{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T01:57:59Z","timestamp":1785463079791,"version":"3.56.0"},"reference-count":40,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2026,5,25]],"date-time":"2026-05-25T00:00:00Z","timestamp":1779667200000},"content-version":"vor","delay-in-days":24,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T00:00:00Z","timestamp":1777593600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100002491","name":"Bristol-Myers Squibb","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100002491","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["isevjournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["J of Extracellular Bio"],"published-print":{"date-parts":[[2026,5]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Imaging flow cytometry (IFCM) has emerged as a powerful method for high\u2010throughput phenotyping of small extracellular vesicles (sEVs) at single\u2010vesicle resolution. However, the reliability and sensitivity of IFCM critically depend on the performance of the antibodies used. In this study, we systematically compared several commercially available anti\u2010PD\u2010L1 antibodies for their ability to detect PD\u2010L1\u2010positive sEVs. Despite being derived from the same clone, antibodies from different manufacturers showed striking differences in labelling efficiency. These findings demonstrate that antibody validation for cellular targets does not ensure suitability for single\u2010EV analysis and underscore the need for rigorous, application\u2010specific qualification. To address this, we developed a robust IFCM workflow in full alignment with MIFlowCyt\u2010EV reporting standards. Using a qualified anti\u2010PD\u2010L1 antibody, we achieved reproducible detection of PD\u2010L1\n                    <jats:sup>+<\/jats:sup>\n                    sEVs directly in unprocessed plasma samples from tumour patients. To assess the quantitative potential of single\u2010EV analysis, we compared this approach with bead\u2010based capture and performed dilution experiments. While bead\u2010based methods yielded a relatively narrow range of mean fluorescence intensities across samples, IFCM revealed distinct PD\u2010L1\n                    <jats:sup>+<\/jats:sup>\n                    sEV levels, supporting its semi\u2010quantitative capability. Finally, we applied this workflow to plasma samples from patients with head and neck, lung, and breast cancer, as well as healthy donors. In subsets of all three cancer cohorts, elevated PD\u2010L1\n                    <jats:sup>+<\/jats:sup>\n                    sEV levels were observed, suggesting potential clinical relevance. While in\u2010depth clinical correlations lie beyond the scope of this study, our findings establish a validated, standardized protocol for IFCM\u2010based single\u2010EV profiling and highlight the central role of antibody quality in ensuring analytical accuracy.\n                  <\/jats:p>","DOI":"10.1002\/jex2.70144","type":"journal-article","created":{"date-parts":[[2026,5,26]],"date-time":"2026-05-26T03:27:16Z","timestamp":1779766036000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Single\u2010EV Analyses Require Rigorous Antibody Qualification: PD\u2010L1 Profiling in Cell Models and Patient Plasma"],"prefix":"10.1002","volume":"5","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8659-8610","authenticated-orcid":false,"given":"Tobias","family":"Tertel","sequence":"first","affiliation":[{"name":"Institute for Transfusion Medicine University Hospital Essen University of Duisburg\u2010Essen  Essen 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