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A solid-phase elisa to detect antibodies bound to the surface of canine platelets (platelet-bound antibodies) is described. Using this assay, the effect of anticoagulant and storage time of anticoagulant blood on the concentration of antibodies bound to the surface of platelets from clinically normal dogs was investigated. Blood from 3 clinically normal dogs was anticoagulated with acid citrate dextrose, Na3 citrate, and aqueous K3 edta and stored on ice for up to 48 hours. Platelet-bound antibody concentration was measured on platelets isolated from anticoagulated blood immediately after venipuncture and subsequent to storage of blood for 24 and 48 hours. Differences in platelet-bound antibody concentrations were investigated among dogs, anticoagulants, and storage times by anova and Bonferroni pair-wise comparison of means. There was no effect of dog on platelet-bound antibody concentration. The effect of time was significant (P < 0.0001), with higher concentration of platelet-bound antibodies detected with increasing storage time. Effect of anticoagulant on platelet-bound antibody concentration was not statistically significant; however, there was a trend to increasing concentration of antibodies bound to platelets isolated from Na3 citrate- and K3 edta-anticoagulated blood. Moreover, there was significant (P = 0.02) interaction between anticoagulant and time. Platelet-bound antibody concentration increased with storage of anticoagulated blood prior to platelet isolation and with use of Na3 citrate and K3 edta anticoagulants. The preferred anticoagulant for platelet-bound antibody measurement is acid citrate dextrose. Platelet-bound antibody concentration should be determined not longer than 24 hours after blood collection.

Free access
in American Journal of Veterinary Research



To develop a flow cytometric assay for detection of platelet-bound IgG in dogs.

Sample Population

Negative-control platelets were obtained from 5 clinically normal Greyhounds. Positive-control platelets were platelets from 1 clinically normal dog, sensitized with dog anti-canine platelet alloantibodies.


Washed platelets were incubated with mouse anti-canine IgG conjugated to fluorescein isothiocyanate and analyzed by flow cytometry. Optimal dilution of antibody reagent and dose-response were determined, as were effects on platelet-bound IgG detection of storage time and temperature of K3EDTA-anticoagulated blood samples, variable platelet numbers, and variable filling of K3EDTA evacuated tubes.


A 1:128 dilution of antibody reagent was optimal. There was a linear increase in platelet-bound IgG when normal canine platelets were incubated with increasing concentrations of positive-control serum. Variable numbers of positive-control platelets tested and variable filling of K3EDTA evacuated tubes had no significant effect on platelet-bound IgG concentration. Platelet-bound IgG concentration increased with storage time at room temperature (P = 0.0003), but not when blood was kept cool. Sufficient platelets for assay were able to be isolated from 3 ml of blood from 5 dogs with < 10,000 platelets/μl.


This assay for platelet-bound IgG in dogs is simple, repeatable, and practical. The assay is not affected by platelet count or variable filling of evacuated tubes, and requires only 3 ml of K3EDTA-anticoagulated blood. Blood samples for testing require packaging on ice and overnight delivery but, after arrival at the laboratory, can be refrigerated and analyzed within 72 hours of collection.

Clinical Relevance

Assays for platelet-bound IgG may help in assessing causes and treatment of thrombocytopenia.

Free access
in American Journal of Veterinary Research


The sensitivity and specificity of 2 antibody tests for diagnosis of idiopathic thrombocytopenic purpura (itp) in dogs were investigated prospectively. An elisa to detect antibodies bound to the surface of platelets from affected dogs (direct test) was performed in 34 dogs with a clinical diagnosis of itp and in 21 dogs with thrombocytopenia attributable to other causes. An elisa to detect platelet-bindable antibodies in serum from affected dogs (indirect test) was performed in 32 dogs with itp and in 15 dogs with other causes of thrombocytopenia. The direct test was positive in 32 of 34 dogs with itp (sensitivity, 94%) and negative in 13 of 21 dogs with other causes of thrombocytopenia (specificity, 62%). Positive direct test results were obtained in 2 dogs with systemic lupus erythematosus, and in 1 dog each with concurrent Ehrlichia canis and Babesia canis infections, dirofilariasis, myelodysplasia, disseminated intravascular coagulation (of unknown cause), and thrombocytopenia subsequent to administration of trimethoprim/sulfadiazine, as well as in 1 dog with thrombocytopenia 14 days after a whole blood transfusion. The indirect test had positive results in 11 of 32 dogs with itp (sensitivity, 34%) and negative results in 12 of 15 dogs with other causes of thrombocytopenia (specificity, 80%). Positive indirect test results were obtained in 1 dog each with systemic lupus erythematosus, concurrent E canis and B canis infections, and thrombocytopenia subsequent to administration of trimethoprim/sulfadiazine. Detection of platelet-bound antibodies was more sensitive than detection of serumplatelet bindable antibodies in confirming a diagnosis of itp in dogs. Neither test was specific for itp. Therefore, a negative test result for platelet-bound antibodies in dogs with thrombocytopenia is helpful in excluding itp as a cause of thrombocytopenia; however, a positive test result is not specific for itp, and other causes of immune-mediated thrombocytopenia must be excluded to establish a diagnosis of itp.

Free access
in Journal of the American Veterinary Medical Association