Resources Microvesicle Analysis Interest Development Group

Microvesicle Analysis Interest Development Group


Mission and Objectives

Welcome to the ISAC Microvesicle Analysis Interest Group web page. Our goal is to provide a forum for information and interaction to enable and standardize the quantitative and multiparameter analysis of small cell-derived membrane vesicles.

Watch ISTH Academy presentation - Microvesicles Webinar Video

Contacts and participants

Francoise Dignat-George - Aix-Marseille University (

Nancy Fisher - University of North Carolina (

Phil Hexley -  University of Nebraska (

Joanne Lannigan - University of Virginia (

Francois Mullier -  Université Catholique de Louvain (

John Nolan - The Scintillon Institute (

Philippe Poncelet - BioCytex (


Cell-derived Membrane Vesicles

Mammalian cells release small membrane vesicles that can carry biological molecules and signals to exert biological effects at a distance. Exosomes are membrane vesicles of intracellular origin that are secreted from the cell by exocytosis. Ectosomes, also commonly referred to as microvesicles or microparticles, are shed from the plasma membrane. These membrane vesicles have been implicated in a wide range of physiological functions, but their very small size makes them a challenge to analyze.


Flow Cytometry of Microvesicles

Several different measurement approaches can be taken for the analysis of cell-derived microvesicles, and each has their advantages and disadvantages. For example, ELISA-type immunoassays can measure the total amount of a microvesicle-associated antigen in a sample, but do not provide single vesicle information. Optical measurements such as nanoparticle tracking analysis can provide information on the size and number of microvesicles, but not information about the amount of an antigen on individual vesicles. Flow cytometry is distinguished by its ability to measure multiple targets on individual particles, but the small size and dim signals from most microvesicles challenges the sensitivity limits of flow cytometry.


Standardization Efforts

Because of the potential importance of microvesicles as diagnostic or prognostic markers, there is significant interest in standardizing sample preparation and measurement approaches for cell-derived microvesicles. Notable efforts have taken place under the auspices of the International Society for Thrombosis and Hemostasis (ISTH), which has sponsored efforts to attempt to standardize flow cytometry measurements of microvesicles, as well as sample collection and processing steps. These continuing efforts have raised several important issues that impact the analysis of cell derived membrane vesicles, including:


  • How do differences in light scatter measurement among different commercial flow cytometers affect the microvesicle measurement?
  • What are the limits of antigen detection on microvesicles using fluorescence-labeled antibodies?
  • What is the appropriate use of microspheres in standardizing and calibrating instrument for use in measure small microvesicles?
  • What are the most appropriate gating strategies for analysis of microvesicles?
  • What is the minimum information that should be reported for a flow cytometry measurement of microvesicles?


Improving the Measurement of Microvesicles

The efforts described above have led to several important insights that will help standardize microvesicle measurements using the current generation of commercial flow cytometers. However, they also highlight the need for new, more sensitive methods for measuring cell-derived vesicles. Toward this end, it is important to address a further set of issues:


  • What are the best controls or reference samples for standardizing the measurement of microvesicles by any method?
  • What are the appropriate validation experiments that can define the performance of any new or existing microvesicle analysis method?


Get Involved

If you are interested the analysis of cell-derived membrane vesicles or other nanoparticles, and would like to participate in efforts to standardize and validate both existing measurement approaches and new technologies, you are welcome to join by sending your contact information and a brief description of your interests and expertise to


More Reading

van der Pol E, Coumans FAW, Grootemaat AE, Gardiner C, Sargent IL, Harrison P, Sturk A, van Leeuwen TG, Nieuwland R. Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J Thromb Haemost 2014; 12: 118292.


Sarah E. Headland, Hefin R. Jones, Adelina S. V. D’Sa, Mauro Perretti & Lucy V. Norling Cutting-Edge Analysis of Extracellular Microparticles using ImageStreamX Imaging Flow Cytometry. Nature Scientific Reports 4 : 5237 DOI: 10.1038 June 10 2014.


Erdbrügger U, Rudy CK, E Etter M, Dryden KA, Yeager M, Klibanov AL, Lannigan J. Imaging flow cytometry elucidates limitations of microparticle analysis by conventional flow cytometry. Cytometry A. 2014 Sep;85(9):756-70. doi: 10.1002/cyto.a.22494. Epub 2014  Jun 5.

