Removal of platelets from blood plasma to improve the quality of extracellular vesicle research

BACKGROUND: Blood plasma is commonly used for biomarker research of extracellular vesicles (EVs). Removing all cells prior to analysis of EVs is essential. OBJECTIVES: We therefore studied the efficacy of the most commonly used centrifugation protocol to prepare cell‐free plasma. METHODS: Plasma was prepared according to the double centrifugation protocol of the International Society on Thrombosis and Haemostasis (ISTH) in three independent studies. The concentrations of platelets, platelet‐derived EVs, and erythrocyte‐derived EVs were measured by calibrated flow cytometry. RESULTS: The mean platelet concentration ranged from 5.1 × 10(5)/ml to 2.8 × 10(7)/ml and differed 55‐fold between studies. Thus, the ISTH centrifugation protocol does not remove all platelets and results in variation between studies. As the concentration of platelet‐derived EVs and platelets correlates linearly (R (2) = .56), and the volume fraction of EVs and platelets in plasma are similar, the presence of platelets affects downstream analysis. To remove platelets a 0.8‐μm polycarbonate filter was used to lower the platelet concentration 146‐fold (p = .0013), without affecting the concentration of platelet‐derived and erythrocyte‐derived EVs (p = .982, p = .742). CONCLUSIONS: To improve the quality of EV research, we recommend (1) measuring and reporting the platelet concentration in plasma used for EV research, or (2) removing platelets by centrifugation followed by filtration.