Michael J. Day, BSc, BVMS(Hons), PhD, DSc, DECVP, FASM, FRCPath, FRCVS
School of Clinical Veterinary Science University of Bristol, Langford, United Kingdom
Immune-Mediated Haemolytic Anaemia (IMHA)
IMHA is one of the most common immune-mediated disorders of the dog and cat. By definition, IMHA involves haemolysis of erythrocytes that is mediated by the action of antibody and complement. Haemolysis may be intravascular when complement fixation progresses to formation of the terminal membrane attack complex with osmotic lysis of the target red cell, or extravascular when IgG and complement C3b coated erythrocytes are removed from the circulation by splenic or hepatic macrophages bearing Fc and C3b receptors. IMHA might be a primary idiopathic (or autoimmune) event in the absence of recognized underlying disease or the presence of trigger factors. Autoimmune haemolytic anaemia (AIHA) is often breed related and associated with the inheritance of specific alleles of genes of the Major Histocompatibility Complex (MHC) in the dog. By contrast, many cases of IMHA are secondary to underlying infectious, inflammatory or neoplastic diseases, or to the administration of drugs or vaccines. In most cases of IMHA the immune system targets circulating erythrocytes, but in some animals the immune attack is focused upon bone marrow erythroid precursors leading to a non-regenerative IMHA (NRIMHA) or pure red cell aplasia (PRCA).
The diagnosis of IMHA is becoming an increasingly complex event as an increasing range of potential triggers must be identified. This presentation reviews the diagnostic approach to IMHA in the dog and cat.
Initial clinical and historical assessments are supported by haematological analysis and bone marrow assessment in non-regenerative forms of disease. Haematological analysis is the most important diagnostic procedure, and key features of the haemogram consistent with IMHA include:
Strongly regenerative, severe haemolytic anaemia (except in cases of NRIMHA/PRCA or peracute onset disease). The reticulocyte count should be determined. In cats the distinction between aggregate and punctate reticulocytes should be made, with release of aggregate reticulocytes indicative of active regeneration. An aggregate reticulocyte count above 50 x 109/l is considered indicative of active regeneration in cats.
Autoagglutination of erythrocytes macro- or microscopically. Macroscopic agglutination should be further investigated by the 'in-saline agglutination test' which will distinguish true agglutinates from rouleaux. This simple, in-house procedure involves placing a drop of EDTA anticoagulated blood onto a microscope slide and adding a drop of saline. Only agglutinates will persist when the blood is so diluted. In cats, additional saline dilution may be required compared with dogs.
Spherocytosis (in dogs, remembering that most laboratories will not attempt to define these cells in cats).
Left shift neutrophilia. This may sometimes be profound and require differentiation from myeloproliferative disease. The major reason for this change is suggested to relate to centrilobular hepatocyte degeneration and necrosis.
Concurrent immune-mediated thrombocytopenia (IMTP) in Evans' syndrome. Concurrent immune-mediated neutropenia (IMNP) may also occur, and various combinations of the three cytopenias are recognized.
Recent studies have also shown a high prevalence of haemostatic abnormalities in dogs with IMHA and suggested that these may underlie the predisposition to development of pulmonary thromboembolism.
The Coombs Test
The identification of erythrocyte-bound antibody and/or complement is based on the Coombs or direct antiglobulin test (DAT) which remains the mainstay for definitive diagnosis and is widely available in a commercial setting, although not all laboratories offer complete testing using multiple antisera that are fully titrated at different incubation temperatures. The Coombs test involves the demonstration of antibody and/or complement on the surface of patient red blood cells, by incubation of a washed red cell suspension with antisera that will cause gross agglutination. The polyvalent Coombs reagent detects all immunoreactants, whilst specific antisera (to IgG, IgM and complement C3) may be used in parallel to further define the nature of the immune reaction. Our most recent study has reported that testing with a panel of reagents is more likely to give a positive diagnosis than testing with polyvalent reagent alone. Moreover, there are distinct differences in the pattern of test reactivity between dogs with primary and secondary disease. Dogs with primary disease are more likely to have IgG antibody and less likely to have IgM antibody compared with dogs having secondary IMHA.
Although other ELISA-based tests for IMHA have been developed, they are more complicated than is required for routine clinical diagnosis. In some countries the osmotic fragility test is favoured as an adjunct diagnostic test. More recently, a flow cytometric method for detection of erythrocyte-bound antibody has been applied to canine samples. This test has greater sensitivity, but less specificity, than the Coombs test and depends on the availability of a flow cytometer. The latest innovation is the development of rapid, in-house, tube-based tests for both dog and cat. These simply determine a positive/negative result using polyvalent antiserum but appear to work well in validation studies conducted in my laboratory. However, in order to use these tests it is necessary to purchase a specific centrifuge from the manufacturer.
It is often stated that the Coombs test is invalid in cats. This suggestion is based on the results of a single, rather old and poorly validated study in which it was reported that 8/20 clinically normal cats tests had a low-titred (2) positive Coombs test using IgM antiserum. In fact, the Coombs test may be performed for cats in exactly the same way as for dogs (although anti-complement reagents are not available for this species) and provide the same useful diagnostic information. We have recently undertaken a large validation study showing good correlation between the feline Coombs test and haemolytic anaemia.
Adjunct Laboratory Testing
The increasing availability of sophisticated laboratory diagnostic tests will mean more ready identification of animals with secondary IMHA. For example, animals with Coombs positive anaemia may be readily screened for microparasitic diseases by PCR using a peripheral blood sample and these methods are far more sensitive than attempts to detect circulating parasitemia by examination of a blood smear. A range of infections are associated with the development of IMHA and many of these involve arthropod transmitted agents (e.g., Babesia, Cytauxzoon, Ehrlichia, Leishmania and Mycoplasma). Testing for FeLV and FIV should be routine in anaemic cats and any cat with haemolytic anaemia should be tested for the three species of feline haemoplasma (Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum and Candidatus Mycoplasma turicensis) by PCR.
Advances in molecular diagnosis may mean that further underlying causes of immune-mediated blood dyscrasias (e.g., subclinical lymphoma by clonality testing) may be more readily identified in the future. Although not widely available, if there is concurrent thrombocytopenia or neutropenia flow cytometric testing has been developed for the detection of platelet-or neutrophil-bound antibodies in such patients. Some animals with immune-mediated cytopenia will have positive serum antinuclear antibody (ANA) and if the cytopenia is part of a multisystemic disease process testing for ANA titre is recommended.
References are available upon request.