Impact of recalcification on procoagulant collagen-and-thrombin-activated (COAT) platelet generation in platelet-rich plasma samples with low platelet counts.

BACKGROUND

Assessment of platelet procoagulant function by flow cytometry is increasingly recognized for diagnosing platelet disorders. Induction and accurate detection of phosphatidylserine exposure on the platelet surface requires proper recalcification of citrated blood samples, particularly in plasma from thrombocytopenic patients, in which residual citrate can limit free extracellular calcium availability.

OBJECTIVES

To define optimal recalcification conditions for reliable measurement of procoagulant Collagen-And-Thrombin-activated (COAT) platelets in platelet-rich plasma (PRP) samples with low platelet counts.

METHODS

Fresh PRP from healthy donors was diluted with autologous platelet-poor plasma to simulate platelet counts from 160 × 109/L to 15 × 109/L. Undiluted PRP (control) and diluted samples were spiked with varying calcium concentrations and stimulated with convulxin and thrombin. Flow cytometry was used to measure annexin V and PAC-1 binding to assess procoagulant platelet generation.

RESULTS

For PRP with platelet counts ≥100 × 109/L, no addition of calcium beyond the 2.5 mM already present in the buffer was required. For platelet counts between 30 and <100 × 109/L, supplementation with 3 mM calcium restored procoagulant platelet generation to 85% to 115% of undiluted PRP control levels. Counts of 20 to <30 × 109/L required 5 mM calcium to achieve an almost comparable restoration. Of note, higher calcium concentrations (>5 mM) impaired platelet function, highlighting the need to avoid excessive recalcification.

CONCLUSIONS

Optimized PRP recalcification prevents underestimation of procoagulant platelet potential in thrombocytopenic samples. This practical approach addresses a key gap identified by the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee and helps laboratories to ensure reliable platelet function testing in PRP samples from thrombocytopenic patients.