Alterations in normal canine platelets during storage in EDTA anticoagulated blood

Abstract: Flow cytometric detection of platelet surface-associated IgG (PSAIgG) can be used to determine whether immunologic factors are contributing to thrombocytopenia in dogs. In vitro alterations in platelet activation and morphology, however, could impact the results of this test. The purpose of this study was to determine whether the PSAIgG test for immune-mediated thrombocytopenia was valid on whole blood in EDTA anticoagulant after 24–72 hours of storage, and to characterize other alterations in canine platelets that could impact immunologic testing. Platelets were harvested and analyzed immediately after blood collection and after 24, 48, and 72 hours of storage at 4°C. Spontaneous and thrombin-induced changes in the following platelet parameters were evaluated using flow cytometric techniques: PSAIgG, platelet microparticle formation, membrane expression of P-selectin and glycoprotein CD61, exogenous IgG binding, surface-exposed phosphatidylserine, and fibrinogen binding. The amount of PSAIgG increased 6-to 9-fold in stored samples compared with fresh samples. Platelet microparticle formation was spontaneous in stored samples and increased significantly over time. Membrane phosphatidylserine, P-selectin, and fibrinogen binding were not altered by storage, indicating that platelet activation was minimal in stored samples. Although storage decreased the percentage of platelets positive for CD61 by 8-to 10-fold compared with fresh samples, activation by high-dose thrombin partially restored the percentage of CD61-positive platelets in 24-hour-old samples. In conclusion, even though platelets stored in EDTA for up to 72 hours remain in a resting state, aged platelets have an increased tendency to form microparticles and have increased surface IgG and decreased surface CD61, which may contribute to false-positive results for tests of immune-mediated thrombocytopenia.     

Equine platelet CD62P (P-selectin) expression: a phenotypic and morphologic study

Acute inflammatory diseases, such as colic, septicemia and endotoxemia are common in equines and have been shown to be correlated to vascular injury and thrombosis. In humans with similar thrombotic conditions, P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1)-mediated platelet-leukocyte adhesion contributes to the pathogenesis of these disorders through the generation of inflammatory mediators and tissue factor. As such, we hypothesized that a P-selectin-PSGL-1 (platelet-leukocyte) interaction, similar to that in humans, may also exist in the horse. The objective of this study was to investigate phenotypic and morphological properties of equine platelet activation with a focus on CD62P (P-selectin) expression and CD62P mediated platelet-leukocyte interactions. To study high levels of platelet activation, we used 1 U/ml thrombin to induce secondary, irreversible aggregation in both human and equine platelets. Addition of glycyl-L-prolyl-L-arginyl-L-proline amide (GPRP) prior to thrombin activation blocked fibrin polymerization, allowing the use of flow cytometry to study alpha-granule expression as a measure of platelet activation. Thrombin activation resulted in high levels of activation, measured as P-selectin expression, in both humans and equines. Interestingly, our research illustrates that in healthy horses, P-selectin is also constitutively expressed on 20-25% of resting platelets. This finding is in direct contrast to humans, in which P-selectin expression is negligible (<5%) in the absence of agonist activation. The high baseline level of P-selectin expression among equine platelets may suggest that they are primed for leukocyte adhesion, possibly resulting in prothrombotic conditions. This phenomenon could be of significant clinical relevance, as it may be related to the rapid clinical decline often seen in equine patients with colic and endotoxemia, where vascular injury and thrombotic complications compromise patient survival. Based on these findings, further investigation into the mechanisms of platelet P-selectin-mediated inflammation and platelet-leukocyte mediated vascular injury in the horse appears warranted.     

