Platelet removal from human blood plasma improves detection of extracellular vesicle‐associated miRNA

Human blood plasma prepared by centrifugation contains not only extracellular vesicles (EVs) but also platelets and erythrocyte ghosts (ery‐ghosts). Here we studied whether analysis of miRNA associated with plasma EVs (EV‐miRNA) is affected by the presence of platelets and ery‐ghosts. EDTA blood was collected from healthy donors (n = 3), and plasma was prepared by the centrifugation protocol recommended by the International Society on Thrombosis and Haemostasis (ISTH), and by a centrifugation protocol from an EV‐miRNA expert lab (non‐ISTH protocol). EVs were isolated from plasma by size‐exclusion chromatography CL‐2B (SEC2B), and concentrations of platelets, activated platelets, ery‐ghosts and EVs (150–1000 nm) were measured by calibrated flow cytometry. Two EV‐associated miRNAs (let7a‐5p and miR‐21‐5p), and one platelet‐associated miRNA (miR‐223‐3p), were measured by qRT‐PCR. Measurements were performed with and without filtration using 0.8 μm track‐etched filters to remove platelets and ery‐ghosts from plasma and EV‐enriched SEC fractions. Plasma prepared by both centrifugation protocols contained platelets and ery‐ghosts, which co‐migrated with EVs into the EV‐enriched SEC2B fractions. Filtration removed platelets and ery‐ghosts (>97%; p ≤ 0.05) and did not affect the EV concentrations (p > 0.17). The miRNA concentrations were 2–4‐fold overestimated due to the presence of platelets but not ery‐ghosts. Thus, filtration of human plasma is expected to improve comparability and reproducibility of quantitative EV‐miRNA studies. Therefore, we recommend to measure and report the plasma concentration of platelets for EV‐miRNA studies, and to filter plasma before downstream analyses or storage in biobanks.