PB0912 - Study to determine the minimum number of platelet events acquisition required for flow cytometry based platelet function testing in healthy controls and patients with thrombocytopenia

Background: There is limited literature assessing the minimum number of platelet events that need to be acquired for flow cytometry based platelet activation/function tests. Various studies have acquired 5,000 to 10,000 platelet events for flow cytometry-based platelet function tests. However, it may be difficult and time-consuming to acquire these many events, particularly in thrombocytopenia patients.

Aims: To assess the minimum number of platelet events acquisition required for flow cytometry-based platelet function/activation test in healthy controls and thrombocytopenia patients

Methods: Healthy controls (n=9) and patients with thrombocytopenia (n=9) were recruited after informed consent. Gating of CD41-positive platelet events on the time-gate was performed to obtain serially decreasing (15000, 10000, 5000, 2500, 1200 and 500) CD41-positive acquired events (Figure 1). Platelet function was assessed for serially decreasing CD41-positive acquisition events as gated on the timeline using activation markers CD62P (0.10 μg/ml) and PAC-1 (0.42 μg/ml) in response to agonist stimulation by ADP (30µM) and convulxin (1µg/mL) respectively.

One-way ANOVA followed by post hoc analysis by Tukey’s Multiple comparison test was performed to compare the statistical difference between the percentage and Median Fluorescence Intensity (MFI) of CD62P and PAC-1 expression upon agonist activation at serially decreasing platelet acquisition events in healthy controls and thrombocytopenia patients. A P-value < 0.05 was considered significant.

Results: Platelet counts of patients with thrombocytopenia ranged from 16000/μL to 83000/μL, median platelet count being 40000/μL. There was no statistical difference in the CD62P and PAC-1 expression (in terms of both percentage and MFI) upon agonist stimulation at serially decreasing platelet events, even up to 500 CD41-positive events. (Figure 2)

Conclusion(s): Acceptable reproducibility of flow cytometry-based platelet function / activation test was observed with the collection of 500 CD41-positive events. Thus, acquisition of even 500 platelet events is sufficient for reliable flow cytometry-based platelet function testing, making this test particularly useful in patients with thrombocytopenia.