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.     

Haemoplasma infection is not a common cause of canine immune-mediated haemolytic anaemia in the UK

Objective : The aim of this study was to investigate whether the two canine haemoplasma species, Mycoplasma haemocanis and “Candidatus Mycoplasma haematoparvum,” are commonly associated with immune-mediated haemolytic anaemia (IMHA) in UK dogs. Methods : Three groups of dogs were recruited to the study: anaemic dogs with primary IMHA (n=37); anaemic dogs not meeting the inclusion criteria for primary IMHA (n=77) and non-anaemic dogs (n=113). DNA was extracted from 100 μl of blood and subjected to real-time quantitative polymerase chain reaction (qPCR) assays for both species of Mycoplasma. Each assay incorporated co-amplification of canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous internal control. Results : Canine GAPDH was successfully amplified by qPCR from all 227 canine blood samples but none contained M. haemocanis or “Candidatus M. haematoparvum” DNA. Clinical Significance : Haemoplasma infection is uncommon in dogs in the UK and no evidence was found that these organisms act as triggers for IMHA.     

Vaccine-Associated Immune-Mediated Hemolytic Anemia in the Dog

Vaccination has been incriminated as a trigger of immune-mediated hemolytic anemia (IMHA) in dogs and in people, but evidence to support this association is lacking. In a controlled retrospective study, idiopathic IMHA was identified in 58 dogs over a 27–month period. When compared with a randomly selected control group of 70 dogs (presented for reasons other than IMHA) over the same period, the distribution of cases versus time since vaccination was different (P < .05). Fifteen of the dogs (26%) had been vaccinated within 1 month (mean, 13 days; median, 14 days; range, 1 to 27 days) of developing IMHA (P < .0001), whereas in the control group no marked increase in frequency of presentation was seen in the first month after vaccination. The dogs with IMHA were divided into 2 groups based on time since vaccination: the vaccine IMHA group included dogs vaccinated within 1 month of developing IMHA; the nonvaccine IMHA group included dogs that developed IMHA more than 1 month after vaccination. The recently vaccinated dogs with IMHA (vaccine IMHA group) had significantly lower platelet counts (P < .05) and a trend towards increased prevalence of intravascular hemolysis and autoagglutination when compared with the nonvaccine IMHA group. Similar mortality rates were seen in the vaccine IMHA group (60%) and the nonvaccine IMHA group (44%), with the majority of fatalities (>75%) occurring in the first 3 weeks after presentation. Persistent autoagglutination was a negative prognostic indicator for survival in both groups (P < .05). Presence of icterus and hyperbilirubinemia were negative prognostic indicators for survival in the nonvaccine IMHA group (P < .0001 and P < .01, respectively) but not in the vaccine IMHA group. In the recently vaccinated dogs, combination vaccines from various manufacturers against canine distemper, adenovirus type 2, leptospirosis, parainfluenza, and parvovirus (DHLPP) were involved in each case. Vaccines against rabies virus, Bordetella spp, coronavirus, and Lyme Borrelia were administered concomitantly to some dogs. This study provides the first clinical evidence for a temporal relationship of vaccine-associated IMHA in the dog.     

Use of thromboelastography in dogs with immune-mediated hemolytic anemia: 39 cases (2000–2008)

Objective – To analyze thromboelastograms (TEGs) of naturally occurring cases of immune-mediated hemolytic anemia (IMHA) in order to identify whether a hypercoagulable state was present and whether its presence was associated with differences in survival.
Design – Retrospective study spanning January 2000 to June 2008. Medical records of dogs were evaluated. Endpoints were considered death or discharge from the hospital.
Setting – Academic teaching hospital.
Animals – Thirty-nine dogs with a diagnosis of IMHA and at least one TEG performed during hospitalization were included.
Interventions – None.
Measurements and Main Results – Four values were evaluated from the TEG: the R time (R), K time (K), alpha angle (α), and maximum amplitude. From these values, a coagulation index (CI) was calculated to classify patients as normocoagulable, hypercoagulable, or hypocoagulable. Thirty-three of 39 patients were hypercoagulable based on the CI. The 6 remaining dogs were normocoagulable. The patients with a normocoagulable CI had an increased mortality rate (100%) when compared with the hypercoagulable patients using Fisher's exact test ( P =0.02). Additionally, prolongation of partial thromboplastin time did not preclude hypercoagulable TEG values.
Conclusions – The majority of dogs with IMHA were hypercoagulable as measured by TEG. A normal CI was associated with a worse outcome in this patient population. TEG may provide additional and complementary information to prothrombin time and partial thromboplastin time relating to coagulation status in dogs with IMHA and may help predict prognosis and potentially guide clinical decisions to utilize anticoagulant drugs.     

