title: Characterization of extracellular vesicles in whole blood: Influence of pre-analytical parameters and visualization of vesicle-cell interactions using imaging flow cytometry.
abstract: Extracellular vesicles are central players in intercellular communication and are released from the plasma membrane under tightly regulated conditions, depending on the physiological and pathophysiological state of the producing cell. Their heterogeneity requires a spectrum of methods for isolation and characterization, where pre-analytical parameters have profound impact on vesicle analysis, particularly in blood, since sampling, addition of anticoagulants, as well as post-sampling vesicle generation may influence the outcome. Here, we characterized microvesicles directly in whole blood using a combination of flow cytometry and imaging flow cytometry. We assessed the influence of sample agitation, anticoagulation, and temperature on post-sampling vesicle generation, and show that vesicle counts remained stable over time in samples stored without agitation. Storage with gentle rolling mimicking agitation, in contrast, resulted in strong release of platelet-derived vesicles in blood anticoagulated with citrate or heparin, whereas vesicle counts remained stable upon anticoagulation with EDTA. Using imaging flow cytometry, we could visualize microvesicles adhering to blood cells and revealed an anticoagulant-dependent increase in vesicle-cell aggregates over time. We demonstrate that vesicles adhere preferentially to monocytes and granulocytes in whole blood, while no microvesicles could be visualized on lymphocytes. Our data underscore the relevance of pre-analytical parameters in vesicle analysis and demonstrate that imaging flow cytometry is a suitable tool to study the interaction of extracellular vesicles with their target cells.
authors.affiliation: Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Health Sciences and Biomedicine, Danube University Krems, Austria.
authors.forename: Birgit
authors.initials: B
authors.lastname: Fendl
authors.affiliation: Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Health Sciences and Biomedicine, Danube University Krems, Austria.
authors.forename: René
authors.initials: R
authors.lastname: Weiss
authors.affiliation: Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Health Sciences and Biomedicine, Danube University Krems, Austria.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Fischer
authors.affiliation: Core Facility Flow Cytometry & Surgical Research Laboratories, Medical University of Vienna, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Spittler
authors.affiliation: Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Health Sciences and Biomedicine, Danube University Krems, Austria. Electronic address: viktoria.weber@donau-uni.ac.at.
authors.forename: Viktoria
authors.initials: V
authors.lastname: Weber
mesh_terms: D001769:Blood; D001773:Blood Cells / Q000166:cytology* / Q000502:physiology*; D001793:Blood Preservation / Q000379:methods; D002448:Cell Adhesion / Q000502:physiology*; D002478:Cells, Cultured; D000067128:Extracellular Vesicles / Q000502:physiology* / Q000648:ultrastructure*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods
keywords: Anticoagulation; Flow cytometry; Imaging flow cytometry; Microvesicles; Platelets; Storage