Effects of short-term racing activity on platelet and neutrophil activation in dogs

OBJECTIVE: To determine whether platelets and neutrophils become activated in dogs during short-distance sled-pulling activity. ANIMALS: 18 physically fit adult Siberian Huskies. PROCEDURE: Dogs were allocated into 2 teams (9 dogs/team). Each team ran a course of approximately 6.4 km while pulling a sled that contained 2 people. Blood samples were collected immediately before and within 10 minutes after completion of sled-pulling activity. Blood was aspirated into sterile syringes and immediately transferred to evacuated tubes containing EDTA solution. Platelet activation status was evaluated by determining cell-surface P-selection expression, number of platelet aggregates and platelet microparticles, mean platelet-component (MPC) concentration, and mean platelet-component distribution width (MPCDW) concentration. Neutrophil activation status was evaluated by determining cell-surface CD11/CD18 expression, neutrophil size, and neutrophil granularity. RESULTS: Short-duration strenuous sled-pulling activity was associated with lower MPC concentration, higher MPCDW concentration, and higher cell-surface P-selectin expression after activation with phorbol myristate acetate. An increase in neutrophil CD11/CD18 expression and a decrease in neutrophil granularity were also observed after exercise. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study provide evidence of priming and activation of platelets and activation of neutrophils after strenuous short-duration sled-pulling activity. Additional studies will be needed to determine whether these changes have adverse effects on animal performance or induce tissue injury.     

Flow cytometric detection of activated platelets in the dog

Background: Platelet activation appears to play a role in a variety of canine thrombotic disorders. At present, tests for the detection of activated platelets are not used routinely in veterinary clinical laboratories. Objective: The purpose of this study was to develop a clinically applicable method to detect activated canine platelets. Methods: A flow cytometric assay was developed to detect activated platelets, platelet aggregates, and platelet microparticles in the dog. Blood was collected from healthy dogs using EDTA or sodium citrate as the anticoagulant, and platelet‐rich plasma was prepared. Platelets were activated by adding phorbol myristate acetate. In some experiments, platelets were fixed by incubation with 0.5% paraformaldehyde. In other experiments, platelets were stored for 4 or 24 hours at 4°C before analysis. Activated platelets were detected by measuring surface expression of P‐selectin and by determining the percentages of platelet aggregates and microparticles using forward‐angle vs side‐angle light scatter plots. Results were analyzed by using 2‐way ANOVA and the SchefféF‐test. Results: Platelets collected in EDTA had minimal expression of P‐selectin, whereas platelets collected in sodium citrate had greater median fluorescence intensity. Fixation with 0.5% paraformaldehyde before labeling platelets with anti‐P‐selectin did not affect antibody binding or the percentages of platelet aggregates and microparticles. Storage of platelet‐rich plasma at 4°C for 4 hours did not affect antibody binding or the percentages of platelet aggregates or microparticles. Activation of platelets ex vivo by addition of 10 ng/mL phorbol myristate acetate resulted in a large increase in expression of P‐selectin but only slight increases in platelet aggregates and microparticles. Conclusion: Determination of platelet P‐selectin expression and percentages of platelet aggregates and platelet microparticles may provide a clinically applicable means for detection of activated platelets in dogs. The capacity to use EDTA‐anticoagulated blood samples and to fix platelets for evaluation at a later time makes the test attractive as a routine diagnostic tool.     

Alterations in bovine platelet function and acute phase proteins induced by Pasteurella haemolytica A1.

Platelet function was assessed by aggregometry in 10 Holstein calves before and after exposure to Pasteurella haemolytica (biotype A, serotype 1) by intrabronchial challenge. At 24 h after exposure the platelets had become more reactive to stimulation with known platelet agonists such as adenosine diphosphate (ADP) and platelet-activating factor (PAF) and the platelet aggregates that formed were more resistant to disaggregation. The activation of platelets was an early response in the challenged calves as platelet function had returned to pretreatment levels 72 h after exposure to the bacteria while the acute phase reactant proteins, haptoglobin and fibrinogen, were approaching their peak values and alpha 2-macroglobulin levels had also risen significantly (P < 0.05) at this time. The plasma levels of these proteins were still elevated and albumin levels were depressed 6 d post-treatment. At post-mortem all calves exhibited pneumonic tissue damage. When P. haemolytica leukotoxin was added directly to bovine platelet suspensions both spontaneous aggregation and an increase in the aggregation response to ADP and PAF stimulation were observed. The morphological appearance of the platelet aggregates exhibited the typical pattern for bovine platelets with 2 distinct zones of cells being visible within each aggregate. One zone contained platelets in which the cytoplasmic granules were still evident and the other zone contained irregularly shaped platelets devoid of granular content. In the latter zone, discrete gaps, or pores, were evident in the plasma membrane of numerous platelets. This pore formation is characteristic of leukotoxin action and is not observed in ADP or PAF induced aggregates.     

