Generation

G. Szabo 1 ; I. Beke Debreceni 1 ; P. Soltesz 2 ; J. Kappelmayer 1

1 University of Debrecen, Faculty of Medicine, Department of Laboratory Medicine, Debrecen, Hungary, 2 University of Debrecen, Faculty of Medicine, Department of Internal Medcine, Debrecen, Hungary

Background: Antiphospholipid syndrome (APS) is characterised by both arterial and venous thrombosis as well as pregnancy morbidity with the presence of antiphospholipid antibodies (aPL).

Aims: The aim of this study was to investigate the effects of aPL on fibrin and thrombin formation.

Methods: Antiphospholipid antibodies were separated from three patients with antiphospholipid syndrome, who showed triple positivity for the laboratory criteria of APS: anti‐β2‐glycoprotein I (aβ2GPI), anti‐cardiolipin (aCL), lupus anticoagulant (LA) and had thrombosis in their history. Separation of aβ2GPI IgG antibodies was performed by a two‐step affinity chromatography system by using Protein G and purified β2GPI. The effect of aPL antibodies was investigated in dilute APTT (dAPTT) and PT (dPT) assays and by the Thrombin Generation Test (TGT), where antibodies were added to pooled normal plasma at concentrations up to 125 U/ml.

Results: The presence of 15 U/ml aβ2GPI, which is below the upper reference limit (20 U/ml), prolonged both the dAPTT and dPT assays by 17% from 68.4±1.4 seconds to 79.9±11.4 seconds and by 31% from 29.3±0.5 seconds to 38.5±11.4 seconds respectively. Elevating the level of aβ2GPI to 125 U/ml increased these values to 27% and 211% in dAPTT and dPT test respectively. Similarly, the time parameters of TGT were prolonged in a dose dependent manner. Using 125 U/ml aβ2GPI in TGT, Lag time and Time to Peak values increased by 23% and 13% respectively. Despite the fact that fibrin formation was delayed at each concentration of antibodies, the Endogenous Thrombin Potential (ETP) was increased already at 31 U/ml by 11% from 1481 nM*min to 1647 nM*min and the Velocity Index was also augmented by 19% from 68.9 nM/min to 82.0 nM/min.

Conclusions: Although purified aβ2GPI prolonged coagulation tests, it was found that more thrombin was generated and faster even at low concentrations of aβ2GPI, which may contribute to their prothrombotic effect.

PB438: Preeclampsia and Antiphospholipid Antibodies

A. Makatsariya; J. Khizroeva ; V. Bitsadze; M. Arslanbekova

I.M. Sechenov First Moscow State Medical University, Department of Obstetrics and Gynecology, Moscow, Russian Federation

Background: The association between antiphospholipid antibodies (aPL) and preeclampsia is not clear, and a causal relationship has not yet been proven.

Aims: The evaluation of spectrum of antiphospholipid antibodies (APA) in women with the history of severe preeclampsia (SP).

Methods: ELIZA‐method was used to measure IgM/IgA APA screen, anti‐b2‐glycoprotein I (GPI), antiannexin V, antiprothrombin in 125 women with severe preeclampsia. Control group was consisted of 60 health women.

Results: APA circulation in patients with history of severe preeclampsia was found in 32%. It is interesting that antibodies to b2‐GPI were prevailed (31.6%). Antibodies to cardiolipin and antibodies to APA subgroups were respectively 21% and 17%. Lupus anticoagulant circulation was in 15.7%. Antiannexin V ‐ in 5.2%, antiprothrombin – 5.2%. Combination of LA, anti‐b2‐GPI, anticardiolipin was in 12.1%, LA, anti‐annexin V and anti‐b2‐GPI ‐ in 13.7%, anti‐prothrombin and anti‐b2‐GP I ‐ in 8.9%, LA, annexin V and b2‐GPI ‐ in 7.9%. The worst clinical picture was observed in women with combination of different APA.

Further we managed the next pregnancy in 54 of these 125 examined women. Most of them (39) were observed from the fertile cycle. 15 other women admitted already being pregnant. All patients were re‐examined for APA circulation. In 3 cases we observed the development of preeclampsia (mild form) and in 1 case ‐ repeated severe preeclampsia in women who applied in III trimester of pregnancy. The development of severe preeclampsia we consider with late admission to the hospital. All women received anticoagulant therapy. No case of severe preeclampsia development in patients with anticoagulant therapy started from the fertile cycle or I trimester of pregnancy.

Conclusions: The presence of history of preeclampsia is the indication for the APA‐testing and the beginning of the anticoagulant therapy from the moment of preparing for pregnancy.

PB439: Direct Oral Anticoagulants in Venous Thromboembolic Disease; Boon or Bane?

A. Ang ; N. Pandit

Tan Tock Seng Hospital, Singapore, Singapore

Background: Much akin to oral contraceptive pill's revolutionising birth control in the 1960's, direct oral anticoagulants (DOACs) have attained popularity in this decade for the treatment of venous thromboembolism (VTE) and stroke prevention in atrial fibrillation. Many patients diagnosed with deep vein thrombosis (DVT) now have treatment initiated either in the emergency department or in the outpatient (clinic) setting before being referred to the specialist for further management.

Since DVT's are the first presentation of up to 55% of antiphospholipid antibody syndrome (APS) cases, many APS patients (unknown at first presentation) with DVTs are treated with DOAC's.

DOAC make it challenging for subsequent serological testing (for APS confirmation) and for convincing patients to switch to warfarin, given the convenience of DOAC's.

Aims: We describe a case of a 30‐year‐old female dental assistant who was diagnosed in the emergency department with proximal lower limb DVT. She was initiated with rivaroxaban and referred to the vascular medicine clinic. Baseline blood tests before commencement of rivaroxaban showed prolonged APTT and low platelets.

Methods: N/A.

Results: Antibody testing for APS include the lupus anticoagulant (LA) anti‐cardiolipin IgG and IgM, and anti‐β2glycoprotein I. Since LA can be falsely positive during use of rivaroxaban, we have to rely on the latter three serologies, making it possible to miss the diagnosis of APS since LA is most strongly associated with APS. Subsequently, once diagnosed with APS (or for that matter even suspected), the physician must discuss switching from rivaroxaban to warfarin since the former is not approved for APS associated VTE.

Conclusions: This case aims to highlight the importance of: ‐ having a high index of clinical suspicion of APS in idiopathic DVT prior to initiation of anticoagulation, ‐ Awareness of the fallacies of serological testing in the presence of a DOAC and how to navigate around them and ‐ briefly discuss treatment of DVT in APS.

PB440: Outcomes in Non‐valvular Atrial Fibrillation Patients with Good and Poor INR Control after Switching to Direct Oral Anticoagulants

B. Haymart 1 ; X. Kong 1 ; E. Kline‐Rogers 1 ; S. Almany 2 ; J. Kozlowski 3 ; G. Krol 4 ; M. Dahu 5 ; J.B. Froehlich 1 ; G.D. Barnes 1 ; S. Kaatz 4

1 University of Michigan, Ann Arbor, United States, 2 Beaumont Hospital, Royal Oak, United States, 3 Cardiology and Vascular Associates, Commerce, United States, 4 Henry Ford Hospital, Detroit, United States, 5 Spectrum Health, Grand Rapids, United States

Background: Several international guidelines recommend direct oral anticoagulants (DOACs) over vitamin K antagonists (VKAs) for stroke prevention in non‐valvular atrial fibrillation (NVAF), and warfarin patients with poor INR control are commonly viewed as ideal patients for switching to DOACs.

Aims: To examine outcomes in NVAF patients with good and poor INR control before and after switching from warfarin to a DOAC.

Methods: Since 2009, abstractors at 6 anticoagulation services have entered patient data into the Blue Cross Blue Shield of Michigan funded Michigan Anticoagulation Quality Improvement Initiative (MAQI 2 ) registry. NVAF patients in the MAQI 2 database were stratified based on time in therapeutic range (TTR): good control (TTR≥60%) and poor control (TTR<60%). Ischemic stroke and major bleeding rates were calculated for both groups before and after switching to a DOAC. Comparisons were made using Poisson and Chi‐square tests and Cox's Proportional Hazard Modeling.

Results: 413 NVAF patients were identified with 212 (51%) having good INR control and 201 (49%) having poor INR control. After switching to a DOAC, patients with good INR control had a significantly higher ischemic stroke rate (2.08 vs. 0.98 per 100 pt‐yr, P<0.001, HR 2.33) and major bleed rate (4.2 vs. 2.4 per 100 pt‐yr, P=0.04, HR 2.15) than while on warfarin. Patients with poor INR control had similar ischemic stroke rates (1.67 vs. 1.88 per 100 pt‐yr, P=0.30, HR 0.79) and major bleed rates (6.7 vs. 5.0 per 100 pt‐yr, P=0.14, HR 1.39) before and after switching. The composite endpoint of ischemic stroke and major bleeding showed a similar pattern (Table 1).

Conclusions: In our MAQI 2 patients with good INR control switched to DOACs, the increased convenience came at a price: double the risk of ischemic stroke and major bleeds. Our poorly controlled warfarin patients, who are frequently viewed as ideal candidates, showed no clinical benefit from switching.

TABLE 1 Event rates before and after switching to DOAC Good control (TTR≥60%) Poor Control (TTR<60%) Before switch After switch P‐value Hazard ratio (after vs. before) Before switch After switch P‐value Hazard ratio (after vs. before) # of patients 212 212 ‐ ‐ 201 201 ‐ ‐ # patient years 408.2 385.4 ‐ ‐ 159.6 298.9 ‐ ‐ # ischemic strokes 4 8 ‐ ‐ 3 5 ‐ ‐ Rate of ischemic stroke (# per 100 pt‐yr, 95% CI) 0.98 (0.88, 1.08) 2.08 (1.93, 2.22) <0.001 2.33 (0.67, 8.10) 1.88 (1.67, 2.09) 1.67 (1.53, 1.82) 0.3 0.79 (0.16, 3.81) # major bleeds 10 16 ‐ ‐ 8 20 ‐ ‐ Rate of major bleeding (# per 100 pt‐yr, 95% CI) 2.4 (2.30, 2.60) 4.2 (3.95, 4.35) 0.04 2.15 (0.92, 5.04) 5.0 (4.66, 5.36) 6.7 (6.40, 6.98) 0.14 1.39 (0.60, 3.23) Composite event rate (# per 100 pt‐yr, 95% CI) 3.4 (3.25, 3.61) 6.2 (5.98, 6.48) 0.006 2.12 (1.04, 4.31) 6.9 (6.48, 7.30) 8.4 (8.03, 8.69) 0.26 1.29 (0.60, 2.74

PB441: PK/PD (Thrombinography and Fibrinography) Response Variability to Rivaroxaban in Patients Aged 80 Years and Older with Atrial Fibrillation: Results of the ADAGE Study

G. Foulon 1,2 ; E. Curis 3,4 ; J. Le Guen 5 ; A.‐C. Martin 1,6 ; C. Lafuente‐Lafuente 7 ; E. Puymirat 8 ; P. Gaussem 1 ; I. Gouin‐Thibault 1 ; E. Pautas 1,9 ; V. Siguret 1,2

1 Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1140, Paris, France, 2 Hôpital Lariboisière (AP‐HP), Hématologie Biologique, Paris, France, 3 Université Paris Descartes, Sorbonne Paris Cité, Biostatistiques, Paris, France, 4 Hôpital Saint‐Louis (AP‐HP), Bio‐Informatique, Paris, France, 5 Hôpital Européen Georges Pompidou (AP‐HP), Gériatrie, Paris, France, 6 Hôpital d'Instruction des Armées Percy, Service de Santé des Armées, Cardiologie, Clamart, France, 7 Hôpital Charles Foix (AP‐HP)‐ UPMC, Gériatrie, Paris, France, 8 Hôpital Européen Georges Pompidou (AP‐HP), Cardiologie, Paris, France, 9 Hôpital Charles Foix (AP‐HP) ‐ UPMC, Unité de Gériatrie aiguë, Paris, France

Background: The number of polypathological and polymedicated patients aged 80 years and over receiving rivaroxaban for atrial fibrillation (AF) is rising steadily. Few specific pharmacokinetics (PK)/pharmacodynamics (PD) data are available among these patients characterized by a basal hypercoagulable state and a high hemorrhagic risk under anticoagulant treatment.

Aims: We sought i/ to determine the PK/PD profiles of rivaroxaban in frail elderly patients; ii/ to assess the relationships between rivaroxaban plasma concentrations and thrombin generation (TG)/fibrinography parameters.

Methods: ADAGE ( NCT02464488 , Ile de France VI ethic committee) is a prospective, observational, academic, multicenter clinical study. We recruited patients aged 80 years or older, receiving rivaroxaban for non‐valvular AF since, at least, 4 days. Each patient had one to five samples performed over a 20‐day period, at different time‐points after rivaroxaban intake. Plasma rivaroxaban anti‐Xa activity (ng/ml) was measured by chromogenic method (Stago, France). Thrombinography and fibrinography parameters were measured using the innovative Thrombodynamics ® system (HemaCore‐Russia).

Results: To date, 57 patients (mean age 86±4 years, mean total CIRS comorbidity score 9.6±3.7, mean creatinine clearance ‐Cockcroft‐ 51±17 ml/min) have been included. A total of 129 samples were collected (Figure 1).

FIGURE 1 Rivaroxaban anti‐Xa activity as a function of time after daily intake

Median peak anti‐Xa activity was 203 ng/ml (interquartile range IQR 122‐301) and 46 ng/ml (IQR 33‐71) at Cmin (15‐mg daily dose). Regarding TG parameters, lag time, time to peak, peak height and endogenous thrombin potential were significantly associated with rivaroxaban concentrations (Figure 2).

FIGURE 2 Relationships between rivaroxaban anti‐Xa activity and the different TG parameters

Among fibrinogram parameters, only lag time was associated with rivaroxaban concentrations (P<10 −4 ).

Conclusions: This is the first study providing new data on both PK and PD evaluated by thrombinography/fibrinography in polypathological and polymedicated very elderly patients receiving rivaroxaban.

PB442: Health‐related Quality of Life after Pharmacomechanical Catheter‐directed Thrombolysis for Proximal Deep Vein Thrombosis

S.R. Kahn 1 ; J.A. Julian 2,3 ; C. Kearon 3,4 ; C.‐S. Gu 2,3 ; D.J. Cohen 5,6 ; E.A. Magnuson 7 ; A.J. Comerota 8 ; M.R. Jaff 9 ; M.K. Razavi 10 ; S.Z. Goldhaber 9,11 ; S. Vedantham 12 ; ATTRACT Study Investigators

1 Jewish General Hospital, Lady Davis Institute, Center for Clinical Epidemiology, Montreal, Canada, 2 McMaster University, Department of Oncology, Hamilton, Canada, 3 Juravinski Hospital and Cancer Centre, Hamilton, Canada, 4 McMaster University, Department of Medicine, Hamilton, Canada, 5 University of Missouri‐Kansas City, Department of Medicine, Kansas City, United States, 6 Saint Luke's Mid America Heart Institute, Cardiovascular Research, Kansas City, United States, 7 Saint Luke's Mid America Heart Institute, Kansas City, United States, 8 Inova Heart and Vascular Institute, Inova Alexandria Hospital, Alexandria, United States, 9 Harvard Medical School, Boston, United States, 10 St Joseph's Hospital, Orange, United States, 11 Brigham and Women's Hospital, Division of Cardiovascular Medicine, Boston, United States, 12 Mallinckrodt Institute of Radiology, Washington University, St. Louis, United States

Background: After acute deep vein thrombosis (DVT), many patients have impaired quality of life (QOL).

Aims: To assess if pharmacomechanical catheter‐directed thrombolysis (PCDT) improves short‐term or long‐term QOL in patients with proximal DVT.

Methods: Secondary analysis of the ATTRACT Trial, a randomized open‐label controlled trial done in 56 U.S. clinical centers. Patients with proximal DVT of the femoral, common femoral, or iliac veins were potentially eligible. Participants were randomised to receive, or not receive, PCDT. All patients received anticoagulation and were asked to wear compression stockings for 24 months. QOL was assessed at 30d and at 6, 12, 18 and 24 m using the VEINES‐QOL disease‐specific QOL measure and the SF‐36 (PCS and MCS summary scores) generic QOL measure. QOL scores at each timepoint and change in scores from baseline to 24 m were compared in the PCDT and No PCDT groups overall and in the iliofemoral (IF) DVT and femoropopliteal (FP) DVT subgroups.

Results: 691 of 692 ATTRACT patients were analysed (mean age 53 years, 62% male, 57% IF DVT). VEINES‐QOL scores were higher (i.e. better) in PCDT versus No PCDT at 30d and at 6 m (Table). VEINES‐QOL scores at 12, 18 and 24 m, SF‐36 PCS and MCS scores at all timepoints, and baseline to 24 m change scores for all QOL measures were similar between groups. Among IF DVT patients, in PCDT versus No PCDT, VEINES‐QOL scores were higher at 30d and at 6 m, but not at 12, 18 and 24 m, and there was greater improvement in baseline to 24 m change scores. In FP DVT patients, scores at all timepoints and baseline to 24 m change scores were similar in the PCDT and No PCDT groups.

TABLE 1 VEINES‐QOL scores by Treatment and DVT Type

Conclusions: PCDT leads to better disease‐specific QOL at 30d and 6 m after proximal DVT, but not later. In patients with iliofemoral DVT, PCDT led to better disease‐specific QOL at 30d and 6 m and also greater improvement in QOL over 24 m follow‐up.

PB443: DTI/DXI Interferences with Global Coagulation Tests in Daily Care Emergency Admissions ‐ Results of the Prospective DRESDEN NOAC REGISTRY ( NCT01588119 )

L. Wunder; L. Tittl; S. Marten; J. Beyer‐Westendorf

University Hospital Carl Gustav Carus, Thrombosis Research Unit, Dresden, Germany

Background: Emergency management of direct oral anticoagulants (DOAC) recipients is challenging because direct thrombin inhibitors (DTI) and direct FXa inhibitors (DXI) may or may not affect prothrombin time (PT), international normalized ratio (INR) or activated thromboplastin time (aPTT), depending on the test assay and the time between last DOAC intake and blood sampling.

Aims: To evaluate impact of DOAC exposure on global coagulation tests.

Methods: Evaluation of first PT, INR or aPTT tests during emergency hospitalizations of DTI/DXI patients in the prospective Dresden NOAC Registry .

Results: We identified 724 emergency admissions (77 DTI; 647 DXI admitted to 56 hospitals using 50 different laboratories). In 490 cases (67.7%), last intake of DTI or DXI was within 12 hours before blood sampling.

In ˜63% of all cases INR and PT were elevated above the upper limit of normal (ULN), of which ˜65% were >20% above ULN. In 43% of all cases, aPTT was elevated above ULN, in 41% >20% above ULN (Table 1)

TABLE 1 Distribution of normal or elevated coagulation test results in relation to last DOAC exposure

Irrespective of the duration between last drug intake and blood sampling (<6 or >24 h) and irrespective of DOAC type a large proportion of patients demonstrated elevated coagulation test values.

In DTI recipients, 68‐89% had aPTT values >ULN (89% still elevated beyond 24 hours) and 42 to 78% had PT and INR values >ULN (Figure 1A‐C).

FIGURE 1 Time‐dependent impact of DTI or DXI on global coagulation tests (n.a. not available)

In DXI recipients, 66‐70% had elevated PT and INR values; this proportion declined to 52% over time. In contrast, only 46% had elevated aPTT shortly after drug intake and this declined to 29% beyond 24 hours (Figure 2A‐C).

DOAC specific testing was performed in 35 DTI (34 thrombin time, 1 ecarin clotting time) and in 18 DXI cases (chromogenic aXa assays) only.

Conclusions: Many DOAC recipients present with elevated PT, INR or aPTT values during emergency admissions but false negative (normal) results within 6 hours of last intake as well as elevated values later than 24 hours after last DOAC intake are common observations. DOAC specific testing is rarely performed in emergency settings.

PB444: Evaluation of the Safety, Tolerability, Pharmacokinetic and Pharmacodynamic Properties of the Anti‐factor XI Antibody, AB023, in Healthy Adult Volunteers

C.U. Lorentz ; E.I. Tucker; M. Wallisch; N.G. Verbout; B. Markway; M.R. Carris; A. Gruber

Aronora, Inc., Portland, United States

Background: Thrombosis remains among the leading causes of morbidity and mortality. Marketed antithrombotics are effective, but all exhibit dose‐limiting anti‐hemostatic toxicity. Activated factor (F) XI is a hemostatic enzyme that also promotes pathologic thrombus formation and inflammation. Since contact activation of FXI by activated FXII (FXIIa) is prothrombotic in animal models, and FXII has no known hemostatic function, we targeted this molecular mechanism using AB023 (xisomab 3G3), a humanized IgG4 antibody that selectively inhibits FXI contact activation.

Aims: To evaluate the safety, tolerability, pharmacokinetic (PK) and pharmacodynamic (PD) properties of AB023 in healthy human volunteers.

Methods: In this randomized, single ascending dose (0.1‐5 mg/kg), double‐blind, placebo‐controlled, phase 1 study, AB023 was administered intravenously to healthy adults. Safety parameters (bleeding and prothrombin times, physical examinations, vital signs, electrocardiograms (ECGs), injection site reactions, immunogenicity, and other adverse events (AE)) were evaluated. PK parameters were determined by measuring plasma AB023 levels and the PD effect of AB023 was assessed using activated partial thromboplastin time (APTT). The study was approved by the local ethics review board and all subjects provided written informed consent.

Results: AB023 demonstrated a favorable safety and tolerability profile, with no instances of abnormal or increased bleeding or prothrombin times. No drug‐related AEs were noted. APTT prolongation was observed in all cohorts after AB023 administration, reaching saturating (maximum anticoagulant) effect at and above the 0.5 mg/kg dose (1.7 to 2.4‐fold APTT prolongation). The duration of saturating anticoagulation was dose‐dependent.

Conclusions: This first‐in‐human study of AB023 did not show demonstrable safety risks or signs of toxicity, even at the highest dose tested. The potential safety profile of AB023 makes it a promising therapeutic agent that warrants progression into phase 2 trials.

PB445: The Indirect Effects of Dabigatran En Rivaroxaban on Fibrinolysis

S. Zwaveling 1 ; S. Bloemen 2 ; J. Konings 3 ; H. Kelchtermans 3 ; C. Hemker 3 ; A. Miszta 4 ; B. de Laat 3

1 Maastricht University, Biochemistry, Maastricht, the Netherlands, 2 Synapse Research Institute, Biochemistry, Maastricht, the Netherlands, 3 Synapse Research Institute, Maastricht, the Netherlands, 4 University of North Carolina, Department of Pathology and Laboratory Medicine, Chapel Hill, United States

Background: The effects of direct oral anticoagulants (DOACs) on fibrinolysis are unclear. Thrombin enhances a denser fibrin network, activates factor XIII and thrombin‐activatable fibrinolysis inhibitor (TAFI) via thrombomodulin (TM), forming a stronger clot. By reducing thrombin levels DOACs may indirectly facilitate clot lysis.

Aims: To study the effect of rivaroxaban (RIV) and dabigatran (DAB) on clot lysis.

Methods: Turbidimetry (405 nm), started with 1pM TF and 100 IU tissue‐type plasminogen activator (t‐PA), was used to evaluate clot lysis time (CLT) of normal pool plasma (NPP) spiked with DAB (300 nM), RIV (750 nm) or without (control). Also, experiments were performed in the presence and absence of 1 nM TM, 1 nM activated protein C (APC) and in TAFI‐deficient plasma. The CLT was defined as the time from half maximal fibrin formation to half maximal degradation.

Results: Both DOACs decreased the CLT significantly (control: 31.3 minutes vs. DAB: 17.0 minutes and RIV: 14.7 minutes). In the presence of TM, the CLT of the control was prolonged (31.3 minutes vs. 83.7 minutes) (Fig 1A), while RIV and DAB affected the CLT differentially (DAB: 82.3 minutes; RIV: 65.7 minutes) (Fig 1A). The addition of APC (to test TAFI dependence) did not change the CLT in NPP, but showed a cumulative anticoagulant effect with DOACs present (Fig 1B). In TAFI‐deficient plasma the increase of the CLT by TM was muted, confirming a TAFI‐dependent reaction (−TM: 18.7 minutes. vs. +TM: 18.3 minutes). Also in the presence of DOACs the TM effect faded (DAB‐TM: 18.0 minutes vs. DAB+TM: 18.7 minutes; RIV‐TM: 15.0 minutes vs. RIV+TM: 15.0 minutes) (Fig 1C).

Conclusions: RIV and DAB, by diminishing thrombin, accelerate fibrinolysis. TM, which enables TAFI‐dependent reactions, prolongs the CLT in the presence of both DOACs. APC adds to the fibrinolytic effect of DOACs. In vivo, TM and protein C, can vary in concentration, depending on the location. Due to the difference in response to TM and APC, further research into the fibrinolytic effects of DOACs is indicated.

FIGURE 1 (A) CLT prolongs with TM; (B) APC enhances anticoagulant effect of DOAC; (C) TAFI deficiency neutralises the prolongation by TM with and without DOAC

PB446: Risk of Thrombosis and Bleeding in Patients Treated with Warfarin or Apixaban for Non‐valvular Atrial Fibrillation: A Large, National, Retrospective Study

M. Wanat 1 ; X. Wang 2 ; S. Abughosh 2 ; H. Chen 2 ; M. Johnson 2 ; M. Fleming 2

1 University of Houston, Department of Pharmacy Practice and Translational Research, Houston, United States, 2 University of Houston, Department of Pharmaceutical Health Outcomes and Policy, Houston, United States

Background: Optimizing anticoagulant therapy in patients with atrial fibrillation (AF) is essential to reduce the risk of thromboembolic events and improve quality of life. Limited real world (phase IV) data is available comparing anticoagulant outcomes.

Aims: To evaluate thrombotic and bleeding endpoints in patients with non‐valvular atrial fibrillation.

Methods: A retrospective cohort study was conducted using a large United States electronic medical record (2012‐2016). Patients aged 18 years with a diagnosis of NVAF and on warfarin or apixaban were included. Select exclusion criteria included previous venous thromboembolism and recent cardiac surgery. The primary endpoint was a composite of stroke (ischemic or hemorrhagic) or systemic embolism. The primary safety endpoint was major bleeding using the ISTH definition. Patients were matched using propensity scoring. Univariate survival analyses were conducted by using the log‐rank test and Kaplan‐Meier survival curves. A Cox Proportional Hazards Model was used to estimate the risk‐adjusted effect of treatment on time until outcome. Outcomes were defined as time to first event.

Results: A total of 332,100 patients with NVAF were identified, of which 31,612 patients met inclusion criteria, and 20,378 were included in the propensity matching analysis. There were no differences in baseline characteristics with respect to age, comorbidities, CHADS2‐Vasc score, and concurrent medications. The primary endpoint occurred in 122 (1.2%) patients in the apixaban group compared to 166 (1.63%) patients in the warfarin group (HR 0.84, P<0.0001). Major bleeding occurred at a lower rate in the apixaban group (n=600, 5.89%) compared to the warfarin group (n=887, 8.71%) (OR 1.54, P<0.00001).

Conclusions: There was a statistically significant reduction in thrombotic and bleeding outcomes seen with apixaban compared to warfarin. Further randomized controlled trials should be performed to assess the real world effectiveness of oral anticoagulants.

PB447: Novel Universal Antidote to Non‐Vitamin K Antagonist Anticoagulants

G. Jourdi 1,2 ; I. Gouin‐Thibault 1,3 ; V. Siguret 1,4 ; S. Gandrille 1,5 ; P. Gaussem 1,5 ; B. Le Bonniec 1

1 Paris Descartes University, Inserm UMR 1140, Paris, France, 2 Cochin Hospital, AP‐HP, Laboratory of Hematology, Paris, France, 3 CHU Pontchaillou, Laboratory of Hematology, Rennes, France, 4 Lariboisiere Hospital, AP‐HP, Laboratory of Hematology, Paris, France, 5 Georges Pompidou European Hospital, AP‐HP, Laboratory of Hematology, Paris, France

Background: Direct Oral Anticoagulants (DOAC) inhibit thrombin (dabigatran) or factor Xa (FXa; xabans).The risk of hemorrhage still persists with DOAC and there is a need for antidote in case of life‐threatening bleeding or urgent surgery. Although an antidote to dabigatran, idarucizumab, has been approved, antidotes targeting xabans are still under evaluation.

Aims: To conceive and evaluate an antidote neutralizing xabans while being neutral with regard to hemostasis. We hypothesized that a complex formed by Gla‐domainless FXa (GDFXa) and α2‐macroglobulin (a2M) would fulfill these specifications.

Methods: Human α2M was purified and incubated with recombinant GDFXa. Kinetics of FXa inhibition, either free or within the GDFXa‐α2M complex, by xabans were determined. GDFXa‐α2M efficacy and neutrality were tested in vitro by clot waveform assay, rotational thromboelastometry and chromogenic assays. Moreover the effect of GDFXa‐α2M was tested on dabigatran, heparin and its derivatives. In vivo efficacy and safety were evaluated in a mouse bleeding model along with D‐dimers and thrombin‐antithrombin complex (TAT) measurement by Elisa.

Results: Affinity of xabans for FXa and GDFXa‐α2M were comparable. In vitro, 1.7 μM GDFXa‐a2M fully neutralized up to 600 ng/ml xabans, 80% of 600 ng/ml dabigatran and 50% of 1 to 1.5 IU/ml heparin or derivatives. In vivo , GDFXa‐a2M significantly decreased bleeding induced by rivaroxaban, dabigatran or enoxaparin without increasing D‐dimer or TAT levels nor affecting ex vivo coagulation assays. Half a mg GDFXa‐a2M injected in mice persisted with a half‐life of 85 minutes, suggesting that anticoagulant neutralization could only require a single IV bolus administration.

Conclusions: We suggest GDFXa‐a2M as a promising antidote of DOAC and heparin derivatives, with neither pro‐ nor anti‐coagulant effect.

PB448: Comparing the Proportions of Patients that Meet Criteria for Extended Prophylaxis with Betrixaban Based on APEX Trial and FDA Indication

S. Kaatz 1 ; A. Conlon 2 ; E. McLaughlin 2 ; S. Flanders 2 ; V. Chopra 2 ; D. Paje 2

1 Henry Ford Hospital, Detroit, United States, 2 University of Michigan, Ann Arbor, United States

Background: Betrixaban is the first anticoagulant approved in the United States for extended venous thromboembolism (VTE) prophylaxis in hospitalized medical patients. The evidence of benefit was based on data from the Acute Medically Ill VTE Prevention with Extended Duration Betrixaban (APEX) trial, which used a study population enriched with patients at high risk for VTE, such as those with elevated D‐dimer or ≥75 years. However, the Food and Drug Administration (FDA) approved indication covers a broader population with less defined risk.

Aims: Compare the proportion of real‐world hospitalized medical patients eligible for betrixaban using APEX trial criteria with those using FDA‐approved indication.

Methods: Trained abstractors collected data from a representative sample of medical patients discharged between January 2012 and November 2017 (prior to betrixaban approval) at 57 hospitals participating in the Michigan Hospital Medicine Safety consortium. Adult, non‐pregnant patients without an initial intensive care unit (ICU) stay or surgery were included in this analysis. Applicable elements of APEX trial eligibility and FDA‐approved indication were mapped to consortium data.

Results: Of 107,803 patients in our multi‐center cohort of hospitalized medical patients, 1499 (1.4%) would be eligible for betrixaban based on APEX selection criteria and 32,012 (29.7%) based on FDA‐approved indication, P<0.001 The proportions of patients meeting each select element of APEX criteria and FDA indication are shown in the Table.

Conclusions: Over a quarter of hospitalized medical patients would be considered for betrixaban extended prophylaxis per FDA approval yet a minority would meet APEX final protocol criteria. Marked differences were seen with age, medical conditions and immobility status. Further risk stratification may be necessary to identify patients that would benefit most from betrixaban. Our analysis is limited by absence of data on D‐dimer results.

TABLE 1 Proportion of patients meeting select elements of APEX criteria and FDA‐approved indication Criteria Element APEX Trial n (%) FDA Indication n (%) P Age ≥75 36,756 (34.1%) Adults (≥18 years) 107,803 (100%) <0.001 Medical condition Heart failure, respiratory failure, acute infection, rheumatic disorder, stroke 43,962 (40.8%) Acute medical illness 107,803 (100%) <0.001 Immobility Severe on day one AND severe or moderate for 3 or more days 10,128 (9.4%) Severe or moderate 37,994 (35.2%) <0.001 Length of stay ≥3 days 81,020 (75.2%) Not specified 107,803 (100%) <0.001 Hemoglobin ≥9.5 89,879 (83.4%) Not specified 107,803 (100%) <0.001 No Renal failure Creatinine clearance ≥15 103,875 (96.4%) Implied not to use with creatinine clearance ≤15 103,875 (96.4%) na No liver failure >3 times upper limit of normal for AST, ALT or alkaline phosphatase or >2 times upper limit of normal for bilirubin, or active liver disease or hepatic dysfunction 106,739 (99.0%) Not recommended in patients with hepatic impairment 106,739 (99.0%) na No dual anti‐platelet No aspirin and P2Y12 inhibitor 99,073 (91.1%) Caution recommended but not precluded 107,803 (100%) <0.001

PB449: Reversal of Direct Factor Xa Inhibitor‐Associated Major Bleeding with Four‐factor Prothrombin Complex Concentrate: A Systematic Review and Meta‐analysis

S. Piran 1 ; R. Khatib 2 ; S. Schulman 1 ; A. Majeed 3 ; A. Holbrook 4 ; D.M. Witt 5 ; R. Nieuwlaat 6

1 McMaster University, Division of Hematology and Thromboembolism, Department of Medicine, Hamilton, Canada, 2 Northwestern University Feinberg School of Medicine, Department of Neurology, Chicago, United States, 3 Karolinska University Hospital and Karolinska Institutet, Department of Medicine, Division of Haematology, Coagulation Unit, Stockholm, Sweden, 4 McMaster University, Division of Clinical Pharmacology and Toxicology, Department of Medicine, Hamilton, Canada, 5 University of Utah College of Pharmacy, Department of Pharmacotherapy, Salt Lake City, United States, 6 McMaster University, Department of Health Research Methods, Evidence and Impact, Hamilton, Canada

Background: A targeted antidote for reversal of direct factor Xa inhibitors is currently not available for clinical use.

Aims: In a systematic review, we evaluated the safety and efficacy of four‐factor PCC in the reversal of direct factor Xa‐associated major bleeding.

Methods: A systematic literature search was conducted using MEDLINE, EMBASE, and the Cochrane Register of Controlled Trials up to January 2018. We pooled the data using a random effects generic inverse variance method. The quality of evidence was assessed using the GRADE approach.

Results: 1470 studies were initially screened and 8 studies with 286 patients were included. Follow‐up duration was median of 9 (6‐28) days in one study, 30 days in two, 90 days in four, and 180 days in one study. Only 2 studies used the International Society on Thrombosis and Haemostasis (ISTH) criteria for assessment of effective management of major bleeding. The pooled proportion of effective management of major bleeding using the ISTH criteria was 0.69 with 95% confidence intervals (CIs) 0.61 to 0.76. The pooled proportion of effective management of major bleeding not employing the ISTH criteria was 0.75 (95% CI, 0.53 to 0.97). The crude mortality pooled proportion was 0.19 (95% CI, 0.07 to 0.31). The crude pooled proportion of thromboembolic events was 0.05 (95% CI, 0.01 to 0.09). There was no statistical heterogeneity between the studies for any outcome (I 2 =0%) except for effective management of major bleeding not employing the ISTH criteria (I 2 =84%) and crude mortality (I 2 =68%). These heterogeneities may be due to differences in baseline characteristics and co‐interventions in the component studies.

Conclusions: Based on a pooled meta‐analysis of single‐arm observational studies at high risk of bias, it is difficult to ascertain whether 4‐factor PCC is more likely to achieve hemostasis than supportive care alone. The rate of adverse events was low.

PB450: Resumption of Anticoagulant Therapy after Anticoagulant‐Related Gastrointestinal Bleeding: A Systematic Review and Meta‐analysis

D. Little 1 ; C. Chai‐Adisaksopha 1 ; C. Hillis 1 ; D. Witt 2 ; M. Monreal 3 ; M. Crowther 1 ; D. Siegal 1,4

1 McMaster University, Hamilton, Canada, 2 University of Utah, Salt Lake City, United States, 3 Universidad Catolica de Murcia, Barcelona, Spain, 4 Population Health Research Institute, Hamilton, Canada

Background: Oral anticoagulation (OAC) is permanently discontinued in up to 50% of patients after gastrointestinal bleeding (GIB). A previous meta‐analysis showed a reduced risk of thromboembolism (TE) and death, and a clinically (but not statistically) significant increased risk of re‐bleeding associated with resumption. Additional data have since become available.

Aims: To determine the risks of TE, recurrent GIB and death in patients who resumed OAC compared to those who did not after OAC‐related GIB.

Methods: We conducted an updated systematic review and meta‐analysis by searching EMBASE, MEDLINE, and Cochrane CENTRAL for randomized controlled trials and cohort studies published from January 2014 to September 2017. Results were combined with the previous meta‐analysis. Risk of bias was assessed using the ROBINS‐I tool. Pooled relative risk (RR) ratios and 95% confidence intervals (CI) were calculated using a random effects model. Heterogeneity was assessed using the I 2 statistic (PROSPERO CRD42017078574).

Results: 2,551 articles were screened in duplicate by title and abstract, and 7 new studies were eligible after full‐text review. Ten observational studies (3,021 patients) were included in the final review. There was a reduced risk of TE (RR 0.35 95% CI 0.15‐0.65, P=0.002, I 2 =56.3%, 8 studies) and death (RR 0.59 95% CI 0.46‐0.75, P<0.001, I 2 =58.2%, 7 studies), and an increased risk of recurrent GIB (RR 1.77 95% CI 1.19‐2.63, P=0.005, I 2 =39.5%, 9 studies) in patients who resumed OAC compared to those who did not. Studies were judged to be at serious risk of bias.

Conclusions: Resuming OAC after OAC‐related GI bleeding appears to be associated with an important decrease in TE (by 65%) and death (by 41%), but an increase in recurrent GIB. The serious risk of bias of included studies precludes firm conclusions regarding benefits and harms. Prospective data are needed to identify patients for whom the net clinical benefit favours OAC resumption and the optimal timing of resumption.

PB451: Development and Characterization of Novel Nanoparticulate Carrier for Oral Delivery of Low Molecular Weight Heparin

S. Bhargava 1 ; V. Bhargava 2 ; A. Jain 3 ; V. Bhargava 4

1 United Institute of Pharmacy, Kanpur, India, 2 KRV Hospital, Kanpur, India, 3 Bhagyodaya Pharmacy College, Sagar, India, 4 GTB Hospital, Kanpur, India

Background: Low Molecular Weight Heparins (LMWH) are administered by parenteral route & repetitive injections are main disadvantages which demand for an improvement of administration strategy by other non‐invasive routes. It can be replaced by oral warfarin for outpatient therapy but has a slow onset & high incidence of drug‐drug interaction. Due to these limitations to oral delivery of LMWH, different approaches have been proposed to enhance oral absorption.

Aims: The objective of the present study was to enhance the oral bioavailability of an anticoagulant drug by polymeric nanoparticulate carrier system.

Methods: Chitosan nanoparticles (NPs) were prepared by ionic gelation of chitosan & coated with alginate solution. NPs were characterized for morphology, particle size, polydispersity index, zeta potential, drug loading & entrapment efficiency using SEM, Zeta‐sizer, FTIR & DSC. The in‐vitro release & permeation study was performed, in‐vivo studies included venous thrombosis model, uptake using fluorescence microscopy & pharmacokinetic studies in rats.

Results: Coating of alginate over the NPs improved the release profile of enoxaparin from the nanoparticles for successful oral delivery. In‐vitro permeation studies elucidated that more amount of drug permeated across the intestinal epithelium due to alginate coating. In‐vivo studies show the enoxaparin loaded nanoparticles exhibit better anticoagulant activity during oral administration. The results of the present study indicated that this system is more stable as compared to other system.

Conclusions: Enoxaparin loaded alginate coated chitosan nanoparticulate carrier was prepared & optimized. The prepared system showed enhanced drug stability, improved availability across the intestine reduced thrombus formation in rat venous thrombosis model along with reduced side effects as compared to the drug solution. These results concluded that the designed nanocarriers as possible candidates for improving the absorption of low molecular weight heparin by oral administration

PB452: Recurrent Thrombosis in Patients with Antiphospholipid Antibodies Treated with Vitamin K Antagonists or Rivaroxaban

M. Abbattista 1 ; P. Bucciarelli 1 ; A. Tripodi 1,2 ; A. Artoni 1 ; F. Gianniello 1 ; C. Novembrino 1 ; F. Peyvandi 1,3 ; I. Martinelli 1

1 A.Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’ Granda ‐ Ospedale Maggiore Policlinico, Milano, Italy, 2 University of Milan, Department of Clinical Sciences and Community Health, Milano, Italy, 3 University of Milan, Department of Pathophysiology and Transplantation, Milano, Italy

Background: Antiphospholipid antibody syndrome (aPS) carries an increased risk of first and recurrent thrombosis despite anticoagulant therapy with vitamin‐K antagonists (VKA). Few and inconsistent data are available on efficacy and safety of direct oral anticoagulant (DOAC) therapy in patients with aPS.

Aims: To investigate the incidence rate of recurrent thrombosis in a cohort of patients with aPS treated with VKA or DOAC.

Methods: Patients referred from Sep 2013 to Dec 2016 to the Angelo Bianchi Bonomi center for a thrombophilia work‐up after an episode of venous thrombosis who were diagnosed with aPS and followed during treatment with VKA or DOAC.

Results: Twenty‐eight patients, 15 on VKA and 13 on the DOAC rivaroxaban, were followed for 23.6 and 19.0 months, respectively. Five patients experienced recurrent thrombosis, one on VKA (incidence rate 2.4, 95%CI 0.2‐11.3 per 100 pts‐yr) and 4 on rivaroxaban (incidence rate 19.4, 95%CI 6.5‐46.2 per 100 pts‐yr) for a hazard ratio of 7.53, 95%CI 0.84‐67.6. Recurrences were arterial in 4 patients (3 acute myocardial infarctions and one cerebral ischemic stroke) and venous in one (cerebral vein thrombosis). All patients with recurrent thrombosis had triple positivity for lupus anticoagulant, anticardiolipin and anti‐β2 glycoprotein I antibodies.

Conclusions: In our cohort, the risk of recurrent thrombosis was increased in patients with triple positivity aPS treated with rivaroxaban compared to those treated with VKA. We suggest caution in prescribing rivaroxaban to patients with triple positivity aPS until data from ongoing randomized controlled trials will be available.

PB453: Measurement of Anti‐FXa Activity of Edoxaban and its Metabolites in Plasma and Consistency with LC:MS/MS Reference Concentrations

C. Dunois ; M. Peyrafitte; C. Amiral; A. Javillier; J. Amiral

HYPHEN BioMed, Research and Validation, Neuville sur Oise, France

Background: Measurement of Edoxaban, a direct FXa inhibitor, and its metabolites (M‐4, M‐6 and M‐8), can be necessary in some clinical situations, although this therapy does not require monitoring.

Aims: To validate the measurement of Edoxaban and its metabolites Anti‐FXa activity in plasma, with automated chromogenic assays.

Methods: One stage (BIOPHEN Heparin LRT) and two stages (BIOPHEN DiXaI) Anti‐FXa chromogenic methods are used in combination with specific Edoxaban calibrators and controls. Low (0 to 120 ng/ml) and standard (0 to 500 ng/ml) calibration ranges are developed on Sysmex CS instruments, STA‐R, or ACL‐TOP for measuring Anti‐FXa activity of Edoxaban and its metabolites in plasmas from healthy subjects or anticoagulated patients. Results are compared with concentrations measured with the reference LC:MS/MS method.

Results: Low within‐run and between‐run coefficients of variation are obtained with both assays on all analyzers (1.1% to 7.0%). Linear range is from 2 to 916 ng/ml for BIOPHEN Heparin LRT and from 7 to 916 ng/ml for BIOPHEN DiXaI. No interference is observed for Heparin up to 2 IU/ml with BIOPHEN DiXaI. The anti‐FXa activities are well correlated between the two chromogenic assays (CS‐5100: y=0.95x ‐ 6.75; r=0.997, P<0.001) and consistent with LC:MS/MS, but concentrations were slightly higher with bioassays, which measure the global activity of all forms (Edoxaban and metabolites). When LC:MS/MS measurement of M4 metabolite was added, a better compliance was noted (BIOPHEN Heparin LRT: y=0.98x + 2.43; r=0.998, P<0.001 and BIOPHEN DiXaI: y=1.00x + 13.10; r=0.997, P<0.001).

Conclusions: Chromogenic assays combined with dedicated calibrators and controls allow measuring Edoxaban activity in citrated plasma, accurately and reliably, and can be used for assessing low concentrations, using any coagulation analyzer, especially before urgent surgery. An excellent correlation is observed with LC:MS/MS measurements of Edoxaban + M4.

PB454: Variable Response in Individuals to Rivaroxaban and Apixaban over Time

S. Zwaveling 1 ; J. Konings 2 ; H. Kelchtermans 2 ; B. de Laat 2 ; C. Hemker 2 ; S. Bloemen 2

1 Maastricht University, Biochemistry, Maastricht, the Netherlands, 2 Synapse Research Institute, Maastricht, the Netherlands

Background: Direct oral anticoagulants (DOACs) are prescribed at fixed dosages due to presumed stable pharmacokinetics and ‐dynamics. The relation between dose and plasma levels, as that between plasma levels and the effect on thrombin generation (TG), were found to be highly variable between persons.

Aims: To study the stability of the individual response to a fixed DOAC concentration over time.

Methods: Twelve consenting, healthy donors were included. Blood was collected every two months to a total of six samples. TG was determined in platelet poor plasma, with or without addition of 300 nM rivaroxaban or 200 nM apixaban using 5 pM tissue factor and 4 μM phospholipids. The DOAC response was stated as % inhibition of endogenous thrombin potential (ETP) or peak relative to the control.

Results: The effect of a fixed concentration of apixaban or rivaroxaban on TG parameters in a single donor was not stable over time (Figures 1 and 2). The inhibition of the ETP showed within‐subjects CVs of 5.8‐35.0% for apixaban and 13.1‐41.2% for rivaroxaban; the peak between 2.7‐15.4%, and 1.1‐6.2%, respectively. The variance between uninhibited TG over time was less, mostly for the ETP (within‐subjects CVs 3.4‐14.2% for ETP and 1.2‐8.5% for peak).

Conclusions: Earlier we found that the in vitro response to a fixed DOAC concentration between persons is very variable (CV up to 30.2%). Here we show that also the in vitro response of the same donor changes over time. The shape of the TG curve in the presence of direct factor Xa inhibitors, makes that, contrary to the ETP, the peak is very sensitive at low, but not at higher concentrations of the inhibitor. Therefore, at an inhibition of the ETP around 40‐80%, the variation in the peak is minor. In vivo the pharmacokinetic variation will add to the pharmacodynamic variance detected here. We hypothesize that patients on DOAC could benefit from personalized treatment.

FIGURE 1 Variation per individual in the effect of rivaroxaban and apixaban on TG parameters over six time points during eleven months (D, donor)

FIGURE 2 Response of 12 donors to rivaroxaban and apixaban. Differences were analyzed using the Friedman test with Dunns posthoc *P<0.05, **P>0.01

PB455: Effect of Anti‐arrhythmic Medication Drug Interactions on Thrombotic and Bleeding Outcomes in Patients with Atrial Fibrillation

M. Wanat 1 ; X. Wang 1 ; S. Abughosh 2 ; H. Chen 2 ; M. Johnson 2 ; M. Fleming 2

1 University of Houston, Department of Pharmacy Practice and Translational Research, Houston, United States, 2 University of Houston, Department of Pharmaceutical Health Outcomes and Policy, Houston, United States

Background: Optimizing anticoagulant therapy in patients with atrial fibrillation (AF) is essential to reduce the risk of thromboembolic events and improve quality of life. No real world studies have addressed the effect of antiarrhythmic medications (AADs) on outcomes in patients with non‐valvular atrial fibrillation (NVAF).

Aims: To evaluate thrombosis and bleeding in patients receiving apixaban versus warfarin, and a concomitant anti‐arrhythmic medication.

Methods: This was a retrospective cohort study using a large electronic medical record (2012‐2016). Patients aged 18 years with a diagnosis of NVAF and on warfarin or apixaban, in addition to an AAD were included. Select exclusion criteria included venous thromboembolism and cardiac surgery during the 12 months prior to the index date. The primary endpoint was a composite of stroke (ischemic or hemorrhagic) or systemic embolism. The primary safety endpoint was major bleeding (ISTH definition). Patients were matched using propensity scoring. Univariate survival analyses were conducted using the log‐rank test and Kaplan‐Meier survival curves. A Cox Proportional Hazards Model was used to estimate the risk‐adjusted effect of treatment on time until outcome.

Results: 3,774 patients met inclusion criteria, and 2,496 were included in the propensity matching. There were no differences in baseline characteristics with respect to age, comorbidities, CHADS2‐Vasc score, and concurrent medications. The most commonly used AAD was amiodarone (54.41% vs. 56.17%, P=0.9062).The primary endpoint occurred in 1.04% (n=13) of patients in the apixaban group compared to 1.36% (n=17) in the warfarin group (HR=0.5, P=0.4236). Major bleeding occurred at a similar between the apixaban group (5.29%, n=66) and the warfarin group (6.89%, n=86) (P=0.0763).

Conclusions: There was no difference in thrombotic or bleeding outcomes in patients with NVAF on apixaban or warfarin, and a concomitant AAD. Further randomized controlled trials should be performed to assess the effect of drug interactions.

PB456: Lean Body Weight is the Best Scale for Venous Thromboprophylaxis Algorithm in Severely Obese Patients Undergoing Bariatric Surgery

B. Gaborit 1 ; P.‐A. Moulin 2 ; T. Bege 3 ; S. Boullu 4 ; C. Vincentelli 5 ; O. Emungania 3 ; P.‐E. Morange 2 ; S. Berdah 3 ; A. Dutour 6 ; C. Frere 7

1 Centre Spécialisé de l'Obésité (CSO) PACA Ouest, Pole ENDO, Assistance Publique ‐ Hôpitaux de Marseille; Inserm U 1062, Inra U 1260, Aix‐Marseille Université, Endocrinology, Metabolic Diseases and Nutrition Department, Marseille, France; 2 Timone Hospital, Assistance Publique ‐ Hôpitaux de Marseille, Aix‐Marseille Université, Hematology Department, Marseille, France; 3 Centre Spécialisé de l'Obésité (CSO) PACA Ouest, Pole ADOU North Hospital, Assistance Publique ‐ Hôpitaux de Marseille, Aix‐Marseille Université, Digestive Surgery Department, Marseille, France; 4 Centre Spécialisé de l'Obésité (CSO) PACA Ouest, Pole ENDO, Assistance Publique ‐ Hôpitaux de Marseille, Aix‐Marseille Université, Endocrinology, Metabolic Diseases and Nutrition Department, Marseille, France; 5 Centre Spécialisé de l'Obésité (CSO) PACA OuestPole ENDO, Assistance Publique ‐ Hôpitaux de Marseille, Aix‐Marseille Université, Endocrinology, Metabolic Diseases and Nutrition Department, Marseille, France; 6 Centre Spécialisé de l'Obésité (CSO) PACA Ouest; Pole ENDO, Assistance Publique ‐ Hôpitaux de Marseille; Inserm U 1062, Inra U 1260, Aix‐Marseille Université, Endocrinology, Metabolic Diseases and Nutrition Department, Marseille, France; 7 Pitié‐Salpêtrière Hospital, Assistance Publique Hôpitaux de Paris, Institute of Cardiometabolism and Nutrition, ICAN, Sorbonne Université, Haematology Department, Paris, France

Background: Severely obese patients undergoing bariatric surgery (BS) are at increased risk for venous thromboembolism (VTE). How standard low molecular weight heparin (LMWH) regimen should be adapted to provide both sufficient efficacy and safety in this setting is unclear.

Aims: We aimed to compare the influence of four body size descriptors (BSD) on peak anti‐Xa levels in BS obese patients receiving LMWH fixed doses to identify which one had the greatest impact.

Methods: One hundred and thirteen BS obese patients [median body mass index (BMI), 43.3 kg/m 2 (IQR, 40.6‐48.7 kg/m 2 )] receiving subcutaneous dalteparin 5,000 IU twice daily were included in this prospective monocenter study. Peak steady‐state anti‐Xa levels were measured peri‐operatively following thromboprophylaxis initiation.

Results: Only 48% of patients achieved target anti‐Xa levels (0.2‐0.5 IU/ml). In univariate analysis, age, gender, total body‐weight (TBW), lean body‐weight (LBW), ideal body‐weight (IBW), BMI and estimated glomerural filtration rate (eGFR) were associated with anti‐Xa levels. The strongest negative association was observed with LBW (r=−0.56, P<0.0001). Receiver operating characteristic curves indicated that among BSD LBW (cut‐off >55.8 Kg) had the highest sensitivity (73%) and specificity (69%) to predict sub‐prophylactic anti‐Xa levels. In multivariate analysis, LBW and eGFR remained associated with anti‐Xa levels (β=−0.47±0.08, P<0.0001 and β=−0.19±0.08; P=0.02, respectively).

Conclusions: In BS morbidly obese patients receiving LMWH for thromboprophylaxis after BS, LBW and eGFR are the main determinants of anti‐Xa level, and could be proposed in LMWH‐based thromboprophylaxis dosing algorithms. The efficacy of a LBW‐scale based dosing algorithm for optimal VTE prevention deserves further prospective randomized trials.

PB457: Long‐term Monitoring of Patients after Stroke on Antiplatelet Agents Allows to Identify the Advantages and Limitations of Impedance Aggregation

T. Vavilova 1,2 ; O. Rodionova 2 ; V. Sorokoumov 2 ; L. Isaeva 2

1 Almazov National Medical Research Centre, St. Petersburg, Russian Federation; 2 City Diagnostic Centre N 1, St. Petersburg, Russian Federation

Background: Patients with previous stroke have a significant variability in the response to antiplatelet therapy. The laboratory method, validated in real clinical practice, has not been determined to date.

Aims: To identify the laboratory and clinical advantages and limitations of impedance aggregation in long‐term setting after stroke under antiplatelet therapy.

Methods: 330 patients after stroke were included: aspirin intake (ASP) – 262, clopidogrel (CLOP) – 51, or dual antiplatelet therapy (DAT) – 17; also – 65 healthy persons. All groups are comparable in age and sex. Platelet activity were assessed by impedance aggregation (CHRONO‐LOG 590, USA), induced by final ADP 10 μM/ml and collagen (Col) 2 μg/ml. LagTime, area under the aggregation curve (AUC) were measured. Special attention was paid to preanalytical stage.

Results: The main shift in patients receiving ASP have affected collagen‐induced tests (LagTime Col extension up to 70 (61; 82) s; AUC Col reduction 53.35 (41.70, 65) ohm*s) with LagTime ADP shortening to 23.50 (12; 37) s. The maximum effect was achieved by DAT with reliable changes in all parameters: LagTime Col to 78.00 (72.00, 86.00) s; amplitude decrease (ADP and Col) to 4.00 (1.00, 6.00) ohms and 13.00 (10.00, 16.00) ohms, respectively, AUC ADP and Col reduction to 15.70 (9.40, 25.40) ohm*s and 36.50 (30.30, 50.90) ohm*s, respectively. The platelet activity study at intervals 10.66±0.85 months showed positive LagTime ADP and AUC Col affect if regular ASP intake. The response to CLOP was more stable. Compliance study after 2‐3 years in 100 patients showed that 13.1% of those taking ASP and 13.8% of those taking CLOP interrupted treatment. In the DAT group, 100% compliance was observed. 16% of patients had recurrent vascular events and 3% – minor bleeding without connection with aggregation data.

Conclusions: Impedance aggregation allows monitoring the effect of antiplatelet therapy in patients after stroke, to evaluate the adherence for therapy, but not to predict recurrent vascular events.

PB458: Post Analytical External Quality Assessment of Anticoagulant Dosing for Warfarin Therapy

D. Kitchen ; S. Munroe‐Peart; L. Brown; I. Jennings; S. Kitchen; T. Woods; I. Walker

UK NEQAS for Blood Coagulation, Sheffield, UK

Background: UK National External Quality Assessment Service for Blood Coagulation (NEQAS BC) has been providing External Quality Assurance (EQA) for haemostasis testing for over 40 years. A study of post analytical assessment of anticoagulant dosing was introduced to centres testing INR within the programmes.

Aims: The aim of this study is to assess the variation within anticoagulant dosing and ultimately to improve patient care.

Methods: Virtual patient scenarios were distributed containing a brief clinical history including previous warfarin dosage, a target INR value and a series of historical INR results, together with the dates of testing. Study participants input these data into their computerised decision support software (CDSS) or chose to dose manually and recorded their recommended dose and recall periods based on these data.

Results: 5 exercises have been completed by 1,610 centres (mean 322 per exercise). The virtual patient scenarios have included; 2 cases of a patient with an increasing INR, a patient with a falling INR, a patient with a rising INR which is now at the top of the therapeutic range and a patient with an unexpected high INR of 7.4. Good agreement has been seen for dosing recommendations amongst the majority of users however for 3 of the 5 exercises some responses were a cause for concern. 1 centre suggested a dose increase for a patient with an increasing INR above the target range. 6 users suggested lowering the dose for a patient with a falling INR below the target range and 3 users suggested a dose increase for the patient with an INR of 7.4. Recall periods for these exercises showed good overall agreement but some variation was seen. For example the centre that suggested increasing the dose for the case with an INR of 7.4 suggested a recall time of 4 weeks.

Conclusions: UK NEQAS BC will continue to provide exercises to assess post analytical issues. Education of centres taking part should improve agreement and ultimately patient care.

PB459: Creation of an Algorithm for Predicting Doses of Warfarin <17.5 mg Using CYP2C9 and VKORC1 Polymorphisms and Non‐Genetic Factors

A.O.M. Mourão 1 ; K.B.G. Borges 2 ; E.A. Reis 3 ; R.P. Souza 4 ; E.I.F. Campos 1 ; D.D. Ribeiro 1 ; M.O.C. Rocha 1 ; M.A.P. Martins 1,2

1 Faculdade de Medicina da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 2 Faculdade de Farmácia da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 3 Instituto de Ciências Exatas da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 4 Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

Background: Warfarin is widely used to prevent thromboembolic events. There is a wide variation in inter‐individual and intra‐individual doses during treatment and warfarin maintenance dose can range 8.1‐245.0 mg/week.

Aims: To evaluate the association of polymorphisms CYP2C9*2 (rs1799853), CYP2C9*3 (rs1075910) and VKORC1‐G1639A (rs9923231) and non‐genetic factors with low doses of warfarin (<17.5 mg/week) and to create an algorithm to predict drug sensitivity.

Methods: This was a retrospective cohort study performed in 2015 at a Brazilian anticoagulation clinic. Indications for oral anticoagulation included atrial fibrillation, mechanical heart valve prosthesis or history of ischemic stroke. We investigated the association of non‐genetic factors and warfarin genetic polymorphisms with low doses of warfarin during 2009‐2015. Data review was performed through multivariate logistic regression and the Receiver Operating Characteristic (ROC) curve of the drug sensitivity prediction was constructed from the model. This project was approved by the University Ethics Committee (code number CAAE/23725213.5.0000.5149). All participants signed an informed consent.

Results: A total of 312 patients with heart diseases were included in the study. The mean age was 60.4 (±13.5) years and 59.9% were female. Overall, 12.8% of patients used weekly doses <17.5 mg of warfarin. In the multivariate regression model, variables associated with warfarin doses <17.5 mg/week were: age [odds ratio (OR) 1.03, 95% confidence interval (CI) 1.01‐1.06; P=0.047], genotype VKORC1 AA (OR 31.61, 95% CI 11.20‐100.15, P<0.001) and genotype CYP2C9 2/2, 2/3 or 3/3 (OR 16.48, 95% CI 3.37‐81.79, P<0.001). The ROC curve showed an area of 82.9% (75.0‐90.7%) and cutoff point of 0.14, according to Youden's criteria, with sensitivity of 67.5% and specificity of 87.5%.

Conclusions: In this study, propensity to warfarin sensitivity can only be identified from age and genetic factors.

PB460: Warfarin Use Increases Bleeding Risk in Hemodialysis Patients with Atrial Fibrillation: Evidence from Meta‐analysis Using Real World Data

S. Hussain 1 ; A.N. Siddiqui 1 ; H. Baxi 2 ; A. Habib 3 ; A.K. Najmi 2

1 Jamia Hamdard (Hamdard University), Department of Pharmaceutical Medicine, New Delhi, India, 2 Jamia Hamdard (Hamdard University), Department of Pharmacology, New Delhi, India, 3 Hamdard Institute of Medical Sciences and Research, Jamia Hamdard (Hamdard University), New Delhi, India

Background: Warfarin is the most widely prescribed anticoagulant to prevent thromboembolic complications in atrial fibrillation patients. Atrial fibrillation is common co‐morbid condition in hemodialysis patients. In the last decade several epidemiological studies linked use of warfarin with the increase risk of bleeding.

Aims: To quantify the bleeding risk associated with the use of warfarin.

Methods: This meta‐analysis was conducted in accordance with the PRISMA guidelines. PubMed, Embase and Cochrane central databases were searched from inception to January 2018. Risk of bias was judged using Newcastle‐Ottawa Scale. Primary outcome was to quantify the bleeding risk associated with the warfarin use. Secondary outcome was to assess the bleeding risk based on subgroups like bleeding sites, study design, study quality and follow‐up period. Based on heterogeneity random effect or fixed effect model was chosen. Review Manager (RevMan) version 5.3 was used for performing statistical analysis.

Results: A total of 13 studies constituting a pooled sample of 53,380 patients (44.77% female) of which 16,671 were warfarin user. Half of the studies were of high quality. More than 50% included studies assessed bleeding risk using HAS‐BLED score. Warfarin use was significantly associated with the bleeding risk with pooled RR 1.17 (95% CI: 1.10 to 1.24, P≤0.00001) (Figure 1).

FIGURE 1 Forest plot showing bleeding risk among warfarin user compared to non‐user

No significant association was observed for major bleeding risk RR 1.02 (95% CI: 0.86 to 1.22, P=0.81). Subgroup analysis revealed significant association of warfarin use with the intracranial haemorrhage in an adjusted analysis (6 studies) with pooled relative risk of 1.54 (95% CI: 1.23 to 1.92, P=0.0001). In contrast no significant association was observed between warfarin use and gastrointestinal bleeding.

Conclusions: The results indicate that warfarin use increases the risk of bleeding in hemodialysis patients with atrial fibrillation.

PB461: Change of Rivaroxaban Anticoagulant Effect during the 24 Hours

I. Geltcer; O. Smirnova; O. Matvienko ; O. Golovina; L. Papayan

1 Russian Research Institute of Hematology and Transfusiology, Laboratory of Blood Coagulation, St. Petersburg, Russian Federation

Background: Practical recommendations for the extended therapy of venous thromboembolism (VTE) include the appointment of rivaroxaban once‐daily.

Aims: The aim of the study was to evaluate the dynamics of thrombin generation inhibition within 24 hours on the treatment of once‐daily rivaroxaban.

Methods: The study involved 35 patients with VTE. Blood was taken at 1.5‐4 hours and 19‐39 hours after the last drug administration. Control group consisted of 28 healthy adults. The thrombin generation (TG) was measured by Calibrated Automated Thrombogram (CAT) according to Hemker et al., with PPP reagent ± TM. We analyzed the following parameters: endogenous thrombin potential (ETP, nM*min), thrombin peak height (Peak, nM). Parameter protein C (PC) system efficacy was estimated as sensitivity to TM – the reducing the values of the ETP and Peak thrombin after the addition of TM (%). Statistica 6.0 package was used. The data were presented as median (Me) and 95% confidence interval (95% CI). P values were calculated using the Mann‐Whitney test.

Results: TABLE 1 Parameters of CAT in patients at 1.5‐4 and 19‐39 hours after rivaroxaban administration (Me, 95% CI) CAT parameters Healthy adults (Me, 95% CI) (n=28) Patient at 1.5‐4 hours after the drug administration (n=35) Patient at 19‐39 hours after the drug administration (n=35) ETP without TM, nM*min 1,756.00 (1,220.55‐2,159.85) 861.6** (482.6‐1,434.3) 1,558.8 (1,230.1‐2,315.4) ETP with TM, nM*min 878.00 (538.75‐1,381.00) 177.4** (73.9‐369.8) 574.1* (262.1‐1,172.0) Peak without TM, nM 293.9 (198.70‐376.17) 42.7** (23.5‐80.3) 164.4** (96.8‐279.1) Peak with TM, nM 174.49 (113.81‐310.00) 22.0** (7.0‐52.8) 96.7** (42.8‐200.8) Sensitivity of ETP to TM, % 51.53 (22.93‐64.40) 81.9** (60.6‐88.8) 67.2* (39.2‐81.7) Sensitivity of Peak to TM, % 35.78 (14.77‐53.10) 49.0* (30.0‐76.3) 38.3 (9.6‐66.5)

All parameters of CAT in patients at 1.5‐4 hours after ingestion rivaroxaban are changed (Table). ETP and Peak without and with TM were significantly reduced compared to the healthy individuals values. ETP and Peak sensitivity to TM were increased. Data of CAT in patients at 19‐39 hours after rivaroxaban receiving are changed too. The ETP was significantly reduced only after addition of TM. Peak thrombin without and with TM was decreased. At the same time, the ETP sensitivity to TM was enhanced with regard to control, the Peak sensitivity to TM was within the normal range. *P<0.00005, **P<0.000001 as compared with healthy adults

Conclusions: Our results show that once‐daily rivaroxaban provides sufficient anticoagulant effect throughout the day. Prolonged anticoagulation in patients with VTE within 24 hours may be associated with the enhancement of PC system efficacy in patients received factor Xa inhibitor.

PB463: Flow Based Analysis of Heparin Inhibition of VWF Dependant Platelet Capture

A. Ranger ; T. Mckinnon; M. Laffan

Imperial College London, Centre for Haematology, London, UK

Background: The principal role of Von Willebrand Factor (VWF) is platelet capture at sites of vascular injury, via interaction of its A1 domain with platelet glycoprotein Ib (GPIb). The GPIb binding site overlaps with a binding site for heparin and previous studies have demonstrated inhibition of platelet binding by heparin. The physiological and clinical relevance of this effect is not known.

Aims: The aim of this study was to determine the ability of different heparin fractions to mediate inhibition of VWF‐dependent platelet capture on a collagen surface in a physiological flow based assay.

Methods: Healthy donor blood was incubated with unfractionated heparin (UFH), low molecular weight heparin (LMWH) (1‐10 units/ml) or Fondaparinux (1‐10 ug/ml). Concentrations correspond to standard therapeutic anticoagulation, cardiopulmonary bypass surgery (CPB) and supratherapeutic levels. Platelets were labelled with DiOC6. Blood was perfused at 1,500/second, through a microfluidic channel coated with Type I Collagen. Platelet capture over time was quantified using an inverted fluorescent microscope and ImageJ software. A Friedman test with Dunn's multiple comparisons was used for statistical analysis of the data.

Results: At 4 min, surface coverage by platelets was reduced in the presence of UFH at 5 U/ml 21.6±9.5 (P=0.0429) and 10 U/ml 15.0±5.0 (P<0.0007) cf. control 43.4±10.1. In the presence of 10 U/ml Tinzaparin platelet capture was statistically reduced compared to control (P<0.0007). No significant difference was seen in platelet capture in the presence of Fondaparinux or Enoxaparin at the doses tested.

Conclusions: UFH inhibits VWF dependent platelet capture under flow. These results suggest the inhibitory effect on platelet capture is size dependent but cannot be mediated by the antithrombin‐binding pentasaccharide alone. It may explain why unpredictable bleeding complications occur during UFH therapy, especially at the high concentrations used during cardiopulmonary bypass, but also with some LMWH.

PB464: Comparative Safety of Co‐prescribed Calcium Channel Blockers and Oral Anticoagulants for Adverse Bleeding Events in Atrial Fibrillation

P. Pham; S. Schmidt; J. Brown

University of Florida College of Pharmacy, Gainesville, United States

Background: Direct oral anticoagulants (DOACs) have fewer drug interactions than VKAs. However, potential interactions with commonly co‐prescribed medications are a safety concern. Verapamil (V) and diltiazem (D) are inhibitors of P‐glycoprotein and diltiazem has CYP3A4 inhibitory activity, interactions which may increase bleeding risk.

Aims: To evaluate the risk of bleeding with co‐prescribed anticoagulants and V/D using an active comparator design.

Methods: This retrospective cohort study utilized claims data from 2010 to 2015. New users of anticoagulants with atrial fibrillation were identified. V and D use was assessed in the 6 months prior to this index date and required minimum 90 days cumulative exposure. Amlodipine (A) was chosen as the active comparator as a similar medication with no interaction profile. Due to small sample size and expected event rates, the V and D groups were combined. Inverse probability of treatment weights (IPTW) were generated to balance the V/D and A groups on measured baseline characteristics stratified by OAC treatment. A proportional hazards model assessed the risk of major, moderate, and overall bleeding over a 1‐year follow‐up.

Results: A total of 15,623 OAC users with V/D or A co‐prescribed including 8,469 warfarin, 2,675 dabigatran, 2,935 rivaroxaban, and 1,545 apixaban users. Primary results showed no increased risk of overall bleeding, moderate, or major bleeding for V/D versus A users for any anticoagulant. Only dabigatran+V/D showed an increased risk (HR=1.45 [1.01‐2.12]) for minor gastrointestinal bleeding compared to dabigatran+A users. Sensitivity analyses showed no variation in results.

TABLE 1 Risk of bleeding verapamil or diltiazem versus amlodipine during anticoagulant treatment in atrial fibrillation Hazard ratio (95% confidence level) Outcome Measure Warfarin DOAC (pooled estimate) Dabigatran Rivaroxaban Apixaban Overall bleeding 1.13 (0.98‐1.30) 1.10 (0.94‐1.28) 1.28 (0.98‐1.66) 0.97 (0.78‐1.22) 1.17 (0.80‐1.71) Overall GI bleeding 1.14 (0.92‐1.41) 0.93 (0.74‐1.18) 1.39 (0.97‐1.99) 0.75 (0.53‐1.07) 0.60 (0.30‐1.20) Major or moderate bleeding 0.96 (0.74‐1.24) 1.13 (0.84‐1.53) 1.34 (0.74‐2.42) 1.02 (0.68‐1.54) 1.29 (0.66‐2.49) Major or moderate GI bleeding 1.27 (0.89‐1.81) 0.65 (0.41‐1.03) 1.30 (0.61‐2.76) 0.54 (0.28‐1.05) 0.27 (0.06‐1.14) Any minor bleeding 1.16 (0.99‐1.34) 1.10 (0.93‐1.29) 1.30 (0.99‐1.70) 0.95 (0.75‐1.20) 1.21 (0.82‐1.78) Minor GI bleeding 1.14 (0.92‐1.42) 0.95 (0.75‐1.21) 1.46 (1.01‐2.12) 0.74 (0.51‐1.07) 0.60 (0.30‐1.20) GI, gastrointestinal; DOAC, direct oral anticoagulant

Conclusions: In this large study, the drug interaction between DOACs and V/D did not show evidence of an increased risk of bleeding when compared to an active comparator group.

PB465: Real‐world Experience of Direct Oral Anticoagulants Use in North Melbourne, Australia

R. Brook; T. Kitipornchai; P. Ho; H.Y. Lim

Northern Health, Haematology, Epping, Australia

Background: There is limited data evaluating the real‐world use of direct oral anticoagulants (DOACs), particularly compliance to prescribing recommendations and safety data.

Aims: To evaluate our real‐world DOAC use, in comparison to clinical trials and other reported real‐world experience.

Methods: Retrospective evaluation of all patients commencing/continuing on DOAC between September 2013 and June 2016 in our hospital including demographics, type of malignancy and safety outcomes.

Results: 884 patients with median age 69 years (range 17‐96) were identified. The average creatinine clearance was 85.9 mL/min, with 10 patients having CrCl<30 mL/min. The most common indication for DOAC was atrial fibrillation (AF) (n=523, 59.1%) with average CHADS2VASC score 4. Of note, we identified 54 cancer patients on DOACs (23 (42.6%) for venous thromboembolism (VTE) and 31 (57.4%) for AF). Overall, there were 27 episodes of clinically significant bleeding (ISTH‐SSC score 3/4) including one death. These patients were older (median age 78, P=0.02) and six patients (22.2%) were on concurrent anti‐platelet therapy, comparable to non‐bleeders (13.2%, P=0.18). Tables 1 and 2 compare our bleeding rates to those previously reported. Ten episodes of ischaemic stroke (1.9%) and 9 VTEs (2.5%) were reported while on DOACs. In the subset of cancer patients, there was an episode of major bleeding (ISTH‐SSC 4) and recurrent VTE respectively in the those prescribed DOACs for VTE‐related reasons, comparable to our warfarin/enoxaparin era data (1/23 (4.3%) versus 9/240 (3.9%), P=0.89) recurrence on therapeutic anticoagulation). No episode of ischaemic stroke was reported in the cancer patients.

TABLE 1 Real‐life DOAC experience compared to clinical trial data in AF AF (n=523) Real Life Data for AF Clinical Trial Data for AF This Study Danish study (Larsen et al) ROCKET‐AF RELY ARISTOTLE Rivaroxaban (45.5%) 2.1% (5/238) 3.6% 3.6% Dabigatran (17.2%) 3.3% (3/90) 2.0% 2.9‐3.3% Apixaban (37.3%) 6.2% (12/195) 2.7% 2.1%

TABLE 2 Real‐life DOAC experience compared to clinical trial data in VTE VTE (n=361) Real Life Data for VTE Clinical Trial Data for VTE This study XALIA EINSTEIN AMPLIFY Rivaroxaban (90.0%) 2.2% (7/325) 0.7% 0.8% Apixaban (9.7%) 0% (0/35) 0.6%

Conclusions: The bleeding and recurrent thrombosis rate in our study is comparable to real‐world and clinical trial data although interestingly, it was higher in AF patients on apixaban. DOACs also appear to be effective and safe for both AF and VTE treatment in cancer patients.

PB466: Relationship Between Dabigratan Concentrations in Plasma and Results of Routine Coagulation Assays PT, APTT, TT and Fibrinogen

S. Margetic ; A. Bronic; I. Celap; I. Vuga

Sestre Milosrdnice University Hospital Center, Department of Clinical Chemistry, Zagreb, Croatia

Background: Routine coagulation tests prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen are affected by dabigatran therapy, but are not standardized for assessment of its anticoagulant effect. However, exact knowledge of its impact on routine coagulation tests is a precondition for their correct interpretation.

Aims: To assess relationship between dabigatran concentrations and results of routine coagulation assays.

Methods: The study included 43 patients on dabigatran therapy. Plasma dabigatran concentrations were determined with DTI Innovance assay (Siemens, Germany) as a part of Croatian Science Foundation project HRZZ‐IP‐2016‐06‐8208. All coagulation assays were determined on BCSXP analyzer using commercial reagents (Table 1).

Results: Dabigatran plasma concentrations ranged from 22 to 401 ng/mL (median 101 ng/mL; 95%CI 59‐157 ng/mL). TT was unmeasurable (>150 s) in all patients with dabigatran levels above 100 ng/ml with significantly higher (P<0.0001) dabigatran levels compared to patients with TT<150s (median 69s). Among all patients, 16/43=0.37 had PT and 5/43=0.12 had APTT results in the reference range while 27/43=0.63 and 38/43=0.88 had prolonged PT and APTT, respectively. PT, APTT and fibrinogen were significantly different between patients with dabigatran levels below and above 100 ng/mL (Table 1).

TABLE 1 Relationship between dabigatran concentrations and results of routine coagulation assays Assay Dabigatran (ng/mL) TT (s) PT (% activity) PT INR APTT (s) APTT (ratio) Fibrinogen (g/L) Commercial reagent (Siemens) Innovance DTI BC Thrombin Innovin Innovin Actin FS Actin FS Multifibren U N=25; Median 54 69 82 1.1 37.5 1.36 4.0 95%CI for median 43‐70 45‐90 64‐90 1.1‐1.3 35‐41 1.3‐1.5 3.1‐5.2 Interquartile range (IQR) 43‐70 45‐90 66‐89 1.1‐1.3 35‐41 1.3‐1.5 3.2‐5.2 N=18; Median 209 >150 50 1.46 65.0 2.35 3.1 95%CI for median 141‐321 ‐‐‐ 44‐55 1.3‐1.6 50‐69 1.8‐2.5 2.3‐3.7 Interquartile range (IQR) 141‐305 ‐‐‐ 44‐54 1.3‐1.6 52‐70 1.9‐2.5 2.3‐3.7 P (N=25 vs. N=18) <0.0001 <0.0001 0.0003 0.0007 0.0001 0.0001 0.0208

Conclusions: The results of routine coagulation tests are strongly dependent on dabigatran plasma concentrations. Due to its high sensitivity, TT could be useful for detecting minimal dabigatran levels in plasma, but is too sensitive for dabigatran levels above 100 ng/mL. PT lacks sensitivity in detecting therapeutic levels of dabigatran but neither the APTT was invariably prolonged in patients with dabigatran levels below 100 ng/mL. In summary, routine coagulation tests PT, APTT and TT, although dependent on dabigatran concentrations, could not be used to assess therapeutic response to dabigatran therapy through a wide range of its concentrations in plasma.

PB467: Measuring Anti‐factor Xa Levels to Monitor Enoxaparin in Pregnant Women: A Prospective Comparative Study

F. Aleidan 1,2 ; A. Aldakhil 3 ; B. Allehyani 3 ; G. Aljarba 3 ; M. Yahia 3 ; N. Alghtani 2 ; A. Alsuhaibani 2 ; A. Alaklabi 2

1 King Saud bin Abdulaziz university for Health Sciences, Riyadh, Saudi Arabia; 2 King Abdulaziz Medical City, Riyadh, Saudi Arabia; 3 Prince Noura University, Riyadh, Saudi Arabia

Background: Enoxaparin is considered safe and efficacious and is a commonly used anticoagulant for pregnant women with appropriate indications.

Aims: To compare the anti‐factor Xa peak levels (therapeutic, subtherapeutic, or supratherapeutic) between pregnant and non‐pregnant women who are receiving therapeutic doses of enoxaparin and to assess the incidence of new thrombosis and bleeding complications.

Methods: A prospective observational cohort study was conducted between July 1st, 2017 and January 31st, 2018 at King Abdulaziz Medical City, Riyadh, Saudi Arabia. Sixty‐eight patients (36 pregnant and 32 non‐pregnant women) who received a therapeutic doses of enoxaparin and had a three steady‐state antifactor Xa peak levels collected during the study period. Institutional review board approval was obtained to collect data from patients′ hospital visit. Enrolled patients were followed for at least three consecutive anti‐factor Xa levels results with an interval duration of 4 weeks and enoxaparin dose adjusted based on levels’ results.

Results: The analysis of anti‐factor Xa levels data at the first measurement showed that only 41.7% of pregnant women after commencing enoxaparin were in therapeutic range compared to 65.6% of non‐pregnant women, the difference was not statistically significant (P=0.120). The second measurement of anti‐factor Xa level showed that pregnant women having a significantly lower proportion when compared to non‐pregnant women (58.3% vs. 86.2%; P=0.010). Twelve weeks after commencing enoxaparin, therapeutic anti‐factor Xa level was statistically significant lower in pregnant women compared to non‐pregnant group (61.1% vs. 81.3%; P=0.021). No major bleeding or new thrombosis was observed.

Conclusions: Based on the results of this study, close and frequent monitoring of anti‐factor Xa levels during pregnancy needs to be considered and can guide enoxaparin adjustment with subtherapeutic or supratherapeutic ranges.

PB468: Direct Oral Anticoagulants in Patients with Nonvalvular Atrial Fibrillation in Clinical Practice: Outcomes from the First Observational and Prospective Study in Spanish Population

M. Cerdá Sabater ; E. Johansson; F. Martínez García; M. Fernández Caballero; A. Varela Magallón; S. Rodríguez López; F. Bosch Albareda; A. Santamaría Ortiz

Hospital Vall d'Hebrón, Barcelona, Spain

Background: The introduction of direct oral anticoagulants (DOACs) in clinical practice has changed the anticoagulation pattern in atrial fibrilation (AF) population. However, the majority of the studies about DOAC have a retrospective design, data come from insurance databases or nationwide/regional registries, clinical characteristics of patients often remain unknown, and a comprehensive analysis of data is not frequently performed. Therefore, studies analyzing real‐life data about the effectiveness and safety of DOACs in prospective cohorts of AF patients are warranted.

Aims: This observational and prospective study was aimed to analyze the effectiveness and safety of the 4 DOACs currently marketed in a cohort of patients with nonvalvular AF attended in clinical practice in the province of Barcelona (Spain).

Methods: Patients were recruited between January 2015 and September 2017. A total of 1,443 patients were included, 663 (46.0%) patients were taking rivaroxaban, 352 (24.4%) dabigatran, 325 (22.5%) apixaban, and 103 (7.1%) edoxaban. Mean age was 77.2±9.7 years, 52.8% were women, 20.0% had prior stroke/transient ischemic attack, 34.4% creatinine clearance <50 ml/min and mean CHA2DS2‐VASc was 4.1±1.5. Compared with patients taking the standard dose of DOACs, those taking the low dose of DOACs were older, and had a worse renal function. See characteristics in Table 1.

Results: After a mean follow‐up of 1.7 years, rates of ischemic stroke, major bleeding and intracranial bleeding were 0.7, 1.3, and 0.2 events/100 patient‐years, respectively. Rates of ischemic stroke and intracranial bleeding were similar among DOACs, but rates of major bleeding were higher with dabigatran and apixaban and lower with rivaroxaban. See Table 2.

Conclusions: In conclusion, this is the first prospective study that have analyzed the management and outcomes of all DOACs currently marketed in AF patients in Spain. Despite differences in the clinical profile, this study showed that the use of DOACs in clinical practice is effective and safe.

TABLE 1 Baseline clinical characteristics of the study population according to the type of DOACs Total (n=1,443) Rivaroxaban (n=663) Apixaban (n=325) Dabigatran (n=352) Edoxaban (n=103) PDOACs Age (years) 77.2±9.7 77.8±9.7 78.9±8.7 73.1±15.2 78.7±7.0 <0.001 Sex, women, n (%) 762 (52.8) 364 (54.9) 157 (48.3) 193 (54.8) 48 (46.6) NS CHA2DS2‐VASCc 4.1±1.5 4.0±1.4 4.4±1.5 3.9±1.5 4.2±1.5 <0.001 Hypertension, n (%) 1,226 (84.9) 564 (85.1) 275 (84.6) 299 (84.9) 88 (85.4) NS Diabetes, n (%) 431 (29.9) 173 (26.1) 129 (39.7) 90 (25.6) 39 (37.9) <0.001 CrCl (ml/min) 59.0±21.3 59.1±19.8 49.8±21.5 66.4±21.7 62.3±18.0 <0.001 CrCl <50 ml/min, n (%) 497 (34.4) 229 (34.5) 177 (54.5) 66 (18.8) 25 (24.3) <0.001 Heart failure, n (%) 264 (18.3) 99 (14.9) 95 (29.2) 38 (10.8) 32 (31.1) <0.001 Prior stroke/TIA, n (%) 289 (20.0) 123 (18.6) 81 (24.9) 68 (19.3) 17 (16.5) NS

TABLE 2 Thromboembolic and bleeding events according to the type of DOACs n (%) Events/100 patient‐years Total (n=1,443) Rivaroxaban (n=663) Apixaban (n=325) Dabigatran (n=352) Edoxaban (n=103) PDOACs Ischemic stroke n (%) Events/100 patient‐years 16 (1.1) 0.7 5 (0.7) 0.5 4 (1.2) 0.9 5 (1.4) 0.6 2 (1.9) 2.4 NS Major bleeding n (%) Events/100 patient‐years 33 (2.3) 1.3 9 (1.3) 0.8 7 (2.2) 1.7 16 (4.5) 1.8 1 (1.0) 1.2 0.009 Intracranial hemorrhage n (%) Events/100 patient‐years 4 (0.3) 0.2 0 0 2 (0.6) 0.5 2 (0.6) 0.2 0 0 NS

PB469: Real‐world Compliance Patterns with Direct Oral Anticoagulants with Acute Venous Thromboembolism

M. Bartlett 1 ; D. Bott Kitslaar 1 ; E. Vargas 2 ; A. Casanegra 1 ; D. Froehling 1 ; D. Hodge 2 ; R. McBane 1 ; W. Wysokinski 1

1 Mayo Clinic, Thrombophilia Center, Rochester, United States; 2 Mayo Clinic, Health Sciences Research, Jacksonville, United States

Background: It is unknown whether the different dosing patterns of apixaban (twice daily) or rivaroxaban (once daily) influence patient compliance in acute venous thromboembolism (VTE).

Aims: To compare patient compliance between apixaban and rivaroxaban in a real‐world setting for the treatment of acute VTE.

Methods: A survey was sent to patients enrolled into the Mayo Thrombophilia Clinic Direct Oral Anticoagulants Registry after July 2016, asking if they had missed one or more treatment doses. Patients diagnosed acute VTE within 1 year of receiving the survey were included. All participants were prospectively followed to evaluate rates of recurrent VTE, major bleeding or death (reported as rate per 100 patient‐years). Patients reporting missing any doses were considered non‐compliant. Differences in compliance rates between apixaban and rivaroxaban were compared using a chi‐square test. Differences in clinical outcomes between compliant and non‐compliant patients were compared using a log rank test.

Results: Three‐hundred and sixty three patients were included in the study group and 185 (51%) responded (129 taking apixaban and 56 rivaroxaban). The mean age was 60.3 years and 45.4% were female. Patients treated with apixaban were more likely to have active cancer (44.2% vs. 17.9%, P‐value <0.001). Fifty patients taking apixaban (38.8%) and twenty patients taking rivaroxaban (35.7%) reported missing one or more doses (p‐value 0.69). There were higher rates of VTE recurrence (6.60 vs. 3.87 per 100 patient‐years) and death (3.23 vs. 1.91 per 100 patient‐years) in non‐compliant patients but this difference was not statistically significant (Table 1).

TABLE 1 Clinical outcomes stratified by compliance pattern Non‐compliant (N=70) P‐value VTE recurrence Rate per 100 person‐years N events Total person‐years 3.87 2 51.74 6.60 2 30.31 0.74 Major bleeding Rate per 100 person‐years N events Total person‐years 3.83 2 52.25 3.23 1 30.98 0.87 Death Rate per 100 person‐years N events Total person‐years 1.91 1 52.40 3.23 1 30.98 0.39 Any major event Rate per 100 person‐years N events Total person‐years 23.26 12 51.59 13.20 4 30.31 0.40

Conclusions: The rate of non‐compliance was similar in patients on apixaban (twice daily) and rivaroxaban (once daily) for acute VTE. Non‐compliance was not associated with different rates of clinical outcomes but small patient numbers preclude a firm statement regarding this effect.

PB470: Multihospital System Implementation of the Anti‐Xa for Titration for Monitoring Unfractionated Heparin (UH) Therapy

P. Lindholm 1 ; K. Zmuda 2 ; E. Short 3 ; R. Sumugod 2 ; A. Shah 4 ; D. Cooke 5

1 Northwestern University, Naperville, United States; 2 Northwestern Medicine HealthCare, Laboratory Medicine, Chicago, United States; 3 Northwestern Medicine HealthCare, Pharmacy, Chicago, United States; 4 Northwestern Medicine HealthCare, Quality Performance Administration, Chicago, United States; 5 Northwestern Medicine HealthCare, CEO Office, Chicago, United States

Background: Northwestern Memorial HealthCare implemented a common electronic medical record for its six acute care hospitals (ranging from a 1,000 bed academic medical center to a 25 bed critical access hospital) in early 2018. Common protocols for unfractionated heparin (UH) dosing and monitoring using the chromogenic anti‐Xa assay were developed.

Aims: To demonstrate the feasibility of simultaneously introducing anti‐Xa monitoring of UH therapy across a multihospital health system and understand in advance the implications of doing so in the management of UH therapy.

Methods: A physician‐led, multidisciplinary team was established to standardize titrated dose UH protocols. Hemostasis testing instrumentation was standardized across hospitals. A single electronic order set containing three dosing protocols provided UH dose titration nomograms driven by anti‐Xa assay results. A pilot study compared anti‐Xa and aPTT results using more than 500 samples during UH therapy over 8 weeks. The central lab provided patient correlation samples with single‐source chromogenic assay and instrument platforms.

Results: The pilot study compared anti‐Xa with aPTT in UH patients using paired samples. Actual dose titration was based on aPTT results only. The anti‐Xa /aPTT relationship showed excellent agreement with the existing hemostasis laboratory data. Despite this fact, we observed titration nomogram category concordance in only 56 percent of samples. Among discordant paired samples, the indicated dose titration varied by two or more nomogram categories in one out of every eight instances.

Conclusions: The concurrent implementation of the anti‐Xa assay for monitoring UH therapy in a multihospital system is feasible and supports standardization of practices within the system. Use of the anti‐Xa assay for UH dose adjustment will result in altered titration in a large proportion of cases.

PB471: Correcting Inappropriate Prescribing of Direct Oral Anticoagulants: A Population Health Approach

T. Dawson; D. DeCamilo; E. Kline‐Rogers; B. Haymart; J. Froehlich; G. Barnes

University of Michigan, Frankel Cardiovascular Center, Ann Arbor, United States

Background: Patients starting on direct oral anticoagulants (DOAC) are frequently prescribed inappropriately according to the US FDA package insert. Methods to correct inappropriate DOAC drug selection and dosing have yet to be fully studied.

Aims: To assess the efficacy a population health approach to inappropriate DOAC prescribing.

Methods: As a part of an IRB‐approved quality improvement project, we randomly selected patients newly starting on DOAC therapy within our health center for manual chart abstraction. Indication of DOAC therapy, drug and dose selection, demographics, comorbidities, laboratory data, and concurrent medication use were obtained by a trained abstractor. We explored the rate of inappropriate DOAC dosing according to the FDA prescribing materials. When inappropriate prescribing was detected in a timely manner, an anticoagulation physician contacted the prescribing provider. We followed all patients to determine if any therapeutic changes were made.

Results: Between August 2016 and December 2017, 340 new DOAC patients were randomly selected for inclusion in the study. DOAC drug selection or dosing was inappropriate in 39/340 (11.5%) patients (Table).

TABLE 1 Inappropriate DOAC Prescribing and Provider Action Dosing Indication and DOAC Selected (n=39) Possible Contraindication* (n=40) VTE 24 (61.5%) Dose Frequency Not Adjusted After Initial Phase in VTE 8 (20.5%) Atrial Fibrillation/Flutter 15 (38.5%) Inappropriate Dose Based on Renal Function 8 (20.5%) Apixaban 14 (35.9%) Atrial Fibrillation Dosing for VTE 6 (15.4%) Dabigatran 1 (2.6%) Bioprosthetic Heart Valve 5 (12.8%) Rivaroxaban 25 (61.5%) Severe Hepatic Impairment 2 (5.1%) Prescribing Provider Contacted (n=19) Concomitant Medication Interaction 3 (7.7%) DOAC Change or DOAC Dose Change 12 (63.2%) *one patient had 2 contraindications Prescribing Provider Aware, No Plans to Change 4 (21.1%) Other 3 (15.8%)

Findings from the 39 patients include: 1) dosing frequency not adjusted after initial phase in VTE (8/39, 20.5%), 2) inappropriate dosing based on renal function (8/39, 20.5%), and 3) atrial fibrillation dosing for VTE (15.4%). The prescribing provider was contacted for 19/39 (48.7%) cases. A drug or dose was changed in 12/19 (63.2%) patients where contact was initiated with the primary DOAC prescriber.

Conclusions: While inappropriate DOAC drug/dose selection remains common, contact between the anticoagulation and prescribing physician frequently lead to changes in drug selection or dose. Further studies are needed to explore how best to scale this approach for large health systems and populations, including the use of non‐physician anticoagulation staff and computerized screening of inappropriate drug/dose selection.

PB472: Impact of Rivaroxban on Platelet Reactivity and von Willebrand Factor Antigen and Activity in Patients with Venous Thrombosis

N. Schultz 1,2,3 ; P.A. Holme 2,4 ; C.E. Henriksson 2,5 ; S. Bjørnsen 1 ; P.M. Sandset 2,4 ; E.M. Jacobsen 4

1 Oslo University Hospital, Research Institute of Internal Medicine, Oslo, Norway; 2 University of Oslo, Institute of Clinical Medicine, Oslo, Norway; 3 Akershus University Hospital, Department of Haematology, Lørenskog, Norway; 4 Oslo University Hospital, Department of Haematology, Oslo, Norway; 5 Oslo University Hospital, Department of Medical Biochemistry, Oslo, Norway

Background: The factor Xa inhibitors are safe and effective alternatives to warfarin in treating venous thrombosis. Several studies, however, indicate that patients treated with rivaroxaban have an increased risk of abnormal uterine bleedings. It is not known why the risk of uterine bleedings increases while the risk of intracranial and gastrointestinal bleedings decreases with the use of rivaroxaban compared to warfarin. Furthermore, the effect of rivaroxaban on the primary haemostasis is poorly understood.

Aims: To investigate the effect of rivaroxaban on platelet function and von Willebrand factor antigen (vWF) antigen and activity.

Methods: Blood from 12 rivaroxaban‐treated patients (due to venous thromboembolic episodes with known reversible causes) was collected before (trough concentration) and 2 hours (peak concentration) after tablet intake, and 4‐6 weeks after discontinuing the treatment (control value). The platelet reactivity was assessed in platelet rich plasma by light transmission aggregometry (LTA) in response to arachidonic acid, ADP, TRAP, adrenalin, ristocetin, collagen and thrombin. Latex immunoagglutination assays were used to measure vWF antigen and vWF activity. Rivaroxaban concentration was measured by an anti‐FXa activity method calibrated for rivaroxaban.

Results: Platelet reactivity measured by LTA was similar before (trough and peak concentration) and after cessation of rivaroxaban. There were also no differences in vWF antigen and activity between the different time‐points. The correlation between the rivaroxaban concentration and platelet reactivity was weak.

Conclusions: In rivaroxaban‐treated patients, we found no differences in platelet reactivity or vWF antigen/activity in blood drawn at trough‐ and peak concentration compared to 6 weeks after discontinuation of rivaroxaban; hence rivaroxaban seems not to influence these parameters.

PB473: Weekly Warfarin Maintenance Dose According to Variations in Four Genes Evaluated in Patients Assisted at a Brazilian Anticoagulation Clinic

E.I.F. Campos 1 ; K.B.G. Borges 2 ; A.O.M. Mourão 1 ; M.O.C. Rocha 1 ; D.D. Ribeiro 1 ; R.P. de Souza 3 ; M.A.P. Martins 1,2

1 Faculdade de Medicina da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 2 Faculdade de Farmácia da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 3 Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

Background: Warfarin is an oral anticoagulant used to treat and prevent thromboembolism. There is evidence that genetic variations on CYP2C9, VKORC1, MDR1 and APOE genes affect warfarin dose and it can be associated with drug sensitivity or resistance.

Aims: To compare weekly warfarin maintenance dose for CYP2C9, VKORC1, MDR1 and APOE genotypes.

Methods: This was a cross‐sectional study, performed in 2015 at an anticoagulation clinic of a Brazilian university hospital. Polymorphisms *1, *2 and * 3 for CYP2C9 gene, −1,639 G> A for VKORC1 gene, 3,435 C>T for MDR1 and *ε2, *ε3 and *ε4 for APOE were genotyped using Real‐Time PCR. We collected data through patient interviews and medical chart review. Weekly warfarin maintenance dose was obtained from the hospital computerized system. Descriptive statistical methods were used for data evaluation. The comparison of weekly warfarin dose and genotypes was performed using ANOVA and a significance level of 0.05. This project was approved by the University Ethics Committee (code number CAAE/08136613.4.0000.5149). All participants signed an informed consent.

Results: Evaluating the 136 study participants, mean age was 60.1±13.6 years and 78 (57.4%) were female. The main indication for warfarin use was atrial fibrillation (n=98; 72.1%). The presence of polymorphic allele ( A ) in VKORC1 and at least one polymorphic allele ( * 2 and/or * 3) in CYP2C9 was associated with a reduced weekly warfarin maintenance dose (P<0.001). Conversely, the presence of * ε2 and/or * ε4 polymorphic alleles in APOE and polymorphic allele (A) in MDR1 was not associated with statistically significant dose modification (Table 1).

