Effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation.

The research explores how formaldehyde fixation impacts equine platelets as measured by flow cytometry, a tool used to analyze the physical and chemical characteristics of particles in a fluid. The study suggests that the use of lower concentrations of formaldehyde should be employed to fix horse platelet samples and these fixed samples should be analyzed within 12 hours to avoid abnormal increases in fluorescence.

Research Methodology

• The investigation included blood samples from 6 Thoroughbreds and analyzed the degree of fluorescence associated with the binding of fluorescein isothiocyanate (FITC)-conjugated anti-human fibrinogen antibody and FITC-annexin V in platelet samples both unactivated and activated by substances such as adenosine diphosphate (ADP), platelet activating factor (PAF), and A23187.
• The samples were divided into two groups – unfixed and fixed with 0.5, 1.0, and 2.0% formaldehyde. Fixation is the process of preserving or hardening biological tissues from decay due to autolysis or putrefaction.
• The fluorescence of these samples was measured using flow cytometry, which is a technique used to measure the physical and chemical characteristics of cells or particles in a solution as they pass through a laser.

Key Findings

• The study found that when FITC-anti-human fibrinogen antibody was added to the samples prior to fixation, a 2.0% formaldehyde resulted in a 30% increase in fluorescence in samples activated by ADP and PAF, and a 60% increase in those activated by A23187.
• If fixation happened for 24 hours before the addition of the antibody, fluorescence in samples containing anti-human fibrinogen antibody reduced for all three concentrations of formaldehyde in PAF-activated samples.
• It was also found that adding any of three concentrations of formaldehyde after incubating with FITC-annexin V resulted in significant increases in fluorescence in both unactivated and activated platelet samples.
• The researchers observed that as the length of fixation time increased, there was a gradual, significant increase in fluorescence, noticeable at 24 hours.

Conclusions

• The study concludes that since the use of 2.0% formaldehyde brings about significant changes in fluorescence in activated platelet samples with anti-fibrinogen antibody, lower concentrations of formaldehyde should be used to fix equine platelet samples.
• In order to avoid artificial increases in fluorescence, formaldehyde-fixed platelet samples should ideally be studied within 12 hours of fixation.
• Fixation of samples that include FITC-annexin V should be avoided because of considerable increases in fluorescence that may negatively affect the interpretation of results.