J Extracell Vesicles. 2014 Dec 22;3:26913. doi: 10.3402/jev.v3.26913. eCollection 2014. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. Lötvall J
J Extracell Vesicles. 2015 Dec 31;4:30087. doi: 10.3402/jev.v4.30087. eCollection 2015.
Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper.
Lener T et. al.
PLoS One. 2016 Feb 22;11(2):e0149866. doi: 10.1371/journal.pone.0149866. eCollection 2016.
Comparative Analysis of Technologies for Quantifying Extracellular Vesicles (EVs) in Clinical Cerebrospinal Fluids (CSF).
Akers JC et. al.
Cytometry A. 2016 Feb;89(2):109-10. doi: 10.1002/cyto.a.22814.
Measurement of extracellular vesicles and other submicron size particles by flow cytometry.
Lannigan J, P Nolan J, Zucker R
Nanomedicine. 2016 Jan 6. pii: S1549-9634(15)00615-2. doi: 10.1016/j.nano.2015.12.370. [Epub ahead of print]
Enumeration of extracellular vesicles by a new improved flow cytometric method is comparable to fluorescence mode nanoparticle tracking analysis.
Pasalic L et. al.
Cytometry A. 2016 Feb;89(2):123-34. doi: 10.1002/cyto.a.22795. Epub 2015 Dec 9.
Analytical challenges of extracellular vesicle detection: A comparison of different techniques.
Erdbrügger U, Lannigan J.
Cytometry A. 2016 Feb;89(2):196-206. doi: 10.1002/cyto.a.22787. Epub 2015 Oct 20.
High sensitivity flow cytometry of membrane vesicles.
Stoner SA et. al.
Cytometry A. 2016 Feb;89(2):184-95. doi: 10.1002/cyto.a.22669. Epub 2015 Apr 9.
Fluorescence triggering: A general strategy for enumerating and phenotyping extracellular vesicles by flow cytometry.
Arraud N et. al.
Cytometry A. 2015 Nov;87(11):1052-63. doi: 10.1002/cyto.a.22649. Epub 2015 Apr 2.
Techniques to improve detection and analysis of extracellular vesicles using flow cytometry.
Inglis HC et. al.
Cytometry A. 2016 Feb;89(2):159-68. doi: 10.1002/cyto.a.22621. Epub 2015 Mar 21.
Super-resolved calibration-free flow cytometric characterization of platelets and cell-derived microparticles in platelet-rich plasma.
Konokhova AI et. al.
Cytometry A. 2016 Feb;89(2):135-47. doi: 10.1002/cyto.a.22644. Epub 2015 Feb 16.
Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry.
Groot Kormelink T et. al.

Curtis, A.M., J.Edelberg, R.Jonas, W. T. Rogers, J.S. Moore, W. Syed and E. R Mohler III Endothelial microparticles: Sophisticated vesicles modulating vascular function. (2013). Vascular Medicine: 18(4) 204–214

Yuana, Y., R. I. Koning, M. E. Kuil, P.C.N. Rensen, A.J. Koster, R.M. Bertina, and S. Osanto. Cryo-electron microscopy of extracellular vesicles in fresh plasma. (2013) J Extracell Vesicles. (2013); 2:10.3402/jev.v2i0.21494.

Zwicker, J.I., et al., Tissue factor-bearing microparticles and thrombus formation. Arterioscler Thromb Vasc Biol, 2011. 31(4): p. 728-33.

Dignat-George, F. and C.M. Boulanger, The many faces of endothelial microparticles. Arterioscler Thromb Vasc Biol, 2011. 31(1): p. 27-33.

Nieuwland, R. and A. Sturk, Why do cells release vesicles? Thromb Res, 2010. 125 Suppl 1: p. S49-51.

Cocucci, E., G. Racchetti, and J. Meldolesi, Shedding microvesicles: artefacts no more. Trends in Cell Biology, 2009. 19(2): p. 43-51.

