BVSc (Hons) MMedVet (Med) PhD Dipl. ECVIM (Internal Medicine)
Bryanston Veterinary Hospital
PO Box 67092, Bryanston, 2021, South Africa
Immune-mediated haemolytic anaemia (IMHA) is characterized by the immune destruction of erythrocytes that have been coated with immunoglobulins, complement, or both, resulting in direct destruction or phagocytosis and removal from systemic circulation. Antibodies may be directed against the normal erythrocyte (primary or idiopathic) or against erythrocytes that have been antigenically altered through interaction with secondary causes. The antibodies are directed against a protein or glycoprotein that is a normal component of the erythrocyte membrane. The term autoimmune disease implies a primary immune response or intolerance to a self-antigen, whereas, immune-directed diseases have either a primary or underlying disease (secondary) responsible for the immune reaction. Thus IMHA more accurately describing a process by which erythrocytes are destroyed and has replaced the term autoimmune haemolytic anaemia.
Immune-mediated haemolytic anaemia is a common and often fatal haemolytic disorder in the dog; whereas it is uncommon in the cat. It is, thus, important that causes for IMHA be aggressively looked for and treated before a diagnosis of primary IMHA is made. A genetic predisposition is evident in dogs such as the cocker spaniel, Springer spaniel, Old English sheepdog, Irish setter, poodle, and dachshund; whereas no breed, gender, or other association has been found in cats. Female dogs also appear predisposed, even when spayed.
Regardless of the underlying cause, IMHA results from a breakdown in immune self-tolerance and arises from development of IgG or IgM antibodies directed against the erythrocyte. The degree of erythrocyte lysis depends on the type and amount of antibody that binds to the erythrocyte membrane and the involvement of complement fixation.
When the IgG molecule attaches to the erythrocyte membrane it is phagocytosed by macrophages possessing multiple receptors for the constant fragment (Fc) portion of the IgG molecule and as these macrophages are found primarily in the spleen, active erythrophagocytosis results in splenomegaly. As the disease progresses immunoglobulin production increases and erythrophagocytosis then occurs within the liver resulting in hepatomegaly.
Once the immunoglobulin is attached, activation of the complement system occurs with binding of complement proteins to the erythrocyte membrane. Complement activation causes immediate intravascular lysis or enhances extravascular lysis. CI, a serine protease from the liver, enters the complement cascade and generates a “membrane attack complex” that attaches to the erythrocyte membrane causing “holes to be punched” in the erythrocyte membrane, allowing an influx of water and electrolytes, cell swelling, and lysis. The membrane complex can also be removed by macrophages within the spleen (partial erythrophagocytosis), producing spherocytes.
Historically, in most dogs with IMHA, no underlying condition was ever identified, and thus it was considered to be primary or idiopathic IMHA However, in later studies, which probably reflected more intense clinical investigations, an underlying disease process or trigger could be identified, including drug exposures, emerging infections, bee stings, neoplastic processes, and other immune disorders. Best exemplified in babesiosis and haemobartonellosis, haemolysis is severely exaggerated by immune processes. Many chronic infections including abscesses, discospondylitis, pyometra, and pyelonephritis can induce secondary IMHA. A temporal association between vaccination and onset of IMHA has also been suggested. In a limited retrospective study, 25% of dogs with IMHA of unknown cause were vaccinated within 1 month of onset of clinical signs. As this correlation was associated with modified and killed vaccines against common infectious diseases from different manufacturers, it appears likely that vaccines may trigger or enhance a smouldering immune process rather than be the underlying cause. The association of IMHA with other immune disorders, including hypothyroidism and immune-mediated thrombocytopenia (ITP), lends support to the hypothesis of a general immune disturbance. In contrast to humans, IMHA in dogs is often associated with inflammation and necrosis, suggesting again an underlying mechanism to trigger an immune response.
Causative factors that can be associated with secondary IMHA in the dog include the following:
- Bacterial endocarditis
- Chronic bacterial infections
- Systemic fungal infections
- Intestinal helminths
- Solid tumours
- Endogenous oestrogen
- Blood transfusions
- Snake bites
- Immune disorders:
- Systemic lupus erythromatosis
- Primary and secondary immunodeficiency
In the cat the majority of cases of IMHA are secondary and are associated with feline leukaemia virus infection, lympho-proliferative disease, haemoplasma infections (formerly Haemobartonella), or drug reactions.
