An enzyme-linked antiglobulin test for the detection of erythrocyte-bound antibodies in canine autoimmune haemolytic anaemia

A method has been developed which allows identification and quantitation of red cell-bound immunoglobulins and complement in canine blood. The technique utilizes ELISA methodology and the assay identifies cases of autoimmune haemolytic anaemia which are negative by the Direct Coombs test. Further, the amount of antibody present on the red cells shows a close correlation to the haemoglobin level; suggesting that the degree of sensitization of the RBCs influences their rate of destruction. Favourable response to treatment correlates with a decrease in the levels of bound antibody and complement.     

Serum cardiac troponin I in dogs with primary immune-mediated haemolytic anaemia

Objectives : The assessment of serum cardiac troponin I concentrations in dogs with a range of nonprimary cardiac illnesses has revealed that cardiac myocyte damage is commonplace in many canine diseases. Whilst it is well established that dogs with fatal immune‐mediated haemolytic anaemia frequently have cardiac pathology based on post‐mortem examinations, there is limited information on the incidence of cardiac myocyte damage in this population of dogs. Methods : Serum cardiac troponin I concentrations were measured in 11 healthy dogs, 27 dogs with primary haemolytic anaemia and 49 hospitalised dogs without primary cardiac or haematological disorders. Results : Dogs with primary haemolytic anaemia have higher serum concentrations of cardiac troponin I than hospitalised ill dogs (P<0.005) and healthy dogs (P<0.01). Using a cut‐off of less than 0.1 ng/mL, 20 of 27 dogs with primary haemolytic anaemia had increased serum cardiac troponin I concentrations, which was a significantly higher proportion compared to the hospitalised ill dogs (P<0.001, 16 out of 49 dogs) and healthy dogs (P<0.05, 3 out of 11 dogs). Clinical Significance : Dogs with primary haemolytic anaemia have a higher incidence of subclinical myocyte damage than healthy dogs or dogs with non‐haematological or primary cardiac illnesses. The prognostic significance of increased serum cardiac troponin I concentrations in dogs with primary haemolytic anaemia merits further investigation.     

Haemolytic anaemia as a complication following colic surgery in a 10‐year‐old Arabian stallion

Immune‐mediated haemolytic anaemia is rare in the horse. This case report discusses the clinical presentation and treatment of this condition in an Arabian stallion following abdominal surgery for large colon volvulus. On Day 10 of hospitalisation the stallion's PCV dropped by 10% whilst his total solids remained stable, consistent with haemolytic anaemia. The stallion was treated with whole blood transfusions and dexamethasone and recovered to be discharged on Day 42. This is an interesting example of immune mediated haemolytic anaemia associated with a complicated gastrointestinal condition. It demonstrates the wide variety of potential aetiologies underlying a haemolytic episode and the necessity for expedient treatment.     

Septic funiculitis caused by Streptococcus equi subspecies equi infection with associated immune‐mediated haemolytic anaemia

A 2‐year‐old Quarter Horse gelding presented for anaemia, icterus, depression and intermittent colic 2 weeks after routine castration. Bilateral septic funiculitis with Streptoccocus equi ssp. equi with secondary immune‐mediated haemolytic anaemia were diagnosed. A blood transfusion was required to facilitate general anaesthesia for surgical excision of the septic funiculitis. Antibiotic therapy was provided initially with chloramphenicol and later enrofloxacin. Immunosuppressive therapy was provided with dexamethasone and later azathioprine. The horse responded well to treatment and was discharged 8 weeks after presentation. Streptococcus equi ssp. equi should be considered in cases with septic funiculitis and the potential for a secondary immune‐mediated haemolytic anaemia exists with this bacterial species.     

Investigation into factors influencing performance of the canine antiglobulin test

Antiglobulin (Coombs') reagents were assessed for their ability to detect immunoglobulin and complement attached to red cells. Polyspecific and monospecific reagents were prepared using a number of immunisation protocols. Performance of these antisera against control red cells was compared, in a direct Coombs' test, with samples from cases of canine autoimmune haemolytic anaemia (AIHA). A combined reagent containing two monospecific antisera (anti-IgG + anti-C3) gave optimum results. Positive control red cells were required to standardise canine Coombs' reagents for the laboratory diagnosis of AIHA. The optimum incubation temperature for the canine Coombs' test was shown to be 37 degrees C.     

