Immune-mediated hemolytic anemia (IMHA) is a common cause of anemia in dogs and a primary idiopathic or autoimmune form of the disease is considered to be the most frequent form of IMHA in this species. Anemia may be severe and recurrent, with a mortality rate up to 70% according to certain publications. Diagnostic trail, appropriate treatment and potential prognosis depend on an understanding of the underlying mechanisms of the disease.

Immunopathogenesis

Immune-mediated hemolytic anemia is a type II immune reaction in which circulating red blood cells' (RBC) destruction is antibody-mediated (cytotoxic). Antibody attachment to erythrocyte surface depends on two main causes:

 In the autoimmune form of the disease, antibodies (auto-antibodies) recognize a self-antigen of the erythrocyte membrane. Autoimmune hemolytic anemia (AIHA) may occur as a single clinical entity (idiopathic AIHA), may be recognized concurrently with autoimmune thrombocytopenia (Evans' syndrome), or may be part of a multisystemic autoimmune disease like systemic lupus erythematosus (SLE). The development of autoimmunity results from a failure of the normal control mechanisms of the immune system. Autoimmune diseases are multifactorial disorders in which clinical expression relies on the presence of an optimum array of predisposing factors. Genetic factors are key determinants of disease susceptibility that explain breed or familial predispositions. Other predisposing factors must be important: hormonal background, age, environmental factors (infectious agents, drugs and chemicals, etc.). The aim in the treatment of AIHA as with all autoimmune diseases is to manipulate (to down regulate or suppress) the immune response that causes the disease so that the process is reduced or abolished.

 In secondary IMHA, antibodies have a specificity for a foreign antigen (an infectious agent or a drug) that is associated with the RBC surface, or for a neo-antigen, which is an RBC determinant modified by a drug, an infectious agent or secondary to a neoplastic phenomenon. Immune-complexes can also be adsorbed at the RBC surface. In this case, RBC destruction is due to bystander hemolysis as the causative antibody is not specific to the normal RBC. The prognosis of secondary IMHA is more closely related to the underlying disorder than the hemolytic anemia. Therapy should be directed to the control of this disease, and the management of the hemolytic process itself.

In literature, the use of the subsequent denominations: idiopathic, primary, autoimmune or secondary IMHA is sometimes confusing:

 Primary or idiopathic IMHA is used when there is no underlying disease or evidence of recent drug administration and is frequently used as a synonym of autoimmune while the presence of true autoantibodies is not verified.

 Secondary IMHA is used when an underlying disease is present (or drug administration), even if anemia is due to true autoantibodies, like in systemic autoimmune disease like SLE, in some neoplasia (especially lymphoproliferative diseases), or after the administration of some drugs (such as α-methyldopa in humans).

Antibody attachment to cell membranes triggers RBC destruction by a number of different mechanisms. This is influenced by many factors including the nature of the antibody itself (IgM, IgG and IgG subclasses), the concentration of antigen sites, complement, and macrophage activity. Cell destruction may be intravascular or extravascular:

 In the presence of a potent complement-fixing antibody and a large number of antigen sites, the complement cascade may proceed to its full amplification with the formation of the destructive terminal attack complex, with resulting intravascular cell lysis.

 More frequently, the surface immunoglobulin and complement interact with immunoglobulin heavy chain (Fc) and complement receptors expressed by macrophages. The macrophages attempt to remove the abnormal protein; the RBC are subjected to destruction by immune phagocytosis in extravascular sites such as the spleen or liver (extravascular hemolysis). During the process, some of the bound cells, now membrane depleted, become detached and re-enter the circulation often as spherocytes. These are then exposed to either mechanical destruction in the circulation or more importantly premature death in the adverse metabolic and osmotic environment of the splenic sinusoids.

 Typically, IMHA is caused by antibodies directed against circulating, mature RBC, with bone marrow mounting a healthy regenerative response (peripheral phenomenon). However, in some dogs (up to about one third), antibodies target erythroid precursors at any stage in their development in the bone marrow instead of, or in addition to, circulating RBC, resulting in defective or ineffective erythropoiesis, and pure red cell aplasia.

