Introduction
The anti-CD38 monoclonal antibody (MoAb) daratumumab (DARA) (Darzalex; Janssen Biotech, Inc.) is highly efficacious in relapsed and refractory multiple myeloma (MM). It is the first anti-CD 38 MoAb which is approved by the US FDA (2015) and TGA Australia (2017).[ 1 , 2 , 3 , 4 ]
MM is a plasma cell neoplasm with a chronic and incurable course requiring successive treatments that prolong and improve the patient’s quality of life.[ 5 ] This drug is an lgG1k CD38-targeted monoclonal antibody (MoAb) which is expressed in myeloma cells; CD38 is an integral transmembrane glycoprotein that is expressed on many cell types and highly expressed on plasma cells but variably and weakly expressed on normal human red blood cells (RBCs)[ 6 , 7 , 8 , 9 ] and consequently, anti-CD38 monoclonal antibodies can bind directly to CD38 expressed on RBCs, causing panreactivity in vitro .[ 10 , 11 ]
The use of DARA and other anti-CD38 therapies leads to interference with routine immunohematological tests representing a new challenge to blood banks worldwide in patients being treated for MM as many will require blood transfusions as part of their support during ongoing disease treatment.[ 10 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ] Panagglutination caused by anti-CD38 may mask the presence of a clinically significant RBC antibody (Ab) in the patient’s plasma, which may be overlooked, particularly in urgent situations, and subsequently, result in an acute or delayed hemolytic transfusion reaction. Although DARA does not interfere with routine ABO/RhD typing, the plasma of DARA-treated patients is pan-reactive in Ab screening, Ab identification, and antiglobulin tests, as a result of binding of DARA to CD38 on reagent erythrocytes.[ 10 , 12 ]
Chemical treatments of panel cells, the use of a cord blood cell panel, and the inactivation of anti-CD38 in the patient’s plasma through the use of soluble human CD38 are the effective strategies issued by AABB to effectively bypass DARA panreactivity.[ 20 ]
Dithiothreitol (DTT), a disulfide-bridging reductant reagent is commonly used in blood banks and has been applied as an affordable and efficient resource to resolve this identified panreactivity through the destruction of antigenic sites for CD38 on RBCs.[ 10 , 13 ] However, DTT is known to denature antigens from the Kell blood group system and other blood group antigens found less commonly in the population.[ 21 ] Other alternative and promising solutions, such as the use of cord blood cells[ 22 ] and neutralization of free DARA in plasma, are not yet widely available.[ 10 , 12 ] To avoid possible predictable transfusion complications, the safe release of blood components is a must.
Aims and objectives
The aim of this study is to investigate the efficacy and role of DTT in resolving pretransfusion immunohematology testing challenges encountered in patients undergoing treatment with the anti-CD38 monoclonal antibody DARA for MM.
Materials and Methods
Patient samples
Pretransfusion samples were collected from three hematological patients diagnosed with MM and undergoing treatment with anti-CD38 drugs, necessitating RBC transfusion. Clinicians provided additional details, including patient gender, age, transfusion history, and the interval between anti-CD38 monoclonal antibody infusion and sample collection for immunohematological serological testing.
Serologic testing
The samples underwent ABO and Rh typing using the Ortho BioVue system through the column agglutination technique. Direct (polyspecific) and antihuman globulin (AHG) crossmatching were conducted using the column agglutination technique in LISS/Coombs’ AHG Gel Card containing IgG and C3d (Bio-Rad, Morat, Switzerland). Ab screening was performed using a 3-cell screening panel SURGISCREEN ® Reagent RBCs based on column agglutination technology (CAT) in an ortho vision swift fully automated analyzer.
Samples giving positive Ab screen either on 3- or 4-cell screening panel were subjected to Ab identification by extended 11-cell panels by CAT (Resolve Panel A, Ortho Clinical Diagnostics, USA) with beads technique (BLISS-Coombs’ AHG Gel Card, BioVue, Ortho Clinical Diagnostics, USA).
Direct antiglobulin test (DAT)-positive samples were additionally tested for monospecific reactivity including IgG, IgM, C3c, C3d, and control using DC screening (Bio-Rad, Morat, Switzerland). Eluates were prepared from patient samples with a positive DAT using the acid elution technique (BAG-Elutions-kit).
Serum samples showing panreactivity with indirect antiglobulin test screening and panel cells were further tested using RBCs treated with a 0.2 M DTT solution.
The 0.2 mol/L DTT solution was prepared by diluting 1 g DTT in 32 mL of phosphate-buffered saline (PBS) with a pH of 8.0. RBCs positive for the K+ and E+ antigens served as positive and negative controls to validate the efficacy of DTT treatment.
