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.     

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.     

Canine idiopathic thrombocytopenia: clinical observations and long-term follow-up in 54 cases

Idiopathic thrombocytopenia purpura (ITP) was diagnosed in 54 dogs. Bleeding was associated with platelet counts less than or equal to 30,000/mm3, and occurred most frequently at mucosal surfaces and in the skin. Other hematologic changes were variable, the most common being regenerative anemia with leukocytosis. Bone marrow examination revealed variable megakaryocyte numbers, but hyperplasia was most commonly observed. Results of the platelet factor 3 test were positive in only 7 of 25 dogs so tested. Treatment included various combinations of corticosteroid, vincristine, cyclophosphamide, and splenectomy. The dogs could be classified into 4 groups on the basis of the course of disease: (1) 14 dogs that died or were euthanatized during the first episode of thrombocytopenia, (2) 17 dogs that recovered after a single episode of thrombocytopenia (acute ITP), (3) 8 dogs in which thrombocytopenia recurred over a period of up to 8 months before recovering (acute, recurrent ITP), and (4) 15 dogs that experienced repeated episodes of ITP for periods of up to 8 years (chronic ITP).     

Flow cytometric detection of platelet-bound antibody in three horses with immune-mediated thrombocytopenia

Immune-mediated thrombocytopenia (IMT) is a sporadic cause of thrombocytopenia in horses for which it is difficult to establish a definitive diagnosis. In this report, we describe 3 horses with severe thrombocytopenia in which flow cytometric analysis of platelets for surface-bound IgG was used in an attempt to substantiate a provisional diagnosis of IMT. A distinct proportion (4.28%, 5.04%, and 7.95%) of platelets with surface-bound IgG was detected in the 3 thrombocytopenic horses, but not in 6 healthy horses (0.03% to 0.15%) or 6 horses with colic (0.00% to 1.21%). These results, in conjunction with elimination of other potential causes of the thrombocytopenia, established a diagnosis of IMT. The horses were treated with glucocorticoids alone or in combination with azathioprine, and the thrombocytopenia gradually resolved. Flow cytometric detection of platelet-bound IgG was readily performed and may be a useful adjunct for the diagnosis of IMT.     

Therapy of Immune Mediated Thrombocytopenia

Fifteen dogs with immune mediated thrombocytopenia (IMT) were studied retrospectively. All dogs had a thrombocyte count below 50,000/microliters when response to therapy was studied. Platelet counts greater than 50,000/microliters were present in all dogs within 2-9 days of initiating medical therapy. Eight dogs experienced a single episode of thrombocytopenia and seven dogs relapsed over the following 5 to 24 months. Clinical parameters from dogs that experienced a single episode of IMT were compared with data from dogs that relapsed to determine whether any information would identify dogs that were prone to relapse. Signalment, severity of thrombocytopenia, and time to achieve a platelet count above 50,000/microliters were found not to differ (P greater than 0.05) between these two groups. Five of the seven dogs with relapsing IMT were splenectomized after 2 to 4 episodes (mean, 2.8 +/- 0.8) of thrombocytopenia over 2 to 14 months. The postoperative progress of these five dogs was followed for 6 to 17 months. Platelet counts were sustained above 200,000/microliters in 4/5 after splenectomy and it was possible to discontinue medical therapy in these dogs. In comparison, the 2 relapsing IMT cases that were not splenectomized continued to require intermittent immunosuppressive therapy. We conclude that signalment and routine pretreatment laboratory test results are not useful in distinguishing dogs with relapsing IMT from those that will experience one episode of IMT. Seemingly, splenectomy is useful in the management of dogs with relapsing IMT.     

Evaluation of a manual technique for detection of neutropenia and thrombocytopenia in dogs receiving chemotherapy

OBJECTIVE: To determine accuracy of a manual technique for detection of neutropenia and thrombocytopenia in dogs receiving chemotherapy. DESIGN: Masked prospective study. ANIMALS: 11 dogs treated with chemotherapy for neoplasia. PROCEDURE: 124 blood samples from dogs being treated with chemotherapy for various neoplasms were processed through an automated cell counter, and results were compared with those obtained by use of a rapid manual technique for estimating neutrophil and platelet concentrations to determine whether the manual technique could accurately detect dogs with neutropenia or thrombocytopenia. RESULTS: By use of automated techniques, neutropenia (< 3,000 cells/microl) was detected in 17 of 124 blood samples, and thrombocytopenia (< 100,000 platelets/microl) was detected in 3 of 124 blood samples. The manual technique correctly identified 16 of 17 (94%) blood samples with neutropenia, with a specificity of 92% (98/107). The manual technique correctly identified 3 of 3 (100%) blood samples with thrombocytopenia, with specificity of 94% (114/121). CONCLUSIONS AND CLINICAL RELEVANCE: Manual estimates of neutrophil and platelet counts are sensitive and specific; however, a full differential cell count is still preferable.     