Diagnosis

Basic Hematological Data and Prognosis Factors

Hematology in patients with IMHA typically reveals a moderate to severe anemia, which is most commonly regenerative, with anisocytosis, polychromasia, a high reticulocyte count (> 120 G/L) and, sometimes, increased numbers of nucleated RBC. Reticulocyte counts can however sometimes be inappropriately low, either because anemia is peracute (since it takes 3 to 5 days for the bone marrow to mount a strong regenerative response), or because antibodies are also directed against RBC precursors. Additionally hematology can often reveal clues that suggest an immunologic mechanism:

 Agglutination: An autoagglutination can be observed in the collection tube containing anticoagulated blood, by placement of a drop of blood onto a slide, or on the blood smear. Autoagglutination may only occur at +4°C, so blood should be refrigerated before making this assessment. True autoagglutination may be grossly distinguished from rouleaux formation by the addition of an equal volume of saline to the drop of blood. Rouleaux is dispersed by this procedure. A positive slide agglutination result is highly suggestive of IMHA diagnosis, and also suggests that the condition is likely to be acute and severe. A negative slide agglutination does not rule out IMHA: incomplete (non-agglutinating, non-hemolytic) antibodies are reported to be the most common antibodies in small animals with IMHA. Recent clinical studies however, report a much higher incidence of positive slide agglutination, perhaps reflecting a referral bias as the result of practitioners tending to refer only the most severe cases.

 Spherocytosis: Spherocytes are small spherical erythrocytes that, when present in high numbers, strongly suggest an IMHA diagnosis.

IMHA patients are at risk for developing thromboembolism and disseminated intravascular coagulation (DIC). Platelet count and a more complete investigation of the coagulation cascade should be considered. Thrombocytopenia is associated with a poor survival prognosis. A consensus seems to be for two other clinicopathological parameters: high serum total bilirubin concentration and leukocytosis (with neutrophilic left shift and toxic changes).

Immunological Testing

Specific immunological testing can be used to support a tentative diagnosis of IMHA, and the definitive diagnostic procedure of IMHA remains the direct Coombs' test.

Direct Coombs' test (direct antiglobulin test, DAT): A full Coombs' test is performed with a polyvalent antiglobulin (a mix of antibodies directed against canine IgG, IgM and complement), and the test should be performed at body temperature (+37°C) and at +4°C. The titer of each positive reaction should be determined. No clear association between the titer of RBC-bound antibody and disease severity exists, although low-titered reactions are more consistent with secondary IMHA than AHAI. Moreover, the titer should be used for disease follow-up and treatment monitoring.

Alternate technologies for antiglobulin tests: Most of them have been designed to either increase the sensitivity of the test or to lessen the subjectivity associated with the assessment of the degree of agglutination (enzyme-linked antiglobulin test, immunofluorescent test and gel test). Gel test is a form of a column agglutination assay, where RBC agglutinates can be trapped by a specific type of matrix. Gel tests were first developed at the Blood center of Lyon (Ètablissement Français du Sang, Lyon, France), then commercially by DiaMed AG (Cressier, Switzerland), and have provided an innovative approach to blood group serology and antiglobulin testing.

Autoantibodies: Autoantibodies eluted from the surface of patient RBC or present in serum (circulating autoantibodies) may be detected using an indirect Coombs' test by incubation with normal erythrocytes. Antinuclear antibodies, and platelet autoantibodies in case of concurrent thrombocytopenia (Evans' syndrome), should be requested in the investigation of primary idiopathic IMHA, presumably autoimmune.

Identification of the Underlying Disease

Since IMHA can be secondary, confirmation of a diagnosis of IMHA is not necessarily the end of the diagnostic trail. Primary IMHA can only be diagnosed with absolute certainty once potential underlying causes have been thoroughly investigated! The influence of secondary factors is now more widely recognized. This reflects the availability of more advanced diagnostic means for detecting the underlying disease, the geographic extension of particular underlying infectious diseases, and the recognition of new trigger factors. Unfortunately, this presents practitioners with a dilemma: although IMHA is unlikely to be a treat unless the underlying causes have been identified and eliminated, a complete search for such causes can be time consuming, expensive, potentially invasive and, in the case of primary IMHA, ultimately fruitless. Therefore a justification for a better characterization of AHMI and rigorous diagnosis based upon the detection of autoantibodies is needed.

