Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration

BackgroundPlasma and other body fluids contain membranous extracellular vesicles (EVs), which are considered to derive from activated or apoptotic cells. EVs participate in physiological and pathological processes and have potential applications in diagnostics or therapeutics. Knowledge on EVs is, however, limited, mainly due to their sub-micrometer size and to intrinsic limitations in methods applied for their characterization. ObjectivesOur aim was to provide a comprehensive description of EVs from plasma of healthy subjects. MethodsCryo-transmission electron microscopy combined with receptor-specific gold labeling was used to reveal the morphology, size and phenotype of EVs. An original approach based on sedimentation on electron microscopy grids was developed for enumerating EVs. A correlation was performed between conventional flow cytometry and electron microscopy results. ResultsWe show that platelet-free plasma samples contain spherical EVs, 30nm to 1m in diameter, tubular EVs, 1-5m long, and membrane fragments, 1-8m large. We show that only a minority of EVs expose the procoagulant lipid phosphatidylserine, in contrast to the classical theory of EV formation. In addition, the concentrations of the main EV sub-populations are determined after sedimentation on EM grids. Finally, we show that conventional flow cytometry, the main method of EV characterization, detects only about 1% of them. ConclusionThis study brings novel insights on EVs from normal plasma and provides a reference for further studies of EVs in disease situations.