TABLE 1 Doses stratification by genotype (n=136) Genes Genotypes (n) Dose/week Anova (P value) VKORC1 GG (66) GA (51) AA (19) 37.5±16.4 26.5±10.4 17.0±6.2 <0.001 <0.001 CYP2C9 *1*1 (95) *1*2 (23) *1*3 (12) *2*2 (3) *2*3 (3) At least one polymorphic allele (*2 and/or *3) (41) 33.8±15.4 25.4±14.0 21.0±6.6 16.9±8.2 16.7±2.7 22.9±11.6 0.0189 0.0056 0.0630 0.0587 <0.001 APOE *ε3*ε3 (84) *ε2*ε3 (16) *ε2*ε4 (1) *ε3*ε4 (33) *ε4*ε4 (2) 31.9±16.3 26.6±11.2 34.6 28.3±14.2 36.2±5.3 0.2164 0.8705 0.2709 0.7159 MDR1 GG (55) GA (57) AA (24) 33.0±15.8 28.3±12.5 29.9±18.9 0.0796 0.4571

Conclusions: Only CYP2C9 and VKORC1 polymorphisms influenced weekly warfarin maintenance dose requirements in the studied population. The knowledge of patient genotypes can help make more accurate dose adjustments and bring more quality to the treatment of patients using warfarin.

PB474: Spontaneous Rectus Sheath Hematoma in Patients on Anticoagulant Therapy: A Systematic Review

S. Khaliq

Fauji Foundation Hospital/Foundation University Medical College Rawalpindi, Rawalpindi, Pakistan

Background: Rectus sheath hematoma is a rare complication of anticoagulant therapy that is often misdiagnosed and can lead to life‐threatening emergency especially in the elderly.

Aims: Systematic review of literature to identify incidence,clinical characteristics, types of anticoagulants, work‐up, management and outcome of patients with rectus sheath hematoma on anticoagulant therapy.

Methods: Literature search (1957‐2017) on spontaneous rectus sheath hematoma in patients on anticoagulant therapy was done using PubMed, Embase and Cochrane. Pooled estimates expressed as percent with 95% Confidence Interval (CI) were generated using inverse‐variance weighted method in random‐effect models.

Results: 50 articles met our inclusion criteria (13 case series, 28 case reports and 9 review articles) 243 patients with spontaneous rectus sheath hematoma on anticoagulant therapy were identified (n=116, 48%) were males (n=127, 52%) were females. Mean age was 64.3 years. Presenting features included abdominal pain (90%, 95% CI=91‐100), abdominal mass (88%, 95% CI=70‐90), radiological workup included ultrasonography and CT scan abdomen (sensitivity 100%). (33%, 95% CI=18‐44) were on low molecular weight heparin, (10%, 95% CI=0‐30) were on unfractionated heparin, (54%, 95% CI=32‐76) were on warfarin (0.8%, 95% CI=0‐10) were on rivaroxaban, (0.8% 95% CI=0‐10) were on apixaban. (67%, 95% CI=46‐78) hematomas resolved after conservative management, (09%, 95% CI=0‐27) required surgical intervention i.e. selective embolization of epigastric arteries, (11%, 95% CI=02‐35) were misdiagnosed and led to unnecessary laparotomy, (13%, 95% CI=05‐40) were fatal. Recurrence over 1 year period was (9%, 95% CI=0‐27).

Conclusions: Rectus sheath hematoma can evolve into a life‐threatening emergency in patients on heparin, warfarin as well as direct acting oral anticoagulants. Rectus sheath hematoma should always be kept in the differential diagnosis of acute abdomen in patients on anticoagulants to avoid misdiagnosis and unnecessary surgical intervention.

PB475: Promising Antithrombotic Peptide Agents

M. Grigorjeva ; L. Lyapina

Moscow State University named after M.V. Lomonosov, Biology, Moscow, Russian Federation

Background: It is known that the regulatory glyproline oligopeptides have anticoagulant, fibrinolytic, antifibrin‐stabilizing and antiplatelet properties in vitro and in vivo. Specified properties of these peptides must provide their antithrombotic effect.

Aims: To study the ability of glyproline oligopeptides to normalize blood coagulation and prevent thrombus formation during venous thrombosis provocation.

Methods: The experiments were carried out on white rats according to the ethical principles of the Helsinki Declaration. Peptides (200 μg/kg) Pro‐Gly (PG), Pro‐Gly‐Pro (PGP), Leu‐Pro‐Gly‐Pro (LPGP), Leu‐Pro‐Gly (LPG) and saline (control) were intranasal entered to rats within 6‐7 days. One hour after the last drugs injection we carried out the formation of blood clots in v.jugularis (Wessler model with our modification) and recorded the time of formation and lysis of thrombus. Total fibrinolytic activity (FA), activity of tissue plasminogen activator (t‐PA) and fibrin‐depolymerization activity (FDPA) of blood plasma were measured by fibrin plate method.

Results: It was established that PG, PGP, LPGP LPG prevented the formation of fibrin clots in the provocation of venous thrombosis. The results obtained can be represented by reducing the antithrombotic effect in the following way: LPGP (96‐100%) > LPG (85‐90%) > PGP (70‐85%) > PG (70‐80%). Besides, administration of peptides before formation of thrombus lead to increase of FA (1.3‐1.5 times), FDPA (1.6‐2.2 times), t‐PA (2.0‐2.5 times) compared with control rats. We have shown that oligopeptides promote release of t‐PA from endothelial cells. Therefore, in these experimental conditions generated in the blood fibrin clots were subjected to lysis as enzyme (plasmin) and by non‐enzymatic (action themselves peptides).

Conclusions: Thus, glyproline oligopeptides activate or normalize the function of the anticoagulant system, participate in the prevention of venous thrombogenesis and promote the manifestation of thrombolytic action of the plasmin.

PB476: Knowledge of, Satisfaction with and Adherence to Oral Anticoagulant Drugs among Patients in Tripoli Medical Center; Tripoli, Libya

M. Abugrin 1 ; A. Ali 1 ; E. Sadouh 1 ; A. Khaled 1 ; H. Ahmed 1 ; M. Elhadi 1 ; T. Emahbes 2 ; A. Elhadi 1 ; A. Saeed 1 ; T. Khaled 1 ; E. Omran 3

1 Tripoli University, Faculty of Medicine, Tripoli, Libyan Arab Jamahiriya; 2 Tripoli University, Department of Community Medicine, Tripoli, Libyan Arab Jamahiriya; 3 Tripoli Medical Center, Anticoagulant Clinic, Tripoli, Libyan Arab Jamahiriya

Background: Whether the level of patient's knowledge about anticoagulant plays any role in maintenance of therapeutic international normalized ratio (INR) is controversial. Also, medication adherence is an important element for effective anticoagulation control due to the narrow therapeutic index of warfarin.

Aims: The objective of the study was to assess patients’ knowledge and measure their adherence to oral anticoagulant treatment and to identify potential predictors of INR control among patients in Tripoli Medical Center (TMC).

Methods: A cross‐sectional study was conducted in anticoagulant clinic at TMC, Tripoli Libya during October 2017‐January 2018, whereby patients on oral anticoagulant treatment were recruited. Data on clinical and demographic characteristics, thrombosis or bleeding events, time in therapeutic range (TTR) was calculated using previous INR values. Oral Anticoagulation Knowledge (OAK) test was administered to measure knowledge about anticoagulant. Also, Morisky Medication Adherence Scale (MMAS‐8) and time in therapeutic range (TTR) were calculated.

Results: Mean (SD) age was 55 (10.23) years, females (82.8%) exceeded males (17.2%), and 86.3% were on warfarin for >4 years. The mean TTR was calculated as 55.34±40.62%. Of the sample patients, 48.3% were in the good control category (TTR>70%), 10.3% were in the intermediate category (50%<TTR<70%), and 41.4% were in the poor control category (TTR<50%). The mean OAK score was 10/20; 58.6% of patients failed the OAK test. More than (34%) of study participants were adherent (MMAS‐8=8). Adherent patients were more likely to have good anticoagulation control compared to non‐adherent. However, passing OAK was not independently associated with low TTR control among the patients.

Conclusions: Knowledge and adherence for anticoagulant therapy can have a substantial effect on anticoagulation control. Thus, an educational program should be accommodated to help patents in improving their medication control and reducing side effects.

PB477: Development, Implementation and Evaluation of Application for Monitoring Oral Anticoagulant Therapy

J.A.Q. Oliveira 1 ; T.Q.V. Sá 2 ; J.O.G. Ramos 2 ; L.B. Ribeiro 2 ; M.A.P. Martins 3 ; A.L.P. Ribeiro 4 ; M.S. Marcolino 4

1 Medical School and Hospital das Clínicas da Universidade Federal de Minas Gerais, Programa de Pós‐Graduação em Infectologia e Medicina Tropical and Telehealth Center, Belo Horizonte, Brazil; 2 Hospital das Clínicas da Universidade Federal de Minas Gerais, Telehealth Center, Belo Horizonte, Brazil; 3 Pharmacy School and Hospital das Clínicas da Universidade Federal de Minas Gerais, Pharmaceutical Products, Belo Horizonte, Brazil; 4 Medical School and Hospital das Clínicas da Universidade Federal de Minas Gerais, Internal Medicine and Telehealth Center, Belo Horizonte, Brazil

Background: Anticoagulation underutilization is a challenge in low and middle‐income countries. Warfarin is still the main anticoagulant used, and health professionals are often insecure to handle this therapy.

Aims: To describe the development, implementation and evaluation of an application for anticoagulation management of patients using warfarin.

Methods: The web application was developed using Java 1.8 and JSF 2.2 interfaces. Its security was based on Spring Security guaranteeing the inviolability of the data. After being tested, it was implemented in a public anticoagulation clinic in Belo Horizonte, Brazil, from October/2016 to June/2017. A usability survey was applied to 10 health professionals.

Results: The application has 4 blocks: identification data; indication of anticoagulation, hemorrhagic and thromboembolic risks; consultation record; quality of anticoagulation, evaluated by time in the therapeutic range (TTR).

Multiple tests allowed adjustments before implementation. In implementation phase, 505 patients were included (57.8% female, median age 63 [interquartile range – IQR, 51‐73] years). In at least one consultation, 44.6% of patients reported irregular consumption of vitamin K sources. During the follow‐up, 27.9% patients had bleeding, most frequently hematoma (45.7%). The median TTR was 60.6% (IQR, 32.8‐79.1%). Most of the healthcare professionals considered the application very easy to use (80%) and 90% declared to be satisfied. For 60% of them, the application has greatly reduced the time of consultations and 40% considered the reduction only moderate.

Conclusions: The application proved to be useful in the systematization of information, enabling the recognition of critical issues for the quality of the treatment. Most users were satisfied and consider the application contributed to reduce time spent in the consultations, which may reflect in increasing the anticoagulation clinic capacity, benefiting more patients needing this care.

PB478: Patient Satisfaction Associated with Warfarin Treatment in Different Settings: A Cross‐sectional Study

N. Riva 1 ; C. Borg Xuereb 2 ; W. Ageno 3 ; M. Makris 4 ; A. Gatt 1

1 University of Malta, Department of Pathology, Faculty of Medicine and Surgery, Msida, Malta; 2 University of Malta, Department of Gerontology, Faculty for Social Wellbeing, Msida, Malta; 3 University of Insubria, Department of Medicine and Surgery, Varese, Italy; 4 University of Sheffield, Sheffield Haemophilia and Thrombosis Centre, Sheffield, UK

Background: Warfarin is monitored through venepuncture and laboratory coagulometers (laboratory INR) or finger‐prick and point‐of‐care (POC INR). So far, the quality of life (QoL) associated with laboratory versus POC INR has never been directly compared using specific psychometric questionnaires.

Aims: To evaluate patient QoL in two different settings (laboratory vs. POC INR monitoring), using Duke Anticoagulation Satisfaction Scale (DASS) and Perception of Anticoagulation Treatment Questionnaire (PACT‐Q2).

Methods: We included adult patients on long‐term warfarin and at least 12 months experience with laboratory or POC INR. Questionnaires (DASS range 25‐175, lower scores higher satisfaction; PACT‐Q2 range 0‐100, higher scores higher satisfaction) were administered at inclusion, after signing the consent form. The study started in July 2017. For the POC INR monitoring group, patients were enrolled from 5 Health Centres in the Maltese island (Rabat, Cospicua, Floriana, Qormi, Mosta). For the laboratory INR monitoring group, recruitment of patients attending the Anticoagulation Clinic at Mater Dei Hospital (Msida, Malta) is ongoing and will be completed by March 2018. We planned a sample size of at least 300 patients.

Results: 160 patients (mean age 70.9±9.3, 58% males) completed the questionnaires for the POC INR monitoring group (response rate 39%). The overall mean DASS score was 50.9 (±17.1), with the following scores obtained in each dimension: “limitations” 16.5 (±8.0); “hassles/burdens” 13.7 (±6.7); “psychological impact” 20.7 (±6.7). Mean scores for the PACT‐Q2 dimensions were: “convenience” 85.9 (±13.8), “treatment satisfaction” 67.6 (±11.8). A strong correlation was found between DASS overall and PACT‐Q2 “convenience” (r=−0.81). Males showed higher satisfaction compared to females (48.1 vs. 54.7 P=0.017).

Conclusions: Warfarin patients monitored with the POC showed good QoL, measured either with the DASS or the PACT‐Q2. This study will also provide information about the comparison between different settings.

PB479: A Multicenter Experience with Idarucizumab “in Real World” as Reversal Anticoagulation of Dabigatrán

M. Villalba Montaner 1,2 ; R. Martínez Fernández 3 ; O. Gavin Sevastián 4 ; I. Cuesta Gallardo 5 ; J.M. Real Campaña 1 ; L.I. Sancho Val 6 ; A.R. Millán Acero 7 ; E. Salvador Rúperez 1 ; S.F. Pinzón Mariño 1 ; B. Sánchez Quiñones 3 ; A.J. Garcia Ortego 8 ; G. Ferrer Garrido 8 ; N. Fernandez Mosteirin 9 ; J.M. Calvo Villas 9 ; P. Delgado Beltrán 8

1 Hospital Miguel Servet, Zaragoza, Spain; 2 Hospital Miguel Servet, Hematology, Zaragoza, Spain; 3 Hospital Clínico Universitario Lozano Blesa, Zaragoza, Spain; 4 Hospital Clínico Universitario Lozano Blesa, Aragon Society of Thrombosis and Haemostasis, Zaragoza, Spain; 5 Hospital Obispo Polanco, Aragon Society of Thrombosis and Haemostasis, Teruel, Spain; 6 Hospital de Alcañiz, Teruel, Aragon Society of Thrombosis and Haemostasis, Zaragoza, Spain; 7 Hospital San Jorge, Huesca, Aragon society of thrombosis and haemostasis, Huesca, Spain; 8 Hospital Miguel Servet, Hematology, ZARAGOZA, Spain; 9 Hospital Miguel Servet, Aragon Society of Thrombosis and Haemostasis, Zaragoza, Spain

Background: Despite of idarucizumab is approved as reversal agent of anticoagulant effect of dabigatran in major bleeding and emergency surgeries, the reported experience of idarucizumab “in real world” is scarce.

Aims: Our aim was to analyze idarucizumab as reversal agent of anticoagulation of dabigatran in current clinical practice in Centers of Aragón (Spain) from 2016 up to now.

Methods: From January‐16 to date, 14 patients were treated with idarucizumab and registered in this prospective/retrospective study. We analyzed clinical variables and laboratory tests; efficacy, safety and thrombogenic capacity of the drug and related mortality with the use of idarucizumab.

Results: The indication of idarucizumab were emergency surgeries in 10 patients, major bleeding in 3 pts and 1 pt with severe renal failure received the drug off‐label. Types of surgery were 1 splenic rupture, 2 aortic dissections, 1 cardiac transplant, 1 cholecystectomy, 1 stroke fibrinolysis, 1 nephrectomy, 2 lower limb thrombectomies, and 1 stroke thrombectomy. One patient was at renal failure (clearance creatinine <30 ml/min). Activated partial thromboplastin time (aPTT) and prothrombin time (PT) were prolonged in 6 patients prior to idarucizumab infusion. Two hemorrhagic complications related with surgery: retrocardiac hematoma and cerebral hemorrhage, were registred. Regarding of major bleeding, 2 were intracerebral hemorrhage and 1 retroperitoneal hemorrhage. aPTT and PT were prolonged and renal function tests were normal prior to idarucizumab. Neither adverse effects nor thrombotic events were identified. Three patients died in relation with intraventricular hemorrhage, heart transplant complications and septic shock.

Conclusions: In our experience idarucizumab reversed the anticoagulation of dabigatran immediately, completely, sustainably and safely. Multidisciplinary protocols for using idarucizumab would optimize its effectiveness in approved indications.

PB480: The Effect of Peptide Selank on the Parameters of the Rat Hemostasis System against the Background of Endothelial Dysfunction

E. Rogozinskaya ; L. Lyapina

Lomonosov Moscow State University Biology Faculty, Moscow, Russian Federation

Background: Glyproline peptides effect a peptidergic regulation in the body. The structure of these oligopeptides contains a sequence of amino acids: PRO‐GLY‐PRO (PGP). Multiple studies have shown that a protein with the structure PGP is present exhibits antiplatelet, anticoagulant and fibrinolytic properties of blood plasma. The addition of the amino acid ARG (R) to the sequence will significantly increase the antithrombotic effects in the body due to the release into the bloodstream of NO.

Aims: Study the effect of the Selank peptide, THR‐LYS‐PRO‐ARG‐PRO‐GLY‐PRO (TKPRPGP) on the parameters of the hemostasis system against the background of endothelial dysfunction.

Methods: Selank peptide solution was used at a concentration of 150 mg/ml, a NO‐synthase inhibitor ‐ L‐name solution at a concentration of 10 mg/ml. We delished the white rats of the Wistar line (300 g) into 4 groups. 3 groups were experienced: rats receiving Selank; L‐name; Selank + L‐name, 1 group ‐ control: NaCl. The substances were introduced for 7 days. Blood was taken from the jugular vein and a series of experiments was conducted to determine the effect of the drugs on the parameters of hemostasis on the 8th day. The studies were performed using the APTT test and thromboelastogram (TEG) parameters, and total fibrinolysis (TF) and tissue plasminogen activator activity (t‐PA) were determined.

Results: It has been established that Selank and Selank + L‐name enhanced the TP in the blood plasma (lysis zones from 60 to 110 mm 2 ) and t‐PA activity (by 40‐60%). In control and L‐name there were practically no lysis zones. The presence of anticoagulant activity of Selank in conditions of endothelial dysfunction was shown according to APTT and TEG parameters.

Conclusions: The arginine‐containing glyproline peptide ‐ TKPRPGP exhibited enzymatic fibrinolytic and anticoagulant activity against the background of endothelial dysfunction caused by L‐name. This indicates not only its prophylactic, but also therapeutic effect of emerging endothelial dysfunction.

PB481: Retrospective Observational Study of Patients Treated with Rivaroxaban in Saudi Arabia

M. Riazuddin ; M. Ahmed; S. Khan; O. Emadi; M. Abufarhaneh; M. Alkasab; H. Alsudairy; K. Alshammari; A. Sheikh; A. Alrashed; I. Butt; A. Alfifi; M. Khan

King Faisal Specialist Hospital & Research Centre, Medicine (Internal Medicine), Riyadh, Saudi Arabia

Background: Atrial Fibrillation (AF) and Venous Thromboembolisms (VTEs) are major causes of morbidity and mortality. Novel oral anticoagulants (NOACs) have been shown to be as effective as vitamin K antagonists (VKAs) with a propensity to cause less major bleeding. Available studies suggest that AF and VTEs are a major burden in the Gulf Region; however there is a paucity of real world data on the use of NOACs in this part of the world.

Aims: The aim of this study is to assess the safety and effectiveness of rivaroxaban in routine clinical practice in a large tertiary referral center in Saudi Arabia.

Methods: A cohort of patients who received rivaroxaban during 2012‐2015 were identified. All adverse events were recorded including major bleeding (using ISTH criteria), non‐major bleeding events, symptomatic thromboembolic events (stroke, VTE and Myocardial Infarction) and all cause death. Statistical analysis were descriptive, exploratory and generally limited to summary statistics (e.g. mean±standard deviation [SD] or median±quartiles).

Results: A total of 508 patients were prescribed rivaroxaban during the study period. The most common indication for rivaroxaban was non valvular AF (65.3% of patient) followed by VTE (34.7%). Mean age of the cohort was 60.4 years (±16.4) and 50.5% were female. For the evaluable patients (489), 17.5% had renal impairment (eGFR<60 ml/min/1.73 m2). For patients on rivaroxaban for non valvular AF the mean CHA2DS2‐VASc score was 3.2 (±1.8). Bleeding occurred in 72 (14.1%) patients out of which 21 (4.1%) were major bleeding incidences. Thrombosis events occurred in 13 (2.5%) patients. Fourteen (2.7%) patients died during the study unrelated to the treatment. One patient died of fatal bleeding.

Conclusions: This is the first study to describe the use of rivaroxaban in a broad patient population in clinical practice in the Middle East. The overall rates of bleeding and thrombosis in this study were comparable to major clinical trials.

PB482: Combined Q‐Switched‐Nd: YAG Laser‐mediated Photomechanic Thrombolytic Therapy Plus Tissue Plasminogen Activator (tPA) Administration Reduce Thrombus Content in the Arterial Atherothrombotic Stenosis

H. Mehrad 1,2 ; S.M. Tabatabaei 3 ; M. Bolourian 4 ; M.R. Moktari 4 ; M.M. Hosseini 4 ; A. Mirzakhani 4 ; M.H. Daghighi 5

1 Islamic Azad University, Tabriz Branch‐Faculty of Basic Sciences, Department of Physics, Tabriz, Iran, Islamic Republic; 2 Tabriz University of Medical Sciences, Neurosciences Research Center, Tabriz, Iran, Islamic Republic; 3 Islamic Azad University, Tabriz Branch‐Faculty of Medical Sciences, Department of Physiology, Tabriz, Iran, Islamic Republic; 4 Islamic Azad University, Tabriz Branch‐Faculty of Mechanics, Department of Bioengineering, Tabriz, Iran, Islamic Republic; 5 Tabriz University of Medical Sciences, Department of Radiology, Tabriz, Iran, Islamic Republic

Background: Cardiovascular disease is the leading cause of morbidity and mortality worldwide. Vascular disease arises through the complications of atherosclerosis, a complex chronic inflammatory condition affecting the arterial circulation. It leads to the development of vascular lesions or atherosclerotic plaques, which manifest as asymmetrical thickenings of the intima of medium to large sized elastic and muscular arteries. Arterial thrombosis on ruptured atherosclerotic plaques can lead to acute events, such as myocardial infarction and ischemic stroke.

Aims: The aim of this study was to generate a rabbit model of common carotid artery atherothrombotic stenosis and the subsequent investigating the feasibility of combined Nd:YAG laser‐ mediated photomechanic thrombolytic therapy plus tPA administration in this model.

Methods: Briefly, New Zealand white rabbits were submitted to common carotid artery atherothrombotic stenosis by primary balloon injury followed 1.5% cholesterol‐ rich diet injury for 12 weeks and finally perivascular severe cold injury. Then treatment group underwent combined Q‐switched Nd:YAG laser (30 W, 5 ns)‐ mediated thrombolytic therapy plus tPA (0.6 mg/kg) administration.

Results: Results from histopathology, B‐mode and color Doppler ultrasonography, showed a significant reduction in the mean value for thrombus content, blood mean velocity and a significant increase in the mean value for blood volume flow at the stenotic region in the treatment group compared with the other groups (P<0.05).

Conclusions: Enhanced anti‐ thrombotic effect of tPA, due to optical cavitation bubbles‐ induced by Q‐ Switched‐ Nd:YAG laser, can cause to reduce the thrombus content and significantly dilate the luminal cross‐sectional area of stenosis and lower treatment time in comparison with conventional tPA thrombolytic therapy.

PB483: Description and Evaluation of the Comparison of Activated Partial Thromboplastin Time Versus Anti‐Factor Xa Activity for Therapeutic Unfractionated Heparin Monitoring in a Tertiary Academic Medical Center Using Three Different Adult Weight‐Based, Nurse Managed Nomogram Protocols

L. Talamo 1 ; D. Chyn 2 ; S. Palkimas 3 ; H. Maitland 1

1 University of Virginia, Hematology/Oncology, Charlottesville, United States; 2 University of Virginia, Statistics, Charlottesville, United States; 3 University of Virginia, Pharmacy, Charlottesville, United States

Background: Unfractionated heparin (UFH) is often used in hospitalized patients. Weight‐based, nurse managed UFH nomograms (N) are effective and safe 1 . The University of Virginia (UVA) historically used activated partial thromboplastin time (aPTT) to monitor UFH. Because aPTT is subject to interference from other sources, UVA now uses anti‐factor Xa (Xa) for monitoring. UVA utilizes three N: standard/full bolus (SN; treatment of thromboses or atrial fibrillation), low‐intensity (LIN; acute coronary syndrome), and delayed therapeutic/no bolus (DTN; high bleeding risk).

Aims: Our goal was to show that patients (Pt) achieve therapeutic (Th) anticoagulation (AC) more rapidly with less dose adjustments (DA) using Xa versus aPTT.

TABLE 1:

Figure 1

Methods: Data on 180 Pt, 30 from each N in both aPTT and Xa groups, who received UFH infusions at UVA from October‐December 2015 (aPTT) or 2016 (Xa) was obtained. Inclusion criteria: Pt over age 18 on UFH with appropriate monitoring. Exclusion criteria: interruption in UFH infusion for any reason, incorrect/change in N use/type.

Results: Xa Pt were found to achieve Th values sooner, with less DA, regardless of N type (Figure 1 a,b). Xa Pt on the SN reached Th values faster than those on LIN/DTN (Tbl1). With SN, the majority of aPTT Pt were supraTh on first check, whereas Xa Pt were mostly at goal (Table 2). There was no difference in bleeding rates when comparing N types, sex, or age (Figure 2).

Conclusions: Use of Xa resulted in less time to Th range, less DA and more Pt reaching goal with the first lab assessment. Though not statistically significant, there was a trend toward achieving Th value within 24 hours with Xa use (Table 3). Xa use lead to less waste/money on excess lab tests and less intravenous punctures; a safer process for Pt as they are at risk for bleed or thrombus for less time, and nurses have time for other Pt care tasks. Pt requiring AC for treatment of thrombotic events should receive SN as this resulted in the most rapid Th levels. Use of SN did not lead to increased risk of bleeding.

PB484: Idarucizumab for Reversing Anticoagulation in Patients Treated with Dabigatran: Experience in our Center

E. Morente Constantin 1 ; P. Romero Garcia 2 ; M. Rivas Luque 1 ; A. Garcia Ruiz 1 ; M.D.P. Garrido Collado 1 ; M. Jurado Chacon 1

1 Hospital Universitario Virgen de las Nieves, Granada, Spain; 2 Complejo Asistencial de Soria, Soria, Spain

Background: Patients who are receiving oral anticoagulant therapy may benefit from anticoagulant reversal if they present life‐threatening bleeding. Idarucizumab is a humanized monoclonal antibody fragment that binds dabigatran. It is indicated patients treated with this anticoagulant when a rapid reversal of its anticoagulant effect is required. The recommended dose is 5 g.

Aims: This is a descriptive study to determine whether darucizumab would be able to reverse the anticoagulant effect of dabigatran in patients who are about to undergo an urgent procedure.

Methods: Two clinical cases are described:170 y‐o woman with abdominal pain. In the previous 24 hours, surgical hysteroscopy had been performed. In CT scan, hemoperitoneum were detected suggesting uterine perforation. The last dosis of Dabigatran was 6 hours ago. 5 g of Idarazizumab was given. Hysterectomy with a double annexectomy was made 12 hours after surgery, anticoagulant treatment with LWH was introduced. She also presented hemorrhagic subhepatic abscess as a surgical complication (not related to dabigatran). 20 days later, Dabigatran was reintroduced.255 y‐o male with pain located in renal fossa, dysuria and pollakiuria. Abdominal US and CT scan were performed, showing complicated renal colic. It was decided to implant double J‐catheter The last dose of Dabigatran was 12 hours ago. 5 g of Idarazizumab was given and the intervention was carried out without incident. 24 hours later, after a good clinical evolution, Dabigatran was restarted with no new complications thus far.

Results: The use of Idarucizumab permitted rapid and safe intervention of patients. Both had normal hemostasis during the procedure. 5 g dose of idarucizumab was sufficient.

Conclusions: Idarucizumab is integrated into protocol for the emergency management of patients on dabigatran. Idarucizumab is effective for dabigatran reversal among patients who have uncontrolled bleeding or will be undergoing urgent surgery. Idarucizumab rapidly, durably, and safely reversed the anticoagulant effect of dabigatran.

PB485: Warfarin Dose Requirements in Patients with Heart Diseases Assisted at an Anticoagulation Clinic in the Southeast Brazil

E.I.F. Campos 1 ; C.M. Bertollo 2 ; A.M.M. Reis 2 ; I.G. Ferreira 2 ; J.C. Sá 2 ; M.O.C. Rocha 1 ; M.A.P. Martins 1,2

1 Faculdade de Medicina da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 2 Faculdade de Farmácia da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

Background: Warfarin is an oral anticoagulant extensively used in patients with risk factors for thromboembolism. Monitoring its use is necessary because of the need to prevent the occurrence of adverse events. In clinical practice, there is a wide variability in warfarin dose‐response.

Aims: To describe weekly warfarin maintenance dose in patients with heart diseases.

Methods: This is an observational descriptive study, developed during 2015‐2016 at a public anticoagulation clinic located in the Southeast Brazil. We reviewed medical charts to collect patient's data. Time in therapeutic range (TTR) was calculated using the Rosendaal method. Warfarin maintenance dose was calculated by adding up the patient's weekly doses and dividing by the number of follow‐up weeks. Patients were allocated into three groups of weekly dose: <17.5 mg (sensitive), 17.5‐70.0 mg, and >70.0 mg (resistant). This project was approved by the University Ethics Committee (CAAE/08136613.4.0000.5149). All participants signed an informed consent.

Results: A total of 247 patients were included in the study. Mean age was 69.0±12.2 years. Overall, 137 (55.5%) patients were female and 159 (64.4%) non‐whites. The monthly income was $243.96 (Quartiles, Q1=$243.96, Q3=$371.52). Mean number of medications taken chronically was 5.9±2.3. Most patients (n=222, 89.9%) had target INR 2.00‐3.00 and the main indication for warfarin use was atrial fibrillation (n=213, 86.2%). TTR was 64,0% (Quartiles, Q1=48,4, Q3=75,1). Weekly maintenance dose was 25.7 mg (Quartiles, Q1=19.9, Q3=33.8). A total of 46 (18.6%) patients used <17.5 mg/week, 199 (80.6%) 17.5‐70.0 mg/week and two patients (0.8%) used >70.0 mg/week.

Conclusions: There was wide variation in warfarin maintenance doses, and most patients were taking intermediate doses of warfarin. Data on warfarin dose requirements is relevant for the elaboration of protocols better adjusted to the target population and to improve estimates of the magnitude of dose adjustments.

PB486: Direct‐acting Oral Anticoagulant Drug Level Monitoring in Clinical Patient Management

A. Rottenstreich; N. Zacks; G. Kleinstern; B. Hirsch Raccah; B. Roth ; Y. Kalish

Hadassah Medical Center, Jerusalem, Israel

Background: The role of drug‐level monitoring among patients using direct‐acting oral anticoagulant (DOAC) is unclear.

Aims: We aimed to investigate ′real‐life′ utilization of DOAC monitoring and its effect on clinical management.

Methods: A review of records of patients who underwent DOAC level testing during 2013‐2017 in a university hospital.

Results: Overall, 212 patients (median age 77 years) underwent 292 DOAC measurements [apixaban (n=147), rivaroxaban (n=102), dabigatran (n=43)]. Monitoring volume increased by 460% during the study period. DOAC level testing was performed during routine follow‐up in 51 (17.5%) cases, whereas the remaining 241 (82.5%) measurements were performed due to selected clinical circumstances, most commonly: bleeding (n=60), perioperative status (n=45), breakthrough thrombosis (n=37) and renal failure (n=35). Drug levels were within the expected range in 210 (71.9%), above the expected range in 62 (21.2%) and lower than the expected range in 20 (6.8%). In multivariate analysis, lower glomerular filtration rate (P=0.001) and lower body mass index (P=0.006) were associated with DOAC levels above the expected range. Clinical decisions were affected by DOAC monitoring following most (140/241, 58.1%) measurements for which we identified an indication for testing; yet only rarely when monitoring was performed during routine follow‐up (7.8%, 4/51) (P<0.0001).

Conclusions: While no benefit of routine DOAC monitoring was observed, drug level measurement has an important role in the management of patients in selected circumstances. Age, body weight and creatinine clearance were found to be significant predictors of drug levels. Future studies are warranted to establish associations between drug levels and clinical outcomes, and better delineate the role of DOAC monitoring.

PB487: Establishment of a Centralized Monitoring System for Patients Receiving Anticoagulant Therapy in the Arkhangelsk Region Russia

N. Vorobyeva ; A. Vorobyeva; A. Schapkov

Nord State Medical University, Haemostasiology, Arckhangelsk, Russian Federation

Background: According to experts, more than 88% of patients with atrial fibrillation (ciliary arrhythmia) have an indication for anticoagulant therapy, and only 23% receive it. One of the factors limiting long‐term use of anticoagulants in outpatient practice is the need to ensure continuous monitoring of the safety and efficacy of antagonist vitamin K therapy (AVK). Clinical practice shows that only a third of the time patients are in the target therapeutic range of INR.

Aims: Is to organize a centralized monitoring of the INR indicator throughout the entire Arkhangelsk region.

Methods: In 28 medical hospitals anti‐thrombotic cabinets are organized. All patients receiving warfarin are included in the regional register (n=5,407). The determination of the INR indicator is performed on the instrument CoaguChek XS Plus, connected to a WEB server. All patients underwent training on the basis of anti‐thrombotic cabinets to improve compliance with warfarin therapy.

Results: The indicator TTR was 73%. Bleeding on the background of therapy AVK amounted to 1.8%, thrombosis – no. Advantages of introducing anticoagulant cabinets in comparison with typical practice: 2 times the time spent in the target range of INR from 34% to 70%; he number of complications and negative clinical outcomes is reduced by an average of 13%; the number of hospitalizations is reduced by an average of 8%; 2 times the number of lives saved from 25 to 50. The number of negative clinical outcomes is predicted to decrease: thromboembolic complications by 11%, strokes by 14%, bleeding by 23%, and mortality by 11%.

Conclusions: The introduction of a system of anticoagulant rooms will allow more than 2 times increase in the number of lives saved, while at the same time ensure a twofold decrease in the cost of preserving life.

PB488: Evaluation of Algorithms for Warfarin Dose Prediction

E.I.F. Campos 1 ; K.B.G. Borges 2 ; J.M. Costa 2 ; M.A.P. Martins 1,2

1 Faculdade de Medicina da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 2 Faculdade de Farmácia da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

Background: Warfarin is the most widely used oral anticoagulant for treatment and prevention of thromboembolic disorders, despite its narrow therapeutic index. To improve quality of therapy, dosing prediction algorithms have been developed and incorporation of genetic factors in these models can bring benefits to treatment.

Aims: To review the use of pharmacogenetic algorithms for warfarin dose prediction.

Methods: This study is a review and studies were searched in PubMed database. We selected studies published during 2003‐2015 in English, Portuguese and Spanish. We used the following search terms: warfarin, algorithms, pharmacogenetics, CYP2C9, VKORC1, blood coagulation. We included studies involving development, validation or evaluation of pharmacogenetic algorithms for warfarin dose prediction.

Results: We selected 19 studies. Polymorphisms in CYP2C9 and VKORC1 accounted for about 10‐45% of the overall variation in warfarin dose, depending on population and genotypes detected. Dosing algorithms have been developed but their potential benefits in warfarin therapy have produced controversial results. Most algorithms explained about 60.0% of variation in warfarin dose, which becomes necessary to include more clinical factors (comorbidities, target INR, concomitant drugs used) and genetic factors (other important genes such as apolipoprotein E, MDR1, CYP4F2). Construction of these models should include specific factors of target population that interfere in warfarin dose as well as frequency of the most common alleles. Validation of algorithms becomes crucial for application in clinical practice.

Conclusions: According to the literature consulted, benefits of genotype‐guided warfarin therapy remain under discussion and further studies are needed.

PB489: Development of an Educational Intervention Focused on Low‐income Population Treated with Warfarin

J.M. Costa 1 ; H.C. Barbosa; 2 ; J.A.Q. Oliveira 2 ; L.R.A. Campos 1 ; E.I.F. Campos 2 ; H.C. Torres 3 ; M.A.P. Martins 1,2

1 Faculdade de Farmácia da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 2 Faculdade de Medicina da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 3 Escola de Enfermagem da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

Background: Warfarin is widely prescribed for primary and secondary prevention of thromboembolism, despite its increased risk of adverse events. Patients with low socioeconomic status may present additional barriers to attain anticoagulation control. Educational interventions may provide better outcomes.

Aims: To create an evidence‐based educational intervention focused on low‐income population at an anticoagulation clinic in Brazil.

Methods: The development of the educational intervention was based on a literature review, in Medline. Inclusion criteria: studies involving well described evidence‐based interventions in patients using warfarin, no language restriction, and year of publication 2007‐2017.

Results: From 577 studies identified, 16 were selected. Studies presented heterogeneous methods to perform educational interventions, six of them involved low‐income patients. The main aspects chosen for the intervention were: group approach ‐ which allows greater interaction among patients; feedback techniques to identify learning; and telephone calls. We proposed an educational intervention consisting of four one‐hour meetings with up to 15 participants, offered fortnightly. Pharmacists, nutritionists, and psychologists will be responsible for conducting the meetings. During the first meeting, the topics to be discussed will involve patients′ feelings about anticoagulation therapy, and patients will be encouraged to set an individual goal to improve treatment outcomes. In the subsequent meetings, the topics discussed include the use of oral anticoagulant, risks and benefits of drug therapy, and drug and food interactions. Interactive techniques will be used. In between meetings, patients will receive a telephone call to clarify doubts and review their individual goals.

Conclusions: Revising the current literature, we proposed an evidence‐based educational intervention to improve anticoagulation control in a low‐income population.

PB490: A Novel Factor Va Inhibitory Antibody Prevents Blood Clots without Increasing Bleeding Risk

J. Wu ; R. Li; Y. Yang; N. Chen; Y. Li; J. He; X. Fan

Southwest Medical University, Luzhou, China

Background: Current anti‐thrombotic drugs, including antiplatelet agents and anticoagulants, are associated with increasing bleeding risk.

Aims: We show an antihrombotic therapy without bleeding risk based on a coagulation protein factor V function‐neutralizing antibody.

Methods: We screened for antibodies against activated factor V (FVa) using phage display and demonstrated that recombinant fully human antibody 1C11 binds into the FVa enzymatic pocket.

Results: 1C11 interfered with FVa‐mediated coagulation, delayed thrombosis after mouse carotid artery and cremaster muscle microvascular arterioles injury without affecting parameters of haemostasis.

Conclusions: These data establish that targeting of FVa is a safe mode of thromboprotection in arterial thrombosis, and provide a clinically relevant anti‐thrombotic therapeutic strategy with reduced bleeding risk.

PB491: Direct Oral Anticoagulant Use in Obese Patients: Preliminary Results of a Pilot Study

L. Talamo 1 ; S. Palkimas 2 ; H. Maitland 1

1 University of Virginia, Hematology/Oncology, Charlottesville, United States; 2 University of Virginia, Pharmacy, Charlottesville, United States

Background: Direct oral anticoagulants (DOACs) are approved for treatment and prevention of venous thromboembolism, and ischemic stroke prevention. No large randomized controlled trial has investigated the efficacy and safety of DOACs in obese patients (OP), who are underrepresented in clinical trials. Subgroup analyses suggest that DOACs are efficacious and safe in OP, however these studies are limited by small numbers. The International Society on Thrombosis and Haemostasis (ISTH) suggests checking a drug‐specific peak and trough level if DOACs are used in patients (Pt) with a body‐mass index (BMI) >40 kg/m 2 or weight >120 kg. A retrospective review suggested that OP on DOACs are at increased risk of bleeding compared to Pt of normal weight (NWP).

Aims: We aimed to determine if fixed dosing of DOACs in OP was sufficient to obtain therapeutic (Th) drug levels. Safety and efficacy outcomes were monitored.

Methods: We plan to collect DOAC levels (liquid chromatography‐mass spectrometry) in 60 OP and 60 NWP (BMI 18.5‐24.9). Pt on standard dosing of DOACs for any indication were included. Trough levels were checked one hour before a dose of apixaban (A) 5 mg twice daily or rivaroxaban (R) 20 mg daily.

FIGURE 1

FIGURE 2

TABLE 1

Results

We have collected data on 27 OP and 10 NWP. Three OP had unusable results. 14 Pt (58%) had levels within the Th range. Nine Pt had supraTh levels, one Pt was subTh. Of the NWP, all took A and 70% were Th. Eight OP and 3 NWP have completed 6 months of follow up. Table 1 lists bleeding events.

Conclusions: Of the evaluable Pt, the majority had Th levels. No Pt in either group had a new thrombus or stroke, despite two Pt having subTh levels. 37.5% of OP had a supraTh level, but none bled. Only three Pt bled; all were minor bleeds and all had Th drug levels. This preliminary data suggests that OP very infrequently have subTh level; concern for new thrombosis due to “underdosing” may be unfounded. The target range of DOAC may be inaccurate, or it may be unnecessary to check drug levels in OP. More information is needed.

PB492: Arterial Thrombosis Depends on Shear Rate More Than Shear Rate Gradient

D. Kim 1 ; C. Lauren 2 ; D. Ku 1

1 Georgia Institute of Technology, Atlanta, United States; 2 Abbott, Abbott Park, United States

Background: Arterial thrombus is concentrated at stenosis. The relative roles of high shear rate and shear rate gradients in short stenoses are often overlapping and difficult to distinguish.

Aims: This study separates the two factors into distinct zones that are spatially resolved in a microfluidic device.

Methods: A specialized test section was designed with long converging and diverging sections that are separate from a zone of uniform high shear rate (Figure a). The channel was split into 5 regions ‐ contraction (1), stenosis (2‐4), divergence (5). CFD was used to quantify the velocity and wall shear rates through the test section. Heparinized whole human blood was perfused into the fibrillar collagen‐coated microfluidic device with a constant pressure head of 65 mm. Test section image was recorded every 0.5 seconds using microcope with CCD camera with a resolution of 10 μm. Thrombus buildup was quantified as intensity level change, binned, and averaged by area for each region of interest.

Results: Wall shear rate in the stenosis (Regions 2‐4) is 3,200/second while the streamwise shear gradient was nil by CFD. Regions 1 and 5 had large wall shear gradients (126,000/s‐cm) in the streamwise direction. Thrombosis was low in regions 1 and 5, while significantly higher in regions 2‐4 (P<0.01). Figure b describes normalized mean intensity level for each region. All 8 experimental results showed highest mean intensity level at 2 (P<0.01). Regions 3 versus 4 or 1 versus 5 were not significant. Regions 3 and 4 may exhibit decreased thrombus formation due to depletion of plasma vWF at the leading edge.

FIGURE 1 (a) Geometry and CFD result of shear rate profile, (b) Mean intensity level at each region (1: contraction, 2‐3‐4: stenosis, 5: divergence, n=8)

Conclusions: Arterial thrombosis over collagen stenosis from the adhesion of vWF and then platelets is correlated to the magnitude of the wall shear stress without the need to invoke the elongational shear gradient. This study definitively identifies high wall shear rate as the dominant factor over shear rate gradients for occlusive thrombosis.

PB493: Increased Platelet Count and Reticulated Platelets in Recently Symptomatic versus Asymptomatic Carotid Artery Stenosis and in Cerebral Microembolic Signal‐Negative Patient Subgroups: Results from the HEIST Study

S. Murphy 1 ; S.‐T. Lim 2 ; J. Kinsella 3 ; D. Murphy 4 ; H. Enright 5 ; D. McCabe 6 ; HEIST Study Group

1 Adelaide and Meath Hospital, Neurology Department, Dublin, Ireland; 2 Adelaide and Meath Hospital, Department of Neurology, Dublin, Ireland; 3 St Vincent's University Hospital, University College, Department of Neurology, Dublin, Ireland; 4 Adelaide and Meath Hospital, Department of Haematology, Dublin, Ireland; 5 Adelaide and Meath Hospital, Department of Haematolog, Dublin, Ireland; 6 School of Medicine, Trinity College, Academic Unit of Neurology, Dublin, Ireland

Background: The pathophysiological mechanisms responsible for the disparity in stroke risk between asymptomatic and symptomatic carotid stenosis are not fully understood. The functionally‐important reticulated platelet fraction (%RPF) and reticulocytes could play a role.

Aims: To compare full blood count parameters and platelet production/turnover/activation markers in asymptomatic versus recently symptomatic moderate (≥50‐69%) or severe (≥70‐99%) carotid stenosis patients.

Methods: Data from 34 asymptomatic patients were compared with 43 symptomatic patients in the ‘early phase’ (≤4 weeks) and 37 patients in the ‘late phase’ (≥3 months) after TIA/ischaemic stroke in this prospective, multi‐centre, observational‐analytical study. Reticulated platelets were quantified by whole‐blood flow cytometry and reticulated platelets and reticulocytes by ‘automated assays’ (Sysmex XE‐2100 ™ ). Bilateral simultaneous transcranial Doppler ultrasound monitoring classified patients as micro‐embolic signal (MES)+ve or MES‐ve.

Results: Mean platelet count was higher in early (216 × 10 9 /L) and late symptomatic (219 × 10 9 /L) than asymptomatic patients (194 × 10 9 /L; P=0.04). Mean platelet volume was higher in early symptomatic than asymptomatic patients (10.8 vs. 10.45 fl; P=0.045). Automated assays revealed higher %RPF in early (5.78%) and late symptomatic (5.11%) than asymptomatic patients (3.48%; P≤0.01). Reticulocyte counts were lower in early (0.92%) and late symptomatic (0.93%) than asymptomatic patients (1.07%, P≤0.036). The automated %RPF was higher in early symptomatic than asymptomatic MES‐ve patients (5.7% vs. 3.55%; P=0.001).

Conclusions: Increased platelet counts and a shift towards production of a population of larger, young, reticulated platelets could contribute to a higher risk of cerebrovascular events in recently symptomatic versus asymptomatic carotid stenosis patients, including those who are MES‐ve.

PB494: The Collagen‐Induced Platelet Aggregation and Artery Status in Patients With Arterial Hypertension of Very High Risk

E. Shorikov 1 ; D. Shorikova 2

1 Bucovinian State Medical University, Internal Medicine and Clinical Pharmacology, Chernivtsi, Ukraine; 2 Bucovinian State Medical University, Chernivtsi, Ukraine

Background: Atherotrombosis is one of the complications of arterial hypertension (AH). The crucial role in the development of it played thrombocyte activation. Different studies have shown controversial results in blood platelet functionality profiles due to concomitant diabetes mellitus of type 2 (DM 2) on the background of cardiovascular pathology according to the state of arteries.

Aims: To set the types and relationship of the thrombocyte activation inducted by collagen in the clinical course of AH with DM 2.

Methods: We have studied 252 patients (155 women and 97 men, age of 34 to 67): 118 people were with AH of high risk and 134 people were with AH with very high risk (+ DM 2). The 3 indexes of collagen‐induced platelet aggregation (the level (LA), time (TA) and rate of aggregation (RA)) and the plasma level of NO‐metabolites, the indexes of systemic vascular resistance, glomerular filtration rate (GFR) were measured.

Results: Using multivariate nonparametric analysis of variance we have set the significant increase of the LA and RA (P<0.05) in the group of AH with the very high risk. There was not significant difference between the TA in all groups (P>0.05). As for the interrelationship between the aggregation and level of NO‐metabolites we have found a reliable negative correlation between the LA and NO‐metabolites (R=−0.31; P=0.045) and RA and NO‐metabolites (R=−0.26; P=0.019). Also we have set the association between the increase of general vascular resistance index and the LA index (R=0.31; P=0.005) and general vascular resistance index and RA (R=0.23; P=0.04). As for GFR we have set weak but reliable interaction between the decrease of GFR and changes of all indexes: LA (R=−0.24; P<0.001), TA(R=0.21; P=0.036) and RA(R=−0.22; P=0.009) accordingly. The independent coefficients for GFR and LA, TA and RA also stayed reliable (P<0.05).

Conclusions: The AH with DM 2 followed by the activation of thrombocytes, which directly deal with changes of functionality of the arteries in different places of riverbed.

PB495: Significance of Single Nucleotide Polymorphisms (SNPs) Within Genes Encoding Platelet Glycoprotein Receptors in Patients With Infective Endocarditis (IE)

M. Sorokina 1 ; A. Andreev 1 ; A. Balatskiy 1 ; A. Pisaryuk 2 ; N. Povalyaev 1 ; E. Kotova 2 ; Y. Karaulova 2 ; A. Milto 3

1 Lomonosov Moscow State University, Moscow, Russian Federation; 2 Peoples’ Friendship University of Russia, Moscow, Russian Federation; 3 V.V. Vinogradov City Clinical Hospital, Moscow, Russian Federation

Background: Embolic events are a main cause of morbidity and mortality in patients with infective endocarditis and are of high prognostic importance. It is tempting to hypothesize that an inherited protrombotic condition may synergize with a predisposing procoagulant status present in patients with IE (inflammation, sepsis, organ dysfunction etc.) and thus increase embolic risk.

Aims: To investigate the assosiation of SNPs in genes of platelet membrane glycoproteins with embolic risk in patients with infective endocarditis.

Methods: 47 patients with verified IE (DUKE 2015), admitted to V.V.Vinogradov city clinical hospital in Moscow, were included in the study. Embolic complications were present in 16 (34%) patients with IE (group 1), rest 31 (66%) had no embolism (group 2). We identified 4 SNPs (rs1126643, rs5918, rs6065, rs1613662) in 4 genes (GP Ia, GP IIIa, GPIba, GP VI), all of which have been implicated as increasing the risk of arterial thromboembolism. Genomic DNA was extracted from EDTA‐stabilized peripheral venous blood using QIAmp DNA Blood Mini Kit and QIAcube ™ automatic station (QIAGEN). Genotyping was performed by DNA‐Technology ® SNP genotyping assays (DNA‐Technology). The statistical analysis was performed by SNPStats online tool.

Results: We did not find statistically significant differences between the groups for rs5918, rs1126643 and rs1613662. Patients with C/T genotype of GP1ba rs6065 demonstrated lower risk of embolism: 10% against 41.7% in patients with genotype C/C and T/T (P=0.044). The whole genotype distributions in two groups are presented in Table 1.

TABLE 1 Association of the polymorphisms within platelet membrane receptors genes with embolism in IE patients SNP Model Genotype IE with embolism IE without embolism OR (95% CI) P value AIC (Akaike information criterion) HWE (Hardy‐Weinberg equilibrium) rs5918 ‐ T/T 6 (46.1%) 19 (65.5%) 1.00 0.24 54.6 0.17 rs5918 ‐ T/C 7 (53.9%) 10 (34.5%) 2.22 (0.58‐8.40) 0.24 54.6 0.17 rs1126643 Dominant C/C 2 (13.3%) 10 (32.3%) 1.00 0.15 60.0 0.99 rs1126643 Dominant C/T + T/T 13 (86.7%) 21 (67.7%) 3.10 (0.58‐16.42) 0.15 60.0 0.99 rs6065 Overdominant C/C + T/T 15 (93.8%) 21 (70.0%) 1.00 0.044 59.4 0.56 rs6065 Overdominant C/T 1 (6.2%) 9 (30.0%) 0.16 (0.02‐1.36) 0.044 59.4 0.56 rs1613662 Overdominant T/T + C/C 14 (87.5%) 24 (77.4%) 1.00 0.39 63.6 0.19 rs1613662 Overdominant T/C 2 (12.5%) 7 (22.6%) 0.49 (0.09‐2.69) 0.39 63.6 0.19

Conclusions: Our results show that no significant association exists between higher prevalence of embolism in IE patients and the four SNPs considered. The current study is underway and we're enrolling more patients in our sample. Further investigations are needed to understand the genetic susceptibility to embolic events in IE patients.

PB496: Risk Factors for Brain Vessels′s Thrombosis/Stenosis in Young Patients With Ischemic Stroke

T. Tsvetanov 1 ; M. Cholakova 2 ; M. Staneva 1 ; P. Antova 1 ; I. Staykov 2

1 Acibadem City Clinic Tokuda Hospital Sofia, Angiology, Sofia, Bulgaria; 2 Acibadem City Clinic Tokuda Hospital Sofia, Neurology, Sofia, Bulgaria

Background: Thrombophilia is defined as a predisposition to form blood clots and is characterized by deficiencies and mutations in endogenous anticoagulants. Thrombophilic polymorphism such as factor V Leiden, MTHFR mutation (A1298C, C677T), prothrombin mutation G20210A, PAI‐1 mutation, antiphospholipid antibodies, Protein S, Protein C are established risk factors for venous thrombosis, but their role in arterial thrombosis is still controversial.

Aims: To investigate genetic and acquired risk factors for carotid or vertebral thrombosis/ stenosis in young patients with ishemic stroke.

Methods: This prospective study included 53 young patients (age 18‐50 years) with ischemic stroke (34 male, 19 female), 19 in vertebrobasilar system, 34 in carotid system. Patients were divided in two groups ‐ with thrombosis and with clinically significant stenosis. All patients underwent ECG, clinical cardiological evaluation, colour‐coded duplex utrasonography of the cerebral vessels, computed tomography/computed tomography angiography of cerebral vessels or magnetic resonance imaging of the head and thrombophilia factors examination.

Results: Twelve patients are with thrombosis, 5 in the vertebrobasilar system and 7 in the carotid system, and one patient with severe carotid stenosis. Eleven of these patients have more than one risk factor for thrombophilia plus dyslipidemia and one patient has only risk factors for thrombophilia. The other 41 patients are with ischemic stroke without clinically significant stenosis or thrombosis. Thirty five of them have acquired risk factors (arterial hypertension, dyslipidemia, diabetes mellitus, atrial fibrillation, obstructive sleep apnea, smoking, oral contraceptives, family history) and hyperhomocysteinemia. The prevalence of acquired risk factors for stroke are significantly higher in ischemic stroke patients without thrombosis and clinically significant stenosis.

Conclusions: We found that thrombophilia could be a risk factor for thrombosis or severe stenosis in young patients with stroke.

PB497: In Vitro Effects of BCR‐ABL Tyrosine Kinase Inhibitors on Endothelial Cells Survival and Functions

H. Haguet 1,2 ; J. Douxfils 1 ; C. Graux 3 ; C. Chatelain 1 ; F. Mullier 2 ; J.‐M. Dogné 1

1 University of Namur, Department of Pharmacy, Namur, Belgium; 2 CHU UCL Namur, Hematology Laboratory, Yvoir, Belgium; 3 CHU UCL Namur, Department of Hematology, Yvoir, Belgium

Background: BCR‐ABL tyrosine kinase inhibitors (TKIs) are the mainstay of the treatment for chronic myeloid leukemia (CML). New generation TKIs (dasatinib, nilotinib, bosutinib and ponatinib) increase the risk of arterial thromboembolism in patients with CML. Clinical data suggest that new generation TKIs might accelerate atherogenesis.

Aims: This research aims to determine the effect of BCR‐ABL TKIs on endothelial cell survival and major functions using an in vitro model (i.e. HUVEC).

Methods: Viability was assessed using MTS and LDH assays following standard protocols. Expression of 3 adhesion molecules (i.e. ICAM‐1, VCAM‐1 and E‐selectin) was quantified by on‐cell ELISA after 4‐hour TNF‐α activation. Endothelial cell migration was evaluated by scratch assays (i.e. monitoring of wound closure of a scratch on confluent monolayer).

Results: At high concentration, dasatinib, nilotinib and ponatinib reduce cell metabolism. Additionally, high‐dose ponatinib induces necrosis as demonstrated by increased LDH release. On‐cell ELISA demonstrates decreased expression of 3 adhesion molecules (i.e. ICAM‐1, VCAM‐1 and E‐selectin) by dasatinib, nilotinib and ponatinib at high concentration. This diminution correlates with the decreased viability of endothelial cell with these 3 treatments. Imatinib and bosutinib have no or little impact on adhesion molecule expression. Finally, scratch assays indicate that high‐dose dasatinib inhibits endothelial cell migration whereas other TKIs do not have any impact.

Conclusions: Over the 5 commercialized BCR‐ABL TKIs, dasatinib, nilotinib and ponatinib possess the most impact on endothelial cells and possibly promote atherosclerosis development through impaired endothelium permeability, enabling migration and trapping of lipoprotein into the intima. Determination of mechanism(s) by which TKIs promote cardiovascular events is required to implement appropriate risk minimization measures and select patients to whom the prescription of these drugs should be avoided.

PB498: Impaired Fibrinolytic Activity in Nigerian Myocardial Infarction Patients

O.I. Ajayi ; F.S. Ozimede

University of Benin, Physiology, Benin City, Nigeria

Background: Myocardial infarction (MI) is referred to as the changes that occur in cardiac muscle and the sudden deprivation of circulating blood leading to necrosis of myocardial tissue. The pathogenesis has been well documented in countries with advanced medical technologies while in developing countries such as Nigeria, MI as an unfolding major cardiovascular events leading to sudden death in most cases.

Aims: This study was designed to ascertain the fibrinolytic patterns in acute MI events with a view to indicate their possible role in diagnosis and management.

Methods: A total of 10 acute myocardial infarction (AMI) patients together with 20 age and sex ‐matched apparently healthy subjects as controls were studied. Blood samples were taken at the point of admission (Day 0), on the 4th and 7th day respectively after treatment has commenced. Fibrinolytic indices such D‐dimer concentration (DDC) and Euglobulin lysis time test (ELT) were measured with standard laboratory methods.

Results: We observed a significantly increase in Euglobin lysis time as well as in increased D‐dimer levels AMI patients on admission compared with controls (P<0.05, respectively). DDC became significantly lowered from the 4th day of admission while all the ELT remained significantly high until the 7th day of admission and treatment (P<0.05, respectively). On the other hand lipids such as Cholesterol and HDL remained relatively unchanged from admission and throughout the duration of the study.

Conclusions: Conclusively, impaired fibrinolytic activity could be a major associated risk of thrombosis in Nigerians with AMI. The role of lipids in AMI pathogenesis in Nigerians seem to be unpronounced.

PB499: Shear Enhances Fibrin‐induced GPVI Shedding and Platelet ADAM10 Function

S.J. Montague 1,2 ; S. Hicks 1,3 ; P.Y. Choi 3,4 ; C.S.‐M. Lee 1 ; X. He 2 ; R.K. Andrews 5 ; W.M. Lee 1,2 ; E.E. Gardiner 1,3

1 The Australian National University, ACRF Department of Cancer Biology and Therapeutics, Canberra, Australia; 2 The Australian National University, Research School of Engineering, College of Engineering and Computer Science, Canberra, Australia; 3 National Platelet Research and Referral Centre, Canberra, Australia; 4 Canberra Hospital, Canberra, Australia; 5 Monash University, Australian Centre for Blood Diseases, Melbourne, Australia

Background: Fibrin, shear stress and active Factor X (FXa) induce glycoprotein (GP) VI shedding independent of direct thrombin activity. AD isintegrin A nd M etalloproteinase (ADAM) 10 is the main sheddase releasing human platelet soluble GPVI ectodomain.

Aims: To assess molecular links between these agonists and ADAM10 activity.

Methods: A cone/plate viscometer applied 300‐7,500/second uniform shear to platelet rich plasma on plates coated with fibrin, fibrinogen or collagen. We monitored platelet CD62P, GPVI and ADAM10 levels by flow cytometry, GPVI shedding by western blotting and platelet ADAM10 activity following shear, fibrin or FXa exposure with an ADAM10 fluorescence resonance energy transfer (FRET) assay.

Results: Platelet surface CD62P increased and GPVI decreased with increasing shear exposure and/or in the presence of fibrin/collagen. Polymerised fibrin (thrombin treatment of fibrinogen) induced GPVI shedding and αIIbβ3‐independent platelet accumulation. Accumulation was reduced 4‐fold when fibrin polymerisation was prevented by GPRP. Platelet ADAM10 activity significantly increased following fibrin exposure compared to fibrinogen (P=0.018, n=3) and untreated platelets (P=0.021). Fibrin‐mediated ADAM10 activity was not diminished by GPRP, Src family kinase inhibitors or GI254023X (ADAM10‐specific inhibitor) inclusion but was inhibited by EDTA.

Conclusions: Fibrin induced platelet accumulation, GPVI release and increased ADAM10 activity. Whilst accumulation and shedding were reduced in monomeric fibrin conditions with GPRP, ADAM10 activity remained elevated, suggesting fibrin‐GPVI interactions enhance ADAM10 activity in a manner which is not fully blocked by GI254023X, implying potential roles for fibrin‐GPVI unrelated to GPVI shedding. Assessing how fluid shear stress, FXa and fibrin cooperate to trigger platelet activation and regulated GPVI shedding/ADAM10 activity will help establish how coagulopathy and platelet activation intersect under different biorheological conditions.

PB500: Resonance Raman Spectroscopy as a Tool to Study Blood Flow in Brain Vessels in vivo

E.I. Nikelshparg 1 ; N.A. Brazhe 1 ; K. Thomsen 2,3 ; M. Lønstrup 2 ; A.R. Brazhe 1 ; G.V. Maksimov 1 ; M. Lauritzen 2,3 ; O. Sosnovtseva 4

1 Lomonosov Moscow State University, Faculty of Biology, Biophysics Department, Moscow, Russian Federation; 2 Copenhagen University, Institute of Neuroscience, Copenhagen, Denmark; 3 Rigshospitalet Glostrup, Department for Clinical Neurophysiology, Glostrup, Denmark; 4 Copenhagen University, Faculty of Health and Medical Sciences, Copengagen, Denmark

Background: Blood oxygenation (sO2), relative amount of hemoglobin (Hb) and Hb functional properties change under systemic blood disorders, global and local ischemia or microthrombosis. sO2 determines functioning of surrounding tissues and in its turn, state of tissue cells affects Hb properties. Therefore, it is important to develop methods to investigate sO2 and Hb properties locally in venules, arterioles and capillaries in vivo .

Aims: We aimed to develop an approach to evaluate sO2 and relative amount of Hb in cortex vessels in vivo using resonance Raman spectroscopy (RRS) and to validate the proposed approach under hypoxia and hyperoxia.

Methods: InVia Raman microspectrometer (Renishaw, UK) with 532 nm laser was used to record RRS spectra from arterioles, venules and capillaries in the sensory whisker barrel cortex of anesthetized mice ventilated with gas mixture containing different oxygen amount.

Results: We performed calibration experiments in vitro and we proposed to use different ratios of oxygen‐sensitive RRS peaks to monitor blood oxygenation. We demonstrated that RRS can be successfully applied for the quantitative evaluation of Hb oxygenation in arterioles, venules and capillaries under normoxia, hyperoxia and hypoxia.

Conclusions: The proposed approach can be applied for the quantitative analysis of blood oxygenation in vessels in brain in vivo . RRS can be a useful tool to study neurogliavascular coupling, to monitor blood flow under ischemia and reperfusion and to trace aggregation of erythrocytes and blood clot formation. EIN, NAB and ARB acknowledge financial support from the Russian Science Foundation (grant №17‐74‐20089). KT, ML and ML acknowledge financial support from Lundbeck Foundation, the NOVO‐Nordisk Foundation and the Danish Medical Research Council.

PB501: Assessing Three‐Dimensional Fibrin Polymerization Dynamics During Heparin Administration and Protamine Reversal in Cardiac Surgical Patients

D.M. Tshikudi 1,2 ; O. Simandoux 1,2 ; D. Kang 3 ; D. Yelin 4 ; S.K. Nadkarni 1,2

1 Massachusetts General Hospital, Boston, United States; 2 Wellman Center for Photomedicine, Boston, United States; 3 College of Optical Sciences, University of Arizona, Tucson, United States; 4 Technion, Israel Institute of Technology, Biomedical Engineering, Haifa, Israel

Background: Fibrin polymerization and structural stability is vital to clot strength. Cardiac surgery with cardiopulmonary bypass (CPB) causes fibrin disruption, which is likely associated with major postoperative blood loss. We investigate a novel approach, spectrally encoded confocal microscopy (SECM), that enables the rapid visualization of fibrin polymerization dynamics and microstructure, potentially enabling the prediction of bleeding after CPB.

Aims: To assess the effect of heparin and protamine administration on clot polymerization and fibrin microstructure in CPB patients using SECM.

Methods: Platelet rich plasma (PRP) from 10 CPB patients was collected at baseline and protamine reversal. To assess the effects of heparin, 6 samples were spiked with varying heparin doses (0.1‐2 USP/mL). All samples were tested using dynamic 3D SECM imaging at intervals of 1 minute during coagulation activation via Kaolin. From the SECM images, clot polymerization parameters including fibrin time (FT), fibrin density (FD) and fibrin length were derived.

Results: SECM permitted 3D visualization of fibrin microstructure with a spatial resolution of ˜1.0 μm over a 400×300 μm field of view and 30 μm depth. During clotting, fibrin fiber distribution was heterogenous with regions of low or high FD. In CPB patients, FD was significantly lower following protamine reversal (P<0.05) compared to baseline despite similar FT, suggesting that fibrin microstructure is not restored immediately following CPB. In the spiked PRP samples, with increasing heparin dose, fibrin polymerization was delayed with higher FT (P<0.05), fibrin strands were visibly thinner and longer and the FD was lower (P<0.05). Like the CPB samples, in spiked samples fibrin density was not restored following protamine reversal (P<0.01).

Conclusions: Visualizing and quantifying fibrin polymerization dynamics and microstructure using SECM provides valuable information to assess clot stability following CPB and will likely improve the capability to predict postoperative bleeding.

PB502: Anticoagulation Monitoring of Pediatric Patients on Life Support Sytems Using a Microfluidic Device That Mimics Complex Vascular Architecture

D. Luna 1 ; N. Pandian 1 ; J. Bui 1 ; P. Gadangi 1 ; T. Mathur 1 ; C. Reyes 1 ; J. Teruya 2 ; A. Jain 1

1 Texas A&M College of Engineering, Department of Biomedical Engineering, College Station, United States; 2 Texas Children's Hospital & Baylor College of Medicine, Division of Transfusion Medicine & Coagulation, Houston, United States

Background: Accurate monitoring of anticoagulant therapy is critical for pediatric patients on life support systems such as Extracorporeal Membrane Oxygenation (ECMO) and Left Ventricular Assist Devices (LVAD), however, there are no reliable coagulation assays that can precisely predict these patient's thrombotic or bleeding risk.

Aims: Here, we show a pediatric shear‐based microfluidic device that incorporates principles of Virchow's triad (Figure 1A‐B) that can reconstitute physiologically‐relevant thrombi formation. We have observed that this device is sensitive to clinically relevant doses of anticoagulant medication, Heparin and Bivalirudin, in healthy patient blood samples (Figure 1 C‐D).

FIGURE 1 Pediatric shear‐based coagulation monitor. (A) Virchow's Triad (B) Shear‐based coagulation monitor (C) Fibrin formation (D) Platelet aggregation

Methods: The microfluidic device contains three distinct regions made from PDMS, the pre‐stenosed, tortuous, and post‐stenosed (channel width and height are 200 μm and 75 μm, respectively). Blood samples were drawn after informed, written consent as per ethical guidelines of Institutional Review Board of Texas A&M University. Recalcified citrated blood was spiked with a clinically relevant dose of either Heparin or Bivalirudin and then perfused at a wall shear stress of 20‐30 dyne/cm 2 and fluorescently labelled platelets and fibrin formation were visualized and quantified.

Results: When healthy patient blood samples were perfused through the device we observed that the blood clotted in the post stenosed region. Next, we found when adding a clinically relevant dose of Heparin and Bilvalirudin (1 IU/mL and 250 ug/mL, respectively) both platelet aggregation and fibrin formation had been reduced, suggesting that the device is sensitive to anticoagulant medication. Thus, the results support the hypothesis that a microfluidic device that contains aspects of Virchow's triad may be used for coagulation monitoring.

Conclusions: This pediatric shear based microfluidic device could lead to better prediction of the effect of anticoagulants delivered to patients on life support systems.

PB503: Ir‐CPI: An Original Inhibitor of Thrombosis and Thrombosis‐associated With Cancer

R. Darbousset 1 ; V. Pireaux 2 ; L. Crescence 3 ; S. Demoulin 2 ; L. Panicot‐Dubois 3 ; E. Godfroid 2 ; C. Dubois 3

1 The University of Sydney, Heart Research Institute, Sydney, Australia; 2 Bioxodes, Marche‐en‐Famenne, Belgium; 3 Aix‐Marseille University, Center for CardioVascular and Nutrition Research, Marseille, France

Background: Ir‐CPI, a protein derived from saliva of the tick Ixodes ricinus , is an inhibitor of the activated form of factor XI (FXIa) and XII (FXIIa). In vitro, this molecule inhibits specifically the contact phase pathway.

Aims: Our aims were to assess the efficacy of Ir‐CPI in the prevention of thrombosis dependent on the Tissue Factor (TF) pathway in mouse models of thrombosis.

Methods: Two thrombosis models dependent on TF activation were studied. First, we used a laser‐injury model on a cremaster muscle arteriole that is strictly dependent on TF and independent of FXII. Second, we used a laser‐induced thrombosis model in mice bearing a pancreatic tumor. This model is mainly dependent on TF expressed by cancer cell‐derived microparticles. As control, we assessed the efficacy of Ir‐CPI in the Deep Vein Thrombosis (DVT) model in which FXII plays a central role in thrombus formation.

Results: As expected, Ir‐CPI significantly reduced thrombus weight in the DVT model. Surprisingly, Ir‐CPI inhibited thrombus formation, platelet accumulation and fibrin generation in the laser‐injury model in normal mice. The effects of Ir‐CPI on thrombosis were confirmed in tumor‐bearing mice despite their procoagulant state. Since neutrophils play a key role in the activation of the TF dependent pathway in the laser model, the presence and activation of neutrophils were compared in control and Ir‐CPI‐treated mice. In vivo, Ir‐CPI significantly inhibited neutrophil accumulation and their secretion of Neutrophil Elastase at the site of injury. In vitro, Ir‐CPI strongly diminished the activation of neutrophils leading to NETs formation.

Conclusions: As expected, Ir‐CPI is an effective antithrombotic in the FXII‐dependent mouse model. Surprisingly, it is effective in a laser‐injury model, even in a prothrombotic tumoral state. The inhibitory effect on neutrophil recruitment in vivo and NETs formation in vitro strongly suggests that Ir‐CPI has additional targets and offers new therapeutic opportunities in thrombosis‐associated diseases.

PB504: The Relation between Coagulant and Vascular Function in Adult Survivors of Childhood Cancer

M. Panova‐Noeva 1,2 ; B. Wagner 1 ; M. Nagler 3 ; S. Eckerle 4 ; H. Spronk 5 ; M.‐A. Neu 4 ; H. Merzenich 6 ; N. Arnold 2,3 ; J. Prochaska 1,2,3 ; A. Schneider 6 ; K. Lackner 7 ; H. ten Cate 5 ; T. Münzel 2,8 ; J. Faber 4 ; P. Wild 1,2,3

1 University Medical Center of the Johannes Gutenberg University, Center for Thrombosis and Hemostasis, Mainz, Germany; 2 University Medical Center of the Johannes Gutenberg University, Center for Translational Vascular Biology (CTVB), Mainz, Germany; 3 University Medical Center of the Johannes Gutenberg University, Preventive Cardiology and Preventive Medicine, Mainz, Germany; 4 University Medical Center of the Johannes Gutenberg‐University Mainz, Department of Pediatric Hematology/Oncology/Hemostaseology, Mainz, Germany; 5 Maastricht University Medical Center, Cardiovascular Research Institute Maastricht (CARIM), Maastricht, the Netherlands; 6 University Medical Center Mainz, Institute of Medical Biostatistics, Epidemiology and Informatics (IMBEI), Mainz, Germany; 7 University Medical Center of the Johannes Gutenberg University, Institute for Clinical Chemistry and Laboratory Medicine, Mainz, Germany; 8 University Medical Center of the Johannes Gutenberg‐University Mainz, Center for Cardiology I, Mainz, Germany

Background: The pathological mechanisms for an increased risk of cardiovascular diseases (CVD) in adult childhood cancer survivors are not well elucidated yet. Endothelial dysfunction has been implicated in these individuals. Thrombin generation in presence of platelets is emerging as potential assay to assess individual risk for CVD.

Aims: To investigate the relation between coagulation potential and vascular function in 200 adult survivors of a childhood cancer, sex specifically.

Methods: Endogenous thrombin potential (ETP), measured by the calibrated automated thrombogram, in platelet rich plasma (PRP) and platelet free plasma (PFP) was correlated to vascular function measurements, reflexion index (RI) and reactive hyperemia index (RHI) assessed by photo and pneumatic plethysmography, respectively. The relation between ETP and vascular function measurements was assessed sex‐specifically by Spearman rank correlation coefficient and multivariable linear regression analysis.

Results: Female cancer survivors presented with higher ETP, measured both in PRP (P<0.001) and PFP (P<0.001), lower RI (P<0.001) and no difference for RHI (P=0.13) compared to male survivors. A negative correlation between RI and ETP (in PRP, R=−0.25; in PFP, R=−0.16) and positive correlation between RHI and ETP in PRP only (R=0.16) was observed. In females only, a linear regression model for ETP adjusted for age confirmed the negative association with RI in PRP (beta estimate: −6.85 [−12.19, −1.51]). After adjustment for each traditional cardiovascular risk factor, the observed association was lost after adjustment for hypertension only. No relevant associations were found for RI in males and for RHI in both males and females.

Conclusions: This study demonstrates a link between platelet‐related hypercoagulability and vascular function of resistance vessels measured by digital volume plethysmography in female cancer survivors, which is potentially mediated by the presence of arterial hypertension.

PB505: Effectiveness and Safety of Rivaroxaban Versus Low Molecular‐Weight Heparin for Treatment of Cancer‐Associated Thromboembolism

C.I. Coleman 1 ; T.J. Bunz 2 ; C.G. Kohn 3 ; J. Beyer‐Westendorf 4

1 University of Connecticut, Suffield, United States; 2 New England Health Analytics LLC, Pharmacoepidemiology, Granby, United States; 3 Hartford Hospital, Evidence‐Based Practice Center, Hartford, United States; 4 University Hospital ‘Carl Gustav Carus’ Dresden, Thrombosis Research Unit, Department of Medicine I, Division Hematology, Dresden, Germany

Background: Trials suggest oral Factor Xa inhibitors (including rivaroxaban) may be non‐inferior to low molecular‐weight heparins (LMWHs) in the treatment of cancer‐associated thrombosis (CAT).

Aims: To assess the effectiveness and safety of rivaroxaban versus LMWH for treatment of CAT in routine practice.

Methods: Within US MarketScan claims data from 1/2012‐12/2016, we identified adults with active cancer, who had ≥1 primary hospitalization/emergency department visit diagnosis code for VTE, received rivaroxaban or a LMWH as their first outpatient anticoagulant within 30‐days of the index VTE and had ≥12‐month continuous insurance coverage prior to the index event (baseline). Rivaroxaban users were 1:1 matched to LMWH users via propensity‐scores, with standardized differences <0.1 for all covariates after matching. The primary endpoint was the composite of recurrent VTE or major bleeding. Patients were followed for 12‐months or until endpoint occurrence, insurance disenrollment or end of follow‐up.

Results: In total, 857 rivaroxaban users were matched to 857 LMWH users. Median (interquartile range) patient follow‐up was 10 (4.12) months. Median age was 61 (54.70) years and pulmonary embolism was present in 48% of patients. Cancer sites included: breast (23%), genitourinary/prostate (20%), lung (15%), colorectal (13%), hematologic (13%), gynecologic (9%), pancreas (6%), stomach/esophagus (4%) and other (47%). Time from diagnosis of active cancer to CAT was ≤3‐months in 29%, 3‐6 months for 17% and >6‐months for 54% of patients, Eastern Cooperative Oncology Group grade was 0‐2 in 94% of patients and 51% of patients had metastatic disease. Median time to first outpatient anticoagulation fill was 2 (1.4) days. Upon Cox regression, treatment with rivaroxaban and LMWH resulted in a similar incidence of the composite endpoint (Table).

TABLE 1 Comparison of Rivaroxaban and Low Molecular‐Weight Heparin for Cancer‐Associated Venous Thrombosis Endpoint N Events Incidence Rate/100PY Rivaroxaban (N=857) Incidence Rate/100PY LMWH (N=857) AIRD/100PY HR (95% CI) P‐value Composite of Recurrent VTE or Major Bleeding 122 10.91 10.88 +0.03 1.04 (0.73‐1.48) 0.84 Recurrent VTE 72 5.61 7.08 −1.47 0.83 (0.52‐1.32) 0.43 Major Bleeding 51 5.23 3.57 +1.66 1.49 (0.85‐2.62) 0.16 AIRD, absolute incidence rate difference; CI, confidence interval; HR, hazard ratio; N, number; PY, person‐years; VTE, venous thromboembolism *Propensity‐scores calculated based upon multivariable logistic regression incorporating using demographics, cancer type, presence of metastatic disease, Eastern Cooperative Oncology Group (ECOG) grade, comorbidities, VTE risk factors and concomitant medications identified during the 12‐month pre‐index VTE baseline period.

Conclusions: No difference in effectiveness and safety was observed between rivaroxaban and LMWHs for CAT treatment.

PB506: Optimizing Therapy for Venous Thromboembolism in Multiple Myeloma: A Systematic Review

S. Abegunde 1 ; I. Ajayi 2

1 National Hospital Orthopaedic Hospital, Lagos, Nigeria; 2 University of Benin, Benin City, Nigeria

Background: Patients with multiple myeloma (MM) are at increased risk of venous thromboembolism (VTE). Recently, questions have arisen about how best to manage this condition in MM especially as regard the use of thrombo‐prophylaxis.

Aims: To examine the current best evidence regarding optimal therapy for VTE in MM.

Methods: The PUBMED, EMBASE, MEDLINE and Cochrane Database were searched for relevant English‐language reports of clinical trials and studies, for the current best evidence regarding the efficacy and safety of the different antithrombotic agents including aspirin, low molecular weight heparin (LMWH), warfarin and direct oral anti‐coagulant (DOAC) used for treating VTE in MM patients. Reference lists of the identified articles were reviewed for additional relevant publications. Studies were eligible for inclusion if they were randomized controlled trials (RCTs) or observational studies comparing thrombo‐prophylaxis in adult MM patients.

Results: Our search yielded 605 potentially eligible studies. Only 3 studies were RCTs, with the remainder being observational studies. Most studies had small sample sizes (n=30‐1,247 patients). Patient populations were heterogeneous and included those with disease and treatment related VTE. There were variations in individual studies with respect to treatment protocol and dosing. One high ‐quality RCT suggests that bortezomib‐based regimens were associated with significantly low VTE rates compared to Immunomodulatory drug based regimens (P<0.05). LMWH was associated with a significant reduction in symptomatic VTE compared with the vitamin K antagonist warfarin (RR 0.33, 95% CI 0.14 to 0.83), while difference between LMWH and aspirin was not statistically significant (RR 0.51, 95% CI 0.22 to 1.17).

Conclusions: There is weak recommendation but moderate quality evidence that supports using LMWH or aspirin for VTE prophylaxis in MM. Given the morbidity and mortality associated with VTE, the need for high‐quality studies evaluating patient‐important clinical outcomes is hereby emphasized.

PB507: Low Molecular Weight Heparin Dose in the Long‐Term Management of Cancer Associated Thrombosis: Single Centre Experience

A. AL Zaki 1 ; R. Schmidt 2 ; N. Desilet 1 ; N. Ratnaweera 1 ; E. Peterson 1 ; A.Y.Y. Lee 1

1 University of British Columbia, Hematology, Vancouver, Canada; 2 University of British Columbia, Internal Medicine, Vancouver, Canada

Background: Venous thromboembolism (VTE) is a known complication of cancer. Long‐term therapy with low molecular weight heparin (LMWH) is the treatment of choice for cancer associated thrombosis (CAT). However, the ideal dose of LMWH has not been thoroughly studied.

Aims: To evaluate the efficacy and safety of continuing with full dose LMWH for the treatment of CAT for 3 months rather than decreasing the dose by 75‐80% after 4 weeks (CLOT protocol).

Methods: We conducted a historical cohort study of adult patients with cancer and acute VTE diagnosed between January 2013 and December 2015. Patients were followed for a minimum of 3 months after the index VTE. The primary outcome was VTE recurrence in patients who received full dose of LMWH compared to those treated as per the CLOT protocol. The secondary outcome included bleeding complications. Demographic characteristics of patients will be summarized in frequency tables and descriptive statistics. The cumulative incidence of recurrent VTE and of bleeding will be determined with corresponding 95% CI

Results: Four hundred eighty‐four patients were included, 78 of these were treated per CLOT protocol. At 3 months 5 of 78 CLOT patients and 23 of 406 full dose patients had experienced a recurrent VTE, the cumulative incidence was 6.5% and 5.8%, respectively (difference=0.7%; 95% CI: −5.3% to 6.7%; P=0.82). Bleeding complications by 3 months were observed in 1 CLOT and 28 full dose patients, the cumulative incidence was 1.3% and 7.0%, respectively (difference=−5.7%; 95% CI: −8.7% to 0.4%; P=0.07). Six CLOT patients and 94 full dose patients had died by 3 months (survival probabilities=92.2% vs. 76.5%; difference=15.7%; 95% CI: 8.4% to 23%, P=0.002).

TABLE 1 Baseline characteristics N (%) All cohort (N=584) Full dose LMWH (N=406) CLOT protocol LMWH (N=78) Age, (mean) years 64 63.1 64 No. of females 289 (49) 204 (50.3) 42 (53.8) Weight, (mean) Kg 74.9 73.7 76.3 metastatic cancer 307 (52.1) 217 (53.4) 42 (53.8) Primary cancer site (%) Brain Lung Breast Upper GI Lower GI GU except prostate Prostate Gynecological Hematological Melanoma Others 14 (2.4) 73 (12.4) 41 (7) 101 (17.2) 87 (14.8) 31 (5.3) 1 (0.2) 63 (10.7) 113 (19.2) 4 (0.7) 32 (5.4) 10 (2.5) 60 (14.8) 28 (6.9) 68 (16.8) 51 (12.6) 21 (5.2) 22 (5.4) 45 (11.1) 74 (18.2) 2 (0.5) 25 (6.2) 4 (5.1) 8 (10.3) 8 (10.3) 9 (11.5) 20 (25.6) 5 (6.4) 2 (2.6) 5 (6.4) 13 (16.7) 1 (1.3) 3 (3.9) Cancer treatment: Chemotherapy Immunotherapy Hormonal therapy Radiation therapy Cancer surgery 311 (52.8) 83 (14.1) 31 (5.3) 87 (14.8) 27 (4.6) 214 (52.7) 60 (14.8) 23 (5.7) 61 (15.1) 17 (4.2) 49 (62.8) 18 (23.1) 4 (5.1) 16 (20.5) 3 (3.9) Recent immobilization 61 (10.4) 43 (10.6) 6 (7.7) Previous history of VTE 25 (4.2) 12 (3) 3 (3.9) Smoking 38 (6.5) 26 (6.4) 6 (7.7)

TABLE 2 Primary outcomes at 3 months Primary outcome N (%) Full dose (N= 406) CLOT protocol (N= 78) P value VTE recurrence 23 (5.8) 5 (6.5) 0.82 Bleeding Major Clinically significant non‐major Minor 28 (7) 17 3 8 1 (1.3) 0 1 0 0.07 Death 94 (23) 6 (7.7) 0.002

Conclusions: In cancer patients with acute VTE, the use of full dose LMWH during the first 3 months was not significantly associated with lower VTE recurrence compared to CLOT protocol. Bleeding complications were not significantly different between the two groups.

PB508: Comparative Efficacy and Safety of Anticoagulation for the Treatment of Cancer‐associated Thrombosis: A Systematic Review and Meta‐analysis of Randomized Controlled Trials

C.I. Coleman 1 ; K. Sasiela 1 ; E. Smith 2 ; S.E. Trexler 1 ; K. Jackman 1 ; W.L. Baker 1

1 University of Connecticut, School of Pharmacy, Storrs, United States; 2 Griffin Hospital, Preventive Medicine, Derby, United States

Background: There is a growing body of randomized controlled trial (RCT) evidence assessing the efficacy and safety of subcutaneous and oral anticoagulation for the treatment of cancer‐associated thrombosis (CAT).

Aims: To evaluate the efficacy and safety of low molecular weight heparins (LMWHs), vitamin K antagonists (VKAs) and direct‐acting oral anticoagulants (DOACs) for the treatment of CAT.

Methods: A search of MEDLINE, CENTRAL and conference abstracts through 12/2017 was performed. RCTs enrolling adults with CAT, comparing ≥2 full dose anticoagulants (LMWH, VKA, DOAC) and evaluating recurrent venous thromboembolism (VTE), major bleeding and/or all‐cause death were included. Pooled relative risks (RRs) with 95% confidence intervals (CIs) were calculated using a random‐effects model and inverse variance weighting. Between‐study heterogeneity was calculated using the Sidik‐Jonkman estimator. Statistical heterogeneity was assessed using the I 2 statistic (>50% representing substantial heterogeneity).

Results: We included 13 RCTs in this meta‐analysis. The risk of recurrent VTE was 39% lower with DOACs compared to LMWHs, although 95% CIs included a RR=1.0 (Table). We observed a 76% greater, yet non‐significant, increased risk of major bleeding with DOACs versus LMWHs. Both DOACs and LMWHs significantly reduced the risk of recurrent VTE compared to VKA (RR=0.54 and 0.53), but did not impact major bleeding (RR=0.61 and 1.01). No differences were seen in favor of any anticoagulant for the death endpoint. I 2 ≤37% for all comparisons/outcomes.

TABLE 1 Results of meta‐analysis Endpoint Comparison a N Studies N Patients RR (95% CI) Recurrent VTE DOAC vs. LMWH 2 1452 0.61 (0.38 to 1.00) DOAC vs. VKA 4 2436 0.54 (0.34 to 0.88) LMWH vs. VKA 7 2096 0.53 (0.37 to 0.76) Major Bleeding DOAC vs. LMWH 2 1452 1.76 (0.72 to 4.31) DOAC vs. VKA 4 2437 0.61 (0.29 to 1.29) LMWH vs. VKA 6 2024 1.01 (0.53 to 1.90) All‐Cause Death DOAC vs. LMWH 2 1452 0.99 (0.90 to 1.09) DOAC vs. VKA 3 1923 0.99 (0.72 to 1.35) LMWH vs. VKA 6 2024 1.01 (0.84 to 1.21) a The second treatment is referent

Conclusions: DOACs may reduce patients’ risk of recurrent VTE compared to LMWHs, but with a potentially higher risk of major bleeding. DOACs and LMWHs both reduce recurrent VTE risk versus a VKA, and do so without increasing major bleeding. The choice of anticoagulation in CAT, particularly between a DOAC and LMWH, should take into account individual patient characteristics including their risk of thromboembolism versus bleeding, likelihood of adherence and patient preference.

PB509: A Monoclonal Antibody to Human Podoplanin Inhibits Human Platelet Aggregation Induced by Tumor Cells

Y. Zhao

The First Affiliated Hospital of Soochow University,, Jiangsu Institute of Hematology, Suzhou, China

Background: Podoplanin (PDPN) is frequently up regulated on many tumors and is involved in tumor metastasis and malignant progression.

Aims: This study is to investigate target therapeutic potentiality for tumor growth and metastasis based on antibody blockage of the interaction between PDPN and CLEC‐2.

Methods: Mouse monoclonal antibodies (mAbs) to human PDPN were developed using standard hybridoma technology. The specificity of mAbs was characterized by flow cytometry and Western blot analysis. The genous PDPN‐expressing in human cancer cells were detected using western‐blot and flow cytometry. Inhibition of mAb on the interaction between PDPN and CLEC‐2 was tested using platelet aggregation induced by cancer cell lines.

Results: Two mAbs named SZ‐161 to SZ‐168 against human PDPN were obtained. Both SZ‐163 and SZ‐168 were identified as IgG1 subtypes and both antibodies bound specifically with the synthetic PDPN peptide. Furthermore, immunoblotting indicated that both SZ‐163 and SZ‐168 bound to the human recombinant protein PDPN‐Fc (67 kDa), and to endogenous PDPN (46 or 36 kDa) in U87 astroglioma cells, NCL‐H226 lung squamous cells, endogenous PDPN (36 or 25 kDa) in human malignant melanoma cell line C8161 and and nasopharyngeal cancer cell line CNE‐2. In addition, flow cytometric analyses indicated that SZ163 and SZ168 recognized PDPN expressed on the tumor cell surface. SZ168, not SZ‐163, significantly inhibited NCL‐H226, C8161 or CNE‐2 induced platelet aggregation in a dose‐dependent manner and the maximal inhibition of SZ168 was 73.9 ± 3.0% on C8161 or 77.1 ± 2.7% on CNE‐2, or 83.9 ± 4.0% on NCL‐H226.

Conclusions: SZ168, a monoclonal antibody to human podoplanin inhibits platelet aggregation induced by cancer cell was developed and SZ168 may be a target therapeutic potentiality for tumor growth and metastasis based on antibody blockage of the interaction between PDPN and CLEC‐2.

PB510: D‐dimer Levels for Prediction of Symptomatic Central Venous Catheter‐related Thrombosis in Breast Cancer Patients

I. Benzidia 1 ; C. Frère 2 ; A. Hij 1 ; M. Espié 3 ; J. Gligorov 4 ; A. Elias 5 ; P.F. Dupré 6 ; S. Villiers 7 ; S. Giacchetti 3 ; P. Debourdeau 8 ; D. Farge 1

1 Saint Louis Hospital, Assistance Publique‐Hôpitaux de Paris, University Paris Diderot, Internal Medicine Unit, Autoimmune and Vascular Diseases, Paris, France; 2 Pitié‐Salpêtrière Hospital, Assistance Publique‐Hôpitaux de Paris, Sorbonne Université, INSERM, UMR_S 1166, Institute of Cardiometabolism and Nutrition, ICAN, Department of Haematology, Paris, France; 3 Saint Louis Hospital, Assistance Publique Hôpitaux de Paris, University Paris Diderot, Breast Cancer Unit, Paris, France; 4 Tenon Hospital, Assistance Publique Hôpitaux de Paris, IUC‐UPMC Sorbonne Université, Francilian Breast Intergroup, APREC, Medical Oncology, Paris, France; 5 Sainte Musse Hospital, Department of Vascular Medicine, Toulon, France; 6 CHRU Brest, Department of Gynecology and Surgery, Brest, France; 7 Saint Louis Hospital, Assistance Publique‐Hôpitaux de Paris, Department of Anesthesiology and Reanimation, Paris, France; 8 Sainte Catherine Institute, Department of Medical Oncology, Avignon, France

Background: Previous studies found an association between high D‐dimer levels and the risk of deep vein thrombosis and/or pulmonary embolism in cancer patients, indicating that D‐dimer might constitute a valuable biomarker to predict venous thromboembolism in this setting. Only small sample size studies have evaluated D‐dimer levels for prediction of central venous catheter (CVC)‐related thrombosis (CRT) yet.

Aims: We assessed whether D‐dimer levels can identify patients at high risk of developing CRT in a large sample size study.

Methods: 490 non‐metastatic invasive breast cancer patients undergoing insertion of a CVC for chemotherapy were included in this prospective multicenter cohort study. At the time of study enrollment, a blood sample was drawn for D‐dimer measurement. D‐dimer levels were measured using an Enzyme Linked Fluorescent Assay. Patients were followed‐up for 6 months after CVC insertion and all symptomatic CRT were objectively confirmed by Doppler‐ultrasonography. Receiver operating characteristic curves were used to calculate the optimal cut‐off value of D‐Dimers levels for CRT prediction.

Results: During the 6 months duration follow‐up, 14 out of 490 patients (2.8%) developed a symptomatic CRT. Baseline D‐dimer levels were significantly higher in patients developing a CRT compared to those without CRT (P=0.0292). A cut‐off value of 353 ng/mL yielded the highest Youden index of 0.36 (sensitivity, 100%; specificity, 35.8%; area under the curve, 0.671). Fourteen out of 321 patients (4.3%) with D‐dimer levels >353 ng/mL developed a CRT while no patient with D‐dimer levels £ 353 ng/mL developed a CRT, which therefore carried positive and negative predictive values of 4.36% and 100%, respectively.

Conclusions: In this analysis of the large sample size CAVECCAS study, D‐dimer levels alone do not appear as a relevant biomarker to identify patients at high risk of developing CRT within 6 months follow up after CVC insertion.

PB511: Hypercoagulation Screening as Tool for Cancer Diagnosis: An Update on Tumor Incidence and Risk Factors in the HYPERCAN Study

S. Gamba 1 ; D. Raffaeli 1 ; M. Marchetti 1 ; L. Russo 1 ; C.J. Tartari 1 ; C. Giaccherini 1 ; C. Verzeroli 1 ; S. Brevi 1 ; V. Milesi 1 ; G. Sampietro 2 ; P. Malighetti 3 ; D. Spinelli 3 ; A. Falanga 1

1 Hospital Papa Giovanni XXIII, Division of Immunohematology and Transfusion Medicine, Bergamo, Italy; 2 ATS Bergamo, Unit of Epidemiology and Statistics, Bergamo, Italy; 3 Università degli Studi of Bergamo, Human Factors and Technology in Healthcare, Bergamo, Italy

Background: Cancer induces a systemic hypercoagulable state that may represent the earliest clinical manifestation of an occult cancer. The HYPERCAN is an ongoing prospective Italian, multicenter, observational study to evaluate the hypercoagulability in healthy and cancer subjects.

Aims: To provide an update on tumor incidence, clinical, biochemical and lifestyle risk factors for cancer in a population of healthy blood donors.

Methods: Blood donors from Bergamo and Milan areas of both gender, age range 35‐65, are enrolled and followed‐up for 5 yrs for cancer occurrence, after informed consent . Whole blood, collected at enrollment and after 6‐12 months, is processed to obtain plasma, DNA, and serum samples. Clinical, hematological and biochemical data are collected at each visit. A lifestyle questionnaire is filled at enrollment. Identification of all malignant tumors is carried out every 6 months.

Results: 8,269 blood donors (71% M) have been recruited between April 2012 and January 2018. Routine biochemical and hematological workups were into the normal range values in more than 90% of subjects. Data from questionnaires show that 57% of the donors are not smoker, 15% are regular smokers, and 49% of them are moderate/low alcohol consumer (≥2 drink/die). After a median follow‐up of 2.9 years, 76 (51M/25F) cancer cases (expected 95 cases) have been recorded. The distribution of tumor types is the same as in the general population, with prostate (27%) and colorectal (16%) cancers being the most frequent tumors in males, and breast cancer (42%) in females. The histologic stage is I‐II in the 55% of cases, and IV in 6%. Interestingly, cancer individuals had lower educational status, higher glycemia and cholesterol levels as compared to the non‐cancer population (P<0.05).

Conclusions: Data show that in our population of blood donors, cancer type diagnosis has the same distribution as in the general population of the same geographic area, although, the incidence is lower than that predicted from epidemiological data.

PB512: Patients Experience of Living with Cancer Associated Thrombosis in New Zealand (PELICANZ)

T. Woulfe 1 ; K. Mann 2 ; D. Pollack 3 ; A. Nelson 4 ; H. Prout 4 ; P. Gee 4 ; S. Noble 4

1 Waitemata District Health Board, Haematology, Takapuna, New Zealand; 2 Auckland District Health Board, Auckland, New Zealand; 3 Mid Central District Health Board, Palmerston North, New Zealand; 4 Cardiff University, Cardiff, United Kingdom

Background: Previous research from Europe has identified areas of unmet clinical and support need for cancer patients diagnosed with venous thromboembolism. It is not known whether such experiences are restricted to those particular countries healthcare systems and/or cultures.

Aims: To evaluate patients’ experience of cancer‐associated thrombosis (CAT) within New Zealand.

Methods: Twenty patients with CAT were recruited across three regional thrombosis services in Waitemata, Auckland and Palmerston North in the North Island of New Zealand. Semi structured interviews were audio recorded and transcribed. Transcripts were coded using Invivo software. Analysis was undertaken using an applied framework matrix with an inductive approach to identify any new themes. This was in order to explicate potential differences that were not apparent in the European datasets.

