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.     

Thrombocytopenia and platelet activity in dogs experimentally infected with Rangelia vitalii

The aim of this study was to evaluate the platelet count, coagulation time and platelet activity in dogs experimentally infected with Rangelia vitalii during the acute phase of the disease. For this study, 12 young dogs (females) were used, separated in two groups. Group A (uninfected control) was composed by healthy dogs (n=5), and group B consisted of R. vitalii-infected animals (n=7). After being inoculated with R. vitalii-infected blood, animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite five days post-inoculation (PI). Blood samples were collected on days 0, 10, 20 and 30 PI. The material collected was placed in tubes containing EDTA for quantification of platelets, citrate anticoagulant platelet aggregation, and measuring the clotting time. Right after blood collection on days 10 and 20 PI, dogs were anesthetized for collecting bone marrow samples. A significant reduction (P<0.01) of the number of platelets was observed in R. vitalii-infected blood, when compared with uninfected dogs on days 10 and 20 PI. Additionally, macro-platelets were observed only in infected dogs. Prothrombin time and activated partial thromboplastin time did not differ between infected and uninfected dogs. The megakaryocyte count increased (P<0.01) significantly in infected dogs when compared with uninfected ones on days 10 and 20 PI. Platelet aggregation decreased (P<0.01) significantly in infected dogs in comparison to the control on days 10 and 20 PI. Therefore, rangeliosis in dogs causes a severe thrombocytopenia during the acute phase of infection. This platelets reduction probably occurred due to splenic sequestration and/or immune-mediated thrombocytopenia.     

Detection of Antiplatelet Antibody With a Platelet Immunofluorescence Assay

An indirect platelet immunofluorescence assay (PIFA) was developed for detection of circulating antiplatelet antibody in dogs with suspected immune-mediated thrombocytopenia (ITP). The PIFA was performed on 10 healthy dogs with normal platelet counts; 76 thrombocytopenic dogs, 20 of which were suspected of having ITP; and 18 dogs with other diseases and normal platelet counts. All normal dogs had negative test results. Fourteen (70%) of 20 dogs suspected of having ITP had positive test results. Fifteen of the remaining 56 thrombocytopenic dogs had positive test results, 9 had cancer and 6 had other immune-mediated diseases including systemic lupus erythematosus (SLE). In this study, the PIFA assay seemed to be more sensitive (70%) than the megakaryocyte immunofluorescence assay (41 %) in the diagnosis of ITP. Of the 9 PIFA-positive dogs with neoplasia, 6 had lymphoproliferative disorders. The PI FA was positive in 5 of 18 diseased dogs with normal platelet counts. There was an inverse relationship between the platelet count and the intensity of fluorescence in the PIFA-positive dogs. We conclude that the PIFA is a sensitive screening method for detecting circulating antiplatelet antibody.     

Platelet aggregation studies in dogs with acute Ehrlichia platys infection

Ten adult dogs (5 Beagles and 5 mixed-breed dogs) were inoculated IV with canine platelets containing Ehrlichia platys. Inclusions and morulae of E platys developed in platelets of infected dogs at 10 to 14 days after inoculation, followed by marked thrombocytopenia at 14 to 21 days. Parasitemia and marked thrombocytopenia recurred at 24 to 28 days after inoculation. Increased numbers of megakaryocytes were observed in marrow aspirate smears from infected dogs, indicative of regenerative thrombocytopenia. Prior to infection, platelet-rich plasma from these dogs was determined to have similar aggregatory response to arachidonate. After infection with E platys, the aggregatory response of platelet-rich plasma to collagen or 3 dilutions of adenosine diphosphate was evaluated. A statistically significant (P less than 0.05) inhibition of platelet aggregatory response to the lowest dilution of adenosine diphosphate was detected for mixed-breed dogs, whereas aggregation responses were unchanged in Beagles. Results indicate that platelet activation may occur in dogs with acute ehrlichial infection.     

