Standardization and reference ranges for whole blood platelet function measurements using a flow cytometric platelet activation test

Abstract

IntroductionPlatelet function testing with flow cytometry has additional value to existing platelet function testing for diagnosing bleeding disorders, monitoring anti-platelet therapy, transfusion medicine and prediction of thrombosis. The major challenge is to use this technique as a diagnostic test. The aim of this study is to standardize preparation, optimization and validation of the test kit and to determine reference values in a population of 129 healthy individuals.MethodsPlatelet function tests with 3 agonists and antibodies against P-selectin, activated αIIbβ3 and glycoprotein Ib (GPIb), were prepared and stored at -20°C until used. Diluted whole blood was added and platelet activation was quantified by the density of activation markers, using flow cytometry. Anti-mouse Ig κ particles were included to validate stability of the test and to standardize results. Reference intervals were determined.ResultsBlood stored at room temperature (RT) for up to 4h after blood donation and preheated/tested at 37°C resulted in stable results (%CV<10%), in contrast to measuring at RT. The intra-assay %CV was <5%. Incubation of anti-mouse Ig κ particles with antibodies stored for up to 12 months proved to give a stable fluorescence. The inter-individual variation measured in the 129 individuals varied between 23% and 37% for P-selectin expression and αIIbβ3 activation, respectively.ConclusionsThe current study contributes to the translation of flow cytometry based platelet function testing from a scientific tool to a diagnostic test. Platelet function measurements, using prepared and stored platelet activation kits, are reproducible if executed at 37°C. The reference ranges can be validated in clinical laboratories and ongoing studies are investigating if reduced platelet reactivity in patients with bleeding complications can be detected.

Adenosine Receptor Agonists Exhibit Anti-Platelet Effects and the Potential to Overcome Resistance to P2Y12 Receptor Antagonists

“…High degrees of inter-individual variability were previously observed for the platelet response to agonists (particularly ADP) and anti-platelet drugs [48][49][50][51]; this was also observed in the present study. Therefore, the effect of AR agonists on platelet aggregation was evaluated in high-and low-responders to P2Y 12 inhibitors using aggregometry: a simple method which allows rapid assessment of platelet response.…”

High prevalence of reduced thrombin generation and/or decreased platelet response in women with unexplained heavy menstrual bleeding

“…All samples were analysed on a FACS Canto flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The CVs of platelet function are below 10% for all platelet activation measurements, except ADP‐induced P‐selection expression, which has a CV below 20% …”

Polyphenols: Modulators of Platelet Function and Platelet Microparticle Generation?

“…However, it has been shown, especially in aggregometry assays, that factors such as gender and race in healthy subjects may affect platelet function results [45]. A study aimed at evaluating the reference intervals of flow cytometry-based platelet function results found that ADP-induced platelet activation was significantly higher in females than males [112]. On the contrary, some studies have demonstrated no significant antiplatelet effect in hypertensive populations [93,94,96] (Table 1).…”