Background/Objective: Extracellular vesicles (EVs) are critical mediators of intercellular communication and hold promise as diagnostic biomarkers. However, the lack of standardized methods for their identification and quantification has hindered their clinical translation. This study aimed to develop and validate a reliable protocol for the direct detection, subtyping, and isolation of EVs from fresh peripheral blood (PB), minimizing pre-analytical manipulation. Methods: A polychromatic flow cytometry (PFC) protocol based on a lipophilic cationic dye (LCD) and phalloidin was optimized to identify intact EVs. Fluorescence-activated cell sorting was employed for isolation. EV subtypes were characterized using specific antibodies (CD45, CD41a, CD31), and their protein cargo analyzed via proteomics. Comparison with ultracentrifugation (UC) and other enrichment techniques was also performed. Results: LCD+/Phalloidin− events were confirmed as intact EVs by NTA, TEM, and ImageStream. The protocol enabled reproducible quantification of leukocyte-, platelet-, and endothelium-derived EVs. EVs sorted by this method exhibited significantly lower contamination from plasma proteins compared to UC-based methods. Proteomic analysis revealed that EVs carry a specific cargo enriched in binding and regulatory proteins.
An optimized flow cytometry method for the analysis of extracellular vesicle from fresh peripheral blood samples
Sabino Porro;Pasquale Simeone;Davide Brocco;Francesca D’Ascanio;Domenico De Bellis;Giulia Colasante;Arianna Aquilini-Mummolo;Tamer Esmail;Ayesha Younas;Mariagiulia Filoso;Camilla Cichella;Maria Concetta Cufaro;Alice Di Sebastiano;Damiana Pieragostino;Michela Battistelli;Piero Del Boccio;Antonella Fontana;Paola Lanuti.
2026-01-01
Abstract
Background/Objective: Extracellular vesicles (EVs) are critical mediators of intercellular communication and hold promise as diagnostic biomarkers. However, the lack of standardized methods for their identification and quantification has hindered their clinical translation. This study aimed to develop and validate a reliable protocol for the direct detection, subtyping, and isolation of EVs from fresh peripheral blood (PB), minimizing pre-analytical manipulation. Methods: A polychromatic flow cytometry (PFC) protocol based on a lipophilic cationic dye (LCD) and phalloidin was optimized to identify intact EVs. Fluorescence-activated cell sorting was employed for isolation. EV subtypes were characterized using specific antibodies (CD45, CD41a, CD31), and their protein cargo analyzed via proteomics. Comparison with ultracentrifugation (UC) and other enrichment techniques was also performed. Results: LCD+/Phalloidin− events were confirmed as intact EVs by NTA, TEM, and ImageStream. The protocol enabled reproducible quantification of leukocyte-, platelet-, and endothelium-derived EVs. EVs sorted by this method exhibited significantly lower contamination from plasma proteins compared to UC-based methods. Proteomic analysis revealed that EVs carry a specific cargo enriched in binding and regulatory proteins.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
