biostudies-literatureUnknown23(1)Cufaro MC<h4>Background</h4>The isolation and proteomics characterization of extracellular vesicles (EVs) from body fluids is challenging due to their vast heterogeneity. We have recently demonstrated that Fluorescence-activated Cell Sorting (FACS) efficiently isolates the whole EV circulating compartment directly from untouched body fluids enabling a comprehensive EV proteomics analysis.<h4>Results</h4>Here, we characterized, for the first time, a single-phenotype EV subset by sorting leukocyte-derived EVs (Leuko EVs) from peripheral blood and tears of healthy volunteers. Using an optimized and patented staining protocol of the whole EV compartment we identified and excluded non-EV particles, debris and damaged EVs. We further isolated, using an anti-CD45 antibody, Leuko EVs (CD45+ EVs), reaching Journal of translational medicine565https://www.ebi.ac.uk/biostudies/studies/S-EPMC12093873biostudies-literatureFACS-Proteomics strategy toward extracellular vesicles single-phenotype characterization in biological fluids: exploring the role of leukocyte-derived EVs in multiple sclerosis.PMC12093873Cufaro MCFontana ACicalini IRispoli MGDe Bellis DVeschi SBrocco DLanuti PDel Boccio PSimeone PTomassini VPiro ADi Sebastiano AKhorooshi RMHFederici LPilato SPieragostino DfalseFACS-Proteomics strategy toward extracellular vesicles single-phenotype characterization in biological fluids: exploring the role of leukocyte-derived EVs in multiple sclerosis.<h4>Background</h4>The isolation and proteomics characterization of extracellular vesicles (EVs) from body fluids is challenging due to their vast heterogeneity. We have recently demonstrated that Fluorescence-activated Cell Sorting (FACS) efficiently isolates the whole EV circulating compartment directly from untouched body fluids enabling a comprehensive EV proteomics analysis.<h4>Results</h4>Here, we characterized, for the first time, a single-phenotype EV subset by sorting leukocyte-derived EVs (Leuko EVs) from peripheral blood and tears of healthy volunteers. Using an optimized and patented staining protocol of the whole EV compartment we identified and excluded non-EV particles, debris and damaged EVs. We further isolated, using an anti-CD45 antibody, Leuko EVs (CD45+ EVs), reaching 2025-01-01T00:00:00Z2025 May2026-06-01T10:22:47.507Z2026-04-08T11:24:10.714ZS-EPMC120938734039463310.1186/s12967-025-06558-4