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.     

Hemostatic abnormalities in dogs with primary immune-mediated hemolytic anemia

Hemostatic parameters were prospectively measured in 20 dogs with primary immune-mediated hemolytic anemia. Eight of 20 dogs had received prior treatment with prednisone. Activated partial thromboplastin time was increased in nine dogs; one-stage prothrombin time was increased in two dogs; fibrinogen concentration was increased in 17 dogs; and antithrombin activity was decreased in 10 dogs. Fibrin(ogen) degradation products concentration was increased in 12 dogs, and D-dimer concentration was increased in 16 dogs. Four or more laboratory criteria of disseminated intravascular coagulation (DIC) were present in nine dogs, and three criteria of DIC were found in four additional dogs. Thromboembolism was the most common finding in the dogs that died. In this study population, mortality was not significantly associated with any clinical finding or laboratory variable.     

Treatment of immune-mediated hemolytic anemia in dogs with cyclophosphamide

A review of 60 cases of immune-mediated hemolytic anemia (IMHA) in the dog was performed in order to characterize the disease and to identify potential prognostic indicators. Dogs ranged in age from 1 to 13 years, with a mean age of 6.5 years. The 2 most commonly affected breeds were Cocker Spaniels and Labrador Retrievers. Fifty-two of the 60 dogs tested (87%) were autoagglutination positive and spherocytes were present in 45 (75%). Forty-one (89%) of 46 patients tested positive for the presence of immunoglobulin on the red blood cell surface (Coombs assay). The most common clinical signs at presentation were lethargy, weakness, pale mucous membranes, icterus, hemoglobinuria, and anorexia. PCV less than 25% was present in 59 (98%) dogs. At the time of presentation, 35 dogs (58%) had a nonregenerative anemia, whereas 25 patients (42%) had a regenerative response. Thrombocytopenia was seen in 41 (68%) dogs. Nine of 34 dogs (26%) had a prolonged prothrombin time, 19 of 34 (56%) had a prolonged activated partial thromboplastin clotting time, and 12 of 34 (35%) had abnormal fibrinogen concentrations. All dogs received prednisone at immunosuppressive doses (2.2-4.4 mg/kg PO as a single or divided dose every 24 hours) and cyclophosphamide as primary therapy. Forty-one dogs (63%) received cyclophosphamide at 50 mg/m2 q24h for 4 days, whereas 9 dogs (15%) received an initial high dose (200 mg/m2) followed by 3 days of a lower dose (50 mg/m2 q24h). No statistical difference in survival times was found for either protocol. Thirteen dogs were treated with azathioprine in addition to cyclophosphamide and prednisone. The median survival time of dogs that received all 3 drugs was 370 days as compared to 9 days for those dogs that were treated with cyclophosphamide and prednisone alone. Thirty-one (52%) dogs died from the disease, 13 (22%) dogs were alive, and 15 (25%) dogs were lost to follow-up. The median length of survival for all dogs was 21 days. Eight dogs that were discharged from the hospital suffered a relapse (PCV < 25%).     

Hemosiderin in leukocytes of dogs with immune-mediated hemolytic anemia

Hemosiderin granules were identified in blood neutrophils and monocytes of three dogs. The brownish granules were 1 to 4 microns in diameter and stained positively for iron with Prussian blue stain. All three dogs had evidence of immune-mediated hemolytic anemia and received whole blood transfusions prior to observation of hemosiderin. The mechanism of hemosiderin accumulation by blood leukocytes from these dogs was undetermined. Iron overload produced by administration of whole blood transfusions during immune-mediated hemolytic anemia was implicated as a causative factor.     

Isotype-specific antibodies in horses and dogs with immune-mediated hemolytic anemia