Neutrophil and platelet activation in equine recurrent airway obstruction is associated with increased neutrophil CD13 expression, but not platelet CD41/61 and CD62P or neutrophil–platelet aggregate formation

Recurrent airway obstruction (RAO) in mature horses is characterized by reversible airway obstruction and neutrophilic inflammation; there is also functional activation of circulating platelets and neutrophils. This study was undertaken to determine if changes in activation marker expression and heterotypic aggregate formation can be used as an indicator of this increased functional responsiveness.In vitro conditions for flow cytometric measurement of CD13, CD41/61 and CD62P expression on activated cells and heterotypic aggregate formation were established. Values were then compared before and after antigen challenge of RAO and healthy horses. Platelet adhesion to serum-coated plastic was measured as a functional marker of platelet activation.In vitro activation resulted in increased expression of neutrophil CD13 and platelet CD41/61 and CD62P. Activation of both cell types caused a significant increase in neutrophil–platelet aggregates.In horses with RAO, but not controls, there was a significant increase in the percentage of CD13 positive neutrophils at 10 h and 24 h and in the mean fluorescence intensity at 10 h. This was accompanied at 24 h by an increased mean platelet side scatter and thrombin-stimulated platelet adhesion.In conclusion, CD13 expression can be used as an indicator of equine neutrophil activation both in vitro and in vivo. Equine platelet activation in vitro can be detected by measuring CD41/61 or CD62P expression, and PAF-activated platelets and neutrophils form aggregates. However, despite evidence of circulating platelet activation, neither a change in expression of platelet activation markers, nor heterotypic aggregate aggregate formation could be detected.     

Evaluation of flow cytometric and automated methods for detection of activated platelets in dogs with inflammatory disease

OBJECTIVE: To evaluate platelet surface-associated P-selectin, mean platelet component concentration (MPC), mean platelet component distribution width (MPCDW), mean platelet volume (MPV), and platelet distribution width (PDW) for detection of activated platelets in dogs with septic and nonseptic inflammatory disease. ANIMALS: 20 healthy dogs and 20 dogs with septic and nonseptic inflammatory disease. Procedures-Platelet surface-associated P-selectin (expressed as the median fluorescence intensity [MFI] of the platelet population), MPC, MPCDW, MPV, and PDW were determined in 20 healthy adult dogs, and reference ranges were calculated. These parameters were also determined in 11 dogs with nonseptic and 9 dogs with septic inflammatory disease and evaluated to determine which parameters were useful for detection of activated platelets. Results-12 dogs with inflammatory disease had P-selectin greater than the upper limit of the reference range, whereas 16 dogs with inflammatory disease had MPC lower than the lower limit of the reference range. All dogs in which P-selectin was greater than the upper limit of the reference range had MPC lower than the lower limit of the reference range. The correlation coefficient for P-selectin and MPC was 0.62. Differences in the MPCDW, MPV, and PDW in most dogs with inflammatory disease (compared with healthy dogs) were found; however, the correlation coefficients for P-selectin and MPCDW, MPV, and PDW were low. CONCLUSIONS AND CLINICAL RELEVANCE: Platelet surface-associated P-selectin and MPC appeared to be useful to detect activated platelets in most dogs with septic and nonseptic inflammatory disease.     

