title: Procoagulant and platelet-derived microvesicle absolute counts determined by flow cytometry correlates with a measurement of their functional capacity.
abstract: BACKGROUND
  Flow cytometry is the most commonly used technology to measure microvesicles (MVs). Despite reported limitations of this technique, MV levels obtained using conventional flow cytometry have yielded many clinically relevant findings, such as associations with disease severity and ability to predict clinical outcomes. This study aims to determine if MV enumeration by flow cytometry correlates with a measurement of their functional capacity, as this may explain how flow cytometry generates clinically relevant results.
  
  
  METHODS
  ONE HUNDRED SAMPLES FROM HEALTHY INDIVIDUALS AND PATIENTS WITH OBSTRUCTIVE SLEEP APNOEA WERE ANALYSED BY CONVENTIONAL FLOW CYTOMETRY (FACSCALIBUR) AND BY THREE FUNCTIONAL MV ASSAYS: Zymuphen MP-activity in which data were given as phosphatidylserine equivalent, STA(®) Phospholipid Procoag Assay expressed as clotting time and Endogenous Thrombin Potential (ETP) reflecting in vitro thrombin generation. Correlations were determined by Spearman correlation.
  
  
  RESULTS
  Absolute counts of lactadherin+ procoagulant MVs generated by flow cytometry weakly correlated with the results obtained from the Zymuphen MP-activity (r=0.5370, p<0.0001); correlated with ETP (r=0.7444, p<0.0001); negatively correlated with STA(®) Phospholipid Procoag Assay clotting time (-0.7872, p<0.0001), reflecting a positive correlation between clotting activity and flow cytometry. Levels of Annexin V+ procoagulant and platelet-derived MVs were also associated with functional assays. Absolute counts of MVs derived from other cell types were not correlated with the functional results.
  
  
  CONCLUSIONS
  Quantitative results of procoagulant and platelet-derived MVs from conventional flow cytometry are associated with the functional capability of the MVs, as defined by three functional MV assays. Flow cytometry is a valuable technique for the quantification of MVs from different cellular origins; however, a combination of several analytical techniques may give the most comprehensive information on the role of MVs in health and disease.
authors.affiliation: Department of Clinical Immunology, Oxford University Hospitals NHS Trust, Oxford, UK.
authors.forename: Lisa
authors.initials: L
authors.lastname: Ayers
authors.affiliation: School of Immunity and Infection, University of Birmingham Medical School, Birmingham, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Harrison
authors.affiliation: Sleep Disorders Centre and Pulmonary Division, University Hospital of Zurich, Zürich, Switzerland.
authors.forename: Malcolm
authors.initials: M
authors.lastname: Kohler
authors.affiliation: Department of Clinical Immunology, Oxford University Hospitals NHS Trust, Oxford, UK.
authors.forename: Berne
authors.initials: B
authors.lastname: Ferry
keywords: clotting assays; conventional flow cytometry; functional assays; microvesicles; thrombin generation