Bone Marrow Analysis

Bone marrow analysis (aspiration cytology) is also indicated in patients suspected to have the non-regenerative forms of IMHA. In pure red cell aplasia, bone marrow evaluation indicates an erythroid maturation arrest (a relative or complete lack of RBC precursors within the marrow). Phagocytosis of erythrocytes and RBC precursors is frequently seen.

Treatment

The imprecision of the diagnosis, the lack of prospective treatment efficacy studies, the poor prognosis associated with the disease, and the high cost of treatment and supportive care contribute to a frustrating task for choosing a treatment regimen for dogs.

Supportive/Ancillary therapy

Patients with severe anemia will benefit from reducing oxygen demand (cage rest). Severely compromised dogs may require transfusion or oxygen-carrying support (Oxyglobin®), yet the use of this type of supportive therapy has been controversial. Since patients are prone to thromboembolism and DIC, particularly those with severe anemia and autoagglutination, preventive therapy could be recommended. A poor response to the prophylactic administration of heparin (unfractionated) as an antithrombotic strategy has been reported. An ultralow-dose aspirin regimen (0.5 mg/kg/day for 2 days) is promising.

Initial Immunosuppressive Therapy

High glucocorticoid doses are the first Line of treatment for arresting RBC hemolysis in IMHA patients. We use an initial infusion of methylprednisolone (2 to 4 mg/kg, once or twice a day at admission) followed by oral prednisone or prednisolone (1 to 2 mg/kg twice daily) for a minimum of 7 to 10 days. IMHA patient should be monitored at least weekly until the anemia resolves. Once hematological and immunological signs are improved (hematocrit above 30% and negative DAT), the dose is then tapered by 50 % every two weeks over a three-month period depending on the initial dosage, hematocrit and severity of side effects.

Specific Treatment

Immunosuppressive therapy: In dogs that have severe, acute-onset intravascular hemolysis or in the case of persistent or relapsing-remitting AHMI, azathioprine (2 mg/kg orally once daily) should be added to glucocorticoids. Azathioprine is relatively inexpensive and usually well tolerated. Potential side effects are rare, and idiosyncratic severe myelosuppression (within a few weeks of commencing therapy) should be reversible if leucopenia is promptly detected. Other approaches to therapy are documented. The benefit of cyclophosphamide is controversial. Cyclosporine is costly, but has been used successfully to treat dogs with refractory IMHA. Another expensive, but promising drug is leflunomide. This inhibitor of pyrimidine biosynthesis is very well tolerated in dogs (4 mg/kg/day). We use also danazol (5 mg/kg orally t.i.d), an impeded androgen, as an adjunctive therapy with glucocorticoids in non-regenerative forms of IMHA in order to reduce the dose of steroid that is needed for long-term therapy.

Other treatment: Since arguably rickettsial agent and related organisms (Bartonella, haemoplasma, etc.) may predispose the animal to secondary IMHA, a treatment based on doxycycline (10 mg/kg/day for 28 days) might be indicated in endemic areas.

References

1.  Carr AP, Panceira DL, Kidd L. Prognostic factors for mortality and thromboembolism in canine immune-mediated hemolytic anemia: a retrospective study of 72 dogs. J. Vet. Intern. Med., 2002, 16: 504-509.

2.  Day MJ. Immune-mediated hemolytic anemia. In Feldman BF, Zinkl JG, Jain NC. Schalm's veterinary hematology, 5th ed. Lippincott, Williams & Wilkins, Philadelphia. 2000, pp. 799-806.

3.  Miller SA, Hohenhaus AE, Hale AS. Case-control study of blood type, breed, sex, and bacteremia in dogs with immune-mediated hemolytic anemia. J. Am. Vet. Med. Ass., 2004, 224: 232-235.

4.  Thompson MF, Scott-Moncrieff JC, Brooks MB. Effect of a single plasma transfusion on thromboembolism in 13 dogs with primary immune-mediated hemolytic anemia. J. Am. Anim. Hosp. Assoc., 2004, 40: 446-454.

5.  Wardrop KJ. The Coombs' test in veterinary medicine: past, present, future. Vet. Clin. Path. 2005, 34: 325-334.

6.  Weinkle TK, Center SA, Randolph JF, Warner KL, Barr SC, Hollis NE. Evaluation of prognostic factors, survival rates, and treatment protocols for immune-mediated hemolytic anemia in dogs: 151 cases (1993-2002). J. Am. Vet. Med. Ass., 2005, 226: 1869-1880.