IMHA may present at any age but is most commonly encountered in young adult to middle-aged dogs. Often the clinical history is brief and vague and occasionally an underlying condition may be identified. A brief episode of vomiting or diarrhoea may precede the typical signs of anaemia (lethargy, weakness, exercise intolerance, pallor) and haemolysis (pigmenturia, icterus). Some animals may be febrile, presumably because of erythrocyte lysis or an underlying infectious or inflammatory disease process. Others develop dyspnoea, indicating pulmonary problems either as cause of the underlying disease or as a thromboembolic complication of IMHA. Clinical examination may also reveal mild splenomegaly and, less commonly, mild hepatomegaly and lymphadenopathy, which again suggest a secondary cause of IMHA. A normal-sized spleen does not rule out a haemolytic process. Furthermore, signs attributable to their underlying disease may predominate, whereas chronic or recurrent signs of IMHA suggest a primary form.
ROUTINE LABORATORY TESTING
IMHA can be a mild to life threatening condition. The haematocrit may drop precipitously at any time because of active haemolysis. Although a regenerative, macrocytic-hypochromic anaemia would be expected, as many as one third of animals with IMHA have a non-regenerative anaemia when first examined. This can be attributed to a peracute disease course for which there has not been time for a regenerative response to occur. Alternatively, antibodies may be directed against the erythroid precursors, thereby removing metarubricytes and reticulocytes, or the IMHA disease process may change the microenvironment of the bone marrow and thereby impair erythropoiesis. Evidence of ineffective erythropoiesis and erythrophagocytosis may be found on bone marrow cytology. Auto-agglutination of erythrocytes and spherocytosis are typical findings on a blood smear examination.
Besides erythroid abnormalities, a leukocytosis is often present, mostly because of a mature neutrophilia, but degenerative left shifts have also been observed. As high leukocyte counts are not generally encountered with other forms of anaemia, this probably reflects a unique cytokine-mediated response to inflammation and necroses specific for IMHA, but concomitant infection and steroid-induced leukocytosis should also be considered. Furthermore, thrombocytopenia related to a concomitant ITP (Evans’ syndrome) or other consumptive processes, such as with disseminated intravascular coagulation (DIC), may occur. Dogs with IMHA often have coagulation test abnormalities with suggesting a DIC like syndrome.
Typical findings on urine analysis are haemoglobinuria and bilirubinuria. There may also be evidence of a bacterial cystitis, which may indicate an underlying infectious disease or may occur secondarily because of immune deregulation or immunosuppressive therapy. Serum biochemistry may show haemoglobinaemia and hyperbilirubinaemia, and the higher the serum bilirubin concentration the poorer the prognosis. Liver enzyme activities and serum urea are often elevated.
DIAGNOSTIC LABORATORY TESTING
A diagnosis of IMHA requires demonstration of accelerated immune destruction of erythrocytes. Thus besides documenting a haemolytic anaemia, a search for antibodies or complement or both directed against erythrocytes is required. To reach a definitive diagnosis of IMHA one or more of the following three hallmarks has to be present: marked spherocytosis, true auto-agglutination, or a positive Coombs’ test.
Spherocytes are spherical erythrocytes that appear microcytic with no central pallor that result from either partial phagocytosis or lysis. Such cells are rigid and extremely fragile in the erythrocyte osmotic fragility test as they cannot expand in hypotonic solution. Large numbers of spherocytes are highly suggestive of IMHA and are present in approximately two thirds of dogs with IMHA, but small numbers may be seen with hypophosphataemia, zinc intoxication, and microangiopathic haemolysis. Hereditary spherocytosis related to various membrane defects in humans has only been reported in one dog. In cats, spherocytes are difficult, if not impossible, to identify owing to the small size and lack of central pallor of normal feline erythrocytes.
IgM and, in large quantities, IgG antibodies may cause direct auto-agglutination. Agglutination may be macroscopic when blood is in an EDTA tube or placed on a glass slide or it maybe microscopic as small clumps of erythrocytes on a stained blood smear or in a saline wet mount. Auto-agglutination has to be distinguished from rouleaux formation, in which erythrocytes stack up on top of each other.