Splenectomy as adjunctive therapy for immune-mediated thrombocytopenia and hemolytic anemia in the dog

Splenectomy was done in 9 dogs having immune-mediated hematologic disorders refractory to medical therapy. These disorders were immune-mediated thrombocytopenia (n = 3), immune hemolytic anemia (n = 3), and Evan's syndrome (n = 3). The diagnoses were based on clinical observations, laboratory test data, and differential of other conditions. In the 12 months after splenectomy was done, the dogs reflected clinical improvement and return of platelet and/or erythrocyte counts to clinically acceptable limits; medical treatment was stopped or reduced in 8 of 9 patients. The exceptional patient had shown clinical improvement without change in the platelet count. At the end of 1 year, survival rate was excellent, and postsplenectomy complications, such as hemobartonellosis, did not appear. It is believed that splenectomy may be useful for treating immune-mediated thrombocytopenia, anemia, and Evan's syndrome that seem refractory to medication.     

Resolution of severe thrombocytopenia in two standard Poodles with surgical correction of splenic torsion

Objective: To describe the diagnosis and treatment of 2 cases of severe thrombocytopenia associated with splenic torsion and to discuss the pathophysiologic mechanisms underlying the thrombocytopenia. Summary: We report 2 cases of severe thrombocytopenia associated with splenic torsion. Each dog presented with non‐specific clinical signs, radiographic evidence of an intra‐abdominal mass, and platelet counts of less than 25,000 platelets/μL. The diagnosis of splenic torsion was made with abdominal ultrasonography and was confirmed during exploratory laparotomy. Both dogs recovered rapidly following splenectomy. The cause of thrombocytopenia associated with splenic torsion is not fully elucidated, but may be because of either platelet sequestration within the torsed spleen, platelet consumption in disseminated intravascular coagulation, or a combination of both. New information provided: This report provides previously unreported evidence that the degree of thrombocytopenia associated with splenic torsion may be of a severity at which primary hemostasis is compromised, and resolution of thrombocytopenia occurs after splenectomy.     

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.     

Clinical and hematologic findings in canine ehrlichiosis

The clinical and hematologic findings for 56 dogs with ehrlichiosis were studied retrospectively. All dogs had a high serum antibody titer to Ehrlichia canis. The frequency of bleeding disorders, thrombocytopenia, and pancytopenia in these dogs was lower than previously described for dogs so affected. A bleeding disorder caused by a suspected qualitative platelet defect was found in some dogs that did not have thrombocytopenia. Megakaryocytic hyperplasia and high numbers of plasma cells were observed frequently on marrow aspirate smears. The clinical and laboratory findings of dogs were variable and were considered nonspecific for ehrlichiosis.     

Platelet-bound antibodies detected by a flow cytometric assay in cats with thrombocytopenia

In cats, primary or secondary immune-mediated thrombocytopenia have rarely been described or characterised. The objective of this study was to determine platelet-bound antibodies (PBA) by a flow cytometric assay in both healthy and thrombocytopenic cats. Direct PBA testing was performed in 42 thrombocytopenic cats (platelet counts 6-179 x 10(9)/l, median 56 x 10(9)/l). Of these 42 cats, 19 had positive PBA test results, 17 of which were considered to have secondary immune-mediated thrombocytopenia (sITP). Underlying diseases included fat necroses (four cases), feline infectious peritonitis (three), feline leukaemia virus (two) or feline immunodeficiency virus (two) infections, lymphoma (two), leukaemia (one), hepatitis (one), pyelonephritis (one), or hyperthyroidism (one). In two cats, no underlying disease was found suggesting a primary immune-mediated thrombocytopenia (pITP). The PBA test was negative in 23 cats diagnosed with varying underlying diseases and in 47 healthy control cats with platelet values within the reference range. Only seven of the 42 cats with thrombocytopenia (platelet count 10-57 x 10(9)/l, median 34 x 10(9)/l) had spontaneous bleeding. This study suggests that immune-mediated destruction of platelets might be an important pathological mechanism for feline thrombocytopenia caused by various underlying diseases. In cats, pITP appears to be rarely diagnosed.     