Results: Twenty patients (10 male, 10 female) aged 51‐83 (mean 69) participated. There were marked commonalities observed between the New Zealand and European patients. Patients felt they had littleunderstanding of the risks of CAT or signs and symptoms, which would necessitate medical attention. They expressed a need for more information at the time of diagnosis and felt the management of their CAT was managed independently of their cancer. Whilst toverall management of CAT was good, they were often unable to identify which clinician or service had overall responsibility for CAT. The general practitioner often took responsibility by default. The level of distress reported by European patients was not as marked in the New Zealand participants.

Conclusions: The PELICANZ study corroborates many data identified in previous PELICAN studies in Europe. This supports a hypothesis that the needs of the CAT patient are similar across the globe with only minor cultural differences. The distress associated with CAT is a common experience but less so in New Zealand. The reason for this is unclear although it may indicate a higher level of resilience in this population.

PB513: Cost‐Effectiveness of Edoxaban Versus Dalteparin for Treatment of Cancer‐Associated Thrombosis

N. Connell 1,2 ; J. Connors 1,2

1 Brigham and Women's Hospital, Hematology Division, Boston, United States; 2 Harvard Medical School, Boston, United States

Background: Low‐molecular weight heparin has been standard treatment for cancer‐associated venous thromboembolism (VTE) since the CLOT study (NEJM 2003) showed dalteparin to be more effective than vitamin K antagonists. The Hokusai Cancer VTE study (NEJM 2017) recently showed the direct oral anticoagulant edoxaban to be non‐inferior to dalteparin for a combined outcome of recurrent VTE and major bleeding, offering a new option for treatment of cancer‐associated VTE.

Aims: Evaluate the cost‐effectiveness of edoxaban and dalteparin for treatment of cancer‐associated VTE from a United States (US) societal perspective.

Methods: A decision model was constructed comparing edoxaban and dalteparin for 6 months of therapy (Figure).

Probabilities for each outcome along with associated quality of life utility estimates were combined with cost data to estimate total cost and quality‐adjusted life years (QALYs) for each anticoagulation strategy. Inputs were taken from previously published literature and costs inflated to 2017 US dollars. Average wholesale medication prices were taken from the Red Book. Sensitivity analyses were performed to assess the impact of point estimate uncertainty on model outcomes. A subgroup analysis of patients with gastrointestinal (GI) malignancies was performed given findings in Hokusai.

FIGURE 1 Graphical representation of the decision model comparing edoxaban to dalteparin for cancer‐associated thrombosis

Results: Results of the model are shown in the Table.

TABLE Results of the cost effectiveness of edoxaban versus dalteparin for cancer‐associated VTE Cost (2017 USD) Effectiveness (QALYs) ICER (2017 USD per QALY) All cancer patients Edoxaban $6,061 0.34 Dalteparin $19,398 0.35 $1,873,534 Gastrointestinal Malignancy Edoxaban * $7,227 0.34 Dalteparin $19,384 0.35 $694,058 *The cost of edoxaban in this strategy includes the increased rate of non‐ICH major bleeding in GI malignancy patients on edoxaban

The 6‐month cost was $6,067 and $19,398 for edoxaban and dalteparin respectively with similar projected utility values. The incremental cost effectiveness ratio (ICER) of dalteparin was over $1.8 million/QALY. Sensitivity analyses did not change results. While edoxaban was more expensive in GI malignancy patients compared to general oncology patients due to the increased rate of bleeding, it remained the preferred strategy over dalteparin (ICER $694,058).

Conclusions: With similar quality adjusted life years and significantly lower cost, edoxaban is cost‐effective for the treatment of cancer‐associated VTE at standard willingness to pay thresholds.

PB514: Non‐phosphorylated and Phosphorylated Signal Transduction Peptide (STP) of Tissue Factor (TF) in Urine Are Potential Biomarkers for Bladder and Colon Cancer

R. Greenfield 1 ; T. Herd 2 ; K. Date 3 ; A. O'Kane 4 ; A. Maraveyas 5

1 C‐Term Diagnostics, Yonkers, United States; 2 C‐Term Diagnostics, Eastleigh, United Kingdom; 3 Hull York Medical School, Joint Centre for Cancer Studies, Hull, United Kingdom; 4 Fusion Antibodies PLC, Belfast, United Kingdom; 5 Hull York Medical SchoolYork Medical School, Joint Centre for Cancer Studies, Hull, United Kingdom

Background: Tissue Factor bound to microvesicles was shown to be elevated in urine of patients with various cancers. Phosphorylation of the TFSTP at Ser253 and Ser258 is important for the formation of TF‐microvesicles. We investigated the marker potential of the TF‐STP forms in urine of cancer patients to quantitate non‐phosphorylated (np) and phosphorylated TF‐STP.

Aims: To develop immunoassays and methods to quantitate np‐TF, TF‐pSer253 and TF‐pSer258 in urine from healthy individuals and patients with various cancer types.

FIGURE 1 Quantitative analysis of TF_pSer258 (A) and TF‐pSer253 (B) in unfractionated (U) urine of healthy and Cancer patients

Methods: Antibodies were raised to phosphorylated and non‐phosphorylated peptides. ELISAs were constructed for the three TF‐STP forms. TF‐STP forms were quantitated in detergent solubilized urine samples from healthy and from bladder, colon, prostate and breast cancer patients.

Results: TF‐pSer258 was significantly elevated in bladder cancer compared to the healthy group (Figure 1A) and TF‐pSer253 was elevated in colon cancer (1B). ROC analysis showed that TF‐pSer258 was a strong biomarker for both early stage and late stage muscle invasive bladder cancers with 86.8 sensitivity and 91.1 specificity (PPV=89.1; NPV=89.1). ROC analysis indicated np‐TF had overall bladder cancer sensitivity of 76% and specificity 77% (PPV=74.3; NPV=79.5) at cut‐off at 3.48 ng/ml. However, non‐phosphorylated TF was a stronger biomarker for the muscle invasive late stage bladder cancers [19/20, 95%]. TF‐pSer253 was significantly elevated only in the colon cancer group. ROC analysis indicated TF‐pSer253 had a sensitivity of 71.4% sensitivity and 69% specificity for colon cancer.

Conclusions: TF‐STP forms may be novel biomarkers differentially expressed in urine of different cancers. TF‐pSer258 may be a sensitive marker for non‐invasive and invasive urothelial malignancies. Np‐TF was preferentially elevated in muscle invasive late stage bladder cancers. TF‐pSer258 in conjunction with np‐TF may provide early diagnostic and staging information for bladder cancer. TF‐pSer253 may be a unique diagnostic marker for colon cancer.

PB516: Leucocyte and Platelet Activation in Patients with BCR‐ABL Negative Myeloproliferative Disorders

C. Portelli 1 ; M.C. Cini 1 ; P. Brincat 2 ; K. Vella 2 ; N. Calleja 3 ; A. Gatt 4

1 University of Malta, Faculty of Health Sciences, Msida, Malta; 2 Mater Dei Hospital, Pathology Department, Msida, Malta; 3 University of Malta, Faculty of Medicine and Surgery, Msida, Malta; 4 University of Malta, Faculty of Medicine and Surgery, Pathology Department, Msida, Malta

Background: Polycythaemia vera, essential thrombocythaemia and primary myelofibrosis are BCR‐ABL negative myeloproliferative disorders (MPDs) characterised by the clonal proliferation of haematopoietic stem cells in the bone marrow. MPD patients have a high risk of suffering from thrombotic events. The pathogenesis of thrombosis is thought to arise in part from the overactivation of blood cells.

Aims: This study aims to establish the differences in leucocyte and platelet activation parameters between MPD patients and a normal cohort, and to correlate these findings with the clinical risk of thrombosis, which is currently the gold standard for decisions related to therapy.

Methods: The degree of activation of platelets and leucocytes was evaluated by flow cytometry in 71 MPD patients attending Sir Anthony Mamo Oncology Centre and 19 healthy controls. CD62P was used as a platelet activation marker, while leucocyte alkaline phosphatase (LAP) and CD11b were used to assess monocyte and neutrophil activation. Patient results were compared to the control group by using the independent sample t‐test. Informed consent was obtained from all subjects after approval by the University of Malta Research Ethics Committee.

Results: MPD patients have a higher percentage of platelets expressing CD62P than controls, as well as a higher percentage of neutrophils expressing LAP and a higher number of CD11b on monocytes. No significant difference was found between the number of CD11b on neutrophils in patients and controls. When divided into JAK2+ , JAK2 − and controls, we found that JAK2+ patients could be differentiated from JAK2 − patients due to higher leucocyte CD11b, whilst JAK2‐ patients had higher CD62P than controls.

Conclusions: These parameters, used in conjunction with other tests, can aid in the diagnosis and prognostic stratification of MPD patients. Since the activation status of blood cells may be linked to thrombotic events, assessing these biomarkers can better direct the treatment approach and possibly predict thrombosis.

PB517: Estimation of Risk Factors for PICC Catheter Related Thrombosis in Patients with Hematologic Malignancies

Z. Weiwei ; F. Yun

Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

Background: There are many reasons that lead to the formation of PICC‐CRT. There are many risk factors for the formation of PICC‐CRT in different diseases in the world. However, the formation of PICC‐CRT in hematological tumors is rarely reported. We found that hematological tumors The occurrence of patients with PICC‐CRT also has its own characteristics.

Aims: To investigate the risk factors of PICC catheter related thrombosis in patients with hematologic malignancies.

Methods: Based on the matching method typed 1:2 of gender, age (±2 years) and disease,collected the clinical data of the hematological tumor patients underwear PICC catheterization in our hospital from January 1, 2015 to June 2017, 102 patients who did not occur PICC‐CRT were selected as controls. A retrospective case control study was conducted. Single factor analysis was performed using hierarchical COX regression analysis, P<0.1 was considered statistically significant; Multivariate analysis was performed using conditional logistic regression analysis , P<0.05 was considered statistically significant.

Results: The median time to PICC‐CRT was 22 (8˜60) days. Single factor analysis showed hs‐CRP (P=0.004) D‐ two dimer (P=0.043), Blood transfusion (P=0.013), Chemotherapy (P=0.014), Inject EPO (P=0.000) compared with statistical significance. Conditional Logistic regression analysis showed: hs‐CRP (OR=0.241, 95% CI: 0.085‐0.687, P=0.008); Chemotherapy (OR=0.203, 95% CI: 0.067‐0.611, P=0.005); Inject EPO (OR=0.218, 95% CI: 0.077‐0.616, P=0.004) compared with statistical significance

Conclusions: hs‐CRP, Chemotherapy, Inject EPO were independent risk factors for PICC catheter‐related thrombosis in patients with hematological malignancies.

PB518: Optimisation of Thrombin Generation as a Predictive Biomarker for Venous Thromboembolism in Gynaecological Malignancies

M.P. Ward 1 ; F. Abu Saadeh 1,2 ; S.A. O'Toole 1 ; Z. Machocki 1,2 ; N. Gleeson 1,2 ; L.A. Norris 1

1 Trinity College Dublin, Obstetrics and Gynaecology, Dublin, Ireland; 2 St James's Hospital, Gynae‐Oncology, Dublin, Ireland

Background: Gynaecological cancers have been associated with high rates of venous thromboembolism (VTE). Thrombin generation (TG) is a novel biomarker that assesses global coagulation activation.

Aims: The aim of this study is to optimise the TG test for prediction of VTE in gynaecological cancer patients post‐surgery.

Methods: 24 gynaecological cancer patients who suffered VTE post‐surgery were matched for histological subtype, disease and treatment with 56 gynaecological cancer patients who remained thrombosis free. TG was measured in blood taken prior to surgery using 1 pM and 5 pM Tissue Factor (TF) ± (10 nM) thrombomodulin (TM). Using the data obtained, the predictive ability of TG in 357 cancer patients was assessed.

Results: 1 pM and 5 pM TF gave significantly higher ETP in the VTE group compared to non VTE (1858 nM/min v 2302 nM/min; P<0.03 and 2173 nM/min v 2744 nM/min; P<0.007). Peak thrombin was also higher in the VTE group (358.2 nM and 424.8 nM; P<0.008) at 5 pM TF but not at 1 pM (299.2 nM and 322.5 nM; P<0.67). Addition of 10 nM TM also showed a significant difference in ETP values between the groups (147.5 nM/min and 381.5 nM/min; P<0.01). The interassay coefficient of variation (CV) was 11.11% (1 pM TF) and 10.10% (5 pM TF) respectively for ETP. Addition of 10 nM TM increased the CV to 43.14% (1 pM) and 50.22% (5 pM TF). TG (5 pM TF) was determined in 357 gynaecological cancer patients prior to surgery of which 32 developed VTE post‐surgery. ETP levels were significantly higher in the VTE group compared with the non VTE group (P<0.01). Patients who had ETP values above the 75th percentile had an increased risk of VTE OR=4.0 (1.693‐9.46 (95% CI) when adjusted for stage and histological subtype. Peak thrombin levels were also higher in the VTE group but this was not significant (P<0.08).

Conclusions: Thrombin generation assay is a useful predictor of VTE in gynaecological cancer patients. 5 pM TF produces reproducible values of ETP which can indicate the patients who are likely to develop VTE post‐surgery.

PB519: Evaluation of Risk Factors and Assessment Models for Predicting Venous Thromboembolism in Lung Cancer Patients

J. Rupa‐Matysek 1 ; M. Lembicz 2 ; E. Rogowska 2 ; L. Gil 1 ; M. Komarnicki 1 ; H. Batura‐Gabryel 2

1 Poznan University of Medical Sciences, Department of Hematology and Bone Marrow Transplantation, Poznań, Poland; 2 Poznan University of Medical Sciences, Department of Pulmonology, Allergology and Respiratory Oncology, Poznań, Poland

Background: The aim of the study was to investigate the prognostic significance of selected risk assessment models (RAMs) for predicting venous thromboembolism (VTE) events in patients undergoing outpatient chemotherapy for lung cancer.

Aims: We evaluated the following VTE‐risk assessment tools: Khorana Risk Score (KRS), PROTECHT (PROpylaxis of ThromboEmbolism during CHemoTherapy) score, CONKO score and COMPASS‐Cancer‐Associated Thrombosis Score (COMPASS‐CAT).

Methods: Patients’ clinical and laboratory data results pre‐chemotherapy were collected.

Results: Retrospective analyses were performed on 118 patients with lung cancer, 20 of whom developed VTE with a median of 2.5 months from diagnosis. Patients receiving gemcitabine‐based regimen (25%) had a greater incidence of VTE than patients undergoing other treatment (45% vs. 16%, P=0.0042). Patients with a history of atrial fibrillation (AF) developed VTE more often than other patients (35% vs. 6%, P=0.0002). Patients with chronic kidney disease (CKD) had more VTE events than patients without CKD (30% vs. 9%, P=0.0109). In the multivariate analysis, high COMPASS‐CAT Score (OR 8.73; 95% CI 1.01‐75.22, P=0.049), gemcitabine chemotherapy (OR 3.37; 95% CI 1.09‐10.39, P=0.035) and AF (OR 7.19; 95% CI 1.89‐27.33, P=0.004) were all significantly associated with VTE development. VTE occurred in; 13% (n=2) of the KRS high‐risk group, 17.7% (n=11) of the PROTECHT high‐risk group, 15% (n=4) of the CONKO high‐risk group and 23.8% (n=20) of the COMPASS‐CAT high‐risk group (n=84). Only the COMPASS‐CAT Score was able to identify 100% of patients who developed VTE, and best discriminated between patients with high and low risk of VTE development (C statistic 0.89). The ROC analysis indicated a cut‐off value of 11 points (95% CI 0.821‐0.962) for COMPASS‐CAT for VTE development in patients with lung cancer.

Conclusions: In conclusion, in our study of all the VTE‐RAMs analyzed, the COMPASS‐CAT model was the most accurate predictor of VTE development in patients with lung cancer.

PB520: Splanchnic Thrombosis Associted to Myeloproliferative Neoplasms: A Cohort of 233 Tunisian Patients

I. Ghachem 1 ; H. Abbassi 2 ; S. Hadhri 2 ; L. Chaouch 2 ; H. Skouri 2,3

1 Laboratory of Hematoalogy, CHU Sahloul Sousse, Sousse, Tunisia; 2 Laboratory of Hematoalogy, Sousse, Tunisia; 3 Sahloul Sousse, Tunisia, Tunisia

Background: Splanchnic vein thrombosis (SVT) is a common and serious complication of overt and latent myeloproliferative neoplasms (MPN) with molecular features such as JAK2V617F, MPL and CALR mutations.

Aims: To assess the prevalence of JAK2V617F, MPL and CALR mutations in patients with SVT and to analyze the relationship between the blood counts and JAK2 mutation status.

Methods: This is a cohort of 233 Tunisian with non‐malignant and non‐cirrhotic SVT between 2007 and 2015, who were received for the screening of JAK2 V617F mutation. MPL and CALR mutations were searched for JAKV617F negative patients.

Results: JAK2 mutation was detected in 20% of SVT. There was a significant higher JAK2 prevalence in BCS compared with PVT (37.5% and 19%; P=0.04). MPL and CALR mutations were not detected in any of the 186 JAK2 negative patients. The frequency of an overt NMP was 64% in SVT, 61% in PVT and 50% in SBC. The frequency of latent NMP was 36% in SVT, 34% in PVT and 50% in SBC. Antiphospholipid antibodies (18.4%) and factor II Leiden (0.9%) were more prevalent in PVT and protein C deficiency was frequent in BCS. JAK2 was associated with significantly higher hemoglobin (Hb) levels, hematocrit (Ht) levels, white blood cell (WBC) and platelet counts in PVT patients. In BCS, JAK2 was associated with a significant higher platelet counts. The frequency of the JAK2 mutation increased progressively with Hb levels, WBC and platelet counts. Patients with Hb level ≥14.5 g/dl for men, Hb level ≥11.4/dl for women, WBC count ≥6,100/mm 3 , and platelet count ≥238,000/mm 3 should be tested for JAK2 mutation. This strategy avoids 89.5% of unnecessary JAK2 mutations testing. A platelet count ≥238 000/mm 3 was an independent factor correlated with the JAK2 mutation and, therefore, a highly predictor of latent MPN (OR=17.3; 95% CI [2.8‐105.1]); P=0.002).

Conclusions: JAK2V617F mutation is a good marker for occult MPN in SVT. The correlation between JAK2 mutation and blood counts could explain the role of JAK2 mutation in the pathogenesis of SVT.

PB521: The Effect of Hydroxyurea on Blood Coagulation in Sickle Cell Disease

M. Borhany ; M. Abid; N. Fatima; T. Shamsi

National Institute of Blood Disease and Bone Marrow Transplantation., Karachi, Pakistan

Background: Sickle cell disease (SCD) is a genetic disorder that is frequently referred to as a hypercoagulable state. Hydoxyurea is known to decrease the frequency of vaso‐occlusive complications and blood transfusions in severely affected individuals. It has been reported to decrease coagulation activation. Previous studies have not evaluated the effect of hydroxyurea on markers of coagulation activation over time.

Aims: To assess the effect of hydroxyurea on markers of fibrinolysis (D‐dimer) and endothelial activation (soluble vascular cell adhesion molecule‐1 [soluble VCAM‐1]) in SCD patients in their non‐crisis in the past 4 weeks, no transfusions in past 3 months, not on aspirin, or anticoagulation.

Methods: Patients (10 years and older) with documented sickle cell anemia (HbSS or HbSb‐thalassemia) eligible for treatment with hydroxyurea were studied in this prospective, observational study. Laboratory investigations were obtained at baseline, prior to commencement of therapy with hydroxyurea, with repeat evaluations at 3 and 6 months of post therapy. Non parametric test was applied to observe the association.

Results: Twenty‐five patients were enrolled (females: 14 [56%]), with a median age of 23 years. Hemoglobin (Hb), lactate dehydrogenase (LDH), VCAM, and D‐dimer at baseline and post hydroxyurea were deranged while TLC, MCV, PT, APTT and platelet count were in normal ranges. Wilcoxon sign rank test was applied to observe the post therapy effect (Table 1).

TABLE 1 Laboratory parameters at baseline and post therapy hydroxyurea Lab Parameters Baseline (Median) Post therapy Hydroxyurea (Median) P‐value VCAM 576.5 526.4 0.381 D‐dimer 1277.6 629.6 0.002 Hemoglobin (Hb) 9.10 9.5 0.74 Total Leukocyte count (TLC) 8.7 7.2 0.01 Platelet 243 196.5 0.157 MCV 74.1 76.5 0.009 LDH 864 567.5 0.102

The difference of coagulation parameters and hemogram in two categories of sickle patients groups was observed by applying Mann Whitney test. No significant results were found except on MCV and D‐dimer at 3 and 6 months post therapy respectively.

Conclusions: This study of SCD patients on hydroxyurea doesn't show significant effect on endothelial activation marker (VCAM), however fibrinolysis marker (D‐dimer) has good correlation. More studies are warranted to see the effect of hydroxyurea on blood coagulation in these patients.

PB522: The Effect of Inflammatory Cytokines IL‐6, IL‐10, IL‐1b and TNFa on Thromboelastography Parameters in Pediatric Patients with Acute Lymphoblastic Leukemia

S. Prasca 1 ; E. Orgel 1,2 ; J.H. Parmentier 1 ; S. Mittelman 3 ; G. Young 1,2

1 Children′s Hospital Los Angeles, Children′s Center for Cancer and Blood Diseases, Los Angeles, United States; 2 University of Southern California, Keck School of Medicine, Los Angeles, United States; 3 UCLA/Mattel Children′s Hospital, Division of Pediatric Endocrinology, Los Angeles, United States

Background: Obesity and cancer are both well‐established hyper‐inflammatory states. While the effect of inflammation on thrombosis has been described, evidence for the impact of inflammation on coagulation assays such as thromboelastography (TEG) is scarce. Thrombosis is an important treatment‐related toxicity in acute lymphoblastic leukemia (ALL). Understanding the effect of inflammation on the TEG assay will support future investigation into ALL treatment‐associated hypercoagulability.

Aims: To assess the effect of pro‐inflammatory cytokines on TEG parameters in ALL pediatric patients during induction therapy.

Methods: A prospective study in newly diagnosed ALL patients <21 years old. The study was IRB approved and informed consent obtained from all participants. Pro‐inflammatory cytokines (IL‐1b, IL‐6, IL‐10 and TNFa) and TEG parameters (R, K, alpha angle and MA) were measured at Day 0 and at end of induction (EOI). Spearman Rho correlation was used to evaluate cytokines’ impact on TEG. All tests were two‐sided; P<0.05 was considered significant.

Results: Thirteen patients were enrolled. 5/13 (38.5%) were female. Six (46.2%) patients had a venous thromboembolism (VTE). Abnormally elevated TNFa and IL‐6 levels were significantly associated with shortened K time (P=0.03) in TEG. Likewise, TNFa was inversely correlated with R time (P=0.02), K time (P=0.002) and showed a trend to increase alpha angle (Spearman Rho: 0.3; P=0.06). Other cytokines did not show effect on TEG. Cytokines or TEG parameters were not associated with VTE due to the small sample size.

Conclusions: In ALL pediatric patients receiving induction therapy, TNFa and IL‐6 elevated in the context of inflammation significantly influence TEG parameters associated with greater hypercoagulability. Pro‐inflammatory cytokines induced changes in TEG parameters and should be incorporated into future studies of hypercoagulability and risk of thrombosis in pediatric ALL.

PB524: Clinical and Global Hemostatic Parameters in Patients with JAK2 V617F Mutation Myeloproliferative Neoplasms (MPNs)

A. Sasakul 1,2 ; P. Chantrathammachart 1 ; S. Chuncharunee 1 ; A. Ungkanont 1 ; P. Angchaisuksiri 1 ; P. Niparuck 1 ; S. Pukiat 1 ; S. Phusanti 1

1 Mahidol University, Division of Hematology, Department of Medicine, Bangkok, Thailand; 2 Chulabhorn Hospital, Department of Hematology, Bangkok, Thailand

Background: Traditional coagulation tests fail to show abnormalities and predict chance of thrombosis in MPNs patients. In this study, global hemostatic tests: viscoelastic hemostatic assay (VHA) and thrombin generation (TG) were used to evaluate hypercoagulability in MPN patients with or without JAK2 V617F mutation.

Aims: To determine the association between JAK2 V617F mutation status and thrombotic complication, and to investigate blood parameters correlate with thrombosis in MPNs patients.

Methods: Data of 296 MPNs patients treated in Ramathibodi hospital from 2012 to 2016 were reviewed. From these patients, blood samples from 56 patients (14 PV, 35 ET, 6 PMF, and 1 MPN‐U) were used for global hemostatic tests.

Results: The total of 296 patients were classified as PV (85), ET (165), PMF (40) and MPN‐U (6). JAK2 V617F mutation were detected in 87%, 75%, 65% and 67% respectively. Arterial thrombosis occurred in 21%, venous thromboembolism in 7%, bleeding in 2% of patients. Thrombotic complication were common in ET and PV. MPN patients with JAK2 V617F mutation have high prevalence of thrombosis [OR=2.77; P=0.02 (95% CI 1.16‐6.57)]. The VHA showed hypercoagulable profile in ET and PV patients characterized by shortening of EXTEM‐clotting time, EXTEM‐clot formation time and higher of EXTEM‐maximun clot firmness compared to control but there was no difference between JAK2 V617F mutation and wild type status. Patients with JAK2 V617F mutation trend to have higher level of soluble p‐selectin compared with JAK2 V617F wide type and control group. In addition, there was no difference in TG parameters from JAK2 V617F mutation, wild type and control plasma.

Conclusions: In MPN patients, JAK2 V617F mutation correlates with the high prevalence of both arterial and venous thrombosis. From our study, hypercoagulable state seem to influence mainly by cellular component.

PB525: Generation of Neo‐epitopes on Hypochlorous Acid‐modified Human Antithrombin: An Immunological Study

P. Ahmad 1 ; I.Q. Tantry 2 ; M.M. Rizwee 2 ; A. Ali 2 ; M.A. Jairajpuri 1

1 Jamia Millia Islamia (A Central University), Biosciences, New Delhi, India; 2 J. N. Medical College, Aligarh Muslim University, Biochemistry, Aligarh, India

Background: Antithrombin (AT) is a glycoprotein with evidence of the presence of cleaved and latent forms. Native form is an anticoagulant, while; latent and cleaved have been shown to have antiangiogenic potential. Hypochlorous acid (HOCl) is a potent oxidant which is produced in vivo during respiratory burst. It can alter a variety of bio‐molecules like DNA and proteins. An excessive or sustained production of HOCl may result in tissue damage leading to progression of various diseases like cancers.

Aims: Effects of HOCl on generation of neo ‐epitopes on AT.

Methods: Isolation of AT and its conversion to various forms were done as per the protocols and subjected to different concentrations of HOCl. To check immunogenicities of unmodified and HOCl‐modified AT, white New Zealand female rabbits were immunized intramuscularly at multiple sites and ELISA was performed. The IgG purification was done by Protein A‐agarose column. This study had bioethical clearance.

Results: Direct binding ELISA showed antibody titre of >1:12,800 in all cases of modified‐AT, while it was <1:6,400 in case of unmodified native‐AT and >1:6,400 in rest conformers. Preimmune sera showed negligible bindings under identical experimental conditions. To detect the specificities of experimentally produced antibodies, competitive inhibition ELISA was done. The result showed that in all cases percent inhibition was more in HOCl‐modified conformers compared to that of unmodified forms. The results of direct binding and inhibition ELISA indicated generation of neo ‐epitopes on AT after HOCl exposure which might be due to oxidation/modification of the side chains of AT.

Conclusions: Structural alterations in AT induced by HOCl caused generation of neo ‐epitopes which were not ordinarily present. We propose that these epitopes may project the physiologic protein as ‘alien’ for the immune system leading to antibody generation with significance in various pathological conditions like cancer.

● Financial support from UGC India is thankfully acknowledged.

PB526: Polymorphism of Folate Protein Genes Metabolism as a Predictor of Thrombosis in Patients with Proliferative Disease and Breast Cancer

A. Markovsky

Chita State Medical Academy, Chita, Russian Federation

Background: In 50% of patients venous thrombosis (VT) identified genetic factors, including mutations enzyme systems folate metabolism.

Aims: Compare the frequency of alleles and genotypes of polymorphisms of genes folate cycle protein and homocysteine (HC) ​​levels in the blood serum in patients with proliferative diseases and breast cancer.

Methods: The study included 35 patients with proliferative breast disease (PZMZH) and 77 ‐breast cancer at the age of 56 ± 10.0 years. The control group consisted of 144 healthy women aged 40.2 ± 9.5 years. Determination of serum HC was performed by high performance liquid chromatography. Genotyping was conducted by RT‐PCR. Statistical analysis was performed using Statistica 6.0. To compare the frequency distributions of genotypes and alleles between treatment groups using χ 2 test. When carrying out descriptive statistics were calculated median and percentiles (25th and 75th). Statistical significance of differences was determined by the Mann‐Whitney test.

Results: HC level in patients with breast cancer ‐ 9.13 [8.4‐9.9] mol/L, and in patients with PZMZH ‐ 8.0 [7.6‐8.8] mol/L (P<0.000005) was higher than in the control group ‐ 7.9 [7.1‐8.7] mol/l. When evaluating the association of serum HC concentration of both patients and healthy with the carriage of SNP of folate metabolism genes have been identified due. However, the greatest increase in HC ​​levels among treatment groups was observed in carriers of the allele in the genome complexes MTHFR677T x MTHFR1298C x MTR2756A x MTRR66A and MTHFR677T x MTHFR1298A x MTR2756G x MTRR66A ‐ 9.6 [8.2‐11.1] mol/L.

Conclusions: Certain combinations of genotypes represented in the following chetyrehlokusnyh allelic patterns, ‐ MTHFR677T x MTHFR1298C x MTR2756A x MTRR66A and MTHFR677T x MTHFR1298A x MTR2756G x MTRR66A, may be associated with a significant increase in serum HC ​​concentration and be a risk factor for disorders of the hemostatic system in patients proliferative diseases and breast cancer.

PB527: The Usefulness of Standard and Derived ROTEM Parameters for DIC Prediction in Septic Patients

E. Scarlatescu 1 ; D.R. Tomescu 1,2

1 Fundeni Clinical Institute, Anaesthesia and Intensive Care III, Bucharest, Romania; 2 University of Medicine and Pharmacy Carol Davila, Bucharest, Romania

Background: Rapid identification of septic DIC patients is crucial for the initiation of early treatment. The JAAM DIC score has a good prognostic value for septic patients, but waiting for lab tests results leads to unwanted delays in DIC diagnosis. DIC prediction using rotational thromboelastometry (ROTEM) would allow for a rapid DIC diagnosis with minimal time delays at the bedside.

Aims: To study and to compare the usefulness of different ROTEM parameters for the prediction of DIC in septic patients.

Methods: Critically‐ill septic patients were included in this retrospective observational study in the first 36 hours after the diagnosis of septic shock or sepsis with at least 2 organ dysfunctions considered to be caused by infection. JAAM score was used for DIC diagnosis and whole‐ blood ROTEM (EXTEM and FIBTEM assays) was performed in each patient at study inclusion.

Results: The JAAM DIC score was positive in 44 patients (45.36%) from the total number of 97 patients included in the study. The mortality at ICU discharge was higher in patients diagnosed with DIC using the JAAM score than in patients without DIC (P=0.005). Coagulation initiation and propagation were delayed, clot firmness, maximum elasticity (MCE) and maximum velocity of clot formation were reduced in DIC patients; clot lysis was similar in patients with and without DIC.

TABLE 1 Standard and derived ROTEM parameters in patients with and without DIC (diagnosed using JAAM DIC score) Parameter DIC patients (n=44) Patients without DIC (n=53) P value EXTEM CT (s) 84 (68) 67 (15) 0.01* EXTEM CFT (s) 143 (200) 96 (58) <0.001* EXTEM MCF (mm) 54.4 (±14.2) 62.8 (±11.6) <0.001* EXTEM ML (%) 3 (5.5) 5 (3) 0.256 FIBTEM MCF (mm) 13.7 (±7.5) 22.8 (±11.1) <0.001* MaxVel (mm*100/s) 13.14 (±6.11) 16.55 (±6.99) 0.001* t‐MaxVel (s) 139 (108.5) 82 (76) <0.001* AUC 5150.9 (±1398.4) 6562(±1101.36) <0.001* MCE 123.2 (±75.46) 229 (±120) <0.001*

TABLE 2 Receiver Operating Characteristics Curve for the prediction of DIC using ROTEM parameters Parameter AUC 95% CI P value CT EXTEM 0.719 [0.609; 0.830] 0.001 CFT EXTEM 0.681 [0.555; 0.807] 0.008 MCF EXTEM 0.773 [0.652;0.893] <0.001 MCF FIBTEM 0.709 [0.578; 0.840] 0.007 MaxVel 0.676 [0.537; 0.815] 0.02 AUC 0.807 [0.711;0.903] <0.001 MCE 0.811 [0.716; 0.905] <0.001

ROC curves revealed that MCE and total thrombus formation (reflected by the area under the velocity curve, AUC) had the best values for DIC prediction.

An AUC value lower than 6175 or MCE value less than 158 had a sensitivity of 73% each and specificity of 76% and respectively 80% for DIC prediction in septic patients.

Conclusions: Clot elasticity and derived parameters from the ROTEM velocity curve could serve as useful tools for rapid detection of DIC in septic patients, allowing the timely identification of patients with higher mortality risk which might benefit from additional therapies. Further studies are needed to assess the clinical relevance of these parameters.

PB528: Recombinant A2 Domain of von Willebrand Factor Attenuates Microthrombi Formation, Organ Injuries and D‐dimer Levels in a Porcine Model of Methicillin‐resistant Staphylococcus Aureus (MRSA) Sepsis‐induced Disseminated Intravascular Coagulation (DIC)

M. Cruz 1 ; J. Marini 2 ; B. Guillory 1 ; M. Martnez‐Vargas 1 ; C. Valladolid 3 ; D. Cohen 4 ; F. Lam 2 ; S. Cirlos 1 ; Q. Da 1 ; B. Stoll 2 ; Y. Yuan 2 ; S. Kaplan 2 ; M. Desai 2 ; V. Vijayan 1 ; T. Nguyen 2

1 Baylor College of Medicine and MEDVAMC, Medicine, Houston, United States; 2 Baylor College of Medicine, Pediatrics, Houston, United States; 3 Baylor College of Medicine and MEDVAMC, Molecular Physiology, Houston, United States; 4 Baylor College of Medicine, Pathology, Houston, United States

Background: Disseminated intravascular coagulation (DIC) is manifested in sepsis, causing microvascular thrombosis that leads to multiple organ dysfunction syndrome (MODS). To date there is no drug for sepsis‐induced DIC. Previously, we demonstrated that the A2 protein (recombinant A2 domain of human von Willebrand factor [VWF]) improved mortality, reversed disseminated microvascular thromboses, and lessened systemic inflammation in mice with endotoxemia‐induced DIC.

Aims: To use a porcine sepsis‐induced DIC model to test the effect of the A2 protein, and to characterize the molecular mechanism by which the A2 protein attenuates DIC.

Methods: Our model used a clinically relevant and prevalent human MRSA strain. We treated the pigs with varying concentrations of A2 protein (1‐3.5 mg/kg) and was administrated (via intravenous) at 24 hours after the bacterial intravenous inoculation. Necropsy was performed at 70 hours and tissues were collected. Two A2 mutants were examined for their binding activity for fibrin and vimentin and for their effect on mice with endotoxemia‐induced DIC.

Results: The A2 protein decreased kidney and liver injuries in a dose dependent manner. The A2 protein diminished fibrin deposition in kidney and significantly reduced plasma D‐dimer level in septic pigs as compared to non A2‐treated sick pigs. Finally, the mutants had normal binding activity for vimentin but had a markedly decreased affinity for fibrin. The two mutants failed to mimic the effects of the WT A2 protein: in preventing fibrin‐rich microthrombi formation in mice with endotoxemia‐induced DIC, and in blocking platelet adhesion to fibrin(ogen) under flow conditions.

Conclusions: The results indicate that the A2 protein effectively reduced formation of microvascular thrombi in bacteria‐induced DIC in a pig model for sepsis. Lastly, the results obtained from using the A2 mutants suggest that the interaction of A2 protein with fibrin appears to be the main mechanism by which A2 protein prevents microvascular thrombosis in vivo.

PB529: The Expression of Tissue Factor between Monocyte Subsets: A Role in Sepsis‐associated Coagulopathy?

K.M. Musgrave 1,2 ; M.‐H. Ruchaud‐Sparagano 1 ; J. Scott 1 ; A.J. Rostron 1 ; A.J. Simpson 1,2

1 Newcastle University, Institute of Cellular Medicine, Newcastle upon Tyne, United Kingdom; 2 Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, United Kingdom

Background: Tissue factor (TF) expression by monocytes contributes to sepsis‐associated coagulopathy. Monocytes are divided into 3 subsets according to CD14 and CD16 expression: classical (CD14++CD16), intermediate (CD14++CD16+) and non‐classical (CD14+CD16++).

Aims: To understand how TF expression varies between monocyte subsets.

Methods: Monocytes isolated from healthy volunteers (n=5) using fluorescence‐activated cell sorting were incubated for 24 hours with 1 ng/mL of lipopolysaccharide (LPS). A paired t test was used to compare TF expression with and without LPS. Monocytes were also collected from a human endotoxaemia model (approved by a medical ethics committee and compliant with the Declaration of Helsinki). Participants (n=5), after giving informed consent, were injected with intravenous LPS (2 ng/kg); blood was taken at baseline, 90 minutes, 4, 6, 10 and 24 hours, and 7 days. TF expression was compared with Friedmann ANOVA. Flow cytometry was used to measure the percentage of TF expression and median fluorescent intensity (MFI).

Results: Healthy monocyte subsets differ in TF expression. Intermediates expressed most TF, but showed no change in expression with LPS. Both the classical and non‐classical increased their TF expression with LPS (classical: median 6% cells TF‐positive without LPS and 28% with, P<0.05; non‐classical: median 2% without and 12% with, P<0.05). MFI showed a similar pattern.

The human endotoxaemia model displays profound monocytopenia at 90 minutes followed by restoration of classical at 10 hours and expansion in intermediate and non‐classical at 24 hours (P<0.05). There is a trend for classical monocytes to decrease their TF MFI post LPS, whereas there is an increase in intermediate (at 4 hours) and non‐classical (at 6 hours) TF. The percentage of TF positive cells was unchanged.

Conclusions: In conclusion, not only does TF expression vary between the monocyte subsets, but the subsets differ in their response to LPS. This adds to our understanding of coagulopathy during sepsis.

PB530: Added Value of the DIC Score and of D‐dimer to Predict Outcome after Successfully Resuscitated Out‐of‐Hospital Cardiac Arrest

N. Buchtele 1 ; C. Schörgenhofer 1 ; A. Schober 2 ; B. Jilma 1 ; M. Schwameis 2

1 Medical University of Vienna, Department of Clinical Pharmacology, Vienna, Austria; 2 Medical University of Vienna, Department of Emergency Medicine, Vienna, Austria

Background: The occurrence of overt disseminated intravascular coagulation (DIC) is associated with poor outcomes and high mortality risk in various medical conditions. So far, the prevalence of DIC in European patients with out‐of‐hospital cardiac arrest (OHCA) has not been assessed.

Aims: We aimed to determine the prevalence of overt DIC, its association with outcome, and the predictive value of d‐dimer in a European collective of OHCA patients. We hypothesized that in a European cohort of resuscitated OHCA patients the prevalence of DIC is substantially lower than a published 30% DIC‐rate in Korean OHCA patients. Furthermore the determination of D‐dimer levels at admission, but not the DIC score, could improve mortality prediction above traditional predictors.

Methods: Data were extracted from a prospective registry comprising OHCA patients admitted between 2006‐2015, who achieved return of spontaneous circulation and had parameters for DIC score calculation available. The primary outcome was the prevalence of overt DIC at admission. Secondary outcomes included the association of overt DIC with 30‐day mortality and the incremental contribution of the DIC score and D‐dimer levels to 30‐day mortality prediction using logistic regression. Three stepwise models were evaluated by receiver‐operating‐characteristic analysis.

Results: Out of 1179 patients 388 were included in the study. Overt DIC was present in 8% of patients and associated with substantial 30‐day mortality (83% vs. 39%). The AUC for model 1, including traditional mortality predictors, was 0.83. While the inclusion of the DIC Score had no effect on mortality prediction (model 2, AUC 0.83). The inclusion of D‐dimer levels significantly improved prognostication above traditional predictors (model 3, AUC 0.89).

Conclusions: Overt DIC was rare in a European cohort of OHCA patients. D‐dimer levels improved 30‐day mortality prediction and provides added value to assess early mortality risk after OHCA.

PB531: Neutrophil Fluorescence: A New Indicator of Cell Activation during Septic Shock‐induced Disseminated Intravascular Coagulation that Could Reflect NETosis

L. Stiel 1,2 ; X. Delabranche 1,3 ; R. Clere‐Jehl 1,3 ; C. Mayeur‐Rousse 4 ; F. Zobairi 1 ; F. Toti 1 ; F. Meziani 1,3 ; L. Mauvieux 4,5 ; J. Helms 3,6

1 Strasbourg University, UMR 1260, French National Institute of Health and Medical Research, Regenerative Nanomedicine, Strasbourg, France; 2 Strasbourg University Hospital, Réanimation Médicale ‐ Hôpital de Hautepierre, Strasbourg, France; 3 Strasbourg University Hospital, Réanimation Médicale ‐ Nouvel Hôpital Civil, Strasbourg, France; 4 Strasbourg University Hospital, Laboratoire d'Hématologie et Hémostase, Strasbourg, France; 5 Strasbourg University, EA 3430, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Strasbourg, France; 6 Strasbourg University, EFS Grand Est, BPPS UMR‐S 949, French National Institute of Health and Medical Research, Strasbourg, France

Background: Septic shock (SS) is the most severe form of host response to infection. It is characterized by vascular cell and coagulation activation eventually leading to disseminated intravascular coagulation (DIC) that increases mortality. Neutrophils thus stand at the interface between hemostasis and immunity, notably through neutrophil extracellular traps (NETs) release.

Aims: This study was designed to assess neutrophil activation for the diagnosis of SS‐induced DIC and to investigate whether NETosis is involved in SS‐induced DIC.

Methods: One hundred adults referred for SS were prospectively enrolled at admission. DIC was diagnosed according to JAAM 2006 score. Blood cell count was performed on Sysmex ™ XN20 flow cytometer, measuring neutrophil‐side‐fluorescence‐light (NEUT‐SFL) by binding of a fluorescent dye to nucleic acids, reflecting chromatin condensation. Leucocyte and neutrophil‐derived microvesicles (MVs) were measured by prothrombinase test. In vivo NETosis was assessed using cytology on blood smears and DNA‐associated myeloperoxidase activity.

Results: Along the hundred patients included, thirty‐five met DIC criteria. A value of NEUT‐SFL above 57.3 arbitrary units had a sensitivity of 90.9% and a specificity of 80.6% for DIC diagnosis. NEUT‐SFL was correlated to the shedding of leucocyte‐ and neutrophil‐derived MVs. In a sample of twenty patients (10 without and 10 with DIC), DIC was correlated with:(i)neutrophil cytological changes,(ii)NEUT‐SFL,(iii)DNA‐associated MPO activity,(iv)neutrophil‐derived MVs.

FIGURE 1 Receiver‐operating characteristic (ROC) curve of NEUT‐SFL in septic shock‐induced DIC

FIGURE 2 Septic shock‐induced DIC is associated with DNA‐associated MPO (NETs) in plasma

Conclusions: SS‐induced DIC is characterized by a significant neutrophil activation, with increased NEUT‐SFL and is correlated to NETosis. NEUT‐SFL reflects the degree of chromatin condensation and could be a reliable indicator of NETosis. Assessment of NEUT‐SFL can be routinely available on blood cell count and may represent an ndicator of neutrophil activation during SS‐induced DIC. NEUT‐SFL could thus be an accurate marker of neutrophil activation and help diagnose DIC during SS.

PB532: Evaluation of the Effectiveness of AT III Concentrate in Acute DIC‐Syndrome

N. Vorobyeva ; A. Vorobyeva

Nord State Medical University, Haemostasiology, Arckhangelsk, Russian Federation

Background: DIC‐syndrome is a monitoring of the activity of AT III. It is important to note that in 2016 Japanese authors included a mandatory laboratory evaluation of the functional activity of AT III in the updated scale of diagnosis of DIC syndrome.

Aims: The aim is to analyze the effectiveness of the use of AT III concentrate in patients with acute DIC syndrome

Methods: A randomized study of the comparative efficacy of treatment of DIC syndrome with AT III concentrate, freshly frozen plasma was performed. The criteria for inclusion in the study are DIC JAAM. 28 patients with acute DIC syndrome and low activity of AT III were included. When using FFP, its transfusion was performed at a dose of 10‐17 ml per kg of body weight no later than 10 minutes after thawing. In the AT III concentrate group, the drug was administered with an infusomat at a rate of 1000 IU / h.

Results: The correlation between the activity of AT III and the level of creatinine (r=−0.6, P=0.06), the number of balls on the SOFA (r=−0.48, P=0.16), the level of urea (mol/l) (r=−0.56, P=0.05) the level of FM (r=0.52, P=0.12), fibrinogen level (r=0.56, P=0.058).The study showed that in patients with DIC syndrome, who had an AT III level of 52.6±9.7% of the norm, the introduction of AT III concentrates before the restoration of the normal level of the inhibitor was significantly more effective than the treatment with fresh‐frozen plasma. The higher efficiency of the administration of small doses of the drug AT III (500 units) is shown several times a day before the single administration of the whole daily dose (1,500 U). In the presence of DIC syndrome, concentrate AT III significantly reduced the lethality by 14.6% (P=0.02).

Conclusions: Our study demonstrated the effectiveness of the use of AT concentrate in DIC syndrome in real clinical practice.

PB533: von Willebrand Factor Interactions with Platelets and Leukocytes is Critical for Obesity‐mediated Venous Thrombosis

A. Michels 1 ; L.L. Swystun 1 ; C. Dwyer 1 ; K. Nesbitt 1 ; C. Kawecki 2 ; P. Lenting 2 ; D. Lillicrap 1

1 Queen's University, Kingston, Canada; 2 Inserm U 1176 ‐ Hopital Bicetre, Paris, France

Background: Diet‐induced obese (DIO) mice have elevated VWF:Ag and increased VWF‐dependent venous thrombogenicity. VWF localizes platelets, NETs and tissue factor (TF)‐positive leukocytes to the thrombus. The anti‐murine VWF A1 domain nanobody (KB‐VWF‐006bi) has been shown to interfere with VWF‐platelet and leukocyte interactions in vivo .

Aims: To characterize mechanisms that regulate VWF:Ag and VWF‐mediated thrombogenicity in a mouse model of DIO deep vein thrombosis (DVT), and to assess the influence of KB‐VWF‐006bi on DVT formation in vivo .

Methods: C57BL/6 wild‐type and VWF KO mice were either fed a high‐fat 60% or 10% standard fat diet for 2 weeks. The half‐life of murine plasma‐derived (pdm) VWF was assessed in DIO and control mice. DVT was induced by IVC ligation and thrombi were weighed and longitudinal sections were analyzed by IHC 24 hour‐post stenosis. DIO mice were infused with KB‐VWF‐006bi or a control nanobody (KB‐VWF‐004bi) (4 mg/kg) immediately prior to DVT induction.

Results: Circulating VWF: Ag (1.37‐fold, P<0.0001) and VWFpp (1.24‐fold, P=0.002) but not angiopoietin‐2 (P=0.92) levels were significantly elevated in DIO mice, while VWFpp/VWF:Ag was not altered (P=0.66). DIO VWF KO mice exhibited slower clearance of pdmVWF compared to controls (5.5 vs. 7.1 hours, P=0.0025). Quantitative IHC of thrombi from DIO and control mice showed comparable TF levels (P=0.46) which did not correlate with thrombus weight (r 2 =0.02, P=0.66) or leukocyte content (r 2 =0.11, P=0.24). Citrullinated histone H3 staining (a hallmark of NET release) was not elevated in thrombi from DIO mice (P=0.38), but significantly correlated with total thrombus weight (r 2 =0.37, P=0.02) and leukocyte content (r 2 =0.56, P=0.002). Thrombus size was reduced by 66% in DIO mice that received KB‐VWF‐006bi compared to controls (P=0.006).

Conclusions: Elevated VWF levels in DIO mice appear to be due to both increased synthesis/secretion and impaired clearance. Targeting the VWF A1 domain is protective against obesity‐mediated thrombogenicity.

PB534: Iron Deficiency Increases Platelet Procoagulant Capacity in a Ferric Chloride Model of Arterial Thrombosis

K. Jimenez 1 ; G. Scharbert 2 ; A. Leitner 1 ; P. Schwabl 1 ; F. Leitner 1 ; R. Evstatiev 1 ; V. Khare 1 ; C. Gasche 1

1 Medical University of Vienna, Internal Medicine III, Division of Gastroenterology and Hepatology, Vienna, Austria; 2 Medical University of Vienna, Department of Anaesthesia, General Intensive Care and Pain Management, Vienna, Austria

Background: Iron deficiency (ID) is globally prevalent and is associated with secondary thrombocytosis. Resultant alterations in thrombotic tendency have not been extensively studied.

Aims: To evaluate thrombotic tendency in a rat model of iron deficiency.

Methods: Sprague‐Dawley rats were fed control (Con, 196 mg iron/kg) or iron deficient chow (Def, < 10 mg iron/kg) for 7 weeks and additionally received 3 doses of either 20 mg ferric carboxymaltose/kg BW i.p. (Def+Fe) or placebo (Pl). Platelet function was evaluated using the PFA‐100, impedance aggregometry (IA), and thromboelastography (TEG). Thrombosis was induced in the right common carotid via application of 10% FeCl3 for 5 minutes. Time to occlusion was assessed with a distally placed ultrasonic‐flowprobe.

Results: Def+Pl rats had microcytic anemia and thrombocytosis, both alleviated by iron therapy in Def+Fe (Table 1). Arterial thrombus dimensions were largest in Def+Pl (Table 1, Figure 1). Occlusion times were similar between groups, with a tendency to have a greater area under the flow curve in Def+Pl rats. PFA‐100 did not occlude in 3 out of 5 Def+Pl animals, and in those that occluded, PFA‐100 was generally longer than in Con+Pl rats. Aggregation to ADP was similar between groups. There was a trend for an increase in aggregation to collagen in Def+Pl animals. Time to initial fibrin formation (R) and kinetics (K) were shortest in Def+Pl, while clot strength (MA) was highest in Def+Pl.

TABLE 1 Animal blood counts and occlusion parameters Parameter Con+Pl (n=9) Def+Pl (n=11) Def+Fe (n=9) Hemoglobin (g/dl) 16.8 (IQR: 16.3‐17) 8.3*# (IQR: 6.9‐9.8) 15.1*# (IQR: 15.0‐15.3) Mean corpuscular volume (fL) 57.6 (IQR: 56.9‐59.1) 35.6*# (IQR: 29.0‐37.4) 53.6*# (IQR: 51.0‐54.0) Platelet (x10^3/µl) 909 (IQR: 835‐1053) 1380*# (IQR: 1261‐1605) 973# (IQR: 937‐1054) Occlusion time (s) 810 (IQR: 710‐890) 750 (IQR: 645‐895) 880 (IQR: 750‐945) Area under the flow curve 5886.8 ± 3078 7019.5 ± 2267.7 5478.32 ± 2143 P<0.05 versus Con+Pl (*) or versus Def+Pl (#)

FIGURE 1 Thrombus size and Platelet function tests

Conclusions: ID leads to thrombocytosis with increased procoagulant capacity. In this model of ferric chloride‐induced arterial thrombosis, ID is associated with increased thrombus size. This is likely attributable to increased platelet aggregability and contribution to clot formation, despite reduced adhesion. Iron replacement therapy reverses this prothrombotic state.

PB535: Dabigatran Enhances the Antibiotic Efficacy and Reduces Sepsis Induced Thrombosis in Severe Staphylococcus aureus Aortic Endocarditis

C.J. Lerche 1,2 ; L.J. Christophersen 1 ; P.Ø. Jensen 1 ; J.P. Gøtze 3 ; P.R. Nielsen 4 ; K. Thomsen 1 ; N. Høiby 1,2 ; H. Bundgaard 5 ; C. Moser 1

1 Copenhagen University Hospital, Rigshospitalet, Department of Clinical Microbiology, Copenhagen, Denmark; 2 Copenhagen University, Institute of Immunology and Microbiology, Copenhagen, Denmark; 3 Copenhagen University Hospital, Rigshospitalet, Department of Clinical Biochemistry, Copenhagen, Denmark; 4 Zealand University Hospital Roskilde, Department of Pathology, Roskilde, Denmark; 5 Copenhagen University Hospital, Rigshospitalet, Department of Cardiology, Copenhagen, Denmark

Background: Invasion of Staphylococcus aureus on cardiac valves facilitates fibrin formation, platelet aggregation and formation of septic thrombosis, which are cornerstones in the pathogenesis of infective endocarditis (IE). S. aureus triggers host defense cells, coagulation system and adhesion of platelets facilitating thrombin generation catalyzing fibrin clot formation on valves. The thrombin inhibitor dabigatran might limit some of above adverse effects.

Aims: To investigate the effect of adjuvant dabigatran treatment in experimental S. aureus IE.

Methods: In male Wistar rats (230 g) high grade S. aureus aortic valve IE was established. Infected rats treated with gentamicin (20 mg/kg/day s.c.) were randomized into two groups, (1) receiving dabigatran etexilate (10 mg/kg i.p. BID) in approximation to human dosing or (2) saline. Infected rats where treated in 2 days and evaluated day 3 post‐infection by blood cultures and quantitative bacteriology of valve vegetations, myocardium, spleen and kidneys. Planimetric assessment of valve vegetation size and inflammatory cell markers was measured.

Results: The frequency of negative blood cultures was found to be highest in the dabigatran group (P<0.02). Reduced bacterial load in the aortic valve vegetations and in the spleen was observed in the dabigatran treated group (n=12) compared to the saline treated group (n=12), (P<0.4, P<0.01). Analysis of valve vegetation size showed significantly reduced vegetation formation in the dabigatran treated group compared to vehicle (mean 1.6 mm 2 ±1.25 vs. 4.0 mm 2 ±2.24, P<0.004) as well as reduced inflammation and cell adhesion markers TIMP‐1, L‐Selectin (P<0.03) and ICAM‐1 (P<0.06).

Conclusions: Dabigatran reduces bacterial load, valve vegetations and inflammatory markers in experimental S. aureus IE. Furthermore, dabigatran reduced the hematogenous dissemination of S. aureus to the spleen. Our results indicate adjuvant dabigatran treatment might be a beneficial strategy to reduce valve inflammation of this severe disease.

PB536: Development of an Easily Reproducible and Inexpensive Experimental Rabbit Carotid Model of Atherothrombotic Occlusion with Morphological Similarities to the Human Disease: Monitored by Color Doppler Ultrasonography and Histopathology

H. Mehrad 1,2 ; L. Zeinalizad 3 ; A. Arshadi 3 ; S. Sarabi 3 ; Z. Pishezari 3 ; M. Haji Sagati 3 ; M. Farhoudi 2

1 Islamic Azad University, Tabriz Branch‐ Faculty of Basic Sciences, Department of Physics, Tabriz, Iran, Islamic Republic of; 2 Tabriz University of Medical Sciences, Neurosciences Research Center, Tabriz, Iran, Islamic Republic of; 3 Islamic Azad University, Tabriz Branch‐ Faculty of Basic Sciences, Department of Bioengineering, Tabriz, Iran, Islamic Republic of

Background: Direct investigation of atherothrombosis in human carotid arteries is by nature difficult to do. As a result, animal models offer an alternative that can replicate the physiopathology aspects of atherothrombotic occlusion while allowing for controllable variables and statistical data in a short period of time. Therefore, an appropriate animal model could prove critical to the research and development of new diagnostic and therapeutic modalities.

Aims: The aim of this study was to generate an easily reproducible and inexpensive experimental rabbit carotid model of atherothrombosis with morphological similarities to the human disease.

Methods: Briefly, New Zealand white rabbits were submitted to right common carotid artery atherothrombotic occlusion by primary balloon injury followed 1.5% cholesterol‐ rich diet injury for 12 weeks and finally perivascular severe cold injury using liquid nitrogen.

Results: Histopathology results showed the formation of advanced atherosclerosis with lipid‐laden cells, atheroma, neovessel ‐ rich plaque and severe stenosis (>70%) in all of the rabbits’ arteries after 12 weeks. Results from color Doppler ultrasonography at the stenotic region showed a significant increase in the mean value for blood mean velocity, wall mean thickness, percentage of luminal cross‐ sectional area of stenosis and formation of intraplaque neovessels and significant reduction in the mean value for blood volume flow in the atherothrombotic group compared with the control group (P<0.05). Four weeks after perivascular severe cold injury results from histopathology and color Doppler ultrasonography showed the atherothrombotic occlusion in all of the rabbits’ arteries.

Conclusions: In conclusion, we successfully produced atherothrombotic occlusion on advanced atherosclerosis in the rabbit carotid artery that is similar to the condition seen in patients. This condition in rabbits can be properly assessed by B‐mode ultrasound image processing.

PB537: Effect of Cooling Environment in the State of Haemostasis System in Rats

N. Lycheva

St. Petersburg Institute of Pharmacy, St. Petersburg, Russian Federation

Background: It is known that in the course of the cooling environment,various pathological states are formed.

Aims: To study hemostatic system in pre‐reactive period of a cold injury that occurred during air and water‐immersion cooling.

Methods: Single air hypothermia was modulated by placing animals into a cooling chamber at a temperature −25°C. The animals stayed in a chamber until rectal temperature was 30°C. Water‐immersion cooling was modulated by placing animals into water at a temperature 5°C and air temperature 7°C. Animals stayed in the water until rectal temperature was 27…30°C. We assessed the state of vascular‐platelet and plasma hemostasis as well as physiological state of anticoagulant and fibrinolytic systems.

Results: We stated that during the immersion in water cooling, experimental animals develop thrombocytosis and activate their aggregation function. Recorded thrombinemia was recorded at the final stage, that is, a significant increase in the concentration of soluble monomeric fibrin complexes and a decrease in the time of their self‐assembly. In addition, inhibition of fibrinolytic plasma activity was observed against the background of a decrease in the concentration of antithrombin III. In this case, one hypothermia of the air also caused significant changes in the hemostatic system. A decrease in platelet aggregation activity, hypocoagulation, and inhibition of fibrinolytic activity in blood plasma were observed.

Conclusions: Thus, rectal temperature +30°C during immersion cooling was accompanied by the expressed activation of coagulation and registration of the state of thrombotic readiness. During air‐cooling when rectal temperature reached +30°C, secondary hypocoagulation shifts were recorded.

The reported study was funded by RFBR, according to the research project № 16‐34‐60054 mol_a_dk.

PB538: Comparative Characteristics of the Hemostasis System State in Rats during Hypothermic and Early Reactive Periods of General Cold Trauma

N. Lycheva

St. Petersburg Institute of Pharmacy, St. Petersburg, Russian Federation

Background: Cold trauma is an important social problem.

Aims: To study the state of hemostatic system in rats during hypothermic and post‐hypothermic periods.

Methods: Male Wistar rats (53 individuals) were used in the study. The animals of the experimental groups underwent single immersion cooling in water at a temperature of 5°C until profound hypothermia was reached. The control group of the animals was placed in water at a temperature of 30°C. In the animals of the first group, blood was taken immediately after reaching profound hypothermia. In the second group blood was taken 24 hours after cooling stopped.

Results: Comparative analysis of the results showed that immediately after the end of a single cold exposure, there was a significant increase in platelet aggregation activity, appearance of thrombinemia markers in the bloodstream and inhibition of fibrinolytic system activity. 24 hours after the experimental exposure, these parameters returned to the initial values. When assessing the activity of external and internal ways of coagulation immediately after the termination of cooling, the development of hypocoagulation was established, both with routine tests and from thromboelastogram data. At the end of a 24‐hour period, hypocoagulation, recorded immediately after reaching the rectal temperature of the sought‐for value, was preserved. Thus, after the end of a 24‐hour period after cold exposure termination, most of the parameters of the hemostatic system that had deviated from the normal level returned to it immediately after the end of the experiment. The delayed effect of hypothermia in this cold exposure regime manifested itself only by reduced coagulation activity at the initial steps of coagulation.

Conclusions: Signs of abnormal hemostasiological blood properties, recorded immediately after the cooling termination, disappear within 24 hours and only hypocoagulation remains in the bloodstream. The reported study was funded by RFBR, according to the research project № 16‐34‐60054 mol_a_dk.

PB539: A Comparative Characteristic of the State of the Hemostasis System in Rats under the Action of a Single Immersion Hypothermia Immediately and a Day after they Reached an Super Deep Degree of Hypothermia

N. Lycheva

St. Petersburg Institute of Pharmacy, St. Petersburg, Russian Federation

Background: Hypothermia is the cause of cold organ damage.

Aims: To study the state of hemostatic system in rats during hypothermic and post‐hypothermic periods.

Methods: Immersion hypothermia was modeled by placing animals in individual cells in water at a temperature of 5°C and air of 7°C. The cooling was stopped when the rectal temperature was 10…16°C, this is called the super deep degree of hypothermia. The cooling lasted 55±5 minutes. Control ‐ the blood of 20 animals,which were placed in water at a temperature of 30°C and air 22°C. All animals were divided into 4 groups. In group 1(control), the blood was taken immediately after extraction from the water. In the animals of the 2nd group (experimental), immediately after reaching the super‐deep degree of hypothermia. In the third group‐the blood was collected 24 hours after extraction from the water (control).In the 4th group,blood was collected 24 hours after the cooling stopped (experimental).

Results: Comparative analysis of the results showed that immediately after the end of a single cold exposure,an increase in platelet aggregation activity and development of hypocoagulation shifts,a decrease in antithrombin III concentration against a background of increased fibrinogen concentration and inhibition of fibrinolytic system activity were observed. This is a distress reaction in response to a single action of the stimulus. One day after cooling stop the hypocoagulation shifts were maintained in the bloodstream and the concentration of fibrinogen increased and thrombinemia markers were recorded. Thus, 1 day after the general supercooling,the risk of developing a state of thrombotic alert remained and the state of distress increased.

Conclusions: Signs of impaired hemostasiological properties of the blood,recorded immediately after the cessation of cooling,are aggravated in 24 hours by the appearance of thrombinemia markers in the bloodstream. The reported study was funded by RFBR, according to the research project № 16‐34‐60054 mol_a_dk.

PB540: Dynamics of Indicators of the Hemostasis System in Rats as the Core Temperature Decreases

N. Lycheva

St. Petersburg Institute of Pharmacy, St. Petersburg, Russian Federation

Background: Hypothermia has a general impact on the organism, being both a natural environmental factor and an artificial factor used in medical practice. Exposure to low temperature can be a disturbing factor, inducing destructive changes of various degrees in tissues. Concerning medical practice, hypothermia is obligatory during open heart surgeryand is an important part of combined therapy of certain medical emergencies, including traumatic brain injuries, ischemic and hemorrhagic brain lesions.

Aims: To study the dynamics of parameters of the hemostasis system with a decrease in core temperature.

Methods: Studies were performed on 76 male rats of the Wistar line. Immersion hypothermia was modeled by placing animals in a container with water to a depth of 4.5 cm at a water temperature of +5°C, air +7°C. Animals were divided into 4 groups: mild degree of hypothermia (rectal temperature +32°C…+35°C); Moderate degree (rectal temperature +27°C…+30°C); Deep degree (rectal temperature +20°C…+23°C); Super deep degree (rectal temperature <20°C). Control animals (n=20) were placed in water at a temperature of +30°C for a time corresponding to the time of the test group.

Results: A mild degree of hypothermia was accompanied by hypoaggregation, hypocoagulation, and activation of fibrinolysis. Moderate degree of hypothermia was accompanied by the development of thrombinemia and inhibition of fibrinolytic activity of blood plasma. Further cooling was accompanied by a consistent inhibition of fibrinolytic system activity against the background of a high concentration of soluble fibrin‐monomer complexes and an increasing amount of fibrinogen.

Conclusions: Deterioration of the hemostatic system is observed as the nucleus decreases.

The reported study was funded by RFBR, according to the research project № 16‐34‐60054 mol_a_dk.

PB541: A Highly Efficient and Adhensive Antibacterial Dextran Derivative for Rapid Hemostasis with Wound Healing

C. Liu 1 ; X. Liu 2 ; G. Sun 2 ; W. Qiao 1

1 Dalian University of Technology, State Key Laboratory of Fine Chemicals, School of Chemical Engineering, Dalian, China; 2 Dalian Medical University, The First Affiliated Hospital, Dalian, China

Background: High‐efficient hemostasis becomes increasingly crucial in civilian and military trauma, however the available hemostats still exist various drawbacks and side‐effects.

Aims: Herein, aldehyde dextran (PDA) prepared by lyophilization was developed for hemorrhage control.

Methods: Structure of PDA was confirmed by 1 H NMR, FT‐IR, SEM, absorption and adhension tests. Biocompatibility of PDA was explored by skin irritation in rabbit, hemolysis, cytotoxity and degradation tests. Moreover, hemostasis property of PDA was evaluated by coagulation tests in rabbit ear vein incision, liver injury and femoral injury. Meanwhile, PDA was applied as wound‐healing dressing to repair rabbit full‐thickness skin defects and antibacterial to act on Staphylococcus aureus ( S. aureus ) and Escherichia coli ( E. coli ) by the halo method. Finally, hemostasis mechanisms of PDA were conducted by cell aggregation and thrombelastogram tests.

Results: PDA with pore size of ˜30‐50 μm could not only absorb blood quickly (62.6 g/g), but also possess higher tissue adhesion (˜100 kPa). Importantly, PDA were verified to be an efficient hemostat in rabbit models. Hemostasis of ear vein injury was attained in 54±6.0 seconds, while that of commercial product (CP) and blank controls was 199±19.8 seconds and 249±4.2 seconds. Significantly, 1 g PDA could promote rapid efficient hemostasis in femoral injury, for which could tightly adhere and seal to the wound site. Besides, it revealed PDA with almost no irritant and good histocompatibility helped heal wounds. Antibacterial activities against E. coli (˜7.50 cm) and S. aureus (˜7.12 cm) also performed well compared with CP (˜0 cm). Essentially, hemostasis mechanisms might be PDA could rapidly aggregate erythrocytes and platelets to activate coagulation system. Notably, PDA with low hemolysis exhibited nearly no cytotoxicity and could completely dissolve just within 9 hours.

Conclusions: Overall, the adhensive, antibiotic and conglutinant PDA would be a promising quick‐hemostatic dressing for uncontrollable hemorrhage.

FIGURE 1 Hemostasis mechanism of PDA

PB542: Rapid Detection and Classification of Direct‐acting Oral Anticoagulants (DOACs) with a Novel Point‐of‐Care Thromboelastography DOAC Cartridge and Algorithm

J.D. Dias 1 ; C. Lopez‐Espina 2 ; J. Ippolito 3 ; H. Hsiao 3 ; F. Zaman 2 ; B. Mathew 2 ; A. Muresan 2 ; M. Walsh 4 ; B. Thurston 5 ; R. Artang 6 ; K. Bliden 7 ; P. Gurbel 8 ; J. Hartmann 3 ; H.E. Achneck 3

1 Haemonetics Corporation, Signy, Switzerland; 2 Haemonetics Corporation, Rosemont, United States; 3 Haemonetics Corporation, Braintree, United States; 4 Memorial Hospital of South Bend, Emergency Medicine, South Bend, United States; 5 Spartanburg Regional Medical Center, General Surgery, Trauma, Spartanburg, United States; 6 Essentia Health St. Mary's‐Heart & Vascular Center, Cardiology, Duluth, United States; 7 Inova Heart and Vascular Institute, Inova Center for Thrombosis Research and Drug Development, Falls Church, United States; 8 Inova Heart and Vascular Institute, Interventional Cardiology and Cardiovascular Medicine Research, Falls Church, United States

Background: The increasing number of patients receiving DOACs has triggered the need for a test that can rapidly detect the presence of a DOAC and classify the type of a DOAC a patient may be on, especially in emergency situations. A novel thromboelastography (TEG) test was created, consisting of a DOAC cartridge and algorithm that work with the TEG analyzer to both detect and distinguish between the two types of DOACs, Anti‐Factor Xa (AFXa) and Direct Thrombin Inhibitor (DTI).

Aims: To establish normal reference ranges for the AFXa and DTI assay parameters and evaluate the sensitivity and specificity of the DOAC cartridge and its algorithm.

Methods: Following IRB approval, blood was collected from consented subjects at 5 US sites in an open label, multi‐center, observational clinical trial with the following cohorts: 160 healthy donors for reference range estimation (RR); 190 subjects taking DOACs (On‐DOAC, 115 subjects on AFXa and 75 on DTI for at least 7 days) for sensitivity assessment and 24 subjects not on DOACs (Non‐DOAC) for assessment of false positives. Normal RRs were estimated using the nonparametric percentile estimation method with imposing data quality and outlier screening. Robustness was evaluated by means of sensitivity analysis.

Results: The sensitivity of detecting the presence of a DOAC in subjects’ blood and correctly classifying as either AFXa or DTI was at least 98% (113/115 subjects for AFX and 75/75 subjects for DTI). We observed 100% specificity. None of the 24 Non‐DOAC control subjects were incorrectly classified. The mean time to results was 3.5 minutes.

Conclusions: The new DOAC cartridge and algorithm were observed to function as a highly sensitive and specific rapid point‐of‐care test that could determine within minutes if a patient is on a DOAC and what type of DOAC.

PB543: Normal to High Anti‐Xa Levels in High Body Weight Patients Using Apixaban and Rivaroxaban

Z. Mahir 1 ; T. Dowling 1 ; V. Collings 1 ; G.W. Moore 2 ; M.P. Crowley 1 ; K. Breen 1 ; B.J. Hunt 1 ; A.T. Cohen 1

1 Guy's and St Thomas’ NHS Foundation Trust, Haemostasis & Thrombosis, London, United Kingdom; 2 Guys and St. Thomas’ NHS Foundation Trust, Haemostasis and Thrombosis, Viapath Analytics, London, United Kingdom

Background: There is limited clinical data available for using direct oral anticoagulants (DOACs) in patients with high body weight, and the available pharmacokinetics/pharmacodynamics evidence suggests that decreased drug exposures may occur. 1 The International Society of Thrombosis and Haemostasis guidelines recommends using anti‐Xa monitoring in patients over 120 kg. 1

Aims: To identify whether high body weight reduces the drug exposure to apixaban and rivaroxaban using anti‐ Xa levels.

Methods: (1) Patients over 120 kg consented to measurement of an anti‐Xa trough level taken (prior to their next dose of DOAC) if on either apixaban or rivaroxaban

(2) Rivaroxaban and apixaban levels were quantified with a chromogenic anti‐Xa assay, STA ® ‐Liquid Anti‐Xa assay (Stago UK, Reading, UK) on a Sysmex CS2000i automated coagulation analyser (Sysmex UK, Milton Keynes, UK). Drug‐specific standards, STA ® ‐Rivaroxaban Calibrator and STA ® ‐Apixaban Calibrator (Stago UK) were employed to calibrate the assays.

(3) Data was limited to include patients seen in 2017.

Results: Trough level data was available in 30 patients.

TABLE 1 Baseline characteristics and findings Age, mean±SD (range) years 61±12 (23‐78) Sex 25 male 5 female Weight, mean±SD (range) kg 137±22 (120‐230) Indication 26 stroke prevention in atrial fibrillation 3 secondary prevention of venous thromboembolism (VTE) 1 VTE treatment DOAC type 19 rivaroxaban 11 apixaban Anti‐Xa trough level Apixaban ( 23‐109 mg/L) Rivaroxaban (19‐60 ng/mL) 22 within therapeutic range 8 above therapeutic range

In addition, there were no new thrombotic events found in any of the groups.

Conclusions: All patients were found to have a normal or supranormal trough level. We found no evidence that high body weight reduces drug exposure in any patients.

References: 1. Use of the direct oral anticoagulants in obese patients: guidance from the SSC of the ISTH K. Martin, J Beyer‐Westendorf B.L. Davidson, M.V Huisman, P.M Sandset and S. Moll Journal of Thrombosis and Haemostasis, 14: 1308‐1313

PB544: Global Coagulation Assay Changes in Patients with Chronic Renal Impairment

H.Y. Lim 1,2 ; M. Munnings 1 ; D. Barit 3 ; H. Nansurkar 2 ; P. Ho 1

1 Northern Health, Haematology, Epping, Australia; 2 Australian Centre for Blood Diseases, Melbourne, Australia; 3 Northern Health, Nephrology, Epping, Australia

Background: Chronic kidney disease (CKD), and haemodialysis in particular, is thought to be a hypercoagulable state, which may contribute to increased cardiovascular risks. However, current available coagulation studies only measures time to start of clot formation and do not accurately assess an individual's thrombotic risks.

Aims: To evaluate the changes of global coagulation assays in CKD patients on haemodialysis.

Methods: Fasting blood samples were collected prior to haemodialysis for full blood evaluation, coagulation studies and experimental testing. Thromboelastography using citrated whole blood was performed with TEG 5000 and platelet‐poor plasma were obtained for assessment of thrombin generation with calibrated automated thrombogram (CAT) and fibrin‐aggregation with overall haemostatic potential (OHP) assay.

Results: Twenty‐one haemodialysis patients were recruited and the results were compared to age‐matched normal controls previously collected. Haemodialysis patients had lower haemoglobin and platelet count with increased fibrinogen, VWF antigen and factor VIII level (P<0.01). They had more prothrombotic TEG profile when compared to normal controls, with increased maximal amplitude (71.1 vs. 60.0 mm, P<0.001) and reduced clot lysis (0.0% vs. 0.5%, P=0.001) although there was no significant difference in thrombin generation. D‐dimer was markedly increased in haemodialysis patients independent of age with only 4 patients found to be within normal range (770 vs. 189 ng/mL, P<0.001) but this did not correlate with fibrin generation parameters.

Conclusions: Haemodialysis patients appear to be more prothrombotic, as characterised by increased fibrinogen, VWF antigen and factor VIII levels, as well as TEG parameters. D‐dimer was markedly increased, which questions the clinical usefulness of D‐dimer in predicting acute venous thromboembolism in these patients. The lack of correlation with the fibrin generation assay may signify reduced renal clearance of D‐dimer.

PB545: Assessment of the Agreement between the International Normalised Ratio (INR) Values Obtained by Applying Two Different Reagent/Instrument Combinations

A.N. Blanco ; L. Remotti; M.F. Ingratti; S.H. Grosso; M.L. Romero; M.G. Agazzoni; M.P. Vera Morandini; A. Sánchez‐Luceros

Academia Nacional de Medicina, IIHEMA, Hemostasia y Trombosis, CABA, Argentina

Background: The INR is assessed to monitor patients on antivitamin K treatment (AVK).

Aims: The agreement between INR values obtained with 2 different thromboplastin/instrument combinations was evaluated.

Methods: INR was determined in 21 normal controls and 245 patients on AVK (acenocoumarol), using either NeoplastineR (NeoR) (Diagnostica STAGO) or ThromborelS (ThrS) (Siemens Health Care Diagnostics) on STA‐Compact (Diagnostica STAGO). Reagents were either pre‐calibrated (NeoR) or locally calibrated (ThrS). NeoR's INR values were considered as reference values and patients were grouped according to them in 4 categories I‐INR2‐4 (therapeutic range), II‐INR>4, III‐INR>5 and IV‐INR>6. In addition, INR values were subjectively considered as: A‐underdose (≤1.9), B‐borderline/low (>1.9‐2.1), C‐in the therapeutic range (>2.1‐3.8), D‐borderline/high (>3.8‐4.2) and D‐overdose (>4.2). Data were analysed applying the Spearman correlation test, the Bland‐Altman plot and the Cohen's Kappa coefficient.

Results: Overall, good correlation between NeoR's INR values and ThrS's INR values were observed (r: 0.94; CI: 0.92‐0.95; P<0.0001). Similar results were obtained for patients in which INR values were in the therapeutic range (r: 0.94; CI: 0.90‐0.96; P<0.0001). However, the correlation was worse for those with INR out of range (INR>4, r: 0.83; INR>5, r: 0.78; INR>6, r: 0.53). The mean of the differences between the INR values for NeoR and ThrS in the different groups, the limits of agreement as well as Cohen′s Kappa coefficients are displayed in the table.

TABLE 1 Differences between the INR values for NeoR and ThrS in the different groups n Mean of the differences Limits of agreement Global % of agreement Kappa 95% Confidence interval Weighted Kappa Normal controls & patients 266 0.954 ‐0.0587 to 2.495 62.78 0.447 Moderate 0.371 to 0.522 0.638 Good Normal controls 21 0.001 ‐0.054 to 0.056 100 ‐‐ ‐‐ ‐‐ INR 2‐4 60 0.346 ‐0.035 to 0.727 78.33 0.116 Poor ‐0.105 to 0.336 0.204 Fair INR>4 193 1.259 ‐0.208 to 2.727 51.30 0.047 Poor 0.014 to 0.079 0.165 Poor INR>5 122 1.491 ‐0.086 to 3.069 70.49 0.000 Poor ‐‐ 0.000 Poor INR>6 51 2.001 0.195 to 3.807 100 ‐‐ ‐‐ ‐‐

Conclusions: The agreement between the 2‐different thromboplastin/instrument combinations was good to classify patients and normal controls. However, the strength of agreement was moderate among patients under anticoagulant treatment and it was poor for those with INRs over the therapeutic range. NeoR's values were commonly higher than ThrS's values, suggesting a possible different sensitivity to coagulation factors or a bias in the calibration procedure.

PB547: The Coagulation System State in Patients with Philadelphia‐negative Myeloproliferative Neoplasms

N. Silina; O. Matvienko ; N. Korsakova; O. Golovina; S. Voloshin; V. Shuvaev; E. Efremova; M. Fominykh

Russian Research Institute of Haematology and Transfusiology, St. Petersburg, Russian Federation

Background: Philadelphia‐negative myeloproliferative neoplasms (MPN) are included clonal hematopoietic diseases such as Primary Myelofibrosis (PMF), essential Thrombocitemia (ET) and Policitemia Vera (PV). PV and ET have some common properties such as relatively normal maturation of cells and similarity of clinical symptoms. PMF has unique symptoms caused by bone marrow fibrosis. Imbalance of coagulation system in MPN patients leads to complications such as thrombosis and bleedings.

Aims: to estimate the coagulation system state in MPN patients (PV, ET and PMF, respectively) with thrombin generation test.

Methods: The study included 25 MPN patients (25‐70 years, median=51). Patients were separated on two groups. The first group involved 14 patients with PV and ET; the second group consisted of 11 PM patients. Thrombin generation was assessed by calibrated automated thrombinography (CAT) according to Hemker et al. Measure was conducted in platelet poor plasma (PPP) with or without presence of thrombomodulin (PPP reagent ± TM). The following parameters were evaluated: endogenous thrombin potential (ETP, nM*min), peak thrombin (Peak, nM), lag‐time (Lag, min), and time to peak (TTP, min). Sensitivity ETP and Peak for TM were calculated as percent of decreasing of these parameters after adding to assay of TM (S ETP, % and S Peak, % respectively). STATISTICA 6.0 was used. Results are given as median (Me) with 95% confidence intervals (CI), P<0.05 was considered statistically significant.

Results: View the Table Parameters of CAT.

TABLE 1 Difference between two groups of patients Parameters of CAT Group 1 (n=14) Group 2 (n=11) ETP, nM*min 1333.67 (1135.76‐1776.12)* 1107.86 (781.6‐1626.6) S ETP (%) 38.7 (17.9‐66.7) 31.45 (2.5‐68.7) Peak, nM 202.165 (113.5‐302.01) 163.26 (94.0‐235.0) S Peak (%) 22.7 (9.9‐52.7) 5.64 (0.01‐48.0) Lag, min 3.55 (2.0‐5.17)* 5.24 (3.09‐7.59) TTP, min 6.58 (4.0‐9.1)* 8.32 (5.67‐11.5)

Conclusions: Patients in group 1 have significant shorter time parameters (Lag, TTP) and higher endogenous thrombin potential. These results may suggest that patients with PV and ET have quicker initiation and intensity of thrombin generation than PMF patients. It might be associated with thrombotic complication frequency, but such conclusions are need more studies.

PB548: Thromboelastographic Changes in Patients with BCR‐ABL Negative Myeloproliferative Disorders

M.C. Cini 1 ; C. Portelli 1 ; K. Vella 2 ; P. Brincat 3 ; P. Gatt 4 ; N. Calleja 5 ; A. Gatt 6

1 Faculty of Health Sciences, University of Malta, Msida, Malta; 2 Coagulation Medicine Laboratory, Department of Pathology, Mater Dei Hospital, Msida, Malta; 3 Haematology Laboratory, Department of Pathology, Mater Dei Hospital, Msida, Malta; 4 Clinical Perfusion Department, Mater Dei Hospital, Msida, Malta; 5 Directorate for Health Information & Research, Guardamangia, Malta; 6 University of Malta, Department of Pathology, Msida, Malta

Background: Patients with BCR‐ABL negative myeloproliferative disorders (MPDs), present with an increased incidence towards thrombosis and bleeding. The mechanisms responsible for the thrombotic risk are still ill‐defined. Several pro and anticoagulant factors have been measured in previous studies but separate measurement of each factor is not sufficient in MPD patients due to complex abnormalities present. A global assay, thromboelastography (TEG) might be more suited to detect the procoagulant imbalance in similar patients.

Aims: To investigate the haemostatic differences between MPD patients and a normal cohort by using TEG, including a novel TEG modification using protein C activator, Protac.

Methods: Thirty‐nine MPD patients were enrolled from Haematology‐Oncology Outpatients at Sir Anthony Mamo Oncology Centre in Malta and 18 normal subjects as controls. After ethics approval and informed consent, citrated whole blood samples were collected. TEG was carried out with and without Protac.

Results: Reaction (R) time was significantly longer in MPD patients than in controls both with and without Protac. There was no significant difference in kinetics (K) time and α‐angle between patients and controls with and without Protac. Maximum amplitude (MA) was significantly higher in patients than in controls without Protac but no significant difference was found in MA with Protac. No significant differences were detected in the MA ratio (MA with Protac/MA without Protac) between controls and patients.

Conclusions: MPD patients show a more prolonged R time and a higher MA. However, using the Protac modification, they did not demonstrate a protein C resistance profile. Further studies are ongoing to try to explain these TEG changes.

PB549: The Impact of Age, Ethnicity and Gender on Global Coagulation Assays

H.Y. Lim 1,2 ; C. Ng 1 ; J. Rigano 3 ; H. Nandurkar 2 ; P. Ho 1

1 Northern Health, Haematology, Epping, Australia; 2 Australian Centre for Blood Diseases, Melbourne, Australia; 3 Austin Pathology, Heidelberg, Australia

Background: Current coagulation studies only measures the time to start of clot formation, which makes up 5% of the thrombotic process, and hence do not reliably evaluate an individual's coagulation profile. Global coagulation assays may be better surrogate measures of an individual's thrombosis risk. However, it is important to evaluate the differences within the normal population prior to the inclusion of these assays into studies on diseased populations.

Aims: To evaluate differences in global coagulation assay parameters within the normal controls.

Methods: Normal controls with no thrombotic/cardiovascular history and no anticoagulant/antiplatelet use were recruited. Thromboelastography using citrated whole blood was performed with TEG 5000S and platelet‐poor plasma were obtained for assessment of thrombin generation with calibrated automated thrombogram (CAT), fibrin‐aggregation (with and without tPA) with overall haemostatic potential (OHP) assay and p‐selectin.

Results: 96 normal controls (median age 44 years) were recruited. Females had prothrombotic TEG and CAT parameters (P<0.01) as well as increased fibrin generation (overall coagulation potential, OCP) and overall fibrinolytic potential (OFP) (Table 1). Normal controls ≥50 years old had more prothrombotic TEG (including reduced clot lysis) with increased fibrin generation and proteolysis while thrombin generation was comparable. East Asians (n=31) had lower endogenous thrombin potential on CAT (P<0.01) compared those of European origin (n=54) despite minimal differences seen on TEG and fibrin generation. Higher P‐selectin is associated with older age, poorer lipid profile (P<0.05), higher maximum amplitude (P=0.01) and paradoxically lower CAT parameters (P<0.05).

TABLE 1 Differences seen in global coagulation assays between gender and age groups Male Female P‐value Age <50 years Age ≥50 years P‐value TEG parameters R time (min) 8.4 6.9 0.03 8.1 6.4 0.01 α‐angle (°) 51.2 59.4 <0.01 51.7 62.8 <0.01 Max amplitude (mm) 54.3 59.5 <0.01 56.1 60.1 0.01 LY30 (%) 1.9 2.1 0.82 3.0 0.0 0.01 CAT parameters ETP (nM.min) 1258.0 1390.2 0.02 1336.2 1362.8 0.62 Peak height (nM) 193.2 236.7 0.11 219.1 227.3 0.55 Vel Index (nM.min) 56.5 80.5 0.01 74.8 70.1 0.54

Conclusions: This study highlights the differences seen in gender, age and ethnicity on global coagulation assays. Further studies are required to assess the clinical significance of these differences.

PB551: Identification of Protein C System‐associated Thrombophilia using an Endothelial Cell‐based Thrombin Generation Assay

E. Coll 1 ; A. Amirkhosravi 1 ; M. Brodie 2 ; P. Martins 2 ; H. Desai 2 ; L. Robles‐Carrillo 2 ; E. Spruill 2 ; J. Francis 2

1 Florida Hospital, Center for Thrombosis Research, Longwood, United States; 2 Florida Hospital, Center for Thrombosis Research, Winter Park, United States

Background: Defects in the Protein C (PC) system are associated with an increased risk of venous thrombosis, due to a failure to regulate thrombin production. The thrombin generation assay (TGA) may therefore be a useful tool to demonstrate the potential hypercoagulable phenotype of patients with such conditions. We previously showed that the introduction of a surrogate endothelial monolayer to the TGA allows the assessment of the endothelial‐dependent PC pathway. Thus, by measuring thrombin generation (TG) kinetics ± these cells, the contribution of the PC anticoagulant system to TG can be assessed in a physiologically relevant manner. PC‐ or S‐depleted (PCd, PSd), as well as factor V Leiden (FVL) plasmas all show an abnormal TG profile in this endothelial‐based TG assay (EcTGA).

Aims: We investigated whether the EcTGA was capable of detecting a TG abnormality in clinical samples having PC/PS deficiency or FVL.

Methods: Residual plasma samples of 151 in‐hospital patients tested for thrombophilic abnormalities in our Hemostasis and Thrombosis Clinical Lab were subjected to EcTGA. Results were expressed as % reduction in TG in the presence of endothelial cells. Normal (n=69), FVL, and PCd/PSd plasmas served as abnormal controls.

Results: The use of therapeutic anticoagulation prior to sample collection precluded the acquisition of meaningful thrombograms in 125 samples. Of the remaining 26, 18 (69%) had a PC system‐related defect. However, other hemostatic abnormalities (commonly an elevated FVIII) were also present, and only 4 (15%) had a unique defect. Abnormal TG profile was observed in 11 of 18 (61%) patients based on a % reduction of TG parameters (PT & ETP) falling below the 10th percentile cut‐off derived from normals.

Conclusions: EcTGA may be a useful global assay to screen for the presence of PC system‐associated thrombophilic abnormalities. However, the frequency of anticoagulation and the “acute‐phase” status of hospitalized patients could preclude analysis or affect assay sensitivity in this population.

PB553: Development of a New Point‐of‐care Test for the Detection of Direct Oral Anticoagulants (DOACs)

G. Frydman 1,2,3 ; F. Ellett 4 ; N. Davis 3 ; J. Jorgensen 4 ; D. Hayden 5 ; E. Van Cott 6 ; H. Dalzell 7 ; D. Padmanabhan 7 ; M. Majmudar 7,8 ; C. Vanderburg 9 ; M. Toner 4 ; J. Fox 1 ; R. Tompkins 2

1 Massachusetts Institute of Technology, Division of Comparative Medicine, Cambridge, United States; 2 Massachusetts General Hospital, Center for Surgery, Innovation & Bioengineering, Boston, United States; 3 Massachusetts Institute of Technology, Department of Biological Engineering, Cambridge, United States; 4 Massachusetts General Hospital, BioMEMS Resource Center, Boston, United States; 5 Massachusetts General Hospital, Department of Biostatistics, Boston, United States; 6 Massachusetts General Hospital, Department of Pathology, Boston, United States; 7 Massachusetts General Hospital, Cardiology Division, Department of Medicine, Boston, United States; 8 Massachusetts General Hospital, Healthcare Transformation Lab, Boston, United States; 9 Massachusetts General Hospital, Harvard NeuroDiscovery Center, Boston, United States

Background: There are currently no FDA‐approved coagulation tests for the detection of direct oral anticoagulants (DOACs) in the bloodstream. The accurate detection of DOACs in the emergency room setting can be potentially life‐saving.

Aims: Evaluation of a new rapid point‐of‐care test in order to both detect the presence of DOACs in patient samples and to identify the specific type of DOAC as the cause of prolonged clotting times.

Methods: Plasma samples spiked with rivaroxaban, apixaban, and dabigatran were tested to establish reference curves. A single‐center study was then conducted (n=100) to determine the sensitivity and specificity of this point‐of‐care test in an acute care setting. Plasma samples from patients admitted to the emergency department (ED) were screened for use of factor Xa inhibitors, including apixaban and rivaroxaban, within the past 24 hours. Test results were then compared to standard coagulation test results, prothrombin time (PT) and international normalized ratio (INR), as ordered by the treating clinicians.

Results: This test was able to detect DOACs in plasma samples, differentiate between Xa and IIa inhibitors, and provide a quantitative read‐out. When evaluating patient samples, the test had a 92.9% sensitivity and 97.9% specificity for the detection of DOACs in samples collected from the ED. Additionally, unlike PT and INR, this test could reliably identify patients that had prolonged clotting times secondary to the presence of a DOAC.

Conclusions: A point‐of‐care test has been developed for the detection of DOACs in whole blood and plasma as well as identify the specific type of DOAC. This test overcomes the current limitations of coagulation testing, and could be useful as a tool for the detection and monitoring of DOACs in emergency and critical care settings. Clinical situations where a patient′s medical record is unavailable and precautions need to be taken prior to invasive interventions are particularly relevant, especially with the development of new DOAC reversal agents.

PB554: A Rapid Fully‐automated Chemiluminescent Immunoassay for the Early Quantification of ADAMTS13 Activity in TTP Diagnosis

C. Valsecchi 1 ; M. Maribet Perez 2 ; S. Faraudo 2 ; S. Blanch 2 ; F. Peyvandi 1

1 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Milano, A. Bianchi Bonomi Hemophilia and Thrombosis Centre, Università degli Studi di Milano, Department of Pathophysiology and Transplantation, Milano, Italy; 2 Biokit Research & Development, Assay Development Department, Lliçà d'Amunt, Barcelona, Spain

Background: Differential diagnosis of thrombotic microangiopathies can be difficult due to similarities in clinical and laboratory features. Thrombotic thrombocytopenic purpura (TTP) is characterized by a severe deficiency of ADAMTS13 activity (<10% of normal), and rapid measurement is pivotal for early diagnosis and guidance of the correct treatment of such a rare and life‐threatening disease.

Aims: The aim of the study was to evaluate the performance of an ADAMTS13 activity assay currently in development (Instrumentation Laboratory, Bedford, MA, USA).

Methods: The assay is a 2‐step, fully‐automated chemiluminescent immunoassay, standardized against the WHO 1st International Standard ADAMTS13 Plasma, with an analytical time of 33 minutes. Magnetic particles are coated with the VWF73 peptide containing the ADAMTS13 cleavage site. Chemiluminescent detection is based on an isoluminol‐labeled monoclonal antibody reacting specifically with the cleaved peptide. A total of 76 samples, including normal donors and patients being evaluated for TTP, were tested with the chemiluminescent immunoassay, an in‐house FRET‐VWF73 assay, and an ADAMTS‐13 Activity ELISA (Technoclone, Vienna, Austria). Agreement between methods was assessed focusing on the severe ADAMTS13 activity deficiency (≤10%).

Results: The study revealed high agreement with the in‐house FRET assay (Kappa=0.97) and the ELISA (Kappa=1.00) in classifying TTP patients with severe ADAMTS13 activity deficiency.

Table 1 and 2.

TABLE 1 Agreement AcuStar ‐FRET. Discrepant sample: FRET=10% ; AcuStar=11.6%) Kappa=0.97 FRET AcuStar ≤10% >10% Total ≤10% 27 0 27 >10% 1 48 49 Total 28 48 76

TABLE 2 Agreement AcuStar‐Elisa Kappa=1 ELISA AcuStar ≤10% >10% Total ≤10% 27 0 27 >10% 0 49 49 Total 27 49 76

Conclusions: In this study, we evaluated a chemiluminiscent ADAMTS13 activity immunoassay, which demonstrated excellent clinical performance by high agreement with severe ADAMTS13 deficiency (≤10% activity) with the in‐house FRET‐VWF73 assay and commercially available ADAMTS‐13 Activity ELISA. This assay could be useful in facilitating the diagnosis of TTP at the day of patient presentation in emergency rooms because it is rapid, fully automated, and does not require specialized laboratory staff.

PB555: Urgent Laboratory Investigation of HIT Syndrome with an Automated Immunoassay in Cardiovascular Patients

G. Soufla; E. Iliopoulou; T. Kanellopoulou; S. Georgantis; M. Katafygioti; T. Kostelidou

Onassis Cardiac Surgery Center, Department of Hematology/Blood Transfusion/Coagulation and Hemostasis, Athens, Greece

Background: Heparin‐induced thrombocytopenia (HIT) is a serious complication of heparin therapy resulting in heparin/platelet‐factor4 complexes (antiPF4/H antibodies‐Abs). Upon clinical suspicion, laboratory evaluation for HIT syndrome should be performed.

Aims: To evaluate the reliability of a rapid, automated assay for HIT diagnosis in urgent cases before cardiac surgery.

Methods: We analysed clinical and laboratory characteristics of 108 patients for HIT syndrome presence in a single cardiac surgery centre. Clinical suspicion was determined by “4T score” (<3 low, ≥3intermediate/high probability). Patients were screened for HIT Abs with both HemosIL HIT‐Ab(PF4‐H) (HIT‐IL) and ELISA‐IgG (EIA). A HIT‐IL value ≥1.0 U/mL was considered positive for HIT Abs. We considered EIA (Asserachrom Stago) OD < 1.0 as negative, OD 1.0‐2.0 as intermediate and OD>2.0 as strongly positive for HIT‐Abs presence. Functional test was performed with heparin‐induced platelet aggregation test (HIPA) in selected cases to confirm a positive result.

Results: Both HIT‐IL and EIA results were statistically associated (P<0.001), as well as with the “4T score” (P<0.001). In the intermediate/high probability group, 83% with a positive HIT‐IL>5 U/mL, had an EIA OD>2.0 (P=<0.001).

FIGURE 1 ELISA OD values in 4T≥3 patients with HIT‐IL>5 U/mL are significantly higher than in patients with HIT‐IL<5 U/mL (P<0.001)

In the same group, a positive HIT‐IL between 1.8‐5 U/mL was correlated with 50% probability of an EIA OD>2.0, whereas HIT‐IL 1‐1.8 U/mL yielded an EIA OD<1.0. Overall in this group HIT‐IL had a 75% positive and a 90% negative predictive value of HIT‐Abs presence. IL positive patients with low HIT probability (4T<3) are most likely to be negative in EIA (75%).

Conclusions: Upon an urgent diagnosis concerning HIT syndrome, the use of a rapid automated assay to override time consuming and expensive tests is an attractive solution. Our results confirmed a strong association of HIT‐IL>5 U/mL with 4T≥3 and EIA OD>2.0 indicating its value in assessing strong probability of HIT. HIT‐IL<5 U/mL should in any case be evaluated with EIA and HIPA.

