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

• Published in: Journal of thrombosis and haemostasis 2014-05, Vol.12 (5), p.614-627
• Main Authors: Arraud, N., Linares, R., Tan, S., Gounou, C., Pasquet, J.‐M., Mornet, S., Brisson, A. R.
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Apoptosis; Bioengineering; blood plasma; Blood Platelets - cytology; cell‐derived microparticles; Cryoelectron Microscopy; cryo‐electron microscopy; Exosomes - chemistry; Flow Cytometry; Glycophorin - metabolism; Gold - chemistry; Hematology; Human health and pathology; Humans; Image Processing, Computer-Assisted; Imaging; immunogold techniques; Life Sciences; Male; Metal Nanoparticles - chemistry; Phenotype; Phosphatidylserines - chemistry; Plasma - metabolism; Plasma - physiology; Platelet Membrane Glycoprotein IIb - metabolism
• ISSN: 1538-7933; 1538-7836; 1538-7836
• DOI: 10.1111/jth.12554

Summary Background Plasma 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. Objectives Our aim was to provide a comprehensive description of EVs from plasma of healthy subjects. Methods Cryo‐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. Results We show that platelet‐free plasma samples contain spherical EVs, 30 nm to 1 μm in diameter, tubular EVs, 1–5 μm long, and membrane fragments, 1–8 μm 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. Conclusion This study brings novel insights on EVs from normal plasma and provides a reference for further studies of EVs in disease situations.