Detection of antiplatelet immunoglobulin in thrombocytopenic dogs

Indirect enzyme-linked immunosorbent assays (ELISA) were used to detect platelet-associated immunoglobulin in sera from dogs with immune-mediated thrombocytopenia (IMT) and/or other autoimmune syndromes. One ELISA, utilizing whole platelets as the antigen substrate, readily detected antibody associated with platelets, either as specific, antiplatelet antibody or as immune complexes. This assay apparently lacked specificity because of the position reactions with sera from dogs with miscellaneous autoimmune disorders and no concurrent thrombocytopenia. Although the second ELISA, utilizing immunoaffinity purified platelet antigens was not influenced as much by immune complexes, absorbance values apparently were slightly increased. However, a small number of dogs with non-thrombocytopenic autoimmune disease tested positive. Immunoadsorption and Western immuno-blotting techniques demonstrated a complex pattern of specificities for antiplatelet antibodies. Clinical significance of these findings is discussed.     

Application of therapeutic plasma exchange in dogs with immune-mediated thrombocytopenia

Therapeutic plasma exchange (TPE) is an emerging treatment for dogs with immune-mediated diseases, but reports for treatment of immune-mediated thrombocytopenia (IMT) are lacking. These case reports illustrate the application of centrifugal TPE in 4 dogs with IMT. All dogs presented with severe hemorrhage requiring ≥1 blood transfusions, were unresponsive to conventional treatment or both. Dogs were treated with 3 sequential centrifugal TPE sessions, totaling 4.0 to 4.9 total plasma volumes exchanged per dog. In 3 dogs, TPE was associated with improvement in clinical manifestations of bleeding and platelet count in combination with immunosuppressive drugs. One dog was euthanized after 3 treatments because of persistent severe thrombocytopenia and hemorrhage. Preliminary observations indicate that TPE is safe and may be a useful adjunct in the management of IMT that is severe or refractory to traditional treatment.     

Mechanisms of platelet destruction in immune-mediated thrombocytopenia: in vitro studies with canine platelets exposed to heterologous and isologous antiplatelet antibodies

Normal canine platelets coated with heterologous or isologous antiplatelet antibody were interacted with viable canine neutrophils in vitro. Platelet phagocytosis was assessed by detecting nitroblue tetrazolium (NBT) reduction, measuring uptake of opsonised 51Cr-labelled platelets, and electron microscopy. The NBT reduction and uptake of 51Cr-labelled opsonised platelets were markedly increased. Electron microscopy revealed phagocytosis of antibody-coated platelets and their degradation intracellularly. Exposure of canine platelets to rabbit anti-canine platelet antibody in vitro produced morphological changes in platelets and caused serotonin release. Serotonin was not released in the absence of antiplatelet antibody or in the presence of normal rabbit gamma-globulin. Morphological changes in the platelets included disappearance of alpha and dense granules and exaggeration of the open canalicular system. These observations indicate that circulating platelets may be vulnerable to an antiplatelet antibody and that antibody-mediated phagocytosis of platelets is an important mechanism in the pathogenesis of immune-mediated thrombocytopenia.     

Epidemiologic survey of thrombocytopenia in dogs: a report on 987 cases

Thrombocytopenia was documented in 987 of 18,910 (5.2%) dogs admitted to North Carolina State University, College of Veterinary Medicine, Veterinary Teaching Hospital, between 1983 and 1989. Classifying thrombocytopenic dogs by etiologic groups revealed the following proportionate ratios: 5% (48/987) immune-mediated thrombocytopenia; 13% (130/987) neoplasia-associated thrombocytopenia; 23% (224/987) inflammatory/infectious thrombocytopenia; and 59% (585/987) miscellaneous thrombocytopenia. Dogs with immune-mediated thrombocytopenia had significantly (P < 0.05) lower platelet counts (mean 36,760 +/- 50,288 microliter) than dogs in the other three groups, and Doberman Pinschers were overrepresented in all groups except the immune-mediated thrombocytopenic group. We conclude that thrombocytopenia is a prevalent and potentially important diagnostic finding in a variety of disease states.     

Hemolytic anemia caused by Babesia gibsoni infection in dogs.

