Unravelling Plasma Extracellular Vesicle Diversity With Optimised Spectral Flow Cytometry
ABSTRACT Extracellular vesicles (EVs) are crucial for intercellular communication and are found in various biological fluids. The identification and immunophenotyping of such small particles continue to pose significant challenges. Here, we have developed a workflow for the optimisation of a next‐ge...
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| Format: | Article |
| Language: | English |
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Wiley
2025-04-01
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| Series: | Journal of Extracellular Biology |
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| Online Access: | https://doi.org/10.1002/jex2.70045 |
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| author | Daniela Boselli Francesca Clemente Simona Di Terlizzi Christina Pagiatakis Laura Papa Genny Del Zotto Chiara Villa Giuseppe Alvise Ramirez Norma Maugeri Angelo A. Manfredi Achille Anselmo |
| author_facet | Daniela Boselli Francesca Clemente Simona Di Terlizzi Christina Pagiatakis Laura Papa Genny Del Zotto Chiara Villa Giuseppe Alvise Ramirez Norma Maugeri Angelo A. Manfredi Achille Anselmo |
| author_sort | Daniela Boselli |
| collection | DOAJ |
| description | ABSTRACT Extracellular vesicles (EVs) are crucial for intercellular communication and are found in various biological fluids. The identification and immunophenotyping of such small particles continue to pose significant challenges. Here, we have developed a workflow for the optimisation of a next‐generation panel for in‐depth immunophenotyping of circulating plasma EVs using spectral flow cytometry. Our data collection followed a multistep optimisation phase for both instrument setup and 21‐colour 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+ 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‐dimensional immunophenotyping in EV research. |
| format | Article |
| id | doaj-art-2b046effe97a4a4b900268db0b3091ce |
| institution | OA Journals |
| issn | 2768-2811 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Extracellular Biology |
| spelling | doaj-art-2b046effe97a4a4b900268db0b3091ce2025-08-20T02:20:05ZengWileyJournal of Extracellular Biology2768-28112025-04-0144n/an/a10.1002/jex2.70045Unravelling Plasma Extracellular Vesicle Diversity With Optimised Spectral Flow CytometryDaniela Boselli0Francesca Clemente1Simona Di Terlizzi2Christina Pagiatakis3Laura Papa4Genny Del Zotto5Chiara Villa6Giuseppe Alvise Ramirez7Norma Maugeri8Angelo A. Manfredi9Achille Anselmo10Experimental Imaging Center, FRACTAL, Flow cytometry Resource, Advanced Cytometry Technical Applications Laboratory IRCCS Ospedale San Raffaele Milan ItalyExperimental Imaging Center, FRACTAL, Flow cytometry Resource, Advanced Cytometry Technical Applications Laboratory IRCCS Ospedale San Raffaele Milan ItalyExperimental Imaging Center, FRACTAL, Flow cytometry Resource, Advanced Cytometry Technical Applications Laboratory IRCCS Ospedale San Raffaele Milan ItalyDepartment of Cardiovascular Medicine IRCCS Humanitas Research Hospital Rozzano Milan ItalyDepartment of Cardiovascular Medicine IRCCS Humanitas Research Hospital Rozzano Milan ItalyDepartment of Research and Diagnostics IRCCS Istituto Giannina Gaslini Genoa ItalyExperimental Imaging Center, FRACTAL, Flow cytometry Resource, Advanced Cytometry Technical Applications Laboratory IRCCS Ospedale San Raffaele Milan ItalyUnit of Immunology, Rheumatology, Allergy and Rare Diseases IRCCS Ospedale San Raffaele Milan ItalyUniversità Vita‐Salute San Raffaele Milan ItalyUniversità Vita‐Salute San Raffaele Milan ItalyExperimental Imaging Center, FRACTAL, Flow cytometry Resource, Advanced Cytometry Technical Applications Laboratory IRCCS Ospedale San Raffaele Milan ItalyABSTRACT Extracellular vesicles (EVs) are crucial for intercellular communication and are found in various biological fluids. The identification and immunophenotyping of such small particles continue to pose significant challenges. Here, we have developed a workflow for the optimisation of a next‐generation panel for in‐depth immunophenotyping of circulating plasma EVs using spectral flow cytometry. Our data collection followed a multistep optimisation phase for both instrument setup and 21‐colour 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+ 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‐dimensional immunophenotyping in EV research.https://doi.org/10.1002/jex2.70045antibodyextracellular vesiclefluorochromeimmunophenotypingplasmaSLE |
| spellingShingle | Daniela Boselli Francesca Clemente Simona Di Terlizzi Christina Pagiatakis Laura Papa Genny Del Zotto Chiara Villa Giuseppe Alvise Ramirez Norma Maugeri Angelo A. Manfredi Achille Anselmo Unravelling Plasma Extracellular Vesicle Diversity With Optimised Spectral Flow Cytometry Journal of Extracellular Biology antibody extracellular vesicle fluorochrome immunophenotyping plasma SLE |
| title | Unravelling Plasma Extracellular Vesicle Diversity With Optimised Spectral Flow Cytometry |
| title_full | Unravelling Plasma Extracellular Vesicle Diversity With Optimised Spectral Flow Cytometry |
| title_fullStr | Unravelling Plasma Extracellular Vesicle Diversity With Optimised Spectral Flow Cytometry |
| title_full_unstemmed | Unravelling Plasma Extracellular Vesicle Diversity With Optimised Spectral Flow Cytometry |
| title_short | Unravelling Plasma Extracellular Vesicle Diversity With Optimised Spectral Flow Cytometry |
| title_sort | unravelling plasma extracellular vesicle diversity with optimised spectral flow cytometry |
| topic | antibody extracellular vesicle fluorochrome immunophenotyping plasma SLE |
| url | https://doi.org/10.1002/jex2.70045 |
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