The direct Coombs’ test is used to detect antibodies and/or complement on the surface of the erythrocyte. The presence of antibodies on erythrocytes, together with species-specific antiglobulins against IgG, IgM, and C3b, allow antibody bridging and thereby agglutination or lysis of coated erythrocytes (or both). Most dogs are IgG positive, some are IgG and IgM positive, and a few are only IgM positive. To reach a definitive diagnosis of IMHA, the direct Coombs’ test should be positive, but this does not discriminate between primary and secondary IMHA.
A small proportion of dogs may have IMHA despite a negative Coombs’ test result. False-negative Coombs’ test results may occur because of insufficient quantities of bound antibodies on erythrocytes and for many technical reasons such as inappropriate reagents or dilutions. Negative results are also obtained for animals in which the disease is in remission; however, a few days of immunosuppressive therapy is unlikely to cause a false-negative result. Some treated animals have a positive Coombs’ test long after the IMHA resolves. A false-positive Coombs’ test is rare and be seen after an incompatible transfusion or because of technical problems. Some animals may have a positive Coombs’ test result without and evidence of haemolysis and anaemia, which has been observed in healthy human blood donors.
Following a diagnosis of IMHA it is important to differentiate primary from secondary as this will impact on the long-term outcome. A detailed history remains the cornerstone for an accurate diagnosis. The animal may have been recently vaccinated, travelled to tick/parasite-infested regions, or given antibiotics. After the initial haematology, serum biochemistry and confirmatory tests for IMHA, further evaluation should include thoracic and abdominal radiographs and abdominal ultrasound. Evaluation of the bone marrow may be also needed, especially if the animal’s anaemia is non-regenerative, a destruction process directed at red cell precursors is suspected, or another cytopenia is present. Bone marrow aspirates may be helpful in diagnosing red blood cell aplasia or hypoplasia, neoplastic infiltration, or immune destruction of erythrocyte precursors.
In summary a full investigation into the aetiology of an animal with IMHA would include a combination of the following:
- Clinical examination.
- Urine and faecal analyses.
- Serum biochemistry.
- Urine and blood culture.
- Serology and/or PCR (leptospirosis, toxoplasmosis, FeLV, FIV, Ehrlichia).
- Survey thoracic radiographs.
- Abdominal ultrasonography.
- Bone marrow aspirate/biopsy.
In both primary and secondary IMHA the erythrocyte destruction needs to be aggressively treated with immunosuppressive therapy such as corticosteroids, azathioprine, cyclophosphamide, cyclosporine, danazol, intravenous immunoglobulins, micophenolate, and leflunonamide. The main goal with immune-suppressive therapy is to control the immune response by reducing phagocytosis, complement activation, and anti-erythrocytic antibody production. If effective there should be an initial stabilisation of the haematocrit, followed by the normalisation of the haematocrit over a period of time. Supportive care includes anticoagulant therapy, gastric protectants, fluid therapy, and blood transfusions.
Because the severity of IMHA ranges from an indolent to a life-threatening disease, therapy has to be tailored for each animal and depends, in part, on whether the IMHA is primary or secondary. Removal of the triggering agent or treatment of the underlying condition can bring the IMHA under control. Thus if the IMHA is thought to be secondary to an infection, treatment with antiprotozoals, antirickettsials, or antibiotics should be instituted. Because of the potential for underlying occult infection and the predisposition to infection related to the immune deregulation associated with IMHA and immunosuppressive therapy, antibiotic therapy is generally indicated. In addition, surgical correction of abscesses or other infections may be considered. Nonessential drugs, particularly those that might cause an immune reaction, should immediately be withdrawn. Despite these interventions, transfusion and immunosuppressive therapy are probably still required in the initial control of secondary IMHA.
Rehydration of the severely ill animal is pivotal to improve organ perfusion even when it lowers the haematocrit. If hypoxia due to severe anaemia and a rapidly dropping haematocrit to critical levels ensues, blood transfusions are beneficial. The increased oxygen-carrying capacity provided by transfused cells may be sufficient to maintain the animal for the few days required for other treatment modalities to become effective. The notion that transfusions are especially hazardous in animals with IMHA has been overemphasized and is not supported by studies in both dogs and humans. This is because the anti-erythrocytic antibody in IMHA is not an alloantibody and thus the destruction of transfused cells is no higher than that of autologous erythrocytes.