Théry, C., M. Ostrowski, and E. Segura, Membrane vesicles as conveyors of immune responses. Nature Reviews Immunology, 2009. 9(8): p. 581-593.

Recent Microvesicle Analysis Standardization Efforts

Cointe S, Judicone C, Robert S, Mooberry MJ, Poncelet P, Wauben M, Nieuwland R, Key NS, Dignat-George F, Lacroix R. Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop. J Thromb Haemost 2016; DOI: 10.1111/jth.13514

Lacroix et al, High-Sensitivity Flow Cytometry Provides Access to Standardized Measurement of Small-Size Microparticles, Arterioscler Thromb Vasc Biol. 2012 Apr;32(4):1054-8.

Robert, S., et al., Standardization of platelet-derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies? J Thromb Haemost, 2009. 7(1): p. 190-7.

Standardization of Flow Cytometry Measurements

Hoffman, R.A., Standardization, calibration, and control in flow cytometry. Curr Protoc Cytom, 2005. Chapter 1: p. Unit 1 3.

Hoffman, R.A. and J.C. Wood, Characterization of flow cytometer instrument sensitivity. Curr Protoc Cytom, 2007. Chapter 1: p. Unit1 20.

Lee, J.A., et al., MIFlowCyt: the minimum information about a Flow Cytometry Experiment. Cytometry Part A, 2008. 73(10): p. 926-930.

Methodological Aspects of Microvesicle Measurements

Ayers,L., P.Harrison, M. Kohler and B. Ferry. Procoagulant and platelet-derived microvesicle absolute counts determined by flow cytometry correlates with a measurement of their functional capacity (2014) Journal of Extracellular Vesicles 2014, 3: 25348

Arraud, N., C. Gounou, R. Linares and A.R. Brisson. A simple flow cytometry method improves the detection of phosphatidylserine-exposing extracellular vesicles. (2014) Journal of Thrombosis and Haemostasis Epub October 2014 PMID: 25348269

Larson, M.C., M.R. Luthi, N. Hogg and C.A. Hillery. 2013 Calcium-Phosphate microprecipitates mimic microparticles when examined with flow cytometry. Cytometry Part A. 83A: 242-250.

Nolan, J.P., and S.A. Stoner. 2013 A trigger channel threshold artifact in nanoparticle analysis. Cytometry Part A. 83A: 301-305.

Van der Pol et al., Single vs. swarm detection of microparticles and exosomes by flow cytometry. J Thromb Haemost, 2012. 10(5):919-30

Lee, R.D., et al., Pre-analytical and analytical variables affecting the measurement of plasma-derived microparticle tissue factor activity. Thromb Res, 2011.

Chandler, W.L., W. Yeung, and J.F. Tait, A new microparticle size calibration standard for use in measuring smaller microparticles using a new flow cytometer. J Thromb Haemost, 2011. 9(6): p. 1216-24.

Ayers, L., et al., Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay. Thromb Res, 2011. 127(4): p. 370-7.

Yuana, Y., R.M. Bertina, and S. Osanto, Pre-analytical and analytical issues in the analysis of blood microparticles. Thromb Haemost, 2011. 105: p. 396-408.

Zwicker, J.I., Impedance-based flow cytometry for the measurement of microparticles. Semin Thromb Hemost, 2010. 36(8): p. 819-23.

van der Pol, E., et al., Optical and non-optical methods for detection and characterization of microparticles and exosomes. J Thromb Haemost, 2010. 8(12): p. 2596-607.

Orozco, A.F. and D.E. Lewis, Flow cytometric analysis of circulating microparticles in plasma. Cytometry A, 2010. 77(6): p. 502-14.

Lacroix, R., et al., Overcoming limitations of microparticle measurement by flow cytometry. Semin Thromb Hemost, 2010. 36(8): p. 807-18.

Jy, W., et al., Measuring circulating cell-derived microparticles. J Thromb Haemost, 2004. 2(10): p. 1842-51.

Other Resources

Vascular Biology Subcommittee of the Scientific Standardization Committee of the ISTH

International Society for Extracellular Vesicles (

ISEV Meetings (

Our vision is to advance the impact of cytometry in meeting current and emerging challenges in the life, biomedical, and physical sciences.

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