PB556: Platelet Hyporeactivity Combined with Enhanced Coagulation in Healthy Term and Preterm Newborns During First Days of Life

E. Koltsova 1,2 ; E. Balashova 3 ; A. Ignatova 1 ; A. Poletaev 1 ; D. Polokhov 1 ; A. Kuprash 1 ; O. Ionov 3,4 ; A. Kirtbaya 3,4 ; A. Lenyushkina 3 ; L. Timofeeva 3 ; V. Zubkov 3,4 ; D. Degtyarev 3,4 ; G. Sukhikh 3 ; F. Ataullakhanov 1,2,5 ; M. Panteleev 1,2,5 ; A. Balandina 1,2

1 Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russian Federation; 2 Center for Theoretical Problems of Physicochemical Pharmacology, Moscow, Russian Federation; 3 National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov of Ministry of Healthcare of Russian Federation, Moscow, Russian Federation; 4 Sechenov First Moscow State Medical University, Moscow, Russian Federation; 5 Moscow State University named after M.V. Lomonosov, Moscow, Russian Federation

Background: Preterm newborns are a group of high risk of bleeding and thrombosis in the early neonatal period. The laboratory diagnostic tools at the disposal are not sufficiently sensitive to the manifestation of impairments in neonates.

Aims: To assess hemostasis in preterm and term newborns using global coagulation testing and flow cytometry characterization of all platelet functional responses.

Methods: Ten preterm neonates (33‐34 gestation weeks) and ten term neonates (>37 gestation weeks) were enrolled. Platelet functional activity was estimated by flow cytometry, coagulation was assessed using integral assay of thrombodynamics and standard coagulation assays (activated partial thromboplastin time (APTT), prothrombin, fibrinogen, D‐dimer, antithrombin III and protein C concentrations). All parents gave their written informed consent; the protocol was approved by ethics committee.

Results: All platelet functions (specifically, lack of all granules, of their release, phosphatidylserine exposure and integrin activation) were profoundly (by 50‐70%) impaired in newborns. Preterm infants did not differ from term in glycoprotein Ib, P‐selectin release or mepacrine uptake/release, but had decreased glycoprotein IIb‐IIIa, lower glycoprotein IIb‐IIIa activation, and phosphatidylserine externalization upon stimulation. The shape change of platelets after activation was smaller in newborns compared to adults and in preterm compared to term. Clot growth rate in thrombodynamics was increased compared to adult values.

Conclusions: Hemostasis of newborns is characterized by pronounced plasma hypercoagulation and reduced functional activity of platelets in comparison with the adults. There is no significant difference in the parameters of coagulation, but some of the platelet functional responses are depressed in preterm newborns compared with terms.

The work was supported by grant №17‐74‐10224 from the Russian Science Foundation.

PB557: Von Willebrand Factor Antigen Levels Predict Major Adverse Cardiovascular Events in Patients with Asymptomatic Carotid Stenosis

K. D. Kovacevic 1 ; F. J. Mayer 2 ; B. Jilma 1 ; C. J. Binder 2 ; A. D Blann 3 ; E. Minar 4 ; M. Schillinger 4 ; M. Hoke 4

1 Medical University of Vienna, Clinical Pharmacology, Vienna, Austria; 2 Medical University of Vienna, Department of Laboratory Medicine, Vienna, Austria; 3 University of Birmingham, Institut for Cardiovascular Sciences, Birmingham, United Kingdom; 4 Medical University of Vienna, Department for Internal Medicine, Vienna, Austria

Background: Von Willebrand factor (vWF) plays an important role in thrombogenesis and mediates platelet adhesion particularly under high shear forces, such as found in stenotic arteries causing myocardial infarction or stroke.

Aims: To study whether serum levels of VWF antigen (VWF:Ag) predict the occurrence of major adverse cardiovascular events in asymptomatic patients suffering from carotid artery stenosis.

Methods: Inclusion and exclusion criteria of the ICARAS design have been published previously. Patients had atherosclerotic carotid artery disease as defined by the presence of nonstenotic plaques or carotid stenosis of any degree. Concentrations of VWF:antigen (VWF:Ag) were measured by enzyme immunoassay (CV% for duplicates <5%).

Results: Before conducting the study we demonstrated the stability of VWF:Ag even after 3 freeze‐thaw cycles, similar concentrations between citrated plasma and serum (±4%) and found an excellent correlation between plasma and serum samples (r=0.95‐0.98). Levels of VWF:Ag predicted future cardiovascular events in 811 patients with asymptomatic carotid stenosis. High levels of vWF:Ag were an independent predictor of major adverse cardiovascular events, as were previous myocardial infarction, family history of cardiovascular disease and high levels of high sensitivity C reactive protein. Patients with VWF:Ag concentrations in the 4th quartile had a 44% event rate after an average 3‐year follow up and a hazard ratio of 2.15 (95% confidence interval of 1.47‐3.13; P<0.001).

Conclusions: High serum concentrations of vWF:Ag predict major cardiovascular events in patients with asymptomatic carotid stenosis, and given their high event rate may be useful for risk stratification of such patients.

PB558: Thrombin Generation in Real‐world Patients Treated with Two Different Doses of Apixaban

M.D.L. Herrera 1 ; S. Pons 1 ; A. Grinspon 1 ; S. Aguirre 1 ; V. Monserrat 1 ; A. Brodsky 2 ; A.S. Scazziota 1

1 Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Hospital de Clínicas, Buenos Aires, Argentina; 2 Universidad de Buenos Aires, Facultad de Medicina, Buenos Aires, Argentina

Background: Apixaban is given at fixed doses but in certain clinical situations it is necessary to evaluate in‐vivo intensity of anticoagulation activity. Coagulation assays are not reliably for determining apixaban effects, chromogenic anti‐Factor Xa activity (aFXa) using specific calibrator is the preferred clinical assay. Nevertheless, to assess the anticoagulant activity, a global test such as Thrombin generation (TG), could be a useful tool.

Aims: To evaluate the anticoagulant effect of apixaban on TG parameters and correlate them with drug concentrations, in patients receiving two different doses of apixaban.

Methods: 20 patients receiving apixaban 2.5 mg (n=10) or 5 mg (n=10) BID for stroke prevention in non‐valvular atrial fibrillation. Two blood samples were taken: at peak (P) and trough (T). Plasmas were tested with apixaban calibrated aFXa. TG was assayed via the calibrated automated thrombogram (Thrombinoscope), activated with tissue factor 5 pM. TG parameters: lag time (LT), time to peak (ttP), thrombin Peak (TP), and endogenous thrombin potential (ETP).

Results: There was a significant difference in apixaban concentrations, between P and T only at apixaban 5 mg (P 0.001). In these patients, aFXa at P was higher and statistically different from that at 2.5 mg dose (P 0.024), although TG parameters didn′t change significantly. Both apixaban doses reached similar aFXa levels at T. Both apixaban doses decreased significantly all TG parameters compared with normal controls, at P such as T. However, TG parameters showed a significant difference between P and T at 5 mg apixaban dose, but not at the lowest dose (Table 1 and 2).

TABLE 1 Apixaban 5 mg BID Trough (T) Peak (P) Control (C) P T vs. C P P vs. C P T vs. P aFXa ng/mL 98±65 338±163 – – – 0.001 LT m 4.2±1.1 7.0±4.1 3.1±1.3 0.001 0.007 0.042 ETP nM/m 1,109±211 846±215 1,365±125 0.004 0.001 0.011 Peak nM 145±80 84±32 232±101 0.05 0.001 0.038 ttP m 8.3±2 6.2±2.1 6.2±2.1 0.01 0.002 0.034

TABLE 2 Apixaban 2.5 mg BID Trough (T) Peak (P) Control (C) P T vs. C P P vs. C P T vs. P aFXa ng/mL 102±74 182±117 – – – 0.007 LT m 6.1±1.9 5.9±1.3 3.1±1.3 0.0001 0.0001 0.99 ETP nM/m 1,025±228 984±324 1365±125 0.0001 0.001 0.74 Peak nM 127±40 94±51 232±101 0.007 0.001 0.11 ttP m 8.9±2 9.1±2 6.2±2.1 0.004 0.004 0.99

Conclusions: Our results suggest that apixaban levels do not necessarily correlate with TG parameters. We found that on the P state, aFXa depends on apixaban dose intaken, nevertheless TG parameters don't show a dose dependent suppression of TG. The measurement of anticoagulation status by TG in adition to aFXa assay, could be a sensitive way of detecting in‐vivo intensity of anticoagulation.

PB559: Specific Point‐of‐Care Testing of Coagulation in Patients Treated with Dabigatran

F. Härtig 1 ; I. Birschmann 2 ; A. Peter 3 ; M. Ebner 4 ; C. Spencer 1 ; M. Gramlich 3 ; H. Richter 1 ; J. Kuhn 2 ; R. Lehmann 3 ; C.S. Zuern 3 ; U. Ziemann 1 ; S. Poli 1

1 University Hospital Tübingen, Department of Neurology & Stroke, Tübingen, Germany; 2 Ruhr University ‐ Heart and Diabetes Centre, Institute for Laboratory & Transfusion Medicine, Bad Oeynhausen, Germany; 3 University Hospital Tübingen, Department of Internal Medicine, Tübingen, Germany; 4 Charité University Medicine, Department of Internal Medicine and Cardiology, Berlin, Germany

Background: Direct oral anticoagulants (DOAC), like dabigatran, are increasingly replacing vitamin K antagonists for prevention of thromboembolism in atrial fibrillation (AF). Despite treatment, stroke rate in DOAC‐treated AF‐patients remains 1‐2% per year. Subsequently, stroke physicians face a growing number of DOAC‐treated patients with acute stroke. Rapid assessment of coagulation in DOAC‐treated patients is vital prior to thrombolysis or reversal therapy, but existing point‐of‐care (POC) testing is suboptimal.

Aims: To evaluate DOAC‐specific POC coagulation testing (SPOCT) and investigate whether POC‐ecarin clotting time (POC‐ECT) accurately reflects dabigatran plasma concentrations.

Methods: Prospective single‐centre observational SPOCT‐DOAC I study enrolling patients receiving a first dose of dabigatran and patients already on dabigatran‐treatment. Subjects receiving other anticoagulants were excluded. Six blood samples were collected from each patient: before drug intake, 30 minutes, 1, 2, 8, and 12 hours after intake. POC‐ECT was performed with whole blood (WB), citrated whole blood (CB) and citrated plasma (CP). Dabigatran plasma concentrations were determined by ultra‐performance liquid chromatography/tandem mass spectrometry.

Results: 240 blood samples from 40 patients were analysed. Dabigatran concentrations ranged from 0 to 275 ng/mL and POC‐ECT with WB/CB/CP from 20 to 186/184/316 seconds. Pearson's correlation coefficient showed a strong correlation between dabigatran concentrations and POC‐ECT with WB/CB/CP (r=0.88/0.95/0.96). Dabigatran concentrations >30 ng/mL (threshold for thrombolysis/surgery according to clinical guidelines) were detected by POC‐ECT with WB/CB/CP (>36/35/45 seconds) with 95.1/97.2/96.5% sensitivity and 81.4/86.6/93.8% specificity.

Conclusions: This is the first study evaluating DOAC‐specific POC coagulation testing. Correlation of POC‐ECT with actual dabigatran concentrations is excellent. POC‐ECT detects clinically relevant drug levels with high sensitivity and specificity.

PB560: Active von Willebrand Factor Levels in Kidney Disease Are Associated with Disease Severity and Type of Dialysis

L.N. van der Vorm 1,2,3 ; R. Visser 4 ; D. Huskens 1,2 ; A. Veninga 2 ; J.A. Remijn 1,3 ; H.C. Hemker 1,2 ; P.L. Rensma 4 ; R. van Horssen 5 ; B. de Laat 1,2,3

1 Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands; 2 Synapse Research Institute, Maastricht, the Netherlands; 3 Gelre Hospitals, Department of Clinical Chemistry and Hematology, Apeldoorn, the Netherlands; 4 Elisabeth‐TweeSteden Hospital, Department of Nephrology, Tilburg, the Netherlands; 5 Elisabeth‐TweeSteden Hospital, Department of Clinical Chemistry and Hematology, Tilburg, the Netherlands

Background: Thrombosis is a common complication in chronic kidney disease (CKD). Previous studies reported increased levels of von Willebrand Factor (VWF) antigen in CKD patients. Pathologically high shear or binding to collagen unfold VWF, exposing the VWF A1 domain to allow platelet binding. Ultralarge VWF multimers, released upon activation or dysfunction of endothelial cells, are also in their active conformation. Active VWF is increased in several conditions with thrombotic complications, but has not been studied in CKD.

Aims: We aimed to assess levels of active VWF in various stages of kidney disease.

Methods: We developed an ELISA assay to quantify active VWF, using a recombinant antibody fragment recognizing a cryptic epitope on the VWF A1 domain (intra‐ and inter‐assay variations were 4.4% (N=11) and 7.3% (N=6), respectively). Total VWF was measured with an ELISA using a polyclonal anti‐VWF antibody. VWF levels were normalized (%) to normal pooled plasma (NPP), and compared using Kruskal Wallis test with post hoc Dunn′s test. We quantified active VWF in plasma samples from a cohort of patients with various stages of kidney disease, without (CKD) or with hemodialysis (HD) or peritoneal dialysis (PD). Informed consent and medical ethics approval were obtained.

Results: Active VWF was elevated in CKD and end‐stage renal disease (ESRD) patients compared to healthy controls, to an extent associated with disease severity and type of dialysis (Figure 1A). Levels of total VWF were also increased in these patients (Figure 1B), as expected since active VWF contributes to the amount of total VWF.

FIGURE 1 Active VWF and total VWF are elevated in CKD and ESRD compared to healthy controls

Conclusions: In conclusion, active VWF levels are increased in patients with CKD and ESRD. We propose that this hemostatically active VWF may be more strongly associated to thromboembolic morbidity and mortality than VWF:Ag levels. Future studies with larger patient numbers and clinical follow‐up are required to unravel the relationship of active VWF with clinical outcome and its potential role in clinical management of CKD.

PB561: Role of Thromboelastography to Assess Thrombin Generation in Acute Coronary Syndrome (ACS)

Q. Lu ; X. Wang

Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Clinical Laboratory, Shanghai, China

Background: The mechanism of ACS involves thrombus development after the rupture of atherosclerotic plaques. Thrombin is generated at blood‐plaque interface, which is a potent natural platelet activator that plays a pivotal role between thrombosis and inflammation. Thromboelastography (TEG) is a global hemostasis test providing information on multiple facets of the clotting process, including thrombin generation by velocity curve parameters, such as maximum rate of thrombus generation (MRTG) and total thrombus generated (TTG).

Aims: To investigate the early prognostic value of MRTG and TTG in patients with ACS.

Methods: A total of 168 ACS patients, 58 patients with stable angina pectoris (SAP) as control were enrolled. Baseline characteristics data of the two groups were recorded. Routine blood test, cardiac markers, routine coagulation tests and TEG were determined. Receiver operating characteristic (ROC) curve was used to evaluate the diagnosis performance of each index. Logistic regression analysis was used to define the independent risk of ACS.

Results: 1. cTNI level in SAP measured were significantly lower than ACS patients (P<0.01), while other biomarkers (MYO and CK‐MB) showed no statistically differences.

TABLE 1 Cardiac markers and Thromboelastography parameters ACS SAP P value MYO (ng/ml) 30.30 (22.55‐41.03) 30.25 (22.88‐40.18) 0.888 CK‐MB (ng/ml) 1.80 (1.10‐2.50) 1.45 (1.10‐2.40) 0.248 cTNI (ng/ml) 0.05 (0.01‐0.31) 0.01 (0.01‐0.03) 0.000** NEUTROPHIL‐TO‐LYMPHOCYTE RATIO(NLR) 2.52 (1.93‐3.55) 1.95 (1.41‐2.47) 0.000** Angle (degree) 71.14±4.60 65.30±4.24 0.000** MA (mm) 70.34±4.55 63.06±3.59 0.000** CI 2.14±1.22 0.71±1.01 0.000** MRTG(mm/min) 15.84 (13.27‐19.33) 11.20 (9.85‐12.86) 0.000** TTG(mm/min) 848.60±54.32 762.66±43.95 0.000**

2. MRTG and TTG were both significantly higher in ACS group (P<0.01). The area under ROC curves of MRTG and TTG were 0.849 and 0.897. The optimal cut‐off value for the diagnosis of ACS was 13.4 mm/min and 805.6 mm/min, while the sensitivity was 73.2%/78.6% and the specificity was 84.5%/87.9%, respectively.

TABLE 2 Receiver operation characteristics (ROC) curve AUC(95% CI) Cutoff value Sensitivity (%) Specificity (%) cTNI (ng/ml) 0.743 (0.677‐0.810) 0.04 57.1 82.8 ANGLE (degree) 0.824 (0.765‐0.883) 69.55 63.1 91.4 MA (mm) 0.900 (0.857‐0.944) 66.60 80.4 87.9 CI 0.813 (0.753‐0.874) 1.35 75.6 75.9 MRTG (mm/min) 0.849(0.793‐0.904) 13.39 73.2 84.5 TTG (mm/min) 0.897(0.853‐0.942) 805.61 78.6 87.9 FIBRINOGEN (g/L) 0.784 (0.718‐0.850) 2.85 76.8 70.7 PLATELET (×10 9 /L) 0.687 (0.614‐0.760) 202.00 45.2 87.9 NLR 0.703(0.629‐0.778) 2.26 61.9 70.7

3. Logistic regression analysis showed that MRTG was an independent risk factor and auxiliary diagnostic indicator for ACS.

4. Significant spearman correlations were found between MRTG with cTNI (r=0.223, P<0.01), and NLR (inflammation marker) (r=0.306, P<0.01).

Conclusions: MRTG could be used as a predictor of thrombin generation involved with infarctions and inflammation, which is eligible to be a new biomarker for early diagnosis of ACS and could provide baseline information for anticoagulation therapy.

PB562: Evaluation of the DOAC‐Stop ® Procedure to Overcome the Effect of Direct Oral Anticoagulants on Several Thrombophilia Screening Tests

J. Favresse 1 ; B. Lardinois 1 ; L. Sabor 1 ; B. Devalet 1 ; J. Vandepapeliere 1 ; M. Braibant 1 ; S. Lessire 1 ; B. Chatelain 1 ; H. Jacqmin 1 ; J. Douxfils 2 ; F. Mullier 1

1 CHU UCL Namur, Yvoir, Belgium; 2 Université de Namur, Namur, Belgium

Background: The impact of direct oral anticoagulants (DOACs) on laboratory assays used for thrombophilia testing is a well‐known issue and may cause false‐positive and negative results.

Aims: To evaluate the efficiency of a new and simple procedure using an absorbent product (DOAC‐Stop ® ; Haematex Research, Hornsby, Australia) for extracting DOACs from plasmas in real‐life settings.

Methods: 71 DOAC‐treated patients (22 apixaban, 13 dabigatran, 15 edoxaban and 21 rivaroxaban) and 10 control patients were enrolled. Protein S (STA ® ‐Liatest ® Free Protein S), protein C (STA ® ‐Stachrom ® Protein C), antithrombin activity (STA ® ‐ Stachrom ® AT III), and APC‐R (Pefakit ® APC‐R Factor V Leiden) were assayed on a STA‐R MAX analyzer (Diagnostica Stago). The dRVVT screen and confirm (STA ® ‐Staclot ® dRVVT Screen and Confirm), and the PTT‐LA were assayed on the KC10 coagulometer (Amelung GmbH). Concentrations of dabigatran were estimated using the STA ® ‐ECA‐II assay and concentrations of other DOACs with the STA‐liquid anti‐Xa ® assay. All these procedures were performed on the STA‐R MAX analyzer (Diagnostica Stago). Results obtained after the DOAC‐Stop ® treatment were compared to the reference (before treatment) with a paired t‐test.

Results: Using our procedures, false positive results due to DOACs were only observed with lupus anticoagulant tests (up to 87.5%). A significant drop in apixaban, dabigatran, edoxaban and rivaroxaban plasma concentrations following the DOAC‐Stop ® treatment was observed (67.6 to 2.9 ng/ml (P<0.0001), 64.2 to 7.4 ng/ml (P=0.009), 102.1 to 8.8 ng/ml (P=0.001) and 141.3 to 0.3 ng/ml (P=0.002), respectively).

FIGURE 1 Impact of the DOAC‐Stop® absorbent treatment on apixaban, dabigatran, edoxaban and rivaroxaban concentrations

Consequently, the DOAC‐Stop ® treatment was mostly effective to overcome the effect of DOACs on PTT‐LA, dRVVT screen and confirm tests.

Conclusions: The DOAC‐Stop ® absorbent procedure appeared to be an effective and simple way to overcome the interference of DOAC on coagulation tests and should facilitate the interpretation of thrombophilia screening tests in patients taking DOACs.

PB563: Laboratory Assessment of Dabigatran Levels and Anti‐Xa Anticoagulant Levels

I. Paskaleva ; D. Dineva; E. Doncheva

National Heart Hospital, Laboratory Department, Sofia, Bulgaria

Background: Direct oral anticoagulants (DOAC) show inter‐ and intra ‐ individual variability in plasma concentrations. Assessment of the individual response to drug treatment and risk balance could require laboratory measurement.

Aims: To evaluate the variation in DOAC drug levels among individual patients.

Methods: A total of 165 patients with non‐valvular atrial fibrillation were enrolled, 60 of them were on dabigatran (150 mg or 110 mg twice daily), 65 patients on apixaban (5 mg or 2.5 mg twice daily), and 40 patients on rivaroxaban (20 mg or 15 mg once daily). Blood was taken at trough level for dabigatran or apixaban and at peak level for rivaroxaban. Diluted‐thrombin‐time (DTI) was performed with Hemoclot (Hyphen BioMed) for dabigatran and anti‐FXa measurments for apixaban and rivaroxaban was calibrated with Hyphen DiXaI on CS 2500 (Sysmex).

Results: Dabigatran given at a dose 150 mg twice daily produced plasma trough level of 85 ng/ml (52‐182 ng/ml) and at 110 mg twice daily was 117 ng/ml (43‐195 ng/ml), expressed as mediana (5‐9th perc). Four patients had values above the upper limit with highest value of 404 ng/ml and three had values below 30 ng/ml. For apixaban 5 mg twice daily the median trough level was 86 ng/ml (55‐141 ng/ml) and for 2.5 mg twice daily 58 ng/ml (21‐141 ng/ml) ‐ two patients had values above the upper limit (up to 680 ng/ml) and three had values below 50 ng/ml. Rivaroxaban administered at a dose 20 mg daily or 15 mg daily produced peak of 215 ng/ml (98‐403 ng/ml) and 267 ng/ml (188‐450 ng/ml), respectively. Five patients with rivaroxaban experienced mucosal bleeding. We observed haematuria in patients on dabigatran treatment.

Conclusions: Fifteen out of 165 measured levels were beyond the expected limits in our outpatient groups, which allows for better assessment of risk balance in such patients.

PB564: Monitoring Treatments by Unfractionated Heparin. Stability of Plasma Anti‐Xa Activity for up to 4 Hours in Citrated Tubes

P. Toulon 1 ; F. Fischer 1 ; A. Appert‐Flory 1 ; S. Buvat 1 ; D. Jambou 1 ; M.‐H. Mahagne 2

1 Pasteur Unversity Hopital, Hematology, Nice, France; 2 Pasteur Unversity Hopital, Neurology, Nice, France

Background: Current guidelines recommend a maximum delay of 2 hours between blood sampling and testing for anti‐Xa activity and aPTT prescribed for monitoring treatments by UFH, when citrated tubes are used. As such a short delay could be an issue, particularly at multisite centers, we evaluated the potential impact of longer delays.

Aims: For that purpose, 2 citrated tubes (0.109 M) were obtained from 57 patients on UFH: one centrifuged and tested within 1 hour after sampling and one stored for 4 hour at room temperature before being centrifuged.

Methods: Anti‐Xa activity was evaluated using 2 reagents [Biophen Heparin LRT (Hyphen Biomed) and HemosIL Liquid Heparin (Werfen)], and aPTT using HemosIL SynthASil (Werfen) automated on an ACL TOP 700 CTS (Werfen).

Results: Anti‐Xa activity was significantly lower after a 4 hour‐delay than after a <1 hour‐delay, but the mean bias calculated according to Bland‐Altman was below 0.05 IU/mL for both reagents (Table), a value lower than the technique imprecision. Considering 0.30 to 0.70 IU/mL as the therapeutic range, there were 3 cases of discrepancy (5.3%) with one reagent, and 11 with the other (19.3%). Most of them were around the lower limit of the therapeutic range and would have no impact on anticoagulation management of the patients. aPTT was significantly shortened (P<0.0001) after a 4 hour‐delay, with a mean bias of ‐8.5 seconds, corresponding to a −0.27 decrease in ratio (Table). If aPTT therapeutic range was calculated to correspond to anti‐Xa activities between 0.30 and 0.70 IU/mL, 12 cases of discrepancy were found, some of them could have induce UFH dosage change.

TABLE 1 Anti‐xa and aPTT test results (median values and ranges) after a <1h‐delay and a 4‐delay between sampling and analysis >1 hour‐delay 4 hour‐delay Analytical comparison Bias (Bland‐Altman) (95% CI) Anti‐Xa Hyphen (IU/mL) 0.45 (0.18 1.20) 0.38 (0.16 1.18) <0.0001 −0.05 (−0.13 +0.03) Anti‐Xa IL (IU/mL) 0.38 (0.13 1.22) 0.32 (0.11 1.21) <0.0001 −0.05 (−0.16 +0.06) aPTT SynthASil (ratio) 1.87 (1.02 >5.0) 1.54 (0.99 >5.0) <0.0001 −0.27 (−0.67 +0.12)

Conclusions: These results suggest that it is safe to extend to 4 hour the delay between blood sampling and measurement of anti‐Xa activity for monitoring UFH treatments, with the tested reagents that contain dextran sulphate, a compound able to bind PF4 with a high affinity and so to displace heparin from its complex with PF4. Changes in aPTT were higher, suggesting a shorter acceptable delay. This point is currently investigated.

PB565: Utility of PF4/IgG ELISA Optical Density Values as an Indicator of Positivity of the Heparin Induced Platelet Activation Test in Laboratory Testing for Heparin Induced Thrombocytopenia (HIT)

M. Byrne ; S. Lawlor; C. Haran; C. Gannon; B. White; K. Ryan; J. O'Donnell; N. O'Connell

National Coagulation Centre, St James's Hospital, Dublin, Ireland

Background: For the diagnosis of HIT, a pre‐test clinical probability (4T) score and test panel, including a rapid test (ID‐PaGIA Heparin/PF4 Antibody Test Biorad), PF4/IgG ELISA test (Immucor) and Heparin Induced Platelet Activation test (HIPA) is employed in our laboratory. Where the 4T score is >3, samples are tested by PaGIA test followed by ELISA. Samples with an ELISA optical density (OD) value >0.400 and demonstrable heparin dependence are positive and sent to a referral laboratory (Universitätsmedizin, Greifswald, Germany) for the identification of platelet activating antibodies by HIPA.

Aims: The review examines the utility of the ELISA OD value as an indicator of positivity in the HIPA test

Methods: A review of samples tested from September 2016 to December 2017 examined the number of positive PaGIA tests; positive ELISA tests and subsequent positive HIPA tests. ELISA OD results were grouped in 3 groups (>0.400<1.000) (>1.000<1.500) (>1.500) to determine likelihood of a positive HIPA test with increasing OD.

Results: 271 samples were tested during this period. 78 samples were positive by PaGIA. When tested by ELISA 42/78 were positive and 36/78 were negative. 193 samples were negative by PaGIA. When tested by ELISA 1/193 was positive and 192/193 were negative. All ELISA positive samples (n=43) were referred for HIPA testing. Where the PaGIA test was positive and OD value was >0.400<1.000 (n=11), 3/11 were positive and 8/11 were negative in the HIPA test. Where the OD value was >1.000<1.500 (n=10), 6/10 were positive and 4/10 negative in the HIPA test. Where the OD value was >1.500 (n=21), 21/21 were positive in the HIPA test. One sample negative by PaGIA test and positive by ELISA (OD=0.503) was negative in the HIPA test.

Conclusions: The ELISA OD value is useful as an indicator of positivity with the HIPA test. The likelihood of a positive HIPA test increases with OD, especially at OD >1.500. The HIPA test detects platelet‐activating clinically significant antibodies, crucial for the diagnosis of HIT.

PB566: Natural Anticoagulant Deficiencies in Thais: A Population‐based Study

P. Rojnuckarin ; R. Settapiboon; B. Akkawat; N. Uaprasert; T. Intragumtornchai

Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Department of Medicine, Bangkok, Thailand

Background: For hereditary thrombophilia, factor V R506Q and prothrombin G20210A are common in Caucasians but anticoagulant protein deficiencies are more prevalent in Asians. However, genetic studies in Thais are lacking.

Aims: To determine the genetic basis of protein C and protein S deficiencies in general Thai population.

Methods: Healthy volunteers were tested for protein C activity and free protein S antigen. Subjects with low values were asked for repeated blood samplings and had acquired causes ruled out. Cases with persistently low protein C or protein S levels were assayed for respective gene mutations using direct sequencing of all exons and regulatory regions. Multiplex ligation‐dependent probe amplification (MPLA) was performed when PROS1 gene mutation was not found.

Results: Protein C activities were assayed in 5234 subjects (Table). The values in men were lower than those of post‐menopausal women (P<0.001) but not different from those of younger women (P=0.08). Among 18 cases with persistently low protein C, 7 types of point mutations in PROC gene were found in 17 subjects (94.4%) and 64.7% of them had R167W mutation. One novel mutation at the splice site in IVS‐4 was discovered.

TABLE 1 Subjects and Laboratory Results Protein C Activity Free Protein S Antigen Total tested subjects 5,234 5,242 Total female subjects 3,094 (59.1%) 3,102 (59.2%) Reference ranges 70‐170 IU/dL 53‐124 IU/dL Levels (Mean ± SD): Men/Pre‐menopausal women/Post‐menopausal women 122.7±26.7/121.5±25.3/138.0±27.8 IU/dL 100.4±25.63/82.35±25.09/88.9±25.36 IU/dL Subjects with low levels 56 (1.07%) 254 (4.85%) Repeatedly low levels/ Second samples available 18/26 (69.2%) 51/110 (46.4%) Genetic tests (%Female) 18 (55.6%) 29 (86.2%) Mutations found 17 (94.4%) 5 (17.2%) Recurrent mutations R147W (N =11) Homozygote (5) Heterozygote (6) IVS‐12 (N = 2)

Protein S levels were measured in 5242 volunteers (Table). The levels were highest in men, followed by post‐menopausal women and followed by pre‐menopausal women, respectively (P<0.001). The majority of repeatedly low protein S was in female (45/51, 88.2%). Using the cut‐points of below 50 IU/dL in male and 45 IU/dL in female (2.5 percentile), 29 subjects were selected for PROS1 gene sequencing. Four types of point mutations were found in 5 subjects (17.2%). Free protein S levels ranged from 5‐27 U/ml in cases with mutations. No large deletion was detectable by MLPA.

Conclusions: In Thai population, protein C deficiency was attributed to PROC mutations, especially R167W. However, low protein S levels were more commonly found in female subjects with undetectable PROS1 mutation.

PB567: Changes in Tissue Factor Concentration and APTT in Haemolysed and Non‐haemolysed Paired Citrate Samples

K. Hickey ; A. Woolley; S. Kitchen

Sheffield Haemophilia and Thrombosis Centre, Sheffield, United Kingdom

Background: About two percent of coagulation requests are rejected in our institution due to in vitro haemolysis. Traumatic phlebotomy is sometimes assumed to stimulate release of tissue factor (TF) into samples, which may explain the changes in APTT observed.

Aims: Evaluate the changes in APTT and TF concentration in a cohort of rejected haemolysed and non‐haemolysed replacement samples.

Methods: Haemolysed samples selected based on the Sysmex CS5100 analyser (Sysmex, UK) HIL flags. Residual plasma from samples taken into BD vacutainer (0.109 M citrate) were frozen (−80°C) in patients where a haemolysed sample (HS) and a subsequent non‐haemolysed sample (nHS) from same donor was received (n=40).

APTT performed using Actin FS (Siemens, UK) on the Sysmex CS5100. Haemoglobin concentrations measured on Sysmex XN9000 (Sysmex, UK). Tissue Factor ELISA performed using ZYMUTEST Tissue Factor assay (Hyphen BioMed, UK).

Results: TF concentrations in sample pairs (n=10) were below the limit of quantification for the assay (3 pg/mL) and values were excluded from the statistical analysis. In the remaining sample pairs (n=30), the haemoglobin concentration in nHS, mean 0.15 g/L (median 0.0 g/L) was significantly different (P=<0.0001) from HS, mean 1.9 g/L (median 1.2 g/L). APTT in nHS, mean 27.4 seconds (median 26.4 seconds) and HS, mean 26.6 seconds (median 25.3 seconds) showed a statistically significant difference (P=0.0108). TF concentration in nHS ranged 3.7‐201.4 pg/mL and HS 3.0‐231.8 pg/mL (r 2 =0.902). TF in nHS, mean 25.4 pg/mL (median 11.1 pg/mL) versus HS mean 26.7 pg/mL (median 11.2 pg/mL) showed no significant difference (P=0.3432). One nHS APTT increased by 7.2 seconds, with 25% decrease in TF, however large TF changes were measured in sample pairs (range −51% to +52%). One nHS APTT decreased by 1.1 seconds with a 49% increase in TF.

TABLE 1 Observed changes in haemolysed and non‐haemolysed sample pairs Group Haemoglobin (g/L) APTT (sec) Tissue Factor (pg/mL) Haemolysed mean (median) 1.9 (1.2) 26.6 (25.3) 26.7 (11.2) Non‐Haemolysed mean (median) 0.15 (0.0) 27.4 (26.4) 25.4 (11.1) P‐value <0.0001 0.0108 0.3432

Conclusions: TF correlated strongly between nHS and HS pairs. No correlation was seen between TF and APTT changes in HS and nHS samples. This suggests that TF is not primary component of APTT change in HS.

FIGURE 1 Tissue Factor concentration in heamoysed and non‐ haemolysed sample pairs

PB568: Antithrombin Deficiency Has Lower Prognostic Value in Comparison with INR in Critically ill Patients with SIRS

E. Neporada 1 ; A. Travkov 1,2 ; S. Astrakov 1,3

1 Novosibirsk State University, Novosibirsk, Russian Federation; 2 Medpraktika Clinic, Novosibirsk, Russian Federation; 3 City Clinical Hospital #25, Novosibirsk, Russian Federation

Background: Low platelet count and increased INR are frequent laboratory findings in patients with systemic inflammatory response syndrome (SIRS) and both tests have prognostic value. Antithrombin (AT) deficiency is reported to be observed early in SIRS, but this test is not used routinely.

Aims: The aim of the study is to determine if AT deficiency is a useful prognostic tool in ICU patients with SIRS.

Methods: 135 consecutive ICU patients with 3 or 4 criteria of SIRS were included. During 4 days after SIRS manifestation patients were assessed for low platelet count <120 cells/mcL or severe platelet decline ≥50% during 24 hours in combination with INR>1.2. Also general condition, APACHE II prognostic score, organ disfunction, clinical and laboratory test results and 30‐days mortality was evaluated.

Results: Abnormal laboratory hemostatic tests’ results (low platelet count in combination with increased INR) were observed in 26.6% of critically ill patients with SIRS. In patients with severe AT deficiency <50% odds ratio for emerging of reduced platelete count together with increased INR was 3.5 (95% CI 1.2; 10.4).

TABLE 1 Variables, predicting abnormal laboratory hemostatic tests’ results (low platelet count in combination with increased INR) Independent variables Regression coefficient P Odds ratio 95% CI for OR Gender (females/males) 1.58 0.007 4.85 1.54; 15.30 AT deficiency <50% 1.26 0.022 3.52 1.20; 10.40 Fibrinolysis time −0.02 0.049 0.98 0.96; 1.00

However AT deficiency did not demonstrate significant prognostic value (P=0.18) in our study, while INR was a significant predictor of outcome (P=0.024). [tab_02]

TABLE 2 Variables, predicting outcome at the 30th day Independent variables Regression coefficient P Odds Ratio 95% CI for OR INR 1.94 0.020 6.92* 1.36; 35.25 APACHE II 0.12 0.014 1.13* 1.02; 1.24 Age (years) 0.03 0.065 1.03 1.00; 1.07 AT deficiency <50% −0.85 0.180 0.43 0.12; 1.48

Conclusions: AT deficiency is a frequent finding in patients with SIRS the same as low platelet count and increased INR. Yet such common laboratory test as INR in comparison with AT deficiency evaluation has higher prognostic value in ICU patients with SIRS.

PB569: DOAC Filter Effect on Thrombin Generation: Pre‐analytical Considerations and Analytical Perspectives

C. Bouvy 1 ; J. Evrard 1 ; R. Siriez 1 ; F. Mullier 2 ; J. Douxfils 1 ; D. Gheldof 1

1 University of Namur, Pharmacy, Namur, Belgium; 2 Université Catholique de Louvain, Hematology Laboratory, Yvoir, Belgium

Background: Thrombin Generation (TG) is known to be highly sensitive to Direct Oral Anticoagulant (DOAC) and this effect is currently under further evaluation with the ST‐Genesia, a new fully automated and standardized TG analyzer. Given the wide inter‐individual variability of DOAC levels and TG profiles over the therapeutic range, one can imagine that the DOAC measurement algorithm could be improved by comparing the TG of a patient under treatment to himself, with DOAC removed from his sample.

Aims: To evaluate the efficiency of a newly designed DOAC filter to remove DOAC from plasma sample. Recovery of baseline TG parameters and percentage of DOAC elimination assessed through evaluation of plasma drug concentrations were used as final outcome.

Methods: Fresh normal pooled plasma from 6 healthy volunteers were mixed with either dabigatran, rivaroxaban or apixaban at 0‐50‐100‐200‐300 ng/ml. Plasmas were tested before and after filtration on DOAC filter, using ST‐Genesia with STG‐DrugScreen application and on STA‐R Max analyzer using calibrated STA‐Liquid anti‐Xa or STA‐ECAII for anti‐Xa and anti‐IIa drugs, respectively (all products from Stago, France).

Results: All DOACs plasma level measurements returned below the LOQ of calibrated assays (Figure 1). Once filtered, all plasma samples had TG parameters within the same range. However, compared to the baseline non‐filtered sample (containing 0 ng/mL of DOAC), filtered samples had a slight increase of the thrombin peak (Figure 1) while lag time, time‐to‐peak, endogenous thrombin potential returned to baseline value.

FIGURE 1 Impact of DOAC filter on DOACs removal and interference with peak height in thrombin generation

Conclusions: DOAC filters efficiently remove apixaban, dabigatran and rivaroxaban from plasma up to 300 ng/ml and allows the recovery of the majority of TG parameters. The peak height increase observed after filtration needs further investigation. Additional studies are required to assess the efficacy and utility of DOAC filters on patients under DOAC treatment, for example in a thrombophilia testing setting.

PB570: Heparin‐induced Thrombocytopenia: An International Assessment of the Quality of Laboratory Testing

Z. Liederman 1 ; E.M. Van Cott 2 ; K. Smock 3 ; P. Meijer 4 ; R. Selby 1

1 University of Toronto, Toronto, Canada; 2 Harvard Medical School, Boston, United States; 3 University of Utah, Salt Lake City, United States; 4 ECAT Foundation, Voorschoten, the Netherlands

Background: Accurate diagnosis of heparin induced thrombocytopenia (HIT) is essential to ensure timely treatment and prevent severe complications. Currently, multiple unique assays are commonly used in practice.

Aims: To use external proficiency testing to assess the accuracy of HIT diagnostic methods.

Methods: From 2013 to 2017, the ECAT (External Quality Control for Assays and Tests) Foundation distributed 10 testing challenges (5 positive patient samples, 5 negative pooled patient samples) internationally to assess HIT assay proficiency. Laboratories submitted qualitative classifications as well as optical densities (OD) if applicable.

Results: In total, 3152 results were submitted from 437 laboratories across North America, Europe, Australia and Israel. Overall, 93% of positive samples and 88% of negative samples were correctly classified. Quantitative immunological assays including ELISA and latex immunoassays, were the most common methods used (63% of responses). Qualitative assays including gel agglutination and immunofiltration accounted for 37% of responses. Over the 5‐year period there was an absolute increase of 8% in qualitative method use and a 35% absolute increase compared to 2012 North American Specialized Coagulation Laboratory Association (NASCOLA) data. Quantitative methods correctly classified 96% of positive samples and 97% of negative samples. There was substantial interassay variation in positive OD's for each sample (35‐50%). Polyvalent assays had a higher false positive rate compared to IgG only assays (2% vs. 1%). Qualitative methods classified 89% of positive samples and 73% of negative samples correctly. However, the false positive rate for negative samples varied widely year‐to‐year.

TABLE 1 Response percentages for positive samples shown by mean and range (in brackets) for each assay class. Assays used by <1% of labs were excluded False negative result Borderline result True positive result Quantitative Assays 2 (0‐16) 2 (0‐47) 96 (47‐100) Qualitative Assays 6 (0‐14) 5 (0‐11) 89 (73‐100)

TABLE 2 Response percentages for negative samples shown by mean and range (in brackets) for each assay class. Assays used by <1% of labs were excluded True negative result Borderline result False positive result Quantitative Assays 97 (63‐100) 1 (0‐13) 1 (0‐25) Qualitative Assays 73 (29‐92) 7 (0‐8) 19 (1‐71)

Conclusions: Quantitative assays are used by the majority of laboratories, and classify samples with >95% accuracy, however agreement on OD is poor. Qualitative method use is increasing despite lower accuracy. Ongoing study is needed to identify sample traits that impact qualitative method specificity.

PB571: A Comparison of Two Point‐of‐Care Testing Platforms (Coaguchek ® Pro II and Coaguchek ® INRange) to Standard Laboratory Measurement of the International Normalised Ratio

S.H. Yeang 1 ; E.J.S. See 1 ; Y.T. Shim 1 ; M.C. Kong 1 ; S.K. Koh 1 ; Y.F. Lai 2 ; C.X.Y. Liew 1 ; W.H. Wong 1 ; H. Kaur 1 ; H.M. Tay 1 ; C.W. Tan 1 ; H.J. Ng 1

1 Singapore General Hospital, Singapore, Singapore; 2 Seng Kang Health, Singapore, Singapore

Background: Point‐of‐care testing (POCT) of the International Normalised Ratio (INR) using CoaguChek ® XS Plus and CoaguChek ® XS Pro platforms are the standard of care for monitoring our outpatients on warfarin. Our previous investigations have shown that there is a loss of concordance with INR performed using venous samples at INR>3.5. Our current test protocol calls for a mandatory venous INR for POCT INR>3.5. CoaguChek ® Pro II and CoaguChek ® INRange (home monitoring), are now available. There is a need to ensure their validity before using them in the clinical setting.

Aims: To compare the performance of the new platforms against venous sample and the current POCT INR tests.

Methods: Patients who attended our anticoagulation clinic and hospitalised patients on warfarin were recruited. After informed consent, 3 ml of venous blood was collected from each patient, followed by one capillary pin prick distributed to CoaguChek ® Pro II, CoaguChek ® INRange and CoaguChek ® XS Pro. Analyses were performed with INR readings performed within 4 hours apart. The primary outcome is the correlation coefficient between POCT and venous INR readings. The secondary outcome is the INR discrepancy between POCT and venous sample.

Results: A total of 142 venous samples and 406 POCT INRs were taken. The Pearson's Correlation coefficient (r) for CoaguChek ® Pro II, CoaguCheck ® INRange and CoaguChek ® XS Pro for INR were 0.976, 0.976 and 0.973 respectively (Figure 1).

FIGURE 1 Correlation between various CoaguChek® platforms and venous INR

Among the 3 machines tested, Coaguchek ® Pro II produced the most reliable results‐ (1) excellent r>0.9; (2) tolerance test for absolute INR difference fell within ±0.5 consistently; and (3) 95% limit of agreement within 0.5 from venous samples up to INR threshold of 4.5 (Figure 2).

FIGURE 2 Bland Altman plots between CoaguChek® Pro II and venous at various INR cut offs

Conclusions: Both CoaguChek ® Pro II and CoaguChek ® INRange have excellent r>0.9. The good degree of concordance with venous INR<4.5 for CoaguChek ® Pro II is higher than its predecessors. We can potentially use a cut‐off of POCT INR=4.5 before mandating venous INR to be taken for patients in the clinical setting.

PB572: Measurement of Coagulation Factors during Rivaroxaban and Apixaban Treatment: Results from Two Crossover Trials

L.J.J. Scheres 1 ; W.M. Lijfering 1 ; S. Middeldorp 2 ; Y.W. Cheung 2 ; S. Barco 3 ; S.C. Cannegieter 1 ; M. Coppens 2

1 Leiden University Medical Center, Department of Clinical Epidemiology, Leiden, the Netherlands; 2 Academic Medical Center, Department of Vascular Medicine, Amsterdam, the Netherlands; 3 University Medical Center of the Johannes Gutenberg University, Center for Thrombosis and Hemostasis, Mainz, Germany

Background: Prediction models for venous thromboembolism recurrence will likely be improved by adding levels of coagulation factors. Risk assessment is ideally performed during anticoagulant treatment, however, the influence of direct oral anticoagulants on coagulation factors is uncertain.

Aims: To assess the influence of rivaroxaban and apixaban on several coagulation factor levels.

Methods: In two crossover trials we assessed the influence of rivaroxaban and apixaban intake on factor (F)VIII, FXI and FXII‐activity and fibrinogen, von Willebrand Factor (vWF:Ag) and D‐dimer levels. At three sessions with a washout period in between, blood was taken from 12 healthy male individuals immediately before intake of rivaroxaban 15 mg twice daily (n=6) or apixaban 10 mg twice daily (n=6) and three hours after the last intake.

Results: Overall, measured levels were lower after rivaroxaban/apixaban intake. The paired mean difference after rivaroxaban intake was −38 IU/dL (95% CI −43; −33) for FVIII:C, −29 U/dL (95% CI −45; −12) for FXI:C, −22 IU/dL (95% CI −43; −1) for FXII:C, −0.11 g/L (95% CI −0.25; 0.03) for fibrinogen, −7 IU/dL (95% CI −18; 3) for vWF:Ag, −27 ng/mL (95% CI −50; −4) for D‐dimer and −0.36 (95% CI −0.57; −0.15) for Ln D‐dimer. After apixaban intake this was −29 IU/dL (95% CI −38; −21) for FVIII:C, −29 IU/dL (95% CI −36; −) for FXI:C, −19 IU/dL (95% CI −24; −15) for FXII:C, −0.18 g/L (95% CI −0.33; 0.03) for fibrinogen, −52 ng/mL (95% CI −100; −4) for D‐dimer, 0.25 (−0.60; 0.09) for Ln D‐dimer and 1 IU/dL (95% CI −7; 9) for vWF:Ag.

Conclusions: FVIII:C, FXI:C, FXII:C and D‐dimer measurements were influenced by rivaroxaban/apixaban intake, while fibrinogen and vWF:Ag were not.

PB573: Stability of Thrombin Generation and Thrombin Dynamics in Healthy Individuals over a One‐year Period

J. Konings 1,2 ; D. Yin 1,2 ; R. Kremers 1,2 ; D. Huskens 1,2 ; S. Bloemen 1,2 ; B. De Laat 1,2 ; H. Kelchermans 1,2

1 Maastricht University Medical Center, Synapse Research Institute, CARIM, Maastricht, the Netherlands; 2 Maastricht University Medical Center, Department of Biochemistry, CARIM, Maastricht, the Netherlands

Background: Epidemiologic studies have shown that thrombin generation (TG) is able to detect hypo‐ and hypercoagulability. To achieve clinical applicability, TG parameters should be relatively stable within one individual. Biological variation in levels of coagulation factors and platelets may impact this stability.

Aims: To determine the stability of TG and thrombin dynamics parameters in healthy individuals over a one‐year period.

Methods: Our protocol was evaluated by the local medical ethical board and all participants gave written informed consent. Blood was collected from 12 healthy individuals once per month during one year. Cell counts were performed. TG was determined in platelet rich plasma (PRP) at 1 pM tissue factor (TF) and in whole blood (WB) at 0.5 pM TF. In platelet poor plasma (PPP) TG and thrombin dynamics were determined at 1 pM and 5 pM TF. TG parameters determined were endogenous thrombin potential (ETP), peak height, lag time and time to peak. Thrombin dynamics parameters determined were thrombin decay capacity (TDC; capacity of the thrombin inhibitors in plasma to inhibit thrombin), total prothrombin conversion (PC) and maximal rate of PC (PC max).

Results: The intra‐individual variation was lowest in PPP at 5 pM TF (Table 1), ranging from 5.2% for the ETP to 11.5% for the PC max. For the lower TF concentrations, variations were comparable in PPP, PRP and WB (between 9.6% and 26.9% for all parameters). Although significant differences were demonstrated between different months, no obvious trend in monthly variation for TG or thrombin dynamics parameters in WB, PPP or PRP could be established (Figure 1). The monthly variation was comparable to the variation observed in platelet and white blood cell count.

Conclusions: In healthy individuals, the monthly variation in TG over a one‐year period was random and comparable to the variation in cell counts. Our data suggest that TG, particularly at 5 pM TF, is sufficiently stable within one individual to be used for clinical applicability.

TABLE 1 Mean intra‐individual coefficient of variation (%CV) for thrombin generation and thrombin dynamics parameters Thrombin generation parameters Thrombin dynamics ETP (%) Peak (%) Lag time (%) Time to peak (%) TDC (%) PC total (%) PC max (%) PPP 6.2 PPP‐1 pM TF 14.1 26.9 9.9 9.6 12.9 26.3 PRP‐1 pM TF 20.6 20.9 16.8 11.9 WB‐0.5 pM TF 18.7 17.1 13.2 12.0 PPP‐5 pM TF 5.2 9.2 7.7 6.3 6.3 11.5

FIGURE 1 Monthly variation in TG. Differences between months were analyzed by Friedman test with Dunn's post hoc test. *P<0.05, **P<0.01, ***P<0.001

PB574: The Association of Hereditary Thrombophilia with Clinically Relevant Hypercogulation State and Free Flap Thrombosis in Microvascular Surgery

K. Drizlionoka 1 ; J. Stepanovs 2 ; J. Krustiņš 3 ; A. Ozoliņa 4 ; L. Ņikitina‐ Zaķe 5 ; B. Mamaja 2

1 Riga Eastern Clinical University Hospital, Anaesthesiology Department, Riga, Latvia; 2 Riga Eastern Clinical University Hospital, Riga, Latvia; 3 The Centre of Plastic and Reconstructive Microsurgery of Latvia, Riga, Latvia; 4 Riga Stradins University, Riga, Latvia; 5 Latvian Biomedical Research and Study Centre, Riga, Latvia

Background: Thrombosis is the subject of concern in microvascular free flap surgery and hereditary thrombophilia could be the topic of interest.

Aims: To assess influence of hereditary thrombophilia on coagulation and thrombosis rate in free flap surgery.

Methods: 100 patients with free flap surgery were included in observational case control study. We selected 9 single nucleotide polymorphisms (SNPs) (rs5361 in SELE, rs2066865 in FGG, rs2227589 in SERPINC1, rs1613662 in GP6, rs13146272 in CYP4V2, rs2289252 in F11, rs1801133 in MTHFR, rs6025 in F5, rs1799963 in F2) reported to be associated with vascular thrombosis. Plasma fibrinogen concentration, antithrombin level, platelet count were analysed. Thromboelastometry (ROTEM) was performed for CT , CFT , MCF Fibtem, Intem and FPR (fibrinogen/platelet ratio) evaluation. Demographic data, cause of tissue defect, history of comorbidities, family history were collected.

Results: Patients with SNP in rs2066865 (G>A) in FGG gene had higher plasma fibrinogen concentration (G/G‐ 3.9 g/L±1.15; G/A‐ 4.75 g/L±1.31; A/A‐ 6.11 g/L±1.41) even in time period overrun 30 days. In 4/20 patients with FGG gene mutation thrombosis with complete flap failure eventuated. In all 20 patients thromboelastometry confirmed increased MCF Fibtem values (G/A‐ 27.77±12.02; A/A‐ 29.75±6.24). Higher rates of flap thrombosis was in group with recent trauma as an external trombogenic factor, SNPs did not significantly differ in both groups, except SNP in rs2066865 (G>A) in FGG gene, which was found in non‐ recent trauma group more often. SNP rs2289252 in F11 was found frequently however no correlation with thrombosis was found, rather indicating high prevalence in population.

Conclusions: Increased plasma fibrinogen concentration and thromboelastometry indicating hypercoagulation were found in patients with single rs2066865 (G>A) in FGG gene, however no clear association with increased risk for thrombosis in free flap surgery was found. Further data and larger sample size are needed.

PB575: Use of a Liquid Recombinant Thromboplastin (DG‐PT RecombiLIQ, Diagnostic Grifols) for Monitoring Vitamin K Antagonist Therapy

A. Woolley ; S. Kitchen

Sheffield Royal Hallamshire Hospital, Haemophilia and Thrombosis Centre, Sheffield, United Kingdom

Background: Liquid formulations for laboratory reagents save staff time and avoid reconstitution errors and consequent out of limits internal quality control results which can delay release of patient results. This means that liquid thromboplastin offers potential quality and patient safety improvements. For any new reagent data are needed to confirm accuracy of results and for comparisons to other reagents to help predict whether changing reagents could affect management of patients.

Aims: We aimed to assess use of DG‐PT RecombiLIQ across a full range of INRs by comparison to reference and widely used recombinant thromboplastins.

Methods: Samples from patients taking warfarin were collected into Vacutainer plus 0.109M citrate tubes. Prothrombin times were determined using DG‐PT RecombiLIQ (Q Smart analyser), Innovin (Sysmex CS 5100 analyser), Recombiplastin 2G (Werfen ACL TOP analyser) and the reference thromboplastin rTF 09 (manual tilt tube). Manufacturers instrument specific ISI were used with a locally determined mean normal PT to derive INRs. The following groups were studied: INRs<2 (n=18); INRs stabilised at 2‐4 (n=41): INRs in the range 4‐8 with rTF 09 (n=23), INRs of >8 with rTF 09 (n=7). Mean results were compared by ANOVA.

Results: The mean INRs of each group are shown in Table 1. For stabilised patients INRs with DG‐PT RecombiLIQ were on average <2% different from those obtained with rTF 09. In over‐anticoagulated patients DG‐PT RecombiLIQ results were approximately 15% lower than with the reference thromboplastin.

TABLE 1 Mean INR results Group DG‐PT RecombiLIQ Recombiplastin 2G Innovin rTF09 ANOVA INRs <2 1.77 1.78 1.72 1.63 P<0.0001 INRs 2‐4 2.69 2.87 2.70 2.66 P<0.0001 INRs 4‐8 5.20 6.30 6.31 6.10 P<0.0001 INRs >8 7.66 9.71 8.91 8.83 P<0.0001 All samples 3.54 4.07 3.93 3.83 P<0.0001

Conclusions: Although some differences were noted at elevated INRs laboratories switching between Innovin, Recombiplastin 2G and DG‐PT RecombiLIQ should not see differences in prescription of vitamin K antagonist drugs. Despite the very low ISI of DG‐PT RecombiLIQ, (0.86 on Q Smart) results in INR were not too prolonged at these INR levels. DG‐PT RecombiLIQ is suitable for routine use in monitoring VKA therapy. Liquid formulation offers advantages over lyophilised.

PB576: Heparin Anti‐Xa Assay versus Activated Partial Thromboplastin Time to Monitor Unfractionated Heparin during Extracorporeal Membrane Oxygenation

D. Jayakody Arachchillage 1,2 ; E. Dhillon 1 ; C. Vandenbriele 1 ; J. Dutton 1 ; N. Bhudia 1 ; M. Laffan 2

1 Royal Brompton & Harefield NHS Foundation Trust, London, United Kingdom; 2 Imperial College London, London, United Kingdom

Background: Bleeding and thrombotic complications are leading causes of morbidity and mortality in patients receiving extracorporeal membrane oxygenation (ECMO). We use an unfractionated heparin [UFH] infusion to achieve anti‐factor Xa (anti‐Xa): 0.2‐0.3 IU/ml to prevent thrombotic complications in the patient or in the ECMO circuit. In house comparisons with an anti‐Xa chromogenic assay showed that an activated Partial Thromboplastin Time (APTT) of 50‐60sec corresponded to the target anti‐Xa 0.2 to 0.3 IU/mL.

Aims: To determine whether the APTT could be used to monitor anticoagulant intensity in these acutely septic patients

Methods: Patients were heparin dosed according to anti‐Xa level (Liquidanti‐Xa) results and APTT (SynthASil) recorded in parallel. Time to therapeutic anticoagulation, thrombotic and bleeding complications were recorded. Clauss fibrinogen level was done using FIB‐C XL.

Results: A total of 79 patients (mean age 46 years, M: F 50:29) were included; 75 (95%) patients achieved a target anti‐Xa within an average of 20.1 hours compared to only 40 (50%) achieving a target APTT within average 48.6 hours (P<0.0001). Thirty nine patients who did not achieve a target APTT had significantly higher fibrinogen level compared 40 patients who did (mean fibrinogen g/L [confidence interval] 5.4 [4.5‐6.9] vs. 3.2 [2.2‐4.1], (P=0.004).The median number of tests prior to target APTT was double that for anti‐Xa 9 vs. 4, (P<0.0001). Bleeding or thrombosis complications were present in 25% (20/79) and 18% (14/79) respectively. Eight of 11 patients with major bleeding had APTT below the target level but anti‐Xa >0.3 IU/ml at the time of bleeding whilst 57% (8/14) of the patients developing thrombosis had APTT>60 seconds but anti‐Xa values below the target (<0.1 IU/ml) at the time of thrombotic events.

Conclusions: APTT levels frequently remained sub‐therapeutic for a considerable time after a therapeutic level of anti‐Xa had been reached, which required fewer tests, correlated better with adverse bleeding and thrombotic events.

PB577: An Automated Modified Anti‐Xa Assay for the Potency Assessment of Bemiparin in Pharmaceutical Formulations

V. Monserrat; S. Aguirre; R. Raimondi; A. Scazziota

Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Hospital de Clínicas “José de San Martín”, Departamento de Bioquímica Clínica, Laboratorio de Hemostasia y Trombosis, Buenos Aires, Argentina

Background: Bemiparin, a second‐generation low‐molecular weight heparin (LMWH), has a mean MW of 3,600 Da and a large anti‐Xa(AXa):anti‐IIa ratio(8:1). Assays based on inhibition of factor Xa activity are used to assess the biological potency of LMWHs, but they were mainly designed for clinical use in first‐generation preparations rather than pharmaceutical formulations.

Aims: To assess the potency of a pharmaceutical sample of a Bemiparin Sodium injection using a modified anti‐factor Xa automated assay.

Methods: Human antithrombin III 10 IU (Chromogenix ® ) was added to classical STA‐liquid Anti‐Xa ® (Stago) to measure AXa activity in a STA Compact Max Analyzer ® (Stago). We reduced the sample dilution and doubled the kinetic method reading times. Independent dilutions of BRP (European Pharmacopoeia Reference Standard, H0185000, 102 IUAXa/vial) and Bemiparin equivalent to 12,500 IU AXa/ml (range 0.025‐0.204 IU AXa) were tested. Repeatability was assessed with 0.025 and 0.204 AXa/ml samples in quadruplicate, intermediate precision (interday) was assessed by analyzing the 0.025 AXa/ml. Accuracy was assessed in 7 assays at a potency level of 100%. We determined the potency of 2 different samples of Bemiparin.

Results: A linear regression line was obtained and values of the determination coefficient (r 2 =0.9269 and r 2 =0.9347) indicate good linearity.The repeatability was 0.13% with bemiparin activities from 11,653.3 to 15,607.9 UI/ml with a value for combined potency of 13,570.1 IU/ml. Interday precision was 8.13% with activities from 11,653.3 UI/ml to 17,691.5 UI/ml. Accuracy was 6.05% with a mean relative potency of 101.44%. Evaluated Bemiparin samples showed potency values of 105.9% and 92.6%.

Conclusions: We adapted an automated AXa assay obtaining good linearity, precision and accuracy. In a near future, robustness and limits of detection and quantitation should be determined in order to validate this proposed assay.

PB579: The Impact Of »In Vitro« Added Idarucizumab On Thrombin Generation

N. Vene ; A. Mavri; M. Bozic Mijovski

University Medical Centre Ljubljana, Department of Vascular Diseases, Ljubljana, Slovenia

Background: Idarucizumab is a highly selective monoclonal antibody fragment that binds a direct thrombin inhibitor dabigatran and fully neutralizes its anticoagulant effect. It is believed that idarucizumab administration does not have procoagulant properties.

Aims: The aim of this study was to assess the influence of “in vitro” addition of idarucizumab on thrombin generation in plasma samples from patients with atrial fibrillation treated with dabigatran.

Methods: Blood was drawn from 44 patients before initiation of therapy with dabigatran (baseline) and then twice at trough and twice at peak dabigatran concentration. In plasma, thrombin generation was measured before (DABI) and after the addition of idarucizumab to plasma (final concentration of 125 mg/L, DABI+I) with Technothrombin TGA RC Low reagent and Technothrombin substrate (both Technoclone, Austria). Lag time, time to peak thrombin (TTP), peak thrombin and area under the curve (AUC) were recorded. Dabigatran concentration was measured with the in‐house modified thrombin time.

Results: The addition of idarucizumab to plasma samples significantly shortened lag time and TTP to the levels comparable to baseline values. Idarucizumab significantly increased the amount of thrombin formed, as AUC was significantly lower in DABI and significantly higher in DABI+I samples compared to baseline. Peak thrombin was similar in DABI and baseline samples, but significantly increased in DABI+I

TABLE 1 Thrombin generation in “ex vivo” samples with dabigatran (DABI), after the addition of idarucizumab (DABI+I) and baseline samples. Means ± standard deviations are shown with ANOVA P values Thrombin generation DABI DABI+I Baseline P Lag time (min) 36.4±9.6 * 16.0±4.2 17.2±3.3 <0.001 Peak Thrombin (nM) 208±102 270±94 * # 203±86 <0.001 TTP (min) 43.3±10.6 * 22.5±5.8 24.6±4.7 <0.001 AUC 2184±1126 * 4138±692 * 3400±726 <0.001 *Post‐hoc P<0.001 compared to baseline values, # post‐hoc P<0.001 compared to DABI

Conclusions: Our study showed significantly increased peak thrombin and AUC of a thrombin generation assay in plasma samples from patients receiving dabigatran after addition of idarucizumab, suggesting an “in vitro” hypercoagulable effect.

PB580: Limits to Sensitivity of 4T Score for Heparin Induced Thrombocytopenia

D. Jayakody Arachchillage 1,2 ; F. Kamani 1 ; S. Fox 1 ; K. Patel 1 ; M. Laffan 2

1 Royal Brompton & Harefield NHS Foundation Trust, London, United Kingdom; 2 Imperial College London, London, United Kingdom

Background: Heparin induced thrombocytopenia (HIT) should be suspected if the 4T pre‐test probability (PTPS) score is high (6‐8) or intermediate (4‐5). However, thrombocytopenia is common in patients following cardiopulmonary bypass or acute sepsis which may reduce the specificity of this score.

Aims: To investigate whether the standard PTPS can identify HIT in a major UK cardiac surgical and extracorporeal membrane (ECMO) centre.

Methods: HIT screening was performed in patients who had received heparin and developed thrombocytopenia with typical pattern of platelet drop for HIT with or without thrombosis. A citrated blood sample and a completed pre‐test probability (4Ts) score were collected from all patients, prior to the HIT screening test. Scores were matched with platelets counts and evidence of thrombosis from radiological reports. HIT antibody testing was performed on an ACL TOP500 analyser using Hemosil HIT‐Ab (PF4‐H) kit (Werfen UK). Those with positive HIT screening underwent confirmatory testing by ELISA (HYPHEN BioMed, France).

Results: In 15 months, 47 patients had HIT screening tests. Median (range) platelet count pre heparin exposure and on the day of HIT screening were 148 (107‐426) × 10 9 /L respectively. Twenty patients (20/47, 42.6%) had a positive screening test and all were confirmed positive by ELISA (100% positive predictive value). The median PTPS of these patients was 5 (3‐7). However, 4/20 (20%) patients had a PTPS of 3. Seventeen of 20 (85%) with positive HIT screen and confirm tests had proven thrombosis including 3 patients with PTPS of 3. The median PTPS of patients with negative HIT screening was 4 (3‐6).

Conclusions: Although the PTPS was good at predicting HIT in the majority of patients, it was not useful in some . Guidelines suggest that a 4T score of 0‐3 represents a low risk for HIT and does not require further testing, but this did not prove correct in our patient group. The 4T score did not always discriminate patients with positive or negative HIT tests.

PB581: External Quality Assessment of Point of Care Testing in Haemostasis

S. Munroe‐Peart ; D. Kitchen; L. Brown; I. Jennings; S. Kitchen; T. Woods; I. Walker

UK NEQAS for Blood Coagulation, Sheffield, United Kingdom

Background: Point of care testing (POCT) in hemostasis has increased in diversity, technologies employed and the number of centres performing POCT. The quality of POCT results need to be assessed in the same way as laboratory results. This should include External Quality Assessment (EQA) using material which is, as far as possible, commutable with patient samples.

Aims: The UK National external assessment service for Blood Coagulation (NEQASBC) aims to provide EQA programmes meeting the requirement for commutable samples for a wide range of hemostasis POCTs. Programmes are provided for INR monitoring for 7 different POCT devices, for ACT+ and ACT LR, thromboelastography (TEG) and thrombelastometry (ROTEM), and D‐dimer on POCT devices.

Methods: EQA samples are distributed to centres for analysis and results returned to UK NEQAS BC. For each exercise, median values are calculated together with a target range. Centres’ results falling outside the target range are deemed “outwith consensus”, indicating a potential problem with the centres’ performance.

Results: EQA exercises demonstrate generally good performance (POCT INR CVs are comparable to CVs in the laboratory INR programme). EQA has highlighted device issues which could have resulted in incorrect patient results being produced, including an INR device found to have electrical corrosion, a Rotem user found to be using expired reagents, and a TEG participant using inappropriate reagents. Inappropriate interpretations of D‐Dimer results have been demonstrated.

Conclusions: These programmes demonstrate the value of EQA in detecting analytical and post‐analytical errors. Importantly, these data reflect the activity only amongst centres enrolled in the UK NEQAS BC POCT programmes. In the absence of a requirement for independent accreditation of POC testers, there is no obligation for centres performing POCT to enrol in an EQA programme, and therefore no assurance of the quality of results used to make clinical decision on patients in centres not performing EQA.

PB582: Clot Waveform Analysis for Measuring and Quantifying Fibrin Clot Formation

J. Evrard 1 ; R. Siriez 1 ; P. Themans 2 ; J. Laloy 1 ; F. Mullier 3 ; J.‐M. Dogné 1 ; J. Douxfils 1

1 Université de Namur, Department of Pharmacy, Namur, Belgium; 2 Université de Namur, Department of Mathematics, Namur, Belgium; 3 Université Catholique de Louvain, CHU UCL Namur, Yvoir, Belgium

Background: Most widely known global coagulation assays are thrombin generation test (TGT) and viscoelastometric assays (ROTEM or TEG). They both analyze an endpoint of the coagulation cascade and allow the detection of several coagulation abnormalities. The fibrin generation can be monitored to provide an analysis of the optical waveform generated during clotting test allows the exploration of the coagulation process in more details.

Aims: This study aims at investigating which test between TGT and fibrin generation test (FGT), defined as the first derivative of the clot waveform analysis, is the more sensitive in regards to the coagulation process.

Methods: Thrombin generation in plasma was monitored during 45 minutes using CAT ® analyser. The fibrin clot generation was monitored during 15 minutes at 671 nm on a TECAN analyser. Normal pooled plasma (NPP) was incubated at 37°C with an inducer of the intrinsic pathway of the coagulation (Actin FS ® ; Siemens) or inducers of the extrinsic pathway (PPP reagent ® or PPP reagent Low ® ; Thrombinoscope BV). Briefly, 20 μL of reagent were mixed with 80 μL of NPP in a 96‐well microplate and incubated for 3 minutes at 37°C. The coagulation process was triggered by the addition of 20 μL of FluCa solution (Thrombinoscope BV ® ) for TGT or 20 μL of 100 mM CaCl2 for FGT, respectively.

The first derivative curves were calculated from sigmoid curves for FGT to obtain a Gaussian curve comparable to thrombin generation curves.

Results: The curves of first derivative of fibrin generation process starts earlier than thrombin generation for all reagents tested. The mean parameters generally measured are different. The time to peak (Ttpeak) is also shorter for fibrin generation. This reflects that fibrin generation occurs before the CAT ® analyser is able to detect the thrombin generation. This supposes FGT has higher sensitivity than TGT.

FIGURE 1 Comparison of fibrin generation test and thrombin generation test in the same conditions

Conclusions: This study unlocks new perspectives about fibrin generation test. Further assays have to be done to improve sensitivity and quality of our tests.

PB583: The Calibrated Automated Thrombography in Patients with Factor XII Deficiency

O. Matvienko; N. Silina ; O. Golovina; L. Papayan

Russian Research Institute of Haematology and Transfusiology, Saint‐Petersburg, Russian Federation

Background: Factor XII deficiency is associated with failure in the system of contact activation. The Calibrated Automated Thrombography (CAT) can be useful in the functional testing of coagulation system of patients with factor XII deficiency.

Aims: To estimate the coagulation system state in factor XII deficiency patients with thrombin generation test.

Methods: The study included 14 patients with factor XII deficiency and thrombotic complications (24‐72 years, median=37.5) and 21 healthy controls (20‐61 years, median=31.5). Factor XII activity was measured with “ACL TOP 300” coagulometer, Instrumentation Laboratory, USA, XIIa‐dependent fibrinolysis ‐ with reagent “RENAM”, Russian Federation. Thrombin generation was assessed in platelet poor plasma by CAT according to Hemker et al. The following parameters were evaluated: endogenous thrombin potential (ETP, nM*min), peak thrombin (Peak, nM), lag‐time (LT, min), and time to peak (TTP, min). STATISTICA 6.0 was used. Results are given as median (Me) with 95% confidence intervals (CI), P<0.05 was considered statistically significant.

Results: View a Table

TABLE 1 Parameters Patients (n=14) Controls (n=21) Factor XII activity, % 30.65 * (0.36‐74.12) 110.00 (75.70‐151.50) XIIa‐dependent fibrinolysis, min 11.00 * (5.30‐31.00) 5.33 (4.15‐7.33) LT, min 3.42 * (2.31‐7.36) 3.00 (2.00‐4.00) TTP, min 6.93 * (4.78‐14.5) 6.00 (4.16‐8.00) *P<0.05 compare to controls.

ETP and Peak values were not differed from controls.

Conclusions: Patients with factor XII deficiency have significant prolongation time of XIIa‐dependent fibrinolysis and time parameters (LT, TTP) of CAT. These results suggest sluggish initiation of thrombin generation because of contact activation system failure in the patients.

PB584: Thrombin Generation Analysis with a New Automated System (ST‐Genesia): Inter‐series Performances during DRIVING Study and Comparison with CAT System

V. Siguret 1,2 ; G. Foulon 1,2 ; J. Abdoul 1 ; A. Carlo 3 ; T. Lecompte 4,5 ; I. Gouin‐Thibault 6,7

1 Université Paris Descartes, Sorbonne Paris Cité, UMR‐S 1140, Paris, France; 2 Hôpital Lariboisière ; AP‐HP , Hématologie Biologique, Paris, France; 3 Diagnostica Stago, Asnières, France; 4 Université de Genève, Genève, Switzerland; 5 Hôpitaux Universitaires de Genève, Genève, Switzerland; 6 Université Paris Descartes, Sorbonne Paris Cité, Paris, France; 7 Hôpital Pontchaillou, Hématologie Biologique, Rennes, France

Background: The DRIVING study aimed at evaluating the PK response variability to direct oral anticoagulants of 60 healthy volunteers (Gouin et al , JTH 2016 ). We also assessed thrombin generation (TG) in different time‐point samples over a 24‐hour period after a single rivaroxaban intake using ST‐Genesia. This is a new fully automated analyzer enabling quantitative standardized TG evaluation using dedicated reagents, calibrator and internal quality controls (IQC). No comparisons of this system with CAT have been published yet.

Aims: To determine ST‐Genesia inter‐series precision using 3 experimental conditions during the experiments conducted for DRIVING study and to perform, in a subset of samples, a comparison with CAT.

Methods: DRIVING samples were tested with ST‐Genesia using two tissue factor concentrations in the picomolar range, higher in STG‐DrugScreen reagent than in STGThromboScreen reagent, the latter with and without thrombomodulin (TM) (Stago, France). For each run, IQC and reference plasma enabling normalization of results (expressed as ratio or %) were tested and coefficients of variation (CVs) calculated. We also tested 47 DRIVING samples covering a wide range of TG profiles with CAT and PPP‐reagent (Stago).

Results: 594 tests were performed with ST‐Genesia over 15 runs. IQC CV results with ThromboScreen without TM are reported on Table 1.

TABLE 1 ST‐Genesia results for the 3 IQC plasmas using STG‐ThromboScreen without TM ‐ ratios and % calculated with reference plasma results TG parameters Low STG‐QualiTest Low STG‐QualiTest Normal STG‐QualiTest Normal STG‐QualiTest High STG‐QualiTest High STG‐QualiTest Mean (n=15) CV (%) Mean (n=15) CV (%) Mean (n=15) CV (%) Lag time (min) 2.12 5.4 1.58 4.5 1.35 5.0 Lag time (ratio) 1.20 6.10 0.90 6.50 0.77 5.20 Peak height (nM) 78.0 10.0 229.0 5.8 413.0 2.8 Peak height (%) 28.0 7.8 82.0 4.3 148.0 5.4 Time to peak (min) 5.0 4.3 3.8 3.8 2.4 3.0 Time to peak (ratio) 1.29 3.90 0.98 2.80 0.61 2.80 Endogenous thrombin potential (nM*min) 485.0 6.3 1189.0 4.2 1346.0 5.5 Endogenous thrombin potential (%) 35.0 4.3 86.0 5.1 98.0 5.3

Satisfying CVs were obtained using the other conditions as well. Comparisons between ThromboScreen without TM with ST‐Genesia and PPP‐reagent with CAT showed limited biases (Figure 1).

FIGURE 1 Agreement between ST‐Genesia (ThromboScreen without TM) and CAT (PPP reagent) results examined using Bland‐Altman difference plots (n=47)

Coefficients of determination (R 2 ) were of 0.894 for lag‐time, 0.918 for time to peak, 0.834 for peak height and 0.615 for endogenous thrombin potential.

Conclusions: This study is the first extensive ST‐Genesia test campaign with samples from anticoagulated subjects.

The good inter‐series precision highlighted the reliability and standardization of TG assessment with this new system.

In addition, we obtained a good agreement between ST Genesia and CAT under our experimental conditions.

PB585: Evaluation of Direct Oral Anticoagulants Assays in External Quality Control Surveys

C. Bon 1 ; R. Meley 2 ; M. Lopez 1 ; C. Sotta 1 ; J.‐C. Eynard 1 ; B. Poggi 3

1 PROBIOQUAL, Lyon, France; 2 Hopital Nord, Laboratoire d'Hématologie, St Etienne, France; 3 Hopital de la Croix‐Rousse, Service de Biochimie et Biologie Moléculaire, Lyon, France

Background: Direct Oral Anticoagulants (DOAC) prescription is continuously increasing and monitoring of their concentration is necessary for certain clinical situations. Commercial specific tests are now available, based on various methodologies.

Aims: The aim of this work is to evaluate the performances of DOAC tests in routine practice by using External Quality Assessment results of Apixaban, Dabigatran and Rivaroxaban assays,obtained from ProBioQual, a French proficiency testing association.

Methods: Participant laboratories (108 in 2017) received during the year, 18 lyophilized human citrated plasmas : 6 spiked with Apixaban,6 with Dabigatran, 6 with Rivaroxaban, at 2 or 3 levels for each drug. Statistical evaluation was performed according to the ISO 13528 guideline by applying robust algorithm A. The consensus value was calculated for all participants together (AP) and for each peer group (PG). The imprecision of the methods was evaluated by the inter‐laboratory coefficient of variation (CV), and the accuracy was quantified as median bias (bias50) and 90th percentile bias (bias90) of laboratory results from AP consensus value.

Results: The inter‐laboratory dispersion is inversely proportional to the DOAC concentration. For Apixaban and Rivaroxaban, the AP CVs decrease respectively from 13.8% to 7.0%, and 10.7% to 6.8%, for a concentration of 80‐300 μg/L. With Dabigatran,the AP CV is 13% for 62 μg/L and 6.9% for 285 μg/L.

For high levels, AP bias90 and bias50 are the same for Apixaban and Rivaroxaban (respectively 12% and 4.5%), whereas for the lowest level, AP bias90 are lower for Rivaroxaban (20% and 6.3%) than for Apixaban (22% and 9.5%). With Dabigatran, AP bias90 vary from 25% to 15%, higher than for the other drugs.

Conclusions: The commercial DOAC tests have satisfactory routine performances for the measurement of Apixaban, Dabigatran and Rivaroxaban concentrations, with acceptable inter‐laboratory variability.

PB586: D‐dimer Measurement: Lipid Interference Removal through High‐speed Centrifugation

F. Estève 1 ; M. Ramzi 1 ; N. Jonathan 1 ; S. Goulet‐Cornilleau 1 ; A. Auby 2 ; A. Carlo 2 ; M. Grimaux 1

1 Stago, Gennevilliers, France; 2 Stago, Asnières‐sur‐Seine, France

Background: D‐dimer immunoturbidimetric assays are based on the change in turbidity of a suspension of microlatex particles as measured by photometry. Plasmas with high lipid levels have an optical aspect that can interfere with D‐dimer measurements for strong cloudy aspects as reported on some product package inserts or in literature (Nagant et al Clin Lab 2016) . CLSI C56 guideline explains that this optical interference is due to elevated levels of lipids in the sample, resulting in a high self‐turbidity. Even if not very frequent (0.02% as per Mainali et al, Practical Laboratory Medicine 2017 ) this self‐turbidity can become an issue for the clinical laboratory. High‐speed micro centrifugation or some samples pretreatment solutions are options proposed to remove this interference on tests (Vermeer HJ et al, Clin Chem Lab Med 2007 and Lippi et al Semin Thromb Haemost 2013 ), but these lack description and are not available in every laboratory.

Aims: Provide guidance on how to overcome easily the lipid interference limitation for D‐dimer level measurement in cloudy plasma samples.

Methods: Patients plasma samples with high lipid contents (triglycerides level >3.93 g/L) were centrifuged at 10 000 g for 1, 2, 3, 4, 5 or 6 minutes and at 5 000 g for 3, 5, 10, 15, 20 minutes. Particular care was taken to avoid re‐suspending the superior lipidic phase formed through centrifugation. Residual OD at 540 nm of the 1/6 diluted milky plasma sample was measured with a spectrophotometer.

Results: High speed centrifugation reduces the self‐turbidity of the samples and an optimal centrifugation duration can be identified to remove the optical interference due to lipids.

FIGURE 1 Effect of centrifugation at 10,000 g of a lipaemic sample

FIGURE 2 Effect of centrifugation at 5,000 g of a lipaemic sample

Conclusions: Our data show that high‐speed centrifugation at least 2 minutes at 10 000 g or 5 minutes at 5 000 g is safe and effective in removing lipid particles as seen through optical density measurements. This lipid particles removal should reduce significantly the optical interference in lipemic patient samples and bring it to an acceptable level to secure D‐dimer assays.

PB588: Development of a HPLC‐UV Method for the Measurement of Dabigatran from Plasma and its Application as Reference Method for the Evaluation of a Novel Chromogenic Dabigatran Assay

A. Kuruczné Kern ; M. Olajos; A. Keczán; J. Antal

Diagon Ltd, Budapest, Hungary

Background: Dabigatran is a direct thrombin inhibitor, the application of which is spreading worldwide. Although dabigatran therapy does not require monitoring, there is a growing demand for new haemostasis assays applicable for the assessment of dabigatran level in certain medical situations like trauma or overdosing. For the evaluation of these tests usually LC‐MS/MS is used as reference method. Since LC‐MS/MS is not commonly available and the quantification requires expensive isotope labelled dabigatran, the development of a HPLC‐UV method for the measurement of dabigatran concentration in patient samples would be advantageous.

Aims: Our aim was to develop a HPLC‐UV method for determination of dabigatran level and to use it as a reference method for the evaluation of our novel chromogenic assay.

Methods: Prior to the HPLC‐UV measurement interfering proteins were eliminated from plasma samples by addition of trifluoro acetic acid, heat treatment, and filtering. Samples were analysed by reversed phase HPLC at 292 nm UV detection. Normal plasma pool spiked with dabigatran at various concentrations was applied for calibration and for the analytical characterisation of the method. External dabigatran control plasmas were applied to validate the method. Plasma samples from patients taking dabigatran were measured by our novel chromogenic dabigatran assay and the developed HPLC‐UV method.

Results: With the developed HPLC‐UV method linear calibration was observed up to 800 ng/ml dabigatran concentration, with LoD of 5 ng/ml and LoQ of 20 ng/ml. The method was appropriate for the determination of the level of dabigatran in control plasma samples. Excellent correlation was observed between the HPLC‐UV method and our novel chromogenic dabigatran assay.

Conclusions: We developed and characterised a HPLC‐UV method for the determination of dabigatran level in plasma samples. Using it as a reference method excellent correlation was observed with our novel chromogenic dabigatran assay on patient samples.

PB589: Fibrinolysis System and Combined oral Contraceptives

N. Vorobyeva ; D. Gamyrkina

Nord State Medical University, Haemostasiology, Arckhangelsk, Russian Federation

Background: According to some researchers, fibrinolytic activity of blood has an increasing tendency on the background of taking hormonal drugs, and this is often explained by the compensatory enhancement of fibrinolysis activity on the background of hypercoagulable development. On the other side, there is the oppression of the fibrinolytic system due to decrease in the synthesis of the tissue activator of plasminogen by endothelial cells and an increase in the level of PAI‐I by hepatocytes. In recent years scientists pay more attention to hereditary factors predisposing to disorders in hemostatic and thrombotic complications among patients taking COCs.

Aims: The objective is to study the effect of genetic polymorphisms on the fibrinolysis system when using COCs.

Methods: A cross‐clinical laboratory study was conducted among 100 women at the average age of 29,0 (23,3‐35,0) years. Depending on the COC's reception mode women were divided into two groups (n=50 in each group). In the process of the study, the fibrinolytic activity of blood plasma was determined using the euglobulin fraction lysis method with streptokinase stimulation. The result data of molecular genetic studies of six polymorphisms (fibrinogen G455A, prothrombin G20210A, clotting factor V G1691A, MTHFR C677T, PAI‐I 675 4G / 5G, platelet receptors GpIIIa T1565C, ApoE E2 / E3 / E4) were analyzed: the linear regression method was used to determine independent factors that affect the level of fibrinogen.

Results: The presence of a homozygous form of fibrinogen polymorphism (‐455 A / A) is the statistically significant factor of inhibition of fibrinolysis in a single‐factor analysis (P=0.045). The significance level was slightly lower than statistically significant in multi‐factor analysis (P=0.054).

Conclusions: In our opinion, the increased expression of the fibrinogen gene among carriers of the “‐455 A/A” allele may cause the inhibition of fibrinolysis.

PB590: A Novel Prothrombin Time (PT) Assay ‐ A Tool for Rapid Screening of All Non‐Vitamin K‐Dependent Oral Anticoagulants (NOACs)

M. Abelius 1 ; E. Theodorsson 2 ; M. Rånby 3 ; T.L. Lindahl 2

1 MediRox AB, Nyköping, Sweden; 2 Linköping University, Department of Clinical Chemistry and Department of Clinical and Experimental Medicine, Linköping, Sweden; 3 Zafena AB, Borensberg, Sweden

Background: Non‐vitamin K‐dependent oral anticoagulants (NOACs) have been shown to interfere with routine and specialty coagulation assays. Thus, an unknown presence of NOACs can result in misdiagnosis. Several assays are currently needed to confirm or exclude the presence of NOACs. This is impractical and costly, a comprehensive screening assay for all NOACs is needed.

Aims: To develop a prothrombin time (PT) assay with sufficiently high sensitivity to NOACs to detect all interfering NOACs when used together with a conventional PT assay (insensitive to NOACs).

Methods: Two PT assays, one sensitive (newly developed prototype) and one insensitive (MRX Owren's PT, MediRox) to NOACs, were calibrated using NOAC‐free plasma samples with known INR values. A PT ratio was calculated by dividing the INR values generated from the NOAC sensitive PT assay with INR values from the NOAC insensitive PT assay. Plasma samples from healthy donors (n=21), patients treated with apixaban (n=40), rivaroxaban (n=16) and dabigatran (n=2) were analysed with the two PT reagents on Sysmex CS 2100i and quantitative assays for NOACs with α‐FXa methods and diluted thrombin time at the central hospital laboratory. The study was done according to Swedish law on biobanking and research ethics.

Results: The presence of NOACs was detected (as a PT ratio above the mean plus 2*SD for the NOAC‐free samples) in all plasma samples from patients treated with rivaroxaban (range 50‐406 μg/L) and dabigatran (range 40‐120 μg/L), and 37 of the 40 samples with apixaban (range 55‐426 μg/L). A linear relationship was shown between the PT ratio, representing the functional effect of the drug on the coagulation, and the concentration of NOAC determined by chromogenic substrate methods, R 2 =0.63 for apixaban and R 2 =0.69 for rivaroxaban.

Conclusions: This novel PT assay can be used to detect dabigatran, apixaban and rivaroxaban in patient plasma, supporting its potential use as a screening assay for detection of all NOACs.

PB591: Reference Change Value for INR: A Simple Tool for Management of Patients on Oral Anticoagulation

S. Aguirre; V. Monserrat; R. Raimondi; A. Scazziota

Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Hospital de Clínicas “José de San Martín”, Departamento de Bioquímica Clínica, Laboratorio de Hemostasia y Trombosis, Buenos Aires, Argentina

Background: Patients on oral anticoagulation with vitamin K antagonists (VKAs) are monitored with the International Normalized Ratio (INR). To decide the dose adjustment it needs to be known whether a difference between two INR values represents a biological change in intensity of treatment or it is only due to inherent sources of variation: intraindividual (CVI), preanalytical (CVP) and analytical imprecision (CVA), which are included in the Reference Change Value (RCV). To be significant, a change between serial results should be greater than the RCV.

Aims: To determine the CVI in patients receiving VKAs and to calculate a RCV for INR for personalized dose adjustment.

Methods: We retrospectively analyzed 15 patients (4 women, 11 men, mean age 76), receiving a constant dose of acenocoumarol (AC) with an INR value between 2 and 3. Treatment period was 6 months or longer with at least 6 consecutive INR measurements within therapeutic range. Prothrombin times (PTs) were measured with STA‐Neoplastin R ® in a STA Compact Max ® (Stago), INRs using the International Sensitivity Index provided by the manufacturer. For each selected patient the total coefficient of variation (CVT) was calculated for the consecutive INR measurements. CVA came from the internal quality control. Mean CVI was calculated as CVI=(CVT 2 ‐CVA 2 ) 1/2 . CVP was considered minimal. RCV was calculated as: 2 1/2 . Z .(CVA 2 +CVI 2 ) 1/2 , where Z is 1.96 for P<0.05.

Results: Mean CVT was 11.18% (range 4.78%‐16.69%), with a CVI of 11.44%. The analytical imprecision observed for this pair reagent‐analyzer was 2.41% and the calculated RCV for INR values was 9.27%.

Conclusions: Current recommendations are given with respect to adjustments of VKAs dosages if the INR is outside the target range. The calculated RCV is a simple tool that could be used when an INR result is in the range limit and it would imply a dose change, as well as in case of an abrupt change even within the INR target range.

PB593: Reproducibility of Rotem ® Device

E. Scalambrino 1,2 ; L. Padovan 1,2 ; V. Chantarangkul 1,2 ; M. Clerici 1,2 ; F. Peyvandi 1,2,3 ; A. Tripodi 1,2

1 IRCCS Cà Granda Foundation, Maggiore Hospital Policlinic, Internal Medicine‐Hemostasis and Thrombosis, Milan, Italy; 2 Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan, Italy; 3 University of Milan, Department of Pathophysiology and Transplantation, Milan, Italy

Background: Rotational tromboelastometry (ROTEM ® ) describes viscoelastic changes occurring during coagulation and provides graphical representation of fibrin polymerization, using whole blood. It is used bedside to quickly monitor haemostasis.

Aims: ROTEM ® results are commonly reported as average of duplicate measurements. This increases the cost and time to get results. On the other hand, data on reproducibility of ROTEM ® are limited especially intra‐run precision. Therefore, we investigated the comparability of duplicate measurements.

Methods: We performed retrospective analyses of the database of 347 duplicate measurements obtained for samples collected from June 2013 to April 2017. Samples included 136 healthy subjects, 51 obese patients, 36 patients with Cushing's syndrome and 124 cirrhotic patients. For each sample, duplicate measurements were taken using ExTem reagents on two different channels by means of a 4‐channels ROTEM ® delta (Werfen). The following parameters were recorded and anlyzed: CT (clotting time); CFT (clot formation time); MCF (maximum clot firmness).

Results: As expected, high correlations were observed between duplicates provided by first and second measurement for all the parameters (R from 0.89 to 0.99). Bland‐Altman analyses for first and second measurement revealed bias (mean of percentage differences) of 2.8% for CT, ‐3.4% for CFT and 1.0% for MCF. The average between‐duplicate coefficient of variation (CV) were 4.4% (for CT), 4.3% (CFT) and 1.2% (MCF).

Conclusions: Results show that duplicate measurements for the same sample are comparable. CVs are smaller than 6%, and bias (mean percentage differences) are smaller than 3.5% for all parameters.

PB594: Taipan Snake Venom Time Detects the Lupus Anticoagulants in Anticoagulated, Triple‐positive Antiphospholipid Syndrome Patients

G.W. Moore 1 ; R.A. Archer 1 ; E.S. Bromidge 1 ; A.P. Culhane 1 ; B.J. Hunt 1,2

1 Guy's and St Thomas’ NHS Foundation Trust, Viapath Haemostasis & Thrombosis, London, United Kingdom; 2 Guy's and St Thomas’ NHS Foundation Trust, Haemostasis & Thrombosis, London, United Kingdom

Background: Patients with antiphospholipid syndrome (APS) are at higher risk of thrombosis, pregnancy morbidity and recurrence if their laboratory profile reveals positivity for all three criteria antibodies, lupus anticoagulants (LA), anticardiolipin antibodies (aCL) and anti‐β2 glycoprotein I antibodies (aβ2GPI). LA are detected in coagulation assays but the mainstay dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) are compromised by various therapeutic anticoagulants. Taipan snake venom time (TSVT) screening test with ecarin time (ET) confirmatory test use venoms insensitive to the effects of vitamin K antagonists (VKA) and direct FXa inhibitors (DFXaI).

Aims: Assess detection of clinically significant antibodies by TSVT/ET

Methods: Our local LA detection strategy employs TSVT/ET in addition to dRVVT/APTT in patients on VKA or DFXaI anticoagulation. Diagnostic data from February 2016‐January 2018 were scrutinised for triple positive profiles in anticoagulated patients, finding 34 from patients with established triple positivity (28 warfarin, 5 rivaroxaban, 1 apixaban) and 23 with established APS from persistent LA and aCL but no prior aβ2GPI analysis (22 warfarin, 1 rivaroxaban).

Results: The LA in 32 of 34 (94.1%) of known triple positives were detected by elevated TSVT ratio (cut‐off 1.14) and significant correction by ET ratio (cut‐off >10%). TSVT ratio ranged from 1.15‐2.54, median 1.61; percent correction ranged from 11.8‐54.3, median 38.4. The two TSVT negative patients were on rivaroxaban and had TSVT ratios of 1.14 and 1.12. The LA in all 23 APS patients with no prior aβ2GPI analysis were detected by TSVT/ET, TSVT ratio ranged from 1.21‐1.95, median 1.67; percent correction ranged from 19.8‐46.3, median 36.0.

Conclusions: TSVT/ET analysis has high sensitivity to the clinically significant LA found in triple positive APS patients with potential to enhance diagnostic decision making in VKA and DFXaI anticoagulated patients where conventional assays may be compromised.

PB595: Rotational Thromboelastometry (ROTEM) as an Alternative Method for Coagulation Assessment in Pediatric Patients Undergonig Invasive Intervention

M. Durila 1 ; M. Durilova 2 ; M. Rygl 2 ; J. Skrivan 3 ; J. Jonas 1 ; T. Vymazal 1

1 Charles University, Department of Anaesthesiology and Intensive Care Medicine, Second Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic; 2 Charles University, Department of Paediatric Surgery, Second Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic; 3 Charles University, Department of Paediatric ENT, Second Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic

Background: Standard coagulation tests (aPTT or PT) are used for assessment of coagulation profile in pediatric patients undergoing invasive interventions such as introducing central venous catheter, tonsillectomy, laparotomy etc. However, these tests do not reflect the profile of whole blood coagulation. Rotational thromboelastometry (ROTEM) as point of care (POC) viscoelastic test may serve as an alternative method periprocedurally. Due to its ability to assess coagulation profile of whole blood, it might yield normal results despite prolonged aPTT/PT results.

Aims: To find out if invasive procedures were performed without nonstandard ‐ increased bleeding if ROTEM test was normal despite prolonged values of aPTT/PT in pediatric patients.

Methods: We retrospectively analyzed data for period of years 2015‐2017 for pediatric patients with prolonged values of aPTT or PT and normal ROTEM tests ‐ INTEM (assessing internal pathway of coagulation) and EXTEM (assessing external pathway of coagulation) and we looked for nonstandard increased bleeding during or after invasive procedures.

Results: In 26 pediatric patients (children from 0 month old to 17 years old) we found that INTEM and EXTEM tests showed normal coagulation despite prolonged values of aPTT‐R (ratio) with median 1.46 (min 1.00 and max 2.05) and PT‐R (ratio) with median 1.3 (min 0.89 and max 2.11). In these patients, no nonstandard increased bleeding was observed during interventions or postoperatively. Types of procedures are shown in Table 1.

Conclusions: Our data show that ROTEM method can serve as an alternative test for assessment of coagulation in pediatric patients undergoing acute surgical or other invasive procedures, especially using it as POC test. It seems that ROTEM assessment of coagulation may lead to decreased administration of fresh frozen plasma or other coagulation factors which would have been given to the patients before interventions to correct pathologic values of aPTT/PT.

PB596: Improving Lupus Anticoagulant Testing with 2 New APTT and dRVVT Reagents

C. Frere 1 ; M. Kurdi 2 ; M. Peyrafitte 3 ; A. Permal 2 ; C. Dunois 3 ; I. Martin‐Toutain 2

1 Pitié‐Salpêtrière Hospital, Assistance Publique Hôpitaux de Paris, Institute of Cardiometabolism and Nutrition, ICAN, Sorbonne Université, Haematology Department, Paris, France; 2 Pitié‐Salpêtrière Hospital, Assistance Publique Hôpitaux de Paris, Haematology Department, Paris, France; 3 Validation et Etudes Cliniques, HYPHEN BioMed, Neuville sur Oise, France

Background: Integrated tests for lupus anticoagulant (LA) detection consist of performing APTT and dRVVT in parallel at low and high phospholipid concentrations. They do not require mixing tests. Results are interpreted according to specific cutoff values by calculating LA ratio. As it has been demonstrated that treatment with oral anticoagulants (OAC) increased the rate of false‐positive (FP) results, LA testing is not recommended in patients treated with OAC.

Aims: To evaluate the performance of different reagents for LA testing in patients treated with OAC.

Methods: Blood samples from 50 healthy volunteers were collected to generate reference‐values. Plasma samples of 45 patients already diagnosed with LA, 11 patients treated with rivaroxaban, 12 patients treated with apixaban, 10 patients treated with dabigatran and 10 patients treated with vitamin K antagonists (VKA, INR>3) were tested for LA screening using 2 combinations (A and B) of APTT and dRVVT integrated tests (combination A: HYPHEN BioMed; combination B: Siemens Healthcare). Normalized ratio of >1.2 were considered positive.

Results: The agreement between the results obtained with the 2 combinations of tests in patients already diagnosed with LA was 100% (κ=1; P<0.0001). In patients treated with rivaroxaban, the combination A resulted in negative LA diagnosis in all cases whereas the combination B resulted in 82% FP. In patients treated with apixaban, the combination A resulted in only 16% FP versus 33% with the combination B. In patients treated with dabigatran, the 2 combinations of tests resulted in 40% FP. In patients treated with VKA, the combination A resulted in negative LA diagnosis in all cases whereas the combination B resulted in 70% FP.

Conclusions: Both combinations of tests demonstrated a high sensitivity and specificity for LA screening. However, combination A (CEPHEN LS/LR and HEMOCLOT LA‐S/LA‐C) decreased the rate of FP in patients treated with OAC, constituting a better candidate.