Reagent and control RBCs (100 µl of a 3%–5% suspension) were washed four times with PBS, pH 7.3. Then, 400 µl of 0.2 mol/L DTT was added to each tube. These RBCs were incubated at 37°C for 30 min with periodic mixing by inversion (three to four times during incubation). The RBCs were washed four times with PBS, pH 7.3, and used for subsequent testing following the DTT treatment method described in the AABB technical manual.[ 23 ] and Mechanism of daratumumab interference in IAT is explained in Figure 1 .
Results
In our study, we examined three cases of MM patients undergoing treatment with DARA. The 1 st patient was a 64-year-old male with anemia (hemoglobin – 3.5 g%) who had previously received one unit of RBC transfusion a year back. The 2 nd patient, a 58-year-old male, had relapsed MM and had no previous transfusions. The 3 rd patient, a 52-year-old male, presented with MM, anemia, and bleeding and had no prior transfusions recorded. The time interval between DARA infusion and sample collection for serological testing was 20 days for the 1 st patient, 2 days for the 2 nd patient, and not known for the 3 rd patient.
Serologic testing revealed varied results across the patients which are mentioned in Table 1 .
Table 1
Pre- and posttransfusion findings of the patients
DTT=Dithiothreitol, DAT=Direct antiglobulin test, DARA=Daratumumab, MM=Multiple myeloma, RBC=Red blood cell, PRBCS=Packed RBCs, Hb=Hemoglobin, Ab=Antibody
| A 64-year-old male with MM undergoing treatment with DARA with anemia (Hb -3.5 g%) | A 58-year-old relapsed MM male patient | A 52-year-old male with MM having anemia and bleeding | |
| Transfusion history | One unit of RBC transfusion a year back | No previous transfusion | No previous transfusion |
| The time between DARA infusion and sample collection | 20 days | 2 days | Not known |
| Previous Ab screen | Negative | Negative | Negative |
| Current ABO/Rh | B positive | A positive | AB positive |
| DAT (polyspecific card) | Negative | Positive (+3) | Positive (+4) |
| DAT (monospecific) | Negative | Ig G and C3d – positive, ctl- negative | IgG, IgM, C3c, C3d – positive, ctl- negative |
| Eluates | Negative | Negative | Negative |
| Ab screening (3-cell panel) | Panreactive (+2) | Panreactive (+3) | Panreactive (+3) |
| Ab identification (11-cell panel) | Panreactive (+2) | Panreactive (+3) | Panreactive (+3) |
| Red cell crossmatch | Incompatible (+2) | Incompatible (+3) | Incompatible (+3) |
| Post-DTT treatment | Ab screen - negative Red cell cross-match compatible with Coomb’s phase | Ab screen - negative Red cell cross-match compatible with Coomb’s phase | Phenotype matched O negative PRBCS were transfused since the patient was bleeding |
| Transfusion | Uneventful | Uneventful | Uneventful |
Following these cases, we modified pretransfusion testing protocol for patients on DARA.
Routine transfusion request
Blood request forms must include compulsory information regarding transfusion, obstetric, and DARA drug history
Conduct ABO/Rh typing
Perform DAT including polyspecific and monospecific testing if polyspecific DAT is positive
Conduct Ab screening panel
Perform elution (acid elution) in cases where DAT is positive
If panagglutination indicates interference with anti-CD38 monoclonal antibody (MoAb) on the Ab screen, conduct an Ab screen using DTT
In the event of a negative Ab screen after DTT treatment, assume no clinically significant red cell alloantibodies. However, it cannot exclude antibodies to antigens denatured by DTT. Transfuse ABO/RhD compatible blood and blood compatible for any significant antigens denatured by DTT, such as the Kell phenotype
If the Ab screen is positive, suggesting the presence of significant alloantibodies, transfuse antigen-negative blood after excluding antibodies to antigens denatured by DTT
Conduct extended RBC phenotype testing, including Rh and Kell
Issue donor RBCs.
Urgent transfusion request
Switch to ABO/RhD and Kell-compatible red cells where applicable
Collect pretransfusion samples for retrospective testing and crossmatching.
Discussion
MM, a malignancy affecting plasma cells, arises when a group of these cells becomes cancerous and proliferates within the bone marrow. This condition can lead to bone damage, compromised immune function, renal impairment, and decreased RBC count.[ 24 ] While treatments such as proteasome inhibitors, immunomodulatory drugs, and hematopoietic stem cell transplantation have shown promise in extending survival, patients still succumb to refractory disease.[ 25 , 26 ] Recent therapeutic advances offer hope for MM patients resistant to standard treatments.
One such advancement involves the development of a novel monoclonal antibody (MoAb) targeting CD38, a protein expressed at elevated levels on malignant plasma cells. This MoAb presents a promising monotherapy option for these patients. However, CD38 expression, albeit at lower levels, also occurs on myeloid and lymphoid cells. Consequently, the interaction of the drug with these cells can disrupt pretransfusion testing protocols. Recognizing this challenge, AABB released guidelines on “Mitigating Anti-CD38 Interference with Serological Testing” on January 15, 2016.