Platelet activation in ponies with airway inflammation

REASON FOR PERFORMING STUDY: Platelet activation occurs in human obstructive airway diseases and in laboratory animal models. However, there is limited evidence that platelets may be involved in equine recurrent airway obstruction (RAO) and other inflammatory diseases. This study investigated whether platelet activation also occurred in RAO. HYPOTHESIS: Platelet function is altered in ponies with active RAO. This alteration can be detected ex vivo by measuring platelet adhesion. METHODS: An in vitro platelet adhesion assay measuring acid phosphatase (AcP) activity colorimetrically was adapted for use with equine platelets and responses to selected agonists were established. Platelet adhesion and aggregation was evaluated in vitro on platelets isolated from 6 ponies with RAO before, during and after a 7 h natural antigen challenge. Three ponies with no history of airway disease were also studied. RESULTS: Adhesion of equine platelets to serum coated plastic was detected at concentrations of 10-100 radicaló 10(9)/l. Adhesion increased in response to stimulation with platelet activating factor and thrombin, but not equine interleukin 8. Prior to the antigen challenge, adhesion of nonstimulated platelets was low and increased significantly (P<0.05) 24 h after initiation of the challenge in RAOs, but not in the normal animals. No changes in platelet aggregation were noted in either group. CONCLUSIONS: The described assay offers an alternative method to evaluate platelet function in healthy and diseased horses and can detect changes not observed using a classic aggregation assay. Circulating platelets are activated 24 h after antigen challenge of ponies with RAO and may play a role in pulmonary inflammation and/or the pathophysiology of RAO. POTENTIAL RELEVANCE: Investigating platelet function in RAO and airway inflammation may reveal new aspects of the pathogenesis of inflammatory lung disease in the horse.     

Flow cytometric evaluation of disseminated intravascular coagulation in a canine endotoxemia model

Sepsis is associated with substantial morbidity and mortality in dogs. Alterations in hemostasis by systemic inflammation play an important role in the pathophysiology of sepsis. To evaluate the functional hemostatic changes in sepsis, we evaluated coagulation profiles and flow cytometric measurement of P-selectin (CD62P) expression on platelets, as well as platelet-leukocyte aggregation from a lipopolysaccharide (LPS)-induced endotoxemia model in dogs (n = 7). A sublethal dose of LPS [1 mg/kg body weight (BW)] induced thrombocytopenia and increased activated partial thromboplastin time (aPTT), prothrombin time (PT), and D-dimer concentrations. Flow cytometry analysis showed a significant increase in P-selectin expression on platelets between 1 and 24 h of a total 48 h of the experiment. In addition, platelet-leukocyte aggregation was significantly increased in the early stage of endotoxemia (at 1 and < 6 h for platelet-monocyte aggregation and at 3 h for platelet-neutrophil aggregation). Our results suggest that CD62P expression on platelets and platelet-leukocyte aggregation, as measured by flow cytometry, can be useful biomarkers of disseminated intravascular coagulation (DIC) in canine sepsis. These functional changes contribute to our understanding of the pathophysiology of hemostasis in endotoxemia.     

Measurement of the activation of equine platelets by use of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody

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.     

Cryopreservation of Canine Platelets

Background: Platelet cryopreservation allows long-term storage and immediate availability of transfusion products.
Hypothesis: The addition of a preparation inhibiting platelet activation (Thrombosol, in 2% dimethyl sulfoxide [DMSO]) will enhance in vitro function and prolong in vivo survival of cryopreserved platelets compared with those preserved in 6% DMSO.
Animals: Thirty-three research dogs.
Methods: Prospective study. Eleven fresh canine apheresis platelet concentrates (PCs) were each split into 3 units: fresh and cryopreserved in 6% DMSO or Thrombosol. Platelet analysis, performed 1–10 weeks postfreezing, included in vitro functional testing and in vivo survival assessed by administration of biotinylated platelets.
Results: Platelet aggregation was diminished in cryopreserved PC. Cryopreserved platelets could be activated, as based on mean thrombin-stimulated P-selectin expression (6% DMSO, 23.0%; Thrombosol, 18.4%), although to a lesser extent than fresh PC (49.1%) ( P < .0001). The mean maximum in vivo platelet recovery for fresh PC was 80.3%, significantly greater than recovery for 6% DMSO (49.2%) and Thrombosol PC (43.7%) ( P ≤ .001). The half-life (days) of fresh PC (3.8 ± 0.4) was significantly ( P < .002) greater than that of 6% DMSO (1.9 ± 1.0) and Thrombosol (2.4 ± 1.1) PC, with no difference ( P = .3) between cryopreserved PC.
Conclusions and Clinical Importance: Cryopreservation of canine platelets using Thrombosol did not provide any advantage over preservation using 6% DMSO. Cryopreserved platelets can be activated in vitro and provide therapeutic benefit when fresh platelets are unavailable. Further studies are needed to assess their in vivo hemostatic function.     