Babesia gibsoni caused severe hemolytic anemia in 11 dogs from southern California. The most common clinical signs of B gibsoni infection were lethargy, anorexia, anemia, and thrombocytopenia. Acute infection with B gibsoni may be misdiagnosed as autoimmune hemolytic anemia. Diagnosis was most reliably determined by identification of the intraerythrocytic parasites on Giemsa-stained blood smears. The pathogenicity of B gibsoni, difficulties in diagnosis, the parasite's resistance to treatment with available drugs, and frequent interstate movement of dogs indicate that this disease may be a serious threat to dogs throughout the United States.     

Measurement of thrombopoietic activity through the quantification of megakaryocytes in bone marrow cytology and reticulated platelets

Reticulated platelets are considered as marker for bone marrow thrombopoiesis. The aim of the study was evaluate the role of reticulated platelets as markers of thrombopoiesis in dogs. Reticulated platelets analysis by flow cytometry and megakaryocyte quantification by bone marrow cytology were determined in 29 healthy adult dogs (control group), 14 dogs with thrombocytopenia without megakaryocytic hypoplasia (group A) and 14 dogs with thrombocytopenia which presented megakaryocytic hypoplasia (group B), detected by bone marrow aspiration cytology. Blood samples were collected and the platelet rich plasma was obtained for reticulated platelets quantification in flow cytometry. Megakaryocytes were quantified in aspiration cytology by two techniques in marrow particles, and correlated to reticulated platelets counts. There are no differences between megakaryocyte quantification. Although there is no correlation between reticulated platelet values and megakaryocyte in bone marrow cytology, the interpretation of reticulated platelet values can be based both on absolute or relative corrected values.     

Plateletcrit is superior to platelet count for assessing platelet status in Cavalier King Charles Spaniels

Background: Many Cavalier King Charles Spaniel (CKCS) dogs are affected by an autosomal recessive dysplasia of platelets resulting in fewer but larger platelets. The IDEXX Vet Autoread (QBC) hematology analyzer directly measures the relative volume of platelets in a blood sample (plateletcrit). We hypothesized that CKCS both with and without hereditary macrothrombocytosis would have a normal plateletcrit and that the QBC results would better identify the total circulating volume of platelets in CKSC than methods directly enumerating platelet numbers.
Objectives: The major purpose of this study was to compare the QBC platelet results with platelet counts from other automated and manual methods for evaluating platelet status in CKCS dogs.
Methods: Platelet counts were determined in fresh EDTA blood from 27 adult CKCS dogs using the QBC, Sysmex XT-2000iV (optical and impedance), CELL-DYN 3500, blood smear estimate, and manual methods. Sysmex optical platelet counts were reanalyzed following gating to determine the number and percentage of normal- and large-sized platelets in each blood sample.
Results: None of the 27 CKCS dogs had thrombocytopenia (defined as <164 × 10⁹ platelets/L) based on the QBC platelet count. Fourteen (52%) to 18 (66%) of the dogs had thrombocytopenia with other methods. The percentage of large platelets, as determined by regating the Sysmex optical platelet counts, ranged from 1% to 75%, in a gradual continuum.
Conclusions: The QBC may be the best analyzer for assessing clinically relevant thrombocytopenia in CKCS dogs, because its platelet count is based on the plateletcrit, a measurement of platelet mass.     

Gold-induced immune thrombocytopenia in the dog

In two seven-year studies with gold compounds in dogs of both sexes, thrombocytopenia was observed after 45 to 72 months of dosing in three of 14 and two of 14 dogs in high-dose groups that received 2.4 to 3.6 mg/kg of auranofin per day orally or 0.5 to 2.0 mg/kg of gold sodium thiomalate intramuscularly once every three days, respectively. An immune basis for the disorder was suggested by the apparent consumptive nature of the thrombocytopenia (increased bone marrow megakaryocytes and large peripheral blood platelets), the response to corticosteroid therapy and the demonstration of increased platelet-associated immunoglobulin. The latter was demonstrated with a solid phase radioimmunoassay and by electron microscopy using a staphylococcal protein A-colloidal gold conjugate. Platelet-associated immunoglobulin decreased as the platelet counts rose, and in one dog monitored over periods of steroid-induced remissions and subsequent relapses, the amount of platelet-associated immunoglobulin G correlated inversely with the platelet count (r = 0.82). These findings suggest that the long-term administration of gold compounds in dogs is associated with a dose-dependent incidence of thrombocytopenia, which is immune-mediated and similar to that associated with parenteral chrysotherapy in man. The application of tests for platelet-associated immunoglobulin to canine patients with immune thrombocytopenia should be useful in the diagnosis of the disorder in clinical practice.     

