Measurement of microvesicle levels in human blood using flow cytometry

Abstract

Microvesicles are fragments of cells released when the cells are activated, injured, or apoptotic. Analysis of microvesicle levels in blood has the potential to shed new light on the pathophysiology of many diseases. Flow cytometry is currently the only method that can simultaneously separate true lipid microvesicles from other microparticles in blood, determine the cell of origin and other microvesicle characteristics, and handle large numbers of clinical samples with a reasonable effort, but expanded use of flow cytometric measurement of microvesicle levels as a clinical and research tool requires improved, standardized assays. The goal of this review is to aid investigators in applying current best practices to microvesicle measurements. First pre-analytical factors are evaluated and data summarized for anticoagulant effects, sample transport and centrifugation. Next flow cytometer optimization is reviewed including interference from background in buffers and reagents, accurate microvesicle counting, swarm interference, and other types of coincidence errors, size calibration, and detection limits using light scattering, impedance and fluorescence. Finally current progress on method standardization is discussed and a summary of current best practices provided. V C 2016 Clinical Cytometry Society

Revealing the diversity of extracellular vesicles using high-dimensional flow cytometry analyses

“…EV levels are modulated in certain pathologies, and studies have established the vast diversity of EVs produced by cells, suggesting that EVs might be used as biomarkers27. Groups of scientists have established the most appropriate pre-analytical conditions for the study of EVs and for the design of modern methodologies for their fine characterization3910111314171826272930484950. Furthermore, efforts have been made in recent years to institute a coherent nomenclature for EVs44.…”

“…For instance, although fluorescence, rather than light scatter, used as trigger greatly improves EV detection, the distinction of EVs from background in FCM is still an obstacle in complex fluids121317. Furthermore, platelets are abundant in blood and represent an important source of EVs; however, they are frequently misinterpreted as EVs given their relatively small dimensions11. The absence of such analytical tools for the comprehension of EV functions prompted our study.…”

“…Furthermore, EVs are induced by cell activation, and EV levels in biological fluids are altered in different pathologies, such as cancer, rheumatic diseases, neurodegenerative disorders, organ damage, and infectious diseases238. Hence, EVs appear to be potent biomarkers, and their detection in blood and other biological fluids is improving with the recent establishment of pre-analytical conditions, standardization of quantification approaches, and development of more efficient means of detection9101112131415.…”

Could Microparticles Be the Universal Quality Indicator for Platelet Viability and Function?

“…It is now well accepted that accurate bead standards with appropriate refractive indices to gate microparticles by flow cytometry still need to be developed [47, 59]. Currently, comparing data from different studies is difficult due to the wide variety of methods for microparticle determination used by different laboratories [60].…”

“…This is possibly due to the use of native or fixed samples without differential centrifugation to remove platelets prior to testing. The much lower microparticle concentrations detected by flow cytometry could be related to (1) beads being inadequate as size standards [47], (2) loss of microparticles below the electronic threshold [44], and (3) limitations such as swarm detection [57, 59]. …”