Treatment of severe immune-mediated thrombocytopenia with human IV immunoglobulin in 5 dogs

BACKGROUND: Glucocorticoids with or without other immunotherapy are the initial treatment of choice for dogs with severe immune-mediated thrombocytopenia (IMT). The majority of treated dogs will have improvements in platelet counts within 5 to 7 days of starting therapy, but complications from hemorrhage often occur before a response is seen. Human IV immunoglobulin (hIVIG) blocks Fc receptors on mononuclear phagocytic cells in dogs; it is used in people with idiopathic thrombocytopenic purpura. HYPOTHESIS: The purpose of this study was to describe adverse effects and benefit of hIVIG in addition to conventional immunosuppressive therapy in dogs with severe IMT. ANIMALS: Five client-owned dogs with severe primary IMT. METHODS: Case series. The hospital database was searched for dogs with primary IMT treated with hIVIG. RESULTS: No adverse effects were noted during or after hIVIG infusion in any treated dog. Over a 6-month follow-up, all dogs were clinically normal when using conventional immunosuppressive therapy. Human IVIG was administered 3 days after initiation of immunosuppressive therapy in 4 dogs, and, after 2 days, in 1 dog. In all dogs, the mean platelet counts pre- and 24 hours post-hIVIG infusion (0.28-0.76 g/kg) were 2,500/pL and 50,600/microL (62,750/microL for the 4 responders), respectively. One dog failed to respond as promptly to hIVIG (0.34 g/kg), and the platelet count increased to 66,000/microL after 9 days of immunosuppressive therapy. The mean duration of hospitalization post-hIVIG in all 5 dogs was 1.8 days (12 hours for responders), and the mean total length of hospitalization was 4.6 days (3.5 days for responders). Active hemorrhage resolved and no packed red blood cell transfusions were required after hIVIG infusion for responders. CONCLUSIONS AND CLINICAL IMPORTANCE: Human IVIG was well tolerated and appeared to be associated with rapid platelet count recovery and amelioration of clinical signs in most dogs with IMT.     

Application of vincristine-loaded platelet therapy in three dogs with refractory immune-mediated thrombocytopenia

Three dogs presented with refractory immune-mediated thrombocytopenia (IMT). All patients failed to respond to prednisone, which is considered a mainstay of immunosuppressive therapy. Vincristine-loaded platelets (VLPs), which act selectively on mononuclear phagocytes,were introduced. After the VLPs were transfused, two dogs responded quickly withimproved clinical signs while the third dogwith recurrent IMT was euthanized due to its deteriorating condition. This case report describesthe efficacy of VLP therapy in refractory IMT patients.     

Development of a sensitive immunoradiometric assay for detection of platelet surface-associated immunoglobulins in thrombocytopenic dogs

OBJECTIVE: To develop a direct assay to measure platelet surface-associated immunoglobulins (PSAIg) in dogs and to determine whether the assay is useful in the diagnosis of immune-mediated thrombocytopenia (IMT). ANIMALS: 20 healthy dogs were used to develop reference intervals, and 23 dogs with IMT and 17 with non-IMT were used to evaluate the clinical use of this assay. PROCEDURE: After optimization of platelet collection and assay conditions, concentrations of PSAIg were measured, using radiolabeled staphylococcal protein A (SpA) and polyclonal antibodies against canine IgG (anti-gamma) and IgM (anti-micro). Concentrations of PSAIg were expressed as the percentage of radiolabeled immunoglobulin detector bound. RESULTS: Cut-off values (mean + 3 SD) were as follows: SpA, 1.1%; anti-gamma, 1.3%; and anti-micro, 3.5%. Values greater than these cut-off values were considered positive. Values determined by use of radiolabeled SpA for all dogs with IMT were greater than the cut-off value; values were considered high positives (> 5 times cut-off value) for 22 of these 23 dogs. Although 9 of 17 dogs with non-IMT also had PSAIg concentrations greater than the cut-off value, values were considered high positives for only 3 of these 9 dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The immunoradiometric assay developed is a reliable and sensitive method to detect PSAIg in dogs. However, to obtain accurate results, optimum temperature, time, and storage conditions must be used. Detection of increased concentrations of PSAIg in dogs presumed to have non-IMT should alert clinicians to reconsider an immune-mediated basis for the thrombocytopenia.     

