School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA
Immune-mediated hemolytic anemia (IMHA) is a common hematological disorder in dogs, may be primary (idiopathic, autoimmune) or occur secondarily to underlying diseases and is often associated with life-threatening complications. The approach to the diagnosis of IMHA is being discussed with emphasis on autoagglutination, spherocytosis, and Coombs' test as well as some controversies surrounding those tests. Moreover, its management with selection of immunosuppression, transfusion support and anti-thrombotic therapy will be discussed highlighting, although there is limited published evidence of their efficacy.
Immune-mediated hemolytic anemia (IMHA) arises when an immune response targets directly or indirectly erythrocytes and hemolytic anemia ensues. In primary IMHA, no inciting cause can be identified, hence the term idiopathic IMHA or autoimmune hemolytic anemia (AIHA). In contrast, secondary IMHA is associated with an underlying condition or triggered by an identifiable agent. Furthermore, alloimmune hemolytic anemias, such as hemolytic transfusion reactions are caused by specific anti-erythrocytic alloantibodies.
In dogs idiopathic IMHA (AIHA) has been considered the most common cause of hemolytic anemias for decades, and many anemic dogs are presumptively managed for AIHA. Although numerous retrospective studies and review articles have been published, much remains anecdotal. Questions that will be addressed include the following:
Is it a Primary or Secondary IMHA?
Many dogs suspected to have idiopathic IMHA present with an acute history of vomiting, diarrhea, lethargy, or anorexia indicating a recent trigger rather than reflecting signs of hemolytic anemia. The various breed and family predilections (about 1/3 of all cases of IMHA are seen in American Cocker Spaniels) suggest strongly the involvement of genetic factors leading to a predisposition to IMHA. There is ample evidence for infectious agents to be associated with IMHA, caused by parasitic (babesiosis, leishmaniasis, ehrlichiosis, anaplasmosis and dirofilariasis), fungal, and bacterial diseases (leptospirosis, chronic abscesses, discospondylitis, pyometra, colitis, and pyelonephritis). The relationship between infection and autoimmunity may be explained by molecular mimicry. Furthermore, in limited surveys, seasonality (summer) and a temporal association between vaccination and onset of IMHA have been suggested, but since no specific vaccine has been implicated, it appears likely that vaccines enhance a smoldering immune process. Many dogs with IMHA have severe leukocytosis (with or without left shift) and also considerable serum liver enzyme elevations (prior to prednisolone) suggestive of serious inflammatory processes, which may enhance the immune destruction by activating the macrophage system and the thrombotic tendency. Also at necropsy there is considerable evidence of inflammation and necrosis in most cases of IMHA. Beside the genetic factors and the infectious/inflammatory triggers, several drugs and toxins (e.g., sulfonamides, bee sting) and neoplastic disease processes have been associated with IMHA. Thus, an extensive search for an underlying disorder or trigger is always warranted.
What is the Clinical Evidence for Erythrocyte Immune Destruction?
A diagnosis of IMHA must demonstrate accelerated immune destruction of erythrocytes. Evidence of a hemolytic anemia is suggested clinically by icterus and a regenerative anemia with hyperbilirubinuria, and the presence of hemoglobinemia and hemoglobinuria refers to an intravascular process. However, the erythroid response in the bone marrow may be blunted by the immune process or the underlying disease, thereby leading to non-regenerative anemias. Besides documenting a hemolytic anemia, one or more of the following three hallmarks must be present to support a diagnosis of immune-mediated hemolysis.
Anti-erythrocytic IgM and in large quantities IgG antibodies may cause direct erythrocyte autoagglutination. Autoagglutination may be seen by naked eye in EDTA tube or on a glass slide or may become apparent microscopically as small clumps of erythrocytes. For yet unexplained reasons, canine erythrocytes have a tendency to unspecifically agglutinate in the presence of plasma and colder temperatures as well as possibly EDTA anticoagulant. Mixing blood with one drop of saline may break up rouleaux formation but not other forms of unspecific red cell agglutinations. It is, therefore, important to determine whether the agglutination persists after 3x "saline washing," which has been coined true or persistent autoagglutination. True autoagglutination is indicative of an immune process, but precludes the performance of Coombs' test or blood typing/crossmatching. If the agglutination breaks up after washing, the Coombs' test is expected to be positive, if it is a case of IMHA.
If erythrocytes are only partially phagocytized or lysed by complement, erythrocytes with reduced surface area to volume ratio, known as spherocytes, are formed. On microscopic examination they appear spherical and microcytic with no central pallor and are extremely fragile. Large numbers of spherocytes are nearly diagnostic for IMHA (hereditary spherocytosis), whereas small numbers may be seen with other conditions including DIC, endotoxemia and zinc intoxication.