Classes of antibody bound to erythrocytes were determined using direct immunofluorescence (DIF) flow cytometry in 3 horses and 12 dogs with immune-mediated hemolytic anemia (IMHA). Background levels of antibody binding were determined in samples from 12 horses and 12 dogs that were free of clinical disease. The range of nonspecific binding of a fluorescein isothiocyanate (FITC)-conjugated goat anti-equine immunoglobulin G (IgG) was 19.9–36.7%, but was eliminated by the use of the F(ab)₂ fragment of FITC-conjugated goat anti-equine IgG. Background binding by other class-specific antibodies to equine and canine erythrocytes was negligible. The DIF results were compared to the direct antiglobulin (Coombs) test in 5 horses and 20 dogs with anemia. The former assay was more sensitive in dogs with IMHA than was the Coombs' test (100% versus 58%). In contrast, the Coombs' test had better specificity than the DIF assay (100% versus 87.5%, respectively). Using clinical parameters or response to therapy as the comparison, the positive and negative predictive values for the DIF test were 92% and 100% compared to the values of the Coombs' test of 100% and 62%. The DIF assay detected low levels of cells bound with antibody (<30%) in 5 dogs that were Coombs' test-negative. For both species, performance of the DIF test was independent of the prozone effect. Five dogs with IMHA had IgG and IgM on erythrocytes, 5 had IgG, and 2 had IgM. Three horses had surface-bound IgG, including a horse with suspected penicillin-induced IMHA, a foal with neonatal isoerythrolysis, and a foal with clostridial septicemia. The DIF method was valuable in monitoring the response to therapy in the foal with neonatal isoerythrolysis.     

Effect of a single plasma transfusion on thromboembolism in 13 dogs with primary immune-mediated hemolytic anemia

Thirteen dogs with primary immune-mediated hemolytic anemia received fresh-frozen plasma within 12 hours of admission, in addition to unfractionated heparin and other therapies, such as prednisone, azathioprine, and packed red blood cell transfusion. Antithrombin activity was quantified prior to transfusion and at 30 minutes and 48 hours after transfusion. Plasma antithrombin activity did not change significantly after a single plasma transfusion. There were no deaths in the first 48 hours of treatment. Thromboembolism was identified at necropsy in six of 10 dogs that died within 12 months of admission. There was no significant difference in the incidence of thromboembolism between the current treatment group and a historical control group.     

Bee sting envenomation resulting in secondary immune-mediated hemolytic anemia in two dogs

Immune-mediated hemolytic anemia secondary to bee envenomation developed in 2 dogs. Clinical signs included lethargy, hematuria, ataxia, and seizures; 1 dog died. Clinicopathologic data included nonregenerative anemia, spherocytosis, positive results for Coombs' test, and occult hematuria. Treatment included oral administration of corticosteroids at immunosuppressive dosages and supportive care. The surviving dog initially responded to corticosteroids, but hemolysis recurred as the dosage was tapered. Hemolysis resolved with prolonged administration of corticosteroids. Bee venom contains hyaluronidase, histamines, and hemolysins that cause toxic and hemolytic effects. Envenomation should be considered in any dog with hemolytic anemia in which other causes are ruled out and exposure to bees is known.     

Perinuclear antineutrophil cytoplasmic autoantibodies in dogs infected with various vector-borne pathogens and in dogs with immune-mediated hemolytic anemia

Objective-To determine the prevalence of perinuclear antineutrophil cytoplasmic autoantibodies (pANCA) in dogs with confirmed or suspected immune-mediated hemolytic anemia (IMHA) or dogs infected with various vector-borne pathogens, including Rickettsia rickettsii, Bartonella henselae, Bartonella vinsonii subsp berkhoffii, Ehrlichia canis, Borrelia burgdorferi, and Leishmania infantum. Animals-55 dogs with confirmed or suspected IMHA, 140 dogs seroreactive for vector-borne pathogens, and 62 healthy dogs and dogs seronegative for vector-borne pathogens. Procedures-Samples were allocated to subgroups on the basis of the health status of the dogs and the degree of seroreactivity against various vector-borne pathogens. Serum samples were tested retrospectively via indirect immunofluorescence assay to determine pANCA status. Results-26 of 55 (47%) dogs with confirmed or suspected IMHA and 67 of 140 (48%) dogs seroreactive for vector-borne pathogens had positive results when tested for pANCA. Serum samples with the highest antibody concentrations against L infantum antigen had the highest proportion (28/43 [65%]) that were positive for pANCA. One of 20 (5%) dogs seronegative for tick-borne pathogens and 8 of 22 (36%) dogs seronegative for L infantum had positive results for pANCA. One of 20 (5%) healthy dogs had serum antibodies against pANCA. Conclusions and Clinical Relevance-pANCA were detected in a high percentage of dogs with IMHA and vector-borne infectious diseases. Therefore, pANCA may be a relatively nonspecific marker for dogs with inflammatory bowel disease, although they could represent a biomarker for immune-mediated diseases and infections.     

