Effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation.
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.
Kingston
Janene K
JK
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman 99164-6610, USA.
Bayly
Warwick M
WM
Sellon
Debra C
DC
Meyers
Kenneth M
KM
Wardrop
K Jane
KJ
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*
D017304:Annexin A5; D005404:Fixatives; D007476:Ionophores; D010972:Platelet Activating Factor; D005557:Formaldehyde; D000001:Calcimycin; D000244:Adenosine Diphosphate