Serial assessment of the coagulation status of dogs with immune-mediated haemolytic anaemia using thromboelastography

This study investigated the coagulation status of dogs with immune-mediated haemolytic anaemia (IMHA) over time. Thirty animals with primary IMHA were blood sampled on three occasions over a 5 day period and assays performed included prothrombin time, activated partial thromboplastin time, D-dimer and fibrinogen concentration, antithrombin activity and recalcified unactivated thromboelastography (TEG). Based on TEG, dogs with IMHA were significantly hypercoagulable vs. controls (P<0.001) and over the 5 day period, 3/4 of the TEG parameters reflected increased clotting kinetics (P ≤ 0.02). The 30 day survival of these patients was 80% and, at hospital admission, the TEG maximum amplitude (MA) was significantly higher in survivors than non-survivors (P=0.015). Each unit increase in MA was associated with an increased odds of 30 day survival of 1.13 (95%; CI 1.02-1.25). Based on TEG, most dogs with IMHA were hypercoagulable on admission and their clotting kinetics increased with time. Relative hypocoagulability identified by TEG at initial assessment was found to be a negative prognostic indicator.     

C‐reactive protein concentration in dogs with primary immune‐mediated hemolytic anemia

Background: Canine primary immune-mediated hemolytic anemia (IMHA) is associated with a high-mortality rate. C-reactive protein (CRP) is the most important acute-phase protein in dogs and may have value as a marker of prognosis or response to treatment in IMHA. Objective: The objectives of this study were to evaluate serum CRP concentration in dogs with primary IMHA at presentation and during treatment, to assess potential differences based on survival time, and to compare CRP with other laboratory parameters of inflammation and prognosis. Methods: Inclusion criteria for primary IMHA were anemia (PCV<0.30 L/L), a positive Coombs' test or persistent autoagglutination of erythrocytes, and the exclusion of underlying diseases by other diagnostic tests. Dogs were divided into 2 groups based on survival: dogs that were still alive 14 days after start of treatment (group 1) and dogs that died or were euthanized before day 14 (group 2). Serum CRP concentration, a CBC, and a biochemistry profile were performed on days 0, 3, 8, and 14. Serum CRP also was determined in 25 clinically healthy dogs. Results: CRP concentration in the 25 clinically healthy dogs ranged from 0–8.9 μg/mL (median 2.2 μg/mL). Thirty dogs were diagnosed with primary IMHA, 24 in group 1 and 6 in group 2. On day 0, CRP concentration in dogs in both groups (median 224 μg/mL) was increased above the reference interval. In group 1 dogs, median CRP concentration was 242 μg/mL on day 0, 69 μg/mL on day 3, 35 μg/mL on day 8, and 2 μg/mL on day 14. In group 2 dogs, median CRP concentration was 194 μg/mL on day 0, 119 μg/mL on day 3, and 41 μg/mL on day 8; only 1 dog in group 2 survived to day 8. There was a significant correlation between CRP and total WBC concentrations on days 0 and 3 (r=−.598, P=.003). Conclusions: Serum CRP concentration was markedly increased in dogs with primary IMHA. CRP concentration did not differ based on patient survival, but might be a marker for long-term monitoring of these patients.     

The use of the rapid osmotic fragility test as an additional test to diagnose canine immune-mediated haemolytic anaemia

Background

Diagnosing canine immune-mediated haemolytic anaemia (IMHA) is often challenging because all currently available tests have their limitations. Dogs with IMHA often have an increased erythrocyte osmotic fragility (OF), a characteristic that is sometimes used in the diagnosis of IMHA. Since the classic osmotic fragility test (COFT) is time-consuming and requires specialized equipment, an easy and less labour-intensive rapid osmotic fragility test (ROFT) has been used in some countries, but its diagnostic value has not yet been investigated.This study aimed to evaluate erythrocyte osmotic fragility in dogs with and without IMHA, to compare results of the classic (COFT) and rapid (ROFT) test and to assess the value of the ROFT as diagnostic test for canine IMHA.Nineteen dogs with IMHA (group 1a), 21 anaemic dogs without IMHA (group 1b), 8 dogs with microcytosis (group 2), 13 hyperlipemic dogs (group 3), 10 dogs with lymphoma (group 4), 8 dogs with an infection (group 5) and 13 healthy dogs (group 6) were included.In all dogs, blood smear examination, in-saline auto-agglutination test, Coombs’ test, COFT and ROFT were performed. In the COFT, OF5, OF50 and OF90 were defined as the NaCl concentrations at which respectively 5, 50 and 90% of erythrocytes were haemolysed.

