title: Flow cytometric detection of circulating platelet-derived microparticles in healthy adult horses.
abstract: OBJECTIVE
  To develop a flow cytometric assay to quantify platelet-derived microparticles (PMPs) in equine whole blood and plasma.
  
  
  METHODS
  Citrate-anticoagulated whole blood from 30 healthy adult horses.
  
  
  METHODS
  Platelet-poor plasma (PPP) was prepared from fresh whole blood by sequential low-speed centrifugation (twice at 2,500 × g). Samples of fresh whole blood and PPP were removed and stored at 4° and 24°C for 24 hours. Platelet-derived microparticles were characterized in fresh and stored samples on the basis of the forward scatter threshold (log forward scatter < 10(1)) and labeling with annexin V (indicating externalized phosphatidylserine) and CD61 (a constitutive platelet receptor). A fluorescent bead-calibrated flow cytometric assay was used to determine microparticle counts. Platelet counts, prothrombin time, and activated partial thromboplastin time were measured in fresh samples.
  
  
  RESULTS
  Significantly more PMPs were detected in fresh whole blood (median, 3,062 PMPs/μL; range, 954 to 13,531 PMPs/μL) than in fresh PPP (median, 247 PMPs/μL; range, 104 to 918 PMPs/μL). Storage at either temperature had no significant effect on PMP counts for whole blood or PPP. No significant correlation was observed between PMP counts and platelet counts in fresh whole blood or PPP or between PMP counts and clotting times in fresh PPP.
  
  
  CONCLUSIONS
  Results indicated that the described PMP protocol can be readily used to quantify PMPs in equine blood and plasma via flow cytometry. Quantification can be performed in fresh PPP or whole blood or samples stored refrigerated or at room temperature for 24 hours.
authors.affiliation: Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
authors.forename: Nora L
authors.initials: NL
authors.lastname: Springer
authors.forename: Eliza
authors.initials: E
authors.lastname: Smith
authors.forename: Marjory B
authors.initials: MB
authors.lastname: Brooks
authors.forename: Tracy
authors.initials: T
authors.lastname: Stokol
mesh_terms: D000818:Animals; D017304:Annexin A5; D001774:Blood Chemical Analysis / Q000379:methods / Q000662:veterinary; D001792:Blood Platelets / Q000166:cytology*; D055252:Cell-Derived Microparticles / Q000502:physiology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary*; D006736:Horses / Q000097:blood*; D008297:Male; D010976:Platelet Count
chemical_list: D017304:Annexin A5