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Here, we have developed a workflow for the optimisation of a next\u2010generation panel for in\u2010depth immunophenotyping of circulating plasma EVs using spectral flow cytometry. Our data collection followed a multistep optimisation phase for both instrument setup and 21\u2010colour panel design, thus maximising fluorescent signal recovery. This spectral approach enabled the identification of novel EV subpopulations. Indeed, besides common EVs released by erythrocytes, platelets, leukocytes and endothelial cells, we observed rare and poorly known EV subsets carrying antigens related to cell activation or exhaustion. Notably, the unsupervised data analysis of major EV subsets revealed subpopulations expressing up to five surface antigens simultaneously. However, the majority of EVs expressed only a single surface antigen, suggesting they may not fully represent the phenotype of their parent cells. This is likely due to the small surface area or the biogenesis of EVs rather than antibody steric hindrance. Finally, we tested our workflow by analysing the plasma EV landscape in a cohort of systemic lupus erythematosus (SLE) patients. Interestingly, we observed a significant increase in CD54<jats:sup>+<\/jats:sup> EVs, supporting the notion of elevated circulating ICAM under SLE conditions. To our knowledge, these are the first data highlighting the importance of a spectral flow cytometry approach in deciphering the heterogeneity of plasma EVs paving the way for the routine use of a high\u2010dimensional immunophenotyping in EV research.<\/jats:p>","DOI":"10.1002\/jex2.70045","type":"journal-article","created":{"date-parts":[[2025,4,25]],"date-time":"2025-04-25T14:10:51Z","timestamp":1745590251000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Unravelling Plasma Extracellular Vesicle Diversity With Optimised Spectral Flow Cytometry"],"prefix":"10.1002","volume":"4","author":[{"given":"Daniela","family":"Boselli","sequence":"first","affiliation":[{"name":"Experimental Imaging Center, FRACTAL, Flow cytometry Resource, Advanced Cytometry Technical Applications Laboratory IRCCS Ospedale San Raffaele  Milan 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