PB597: Performance Verification of the ZL 6000i Cone Plate Rotational Viscometer

D.W. Yoo ; I.‐S. Kim

Pusan National University School of Medicine, Pusan National University Yangsan Hospital, Department of Laboratory Medicine, Yangsan, Korea, Republic of

Background: Whole blood viscosity (WBV) tends to increase in patients with hyperviscosity syndrome, cardiovascular disease, and peripheral vascular disease. Blood viscosity measurements are useful for patients with vascular disease because clinician can treat disease and predict prognosis. The ZL 6000i (ZONCI Technology, Beijing, China) is a fully automated blood viscosity measuring instrument using the new cone plate rotational method.

Aims: In this study, we were able to demonstrate the stability of the blood viscosity measured by the method.

Methods: Precision and reference verification were evaluated according to guideline of the Clinical Laboratory and Standards Institute (CLSI). Precision was measured with high and low level of materials 5 times on the first day and 3 times on the second day. The samples for the reference verification were selected from Health Promotion Center (HPC) at our hospital. The mean, standard deviation (SD) and coefficient of variation (CV) were calculated using the IBM SPSS statistics 21.0 (IBM Corp., Armonk, NY, USA) program.

Results: We verified the reference range of the manufacturer, 36 of the 39 males and all 31 females were within the reference range. To compare the means of both male and female, an independent T‐test was performed and there was statistically different in all shear rates (P values <0.01). The new range of reference values ​​is shown in Table 1. Table 2 shows less than 5% CV in all laboratory precision. Carry over results were less than 1% at 5s and 300s.

Conclusions: This study proved the accuracy and precision of ZL6000i instrument using the cone plate rotational method. We verified of manufacturer′s WBV reference range, 10% is out of the reference range, and if necessary, a clinical laboratory can use this range. However, the mean of WBV is statistically different between men and women, the male and female subgroups must be separated to set the reference range.

PB598: Validation of an Automated Algorithm for Interpretation of Lupus Anticoagulant Testing on Stago STA‐R Max

L. Florin ; K.M.J. Devreese

Ghent University Hospital, Coagulation Laboratory, Department of Laboratory Medicine, Ghent, Belgium

Background: Lupus anticoagulant (LAC) testing is a multistep procedure including screening, mixing and confirmation tests. STA Coag Expert is a software module for STA Max analyzers (Stago, Asnières, France) which includes LAC algorithms, based on ISTH guidelines, for automatic interpretation, calculation and launch of assays in LAC interpretation.

Aims: Evaluation of the automated LAC algorithms.

Methods: 194 patient samples were analysed in parallel on STA‐R Evolution and STA‐R Max and interpreted manually (daily practice) and automated by LAC algorithms (STA Coag Expert), respectively. LAC algorithms use identical flowcharts and cut‐off values as in daily practice. Differently, LAC algorithms only use index of circulating anticoagulant (ICA), whereas in routine also normalized ratios were assessed for interpretation of mixing tests. Interpretation of dRVVT and aPTT pathways and final conclusions were compared between both approaches.

Results: Compared to routine interpretation, LAC algorithm showed a sensitivity of 80% and a specificity of 100% for LAC detection. 11 samples differed in final conclusion: 3 false negatives due to different interpretation of dRVVT mixing test, 8 false negatives due to discrepant clotting times between both analyzers (7 in dRVVT, 1 in aPTT). Excluding these, sensitivity of LAC algorithm increased to 94%. Discrepancies in interpretation of the aPTT mixing test (n=11) did not result in discrepant final LAC result, all having negative confirmation tests. No false positives were observed.

Conclusions: The Lupus Anticoagulant algorithm of the STA‐Coag Expert shows good comparability to the manual interpretation of LAC. LAC expert rules may be a tool to assist laboratories in automatic launching of additional tests and in interpretation of LAC according to ISTH guidelines. This way, the STA‐Coag Expert LAC algorithm benefits in the harmonization of LAC interpretation and hence may improve inter‐laboratory comparability of LAC results.

PB599: Thrombin Generation Test in Detection of Prothrombotic Phenotype in Asymptomatic Carriers of the FV G1691A or/and FII G20210A Mutations

S. Karpich; V. Shmeleva; S. Kapustin; O. Golovina; O. Smirnova; L. Papayan; O. Matvienko

Institute of Hematology and Transfusiology, Saint Petersburg, Russian Federation

Background: Mutations in genes of factor II (G20210A) and V (G1691A) are well known determinants of hereditary thrombophilia, but their presence, especially in the heterozygous form, does not always lead to the development of thrombotic complications. This indicates the importance of objective methods for the detection of prothrombotic phenotype in carriers of the above mutations. Test of thrombin generation (TGT) demonstrate the state of the individual's hemostasis in the interaction of procoagulant and anticoagulant mechanisms and allows to detect hypercoagulability.

Aims: The aim of our study was to assess the hemostatic potential in asymptomatic carriers of the FV G1691A or/and FII G20210A mutations by TGT.

Methods: The study involved 42 asymptomatic carriers of the factor V Leiden and/or G20210A prothrombin gene mutation (M/F 14/28, mean age 37.0±15.0 years). TGT was done in platelet poor plasma according to Hemker et al. The following parameters were derived from the TGT curves: Lag time, endogenous thrombin potential (ETP), peak thrombin (PT), time to peak, rate of thrombin generation (V). The normal ranges for these parameters were calculated in 34 controls. STATISTICA 6.1 was used.

Results: Our data suggest that heterozygous carriership of the factor V Leiden and/or G20210A prothrombin gene mutation may be associated with both prothrombotic and normal phenotype. In asymptomatic carriers of the FV G1691A mutation elevated values of ETP and PT were detected in 44% of cases, while resistance to activated protein C ‐ in 67% of cases. The majority of the FII G20210A mutation carriers demonstrated increased values of ETP and PT (71% and 57%, respectively). The rate of thrombin generation exceeded normal values in 57% and 64% of carriers of the FV G1691A and FII G20210A mutation, respectively.

Conclusions: TGT is a promising method for creating an individual risk profile in asymptomatic carriers of mutations associated with hereditary thrombophilia.

PB600: Management of Direct Oral Anticoagulants for Low‐bleeding Risk Surgery: The Example of Cataract Surgery

A. Godier 1,2 ; A.‐C. Martin 2,3 ; S. Lessire 4 ; F. Mullier 5 ; A. Radu 6 ; M. Vasse 7 ; I. Leblanc 1,8 ; I. Gouin‐Thibault 2,9

1 Fondation Adolphe de Rothschild, Service d'Anesthésie‐Réanimation, Paris, France; 2 Université Paris Descartes, Inserm UMR‐S 1140, Paris, France; 3 Hôpital d'Instruction des Armées Percy, Service de Santé des Armées, Service de Cardiologie, Clamart, France; 4 Université catholique de Louvain, CHU UCL Namur, Department of Anesthesiology, Yvoir, Belgium; 5 Université catholique de Louvain, CHU UCL Namur, Namur Thrombosis and Haemostasis Center, Hematology Laboratory, Yvoir, Belgium; 6 Hôpital Foch, Service d'Anesthésie, Suresnes, France; 7 Hôpital Foch, Service de Biologie Clinique, Suresnes, France; 8 Institut Mutualiste Montsouris, Service d'Anesthésie, Paris, France; 9 Hôpital Pontchaillou, Laboratoire d'Hématologie, Rennes, France

Background: For patients receiving direct oral anticoagulants (DOAC), guidelines recommend not to perform elective low bleeding risk procedures at DOAC peak concentration (Cmax) and propose various protocols of DOAC discontinuation, including 1) withholding DOAC intake the morning of surgery and the previous evening, 2) the whole day before surgery or 3) taking the last dose 12‐24 hours before surgery. The impact of these protocols on pre‐procedural DOAC concentrations ([DOAC]) has not been assessed.

Aims: To determine the proportions of patients with pre‐procedural [DOAC] ≤Cmax and to investigate the impact of the 3 recommended protocols of DOAC discontinuation on [DOAC].

Methods: Pre‐procedural [DOAC] were measured in 116 DOAC‐treated patients planned for cataract surgery and interpreted according to the respective Cmax reported in the summary of product characteristics of each DOAC. Post hoc analysis of the CORIDA study, approved by the research ethics boards (patient information and signed non‐opposition form). NCT02643992 .

Results: The duration of DOAC discontinuation ranged from 7 to 126 hours and [DOAC] ranged from ≤30 to 456 ng/mL, with 97% of the measurements ≤Cmax. Withholding DOAC intake either the morning of surgery and the previous evening or the day before surgery resulted in all [DOAC] ≤Cmax. The second protocol led to lower [DOAC] but longer durations of DOAC discontinuation, up to 58 hours. Conversely, 12‐24 hours DOAC discontinuation resulted in 10% of [DOAC] >Cmax. Neither major bleeding nor thrombotic event was reported.

Conclusions: For DOAC‐treated patients facing elective low bleeding risk procedures, a 12‐24 hours DOAC discontinuation did not guarantee pre‐procedural [DOAC] ≤Cmax. Withholding DOAC intake the morning of surgery and the previous evening is the best option, resulting in all [DOAC] ≤Cmax after a shorter duration of discontinuation than withholding DOAC the whole day before surgery. Larger studies would confirm these results according to bleeding and thromboembolic outcomes

PB601: Using Drug Levels to Guide Peri‐operative Management of Direct Oral Anticoagulants Prior to Emergency Orthopaedic Surgery; Experience from a Large Teaching Hospital within the UK

E. Millen 1 ; L. Sanders 1 ; R. Gooding 1 ; L. Athanatos 2 ; D. Morfey 3

1 University Hospitals of Leicester, Haematology, Leicester, United Kingdom; 2 University Hospitals of Leicester, Trauma and Orthopaedics, Leicester, United Kingdom; 3 University Hospitals of Leicester, Anaethestics, Leicester, United Kingdom

Background: In 2016, a protocol for the peri‐operative management of patients on direct oral anticoagulants (DOACs) requiring emergency orthopaedic surgery was put in place. This was predominantly for patients with a fractured neck of femur requiring urgent surgery. A pre‐operative drug level was obtained prior to a decision regarding timing of surgery.

Aims: Levels of less than 30 ng/ml were deemed safe to proceed and levels of over 100 ng/ml resulted in surgery being delayed. DOAC levels between these figures were left to the individual surgeon's discretion. Bleeding outcomes were reviewed post‐operatively.

Methods: In a 13 month period, 14 patients on DOACs presented requiring emergency orthopaedic surgery; 11 of these patients were on Rivaroxaban and three on Apixaban. 13 patients presented with a fractured neck of femur and one presented with a pathological fracture requiring surgery.

Results: Six patients had a DOAC levels below 30 ng/ml. Of these, two were operated on the same day the level was obtained. The maximum delay to surgery in this group was 2 days. None of these patients had excess bleeding reported by the surgeon and none required blood transfusion.

One patient had a DOAC level of above 100 ng/ml and surgery was delayed for 2 days. No excess bleeding was reported and the patient didn't require a blood transfusion. Of the remaining seven patients with levels between 30 and 100 ng/ml, five were operated on the same day as the level was taken. No excess bleeding was reported in these patients but two did require a blood transfusion. One patient was delayed by one day with no bleeding or transfusions reported. The final patient's surgery was delayed by seven days due to pneumonia, rather than haemostatic concerns.

Conclusions: Our initial experience using DOAC levels to aid decision making for patients on DOACs requiring emergency orthopaedic surgery, may help us decide which patients would benefit from a specific reversal agent once this becomes available. Further cases will be available by ISTH SSC.

PB603: Antithrombin Levels Less than 60% Lead to Reduced Anti‐Xa Levels

C. Pillion 1 ; M. Riordan 2 ; I. Regan 3

1 Our Lady's Children's Hospital Crumlin, Coagulation/Haematology, Dublin, Ireland; 2 Our Lady's Children's Hospital Crumlin, Children's University Hospital, Nephrology Department, Dublin, Ireland; 3 Our Lady's Children's Hospital Crumlin, Coagulation Department, Dublin, Ireland

Background: Antithrombin (AT; antithrombin III) is a natural anticoagulant that is involved in haemostasis by inhibiting thrombin and factor Xa and thus accelerating the neutralisation of coagulation proteins. LMWH is responsible for accelerating AT interaction with thrombin and Xa. With this knowledge in mind, AT deficiency in patients is capable of reducing the ability to achieve the desired anticoagulant effect using LMWH. The anti‐Xa test measures the activity of heparin/LMWH against the activity of activated coagulation factor X; significant variability of anti‐Xa levels in common clinical scenarios has been observed.

The level of AT in vivo and its implications for the measurement of LMWH was investigated in the laboratory.

Aims: The aims of this study were to; Determine the cut‐off concentration at which the AT has an effect on the Anti‐Xa levels by performing in vitro heparin recovery. Determine if in vivo AT level has an effect on the anti‐Xa levels using assay kits that measure Anti‐Xa level, with AT supplementation (AT+) and without (AT−).

Methods: 54 samples were analysed to evaluate LMWH (AT+ and AT−) and AT levels. In vitro plasma samples were prepared by spiking with heparin and AT.

Results: 54 patients receiving LMWH therapy showed that the LMWH assay (AT+) had significantly greater heparin recovery compared with the LMWH (AT−) assay. The in vitro analysis of AT deficient plasma spiked with heparin showed that the heparin recovery was dependent on AT for both types of Heparin (anti Xa) Assays. There was a reduction in the amount of heparin recovered when AT levels fell below 60%.

Conclusions: The study has shown that anti‐Xa requires the presence of AT to carry out its mode of action which may be limiting in determining anti‐Xa levels in AT deficient patients. The cut‐off concentration at which AT had an effect on Anti‐Xa levels was 60%. This finding has implications for measuring LMWH effect in patients that have AT levels below 60%.

PB604: Direct Oral Anticoagulants (DOACs) for the Management of Venous Thromboembolism in Patients Aged Less than 18 Years

U. Faruqi ; J. Alamelu; M. Pelidis; V. Collings; Z. Mahir; E. Williams; N. Husain; T. Dowling; K. Breen; B. Hunt; M. Crowley

Guy's and St Thomas’ NHS Foundation Trust, London, United Kingdom

Background: The incidence of venous thromboembolism in children is increasing and can be up to 57 per 100,000 in those hospitalised. Line‐associated thrombosis is partly responsible for this increment reflecting improvements in the management of chronic conditions. The most commonly used anticoagulants in paediatric practice are low molecular weight heparin (LMWH) and vitamin K antagonists (VKA). The daily injections with LMWH can be distressing particularly in the context of needle phobias. INR monitoring required with warfarin is disruptive to schooling and an unregulated diet can result in labile readings. The use of DOACs is currently off license in those under 18 years old due to limited evidence and experience.

Aims: To review the outpatient prescription of DOACs in teenagers in our tertiary centre.

Methods: A retrospective study assessing use of DOACs in teenagers, reasons for use, side effects and adverse events.

Results: Thirteen teenagers diagnosed from 2013 onwards were included. There were two patients with a thrombus site classed as “other”; one venous sinus thrombosis with antithrombin deficiency and one thrombotic microvascular angiopathy with Behcets disease. There were no recurrent events in those receiving DOACs. Five patients continue on lifelong treatment and in four, duration is unconfirmed.

The reasons for DOAC selection were: less disruption to school; needle phobias; improved compliance and convenience. Some patients were converted to DOACs from other anticoagulation for lifestyle convenience particularly those requiring lifelong treatment. Anti‐Xa monitoring was not routinely performed. TABLE 1 Baseline Characteristics and Findings Age, mean (range) years 15 (13‐17) Sex 7 male 6 female Weight, mean (range) kg 54 (43.5 ‐ 99.7) Height mean (range) cm 170 (162 ‐ 180) Cause 11 provoked 2 unprovoked 11 provoked 2 unprovoked DOAC type 11 rivaroxaban 2 apixaban Site 7 DVT 3 PE 1 both 2 other Treatment 10 primary treatment 3 prophylaxis Diagnosed thrombophilia 2 antiphospholipid syndrome 1 antithrombin deficiency Side effects 2 menorrhagia 1 epistaxis 1 transient haematuria

TABLE 2 Patients with provoked events and finite treatment duration VTE Event Provoking factors Primary treatment or prophylaxis Duration of treatment (months) Time (months) since treatment stopped PE Combined oral contraceptive pill, Prothrombin 20210 Primary 29 22 DVT Anatomical anomaly with venous stenosis Primary 20 3 DVT Thoracic outlet syndrome Prophylaxis after 9 months 6 6 DVT Line provoked Primary 2 25

Conclusions: Our experience of DOACS in teenagers has shown that fixed adult dosing regimes in those with comparable growth indices to adults was efficacious with no major side effects. Rivaroxiban was preferred due to its once daily administration. Whilst there are trials ongoing, all cases should be reviewed individually. DOACs appear to present a viable anticoagulation option in this patient population.

PB605: Inpatient to Outpatient Transition of Care for Pediatric Patients with Venous Thromboembolism

J. Munn 1 ; M. Rajpurkar 2 ; K. Patel 3 ; J. Journeycake 4 ; A. Sharathkumar 5 ; M. Wang 6 ; C. Watson 7 ; E. McCarthy 8 ; D. Cheng 7 ; M. DeSancho 9 ; J. Jaffray 10

1 University of Michigan, Michigan Medicine Hemophilia and Coagulation Disorders Program, Ann Arbor, MI, United States; 2 Wayne State University, Children's Hospital of Michigan, Detroit, MI, United States; 3 Emory University School of Medicine, Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Atlanta, GA, United States; 4 University of Texas Southwest Medical Center, Dallas Children's Hospital, Dallas, TX, United States; 5 University of Iowa, Stead Family Children's Hospital, Iowa City, IA, United States; 6 University of Colorado, Anschutz Medical Campus, Aurora, CO, United States; 7 American Thrombosis and Hemostasis Network, Rochester, NY, United States; 8 American Thrombosis And Hemostasis Network, Rochester, NY, United States; 9 Weill Cornell Medicine, New York Presbyterian Hospital, New York, NY, United States; 10 University of Southern California, Keck School of Medicine, Children's Hospital Los Angeles, Los Angeles, CA, United States

Background: Pediatric venous thromboembolism (VTE) is associated with increased costs, length of stay and hospital readmissions due to recurrence or complications of anticoagulation. Addressing transition of care (TOC) at discharge may help improve outcomes.

Aims: To describe VTE characteristics and anticoagulation knowledge for children enrolled in the American Thrombosis and Hemostasis Network (ATHN) 4 VTE Project.

Methods: Pediatric and adult hospitalized patients with a 1 st episode VTE requiring anticoagulation are eligible to participate in ATHN 4, a multi‐center quality intervention (QI) project being conducted by ATHN. The project has 2 phases: a pre‐intervention (PI) phase with no change in standard practice, and a QI phase consisting of enhanced education and additional follow‐up. Demographics, disease and treatment characteristics and outcomes are collected. Caregiver knowledge and feedback questionnaires are administered at 30 days post‐discharge. Preliminary pediatric data from the PI phase are presented.

Results: 93 children were enrolled in the PI phase (Table 1). 35% of VTEs were in lower extremities or pelvis. 72 (77.4%) patients had a provoked VTE. 59 (63.4%) and 51 (54.8%) had at least one medical or environmental risk factor, respectively. 76 knowledge questionnaires indicated 68 (89.5%) caregivers could recall symptoms to watch for; 77 of 78 (98.7%) could recall the name of the prescribed anticoagulant. 82.9% and 4% were discharged on low molecular weight heparins (LMWH), or direct oral anticoagulants, respectively.

TABLE 1 Patient Characteristics, VTE Risk Factors and Location (N = 93) Age Range # % # % Age (years) 0‐1 34 37% Sex Female 39 42% 2‐5 13 14% Male 54 58% 6‐10 5 5% Ethnicity Non‐Hispanic 79 85% 11‐15 19 20% Hispanic 11 12% 16‐18 22 24% Other/Unknown 3 3% VTE Risk Factors Reported: Number of Risk Factors, Most Frequent Type and Location Medical Risk Factors Pregnancy, trauma, sepsis, obesity, prolonged hospital stay # of Patients with ≥ 1 risk factor 59 63% Hospital Stay > 7 days 36% Environmental Risk Factors Immobility, smoking, surgery, travel, venous catheters # of Patients with ≥ 1 risk factor 51 55% Central Venous Catheter 45% VTE Location Lower Extremity or Pelvis 35% Upper Extremity or Thorax 26%

Conclusions: This study confirms previous bimodal distribution and provoked etiology of pediatric VTE. LMWH is the most commonly prescribed anticoagulant.Current TOC led to retaining knowledge of anticoagulation medication for at least a month. These preliminary findings suggest that approximately 10% of pediatric patients with VTE may benefit from additional discharge education. Patient characteristics and interventions affecting adherence and outcomes continue to be studied.

PB606: Cerebral Sinovenous Thrombosis (CSVT) in Children: 25 Years Single‐center Prospective Pediatric Cohort in Argentina

G. Sciuccati ; C. Cervio; M. Hepner; G. Pieroni; E. Annetta; J.P. Frontroth; R. Romina; C. Pepe; A. Feliú Torres; M. Bonduel

Hospital de Pediatría ‘Prof Dr J P Garrahan’, Hematología y Oncología, Buenos Aires, Argentina

Background: CSVT is increasingly recognized in children. Scarce anticoagulation therapy (ACT) data and variable morbidity and mortality rates were reported.

Aims: To describe the diagnosis, management and outcomes of a pediatric cohort with CSVT registered at a single referral tertiary center.

Methods: From May 1992 to November 2017, consecutive children <18 years old with objectively confirmed CSVT were registered. Newborns were excluded. Clinical features, family history of thrombosis (FHT), imaging findings, thrombophilia, ACT and outcomes were recorded.

Results: Of 155 pts evaluated, 98 males (63%), median age (range) 8 years (0.1‐17.9). Incidence of CSVT was 3.1 per 10,000 hospital admission/year. Risk factors were found in 150 pts (97%): head/neck infections 62 pts (39%), acute lymphoblastic leukemia (ALL) 35 pts (23%)/lymphoma 8 pts (5%) with L‐asparaginase (ASP). Incidence of CSVT in ALL pts was 2.1% (95% CI: 1.6‐3.7%). Clinical data: headache 8 5 pts (49%), seizures 52 pts (33%). Eight pts (5%) had FHT. Location: superficial 125 pts (81%), deep 11 pts (7%) and both 19 pts (12%). Venous infarct occurred in 60 pts (39%), 41 (68%) of them were hemorrhagic. 98 pts were screened for thrombophilia: inherited 5 pts (5%), acquired 41 pts (42%) and combined 7 pts (7%). 143 pts (92%) received at diagnosis enoxaparin followed by vitamin K antagonists in 83 pts (54%), median (range) ACT duration: 5 months (1‐75). AT replacement was used in 27 pts with ALL. One pt had increased hemorrhagic infarction during ACT. No recurrence or mortality attributable to CVST were found. Four untreated pts had thrombosis progression. Among 88 pts with follow‐up imagining, recanalization was complete in 49 pts (56%) and partial 31 pts (35%). 39 pts (44%) had sequelae

Conclusions: This is the largest pediatric CSVT cohort reported to date. In pts with ALL and acquired AT deficiency, AT replacement allowed to continue with ASP.

ACT used at initial diagnosis may contribute to thrombosis resolution and appears to be safe. Lower mortality and morbidity rates were found compared to the other studies.

PB607: Significance of GPIbα ‐5T>C, GPIbα VNTR, GPVI T13254C Polymorphisms and HPA‐2 in the Etiology of Pediatric Arterial Ischemic Stroke

A. Ceri 1 ; D. Coen Herak 2 ; D. Herak 3 ; R. Zrinski‐Topic 4 ; J. Lenicek Krleza 4 ; I. Horvat 2 ; V. Djuranovic 5 ; N. Barisic 6 ; R. Zadro 1,2

1 University of Zagreb, Faculty of Pharmacy and Biochemistry, Department of Medical Biochemistry and Hematology, Zagreb, Croatia, 2 University Hospital Centre Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia, 3 University Hospital Centre Sestre Milosrdnice, Clinical Institute of Chemistry, Zagreb, Croatia, 4 Children's Hospital Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia, 5 Children's Hospital Zagreb, Departrment of Neuropediatrics, Zagreb, Croatia, 6 University Hospital Centre Zagreb, Department of Pediatrics, Division of Neuropediatrics, Zagreb, Croatia

Background: The etiology of pediatric arterial ischemic stroke (AIS) has still not been fully elucidated. The role of platelet glycoprotein (GP) Ibα polymorphisms: ‐5T>C, variable number of tandem repeats (VNTR) and human platelet antigen (HPA) ‐2, which was found to be relevant for the expression and function of GPIbα, has been investigated and associated with increased risk for stroke in adults only. The minor GPVI T13254C allele has been associated with decreased platelet activation. To date, significance of these polymorphisms in pediatric AIS has not been examined.

Aims: To investigate the significance of individual polymorphisms GPIbα ‐5T>C and VNTR, HPA‐2 and GPVI T13254C, together with the haplotype (HPA‐1/‐2/‐3 and GPIbα ‐5T>C) in pediatric AIS and its subtypes, perinatal and childhood AIS.

Methods: Polymorphisms were investigated in 123 children with perinatal (N=58) and childhood AIS (N=65), and in 113 sex‐ and age‐matched controls. Genotyping was performed as follows: GPIbα ‐5T>C by real‐time PCR and melting curve analysis (Ulehlova et al., 2014), GPIbα VNTR using PCR followed by PCR product analysis by electrophoresis on 2% agarose gel (Jilma‐Stohlawetz et al., 2003), HPA‐1, ‐2 and ‐3 by real‐time PCR using TaqMan technology (Ficko et al., 2004) and GPVI T13254C using real‐time PCR followed by high resolution melting analysis.

Results: Increased risk for childhood AIS was found in carriers of VNTR B allele (OR: 2.22; 95% CI: 1.14‐4.32). Statistically significantly (P=0.030) higher HPA‐1a/2b/3a/GPIbα ‐5T haplotype frequency (0.099) was observed in AIS, compared to controls (0.026), resulting in OR of 3.60 (95% CI: 1.14‐11.35). This association was even stronger in children with childhood AIS (OR: 5.48; 95% CI: 1.62‐18.49, P=0.007), but not present in children with perinatal AIS (P=0.960).

Conclusions: The obtained results show that GPIbα polymorphisms increase the risk solely for childhood AIS, whereas their role does not seem to be important in the etiology of perinatal AIS.

PB610: D Dimer Levels and Thrombosis in Children with Systemic Lupus Erythematosus

S. Thakur 1 ; J. Ahluwalia 2 ; N. Kumar 2 ; S. Singh 3 ; A. Jindal 3 ; R. Pilania 3

1 Postgraduate Institute of Medical Education & Research, Chandigarh, India, 2 Postgraduate Institute of Medical Education & Research, Hematology, Chandigarh, India, 3 Postgraduate Institute of Medical Education & Research, Pediatrics, Chandigarh, India

Background: Pediatric Systemic Lupus Erythematosus (pSLE) is complicated by thrombosis in nearly 10% of patients. Thrombosis is often associated with persistent positivity for one or more of the antiphospholipid antibodies (APLA) ‐Lupus anticoagulant (LA), Anticardiolipin antibody (ACA) and anti Beta 2 Glycoprotein 1 antibody (aβ2GP1ab). Lysis of the thrombus is accompanied by a rise in D dimer levels. High D dimer levels may be associated with thrombosis in pSLE with APLA positivity.

Aims: To study the association between D dimer levels antiphospholipid antibodies, thrombosis and clinical presentation.

Methods: This was a prospective, cross sectional, hospital based study. Children aged less than 12 years (when diagnosed to have pSLE) meeting the ACR criteria for SLE were included. Clinical details including that of thrombotic events was recorded. Citrated sample remaining after performing LA testing, was used for D Dimer assay. LA was tested by DRVVT and Silica Clotting Time. ACA and aβ2GP1ab were tested by ELISA kits (Hycor USA). Patients were grouped as those with and without thrombosis and compared for D‐ dimer levels using the Mann Whitney U test at a significance level of P<0.05.

Results: Data on 80 children were available for analysis. The median age was 12 years (2‐27), with a female preponderance. Nine (11%) patients had history of thrombosis. D dimer levels were elevated in 38% cases. The median D dimer levels were not significantly different in patients with and without thrombosis (139 vs. 174 ng/ml, P ‐ 0.89), nor were they associated with clinical manifestations, APLA positivity or drug therapy. Higher median D Dimer levels seen in new patients when compared to older cases (707.5 vs. 122, P‐0.05).

Conclusions: D dimer levels in children with pSLE are not associated with any particular clinical presentation, thrombosis, positivity for APLA or therapy and are likely to be of limited value in predicting risk for thrombosis in these patients.

PB611: Anti‐factor H Autoantibody‐associated Hemolytic Uremic Syndrome in an Argentinean Pediatric Cohort

C. Dos Santos 1 ; N. Mogollon Molina 2 ; M.F. Alberto 2 ; M.L. Romero 2 ; A. Sanchez‐Luceros 1,2

1 IMEX‐CONICET‐Academia Nacional de Medicina, Hemostasis and Thrombosis, Ciudad Autonoma de Buenos Aires, Argentina, 2 Academia Nacional de Medicina, IIHEMA, Hemostasis and Thrombosis, Ciudad Autonoma de Buenos Aires, Argentina

Background: The presence of autoantibodies directed against Factor H (FH), regulator of the complement alternative pathway, is associated with atypical Hemolytic Uremic Syndrome (aHUS) manifesting microangiopathic hemolytic anemia, thrombocytopenia and kidney injury. Affecting mainly children, this autoimmune form was detected in 5‐25% of aHUS pediatric cases in Europe, 30% in Korea and 56% in India.

Aims: To describe for the first time in Argentina the incidence of anti‐Factor H autoantibody‐associated aHUS.

Methods: Our retrospective study, realized with patients’ informed consent, was approved by the institutional ethical committee. Standard and positive control for detection of anti‐FH antibodies by ELISA were kind gifts from Dr Dragon‐Durey (Hôpital Européen G. Pompidou, France).

Results: Anti‐FH autoantibodies were screened in 113 patients (59 adults and 54 children) presenting clinical features of aHUS. Renal biopsy oriented the diagnosis of 3 patients towards C3 glomerulonephretis, a chronic kidney disease defined by abnormalities of the complement alternative pathway. Positive titers were found exclusively in 8 pediatric patients with suspicion of aHUS. Laboratory features and clinical manifestations of anti‐FH positive aHUS cases were compared to subjects with no autoantibodies detected (Table 1). Patients with uncomplete clinical history or studied outside the acute phase (n=19) were excluded from the analysis. Although our study is sample size limited, LDH appeared to be increased in patients presenting anti‐FH antibodies (Mann‐Whitney U‐test P=0.05). No other laboratory test showed significant difference between aHUS groups. Occurrence of extrarenal manifestations was slightly higher in patients with anti‐FH antibodies.

TABLE 1 Clinical and laboratory characteristics at onset in an Argentinean pediatric cohort with or without anti‐FH autoantibody‐associated aHUS Anti‐FH autoantibody‐associated aHUS (n=6) aHUS negative for anti‐FH autoantibody (n=29) Age (years‐old) 7 (5‐13) 6.7 (0‐18) Male 3 (50%) 10 (35%) Anti‐FH autoantibodies titer (AU) (negative if <100 AU) 1,797 (179‐4,361) 3.6 (0‐27) Gastrointestinal symptoms Diarrhea Vomiting 4 (67%) 1 (17%) 3 (50%) 25 (86%) 16 (55%) 9 (31%) Hypertension 3 (50%) 7 (24%) Neurological symptoms Seizure Cerebral infarct 3 (50%) 2 (33%) 1 (17%) 9 (31%) 8 (28%) 1 (3%) Laboratory results Hemoglobin (g/dL ±SD) Platelets count (×10 9 /L ±SD) LDH (IU/L ±SD) Creatinine (mg/dL ±SD) 7.9±2.2 (5.6‐10.3) 45.4±21.4 (28‐80) 4,687±2,550 (2,321‐9,536) 3.5±1.8 (1.5‐6.25) 9.3±4.6 (4‐30) 56.7±34.1 (5‐137) 3,131±2,651 (500‐11,300) 4.4±2.9 (1.2‐11.6) Received treatments Dialysis Corticoids Transfusion (RBC, plasma, or platelets) Plasmapheresis 4 (67%) 4 (67%) 5 (83%) 4 (67%) 16 (55%) 4 (14%) 20 (69%) 10 (35%)

Conclusions: The first study detecting anti‐FH autoantibodies‐associated aHUS in Argentina showed a frequency of 14.8% cases in pediatric cohort. Sample size needs to be increased to compare prognosis between aHUS patients with anti‐FH antibodies and patients with other aHUS forms.

PB613: Prosthetic Valve Thrombosis in Children

P. Mugaba 1 ; B.‐J. Martin 2 ; P. Kewada 3 ; D. Ross 4 ; M. Bauman 5 ; M.P. Massicotte 6 ; A. Mackie 1 ; P. Kawada 7

1 University of Alberta, Pediatrics/Cardiology, Edmonton, Canada, 2 Stanford, Cardiac Surgery, Stanford, United States, 3 University of Alberta, Edmonton, Canada, 4 University of Alberta, Cardiac Surgery, Edmonton, Canada, 5 University of Alberta, Pediatrics/KIDClot Thrombosis, Edmonton, Canada, 6 University of Alberta/Edmonton Clinic Health Academy, Pediatrics/KIDclot Thrombosis, Edmonton, Canada, 7 University of Alberta, Pediatrics, Edmonton, Canada

Background: Prosthetic valve thrombosis (PVT) is associated with significant mortality and morbidity. Children may be at increased risk for PVT due to the challenges associated with pediatric mechanical heart valves in addition to anticoagulation. Evidence based guidelines demonstrating safety and efficacy for treatment of PVT in children are lacking.

Aims: Review of a single center experience in treatment of PVT in children.

Methods: Data on children ages <18 years with a confirmed diagnosis of mechanical PVT and managed at the Stollery Hospital from 2006 to 2015 was collected including: age, sex, weight, cardiac diagnoses, previous cardiac surgery, indication for valve, type and site of valve, anticoagulation and adherence to anticoagulation. In addition, treatment, outcomes and complications were collected.

Results: From 2006 to 2015, 6/50 children (5 males) with median ages 17 months (range 1‐168 months) and weight 8.9 kg (range 3.4 ‐62 kg) with mechanical valve placement developed PVT. There were 4 St Jude's valves (mitral=3, pulmonary=1) and 2 OnX valves (mitral=1, aortic=1). Initial therapy included thrombectomy (n=1), valve replacement (n=1) and fibrinolytic therapy (tPA) (n=3) with no major adverse events. Recurrence of PVT occurred in n=3 (all tPA) at <3 months and n=1 (valve replacement) at 8 months post treatment. All recurrences were treated with tPA again with 3 requiring further therapy, thrombectomy n=1, or valve replacement (n=2 bioprosthetic valve placed). There was one death not related to bleeding.

Conclusions: Although PVT is rare, it is life threatening. Clinical presentation of PVT can be subtle and is related to the resultant degree of valvular obstruction. This study suggests that considering medical therapy (fibrinolytic therapy, tPA) as first line treatment or as a temporizing measure prior to thrombectomy or valve replacement is reasonable. Prospective studies are needed.

PB614: Towards a Consensus for Standardized Assessment of Pediatric Patients with Vascular Malformations (VM) at Risk for Coagulopathy

M.L. Avila 1 ; C.M. Lumia 1 ; M.I. Montoya 1 ; C.C. Trenor 2 ; D. Adams 2 ; F. Blei 3 ; B. Feldman 1 ; L. Brandao 1 ; on behalf of the Special Interest Group on Vascular Anomalies for the American Society of Pediatric Hematology Oncology

1 The Hospital for Sick Children, Department of Paediatrics, Toronto, Canada, 2 Boston Children's Hospital/Harvard Medical School, Boston, United States, 3 Lenox Hill Hospital, New York City, United States

Background: Coagulopathy is a known complication associated with vascular malformations (VM) in children. To date, no specific guidelines exist in the management of this complication.

Aims: To define risk factors for coagulopathy and to generate management recommendations of high‐risk patients in the peri‐procedural setting or for asymptomatic patients presenting with isolated D‐dimer derangements.

Methods: A committee of North American pediatric hematologists with special interest in vascular anomalies (VA) participated in a 2‐step consensus process. Step 1 defined the most important risk factors and most common clinical scenarios related to pediatric VM patients with coagulopathy by means of a systematic literature review and a survey. Step 2 generated recommendations on risk stratification and on those cases following a modified nominal group technique.

Results: Participation in all steps included ˜70% of the 38 invited respondents, who represented most of the centers in the US/Canada. Around 2/3 of participants had ≥10 years of practice and ˜80% worked in a multidisciplinary vascular clinic. The risk factors and cases identified in step 1 were used to inform the participants of the nominal group. In step 2, a set of recommendations was generated. Table 1 shows the 3 more frequent recommendations for risk stratification and for the management of 2 distinct clinical scenarios. Changes of opinion between voting sessions are also shown.

TABLE 1 Risk stratification for coagulopathy and discussion of management of 1 high and 1 low coagulopathy risk case Clinical Question First Round Voting (% of participants electing the option) Second Round Voting (% of participants electing the option) Risk stratification for coagulopathy in patients with VM Low fibrinogen (31%) Low fibrinogen (16%) D‐dimer >5× normal (16%) Personal thrombosis history (12%)* Thrombocytopenia without any other explanation (14%) Low fibrinogen AND thrombocytopenia AND elevated d‐dimer (10%)* High risk VM patient, peri‐procedural management Low Molecular Weight Heparin (46%) Supportive clinical care (43%)* No treatment (46%) No treatment (30%) Other¶ (8%) Low Molecular Weight Heparin (13%) Asymptomatic VM patient with isolated, elevated d‐dimer No treatment (60%) No treatment (41%) Other¶ (32%) Compression garments (39%)* Acetyl salicylic acid (6%) Supportive clinical care (18%)* Legend: (*) Asterisk denotes option choice created only during the discussion process. ¶ Others represents no use of anticoagulants AND no use of antiplatelet therapy.

Conclusions: A panel of pediatric hematology experts recommends using acquired hypofibrinogenemia, personal thrombosis history and a composite of low fibrinogen/platelets and D‐dimer elevation to identify children at high risk for coagulopathy. There is uncertainty of whether anticoagulation should be used, even in high risk patients. Further studies on the management of coagulopathy in children with VM are required.

PB615: Otogenic Lateral Sinus Thrombosis in Children: Are There Prognostic and Predictor Factors? Experience of 25 Cases and a Proposal of a Management Protocol

M. Luciani 1 ; A. Scorpecci 2 ; S. Giannantonio 3 ; M. Massoud 1 ; P. Zangari 4 ; S. Foligno 1 ; G. Di Felice 5 ; A. Minozzi 5 ; P. Marsella 2

1 Bambino Gesù Children's Hospital, IRCCS, Department of Hematology Oncology and Transfusion Medicine, Rome, Italy, 2 Bambino Gesù Children's Hospital, IRCCS, Department of Audiology and Otosurgery Unit, Rome, Italy, 3 Bambino Gesù Children's Hospital, Department of Audiology and Otosurgery Unit, Rome, Italy, 4 Bambino Gesù Children's Hospital, Department of Immmuninfection Diseases, Rome, Italy, 5 Bambino Gesù Children's Hospital, IRCCS, Department of Laboratories, Rome, Italy

Background: Otogenic lateral sinus thrombosis (OLST) is a rare but potentially fatal disease affecting the pediatric population, generally occurring as a complication of otitis media and mastoiditis. No specific guidelines for OLST treatment have been published.

Aims: Our aim is describe from our experience the prevalent clinical, laboratory and radiological features of OLST in children and to find a possible clinical predictors of neuroradiological outcome, proposing a management algorithm derived from our experience.

Methods: A retrospective review was conducted of the clinical records of patients affected by OLST, including followed criteria: pediatric age (0‐16 years) and definite OLST diagnosis confirmed by a pre‐ and post‐contrast CT or venography‐MRI (vMRI) scan.

Results: 25 patients (8 females, 17 males, mean age=6±3 years) were included. A genetic abnormality associated with thrombophilia was found in 23 (92%) patients, the most frequent conditions being heterozygous or homozygous C677T Methyl tetrahydro folate reductase and factor V Leiden mutations. At diagnosis, anticoagulant treatment with low molecular weight heparin (LMWH) was started in all patients, while surgical treatment, consisting of mastoidectomy and tympanostomy tube insertion, was performed in 16/25 (64%) subjects. Follow‐up neuroimaging showed lateral sinus recanalization in 12/25 (48%) of patients 1‐2 months after diagnosis and in 17/25 (68%) after 6‐months. At multivariate logistic regression analysis, no significant predictor of early and late neuroradiological outcome was found.

Conclusions: All children with OLST should be screened for thrombophilia in order to establish risk of recurrences, discontinuation of anticoagulant therapy and a future anti‐thrombotic prophylaxis. Immediately after OLST diagnosis, anticoagulant treatment with LMWH should be started according to international guidelines. Our experience suggests that surgical treatment should not be indicated in all patients, but decided on a case‐to‐case basis.

PB616: Dosing, Safety and Efficacy of Therapeutic and Prophylactic Low Molecular Weight Heparin in the Pediatric Population: A Systematic Review

I.L.M. Klaassen 1,2 ; J.J. Sol 3 ; M.H. Suijker 1 ; K. Fijnvandraat 1 ; M.D. van deWetering 4 ; C.H. vanOmmen 3

1 Academic Medical Center, Pediatric Hematology, Amsterdam, the Netherlands, 2 Academic Medical Center, Vascular Medicine, Amsterdam, the Netherlands, 3 Erasmus MC‐Sophia, Pediatric Hematology, Rotterdam, the Netherlands, 4 Academic Medical Center, Pediatric Oncology, Amsterdam, the Netherlands

Background: The incidence of venous thromboembolism (VTE) in children is rising. Hence, there is an increasing off‐label use of low‐molecular‐weight heparin (LMWH). There is little data about therapeutic and prophylactic LWMH dosages, and their safety and efficacy.

Aims: To review the therapeutic and prophylactic dosages of LMWH required to reach therapeutic and prophylactic target ranges, and, the safety and efficacy of LMWH, in terms of bleeding complications, achieving therapeutic and prophylactic anti‐factor Xa levels, development of (recurrent) VTE and cloth resolution.

Methods: A literature search was conducted in Medline, including all published studies between 1980 and October 2017 concerning dosage, safety or efficacy of LMWH used for therapeutic or prophylactic purposes in neonates and children to 19 years of age.

Results: A total of 49 studies were included, encompassing 3,101 patients. Initial weight‐adjusted dosages to reach therapeutic or prophylactic target ranges decreased with age. In children with therapeutic use of LMWH, major bleeding complications occurred in 1.8% (95% CI: 1.1‐2.5%) of the patients, a mean of 79.9% (95% CI: 77.5‐82.3%) of the children achieved the target range with or without dosage adjustments, recurrent VTE occurred in 3.2% (95% CI: 2.1‐4.3%) and thrombus resolution in 63.5% (96% CI: 60.2‐66.8%) of the patients.. In children with prophylactic LMWH, major bleedings occurred in 0.6% (95% CI: 0.2‐1.0%) of the patients, a mean of 90.4% (95% CI: 84.6‐96.2%) of the children achieved the target range, and 2.2% (95% CI: 1.3‐3.1%) experienced a new VTE.

Conclusions: A higher initial dosage of LMWH was needed in comparison to advised dosages, to achieve target range, especially in neonates and children <5 years. LMWH appeared to be safe and effective for therapeutic and prophylactic treatment of VTE in children.

PB617: Exploring Adolescent's Perceptions and Emotional Reaction to Thrombosis and Anti‐coagulant Therapy: A Qualitative Study

T. Biss 1 ; R. Struthers 2 ; V. Araujo‐Soares 3 ; B. Arnott 3

1 The Newcastle upon Tyne Hospitals NHS Foundation Trust, Haematology, Newcastle upon Tyne, United Kingdom, 2 Newcastle University, Psychology, Newcastle upon Tyne, United Kingdom, 3 Newcastle University, Institute of Health and Society, Newcastle upon Tyne, United Kingdom

Background: Although prevalent in adults, thrombosis occurs less often in young people. Thrombosis may be spontaneous or due to an underlying health condition. Anticoagulant therapy can be intrusive, cause risk of bleeding and involve regular monitoring. Lifestyle changes may be imposed, e.g. sports, contraception, diet and social activities. Studies have explored the impact of adolescent thrombosis on quality of life but not their illness perceptions and emotional impact. The Common Sense Model of Self‐Regulation (CSM) suggests that how someone perceives and interprets their condition may affect their ability to cope, manage and adapt.

Aims: The aim was to explore adolescent's perception of their illness and emotional impact using the CSM.

Methods: A semi‐structured interview was used. Data analysis used the Framework approach to determine the appropriateness of the CSM in this setting.

Results: Ten young people aged 13‐19 years, including 7 females, gave informed consent to participate in the study. Six had cancer associated thrombosis (CAT). Adolescents with thrombosis have social and cognitive reactions to the illness resulting in restrictions in social interactions and illness related anxiety. They identified strong emotional reactions to diagnosis, namely initial and delayed shock. The emotive reaction centred on fear, anxiety and sadness. Support and reassurance from clinicians was important in reducing fear of recurrence. They identified a positive outlook on life following their thrombosis. Variation existed between those with CAT and spontaneous thrombosis; those with CAT had less thrombosis related anxiety and more generalised health related anxiety.

Conclusions: The CSM has identified themes in relation to the emotional impact of thrombosis in adolescence. Response to CAT and spontaneous thrombosis differs. To aid reduction in illness related anxiety clinicians should provide reassurance, education, and support following diagnosis.

PB618: Peripheral Gangrene in a Child: An Uncommon Initial Presentation of Pediatric Lupus

S. Singh 1 ; A. Singh 1 ; J. Ahluwalia 2

1 Post Graduate Institute of Medical Education and Research, Advanced Pediatrics Centre, Chandigarh, India, 2 Post Graduate Institute of Medical Education and Research, Department of Hematology, Chandigarh, India

Background: Systemic Lupus Erythematosus (SLE) is a well recognized multisystem disease. Lupus is usually more severe in children. Peripheral gangrene as the initial presenting symptom is extremely uncommon, especially in children.

Aims: We report a young girl who presented with gangrene of digits and on evaluation was found to have SLE.

Methods: A 12‐year‐old‐girl presented with gangrene of right great toe. She had complained of recurrent Raynaud's phenomenon in the past but had not sought medical consultation for this complaint. On examination she had pallor, hypertension, dry gangrene of right great toe and splenomegaly. Investigations revealed hemolytic anemia, with Coombs test positivity, positive ANA, anti‐dsDNA and antiphospholipid antibodies (viz. anticardiolipin and anti‐beta2 glycoprotein I antibodies). She also had a false positive rapid plasma reagin test. Lupus anticoagulant test was negative. She was found to be heterozygous for Factor V Leiden. Urine examination was unremarkable. Ultrasound Doppler studies for renal and lower limb vessels were normal. CT angiography was also normal. Skin biopsy was suggestive of small vessel vasculitis. In view of autoimmune hemolytic anemia with positive ANA, anti‐ds DNA and antiphospholipid antibodies, a diagnosis of SLE was proffered.

Results: She was initiated on prednisolone and hydroxychloroquine. Nifedipine was added for Raynaud's phenomenon. Anticoagulant therapy with low molecular weight heparin was also added. She showed gradual clinical improvement and was discharged on oral aspirin, prednisolone, nifedipine, hydroxychloroquine and warfarin. She remained well on follow‐up.

Conclusions: Childhood lupus can occasionally present as gangrene of extremities, especially in context of antiphospholipid antibodies. Early recognition and prompt treatment can halt further progression.

PB620: Long‐term Protein C Prophylaxis in Conjuction with Dabigatran Etexilate Treatment in a Case with Late Onset Homozygous Protein C Deficiency

S. Aytac 1 ; G. Skretting 2 ; I. Eker 3 ; F. Gümrük 1 ; M. Çetin 1

1 Hacettepe University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey, 2 Oslo University, Ulleval Hospital, Oslo, Norway, 3 Afyon Kocatepe University, Faculty of Medicine Department of Pediatric Hematology, Afyon, Turkey

Background: Homozygotes with extremely low levels of protein C usually develop life threatening thrombosis however some of those cases may not experience any thrombotic event during the neonatal period and present with a late onset of clinical symptomps and diagnosed later in their lives.

Aims: Here, we want to present an adolescent boy who was treated successfuly with low dose protein C prophylaxis in conjuction with new oral anticoagulants.

Methods: A case present.

Results: A 13 years old boy was referred to our hospital for recurrent deep venous thrombosis since 2 years. He was treated with coumarin and he had a history of skin necrosis as well. His protein C activity was found to be decreased (pro c activity level: 6% (normal range 70‐130%). There was a consanguinity between mother and father (3rd degree cousins) and his parents level were also found to be slightly decreased (mother pro C activity level :57%, father: 64%, brother: 37%, respectively).Mutational analysis revealed a homozygous mutation on the coding gene which was previously described. He was treated with FFP (twice a week) and LMWH (twice a week) since he experienced recurrent deep venous thrombosis (5 times) and pulmonary thromboembolism (2 times) during the 5 years follow up period. During the acute thromboembolic event he was treated with a full‐dose anticoagulation with LMWH and daily FFP. Protein C concentrate was available since 2017 and he was given a prophylaxis with a dose of 25 IU/kg IV protein C concentrate (Ceprotin ® ) twice weekly and dabigatran etexilate (2 × 150 mg/daily PO). Post infusion protein C activity level was found to be around 10% even after 42 hours. no adverse event and no recurrence was observed under this treatment and he was receiving this treatment for 8 months.

Conclusions: Long term low dose prophylaxis with IV protein C concentrate in patients with protein C deficiency who experince recurrent thromboembolic events seems to be effective in conjuction with new oral anticoagulants.

PB621: How do Adolescents Experience Anticoagulant Adherence? A Qualitative Exploration of the Barriers and Facilitators

K.A. Rooney 1 ; T. Biss 2 ; B. Arnott 1 ; V. Araújo‐Soares 1

1 Newcastle University, Newcastle, United Kingdom, 2 Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle, United Kingdom

Background: Adherence to anticoagulant therapy (AT) is essential to ensure safety and efficacy but is frequently suboptimal within an adolescent population. Knowledge of the barriers, which make adherence to AT more challenging, and facilitators, which support greater adherence to AT, would provide an enhanced understanding of AT as experienced by adolescents. No previous studies using qualitative methods have explored adherence to AT from the adolescent perspective.

Aims: To explore barriers and facilitators to treatment adherence in adolescents receiving AT using qualitative methods.

Methods: Seven participants, median age 17 years (range: 11‐19), including 4 males, were recruited through purposive sampling from regional thrombosis and anticoagulation clinics and consented to participate. All had been taking AT for a minimum of 6 months. (warfarin (4), dabigatran (2) & rivaroxaban (1)). Each participant engaged in a semi structured interview, which was audio recorded and transcribed. To enhance trustworthiness, each participant reviewed their transcript to ensure the data was reflective of their experiences. The transcripts were uploaded to NVivo software and analysed using framework analysis, and reviewed by a second coder, with the Theoretical Domain Framework (TDF) as the a‐priori framework. The TDF aggregates key determinants of behaviour from different theories. Data were also explored inductively and any additional themes out with the TDF were assembled outside the framework.

Results: Preliminary analysis of the data highlighted the following barriers and facilitators in relation to the TDF:

FIGURE 1 Framework analysis output

Conclusions: Our findings have explored for the first time, from the adolescents’ experience, the mechanisms that make AT adherence more or less difficult to enact. The development of interventions which build upon the facilitators and address the barriers to AT adherence will have great clinical utility in providing safe and efficacious treatment to an adolescent population.

PB622: IgG4 Related Disorder: A Clinical Possibility in Children with Peripheral Gangrene

S. Singh 1 ; A. Singh 1 ; J. Ahluwalia 2

1 Post Graduate Institute of Medical Education and Research, Advanced Pediatrics Centre, Chandigarh, India, 2 Post Graduate Institute of Medical Education and Research, Department of Hematology, Chandigarh, India

Background: An IgG4 related disorder in context of peripheral gangrene in a child is extremely unusual. We report one such case.

Aims: To describe clinical presentation of a child with digital gangrene who had an IgG4 related disorder.

Methods: 11‐year‐old boy was admitted with diarrhea, gastrointestinal bleed and gangrene in toes, He had pallor, dry gangrene of both feet and hepatosplenomegaly. Peripheral pulses were palpable and blood pressure was normal. Work‐up for antiphospholipid antibodies (ACL, Beta2GPI, LA) was noncontributory. ANA and ANCA were negative. CT abdomen and angiography was normal. Diarrhea and gastrointestinal bleed gradually resolved. He was discharged on oral warfarin. One month later he presented again with gastrointestinal bleed. Possibility of warfarin induced bleeding was considered as INR was 7. However, gastrointestinal bleeding persisted despite vitamin K and FFP administration. He had anemia, thrombocytosis, polymorphonuclear leucocytosis and eosinophilia, Inflammatory parameters (ESR=77 mm/1st hour, CRP=17 mg/L) were elevated. Ultrasound and CT abdomen revealed dilated intrahepatic biliary radicals, with dilation of splenoportal axis and bulky pancreas. Colonoscopy revealed confluent superficial ulcers and friable mucosa. Sigmoid and rectal biopsy showed plasma cells and eosinophilic infiltrates in lamina propria and foci of eosinophilic cryptitis. MRCP revealed nodular dilatation of intahepatic biliary duct suggestive of sclerosing cholangitis. Serum IgG was elevated (2,504 mg/dl; N: 540‐1,610). Considering this multisystem involvement he was evaluated for IgG4 related disease and Ig G4 levels were raised (94 mg/dl; N: 6‐28).

Results: In view of peripheral gangrene, with evidence of inflammation, peripheral and tissue eosinophilia, involvement of pancreas and bile duct with elevated Ig G4 levels, a diagnosis of IgG4 related disorder was proffered.

Conclusions: Children presenting as peripheral gangrene with multisystem involvement should be investigated for IgG4 related disorder.

PB623: Deep Venous Thrombosis with Osteomyelitis in a Child – A Rare Presentation

S. Singh 1 ; A. Singh 1 ; J. Ahluwalia 2

1 Post Graduate Institute of Medical Education and Research, Advanced Pediatrics Centre, Chandigarh, India, 2 Post Graduate Institute of Medical Education and Research, Department of Hematology, Chandigarh, India

Background: Deep Venous thrombosis (DVT) associated with osteomyelitis is very rare in children. A high index of suspicion is needed in such cases to address both issues simultaneously.

Aims: To describe the case of a young boy who had popliteal vein thrombosis associated with osteomyelitis.

Methods: A 12‐year‐old boy presented with fever for 10 days and pain in the left calf region since Day 2 of fever. There was no history of trauma or prolonged immobilization. On examination he was febrile with swelling and tenderness over left calf. Homan′s sign was positive. Initial clinical possibility was DVT of popliteal vein. Investigations revealed anemia, neutrophilic leucocytosis and thrombocytosis. Inflammatory markers (ESR and CRP) were elevated. Compression ultrasound showed left popliteal vein thrombosis. He was started on LMWH. As there was persistent fever and tenderness over the shin, MRI scan of leg was carried out. This showed proximal left tibial metaphyseal and diaphyseal osteomyelitis with features of myositis involving the posterior compartment of leg. He was started on intravenous cloxacillin.

Results: Procoagulant workup showed normal ACL levels and there was no mutation in Factor V Leiden. He had hyperhomocystinemia (19.38 micmol/L) with normal B12 (365 pg/ml) and folate (5.77 ng/ml) levels. HPLC was normal and flow cytometry for PNH was negative. CMV and EBV IgM serology were negative. Basic immunological workup showed hypergammaglobulinemia (IgG‐2311, IgA‐302, IgM‐121) and elevated C3 (180 mg/dl) and C4 (46 mg/dl). IgG subsets were also elevated (IgG2 – 668, IgG3 – 120). Lymphocyte subsets were normal. He was incidentally found to be seropositive for Hepatitis E but there was no jaundice. He became afebrile after treatment and pain also resolved.

Conclusions: The possibility of an underlying osteomyelitis must always be considered in children presenting with isolated DVT especially when there is associated fever.

PB624: Spontaneous Thrombosis in a Patient With Factor XI Deficiency Homozygous for the p.Cys398Tyr Mutation

U. Aygüneş 1 ; S. Caki Kilic 2 ; E. Berber 3

1 Faculty of Medicine, Pediatric Hematology‐Oncology, Sivas, Turkey, 2 Göztepe Medical Park Hospital, Pediatric Hematology, Istanbul, Turkey, 3 Faculty of Medicine, Arel University, Molecular Biology and Genetics, Istanbul, Turkey

Background: Factor XI (FXI) is a haemostatic plasma glycoprotein which circulates as a serine protease zymogen of activated FXI.

Aims: FXI deficiency is a rare bleeding disorder. Bleeding and paradoxically thrombotic complications have also been described.

Methods: We describe here the case of a patient with FXI deficiency who had a spontaneous cerebral dural thrombosis at the age of 2 years.

Results: The patient, individual II.3 in Figure 1, was 2 years old and had a history of weakness of her right arm and leg, and ptosis. The patient had a prolonged activated partial thromboplastin time of 120 seconds, her prothrombin time was 10 seconds and a mixing test for coagulation factor inhibitor detection was negative. She had decreased level of FXI (1.5%). Magnetic resonance angiography revealed that she had a subacute infarction in the posterior limb of the left internal capsule. DNA sequence analysis of the promoter, 15 exons and their boundaries of the F11 gene revealed that the girl was homozygous for a G to A change at nucleotide c.1247. The patient was not given anticoagulant therapy because of severely deficient FXI:C levels when she was first diagnosed with thrombosis. However, she returned to our clinic 2 months later with left hemiparesis. Cranial MR revealed that she had thrombosis at the left transverse and sigmoid sinus. Enoxaparin and fresh‐frozen plasma prophylactically initiated. Her clinical symptoms regressed. After 10 years, she returned to our outpatient clinic with weakness of her left arm and leg again. Cranial MR revealed that she had microinfarcts, cytotoxic edema in the anterior crus of mesencephalon. We evaluated this condition as recurrent arterial trombus and initiated aspirin. After discharge, she was followed up over 1 month, her complaints regressed but did not totally dissolve.

FIGURE 1 The family pedigree and laboratory data

Conclusions: Our case adds to the small number of patients who spontaneously developed cerebral venous thrombosis despite being homozygous for a severe FXI deficiency mutation.

PB625: Plasminogen Activator Inhibitor‐1 and Thrombosis at Childhood

H. Kizilocak; G. Dikme; B. Koc; T. Celkan

Istanbul University, Cerrahpasa Faculty of Medicine, Department of Pediatric Hematology Oncology, Istanbul, Turkey

Background: A number of prothrombotic and fibrinolytic disorders may lead to venous thrombosis. Fibrinolytic system plays a major role in hemostasis. An impaired fibrinolytic system may lead to VTE and the impairments occur due to mainly 2 reasons: (a) low concentration of tissue plasminogen activator (tPA) or (b) increased concentration of its inhibitor, plasminogen activator inhibitor‐1 (PAI‐1), in plasma. Although low plasminogen levels do not correlate with thrombophilia in the literature.

Aims: PAI‐1, a specific plasminogen activator inhibitor, is a 55‐kd glycoprotein synthesized by endothelial cells, hepatocytes, and megakaryocytes. Raised levels of PAI‐1 in plasma may lead to a hypoactive fibrinolytic pathway creating conditions that might lead to VTE.

Methods: A retrospective review of patients with PAI‐1 mutations aged 2 months to 17 years with VTE was done. Data was collected on demographics, risk factors, thrombophilia work‐up, treatment, and relapse. Intracranial thrombosis was diagnosed with MRI and venous thrombosis was diagnosed with doppler USG.

Results: Nine pediatric patients (7 male, 2 female) with VTE were recruited. Median age at first thromboembolism onset was 11 years (range 2 month to 17 years). Six patients had intracranial thrombosis and three had venous thrombosis. Five of the nine PAI‐1 mutations were homozygous with three showing arterial thrombosis, whereas the remaining was heterozygous. Seven patients had positive congenital thrombophilia risk factors other than PAI‐1 mutations (MTHFR, FV Leiden, B‐fibrinogen mutations). Two of the patients had recurrent thrombosis. All of the patients were treated with low molecular weight heparin except one.

Conclusions: We can conclude from our results that PAI‐1 mutations may have a role in the development of VTE. This needs to be further confirmed in a larger study. However, we did not evaluate our patients plasminogen levels. Moreover, simultaneous analysis of PAI‐1 levels will also make genotype phenotype correlations clearer.

PB626: The Joint Effect between Different Types of Combined Oral Contraceptives and Genetic Risk Factors on Venous Thrombosis Risk: Results from the MEGA Case‐control Study

D. Khialani ; Clinical Epidemiology

Leiden University Medical Center, Clinical Epidemiology, Leiden, the Netherlands

Background: The World Health Organization does not recommend the use of combined oral contraceptives (COCs) in women with inherited thrombophilia. This was recently challenged by authors who suggested that COCs could be prescribed to women with Factor V Leiden (FVL) or FII 20210A (F2) mutation when alternative forms of contraception are not well tolerated.

It is yet unknown whether the synergistic effect varies between different types of progestogens and thrombophilia.

Aims: Therefore, we investigated the joint effect between FVL, F2, or fibrinogen gamma (FGG) mutation and COCs on venous thrombosis risk; both in general and for the different progestogen types.

Methods: Data from female participants of the MEGA study, a large population based case‐control study were analyzed. A case‐only analysis was performed to calculate the synergy index (SI) for the joint effects between FVL/F2/FGG, FVL/F2 and FVL and COC in general, and subsequently stratified for different progestogens: levonorgestrel, desogestrel, gestodene or cyproterone acetate. The SI was multiplied with the individual effects of the genetic risk factor(s) and COC(s), obtained by the case‐control analysis, to estimate the joint effect. Additionally, a sensitivity analysis in women using COCs containing 30 microgram of estrogen only and in women without family history of VTE was performed.

Results: The joint effect of FVL/F2/FGG and COC in general was 10.5. When we stratified for the different progestogens, the joint effect of levonorgestrel and genetic risk factor(s) was lower compared with the joint effect of the other three progestogens and genetic risk factor(s) (see Table 1).

Similar results were obtained for the other two analysis (i.e. FVL/F2 and FVL) and the sensitivity analyses.

TABLE 1 FVL/F2/FGG analysis: Individual effects of genetic risk factor(s) and progestogen types and the joint effect FVL/F2/FGG COC COC (general) Levonorgestrel Desogestrel Gestodene Cyproterone − − 1 1 1 1 1 + − 1.6 (1.2‐2.2) 1.6 (1.2‐2.2) 1.6 (1.2‐2.2) 1.6 (1.2‐2.2) 1.6 (1.2‐2.2) − + 5.8 (4.3‐7.8) 4.3 (3.0‐6.1) 9.9 (6.1‐16.0) 6.4 (3.5‐11.7) 4.8 (2.5‐9.3) + + 10.5 * 8.5 * 15.0 * 11.8 * 15.3 * *Joint effect of genetic risk factors and progestogen types, calculated by multiplying the individual effects with the synergy index (not shown).

Conclusions: In women with inherited thrombophilia, COC containing the progestogen levonorgestrel is associated with the lowest risk of VTE.

PB627: Risk of Recurrent Venous Thromboembolism in Pregnancy and Postpartum Period: Results from the MEGA Follow‐up Study

L.J.J. Scheres 1 ; C.W. van Klaveren 1 ; B. Nemeth 1 ; S. Middeldorp 2 ; S.C. Cannegieter 1

1 Leiden University Medical Center, Department of Clinical Epidemiology, Leiden, the Netherlands, 2 Academic Medical Center, Department of Vascular Medicine, Amsterdam, the Netherlands

Background: Pregnancy increases the risk of first venous thromboembolism (VTE). Data on the risk of recurrence during pregnancy and postpartum period in patients with a history of VTE are scarce.

Aims: To assess the risk of recurrent VTE during pregnancy and postpartum period in patients with a history of VTE.

Methods: The MEGA follow‐up study, in which 3,750 patients with a first VTE were followed for recurrence, was linked to the Dutch hospital data registry and Dutch Foundation for Pharmaceutical Statistics database, which contain detailed information on hospital deliveries and anticoagulant use, respectively. Cox‐regression models with pregnancy and postpartum period as time‐dependent covariate were used to estimate hazard ratios (HR) with 95% confidence intervals (95% CI) for recurrent VTE. We investigated this risk assuming different durations of the postpartum period, 1, 3, 6 and 12 months. In an additional sensitivity analysis oral contraceptives users were excluded.

Results: Of all women (n=967), aged <50 years with a first VTE, 98 (10.1%) developed a recurrent VTE after a total follow‐up of 5,581 person years. During follow‐up there were 138 pregnancies. Eleven events occurred during pregnancy or a postpartum period of 3 months (11/138, 8.0%). The incidence rate of a VTE recurrence in this period was 5.3/100py (95% CI: 2.9‐9.6) compared with 1.6/100py (95% CI: 1.3‐2.0) in the non‐pregnant situation, HR 3.0 (95% CI: 1.5‐6.0). After exclusion of oral contraceptive users this HR increased to 4.0 (95% CI: 1.7‐9.3). Of the pregnancies for which this information was available, women who used thromboprophylaxis for at least 3 days during pregnancy or postpartum period, 1/29 (3.4%) had a recurrence, which was the case in 6/98 (6.1%) recurrences in women who did not use thromboprophylaxis.

Conclusions: Women with a VTE history are at increased risk of a recurrent event during and shortly after pregnancy compared with the non‐pregnant situation.

PB628: Misuse of Low Molecular Heparin in Pregnancy

J. Samonikov Tosevska ; T. Makarovska Bojadzieva; M. Blagoevska; I. Nikoloska

Institute of Transfusion Medicine, Blood Coagulation Testing, Skopje, Macedonia, the Former Yugoslav Republic of

Background: Normal pregnancy is associated with hypercoagulability of blood and increased fibrinolytic activity. In spite of that, we noticed a growing tendency towards coagulation testing in pregnant women with no history of recurrent abortions or thrombotic complications.

Aims: To investigate the indications for coagulation testing and for low molecular weight heparin (LMWH) prescribed by the gynecologists in pregnancy.

Methods: Screening haemostasis (PT, aPTT, TT) and D‐dimer were performed in 2,500 women with normal pregnancy (22‐36 gestation weak) and with no history of recurrent abortions or thrombotic complications. We determined the mean value and the standard deviation of the platelet count (150‐450 × 10 9 /L), activated partial thromboplastin time (aPTT) (28‐38 seconds) and D‐dimer (<500 ng/mL). According to the indication for testing women were divided in Group 1 (G1) – routine check, G2 – placental insufficiency and G3‐low fetal growth.

Results: Mean values for the second (14‐28 gestation weak) and third (29‐42 g. w.) trimester were 274±59.7 and 246 ±74.7 for the platelets, 26±2.4 and 25±2.6 for aPTT and 1,460±580 and 1,890±970 for D‐dimer respectively. The proportion of women who received LMWH according to the indication for coagulation testing is given on Table 1.

TABLE 1 Indications for coagulation testing and number of women receiving LMWH Indication for testing Women tested No (%) Women given LMWH No (%) G1‐Routine check 1,745 (69.8) 1,024 (58.6) G2‐Placental insufficiency 633 (25.3) 380 (60.0) G3‐Low fetal growth 122 (4.8) 65 (53.0) Total 2,500 (100.0) 1,469 (58.7)

The total number of 1,469 (58.7%) were given LMWH with no significant difference between the groups. In most of the women (69.8%) coagulation testing was ordered as a routine check and 58.6% of them were given enoxaparine 40 mg/day in the period from 15 to 60 days depending on the monitored D‐dimer levels. D‐dimer showed insignificant variations during the anticoagulation treatment with the trend of increment according to the gestation age.

Conclusions: The indications for LMWH were not clearly defined nor evidence supported. Routine screening haemostasis and D‐dimer testing during pregnancy is not justified. Education is necessary to set standards identifying the women who would benefit from coagulation testing as well as from LMWH.

PB629: Written Alert System Improves Thromboprophylaxis Compliance in Caesarean Women

L. Maggiari; I. Morales; J. Ojeda; B. Perez; I. Perez; V. Ribero; S. Grille; C. Guillermo

Universidad de la Republica., Catedra de Hematologia, Montevideo, Uruguay

Background: Venous thromboembolism (VTE) remains one of the leading causes of maternal death. Risk stratification is difficult and prophylaxis recommendations have low grade of evidence. Risk factors for VTE and prophylaxis guidelines have been highlighted by the 2009 Royal College of Obstetricians and Gynaecologists (RCOG). Caesarean is an important VTE risk factor in this population. Thromboprophylaxis is a cost‐effective way to reduce VTE morbi‐mortality, especially in caesarean women. The use of alerts may help clinicians to identify women at risk of VTE and improve the rational use of thromboprophylaxis.

Aims: To assess compliance to RCOG guidelines in caesarean women and compare the adherence with and without the use of a written alert.

Methods: We conducted a retrospective study at Hospital de Clinicas, Montevideo, Uruguay between 1/2016 to 1/2017. 233 caesarean women were enrolled and stratified in high, intermediate or low risk for VTE as suggested by RCOG guidelines. We evaluated prophylaxis indication with low‐molecular‐weight heparin (LMWH).

Results: Of 233 caesarean women 41 (17.6%) had a written alert and 192 (82.4%) did not. Median age: 25 years (25‐29). Low risk: 3.0% (n=7), high risk: 2.6% (n=6), intermediate risk: 94.4% (n=220). High‐risk women received adequate pharmacological prophylaxis with LMWH in 100%. Of the intermediate‐risk women, only 25.5% (n=56) received adequate LMWH prophylaxis. Neither VTE nor bleeding complications were developed. 61% of women with a written alert had adequate LMWH prophylaxis versus 22.4% of women without it.

Conclusions: Awareness of the thrombotic risk, as conferred by an easy and suitable risk assessment has the potential to improve VTE prophylaxis in caesarean women. In our department, we improved compliance with RCOG guidelines in a 39% with the used of a written alert.

PB631: Management of Venous Thromboembolism (VTE) during Pregnancy and Post‐partum: Findings from the Indian Subset of an International, Observational Study (SAVE)

R.M. Tiwari 1 ; J. Aoun 2 ; J.‐C. Gris 3

1 Sanofi, Medical Affairs, Mumbai, India, 2 Sanofi International Region, Antony, France, 3 University Hospital, Nîmes and University of Montpellier, Department of Haematology, Montpellier, France

Background: Guidelines‐based clinical management of pregnant and post‐partum women with risk factors for VTE in India requires critical evaluation.

Aims: To identify clinical management practices for VTE in India and their concurrence with ACCP/RCOG guidelines.

Methods: Data was assessed from the Indian subset of an international, observational, cross‐sectional study (SAVE) performed between April‐September 2015. Investigators were randomly contacted and included in the study based on their interest. Pregnant women consulting during ante‐ and post‐partum period were recruited consecutively. Women were excluded if they had experienced VTE within 4 months of, or were receiving antithrombotic medication, at enrolment. Assessments were made using data collected through investigator questionnaires and case report forms.

Results: More than 90.0% (n/N=17/18) investigators were aware of the guidelines, out of which, about 88.0% (n/N=15/17) were following them. Prophylactic treatment was prescribed in 82.7% (n/N: 43/52) of women judged to be at‐risk of VTE. The most common intervention during pregnancy was pharmacological treatment (n/N=15/35, 42.9%) with low/intermediate dose low molecular weight heparin (LMWH; 67.9%) and/or aspirin (53.6%). Prophylaxis during post‐partum was largely through LMWH. When physicians had knowledge of the guidelines, 78% (n/N=32/41) of at‐risk women received prophylaxis for VTE. The reported reason for not prescribing VTE prophylaxis against VTE was the perceived lack of positive risk‐to‐benefit ratio.

Conclusions: A high level of awareness of and adherence to guidelines possibly reflects the larger proportion of at‐risk women in India receiving VTE prophylaxis than in South Asia (77.0%). But this was substantially below the global rate (92.5%) observed in the SAVE study. Of note, prescription of aspirin was not as per guidelines and need to be addressed for universal recommendation‐based practice.

PB632: Thrombin Generation and Adverse Pregnancy Outcomes

I. Gribkova 1 ; N. Koroleva 2 ; M. Davydovskaya 1,3 ; A. Murashko 2

1 Clinical Trials and Healthcare Technology Assessment Centre of Moscow Department of Healthcare, Moscow, Russian Federation, 2 Federal State Autonomous Educational Institution of Higher Education I.M. Sechenov First Moscow State Medical University, Moscow, Russian Federation, 3 Neurosurgery and Medical Genetics of Pirogov Russian National Research Medical University, Moscow, Russian Federation

Background: Pregnancy is a well‐established risk factor for thrombosis, and is associated with increased thrombin generation. According to the literature, thrombosis is considered to be one of the mechanisms of adverse pregnancy outcomes. However, currently, there is a lack of consensual data of thrombin generation during complicated pregnancy.

Aims: To evaluate the effectiveness of the thrombin generation test in the determination of thrombotic risk in women with a complicated course of pregnancy.

Methods: The institutional ethics committee approved this study. We examined 32 pregnant women. 22 women with a complicated course of pregnancy made up the study group, 10 practically healthy women – the comparison group. The patients from the study group were divided into a subgroup with a favorable pregnancy outcome (n=15) and a subgroup with an adverse outcome (n=7). The study of the state of the hemostasis system in patients was carried out using standard coagulation tests (prothrombin index, fibrinogen concentration, platelet aggregation), and also with the thrombin generation test (TGT).

Results: A statistically significant increase in the TGT parameter: endogenous thrombin potential (ETP) in pregnant women compared with non‐pregnant women (2,300±400 and 1,700±400, respectively, P<0.005) was shown. A statistically significant increase of ETP in the group of patients with an adverse outcome of pregnancy was shown compared to the group with a favorable outcome (2,700±600 and 2,300±300, respectively, P<0.005). The results of standard coagulation tests were within the normal values in both groups.

Conclusions: TGT could be used to predict adverse pregnancy outcomes. High ETP values are associated with adverse pregnancy outcomes.

PB633: The Management of Pregnancy and Successful Delivery of a Patient with Bland‐White‐Garland Syndrome and a Prosthesis Mitral Valve

A. Makatsariya ; J. Khizroeva; V. Bitsadze; M. Arslanbekova

I.M. Sechenov First Moscow State Medical University, Department of Obstetrics and Gynecology, Moscow, Russian Federation

Background: The management of pregnant women with artificial cardiac valves is rather problematic, due to the fact that these patients traditionally constitute a high‐risk group for the development of thromboembolic disorders. It should be kept in mind that outside of pregnancy, artificial valves are a direct indication to for a lifelong anticoagulant therapy.

Aims: To provide adequate anticoagulant therepy in pregnant womean with a prosthesis mitral valve.

Methods: This article describes the clinical case. The patient was observed at the medical center for women.

Results: The patient is 32 years old with diagnosis: “BWG syndrome. Prosthesis of a mechanical prosthetic mitral valve Sorin 27, reimplantation of the left coronary artery in the ascending aorta with the plasty of the pulmonary artery in 2011. Cardial dysrhythmia by the ventricular extrasystole, paroxysmal form of supraventricular tachycardia. Heart failure (HF) II functional class (FC) NYHA.” The entire pregnancy was given warfarin under the control of MHO. There were repeated hospitalizations due to complaints of paroxysmal atrial flutter and routine examination during pregnancy. At 38 weeks the patient had given birth to a healthy full‐term baby girl weighing 2,750 grams, 47 cm long, and an Apgar score of 8/9 by cesarean section as planned. She was discharged with the child from the hospital in a satisfactory condition on the 8th day.

Conclusions: This clinical case shows both the complexity as well as the possibility of managing pregnancy with the help of various anticoagulant therapy schemes and the successful delivery of a patient with congenital heart disease, BWG syndrome, condition after mitral valve prosthesis, the outcome of which is the birth of a healthy full‐term baby. The most important role in preparation, management of pregnancy in patients with artificial valves, delivery and in the postpartum period has adequate anticoagulant therapy to prevent thrombohemorrhagic disorder.

PB634: Reduced Protein C Global Assay Level in Infertile Women with IVF Failure: A Pilot Study

H. Azzam 1 ; S. El Masry 2 ; H. Youssef 3 ; M. Othman 4 ; M. Awad 3

1 Faculty of Medicine, Mansoura University, Clinical Pathology Dept, Mansoura, Egypt, 2 Dakahlia Psychiatric Hospital, Mansoura, Egypt, 3 Faculty of Medicine, Mansoura University, Mansoura, Egypt, 4 Faculty of Medicine, Queen's University, Ontario, Canada

Background: Protein C (ProC) Global is a global dotting assay that evaluates abnormalities in the protein C anticoagulant pathway. Few studies examined this assay in relation to assisted reproductive therapy (ART) but its role in infertile women with in vitro fertilization failure (IVF) remains unclear.

Aims: In this study, we assessed ProC in infertile women with history of IVF failure and are undergoing ART.

Methods: We examined 45 healthy fertile women conceiving naturally, and 45 infertile women with 2 or more implantation failure undergoing ART. Both ProC and activated protein C resistance (APC‐R) were evaluated.

Results: The results showed that mean ProC expressed as normalized ratio (PCAT‐NR), was significantly lower in the study compared to control group; 0.76±0.15; 0.91±0.14,respectively ( P =0.0001). Follow up on ART outcomes, women who failed ART had significantly lower PCAT‐NR compared to successful ones. PCAT‐NR did not correlated with APC‐R levels in study (r=0.125, P <0.5) or failed ART subgroup. Using logistic regression analysis, patients with lower PCAT‐NR levels showed elevated risk of implantation failure ( P =0.04, OR=0.50, 95% CI=0.26‐0.84).

Conclusions: In conclusion, Pro C global assay may play a role in the etiology of IVF failure which may be independent of APC‐R. Larger studies are encouraged to validate these findings and explore the underlying pathophysiological mechanisms.

PB635: Hereditary Thrombophilia Assessment in Pregnancy

M. Blagoevska 1 ; J. Samonikov Tosevska 1 ; T. Makarovska Bojadzieva 1 ; A. Hristova Dimceva 2 ; L. Gorcaj 3

1 Institute of Transfusion Medicine, Blood Coagulation Testing, Skopje, Macedonia, the Former Yugoslav Republic of, 2 Institute of Transfusion Medicine, Molecular Biology, Skopje, Macedonia, the Former Yugoslav Republic of, 3 Institute of Transfusion Medicine, Imunohaematology, Skopje, Macedonia, the Former Yugoslav Republic of

Background: Abnormalities of haemostasis are involved in more than 60% of recurrent pregnancy loss (RPL) cases from which only 2% because of haemorrhagic disorders. Data suggest that women with thrombophilia have an increased risk of obstetric complications.

Aims: The aim of this study is to evaluate the association between hereditary thrombophilia and RPL.

Methods: In 76 women with RPL and in 80 pregnant women with no history of pregnancy loss genetic markers for thrombophilia such as factor V Leiden (FVL), methylenetetrahydrofolate reductase mutation (MTHFR, C677T) and prothrombin gene mutation (FII, G20210 ) were determined. Coagulation activity of antitrombin (AT), Protein C (PC) and Protein S (PS) was also measured.

Results: Heterozygous mutation for MTHFR was found in 21 (26.2%) of the pregnant women with no history of pregnancy loss. There were no abnormal results in the other markers. Thrombophilic factors were determined in 36 (47.3%) out of 76 women with RPL from which 8 (10.5%) were heterozygous for FVL, 23 (30.2%) were heterozygous for MTHFR, 3 (3.9%) had decreased activity of PC (30‐40% of the normal) and 2 (2.6%) had decreased PS (15% and 20% of normal) activity. The difference in the frequency of heterozygous MTHFR mutation between the RPL women and the control group is not significant.

Conclusions: FVL is significant and independent risk factor for RPL which is not the case with MTHFR. Hereditary thrombophilia is associated with RPL but not all of the thrombophilic factors contribute equally. There is still much to be investigated about their thrombogenic effect which has to be considered in the evaluation of thrombophilia associated RPL and consecutive LMWH thromboprophylaxis.

PB636: Localization and Massiveness of Thrombosis in Women with Pregnancy Related Thrombosis Compared to Women with Unprovoked Venous Thromboembolism

J. Bodrozic 1 ; P. Miljic 1,2 ; D. Lekovic 1,2 ; D. Vasic 3 ; M. Gonic 2,4

1 Clinic of Hematology, Clinical Centre of Serbia, Belgrade, Serbia, 2 University of Belgrade, Faculty of Medicine, Belgrade, Serbia, 3 Clinic for Vascular and Endovascular Surgery, Clinical Centre of Serbia, Belgrade, Serbia, 4 Clinic for Gynecology and Obstetrics, Clinical Centre of Serbia, Belgrade, Serbia

Background: Pregnancy related thrombosis differs from the unprovoked one in the incidence, risk factors and recurrence. It is not known either they also differ in the distribution of thrombosis.

Aims: To investigate either there is a difference between the massiveness of VTE during pregnancy and puerperium compared to non‐pregnant women.

Methods: We conducted a retrospective analysis of 170 women with unprovoked and 202 women with pregnancy related deep vein thrombosis of lower extremities (LE) or pulmonary embolism (PE) who were consecutively referred to our institution from January 2005 to December 2016. When thrombosis of LE was present with PE, the event was accounted as PE. Thrombosis was classified as non‐massive if occurred in either proximal or distal veins. Whole leg or both legs thrombosis was considered as massive. Thrombosis of superficial veins was excluded. All thrombotic episodes were confirmed with duplex ultrasonography and CT pneumoangiography. Descriptive statistics and chi square test were used for statistical analysis.

Results: Out of 202 women with pregnancy related thrombosis, 32 (15.8%) developed PE, 78 (38.6%) developed massive thrombosis while in 92 women (45.5%) thrombosis was non massive. In 170 women with unprovoked thrombosis PE occurred in 60 (35.3%) women, massive thrombosis in 44 (25.9%) and non‐massive in 66 (38.8%). There was no statistically significant difference between massiveness of thrombosis between women with unprovoked thrombosis and those with pregnancy related one (P>0.005). PE occurred more often in woman with unprovoked thrombosis than in those with pregnancy related one (P<0.005).

Conclusions: Two times higher prevalence of PE in women with pregnancy related thrombosis may be a consequence of under diagnosing this complication (reluctance to use CT angiography or perfusion scintigraphy) during pregnancy although we cannot exclude the real difference in prevalence of thrombosis during pregnancy compared to non‐pregnant women.

PB637: Prospective Study of Changes in Haemostasis in High‐risk Pregnancy

L. Stanciakova 1 ; M. Dobrotova 1 ; P. Holly 1 ; Z. Jedinakova 2 ; I. Skornova 1 ; J. Zolkova 1 ; L. Vadelova 1 ; J. Danko 3 ; J. Stasko 1 ; P. Kubisz 1

1 Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, University Hospital Martin, National Centre of Thrombosis and Haemostasis, Department of Haematology and Transfusiology, Martin, Slovakia, 2 Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, University Hospital Martin, National Centre of Thrombosis and Haemostasis, Department of Haematology and Transfusiology, Center of Hemostasis and Thrombosis, HEMO MEDIKA spol. s r.o., Martin, Slovakia, 3 Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, Department of Gynaecology and Obstetrics, Martin, Slovakia

Background: Pre‐existing risk factors combined with acquired physiological prothrombotic changes in the course of pregnancy and during the postpartum period contribute to the increased risk of venous thromboembolism (VTE) and pregnancy complications. These events may lead to maternal morbidity and mortality. They share the common characteristic that is the increased formation of thrombin, which may lead to the production of microthrombi in the implantation site, or later to the disorder of uteroplacental blood flow. In such high‐risk pregnant patients, current guidelines prefer the prevention of thrombotic events using the low‐molecular‐weight heparin (LMWH).

Aims: To evaluate the changes of haemostasis during pregnancy and postpartum period in patients with high‐risk pregnancy with the use of standard and “point‐of‐care” methods.

Methods: In high‐risk pregnant women with previous thromboembolic event, repeated pregnancy loss or other pregnancy complications on anticoagulant thromboprophylaxis, the changes of haemostasis were monitored using conventional coagulation tests and so‐called “point‐of‐care” methods.

Results: In high‐risk pregnant women with recurrent pregnancy loss and thrombophilic state, LMWH subcutaneously once daily was used. We evaluated haemostatic parameters at 10‐12, 16‐18, 26‐28, 35‐36 gestational weeks and 6‐8 weeks following delivery.

In spite of the use of adequate anticoagulation, our results point out to the progression of the hypercoagulant state developed in pregnancy.

Conclusions: Evaluation of the changes of haemostasis in the high‐risk pregnant women may represent key function in the targeted anticoagulant thromboprophylaxis. The study complies with the Declaration of Helsinki and from each patient, the informed consent was obtained and the study was approved by a recognized Medical Ethics Committee.

Acknowledgement: We would like to thank the support of projects of Scientific Grant Agency Vega 1/0168/16 and Agency for the Support of Research and Development APVV‐16‐0020.

PB638: Risk Assessment of Venous Thromboembolism (VTE) during Pregnancy and Post‐partum: Findings from the Indian Subset of an International, Observational Study (SAVE)

R. Tiwari 1 ; J. Aoun 2 ; J.‐C. Gris 3

1 Sanofi, Medical Affairs, Mumbai, India, 2 Sanofi International Region, Antony, France, 3 University Hospital, Nîmes and University of Montpellier, Department of Haematology, Montpellier, France

Background: The clinical burden of VTE in pregnant and post‐partum Indian women is not well documented in India.

Aims: To assess the risk of VTE during pregnancy/post‐partum in India.

Methods: Data was analysed from the Indian subset of an international, observational, cross‐sectional study (SAVE) performed between April‐September 2015. Pregnant women consulting during ante‐ and post‐partum period were recruited consecutively. Women were excluded if they had experienced VTE within 4 months of or were receiving antithrombotic medication, at enrolment. Assessments were made using data collected through investigator questionnaires and case report forms.

Results: Of the 503 eligible women, 470 (93.4%) consulted during pregnancy and mostly during the third trimester (N=201, 40.0%). Previous abnormal pregnancies were reported in 84 (16.7%) women, mostly (n=55) due to miscarriage at<10 weeks of gestation. Nearly 11% women (n/N=56/503) had prior history of other pre‐existing risk factors for VTE. Based on the study investigators’ assessment, 52 (10.3%; 95% CI: 7.8‐13.3] enrolled women were at risk of VTE, the majority of whom were estimated to be at mild (69.2%) or moderate (26.9%) risk. The proportion of at‐risk subjects was higher at post‐partum period than in pregnancy (18.2% vs. 9.8%).

Conclusions: This study shows that the estimated VTE risk in pregnant/post‐partum women in India is lower than regions outside the Western world (51.4%) and overall South Asian (19.9%) population. The proportion of women in India with mild‐to‐moderate risk of VTE during pregnancy/post‐partum is similar to that reported for South Asian region in the SAVE study. Of note, the higher proportion of at‐risk women during post‐partum could be attributed to the lower number of women in the group.

PB639: Genetic Risk Factors of Endothelial Dysfunction in Pregnant Women

N. Korsakova 1 ; S. Kapustin 1 ; N. Silina 1 ; O. Matvienko 1 ; O. Golovina 1 ; A. Nikolaeva 2 ; L. Papayan 1

1 Russian Research Institute of Haematology and Transfusiology, St. Petersburg, Russian Federation, 2 Antenatal Clinic 22, St. Petersburg, Russian Federation

Background: Normal vascular system function is of highest importance in pregnancy. Endothelial dysfunction (ED) is an early nonspecific vascular trouble mark, that can lead eventually to different pathologies including thrombosis. At present, technology of noninvasive quantitative ED evaluation by means of EndoPAT device is developed, that gives the value of Reactive Hyperemia Index (RHI).

Allelic polymorphisms of genes participating in vascular tone and lipid metabolism regulation might influence ED development, predisposing progression of states involved in ED pathogenesis, e.g. arterial hypertension and dyslipidemia.

Aims: Evaluation of genetic polymorphisms impact in ED development in pregnant women.

Methods: The study included 18 pregnant women with confirmed diagnosis of arterial hypertension, gestational or type II diabetes mellitus or/and obesity. ED evaluation was performed with use of EndoPAT 2000 device. RHI value of ≤1.67 was considered as ED mark. Control group consisted of 200 healthy persons.

Molecular genetic typing was performed by PCR for following genetic polymorphisms: angiotensinogen (AGT) T704C, angiotensin‐converting enzyme (ACE) Ins/Del, angiotensin II receptor type I (ATGR1) A1166C, G‐protein β3 subunit (GNB3) C825T and apolipoprotein E (ApoE) E2/E3/E4.

STATISTICA 6.0 was used. Median (Me) and 95% confidence interval (CI) for RHI were calculated. Fisher exact test was used to assess the differencies in allele and genotype frequencies. Odds ratios (OR) and their 95% CI: were calculated.

Results: The patients had the following RHI values: Me 1.4; 95% CI: 1.1‐1.8, that confirms ED presence. Genotype and allele frequences in pregnant women with ED are represented in Tables 1 and 2.

TABLE 1 Genotype frequences in pregnant women with ED Pregnants with ED, % (n=18) Controls,% (n=200) OR; 95% CI Pregnants with ED,% (n=18) Controls,% (n=200) OR; 95% CI AGT 704 C/C 38.9 25.5 1.9; 0.7‐5.1 GNB3 825 T/T 11.1 11.0 1.0; 0.2‐4.7 AGT 704 T/C 33.3 46.0 0.6; 0.2‐1.6 GNB3 825 C/T 66.7 44.5 2.5; 0.9‐6.9 AGT 704 T/T 27.8 28.5 1.0; 0.3‐2.8 GNB3 825 C/C 22.2 44.5 0.4; 0.1‐1.1 ACE Del/Del 50.0 * 26.5 2.8; 1.0‐7.4 Apo E3/E4 38.9 * 17.0 3.1; 1.1‐8.6 ACE Ins/Del 27.8 48.5 0.4; 0.1‐1.2 Apo E2/E4 5.6 1.0 5.8; 0.5‐67.6 ACE Ins/Ins 22.2 25.0 0.9; 0.3‐2.7 Apo E3/E3 50.0 61.0 0.6; 0.2‐1.7 ATGR1 1166 C/C 5.6 7.0 0.8; 0.1‐6.3 Apo E2/E3 5.6 18.5 0.3; 0.03‐2.0 ATGR1 1166 A/C 38.9 37.0 1.1; 0.4‐2.9 Apo E2/E2 0 0.5 0 ATGR1 1166 A/A 55.6 56.0 1.0; 0.4‐2.6 Apo E4/E4 0 2.0 0 *Significant difference, P<0.05.

TABLE 2 Allele frequences in pregnant women with ED Pregnants with ED,% (n=36) Controls,% (n=400) OR; 95% CI Pregnants with ED,% (n=36) Controls,% (n=400) OR; 95% CI AGT 704 C 55.6 48.5 1.3; 0.7‐2.6 GNB3 825 T 44.4 33.3 1.6; 0.8‐3.2 AGT 704 T 44.4 51.5 GNB3 825 C 55.6 66.8 ACE Del 63.9 50.8 1.7; 0.8‐3.5 Apo E4 22.2* 11.0 2.3; 1.0‐5.4 ACE Ins 36.1 49.3 Apo E2 5.6 10.3 0.5; 0.1‐2.2 ATGR1 1166 C 25.0 25.5 1.0; 0.4‐2.1 Apo E3 72.2 78.8 0.7; 0.3‐1.5 ATGR1 1166 A 75.0 74.5 *Significant difference. P<0.05.

Conclusions: Pregnants with ED had significantly higher ACE Del/Del and ApoE3/E4 genotype frequences, OR values indicate the direct correlation of these genotypes with ED development in pregnant women. Also such a correlation was shown for allele ApoE4 confirming its role in ED development.

PB640: Characterization and Outcomes of aTTP Patients with Initial or Recurrent Disease: Experience from the Phase III HERCULES Trial of Caplacizumab

M. Scully 1 ; S. Cataland 2 ; F. Peyvandi 3 ; P. Coppo 4 ; P. Knöbl 5 ; J.A. Kremer Hovinga 6 ; A. Metjian 7 ; J. de laRubia 8 ; K. Pavenski 9 ; F. Callewaert 10 ; D. Biswas 10 ; H. De Winter 10 ; R.K. Zeldin 10

1 University College London Hospitals NHS Trust, Department of Haematology, London, United Kingdom, 2 The Ohio State University Wexner Medical Center, Division of Hematology, Department of Internal Medicine, Columbus, United States, 3 Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy, 4 Saint‐Antoine University Hospital, Department of Hematology, Paris, France, 5 Vienna University Hospital, Department of Medicine 1, Division of Hematology and Hemostasis, Vienna, Austria, 6 Bern University Hospital, Inselspital, University Clinic of Hematology and Central Hematology Laboratory, Bern, Switzerland, 7 Duke University School of Medicine, Division of Hematology, Durham, United States, 8 Universidad Católica de Valencia Hospital Dr. Peset, Hematology Department, Valencia, Spain, 9 St. Michael's Hospital/Research Institute, Department of Laboratory Medicine and Pathobiology, Toronto, Canada, 10 ABLYNX NV, Clinical Development, Zwijnaarde, Belgium

Background: Acquired thrombotic thrombocytopenic purpura (aTTP) is a life‐threatening autoimmune blood clotting disorder. Patients are at risk for significant morbidity and death during each episode. Efficacy and safety data were obtained in the Phase III HERCULES study with caplacizumab in patients with aTTP (Scully et al ., Blood 2017 130:LBA‐1).

Aims: Characterization of disease presentation and evaluation of treatment outcomes in patients enrolled in the HERCULES study with an initial or recurrent aTTP episode.

Methods: Demographics, baseline disease characteristics, and treatment outcomes (time to platelet count response, mortality, recurrence, major thromboembolic (TE) events and refractoriness) were evaluated for both subgroups using descriptive summaries.

Results: 145 patients were randomized, 82 with an initial aTTP episode and 63 with recurrent disease. Demographics were generally balanced between groups, whereas baseline disease characteristics were more severe in initial versus recurrent episodes: mean platelet count (28.8 × 10 9 /L vs. 44.4 × 10 9 /L), mean LDH (598U/L vs. 523U/L) and median cardiac Troponin‐I (0.119 μg/L vs. 0.036 μg/L). The time from first symptoms until diagnosis was also longer in those experiencing an initial episode (6.5 days) versus a recurrent one (3.9 days). More patients in the caplacizumab group had their first aTTP episode (66.7%) versus the placebo group (46.6%). Treatment with caplacizumab improved outcomes [i.e., faster time to platelet count response, lower proportion of patients with either death, recurrence or a major TE event during the treatment period, lower recurrence rate during the overall study period, and prevention of refractoriness] in both subgroups compared to placebo (see Table 1 for detailed results).

TABLE 1 Efficacy outcomes by previous TTP episode (Intention‐to‐treat population) Efficacy outcomes Initial episode Recurrent episode Caplacizumab N=48 * Placebo N=34 Caplacizumab N=24 Placebo N=39 Time to platelet count response: Platelet count normalization rate ratio (95% CI) 1.67 (1.025‐2.722) 1.64 (0.951‐2.818) TTP related death, recurrence of TTP or a major TE event during the study drug treatment period – n (%) 6 (12.8) 19 (55.9) 3 (12.5) 17 (43.6) Recurrence of TTP during Overall Study period – n (%) 6 (12.8) 15 (44.1) 3 (12.5) 13 (33.3) Refractory TTP – n (%) 0 1 (2.9) 0 2 (5.1) *Percentages are based on 47 patients (1 subject withdrew consent prior to study drug treatment).

Conclusions: Patients with an initial aTTP episode have a delayed presentation and more severe disease at baseline than those with recurrent disease. Treatment with caplacizumab improves outcomes in both subgroups.