Anti-erythrocyte antibodies and disease associations in anemic and nonanemic dogs

BACKGROUND: Flow cytometry has been used to detect anti-red blood cell (RBC) antibodies in dogs with immune-mediated hemolytic anemia (IMHA), but the prevalence of anti-RBC antibodies in anemic and nonanemic dogs with a variety of different diseases has not been assessed previously. HYPOTHESIS: We hypothesized that anti-RBC antibodies would be more common in anemic dogs and in dogs with immune-mediated disorders and cancer. ANIMALS: Blood samples from 292 dogs were analyzed prospectively by flow cytometry for anti-RBC antibodies. METHODS: Blood samples from 147 anemic and 145 nonanemic dogs were evaluated by flow cytometry to detect surface-bound immunoglobulin (Ig) G and IgM antibodies on RBC. Disease associations with RBC antibodies were determined, as was the correlation between disease status and the percentage of Ig(+) RBC. The specificity and sensitivity of flow cytometry and clinical variables for the diagnosis of IMHA were compared by Bayesian analysis. RESULTS: Anemic dogs were significantly more likely to be positive for anti-RBC antibodies (IgG, IgM, or both) than nonanemic dogs. Anemic dogs also had significantly higher percentages of Ig(+) RBC than nonanemic dogs, whereas dogs with IMHA had significantly higher percentages of Ig(+) RBC than dogs with all other diseases. Dogs with IMHA, infectious diseases, and immune-mediated thrombocytopenia were significantly more likely to have anti-RBC antibodies than dogs with other medical or surgical diseases. CONCLUSIONS: Anemic dogs with immune-mediated diseases and infectious diseases were at the highest risk for the development of anti-RBC antibodies, and flow cytometry for the detection of IgG on RBC was highly sensitive and specific for the diagnosis of IMHA.     

Immune-mediated thrombocytopenia associated with angiostrongylus vasorum infection in a Jack Russell terrier

A twenty-month-old Jack Russell terrier was presented with a four-day history of thrombocytopenia, echymotic inguinal haemorrhages, coughing and reduced exercise tolerance. Clinical examination revealed several petechial haemorrhages on the gingivae and small echymotic haemorrhages in the inguinal region, along with mild bilateral epistaxis. Haematology confirmed a platelet count of 1.0 × 10/L. Thoracic radiographs revealed a wide-spread mixed alveolar-interstitial lung pattern, apparent throughout the entire lungfield, but particularly marked within the left lung lobes. A presumptive diagnosis of immune-mediated thrombocytopenia was made and the dog was treated with vincristine and immunosuppressive doses of prednisolone. Initially anaemia developed following gastrointestinal haemorrhage; however, after symptomatic treatment the dog showed a marked clinical improvement. Evaluation for an underlying cause of the disease revealed Angiostrongylus vasorum L1 larvae on faecal analysis and treatment with fenbendazole was commenced. The dog made a full clinical recovery with all treatment was withdrawn within five weeks of diagnosis. This is the second report of immune-mediated thrombocytopenia associated with Angiostrongylus vasorum infection and it is the first to be successfully managed. The report highlights that Angiostrongylus vasorum should be considered in young dogs presented with thrombocytopenia.     

Thrombocytopenia associated with administration of trimethoprim/sulfadiazine in a dog.

A diagnosis of trimethoprim/sulfadiazine-induced, immune-mediated thrombocytopenia in a dog was made, using a novel in vitro assay for thrombolytic activity. The assay quantifies thrombolytic activity by measuring the amount of platelet fragments in normal canine platelets before and after incubation with plasma from the thrombocytopenic dog. This report confirms previous reports of the development of thrombocytopenia after administration of trimethoprim/sulfadiazine, and describes a new assay that, after further validation, may be useful in the diagnosis of immune-mediated thrombocytopenia when an adequate sample of platelets cannot be obtained for quantification of platelet-associated IgG.