{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T16:19:22Z","timestamp":1791389962674,"version":"4.3.3"},"reference-count":58,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T00:00:00Z","timestamp":1768435200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Extracellular vesicles (EVs) from human body fluids are valuable tools for biomarker discovery and for exploring the mechanisms underlying various pathologies, including infectious diseases. The translation of EV research into clinical practice is however hindered by the variability in EV pre-clinical investigations. Therefore, standardisation of analytical procedures and reporting policies is essential. Human serum is a key biological matrix for biomarker discovery and is commonly stored within biobanks. Here, we investigated different strategies for EV enrichment from small volumes of biobanked serum and evaluated their impact on EVs\u2019 downstream analyses. EVs were obtained from 250 \u03bcL of biobanked serum using ultracentrifugation (UC), size-exclusion chromatography-based methods (ExoSpin-ES, qEV1-35 nm, and qEV1-70 nm), or ExoRNeasy (ER). The resulting EVs were subsequently characterised for morphology, concentration, surface phenotype, and multi-omics profiles. All methods successfully enriched small EVs expressing tetraspanins on their surface, although at different concentrations. The most efficient method for proteomics analyses was qEV1-70 nm, followed by ES, which was more susceptible to contamination by serum proteins. EV-miRNA cargo was effectively profiled in UC-, ES-, and ER-EVs, with the latter providing the broadest miRNA coverage. Our results support the feasibility of using biobanked serum for EV-based research and further highlight the importance of selecting appropriate EV enrichment methods, since they influence both miRNA and protein cargo characterisation.<\/jats:p>","DOI":"10.3390\/ijms27020892","type":"journal-article","created":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T16:21:13Z","timestamp":1768494073000},"page":"892","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Molecular Evaluation of Different Enrichment Methods for Extracellular Vesicles from Healthy Subjects\u2019 Biobanked Serum"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7595-2346","authenticated-orcid":false,"given":"Michela","family":"Deiana","sequence":"first","affiliation":[{"name":"Department of Infectious, Tropical Diseases and Microbiology, IRCCS Sacro Cuore Don Calabria Hospital, 37024 Negrar di Valpolicella, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-8395-1601","authenticated-orcid":false,"given":"Elisabetta","family":"Vezzelli","sequence":"additional","affiliation":[{"name":"Department of Infectious, Tropical Diseases and Microbiology, IRCCS Sacro Cuore Don Calabria Hospital, 37024 Negrar di Valpolicella, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4561-3447","authenticated-orcid":false,"given":"Cristina","family":"Mazzi","sequence":"additional","affiliation":[{"name":"Clinical Research Unit, IRCCS Sacro Cuore Don Calabria Hospital, 37024 Negrar di Valpolicella, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Denise","family":"Lavezzari","sequence":"additional","affiliation":[{"name":"Department of Infectious, Tropical Diseases and Microbiology, IRCCS Sacro Cuore Don Calabria Hospital, 37024 Negrar di Valpolicella, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0632-5618","authenticated-orcid":false,"given":"Marcello","family":"Manfredi","sequence":"additional","affiliation":[{"name":"Department of Translational Medicine, University of Piemonte Orientale, 28100 Novara, Italy"},{"name":"Institute for Molecular and Translational Cardiology (IMTC), IRCCS Policlinico San Donato, 20097 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesca","family":"Moretta","sequence":"additional","affiliation":[{"name":"Clinical Analysis Laboratory and Transfusional Medicine, Clinical Pharmacology, IRCCS Sacro Cuore Don Calabria Hospital, 37024 Negrar di Valpolicella, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5739-3795","authenticated-orcid":false,"given":"Chiara","family":"Piubelli","sequence":"additional","affiliation":[{"name":"Department of Infectious, Tropical Diseases and Microbiology, IRCCS Sacro Cuore Don Calabria Hospital, 37024 Negrar di Valpolicella, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Federico Giovanni","family":"Gobbi","sequence":"additional","affiliation":[{"name":"Department of Infectious, Tropical Diseases and Microbiology, IRCCS Sacro Cuore Don Calabria Hospital, 37024 Negrar di Valpolicella, Italy"},{"name":"Department of Clinical and Experimental Sciences, University of Brescia, 25121 Brescia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2981-5233","authenticated-orcid":false,"given":"Natalia","family":"Tiberti","sequence":"additional","affiliation":[{"name":"Department of Infectious, Tropical Diseases and Microbiology, IRCCS Sacro Cuore Don Calabria Hospital, 37024 Negrar di Valpolicella, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e12144","DOI":"10.1002\/jev2.12144","article-title":"A brief history of nearly EV-erything\u2014The rise and rise of extracellular vesicles","volume":"10","author":"Couch","year":"2021","journal-title":"J. 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