Impact of recalcification on procoagulant COAT platelet generation in PRP samples with low platelet counts

AbstractBackground Assessment of platelet procoagulant function by flow cytometry is increasingly recognized for diagnosing platelet disorders. Induction and accurate detection of phosphatidylserine exposure on platelets' surface requires proper recalcification of citrated blood samples, particularly in thrombocytopenic patients, where residual citrated plasma can limit free extracellular calcium availability. Objectives To define optimal recalcification conditions for reliable measurement of procoagulant 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 down 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 PAC1 binding to assess procoagulant platelet generation. Results For PRP with platelet counts ≥100×109/L, no additional calcium beyond buffer levels was required. For platelet counts between 30–9/L, supplementation with 3 mM calcium restored procoagulant platelet generation within 85–115% of undiluted PRP control levels. Counts of 20–9/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 ISTH SSC and helps laboratories to ensure reliable platelet function testing in PRP samples from thrombocytopenic patients.