Chapter 1 14 Third, apart from their biological diversity and their large overlap in biophysical properties with other entities, a lack of robust EV detection methods and ambiguities in how data should be interpreted for EV analysis makes interpretation between studies challenging 46, 47. Currently, the gold standard approach for EV analysis is based on the isolation or concentration of EVs. Ultracentrifugation, density-gradient, and size exclusion chromatography are the most widely used EV isolation techniques 48, despite yielding low-purity EV samples due to the coisolation of non-desired molecules such as lipoproteins 11, 45. Additionally, a variety of analytical platforms are available. Nanoparticle tracking analysis (NTA) allows the determination of the size distribution and a rough indication of the concentration of individual nanoparticles in suspension49, but provides limited phenotyping capabilities. In turn, transmission electron microscopy (TEM) is able to image particles <1 nm, but is time consuming and not suitable for looking at shifts in EV populations. Other methods, such as ELISA and Western blot analysis, offer bulk phenotyping abilities but lack quantification 9, 15, 50, 51. Thus, a tool for the accurate determination of the concentration and phenotyping of single EVs in complex samples such as plasma represents an unmet need. The holy grail: direct detection of single EVs in complex samples The only technique that has the potential to detect, size, and phenotype thousands to millions of EVs per minute is flow cytometry (FC) 52. However, most clinical flow cytometers, and their corresponding assays, are designed for cell measurements and are not readily adapted to measure EVs; as the majority of EVs are <300 nm in diameter, conventional FCs struggle to discriminate these particles from background signals 11, 53, 54. Another problem with flow cytometry is that the generated signals are expressed in arbitrary units, which hinders comparison of results between different instruments 47. To address these issues, more sensitive instruments are introduced into the field, and guidelines regarding methods and data reporting are being developed for both flow cytometry (Minimum Information about a Flow Cytometry experiment, MIFlowCyt) and EV research 47. In summary, the ideal EV analysis platform would be able to 1) detect and discriminate single EVs <300 nm in diameter above background signals of the instrument, 2) determine the size, concentration and phenotype of single EVs, 3)
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