A simple flow cytometry method improves the detection of phosphatidylserine‐exposing extracellular vesicles

Summary Background Plasma contains cell‐derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as disease biomarker. However, the enumeration of EVs faces major problems, due to their sub‐micrometer size and to intrinsic limitations in...

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Bibliographic Details
Published in:Journal of thrombosis and haemostasis Vol. 13; no. 2; pp. 237 - 247
Main Authors: Arraud, N., Gounou, C., Linares, R., Brisson, A. R.
Format: Journal Article
Language:English
Published: England Elsevier Limited 01.02.2015
Wiley
BlackWell Publishing Ltd
Subjects:
Annexin A5
Biochemistry
Biochemistry, Molecular Biology
Bioengineering
Biomarkers - blood
blood plasma
Cell-Derived Microparticles - chemistry
Cell-Derived Microparticles - ultrastructure
cell‐derived microparticles
Coagulation
electron microscopy
Flow Cytometry
Fluorescent Dyes
Humans
Life Sciences
Male
Microscopy, Electron
Phenotype
phosphatidylserines
Phosphatidylserines - blood
Predictive Value of Tests
electron microscopy
phosphatidylserines
flow cytometry
cell-derived microparticles
blood plasma
ISSN:1538-7933, 1538-7836, 1538-7836
Online Access:Get full text
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Summary:Summary Background Plasma contains cell‐derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as disease biomarker. However, the enumeration of EVs faces major problems, due to their sub‐micrometer size and to intrinsic limitations in methods of characterization, mainly flow cytometry (FCM). Objectives Our objective is to enumerate EVs in plasma, by taking as the prototype the population of phosphatidylserine (PS)‐exposing EVs, which constitute one of the major EV populations and are responsible for thrombotic disorders. Methods The concentration of PS‐exposing EVs in platelet‐free plasma (PFP) of healthy subjects was measured by FCM using either light scattering or fluorescence as the trigger and fluorescent Annexin‐5 (Anx5) as the specific label. In addition, PS‐exposing EVs were enumerated by electron microscopy (EM) after labeling with Anx5 gold nanoparticles and sedimentation on EM grids. Results We show that about 50× more Anx5‐positive EVs are detected by FCM when detection is triggered on fluorescence as compared with light scattering. By fluorescence triggering, concentrations of 22 000–30 000 Anx5‐positive EVs per μL PFP were determined, using two different flow cytometers. The limit of detection of the fluorescence triggering method was estimated at about 1000–2500 Anx5 molecules. Results from EM suggest that EVs down to 100–150 nm diameter are detected by fluorescence triggering. Conclusion This study presents a simple method for enumerating EVs. We believe that this method is applicable in a general context and will improve our understanding of the roles of EVs in pathophysiological situations, which will open avenues for the development of EV‐based diagnosis assays.
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Manuscript handled by: C. Gachet
Final decision: P. H. Reitsma, 16 October 2014
ISSN:1538-7933
1538-7836
1538-7836
DOI:10.1111/jth.12767