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

Abstract

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 × 105/ml to 2.8 × 107/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 (R2 =.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.