title: Peripheral blood microvesicles secretion is influenced by storage time, temperature, and anticoagulants.
abstract: Microvesicles (MVs) are small membrane bound vesicles released from various cell types after activation or apoptosis. In the last decades, MVs received an increased interest as biomarkers in inflammation, coagulation and cancer. However, standardized pre-analytical steps are crucial for the minimization of artifacts in the MV analysis. Thus, this study evaluated the MV release in whole blood samples under the influence of different anticoagulants, storage time and various temperature conditions. Samples were collected from healthy probands and processed immediately, after 4, 8, 24 and 48 hours at room temperature (RT) or 4°C. To identify MV subpopulations, platelet free plasma (PFP) was stained with Annexin V, calcein AM, CD15, CD41 and CD235a. Analysis was performend on a CytoFLEX flow cytometer. Procoagulatory function of MVs was measured using a phospholipid dependent activity and a tissue factor MVactivity assay. Without prior storage, sodium citrate showed the lowest MV count compared to heparin and EDTA. Interestingly, EDTA showed a significant release of myeloid-derived MVs (MMVs) compared to sodium citrate. Sodium citrate showed a stable MV count at RT in the first 8 hours after blood collection. Total MV counts increased after 24 hours in sodium citrated or heparinzed blood which was related to all subpopulations. Interestingly, EDTA showed stable platelet-derived MV (PMV) and erythrocyte-derived MV (EryMV) count at RT over a 48 h period. In addition, the procoagulatory potential increased significantly after 8-hour storage. Based on both, this work and literature data, the used anticoagulant, storage time and storage temperature differently influence the analysis of MVs within 8 hours. To date, sodium citrated tubes are recommended for MV enumeration and functional analysis. EDTA tubes might be an option for the clinical routine due to stable PMV and EryMV counts. These new approaches need to be validated in a clinical laboratory setting before being applied to patient studies. © 2016 International Society for Advancement of Cytometry.
authors.affiliation: Department of Paediatrics and Adolescent Medicine, Division of Neonatology, Paediatric Intensive Care & Neuropaediatrics, Medical University of Vienna, Währinger Gürtel 18-20, Vienna, 1090, Austria.
authors.forename: Lukas
authors.initials: L
authors.lastname: Wisgrill
authors.affiliation: Department of Surgery, Research Labs, Medical University of Vienna, Lazarettgasse 14, Vienna, 1090, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Lamm
authors.affiliation: Department of Surgery, Research Labs, Medical University of Vienna, Lazarettgasse 14, Vienna, 1090, Austria.
authors.forename: Julia
authors.initials: J
authors.lastname: Hartmann
authors.affiliation: Department of Surgery, Research Labs, Medical University of Vienna, Lazarettgasse 14, Vienna, 1090, Austria.
authors.forename: Falk
authors.initials: F
authors.lastname: Preißing
authors.affiliation: Department of Surgery, Research Labs, Medical University of Vienna, Lazarettgasse 14, Vienna, 1090, Austria.
authors.forename: Klaus
authors.initials: K
authors.lastname: Dragosits
authors.affiliation: Department of Surgery, Research Labs, Medical University of Vienna, Lazarettgasse 14, Vienna, 1090, Austria.
authors.forename: Annica
authors.initials: A
authors.lastname: Bee
authors.affiliation: Clinical Division of Haematology and Haemostaseology, Department of Medicine I, Medical University of Vienna, Vienna, Währinger Gürtel 18-20, Austria.
authors.forename: Lena
authors.initials: L
authors.lastname: Hell
authors.affiliation: Clinical Division of Haematology and Haemostaseology, Department of Medicine I, Medical University of Vienna, Vienna, Währinger Gürtel 18-20, Austria.
authors.forename: Johannes
authors.initials: J
authors.lastname: Thaler
authors.affiliation: Clinical Division of Haematology and Haemostaseology, Department of Medicine I, Medical University of Vienna, Vienna, Währinger Gürtel 18-20, Austria.
authors.forename: Cihan
authors.initials: C
authors.lastname: Ay
authors.affiliation: Clinical Division of Haematology and Haemostaseology, Department of Medicine I, Medical University of Vienna, Vienna, Währinger Gürtel 18-20, Austria.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Pabinger
authors.affiliation: Department of Paediatrics and Adolescent Medicine, Division of Neonatology, Paediatric Intensive Care & Neuropaediatrics, Medical University of Vienna, Währinger Gürtel 18-20, Vienna, 1090, Austria.
authors.forename: Angelika
authors.initials: A
authors.lastname: Berger
authors.affiliation: Department of Surgery, Research Labs, Medical University of Vienna, Lazarettgasse 14, Vienna, 1090, Austria.|Core Facility Flow Cytometry, Medical University of Vienna, Lazarettgasse 14, Vienna, 1090, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Spittler
mesh_terms: D001793:Blood Preservation / Q000379:methods* / Q000592:standards*; D055252:Cell-Derived Microparticles; D006801:Humans
keywords: extracellular vesicles; peripheral blood microvesicles; pre-analytics