Results

Compared with healthy dogs, OF5 and OF50 were significantly higher in group 1a (P < 0.001) and OF5 was significantly higher in group 3 (P = 0.0266). The ROFT was positive in 17 dogs with IMHA, 10 hyperlipemic dogs, one anaemic dog without IMHA and one healthy dog.

Conclusions

Osmotic fragility was increased in the majority of dogs with IMHA and in dogs with hyperlipidemia, but not in dogs with microcytosis, lymphoma or an infection. Although more detailed information was obtained about the osmotic fragility by using the COFT, the COFT and ROFT gave similar results. The ROFT does not require specialized equipment, is rapid and easy to perform and can be used easily in daily practice. Although, the ROFT cannot replace other diagnostic tests, it may be a valuable additional tool to diagnose canine IMHA.     

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.     

Canine idiopathic immune-mediated haemolytic anaemia: a review with recommendations for future research

Idiopathic immune-mediated haemolytic anaemia (IMHA) is one of the most common immune-mediated diseases of dogs. The aim of this article is to review current knowledge of canine IMHA, its etiology, clinical presentation, diagnosis, complications, and treatment, in an attempt to establish why its outcome is still so poor. Clinical signs of anaemia develop within 3 days and dogs present with a median haematocrit of 13%, leucocytosis, a left shift, and reticulocytosis. Coagulation test results support the presence of disseminated intravascular coagulation. About 50% of dogs die in the first 2 weeks after presentation, and analysis of risk factors suggests that mortality is associated with hypercoagulability, inflammatory response, and liver and kidney failure. A positive direct agglutination test, spherocytosis, and true autoagglutination are widely accepted tests to demonstrate anti-erythrocyte antibodies, but are not yet standardized. To date, there is no evidence to support the efficacy of immunomodulators in addition to corticosteroids in the treatment of IMHA. Despite numerous investigations, the prognosis of IMHA remains dismal. There is an urgent need to validate and standardize diagnostic tests and criteria, and clinical trials might benefit from stratifying dogs by mortality risk. Analysis of samples from well-defined cases of canine IMHA might provide insight into the aetiology and pathophysiology of IMHA.     

ACVIM consensus statement on the diagnosis of immune‐mediated hemolytic anemia in dogs and cats

Immune‐mediated hemolytic anemia (IMHA) is an important cause of morbidity and mortality in dogs. IMHA also occurs in cats, although less commonly. IMHA is considered secondary when it can be attributed to an underlying disease, and as primary (idiopathic) if no cause is found. Eliminating diseases that cause IMHA may attenuate or stop immune‐mediated erythrocyte destruction, and adverse consequences of long‐term immunosuppressive treatment can be avoided. Infections, cancer, drugs, vaccines, and inflammatory processes may be underlying causes of IMHA. Evidence for these comorbidities has not been systematically evaluated, rendering evidence‐based decisions difficult. We identified and extracted data from studies published in the veterinary literature and developed a novel tool for evaluation of evidence quality, using it to assess study design, diagnostic criteria for IMHA, comorbidities, and causality. Succinct evidence summary statements were written, along with screening recommendations. Statements were refined by conducting 3 iterations of Delphi review with panel and task force members. Commentary was solicited from several professional bodies to maximize clinical applicability before the recommendations were submitted. The resulting document is intended to provide clinical guidelines for diagnosis of, and underlying disease screening for, IMHA in dogs and cats. These should be implemented with consideration of animal, owner, and geographical factors.     

ACVIM consensus statement on the treatment of immune‐mediated hemolytic anemia in dogs

Immune‐mediated hemolytic anemia (IMHA) causes severe anemia in dogs and is associated with considerable morbidity and mortality. Treatment with various immunosuppressive and antithrombotic drugs has been described anecdotally and in previous studies, but little consensus exists among veterinarians as to the optimal regimen to employ and maintain after diagnosis of the disease. To address this inconsistency and provide evidence‐based guidelines for treatment of IMHA in dogs, we identified and extracted data from studies published in the veterinary literature. We developed a novel tool for evaluation of evidence quality, using it to assess study design, diagnostic criteria, explanation of treatment regimens, and validity of statistical methods. In combination with our clinical experience and comparable guidelines for humans afflicted with autoimmune hemolytic anemia, we used the conclusions of this process to make a set of clinical recommendations regarding treatment of IMHA in dogs, which we refined subsequently by conducting several iterations of Delphi review. Additionally, we considered emerging treatments for IMHA in dogs and highlighted areas deserving of future research. Comments were solicited from several professional bodies to maximize clinical applicability before the recommendations were submitted for publication. The resulting document is intended to provide clinical guidelines for management of IMHA in dogs. These guidelines should be implemented pragmatically, with consideration of animal, owner, and veterinary factors that may vary among cases.     