Coombs’, haemoplasma and retrovirus testing in feline anaemia

Objective : To investigate the associations between Coombs’ testing, haemoplasma and retroviral infections, and feline anaemia. Methods : Haematology, Coombs’ testing (including assessment of persistent autoagglutination) and selected infection testing (haemoplasma, feline leukaemia virus/feline immunodeficiency virus provirus) were performed in blood samples collected from 60 anaemic and 60 non-anaemic cats. Results : No association between infection and anaemia or Coombs’ positivity existed. Anaemic cats (21.7%) were significantly more likely than non-anaemic cats (0%) to have cold autoagglutination (P<0.0001), but significance (set at ≤0.0025 due to multiple testing) was not quite reached when Coombs’ positivity was compared between anaemic (40.4% and 21.7% positive at 4°C and 37°C, respectively) and non-anaemic (20% and 3.3% positive, P=0.021 and P=0.004, at 4°C and 37°C, respectively) cats. Cats with immune-mediated haemolytic anaemia were significantly more likely to have persistent cold autoagglutination (P<0.0001) and be Coombs’ positive at 37°C with polyvalent (P<0.0001), immunoglobulin (Ig)G (P<0.0001) or any antiserum (P<0.0001). Haemoplasmas and retroviruses were uncommonly detected. Clinical Significance : Cats suspected of having immune-mediated haemolytic anaemia should be evaluated for persistent autoagglutination at 4°C as well as performing Coombs’ testing at 37°C, but positive results may occur in with other forms of anaemia. Testing for erythrocyte-bound antibodies should always be interpreted in parallel with documentation of haemolysis in anaemic cats.     

Auto-immune haemolytic anaemia in a horse

A case of auto-immune haemolytic anaemia (AHA) in a 7-year-old, pony gelding is described. Diagnosis was based on a strong, positive reaction to the direct Coombs antiglobulin test and the presence of erythrophagocytosis and spherocytosis in peripheral blood smears. The condition was complicated by an infected haematoma, posterior paresis and urinary incontinence. The triggering mechanism behind the antibody response against the red cells is unknown.     

Flow cytometric evaluation of bound IgG on erythrocytes of anaemic dogs

The detection of red blood cell (RBC)-bound immunoglobulins in case of anaemia with the direct agglutination test (DAT or Coombs test) has been reported to be of low sensitivity. We therefore tested the applicability of flow cytometry for the detection of canine IgG on RBC using two different IgG-specific secondary reagents: goat-anti-dog IgG (GalphaD-IgG) and rabbit-anti-dog IgG (RalphaD-IgG). Membrane staining RBC samples were performed at 4 degrees C. Comparisons of agglutination test at 37 degrees C and 4 degrees C showed, that binding of the secondary antibodies at 4 degrees C was more sensitive compared to agglutination at 37 degrees C and the two antisera differed considerable in their agglutination activity. Binding of GalphaD-IgG and RalphaD-IgG to RBC of healthy dogs (n=15) was low and mean fluorescence intensities were taken to calculate thresholds above which RBC of patients were judged positive. As in agglutination tests, both secondary antisera displayed considerable differences (concentration-dependent binding and histogram profiles) after flow cytometric analysis. Using flow cytometry, with GalphaD-IgG 8 of 17 agglutination-negative patients were positive and RalphaD-IgG was positive with 3 of 3 agglutination-negative RBC samples. Thus, flow cytometric analysis of proved to be a sensitive technique, detecting RBC-bound canine IgG of DAT-negative patients. The results of both techniques, however, are significantly influenced by the used IgG-specific polyclonal reagents.     

Haemolytic anaemia and bilateral uveitis associated with leptospirosis in a 6‐year‐old Quarter Horse gelding

A 6‐year‐old Quarter Horse gelding was presented for bilateral uveitis resulting in vision loss as well as icterus. Anaemia with autoagglutination was consistent with a presumptive immune‐mediated haemolytic anaemia. Urinary PCR was positive for Leptospira spp. and microscopic agglutination test (MAT) titres were elevated to multiple serovars supportive of a diagnosis of leptospirosis. Treatments included broad spectrum antibiotics and aggressive anti‐inflammatory medications. While the horse was hospitalised, the development of bilateral corneal ulcers precluded the use of topical ophthalmic anti‐inflammatories for a number of days. The corneal ulceration resolved, vision returned in both eyes and the immune‐mediated haemolytic anaemia resolved. After 9 days of hospitalisation, oral minocycline was administered for 2 weeks at home as well as low dose oral flunixin meglumine and topical ophthalmic diclofenac and atropine. This case represents the first published case of haemolytic anaemia associated with leptospirosis in a horse.     