Before initiating treatment with the anti-CD38 MoAb (DARA), several precautions are recommended:
Conduct baseline type and screen assessments
Perform baseline phenotype/genotype evaluations
Implement protocols to promptly inform transfusion services on treatment initiation.
Failure to address these concerns may result in missed detection of significant alloantibodies, leading to acute or delayed hemolytic transfusion reactions and delayed blood product issuance. However, pivotal phase III studies, CASTOR and POLLUX, have not reported any hemolytic transfusion reactions attributable to DARA interference with pretransfusion testing.[ 27 , 28 , 29 , 30 ]
Conclusion
We have demonstrated that pretreatment of erythrocytes with DTT effectively deactivates the CD38 antigen, eliminating reactivity with patient serum treated with DARA. This technique offers a simple and accessible solution to mitigate anti-CD38 drug interference in pretransfusion tests. In resource-constrained settings, prolonged incubation techniques can also prove effective.
In the era of monoclonal antibody therapy revolutionizing oncology, effective communication between oncologists and transfusion services is paramount. Utilizing DTT and ensuring early notification to transfusion services about DARA treatment enable timely interventions and uphold transfusion safety standards. It is imperative for transfusion services to inform oncologists about DARA’s interference with pretransfusion tests and recommend conducting type and screen evaluations before initiating treatment.
Conflicts of interest
There are no conflicts of interest.
Funding Statement
Nil.
References
1. Usmani SZ, Weiss BM, Plesner T, Bahlis NJ, Belch A, Lonial S, et al. Clinical efficacy of daratumumab monotherapy in patients with heavily pretreated relapsed or refractory multiple myeloma. Blood. 2016;128:37–44. doi: 10.1182/blood-2016-03-705210.
2. Lokhorst HM, Plesner T, Laubach JP, Nahi H, Gimsing P, Hansson M, et al. Targeting CD38 with daratumumab monotherapy in multiple myeloma. N Engl J Med. 2015;373:1207–19. doi: 10.1056/NEJMoa1506348.
3. Darzalex Package Inserts. Horsham (PA): Janssen Biotech, Inc; 2015.
4. McKeage K. Daratumumab: First global approval. Drugs. 2016;76:275–81. doi: 10.1007/s40265-015-0536-1.
5. Moreau P, Touzeau C. Multiple myeloma: From front-line to relapsed therapies. Am Soc Clin Oncol Educ Book. 2015;35:e504–11. doi: 10.14694/EdBook_AM.2015.35.e504.
6. Plsener T, Lokho M, Gimsing P. Daratumumab, a CD38 monoclonal antibody in patients with multiple myeloma data from a dose escalation phase I/II study. ASH Annual Meeting Abstracts. 2012;120:73.
7. Lokhorst HM, Plesner T, Gimsing P. Phase I/II dose-escalation study of daratumumab in patients with relapsed refractory multiple myeloma. ASCO Meeting Abstracts. 2013;8512;31(Suppl. 15)
8. Dimopoulos MA, Oriol A, Nahi H, San-Miguel J, Bahlis NJ, Usmani SZ, et al. Daratumumab, lenalidomide, and dexamethasone for multiple myeloma. N Engl J Med. 2016;375:1319–31. doi: 10.1056/NEJMoa1607751.
9. Palumbo A, Chanan-Khan A, Weisel K, Nooka AK, Masszi T, Beksac M, et al. Daratumumab, bortezomib, and dexamethasone for multiple myeloma. N Engl J Med. 2016;375:754–66. doi: 10.1056/NEJMoa1606038.
10. Chapuy CI, Nicholson RT, Aguad MD, Chapuy B, Laubach JP, Richardson PG, et al. Resolving the daratumumab interference with blood compatibility testing. Transfusion. 2015;55:1545–54. doi: 10.1111/trf.13069.
11. Zocchi E, Franco L, Guida L, Benatti U, Bargellesi A, Malavasi F, et al. Asingle protein immunologically identified as CD38 displays NAD +glycohydrolase, ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities at the outer surface of human erythrocytes. Biochem Biophys Res Commun. 1993;196:1459–65. doi: 10.1006/bbrc.1993.2416.
12. Oostendorp M, Lammerts van Bueren JJ, Doshi P, Khan I, Ahmadi T, Parren PW, et al. When blood transfusion medicine becomes complicated due to interference by monoclonal antibody therapy. Transfusion. 2015;55:1555–62. doi: 10.1111/trf.13150.