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.     

Use of human intravenous immunoglobulin in dogs with primary immune mediated hemolytic anemia

Immune mediated hemolytic anemia (IMHA) in dogs is a severe disease with a high mortality rate. As human immunoglobulin (HIG) was reported to be beneficial for the treatment of IMHA in dogs we examined the influence of HIG on the course of the disease in our dogs with IMHA. Of 22 dogs with primary IMHA 9 dogs received in addition to routine immunosuppressive therapy HIG at a dose of 0.19 to 0.68 g/kg (median 0.35 g/kg), 13 dogs did not receive HIG (-HIG group). Both groups were similar in terms of age, weight, the presence of autoagglutination, spherocytosis, positive Coombs' test, icterus and pigmenturia. The lowest hematocrit measured during the disease was significantly lower in the +HIG group compared to the -HIG group and dogs in the +HIG group received significantly more transfusions than those of the -HIG group. This is an indication for more severe disease signs of the +HIG group dogs. Although mortality during hospitalization and the time from hospital admission to release or death was not significantly different between the two groups, we interpret this similar course of the IMHA despite more severe signs of the +HIG group dogs as a potential positive effect of the HIG therapy.     

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.     

Application of a direct flow cytometric erythrocyte immunofluorescence assay in dogs with immune-mediated hemolytic anemia and comparison to the direct antiglobulin test.

A direct flow cytometric erythrocyte immunofluorescence assay (FC) was developed and compared with the direct antiglobulin test (DAT) for detection of erythrocyte-bound immunoglobulin (IgG and IgM) and complement (C3) in dogs with immune-mediated hemolytic anemia (IMHA). Tests were performed on erythrocytes from 13 healthy nonanemic dogs and from 13 anemic dogs with IMHA. The FC and DAT were negative for erythrocyte-bound immunoglobulin in all healthy dogs. The FC was negative for erythrocyte-bound C3 in 12 healthy dogs and positive in 1 healthy dog, and the DAT was negative for C3 in all healthy dogs. Of the 13 IMHA dogs tested for erythrocyte-bound IgG, 12 were positive using the FC and 7 were positive using the DAT. Sensitivity for the detection of erythrocyte-bound IgG in the 26 dogs was 92% for FC and 53% for DAT. Specificity for detection of erythrocyte bound IgG for FC and DAT was 100%. The addition of IgM and/ or C3 did not increase the sensitivity for FC or DAT. In this group of dogs, the FC provided a more rapid, cost-effective, sensitive, objective method to quantitate erythrocyte-bound immunoglobulin and/or complement compared with the currently used DAT.     

High intravascular tissue factor expression in dogs with idiopathic immune-mediated haemolytic anaemia

A high mortality occurs in dogs with idiopathic immune-mediated haemolytic anaemia (IMHA) during the first 2 weeks after the diagnosis. The aim of this study was to investigate the inflammatory response and coagulation abnormalities in dogs with IMHA in relation to the prognosis and to establish the contribution of whole blood tissue factor (TF) and IL-8 gene expressions. Gene expressions in dogs with IMHA were compared to healthy dogs, dogs with DIC, dogs with sepsis, and in two groups of dogs that underwent intensive care treatment but had no evidence for either DIC or sepsis. The whole blood TF and IL-8 expressions were up regulated in all non-IMHA groups. Similarly, the TF expression in IMHA dogs was high, but the intravascular IL-8 expression was not increased. The dogs with IMHA had a pronounced inflammatory response that included a high WBC, left shift and monocytosis in comparison to the other disease groups. Coagulation factor activities in IMHA dogs were decreased fitting consumptive coagulopathy and the acute phase proteins FVIII and fibrinogen were increased. The platelet parameters suggested platelet activation and high platelet turnover in IMHA dogs. The model that best explained mortality contained monocytosis, increased activated partial thromboplastin time and elevated creatinine. Whole blood TF gene expression is up regulated and may contribute to consumptive coagulopathy in dogs with IMHA. Increased TF expression by activated platelets is an alternative explanation and should be investigated.     

Immune-mediated hemolytic anemia: 70 cases (1988-1996).