Investigation of a novel, heritable bleeding diathesis of Thoroughbred horses and development of a screening assay

BACKGROUND: Bleeding in racing horses associated with exercise appears to be multifactorial, and clinical investigation into severe cases rarely occurs. Previously, we reported a severe bleeding diathesis in a Thoroughbred mare. Herein, we describe the cellular physiology of this defect, provide a diagnostic tool for identifying it, and demonstrate that the dysfunction is heritable. HYPOTHESIS: The subject has a heritable defect in platelet secretion that reduces thrombin generation in the absence of additional plasma factors and delays the onset of thrombin production even in the presence of these factors. ANIMALS: The study included 3 clinically normal Thoroughbred horses: the subject and her offspring. METHODS: Washed platelets were examined for their ability to (1) translocate phosphatidylserine to the outer leaflet of the platelet membrane as determined by annexin-V binding, (2) generate thrombin as assessed by the activity of the prothrombinase enzyme complex, and (3) bind fibrinogen and form aggregates as determined by flow cytometry. RESULTS: Subject and offspring platelets created procoagulant surfaces by translocating phosphatidylserine. The subject's platelets demonstrated reduced prothrombinase activity, resulting in decreased production of thrombin relative to control platelets. Subject and offspring platelets bound less fibrinogen than control platelets when stimulated with thrombin. CONCLUSIONS AND CLINICAL IMPORTANCE: The subject mare has a transmissible defect that involves reduced generation of thrombin by activated platelets, resulting in decreased aggregation and ineffective clotting. A flow cytometric assay of fibrinogen binding to washed platelets discriminates individuals with this platelet dysfunction and may be useful for discerning subclinical congenital or acquired platelet dysfunctions.     

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. 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 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.     

Detection of activated platelets in dogs with primary immune-mediated hemolytic anemia

Thromboembolism is a major cause of morbidity and mortality in dogs with immune-mediated hemolytic anemia (IMHA). To the authors' knowledge, the role of platelets in thromboembolic events associated with IMHA has not been extensively investigated. In the study reported here, we evaluated cell membrane expression of P-selectin with flow cytometry to determine whether platelets circulate in an activated state in association with primary IMHA. Median P-selectin expression for 20 dogs with primary IMHA was 8.1-fold greater, compared with values for 20 healthy dogs. Fifteen of 20 dogs (75%) with IMHA had P-selectin median fluorescence intensity (MFI) values that exceeded the reference interval for healthy dogs. Additionally, P-selectin MFI after activation of platelets with phorbol myristate acetate was 2.1-fold greater for dogs with IMHA than for healthy control dogs. Despite treatment of all dogs with immunosuppressive therapy and 18 dogs with subcutaneously administered low-dose unfractionated heparin, 7 dogs developed clinical signs consistent with thromboembolism. These data provide support for the hypothesis that platelets circulate in an activated state in many dogs with IMHA.     

