title: Revealing the diversity of extracellular vesicles using high-dimensional flow cytometry analyses.
abstract: Extracellular vesicles (EV) are small membrane vesicles produced by cells upon activation and apoptosis. EVs are heterogeneous according to their origin, mode of release, membrane composition, organelle and biochemical content, and other factors. Whereas it is apparent that EVs are implicated in intercellular communication, they can also be used as biomarkers. Continuous improvements in pre-analytical parameters and flow cytometry permit more efficient assessment of EVs; however, methods to more objectively distinguish EVs from cells and background, and to interpret multiple single-EV parameters are lacking. We used spanning-tree progression analysis of density-normalized events (SPADE) as a computational approach for the organization of EV subpopulations released by platelets and erythrocytes. SPADE distinguished EVs, and logically organized EVs detected by high-sensitivity flow cytofluorometry based on size estimation, granularity, mitochondrial content, and phosphatidylserine and protein receptor surface expression. Plasma EVs were organized by hierarchy, permitting appreciation of their heterogeneity. Furthermore, SPADE was used to analyze EVs present in the synovial fluid of patients with inflammatory arthritis. Its algorithm efficiently revealed subtypes of arthritic patients based on EV heterogeneity patterns. Our study reveals that computational algorithms are useful for the analysis of high-dimensional single EV data, thereby facilitating comprehension of EV functions and biomarker development.
authors: Centre de Recherche du Centre Hospitalier Universitaire de Québec, Faculté de Médecine de l'Université Laval, Département de microbiologie et immunologie, Québec, QC, Canada.
authors: Geneviève
authors: G
authors: Marcoux
authors: Centre de Recherche du Centre Hospitalier Universitaire de Québec, Faculté de Médecine de l'Université Laval, Département de microbiologie et immunologie, Québec, QC, Canada.
authors: Anne-Claire
authors: AC
authors: Duchez
authors: Centre de Recherche du Centre Hospitalier Universitaire de Québec, Faculté de Médecine de l'Université Laval, Département de microbiologie et immunologie, Québec, QC, Canada.
authors: Nathalie
authors: N
authors: Cloutier
authors: Centre de Recherche du Centre Hospitalier Universitaire de Québec, Faculté de Médecine de l'Université Laval, Département de microbiologie et immunologie, Québec, QC, Canada.
authors: Patrick
authors: P
authors: Provost
authors: Department of Medicine, Division of Rheumatology Immunology and Allergy, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
authors: Peter A
authors: PA
authors: Nigrovic
authors: Centre de Recherche du Centre Hospitalier Universitaire de Québec, Faculté de Médecine de l'Université Laval, Département de microbiologie et immunologie, Québec, QC, Canada.
authors: Eric
authors: E
authors: Boilard
mesh_terms: D000465:Algorithms; D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000473:pathology; D015415:Biomarkers / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D004912:Erythrocytes / Q000378:metabolism / Q000648:ultrastructure; D000067128:Extracellular Vesicles / Q000145:classification* / Q000378:metabolism / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D066298:In Vitro Techniques; D010316:Particle Size; D013582:Synovial Fluid / Q000166:cytology / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
chemical_list: D015415:Biomarkers; D013917:Thrombin