Survival times and mortality rates in dogs with idiopathic immune-mediated hemolytic anemia (IMHA) have been infrequently reported in the literature. This study evaluates survival and mortality in a large group of dogs with IMHA. The association of age, sex, and breed with IMHA was evaluated by comparing affected dogs to control dogs admitted to the hospital during the same time period. Treatment regimens were reviewed to determine the effects of different agents upon survival of dogs with IMHA during hospitalization and after discharge. Median survival times for each treatment group were 57 days (prednisone), 28 days (prednisone, cyclophosphamide), 974 days (prednisone, azathioprine), 15 days (prednisone, cyclophosphamide, azathioprine), and one day (no treatment). Overall mortality rate in the population of dogs studied was 70%. Twenty-nine (41.4%) dogs either died or were euthanized while hospitalized. Forty-one (59%) dogs were discharged from the hospital. Of the dogs discharged, 10 died within the first month, another five died within three months, and another five died within a year of discharge due to assumed complications of therapy or relapses of IMHA.     

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.     

Neutrophil Extracellular Traps in Plasma from Dogs with Immune‐mediated Hemolytic Anemia

Background

Neutrophil extracellular traps (NETs) are part of the innate immune response and are essential in local pathogen control, but are associated with pathological inflammation, organ damage, autoimmunity, and thrombosis. Immune‐mediated hemolytic anemia (IMHA) is a pro‐inflammatory, prothrombotic disease associated with high mortality.

Hypothesis/Objectives

Neutrophil extracellular traps (NETs) are a feature of the inflammatory process in dogs with IMHA. The objective of the study was to evaluate plasma from dogs with IMHA for the presence of 2 indirect markers and 1 direct marker of NETs.

Animals

Healthy client‐owned dogs (56) and hospitalized dogs with IMHA (n = 35).

Methods

Prospective study. Plasma samples for all dogs were evaluated for cell‐free DNA using a fluorescence assay, histone‐DNA (hisDNA) complex using an ELISA, and citrullinated histone H3 (specific for NETosis) using Western blot. Reference intervals were generated using plasma from healthy dogs.

Results

In dogs with IMHA, cell‐free DNA concentration was above the reference interval in 17% of samples with a median (range) of 1.0 μg/mL (0.1–17.3), and hisDNA concentration was above the reference interval in 94% of samples with a median (range) of 30.7 × pooled normal plasma (PNP; 0.6–372.1). Western blot for citrullinated histone H3 identified detectable bands in 84% samples from dogs with IMHA.

Conclusions and Clinical Importance

The assay for cell‐free DNA detected evidence of NETs in fewer dogs than did the other approaches. Excessive NETs appears to be a feature of IMHA in dogs and contributions to the prothrombotic state deserve further study.     

Primary immune-mediated hemolytic anemia in 19 cats: diagnosis, therapy, and outcome (1998-2004)

Immune-mediated hemolytic anemia (IMHA) occurs less frequently in cats than in dogs. The value of the Coombs' test (CT) has been questioned, but detailed surveys of its use are lacking. The objective of this study was to describe 19 cats with primary IMHA (pIMHA) and to examine the diagnostic value of the direct CT. The CT was performed in 92 cats; it was negative in 5 healthy, in 9 sick nonanemic, and in 55 cats with different types of anemia. The CT was positive in 18 anemic cats (2 feline leukemia virus (FeLV) positive, 1 with cholangiohepatitis, 15 with no underlying disease). Moreover, agglutination persisted after saline washing in 5 anemic cats (1 lymphoma, 4 pIMHA). Inclusion criteria for pIMHA were a positive CT (15) or persistent agglutination (4), and the exclusion of other diseases. The age of the 19 cats ranged from 0.5 to 9 years (median, 2 years); male cats were overrepresented. The PCV on admission was 6-22% (median, 12%). The anemia was nonregenerative in 11 cats. Additional abnormal laboratory results were leukocytosis (2), lymphocytosis (6), hyperbilirubinemia (13), hyperglobulimemia (10), and increased liver enzyme activities (10). Initial treatment consisted of blood transfusions (10), crystalloids (11), prednisolone (19), antibiotics (19), and H2-blockers (11). Four of 17 cats were euthanized 9, 63, 240 and 2,160 days after initial presentation (mortality rate, 23.5%). Relapses were reported in 5 of 16 cases (31%). Thus, pIMHA appears to occur more frequently than recognized previously, with a more favorable prognosis in cats than in dogs. The CT was useful in identifying immune-mediated pathogenesis.