Comparison of three monoclonal and three polyclonal antibodies in the immunohistochemical diagnosis of canine autoimmune skin diseases

The aim of this study was to evaluate the utility of three monoclonal antibodies (mAbs), and two anticanine IgG and one anticanine IgM polyclonal antibodies (pAbs) for the immunohistochemical diagnosis of canine autoimmune skin diseases. Skin biopsies from 11 cases of pemphigus (7 foliaceus, 3 vulgaris and 1 erythematosus), 12 cases of discoid lupus erythematosus (DLE) and 12 cases of chronic hyperplasic dermatitis were used. The CA4E7 mAb (IgG1 + IgG2) showed similar sensitivity, but higher specificity and lower background than the two anti-IgG pAbs for the immunohistochemical diagnosis of pemphigus and DLE. The CA4F1 mAb (IgG2) and CA3H1 mAb (IgG2) showed moderate and low interepithelial reactivity, respectively, in autoimmune skin diseases, but strong staining of the cytoplasm of plasma cells of the inflammatory infiltrates. These results suggest that the CA4E7 mAb may be valuable in the immunohistochemical diagnosis of such disorders.     

Comparative functional characterization of canine IgG subclasses

To date, very little is known about the functional characteristics of the four published canine IgG subclasses. It is not clear how each subclass engages the immune system via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC), or how long each antibody may last in serum. Such information is critical for understanding canine immunology and for the discovery of canine therapeutic monoclonal antibodies. Through both in vitro and ex vivo experiments to evaluate canine Fc's for effector function, complement binding, FcRn binding, and ADCC, we are now able to categorize canine subclasses by function. The subclasses share functional properties with the four human IgG subclasses and are reported herein with their function-based human analog. Canine Fc fusions, canine chimeras, and caninized antibodies were characterized. Canine subclasses A and D appear effector-function negative while subclasses B and C bind canine Fc gamma receptors and are positive for ADCC. All canine subclasses bind the neonatal Fc receptor except subclass C. By understanding canine IgGs in this way, we can apply what is known of human immunology toward translational and veterinary medicine. Thus, this body of work lays the foundation for evaluating canine IgG subclasses for therapeutic antibody development and builds upon the fundamental scholarship of canine immunology.     

A latex test for canine rheumatoid factor

Canine rheumatoid factor (RF) has been reported in several canine diseases, particularly in arthritis. Although RF can be assayed using IgG sensitized erythrocytes, the test has a number of disadvantages. As an alternative, latex sensitized with canine IgG was investigated as an assay of canine RF. The canine IgG-latex could be easily produced, was stable, and could be standardized with commercial antisera. The reagent detected RF of the IgM anti-canine-aggregated-IgG type. A comparison of the titres obtained using the canine IgG-latex reagent with those obtained using a rabbit IgG-erythrocyte reagent showed no correlation, suggesting that the two assays may detect RF of different specificities.     

Immune-based non-erosive inflammatory joint disease of the dog. 1. Canine systemic lupus erythematosus

The features of 13 dogs with systemic lupus erythematosus (SLE) are described. Canine SLE is a multisystemic disease characterized by autoimmunity and immune complex hypersensitivity. The presence of antinuclear antibody in the blood is an important diagnostic feature. All 13 cases had a non-erosive symmetrical polyarthritis. Autoimmune haemolytic anaemia was seen in five cases, thrombocytopenia in three, skin lesions in four; neurological involvement in one; and gastrointestinal signs in one. Treatment was with cytotoxic drugs(cyclophosphamide initially) and prednisolone.     