13. Chapuy CI, Aguad MD, Nicholson RT, AuBuchon JP, Cohn CS, Delaney M, et al. International validation of a dithiothreitol (DTT)-based method to resolve the daratumumab interference with blood compatibility testing. Transfusion. 2016;56:2964–72. doi: 10.1111/trf.13789.
14. Moreau P, van de Donk NW, San Miguel J, Lokhorst H, Nahi H, Ben-Yehuda D, et al. Practical considerations for the use of daratumumab, a novel CD38 monoclonal antibody, in Myeloma. Drugs. 2016;76:853–67. doi: 10.1007/s40265-016-0573-4.
15. Anani WQ, Duffer K, Kaufman RM, Denomme GA. How do I work up pretransfusion samples containing anti-CD38? Transfusion. 2017;57:1337–42. doi: 10.1111/trf.14144.
16. Anani WQ, Marchan MG, Bensing KM, Schanen M, Piefer C, Gottschall JL, et al. Practical approaches and costs for provisioning safe transfusions during anti-CD38 therapy. Transfusion. 2017;57:1470–9. doi: 10.1111/trf.14021.
17. Hannon JL, Clarke G. Transfusion management of patients receiving daratumumab therapy for advanced plasma cell myeloma. Transfusion. 2015;55:2770. doi: 10.1111/trf.13267.
18. Mitigating the Anti-CD38 Interference with Serologic Testing. AABB Association Bulletin #16–02. Bethesda (MD): AABB; 2016. [[Last accessed on 2016 May 29]]. Available from: https://www. AABB.org/programs/publications/bulletins/Documents/ab16-02.pdf .
19. De Vooght KM, Oostendorp M, van Solinge WW. Dealing with anti-CD38 (daratumumab) interference in blood compatibility testing. Transfusion. 2016;56:778–9. doi: 10.1111/trf.13474.
20. Mitigating the Anti CD38 Interference with Serologic Testing. AABB Association Bulletin #16-03. Bethesda (MD): AABB; 2016.
21. Walker P. Identification of antibodies to red cell antigens. In: Fung M, editor. AABB Technical Manual. Bethesda, MD: Association for the advancement of Blood &Biotherapies; 2014. pp. 405–7.
22. Schmidt AE, Kirkley S, Patel N, Masel D, Bowen R, Blumberg N, et al. An alternative method to dithiothreitol treatment for antibody screening in patients receiving daratumumab. Transfusion. 2015;55:2292–3. doi: 10.1111/trf.13174.
23. Roback JD, Grossnan BJ, Harris T, Hillver CD, editors. Technical Manual. 17th ed. Bethesda, MD: American Association of Blood Banks; 2011.
24. Kyle RA, Rajkumar SV. Multiple myeloma. Blood. 2008;111:2962–72. doi: 10.1182/blood-2007-10-078022.
25. Kumar SK, Rajkumar SV, Dispenzieri A, Lacy MQ, Hayman SR, Buadi FK, et al. Improved survival in multiple myeloma and the impact of novel therapies. Blood. 2008;111:2516–20. doi: 10.1182/blood-2007-10-116129.
26. Turesson I, Velez R, Kristinsson SY, Landgren O. Patterns of improved survival in patients with multiple myeloma in the twenty-first century: A population-based study. J Clin Oncol. 2010;28:830–4. doi: 10.1200/JCO.2009.25.4177.
27. Lonial S, Weiss BM, Usmani SZ, Singhal S, Chari A, Bahlis NJ, et al. Daratumumab monotherapy in patients with treatment-refractory multiple myeloma (SIRIUS): An open-label, randomised, phase 2 trial. Lancet. 2016;387:1551–60. doi: 10.1016/S0140-6736(15)01120-4.
28. Chanan Khan A, Lentzch S, Quach H, Horvath N, Capra M, Ovilla R, et al. Daratumumab, Bortezomib, and Dexamethasone Versus Bortezomib and Dexamethasone Alone for Relapsed or Refractory Multiple Myeloma Based on Prior Treatment Exposure: Updated Efficacy Analysis of CASTOR. San Diego: American Society of Hematology; 2016.
29. Mateos MV, Estell J, Barreto W, Corradini P, Min CK, Medvedova E, et al. Efficacy of Daratumumab, Bortezomib, and Dexamethasone versus Bortezomib and Dexamethasone in Relapsed or Refractory Multiple Myeloma Based on Prior Lines of Therapy: Updated Analysis of CASTOR. San Diego: American Society of Hematology; 2016.
30. Moreau P, Kaufman JL, Sutherland H, Lalancette M, Magen H, Iida S, et al. Efficacy of daratumumab, lenalidomide, and dexamethasone versus lenalidomide and dexamethasone alone for relapsed or refractory multiple myeloma among patients with 1 to 3 prior lines of therapy based on previous treatment exposure: Updated analysis of POLLUX. Blood. 2016;128:489.