Extracellular Vesicle Detection and Analysis via Flow Cytometry

Over the past decade, there has been a rapid growth in studies of secreted membrane vesicles, collectively called extracellular vesicles (EVs). (1) The release of EVs has been reported in the pathologies of cancer (2-5), neurological, hematological (6), cardiovascular (7), autoimmune and rheumatologic diseases (8), and viral infections such as malaria (9).  The study of EVs is gaining interest within both the medical and scientific communities due to the diagnostic and therapeutic possibilities. However, the identification and classification of EVs has been problematic.  Although advances in various fields, including microscopy, have addressed some of the preliminary hindrances, flow cytometry remains the dominant approach for the characterization of submicron cell- derived particles. The primary hurdle in analyzing particles at the submicron level has been to accurately represent their size distribution and light scatter profiles. Instrumentation thresholds were originally designed using whole blood as the standard, thereby excluding cellular measurement below 3mm. Recently, flow cytometric technology has been developed to distinguish populations spanning the <400nm to 1mm range.  In this independent study, several of those technologies are evaluated and compared. As most of the hardware adjustments are accomplished by enhancements to the FSC parameter (Beckman Coulter’s MoFlo Astrios EQ and MoFlo XDP with NanoView module), the study will also evaluate the use of Violet SSC on Beckman Coulter’s CytoFLEX as a novel approach to small particle detection. According to Mie theory, it is hypothesized that Violet SSC will give comparable results, as the lower wavelength will allow for detection of smaller particles.
 
Part 1—Using flow cytometry for vesicles FL and scatter
Part 2--- Flow cytometric analysis after sorting
 

1. Jan Lötvall, Andrew F. Hill, Fred Hochberg, Edit I. Buzás, Dolores Di Vizio, Christopher Gardiner, Yong Song Gho, Igor V. Kurochkin, Suresh Mathivanan, Peter Quesenberry, Susmita Sahoo, Hidetoshi Tahara, Marca H. Wauben, Kenneth W. Witwer, Clotilde Théry .  Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. Journal of Extracellular Vesicles. 2014, 3: 26913.
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3. Rabinowits G, Gercel-Taylor C, Day JM, Taylor DD, Kloecker GH.  Exosomal microRNA: a diagnostic marker for lung cancer. Clin Lung  Cancer 2009;10(1):42-6 556 8.
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5. Toth B, Liebhardt S, Steinig K, et al. Platelet-derived microparticles and coagulation activation in breast cancer patients. Thromb Haemost 562 2008;100(4):663-9.
6. Toth B, Liebhardt S, Steinig K, et al. Platelet-derived microparticles and coagulation activation in breast cancer patients. Thromb Haemost 2008;100(4):663-9.
7. Kirsty M. Danielson and Saumya Das. Extracellular Vesicles in Heart Disease: Excitement for the Future?. Exosomes Microvesicles, 2014, 2:1. doi: 10.5772/58390.
8. Sellam J, Proulle V, Jungel A, et al. Increased levels of circulating microparticles in primary Sjogren's syndrome, systemic lupus erythematosus and rheumatoid arthritis and relation with disease activity. Arthritis Res Ther 2009;11(5):R156.
9. Pierre-Yves Mantel, Anh N. Hoang, Ilana Goldowitz, Daria Potashnikova, Bashar Hamza, Ivan Vorobjev, Ionita Ghiran, Mehmet Toner, Daniel Irimia, Alexander R. Ivanov, Natasha Barteneva, Matthias Marti.  Malaria-Infected Erythrocyte-Derived Microvesicles Mediate Cellular Communication within the Parasite Population and with the Host Immune System. Cell Host & Microbe, Volume 13, Issue 5, 15 May 2013, Pages 521-534.

Learning Objectives:

• Understand how flow cytometric technology has recently been developed to distinguish extracellular vesicle (EV) populations spanning the <400nm to 1mm range
• See an independent study comparing several technologies are evaluated and compared