Cutaneous Alternaria infectoria infection in a dog in association with therapeutic immunosuppression for the management of immune‐mediated haemolytic anaemia

A 4‐year‐old, ovariohysterectomized, English springer spaniel on immunosuppressive therapy was re‐examined for the review of its immune‐mediated haemolytic anaemia and the recent development of skin lesions. For the 3 months since hospital discharge, the dog had been receiving 1.3 mg/kg prednisolone and 2.6 mg/kg ciclosporin, both administered orally twice daily. Physical examination revealed hepatomegaly and multiple, purulent, crusting, erosive to ulcerative lesions over different body areas. Onychorrhexis had occurred on one digit and the underlying corium had blackened. There were two proliferative and one plaque‐like lesions in the mouth. Thick walled fungal hyphae were detected in impression smears from all skin lesions and staining with periodic acid–Schiff’s stain confirmed the presence of multiple fungal hyphae and spores in all biopsies examined. Fungal culture isolated a heavy, pure growth of an Alternaria sp. which was identified as A. infectoria by sequencing the internal transcribed spacer 1 region of the rRNA gene. The animal’s condition prevented detailed investigation of the oral lesions. Withdrawal of the ciclosporin and reduction of the prednisolone dosage resulted in spontaneous resolution of the skin lesions within 40 days. Further gradual decrements in the prednisolone dosage to zero were carried out without recurrence of the immune‐mediated haemolytic anaemia. After 12 months, there has been no recurrence of either the skin lesions or the anaemia. To the authors’ knowledge, this is the first reported case of A. infectoria infection in a dog.     

Evaluation of platelet activation in canine immune-mediated haemolytic anaemia

Objectives : To establish whether heightened platelet activation is a common feature of canine immune-mediated haemolytic anaemia, and to evaluate the hypothesis that platelet activation plays a role in the pathogenesis of thromboembolism. Methods : Using whole-blood flow-cytometric analysis, the proportion of activated platelets and platelet-leucocyte aggregates in blood samples from 14 dogs with immune-mediated haemolytic anaemia and 14 healthy dogs was calculated. General linear models with binomial errors were used to compare groups. Results from the immune-mediated haemolytic anaemia-affected dogs were then correlated with established risk factors for thromboembolism in canine immune-mediated haemolytic anaemia, D-dimer concentration and antithrombin activity. Results : There was a strong correlation between platelet activation and severe thrombocytopenia, with heightened platelet activation being observed predominantly in severely thrombocytopenic dogs. Clinical Significance : Dogs with immune-mediated haemolytic anaemia, particularly those with concurrent severe thrombocytopenia, are likely to have heightened platelet activation, which may play a role in the pathogenesis of thromboembolism.     

Heinz body haemolytic anaemia in a dog secondary to ingestion of a zinc toy: a case report

A 6-year-old Labrador retriever was referred for investigation of severe lethargy and suspected immune-mediated haemolytic anaemia. Clinical examination revealed pale mucous membranes and jaundice. Haematology demonstrated large numbers of Heinz bodies and a marked anaemia, which was strongly regenerative. Serum zinc concentrations were markedly elevated. Analysis of a metal toy vomited by the dog 3 days prior to presentation revealed it to be composed of almost pure zinc. A diagnosis of haemolytic anaemia secondary to acute zinc toxicity was made and supportive therapy instigated. There was a subsequent decrease in numbers of Heinz bodies and a rise in the haematocrit, and the dog made an uneventful recovery. Acute zinc toxicity resulting in haemolytic anaemia is rarely observed, and this case was also unusual in that the main clinicopathological finding was the presence of numerous Heinz bodies without other evidence of oxidative damage to red blood cells.     

Cerebral haemorrhage in a pregnant Standardbred mare with Evan's syndrome

A 3‐year‐old pregnant Standardbred mare was treated at the University of Helsinki Equine Teaching Hospital for severe idiopathic immune‐mediated thrombocytopenia and haemolytic anaemia (Evan's syndrome). Despite initial improvement with supportive care and immunosuppressive doses of corticosteroids and azathioprine, the mare died from a fatal cerebral haemorrhage on Day 21 of treatment.     

