Michael J. Day, BSc, BVMS(Hons), PhD, FASM, DECVP, MRC Path, FRCVS
Antibody and/or complement-mediated destruction of circulating red blood cells (RBC), platelets or neutrophils is known as immune-mediated haemolytic anaemia (IMHA), immune-mediated thrombocytopenia (IMTP), or immune-mediated neutropenia (IMNP) respectively. In some animals, all three conditions may occur concurrently. Occasionally the bone marrow precursors of these cells are targeted instead of (or in addition to) the circulating cells. The immunological destruction of the RBC, platelets or neutrophils occurs by extravascular phagocytosis, or by intravascular osmotic lysis following the generation of terminal membrane attack complexes of the complement pathway.
The diagnostic labels IMHA, IMTP and IMNP in fact cover a wide spectrum of clinical disease. The basic distinction is between primary and secondary disease. In primary IMHA, IMTP or IMNP no underlying cause for the presence of cell-associated antibody can be identified on thorough clinical and laboratory investigation. Primary IMHA, IMTP or IMNP may therefore be considered as a true idiopathic, autoimmune disease and the diagnostic labels autoimmune haemolytic anaemia (AIHA), autoimmune thrombocytopenia (AITP), or autoimmune neutropenia (AINP) applied. AIHA and AITP may occur concurrently (Evans' syndrome) or may be part of a multisystemic autoimmune syndrome (e.g., systemic lupus erythematosus). By contrast, in secondary IMHA, IMTP or IMNP the presence of erythrocyte-or platelet-associated antibody is secondary to an underlying cause--most commonly neoplastic or infectious disease. Although these disorders are recognised in both the dog and cat, this lecture will be restricted to discussion of the canine diseases. IMHA and IMTP are relatively well documented in the dog1,2,3, but the literature concerning IMNP is very limited. The recent increasing reports of canine IMNP suggest that the disease may actually be more common than currently appreciated.4,5,6
In primary, idiopathic AIHA, AITP or AINP the specificity of the autoantibodies is for an autoantigen--in this context a protein or glycoprotein that is a normal component of the erythrocyte, platelet or neutrophil cell membrane. The target membrane antigens in canine AIHA have been defined, and include the anion exchange molecule (band 3), the cytoskeletal molecule spectrin, and a series of membrane glycoproteins (glycophorins)7. The target membrane antigens in canine AITP are the platelet membrane glycoproteins GPIIb and GP111a8. No studies have yet documented the specificity of anti-neutrophil antibodies in the dog.
The production of autoantibodies involves the same series of stages that characterise the synthesis of any immunoglobulin. The key event in the initiation of the autoimmune response is the activation of the autoreactive T lymphocyte. Although the majority of self reactive T cells are deleted during development in the thymus, a proportion escapes this control point and is released into the circulation. Normal humans and animals (including dogs) have circulating T lymphocytes with specificity for erythrocyte, platelet or neutrophil-derived autoantigenic peptides, however these cells are normally kept in check so that autoimmunity does not develop.
A high proportion of dogs with IMHA or IMTP are believed to have true primary, idiopathic AIHA or AITP respectively. Published studies of dogs with thrombocytopenia suggest that up to 70% of cases may have an autoimmune aetiology. Further evidence for the existence of primary AIHA or AITP in the dog comes from the strong breed and familial associations that are documented. Pedigrees of cocker spaniel and old English sheepdogs have been extensively studied.
There have been too few studies of canine AINP to derive prevalence data or suggest any breed predisposition. Autoimmunity is multifactorial in nature, and may require a combination of predisposing factors (e.g., stress, oestrus, whelping, infection), acting on an appropriate genetic background, to upset the normal homeostatic regulation of autoreactive T cells and induce clinical autoimmune disease.
