Accurate characterization of extracellular vesicles (EVs) is critical to explore their diagnostic and therapeutic applications. As the EV research field has developed, so too have the techniques used to characterize them. The development of reference materials are required for the standardization of these techniques. This work, initiated from the ISEV 2017 Biomarker Workshop in Birmingham, UK, and with further discussion during the ISEV 2019 Standardization Workshop in Ghent, Belgium, sets out to elucidate which reference materials are required and which are currently available to standardize commonly used analysis platforms for characterizing EV refractive index, epitope abundance, size and concentration. Due to their predominant use among EV researchers, a particular focus is placed on the optical methods nanoparticle tracking analysis and flow cytometry.

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 erythrocytederived 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 0.8μm 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.

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.