title: Effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation.
abstract: OBJECTIVE
  To investigate the effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation.
  
  
  METHODS
  Blood samples from 6 Thoroughbreds.
  
  
  METHODS
  The degree of fluorescence associated with binding of fluorescein isothiocyanate (FITC)-conjugated anti-human fibrinogen antibody and FITC-annexin V in unactivated and adenosine diphosphate (ADP)-, platelet activating factor (PAF)-, and A23187-activated platelet samples in unfixed and 0.5, 1.0, and 2.0% formaldehyde-fixed samples was assessed by use of flow cytometry.
  
  
  RESULTS
  In samples incubated with FITC-anti-human fibrinogen antibody prior to fixation, addition of 2.0% formaldehyde resulted in a 30% increase in total fluorescence in ADP- and PAF-activated samples and a 60% increase in A23187-activated samples. Fixation for 24 hours prior to addition of antibody resulted in reduced fluorescence of samples containing antihuman fibrinogen antibody for all 3 concentrations of formaldehyde in PAF-activated samples. The addition of all 3 concentrations of formaldehyde after incubation with FITC-annexin V resulted in significant increases in fluorescence in unactivated and activated platelet samples. As length of fixation time increased, there was a gradual increase in fluorescence that was significant at 24 hours.
  
  
  CONCLUSIONS
  Because fixation with 2.0% formaldehyde results in significant changes in fluorescence in activated platelet samples containing anti-fibrinogen antibody, lower concentrations of formaldehyde should be used to fix equine platelet samples. Formaldehyde-fixed platelet samples should be analyzed within 12 hours of fixation to avoid artifactual increases in fluorescence. Fixation of samples containing FITC-annexin V should be avoided because of significant increases in fluorescence that may interfere with interpretation of results.
authors.affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman 99164-6610, USA.
authors.forename: Janene K
authors.initials: JK
authors.lastname: Kingston
authors.forename: Warwick M
authors.initials: WM
authors.lastname: Bayly
authors.forename: Debra C
authors.initials: DC
authors.lastname: Sellon
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Meyers
authors.forename: K Jane
authors.initials: KJ
authors.lastname: Wardrop
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D017304:Annexin A5 / Q000737:chemistry; D001792:Blood Platelets / Q000378:metabolism*; D000001:Calcimycin / Q000378:metabolism; D005404:Fixatives / Q000494:pharmacology*; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary; D005557:Formaldehyde / Q000494:pharmacology*; D006736:Horses / Q000097:blood*; D007476:Ionophores / Q000378:metabolism; D010972:Platelet Activating Factor / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects*
chemical_list: D017304:Annexin A5; D005404:Fixatives; D007476:Ionophores; D010972:Platelet Activating Factor; D005557:Formaldehyde; D000001:Calcimycin; D000244:Adenosine Diphosphate