Isolation and characterization of circulating small extracellular vesicles: a step towards method validation and clinical biomarker application.

Abstract

This study aims to simplify the diagnosis or monitoring of diseases by analyzing small extracellular vesicles (sEVs) in the blood rather than relying on biopsy or cerebrospinal fluid sampling. Secreted by cells, sEVs reflect to some extent the molecular composition of the originating cell. Different methods for isolating sEVs from plasma are compared and evaluated (1), including size exclusion chromatography, differential centrifugation, bead-based immunoprecipitation, polymer-based precipitation, and membrane affinity isolation. The yield and purity of each isolation method are assessed by investigating the presence of typical EV markers, depletion of plasma contaminants, and particle quantity and size distribution. Further, selected isolation methods were assessed in a validation-like setting to show their reproducibility (2). In the following, the interindividual variation (3) has been evaluated for one chosen isolation method. 1) All sEV isolation methods used are suitable for purification from plasma, but the isolates differ significantly in terms of yield and purity. Particularly sEV-rich purifications were chosen to further evaluate the feasibility of clinical sample analysis. 2) The selected methods, based on ultracentrifugation and size-exclusion chromatography, were shown to be reproducible, with less than 30% deviation from the mean for 2/3 of the analyzed samples. Even though the size exclusion chromatography method qEVsingle 35 Tween shows the highest deviations, low levels of total protein and HDL contaminations were shown. 3) The test for inter-individual variability by purifying and characterizing 10 individual human plasma samples found significant variation in the results obtained, verified with different characterization methods. Further, the technical requirements and throughput of the different isolation and characterization methods were assessed. Overall, the study concludes that sEV isolation can support biomarker discovery. Even though all methods used in the study are suitable for purifying sEVs from plasma, the isolates differ significantly in terms of yield and purity. Some isolation methods are more reproducible than others which should be considered in early biomarker projects. The preferred method, qEVsingle 35 Tween, can reveal interindividual differences in sEV amount and phenotype. That has been proven by an sEV characterization workflow that was implemented to get a comprehensive sEV description from 150 µl of plasma. The characterization methods were compared, and their strengths and weaknesses were listed against each other. It is shown which methods are suitable to support biomarker research and clinical applications.