Objective—To evaluate whether markers of platelet activation, including P-selectin expression, phosphatidylserine exposure, platelet-leukocyte aggregates, and microparticle formation, could be measured in nonstimulated and stimulated canine blood samples and develop a standardized protocol for detection of activated platelet markers in canine blood.
Sample population—Blood samples from 10 dogs.
Procedure—Platelet activation was determined by flow cytometric measurement of platelets with P-selectin expression, platelet-leukocyte aggregates, platelet microparticles, and platelets with phosphatidylserine exposure. Changes in specific markers of platelet activation in nonstimulated versus stimulated samples were assessed by use of varying concentrations of 2 platelet agonists, platelet-activating factor (PAF) and adenosine diphosphate. Flow cytometry was used to detect platelet CD61 (glycoprotein IIIa), CD62P (P-selectin), and the leukocyte marker CD45. Annexin V was used to identify exposed phosphatidylserine.
Results—A significant difference was detected in the percentages of platelets with P-selectin, plateletleukocyte aggregates, microparticles, and platelets with annexin V exposure (phosphatidylserine) in samples stimulated with 10nM PAF versus the nonstimulated samples, with platelet-leukocyte aggregates having the greatest difference.
Conclusions and Clinical Relevance—Platelet activation is essential for thrombus formation and hemostasis and may be potentially useful for evaluation of dogs with suspected thromboembolic disease. Prior to development of a thrombotic state, a prothrombotic state may exist in which only a small number of platelets is activated. Identification of a prothrombotic state by use of activated platelets may help direct medical intervention to prevent a thromboembolic episode.
Objective—To determine the characteristics of an automated canine C-reactive protein (CRP) assay and evaluate 2 human CRP assays for use in dogs.
Animals—56 client-owned dogs with pyometra and 11 healthy control dogs.
Procedures—Samples from 11 dogs with high (> 100 mg/L) or low (< 10 mg/L) CRP concentrations (determined by use of a canine ELISA) were evaluated by use of the automated canine CRP assay. Intra- and interassay imprecision was determined (by use of those 2 plasma pools), and assay inaccuracy was assessed by use of logistic regression analysis of results obtained via ELISA and the automated canine CRP assay. Two automated human CRP assays were used to measure plasma CRP concentration in 10 dogs.
Results—By use of the ELISA, mean ± SD plasma CRP concentration was 96.1 ± 38.5 mg/L and 10.1 ± 23.2 mg/L in dogs with pyometra and control dogs, respectively. The automated canine assay had intra-assay coefficients of variation (CVs) of 7.8% and 7.9%, respectively, and interassay CVs of 11.1% and 13.1%, respectively. Results from the automated assay were highly correlated with results obtained via ELISA. The human assay results did not exceed 0.4 mg/L in any dog.
Conclusions and Clinical Relevance—The automated canine CRP assay had less interassay imprecision, compared with the ELISA. The 2 human CRP assays were not suitable for analysis of canine plasma samples. The automated canine CRP assay was more precise than the ELISA for serial evaluations of plasma CRP concentration in dogs.
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
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
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
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)
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
Sample Population—Blood samples obtained from
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;
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
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
Objectives—To assess safety and determine effects
of IV administration of formaldehyde on hemostatic
variables in healthy horses.
Animals—7 healthy adult horses.
Procedure—Clinical signs and results of CBC, serum
biochemical analyses, and coagulation testing including
template bleeding time (TBT) and activated clotting
time (ACT) were compared in horses given a
dose of 0.37% formaldehyde or lactated Ringer’s
solution (LRS), IV, in a 2-way crossover design. In a
subsequent experiment, horses received an infusion
of 0.74% formaldehyde or LRS. In another experiment,
horses were treated with aspirin to impair
platelet responses prior to infusion of formaldehyde
Results—Significant differences were not detected in
any variable measured between horses when given
formaldehyde or any other treatment. Infusion of
higher doses of formaldehyde resulted in adverse
effects including muscle fasciculations, tachycardia,
tachypnea, serous ocular and nasal discharge, agitation,
Conclusions and Clinical Relevance—Intravenous
infusion of formaldehyde at doses that do not induce
adverse reactions did not have a detectable effect on
measured hemostatic variables in healthy horses.
(Am J Vet Res 2000;61:1191–1196)