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

## TL;DR

This study shows how adding the right amount of calcium improves the accuracy of measuring platelet function in blood samples with low platelet counts.

## Contribution

The study identifies optimal calcium concentrations for reliable procoagulant platelet assessment in thrombocytopenic samples.

## Key findings

- For platelet counts ≥100 × 109/L, 2.5 mM calcium in the buffer is sufficient.
- Supplementing 3 mM calcium for counts between 30 and <100 × 109/L restores 85-115% of control levels.
- Excessive calcium (>5 mM) impairs platelet function, emphasizing the need for precise recalcification.

## Abstract

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.

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.

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.

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.

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.

•Adequate extracellular calcium is required for accurate procoagulant Collagen-And-Thrombin–activated (COAT) platelet generation.•Optimal recalcification for procoagulant COAT platelet formation was assessed by flow cytometry.•Excessive remnant citrated plasma in low-count platelet-rich plasma samples binds calcium, causing false results.•Proper recalcification prevents underestimating true procoagulant potential in thrombocytopenia.

Adequate extracellular calcium is required for accurate procoagulant Collagen-And-Thrombin–activated (COAT) platelet generation.

Optimal recalcification for procoagulant COAT platelet formation was assessed by flow cytometry.

Excessive remnant citrated plasma in low-count platelet-rich plasma samples binds calcium, causing false results.

Proper recalcification prevents underestimating true procoagulant potential in thrombocytopenia.