PB641: Efficacy and Safety of Open‐label Caplacizumab in Patients with Exacerbations of Acquired Thrombotic Thrombocytopenic Purpura during the Phase III HERCULES Study

M. Scully 1 ; S. Cataland 2 ; F. Peyvandi 3 ; P. Coppo 4 ; P. Knöbl 5 ; J.A. Kremer Hovinga 6 ; A. Metjian 7 ; J. de laRubia 8 ; K. Pavenski 9 ; F. Callewaert 10 ; D. Biswas 10 ; H. De Winter 10 ; R.K. Zeldin 10

1 University College London Hospitals NHS Trust, Department of Haematology, London, United Kingdom, 2 The Ohio State University Wexner Medical Center, Division of Hematology, Department of Internal Medicine, Columbus, United States, 3 Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy, 4 Saint‐Antoine University Hospital, Department of Hematology, Paris, France, 5 Vienna University Hospital, Department of Medicine 1, Division of Hematology and Hemostasis, Vienna, Austria, 6 Bern University Hospital, Inselspital, University Clinic of Hematology and Central Hematology Laboratory, Bern, Switzerland, 7 Duke University School of Medicine, Division of Hematology, Durham, United States, 8 Universidad Católica de Valencia Hospital Dr. Peset, Hematology Department, Valencia, Spain, 9 St. Michael's Hospital/Research Institute, Department of Laboratory Medicine and Pathobiology, Toronto, Canada, 10 ABLYNX NV, Clinical Development, Zwijnaarde, Belgium

Background: The efficacy and safety of caplacizumab in patients with acquired Thrombotic Thrombocytopenic Purpura (aTTP) have been demonstrated in a single‐blind phase 2 study (Peyvandi et al ., N Engl J Med 2016, 374(6):511‐522) and confirmed in a double‐blind phase 3 study (HERCULES; Scully et al ., Blood 2017 130:LBA‐1).

Aims: Herein we present the efficacy and safety results of patients who received open‐label (OL) caplacizumab in the HERCULES study.

Methods: In case of a recurrence during the double‐blind (DB) treatment period, patients were switched to OL caplacizumab, together with re‐initiation of daily plasma exchange (PEX) and immunosuppression, while maintaining the blind for the initial treatment allocation.

Results: 31 patients experienced an exacerbation during the DB period, 28 in the placebo group and 3 in the caplacizumab group. Twenty‐eight of them were switched to OL treatment with caplacizumab (26 of the 28 placebo‐treated patients and 2 of the 3 caplacizumab‐treated patients). By day 6, 81% of patients receiving OL caplacizumab achieved a confirmed platelet count response (i.e., platelet count ≥150×10 9 /L confirmed by stop of daily PE within 5 days). There were no deaths. One patient (3.6%) experienced a TTP exacerbation, and 1 patient (3.6%) experienced a major thromboembolic event (vena cava thrombosis) during the OL treatment period. After the end of OL treatment, 3 patients had a recurrence of TTP and all 3 had ADAMTS13 activity<10% when the treatment was stopped. The safety profile in the OL caplacizumab group was consistent with that observed in the DB caplacizumab group. In total, 25 patients (89.3%) were reported with at least one treatment‐emergent adverse event.

Conclusions: Open‐label therapy with caplacizumab was efficacious in HERCULES study patients who experienced an exacerbation of aTTP during the double‐blind treatment period. The safety profile was consistent with that observed in the double‐blind period.

PB642: Treatment of Acquired TTP with the Anti‐vWF Nanobody, Caplacizumab, Results in a Significant Reduction in Healthcare Resource Utilization – Data from the Phase III HERCULES Trial

M. Scully 1 ; S. Cataland 2 ; F. Peyvandi 3 ; P. Coppo 4 ; P. Knöbl 5 ; J.A. Kremer Hovinga 6 ; A. Metjian 7 ; J. de laRubia 8 ; K. Pavenski 9 ; F. Callewaert 10 ; D. Biswas 10 ; H. De Winter 10 ; R.K. Zeldin 10

1 University College London Hospitals NHS Trust, Department of Haematology, London, United Kingdom, 2 The Ohio State University Wexner Medical Center, Division of Hematology, Department of Internal Medicine, Columbus, United States, 3 Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy, 4 Saint‐Antoine University Hospital, Department of Hematology, Paris, France, 5 Vienna University Hospital, Department of Medicine 1, Division of Hematology and Hemostasis, Vienna, Austria, 6 Bern University Hospital, Inselspital, University Clinic of Hematology and Central Hematology Laboratory, Bern, Switzerland, 7 Duke University School of Medicine, Division of Hematology, Durham, United States, 8 Universidad Católica de Valencia Hospital Dr. Peset, Hematology Department, Valencia, Spain, 9 St. Michael's Hospital/Research Institute, Department of Laboratory Medicine and Pathobiology, Toronto, Canada, 10 ABLYNX NV, Clinical Development, Zwijnaarde, Belgium

Background: The efficacy and safety of caplacizumab, an anti‐von Willebrand Factor (vWF) Nanobody, for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) were evaluated in the HERCULES study (Scully et al ., Blood 2017 130:LBA‐1).

Aims: To investigate the effect of treatment with caplacizumab on healthcare resource utilization: plasma exchange (PE) parameters, days spent in the hospital and in the intensive care unit (ICU).

Methods: Volume and days of PE, days in hospital and ICU were summarized and compared between the caplacizumab and placebo arms for the overall study drug treatment period, using a normal approximation to the Wilcoxon Rank Sum test.

Results: 145 patients were randomized, 73 to placebo and 72 to caplacizumab. Treatment with caplacizumab led to faster normalization of platelet counts, prevented exacerbations, and prevented patients from becoming refractory to treatment. This was reflected in a 38% reduction in mean (±SE) number of PE days in the caplacizumab (n=71) versus placebo group (n=73): 5.8 (±0.51) days versus 9.4 (±0.81) days (P<0.001). The mean (±SE) total volume of plasma exchanged was similarly reduced by 41%: 21.3 (±1.6) L versus 35.9 (±4.2) L (P<0.001). Average duration of hospitalization (±SE) was decreased by 31% in the caplacizumab (n=71) versus placebo group (n=73): 9.9 (±0.7) days versus 14.4 (±0.7) days (P=0.0025).

A third of the patients were admitted to the ICU (28 patients in the caplacizumab group and 27 patients in the placebo group). In the caplacizumab group, the mean (±SE) number of days spent in the ICU was reduced by 65%: 3.4 (±0.4) days versus 9.7 (±2.1) days (P=0.0098).

Conclusions: Caplacizumab, through rapid blocking of vWF‐mediated platelet adhesion, represents a novel treatment for aTTP. Treatment with caplacizumab results in improved outcomes as reflected by meaningful reductions in healthcare resource utilization.

PB643: Targeted Thrombolytics for Thrombotic Thrombocytopenic Purpura

C. Clark 1 ; M. Waning 1 ; A. Barendrecht 1 ; P. Lenting 2 ; C. Maas 1 ; S. deMaat 1

1 University Medical Center Utrecht, Clinical Chemistry and Haematology, Utrecht, the Netherlands, 2 INSERM, Université Paris‐Sud, Université Paris‐Saclay, Unité Mixte de Recherche, Paris, France

Background: Patients with thrombotic thrombocytopenic purpura (TTP) experience attacks of microvascular thrombosis, when platelets form complexes with ultra‐large von Willebrand Factor (VWF). This is the result of severely decreased ADAMTS13 activity, which normally regulates the thrombogenicity of VWF. Current therapy involves extensive plasma exchange to restore ADAMTS13 activity. However, persistent autoantibodies impede elimination of microthrombi. We previously reported that systemic plasminogen activation (with streptokinase) was therapeutic in a mouse model for TTP, suggesting that plasmin can act as a functional alternative to ADAMTS13. Although plasmin(ogen) can directly bind to VWF, natural plasminogen activators (tPA, uPA) cannot. Furthermore, microthrombi are fibrin‐poor. For optimal therapeutic efficacy and safety, it is wishful to localize plasminogen activation to sites of microvascular occlusion.

Aims: To develop recombinant fusion proteins for targeted delivery of plasminogen activators to platelet‐VWF complexes.

Methods: We fused the catalytic domain of uPA to nanobodies directed against VWF or platelet glycoprotein 1b (GP1b). As a control, we generated a non‐targeting nanobody‐uPA fusion protein. Proteins were expressed in HEK293 cells and showed no spontaneous activity after isolation.

Results: We confirmed binding to VWF by ELISA (affinities of 30 pM, 330 pM and 12.8 nM for three constructs) and binding to GPIb by FACS. All constructs were activatable into active enzymes by plasmin and had similar plasminogen‐activating potential. In the case of VWF‐binding fusion proteins, VWF acts as a scaffold for accelerated plasminogen activation (up to 6‐fold). We next performed functional studies. Fusion proteins that target components of platelet‐VWF complexes accelerate destruction of microthrombi in agglutination experiments and under flow on activated endothelial cells.

Conclusions: Targeted delivery of plasminogen activators to microthrombi is a feasible and novel approach for the treatment of TTP.

PB644: Initial Clinical Presentation of Thrombotic Thrombocytopenic Purpura with Focal Stroke: A Series of Patients from the French National Reference Center Registry for Thrombotic Microangiopathies

M. Delrue 1,2 ; A. Wynckel 3 ; L. Galicier 4 ; E. Mariotte 5 ; P. Poullin 6 ; S. Canet 7 ; A. Veyradier 1,2 ; P. Coppo 8,9 ; A. Stépanian 1,2

1 Lariboisière Hospital, Assistance Publique‐Hôpitaux de Paris, Hematology Department, Paris, France, 2 EA 3518, Institut Universitaire d'Hématologie, Saint Louis Hospital, Paris, France, 3 CHU de Reims, Nephrology Department, Reims, France, 4 Saint Louis Hospital, Assistance Publique‐Hôpitaux de Paris, Clinical Immunopathology Departement, Paris, France, 5 Saint Louis Hospital, Assistance Publique‐Hôpitaux de Paris, Intensive Care Unit, Paris, France, 6 La Conception Hospital, Assistance Publique‐Hôpitaux de Marseille, Hematology Department, Marseille, France, 7 Hospital of Perpignan, Nephrology Department, Perpignan, France, 8 Saint‐Antoine Hospital, Assistance Publique‐Hôpitaux de Paris, Hematology Department, Paris, France, 9 Inserm U1170, Institut Gustave Roussy, Villejuif, France

Background: Thrombotic thrombocytopenic purpura (TTP) is a rare disease associated with ADAMTS13 activity deficiency (<10 IU/dL), with accumulation of ultra‐adhesive von Willebrand factor multimers, resulting in formation of thrombi in microcirculation. Subsequently, various clinical manifestations include heart or renal failure, neurological deficit and abdominal signs. In TTP, neurological presentation is mainly described as disseminated microvascular damage, but more rarely it can be focal, affecting large vessels and single cerebral vascular territory. This latter condition can potentially lead to misdiagnosis and be life‐threatening.

Aims: To determine the frequency of stroke at presentation in TTP patients and to characterize their clinical features.

Methods: Retrospective study (2001‐2017) using the French National Reference Center for thrombotic microangiopathies registry.

Results: Among 1354 TTP patients, 53 had a stroke at initial presentation, resulting in a prevalence of 4%. In these 53 patients, median age was 48 years old (IQR 33‐58) and 79% were women. All were acquired TTP, with anti‐ADAMTS13 antibodies identified in 60% of cases; TTP was inaugural in 60% of cases. In 50% of cases, cerebral infarction and/or neurological deficit were focal and systematized. Moreover, there was no other organ failure except neurological manifestations in 55% of cases, whereas the remaining patients presented also renal or cardiac failure, or abdominal signs. Among the 55% patients group, a unique cerebral artery territory was involved in 41% cases.

Conclusions: Stroke is a rare condition during TTP. Neurological manifestations are often unspecific, but cerebral damages can also be focal and involve macrovessels, like in classical stroke, then involving one cerebral vascular territory. Clinicians managing patients with stroke should exclude TTP with routine blood tests, including platelet count ± hemolysis parameters, since TTP is fatal without specific treatment in emergency.

PB645: Provision of an External Quality Assessment for ADAMTS13

I. Jennings ; S. Kitchen; D. Kitchen; S. Munroe‐Peart; L. Brown; T. Woods; I. Walker

UK NEQAS for Blood Coagulation, Sheffield, United Kingdom

Background: ADAMTS13 assays are vital for diagnosis of thrombotic thrombocytopenic purpura (TTP). The majority of TTP is acquired secondary to antibodies and is usually treated with urgent plasma exchange. TTP is often excluded based on normal ADAMTS13 activity. Misdiagnosis can have fatal consequences so accurate assays are essential for safe management. ADAMTS13 assays can also be used to monitor treatment and identify family members with ADAMTS13 deficiency. Assays are only available in specialist centres, but nonetheless these assays require external quality assessment (EQA) to ensure accurate and precise results.

Aims: UK NEQAS for Blood Coagulation (BC) introduced a programme for ADAMTS13 in 2013. 3 EQA exercises have been completed with 6 samples provided.

Methods: Samples were obtained from patients undergoing plasma exchange for (TTP). For each exercise centres were asked to perform antigen, activity or antibody assays. Median result, range and CV were determined for all results returned.

Results: Activity assay results are shown in Table 1. Antigen assays were performed by 5 centres in exercise 1, 4 centres in exercise 2 and no centres performed antigen assays in exercise 3. Antibody assays were performed by 8 centres in exercise 1 with CVs ranging from 5‐42.6% across the three samples. Antibody assays were performed by 10 centres in exercise two with a CV of 33.5%, and 12 centres in exercise 3 when no analysis could be performed due to the variety of methods used to report results. Results obtained by Collagen Binding assay were lower than those obtained by FRET based and Technozym assays. Since<10% activity is widely used as indicative of TTP it is of particular concern that one centre reported a result of 37% for a known TTP sample with<3% activity.

TABLE 1 Activity assay results Sample Median activity % n CV Range of results 12:01 78.0 22 20.6% 25.4‐104 12:02 60.5 22 17.1% 33‐71.5 12:03 <3.0 22 – 0‐37.1% 16:07 70.0 17 15.9% 43.7‐83 17:01 91.0 15 13.3% 67.8‐108 17:02 92.0 15 16.9% 58.8‐109

Conclusions: These exercises demonstrate generally good agreement between centres performing activity assays, although the range of reported results indicate the need for EQA to ensure the quality of results reported by centres performing ADAMTS13 assays.

PB646: Evaluation of the Fully Automated HemosIL ® Acustar ADAMTS13 Activity Assay

J. Favresse ; B. Lardinois; B. Chatelain; H. Jacqmin; F. Mullier

CHU UCL Namur, Yvoir, Belgium

Background: Thrombotic thrombocytopenic purpura (TTP) is a life‐threatening disease caused by severe ADAMTS13 deficiency leading to the formation of platelet‐rich microthrombi responsible for the destruction of platelets and erythrocytes. It has been shown that rapid ADAMTS13 measurement leading to immediate treatment will delay or prevent relapse. Therefore, any delay in ADAMTS13 results will have a negative impact in the diagnosis, treatment and prognosis of the patient.

Aims: To compare our Technozym ® chromogenic ELISA assay (Technozym ® , Technoclone, Vienna, Austria) with the new and first fully automated HemosIL ® AcuStar ADAMTS13 activity assay (Instrumentation Laboratory, Bedford, MA, USA).

Methods: The AcuStar assay is a two‐step immunoassay also using the GST VWF73 substrate but with magnetic particles and chemiluminescence technology with a release of first results within 30 minutes, in comparison to >3.5 hours for the Technozyme ® assay. Citrated plasma from 38 patients including 8 TTP patients, 2 patients with moderate ADAMTS13 activity (>10‐40%) and 28 patients with normal ADMST13 activity level (>40%) based on our routine assay (Technozyme ® ) were used for the method comparison.

Results: The absolute mean bias on Bland‐Altman plot was 4.8%, but was not constant (95% CI, ‐0.27% to 9.83%) and the limits of agreement (95% CI) were ‐25.30% and +34.90%, reflecting a high variability between both assays.

FIGURE 1 Bland‐Altman plot of ADAMTS13 activity (%) according to the Technozym® chromogenic ELISA and the HemosIL® AcuStar ADAMTS13 activity assay

Based on these results, the same method should be used for monitoring of ADAMTS13. More important to notice is that based on the well acceptable TTP diagnosis cutoff (<10%), no misclassification was pointed out. Consequently, both methods appeared to be effective to identify acute TTP.

Conclusions: Our data suggests that the new AcuStar ® ADAMTS13 activity assay is a valuable tool to diagnose acute TTP. The inherent advantages of automation, the widespread availability 24 hours/24, no need of highly specialized personnel make from the AcuStar ® a platform of choice for haemostasis laboratories.

PB647: Pre‐test Likelihood of ADAMTS13 Severe Deficiency in the Context of Thrombotic Microangiopathies: Validation of the PLASMIC Score in a Southern Italy Hospital

G. Tiscia 1 ; F. Cappucci 2 ; P. Scalzulli 2 ; C. Battista 3 ; F. Aucella 2 ; A. Antonio 4 ; C. Buquicchio 4 ; M. Brigante 5 ; G. D'Andrea 6 ; G. Giordano 5 ; B. Infante 6 ; P. Ranieri 3 ; L. Tullo 6 ; A. Ostuni 3 ; E. Grandone 2

1 Fondazione IRCCS Casa Sollievo della Sofferenza, San Nicandro Garganico, Italy, 2 Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy, 3 Azienda Ospedaliero‐Universitaria Consorziale Policlinico di Bari, Bari, Italy, 4 Ospedale ‘Mons. Dimiccoli’, Barletta, Barletta, Italy, 5 Ospedale ‘A. Cardarelli’, Campobasso, Campobasso, Italy, 6 Azienda Ospedaliera Universitaria Riuniti, Foggia, Foggia, Italy

Background: The PLASMIC score was proposed to predict severe ADAMTS13 deficiency (activity<10%), to stratify patients in the context of Thrombotic MicroAngiopathies (TMAs).

Aims: To assess the PLASMIC score in our hospital.

Methods: We assessed the score in 27 of 42 ADAMTS13 tested (2012‐2017) patients. From electronic records we extracted clinical/laboratory data obtained to time of ADAMTS13 testing. The score has 7 parameters (1 point each):‐platelet count<30 × 10 9 /L, ‐hemolysis variables (reticulocyte count >2.5%, undetectable haptoglobin, or indirect bilirubin>2 mg/dL), ‐no active cancer, ‐no history of organ/cell transplant, ‐mean corpuscular volume (MCV)<90 fL, ‐INR<1.5, and ‐creatinine levels<2 mg/dL. Scoring system defines the low (scale 0‐4), intermediate (5), and high (6 or 7) risk of ADAMTS13<10%. Relevant clinical data on therapeutic procedures and drug use, were collected. To test the discrimination value of the score, a ROC curve was generated and the Area Under Curve (AUC) was calculated.

Results: We assigned 6 patients to the low risk group, 4 and 17 to the intermediate and high‐risk ones, respectively. No severe deficiency was found in the low‐risk group, whereas a severe deficiency was found in 2/4 at intermediate‐risk patients and in 16/17 high‐risk patients. The AUC was equal to 0.86 (95% CI 0.71‐1.00; P=0.015), denoting a good discriminatory performance of the PLASMIC score. In the low‐intermediate risk group (0‐5), we observed 2 short‐term deaths, in patients with severe sepsis. All 27 patients were treated by Plasma EXchange (PEX) and steroids. Seven patients [4 and 3 with a refractory and relapsed TTP were also treated by rituximab. A relapsing TTP was found in 3 (1 with score=5) severely‐deficient patients: in 2 of them (score=6), having a cancer diagnosis (pancreatic cancer and myeloproliferative neoplasm), a long‐term death occurred.

Conclusions: In our setting, PLASMIC score may improve diagnostic approaches in TMAs patients. Further research is needed to confirm present data.

PB648: “How we Treat”: Recurrent TTP Patients Undergoing Elective Surgery

S. Arcudi 1,2 ; B. Ferrari 1,2 ; S. Pontiggia 1 ; I. Mancini 1 ; F. Peyvandi 1,2

1 Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy, 2 University of Milan, and Fondazione Luigi Villa, Department of Pathophysiology and Transplantation, Milan, Italy

Background: Severe ADAMTS13 deficiency has been recognized as the main risk factor for recurrence of thrombotic thrombocytopenic purpura (TTP). Several conditions, including surgery, may influence ultra large Von Willebrand factor and ADAMTS13 levels and act as a trigger for acute TTP episodes.

Aims: Description of management of five patients affected with severe recurrent TTP who underwent elective major surgeries.

Methods: Five patients followed in our center for acquired recurrent TTP with undetectable ADAMTS13 activity during remission (Table 1), were candidates for elective surgery (inguinal hernioplasty, cholecystectomy, laparoscopic hysterectomy and ovarian dissection, total hip arthroplasty).

Results: Three of those five patients were treated with prophylactic plasma exchange (PEX) immediately before surgical procedures, increasing ADAMTS13 levels from<3% to 25%, 40% and 50% respectively. One of those five patients underwent two surgical procedures, the first after 3 PEX and the second prepared with rituximab 18 months before surgery, obtaining partial correction of ADAMTS13. One PEX was also performed the day of surgery, increasing her ADAMTS13 activity level up to 45%. The fifth patient experienced seven acute TTP episodes. After the seventh episode and failure with rituximab, treatment with azathioprine was started and 17 months later her level achieved 42%, allowing her to undergo total hip arthroplasty. All surgeries were successful with no complications, and no relapses occurred in at least 7 months after surgery.

TABLE 1 Patients’ characteristics Patient Number of TTP episodes before surgery ADAMTS13 (FRET) before Prophylactic treatment ADAMTS13 (FRET) after Surgery 1 5 <3% 1 PEX 25% Inguinal hernioplasty 2 (first surgery) 5 <3% 3 PEX 38% Cholecystectomy 2 (second surgery) 5 <3% RITUXIMAB + PEX 45% Total hip arthroplasty 3 4 <3% 1 PEX 50% Total laparoscopic hysterectomy 4 3 <3% 2 PEX 40% Ovarian laparoscopic dissection 5 7 <3% AZATHIOPRINE 42% Total hip arthroplasty

Conclusions: We report the results of our practice on five complicated TTP cases with successful surgical procedures. Our clinical experience suggests that such patients can undergo elective major surgery after achieving an ADAMTS13 activity level of safety, which in our practice is above 25%. In the absence of a randomized clinical trial, prophylactic PEX immediately before surgery, rituximab or azathioprine during the previous months could be reasonable protocols to prevent relapse.

PB649: On the Edge of Thrombotic Thrombocytopenic Purpura: Pathogenic ADAMTS13 Mutations in a Patient with HELLP Syndrome

G. Sinkovits 1 ; D. Csuka 1 ; Á. Szilágyi 1 ; A. Molvarec 2 ; M. Réti 3 ; Z. Prohászka 1

1 Semmelweis University, 3rd Dept. of Internal Medicine, Research Laboratory, Budapest, Hungary, 2 Semmelweis University, 1st Department of Obstetrics and Gynecology, Budapest, Hungary, 3 South‐Pest Hospital Center, Institute of Hematology and Infectology, Dept. of Haematology and Stem Cell Transplantation, Budapest, Hungary

Background: HELLP syndrome is a severe complication of pregnancy, characterized by microangiopathic hemolysis, elevated liver enzymes, and low platelet count. The congenital form of thrombotic thrombocytopenic purpura (TTP), caused by damaging mutations in the ADAMTS13 gene, often presents during pregnancy, with microangiopathic hemolytic anemia, thrombocytopenia, and deficient ADAMTS13 activity without detectable anti‐ADAMTS13 inhibitors.

Aims: Our aim is to present the case of a patient with HELLP syndrome, who was found to be compound heterozygous for deleterious ADAMTS13 mutations.

Methods: The 37‐year‐old female patient was hospitalized because of hypertension and mild edema on the 26th week of gestation, following an episode of nausea and vomiting a week earlier. She had hemolytic anemia, profound thrombocytopenia, elevated liver enzymes, and normal clotting parameters. HELLP syndrome was diagnosed and caesarean section was performed. After the delivery, laboratory parameters spontaneously improved without plasma therapy. However, ADAMTS13 activity was deficient during the episode (initially 10%, later 8%) without anti‐ADAMTS13 inhibitors, and remained severely decreased but measurable (16%‐19%) in remission. To screen for variations in the ADAMTS13 gene, direct DNA sequencing was done.

Results: The patient was found to be heterozygous for the c.3283C>T mutation, causing an R1095W amino acid change, which had previously been described in a patient with congenital TTP in compound heterozygous form. The patient also carries the c.1261C>T mutation, leading to an R421C amino acid substitution. The latter variation has not been described in congenital TTP, but it results in decreased secretion (22%) of the mutant protein according to previous functional studies.

Conclusions: We found two damaging ADAMTS13 mutations in a patient diagnosed with HELLP syndrome that was ADAMTS13 deficient during acute disease and had measurable residual activity in remission.

PB650: Plasmic Score for the Initial Evaluation of Patients with Microangiopathic Hemolytic Anemia and Thrombocytopenia

E. Umit; H. Goze; M. Baysal; M. Demir

Trakya University Faculty of Medicine, Hematology, Edirne, Turkey

Background: Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy (TMA) caused by severely decreased activity of the von Willebrand factor‐cleaving enzyme ADAMTS13. It is characterized by platelet‐rich thrombi in the microvasculature that cause thrombocytopenia, fragmentation hemolysis. TTP is a medical emergency that is almost always fatal if not treated rapidly. The incidence of TTP is 232‐3,2 cases per million population.

Aims: We aimed to evaluate the value of the PLASMIC score, derived to estimate the probability of TTP diagnosis in patients with TMA in regard to diagnosis as well as predicting relapse.

Methods: We evaluated the data of 50 patients presented with TMA symptoms and calculated the PLASMIC score in a retrospective manner. Demographic and laboratory data were collected from the files.

Results: Within the patients 72% were TTP, 7 were complement related TMA, 3 vasculitis, 2 uncontrolled hemolytic anemia, 2 disseminated intravascular coagulopathy. Plasmic score was low in 78%, intermediate in 11 of the patients. ADAMTS13 activity was less than 10% in 15 of the patients with low PLASMIC score and 9 of the patients with intermediate PLASMIC score, which is far from being useful as a predictive scoring system.

Conclusions: As ADAMTS13 enzyme activity determination is almost never an in‐home test throughout the world, a more useful and practical scoring system with strong predictivity is needed. Though our patient group is not large, we did not observe the strength of PLASMIC score as expected.

PB651: Patient Experiences with Acquired Thrombotic Thrombocytopenic Purpura (aTTP): A Conceptual Model

A.O. Oladapo 1 ; D. Ito 1 ; C. Hibbard 1 ; R.N. Krupnick 2 ; T.M. Hare 2 ; B.M. Ewenstein 1

1 Shire, Cambridge, United States, 2 IQVIA, Consulting Services, Cambridge, United States

Background: Acquired thrombotic thrombocytopenic purpura (aTTP) arises from development of autoantibodies to the VWF‐cleaving protein ADAMTS13 and is characterized by microangiopathic hemolytic anemia (MAHA), thrombocytopenia, and consequent organ injury and neurological impairment. Treatment includes plasma exchange and immunosuppression, with management of potential relapse and long‐term sequelae. Better understanding of the patient experience could be helpful to understand treatment needs and meaningful patient outcomes.

Aims: To gain in‐depth understanding of the most salient symptoms and impacts of aTTP and develop a conceptual model of the burden of illness.

Methods: A previously developed conceptual model of symptoms and impacts of the congenital form of TTP (cTTP) ( Res Pract Thromb Haemost . 2017;1[Suppl. 1]:1327) was modified based on review of medical literature, patient blogs and interviews with 5 hematologists treating TTP in the United States (US), United Kingdom, and Austria. Semi‐structured concept elicitation interviews were then conducted with 10 US adults with aTTP to reflect relevant symptoms and impacts of the patient experience with aTTP and revise this model.

Results: aTTP patients experience high disease burden beyond the acute phase. Similar to cTTP, the most salient symptoms and impacts of aTTP include fatigue, headache, bruising, feeling anxious, and inability to work/study. These experiences also are prominent in the conceptual model for patients with cTTP. In addition, characteristics unique to aTTP include dizziness, tingling/numbness, sleep disturbance, and fear of relapse.

Conclusions: While signs, symptoms and impacts of aTTP were largely similar to those of cTTP, a number of unique characteristics were identified for inclusion in the adapted conceptual model. This model will be tested in a broader population of aTTP patients to inform development of a patient‐reported outcome tool to assess aTTP‐specific disease burden and treatment outcomes.

PB652: Identification of Novel ADAMTS13 Gene Mutation in a Patient with Congenital Thrombotic Thrombocytopenic Purpura

A. Rashid 1 ; K. D Friedman 2 ; N. Mushtaq 1

1 Aga Khan University Hospital, Karachi, Pakistan, 2 BloodCenter of Wisconsin, Milwaukee, United States

Background: Congenital thrombotic thrombocytopenic purpura (TTP) is an autosomal recessive disorder due to ADAMTS13 deficiency characterize by thrombocytopenia, microangiopathic hemolytic anemia and thrombosis. Causative mutations have been identified throughout the coding sequence but no specific mutation appears to predominate.

Aims: To evaluate ADAMTS13 gene mutation in a patient presenting with thrombotic thrombocytopenic purpura.

Methods: ADAMTS13 antigen was measured by ELISA and genetic study was done by next generation sequencing.

Results: A male child presented with hyperbillirubinemia within 24 hours of birth but did not require exchange transfusion. He then had fever at 7 weeks of age. Laboratory evaluation showed schistocytic hemolytic anemia and thrombocytopenia. Hemoglobin 7.7 g/dl (11.5‐15.5); Platelets 40 × 10 9 /L (150‐400); Reticulocytes 5.4% (0.8‐2.1); PT 10.6 seconds (9‐13), APTT 28.7 seconds (22.9‐34.5); BUN 23 mg/dl (6‐20); Creatinine 0.3 mg/dl (0.3‐0.7); LDH 1,383 IU/L (208‐378). Blood culture was positive for Streptococcus pneumonia. Anemia and thrombocytopenia continued despite antibiotic treatment although he remained asymptomatic. ADAMTS13 antigen was 40 ng/ml (630‐850).Since ADAMTS13 inhibitor assay was not available a trial of plasma infusions, immune modulation and steroids was attempted. Immune modulation was complicated by hypertension, hematuria, proteinuria and bicytopenia persisted. Genetic analysis detected a novel homozygous frameshift mutation: ADAMTS13 c.3033delC, p.Cys1012AlafsX109 in exon 23. Both parents were found to be heterozygous for the same mutation. Patient is now 8 years old, asymptomatic receiving periodic plasma infusions, has normal creatinine and improving blood counts.

Conclusions: This case revealed a novel ADAMTS13 mutation in a Pakistani child with congenital TTP. To date this variant has not been reported in the literature. The case supports ADAMTS c.3033delC, p.Cys1012AlafsX109 in exon 23 as a pathognomic variant in regards to severe congenital ADAMTS13 deficiency.

PB654: Evaluation of Hospital Readmissions among Patients Hospitalized for Acute Medical Illness and at Risk for Venous Thromboembolism in the US

A. Amin 1 ; R. Neuman 2 ; M. Lingohr‐Smith 3 ; B. Menges 3 ; J. Lin 3

1 University of California, Irvine, School of Medicine, Irvine, United States, 2 Portola Pharmaceuticals, San Francisco, United States, 3 Novosys Health, Green Brook, United States

Background: Patients hospitalized for acute medical illnesses have an elevated risk for venous thromboembolism (VTE).

Aims: To determine the quality of VTE prophylaxis and the frequency of hospital readmissions among such hospitalized patients.

Methods: Acutely ill medical patients were identified from the MarketScan Commercial and Medicare databases from 1/1/2012 to 6/30/2015. Hospitalized patients with acute medical conditions of heart failure, respiratory diseases, ischemic stroke, cancer, infectious diseases, and rheumatic diseases were included. The proportions of patients with all‐cause and VTE‐related readmissions during the 6‐month follow‐up period were evaluated.

Results: Of the population hospitalized for acute medical illnesses (n=17,895; mean age: 58.4 years; 40% ≥65 years; 55.4% female), most patients were hospitalized for infectious diseases (40.6%), followed by respiratory diseases (31.0%), cancer (10.7%), heart failure (10.4%), ischemic stroke (6.4%), and rheumatic diseases (0.9%). In total, 59.1% (n=10,581) of patients did not receive any VTE prophylaxis, and 7.1% (n=1,267) received both inpatient and outpatient VTE prophylaxis. During the 6‐month follow‐up period, 26.8% (n=4,790) of the study population had ≥1 hospital readmission for any cause; 7.0% (n=336) were VTE‐related. Of all readmissions, 29.4% and 33.3% occurred within 30 days of hospital discharge. VTE‐related hospital readmissions were most frequent among patients with cancer (13.7%) and rheumatic diseases (11.5%). Mean all‐cause and VTE‐related hospitalization lengths of stay were 12.1 and 13.1 days, respectively.

Conclusions: The majority of patients hospitalized for acute medical illnesses did not receive any VTE prophylaxis, and hospital readmissions, including those related to VTEs, remained a substantial burden. Thus, better strategies for VTE prophylaxis from the hospital admission through post‐discharge may help reduce the burden of hospital readmissions.

PB655: Comparison of the Risk of Recurrence in Patients with Venous Thromboembolism (VTE) Provoked by Major and Minor Surgery and Trauma

T. Gregorio 1 ; M. Santos 2 ; L. Pagliani 3 ; R. Providencia 4 ; C. Buzea 5 ; A. Katholing 6 ; J. Weitz 7 ; C. Martinez 6 ; A. Cohen 8

1 Vila Nova de Gaia Hospital Centre, Department of Internal Medicine, Vila Nova de Gaia, Portugal, 2 Faculty of Medicine, University of Porto, Porto, Portugal, 3 University of Padova, Department of Medicine, Padova, Italy, 4 University College of London, London, United Kingdom, 5 ′Carol Davila′ University of Medicine and Pharmacy, Bucareste, Romania, 6 Institute for Epidemiology, Statistics and Informatics GmbH, Frankfurt, Germany, 7 McMaster University, Department of Medicine, Hamilton, Canada, 8 King's College London, School of Medicine, London, United Kingdom

Background: VTE is common after surgery and trauma. Rates of recurrence are low with provoked VTE and a 3‐month course of anticoagulation is recommended. However, the risk of recurrence with VTE provoked by minor surgery or trauma relative to that provoked by major surgery or trauma is unknown.

Aims: To test the hypothesis that the risk of recurrence is higher with VTE provoked by minor surgery or trauma than with major surgery or trauma.

Methods: The UK primary care Clinical Practice Research Datalink was interrogated using additional information on hospitalisations and causes of death between 2008 and 2015. Using a validated algorithm, we included patients with VTE that occurred within 90 days after surgery or trauma. Patients with a prior history of VTE were excluded. Cases (patients with recurrent VTE) were matched with up to 5 controls without recurrence for date of first VTE, year of birth, gender and presentation of VTE (primary or secondary care).

Results: Of a total of 1,293 patients with provoked VTE, 964 were provoked by surgery and 329 by trauma. Recurrent VTE occurred in 319 patients. Compared with VTE provoked by major surgery, minor surgery was an independent risk factor for recurrence (adjusted odds ratio [OR] 1.80, 95% confidence interval [CI] 1.23‐2.61). In contrast, there was no significant association between the severity of trauma and the risk of recurrence (OR=1.43, 95% CI: 0.63‐3.23).

Conclusions: Patients with VTE provoked by minor surgery had a stronger association with recurrence than those whose VTE was provoked by major surgery. These patients may benefit from more prolonged anticoagulant therapy.

PB656: Comparative Thrombosis Risk of Vascular Access Devices among Critically Ill Medical Patients

S.M. Stevens 1,2 ; D. White 1 ; S.C. Woller 1,2 ; D.S. Collingridge 3 ; V. Chopra 4 ; G.V. Fontaine 5

1 Intermountain Healthcare, Medicine, Murray, United States, 2 University of Utah, Internal Medicine, Salt Lake City, United States, 3 Intermountain Healthcare, Office of Research, Murray, United States, 4 University of Michigan, Internal Medicine, Ann Arbor, United States, 5 Intermountain Healthcare, Pharmacy, Murray, United States

Background: Central venous catheters (CVC) and peripherally inserted central catheters (PICCs) are vascular access devices (VADs) that facilitate medication delivery and invasive monitoring in critically ill patients. Both increase the risk of venous thromboembolism (VTE). The comparative risk of VTE between these two catheter types is not well defined.

Aims: This study aimed to report the rate of VTE in intensive care unit (ICU) medical patients receiving a PICC, a CVC or both.

Methods: We conducted a single‐center, retrospective cohort study of patients admitted to the medical ICU between November 2007 and November 2013 who received CVC, PICC, both, or neither. The primary outcome was the rate of 30‐day VTE. Secondary outcomes included VTE (in upper extremities, lower extremities, or lungs), major bleeding, and all‐cause mortality.

Results: From 11,111 patients, 5,788 (52%) met inclusion criteria. VADs were placed in 2,403 (42%) patients (PICC, n=816; CVC, n=1,153; both, n=434). Compared with no VAD, the hazard ratio (HR) for 30‐day VTE was 1.8 (95% CI: 1.5‐2.2) for any VAD, 1.9 (95% CI: 1.5‐2.4) for PICC, 1.6 (95% CI: 1.3‐2.0) for CVC, and 2.7 (95% CI: 2.1‐3.5) for patients with both PICC and CVC. PICCs had a non‐significantly higher HR for VTE compared with CVC (1.3; 95% CI: 0.9‐1.6). For patients with both a CVC and PICC the HR for VTE was 1.7 times that of a solitary VAD of either type (95% CI: 1.3‐2.2). With the use of any VAD the HR for upper‐extremity VTE was 6.5 (95% CI: 3.9‐10.9), lower‐extremity VTE was 1.5 (95% CI: 1.2‐1.8), and PE was 1.3 (95% CI: 0.8‐2.1). The HR for all‐cause mortality was 2.1 (95% CI: 1.7‐2.5) and the odds ratio for major bleeding was 1.2 (95% CI: 0.8‐1.7).

Conclusions: Among critically ill patients, PICCs and CVCs were associated with increased risk of VTE. PICCs were associated with a non‐significant trend towards greater risk of thrombosis. Placement of two VADs conferred significantly greater risk of VTE compared than either device alone.

PB657: Cost‐effectiveness of Betrixaban Compared with Enoxaparin for Primary Venous Thromboembolism Prophylaxis in Hospitalised Acute Medically Ill Patients in the United States

V. Laskier 1 ; H. Guy 1 ; M. Fisher 1 ; A. Hafeman 2 ; R. Neuman 2 ; S. Deitelzweig 3 ; A. Cohen 4

1 FIECON, St Albans, United Kingdom, 2 Portola Pharmaceuticals, Inc., San Francisco, United States, 3 Ochsner Health System, New Orleans, United States, 4 Guy's and St. Thomas’ Hospitals, London, United Kingdom

Background: Studies show that the risk of venous thromboembolism (VTE), including VTE related death, continues following discharge in hospitalized acute medically ill (HAMI) patients who receive short‐duration VTE prophylaxis. Betrixaban is the first FDA approved anticoagulant indicated for extended‐duration (ED) VTE prophylaxis.

Aims: To estimate the cost‐effectiveness of betrixaban compared with enoxaparin for primary VTE prophylaxis in adult HAMI patients, who require ED VTE prophylaxis, in the United States (US).

Methods: A cost‐effectiveness analysis was conducted to estimate the cost per quality‐adjusted life‐year (QALY) gained for ED betrixaban (35‐42 days) compared to standard‐duration enoxaparin (6‐14 days) from a US payer perspective over a lifetime horizon. In the first 3 months, a decision tree structure was used to estimate primary events and treatment complications (TCs) based on efficacy and safety data from the Phase 3 APEX study. The decision tree structure included primary events and TCs (VTE, myocardial infarction, ischemic stroke, death, bleeding and thrombocytopenia). After the first 3 months, patients entered a six‐health state Markov model and were at risk of recurrent events and long term complications. Costs captured the treatment and management of primary events, TCs, recurrent events and primary event complications. Published literature identified the risk of recurrent events and long term complications, EQ‐5D utility data and costs. A 3% discount rate per annum was used. Uncertainty was explored through deterministic and probabilistic sensitivity analysis.

Results: Betrixaban dominated enoxaparin, leading to cost savings of $780 and increased QALYs of 0.017 per patient. Furthermore, betrixaban dominated enoxaparin across all sensitivity analyses.

Conclusions: Because of betrixaban's estimated potential cost savings and its demonstrated efficacy over enoxaparin, it can be considered a cost‐effective treatment for adult HAMI patients at risk of VTE, who require ED VTE prophylaxis.

PB658: Venous Thromboembolism Clinical Risk Assessment Model for Patients with a Lower Limb Trauma Requiring Immobilization: The TIP Score

D. Douillet 1 ; A. Penaloza 2 ; T. Moumneh 1 ; P.‐M. Roy 1

1 University of Angers, Angers, France, 2 University of Bruxelles, Bruxelles, Belgium

Background: Thromboprophylaxis for patients with nonsurgical isolated lower limb trauma requiring immobilization is matter of debate. A risk stratification model is warranted in order to base preventive treatment decision on an individual risk assessment.

Aims: Our aim was to develop and assess the potential impact of a clinical risk stratification model taking into account all risk factors from charactertictics of T rauma, I mmobilization and P atients (the TIP score).

Methods: The TIP score criteria were selected by consensus of international experts (n=27) using the Delphi method. Its potential impact was evaluated in a prospective observational study. The primary endpoint was the thromboprophylaxis prescription rate in current practice compared to the theoretical prescription rate with TIP score. The seconday endpoint was the 3‐month rate of symptomatic venous thromboembolism (VTE) (clinicaltrials.gov: NCT03089255 ) .

Results: After 4 successive rounds, 30 items constituting the TIP score were selected, with an absolute (>90%) or strong consensus (>75%) for all. Thirteen items for trauma, 3 for immobilization and 14 for patient characteristics were selected, each ranging from a scale of 1 to 3. The threshold for anticoagulation was defined as >4.

Among 196 patients included in the prospective study, 31 patients had a TIP score >4 and 165 a TIP score<4 for whom 17 and 55 received preventive treatment, respectively. The application of the TIP score may allow a significant reduction of the prescription rate by more than 20% (Table) . The 3‐month VTE rate was 0.53% (n=1/190). One patient (TIP score=5, no treatment) had symptomatic VTE.

Conclusions: The TIP score allows clinical VTE risk stratification for patients with lower limb trauma and orthopedic immobilization. It may lead to a significant reduction in the prescription rate of preventive anticoagulation by targeting safely the high risk patients; validating its major impact in medico‐economic terms. A multicentric validation study is forthcoming.

PB659: Association between Antineoplastic Therapies and Venous Thromboembolism in Patients with Active Cancer

M. Giustozzi 1 ; A. Curcio 2 ; S. Sudikas 3 ; B. Weijs 4 ; T. Field 5 ; A. Katholing 6 ; C. Wallenhorst 6 ; J.I. Weitz 7 ; C. Martinez 6 ; A.T. Cohen 8

1 Università degli Studi di Perugia, Department of Internal and Cardiovascular Medicine, Stroke Unit, Perugia, Italy, 2 Division of Cardiology, Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy, 3 Kantons Hospital Winterthur, Department of Surgery, Zurich, Switzerland, 4 Maastricht University Medical Center and Cardiovascular Research Institute Maastricht, Department of Cardiology, Maastricht, Netherlands Antilles, 5 Vancouver Stroke Program, Djavad Mowafaghian Centre for Brain Health, Division of Neurology, Vancouver, Canada, 6 Institute for Epidemiology, Statistics and Informatics GmbH, Frankfurt, Germany, 7 McMaster University, Thrombosis & Atherosclerosis Research Institute, Hamilton, Canada, 8 Guy's and St Thomas’ NHS Foundation Trust, King's College London, Department of Haematology, London, United Kingdom

Background: Venous thromboembolism (VTE) is a frequent cause of morbidity and mortality in patients with malignancy, particularly in the setting of medical and surgical cancer treatments. The relationship between types of cancer treatment and risk of VTE is not well understood.

Aims: To evaluate the association between cancer therapies and risk of VTE.

Methods: We conducted a cohort study of patients with a first cancer diagnosis between 2008 and 2016 using data from the UK primary care database Clinical Practice Research Datalink, with additional information on hospitalisations and cause of death. The primary study outcome was active cancer‐associated VTE defined as VTE occurring within 180 days after first cancer diagnosis, another primary or secondary hospital discharge diagnosis for cancer, or cancer treatment. Adjusted hazard ratios of the association between type of cancer therapy and VTE events during the first active cancer episode were derived from Cox regression models.

Results: We identified a study cohort of 75,913 patients with a first cancer diagnosis (mean age 66.1±14.9 years, 50% females). Active cancer‐associated VTE occurred in 2,897 patients (3.8%), including 1,038 patients with deep vein thrombosis (DVT) and 1,859 with pulmonary embolism ± DVT. During a mean of 2.0 years, 14,189 of the 75,913 patients received chemotherapy only (18.7%), 6,992 hormonal therapy only (9.2%), 4,158 radiation only (5.5%), 193 immunotherapy only (0.3%), 136 cytotoxic drugs only (0.2%) and 12,269 (16.2%) were treated with combined therapy. The risk of VTE with different therapies is shown in Table 1. Figure 1, shows the instantaneous risk of VTE overtime by type of cancer therapy.

TABLE 1 Predictors for VTE in patients with active cancer (analysis restricted to first active cancer episode) Type of anticancer treatment Cases of VTE Crude HR (95% CI) Adjusted* HR (95% CI) No current anticancer treatment 1,458 (56.2) 1 1 Chemotherapy only 949 (36.6) 3.39 (3.05‐3.77) 2.51 (2.24‐2.82) Radiation only 106 (4.1) 1.95 (1.57‐2.42) 1.41 (1.13‐1.76) Immunotherapy only 8 (0.3) 0.90 (0.42‐1.89) 0.73 (0.35‐1.54) Hormonal therapy only 173 (6.7) 0.44 (0.36‐0.54) 0.97 (0.77‐1.23) Chemotherapy combination 175 (6.7) 2.63 (2.18‐3.18) 2.09 (1.72‐2.53) Radiation combination 25 (1.0) 0.74 (0.49‐1.11) 1.58 (1.03‐2.40) CI: Confidence interval; HR: Hazard ratio; VTE: Venous thromboembolism. *Adjusted for age as a time dependent covariate, gender, BMI, socioeconomic status, drinking status, smoking status, number of previous active cancer episodes, medical illness, trauma, surgical procedure, pregnancy, cancer type, presence of metastases, blood transfusion, corticosteroids and length of hospitalization.

FIGURE 1 Instantaneous risk of VTE per 100 person‐years by time since start of active cancer episode (analysis restricted to first active cancer episode)

Conclusions: Chemo‐ and radiation therapies, alone or in combination with other anticancer therapies, are associated with an increased risk of VTE. Immunotherapy and hormonal therapy were not associated. This information helps to identify cancer therapies in patients who may or not may benefit from primary prophylaxis.

PB660: A Single Center Analysis of Pre‐, Intra‐ and Post‐operative Thrombotic Events in a Cohort of 470 Orthotopic Whole Liver Transplantations

N. Riva 1 ; A. Gatt 1 ; R.K. Pruthi 2,3,4 ; W.L. Nichols 2,3,4 ; J.Y. Findlay 5 ; C.B. Rosen 6 ; P.S. Kamath 7 ; A.A. Ashrani 2

1 University of Malta, Department of Pathology, Faculty of Medicine and Surgery, Msida, Malta, 2 Mayo Clinic, Division of Hematology, Department of Internal Medicine, Rochester, United States, 3 Mayo Clinic, Special Coagulation Laboratory, Coagulation Clinic & Comprehensive Hemophilia Center, Rochester, United States, 4 Mayo Clinic, Hematopathology & Laboratory Medicine, Rochester, United States, 5 Mayo Clinic, Department of Anesthesiology and Perioperative Medicine, Rochester, United States, 6 Mayo Clinic, William J. von Liebig Center for Transplantation and Clinical Regeneration, Rochester, United States, 7 Mayo Clinic, Division of Gastroenterology and Hepatology, Rochester, United States

Background: Thrombotic events are common in liver disease and might cause graft failure after liver transplantation. However, there is scarce information on the risk of thrombosis in these patients.

Aims: Evaluate the incidence of arterial and venous thrombosis pre‐, intra‐ and post‐liver transplantation in patients with chronic liver disease.

Methods: Retrospective data collection on adult patients undergoing whole liver transplantation at Mayo Clinic (Rochester, MN) (2006‐2012). Exclusion criteria: multiple procedures, living partial donor transplant, deceased partial transplants, second transplants. Objectively confirmed thrombosis were classified as venous thromboembolism [VTE], arterial thrombotic events [ATE], other liver‐related thrombosis (hepatic artery thrombosis [HAT], graft thrombosis). Patients were followed up to the first event, re‐transplant, death or 1 year after transplant, whichever came first.

Results: We included 470 consecutive patients (median age 56; 62% males; 29% hepatocellular carcinoma; 29% alcoholic liver disease; 25% hepatitis C virus). Eighty‐nine (18.9%) had pre‐operative history of one or more thrombotic events (74 VTE, 19 ATE, 2 HAT). Seventy‐four had intra‐operative findings of portal vein thrombosis (n=69), right atrial clot (n=2), HAT (n=4). Seventy‐one developed post‐operative thrombosis: 49 VTE, 12 ATE, 14 liver‐related thrombosis (incidence rates: 12.7, 95% CI 9.6‐16.8; 3.1, 95% CI 1.8‐5.5; 3.7, 95% CI 2.2‐6.1, per 100 patient‐years, respectively). Thrombotic events were more common in the first 3 months (79%). At multivariate analysis, cholangiocarcinoma (P=0.008) and red blood cell [RBC] transfusion (P=0.001) were associated with increased risk of post‐operative thrombosis; while elevated bilirubin seemed protective (P=0.002). Thrombotic events increased with the units of RBC transfused (P=0.028).

Conclusions: The results of our study are consistent with an increased risk of thrombotic events in liver transplant recipients, especially in the first 3 months after transplantation.

PB661: miRNA Expression Profile in Venous Thrombosis Disease: Results from the Genetic Analysis of Idiopathic Thrombophilia Project

A. Rodriguez‐Rius 1 ; S. Lopez 1 ; A. Martinez‐Perez 1 ; M. Sabater‐Lleal 2 ; C. Muñoz 1 ; A. Hamsten 2 ; J.C. Souto 3 ; J.M. Soria 1

1 Unit of Genomic of Complex Diseases, IBB Sant Pau, Research Institute of Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 2 Cardiovascular Medicine Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden, 3 Haemostasis and Thrombosis Unit, Department of Hematology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain

Background: The interest of micro‐RNAs (miRNAs) as potential epigenetic biomarkers of diseases has dramatically increased in the last years. However, only few studies have been carried out in venous thrombosis (VT).

Aims: The objective of this study was the establishment of a potential miRNA profile associated with VT.

Methods: For that, 52 VT patients and 52 healthy controls, not genetically related, were selected from the “Genetic Analysis of Idiopathic Trombophilia (GAIT2)” project, a family‐based study with 935 subjects from 35 extended Spanish families. Briefly, miRNAs were isolated from citrated plasma (miRCURY ™ RNA, Exiqon), reverse transcribed to cDNA (Universal RT – cDNA, Exiqon) and 752 miRNAs were measured in each of the 104 individual by qPCR in miRNome Panels (Exiqon). qPCR values were corrected by inter‐plate calibrators, normalized by the global mean and corrected by the first four principal components.

Results: Of the 752 miRNAs, 103 miRNAs were expressed at least in the 90% of the sample (94 subjects) and were tested for associations with VT, intermediate phenotypes and ˜10M imputed variants (SNVs) along the genome. Statistically significant associations (P‐value <0.05) with VT were obtained for 11 miRNAs, of which only 4 have been previously suggested to be related to VT. Most of these 11 miRNAs show also, significant statistic associations with intermediate phenotypes involved in VT. Furthermore, we identified other 7 miRNAs significantly associated with intermediate phenotypes (i.e., Prothrombin time, P‐value=2e‐03). In addition, significant associated loci have been found in 4 of these miRNAs (P‐value <10e‐08), suggesting that genetic variants regulate levels of these miRNAs.

Conclusions: Based on these results, we have selected a miRNA expression profile that shows significant association with VT and/or intermediate phenotypes of VT. This expression profile will be analyzed in the whole GAIT2 (N=935) and validated in other populations.

Financial support: PI14/00582, STAGO, 2018FI00227.

PB662: Clinical Characteristics and Outcome of Patients with Subsegmental Pulmonary Embolism

C. Fernandez‐Capitan 1 ; D. Jimenez 2 ; O. Madridano 3 ; M. Ciammaichella 4 ; E. Usandizaga 5 ; R. Otero 6 ; A. Bura‐Rivière 7 ; F.J. Vazquez 8 ; F. Moustafa 9 ; M. Monreal 10 ; RIETE Investigators

1 Hospital Universitario La Paz, Universidad Autonoma de Madrid, Madrid, Spain, 2 Hospital Universitario Ramón y Cajal, Madrid, Spain, 3 Hospital Infanta Sofía, Madrid, Spain, 4 Ospedale St. John, Rome, Italy, 5 Hospital Sant Joan Despí‐Moises Broggi, Barcelona, Spain, 6 Hospital Universitario Virgen del Rocío, Sevilla, Spain, 7 Hôpital de Rangueil, Toulouse, France, 8 Hospital Italiano de Buenos Aires, Buenos Aires, Brazil, 9 CHU Clermont‐Ferrand, Clermont‐Ferrand, France, 10 Hospital Germans Trias i Pujol, Barcelona, Spain

Background: It is controversial whether patients with acute, subsegmental pulmonary embolism (PE) should be treated with anticoagulants.

Aims: We aimed to compare the rate of symptomatic PE recurrences appearing during the course of anticoagulation in patients with subsegmental PE, segmental PE or more proximal PE.

Methods: We used the RIETE (Registro Informatizado Enfermedad Trombo Embólica, NCT02832245 ) database. All RIETE patients with acute, symptomatic PE diagnosed by helical‐CT scan were included, from March 2001 to August, 2016. We excluded participants without information on PE localization or with prior VTE.

Results: Among 8294 patients with a first episode of PE, 471 (5.7%) had subsegmental PE, 1992 (24%) segmental and 5831 (70%) had central PE. Of these, 100%, 99.8% and 99.8% respectively received anticoagulant therapy (median duration: 173, 179 and 197 days, respectively). During the course of therapy, the rate of PE recurrences was: 1.89 (95% CI: 0.77‐3.93) events per 100 patient‐years in patients with subsegmental PE; 1.02 (95% CI: 0.58‐1.67) in those with segmental PE and 1.32 (95% CI: 1.03‐1.68) in those with central PE. The rate of fatal PE was: 0.62 (95% CI: 0.10‐2.06); 0.94 (0.52‐1.57) and 1.15 (0.88‐1.48) deaths per 100 patient‐years, respectively. When subsegmental PE was stratified in 3 categories according to ultrasound imaging in the lower limbs, the rate of PE recurrences was: zero events per 100 patient‐years in patients with confirmed deep vein thrombosis (DVT); 4.58 (95% CI: 1.68‐10.1) in those with no DVT and 0.79 (95% CI: 0.04‐3.88) in those with no ultrasound imaging.

TABLE 1 Clinical outcomes during the course of anticoagulant therapy Subsegmental PE Segmental PE Central PE N Events per 100 patient‐years N Events per 100 patient‐years N Events per 100 patient‐years Patients, N 471 1992 5831 Duration of therapy, Median days (IQR) 171 (99‐315) 178 (99‐302) 196 (106‐365) Events Recurrent PE 6 1.89 (0.77‐3.93) 14 1.02 (0.58‐1.67) 65 1.32 (1.03‐1.68) Recurrent DVT 3 0.94 (0.24‐2.55) 11 0.80 (0.42‐1.39) 46 0.94 (0.69‐1.24) Major bleeding 17 5.41 (3.26‐8.49) 72 5.31 (4.18‐6.64) 199 4.07 (3.53‐4.66) Death 42 13.1 (9.57‐17.6) 205 14.8 (12.9‐17.0) 604 12.1 (11.2‐13.1)

Conclusions: During the course of anticoagulant therapy, patients with subsegmental PE had a similar rate of PE recurrences and fatal PE events than those with more central PE. Our findings do not support that they should be treated differently, as suggested in the guidelines.

PB663: Assessment of General Awareness of Thrombosis and How Best to Communicate this Information

A.M. O'Neill ; K. Collins; S. Bourke

Thrombosis Ireland, Dublin, Ireland

Background: Thrombosis Ireland was founded in 2016 by patients to raise awareness and understanding of blood clots. Our research is all about how best to connect and convey this information to the public.

Aims: Our aims were to identify the current level of awareness about thrombosis among the general public, establish what information they want and determine the best brand and social media platforms to develop and engage with users.

Methods: We designed an online survey which we circulated through social media. We did quantitive research involving 105 participants and an extensive review of our existing social media platforms.

Results: 59% of those surveyed, did not know what can increase their risk of thrombosis. 61% would look for signs and symptoms first if searching for information on thrombosis and 30% said they would look for causes of thrombosis. When asked where would they look for this information, 74% said they would google and 15% said they would ask their doctor. 66% said they would follow thrombosis content on Facebook and 33% said they would watch thrombosis content on YouTube.

Conclusions: Our research confirmed an immediate need for a national thrombosis awareness campaign. It highlighted the need to form partnerships with GP′s, doctors and nurses to encourage dialogue between them and their patients about blood clots. Clear information on the signs and symptoms and the risk factors is crucial, through all platforms of communication because being aware of these facts can save lives.

PB664: Postoperative Venous Thromboembolism Prophylaxis in Perioperative Trials of Tranexamic Acid: A Systematic Review and Meta‐analysis

J. Taylor 1 ; I. Perelman 1 ; J. Yates 2 ; S. Khair 1 ; J. Lampron 2 ; A. Tinmouth 2 ; A. Davis 2 ; E. Saidenberg 2

1 Ottawa Hospital Research Institute, Ottawa, Canada, 2 The Ottawa Hospital, Ottawa, Canada

Background: The efficacy of Tranexamic acid (TXA) in the reduction of perioperative bleeding has been proven in numerous studies. Previous studies have indicated that there is no increased rate of VTE in patients treated with perioperative TXA as compared to placebo or other therapies.

Aims: We aimed to assess whether the use of postoperative VTE prophylaxis and the type of therapy selected are factors in determining the risk of postoperative VTE in patients treated with perioperative TXA.

Methods: We searched electronic databases (MEDLINE, Embase, CENTRAL, clinicaltrials.gov) from inception through September 2017. Randomized control trials of perioperative TXA involving a comparator group and reporting on postoperative VTE events were included. Study characteristics, results of VTE‐related outcomes, and VTE prophylactic regimens were abstracted and meta‐analyzed using a Der Simonian and Laird random effects model.

Results: 198 trials were identified, of which 86 used postoperative VTE prophylaxis measures. Meta‐analysis showed that TXA was not associated with an increased risk of postoperative VTE for all surgery types and for orthopedic surgeries specifically (P>0.05), regardless of whether VTE prophylaxis was provided to patients. In studies using VTE prophylaxis, the risk of VTE in patients treated with perioperative TXA was not significantly (P>0.05) different for the various pharmaceutical and physical VTE prophylaxis methods employed.

Conclusions: VTE is uncommon in patients treated with TXA perioperatively, especially when proper postoperative prophylactic methods are employed. Additionally, the type of VTE prophylaxis does not seem to impact the already low risk of VTE in this patient population. Future studies are required to assess the use of VTE prophylaxis in patients with a prior history of VTE who are treated with perioperative TXA. However, for other patients, risk of VTE should not be considered a barrier to the use of TXA to minimize blood loss and transfusion need.

PB665: Testosterone Therapy and Venous Thromboembolism: A Systematic Review and Meta‐analysis

D.E. Houghton 1 ; M. Alsawas 2 ; L.J. Prokop 2 ; P. Barrioneuvo 2 ; B. Beuschel 2 ; W. Farah 2 ; M. Tello 2 ; J.B. Layton 3 ; M.H. Murad 2 ; S. Moll 4

1 Mayo Clinic, Rochester, United States, 2 Mayo Clinic, Evidence‐Based Practice Center, Mayo Clinic Robert D. and Patricia E. Kern Center for the Science of Health Care Delivery, Rochester, United States, 3 RTI Health Solutions, Research Triangle Park, United States, 4 University of North Carolina, Department of Medicine, Division of Hematology/Oncology, Chapel Hill, United States

Background: Regulatory bodies have warned about increased venous thromboembolism (VTE) in patients on testosterone therapy, but published clinical data supporting an increased risk for VTE is limited.

Aims: Systematically review current literature to examine the association between exogenous testosterone use and VTE.

Methods: A comprehensive search of several databases from inception to October 31st, 2017 was conducted. The databases included Epub Ahead of Print, Medline In‐Process & Other Non‐Indexed Citations, MEDLINE, and EMBASE, Ovid Cochrane Database of Systematic Reviews, and Scopus. We included randomized control trials (RCTs) and observational studies that evaluated the association between testosterone therapy in men and VTE. Study selection and data extraction were performed by two independent investigators. Random‐effect model meta‐analyses were used to estimate pooled OR and 95% confidence intervals (CIs). Heterogeneity among studies was evaluated by the I 2 statistic.

Results: 6 RCTs (n=2,223) and 5 observational studies (n=1,249,640) were included. Five RCTs were performed in men with documented hypogonadism. The observational studies included: 2 case‐control studies, 2 retrospective cohorts, and 1 retrospective cohort with a nested case‐control study. No statistically significant association between VTE and testosterone use was found (OR 1.34, 95% CI 0.92‐1.97; Figure 1). Heterogeneity was high (I‐squared=84.4%). No significant differences were found when the analysis was stratified by study design: RCTs (1.48, 95% CI 0.57‐3.89), observational studies (cohort: 1.06, 95% CI 0.85‐1.33 and case‐control studies; 1.34, 95% CI 0.78‐2.28).

Conclusions: The best available evidence to date does not support an association between testosterone use and VTE. However, variable methodologies among the included studies limit interpretation of the results.

PB666: Homozygotes in FXI rs 2289252 and ABO rs 8176719 Have a Low Risk of VTE Recurrence

J. Kvasnicka 1 ; P. Bobcikova 1 ; J. Hajkova 1 ; Z. Krska 2 ; M. Sevcik 1 ; T. Kvasnicka 1

1 General Univ. Hospital, Thrombosis Centre, Praha 2, Czech Republic, 2 General Univ. Hospital, 1st Department of Surgery, Praha, Czech Republic

Background: It is known that even the recurrence of venous thromboembolism (VTE) can be genetically dependent.

Aims: The aim of this study was to assess whether genetic variants predict this risk.

Methods: The genetic variants in F5 rs6025, F2 rs1799963, ABO rs8176719, FGG rs6536024 and F11 rs2289252 were tested in 613 patient with recurrent VTE and in 2017 VTE patients without its recurrence in the 5‐year follow‐up observation. The polymorphisms were determined using PCR by FRET process.Specific primers and fluorescently labelled probes were designed in cooperation with TIB MOLBIOL (Berlin, Germany). Chi‐square and logistic procedure was performed using the SAS 9.4, SAS/Genetics 13.1 (Cary, NC USA), and OR above 1.5 was considered as clinically significant. The study was performed in project “RVO VFN 64165” which has been approved by local ethics committee.

Results: Only results with OR above 1.5 are presented:

Odds ratio 95% CI chi‐square test

FXI rs2289252 TT versus CC 1.682 1.304 ‐2.171 P<0.0001

AB0 rs8176719 GG versus ‐ ‐ 1.529 1.148‐2.036 P 0.0135

Conclusions: Only homozygous variants of FXI (rs2289252) and AB0 (rs8176719) determined the VTE recurrence risk with OR above 1.5 in this study.

PB667: Risk of Pulmonary Embolism after Surgical Resection of Meningioma

A. Artoni 1 ; M. Abbattista 1 ; G. Carrabba 2 ; M. Capecchi 1 ; P. Bucciarelli 1 ; F. Gianniello 1 ; A. Lecchi 1 ; M. Castellani 3 ; F. Peyvandi 4,5 ; I. Martinelli 6

1 A.Bianchi Bonomi Hemophilia and Thrombosis Center Fondazione IRCCS Ca’ Granda Ospedale Maggiore, Milano, Italy, 2 Fondazione IRCCS Ca’ Granda Ospedale Maggiore, Division of Neurosurgery, Milano, Italy, 3 Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Nuclear Medicine Department, Milano, Italy, 4 Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, A.Bianchi Bonomi Hemophilia and Thrombosis Center, Milano, Italy, 5 University of Milan, Department of Pathophysiology and Transplantation, Milano, Italy, 6 A.Bianchi Bonomi Hemophilia and Thrombosis Center Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milano, Italy

Background: Patients undergoing meningioma resection are at increased risk of pulmonary embolism (PE). The incidence of post‐operative PE is not well known, as well as predictive variables of the disease.

Aims: To estimate the incidence of PE in a cohort of patients undergoing meningioma resection and the role of D‐dimer in identifying patients at risk.

Methods: Consecutive patients who underwent meningioma resection between 2012 and 2016 were included. A Q‐scan was performed at baseline (day 0, before surgery) and at day 2 after surgery. Results of Q‐scan were used to estimate the incidence of PE, reported as incidence risk and relative 95% confidence interval (CI). D‐dimer was measured at day 0, immediately post‐surgery and at day 2. Patients who developed PE were compared to patients who did not for D‐dimer levels in term of odds ratio. D‐dimer cut‐off levels were set at 75 th percentile of the distribution of levels of patients without PE.

Results: 93 patients (71% women, mean age 60 years) undergoing meningioma resection were included in the study. All of them had a normal Q‐scan at day 0 and 37 had PE revealed by Q‐scan at day 2, for an incidence risk of 39.8% (95% CI, 30‐50%). All patients remained asymptomatic. D‐dimer levels were increased post‐surgery in patients who developed PE (odds ratio 3.0, 95% CI 1.0‐8.6). The increased risk was maintained up to day 2 (odd ratio 5.7, 95% CI 2.1‐15.5), even after adjustment for sex, age, tumor dimension and duration of surgery (odd ratio 3.5, 95% CI 1.0‐12.4).

Conclusions: Patients with meningioma have a 39.8% incidence rate of PE after resection. D‐dimer measured post‐surgery and after 2 days may help to identify patients at increased risk of PE, susceptible to receive an early post‐operative antithrombotic prophylaxis.

PB668: Lower‐leg Injury Leads to a Procoagulant State

C.E. Touw 1 ; B. Nemeth 1 ; I.B. Schipper 2 ; R.G.H.H. Nelissen 3 ; S.C. Cannegieter 1

1 Leiden University Medical Center, Clinical Epidemiology, Leiden, the Netherlands, 2 Leiden University Medical Center, Surgery, Leiden, the Netherlands, 3 Leiden University Medical Center, Orthopaedics, Leiden, the Netherlands

Background: Patients with lower‐leg injury treated with cast immobilization have a 56‐fold increased risk of venous thrombosis (VT) in the first three months, compared to the general population. A possible mechanism of this increased risk could be a procoagulant state as a reaction to the injury.

Aims: To describe the coagulation profile of patients with lower leg injury and compare to controls without trauma.

Methods: Patients with lower‐leg injuries who participated in the POT‐CAST trial (Prevention Of Thrombosis following CAST immobilization) were included for the current analyses. They provided a blood sample at the emergency department immediately after the injury. Patients were compared to subjects without injuries, derived from healthy controls from the Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis (MEGA) study, a population‐based case‐control study. Factors (F)VIII, and XI activity, fibrinogen (FG) and von Willebrand Factor activity (vWF) were measured. Information on risk factors for VT was derived from questionnaires. Linear regression models were used to estimate the mean difference of coagulation factor levels of patients compared to controls with 95% confidence intervals (95% CI), adjusting for sex, age, body mass index, comorbidity, malignancy, and use of oral contraceptives.

Results: TABLE 1 Differences in coagulation factor level between patients with lower‐leg injury and controls without injury Coagulation factor Patients with injury (N=1204), mean (SD) Controls without injury (N=2921), mean (SD) Mean difference (95% CI) Adjusted mean difference (95% CI) Factor VIII activity (%) 148.28 (40.75) 112.75 (44.37) 35.53 (32.62‐38.44) 39.22 (36.21‐42.24) Factor XI activity (%) 117.06 (23.66) 100.29 (19.18) 16.77 (15.27‐18.28) 18.40 (17.01‐19.80) Fibrinogen (mg/dL) 335.68 (94.81) 330.69 (66.66) 4.98 (−0.90‐10.87) 11.64 (6.72‐16.56) Von Willebrand Factor (%) 156.76 (53.71) 111.62 (46.53) 45.14 (41.64‐48.63) 49.35 (46.05‐52.66)

A total of 4209 persons (1204 patients, 2921 controls) were eligible for analyses. Injuries involved lower‐leg fractures (89.4%), Achilles’ tendon rupture (6.9%), ankle distortion (2.4%), and contusion (1.1%). Lower‐leg injury was associated with strongly increased levels of FVIII, FXI and vWF (Table 1). After adjustment for possible confounding factors we also found an association for FG. Of all injuries, tarsal fractures accompanied by a second foot fracture were associated with the highest mean levels and phalanx fractures with the lowest.

Conclusions: Lower‐leg trauma led to elevated levels of FVIII, FXI, FG and vWF, which seemed correlated to the severity of the injury.

PB669: Evaluation of Inter‐rater Agreement of a Risk Assessment Model for Thromboprophylaxis in Medical Hospitalised Patients: Preliminary Results of a Cohort Study

C. Ferreira 1,2 ; R. Avelino Mancini 2 ; M. Monique Lana Diniz 2 ; S. Miriam Paulinelli G Alves 3 ; M. de Bastos 4 ; S. Meireles Rezende 5

1 Hospital Governador Israel Pinheiro, Farmácia Hospitalar, Belo Horizonte, Brazil, 2 Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 3 Hospital Governador Israel Pinheiro HGIP, Farmacia Hospitalar, Belo Horizonte, Brazil, 4 Hospital Governador Israel Pinheiro HGIP, Belo Horizonte, Brazil, 5 Universidade Federal de Minas Gerais, Clinica Medica, Belo Horizonte, Brazil

Background: The International Medical Prevention Registry On VenousThromboembolism risk assessment model (IMPROVE RAM ) evaluates hospital‐acquired venous thrombosis (VT) risk by categorizing seven clinical variables (IMPROVE7). Assessment of inter‐rater agreement on the collection of the variables has not been explored.

Aims: Since data reliability may be an issue in some hospitals during VT RAM Implementation, we aimed to determine inter‐rater agreement on the IMPROVE7 RAM variables in our hospital.

Methods: We included adult non‐surgical inpatients and excluded pregnant, psychiatric or intensive care patients. We also excluded inpatients already under anticoagulation at admission or with a previous VT event or those with VT diagnosed within 48 hours of admission. We used the IMPROVE7 RAM VT risk score calculator. We evaluated agreement between dat collected by a trained thromboprophylaxis team versus the attending physician at hospital admission by using Kappa statistics. We classified Kappa statistics (FLEISS Classification) as poor, fair, moderate, good and very good if <0.20, 0.21‐0.40, 0.41‐0.60, 0.61‐0.80 and 0.81‐1.00, respectively.

Results: We evaluated 887 inpatients from 08/20/2017 to 12/31/2017. Variables showed the following agreement by Kappa: “age>60 years” (0.48; 95% confidence interval [CI], 0.42‐0.55); “immobilization ≥7 days” (0.43; 95% CI, 0.35‐0.51); “Intensive Care Unit or Coronary Care Unit admission” (0.42; 95% CI, 0.25‐0.60); “Paralysis in lower limbs” (0.62; 95% CI, 0.50‐0.74); “Thrombophilia” (0.44; 95% CI, 0‐0.93); “Previous VT” (0.51; 95% CI, 0.36‐0.67) and cancer (0.35; 95% CI 0.23‐0.48).

Conclusions: In this study, the inter‐rater agreement of IMPROVE variables were mostly moderate satisfactory. This highlights the importance of assessing the realibility of data collection by different teams in a hospital during TP implementation as well as periodically.

PB670: The Effect of Alpha‐2 Plasmin Inhibitor Activity and Antigen Levels and p.Arg6Trp Polymorphism on the Risk of Venous Thromboembolism

É. Katona ; R. Bogáti; T. Miklós; R. Gindele; M. Speker; V. Fedoriska; A. Teráz‐Orosz; Z. Mezei; Z. Bereczky

University of Debrecen, Faculty of Medicine, Department of Laboratory Medicine, Division of Clinical Laboratory Science, Debrecen, Hungary

Background: Alpha‐2 plasmin inhibitor (α2PI), the primary inhibitor of plasmin, is modified in the circulation by proteolytic cleavages. Native α2‐PI (Met‐α2PI) is shortened by 12 amino acids at the N‐terminus resulting the Asn‐α2PI form, which is cross‐linked more effectively to fibrin. The p.Arg6Trp polymorphism affects the rate of this cleavage as antiplasmin cleaving enzyme cleaves Met‐α2PI(Arg6) 8‐fold faster than Met‐α2PI(Trp6).

Aims: In this case‐control study we investigated the association of α2PI p.Arg6Trp polymorphism and α2PI activity and antigen levels with the risk of venous thromboembolism (VTE).

Methods: 218 VTE patients and 218 age and sex matched healthy controls (HC) were enrolled in the study. The study was approved by the Regional Ethics Committee and fully complied with the Declaration of Helsinki. α2PI activity was determined by the Berichrom assay, total α2PI antigen levels were determined by a sandwich type ELISA and α2PI Arg6Trp genotype was determined by RT‐PCR.

Results: Genotype distributions were consistent with the Hardy‐Weinberg distribution. The minor allele frequency did not differ significantly between HC and VTE (0.21 and 0.20 respectively; P=0.841). Possession of the Trp allele did not influence the risk for VTE (OR: 0.895, 95% CI: 0.606‐1.321). α2PI activity (129±1.7% vs. 115±2.0%, P<0.001) and total α2PI antigen levels (72.8±9.2 mg/L vs. 64.6±9.7 mg/L P<0.001) were significantly elevated in VTE. The correlation of α2PI activity and antigen levels was stronger in patients (r=0.773, P<0.001) than in controls (r=0.454, P<0.001). Elevated α2PI activity and antigen levels (above 128% and 70.8 mg/L, respectively) increased the risk of VTE (OR (95% CI): 1.750 (1.158‐2.645) and 4.301 (2.848‐6.495), which remained significant after adjustment to other risk factors.

Conclusions: In our study, the α2PI p.Arg6Trp polymorphism had no effect on the risk of VTE, however α2PI levels in the upper quartile resulted in a significant risk enhancement.

Funding: NKFI K120633 and K116228.

PB671: Risk Stratification for Prevention of Venous Thromboembolism in Hospitalized Medical Patients: Can Thromboprophylaxis in Hospitalized Medical Patients Be Safely Restricted to Reduce Costs?

A.M. Bond 1,2 ; S.E. Walker 1,2 ; A. Diamantouros 1,2 ; W.H. Geerts 2,3

1 University of Toronto, Leslie Dan Faculty of Pharmacy, Toronto, Canada, 2 Sunnybrook Health Sciences Centre, Toronto, Canada, 3 University of Toronto, Faculty of Medicine, Toronto, Canada

Background: The benefit‐to‐risk ratio for thromboprophylaxis in hospitalized medical patients is poorly defined. The Padua Prediction Score (PPS) and the Improve Risk Score (IRS) are risk assessment models (RAMs) developed to identify hospitalized medical patients at low risk of hospital‐associated venous thromboembolism (HA‐VTE) and who may not require thromboprophylaxis. The thromboprophylaxis rate in medical patients at Sunnybrook Health Sciences Centre (SHSC) in 2015 was roughly 95%, possibly representing over‐treatment.

Aims: The aims of this analysis were to estimate clinical and economic outcomes of using RAMs to support decision‐making on thromboprophylaxis at SHSC compared to current standard‐of‐practice (SOP).

Methods: RAMs were applied to 199 patients during three samples of consecutive one week admissions to the general internal medicine service at SHSC. A decision tree model using Treeage software was populated using patients identified as low risk of HA‐VTE. Rates of HA‐VTE were estimated using event rates from RAM studies and the MEDENOX trial. Quality of life estimates for VTE and bleeding events were estimated from published literature.

Results: The predicted rate of symptomatic HA‐VTE in hospitalized medical patients at low risk of HA‐VTE at SHSC after one year was very low, and was not significantly different with RAM‐directed thromboprophylaxis compared to SOP‐directed thromboprophylaxis. Accordingly, there was no significant difference in quality of life. RAM‐directed thromboprophylaxis is predicted to have significant cost‐savings compared to SOP‐directed thromboprophylaxis; with predicted annual drug cost savings over $100,000, and total annual cost savings over $200,000.

TABLE 1 Base case estimates comparing RAM‐directed care vs SOP‐directed care after 1 year PPS SOP Difference IRS SOP Difference Low risk patients (N) 2565 2565 ‐ 3155 3155 ‐ Symptomatic VTE events (N) 7 5 +2 22 16 +6 Any bleeding events (N) 32 47 ‐15 40 58 ‐18 QALYs 2564.14 2563.89 +0.25 3153.67 3153.51 +0.16 Drug costs (CAD$) $0 $114,202 ‐$114,202 $0 $158,883 ‐$158,883 Total costs (CAD$) $339,541 $546,954 ‐$207,413 $507,055 $730,969 ‐$223,914 Incremental cost per QALY gained (ICER) PPS‐directed care dominates IRS‐directed care dominates

FIGURE 1 ICER scatterplots for PPS‐directed care (top) and IRS‐directed care (bottom) compared to SOP‐directed care

Conclusions: The use of RAMs may not significantly affect HA‐VTE or patient quality of life, but may result in significant cost savings. Prospective evaluation is required to establish the feasibility of RAMs and ensure the safety of reducing the rate of thromboprophylaxis.

PB672: Predictors of Venous Thromboembolism in Patients Undergoing Neurosurgery for Glioma

M. Capecchi 1 ; P. Bucciarelli 1 ; A. Artoni 1 ; M. Abbattista 1 ; F. Gianniello 1 ; A. Lecchi 1 ; G. Carrabba 2 ; M. Castellani 3 ; F. Peyvandi 4,5 ; I. Martinelli 1

1 A.Bianchi Bonomi Hemophilia and Thrombosis Center Fondazione IRCCS Ca’ Granda Ospedale Maggiore, Milano, Italy, 2 Fondazione IRCCS Ca’ Granda Ospedale Maggiore, Division of Neurosurgery, Milano, Italy, 3 Fondazione IRCCS Ca’ Granda Ospedale Maggiore, Nuclear Medicine Department, Milano, Italy, 4 University of Milan, Department of Pathophysiology and Transplantation, Milano, Italy, 5 A.Bianchi Bonomi Hemophilia and Thrombosis Center Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano, Italy

Background: Patients with glioma undergoing resection have a high post‐operative risk of pulmonary embolism (PE) together with intracranial bleeding. Therefore an optimal stratification of the risk of PE is warranted before prescribing post‐operative antithrombotic prophylaxis.

Aims: To identify individual variables predicting the risk of PR after glioma resection.

Methods: Our study population includes consecutive patients undergoing glioma resection at Ospedale Maggiore Policlinico of Milan between 2012 and 2016. A Q‐scan was performed at baseline (day 0, before tumor resection) and the day after surgery (day 1). Results of Q‐scans were used to estimate the incidence of early asymptomatic pulmonary embolism, reported as incidence risk and its 95% confidence interval (CI). Blood samples for D‐dimer was collected at baseline and at post‐surgery. A multivariate logistic regression model was fitted with sex, age, BMI, tumor volume, duration of intervention, D‐dimer (categorized at the 50th percentile [i.e., 627 ng/mL]) and histologic grade (high vs. low) as predictors of PE. The predictive capability of the model was measured as the area under the ROC curve (AUC) with its 95% CI.

Results: 59 patients were included in the study, 33 (56%) men and 26 (44%) women, with a median age at surgery of 57.5±13.7 years. All patients had a negative Q‐scan at day 0. 19 patients (32%) developed PE at day 2, for an incidence risk of 32.2% (95% CI 21.7‐44.9%). The variables mostly associated with the risk of PE were D‐dimer, (adj. odds ratio 3.0, 95% CI 0.7‐12.7), histologic “high malignancy grade” (2.9, 0.3‐28.4) and BMI (2.0, 0.7‐5.2 for every 5 kg/m 2 increase). The AUC of the ROC curve of the whole predictive model was 0.7 (95% CI 0.6‐0.9) (Figure).

FIGURE 1 ROC curve analysis for the predictive capability of the model

Conclusions: We found a 32% incidence of an early PE after glioma resection. A predictive model including D‐dimer, histologic grade and BMI showed a promising capability of identifying patients at risk of PE who may need stronger antithrombotic prophylaxis.

PB673: IVSS Safety Zone Protocol versus Padua Score in the Prevention of Thromboembolic Disease in Medical Patients: A Comparative Prospective Study

J. Serrano 1 ; B. Rodriguez 2 ; M.C. Chindamo 3

1 Venezuelan Society of Hematology, Presidency, Caracas, Venezuela, 2 Hospital del Seguro Social Patrocion Peñuela Ruiz, Internal Medicine, San Cristobal, Venezuela, 3 Hospital Barra D'Or, Internal Medicine, Rio de Janeiro, Brazil

Background: Prophylaxis of venous thromboembolism (VTE) in hospitalized medical patients is largely underused. We evaluate a simple and effective model (Safety Zone) for identifying the risk of VTE in medical cases.

Aims: To compare thrombotic and bleeding outcomes in two prediction scores for risk of VTE.

Methods: In prospective study with 393 patients admitted to the Medical ward at IVSS San Cristobal, may ‐ dec 2017, that were randomly classified as low or high risk of VTE according of two prediction scores for risk of VTE; Padua Score or Safety Zone, with follow‐up of 90 days after discharge to assess primary end points in both groups: 1.‐ Incidence of symptomatic VTE (confirmed by duplex ultrasound and angioCT) 2.‐ Major bleeding associated with thromboprofilaxis. The prevalence of risk factors for VTE in our population was specified.

Results: Padua group (197 cases) and safety zone group (196 cases) were stratified as high‐risk in 51.27% and 75.51% respectively. The risk factors most prevalent were: Infectious diseases 30.28%, Neoplasms 13%, and CVD, central catheters 12 and 7% each. During hospital stay 1.55% of cases in Padua group developed VTE, while no cases in Safety Zone group at discharge. Follow up at 30 and 90 days showed no VTE events in either group. One case of major bleed with enoxaparin (0.51%) in Padua group. Minor bleeding was comparable for both groups <2%.

Conclusions: Safety Zone score showed similar outcomes for VTE and bleed rates regarding Padua, a trend to higher inclusion of high risk cases for VTE was found in Safety Zone.

PB675: Prophylaxis of Venous Thromboembolism in an Internal Medicine Department

M. Calçada 1 ; I. Neto 1 ; A.R. Freitas 1 ; M. Santos 1 ; C. Bastos 2

1 Centro Hospitalar de Entre o Douro e Vouga, Internal Medicine Department, Santa Maria da Feira, Portugal, 2 Centro Hospitalar de entre o Douro e Vouga, Santa Maria da Feira, Portugal

Background: Prevention of venous thromboembolism (VTE) is one of the most cost‐effective measures. In our Internal Medicine Department (IMD) there is a guide, based on recommendations of the Center for the Study of Pulmonary Vascular Disease of the Portuguese Society of Internal Medicine, established as a quality indicator since 2010, with a target of 90% compliance.

Aims: The aim of the study is to evaluate compliance with VTE prophylaxis in hospitalized patients at an IMD on day of admission (D0) and the third day of hospitalization (D3).

Methods: This cross‐sectional observational study was conducted between January 1 and July 31, 2017. Inclusion criteria were all patients hospitalized during all the Friday (24 hours) in the IMD. Exclusion criteria were hypo‐coagulated or absent patients from the hospital on the third day of hospitalization.

Results: Of a total of 324 patients, 220 were included and 104 were excluded (hypocoagulation or absence of IMD to D3). The risk of VTE was assessed in 97.7% of patients. Of the 220 patients, 95.5% were eligible for prophylaxis. In D0, 13 prescription errors occurred, with a prophylaxis adequacy rate of 95.4%. In D3 we verified the persistence of 7 errors and 6 new errors, translating an adequacy rate of 93.7%.

Conclusions: In the first half of 2017 the risk assessment was recorded on admission to a high percentage of patients; the established target was achieved in majority of patients at risk with adequate prophylaxis. The absence of dose adjustment of low molecular weight heparin to renal function was the most frequent error in D0; the lack of therapeutic review seems to determine the adequacy rate for D3. The establishment of clinical guidelines and their monitoring make it possible to understand and develop good clinical practice.

PB676: Clinical Profile of Acutely‐ill Medical Hospitalized Patients: Preliminary Results of a Cohort Study on Thromboprophylaxis

M.M. Lana Diniz 1 ; R.A. Mancini 1 ; C. Rodrigues Lima Ferreira 2 ; S.M. Paulinelli Garcia Alves 2 ; M. deBastos 2 ; S. Meireles Rezende 3

1 Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 2 Hospital Governador Israel Pinheiro, Belo Horizonte, Brazil, 3 Universidade Federal de Minas Gerais, Departamento de Clínica Médica, Belo Horizonte, Brazil

Background: Venous thromboembolism (VTE) is a life threatening condition occurring in 10%‐26% of clinical hospitalized patients. The main risk factors are older age, intensive care unit (ICU) stay, immobilization, lower limb paralysis, active cancer, previous VTE and thrombophilia. Few studies have investigated risk factors of VTE in clinical hospitalized patients in developing countries using a validated score.

Aims: To describe the baseline characteristics of patients admitted to a teaching hospital and enrolled in a cohort study to evaluate thromboprophylaxis (TP) using IMPROVE7 score.

Methods: Inclusion criteria were non‐surgical, non‐psychiatric, non‐pregnant clinical inpatients older than 18 years, not previously anticoagulated and not diagnosed with VTE until 48 hours after hospital admission. Exclusion criteria were diagnosis of illness in which anticoagulant therapy is needed, surgery during hospitalization. Socio‐demographic and clinical variables were obtained through electronic data record and interview using standardized forms. Number of events and respective percentages were calculated for the categorical variables and the median and interquartile range (IQR) for the continuous variables. All patients signed informed consent forms. The study was approved by ethical committees of involved institutions.

Results: A total of 887 patients were enrolled from 08/20/2017 to 12/31/2017 of whom most (55.9%) are female, median age 67 (IQR, 59‐79) years. The frequency and distribution of VTE risk factors is shown on Table 1. Age>60 years, immobilization >7 days and active cancer were the most frequent risk factors. VTE risk was predominantly low (80.2%) [IMPROVE score ≤2].

TABLE 1 Characteristics of patients included in the cohort Characteristics n (%) Female 496 (55.9) Age>60 years 643 (72.5) ICU stay 37 (4.2) Active cancer 111 (12.5) Lower limb paralysis 49 (5.5) History of thrombophilia 5 (0.6) Previous VTE 52 (5.9) Immobilization>7 days 171 (19.3) Low VTE risk (IMPROVE7 score ≤2) 711 (80.2)

Conclusions: In this cohort, older age, active cancer and immobilization were the main risk factors for VTE. About 20% of patients were high risk for VTE according to the IMPROVE7 score. A follow‐up analysis is underway and will focus on the outcome of VTE and bleeding according to the TP used.

PB680: 25 mmHg versus 35 mmHg Graduated Elastic Compression Stockings to Treat Acute Symptoms of Deep‐Vein Thrombosis: The CELEST Double‐blind Trial

J.‐P. Galanaud 1,2 ; S. Blaise 3 ; C. Vermorel 4 ; C. Rolland 4 ; F. Verriere 5 ; I. Bertaina 5 ; H. Guenneguez 6 ; J.L. Bosson 4

1 Toronto, Medicine, Toronto, Canada, 2 Montpellier, Medicine, Montpellier, France, 3 Grenoble, Vascular Medecine, Grenoble, France, 4 Grenoble, CNRS, Laboratoire TIMC‐Imag, Grenoble, France, 5 Innothera, Arcueil, France, 6 Saint Aubin, Saint Aubin, France

Background: Effectiveness of elastic compression stockings (ECS) to prevent post‐thrombotic syndrome after a lower limb deep vein thrombosis (DVT) is debated and their use to prevent PTS is no more suggested in International Guidelines. However, guidelines also state that the use of ECS can be useful at the acute phase of DVT in a symptom relief perspective. Optimal strength of ECS needs to be determined and particularly the effectiveness of lightest strength of ECS (25 mmHg).

Aims: To compare the effectiveness of 25 mmHg versus 35 mmHg ECS to treat acute symptoms after a proximal lower limb DVT.

Methods: Multicentre double‐blind randomized CELEST study (Sponsor Innothera) comparing the effectiveness of 25 mmHg versus 35 mmHg graduated ECS after a first proximal lower limb DVT. We compared, in a double‐blind way, evolution of pain and oedema (assessed by patient with visual analog scale (VAS) ranging from 0 to 100) from 1 week after randomization (i.e. time when study's stockings started to be worn) to 3 months in each group.

Results: 350 patients were randomized; 68% (n=238) were men and mean age was 57.5 years. In 58% (n=202) of cases DVT involved left lower limb and extended up to the iliac veins in 15% (n=52) of cases. Symptoms intensity was not statistically different between groups at 1 week and decreased similarly in both groups (NS) : From 1 week to 1 month and then to 3 months, pain intensity decreased from 28 interquartile range [10‐62] to 13 [8‐33] and then to 9.5 [4‐17] in group A and from 19 [9‐56] to 12 [7‐28] and then to 8.5 [4‐13] in group B; Oedema intensity decreased from 24 [10‐57] to 14 [6‐35] and then to 9 [4‐16] in group A and from 14 [7‐53] to 10 [5‐19] and then to 8 [4‐13] in group B,. At 3 months, a quarter of patients had a VAS ≥15/100 for either edema or pain symptoms.

Conclusions: After a proximal lower limb DVT, 25 mmHg and 35 mmHg ECS are similarly effective to treat acute DVT symptoms. Given that they are easier to put on, 25 mmHg ECS could consitute a first line treatment in this indication.

PB681: Individualized Prediction Model for Risk of First DVT Using Clinical Assessment and D‐dimer

S. Parpia 1 ; L.‐A. Linkins 1 ; J. Julian 1 ; G.‐J. Geersing 2 ; K. deWit 1 ; C. Kearon 1

1 McMaster University, Hamilton, Canada, 2 University Medical Center Utrecht, Utrecht, the Netherlands

Background: Diagnosis of deep vein thrombosis (DVT) historically begins with assessing clinical pre‐test probability category (e.g. using the Wells score), followed by D‐dimer testing. Combining all clinical and D‐dimer information may lead to a better individualized estimate of DVT probability that facilitates management.

Aims: Our aim was to develop a prediction model that would estimate individual risk for DVT using clinical information and D‐dimer levels.

Methods: The model was developed using multivariable logistic regression of data derived from a published DVT diagnosis study. Pre‐defined variables were: components of the Wells score, age, sex and d‐dimer level. Ultrasound confirmed DVT was the dependent variable. Continuous variables were fitted using restricted cubic splines to relax the linearity assumption. Fast backward selection at P<0.5 without refitting was done to reduce the model. Interactions between variables were assessed. Internal validation to obtain unbiased estimation of model performance was done using bootstrapping, and predictive performance was evaluated using discrimination (c‐statistic) and calibration plots. Finally, we generated a nomogram to predict individual risk of DVT.

Results: A total of 1,535 patients, of whom 6.0% had DVT, were analyzed. Age had a non‐linear relationship with the presence of DVT (increasing risk till 65 years followed by decreasing risk). Five variables formed the final model: d‐dimer level, age, sex, calf swelling, no more likely alternative diagnosis and being bedridden. The bootstrap bias‐corrected c‐statistic was 0.89. Calibration plots showed the model to have good calibration. The nomogram for prediction of DVT is shown in Figure 1.

FIGURE 1 Diagnostic nomogram for predicting DVT

Conclusions: We developed an individualized prediction model for DVT with excellent discriminative performance. The model, which includes D‐dimer level, has four fewer clinical variables than the Wells model. External validation is required before the model can be used in clinical practice.

PB682: Long‐term Risk of Post‐thrombotic Syndrome after Symptomatic Distal Deep‐vein Thrombosis: The CACTUS‐PTS Study

J.‐P. Galanaud 1,2 ; M. Righini 3 ; L. Lecollen 1 ; A. Douillard 4 ; H. Robert‐Ebadi 3 ; D. Pontal 1 ; D. Morrison 5 ; M. Carrier 6 ; I. Quéré 1 ; S.R. Kahn 5,7

1 University Montpellier, Vascular Medicine, Montpellier, France, 2 University Toronto, Medicine, Toronto, Canada, 3 Geneva University Hospitals, Vascular Medicine, Geneva, Switzerland, 4 University Montpellier, Biostatistics, Montpellier, France, 5 Lady Davis Jewish General Hospital, Clinical Epidemiology, Montreal, Canada, 6 Ottawa Hospital Research Institute, Hematology, Ottawa, Canada, 7 University McGill, Medicine, Montreal, Canada

Background: After a proximal lower limb deep‐vein thrombosis (DVT) involving the popliteal vein or above, up to 40% of patients develop the post‐thrombotic syndrome (PTS) as assessed by the Villalta scale (VS). Poor initial anticoagulant treatment is a known risk factor for PTS. The risk of developing PTS after isolated distal DVT (infra‐popliteal DVT without pulmonary embolism), and the impact of anticoagulant treatment on this risk, are unknown.

Aims: To assess long‐term risk of PTS and impact of anticoagulant treatment on this risk after distal DVT.

Methods: Long‐term follow‐up of CACTUS double‐blind trial comparing 6 weeks of sc nadroparin (171 IU/kg/day) versus sc placebo, in addition to elastic compression stockings, for a first symptomatic isolated distal DVT. At least 1 year after randomization, patients had a PTS assessment in clinic or by phone using the VS. PTS was considered present if VS was ≥5 in the leg ipsilateral to DVT.

Results: After a mean 6.0 years after the index DVT event, 179 patients had a PTS assessment (in clinic in 111 patients); 52% were male and mean age and body mass index were 58.3 years and 25.0 Kg/m 2 respectively. Overall, PTS was present in 29% of patients (N=54) and was moderate or severe in 24% of cases (n=13). There was no statistically significant difference in rates of PTS in the nadroparin versus placebo groups (28% vs. 32%, P=0.6) overall, but the rate of PTS was significantly lower in the nadroparin group in the subgroup of patients without evidence of prior chronic venous insufficiency, as assessed by VS <5 in the leg contralateral to DVT (8% vs. 24%, P=0.04).

Conclusions: After a first isolated distal DVT, risk of PTS appears to be about 30%, which is substantial but somewhat lower than that reported after proximal DVT. Anticoagulant treatment for 6 weeks with nadroparin, a low molecular weight heparin, does not provide any clear benefit to prevent PTS compared with placebo, except in patients without pre‐existing chronic venous insufficiency.

PB683: Age‐adjusted versus Clinical Probability‐adjusted D‐dimer to Exclude Pulmonary Embolism

S.M. Stevens 1,2 ; S. Takach Lapner 3 ; S.C. Woller 2,4 ; G. Snow 5 ; C. Kearon 6

1 Intermountain Healthcare, Department of Medicine, Murray, United States, 2 University of Utah, Internal Medicine, Salt Lake City, United States, 3 University of Alberta, Division of Hematology, Edmonton, Canada, 4 Intermountain Healthcare, Medicine, Murray, United States, 5 Intermountain Healthcare, Statistical Data Center, Murray, United States, 6 McMaster University, Medicine, Hamilton, Canada

Background: A low D‐dimer can exclude suspected pulmonary embolism (PE) without imaging in cases at low or moderate clinical probability (CP) for disease. Yet D‐dimer is nonspecific, so many cases without PE still undergo imaging. D‐dimer's specificity is improved by increasing the threshold of a positive test according to age (age‐adjusted; age × 10 ng/mL; AADD) or the CP of PE (CP‐adjusted; 500 ng/mL if low and 1,000 ng/mL if moderate CP; CPADD). It is unclear which adjustment approach is preferable.

Aims: We report the sensitivity, specificity and negative predictive value of AADD compared to CPADD in suspected PE.

Methods: A retrospective cohort of 3,500 consecutive cases imaged for suspected PE at two U.S. emergency departments was assembled. We analyzed cases who were either at low or moderate CP for PE according to the Revised Geneva Score (RGS) and had a D‐dimer. The outcome was acute PE on imaging at presentation or during a 3‐month follow‐up. Specificity and negative predictive value were determined for each D‐dimer strategy.

Results: Of the 3,500 cases, 1,745 met eligibility criteria for this analysis. 646 (37%) were low, and 1,099 (63%) were moderate CP for PE. PE occurred in 145 (8.3%) cases; 6.2% at low, and 9.6% at moderate CP. Sensitivity of the CPADD strategy was 87.5% versus 96.6% for the AADD (difference 9.1%; 95% CI: 4.3% to 14.0%). Negative predictive value of CPADD was 97.1% versus 99.0% for the AADD (difference 1.9%; 95% CI, 0.7% to 3.1%). Specificity of PADD was 37.5% versus 30.2% for the AADD (difference ‐7.3%; 95% CI: ‐9.4% to ‐5.1%). D‐dimer was negative in 35.4% of cases using the PADD strategy versus 28.0% using AADD.

