Comprehensive Comparison of Methods for Isolation of Extracellular Vesicles from Human Plasma

Extracellular vesicles (EVs) are a vital component in cell–cell communication and hold significant potential as biomarkers and therapeutic carriers. Having a reproducible and simple EV isolation method for small volumes of human plasma is essential for biomarker discovery. Although combining multiple methods has been a recent trend in its ability to minimize contamination, it is not ideal for clinical specimens due to the large sample number and small sample volume. This study compared EVs isolated from 100 μL of plasma by nine commonly used methods based on different principles, including centrifugation, polymer precipitation, size exclusion, electrostatic interaction, and affinity enrichment. The isolated EVs were characterized by particle size and number using nanoparticle tracking analysis, purity, and contaminants using Simple Western and overall proteomic profiles using bottom-up proteomics. Despite the same EV enrichment principle, individual methods isolated EVs exhibited distinct characteristics, likely due to variations in the physicochemical properties of materials used and specific protocols. Overall, all of the methods evaluated are reproducible. MagNet and MagCap methods result in purer EVs with the narrowest size distribution and the highest proteome coverage but modest yield. This is the first report on isolating EVs from 100 μL of plasma using nine different methods with detailed characterization.