Quality and efficiency assessment of six extracellular vesicle isolation methods by nano-flow cytometry

Extracellular vesicles (EVs) have sparked tremendous interest owing to their prominent potential in diagnostics and therapeutics. Isolation of EVs from complex biological fluids with high purity is essential to the accurate analysis of EV cargo. Unfortunately, generally used isolation techniques do not offer good separation of EVs from non-EV contaminants. Hence, it is important to have a standardized method to characterise the properties of EV preparations, including size distribution, particle concentration, purity and phenotype. Employing a laboratory-built nano-flow cytometer (nFCM) that enables multiparameter analysis of single EVs as small as 40 nm, here we report a new benchmark to the quality and efficiency assessment of EVs isolated from plasma, one of the most difficult body fluids to work with. The performance of five widely used commercial isolation kits was examined and compared with the traditional differential ultracentrifugation (UC). Two to four orders of magnitude higher particle concentrations were observed for EV preparations from platelet-free plasma (PFP) by kits when compared with the EV preparation by UC, yet the purity was much lower. Meanwhile, the particle size distribution profiles of EV preparations by kits closely resembled those of PFP whereas the EV preparation by UC showed a broader size distribution at relatively large particle size. When these kits were used to isolate EVs from vesicle-depleted PFP (VD-PFP), comparable particle counts were obtained with their corresponding EV preparations from PFP, which confirmed again the isolation of a large quantity of non-vesicular contaminants. As CD9, CD63 and CD81 also exist in the plasma matrix, single-particle phenotyping of EVs offers distinct advantage in the validation of EVs compared with ensemble-averaged approaches, such as Western blot analysis. nFCM allows us to compare different isolation techniques without prejudice.