title: Detection of circulating microparticles by flow cytometry: influence of centrifugation, filtration of buffer, and freezing.
abstract: The clinical importance of microparticles resulting from vesiculation of platelets and other blood cells is increasingly recognized, although no standardized method exists for their measurement. Only a few studies have examined the analytical and preanalytical steps and variables affecting microparticle detection. We focused our analysis on microparticle detection by flow cytometry. The goal of our study was to analyze the effects of different centrifugation protocols looking at different durations of high and low centrifugation speeds. We also analyzed the effect of filtration of buffer and long-term freezing on microparticle quantification, as well as the role of Annexin V in the detection of microparticles. Absolute and platelet-derived microparticles were 10- to 15-fold higher using initial lower centrifugation speeds at 1500 × g compared with protocols using centrifugation speeds at 5000 × g (P < 0.01). A clear separation between true events and background noise was only achieved using higher centrifugation speeds. Filtration of buffer with a 0.2 μm filter reduced a significant amount of background noise. Storing samples for microparticle detection at -80°C decreased microparticle levels at days 28, 42, and 56 (P < 0.05 for all comparisons with fresh samples). We believe that staining with Annexin V is necessary to distinguish true events from cell debris or precipitates. Buffers should be filtered and fresh samples should be analyzed, or storage periods will have to be standardized. Higher centrifugation speeds should be used to minimize contamination by smaller size platelets.
authors.affiliation: Division of Nephrology, Department of Medicine, Hannover Medical School, Hannover, Germany. emily.deyhazra@googlemail.com
authors.forename: Emily
authors.initials: E
authors.lastname: Dey-Hazra
authors.forename: Barbara
authors.initials: B
authors.lastname: Hertel
authors.forename: Torsten
authors.initials: T
authors.lastname: Kirsch
authors.forename: Alexander
authors.initials: A
authors.lastname: Woywodt
authors.forename: Svjetlana
authors.initials: S
authors.lastname: Lovric
authors.forename: Hermann
authors.initials: H
authors.lastname: Haller
authors.forename: Marion
authors.initials: M
authors.lastname: Haubitz
authors.forename: Uta
authors.initials: U
authors.lastname: Erdbruegger
mesh_terms: D017304:Annexin A5; D001792:Blood Platelets / Q000648:ultrastructure; D055252:Cell-Derived Microparticles; D002498:Centrifugation; D015925:Cryopreservation; D005260:Female; D005374:Filtration; D005434:Flow Cytometry / Q000379:methods*; D005615:Freezing; D006801:Humans; D007962:Leukocytes / Q000648:ultrastructure; D008297:Male; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000032:analysis
chemical_list: D017304:Annexin A5; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D019038:Platelet Glycoprotein GPIb-IX Complex