SECONDARY IMMUNE-MEDIATED DISEASE
By current clinical definitions, many animals with IMHA, IMTP or IMNP have primary, idiopathic autoimmune disease that responds to immunosuppressive therapy. By contrast, there are a proportion of cases in which the immune-mediated blood dyscrasia is clearly secondary to an underlying cause, particularly:
1. Neoplasia (e.g., lymphoma or haemangiosarcoma with concurrent IMHA or IMTP)
2. Chronic inflammatory disease (e.g., inflammatory bowel disease with concurrent IMTP)
3. Exposure to an infectious agent (e.g., distemper virus, Dirofilaria, Angiostrongylus, arthropod-borne microparasites such as Babesia, Ehrlichia, Anaplasma, Leishmania, Rickettsia)
4. Recent administration of a vaccine (polyvalent, modified-live, adjuvanted vaccines are generally incriminated)9
5. Drug therapy, the best example of which involves administration of sulphonamide-trimethoprim to Doberman pinschers or a range of other dog breeds10
The diagnostic approach to cases of IMHA or IMTP has not changed in many years. Initial clinical and historical assessments are supported by haematological analysis and perhaps bone marrow assessment in nonregenerative forms of disease. The identification of erythrocyte bound antibody is based on the Coombs' or direct antiglobulin test which remains the mainstay for definitive diagnosis and is widely available in a commercial setting. Although other ELISA-based tests for IMHA have been developed, they are more complicated than is required for routine clinical diagnosis. More recently, a flow cytometric method for detection of erythrocyte-bound antibody has been applied to canine samples11. This test has greater sensitivity, but less specificity, than the Coombs' test. There have also been significant advances in diagnostic testing for the presence of platelet bound antibody. Both direct (using patient platelets) and indirect (using patient serum and substrate platelets) testing by flow cytometry has been validated, although direct testing is the more sensitive method. Unfortunately this is not routinely available outside specialist referral centres, and confirming a diagnosis of IMTP remains problematic. Although anti-neutrophil antibody has been demonstrated in animals with IMNP, the methodology is not widely available, so the diagnosis of IMNP remains one of exclusion.
The increasing availability of sophisticated laboratory diagnostic tests will mean more ready identification of animals with secondary IMHA, IMTP or IMNP. For example, animals with Coombs' positive anaemia, IMTP or IMNP may be readily screened for microparasitic diseases by PCR using a peripheral blood sample. Advances in molecular diagnosis may mean that further underlying causes of immune-mediated blood dyscrasias (e.g., subclinical lymphoma) may be more readily identified in the future.
The therapeutic approach to IMHA, IMTP and IMNP is similar. For those cases in which the blood dyscrasia is secondary to a defined underlying cause, appropriate therapy for the primary disease must be administered. Adjunct therapy for the anaemic or thrombocytopenic state may also be considered, particularly transfusion of whole blood, packed erythrocytes or platelet rich plasma. Administration of heparin may be considered for those animals with IMHA at risk from pulmonary thromboembolism.12
The specific therapy for these disorders still involves administration of immunosuppressive doses of glucocorticoids, with major effect of downregulating Fc receptor expression by phagocytic cells. A range of other drugs (e.g., danazol, azathioprine, cyclophosphamide, cyclosporin A) has been co-administered with glucocorticoids in various regimes to reduce the likelihood of glucocorticoid side effects ('steroid sparing') or have additional effects on the lymphoid cells that initiate the disease. Despite the widespread use of such protocols, there is no clear consensus as to which (if any) combination is most efficacious, and which individual cases are the most likely candidates for combined therapy. In fact, some studies suggest that the use of drugs such as cyclophosphamide or cyclosporin A have no added value, or result in a less successful clinical outcome than use of glucocorticoid alone.13,14 Vincristine has been used in dogs with severe IMTP as it enhances thrombopoiesis in addition to decreasing platelet phagocytosis via effects on macrophages. Blockade of Fc receptors by administration of human gammaglobulin has proven useful in both diseases, but the expense and availability of this product generally precludes its widespread application15.
IMHA and IMTP may have severe clinical presentation and a proportion of dogs will die despite therapy. IMHA with evidence of intravascular haemolysis, autoagglutination, targeting of bone marrow precursors or development of pulmonary thromboembolism carries a guarded prognosis. Death from IMTP is most likely to occur secondary to extensive gastrointestinal haemorrhage. Those animals that recover from the initial disease episode are at risk for relapse, often months or years later. Immunosuppressive therapy should be tapered slowly and maintained for at least 3-6 months, and some dogs may require continual maintenance therapy. Careful monitoring should be performed, most simply by haematological analysis, for at least 6 months after an episode of disease16. The few documented cases of IMNP for which therapy and followup have been discussed would appear to have had a favourable outcome. There is a relatively rapid rebound of circulating neutrophil counts after initiation of glucocorticoid therapy in dogs with primary IMNP6.
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