Conclusions: Use of the CPADD strategy modestly improved the specificity of D‐dimer to exclude PE, but had a lower negative predictive value than the AADD strategy. The AADD strategy appears preferable in the population studied.

PB684: Vena Caval Filters in Patients Presenting with Major Bleeding during Anticoagulation for Venous Thromboembolism

M. Mellado 1 ; J. Trujillo‐Santos 2 ; B. Bikdeli 3 ; D. Jimenez 4 ; M.J. Nunez 5 ; M. Ellis 6 ; P.J. Marchena 7 ; J.R. Vela 8 ; A. Clara 1 ; F. Moustafa 9 ; M. Monreal 10 ; RIETE Investigators

1 Hospital del Mar, Barcelona, Spain, 2 Hospital General Universitario de Santa Lucía, Murcia, Spain, 3 Columbia University Medical Center/New York‐Presbyterian Hospital, New‐York, United States, 4 Hospital Universitario Ramón y Caja, Madrid, Spain, 5 Complejo Hospitalario de Pontevedra, Pontevedra, Spain, 6 Meir Hospital, Kfar Saba, Israel, 7 Parc Sanitari Sant Joan de Deu‐Hospital General, Barcelona, Spain, 8 Hospital Universitario Miguel Servet, Zaragoza, Spain, 9 CHU Clermont‐Ferrand, Emergency Medicine, Clermont‐Ferrand, France, 10 Hospital Germans Trias i Pujol, Barcelona, Spain

Background: Impact of inferior vena caval filters (IVC) on outcomes of patients presenting with major bleeding during anticoagulation for venous thromboembolism (VTE) has not been thoroughly investigated.

Aims: We aimed to assess the impact of IVC filter insertion on outcome within the first 30 days in patients presenting with major bleeding during the first 3 months of anticoagulant therapy for VTE.

Methods: We used the RIETE (Registro Informatizado Enfermedad Trombo Embólica, NCT02832245 ) database. From January 2001 through September 2016, we included patients who presented with a major bleeding event during the first 3 months of anticoagulant therapy. We excluded patients with a pre‐existing IVC filter, those undergoing IVC filter placement beyond the first 48 hours after major bleeding and those who were not receiving anticoagulant therapy when the index bleeding event prior to inclusion occurred.

Results: 1,065 patients with VTE had major bleeding during the first 3 months. Of these, 122 (11%) received an IVC filter. Patients receiving a filter restarted anticoagulation later (median, 4 vs. 2 days) and at lower doses (95±52 vs. 104±55 IU/kg/day of low‐molecular‐weight heparin) than those not receiving a filter. During the first 30 days after bleeding, 63 patients (5.9%) re‐bled, 19 (1.8%) had recurrent pulmonary embolism (PE) and 283 died (129 died of bleeding, 14 of PE). On propensity score analysis, patients receiving a filter had a lower risk of fatal bleeding (hazard ratio [HR]: 0.14; 95% CI: 0.05‐0.45) and all‐cause death (HR: 0.45; 95% CI: 0.27‐0.74), similar risk of any major re‐bleeding (HR: 0.53; 95% CI: 0.21‐1.37) or PE recurrences (HR: 1.59; 95% CI: 0.40‐6.37), and higher risk of DVT (HR: 3.08; 95% CI: 0.99‐9.54) than those not receiving it.

TABLE 1 Clinical outcomes occurring during the first 30 days after bleeding, before and after propensity score matching Before matching (univariate analysis) After matching with a ratio 4:1 Hazard ratio (95% CI) P‐value Hazard ratio (95% CI) P‐value Patients, N 122 vs. 943 122 vs. 429 Recurrent PE 1.31 (0.38‐4.49) 0.67 1.59 (0.40‐6.37) 0.51 Recurrent DVT 2.78 (1.08‐7.18) 0.03 3.08 (0.99‐9.54) 0.052 Re‐bleeding 0.61 (0.24‐1.51) 0.28 0.53 (0.21‐1.37) 0.19 Overall death 0.45 (0.28‐0.74) 0.002 0.45 (0.27‐0.74) 0.002 Fatal PE Coefficients did not converge Coefficients did not converge Fatal bleeding 0.17 (0.06‐0.54) 0.003 0.14 (0.05‐0.45) 0.001

Conclusions: In patients with VTE experiencing major bleeding during the first 3 months of therapy, use of IVC filters was associated with later restarting of anticoagulation, reduced fatal bleeding and mortality.

PB685: Whole Leg Ultrasound for the Diagnosis of Deep Vein Thrombosis Recurrence

M. Sartori ; E. Favaretto; L. Valdrè; G. Guazzaloca; A. Amato; B. Cosmi

Angiology and Blood Coagulation Unit, S. Orsola‐Malpighi University Hospital, Bologna, Italy

Background: There is no gold standard for the diagnosis of deep vein thrombosis (DVT) recurrence. Ultrasound is the most used test for the diagnosis of recurrent DVT, but well designed management diagnostic studies that focus mainly on patients with prior DVT are lacking.

Aims: To assess the safety of withholding anticoagulation from patients presenting with suspected recurrent DVT of lower extremity on the basis of whole leg ultrasound.

Methods: A management study in outpatients referred by the emergency department or by a primary care physician to our department for clinically suspected recurrent DVT. All patients underwent a B‐mode and color Doppler ultrasonography examination of both legs. Recurrent DVT was diagnosed in case of either(i) lack of compressibility of a previously compressible vein, or(ii) at least a 3 mm increase in the diameter of a previously non‐compressible vein (when thrombus diameter changed between 1.1 and 2.9 mm, the examination was repeated 5‐7 days later) or(iii) on thrombus echogenicity, vein wall appearance and vein lumen diameter when a diameter of a previously non‐compressible vein was not available or when thrombus diameter changed between 1.1 and 2.9 mm. All the patients were followed up for 3 months to document the occurrence of symptomatic superficial and deep vein thrombosis or pulmonary embolism.

Results: 247 patients (female: 45.7%; age 66.1±18 year) were enrolled. The diagnostic work‐up showed a recurrent proximal DVT in 28 (11.3%) patients, an isolate distal DVT in 25 (10.1%), and a superficial vein thrombosis (SVT) in 9 (3.6%) patients. Of the 185 patients with negative whole leg ultrasound for DVT recurrence, two (1.08%, 95% CI: 0.13‐3.85) experienced symptomatic venous thromboembolic events (one DVT and one non fatal pulmonary embolism) and two SVTs.

Conclusions: These hypothesis‐generating data suggest that anticoagulant therapy could be withheld for clinically suspected recurrent DVT after one single whole leg ultrasound and justify larger studies.

PB686: Polymorphisms in PARK2 and MRPL37 Are Associated with Higher Risk of Recurrent Venous Thromboembolism in a Sex‐specific Manner

K. Sundqiuist 1,2 ; A. Ahmad 1 ; P.J. Svensson 3 ; B. Zoller 1 ; J. Sundqiuist 1 ; A. Memon 1

1 Lund University, Clinical Sciences, Malmo, Sweden, 2 Icahn School of Medicine at Mount Sinai, Department of Family Medicine and Community Health, Department of Population Health Science and Policy, New York, United States, 3 Lund University, Department of Coagulation Disorders, Malmo, Sweden

Background: Recent studies indicate that mitochondrial DNA (mtDNA) dysfunction is a biomarker of oxidative stress and can predict the risk of cardiovascular diseases (CVDs). Genetic variants in PARK2 (rs4708928) and MRPL37 (rs10888838) genes have been shown to be associated with altered levels of mtDNA in a sex‐specific manner. However, the role of these genetic variants in risk assessment of recurrent venous thromboembolism (VTE) is unknown.

Aims: Aim of our study was to investigate the role of these polymorphisms in recurrent VTE.

Methods: VTE patients from Malmö thrombophilia study (MATS, n=1,465) were included in this study. VTE patients were followed for ˜10 years (1998‐2008). Genotyping was performed by Taqman PCR.

Results: Female patients with PARK2 polymorphism had significantly higher risk of VTE recurrence (Hazard ratio [HR] =2.39, 95% confidence interval [CI] =1.09‐5.24) and male patients with MRPL37 polymorphism had a significantly higher risk of VTE recurrence (HR=1.79, 95% CI=1.01‐3.17) on multivariate Cox regression analysis. Combined analysis of these polymorphism with factor V Leiden (FVL) showed that female patients with both, FVL and PARK2 polymorphism had even higher risk of VTE recurrence (HR=4.49, 95% CI=1.58‐12.75) compared to FVL or PARK2 polymorphism alone or both wild‐type (reference). Similarly, male patients with both FVL and MRPL37 polymorphism had significantly higher risk of VTE recurrence (HR=2.97, 95% CI=1.45‐6.08) compared to those with FVL or MRPL37 polymorphisms alone or the reference group.

Conclusions: Polymorphisms in nuclear genome regulating mtDNA together with FVL may be promising biomarkers for predicting VTE recurrence in a sex specific manner.

PB687: The safety of Low Molecular Weight Heparin, Direct Oral Anticoagulants, & Warfarin in Patients with Chronic Liver Disease; Analysis of 932 Patients from the RIETE Database

J. Shatzel 1 ; R. Chandran 1 ; S. Gardiner 1 ; V. Rosa 2 ; A. Lorenzo 3 ; L. Mazzolai 4 ; A. Villalobos 5 ; R. Quintavalla 6 ; P. Verhamme 7 ; M. Monreal 8 ; the RIETE Investigators

1 Oregon Health & Science University, Portland, United States, 2 Department of Internal Medicine. Hospital Universitario Virgen de Arrixaca, Murcia, Spain, 3 Hospital Universitario de La Paz, Madrid, Spain, 4 Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland, 5 Department of Internal Medicine. Hospital Regional Universitario de Málaga, Malaga, Spain, 6 Azienda Ospedaliera Universitaria, parma, Italy, 7 University of Leuven, Leuven, Belgium, 8 Hospital Germans Trias i Pujol, Badalona, Spain

Background: Patients with chronic liver disease (CLD) are at risk of bleeding. The safety of anticoagulants in CLD patients with venous thromboembolism (VTE) is uncertain.

Aims: Describe the bleeding rates of low molecular weight heparin (LMWH), direct oral anticoagulants (DOAC) & warfarin (VKA) in patients with CLD.

Methods: We selected patients with biopsy proven cirrhosis & patients with steatosis or chronic liver disease without biopsy in the RIETE registry to evaluate bleeding. Cox proportional hazards survival analysis was used to determine predictors of bleeding.

Results: From 2001 to 2017, 932 patients in the RIETE registry had CLD treated with one of the 3 included anticoagulants (Table 1). 200 had biopsy proven cirrhosis. Patients given LMWH had higher rates of cancer & esophageal varices. Bleeding was more common with LMWH than VKA & DOACs. Analysis of patients without cancer found decreased bleeding with VKA as compared to LMWH (HR 0.343, P=0.03) & similar bleeding with DOACs (P=0.86). We performed a separate analysis of the 200 patients with cirrhosis. (Table 2). Cirrhosis patients on VKAs still had less bleeding than LMWH (hazard ratio 0.2893, P=0.01) while bleeding with DOACs was not significantly different (P=0.99). Multivariable analysis found no difference in bleeding between cirrhosis patients and all others P=0.778. However, cirrhosis patients bleed sooner (HR 1.9, P=0.007.). Multivariate analysis confirmed decreased bleeding with VKA (HR 0.32, P=0.00003), & DOACs (0.29, P=0.041) as compared to LMWH. Esophageal varices (HR 2.587, P=0.0014) & male gender (HR 2.0, P=0.0087) predicted bleeding.

TABLE 1 All patients with chronic liver disease

TABLE 2 Patients with biopsy proven cirrhosis

Conclusions: LMWH increased bleeding in CLD patients compared to VKA & DOAC. In patients with cirrhosis, VKAs led to less bleeding than LMWH. DOACs were not associated with a difference in bleeding, although the number of patients on DOACs was small. While unable to precisely define the level of liver disease in each group, this analysis suggests DOACs to be comparatively safe in select CLD patients.

PB688: Clinical Predictors of Right Ventricular Dysfunction in Acute Pulmonary Thromboembolism

J. Bonorino 1 ; J. Bilbao 1 ; H. Fernandez 1 ; J. Santucci 1 ; J. Aboy 1 ; P. Alarcon 1 ; N. Torres 1 ; E. Martino 1 ; G. Moles 1 ; V. Martin Napoli 1 ; P. Colimodio 2 ; R. Melchiori 1 ; M.E. Aris Cancela 2 ; S. Baratta 1 ; A. Hita 1

1 Hospital Universitario Austral, Cardiology, Buenos Aires, Argentina, 2 Hospital Universitario Austral, Hematology, Buenos Aires, Argentina

Background: The ability of certain clinical predictors to identify patients with right ventricular dysfunction (RVD) in acute pulmonary embolism (PE) is not well established.

Aims: Assess the prevalence and prognosis of RVD and identify associated clinical variables in patients with PE.

Methods: Single‐center, observational, cohort study, conducted at a University Hospital. Patients (p) admitted with PE were included. The presence of RVD was assessed by echocardiography and their relation with in‐hospital clinical course was analized. Variables were chosen because they could easily be obtained and predict the presence of PE. An in‐hospital event (IE) was defined as the combination of death, need for mechanical ventilation, ventricular assistance or use of vasoactive drugs.

Results: We included a total of 158 consecutive patients that were admitted between 2006 and 2016. RVD was identified in 57 p (36%) and its finding correlated with a higher rate of in‐hospital events (IE), 14/57 (24.5%) vs. 5/101 (4.9%); RR 1.2, 95% CI: 1.0‐1.4, P=0.001; as well as a significant increase in mortality rate: 6/57 (10.5%) vs. 2/101 (1.9%), RR: 8.5, 95% CI: 1.1‐16, P=0.02. On univariate analysis, predictors of RVD were identified (Table 1). On logistic regression analysis, the following variables persisted as independent predictors: simplified PESI (simplified Pulmonary Embolism Score Index)>0, negative T waves in V1‐V4 on ECG, chest pain at presentation and elevated troponins (Table 2). The final model discriminated variables adequately, with an area under the curve (AUC) of 0.87.

Conclusions: RVD was confirmed in 1 of every 3 patients with PE and was associated with a higher incidence of IE and death. The presence of independent predictive variables, such as sPESI>0, negative T waves in V1‐V4, chest pain at presentation and elevated troponins helped to predict RVD. Such approach could allow to identify patients at higher risk who should undergo urgent echocardiography, since they might require reperfusion therapy.

PB689: Individualized Thromboprophylaxis in Patients with Lower‐leg Cast Immobilization – A Validation and Subgroup Analysis in the POT‐CAST Trial

B. Nemeth ; S. le Cessie; S.C. Cannegieter; Pot‐(k) cast Study Group

Leiden University Medical Center, Department of Clinical Epidemiology, Leiden, the Netherlands

Background: Patients treated with lower‐leg cast immobilization are at risk for developing venous thromboembolism (VTE). New treatment strategies should be established in order to prevent VTE after plaster casts.

Aims: This study aimed to 1. assess the absolute risk of VTE in low‐ and high‐risk groups within the POT‐CAST trial and 2. determine the effectiveness of low molecular weight heparin (LMWH) therapy in these low‐ and high‐risk groups.

Methods: In the POT‐CAST trial, 1,435 with lower leg plaster cast were included in the intention‐to‐treat population. VTE occurred in 10 of the 719 patients (1.4%) in the treatment group (LMWH during the period of immobilisation) and in 13 of the 716 patients (1.8%) in the control group (relative risk, 0.8; 95% CI, 0.3‐1.7).

Results: In the POT‐CAST the overall VTE risk was (Absolute Risk (AR) 1.6% (n=1,435). Men had a higher risk than women (AR 1.9% [95% CI 1.1‐3.3] versus AR 1.3% [95% CI 0.6‐2.4]), although women using oral contraceptives had a similar risk to that of men (AR 1.9% [95% CI 0.2‐6.7]). Patients with classical VTE risk factors such as a body mass index above 30 kg/m 2 or a positive family history had an increased risk, i.e. AR 3.9% (95% CI 0.7‐7.6) and AR 3.3% (95% CI 0.9‐8.1), respectively. Patients with an Achilles tendon rupture developed VTE in 8.5% (95% CI 3.7‐16.1) whereas fractures were associated with lower risks. Surgically treated injuries led to a higher VTE risk than conservatively treated injuries, i.e., AR 3.5% (95% CI 1.3‐7.5) and AR 1.3% (95% CI 0.8‐2.1), respectively. There were no risk groups in which thromboprophylaxis significantly reduced VTE. Relative risks of prophylaxis versus no treatment ranged from 0.3 (95% CI 0.1‐1.3) in women to 2.3 (95% CI 0.6 to 8.9) in patients with an Achilles tendon rupture.

Conclusions: There were no subgroups in which thromboprophylaxis was effective for VTE prevention. High risk patients with lower‐leg cast can be identified based on injury type or some classical VTE risk factors.

PB690: Prevalence of Pulmonary Embolism in Patients Presenting to the Emergency Department with Syncope in an Integrated Healthcare System

S.M. Stevens 1 ; J. Bledsoe 2 ; J. Jacobs 3 ; A. Frizell 4 ; J. Quinn 5

1 Intermountain Healthcare, Internal Medicine‐ Thrombosis Clinic Director, Murray, United States, 2 Intermountain Healthcare, Emergency Medicine, Murray, United States, 3 Intermountain Healthcare, Pulmonary Critical Care Research, Murray, United States, 4 University of Utah, Surgery, Division of Emergency Medicine, Salt Lake City, United States, 5 Stanford University, Emergency Medicine, Palo Alto, United States

Background: Syncope occurs in 1 in 4 people during their lifetime and accounts for a reported 1‐1.5% of all emergency department (ED) visits. Most causes of syncope are benign. Less than 2% of the time causes can be life threatening such as cardiac arrhythmias. Another recognized life‐threatening cause is pulmonary embolism (PE). A recent publication reported the prevalence of PE in patients with syncope to be over 20%.

Aims: We sought to determine the prevalence of PE in patients presenting to the ED with syncope in our large integrated healthcare system.

Methods: We performed a retrospective 180 day, longitudinal study of patients who presented with syncope (ICD‐9: 780.2, ICD‐10: R55) to emergency departments within a 21‐hospital integrated healthcare system from 2010 to 2015 to find the incidence of PE in patients with syncope at index ED visit and within 180 days. For patients with multiple ED admissions in the study period, we considered the first episode to be the index encounter.

Results: We screened 2,749,371 ED encounters to find 32,452 (1.2%) with syncope. Median age was 52 (IQR: 31‐71), 57.5% were female, and 90% were Caucasian. PE was diagnosed on the index ED encounter in 259 (0.8%, 95% CI=0.5‐1.2%), 5,091 (15.7%) had a d‐dimer, and 2,338 (7.2%) had a CTPA. At 180 days following the index encounter, 465 (1.4%, 95% CI=1.1‐1.9%) patients were subsequently diagnosed with a PE and 1,051 (3.2%, 95% CI=2.7‐3.9%) patients died.

Conclusions: An acute PE diagnosis at the index ED encounter and within 180 days of presenting to the ED with syncope is rare across a large integrated healthcare system.

PB692: Efficacy and Safety of Rivaroxaban in Morbid Obesity: A Retrospective Analysis of over 600 Patients with BMI ≥40

D. Rao 1 ; S.S. Tolu 1 ; Y. Choi 1 ; J. Gao 2 ; H.H. Billett 1 ; M. Kushnir 1

1 Montefiore Medical Center of the Albert Einstein College of Medicine, Bronx, United States, 2 Albert Einstein College of Medicine, Bronx, United States

Background: Venous thromboembolism (VTE) and atrial fibrillation (AF) are the most common indications for anticoagulation. Although rivaroxaban is an approved treatment option, pivotal studies that demonstrated its efficacy and safety had limited representation of patients with BMI ≥40, raising concern about its use in this population.

Aims: To assess clinical outcomes of VTE recurrence, stroke and bleeding in morbidly obese patients on rivaroxaban compared to warfarin.

Methods: All adult patients at Montefiore Medical Center with BMI ≥40, initiated on anticoagulation with rivaroxaban or warfarin, between March 1, 2013 and March 1, 2017, were identified using institutional data‐mining software. 942 charts were assessed for eligibility. Selection was performed as shown in Fig 1. Demographic information, recurrent thrombotic and bleeding events while on anticoagulation were recorded. Summary statistics and chi‐square tests were used to assess statistical significance of the differences between the groups.

Results: Data on 659 patients, with a mean age of 59.7 and a mean BMI of 47.3 were collected. Mean follow‐up time was 413 days. Among patients with history of VTE, 2.6% (4/153) of patients on rivaroxaban had recurrent VTE while 1.7% (3/177) had recurrent VTE on warfarin (P=0.56). In patients with atrial fibrillation, CVA rate was 2.8% (5/176) on rivaroxaban, compared to 1.2% (2/162) on warfarin (P=0.3). Major bleeding occurred in 2.1% (7/328) of patients on rivaroxaban and 3.6% (12/331) on warfarin (P=0.25). In a subgroup of patients with BMI ≥50, none had VTE recurrence or stroke on rivaroxaban but 3.6% (2/56) of patients on warfarin had VTE and 2.1% had CVA (1/47).

Conclusions: Although the study was retrospective, the relatively low rates of CVA and recurrent VTE in this large population of morbidly obese patients on rivaroxaban are reassuring.

FIGURE 1 Patient selection

TABLE 1 Results Rivaroxaban n (%) Warfarin n (%) Total n (%) P‐value BMI ≥40 328 (49.8) 331 (50.2) 659 Stroke in patients with AF (n=338) 5/176 (2.8) 2/162 (1.2) 7/338 (2.1) 0.30 Recurrent VTE in patients with prior VTE (n=330) 4/153 (2.6) 3/177 (1.7) 7/330 (2.1) 0.56 Major bleeding (n=659) 7/328 (2.1) 12/331 (3.6) 19/659 (2.9) 0.25 BMI ≥50 67 (40.1) 100 (59.9) 167 Stroke in patients with AF (n=84) 0/37 (0) 1/47 (2.1) 1/84 (1.2) <0.1 Recurrent VTE in patients with prior VTE (n=86) 0/30 (0) 2/56 (3.6) 2/86 (2.3) <0.1 Major bleeding (n=167) 2/67 (3) 6/100 (6) 8/167 (4.8) 0.37

PB693: Assessment of D‐dimer Value at First Venous Thromboembolism and Risk of Recurrence

R. Vidal 1 ; D. Velasco‐Rodríguez 1 ; A. Penedo 2 ; A. García‐Raso 1 ; J.M. Alonso‐Domínguez 1 ; P. Llamas Sillero 1

1 Hospital Universitario Fundación Jiménez Díaz, Hematology, Madrid, Spain, 2 Hospital AmpangUniversitario, Hematology, Madrid, Spain

Background: Venous thromboembolism (VTE) has a high rate of recurrence. Duration of anticoagulation remains controversial in certain cases. AAS has been reported to reduce recurrences. Recent evidence suggests that D‐dimer ≤1,500 μg/L at the time of first VTE can predict low risk of recurrence.

Aims: 1) To evaluate the association between D‐dimer at first VTE diagnosis and risk of first recurrence.

2) To analyze whether secondary prevention with AAS reduced recurrences.

Methods: 557 patients with a first VTE diagnosed between 2006 and 2016 were evaluated. Patients with missing D‐dimer values at diagnosis and those on indefinite anticoagulation were excluded. Selected patients started AAS after stopping anticoagulation. D‐dimer was assayed with the ACL TOP analyzer (HemosIL DDimer, Werfen). The area under the curve (AUC) of D‐dimer in patients without AAS was calculated. To evaluate the contribution to VTE recurrence of D‐dimer, sex, duration of anticoagulation, and the use of AAS, a multivariate analysis was performed. P Values <0.05 were considered to be statistically significant. All analyses were performed using SPSS version 19.0 for Windows (SPSS, Chicago, IL, USA).

Results: 132 eligible patients: 76 deep vein thrombosis (DVT) (57.6%), 48 pulmonary embolism (PE) (36.4%), 8 unusual site (6%). Median follow‐up 4.5 years. Recurrent VTE: 31 (23.48%). Treated with AAS: 44 (33.33%). D‐dimer had a poor AUC (0.606) to predict VTE recurrence. D‐dimer ≤1,500 μg/L was associated with significant lower risk of recurrence (P=0.049). Lower risk of recurrence was seen in DVT patients on AAS (P=0.028), but this result was not reproduced in PE. A lower risk of recurrence was seen in PE patients on anticoagulation for at least 1 year (P=0.036), not reproduced in DVT patients.

Conclusions: Low D‐dimer levels at diagnosis of first VTE seem to predict a lower risk of recurrence. AAS reduced risk of recurrence in DVT but not in PE patients. VTE recurrence was lower in PE patients on anticoagulation for at least 1 year.

PB694: Patients Experience of Living with Cancer Associated Thrombosis in Canada (PELICANADA)

S. Noble 1 ; A. Nelson 1 ; J. Scott 2 ; A. Berger 2 ; K. Schmidt 2 ; H. Prout 1 ; P. Gee 1 ; A. Lee 2

1 Cardiff University, Cardiff, United Kingdom, 2 University British Columbia, Vancouver, Canada

Background: Previous research from the UK and Spain has identified several areas of unmet clinical and support need for cancer patients diagnosed with venous thromboembolism (VTE). It is not known whether such experiences are restricted to those countries healthcare systems and culture.

Aims: To evaluate patients’ experience of cancer‐associated thrombosis (CAT) within a Canadian setting.

Methods: Following ethical approval, twenty consecutive patients attending a regional thrombosis clinic in Vancouver consented to be interviewed about their experiences of living with CAT. Semi structured interviews were audio recorded and transcribed. Transcripts were coded using Invivo software. Analysis was undertaken using an applied framework matrix with an inductive approach to identify any new themes. This was in order to explicate potential cultural and operational differences that were not apparent in the UK and Spanish datasets.

Results: Twenty patients (10 male, 10 female) aged 39‐74 (mean 63) participated. Commonalities between the UK and Canadian patients included the traumatic nature of experiencing CAT, the need for information and adaptive behaviors through ritualization. Three new themes were identified:

1. Patients with incidental thrombosis were usually informed of the diagnosis over the telephone.

2. Early referral to specialist services allowed fast access to information and support, thereby reducing distress.

3. Cost implications of accessing low molecular weight heparin (LMWH) varied according to insurance cover.

Patients were sometimes converted to warfarin for financial reasons.

Conclusions: Many experiences of CAT are similar across countries. However, PELICANADA offers several new insights. The distress associated with CAT is a common experience but may be reduced with early access to specialist services, information and support. Improving information giving to patients with incidental VTE will minimize avoidable distress.

PB695: A Systematic Review to Determine Patient Preferences to Receive Thromboprophylaxis to Prevent Deep Vein Thrombosis and Pulmonary Embolism

N. Dembrey ; M. Grainge

The University of Nottingham, Division of Epidemiology and Public Health, Nottingham, United Kingdom

Background: Pharmacological thromboprophylaxis reduces the risk of venous thromboembolism (VTE) but may be considered undesirable due to the increased risk of bleeding and the burden of therapy. International guidelines frequently advise on risk stratification for receipt of prophylaxis among high risk groups (pregnant women, patients with cancer, orthopaedic patients and patients admitted to hospital). These guidelines however have tended to overlook the importance of patient choice and systematic reviews summarising the research evidence on this topic are lacking.

Aims: The primary aim of this review was to assess how the balance of the risks of clotting and bleeding influence a patient's decision to receive prophylaxis. The secondary aim was to assess how additional factors influence this trade‐off between risk and harm.

Methods: A systematic search of the current literature was performed using Medline, CINAHL, psycINFO and Google scholar. We included studies focussing on pregnancy/child birth, patients admitted to hospital (including medical, surgical and orthopaedic) and patients with cancer. All studies were quality appraised and results synthesised narratively.

Results: Ten studies were included in this review. There was high heterogeneity within studies and between studies in terms of participants and study design. Two studies on the same patient group comprising pregnant women with a previous VTE provided the highest quality data (See Table 1). Across patient groups participants tended to perceive pulmonary embolism (PE) as a more detrimental outcome than serious bleeding and participants preferred an oral route of administration where possible.

TABLE 1 Summary of results by patient group Patient group Number of Studies Summary of results Pregnant women 3 From a probability trade‐off it was reported that women were willing to accept prophylaxis at a median 3% reduction in VTE based on a risk of 16% without prophylaxis. Out of 72 women for whom a mathematical predictive model (assessing harms vs. risks) recommended against prophylaxis, 72% chose to receive this in a discrete choice exercise using the same input values. Patients with cancer 2 Palliative care (PC) cancer patients perceived thromboprophylaxis to be much more acceptable than ambulatory cancer patients. Overall, PC patients considered this a trivial intervention with the perceived benefits far outweighing the harms. General Medical/Surgical patients 2 The mode of administration was a highly influential factor with oral methods of thromboprophylaxis favoured over injectable. Younger participants tended to have a more favourable opinion of injectable thromboprophylaxis than older patients. Orthopaedic patients 3 Participants tended to perceive VTE as a more detrimental health outcome than serious bleeding. Participants showed a preference for oral prophylaxis.

Conclusions: There is currently not a wide enough evidence base for the preferences of these patient groups to be incorporated into clinical guidelines.

PB696: Efficacy and Safety of Rivaroxaban and Apixaban in Relation to Low Molecular Heparin and Vitamin K Antagonists in Acute Venous Thromboembolism – A Single Center Prospective Study

W. Wysokinski 1 ; D. Bott‐Kitslaar 2 ; A. Casanegra 1 ; D. Froehling 2 ; M. Bartlett 3 ; C. Lenz 2 ; D. Houghton 2 ; D. Hodge 4 ; E. Vargas 4 ; R. McBane 2

1 Mayo Clinic, Cardiovascular Diseases, Rochester, United States, 2 Mayo Clinic, Rochester, United States, 3 Internal Medicine, Rochester, United States, 4 Mayo Clinic Florida, Jacksonville, United States

Background: Data outside of randomized controlled trials comparing outcomes of apixaban and rivaroxaban to vitamin K antagonist (VKA) or low molecular weight heparin (LMWH) in patients with acute venous thromboembolism (VTE) is limited.

Aims: To compare VTE recurrence, major bleeding, and clinically relevant non‐major bleeding (CRNMB) rates between rivaroxaban, apixaban, VKA and LMWH therapies for acute VTE in a standardized, evidence‐based clinical practice with anticoagulant selection based on patient characteristics.

Methods: Consecutive patients enrolled in the Mayo Thrombophilia Clinic Anticoagulants Registry between March 1, 2013 and January 30, 2018 with VTE were followed prospectively. Patient status was assessed in person, by mailing a written questionnaire, or by a scripted phone interview.

Results: Among 1,605 patients with VTE in the registry, 896 had acute disease treated with study anticoagulant within the first 14 days of diagnosis, and for at least 3 months or until a study event. There were 293 patients treated with rivaroxaban, 281 with apixaban, 251 with LMWH, and 71 patients receiving VKA. Patients treated with rivaroxaban were younger than patients receiving apixaban, or LMWH. The prevalence of cancer was highest in LMWH group and lowest in VKA group (Table 1). Treatment with rivaroxaban was associated with a lower rate of VTE recurrence compared to the LMWH and VKA group (Table 2). The rate of major bleeding was not different amongst groups but CRNMB rate was lower in apixaban group compared to rivaroxaban. Higher mortality rate in LMWH treated patients compared to apixaban and to rivaroxaban group reflected higher proportion of patients with cancer.

TABLE 1 Patient characteristics Apixaban (N=281) Rivaroxaban (N=293) LMWH (N=251) VKA (N=71) Apixaban/ Rivaroxaban P‐value Apixaban/ LMWH P‐value Apixaban/ VKA P‐value Rivaroxaban/ LMWH P‐value Rivaroxaban/ VKA P‐value Age, mean (SD) 63.0 (14.1) 58.3 (14.2) 62.5 (12.9) 62.5 (14.5) <0.001 0.71 0.79 <0.001 0.029 Female, n (%) 110 (39.1) 141 (48.1) 101 (40.2) 28 (39.4) 0.030 0.80 0.96 0.065 0.19 Cancer, n (%) 135 (48.0) 120 (41.0) 214 (85.3) 18 (25.4) 0.088 <0.001 <0.001 <0.001 0.015

TABLE 2 Clinical outcome VTE Recurrence Major bleeding CRNMB Death Apixaban N events/rate 100 person‐ys 7/5.05 10/7.10 3/2.12 17/11.98 Rivaroxaban N events/rate 100 person‐ys 4/1.96 9/4.45 23/12.01 28/13.66 LMWH N events/rate 100 person‐ys 10/7.63 10/7.66 8/6.06 47/35.2 VKA N events/rate 100 person‐ys 5/15.08 5/15.02 0/0 4/11.94 Apixavan/Rivaroxaban HR (95% CI) 2.02 (0.59,6.93) 1.27 (0.51, 3.12) 0.14 (0.04, 0.47) 0.90 (0.49, 1.67) Apixaban/LMWH HR (95% CI) 0.61 (0.23, 1.60) 0.88 (0.37, 2.12) 0.34 (0.09, 1.29) 0.32 (0.19, 0.56) Apixaban/VKA HR (95% CI) 0.34 (0.11, 1.08) 0.45 (0.15, 1.33) N/A 0.93 (0.31, 2.77) Rivaroxaban/LMWH HR (95% CI) 0.29 (0.09, 0.92) 0.71 (0.29, 1.76) 2.31 (1.03, 5.16) 0.39 (0.24, 0.62) Rivaroxaban/VKA HR (95% CI) 0.16 (0.04, 0.62) 0.39 (0.13, 1.16) N/A 0.97 (0.34, 2.80)

Conclusions: In a setting of standardized, evidence‐based clinical practice, treatment with rivaroxaban is associated with the lower rate of VTE recurrence compared to LMWH and VKA and higher rate of CRNMB compared to apixaban group.

PB697: Circulating Endothelial Cells as a New and Early Candidate Biomarker of Residual Pulmonary Vascular Obstruction after Pulmonary Embolism

B. Planquette 1,2,3 ; N. Gendron 2,3,4 ; J. Peron 5,6 ; A. Roche 1,2,3 ; M. Barritault 7 ; P. Gaussem 2,3,4 ; O. Sanchez 1,2,3 ; D.M. Smadja 2,3,4

1 AP‐HP, Hôpital Européen Georges Pompidou, Pneumology Department, Paris, France, 2 Université Paris Descartes, Sorbonne Paris Cité, Paris, France, 3 Inserm UMR‐S 1140, Paris, France, 4 AP‐HP, Hôpital Européen Georges Pompidou, Hematology Department, Paris, France, 5 Centre Hospitalier Lyon Sud, Institut de Cancérologie des Hospices Civils de Lyon, Pierre‐Bénite, Medical Oncology Department, Lyon, France, 6 CNRS, UMR 5558, Laboratoire de Biométrie et Biologie Evolutive, Equipe Biostatistique‐Santé, Université Lyon 1, Villeurbanne, France, 7 Groupe Hospitalier Est, Hospices Civils de Lyon, Department of Molecular Biology, Lyon, France

Background: Pulmonary embolism (PE) is a frequent and life‐threatening disease. Third to half of patient's present residual pulmonary vascular obstruction (RPVO) that is now considered as a PE chronic complication.

Aims: The main objective of the present study is to evaluate the prognosis value of CEC in patients with a first acute PE.

Methods: This study was a single‐center, prospective, observational cohort study. Consecutive patients aged over 18 years who present a first documented episode of acute PE confirmed by ventilation perfusion scintigraphy (V/Q lung scan) confirmed by (1) spiral computed tomography showing at least multiple sub‐segmental PE, or (2) high probability V/Q lung scan according to the PIOPED criteria, or (3) proximal lower limb DVT on compression ultrasonography. CECs were isolated by immunomagnetic separation as previously described. CEC levels were performed at basal evaluation at day 0 and during follow‐up and at 6 months for PE patients and only once for controls and CTEPH patients.

Results: From January 2014 to May 2015, 56 PE patients were analyzed in the present study. At total, twenty healthy volunteers and nine CTEPH patients were sampled. At PE diagnosis, the mean CEC count in PE patients was 16.54 ± 35 CEC/mL. In healthy volunteers, mean CEC count was lower than in PE patients: 3.17 ± 2.6 CEC/mL (P=0.043). CEC count<5 CEC/mL at PE diagnosis was associated with increased RPVO in univariate analysis (P=0.037). The association remained significant after adjustment on the level initial vascular obstruction (P=0.041). At the moment of RPVO assessment, CEC count<5 CEC/mL was not any more associated with increased RPVO. Furthermore, RPVO was not predicted by neither increase nor decrease in CEC count from diagnosis to moment of treatment withdrawal.

Conclusions: At PE diagnosis a cutoff of 5 CEC/mL could predict PE chronic complications, in particular RPVO.

PB698: Thrombophlebitis Post Varicose Vein Surgery: To Treat or Not to Treat?

H.Y. Lim 1 ; I. Bayat 2 ; P. Ho 1 ; F. Hong 1

1 Northern Health, Haematology, Epping, Australia, 2 Northern Health, Vascular Surgery, Epping, Australia

Background: Great saphenous vein (GSV) incompetence is the most common cause of chronic venous insufficiency. Post‐operatively, patients may report limb oedema, lump and/or pain and present to their local general practitioners (GPs) or emergency departments. In our practice, we noted that there is significant heterogeneity in the management of these patients.

Aims: To determine the management approach of clinicians to patients suffering from limb oedema and/or pain following varicose vein surgery.

Methods: A case study of a patient presenting with left leg oedema and pain 6 days following varicose vein surgery (left saphenofemoral junction ligation, GSV stripping and stab avulsions) with a Doppler ultrasound reported as extensive superficial thrombophlebitis throughout the course of left GSV was sent as a survey to GPs, emergency medicine physicians and haematologists (including registrars). The survey was conducted both electronically using a survey platform and by paper. The clinicians were requested to choose from 5 possible management options, from no anticoagulation to full anticoagulation.

Results: There were 115 responses from 52 GPs (45%), 31 emergency medicine physicians (27%) and 32 haematologists (28%). There is marked heterogeneity in the responses: 26 (23%) chose prophylactic anticoagulation, 30 (26%) full anticoagulation, 8 (7%) aspirin while 32 (including 5 haematologists) chose no anticoagulation (28%) and the remaining 19 (17%) were unsure.

Conclusions: Limited superficial phlebitis occurring in the operated vein or the associated trajectory soon after varicose vein surgery is a common (and often normal) post‐operative phenomenon. There is usually no role for anticoagulation unless there is evidence of deep venous thrombosis. Our survey showed that this is not well known in the medical community, which leads to unnecessary treatment with anticoagulation (50% in our survey). Further education and awareness amongst clinicians is required for patient safety.

PB699: Comparison of Risk Assessment Methods to Prevent Venous Thromboembolism (VTE) in Surgical Patients with Short Length of Hospital Stay

M.C. Chindamo 1,2 ; G.G. Sardinha 3 ; D. Mousinho 4 ; W. Schiavini 4 ; A.B. Nunes 4

1 Hospital Barra D'Or, Internal Medicine, Rio de Janeiro, Brazil, 2 Universidade Federal do Rio de Janeiro, internal Medicine, Rio de Janeiro, Brazil, 3 Hospital Barra D'or, Pharmacy Department, Rio de Janeiro, Brazil, 4 Hospital Barra D'Or, Quality Team, Rio de Janeiro, Brazil

Background: Surgical procedures represent an important risk factor for VTE. Despite the availability of VTE risk assessment tools and prevention guidelines, adherence to pharmacological prophylaxis is suboptimal in the setting of early postoperative discharge.

Aims: To compare the performance of Pannucci score versus 9th ACCP guideline and Caprini score (used as reference tools) to predict VTE risk in patients discharged within 24 hours after surgery.

Methods: We included patients eligible for pharmacological prophylaxis according to 9th ACCP and Caprini score (intermediate or high risk), discharged within 24 hours after surgery without prophylaxis prescription. These cases were reclassified using Pannucci score as very high, high, intermediate or low risk for VTE. Pharmacological prophylaxis was indicated for very high risk score. Symptomatic VTE events were assessed by a phone call interview within 90 days after surgery.

Results: 175 patients nonadherent to pharmacological prophylaxis were included: mean age 51 years, 58% female, mean surgical time ‐102 minutes. Surgical procedures were as follows: 27% no major orthopedic surgery, 26% videolaparoscopy, 19% head and neck, 10% gynecological surgery. VTE risk according to 9th ACCP, Caprini and Pannucci scores was respectively: low – 6 (3.4%), 3 (1.8%) and 128 (73.2%); intermediate – 97 (55.4%), 146 (83.4%) and 34 (19.4%); high risk‐ 72 (41.2%), 26 (14.8%) and 11 (6.3%); very high (only for Pannucci score)‐ 2 (1.1%). Indication of pharmacological prophylaxis according to 9th ACCP and Caprini score (intermediate and high risk) was as follows: 169 (96%) and 172 (98%); Pannucci score – 2 (1.2%). Two VTE events (1.1%) occurred during follow‐up (1 cholecystectomy and 1 knee arthroscopy).

Conclusions: 9th ACCP guideline and Caprini score can lead to an overestimation of VTE risk in surgical patients with very short length of hospital stay. It is necessary to review VTE risk assessment tools in early discharged surgical patients for an adequate indication of pharmacological prophylaxis.

PB700: Peripherally Inserted Central Catheter Associated Upper Extremity Thrombosis (PICC‐UET): A Single Centre Retrospective Study

C. Delaney ; S. Kelliher; K. Saeed; B. Kevane; A. Fortune; M. Fay; F. Ni Ainle

Mater Misericordiae University Hospital, Haematology, Dublin, Ireland

Background: Peripherally Inserted Central Catheter‐Associated Upper Extremity Thrombosis (PICC‐UET) is more common in cancer than non‐cancer patients. Predictors of PICC‐UET in this group are not well established. The Michigan Risk Score was developed to estimate the risk of PICC‐UET and internally validated in medical patients admitted to general wards or ICU settings.

Aims: In this retrospective cohort study we aimed to assess the risk of PICC‐UET in adult haematology patients who underwent PICC insertion. We also aimed to assess whether the recently published Michigan Risk Score (MRS) might be a useful risk stratification tool in our patient cohort.

Methods: Electronic database search revealed 275 PICC insertions between 17/05/2012 and 16/01/2018. Subsequent upper extremity ultrasound scan reports were assessed for evidence of PICC‐UET. Association between PICC‐UET and age, gender, haematologic diagnosis, white cell count, and Michigan Risk Score was assessed.

Results: Of 275 PICC placements, 22 (8%) resulted in symptomatic PICC associated DVT. All episodes of PICC‐UET occurred in patients with a MRS of ≥3. One of the MRS components is a WCC >12 × 10 9 /L. Of 43 patients with white cell count (WCC)>12 × 10 9 /L at PICC insertion, 6 (14%) developed PICC‐UET. This compared with 16 of 232 patients (6.9%) with WCC<12 × 10 9 /L (OR 2.189; 95% CI: 0.805‐5.957).

Conclusions: A high risk of PICC‐UET was observed in our group compared with that observed on internal validation of the MRS in a general medical/intensive care unit population. All episodes of PICC‐UET occurred in patients with lymphoma or acute leukaemia. PICC insertion risk may be more acceptable in patients with myeloma, as a lower PICC‐UET rate was observed in this group, although this may reflect specific thromboprophylaxis measures. WCC>12 × 10^9/L may be a useful predictor of PICC‐UET. The MRS underestimates risk in this patient cohort but does show a trend towards definition of a very high risk patient subset.

PB701: Effectiveness and Safety of Outpatient Rivaroxaban versus Warfarin in Patients with Provoked Acute Venous Thromboembolism

C.I. Coleman 1 ; T.J. Bunz 2 ; J. Jan Beyer‐Westendorf 3

1 University of Connecticut, Suffield, United States, 2 New England Health Analytics LLC, Pharmacoepidemiology, Granby, United States, 3 University Hospital ‘Carl Gustav Carus’ Dresden, Thrombosis Research Unit, Department of Medicine I, Division Hematology, Dresden, Germany

Background: Patients with provoked or unprovoked venous thromboembolism (VTE) may respond differently to oral anticoagulation due to varying thromboembolic and bleeding risk profiles.

Aims: To compare the effectiveness and safety of outpatient rivaroxaban and warfarin in patients with provoked acute VTE.

Methods: Using US MarketScan claims data from 1/2012 to 12/2016, we identified adults with a primary diagnosis of VTE and a provoking risk factor, newly‐initiated on rivaroxaban or warfarin within 30‐days of the index VTE and ≥12‐months of insurance benefits prior to the index event (baseline). Provoking factors included cancer, hospital admission for ≥3‐consecutive days over the prior 3‐months, major surgery, fracture or trauma within 90‐days or pregnancy within 42‐weeks of the incident VTE. Differences in baseline covariates between cohorts were adjusted for using inverse probability of treatment weights based on propensity‐scores (residual standardized differences<0.1 achieved for all covariates). Cox regression was used to compare the incidence of a composite endpoint of recurrent VTE or major bleeding at 3‐ and 6‐months post‐VTE using an intention‐to‐treat approach.

Results: In total, 4,454 rivaroxaban and 13,164 warfarin users with provoked VTE were included. Median age was 58 (48, 68) years, 49% were men and 44% had pulmonary embolism. Common provoking factors (not mutually exclusive) included recent hospitalization (46%), cancer (29%), major surgery (19%) and fracture (11%). At 3‐ and 6‐months of follow‐up, rivaroxaban use was associated with a reduced hazard of the composite endpoint and recurrent VTE alone versus warfarin (Table). Major bleeding was numerically less frequent at 3‐months and significantly reduced at 6‐months with rivaroxaban versus warfarin.

TABLE 1 Comparison of IPTW‐weighed cohorts of Rivaroxaban and Warfarin in provoked venous thromboembolism patients Endpoint Rivaroxaban N=4,454 Warfarin N=13,164 HR (95% CI) 3‐months Composite 3.05% 4.32% 0.72 (0.61‐0.84) Recurrent VTE 2.30% 3.33% 0.70 (0.59‐0.84) Major Bleeding 0.77% 1.02% 0.77 (0.57‐1.06) 6‐months Composite 3.56% 5.21% 0.69 (0.60‐0.80) Recurrent VTE 2.56% 3.66% 0.71 (0.60‐0.84) Major Bleeding 1.07% 1.62% 0.68 (0.53‐0.88) CI, confidence interval; HR, hazard ratio; n, number; N, number.

Conclusions: Our study suggests that compared to warfarin, rivaroxaban has the potential to reduce the risk of recurrent VTE and major bleeding when used to treat acute provoked VTE in routine practice.

PB702: Rivaroxaban and Apixaban for Treatment of Acute Isolated Calf Deep Vein Thrombosis – A Single Center Prospective Study

R. McBane 1 ; D. Bott‐Kitslaar 1 ; A. Casanegra 1 ; D. Froehling 1 ; M. Bartlett 1 ; C. Lenz 1 ; D. Houghton 1 ; D. Hodge 2 ; E. Vargas 2 ; W. Wysokinski 1

1 Mayo Clinic Rochester, Rochester, United States, 2 Mayo Clinic Florida, Jacksonville, United States

Background: Guideline recommendations for managing isolated distal deep vein thrombosis (dDVT) include a preference for serial imaging for 2 weeks over anticoagulation to avoid excess bleeding. The low bleeding rates with direct oral anticoagulants (DOACs) however bring into question this recommendation. Moreover, dDVT excluded from randomized controlled trials of DOACs.

Aims: Venous thromboembolism (VTE) recurrence, major bleeding, and clinically relevant non‐major bleeding (CRNMB) rates in patients with dDVT treated with rivaroxaban or apixaban were compared to patients with proximal DVT (pDVT) with or without distal extension.

Methods: Consecutive patients with acute VTE enrolled in the Mayo Thrombophilia Clinic Anticoagulants Registry (3/1/2013‐1/30/2018) were followed forward in time. Patient status was assessed in person, by mailing a written questionnaire, or by a scripted phone interview. Patients were included if the had study anticoagulants started within the first 14 days of diagnosis and completed at least 3 months of therapy.

Results: Among 1,605 VTE patients, 149 patients had isolated dDVT (mean age 60±14; 46% women; 30% with cancer) treated with rivaroxaban (n=75) or apixaban (n=74). Outcomes were compared to 213 patients with pDVT (mean age 61±14; 41% women; 37% with cancer) treated with rivaroxaban (n=119) and apixaban (n=94). VTE recurrence, major bleeding, CRNMB, and death (per 100 person‐years) were similar between groups or treatment allocation (Table).

Outcomes DOACs dDVT (n=149) DOACs pDVT (n=213) P‐value Apixaban dDVT (n=74) Apixaban pDVT (n=94) P‐value Rivaroxaban dDVT (n=75) Rivaroxaban pDVT (n=119) P‐value VTE recurrence Rate/100 person‐years 1.41 2.01 0.60 3.22 3.93 0.85 0.0 1.01 0.43 VTE recurrence, n 1 3 1 2 0 1 Major bleeding Rate/100 person‐years 4.25 4.69 0.66 6.47 5.74 0.97 2.52 4.12 0.52 Major bleed, n 3 7 2 3 1 4 CRNMB Rate/100 person‐years 7.29 7.56 0.59 0 3.85 0.30 13.32 9.63 0.98 CRMB; n 5 11 0 2 5 9 Death rate/100 person‐years 9.90 8.58 0.67 9.66 5.72 0.49 10.1 10.1 0.89 Deaths; n 7 13 3 3 4 10

Conclusions: In the era therapy, clinical outcomes did not differ by thrombus proximity. These data do not support the opinion that dDVT are a benign entity.

PB703: Simulation in VTE Treatment: Improving the Use of Guideline‐directed Care

J. Spyropoulos ; M. Warters; D. Blevins

Medsacape LLC, New York, United States

Background: Guideline‐recommended anticoagulant therapy is widely underused in the setting of venous thromboembolism (VTE).

Aims: To determine if an online, virtual patient simulation (VPS)‐based continuing medical education (CME) intervention could improve performance of hematologists/oncologists (hem/onc) and cardiologists in managing patients with VTE.

Methods: The CME intervention consisted of two cases presented in a VPS platform. Clinical decisions made by the learners using open field entries within an EHR interface were analyzed using an artificial intelligence engine and, after each decision, tailored clinical guidance (CG) was provided based on current recommendation. Learner decisions were collected post‐CG and compared with each user's baseline (pre‐CG) data using a 2‐tailed paired T‐test to determine P values.

Results: Significant absolute improvements were observed:

Case 1 – patient with newly‐diagnosed PE (n=84 hem/onc; n=123 cardiologists):•Order coagulation studies: 16% improvement among hem/onc (65% pre‐CG vs. 81% post‐CG; P=0.01) and 12% improvement among cardiologists (55% pre‐CG vs. 67% post‐CG; P=0.026)•Order CrCl: 26% improvement among hem/onc (38% pre‐CG vs. 64% post‐CG; P<0.001) and 20% improvement among cardiologists (34% vs. 54%; P<0.001)•Initiate appropriate anticoagulant therapy: 42% improvement among cardiologists (23% pre‐CG vs. 25% post‐CG; P<0.001) and 40% post‐CG (33% pre‐CG vs. 73% post‐CG; P<0.001)

Case 2 – patient with VTE and cancer (n=54 hem/onc; n=94 cardiologists):•Discontinue LMWH: 20% improvement among hem/onc (36% pre‐CG vs. 56% post‐CG; P=0.015) and 15% improvement among cardiologists (38% pre‐CG vs. 54% post‐CG; P=0.014)•Initiate NOAC therapy: 35% among hem/onc (38% pre‐CG vs. 7% post‐CG; P<0.001) and 34% improvement among cardiologists (42% pre‐CG vs. 76% post‐CG; P<0.001)

Conclusions: This study demonstrated that VPS‐based CME can improve evidence‐based practices of cardiologists and hem/oncs, and suggests that this type of intervention can improve outcomes for patients with VTE.

PB704: The Relationship between Neutrophil Lymphocyte Ratio and Deep Vein Thrombosis in Unilateral Limb Edema in Suspected Deep Vein Thrombosis Patients

I. Rinaldi 1 ; R. Hamonangan 2 ; R. Cahyanur 2 ; M.S. Azizi 2 ; A.I. Fatya 3 ; F. Wirawan 3

1 Dr. Cipto Mangunkusumo National General Hospital, Hematology Oncology Division, Internal Medicine Department, Jakarta, Indonesia, 2 Dr. Cipto Mangunkusumo National General Hospital, Cardiology Division, Internal Medicine Department, Jakarta, Indonesia, 3 Dr. Cipto Mangunkusumo National General Hospital, Jakarta, Indonesia

Background: In deep vein thrombosis (DVT), neutrophil undergo adhesion to the blood vessel wall and furthermore induce thrombosis. Neutrophil is suspected to have pro‐ and anti‐ fibrinolysis effect. In line with D‐Dimer, a marker of thrombosis and fibrinolysis, the possibility of neutrophil to be the marker of DVT through the studied neutrophil lymphocyte ratios (NLR) can predict the extent of DVT anatomy and treatment response.

Aims: To discover the relationship between NLR and DVT.

Methods: The study was conducted in cross‐sectional to unilateral limb edema patients suspected with DVT who had undergone Doppler Ultrasonography (USG) through January‐December 2014 in Dr. Cipto Mangunkusumo Hospital. The study was done by recording medical records data such as age, gender, anamnesis and physical examination associated with Wells score, laboratory findings of hemoglobin, hematocrit, leukocyte, thrombocyte, basophil, eosinophil, neutrophil, lymphocyte, monocyte, D‐Dimer, and Doppler Ultrasonography. Statistical analysis was performed with unpaired t test, followed by ROC analysis and determined the optimal cut‐off point of NLR.

Results: The subjects of this study were 118 patients, 50% of which are men, with median age 54.78. Most of the subjects were included in the clinically likely criteria or probable DVT criteria, which are 96.6% (moderate and high pretest probability), with the most comorbidities were cancer 29.7%. This study had found a statistically significant relationship between NLR and DVT with P<0.05 and CI 95% 1.83 (1.34‐2.48). Through the ROC curve, the value of AUC 72.6% was obtained with CI 95% (0.634‐0.818), and the optimal cut‐off point of NLR ≥5.1246 with 67.7% sensitivity and 67.9% specificity.

Conclusions: There is a statistically significant relationship between NLR and DVT and the optimal cut‐off point of NLR ≥5.1246 could determine the presence of DVT with sensitivity of 67.7% and specificity of 67.9%. These results may need to be combined with other existing devices to diagnose DVT.

PB705: Application of an Explanatory Model for Emergency Physician Pulmonary Embolism Testing in a National Cohort

K. deWit; S. Zarabi ; M. Mercuri; T. Chan

McMaster University, Hamilton, Canada

Background: Pulmonary embolism (PE) can be excluded without need for diagnostic testing with clinical probability and D‐dimer blood in low risk patients. The Choosing Wisely Campaign has promoted this approach to rationalize CT scanning for PE in the emergency department. In preparation for an implementation study, we recently developed a theoretical model to explain emergency physician PE testing choices in three hospitals of a Canadian city.

Aims: To test whether this theoretical model can explain PE testing behaviour of emergency physicians across Canada.

Methods: The theoretical model was derived from interviews with 16 emergency physicians from 3 emergency departments in Hamilton, Canada. The model includes environmental influences, Gestalt overestimation of pretest probability, confidence in each test and patient expectations. We conducted interviews with Canadian emergency physicians from 4 cities in 3 provinces. The interviews consisted of

1). recalling a recent patient tested for PE,2). mind mapping to describe testing for PE after watching a simulated video and3). a knowledge test on tests for PE diagnosis.

The transcribed interviews and mind maps (30 to date, estimated sample=50) are undergoing constant comparative framework analysis by two researchers, to map findings from the Canada‐wide interviews to our existing framework. The results will be available in June 2018.

Results: Preliminary results show common themes in PE testing across Canada, including use of Gestalt, over‐estimation of Gestalt risk for PE, concern when no other diagnosis is apparent (despite low risk for PE), parallel testing for multiple pathologies, variable confidence in rule‐out tests, time restrictions around testing and patient preference for CT.

Conclusions: This is the first study to map the barriers and opportunities for evidence‐based PE testing in the emergency department. The results will enable creation of an evidence‐based implementation method for future study across Canada.

PB706: Deep Dive into DVTs: A 3 Year Retrospective Review of Patients with Deep Vein Thrombosis (DVT) on an Ambulatory Integrated Care Pathway (ICP)

V. Graham 1 ; B. White 1 ; K. Ryan 1 ; J.S. O'Donnell 1,2 ; M. Lavin 1,2 ; M.P. Colgan 3 ; G. McMahon 4 ; N. O'Connell 1

1 St Jawmes's Hospital, National Coagulation Centre (NCC), Dublin, Ireland, 2 Royal College of Surgeons in Ireland, Irish Centre for Vascular Biology, Dublin, Ireland, 3 St James's Hospital, Vascular Lab, Dublin, Ireland, 4 St James's Hospital, Emergency Department, Dublin, Ireland

Background: The ICP for ambulatory management of DVT and thrombophlebitis was devised in 2010 as a collaborative initiative between the Emergency department, the Vascular lab and the National Coagulation Centre (NCC). Following an initial pilot, full implementation of the pathway was undertaken in 2011 and from 2011 to 2017, 384 patients have been managed on this pathway. The most recent service development has been the introduction of DOAC starter packs.

Aims: To confirm the safety and efficacy of the ambulatory ICP for DVT.

Methods: A retrospective review of Doppler scan reports and clinical notes was conducted for all patients on the ICP for DVT Ambulatory care between 2015 and 2017. Data collected included patient demographics, location and extent of thrombosis, PTS symptoms and recurrence.

Results: 196 patients were identified, of whom 124 had DVT and 72 had thrombophlebitis. The VTE was unprovoked in the majority of patients (61%). 43 patients had a prior history of VTE. A total of 9 out of 196 patients (5%) had a recurrence after the index event. Nine patients experienced bleeding with two major bleeding events (by ISTH criteria). 25 patients were documented as having PTS symptoms but PTS was not evaluated systematically.

Conclusions: Ambulatory management of acute DVT and thrombophlebitis within a structured ICP is safe and effective as demonstrated by low rates of major bleeding and recurrence.

PB707: The Association of Characteristics of Central Venous Catheters with Venous Thrombotic Events in Pediatric Patients Undergoing Fontan Surgery

V. Kovalik 1 ; A. Hussain 2 ; T. MacDonald 1 ; K. Kulkarni 1

1 IWK Health Centre, Division of Hematology Oncology, Department of Pediatrics, Halifax, Canada, 2 IWK Health Centre, Division of Cardiology, Department of Pediatrics, Halifax, Canada

Background: Fontan surgery (FS) is the palliation for children born with univentricular heart defects. Central venous catheters (CVCs) are frequently placed to facilitate care during and after FS. Although venous thrombotic events (VTE) is a well‐recognized complication in these patients, little is known about its association with CVC characteristics.

Aims: The aim of this study was to assess the association of characteristics of CVCs with VTE in patients who underwent FS.

Methods: A retrospective cohort study was conducted at the IWK Health Centre to review patients who underwent FS between October 2000 and March 2016. After ethics approval, information on demographics, type of FS, presence of thrombosis, number, location and duration of CVC was retrieved. SPSS version 24 was used for statistical analysis.

Results: 64 patients were included in this study. The male to female ratio was 57:43. The mean age at FS of 3.41 years. One, 2 or more than 2 CVCs were required in 22 (42.3%), 25 (48.1%) and 5 (10%) patients respectively. VTE was observed in 19 patients (29.7%). VTE were located in the upper venous system in 6 (31.5%) while intra‐cardiac in 5 (26.3%) of the patients. 12 patients had FS outside of IWK and complete data on CVC was not available. The mean duration of in‐situ CVC was significantly longer in patients with VTE compared to those who did not develop VTE (14±3.8 days vs. 6.3±1.3 days, P=<0.004). VTE was not significantly associated with gender (P=0.786), age (P=0.09), number (P=0.07) and location of CVC (P=0.517) and presence of chylothorax (0.560).

Conclusions: The present study demonstrates that longer duration of in‐ situ CVC is associated development of VTE after FS. Majority of the VTE were located in the upper venous system or intra‐cardiac. Although further confirmation with larger studies adjusting for surgical confounding factors and complications are needed, our data support consideration for early removal of CVCs in patients undergoing Fontan surgery.

PB708: Implementation of an Acute DVT Ambulatory Care Pathway in a Large Urban Centre: Current Challenges and Future Opportunities

S. Kelliher 1 ; B. Kevane 1,2 ; D. Dinu 3 ; F. NiAinle 1,2,4 ; T. Breslin 3

1 Mater Misericordae University Hospital, Haematology, Dublin, Ireland, 2 Rotunda Hospital, Haematology, Dublin, Ireland, 3 Mater Misericordae University Hospital, Emergency Department, Dublin, Ireland, 4 University College Dublin, Conway Institute Sphere Research Group, Dublin, Ireland

Background: Ambulatory management of isolated acute deep venous thrombosis (DVT) is safe and feasible in selected populations. However, in practice a significant number of patients (>50%) continue to be managed as in‐patients, including those potentially suitable for outpatient management.

Aims: To evaluate acute DVT treatment pathways in our emergency department (ED) and to identify barriers to outpatient management.

Methods: This study was an analysis of prospectively collected data pertaining to consecutive patients presenting to the ED of a large city centre teaching hospital in Dublin (Mater Misericordiae University Hospital) between 16/10/2014 and 02/09/2015 who were diagnosed with DVT. Data was recorded at the time of diagnosis in a local hospital database. Variables included age, treatment prescribed, provoking factors, CTPA result, previous VTE history and whether outpatient management was feasible.

Results: 78 patients presented with acute DVT. The majority of patients were male (59%; 46/78). 66.67% (50/78) fulfilled criteria for outpatient care (62% (31/50) were treated with rivaroxaban while the remaining 38% (19/50) were treated with LMWH). Of those that were admitted to hospital, 42.3% (11/26) reported intravenous drug use (IVDU), 42.3% (11/26) had pulmonary embolus and 11.5% (3/26) had significant medical co‐morbidities.

TABLE 1 Patient demographics Demographics N (%) N=241* Age (SD) 58.7 (15.4) Gender (male) 138 (57) Diagnosis Distal DVT 114 (47) Proximal DVT 68 (28) PE 59 (25) Personal history of VTE No history 198 (82) Previous VTE 43 (18) SD, Standard deviation; VTE, Venous Thromboembolism; DVT, Deep Vein Thrombosis; PE, Pulmonary Embolism *18 patients did not answer sufficient questions for ACTS analysis to take place. A total of 223 questionnaires are reported below.

TABLE 2 Summary of the patient‐reported treatment satisfaction results from ACTS from the FIRST Registry All patients who have completed ACTS at 1 month and end of treatment All patients who are on long‐term treatment who have completed ACTS at 1 month and after 1 year Subscale * Participants Median Score (IQR) Median Score (IQR) P value Participants Median Score (IQR) Median Score (IQR) P value n One month End of treatment † n One month One year ‡ Burdens 179 57.0 (53.0‐59.0) 57.0 (54.8‐60.0) <0.05 44 57.0 (55.0‐59.0) 57.0 (54.0‐59.0) NS DVT 144 56.5 (53.0‐59.0) 57.8 (55.0‐60.0) <0.05 25 56.5 (55.8‐58.3) 57.0 (56.0‐59.0) NS PE 35 58.0 (53.3‐59.0) 56.7 (52.0‐58.0) NS 19 57.0 (52.5‐60.0) 57.0 (52.0‐59.0) NS Benefits 178 12.0 (10.0‐14.0) 12.0 (10.0‐14.8) NS 44 12.0 (10.0‐12.0) 12.0 (10.0‐15.0) <0.05 DVT 144 12.0 (10.0‐14.0) 12.0 (10.0‐15.0) NS 25 12.0 (10.0‐12.3) 12.5 (11.3‐15.0) <0.05 PE 34 12.0 (11.0‐15.0) 12.0 (9.5‐14.0) NS 19 12.0 (9.0‐12.5) 12.0 (9.0‐14.0) NS ACTS, Anti‐Clot Treatment Scale; IQR, Interquartile range; DVT, Deep vein thrombosis; PE, Pulmonary embolism. *Higher scores indicate greater treatment satisfaction; ACTS Burdens maximum score 60, ACTS Benefits maximum score 15. †Mean number of days 151 (SD 81.5) for patients completing questionnaire at end of treatment. ‡Mean number of days 460 (SD 125.89) for patients completing questionnaire after 1 year.

Conclusions: Implementation of an outpatient care pathway led to the majority of subjects presenting with DVT being treated without hospital admission. Interestingly, within our cohort (primarily consisting of patients from an urban, inner‐city population), the majority of patients were male and, moreover, IVDU represented a key barrier to successful implementation of outpatient management. Directing resources towards strategies which facilitate outpatient DVT treatment among vulnerable patient groups could represent a means of reducing hospital admissions for DVT in urban centres and, ultimately, lead to health care savings.

PB709: Treatment Satisfaction with Rivaroxaban for the Management of Venous Thromboembolism (VTE) – Early Results from the ‘Follow‐up in Rivaroxaban Patients in Setting of Thromboembolism’ (FIRST) Registry

V. Speed 1,2 ; V. Auyeung 2 ; J. Patel 1,2 ; T. Jones 1 ; J. Samuel 1 ; L. Roberts 1 ; R. Patel 1 ; R. Arya 1

1 King's College Hospital NHS Foundation Trust, London, United Kingdom, 2 King's College London, Institute of Pharmaceutical Sciences, London, United Kingdom

Background: Medication non‐adherence is linked to treatment satisfaction. Patient perspective is increasingly recognised as influencing clinical outcomes; anticoagulation for the treatment of acute VTE and secondary prevention being no exception.

Aims: To evaluate patient satisfaction with rivaroxaban at the start and at the end of treatment for the management of VTE and in those continuing rivaroxaban for secondary VTE prevention.

Methods: FIRST is a UK non‐interventional registry investigating the incidence of long‐term complications of VTE treated with rivaroxaban without bridging heparin in routine practice. Following written informed consent patients were administered the Anti‐Clot Treatment Scale (ACTS), which comprises 15 questions of 2 subscales: burdens (12 items) and benefits (3 items). Questionnaires were completed by patients at 1 month, end of treatment or annually for those on long‐term therapy.

Results: Early results suggest that satisfaction with rivaroxaban is high during the first month and this is sustained at 3‐6 months with burden of treatment lessening over time (P=<0.05). Those patients on long‐term treatment maintain their satisfaction levels beyond 1 year, and encouragingly after 1‐year patient reported benefits appear greater (P=<0.05). Our findings are consistent with trends reported in the Einstein PE and DVT subgroup analyses.

Conclusions: Patient satisfaction with rivaroxaban is high during the acute management of VTE, as measured by the ACTS, and is sustained over time for patients prescribed both short and long‐term treatment for the secondary prevention of VTE. Our registry has previously reported good adherence to rivaroxaban therapy in the context of VTE treatment, which could be associated with the observed high levels of treatment satisfaction reported here. This will be explored further, with greater patient numbers as recruitment to the FIRST registry continues.

PB710: Elevated Levels of Resistin and Visfatin Are Associated with Platelet Activation and Blood Coagulation in Type 2 Diabetic Patients with Venous Thromboembolism

O. Korzh ; E. Lavrova; S. Krasnokutskiy

Kharkiv Medical Academy of Postgraduate Education, Kharkiv, Ukraine

Background: Proteins secreted from adipocytes ‐ so‐called adipocytokines ‐ influence metabolic and vascular function. Resistin and visfatin have been linked to obesity, type 2 diabetes mellitus, inflammation and atherosclerosis. Recent data suggest that various adipocytokines are dysregulated in type 2 diabetes mellitus and might be of pathophysiological and prognostic significance in cardiovascular complications.

Aims: The purpose of this study was to investigate the potential roles of resistin and visfatin in patients with type 2 diabetes mellitus who developed venous thromboembolism (VTE) and to evaluate the correlation between the adipocytokines and markers of coagulative activation.

Methods: We consecutively evaluated 211 type 2 diabetic patients patients with validated VTE. Baseline concentrations of circulating levels of resistin, visfatin, von Willebrand Factor (VWF), factor (F)VIIa, prothrombin fragment 1 + 2 (F1+2), 11‐dehydro‐thromboxane B2 (TXM), activated protein C, and soluble thrombomodulin (TM) were determined by enzyme‐linked immunosorbent assay (ELISA).

Results: Type 2 diabetic patients had significantly higher levels of resistin, visfatin, VWF, FVIIa, F1+2 compared with healthy men. By contrast, anticoagulant markers, such as activated protein C, and TM were depressed in type 2 diabetes. Plasma levels of resistin and visfatin positively correlated with TXM and prothrombin F1+2. Plasma levels of both adipokines have shown an inverse correlation with markers of anticoagulation such as activated protein C and soluble thrombomodulin.

Conclusions: Increased concentrations of resistin and visfatin were associated with type 2 diabetes mellitus. Intriguingly, high plasma levels of both adipokines seem to modulate platelet activation and coagulation. Further studies should focus on VTE prevention in this vulnerable population.

PB711: Evaluation of an Educational Video for Patients Taking Warfarin

J.M. Costa 1 ; R.C.d.S. Groia 1 ; E.I.d.F. Campos 1 ; M.A. Parreiras Martins 2 ; A.M.M. Reis 2

1 Universidade Federal de Minas Gerais, Pharmacist, Belo Horizonte, Brazil, 2 Universidade Federal de Minas Gerais, Pharmacist Products, Belo Horizonte, Brazil

Background: Despite the new oral anticoagulants, warfarin is commonly used for primary and secondary prevention of thromboembolism, especially in developing countries. Patients′ lack of knowledge of warfarin therapy may lead to clinical complications. The use of an educational video can help improve patient behavior towards warfarin therapy.

Aims: To evaluate patients’ knowledge on anticoagulant therapy before and after watching an educational video produced at a Brazilian anticoagulation clinic.

Methods: Educational session consisted of a five‐minute video and a 20‐minute discussion. Topics covered knowledge on patients′ health problems, risks and benefits of warfarin use, warfarin‐drug and warfarin‐food interactions and the need of seeking for health professionals help in case of doubts. Patients’ knowledge was evaluated after applying a questionnaire with six questions, including he question about warfarin indication in order to evaluate patients’ acquired knowledge. The percentage of correct answers was calculated. Cutoff for adequate questionnaire response was ≥60%. response rates were calculated before and after presenting the video to patients. T. We divided patients into two groups: treatment duration<or ≥6 months. Statistical analysis was performed using MacNemer and Mann‐Whitney tests and a significance level of 0.05. The study was conducted in accordance with the Declaration of Helsinki.

Results: A total of 62 patients were included. Mean age was 57.8 years and 35 (56.5%) were female. Overall, 41 (66.1%) patients had treatment duration<6 months. Only knowledge of warfarin indication increased after video (P=0.006). There was no significant difference between the percentage of correct answers into groups with treatment duration>or ≥6 months (P=0.775). The video helped clarify doubts about anticoagulation in 98.4% of the patients.

Conclusions: Results suggest audiovisual material can contribute to increase patient knowledge of warfarin therapy.

PB712: Subsegmental Pulmonary Embolism: An Innocent Bystander?

M.C. Gerritsen 1,2 ; F.A. Mohamed Hoesein 3 ; P.A. deJong 3 ; K.A.H. Kaasjager 1 ; M. Nijkeuter 1

1 University Medical Center Utrecht and Utrecht University, Acute Internal Medicine, Utrecht, the Netherlands, 2 Diakonessenhuis, Internal Medicine, Utrecht, the Netherlands, 3 University Medical Center Utrecht and Utrecht University, Radiology, Utrecht, the Netherlands

Background: Little is known about the clinical characteristics of patients with subsegmental pulmonary embolism (SSPE).

Aims: This study describes a population with SSPE and investigates whether patients with untreated SSPE differ from patients with treated SSPE.

Methods: All Computed Tomographic Pulmonary Angiography and chest CT radiology reports, made from January 2013 until December 2016, were retrospectively reviewed for SSPE diagnosis. Charts were reviewed for characteristics and outcomes, both were compared between treated and untreated SSPE patients.

Results: SSPE was reported in 136 of 6,387 scans (2.1%). Thirty‐eight patients were excluded based on a priory set exclusion criteria. Of 98 patients with SSPE, 16 patients were left untreated (16%). In treated as well as untreated SSPE patients, conventional PE symptoms and clinical signs were seen in the majority of patients (70% and 65%, respectively). A Wells score >4 was found in 52% of both treated and untreated SSPE. SSPE as an incidental finding was rarely described (n=5, 5%). Regarding outcomes, one of 16 untreated patients (7%, 95% CI: 0‐24%) and two of 82 treated patients (2%, 95% CI: 0.2‐8%) showed a recurrent VTE in 3 months of follow‐up. No mortality ‘likely due to PE’ was reported.

Conclusions: Based on our findings, SSPE does not seem to be an innocent finding. Treated SSPE patients do not have more comorbidities, neither more VTE risk factors or a more serious presentation than untreated patients. Management of SSPE seems to be based on clinical assessment of the likelihood of PE rather than CTPA/chest CT results.

PB713: Evaluation of Unmet Clinical Needs in Prophylaxis and Treatment of Venous Thromboembolism in High‐risk Patient Groups

B. Brenner 1 ; J. Beyer‐Westendorf 2,3 ; I. Elalamy 4 ; R. Hull 5 ; D. Imberti 6 ; R. Arya 7

1 Ramben Health Care Campus, Department of Hematology and Bone Marrow Transplantation, Haifa, Israel, 2 The University Carl Gustav Carus, Dresden, Germany, 3 King's College London, Department of Haematology, London, United Kingdom, 4 Tenon University Hospital, Hematology and Thrombosis Center, Paris, France, 5 University of Calgary, Foothills Medical Centre and Thrombosis Research Unit, Calgary, Canada, 6 Hospital of Piacenza, Haemostasis and Thrombosis Centre, Piacenza, Italy, 7 King's College Hospital Foundation NHS Trust, Department of Haematological Medicine, London, United Kingdom

Background: Risk of primary and recurrent venous thromboembolism (VTE) has been associated with several medical conditions, such as critically ill, obese, elderly, patients with cancer, and pregnant women. Initial VTE management is the most acute period regarding prevalence and burden of thrombosis recurrence, hemorrhage, and mortality in these patient populations. While optimal anticoagulation treatment of acute VTE is well‐established, the optimal strategy for primary prophylaxis to prevent VTE is less clear. Further research and consolidation of existing evidence is urgently needed to identify patient groups with insufficient prophylaxis and non‐optimal dosing regimens.

Aims: To design and implement a novel methodology to identify gaps in prophylaxis and treatment of VTE in specific patient populations.

Methods: VTE prophylaxis and treatment gaps were assessed through a questionnaire to collect insights not available in the public domain from 44 international practicing key thought leaders through telephone interviews. Answers were consolidated into novel insights then complemented by an extensive review of current guidelines and published studies collected from PubMed and Cochrane libraries from 2015 to 2017 to validate the emerging gaps.

Results: The gap analysis methodology has identified subpopulations of patients with cancer and pregnant women, where prophylactic anticoagulant regimens may be of benefit. In obese patients, lack of anticoagulant dose adjustment to body weight has emerged as a major obstacle in effective prophylaxis and treatment. Influences on prophylaxis in the critically ill patients are also discussed. The benefit of VTE prophylaxis against the bleeding risk is considered in all populations, especially in the elderly.

Conclusions: This methodology has identified patient subpopulations in whom suboptimal prophylaxis is suspected and has highlighted the need to tailor treatment regimens to specific, more vulnerable, patient groups.

PB714: Outpatient Management of Deep Vein Thrombosis using Direct Oral Anticoagulants is Safe and Cost‐effective

P. Lambros 1 ; S. Savage 1 ; V. Chellaram 1 ; P. Ho 2 ; H.Y. Lim 2

1 Northern Health, Pharmacy, Epping, Australia, 2 Northern Health, Haematology, Epping, Australia

Background: Direct oral anticoagulant (DOACs) is increasingly the treatment of choice for venous thromboembolism (VTE) given its convenience compared to warfarin. To ensure safe prescribing of DOAC and prevent delay of supply for dischargeable patients with deep venous thrombosis (DVT) presenting to Emergency Department (ED) outside pharmacy hours, we implemented a “Rivaroxaban after‐hours pack” system, which requires prescribers to obtain approval through GuidanceMS prior to pack provision.

Aims: To review the safety and cost‐effectiveness of this program.

Methods: A retrospective evaluation of DVT patients discharged from ED with Rivaroxaban after‐hours pack from April 2015 to December 2016, with minimum follow‐up of 6 months.

Results: Seventy‐seven patients with newly diagnosed DVT (median age 52 years (22‐84)) were discharged from ED with Rivaroxaban after‐hours pack. 98.7% of the patients met the weight criteria of safe prescribing (50‐120 kg) and all patients had eGFR >30 mL/min. 67 patients (87.0%) had liver function tests (LFT) performed, of which one had deranged LFT exceeding over 3× upper limit normal. Two patients (2.6%) were known to have active malignancy and no patients had mechanical valve or antiphospholipid syndrome. All patients received pharmacist follow‐up via telephone conversation for education with referral to Thrombosis Outpatients. One patient re‐presented with clinically significant bleeding (1.3%) and two patients had clot extension (2.6%) while on rivaroxaban despite reportedly good compliance. There was marked improvement in length of stay compared to DVT patients audited during the warfarin era (8.0 hours vs. 5.2 days, P<0.001) with 86% associated reduction in cost.

Conclusions: All DVT patients were safely prescribed and given Rivaroxaban through this system, with significant reduction in length of stay in hospital. This system is efficient and cost‐effective in ensuring safe provision of Rivaroxaban outside of pharmacy hours.

PB715: Knowledge Derived from a Hospitalar Thromboprophylaxis Program

M.C. Chindamo 1,2 ; G.G. Sardinha 3 ; W. Schiavini 1 ; D. Mousinho 1 ; A.B. Nunes 1

1 Hospital Barra D'Or, Rio de Janeiro, Brazil, 2 Universidade Federal do Rio de Janeiro, Internal Medicine, Rio de Janeiro, Brazil, 3 Hospital Barra D'Or, Pharmacy Department, Rio de Janeiro, Brazil

Background: Venous thromboembolism (VTE) is a major problem for hospitalized patients. Nevertheless, adherence to VTE prophylaxis guidelines is still suboptimal.

Aims: To analyze VTE events occurred in hospitalized patients and potential opportunities to improve VTE prevention practices.

Methods: In a general hospital with a well‐structured thromboprophylaxis program we analyzed all patients presenting symptomatic VTE events during hospitalization or readmitted within 90 days after discharge due to a VTE event. Patients were classified as medical or surgical and according to initial allocation in intensive care unit (ICU) or floor considering severity of the case. Rates of VTE and hospital length of stay were evaluated in each group.

Results: 6,886 patients at risk for VTE were included: 4,743 medical and 2,143 surgical patients. Global VTE rate was 0.45%: 0.27% in medical and 0.84% in surgical patients. The mean hospitalization time of each group was as follows: 4 days for medical patients admitted on floor; 8 days for medical patients initially admitted in ICU; 2 days for surgical noncritical patients and 7 days for patients who performed immediate postoperative treatment in ICU. VTE cases were distributed as follows according to severity of patients (Table 1):

TABLE 1 VTE cases classification Events Medical non critical Medical critical Surgical non critical Surgical critical VTE rates 3/1,121=0.26% 10/3,622=0.28% 11/1,602=0.68% 7/541=1.30% VTE after hospital discharge 2/3=67% 1/10=10% 9/11=82% 1/7=14%

Higher rates of VTE were found in surgical patients in comparison to medical (0.27% vs. 0.84%); critical surgical patients presented 4× more events than medical patients as a whole. VTE events were more frequent in noncritical patients after hospital discharge, especially in surgical patients.

Conclusions: A VTE prophylaxis program significantly reduces rates of symptomatic VTE in hospitalized patients. The incidence of symptomatic VTE was higher in surgical patients especially after discharge. Strategies to continue domiciliary prophylaxis in high‐risk surgical patients with short length of hospital stay must be prioritized.

PB716: Quality Assessment of Oral Anticoagulation by Time in Therapeutic Range in Patients with Venous Thromboembolism

H.‐G. Hwang 1 ; S.‐M. Koo 2 ; S.‐T. Uh 2 ; Y.‐K. Kim 2

1 Soonchunhyang University Gumi Hospital, Department of Internal Medicine, Gumi, Korea, 2 Soonchunhyang University Seoul Hospital, Department of Internal Medicine, Seoul, Korea

Background: The ability of the health‐care provider to make appropriate dosage and follow‐up decisions can have a major impact on therapeutic effectiveness. Time in Therapeutic Range (TTR) is used to assess the quality of anticoagulation. A strong relationship between TTR and bleeding or thromboembolic rates has been observed across studies. But it is not well known in clinical practice of Korea.

Aims: This study is designed to assess the efficacy and safety of oral anticoagulation with warfarin by time in therapeutic range in patient with venous thromboembolism.

Methods: This was a retrospective, single‐center study that evaluated patients diagnosed with venous thromboembolism (VTE) and managed to adjust dose of warfarin over two‐thirds of follow‐up period in Thrombosis Clinic between February 2009 and August 2010.

The patients who exceed 56 days between two consecutive INRs were excluded from statistical analysis. Patients who had available data over at least 2 months were included to analyze. Validated dosing algorithm was used to manage the INR.

Results: Out of 76 patients with VTE, 47 (23 men; mean age 64.9 years; range, 27—86) were analysed. Approximately two‐thirds of the patients (31/47) were warfarin‐ naïve . Total number of admission were 84. Expected duration of anticoagulation was 6 months in 22 patients (47%), 12 months in 1 (2%), and indefinite in 24 (51%). There were various comorbidities, including active malignancy in 7 patients (15%), mycobacterium tuberculosis on rifampicin in 4 (8.5%), chronic kidney disease in 4 (8.5%), and hemorrhagic cerebral infarct in 1 (2%). There was no patient who exceeds 56 days between two consecutive INRs. Overall TTR was 52.4%. Most proportion of out‐of‐range were below the therapeutic range (29.5%). There was no thromboembolic complication, but 2 cases of major bleeding complications were observed.

Conclusions: The validated dosing algorithm is effective in managing INRs in patients with venous thromboembolism in Korea.

PUBLICATION ONLYBLEEDING DISORDERS

PO001: Study of the Hemostatic System in Children in the Republic of Uzbekistan

I. Berger; A. Makhmudova

Scientific Research Institute of Hematology and Blood Transfusion of the Ministry of Health of the Republic of Uzbekistan, Tashkent, Uzbekistan

Background: Hemostasis disorders in childhood occupy an important place in the general pathology, as they are one of the most frequent pathological conditions encountered in practical medicine and are characterized by an extremely high potential hazard.

Aims: To study the hemostatic system in children with bleeding.

Methods: According to the archival data of the Research Institute of Hematology and Blood Transfusion from 2015 to 2016 were examined 3,000 children and adolescents, aged 0‐20 years. All patients were tested in ambulance department of Scientific Research Institute of Hematology for followed indicators: platelet count, platelet aggregation with ADP, plasma factors (APTT), prothrombin complex. A total of 2,476 coagulograms were performed.

Results: According to the received data, 90% of the children who were treated identified abnormalities in the hemostatic system. Aggregation capacity of platelets was reduced in a significant number of patients – 59%. In most cases it was related with the use of non‐steroidal anti‐inflammatory drugs that have anti‐aggregation effects. A part of the patients showed hypocoagulation in the plasma hemostasis (8%), while 101 children were diagnosed with hemophilia A light form, 157 middle and 46 severe form. In another group of patients (3%), there was a deficit of factors 1,2,5,7 (prothrombin complex). Hypercoagulation shifts during the inflammatory process were determined in 3% of patients (platelet hyperaggregation, shortening of APTT and PV, hyperfibrinogenemia and elevated RFMC level).

Conclusions: Thus, after detailed study of the hemostasis system, 75% of cases reveal abnormalities in the functioning of the coagulation system. According to the results of the survey were developed practical recommendations to pediatricians, surgeons and therapists on the treatment and management of patients in the preoperative and postoperative period, which reduces the risk of hemorrhagic complications in children.

PO002: Total Knee Arthroplasty after Knee Fusion in Severe Hemophilia A

P. Beurrier 1 ; N. Stieltjes 2 ; Y. Ronzi 3 ; M. Hamadouche 4 ; P. Bizot 5

1 CHU Angers, Hemophiliac Treatment Center, Angers, France, 2 Cochin Hospital, AP‐HP, Hemophiliac Treatment Center, Paris, France, 3 Physical and Rehabilitation Medicine Center Les Capucins, Angers, France, 4 Cochin Hospital, AP‐HP, Department of Orthopedic Surgery, Paris, France, 5 Lariboisière Hospital APHP, Department of Orthopedic Surgery, Paris, France

Background: Hemophilia affects joints with destructive lesions, leading sometimes to arthrodesis. Knee arthrodesis was believed to be a definitive statement but, nowadays, “de arthrodesis‐TKA” may be indicated to improve patient's function. Only 2 cases has been reported for hemophilia in literature.

Aims: A man, 42‐year‐old, with severe hemophilia had his first knee hemarthrosis at 14 months and further developed an arthropathy. At 12 years his joint deteriorated and at 15 a knee fusion was done. Daily discomfort was tolerable but absence of motion has never been fully accepted. Moreover, he had many other orthopaedics challenges

. Because of the major functional impairment related to the arthrodesis, the question of knee replacement was considered 17 years later. The aim was to get a flexion of 90° allowing many tasks in familial and professional life. He was informed of the difficulties and potential complications of the procedure.

Methods: All the conditions were fulfilled: patient's motivation, control of HIV infection, no bone defect, good skin quality, acceptable quadriceps muscle strength and continuity of the extensor system. Surgery was performed under general anaesthesia and a tourniquet.

The steps included removal of the former material, quadriceps muscle liberation, patellar and femoro‐tibial osteotomies allowing 90°of flexion, and insertion of a Legacy standard cemented constrained Condylar knee prosthesis with intramedullary stems. During the procedure, the tibial insertion of patellar tendon has been weakened and protected by cerclage.

Results: 562 UI/Kg rF8Fc have been used during 19 Hospital stay days. It seems to be less than usual, FVIII consumption required for such procedures.

Rehabilitation began at day 1, allowing 80° of flexion and full weight bearing. No complication occurred.

Conclusions: Knee arthrodesis is a very disabling condition in severe hemophilia with social and psychological consequences. We can now improve this by a TKA at comparable cost to that of primary TKA in hemophilia.

PO003: Autoimmune Acquired von Willebrand Syndrome with Short von Willebrand Factor Half‐life

E. Salazar ; L. Rice

Houston Methodist Hospital, Houston, United States

Background: Acquired Von Willebrand syndrome (AVWS) is a bleeding disorder characterized structural or functional defects in Von Willebrand Factor (VWF) that arise in the setting of autoimmune, lymphoproliferative or myeloproliferative, malignant, or cardiovascular disorders. Often, high molecular weight multimers are mild to moderately decreased due to increased shear stress conditions such as cardiac valvular abnormalities or ventricular assist devices. AVWS can also arise in the setting of antibody‐mediated functional inhibition or antibody‐mediated clearance of VWF multimers.

Aims: We present a case of autoimmune AVWS due to rapid antibody‐mediated clearance of VWF multimers.

Methods: A full clinical workup for AVWS was conducted.

Results: A 59 year old male with 3 year history of multiple gastrointestinal bleeds was admitted to Houston Methodist Hospital for melena and symptomatic anemia. There was no significant bleeding history in childhood or after previous surgeries. No cardiac or major vascular abnormalities were identified. Von Willebrand studies showed decreased Factor VIII (32%), VWF Antigen (23%), VWF immunofunction (10%), collagen binding (7%), and decreased high and intermediate weight multimers. Serum Protein electrophoresis revealed a monoclonal IgG Kappa (0.2 g/dL). VWF propeptide was 98 IU/dL with a ratio of propeptide to antigen of ˜4. An antibody leading to rapid clearance of VWF was suspected. The patient was administered 4,000 units of Humate‐P with serial measurements of VWF antigen, activity, and multimers at 0, 30 minutes, 3 hours, 6 hours, 12 hours, and 24 hours after infusion. VWF Antigen, activity, and multimers spiked to normal levels within 30 minutes after infusion with rapid decrease to baseline levels and a half‐life of ˜2.5 hours.

Conclusions: Timed VWF studies after Humate‐P infusion demonstrated rapid clearance of VWF in a patient with autoimmune acquired vWF. The patient was successfully treated with prednisone and rituximab.

PO004: Spectrum of Mutations: An Identified Locus of Point Missense Mutation in Fibrinogen Gene Alpha of Inherited Factor 1 Deficiency Patients

T. Khan 1 ; A. Naz 2 ; A. Biswas 3 ; A. Goodeve 4 ; N. Ahmed 5 ; S. Ahmed 6 ; T. Shamsi 7 ; J. Oldenburg 8

1 National Institute of Blood Disease & Bone Marrow Transplantation, Coagulation and Hemostasis, Karachi, Pakistan, 2 National Institute of Blood Disease & Bone Marrow Transplantation, Coagulation, Karachi, Pakistan, 3 Institute of Experimental Hematology and Transfusion Medicine, Bonn, Germany, 4 Sheffield Haemophilia and Thrombosis Centre, Sheffield, United Kingdom, 5 Children Hospital, Lahore, Pakistan, 6 National Institute of Blood Disease & Bone Marrow Transplantation, Molecular Genetics, Karachi, Pakistan, 7 National Institute of Blood Disease & Bone Marrow Transplantation, Medical Director, Karachi, Pakistan, 8 Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany

Background: Inherited Factor 1 deficiency is transmitted as an autosomal recessive disease with an absent level of activated fibrinogen protein in plasma. This disorder has manifested its strong association with cousin marriage in our set of population with rapidly increasing number of new cases each year.

Aims: The aim of this study was to search out for spectrum of mutation in previously identified mutations detected in all three genes of fibrinogen (FGA alpha, beta and gamma).

Methods: This descriptive cross sectional study in concordance with declaration of Helsinki 2000 was conducted in Karachi, Pakistan. All diagnosed cases of inherited factor 1 deficiency were included (fibrinogen functional assay, Laboratoire Stago, Asnieres, France).

Results: Total 11 patients were included in this study. Out of 11 subjects 6 patients were shown to have similar point missense mutation in same exon of similar fibrinogen gene alpha. This gene is also reported as the mostly frequently affected gene with larger number of identified mutations in global data so far.

Conclusions: Rarity of inherited Factor 1 absence is debatable now due to its swiftly growing incidence especially in our region where cousin marriages are accustomed. Although fibrinogen gamma gene is the largest amongst all three genes of fibrinogen protein with 10 exons but spectrum of mutations in fibrinogen alpha gene has proven its frequent involvement and recognized as a major contributor in causing and exhibiting manifestation of disease.

PO005: Response to Recombinant FVIII(C) Therapy in a Young Haemophilia A Patient with Spiral Fracture of the Left Femur

W. Shokunbi ; T. Akingbola; K. Shonde‐Adebola; T. Adeoye

University College Hospital, Ibadan, Haematology, Ibadan, Nigeria

Background: In Nigeria, management of Haemophiliac bleeds have been with blood products until recently that recombinant FVIII(C) is being used in some centres. DC is an 8 years old Haemophilia A patient with FVIII(C) level of 18%, who sustained a spiral fracture of the left femur following a domestic fall. This was complicated by a huge hematoma at the fracture site, requiring blood transfusion on account of PCV of 18%. The Prothrombin Time was 15 (control 15) seconds and APTT of 95 (control APTT 35) seconds. He initially had 7 units of FFP due to non‐availability of cryoprecipitate. Bleeding at the fracture site persisted as evidenced by increase in size of the haematoma at the fracture site. He subsequently had POP cast applied and was commenced on recombinant FVIII(C) infusion (obtained from Haemophilia foundation of Nigeria, an NGO, at a subsidized rate).

Aims: To highlight the response of a Haemophilia A patient with left femoral fracture to rFVIII(C).

Methods: Manual FVIII(C) assays were done on citrated samples taken 2 hours after infusion of rFVIII concentrate (at 3,000 iu/day in two divided doses) by APTT method using Teclot APTT reagent.

Results: Patient had a total of 108,500 units of rFVIII infusion over 46 days. He initially responded to the therapy, with correction of the APTT as compared to the pre‐treatment value (95 sec vs. 45 sec). He however showed a declining response to subsequent infusions of rFVIII concentrate, as evidenced by the gradual reduction in the factor VIII(C) activity, from 22%, to 12%, 8% and 6%.

Conclusions: This patient showed a declining response to therapy with rFVIII over time, likely due to the development of inhibitors. We recommen serial monitoring using dose response curve and FViiiC assay, particularly when managing patients with major and life threatening haemorrhages.

PO008: A Study of the Peculiarities of Hematomesenchymal Dysplasia in Patients with Hemophilia

A. Makhmudova ; I. Berger; M. Makhmudova; M. Nabieva

Scientific Research Institute of Hematology and Blood Transfusion of the Ministry of Health of the Republic of Uzbekistan., Tashkent, Uzbekistan

Background: HMD is the most common pathology in patients with hemorrhagic diathesis and less studied.

Aims: The purpose of this work is to study the specific features of hematomesenochial dysplasia in patients with hemophilia.

Methods: 396 patients were examined. Of these, 170 (43%) with hemophilia, Willebrand disease 60 (15.2%), thrombocytopathy 166 (41.8%). Of the 396 patients, a group of hematomesenchymal dysplasia (HMD) was isolated – 145 patients, which was 37%.

Results: The majority of subjects met from 3 to 5 signs of HMD in different combinations: two ‐ in 79%, three ‐ in 56% and four ‐ in 48%. 38.6% of patients with hemophilia had symptoms of MD, 15.8% had von Willebrand disease, and 66 (45.6%) had thrombocytopathy. In 251 (63.3%) patients, the presence of MD symptoms was not detected. The average level of factor VIII in plasma in patients with hemophilia was 2.1±1.7, and factor IX – 15.2±2.8; and in the group with HMD 1.9±1.4 (P<0.5) factor VIII and 11.8±2.6 (P<0.05), which indicates a more serious course of the process. A direct correlation was found between the severity of complications in the form of arthropathy with levels of factors VIII, IX and the frequency of external phenotypic DST markers. Patients had: asthenic constitution ‐ in 46 (31.8%), stoop – 36 (24.8%), hypermobility ‐ in 21 (14.4%), flat foot 19 (13.1%), elongated fingers in 16 (11%), myopia ‐ in 8 (5.7%). According to ultrasound and ECG findings, congenital heart defects in 2 (1.4%), gallbladder inflection in 15 (10.3%), and nephroptosis in 1 (0.6%) were detected. A study of the hemostasis system showed that the total number of platelets and their morphometric parameters were normal, the platelet aggregation activity was reduced, an increase in bleeding duration was recorded only in 5.8% of cases. Level 3 of the platelet factor was 1.6 times lower compared to the control values.

Conclusions: The findings suggest that hemophilia in combination with MD is more severe and with the greatest and longest complications.

PO009: Near‐fatal Acquired Inhibitor of Human Thrombin

E. Salazar ; L. Rice

Houston Methodist Hospital, Houston, United States

Background: While acquired inhibitors of some coagulation factors, such as factor VIII are relatively commonly identified, inhibitory antibodies against thrombin occur very infrequently. Historically, these antibodies were associated with exposure to topical bovine thrombin used in the surgical setting; however, rare spontaneous autoimmune thrombin inhibitors do occur.

Aims: We describe a case of acquired inhibitor of human thrombin leading to recurrent near‐fatal hemorrhage.

Methods: A full clinical coagulation workup was performed.

