title: Correlative analysis of nanoparticle tracking, flow cytometric and functional measurements for circulating microvesicles in normal subjects.
abstract: BACKGROUND
  Circulating microvesicles (MV) can be analysed using a number of different techniques. The aim of this study was to evaluate the correlation between functional procoagulant based assays including thrombin generation, factor Xa activation test (XaCT), and phosphatidylserine factor Xa-activity by ELISA with optical MV enumeration by flow cytometry and nanoparticle tracking analysis.
  
  
  METHODS
  Citrated blood samples were collected from 60 healthy volunteer blood donors after informed consent. Platelet free plasma was prepared using a standardized published protocol. MV subsets were enumerated by flow cytometry (BDFACS Canto) after staining with specific antibodies for platelets (CD41), endothelial cells (CD105), red cells (CD235) monocytes (CD14), tissue factor (CD142) and for phosphatidylserine expression by binding to annexin V. A standardized protocol using counting beads was employed. Nanotracking analysis was performed on both scatter and fluorescent settings after MV staining with quantum dot stain, Qdot 655. Procoagulant function was assessed by the XaCT assay on an automated coagulation analyser and by thrombin generation assay measuring endogenous thrombin potential (ETP), lagtime, peak (PEAK) and time to peak (ttPEAK) using a Calibrated Automated Thrombogram (CAT). The statistical analysis was carried out with Statistica 12 software using non-parametric tests (Spearman rank order correlations, with significance set at p<0.05).
  
  
  RESULTS
  In normal healthy subjects, thrombin generation parameters correlated with levels of MV measured by flow cytometry. ETP, lagtime, ttPEAK and PEAK correlated with MV expressing phosphatidylserine (rs, Spearman rank order correlation was 0.29, 0.40, 0.31 and 0.34 respectively, p<0.05), and MV expressing tissue factor (rs was 0.29, 0.40, 0.31 and 0.34 respectively, p<0.05), whilst red cell derived MV correlated with lagtime, ttPEAK and PEAK (rs, was 0.35,0.30 and 0.3, respectively, p<0.05). Lagtime and ttPEAK negatively correlated with the clot based XaCT test (rs, was -0.34 and -0.30 respectively, p<0.05) and positively correlated with the ELISA MP-activity assay (rs=0.42 for both, p<0.05). In addition, endothelial MV levels weakly correlated with white cell counts (rs = 0.27, p<0.05).
  
  
  CONCLUSIONS
  Thrombin generation and flow cytometry for phosphatidylserine or tissue factor expressing MV correlate well as markers for procoagulant activity. A combination of optical or non-optical enumeration as well as functional methods may be required for a complete profiling of circulating MV.
authors.affiliation: Haematology Department, Calvary Mater Newcastle, Australia; School of Medicine and Public Health, University of Newcastle, Australia; Pathology North-Hunter, New Lambton, NSW, Australia; Hunter Medical Research Institute, New Lambton, Australia; Hunter Cancer Research Alliance, Calvary Mater Newcastle, Waratah, NSW, Australia. Electronic address: Anoop.Enjeti@calvarymater.org.au.
authors.forename: Anoop K
authors.initials: AK
authors.lastname: Enjeti
authors.affiliation: Haematology Department, Calvary Mater Newcastle, Australia.
authors.forename: Anita
authors.initials: A
authors.lastname: Ariyarajah
authors.affiliation: Haematology Department, Calvary Mater Newcastle, Australia.
authors.forename: Angel
authors.initials: A
authors.lastname: D'Crus
authors.affiliation: Haematology Department, Calvary Mater Newcastle, Australia; School of Medicine and Public Health, University of Newcastle, Australia; Pathology North-Hunter, New Lambton, NSW, Australia.
authors.forename: Michael
authors.initials: M
authors.lastname: Seldon
authors.affiliation: Haematology Department, Calvary Mater Newcastle, Australia; Hunter Medical Research Institute, New Lambton, Australia; Hunter Cancer Research Alliance, Calvary Mater Newcastle, Waratah, NSW, Australia; School of Biomedical Sciences and Pharmacy, University of Newcastle, Australia.
authors.forename: Lisa F
authors.initials: LF
authors.lastname: Lincz
mesh_terms: D055252:Cell-Derived Microparticles / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D053758:Nanoparticles / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*
chemical_list: D013917:Thrombin
keywords: Flow cytometry; Microvesicles; Nanoparticle tracking analysis; Thrombin generation