Characterization of platelet and soluble-porcine P-selectin (CD62P)

P-selectin (CD62P), an adhesion molecule expressed on activated endothelial cells and platelets, mediates the initial attachment of leukocytes to the stimulated endothelium upon inflammation and the interaction between leukocytes and platelets. A soluble form of P-selectin is present in the serum of healthy individuals as a circulating protein and high levels have been described in various pathological situations. The aim of this study was to characterize P-selectin on porcine platelets and investigate the soluble form of this protein, which are uncharacterized in several animal species including pigs. A new monoclonal antibody (mAb) (SwPsel.1.9) against porcine P-selectin was produced using a mouse cell line transfected with pig P-selectin cDNA. This mAb together with a previously described mAb (P-sel.KO.2.5), produced in our laboratory, was used to develop an ELISA to quantify porcine P-selectin. No significant levels of soluble-porcine P-selectin were observed in healthy animals. However, the total amount of P-selectin measured in porcine platelets was similar to that found in humans. Increased levels of this circulating protein were detected in the plasma from pigs after allograft implantation. In vitro, P-selectin expression on platelet membrane was rapidly induced by PMA and thrombin, as assessed by flow cytometry. However, these activators did not stimulate the release of soluble P-selectin. Analysis of the proteolytic cleavage of this protein from COS-transfected cells revealed that PMA treatment failed to cause the shedding of membrane-bound P-selectin. These data suggest that porcine P-selectin is a suitable marker for inflammation and that the mechanism involved in the generation of circulating P-selectin is not proteolytic release.     

A comparison of platelet parameters in EDTA- and citrate-anticoagulated blood in dogs

BACKGROUND: Platelet aggregates are a common artifact in canine blood. Aggregates may affect the accuracy of platelet counts, with important consequences for patient care. OBJECTIVES: The purpose of this study was to determine if platelet counts in dogs were more accurate if blood was collected into citrate instead of EDTA as an anticoagulant. METHODS: Blood was collected from 50 dogs with neoplasia admitted to the oncology service at Cornell University. EDTA and citrate Vacutainer tubes were filled with blood in random order. Platelet counts and parameters (mean platelet volume [MPV], platelet distribution width [PDW], mean platelet component concentration [MPC], platelet component distribution width [PCDW], and automated platelet clump count [APCC]) were determined using an optical-based hematology analyzer (ADVIA 120). Blood smears from each anticoagulated sample were scored visually for platelet aggregates. RESULTS: The median platelet count was significantly lower (median decrease, 27 x 10(9)/L) in citrate-anticoagulated blood compared with EDTA-anticoagulated blood. This was attributed to platelet activation and aggregation: significantly more aggregates were seen in smears of citrate- than of EDTA-anticoagulated blood. Aggregates were typically small and not detected by the analyzer. Also, the MPV and MPC (or density) were significantly higher (median increase, 3 fL) and lower (median decrease, 33 g/L) in citrate-anticoagulated samples, respectively. CONCLUSIONS: Platelets aggregate, likely from activation, when blood from dogs with neoplasia is anticoagulated with citrate for hematology testing, resulting in lower platelet counts. Citrate also yields inaccurate results for MPV and MPC, likely because of inadequate sphering of platelets. Thus, we recommend that citrate not be used as an anticoagulant when accurate platelet counts are desired in dogs.     

Effects of physiologic agonists on canine whole blood flow cytometry assays of leukocyte-platelet aggregation and platelet activation

Platelets play a role in both the innate and adaptive immune systems. Methods for detecting activated platelets and leukocyte-platelet aggregates (LPAs) are useful for basic and applied research concerning the role of platelets in inflammation and immune disorders. The aim of the study was to develop flow cytometric assays for detection of platelets binding to monocytes and neutrophils and for activated platelets in canine whole blood and to investigate the effect of physiologic agonists. Citrate anticoagulated whole blood was incubated with monoclonal antibodies against CD14 and CD61 for detection of LPAs, and the effect of various agonists was investigated. For detection of activated platelets, whole blood was incubated with monoclonal antibodies against CD62P and against a receptor-induced binding site on fibrinogen (CAP1) with CD61 as a platelet identifier. Isotype controls were prepared in parallel. The individual physiologic agonists ADP, collagen and epinephrine increased LPAs, CD62P and CAP1 binding only modestly. However, combinations of agonists gave more substantial increases. A dose-response relationship was seen using alpha- and gamma-thrombin, and ADP as agonists. In conclusion, we have developed flow cytometry assays to measure LPAs and platelet activation in canine whole blood, and have explored the effect of various physiologic agonists at different concentrations.     