Results: A 39 year old female with no significant past medical, bleeding, or surgical history presented to Houston Methodist Hospital with a large left‐sided subdural hematoma after light trauma. Severe brain compression required emergency craniotomy and evacuation. Coagulation workup revealed persistently prolonged prothrombin time (PT) (19.4 seconds), partial thromboplastin time (PTT) (43.9 seconds), and thrombin time (TT) (65.4 seconds). Fribinogen level was normal at 240 mg/dL, and reptilase time was normal. 1:1 mixing studies with normal plasma showed lack of correction of PT, PTT, or TT. The patient had not received any anticoagulants. Anti‐Xa level was<0.1 IU/mL, and treatment with heparinase had no significant effect on the PT, PTT, or TT. Factors 2, 5, 7, 9, 10, 11 levels were relatively normal, but showed nonparallelism, consistent with an inhibitor pattern. Serum protein electrophoresis revealed a prominent IgG Kappa in the beta region, raising the possibility that an IgG antibody against thrombin was present. Total beta globulins were 1.8 g/dL. Incubation of the patient's sample with protein a/g agarose beads led to partial correction of the thrombin time. After hematoma evacuation no spontaneous bleeding occurred. The patient was discharged with plans for outpatient follow‐up, but was readmitted with a new right‐sided subdural hematoma after light trauma.

Conclusions: An antibody against human thrombin with near fatal consequences was identified in a young patient.

PO010: The Radiologist: A Key Member in the Diagnose of a Patient with Hemophilia A

J. Restrepo‐Avenia 1 ; J. Echeverri 1,2 ; J. Duque‐Gil 1 ; J. Ramirez‐Varela 1

1 Centro Médico Imbanaco, Hemophilia Treatment Center, Cali, Colombia, 2 Centro Médico Imbanaco, Radiology Department, Cali, Colombia

Background: Treatment people with hemophilia A ‐PwHA‐ in an integrative approach to prevent and to treat bleeding episodes is one of the objectives of the World Federation of Hemophilia. Radiologists play an important role in the diagnostic and treatment of this population.

Aims: ‐

Methods: We present a 28‐years‐old man who suffered a left distal femur fracture with the intra‐articular involvement of the knee while working on construction. He underwent total knee replacement surgery in a local institution, but he became unstable at the recovery room, with severe pain, and hypovolemic shock status was detected; 3 gr/dL of hemoglobin level were reported; He was politransfused and stabilized, but due to persistent knee bleeding, he was referral to our institution to rule out vascular injury; An arteriography of the lower left limb was performed and was negative, so he was contra‐referral to his local institution, but 24 hours later, he was re‐admitted at our institution due to an inguinal hematoma at angiography puncture site; An abdominal CT Scan reveled a superficial hematoma, but the radiologist also reported: “Thick calcifications are identified towards the iliopsoas insertion bilaterally, mainly on the left side, most likely due to sequelae of already calcified hematomas. These findings can be observed in patients with a history of hemophilia”, so our hemophilia treatment center received the alert, and after a full evaluation and proper care, a moderate hemophilia A diagnostic was made, and the patient underwent intermittent prophylaxis until full rehabilitation was documented.

Results: ‐

Conclusions: We consider expertise is key in the management of PwHA and radiologists make a Hemophilia Treatment Center facility more integrative as they bring insights on both diagnosis and therapeutic options for PwHA. Without our radiologist′s approach, a diagnostic of post‐operative bleeding episode secondary to an undiagnosed congenital bleeding disorder was not be possible.

PO011: A Multinational Prospective, Non‐interventional, Post‐marketing Surveillance Study to Observe the Real‐world Use of Recombinant Factor IX Albumin Fusion Protein (rIX‐FP) in Patients with Hemophilia B

G. Castaman 1 ; J. Oldenburg 2 ; B. Pan‐Petesch 3 ; V. Jiménez‐Yuste 4

1 Careggi University Hospital, Center for Bleeding Disorders, Florence, Italy, 2 University Clinic Bonn, Institute of Experimental Haematology and Transfusion Medicine, Bonn, Germany, 3 Centre Hospitalier Universitaire Brest, Hématologie‐Hémostase, Brest, France, 4 Hospital Universitario La Paz, Unidad de Coagulopatías, Servicio de Hematología, Universidad Autonoma de Madrid, Madrid, Spain

Background: rIX‐FP is a fusion protein genetically linking recombinant human coagulation FIX with recombinant human albumin. rIX‐FP provides patients with low annualized bleeding rates with 7‐, 10‐ and 14‐day prophylaxis and also shows favorable hemostatic efficacy in the treatment of bleeding events and surgical hemostasis, as demonstrated in clinical studies. These results need to be confirmed in routine clinical practice and long‐term data gathered on the effectiveness and tolerability of rIX‐FP.

Aims: Prospective, non‐interventional, multicenter studies have been established to gather effectiveness and safety data on the use of rIX‐FP in real‐world clinical practice in Europe.

Methods: Five studies will be conducted in 6 European countries (Belgium, Germany, France, Italy, Spain and Portugal). We aim to enroll over 300 patients of any age, with mild to severe hemophilia B from hemophilia treatment centers. Patients will be followed for 2 to 3 years or until 100 exposure days have been reached. Patients will be routinely monitored with visits every 3 to 12 months (depending on local practice). Bleeding rates in patients on prophylaxis and effectiveness in treating bleeds will be assessed as well as FIX consumption, infusion frequency and dosing. Patients will also be monitored for adverse reactions and inhibitor development. Impact on joint scores, patient satisfaction, bleeding‐related absenteeism and health‐related quality of life will also be collected, if possible.

Results: To date, 12 patients have been recruited from two countries (Belgium and Italy). Study methodology will be described in detail, and study status updates provided.

Conclusions: These prospective, non‐interventional, multicenter studies will provide data on the real‐world use of rIX‐FP in Europe to confirm the favorable efficacy and safety of rIX‐FP seen in clinical studies.

PO012: A Clinical Audit of Low Dose Prophylaxis Programme for Nigerian Children with Haemophilia

T. Nwagha 1 ; T. Adeyemo 2 ; C. Udo 3 ; S. Yuguda 4 ; K. Korubo 5 ; H. Okoye 1 ; Nigeria Low Dose Prophylaxis Programme

1 University of Nigeria Teaching Hospital, Haematology & Immunology, Enugu, Nigeria, 2 Unigersity of Lagos Teaching Hospital, Haematology & Blood Tansfusion, Lagos, Nigeria, 3 National Hospital Abuja, Haematology, Abuja, Nigeria, 4 Federal Medical Center Gombe, Haematology, Gombe, Nigeria, 5 University of Portharcourt Teaching Hospital, Haematology, Portharcourt, Nigeria

Background: Prophylaxis is considered standard of care for haemophilia patients. The evidence of benefits for prophylaxis especially low dose prophylaxis is incontestable yet most children in developing countries as Nigeria do not have access to this treatment protocol.

Aims: To audit the low dose prophylaxis treatment programme for Nigerian children with haemophilia.

Methods: A multicentre clinical audit of four haemophilia treatment centre in Nigeria; University of Nigeria Teaching hospital Enugu, Lagos University Teaching hospital, National hospital Abuja and Federal Teaching Hospital Gombe. Eighteen children with mild to severe haemophilia were enrolled into low dose prophylaxis treatment programme The factor concentrates used were from the donations from the WFH Humanitarian Aid Program. The reduction of joint bleeding, improvement of joint function and QoL were outcome measures.

Results: In total 18 children,17males and 1 female, median age of 8 (range 2‐16 years) were enrolled. The median duration of observation was 7 months (range 3‐15 months). Seven of children were on primary prophylaxis (41%) while 11 of the children (61%) were on secondary prophylaxis. The median ABR before and after prophylaxis was 10 {range 0‐20, 95% CI,6.7‐13.5, P value 0.62} and 3.0 {range 0‐7, 95% CI: 1‐4‐4.6, P value 0.06} respectively. Mann Whitney analysis of the medians showed a significant P value 0.002. There was a 74% (120/162) reduction in joint bleed. School and sports participation improved.

Conclusions: Low dose prophylaxis treatment programme was beneficial in reduction of joint bleeds, improvement in joint function and Qol of Children with haemophilia in Nigeria.

PO013: Which Is the Best Coagulometer on the Market?

D. Sánchez Argüello ; C. De Brabandere; M. Sarasa Valdes; L. Guerrero Fernandez; C. Fernandez Canal; S. Sánchez Matías; J. Torres Varona; P. Chamorro Chamorro; A. Fernandez Gonzalez; V. Robles Marinas

Hospital de Cabueñes, Gijón, Spain

Background: Nowadays there is a wide range of coagulometers available, making it increasingly difficult to choose the most appropriate according to the needs of each laboratory. There is lack of scientific evidence in this regard, so we decided to compare two automated coagulometers of two different commercial houses.

Aims: To compare the results of Sysmex‐CS‐5100 (Siemens) and ACL‐TOP‐750‐CTS (Werfen) in routine coagulation study (prothrombin time ratio, activated partial thromboplastin time ratio and fibrinogen) using the results in our extern quality control (EQC) program.

Methods: During the EQC program, we receive twelve annual shipments of two samples each. The deviation indexes (DI) of both our equipment were compared with the results obtained in all EQC participating centers from Jan‐2016 to Jan‐2017.

All the testing was made on the same day of each month along with the same technical analyst for both types of equipment.

We classified the results, according to their DI into: excellent (DI 0‐05), good (DI 0.5‐1), satisfactory (D I1‐2) and non‐acceptable (DI>2).

Results: We divided the results into two semesters (fig. 1):

During the first semester, both equipment were similar, being 53% the sum of excellent and good with the Sysmex and 73% with the ACLTOP, although the Sysmex had more unacceptable results (6.67% vs. 3.33%).

During the second semester the results of the ACL‐TOP were superior, since the sum of excellent and good was 73.33% compared to the 26.66% of the Sysmex, which also had more unacceptable results.

Overall, the sum of excellent and good was 40% in the Sysmex and 73.33% in the case of ACL‐TOP.

Conclusions: The results obtained by the ACL‐TOP‐750‐CTS were superior to those of the Sysmex‐CS‐5100. These differences cannot be attributed to human factor, since the laboratory staff stayed the same.

The improvement of the results over time demonstrates the importance of the learning curve when introducing new equipment to a laboratory.

PO015: The FORMA‐07 Study: A Post‐marketing, Phase IV, Observational Study Evaluating the Safety and Efficacy of a New Human Fibrinogen Concentrate in Congenital Fibrinogen Deficiency

C. Solomon 1 ; F. Peyvandi 2 ; B. Schwartz 3

1 Octapharma AG, Lachen, Switzerland, 2 Angelo Bianchi Bonomi Hemophilia and Thrombosis Centre, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico and Luigi Villa Foundation, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy, 3 Octapharma USA, Hoboken, United States

Background: Fibrinogen concentrate is used to treat congenital fibrinogen deficiencies such as afibrinogenemia and hypofibrinogenemia. A new highly purified, double virus inactivated fibrinogen concentrate (Fibryga, Octapharma AG, Switzerland) was licensed in the US, Canada and Europe in 2017 for the treatment of bleeding events (BEs) in these patients. Further data on safety and efficacy are needed in this ultra‐rare disease.

Aims: To collect efficacy data and assess safety of the new fibrinogen concentrate by characterizing risks of thrombosis and anaphylaxis following on‐demand treatment of BEs in adults and adolescents with congenital fibrinogen deficiency ( Table 1 ).

TABLE 1 FORMA‐07 study objectives Primary 1. Characterize incidence of thromboembolic ADRs in on‐demand treatment of major BEs Secondary 1. Describe incidence of anaphylaxis in on‐demand treatment of major BEs 2. Characterize ADRs, serious ADRs, and ADRs of special interest (thromboembolic ADRs, allergic reactions, including anaphylaxis) in on‐demand treatment of BEs 3. Describe objective thromboembolic monitoring in on‐demand treatment of bleeding 4. Describe hemostatic efficacy for on‐demand treatment of BEs

Methods: The post‐marketing, multicenter, open‐label, phase IV, observational FORMA‐07 study will enroll up to 25 patients (≥12 years) with congenital afibrinogenemia and hypofibrinogenemia treated with fibrinogen concentrate for on‐demand treatment of acute BEs. The aim is to describe safety and efficacy for a total of 105 BEs, including ≥10 major BEs in 10 patients. Exclusion criteria include: other bleeding disorders; acquired fibrinogen deficiency; dysfibrinogenemia; anti‐fibrinogen inhibitor; participation in concurrent clinical studies. Fibrinogen concentrate will be individually dosed as per locally approved package insert (target fibrinogen level in the US of 100 mg/dL for minor bleeding and 150 mg/dL for major bleeding). Primary endpoint is the incidence of thromboembolic adverse drug reactions and focus on treatment of major BEs. Secondary endpoints are shown in ( Table 2 ). Safety will be assessed descriptively (incidence, exploratory confidence intervals), and efficacy using an objective 4‐point hemostatic scale.

TABLE 2 Secondary endpoints for on‐demand treatment of BEs in the FORMA‐07 study Hemostatic efficacy: Efficacy assessed by the investigator within 24 hours using an objective 4‐point scale: Excellent, Good, Moderate, None Safety: ADRs, serious ADRs, and ADRs of special interest (allergic reactions, including anaphylaxis) Thromboembolic monitoring: Wells score for pulmonary embolism/deep vein thrombosis Additional BE data: Type, site, and severity; start, end, and duration of BE; dates and times of infusions; drug dose and batch number

Results: The study will start in early 2018; planned duration is ≤6 years. The study will be considered complete when ≥10 patients have ≥1 documented major BE.

Conclusions: Real‐world evidence from routine clinical use will further support clinical study data on the use of Fibryga for the treatment of congenital fibrinogen deficiency.

PO016: Severe Acquired von Willebrand Syndrome (Type 3‐like) in a Patient Affected with Systemic Lupus Erythematosus (SLE)

F. Stufano 1 ; L. Baronciani 1 ; E. Biguzzi 1 ; G. Cozzi 1 ; P. Colpani 1 ; M. Chisini 1 ; F. Peyvandi 1,2

1 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico and Luigi Villa Foundation, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico and Luigi Villa Foundation, Milan, Italy, 2 Università degli Studi di Milano, Department of Pathophysiology and Transplantation, Milan, Italy

Background: AVWS arises in individuals with no previous personal or family history of bleeding. In the past, this condition rarely has been associated with SLE.

Aims: We report a case of a 19‐year‐old woman who presented with spontaneous bruising, severe bleeding from the oral cavity and fatigue.

Methods: Basic coagulation tests were all normal except a prolongation of aPTT with a ratio of 1.51 (normal‐range 0.86‐1.20). Low values of FVIII:C (3 IU/dL) and almost undetectable of VWF parameters (VWF:Ag=2 IU/dL; VWF:RCo <0.5 IU/dL) (Figure, Baseline), with a barely detectable VWF multimer pattern lead to the diagnosis of VWD type 3. The VWF propeptide to VWF:Ag (VWFpp/VWF:Ag) ratio was 74 (normal‐range 0.6‐1.6), indicating a markedly increased plasma VWF clearance, typical of AVWS. This finding was confirmed by the normal intraplatelet VWF content. The Bethesda method, using both VWF:Ag and VWF:RCo assays, showed no evidence of an inhibitor against VWF in the patient plasma. On the contrary, assays for anti‐VWF non‐neutralizing antibodies demonstrated a high titer of IgG/IgM (Figure). An autoimmune disease was hypothesized. Further laboratory investigations revealed the presence of high titer antinuclear antibody (1:1,280), anti‐native DNA antibody (1:40) and anti‐double stranded DNA antibody (311 IU/mL), consumption of complement fraction (C3 and C4) with mild anemia and thrombocytopenia.

Results: Given all the biochemical values and clinical manifestations a diagnosis of AVWS secondary to SLE was made. The patient was treated daily with prednisone, azathioprine and hydroxychloroquine sulfate. After 2 months of therapy, a progressive increment of FVIII and VWF values with concomitant reduc FVIII:C, FVIII …tion of anti‐VWF IgG/IgM antibodies titer was observed.

Figure 1 FVIII:C, FVIII coagulant activity; VWF:Ag, VWF antigen; VWF:RCo, VWF ristocetin cofactor activity;*VWFpp/VWF:Ag, VWFpp to VWF:Ag ratio (not expressed)

Conclusions: Here we describe the laboratory features of an uncommon type 3‐like AVWS patient associated to SLE, reporting the restoring of the VWF levels along with the decrease of anti‐VWF IgG/IgM antibodies titer after immunosuppressive therapy.

PO018: Real‐world Use of BAY 81‐8973 for Routine Prophylaxis in Colombia: A Preliminary Report

G.E. Mora Figueroa 1 ; A. Plata García 2 ; R. Camacho Grimaldos 1

1 Hematologia y Oncologia del Oriente, Villavicencio, Colombia, 2 Oncoriente, Villavicencio, Colombia

Background: BAY 81‐8,973 (Bayer) is a full‐length, unmodified recombinant human factor VIII (FVIII) developed for the treatment of hemophilia A. The safety and efficacy of BAY 81‐8,973 for on‐demand treatment and control of bleeding episodes, perioperative management of bleeding, and routine prophylaxis in patients with severe hemophilia A (<1% FVIII) was evaluated in 3 international clinical studies (LEOPOLD program). However, observed treatment patterns and outcomes in clinical trials conducted under close supervision and with clearly defined eligibility criteria may not adequately represent patient outcomes for treatment outside the clinical research setting.

Aims: Here, we report a limited experience from patients treated in Colombia with BAY 81‐8,973, including a description of weekly prophylaxis dosing regimens with BAY 81‐8,973 used in routine clinical practice.

Methods: A data capture tool was used to obtain clinical information about their patients using BAY 81‐8,973 (n=9). Data were analyzed using descriptive statistics.

Results: All patients on prophylaxis are males aged 11 to 38 years with severe hemophilia A (n=7) or moderate hemophilia A with severe phenotype (n=2). Eight patients have hemophilic arthropathy. Before switching to BAY 81‐8,973, nearly all patients received plasma‐derived FVIII. Mean (range) exposure to BAY 81‐8,973 was 15 (8‐20) weeks. Median (range) dose per infusion of BAY 81‐8,973 was 34 (24.5‐55.5) IU/kg twice or three times per week. Pharmacokinetic data for 5 patients are summarized in the Table. No patients had adverse events, and no FVIII inhibitors were identified. Nearly all patients had 100% treatment adherence. Median total annualized bleeding rate (ABR) was 1; ABRs for spontaneous, traumatic, and joint bleeds were all 0.

TABLE 1 BAY 81‐8,973 Dose and FVIII Levels Patient Age, y Dose, IU/kg Baseline FVIII Level, IU/dL FVIII Levels Postinfusion, IU/dL 4 hours 24 hours 48 hours 72 hours 1 38 28.5 * 1.5 65 11 2.9 2.5 2 25 34 * 0.8 NA NA 2.3 3 3 16 24.4 * 0.7 20.6 14.7 3.4 1 4 25 26.5 * 0.2 38.3 3.9 0.4 NA 5 24 28.5 † 0.2 41 36.6 3.2 0.3 Mean 25.6 28.4 0.7 41.2 16.6 2.4 1.7 *Patients received BAY 81‐8,973 prophylaxis twice weekly. †Patient received BAY 81‐8,973 prophylaxis 3 times per week. NA, not available.

Conclusions: BAY 81‐8,973 routine prophylaxis (administered twice or three times weekly) has been effective and safe in this Colombian population of patients with hemophilia A in a real‐world setting.

PO019: The First Clinical Use of a von Willebrand Factor Concentrate with a Low FVIII Content for the Long‐term Prophylaxis of Severe von Willebrand Disease in Slovakia

T. Simurda ; M. Dobrotova; J. Sokol; J. Stasko; P. Kubisz

Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, University Hospital Martin, National Centre of Hemostasis and Thrombosis, Department of Hematology and Transfusiology, Martin, Slovakia

Background: von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by a quantitative and/or qualitative abnormality in the adhesive plasma protein von Willebrand factor (VWF). Individuals presenting with clinical manifestations of VWD may have a mild, moderate or severe bleeding tendency since childhood, usually proportional to the degree of the VWF deficiency/defect. There are two principle treatments of choice in VWD, i.e. desmopressin and substitution therapy with blood products.

Aims: We therefore wish to report the first successful use of a highly‐purified plasma VWF concentrate with a low FVIII content in the long‐term prophylaxis.

Methods: We present a now 34‐year‐old man with VWD type 3. Early after birth, several spontaneous bleeding manifestations presented, which over subsequent years, continued with many mucosal and joints bleeding episodes. From December, 2015, he was switched from a concentrate containing both VWF and FVIII to a highly‐purified plasma VWF concentrate with a low FVIII content.

Results: In our patient, reporting a personal history of serious hemorrhage, the prophylactic administration of FVIII/VWF concentrates has been changed to that of highly purified plasma VWF concentrate with a low FVIII content 32 IU/kg three times weekly from December 2015. Since then, he noticed a significant reduction of the frequency and intensity of spontaneous bleeding. Patient's quality of life improved in comparison with prophylactic administration of VWF/FVIII concentrates. Nevertheless, we report the first prophylactic regimen of this concentrate used for patient with VWD in Slovakia.

Conclusions: Secondary long‐term prophylaxis with VWF concentrate containing little FVIII appears to reduce the frequency and intensity of bleeds in patients with VWD type 3. However, experience with long‐term VWF concentrate prophylaxis is limited and the optimal dosage schedule has yet to be determined. Further prospective studies are necessary to investigate these issues.

PO021: Outcomes of Splenectomy in Patients with Non‐Hodgkin Lymphomas Ccomplicated by Immune Thrombocytopenia

Y. Yevstakhevych 1 ; I. Yevstakhevych 1 ; Y. Vyhovska 2 ; M. Semerak 1 ; O. Petronchak 3 ; V. Loginsky 4

1 SI ‘ Institute of Blood Pathology and Transfusion Medicine NAMS of Ukraine’, General and Heamatology Surgery, L'viv, Ukraine, 2 SI ‘ Institute of Blood Pathology and Transfusion Medicine NAMS of Ukraine’, Heamatology, L'viv, Ukraine, 3 Lviv State Oncology Regional Treatment and Diagnostic Center, Pathology, L'viv, Ukraine, 4 SI ‘ Institute of Blood Pathology and Transfusion Medicine NAMS of Ukraine’, Genetics of Blood Tumors, L'viv, Ukraine

Background: Diffuse large B‐cell lymphoma (DLBCL) and splenic marginal zone lymphoma (SMZL) can be associated with immune thrombocytopenia (ITP) and massive splenomegaly. If chemotherapy is ineffective in such patients, splenectomy is indicated.

Aims: To analyze the efficacy of slenectomy in patients with DLBCL and SMZL complicated by ITP.

Methods: 31 patients with DLBCL and 44 patients with SMZL underwent splenectomy. Among them, 10 (32.2%) patients with DLBCL and 21 (47.7%) patients with SMZL had a concomitant ITP (platelet count ranging from 10.0‐100.0 × 10 9 ).

Results: Spelenectomy proved to be effective in all patients SMZL with ITP: normalization of platelet count and a complete remission were reached. A post‐surgical survival for more than 3 years was stated in 5 (23.8%) patients with SMZL and ITP, and a survival for more than 5 years ‐ in 11 (52.4%) patients. Spelenectomy was effective in 5 (45.4%) patients with DLBCL and ITP. One patient died on the 4th day following surgery due to acute cardiovascular insufficiency. In 3 (27.3%) patients with DLBCL and ITP, spelenectomy was ineffective: a relapse of lymphoma and thrombocytopenia occurred within 1 month after surgery. The 3‐year post‐surgical survival was observed in one case only.

Conclusions: 1. Splenectomy is an effective treatment option in SMZL complicated by ITP.

2. Short‐term and long‐term surgical outcomes in DLBCL with ITP are less promising so that indications for surgery need to be more restricted.

PO023: Deficiency Acquired in Factor X Revealing an AL Amyloidosis

Y. Ouarhlent ; H. Hamza

Faculty of Medicine, Heamatology, Batna, Algeria

Background: Deficiency Acquired in factor X is manifested by sometimes severe bleeding: epistaxis, haemarthrosis and gastrointestinal bleeding. The circulating FX level seems to be correlated with hemorrhagic symptomatology the acquired deficit in FX is rare and usually related to the presence of amyloidosis. The acquired FX deficiency occurs in about 13% of patients with mild‐chain amyloidosis (AL amyloidosis).

Aims: To think about amyloidosis with a factor X deficiency.

Methods: We report a case of Factor X deficiency diagnosed in anesthesia consultation.

Results: A 59‐year‐old woman was hospitalized in November 2017 for haemorrhages on uterine fibroma The clinical examination revealed a cutaneous paleness with metrorgias. microcytic anemia at 8 g / dL hemoglobin, mean median volume at 56 fl, hyperplatelet at 611 G / L, ferritin at 4 ng / L, prothrombin (PT) at 18% She received vitamin K for 3 days, assay factors VII 74% and ×5%. The serum albumin was 19 g / L, with electrophoresis of the protins a monoclonal peak in the beta‐globulin zone immunofixation monoclonal immunoglobulin IgA lambda (blood level of IgA at 15 g / L). 24‐hour proteinuria 2.5 g without Bence Jones proteinuria, myelogram, 7% plasma cells, salivary gland biopsies, congo red staining showed amyloid deposits there was no clinical, biological or radiological manifestation in favor of the involvement of other organ The diagnosis was AL amyloidosis with factor X deficiency.

Conclusions: FX deficiency in patients with Amyloidosis may be associated with bleeding that can compromise prognosis vital.

PO024: An Audit of Acquired Clotting Defects in Hematology Clinics in Karachi

A. Naz 1,2 ; F. Ahmed 1 ; T.S. Shamsi 3

1 National Institute of Blood Disease & Bone Marrow Transplantation, Thrombosis and Hemostasis, Karachi, Pakistan, 2 Liaquat Unversity of Medical and Health Sciences, Thrombosis and Hemostasis, Karachi, Pakistan, 3 N. M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Clinical Hematology, Karachi, Pakistan

Background: Coagulation tools are widely applied in the inherited bleeding disorders in Pakistan. Many acquired bleeding disorders are associated with different diseases according to published literature but very few reports are documented acquired clotting defects in hematological disorders which cause sometimes life threatening or death.

Aims: To investigate the acquired clotting defects in hematological disorders.

Methods: The data was collected through patient's record by predesigned questionnaire after approval of IRB and patient's consent in this cross sectional study of last year, 2017 from NIBD And BMT, Karachi. Total 340 Symptomatic like bruises and bleeding, admitted and out Patient's were screened for coagulation screening.

Results: Mostly were adult with age range (2.0‐80 Years) and males (194) predominant. Acute Myeloid Leukemia (148), Acute Lymphoblastic leukemia (49), Acute Promyelocytic Leukemia (23), Acute leukemia(10), Beta Thalassaemia Major(22), Idiopathic Thrombocytopenic Purpura (20), Chronic Myeloid leukaemia(16), Fanconi's anaemia(11), Lymphoma(8), Multiple Myeloma(03), Myelodysplastic Syndrome(07), Post Bone Mone Marrow Transplantation(8), others included Chronic Liver Disease, Paroxysmal Nocturnal Hemoglobinuria, Essential Thrombocythemia, Myelofibrosis, Budd chiari syndrome and Pure Red Cell Aplasia(15) were labeled as coagulation defects.

Conclusions: Mostly were adult with age range (2.0‐80 years) and males (194) predominant. Acute Myeloid Leukemia (148), Acute Lymphoblastic leukemia (49), Acute Promyelocytic Leukemia (23), Acute leukemia(10), Beta Thalassaemia Major(22), Idiopathic Thrombocytopenic Purpura (20), Chronic Myeloid leukaemia(16), Fanconi's anaemia(11), Lymphoma(8), Multiple Myeloma(03), Myelodysplastic Syndrome(07), Post Bone Mone Marrow Transplantation(8), others included Chronic Liver Disease, Paroxysmal Nocturnal Hemoglobinuria, Essential Thrombocythemia, Myelofibrosis, Budd chiari syndrome and Pure Red Cell Aplasia(15) were labeled as coagulation defects.

PO025: A Rare Case of Factor II Deficiency Overlooked due to Anaemia in Hematology Clinic

A. Naz 1,2 ; T. Shamsi 3

1 National Institute of Blood Disease and Bone Marrow Transplantation, Thrombosis and Hemostasis, Karachi, Pakistan, 2 Liaquat Unversity of Medical and Health Sciences, Thrombosis and Hemostasis, Hyderabad, Pakistan, 3 National Institute of Blood Disease and Bone Marrow Transplantation, Clinical Hematology, Karachi, Pakistan

Background: Anaemia in adult male is most common symptom which is dealing in haematology clinic. Betel‐nuts, betel leaf and Gutka is common in adult males in Karachi.

Aims: To investigate the cause of bleeding after bone marrow biopsy for the workup of long standing chronic anaemia.

Methods: A 24 years old male presented with complains of long standing chronic anaemia. According to his social history, he was addict of gutka, betel nuts and leaf. Clinician prescribed routine workup for anaemia and bone marrow biopsy. After 1 day patient patient came again with bleeding at the site of bone marrow biopsy and hematoma. Clinician examined his wound and investigated for the bleeding complains.

Results: His lab investigations were; Hemoglobin (7.0 g/dl), Red Cell Count (2.6 × 10 12 /L), rest of the red cell indices (Normal), markedly prolonged Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT).After mixing studies PT of patient's plasma with normal and aged plasma was corrected. Factor II level was 9%. No family history or previous bleeding complains were recorded in his file.

Conclusions: Factor II deficiency is a very rare blood clotting disorder. Different types of anaemia are common in Pakistan due to socioeconomic and hygienic constraints.

PO027: Challenges and Key Learnings in the Design and Implementation of an International Hemophilia Registry with Pharmaceutical Industry Support

C.R.M. Hay 1 ; M. Shima 2 ; M. Makris 3 ; V. Jiménez‐Yuste 4 ; J. Oldenburg 5 ; K. Fischer 6 ; A. Iorio 7 ; M.W. Skinner 8 ; E. Santagostino 9 ; S. von Mackensen 10 ; N. Church 11 ; S. Rauchensteiner 12 ; P. Mathew 11 ; S. Krassova 12 ; C.M. Kessler 13

1 Manchester University Department of Haematology, Manchester, United Kingdom, 2 Nara Medical University, Kashihara, Japan, 3 Department of Infection, Immunity, and Cardiovascular Disease, University of Sheffield, Sheffield, United Kingdom, 4 Autónoma University Madrid and La Paz University Hospital, Madrid, Spain, 5 Department of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany, 6 Van Creveldkliniek, University Medical Center Utrecht, Utrecht, the Netherlands, 7 Department of Health Research Methods, Evidence, and Impact and Department of Medicine, McMaster University, Hamilton, ON, Canada, 8 Institute for Policy Advancement, Ltd, Washington, DC, United States, 9 IRCCS Ca’ Granda Foundation, Maggiore Hospital Policlinico, Milan, Italy, 10 University Medical Centre Hamburg‐Eppendorf, Hamburg, Germany, 11 Bayer, Whippany, NJ, United States, 12 Bayer, Basel, Switzerland, 13 Georgetown University Medical Center, Washington, DC, United States

Background: The Expanding Communications on Hemophilia A Outcomes (ECHO) study was initiated as a prospective, 5‐year, observational cohort study to explore the association of clinical and sociodemographic aspects with patient‐reported outcomes (PROs) in patients aged ≥16 years with moderate/severe hemophilia A irrespective of factor VIII products used. The study was terminated at the end of 2017.

Aims: We report challenges encountered and lessons learned from the development and implementation of this international registry.

Methods: ECHO was planned to collect data from 2000 patients in 9 countries, at baseline and annually for ≥2 years. Clinical data were collected by site staff and 13 PRO instruments (standardized or ad hoc patient surveys) were completed by patients with hemophilia A. The Steering Committee and Project Team analyzed causes for the delayed start and early study closure.

Results: 271 patients enrolled from 1/12/2015 (UK, n=146; Japan, n=76; Spain, n=25; US, n=24). Study initiation was delayed by complex negotiations with contracting parties (eg, need for PRO licenses), long local institutional approval processes, and diversity of bleeding diaries. The pharmacovigilance required of a commercial sponsor, as opposed to a commercially funded investigator‐led study, added to the issues. Investigators and patients objected to the burden of long clinician and patient case report forms. Some ethics committees classified the study as interventional (Figure). Competing studies also adversely affected recruitment. A low return rate of PRO instruments, missing in ˜40% of patients, did not improve when completion time increased to 6 months.

Conclusions: The study was terminated early due to administrative delays in starting it, challenges posed by its global reach, the burden of 13 PROs, and competing studies. An investigator‐led study with narrower research objectives, 1 patient diary, fewer PROs, and earlier and wider patient involvement in the study design, may have been more successful.

FIGURE 1 Major Challenges Faced in the ECHO Study

PO028: Hereditary Afibrinogenemia in Children: A Single Center Experience

M. Söker 1 ; H. Uzel 1 ; K. Öncel 1 ; S. Söker 2

1 Dicle University, Pediatric Hematology and Oncology, Diyarbakır, Turkey, 2 Dicle University, Histology and Embriology, Diyarbakır, Turkey

Background: Fibrinogen is the key glycoprotein for maintaining hemostatic balance and providing coagulation. Congenital afibrinojenemia is a rare autosomal recessive hemorrhagic diathesis characterized by the absence of fibrinogen of very low fibrinogen values below detectable levels. Its clinical manifestations vary according to severity. The most frequent hemorrhages associated with severe fibrinogen deficiency are umbilical cord bleeding, hemarthrosis, mucosal bleeding, muscle hematoma, gastrointestinal bleeding, epistaxis and menorrhagia.

Aims: In this article, four cases of congenital afibrinogenemia were presented. They are discussed according to rarity of disease, clinical, laboratory and treatment approaches of afibrinogenemia.

Methods: The diagnosis ages of our cases were 2 days, 11 months, 7 years and 12 years. Two of them were brothers. The gender of our 11‐month‐old patient and 12‐year‐old patients are female. All patients’ parents were first degree consanguineous union. Complaints during the admission were umbilical cord bleeding, bleeding after head trauma and mucosal bleeding. They were found to have a prolonged prothrombin time and activated partial thromboplastin time with undetectable low levels of fibrinogen. By excluding other causes, they were diagnosed as congenital afibrinogenemia.

Results: They were treated with plasma‐derived fibrinogen concentrate (Haemocomplettan)(50 mg/kg) or fresh frozen plasma. In our cases, three of them are on demand treatment and one of them is prophylaxis treatment.

Conclusions: Congenital afibrinogenemia is an uncommon coagulation disorder usually discovered during childhood. Congenital afibrinogenemia is associated with mild‐to‐severe potentially life‐threatening bleeding. Replacement therapy is effective in treating bleeding episodes, and the aim is to increase the fibrinogen level to 1 g/L. Secondary prophylactic treatment may be concidered after life‐threatening bleeding.

PO029: Paradoxical Thrombotic Events in Bleeding Disorders

L. Stanciakova 1 ; M. Dobrotova 1 ; P. Holly 1 ; J. Ivankova 1 ; M. Samos 2 ; T. Bolek 2 ; P. Kubisz 1 ; J. Stasko 1

1 Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, University Hospital Martin, National Centre of Thrombosis and Haemostasis, Department of Haematology and Transfusiology, Martin, Slovakia, 2 Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, University Hospital Martin, Department of Internal Medicine I, Martin, Slovakia

Background: In spite of the haemorrhagic symptoms of bleeding disorders of haemostasis, they do not prevent the effected individuals from the risk of the thrombosis. Dominant factors leading to the development of thrombosis in these patients include the traditional risky clinical states and characteristics, such as arterial hypertension, smoking, hyperlipidaemia, prolonged immobilization, surgery, advanced age, pregnancy, and last but not least the coexistence of the thrombophilic state, substitution treatment and the administration of by‐passing agents.

Aims: To present the cases of the patients with bleeding disorder and development of thrombotic events. Simultaneously, we show the successful clinical management of their challenging clinical situation.

Methods: We describe clinical characteristics, results of laboratory parameters and treatment in the subjects with bleeding coagulopathy experiencing the thromboembolic events.

Results: In presented cases, the treatment with antithrombotic drugs was used without any complications.

Conclusions: Thrombosis may be developed in the patients with bleeding disorder of any age, as reported in the literature. In the case of the concomitant presence of several prothrombotic risk features, the risk of thrombosis increases. Unfortunately, in general, large studies supporting the development of universal guidelines for such clinical states are missing. Thus, the combined use of the substitution and antithrombotic treatment ought to be individualized.

The project complies with the Declaration of Helsinki and the informed consent in each patient is obtained. The study was approved by a recognized medical ethics committee.

Acknowledgements: We would like to thank the support of projects of Scientific Grant Agency Vega 1/0168/16 and Agency for the Support of Research and Development APVV‐16‐0020.

COAGULATION FACTORS

PO030: Study the Polymorphism of Tumor Necrosis Factor Alpha (TNF‐α) Gene and the Risk of Development of FVIII Inhibitors in Egyptian Hemophilia A Patients: A Single Center Experience

D. El Demerdash 1 ; A. Ayad 1 ; D. Mohamed 2 ; M. Mattar 1 ; H. Sadek 2

1 Cairo University, Kasr Al Ainy School of Medicine, Internal Medicine, Clinical Hematology Unit, Cairo, Egypt, 2 Cairo University, Kasr Al Ainy School of Medicine, Clinical and Chemical Pathology, Cairo, Egypt

Background: The Development of inhibitors against Factor VIII in hemophilia A patients is the most sever and challenging complications to occur, which exponentially increases the treatment cost. Many risk factors have been studied to predict risk of inhibitor development. polymorphism in many immune related genes were thought to increase the risk of factor VIII Inhibitor development. one of them is TNF‐α gene polymorphism (‐308G/A) which has been reported in many disorders such as autoimmune disease and malignancies.

Aims: That's way we aimed for studying risk factors associated with FVIII inhibitors development including the TNF‐α gene polymorphism in cohort of Egyptian hemophilia patients.

Methods: We investigated the TNF‐α gene polymorphism (‐308G/A) in Egyptian adults with moderate and sever hemophilia ( n :50) with long history of FVIII replacement therapy through history, physical examination, laboratory tests including CBC, PTT, FVIII assay, FVIII assay by original Bethesda method, genotyping of TNF‐α gene polymorphism (‐308G/A) bt PCR‐RFLP.

Results: We found FVIII inhibitors in 50% (n:25) of our studied cases, also we found mutant TNF‐α gene GG/AG in 58% in our hemophilia patients (n;29). But, We didn't find any significant correlation between development of factor VIII inhibitor which found in (50% of our studied cases) and other studied risk factors especially (age of 1st exposure, family history of FVIII inhibitors, severity of FVIII deficiency, frequency of exposure, prophylaxis strategy, type of replacement FVIII and genotyping of TNF‐α gene polymorphism (‐308G/A).

Conclusions: Our observations suggest TNF‐α gene polymorphism (‐308G/A) has no relation with the development of FVIII inhibitors in Egyptian patients with hemophilia A.

PO031: Assessment of Some Markers of Thrombosis in a Nigerian Population of Normal Pregnant Women

H. Okoye 1 ; T. Nwagha 1 ; A. Ugwu 1 ; L. Eweputanna 2 ; V. Ugwu 3

1 University of Nigeria/University of Nigeria Teaching Hospital, Haematology and Immunology, Enugu, Nigeria, 2 Abia State University Teaching Hospital, Radiology, Aba, Nigeria, 3 University of Nigeria/University of Nigeria Teaching Hospital, Obstetrics and Gynaecology, Enugu, Nigeria

Background: There is increasing evidence of thromboembolic events in pregnancy in the Nigerian population. With physiologic changes in pregnancy favoring thrombosis, there is need to document levels of these markers in our normal pregnant women for further evaluation and early intervention if need be.

Aims: To determine the plasma concentrations of protein S and protein during pregnancy and correlate values with age, gestational age (GA) and blood group types.

Methods: A cross‐sectional study of normal pregnant women who had their ABO and Rh D blood groups determined and then plasma concentrations of free protein S (fPS), protein C antigen (PCAg) and protein C activity (PCAc) measured using ELISA and Protac methods respectively. Data was analysed using SPSS version 21.

Results: Eighty subjects at a GA of 25‐42 weeks (mean 35.4 ±5.2) were recruited with mean age, systolic and diastolic blood pressures of 30.4 ±5.1, 110.8±12.5 mmHg and 66.6±8.7 mmHg respectively. Forty eight (60.0%) and 32 (40.0%) of the women were of O and non‐O blood group, 76 (95.0%) and 4 (5.0%) were Rh D positive and negative respectively. The mean levels (and range) of fPS, PCAg and PCAc were 47.2 ±10.3 (27.1‐63.1), 77.5 ±23.2 (30.9‐131.9) and 110.4 ±27.6 (58.9‐177.1) respectively. There was no difference in the concentrations of these proteins when compared between the blood group types. PCAg and PCAc correlated significantly with GA groups (r=0.229, P=0.041; r=0.223, P=0.046) while there was a significant correlation between fPS and age (r=0.254, P value=0.023), as shown in Tables 1 & 2.

Conclusions: Although PCAg and PCAc levels increased with increasing GA groups, their mean concentrations in pregnancy remained within normal reference range while fPS levels in pregnancy were below the normal reference value but increased significantly with maternal age. Hence GA and maternal age should be considered when evaluating PCAg, PCAc and fPS levels in pregnant women for increased risk of thromboembolic events.

PO032: Evaluation of Morbidity and Mortality among Patients with Hereditary Bleeding Disorders in Southern Iran

M. Karimi ; A. Farjami; S. Hagpanah; S. Parand

Shiraz University of Medical Sciences, Hematology Research Center, Shiraz, Iran, Islamic Republic of

Background: Advance in medical care of hereditary bleeding disorders (HBD) especially factor replacement therapy and management of disease led them to increase life expectancy, so they experience variety of age‐related morbidity.

Aims: Morbidity and mortality rates were evaluated in patients with HBD in a sample of the Iranian population in one of the largest referral centers in Southern Iran.

Methods: In this cross‐sectional study, 649 patients with HBD were evaluated during 1996‐2016 (a 20 year experience). Aside to baseline characteristics and type of factor deficiency, associated morbidities including viral infections, neurological disorders, asthma, thalassemia, G6PD deficiency, diabetes, hypertension cardiac and renal disease were evaluated. Furthermore, among patients who died, underlying disease and etiology of death were also evaluated.

Results: Mean age of the patients was 24.4±13.5 years old. Factor VIII deficiency was the most prevalent type (50.4%) of coagulation factor deficiency and FVII deficiency (23%) was the most prevalent of rare bleeding disorders.

A total of 0.5% had hepatitis B and 11.5% had hepatitis C. Cardiac disease was seen in 1.5%, hypertension in 0.2%, renal disease in 0.2%, and diabetes in 1.3% of patients. In total, 5.2% of patients had intra cranial hemorrhage, 2.1% had epilepsy and 0.8% had mental retardation. Both asthma and G6PD deficiency were observed in 0.5% of patients. During the 20 year study period, 25 patients died and car accident was the leading cause of death in this population.

Conclusions: Prevalence rate of morbidities was 24.3% in the patients with hereditary bleeding disorders. The most prevalent morbidity was HCV infections (11.5%) underscoring viral monitoring must be performed precisely. Mortality rate among patients with HBD was 3.8% and the most common cause of death was accidents, which is similar to that of the normal Iranian populations showing road safety is a crucial issue in Iran.

PO033: Acquired Deficit in Coagulation Factors and Head Trauma

Y. Ouarhlent ; H. Hamza

Faculty of Medecine, Haematology, Batna, Algeria

Background: Acquired factor VII deficiency is a rare condition whose cause often remains unclear even if certain circumstances of occurrence are well established. The clinical manifestations are very heterogeneous with asymptomatic forms or on the contrary associated with severe haemorrhagic complications for the same level of factor VII. Repeated dosing of coagulation parameters during head trauma may reveal an isolated deficit in coagulation factor and therefore early therapeutic management.

Aims: The aim is to make general practitioners and specialists aware of the importance of determining coagulation factors in the event of abnormalities in the assessment of hemostasis.

Methods: We report a case of acquired, transient and isolated factor VII deficiency (FVII).

Results: We report a case of acquired, transient and isolated factor VII deficiency (FVII) in a polytraumatic. after a fall from a height of 25metres. the scaner had shown a 3 cm intracerebral hematoma with multiple furrows (costal, tibial and ulnar‐radial). on admission a hemostasis report was made among others The prothrombin rate which was normal. Within a few hours, deep abnormalities of coagulation appeared low TP 15%, coagulation factor assays Factor VII at 2% No inhibitor of FVII, the patient received blood transfusions erythrocyte pellets, fresh frozen plasma and vitamin K but without improvement. The deficit was successfully managed by the daily administration of recombinant FVII, for 2 weeks the control hemostasis balance showed a normalization of coagulation parameters.

Conclusions: In front of a disturbed hemostasis assessment in a traumatized person; think about the acquired deficiencies in coagulation factors and consequently the dosage of these.

PO034: Methods of Preparation of Blood Coagulation Factor VIII Concentrate

N. Shurko 1 ; T. Danysh 1 ; V. Novak 2

1 State Institution ‘Institute of Blood Pathology and Transfusion Medicine NAMS of Ukraine’, Laboratory Biochemistry of Blood, Lviv, Ukraine, 2 State Institution ‘Institute of Blood Pathology and Transfusion Medicine NAMS of Ukraine’, Hematology, Lviv, Ukraine

Background: The all bleeding disorders are due to defects in the blood vessels, the coagulation mechanism, or the blood platelets and is a complication of many hematologic diseases. Hemophilia A is recessive disorder caused by deficiency of functional plasma clotting factor VIII (FVIII). The treatment of which consists in the introduction of concentrates of FVIII.

Aims: To create a scheme for obtaining a highly purified concentrate of the blood coagulation FVIII.

Methods: Adsorption/precipitation, ion‐exchange chromatography on DEAE‐ Sepharose, affinity chromatography on the Diasorb‐aminopropyl matrix with Active purple 4GT as ligands in combination methods of antiviral treatment.

Results: It was determined that the pre‐fractionation with barium citrate, aluminum hydroxide (III), and PEG‐4000, provides removal of factors of prothrombin complex, fibrinogen, fibronectin, lipoproteins, denatured proteins, albumin etc. At this stage, we have reached hundredfold purification of the factor VIII preparation (from 0.017±0.001 IU/mg protein to 1.88±0.11 IU/mg protein).

It has also been demonstrated that the use of ion‐exchange chromatography on DEAE‐Sepharose and affinity chromatography allows obtaining FVIII from Cryoprecipitate with a degree of purification to 242 times and preservation of up to 73% of the initial procoagulant activity. The main losses from the initial activity of FVIII and the von Willebrand factor (up to 27%) occurred at the stage of ion‐exchange chromatography. The phenomenon of negative affinity sorption provides almost 100.0% (96.34%) yield of the product.

It has been found that the combination of pre‐fractionation, ion‐exchange and affinity chromatography stages provides a purification rate of 700.0 times.

Conclusions: We developed schemes of plasma fractionation and reached a high degree of purification of FVIII coagulation.

PO035: Experience in our Center of Anticoagulant Therapy with Tinzaparina Sodique in Patients with Cancer, Renal Insufficiency and / or Intolerance to Antivitamin K

M.A. Garcia Ruiz 1 ; E. Morente Constantin 1 ; P. Romero Garcia 2 ; M. Rivas Luque 1 ; P. Garrido Collado 1 ; M.D. Fernandez Jimenez 1 ; M. Jurado Chacón 1

1 Hospital Universitario Virgen de las Nieves, Granada, Spain, 2 Complejo Asistencial de Soria, Soria, Spain

Background: The low molecular weight heparins (LMWH) are typically administered as thromboprophylaxis or as therapy. Monitoring could be considered in certain situations (renal failure, pregnant women). The anti‐factor Xa activity (a‐f Xa) has been proposed to monitor its effect. The therapeutic range of a‐f Xa is between 0.6 and 1 IU/mL when LMWH is administered every 12 hours. At single daily it seems that lies above 1 IU/mL.

Aims: The main aim is to evaluate the efficacy of tinzaparin sodium at therapeutic doses in preventing VTE in patients with renal failure, cancer and/or intolerance to antivitamin k (AVK). Hemorrhagic or thrombotic complications and other possible side effects have been assessed.

Methods: We have analyzed 120 patients, 67 men and 53 women, aged between 27 and 95 y.o. which have received tinzaparin sodium. The main reasons of anticoagulation are atrial fibrillation/flutter, VTE, stroke/TIA and prosthetic valves. There were 5 resistances and 5 allergic reactions to AVK. 10 were pregnant and 23 had renal failure. 42 patients had active cancer or were in remission. 1 with MDS was treated with LMWH because he had intra‐extrahepatic portal vein thrombosis. All the patients had received 175 IU/Kg of Tinzaparin Sodium once a day as initial dose, then the dose was adjusted according to the a‐f Xa levels.

Results: In terms of side effects, 20 patients presented complications: 6 mucosal bleeding, 4 strokes, 4 hemoptysis, 2 DVT and 4 bleeding at the puncture site. When these complications occurred, we proceeded to the corresponding dose adjustment based on new determinations of a‐f Xa.

Conclusions: The results obtained confirm the efficacy, safety and cost‐effectiveness of the use of LMWH. Determination of a‐f Xa levels are considered very useful for dose adjustment and tinzaparin sodium has proved to be effective in preventing VTE. The results obtained have demonstrated that tinzaparin is safe and further studies will confirm our results.

PO036: Diagnosis of a Transient Ischemic Attack (TIA) from Elevated Coagulation Inhibitors (A Case Report)

E. Vagdatli ; M. Issa; K. Bani; K. Tsioni; K. Karantani; A. Diamantopoulou

Hippokration General Hospital, Laboratory of Biopathology, Thessaloniki, Greece

Background: During thrombophilia testing the natural coagulation inhibitors deficiencies (Antithrombin III‐ATIII, Protein C‐PC, Protein S‐PS) are usually evaluated, while their high levels are neglected.

Aims: To report a case in which the observation of high levels of ATIII, PC and PS led to the diagnosis of a mild TIA.

Methods: A 52‐year‐old woman presented at the hospital with a temporary loss of consciousness and dizziness. Complete blood cell count (CBC), biochemical routine examination, prothrombin time (PT) and a thrombophilia screening test were ordered.

Results: The CBS was normal. The biochemical examination showed an abnormal lipid profile (Table I). PT‐Ratio was 0.86. The thrombophilic profile is shown in Table II. Particular emphasis was provided to the elevated levels of ATIII, PC and PS which led to the thought that the patient was trying to cope with its coagulation cascade activation. Thus, the laboratory proceed to the measurement of activated partial thromboplastin time (APTT), fibrinogen (Fb) and DDimers, the values of which indicated coagulation activation: APTT‐ratio:0.82, Fb:561 mg/dl and DDimers:559 ng/ml (n.v.<500). To exclude an increase in the fibrinogen level due to a potential inflammation, CRP was measured, which was within normal ranges (1.1 mg/l). These laboratory findings revealed an activation of the patient's coagulation. A brain M.R.I and a carotid artery triplex ultrasound were performed. No pathological findings were presented. The symptoms were attibuted to a TIA and the patient started a hypo‐lipidemic diet and was treated with folic acid.

TABLE 1 The patient's lipid profile Cholesterol Triglycerides HDL LDL 256 mg/dl 206 mg/dl 53 162

TABLE 2 The patient's thrombophilia screening test ATIII (n.r. 75‐125%) PC (n.r. 70‐140%) PS (n.r. 60‐114%) Factor V‐Leiden mutation Lupus anticoagulant Serum homocysteine (n.v.<13 µmol/L) >122% >150% >149% Not detected Not detected 25 µmol/L

Conclusions: The elevated levels of the coagulation inhibitors are as important as reduced ones. Therefore, their measurement has a dual utility and clinical importance: 1) to detect an inherited deficiency and 2) to be used as coagulation activation markers, the elevated levels of which suggest a state of coagulation activation by triggering a cascade of events.

PLATELETS

PO039: Platelet Transfusion Triggers in Thrombocytopenic Patients in a Tertiary Care Hospital

R. Akbar 1 ; A. Ahmad 1 ; N. Akbar 2 ; S. Yunis 3

1 Health Ways Laboratories Pakistan, Haematology, Rawalpindi, Pakistan, 2 PAF Hospital Faisal base Karachi, Pathology, Karachi, Pakistan, 3 Fauji Foundation Hospital, Haematology, Rawalpindi, Pakistan

Background: The platelet transfusion therapy plays an important role in the support of haematological, oncological, surgical and transplant patients. Prophylactic platelet transfusions are the standard of care for malignancy or chemotherapy/radiation‐induced thrombocytopenia while therapeutic platelet transfusions are commonly used in the postoperative period after bypass heart surgery for platelet dysfunction. Various guidelines are available for optimal platelet transfusion triggers in medical and surgical patients.

Aims: This study aimed at defining the platelet transfusion triggers in thrombocytopenic patients in medical setting in a tertiary care hospital.

Methods: The study was conducted at the Department of Hematology, Fauji Foundation Hospital Rawalpindi Pakistan. A total of 93 were included in the study, of which 57 (61%) were females and 36 (38%) were males. Patients receiving platelet transfusions in surgical setting were excluded from this study. Prior written informed consent was taken and patient characteristics were registered on the proforma.

Results: A total of 225 random donor platelet transfusions were given to 93 patients. Leukaemia being the most common cause of thrombocytopenia counted for 54.83% (n=51) of patients, while rest of them had dengue, decompensated chronic liver disease and other medical conditions. In 74% (n=69) patients who received prophylactic platelet transfusion the pre transfusion platelet count was<20,000/cmm while 26% (n=24) patients who had therapeutic platelet transfusions had platelet counts between 20,000‐50,000/cmm.

Conclusions: A careful clinical approach tailored for individual patient should be adapted with particular attention to the instant availability of good quality platelet products in emergencies.

THROMBOTIC DISORDERS

PO043: Association of Pregnancy Loss with Some Thrombogenic Markers

H. Okoye 1 ; J. Chinawa 2 ; E. Aniwada 3 ; L. Eweputanna 4 ; T. Nwagha 1 ; U. Nwagha 5

1 University of Nigeria/University of Nigeria Teaching Hospital, Haematology and Immunology, Enugu, Nigeria, 2 University of Nigeria/University of Nigeria Teaching Hospital, Paediatrics, Enugu, Nigeria, 3 University of Nigeria/University of Nigeria Teaching Hospital, Community Medicine, Enugu, Nigeria, 4 Abia State University Teaching Hospital, Radiology, Aba, Nigeria, 5 University of Nigeria/University of Nigeria Teaching Hospital, Obstetrics and Gynaecology, Enugu, Nigeria

Background: Unplanned miscarriages or pregnancy losses (PLs) are usually a source of pain and psychological stress to the expectant couple. Controversies exist as to the contribution of thrombophilia to pregnancy loss and little is known of the association of ABO blood group and deficiencies of protein S and C with PL among Nigerian women.

Aims: This study investigates the association of non‐O blood group, deficiencies of protein S (PS) and protein C (PC) with PL.

Methods: This was a descriptive study where 170 consenting women were recruited and grouped into 3 groups (A, B and C) of women with 0, 1 and >1 history of clinically‐ or ultrasonographically recognisable 1ST/2nd trimester PL (respectively). For all participants, we determined their ABO blood group phenotype using the tile method, plasma concentrations of free protein S (fPS) antigen and protein C antigen (PCAg) by ELISA‐based method and PC activity (PCAc) by PROTAC method.

Results: Although 10 (35.71%) of group C participants were of non‐O blood group, 1 (3.57%), 8 (28.57%), and 1 (3.57%) had deficiency of fPS, PCAg and PCAc respectively, there were no statistically significant associations of blood group (χ 2= 0.534, P=0.465), fPS (FT, P=0.123) PCAg (χ 2= 1.717, P=0.674), and PCAc (FT, P=0.749) with PL (table 1). There was however a poor positive correlation of PL with age (rho=0.08) and parity (rho=0.05), as shown in table 2.

Conclusions: This study suggests ABO blood group phenotypes, fPS, PCAg or PCAc may not play any role in PL. However PL has some positive correlation with age, parity and gravidity. We however recommend this result is confirmed on a larger population of women with PL.

PO044: Transition of Care for Adult Patients with Venous Thromboembolism

M. DeSancho 1 ; M. Malone 2 ; C. Holmes 3 ; H. Billett 4 ; T.‐F. Wang 5 ; D. Cheng 6 ; E. McCarthy 6 ; C. Watson 6 ; M. Rajpurkar 7

1 Weill Cornell Medicine, New York Presbyterian Hospital, New York, United States, 2 Dartmouth‐Hitchcock Medical Center, Lebanon, United States, 3 University of Vermont Medical Center, Division of Hematology and Oncology, Burlington, United States, 4 Montefiore Medical Center of the Albert Einstein College of Medicine, New York, United States, 5 Ohio State University Wexner Medical Center, Division of Hematology, Columbus, United States, 6 American Thrombosis and Hemostasis Network, Rochester, United States, 7 Wayne State University, Children's Hospital of Michigan, Detroit, United States

Background: Transition of care (TOC) from inpatient to outpatient environments for patients with a new diagnosis of venous thromboembolism (VTE) is crucial to minimize anticoagulation errors. The American Thrombosis and Hemostasis Network (ATHN) is conducting a multi‐center quality intervention (QI) project assessing the TOC for patients being discharged after hospitalization with a first diagnosis of VTE.

Aims: To describe VTE characteristics and anticoagulation knowledge for adults enrolled in the ATHN 4 VTE TOC project.

Methods: Adult and pediatric patients hospitalized with 1st episode VTE requiring anticoagulation were eligible to participate. The project has two phases: a pre‐intervention (PI) phase with no change in standard practice, and a QI phase providing enhanced education followed by a phone call 1 week later. Demographics, disease and treatment characteristics and outcomes are collected. Patient knowledge and feedback questionnaires are administered at 30 days post‐discharge. Preliminary adult data from the PI phase are presented.

Results: 129 patients were enrolled. VTE was most frequently diagnosed in patients 41 to 65 years old. The majority of patients 105 (81.4%) were non‐Hispanic. Obesity and immobility were the most common risk factors. Pulmonary embolism (PE) was the most common type of VTE diagnosed in 41/92 patients (44.6%). 50 patients (55%) were discharged on direct oral anticoagulants (Table 1). The majority of patients 82/101 (82.2%) could recall VTE symptoms to watch for, and almost all 95/101 (94%) reported 100% adherence to anticoagulation. Five patients (5%) returned to the emergency department and 7 (7%) were re‐admitted within 30 days of discharge.

TABLE 1 VTE risk factors and anticoagulant therapy prescribed VTE risk factors (Most frequently reported) [N=129] # % Obesity (BMI >30 kg/m 2) 39 / 129 30% Immobility (>72 hours and within 30 days) 15 / 129 12% Surgical Interventions within 30 days 14 / 129 11% Anticoagulant therapy [N=91] Direct Oral Anticoagulant (DOAC) 50 / 91 55% Apixaban 26 / 91 29% Rivaroxaban 20 / 91 22% Dabigatran 4 / 91 4% Non‐DOACs (Warfarin, LMWH, Fondaparinux) 41 / 91 45%

Conclusions: Preliminary findings show that obesity is a major risk factor for VTE and that PE was the most prevalent type of VTE. Although recall of VTE symptoms and adherence were high, 7% of patients were readmitted within 30 days of discharge. Patient characteristics, interventions and relationships to outcomes continue to be studied.

PO045: Janus Kinase 2 (JAK2) and Calreticulin (CALR) Mutations in Pakistani Myeloproliferative Neoplasms Patients: Reporting Two Novel CALR Mutations

M. Rashid ; R. Ahmed; U. Zaidi; T. Shamsi

National Institute of Blood Disease & Bone Marrow Transplantation, Karachi, Pakistan

Background: When evaluating classical MPNs, the detection of JAK 2, CALR , or Myeloproliferative Leukemia Virus Oncogene ( MPL ) mutations confirms the presence of an underlying MPN but their absence does not rule out the possibility.

Aims: The primary aim of this study was to analyze the prevalence of JAK 2 and CALR gene mutations in patients with MPN; secondary was to evaluate the impact of gene mutations on clinical features of MPN at diagnosis.

Methods: Our study population consisted of 90 patients with clinically suspected as MPN (also on BM morphology), 30 PV, 36 ET, 4 PMF and 9 unclassified MPN. Male predominance was found in PV patient 76.6%, ET patient were found in young age group (mean 45 years) and more splenomegaly was observed in PMF patient. JAK 2V617F mutation was analyzed by ARMS‐PCR. CALR mutations were identified by bi‐directional Sanger sequencing.

Results: CALR mutations was not found in PV, in ET group frequency of CALR and JAK 2 mutations were same 24.2%. CALR mutant ET was associated with younger age (mean 45 year), higher platelet counts (mean=1,251 × 10 9 /L), lower leukocyte counts (mean=11 × 10 9 /L) and also lower haemoglobin (median=1.7 g/dL) in comparison with JAK2 V617F‐positive ET. More splenomegaly was also observed in CALR mutated ET. In this series11 patient had both coexistence of CALR exon 9 mutation c.1214_1225;p.E405_D408del with JAK2V617F in ET.21 patient's marrow status show myelofibrosis, BCR/ABL gene fusion by Fluorescence in‐situ hybridization (FISH) was positive in 20% cells and CALR sequenced show 52 bp deletion.31 patient was presented with pancytopenia and on marrow diagnosed as PMF show a novel homozygous mutation c.1139delA;p.E380 fs50*.41 patient of ET shows 10 bp deletion (c.1120_1129del) along with 28 bp deletion. This patient was 60 years old male but show clinically worst prognosis and expired within 2 years of diagnosis.

TABLE 1 Total MPN patients 90 PV (30) ET (36) PMF (15) U‐MPN (9) Age at diagnosis in years, (mean + SD) 23 (76.6) 45±17 45 ±19.1 53±20.4 Haemoglobin (g/L), (mean + SD) 17±2.5 45±17 45 ±19.1 53±20.4 White Cell Count (×109/L), mean 17.9±12.6 12.4±6.6 9.8±7.7 36.2±30.5 Platelet count (×109/L), (mean +SD) 458±2.5 1,148±439 112±92 695±506 Splenomegaly, (%) 5/30 (16.6) 10/35 (28.5) 12/13 (92) 6/9 (66.6) Hepatomegaly, (%) 2/30 (6.6) 3/35 (8.5) 3/13 (23) 3/9 (33.3) JAK2V617F mutation, Mutation‐positive/Tested (%) 25/30 (83.3) 8/33 (24.2) 2/9 (22.2) 4/6 (66.6) CALR mutation, Mutation‐positive/Tested (%) 0/29 (0%) 8/33 (24.2) 3/15 (20) 1/9 (11.1) BCR‐ABL1 mutation, Mutation‐positive/Tested (%) 2/15 (13.3) 1/19 (5.2) 2/13 (15.3) 0/6 (0)

Conclusions: CALR positive patients are phenotypically distinct from JAK 2 positive patients. There is need to do allele burden in our cohort.

PO046: Awareness Session for Pharmacist on World Thrombosis Day 13th October 2017 in Hospital of Developing Country Karachi, Pakistan

S. Khan 1 ; S.S. Raza 2 ; U. Rani 1

1 The Aga Khan University Hospital, Pharmacy, Karachi, Pakistan, 2 The Aga Khan University, Karachi, Pakistan

Background: Pakistan is developing country with fragile health system. Knowledge about thromboprophylaxis is limited among pharmacist. Previous surveys about knowledge and awareness about thromboprophylaxis suggest that there is need to create awareness among pharmacist for their valuable contribution in health care.

Aims: To create awareness about significance of Thrombosis and Risk in hospital settings and role of health care provide in prevention of venous thromboembolism.

Methods: Department of Continuing Professional Education (DCPE) of hospital was contacted to plan this activity. Also approval gained from American association continuing medical education AACME to celebrate world thrombosis day and CME credit hours provided to all physicians who attended. One hour CME arranged with questions and answer session.

Results: Session was well attended by Pharmacist, nurses and physicians. Session was appreciated by hospital Pharmacist.

Conclusions: More session of such nature will be planned in future to arrange in different hospital and pharmacy school to create awareness among professional and ungraduated students.

PO047: A Quality Audit on Appropriate Dosing of Rivaroxaban in Acute Care Hospital

S. Khan 1 ; B. Moiz 2 ; A. Khalil 1 ; A. Bilal 1

1 Aga Khan University Hospital, Pharmacy, Karachi, Pakistan, 2 Aga Khan University Hospital, Heamatology, Karachi, Pakistan

Background: Rivaroxaban is the only registered newer oral anticoagulant in Pakistan. It is recently added to the hospital formulary at Aga Khan University Hospital. Despite this, hospital pharmacy had a tablet size of 10 mg only. During routine clinical rounds by pharmacist, it was observed that 15 mg dose was administered by splitting 10 mg film‐coated tablet. Film coated tablets cannot be spilt as per pharmacokinetic of drug formulation. This unsafe practice was placing patients for over or under‐dosing with potential risk of bleeding or thrombosis.

Aims: This was a clinical audit that aimed at observing the unsafe dosing practices of splitting film‐coated Rivaroxaban to admitted or discharged patients.

Methods: Six months data (during July to December 2017) was evaluated retrospectively for appropriate dosing practices. Information was retrieved from computerized physician order entry system (CPOE). Quality improvement tool (PDSA cycle) was applied to review the data. Cause and effect diagram was made to identify possible causes of audit findings and to improve usage of Rivaroxiban.

Results: A total 526 Rx orders for 172 patients for Rivaroxaban was reviewed by a team of hospital pharmacists. Of 172 patients, 128 (74%) were on generic Rivaroxaban and remaining (26%) received Bio similar. Of 128 patients on generic brand, 8, 86, 32 and 2 patients received 20, 15, 10 and 5 mg respectively. Team noticed that for dispensing 10 mg dose, the tablet was split into half to make up the dose of 15 mg. Moreover, 60 of 80 patients (75%) were discharged with instruction of splitting Rivaroxaban at home. Based on above findings, hospital pharmacy initiated procuring and dispensing of 15 mg Rivaroxaban for patients. A re‐audit is needed to observe consistency in practices.

Conclusions: Splitting of film‐coated tablets is not allowed as it will decrease efficacy and potency of drug. High‐risk drugs should be cautiously administered during hospitalization or at the time of discharge.

PO048: Platelet Count and Mean Platelet Volume of Diabetic Clients Attending Nigeria Navy Hospital Calabar, Nigeria

D.C. Okpokam 1 ; C.O. Opara 1 ; O.E. Okpokam 2 ; A.O. Emeribe 3

1 University of Calabar, Medical Laboratory Science (Haematology Unit), Calabar, Nigeria, 2 FHI 360 International, Prevention Care and Treatment, Uyo, Nigeria, 3 Medical Laboratory Science Council of Nigeria, Abuja, Nigeria

Background: Diabetes mellitus (DM) is a group of diseases characterized by an elevated blood glucose level resulting from defects in insulin secretion, in insulin action, or both. The prevalence of DM in Nigeria is estimated of 8%‐10% and 6.5% in Cross River state.

Aims: This was a cross‐sectional study undertaken to evaluate mean platelet volume (MPV) and platelet count (PLT) in patients with diabetes mellitus.

Methods: A total of hundred (100) subjects (males and females) comprising of both diabetic and non diabetic (controls) subjects who were within the ranges of 20‐55 years of age were enrolled in this study. Full automatic blood cell counter and Glucose oxalate method were used to carry out FBC and FBS respectively. Data were analysed using Student T Test using SPSS software (version 22) and Microsoft excel statistical tools for Pearson's correlation coefficient. Significant was expressed as P≤0.05 in compared statistic.

Results: Results showed decrease in PLT (236±9.05 × 10 9 /L vs. 268±5.72 × 10 9 /L), MPV (8.8±1.74 fl vs. 11.0±1.84 fl) as FBS increases (12.2±0.32 mmol/l vs. 4.6±0.06 mmol/L) in diabetics when compared with control subjects and it also revealed in diabetics age range (33‐42) a rise in PLT and MPV when compared with ages 23‐32. The value of PLT (250±8.29 × 10 9 /L vs. 221±16.24 × 10 9 /L) was significantly increased in males compared with the females (P<0.05). A weak correlation was seen in PLT and MPV (r=0.143, 0.125) of diabetic subject when related to FBS while a strong correlation was observed between MPV and PLT (r=0.941) of diabetic subjects (P<0.05).

TABLE 1 Comparison of PLT, MPV and FBS of diabetes subject and control subject Parameters Diabetes (n=50) Control (n=50) T Value P value Platelet (×109/L) 236±9.05 268±5.72 0.004 P<0.05 Mean Platelet Volume (fl) 8.8±1.74 11.0±1.84 0.000 P<0.05 FBS (m mol/L) 12.2±0.32 4.6±0.06 0.000 P<0.05

TABLE 2 PLT, MPV and FBS of Diabetic subject based on gender Parameters Male (n=26) Female (n=24) T Value P Value Platelet (×109/L) 250±8.29 221±16.24 0.012 P<0.05 Mean Platelet Volume (fl) 9.4±1.40 8.6±2.03 0.287 P>0.05 FBS (m mol/L) 12.7±0.48 11.7±0.42 0.163 P>0.05

Conclusions: MPV and PLT were significantly decrease in diabetic patients when compared with the non diabetic (control) and an increase in PLT of male compared with female diabetic subjects. A strong association was observed between MPV and PLT. It is important that MPV and PLT be included as a regular routine screening investigation to monitor for macrovascular diesase in diabetic patients since both test are cheap and less cumbersome.

PO049: Management of Thrombophilia in Pregnancy ‐ Case Report

V. Emilija 1 ; V. Dejanova‐Ilijevska 2 ; V. Neceva 3 ; T. Makarovska‐Bojadzieva 4 ; M. Sorova 3 ; T. Uzunovska 3 ; I. Nikoloska 3

1 Institute of Transfusion Medicine of Macedonia, Hemostasis and thrombosis, Skopje, Macedonia, the Former Yugoslav Republic of, 2 Institute of Transfusion Medicine of Macedonia, Hemophilia, Skopje, Macedonia, the Former Yugoslav Republic of, 3 Institute of Transfusion Medicine of Macedonia, Skopje, Macedonia, the Former Yugoslav Republic of, 4 Institute of Transfusion Medicine of Macedonia, Imunohematology, Skopje, Macedonia, the Former Yugoslav Republic of

Background: There is a link between thrombophilia and pregnancy loss, as well as other gestational vascular complications.

Aims: Management of the complications related to the heritable thrombophilias during pregnancy.

Methods: A pregnant woman having three unsuccessful pregnancies and one dead fetal delivery in its 33 g.w.

Results: The research displayed mutations: homozygotes for factor V Leiden, heterozygosity for prothrombin G20210A variant and homozygosty for MTHFR. Therapy had been initiated since the 3rd g.w. ‐ LMWH, Aspirin and Folic acid. The therapy was ordinated according the results from the PT,a PTT,TT,D‐Dimers,Platelet aggregation with ADP and Colagen and a continuous ultrasound monitoring of the fetus. In the 30th g.w. the results of the aPTT had abruptly started to deteriorate. The patient was under a daily ultrasound monitoring showing good results regarding the fetus, amniotic water and placenta. Because of an emphasized hyperthrombotic condition we started a therapy with Corticosteroides. In its 32nd g.w. the values of aPTT decreased on 21 sec (n.r. 33 sec), even besides enlarged dosage of LMWH which were in correlation with anti X‐a. The dosage of LMWH was from 40 to 12‐mg. daily. It was fascinating that the ultrasound monitoring of the fetus remained within the normal values, but because of the extreme decrease of aPTT. An urgent Sectio Cesarea had been executed and delivered a healthy newborn. The placenta had no specific pathogenesis, but there was a coagulum along the entire umbilical vein which would have probably caused a dead fetal delivery within several hours.

Conclusions: The doctors should not exclusively be driven by certain facts tightly connected to their specialization. The most important thing is to establish good team work and close cooperation between the obstetrician and the transfusiologist that would simultaneously bring the final decision about the pregnancy termination in benefit of the pregnant women and delivery of a healthy newborn.

PO050: When to Test for Thrombophilia

F. Martins Pereira ; A. Brito; A.P. Amaral; F. Lobo; M. Costa

Centro Hospitalar Tondela‐Viseu, Viseu, Portugal

Background: Factor V Leiden mutation (FVLM) and prothrombin gene mutation (PGM) are common causes of thrombophilias, occurring in about 5% and 4% of Europeans respectively. Individuals with these two mutations have an increased risk of developing venous thromboembolism (VTE). Patients who are heterozygous for FVL have a 5 to 7‐fold increased risk of VTE, whereas the risk in homozygous individuals is increased up to 80‐fold. Heterozygous individuals for both mutations have an increased risk up to 84‐fold.

Aims: The objective is to present a relevant case from our clinical practice.

Methods: Patient clinical records were accessed.

Results: A 25‐years‐old female was admitted in our thrombosis and hemostasis department 3 months after having an intermediate‐low risk pulmonary embolism (PE). PE was secondary to a deep vein thrombosis (DVT) in the distal portion of left femoral vein. Patient was a smoker since she was 13 years old. At time of PE patient was taking oral contraceptive. From her familiar history she only refers two myocardial infarctions on her grandfather from her father side. No further relevant medical findings were noted. Taking in consideration the patient age and clinical presentation of the first VTE episode, even though current guidelines don't recommend thrombophilia study in patients with PE secondary to a reversible risk factor, a full thrombophilia workup including protein C, protein S, antithrombin and antiphospholipid antibody, as well as FVLM and PGM screen was completed. Tests showed a homozygous FVL and heterozygous PGM, leading to prescription of a vitamin k antagonist ad aeternum .

Conclusions: Since diagnosis and treatment, patient hasn't had any new VTE. Despite actual guidelines don't recommend preforming thrombophilia testing in patients with “provoked” PE, it's important to evaluate every case individually. As presented a young woman could remain undiagnosed and untreated, with an increased risk of VTE recurrence and pregnancy loss.

PO051: Dramatic Deep Vein Thrombosis Presentation in Young and Healthy Patients: A Report of 3 Cases

C.E. García Einschlag 1 ; M.M. Szelagowski 1 ; M. Giusti 2 ; F. Giachello 3 ; J.H. Milone 1 ; D. Aguerre 4

1 Hospital Italiano, La Plata., Hematology, La Plata, Argentina, 2 Hospital Italiano, La Plata., Laboratory, La Plata, Argentina, 3 Hospital Italiano, La Plata., Intervention Cardiology, La Plata, Argentina, 4 Hospital Italiano de La Plata, Department of Radiology, La Plata, Argentina

Background: Inferior Vena Cava Malformations (IVCM) are rare congenital anomalies with nonspecific symptoms, such as abdominal or lower back pain, or Deep Vein Thrombosis (DVT). IVCM are found in approximately 5% of cases of unprovoked DVT, mostly occurring before the fourth decade of life.

Aims: To describe 3 cases of IVCM and extensive DVT presentation.

Methods: Medical records between, January 2015 and December 2017, were retrospectively analyzed. We found 206 cases of DVT, 3 patients showed an extensive DVT of Inferior Vena Cava (IVC) and lower limbs, with a subsequent diagnosis of IVCM.

Results: Patient 1: a 38‐year‐old man presented with back pain, lower limbs swelling and walking impairment. Angio‐CT revealed DVT of IVC and both iliac and femoral veins. It was unprovoked. Neoplastic and thrombophilia causes were ruled out and later CT venography confirmed IVCM. Patient 2: a 46‐year‐old man referred to our hospital with extensive DVT. He had been previously immobilized; angio‐CT showed DVT of IVC, external iliac and left femoral veins and IVCM was confirmed. Thrombophilia studies were performed a few months later showing a positive lupus anticoagulant. Patient 3: 38‐year‐old woman presented bilateral lower limb painful swelling. CT evidenced an extensive thrombosis of IVC, right femoral and iliac veins. A later angio CT showed IVCM. All three patients were on started low molecular weight heparin, followed by long term vitamin K antagonist in patients 1 and 3, and long term Direct Oral Anticoagulants in patient 2. Patients 1 and 3 had a favorable outcome, whereas patient 2 developed a severe post thrombotic syndrome.

Conclusions: Along the last 2 years, 1.4% of our DVT population was associated with IVCM. This fact underscores the importance of having a high suspicion level for this entity in young and otherwise healthy patients presenting with extensive DVT. A thoughtful evaluation is needed in order to confirm IVCM. We propose long‐term anticoagulant therapy to reduce the risk of recurrent DVT.

PO053: Effectiveness of Direct Oral Anticoagulants (DOAC) in Patients Submitted to Digestive Surgery. Case Report

D. Sánchez Argüello 1 ; C. De Brabandere 1 ; M. Sarasa Valdes 1 ; P. Chamorro Chamorro 1 ; L. Guerrero Fernández 1 ; C. Fernandez Canal 1 ; S. Sánchez Matías 1 ; J. Torres Varona 1 ; S. Ortiz saez 2 ; V. Robles Marinas 1

1 Hospital de Cabueñes, Gijón, Spain, 2 Hospital de jarrio, Gijon, Spain

Background: Although every day there is more information regarding DOAC the management of patients with special characteristics, usually not included in clinical trials, is a challenge.

Aims: We present a case of a patient with a history of Roux‐en‐Y gastric bypass (RYGB), candidate for treatment with DOAC.

Methods: Male, 61 years old with hypertension, diabetes mellitus and hypercholesterolemia. Non valvular atrial fibrillation treated with Acenocoumarol, time in therapeutic range (TRT) 50%. Intervened of RYGB 14 months ago.

Due to the TRT being low despite good compliance, we decided to start treatment with DOAC. Risk of hemorrhage was low (HAS‐BLED 2). Treatment with Dabigatran was started at full dose (150 mg/12 hours).

Dabigatran is administered in the form of a prodrug requiring acid environment for its absorption which was not certain, therefore close monitoring was decided.

Results: One day after starting treatment we determined biological activity of the drug in the blood 3 hours after its administration. Result was 9.33 ng/ml, less than the peak reference levels in our center (64‐443 ng/ml). We concluded that the patient's surgery did not allow the absorption of the drug and therefore decided to change treatment to Apixaban (5 mg/12 hours). Again, we measured the activity 3 hours after its administration, which was 80.3 ng/ml (reference levels 59‐321 ng/ml), within normal ranges.

Suspecting that the absorption could be erratic, after 2 weeks we analyzed levels, being 110.6 ng/ml.

The patient has currently been 1 year on treatment without any complication.

Conclusions: Not all DOAC are eligible as treatment in patients with alterations in digestive absorption.

Determination of biological activity of the drug should be carried out in patients in whom an absorption defect is suspected.

In our experience Apixaban is effective in patients with RYGB.

Dabigatran is not effective in these patients, probably due to the need of acid environment for its absoption. This is consistent with other cases reported in the literature.

PO054: Analysis of the Causes of Bleeding during Therapy with Anticoagulants

N. Vorobyeva ; A. Schapkov; L. Melnichuk

Nord State Medical University, Haemostasiology, Arckhangelsk, Russian Federation

Background: Currently, for prolonged anticoagulant therapy (PACT) is widely used warfarin, and direct oral anticoagulants (DOAC), which also increase the risk of hemorrhagic complications. The development of serious bleeding in patients on the background of the anticoagulant therapy (ACT) can cause not only overdose, but also comorbidities.

Aims: Aims ‐ analysis of reasons for the development of hemorrhagic complications on the PACT.

Methods: Retrospective study ‐ analysis of case histories of 33 patients with hemorrhagic neurological complications on the PACT and admitted to GBUZ AO “the First clinical hospital named. E. E. Volosevich” of Arkhangelsk in the period 2014‐2016.

We studied the presence of the causes of complications, outcomes, complications, their frequency, indications for use PACT, comorbidities, level of INR (international normalized ratio) and BP (blood pressure) during hospitalization.

Results: 33 patients (15 women and 18 men), aged Me=66 [49; 83] years, receiving PACT, were hospitalized with diagnosis of “hemorrhagic stroke/intracranial bleeding”, confirmed clinically and on CT scan. Lethal outcome was noted in by 45.45% (n=15) patients. 30 patients (90.9%) had taken warfarin, 3 patients‐ DOAC (9.1%). 63.6% of patients were out of school “Warfarin‐therapy” (n=21). The abnormal renal function was identified in 33.3% (n=11) patients, liver dysfunction ‐ in 21.2% (n=7). 93.9% of patients had arterial hypertension (90.9% had hypertension grade 3 at admission), which is a major cause of hemorrhagic stroke. At the time of hospitalization, the INR value of 3.5‐4.0 had a 9.8% (n=3) patients, INR greater than 4.0 is 18.8% (n=6), it was indicating an excessive level of medication hypocoagulation.

Conclusions: Reasons that cause hemorrhagic complications of PACT are: the exceeding the target INR more than 3.5, the accompanying pathology of liver, kidney and arterial hypertension. These factors had a place in the history of the studied group of patients, who had serious.

PO055: Antithrombotic and Thrombolytic Effects of Heparinoid from the Pion

L. Lyapina 1 ; M. Lyapina 2 ; M. Kalugina 1

1 Moscow State University named after M.V. Lomonosov, Moscow, Russian Federation, 2 Moscow State University named after M.V. Lomonosov, Biological Faculty, Moscow, Russian Federation

Background: The heparinoid from pion of root (Paeonia anomala) has anticoagulant and fibrinolytic activity.

Aims: The study to effectiveness of the use of heparinoid from pion Marjin root (Paeonia anomala) in the processes of polymerization of fibrin and prevention of thrombosis.

Methods: Experiments were carried out on male Wistar rats in accordance with the ethical principles of the Helsinki Declaration. Scheme of the experiment: experimental animals were injected three times with intranasal introduction heparinoid from pion root. One hour after the last administration of heparinoid, fibrin polymerization was initiated with subsequent thrombus formation by intravenous injection of 0.4 ml of 1% tissue thromboplastin (thrombosis model). Control animals were injected with physiological saline instead of plant heparinoid. Thirty minutes after thromboplastin administration, formation of thromb was observed. The fibrinolytic and anticoagulant blood activity of the experimental rats was determined.

Results: It is established in vitro that the heparinoid preparation from P.anomala has anticoagulant and intrinsic fibrin‐de‐polymerization activity. In the thrombus formation model, a normal or even elevated background of anticoagulant and fibrinolytic activity of blood was noted in the experiment, while in the control animals there was a sharp decrease in the anticoagulant properties of the blood. In addition, a 30% death of rats was observed in the control (6 out of 9 rats survived). In the experiment all the rats were alive.

Conclusions: For the first time established model experiments of thrombus formation, that the preparation of heparinoid from P.anomala prevents the development of thrombosis, and at the initial stages of thrombogenesis promotes dissolution of fibrin clots. The study can serve as an experimental justification for the use of heparinoid drugs in the prevention of development of thrombotic complications.

PO056: Simple Method for Removal of Doacs from Plasma Samples

T. Exner 1 ; N. Michalopoulos 2 ; J. Pearce 2 ; R. Xavier 3 ; M. Ahuja 1

1 Haematex Research Pty Ltd, Laboratory, Sydney, Australia, 2 Queen Elizabeth II Medical Centre, Pathwest, Perth, Australia, 3 Queensland Medical Laboratories, Haematology, Brisbane, Australia

Background: DOACs occur increasingly in patient test plasmas and interfere with most clotting test methods such as assays for clotting factors, heparins and lupus anticoagulants.

Aims: To evaluate a simple method using an adsorbent product (DOAC Stop) for extracting direct oral anti‐coagulants (DOACs) from plasmas.

Methods: DOAC Stop was tested on normal and a range of abnormal plasmas initially using activated partial thromboplastin time (APTT) tests and a more DOAC‐sensitive Russells viper venom‐based clotting test (DOAC Test). Further tests for prothrombin time/International Normalized Ratio (PT/INR), lupus anticoagulants, activated protein C (APC) resistance, antithrombin, plasminogen, protein C and S were carried out on various patient samples.

Results: DOAC Stop was found to remove all types of DOACs including dabigatran, apixaban, rivaroxaban and edoxaban from test plasmas with minimal effect on any of the (mainly clotting) tests considered in this study.

Conclusions: DOAC Stop can be used to identify plasmas containing DOAcs using simple clotting tests. It reduces the false positivity for lupus anticoagulants observed in dilute Russells viper venom time (dRVVT) tests on DOAC‐containing plasmas and could be useful for eliminating unwanted effects of DOACs on routine coagulation testing.

PO058: Thromboprophylaxis in Hereditary Thrombophilia ‐ A Case Report

T. Makarovska Bojadzieva 1 ; M. Blagoevska 2 ; J. Samonikov Tosevska 2 ; I. Nikoloska 2 ; R. Apostolovska 2

1 Institute of Transfusion Medicine, Immunohematology, Skopje, Macedonia, the Former Yugoslav Republic of, 2 Institute of Transfusion Medicine, Skopje, Macedonia, the Former Yugoslav Republic of

Background: Hereditary thrombophilia increases the risk for unprovoked thrombosis in younger people usually presenting with deep vein thrombosis (DVT) and pulmonary embolism (PE) with likelihood of recurrence.

Aims: To show how laboratory and clinical findings worked together in the choice of anticoagulation treatment and the duration of secondary thromboprophilaxis in a patient with PE.

Methods: Case presentation: Computer tomography confirmed the diagnosis of PE in a 32‐year‐old man who was admitted to the cardiology emergency department with D‐dimer level of 5,980 ng/mL after an episode of syncope. After the initial anticoagulation with UF Heparin 30.000i.e./24 hours, enoxaparin 80 mg/12 hours and acenocoumarol were introduced. The therapeutic INR rang (2‐3) could not be achieved so the acenocumarol was switched to rivaroxaban 2 × 15 mg/day. One year later the anticoagulation with rivaroxaban 20 mg/day was discontinued. Thrombophilia testing included: prothrombin (PTB), FVLeiden and methylene tetrahydrofolate reductase (MTHFR) gene mutation, as well as antiphospholipid antibodies, antithrombin, protein C and S.

Results: Thromofilia testing results were normal exept that the patient was homozygous for PTB gene mutation. His parents were heterozygous for PTB; his mother was heterozygous for MTHFR. His brother was compound heterozygote for PTB and MTHFR and his sister was heterozygous for the PTB. Coagulation status monitoring showed hipercoagulability (APTT was 24‐26 seconds) and increasement of D‐dimer (2,100‐2,400 ng/ml) every time when rivaroxaban was discontinued and normal APTT (28‐38 seconds) and D‐dimer (<500 ng/mL) when it was reintroduced (Table 1).

TABLE 1 APTT and D‐dimer test results according to the anticoagulant thromboprofilaxis Rivaroxaban 20 mg/day Duration (months) APTT (sec) D‐dimer (ng/ml) No 3 26.5 2,418 Yes 4 27.6 465 Yes 4 30.0 195 No 3 24.7 2,144 Yes 4 28.3 366

Conclusions: According to the laboratory and having in mind that this was a second episode of a thrombotic event, we decided for an extended thomboprofilaxis. Although extended anticoagulation sometimes implies that it will be continued life‐long we consider worthwhile to apply patient‐oriented approach to the decision when and whether to terminate anticoagulation.

PO059: Comparison of Two Thromboplastin Reagents for Prothrombin Time (PT) Measurement

R. Perez Montes 1 ; E. Martin Martin 1 ; F. Depasse 2

1 University Hospital Marqués de Valdecilla, Santander, Spain, 2 Diagnostica Stago, Asnièers sur Seine, France

Background: Different types of PT reagents exist to address the laboratory needs. STA ® ‐ NeoPTimal (Stago) is a new precalibrated rabbit brain thromboplastin reagent with an ISI close to 1.0.

Aims: Evaluate the new PT reagent STA ® ‐NeoPTimal (Diagnostica Stago, Asnieres sur Seine, France) for PT measurement expressed in seconds and % in comparison with the locally routinely used reagent Thromborel ® S (Siemens, Marburg, Germany), a human placental thromboplastin with an ISI close to 1.0.

Methods: The study was approved by the Ethics Committee of Valdelcilla Hospital (Spain). A local MNPT was determined in 21 plasma samples obtained from normal subjects with STA ® ‐ NeoPTimal on STA‐Compact Max 2 (Stago) and with Thromborel ® S on CA1500 (Siemens) according to the supplier's instructions for use. Local MNPT was used to calculate PT results expressed in %. Consecutive patients referred to the laboratory for a PT screening test or for VKA treatment monitoring were recruited for the evaluation. Plasma samples were tested fresh or frozen.

The statistical analysis was performed according to the CLSI guideline EP09‐A3. Results expressed in percentage where capped at 100% and excluded from the statistical analysis as per study protocol requirement.

Results: 133 patient's samples were included in the study.

Results are summarised in Table I.

TABLE I Comparison of results obtained with STA® ‐ Neoptimal reagent versus Thromborel S Results in seconds Passing‐Bablok regression Results in % Weighted‐Deming regression n 133 74 Slope (95% CI) 1.31 (1.26‐1.37) 1.12 (1.08‐1.17) Intercept (95% CI) −1.84 (−2.61 to −1.14) −1.22 (−2.56‐0.11) Coefficient of correlation 0.989 0.981

Conclusions: STA‐NeoPTimal, precalibrated extraction thromboplastin ISI=1 reagent, well correlates to the routine method used in the lab and can be easily implemented in the laboratory routine testing.

PO061: Thrombophilia in Children and Adolescent: Phenotype ‐ Genotype Relationship

U. El Safy 1 ; M. Fathy 1 ; M. Zakria 1 ; M. Besheer 1 ; T. Hassan 1 ; N. Rafaat 2 ; S. Aush 3

1 Zagazig University, Pediatric Department, Zagazig City, Egypt, 2 Zagazig University, Biochemistery and Molecular Biology Department, Zagazig, Egypt, 3 Zagazig University, Clinical Pathology Department, Zagazig City, Egypt

Background: Hereditary thrombophilia is associated with a higher risk of thrombosis (mainly venous thrombosis) thrombophilia is mainly an autosomal dominant.

Aims: This study aim to correlate of the type of genetic mutation to clinical data, response to therapy and outcome.

Methods: This cross sectional study was carried out Pediatric Hemat&Onco ‐Zagazig University Hospital, from 2010 t0 2017, on twenty patients with thrombosis. All patients were subjected to clinical, full laboratory screening for thrombophilia and radiologic evaluation. The Procedure of gene screening of thrombophilia includes single multiplex PCR for the amplification,followed by reverse hybridization of biotinylated amplification products on test strip. The assay covers the following 12 mutations: FV G1691A (Leiden), FV H1299 R(R2), Prothrombin G20210A, Factor XIIIV34L,B‐Fibrinogen‐455G>A,PAI‐14G/5G,GPIIIL33P(HPA‐1), MTHFRC677T,MTHFRA1298C, ACE I/D, Apo B R3500Q, and Apo E22/E3/E4.

Results: Deep Venous thrombosis was detected in 9 cases. cerebral venous thrombosis [sinus] was in 6 cases, While portal vein thrombosis in 5 cases. All twenty cases of thrombosis revealed abnormal gene mutations in heterozygous form either in a single gene or multiplex. The frequent gene mutation was MTHFRA1298C in 12 cases,,PAI‐1 4G/5G,in 11 cases APO E22/E3/E4 in 11 cases and B‐Fibrinogen‐ 455G in 7 cases. One heterozygous gene was detected in 4 cases. Double heterozygous gene in 4 cases, Three heterozygous gene was detected in 8 cases. Fibrinogen‐ 455G gene mutation in deep venous thrombosis. MTHFRA1298C associated with portal vein thrombosis. While XIIIV34 in cerebral venous thrombosis, Fibrinogen‐ 455G > lacked response to oral anticoagulant, Two cases of mutation of Fibrinogen‐ 455G > developed malignancy.

Conclusions: Thrombophilia gene screening is important for diagnosis and for therapeutic decision and outcome expectation.

PO062: Two Novel Variants in ADAMTS13 Gene and Clinical Relation between Phenotype/Genotype in Congenital Thrombotic Thrombocytopenia Purpura (TTP)

J. Paiva 1 ; A.C. Kempfer 2 ; A.I. Woods 2 ; M.M. Casinelli 1 ; A. Sánchez‐Luceros 1,2 ; M.A. Lazzari 2

1 Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Buenos Aires, Argentina, 2 IMEX‐CONICET‐Academia Nacional de Medicina, Buenos Aires, Argentina

Background: TTP is a rare disorder characterized by microangiopathic hemolytic anemia and thrombocytopenia. Congenital TTP (cTTP) is caused by homozygosity or compound heterozygosity for mutations in the ADAMTS13 gene. We report here two novel variations in heterozygous state in two patients (P) and the relation between the phenotype and genotype.

Aims: To increase the knowledge on hereditary TTP and the relationship between the clinical severity and genotypic.

Methods: P1: female that presented her first episode at 24 years old during the pregnancy, with thrombocytopenic, anemia, abdominal pain without thrombotic antecedents. P2: male that presented his first episode at 10 month of age with thrombocytopenia, petechial and exchange transfusion. ADAMTS13 activity, IgG anti‐ADAMTS13 antibody were evaluated by ELISA and ultralarge von Willebrand factor multimers (ULVWFM) by SDS‐agarose gel electrophoresis. Sequencing was done by ABI 310 genetic analyzer. Different programs were used for in silico analysis. Informed consent and ethical approval were obtained.

Results: Both P showed low ADAMTS13 activity, absence of anti‐ADAMTS13 antibodies, and >15% of ULVWFM. Two heterozygous variations were observed in every P (Table 1). P1 presented a novel variation c.436A>T (p.N146Y) and c.3178C>T (p.R1060W) (Tao Z, 2006), in metalloprotease (MP) domain and TSP1‐7 respectively (Figure 1‐A). P2 presented the variant c.577C>T (p.R193W) and a novel variation c.1851C>G (p.Y617*), that affect the MP domain (Matsumoto M, 2004) and the spacer domain respectively (Figure 1‐B). In silico analysis showed scores corresponding to variations deleterious.

TABLE 1 ADAMTS13 results and genetic variations found in the patients Patients ADAMTS13 Activity (NV:40‐130 %) IgG anti‐ADAMTS13 antibody (NV: ≤15U/mL) ULVWFM (NV: ≤15%) Genetic variation in Allele 1 Genetic variation in Allele 2 Exon affected in allele 1 Exon affected in allele 2 Affected domain in allele 1 Affected domain in allele 2 P1 6 12 30 c.436 A›T (p. N146Y) c.3178 C›G (p.R1060W) Exon 5 Exon 24 MP TSP1‐7 P2 <5 2.5 22 c.577 C›T (p.R193W) c.1851 C›G (p.Y617*) Exon 6 Exon 16 MP Spacer

FIGURE 1 Schematic diagram of ADAMTS13 domains. A) Genetic variations in P1. B) Genetic variations in P2

Conclusions: p.N146Y and p.Y617* appear to modify the MP and spacer domain, assuming these are on a different allele to p.R1060W and p.R193W, their combined effect would be responsible to reduce ADAMTS13 activity; likewise, the combination of p.R193W and p.Y617* would produce more severity TTP phenotype because of quantitative and/or qualitative defects of ADAMTS13 would be manifest at an earlier age.

PO063: False Hypercoagulability Possibly Induced by the in vitro Accidental Presence of Tissue Factor

C. Delianu 1 ; C. Tuchilus 2,3 ; L. Foia 4,5

1 Emergency County Hospital “Sf. Spiridonˮ, Hematology, Iasi, Romania, 2 Emergency County Hospital “Sf. Spiridonˮ, Central Laboratory, Iasi, Romania, 3 University of Medicine and Pharmacy “Grigore T. Popa”, Microbiology, Iasi, Romania, 4 Emergency County Hospital “Sf. Spiridonˮ, Biochemistry, Iasi, Romania, 5 University of Medicine and Pharmacy “Grigore T. Popa”, Biochemistry, Iasi, Romania

Background: The eventual prolonged stasis following venipuncture can lead to plasma release of cell lysate as well as tissue factor, impacting the quality of the coagulation tests. The accidental presence of tissue factor in vitro can trigger the coagulation mechanism, generating a false decrease in the prothrombin time‐PT test.

Aims: The prospective observational study was performed in the “Sf. Spiridonˮ University Hospital (Romania) and targeted identification of short PT results that could suggest a hypercoagulability. The purpose of this study was to compare the results of the PT short tests at the first determination with their corrective values following duplication of the PT analysis from the same sample.

Methods: Over a period of 4 months during 2016, we analyzed 544 PT samples with abnormal higher values (hypercoagulability) before, and after repeat determination.

Results: Out of a total of 544 PT samples with abnormal higher values,duplication of the determination from the same sample identified 200 (36.76%) corrected tests with PT values within the normal reference range (P=0.001) and 344 (63.23%) tests suggesting a hypercoagulability and therefore correlated with a possible risk of thrombosis.

Conclusions: Our results imply that the coagulation in vitro disturbance might be over estimated as the duplication of the PT from the same sample revealed laboratory values within normal range. The discrepancy might be explained by several preanalytical grounds, and, as long as PT results should be reliable for monitoring of therapy, we suggest that duplication of PT determination from the same sample could improve the accuracy of results reporting, as they should be consistent with the clinical actual patient status.