PB0823 - Development and optimisation of a novel 9-parameter flow cytometry panel for human platelet phenotyping

Background: Multiparameter flow cytometry is an invaluable technique for platelet phenotyping as it allows the simultaneous evaluation of multiple platelet markers as proxies for distinct platelet functions, facilitating the examination of platelet activators and inhibitors and the status of platelets in disease.

Aims: To design and optimise a novel whole blood (WB) multiparameter flow cytometry panel for platelet phenotyping.

Methods: We assessed the expression patterns of 9 platelet surface receptors in healthy adult volunteers using fluorescence flow cytometry.

Results: We tested a 9-parameter flow cytometry panel which simultaneously analysed the expression of CD62P, CD42b, αIIbβ3 activation, CD63, CD40L, CD36, CD147, CXCR4 and FcγRIIa. The assay performed equally well in WB and washed platelets. Focusing on WB, we found the assay detected differential changes in receptor expression of all 9 receptors in response to multiple agonists. To assess the robustness and accuracy of the panel, we tested inhibitors of specific platelet activation pathways, including prostacyclin (PGI2), dasatinib, ticagrelor and vorapaxar. We found that PGI2 and ticagrelor targeted ADP-, SFLLRN- and convulxin-mediated platelet activation. In contrast, dasatinib mainly targeted ADP- and convulxin-mediated platelet activation. Vorapaxar only targeted SFLLRN-mediated platelet activation. The assay revealed that the expression of CD62P, CD40L, CD63 and αIIbβ3 activation were more sensitive to inhibition than CD42b, FcγRIIa, CD36, CXCR4, and CD147. The assay performed optimally when fixed samples were analysed immediately ( < 1hour post-fixation), but storage at 4ºC or -80ºC for 24 hours did not significantly affect performance, allowing for remote sample analysis.

Conclusion(s): We have developed a robust 9-parameter flow cytometry panel that accurately detects changes in the expression of multiple platelet surface receptors in response to both agonists and inhibitors. The assay can be used for high-throughput evaluation of the effects of both pathological ligands and new pharmacological agents.