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  • Author or Editor: Kenneth M. Meyers x
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Abstract

Objective—To investigate the effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation.

Sample Population—Blood samples from 6 Thoroughbreds.

Procedure—The degree of fluorescence associated with binding of fluorescein isothiocyanate (FITC)-conjugated anti-human fibrinogen antibody and FITCannexin 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.

Conclusion and Clinical Relevance—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. (Am J Vet Res 2002;63:840–844)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To investigate the potential use of fluorescent- labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody for detection of the activation of equine platelets by use of flow cytometry.

Sample Population—Platelets obtained from 6 Thoroughbreds.

Procedure—Flow cytometry was used to assess platelet activation as indicated by detection of binding of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-thrombospondin antibody to unactivated and ADP-, collagen-, platelet activating factor (PAF)-, and A23187-activated equine platelets. Human platelets were used as control samples. Determination of 14C-serotonin uptake and release was used to assess the extent of platelet secretion.

Results—Anti-human thrombospondin antibody failed to bind to equine platelets. Annexin V bound to platelets activated with PAF or A23187 when platelets had undergone secretion. Anti-human fibrinogen antibody bound to ADP-, PAF-, and A23817- activated platelets, but binding was not dependent on platelet secretion. The extent of binding of anti-fibrinogen antibody was less in equine platelets, compared with that for human platelets, despite maximal stimulation.

Conclusions and Clinical Relevance—Activation of equine platelets can be detected by use of fluorescent- labeled annexin V and anti-human fibrinogen antibody but not by use of anti-human thrombospondin antibody. These flow cytometric techniques have the potential for detection of in vivo platelet activation in horses at risk of developing thrombotic disorders. (Am J Vet Res 2002;63:513–519)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To investigate the effects of sodium citrate, low molecular weight heparin (LMWH), and prostaglandin E1 (PGE1) on aggregation, fibrinogen binding, and enumeration of equine platelets. Sample Population—Blood samples obtained from 4 Thoroughbreds.

Sample Population—Blood samples obtained from 4 Thoroughbreds.

Procedure—Blood was collected into syringes in the ratio of 9 parts blood:1 part anticoagulant. Anticoagulants used were sodium citrate, LMWH, sodium citrate and LMWH, or 300 nM PGE1/ml of anticoagulant. Platelet aggregation in response to ADP, collagen, and PGE1 was assessed, using optical aggregometry. Platelet activation was evaluated, using flow cytometry, to detect binding of fluorescein- conjugated anti-human fibrinogen antibody. Plasma concentration of ionized calcium was measured, using an ion-selective electrode.

Results—Number of platelets (mean ± SEM) in samples containing LMWH (109.5 ± 11.3 × 103 cells/µl) was significantly less than the number in samples containing sodium citrate (187.3 ± 30.3 × 103 cells/µl). Increasing concentrations of sodium citrate resulted in reductions in platelet aggregation and plasma concentration of ionized calcium. Addition of PGE1 prior to addition of an agonist inhibited platelet aggregation in a concentration-dependent manner, whereas addition of PGE1 4 minutes after addition of ADP resulted in partial reversal of aggregation and fibrinogen binding.

Conclusion and Clinical Relevance—A high concentration of sodium citrate in blood samples decreases plasma concentration of ionized calcium, resulting in reduced platelet aggregation and fibrinogen binding. Platelets tend to clump in samples collected into LMWH, precluding its use as an anticoagulant. Platelet aggregation and fibrinogen binding can be reversed by PGE1, which may result in underestimation of platelet activation. (Am J Vet Res 2001; 62:547–554)

Full access
in American Journal of Veterinary Research