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Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration
Author(s) -
Arraud N.,
Linares R.,
Tan S.,
Gounou C.,
Pasquet J.M.,
Mornet S.,
Brisson A. R.
Publication year - 2014
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/jth.12554
Subject(s) - nanoparticle tracking analysis , extracellular vesicles , flow cytometry , phosphatidylserine , vesicle , transmission electron microscopy , biophysics , microvesicles , chemistry , phenotype , morphology (biology) , electron microscope , nanotechnology , microbiology and biotechnology , materials science , membrane , biology , biochemistry , microrna , physics , phospholipid , gene , genetics , optics
Summary Background Plasma and other body fluids contain membranous extracellular vesicles ( EV s), which are considered to derive from activated or apoptotic cells. EV s participate in physiological and pathological processes and have potential applications in diagnostics or therapeutics. Knowledge on EV s 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 EV s 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 EV s. An original approach based on sedimentation on electron microscopy grids was developed for enumerating EV s. A correlation was performed between conventional flow cytometry and electron microscopy results. Results We show that platelet‐free plasma samples contain spherical EV s, 30 nm to 1 μm in diameter, tubular EV s, 1–5 μm long, and membrane fragments, 1–8 μm large. We show that only a minority of EV s 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 EV s from normal plasma and provides a reference for further studies of EV s in disease situations.