Preanalytical conditions for multiparameter platelet flow cytometry

BACKGROUND: Flow cytometry is an important technique for understanding multiple aspects of blood platelet biology. Despite the widespread use of the platform for assessing platelet function, the optimization and careful consideration of preanalytical conditions, sample processing techniques, and data analysis strategies should be regularly assessed. When set up and designed with optimal conditions, it can ensure the acquisition of robust and reproducible flow cytometry data. However, these parameters are rarely described despite their importance. OBJECTIVES: We aimed to characterize the effects of several preanalytical variables on the analysis of blood platelets by multiparameter fluorescent flow cytometry. METHODS: We assessed anticoagulant choice, sample material, sample processing, and storage times on 4 distinct and commonly used markers of platelet activation, including fibrinogen binding, expression of CD62P and CD42b, and phosphatidylserine exposure. RESULTS: The use of suboptimal conditions led to increases in basal platelet activity and reduced sensitivities to stimulation; however, the use of optimal conditions protected the platelets from artifactual stimulation and preserved basal activity and sensitivity to activation. CONCLUSION: The optimal preanalytical conditions identified here for the measurement of platelet phenotype by flow cytometry suggest a framework for future development of multiparameter platelet assays for high-quality data sets and advanced analysis.