Evaluation of platelet activation in canine immune-mediated haemolytic anaemia

Objectives : To establish whether heightened platelet activation is a common feature of canine immune-mediated haemolytic anaemia, and to evaluate the hypothesis that platelet activation plays a role in the pathogenesis of thromboembolism. Methods : Using whole-blood flow-cytometric analysis, the proportion of activated platelets and platelet-leucocyte aggregates in blood samples from 14 dogs with immune-mediated haemolytic anaemia and 14 healthy dogs was calculated. General linear models with binomial errors were used to compare groups. Results from the immune-mediated haemolytic anaemia-affected dogs were then correlated with established risk factors for thromboembolism in canine immune-mediated haemolytic anaemia, D-dimer concentration and antithrombin activity. Results : There was a strong correlation between platelet activation and severe thrombocytopenia, with heightened platelet activation being observed predominantly in severely thrombocytopenic dogs. Clinical Significance : Dogs with immune-mediated haemolytic anaemia, particularly those with concurrent severe thrombocytopenia, are likely to have heightened platelet activation, which may play a role in the pathogenesis of thromboembolism.     

Platelet size,platelet surface-associated IgG,and reticulated platelets in dogs with immune-mediated thrombocytopenia

Abstract: Immune-mediated thrombocytopenia (IMT) is a disorder in which bound IgG on the surface of platelets results in platelet removal and alterations in mean platelet volume. Using flow cytometry, alterations in platelet size, platelet surface-associated IgG (PSAIgG), and numbers of reticulated platelets were determined in 13 dogs with primary IMT and 4 dogs with secondary IMT induced by experimental infection with Babesia gibsoni . Effects of sample age on platelet parameters also were determined, using samples from 20 dogs with normal platelet counts analyzed within 4 hours and after 24, 48, and 72 hours of storage in EDTA. No significant changes in platelet count, platelet size, or reticulated platelet percentage were observed in samples assayed within 4 and 24 hours of blood collection; whereas PSAIgG values increased 3 to 7 fold in samples stored for 24–72 hours. Using reference values for freshly collected or 24-hour-old samples, 10 of 13 (77%) dogs with primary IMT and all B gibsoni-inf ected dogs had increased PSAIgG levels. In 12 (75%) of the 16 dogs with thrombocytopenia the percentage of reticulated platelets was increased; however, absolute numbers of reticulated platelets were within reference values. Moreover, PSAIgG level and the percentage of reticulated platelets were not always increased concurrently in dogs with primary and secondary IMT. Platelet microparticles were detected in all B gibsoni-infected dogs, 8 of 13 (62%) dogs with primary IMT, and transiently in a dog that responded to immunosuppressive treatment. The results of this study indicate that sample age and time of sampling during disease affect interpretation of platelet parameters in dogs with IMT.     

Actions and interactions of ADP, 5- HT, histamine and PAF on equine platelets

Platelets are thought to play a role in equine diseases such as acute laminitis and verminous arteritis and may be involved in allergic disease. Mediators implicated in the pathophysiology of these conditions activate platelets and responses may be enhanced by interactions between mediators. The present study compared platelet aggregation, thromboxane production and release of radiolabelled [(3)H]5- HT in response to 5- HT, histamine, ADP and PAF alone and in combination in vitro.PAF caused concentration-related aggregation, [(3)H]5- HT release and thromboxane production. In contrast, ADP caused aggregation and 5- HT induced the release of [(3)H]5- HT with little effect on other platelet functions. Histamine had little or no effect on equine platelets. Addition of 5- HT (10 microM) prior to ADP significantly displaced the aggregation response curve to the left.The profile of responses to PAF, ADP and 5- HT suggests differential activation of intracellular signalling pathways regulating these events. The enhanced response to ADP in the presence of 5- HT may have implications in thromboembolic disease in the horse.