Case-control study of blood type, breed, sex, and bacteremia in dogs with immune-mediated hemolytic anemia

OBJECTIVE: To determine whether blood type, breed, or sex were risk factors for immune-mediated hemolytic anemia (IMHA) in dogs and whether bacteremia was common in dogs with IMHA. DESIGN: Case-control study. ANIMALS: 33 dogs with IMHA, 1,014 dogs without IMHA for which blood type (dog erythrocyte antigens 1.1, 1.2, 3, 4, 5, and 7) was known, 15,668 dogs without IMHA for which breed was known, and 15,589 dogs without IMHA for which sex was known. PROCEDURE: Blood type, breed, and sex distribution of dogs with IMHA were compared with data for control dogs with Fisher exact tests and by calculating odds ratios (ORs). Results of bacterial culture of blood samples were documented for dogs with IMHA, when available. RESULTS: Dog erythrocyte antigen 7 was associated with a significant protective effect (OR, 0.1) in Cocker Spaniels with IMHA (n = 10), compared with control dogs. Cocker Spaniels, Bichon Frise, Miniature Pinschers, Rough-coated Collies, and Finnish Spitz had a significantly increased risk of IMHA, as did female dogs (OR, 2.1). Blood samples from 12 dogs with IMHA were submitted for bacterial culture, and none had bacteremia. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that blood type, breed, and sex may play a role in IMHA in dogs.     

Assessment of lipid peroxidation and serum vitamin E concentration in dogs with immune-mediated hemolytic anemia

OBJECTIVE: To determine plasma malondialdehyde (MDA) and serum vitamin E concentrations in dogs with immune-mediated hemolytic anemia (IMHA) and healthy control dogs. SAMPLE POPULATION: Serum and plasma samples from 36 dogs with IMHA and 40 healthy control dogs. PROCEDURE: Blood samples were collected from all study dogs. Plasma MDA concentrations were measured by use of a commercial colorimetric assay, and serum vitamin E concentrations (alpha-, gamma, and delta-tocopherol concentrations) were measured via high-performance liquid chromatography. RESULTS: Plasma MDA concentrations were significantly higher in the dogs with IMHA than in the control dogs. Compared with control dogs, serum alpha-, gamma-, and &tocopherol concentrations were significantly lower in the IMHA-affected dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated a state of oxidative stress and reduced antioxidant reserve in dogs with IMHA; this finding provides support for further investigation of the potential benefits of antioxidant treatment in dogs with this disease.     

Idiopathic immune-mediated hemolytic anemia: treatment outcome and prognostic factors in 149 dogs

BACKGROUND: Canine idiopathic immune-mediated hemolytic anemia (IMHA) is associated with a high mortality, especially in the 1st 2 weeks after diagnosis despite treatment. OBJECTIVES: To determine treatment outcome and identify prognostic variables in order to define areas of future research. ANIMALS: One hundred forty-nine dogs with hematocrit <30% and either a positive Coombs' test or spherocytosis and with no evidence of disease that can trigger IMHA were included. METHODS: Retrospective cohort study. All dogs were treated with prednisolone and azathioprine according to a standard protocol. Survival analysis was performed by the Kaplan-Meier method. Variables recorded at the time of diagnosis were tested as possible prognostic variables in a univariate and multivariate Cox proportional hazard model. RESULTS: The main predictors for mortality in dogs with idiopathic IMHA are the presence of increased plasma urea concentration, bands, thrombocytopenia, and petechiae at the time of diagnosis. The estimated Kaplan-Meier half-year survival was 72.6% (95% confidence interval [CI]: 64.9-81.3%). Mortality occurred mostly within the 1st 2 weeks. Cox proportional hazards analysis indicated that increased plasma urea concentration, icterus, and petechiae were the major independent predictors of mortality in the 1st 2 weeks. In most dogs that survived IMHA, a 3-month protocol of azathioprine with prednisolone maintained clinical remission. The estimated half-year survival for dogs that survived the 1st 2 weeks was 92.5% (95% CI: 86-99.3%). CONCLUSIONS AND CLINICAL IMPORTANCE: If the dogs survived IMHA, a 3-month protocol of prednisolone and azathioprine was effective with regard to survival and clinical outcome. Future research should be directed at identifying whether thrombotic tendency in dogs with IMHA is the main contributor to the development of increased plasma urea concentration, icterus, thrombocytopenia, and petechiae.     