Canine distemper virus-induced thrombocytopenia

Effects of canine distemper virus (CDV) infection on circulating platelet values were studied in gnotobiotic dogs inoculated with R252-CDV. Thrombocytopenia (less than 200,000 platelets/microliter) was present on postinoculation day (PID) 5 and persisted through PID 15. Peak thrombocytopenia occurred on PID 10 (less than 85,000 platelets/microliter). Thrombocytopenia was accompanied by lymphopenia, neutropenia, and monocytopenia. Platelet membrane-bound CDV antigen and IgG were present from PID 7 onward; neither the third component of complement nor IgM was detected on platelets from CDV-inoculated dogs. The mean number of megakaryocytes per unit of bone marrow surface area was unchanged. Megakaryocyte infection was present in dogs euthanatized on PID 4 (0.33%), increased slightly in dogs euthanatized on PID 8 (3%), and increased sharply in dogs euthanatized on PID 9 and 10 to 17.8% and 8.3%, respectively. Phagocytosis of platelets by stellate reticuloendothelial (Kupffer's) cells in the liver was prominent in dogs euthanatized from PID 5 onward. Seemingly, CDV-induced thrombocytopenia was mediated by virus-antibody immune complexes on platelet membranes. Decreased platelet production after PID 8 resulting from direct viral infection of megakaryocytes was a likely contributing factor and occurred against a background of profound virus-induced dysfunctions of all hematopoietic cellular elements.     

Presumed primary immune-mediated thrombocytopenia in four cats

Feline primary immune-mediated thrombocytopenia (pIMT) is a rare hematological disorder. Platelet-bound antibody assays for cats have variable specificity and sensitivity and are not widely available. Diagnosis of pIMT is made on the basis of exclusion of other identifiable causes of thrombocytopenia and the response to immunosuppressive therapy. This report describes four cats with severe thrombocytopenia and no detectable underlying disease. One cat was euthanased because of pulmonary hemorrhage, while the other cats had frequent relapses, two of these cats developed diabetes mellitus due to long-term corticosteroid therapy. In these cats IMT had a chronic course and responded poorly to therapy with prednisolone. Alternative immunomodulatory drugs may be considered in the treatment of feline IMT.     

Packed red blood cell transfusions in dogs with gastrointestinal hemorrhage: 55 cases (1999-2001)

Fifty-five dogs received packed red blood cell (PRBC) transfusions for gastrointestinal (GI) hemorrhage during a 26-month period (1999 to 2001), accounting for 11.7% of the PRBC transfusions in that time. Thirty-nine (61%) dogs had an intestinal pathology (primary or secondary) as the cause of GI hemorrhage, including intestinal masses, gastroenteritis, hepatic disease, and renal disease. Nonsteroidal and steroidal anti-inflammatory drug use was found frequently in dogs with GI hemorrhage. Sixteen (39%) dogs were identified as having immune-mediated thrombocytopenia (IMT) and associated GI hemorrhage. Dogs with IMT received more transfusions of PRBC than nonIMT dogs (P<0.03) and received a significantly larger total volume of PRBC (P<0.01) during hospitalization.     

Primary immune-mediated thrombocytopenia in 30 dogs (1997-2003)

During a 6-year period, primary (idiopathic) immune-mediated thrombocytopenia was retrospectively evaluated in 30 dogs. Ages of the dogs ranged from 3 months to 10 years (median 4 years); female dogs were markedly overrepresented with 73%. Clinical examination revealed hemorrhages in 70% of the dogs. Platelet counts ranged from 0 to 111,000/microL (median 8000/microL); 77% of the dogs had platelet counts <30,000/microL. Seventeen dogs were anemic (hematocrit 9% to 36%; median 31%). Immunosuppressive therapy was performed in all but one dog. The recurrence rate of 19 dogs that were followed over an extended period (112 to 1684 days; median 340 days) was 26%. Twenty-nine (97%) dogs survived 14 days after initial presentation, and 27 (93%) dogs survived at least the following 15 to 1684 days (median 220 days).     

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.     

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.     

Comparison of platelet count recovery with use of vincristine and prednisone or prednisone alone for treatment for severe immune-mediated thrombocytopenia in dogs

OBJECTIVE: To evaluate the effect of prednisone alone, compared with a combination of prednisone and vincristine, on platelet counts in bleeding dogs with severe primary immune-mediated thrombocytopenia (IMT). DESIGN: Prospective case study. ANIMALS: 24 dogs with severe primary IMT PROCEDURE: All dogs received immunosuppressive doses of prednisone (1.5 to 2 mg/kg [0.7 to 0.9 mg/lb] of body weight, PO, q 12 h). In addition, 12 dogs received a single dose of vincristine (0.02 mg/kg [0.01 mg/lb], IV). Platelet count, transfusion requirement, and outcome were monitored. A response was defined as an increase in platelet count to > or = 40,000/microl. Dogs in the prednisone group that failed to respond received 1 dose of vincristine on day 7. RESULTS: Dogs that received prednisone and vincristine had a significantly faster increase in platelet count to > or = 40,000 platelets/microl than dogs that received prednisone alone (mean +/- SD, 4.9 +/- 1.1 vs 6.8 +/- 4.5 days, respectively). A similarly rapid response was observed in dogs that received vincristine on day 7 after treatment with prednisone alone failed. Furthermore, duration of hospitalization was reduced in the vincristine group, compared with the prednisone group (5.4 +/- 0.3 vs 7.3 +/- 0.5 days, respectively). No adverse effects attributable to vincristine were observed in any dog. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of combined vincristine and prednisone is associated with more rapid increase in platelet numbers and shortened duration of hospitalization in dogs with IMT, compared with use of prednisone alone. Early use of vincristine seems warranted in dogs with severe primary IMT.     

