title: Pre-analytical and analytical variables affecting the measurement of plasma-derived microparticle tissue factor activity.
abstract: BACKGROUND
  Elevated levels of tissue factor positive (TF(+)) microparticles (MPs) are observed in plasma from a variety of patients with an increased risk of thrombosis. We and others have described the measurement of TF activity in MPs isolated from plasma. The aim of this study was to investigate the effects of pre-analytical and analytical variables on TF activity of MPs isolated from blood of healthy volunteers either untreated or treated ex vivo with bacterial lipopolysaccharide.
  
  
  METHODS
  We evaluated the following parameters: use of different centrifugation speeds to isolate the MPs; comparison of TF activity of MPs isolated from platelet poor plasma versus platelet free plasma; effect of freeze/thaw on MP TF activity; and comparison of the MP TF activity assay with the measurement of TF protein by ELISA or flow cytometry.
  
  
  RESULTS
  MPs prepared from platelet poor plasma by centrifugation at 20,000×g or 100,000×g for 15 minutes had similar levels of TF activity. However, significantly less TF activity was found in MPs isolated from platelet free plasma compared with platelet poor plasma. Interestingly, freeze/thawing of the plasma showed donor to donor variation in MP TF activity, with a moderate increase in some individuals.
  
  
  CONCLUSIONS
  TF(+) MPs can be quantitatively isolated from platelet poor or platelet free plasma by centrifugation at 20,000×g for 15 minutes. Measurement of MP TF activity in plasma may be used to detect a prothrombotic state in patients with various diseases.
authors: Division of Hematology/Oncology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors: R D
authors: RD
authors: Lee
authors: D A
authors: DA
authors: Barcel
authors: J C
authors: JC
authors: Williams
authors: J G
authors: JG
authors: Wang
authors: J C
authors: JC
authors: Boles
authors: D A
authors: DA
authors: Manly
authors: N S
authors: NS
authors: Key
authors: N
authors: N
authors: Mackman
mesh_terms: D000293:Adolescent; D000328:Adult; D015415:Biomarkers / Q000097:blood; D055252:Cell-Derived Microparticles / Q000737:chemistry* / Q000187:drug effects; D002498:Centrifugation; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D005615:Freezing; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000737:chemistry* / Q000187:drug effects; D009657:North Carolina; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D013925:Thromboplastin / Q000032:analysis*; D013927:Thrombosis / Q000097:blood / Q000175:diagnosis*; D013997:Time Factors; D055815:Young Adult
chemical_list: D015415:Biomarkers; D008070:Lipopolysaccharides; C440499:lipopolysaccharide, Escherichia coli O111 B4; D013925:Thromboplastin