Equine immune-mediated hemolytic anemia associated with Clostridium perfringens infection

Background: Previous studies have suggested an association between equine immune-mediated hemolytic anemia and clostridial infections or neck abscesses.
Objective: The purpose of this report was to describe and characterize the hematologic abnormalities in a horse with Clostridium -associated immune-mediated hemolytic anemia. We also retrospectively evaluated hematologic abnormalities in 8 horses with clostridial myositis or subcutaneous emphysema.
Methods: A 7-year-old Foreign Warm-Blood gelding was evaluated for anemia and a cervical abscess. CBCs and reticulocyte counts were obtained using an Advia 120 analyzer and evaluation of Wright's-stained smears. All cases of equine Clostridium spp. myositis or subcutaneous emphysema over a 7-year period were identified in a retrospective search of the University of Minnesota Veterinary Teaching Hospital database. Clinical, hematologic, and microbiological findings were recorded.
Results: Clostridium perfringens genotype A was isolated from the neck wound of the gelding. The CBC was characterized by severe regenerative anemia, intravascular hemolysis, and RBC agglutination. A direct Coombs' test was positive. Moderate numbers of spheroechinocytes were observed. The total automated reticulocyte count was 4.5% (56,700/μL), with most reticulocytes having low absorbance (mature reticulocytes). The anemia responded to penicillin and steroidal and nonsteroidal immunosuppressive drugs. Of 8 horses with myositis, all of which involved the cervical region, 5 were anemic, 1 had a positive direct Coombs' test, and 2 had increased numbers of type III echinocytes and spheroechinocytes.
Conclusions: The presence of type III echinocytes or spheroechinocytes may be helpful in diagnosing immune-mediated hemolytic anemia associated with clostridial infections in horses. Automated reticulocyte counts may detect very low levels of reticulocytosis in hemolytic anemia in horses.     

Prognostic factors for mortality and thromboembolism in canine immune-mediated hemolytic anemia: a retrospective study of 72 dogs

Medical records of 72 dogs diagnosed with immune-mediated hemolytic anemia (IMHA) were reviewed to find risk factors for the disease, for mortality, and for thromboembolism. Coagulation data of 32 patients were evaluated for mortality or thromboembolism risk factors. Cocker Spaniels were at increased risk for IMHA (P = .012). Timing of vaccination was not associated with development of IMHA. PCV ranged from 5 to 33%, with a mean of 16 +/- 5%. Autoagglutination was present in 42% of the dogs. Platelet counts (n = 60) varied from 3,000 to 793,000/microL (mean, 160,117 +/- 133,571; median, 144,000). Thrombocytopenia (platelet count, <200,000/microL) was present in 70% of the dogs, with severe thrombocytopenia (platelet count, <50,000/microL) being present in 22%. One-step prothrombin time (OSPT) was prolonged in 28% of the dogs tested, and activated partial thromboplastin time (APTT) was prolonged in 47% of the dogs tested. Fibrin(ogen) degradation products (FDPs) were detected in 16 of 28 dogs tested (57%). Disseminated intravascular coagulation (DIC) was diagnosed in 10 of 31 (32%) dogs and was suspected in 8 dogs. Thromboemboli were found in 20 of 25 dogs given postmortem examinations. Mortality rate was 58%. Thrombocytopenia (P = .008) and serum bilirubin concentration of >5 mg/dL (P = .015) were risk factors for mortality, and hypoalbuminemia approached significance (P = .053). Severe thrombocytopenia (P = .046), serum bilirubin concentration of >5 mg/dL (P = .038), and hypoalbuminemia (P = .016) were risk factors for thromboembolism. On evaluation of continuous data, decreased platelet count (P = .057), increased bilirubin (P = .062), and decreased albumin (P = .054) approached significance for decreased survival. A higher risk for thrombosis was found with increased alkaline phosphatase (ALKP) (P = .042), increased bilirubin (P = .047), and decreased albumin (P = .012).     

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.