Extracellular vesicles: Great potential, many challenges.

Abstract

Extracellular vesicles (EVs) are small membranous nanoparticles that are released from cells and found in various biofluids including blood, urine, and saliva 1, 2. EVs can be released by secretion from intracellular compartments (exosomes), shed from the cell surface (ectosomes, aka microvesicles, or microparticles), as a consequence of a variety of cellular processes such as activation, apoptosis, or other forms of cell death. EVs carry cargo derived from their cell of origin and may be major players in intercellular communication, as well as to be potential biomarkers for both health and disease. Because EVs originate from many different cell types and via several different mechanisms, EVs found in biofluids are very heterogeneous. This heterogeneity, compounded with their small size makes them very difficult to study. Single particle multiparameter measurement techniques such as flow cytometry would appear to be well suited to the characterization of EVs, but conventional commercial instruments designed for lymphocyte analysis are challenged to measure these small, dim particles. The review by Chandler in this issue summarizes the recent literature describing attempts to measure EVs using commercial flow cytometers. Details of biofluid sample collection, preparation, and storage are expected to have major impacts on downstream analyses. Choices about how to trigger detection of EVs, for example, using light scatter or fluorescence, can have a major impact on measurement results. Interpreting light scatter and fluorescence measurements of detected events in terms of particle size and antigen abundance are extremely challenging and the subject of many misconceptions. The review by Chandler highlights many of these difficulties and misconceptions, and conveys an appropriately cautionary view to the interpretation of the published literature. The interested reader is recommended to also review the recent position papers 3, 4 produced by the International Society for Extracellular Vesicles (ISEV) outlining the current consensus best practices for isolating and characterizing EVs. The recent special issue in Cytometry Part A on the measurement of EVs and other submicron sized particles also provides discussions of the strengths and limitations of conventional methods for measuring EVs 5-7 and for new approaches that may offer advantages over current methods 8, 9. Looking forward, we can expect that the great potential of EVs as biomarkers, diagnostics, and even therapeutics will inspire the development of new instruments, reagents, and assays with improved sensitivity, specificity, and resolution. The development, validation, and adoption of such new methods will depend upon the appropriate calibration and standardization methods to characterize small, dim particles. The flow cytometry community has a long history of collaboration between researchers and instrument and reagent manufacturers to develop standards and calibration approaches to enable quantitative cytometry. To this end, the International Society for Advancement of Cytometry (ISAC), the International Society for Thrombosis and Haemostatis (ISTH), and ISEV has formed a joint working group to collaborate on the evaluation of approaches to standardize the calibration and reporting of EV analysis results that are instrument-independent. Such inter society cooperation and collaboration is critical to assure that data collected across laboratories can be compared and to pave the way for the expansion of flow cytometry into the submicron world not only for research, but for potential clinical utility. John P. Nolan* The Scintillon Institute San Diego, California 92121 Jonni Moore Department of Pathology and Laboratory Medicine Perelman School of Medicine of the University of Pennsylvania Philadelphia, Pennsylvania