General Discussion and Future Perspectives 8 185 Controls are key Omitting EV isolation requires an analysis platform that is able to 1) detect and discriminate EVs from contaminating components in molecularly complex samples, and 2) report the results in a standardized manner (e.g. according to the international system of units). In this thesis, we identified Imaging Flow Cytometry (IFCM) as a suitable technique that can detect EVs, discriminate distinct EV subpopulations, and distinguish EVs from non-EV particles in complex biofluids without prior EV isolation (chapters 3 and 4). The protocols as developed and described in this thesis are compliant with MIFlowCyt-EV 9 - a reporting framework for single EV flow cytometry designed by members of MISEV. To ensure the identification and analysis of single EVs, we performed a strict set of assay controls designed to prove the detection of single EVs, and discriminate them from interfering components such as e.g. lipoproteins or protein aggregates (highly abundant in platelet-poor plasma 4). These controls included buffer only, buffer with reagents, unstained controls, isotype controls, single-stained controls, and procedural controls. Additionally, serial dilution and detergent-treated EV samples are used throughout this thesis. Serial sample dilution assists in evaluating whether EVs are detected as single particles, or to identify potential coincidence detection - also known as “swarm detection” 10. Obtaining a linear particle count consistent with the dilution factor, and maintaining a stable fluorescence and/or light scatter signal are indicative of single particle analysis 9-11. Using the imaging capabilities of IFCM, we designed a gating strategy which allows the simultaneous (selective) analysis of both single and multiple fluorescent events, thereby providing insight into the degree of coincidence detection for any given sample. This is (currently) a unique feature for IFCM which may prove to be beneficial in identifying highly concentrated samples – which consequently need to be further diluted to reduce/eliminate coincidence detection. Detergent controls aid in determining whether detected events represent membrane-enclosed particles. Following incubation with a detergent, the membranous structures of EVs are lysed – reducing their numbers and signals – whilst protein complexes or other particles will persist 9, 12, 13. In this thesis, in all biofluids assessed, we identified double-positive fluorescent events to be largely comprised of EVs as a ≥ 94% reduction in concentration was observed following
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