Canine Dal blood type: A red cell antigen lacking in some Dalmatians

BACKGROUND: Based upon alloantibodies produced after sensitizing dogs with transfused blood, more than a dozen blood group systems have been recognized thus far, and some have been classified as dog erythrocyte antigens (DEA). HYPOTHESIS: A new canine red cell antigen was suspected, based on the development of specific alloantibodies in a Dalmatian previously sensitized by blood transfusions. ANIMALS: Twenty-six Dalmatians (including 1 Dalmatian in need of blood compatibility studies); 55 canine blood donors. METHODS: Serologic tests, including blood typing, crossmatching, and direct Coombs' test were performed by standard tube techniques and a novel gel column technology adapted from human blood banking. RESULTS: By day 40 after transfusion of an anemic Dalmatian, all major crossmatch tests to 55 non-Dalmatian dogs were incompatible. The 2 initial donors, who were compatible before transfusion, were also now incompatible, suggesting the development of an alloantibody to a common red cell antigen. No siblings were available, but 4 of 25 unrelated Dalmatians were crossmatch compatible, suggesting that they were missing the same red cell antigen. The patient was blood typed DEA 1.1, 3, 4, and 5 positive, but DEA 7 negative. Further blood typing and crossmatching results did not support an association to any of these known blood types. The alloantibodies produced were determined to be of the immunoglobulin G class. CONCLUSIONS AND CLINICAL IMPORTANCE: Based upon the identification of an acquired alloantibody in a Dalmatian, a presumably new common blood type named Dal was identified. Dalmatians lacking the Dal antigen are likely at risk of delayed and acute hemolytic transfusion reactions.     

Canine heterophilic antibodies as a source of false-positive B-type natriuretic peptide sandwich ELISA results

BACKGROUND: Interference by heterophilic antibodies is a well-known cause of false-positive sandwich ELISA results in human medicine. They are considered rarely in veterinary species and have not been characterized but could become important as newer, highly sensitive sandwich immunoassay technologies are developed. OBJECTIVES: The goals of this study were to use a B-type natriuretic peptide (BNP-32) sandwich ELISA to determine the effect of heterophilic antibodies on test performance; to characterize canine heterophilic antibodies; and to develop and test a method for heterophilic antibody removal. METHODS: A sandwich ELISA was developed using a mouse IgG(1)K monoclonal and a rabbit polyclonal antibody to two synthetic peptides of canine BNP-32. The effects on false-positive results of heterophilic antibody depletion and blocking by various techniques were compared. The titers of canine heterophilic antibodies were compared with various blood antigens from other species and the relative amount of canine IgG was compared with that of IgM heterophilic antibody. RESULTS: Heterophilic antibodies in dog plasma were shown to be capable of causing false-positive ELISA results. They reacted with blood proteins from a variety of animal species at relatively low titers and consisted of both IgG and IgM. Protein A agarose antibody precipitation, in conjunction with mouse IgG(1)K blocking antibody, was effective in eliminating false-positive sandwich ELISA results while retaining adequate test performance. CONCLUSIONS: Canine heterophilic antibodies can interfere with sandwich ELISA assays and cause false-positive test results. An effective technique for their removal that has a potentially broad application was developed, and allows measurement of canine blood constituents at low picomolar concentrations.     

Pattern of Coombs' test reactivity has diagnostic significance in dogs with immune-mediated haemolytic anaemia

OBJECTIVES: To investigate the clinical significance of the pattern of Coombs' test reactivity in dogs with immune-mediated haemolytic anaemia. METHODS: Sixty-five anaemic dogs with a positive Coombs' test were included. Coombs' testing was performed at 4 and 37 degrees C with polyvalent canine Coombs' reagent and antisera specific for each of canine immunoglobulin G, immunoglobulin M and complement factor C3. The impact of performing testing with only polyvalent antiserum at 37 degrees C was assessed. Chi-squared tests were used to compare Coombs' test reactivity in dogs with primary immune-mediated haemolytic anaemia (group A) and in dogs with concurrent/underlying disease (group B). Following Bonferroni correction, significance was set at P < or = 0.003. RESULTS: Eleven dogs would have been regarded as Coombs' negative had they been tested with polyvalent antiserum at 37 degrees C alone. Group A dogs were significantly more likely to be positive with polyvalent antiserum and/or anti-dog immunoglobulin G at 4 and/or 37 degrees C (P < or = 0.001) and tended to be less likely to be positive with anti-dog immunoglobulin M at 4 degrees C (P=0.040). CLINICAL SIGNIFICANCE: Testing of anaemic dogs with polyvalent Coombs' reagent at 37 degrees C was less sensitive than testing with monovalent reagents at 4 and 37 degrees C. The pattern of Coombs' test reactivity differed significantly between dogs with primary immune-mediated haemolytic anaemia and those with concurrent/underlying disease.