Oxygen inhalation therapy is of little benefit unless the animal is suffering from pulmonary disease such as pulmonary thromboemboli.
Corticosteroids are the initial treatment of choice for IMHA. They interfere with both the expression and function of macrophage Fc receptors and thereby immediately impair the clearance of antibody-coated erythrocytes by the macrophage system. In addition, corticosteroids may reduce the degree of antibody binding and complement activation on erythrocytes and, after weeks, diminish the production of auto-antibodies. Prednisolone (1-2 mg/kg bid) is the mainstay treatment. Dexamethasone can be used but is probably not more beneficial. A response reflected by a stabilized or even rising haematocrit, appropriate reticulocytosis, and less auto-agglutination and spherocytes can be expected within days. Once improvement occurs the initial dose is then tapered by reducing the amount by approximately 25% every 7-14 days. Generally the faster the response to treatment the more rapidly the tapering is done. Within weeks to months, a low-dose alternate-day therapy may be reached with minimal steroid side effects. In secondary IMHA with appropriate control of the underlying disease, the tapering can be accomplished more rapidly.
Other immunosuppressive therapy is warranted when prednisolone fails, controls the disease only at persistently high doses, or causes unacceptable side effects. Cytotoxic drugs are usually given with prednisolone but may eventually be used independently. These cytotoxic drugs inhibit lymphocytes and thereby suppress the anti-erythrocyte antibody production only after weeks. They are therefore probably not effective in the acute management of IMHA but may have a place in the long-term control of refractory and relapsing cases.
Cyclophosphamide (2mg/kg oid) has been advocated in cases of fulminant IMHA, a randomized clinical trial did not show any beneficial effects of cyclophosphamide in the acute management of IMHA. Cyclosporine (5 mg/kg oid) is very beneficial in controlling the immune response in dogs with IMHA. Leflunomide and mycophenolate are in a class of agents similar to that of cyclosporine and have been used with some success. Danazol (5mg/kg oid), an androgen derivative, may inhibit binding of antibodies and phagocytosis. However, a retrospective study in dogs with IMHA was disappointing. Intravenous human immunoglobulin (IVIG) may be helpful in the short-term treatment of dogs with IMHA. IVIG can block Fc receptors on macrophages, which reduce Fc-mediated phagocytosis of IgG-coated erythrocytes, interfere with complement action, and suppress antibody production. A single IVIG dose of 0.25 to 1 g/kg has been beneficial in some refractory cases as indicated by a rising haematocrit and reticulocytosis within days, but the response has often been only temporary.
Splenectomy may be considered in cases that do not respond to immunosuppressive therapy, that require long-term high-dose therapy to remain in remission, or that have intractable drug-induced side effects. The spleen is a major site of autoantibody production, as well as sequestration and destruction of erythrocytes coated with IgG, but probably does not directly affect the clearance of IgM-coated cells. In addition, histological examination of the spleen may provide evidence of an underlying disease. A risk exists of developing overwhelming infections after splenectomy. Splenectomy should not be considered in animals receiving immunosuppressive therapy aside from prednisolone.
Thromboembolic and DIC-like syndromes are serious complications of IMHA that greatly contribute to morbidity and mortality. Although the pathogenesis remains unknown, venipuncture, catheters, and corticosteroid therapy represent potential predisposing conditions. Predisposing factors should, whenever possible, be limited. Adequate perfusion and oxygenation of tissue should be provided with fluids and transfusions. Anticoagulant therapy is instituted only after some evidence or suspicion of thromboembolism exists. Heparin at a dose of 5 to 250 IU/kg subcutaneously every 6 hours or by continuous infusion is the most commonly used drug.
Despite appropriate therapy mortality rate remains high, ranging from 20% to 75%. Negative prognostic indicators are a rapid drop in haematocrit, highly elevated serum bilirubin concentrations, non-regenerative anaemia, intravascular haemolysis, persistent auto-agglutination, and thromboembolic complications.