General Discussion and Future Perspectives 8 187 Single-EV FC Strategies The ideal platform for single EV analysis would 1) be widely available in clinical laboratories, 2) be technically validated, 3) have a high throughput, 4) have high reproducibility, and 5) be able to reveal EV subpopulations via phenotyping 17. Consequently, bulk EV analysis methods – which report only average properties of an EV preparation – such as Western Blot, ELISA/TR-FIA, or mass-spec proteomics are limited in their usefulness 13 when aiming to examine the diagnostic potential of EVs. Flow Cytometry (FC) as an analysis platform fits all the above criteria, but – as described in chapter 1 - most FCs are designed for cell measurements and are not readily adapted to measure EVs 4, 18, 19. To address this issue, several different strategies have been introduced to the field. For example, using an optimized configuration of the commercially available BD Influx FC, Vlist et al have demonstrated the detection of isolated cell supernatant-derived EVs ~100 nm in diameter 20. However, applying these configurations to the FC requires an experienced operator with technicalexpertise. Additionally, such modifications will turn the FC into a dedicated EV detection platform, thus ‘losing’ the ability to be used for cell analysis. Another published FC-based method, performed on a commercially available Beckman Coulter CytoFlex, describes a strategy to directly measure EVs in plasma by labelling the EVs with a fluorescent lipid probe (vFRed) in combination with CFDASE or an anti-tetraspanin mixture 21. In this study, rather than relating SSC signals into particle size through Mie calculations, EV size (surface area) is determined by calibration of EV membrane fluorescence by using a synthetic vesicle size standard, as provided in the vFC EV Analysis kit from Cellarcus Biosciences. Staining with the lipid membrane dye should be consistent for applicability, meaning that either the amount of dye needs to be approximately matched to the number of EVs (which is unknown before analysis), or an excess of dye should be used so that the membrane becomes saturated with dye 22. An excess of dye will, however, lead to increased background signals, which, in turn, will hinder discrimination of EVs above background. These two examples highlight the current status of the EV FC field, where either dedicated EV-detection platforms are being developed, or existing platforms are tested for their sensitivity to detect sub-micron particles. The development of the IFCM-methodology as described in the first part of this thesis falls into the second
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