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.     

Hematological changes during the course of canine babesiosis caused by large Babesia in domestic dogs in Warsaw (Poland)

In the presented study we evaluated the hematological changes in samples of blood obtained from 248 dogs naturally infected with large Babesia. The evaluation included red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), leucocyte counts, thrombocyte counts, mean platelet volume (MPV), morphology of erythrocytes and leucogram. The most common disorders in affected dogs were thrombocytopenia and anisocytosis. The count of erythrocytes below reference values was detected in 26.2% of dogs and 31.4% of affected animals presented hematocrit below the reference values. Hemoglobin concentration below the reference values was noted in 29% of dogs, an increase of MCHC above normal values was detected in 21% of examinated dogs and MCV below normal values was recognized in 2% of dogs. 60.5% of dogs presented anisocytosis, 25% poikilocytosis, 23.8% polychromasia, 19.7% hypochromia and 4.4% erythroblastosis. Thrombocytopenia was detected in 99.5% of dogs, but only 15.3% of examined animals showed increase of MPV, which suggests a response of the bone marrow. 36.3% of dogs had neutropenia, and 21.8% presented a left shift, 14.9% had the lymphocytosis and 7.2% lymphopenia.     

Concurrent immune-mediated haemolytic anaemia and severe thrombocytopenia in 21 dogs

The medical records of 21 dogs with concurrent immune-mediated haemolytic anaemia (imha) and severe thrombocytopenia (defined as an automated platelet count of less than 50x10(9)/l, confirmed by the examination of a blood smear) were reviewed. Their mean (sd) age was 5.8 (2.5) years. When compared with the 24,759 dogs in the hospital population for the same period Airedale terriers and dobermanns appeared to be over-represented with odds ratios of 22.5 (95 per cent confidence interval [ci] 5.2 to 97.9) and 7.6 (95 per cent ci 1.8 to 32.7) respectively. The median duration of the dogs' clinical signs was seven days, with a range from one to 17 days. Eleven of the dogs had a history of a tendency to bleed, and 15 had evidence of bleeding when examined. Twenty of the 21 dogs had been treated with glucocorticoids, nine with vincristine, and seven with azathioprine. Their median stay in hospital was four days, with a range from one to 17 days. The median period for which they survived after admission to hospital was five days, with a range from one to 558 days, and 16 of the 21 dogs had died or been euthanased within 30 days of their admission.     

Role of platelet activating factor in development of thrombocytopenia and neutropenia in dogs with endotoxemia

OBJECTIVE: To determine the role of platelet activating factor (PAF) in lipopolysaccharide (LPS)-induced thrombocytopenia and neutropenia in dogs. ANIMALS: 42 dogs. PROCEDURES: Blood samples were obtained from dogs given LPS (40 microg/kg of body weight; n = 16), PAF (1 microg/kg; 6), PAF (5 microg/kg/h for 90 minutes; 4), or physiologic saline (0.9% NaCl) solution (0.1 ml/kg/h for 90 minutes; 3) IV to monitor changes in blood cell counts, using automated counters and blood smears stained with Giemsa. Blood samples were also obtained from dogs given LPS (40 microg/kg) that had (n = 5) or had not (6) been treated beforehand with TCV-309, a potent PAF antagonist. Concentration of PAF in blood was determined by use of 125I-radioimmunoassay in dogs given LPS at 1 mg/kg (n = 3) and 40 microg/kg (9). RESULTS: Thrombocytopenia and neutropenia were found in all dogs except those given saline solution. The LPS-induced thrombocytopenia was significantly suppressed by prior treatment with TCV-309. The PAF concentrations increased markedly 1 hour after injection of 1 mg/kg of LPS and increased slightly but significantly 10 minutes after injection of 40 microg/kg of LPS. CONCLUSION AND CLINICAL RELEVANCE: PAF plays an important role in the development of LPS-induced thrombocytopenia and neutropenia in dogs. Control of PAF production, PAF-induced effects, or both may be important in the treatment of dogs with gram-negative bacterial infections and associated thrombocytopenia and neutropenia.