Use of a direct enzyme-linked antiglobulin test for laboratory diagnosis of immune-mediated hemolytic anemia in dogs.

Detection of autoantibody, complement, or both bound to RBC is an essential requirement for unequivocal diagnosis of immune-mediated hemolytic anemia in dogs. An enzyme-linked antiglobulin test was adapted for laboratory diagnosis of this disease. The refinement and routine use of this assay have allowed further observation of the pathogenesis of the disease process. In particular, degree of hemolysis can be related to the degree of RBC sensitization associated with primary immune-mediated hemolytic anemia, and this correlation is highest for IgG autoantibody. Results indicate that autoantibody isotype might have an important role in the hemolytic process.     

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.     

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.     

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.     

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.     

Penicillin-induced immune-mediated hemolytic anemia in a horse.

A 5-year-old female American Quarter Horse was determined to have immune-mediated hemolytic anemia after detection of a positive response to a direct Coombs' test. Penicillin-induced immune-mediated hemolytic anemia was confirmed via a direct antiglobulin test, using penicillin-coated RBC. The horse was clinically improved and the anemia resolved in response to supportive care and discontinuation of penicillin treatment. Penicillin should be considered a possible cause of immune-mediated hemolytic anemia in horses.     

Idiopathic immune-mediated hemolytic anemia in a calf.

Severe anemia was found in a 4-month-old heifer, which was admitted with a 1-day history of anorexia, signs of depression, and recumbency. A diagnosis of immune-mediated hemolytic anemia (IHA) was made on the basis of a Coomb's titer of 1:128 and decreased resistance to osmotic stress, as determined by an RBC fragility test. Anaplasmosis and leptospirosis were ruled out as possible causes of the IHA. Other causes of hemolytic anemia, including intoxication by copper, water, Brassica spp, or drugs were ruled out. Therefore the IHA was considered idiopathic. Treatment consisted of supportive therapy, oxytetracycline, and dexamethasone. After 60 days of treatment, CBC, Coomb's test result, and RBC fragility were within normal limits.     

A prospective study of clopidogrel therapy in dogs with primary immune-mediated hemolytic anemia

Background: A major cause of death in dogs with primary immune‐mediated hemolytic anemia (pIMHA) is thrombotic disease. Ultralow‐dose aspirin (ULDA) is commonly used to prevent thrombosis in dogs with pIMHA; however, the efficacy of antiplatelet agents in dogs with pIMHA is unknown. Hypothesis: The use of clopidogrel (CL), alone or in combination with ULDA, would improve survival to discharge and at 90 days without important adverse effects compared with ULDA alone in dogs with pIMHA treated with standard immunosuppressive therapy. Animals: Twenty‐four client‐owned dogs with pIMHA. Methods: Prospective, positive‐controlled, unmasked clinical trial with dogs randomized in 3 treatment groups to receive PO ULDA or CL or both. Results: There was no identifiable adverse reaction, evidence of hemorrhage, or increase in transfusion requirements associated with CL therapy, either alone or combined with ULDA, compared with ULDA alone. There was no significant difference between treatment groups with respect to survival to discharge and at 90 days. Conclusions and Clinical Importance: This study suggests that CL therapy, alone or in combination with ULDA, was safe and had similar short‐term survival compared with ULDA alone in a small group of dogs with pIMHA able to tolerate oral medications and treated with standard immunosuppressive treatment.     

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.     

Evaluation of a standard immunofluorescence assay and a new flow cytometric method for the detection of autoreactive antibodies in dogs with tumours

The major purpose of the presented study was to develop and to evaluate a flow cytometry-based assay (IIFC) for the determination of autoreactive antibodies in sera from canine cancer patients. A blinded study demonstrated the poor reproducibility of the standard, slide-based and microscopically evaluated indirect immunofluorescence test (IIF), especially with sera displaying a cytoplasmic reactivity. In the IIFC, the intra assay coefficient of variance ranged between 5% and 11%, the inter assay variance between 8% and 25%. The IIFC resulted in significantly less positive results among canine cancer patients (16%) than the IIF (40%). The latter results were due to low titered sera indicating that the standard assay may lead to a high proportion of false positive results. The limitation of the IIFC is that no conclusions can be made about the sub cellular localization of the fluorescence. However, this cytometry-based assay makes a more objective and standardized detection of canine autoreactive antibodies possible.     

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.     

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.     

Use of human immunoglobulin in addition to glucocorticoids for the initial treatment of dogs with immune-mediated hemolytic anemia

Objective – To determine the utility of human intravenous immunoglobulin (hIVIG) for the initial treatment of canine immune-mediated hemolytic anemia (IMHA).
Design – Blinded, randomized, clinical trial.
Setting – Veterinary teaching hospital.
Animals – Twenty-eight, client-owned dogs with primary IMHA.
Interventions – At enrollment, after diagnosis of IMHA, dogs were randomly assigned to receive either hIVIG or placebo, in a blinded fashion. For the next 14 days, all dogs received glucocorticoids as the sole immunosuppressant agent. All dogs received low-molecular-weight heparin as an anticoagulant. D-dimer concentrations were evaluated at the beginning and end of the study protocol to monitor for thromboembolic complications.
Measurements and Main Results – Twenty-five of 28 dogs (89%) were discharged from the hospital. Thirteen of those received hIVIG and 12 received placebo. Twenty-four dogs (86%) were alive 14 days after enrollment, and of these 13 received hIVIG and 11 received placebo. D-dimer concentrations were elevated in 86% of all dogs at the time of diagnosis.
Conclusions – For initial treatment of dogs with IMHA, the addition of hIVIG to corticosteroid treatment did not improve initial response, nor did it shorten hospitalization.