Positive Direct Coombs' Test Result
The direct Coombs' test is also known as direct antiglobulin test and is used to detect antibodies and complement on the surface of erythrocytes when the anti-erythrocyte antibody strength or concentration is too low to cause spontaneous agglutination (so-called subagglutinating titer). Separate canine-specific anti-IgG, IgM, and C3b antibodies as well as polyvalent Coombs' reagents are available and laboratory (tube and microtiter plate) and in-clinic tests (gel column no more available; capillary tube, and strip method) have been or are being developed. Although many commercial laboratories offer this Coombs' test for dogs, clinicians have questioned the tests sensitivity. In our recent prospective study the Coombs' test methods used performed extremely well. Thus, dogs with negative Coombs' test results should be reevaluated for other causes of hemolytic anemia. Negative results may be seen because of technical reasons, insufficient quantities of bound antibodies, the presence of weakly bound antibodies, or the disease in remission. A few days of immunosuppressive therapy will not reverse the Coombs' test results, as unlikely a transfusion would cause a positive Coombs' test result.
An increased erythrocytic osmotic fragility has been proposed by few clinicians, albeit there are many other reasons that affect the osmotic fragility test including quality of sample. For this and high variability in test performance in clinical practice, we do not recommend the osmotic fragility test for the diagnosis of IMHA.
Some dogs with IMHA show poor bone marrow regeneration (no or low reticulocyte counts), which is considered to be caused by immune-targeting of precursor cells (no direct evidence) or direct marrow damage. As mentioned above, this may also be due to the yet to be explained massive inflammatory and necrotic response in dogs with IMHA. It is not uncommon to also observe thrombocytopenia in a dog with IMHA. This may be due to an underlying disease such as babesiosis (secondary IMHA), another immune process (Evans' syndrome) or a thrombotic tendency. Some dogs with IMHA also have a positive serum ANA titer, polyarthropathy with a positive RF or immune-mediated hypothyroidism. Thus, it is advisable to assess any IMHA dog thoroughly for other immunopathies, thrombotic tendencies, infectious diseases (PCR and serology), as well as underlying disorders or triggers.
What Fluids and Should Oxygen be Given?
Restoration and maintenance of tissue perfusion with crystalloid fluids is important, even when it results in further lowering of the hematocrit. When severe anemia and a dropping hematocrit lead to signs of tissue hypoxia, packed red blood cell transfusions appear beneficial. The notion that transfusions pose an increased hazard to animals with IMHA has been overemphasized and is not supported by retrospective clinical studies. However, the common occurrence of autoagglutination may make blood typing and crossmatching of the patient impossible. In these cases DEA 1.1 negative blood should be transfused and if the dog was previously transfused (> 4 days) a crossmatch test should also be performed. The bovine hemoglobin solution Oxyglobin is currently no more available.
In contrast to blood, oxygen inhalation therapy is of little benefit, unless the animal is suffering from pulmonary disease such as pulmonary thromboemboli. Furthermore, adequate hydration will reduce, while catherization and steroids may increase the thrombotic tendency. Thanks to adequate transfusion support, animals with IMHA rarely die because of anemia, but because of secondary complications such as thromboemboli, infections and necrosis.
Which Immunosuppressive Therapy?
The insufficient understanding of the pathogenesis, the generally guarded prognosis, the lack of good therapeutic trials, the serious drug side effects, and the high costs of intensive care greatly hamper the successful management of dogs with IMHA. The main goal of immunosuppressive therapy is to reduce phagocytosis, complement activation, and anti-erythrocytic antibody production. Glucocorticoids are the initial treatment of choice for canine and human 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, glucocorticoids may reduce the degree of antibody binding and complement activation on erythrocytes, but only after weeks, diminish the production of autoantibodies. Thus, oral prednisolone at a dose of 1–2 mg/kg twice daily is the mainstay treatment. Alternatively, oral or parenteral dexamethasone at an equipotent dose of 0.6 mg/kg once daily can be applied, but is likely not more beneficial. A pulse glucocorticosteroid therapy may also be considered initially. It should be noted that the markers of immune destruction such as autoagglutination, spherocytosis and positive direct Coombs' test results should be monitored and will only slowly disappear following treatment.
Additional immunosuppressive therapy is warranted when prednisone fails, only controls the disease at persistently high doses, or when it causes unacceptable side effects. They are generally used together with prednisolone, but may eventually be used independently. Historically, cytotoxic drugs such as cyclophosphamide were added, however, surveys failed to show any beneficial effects. Retrospective studies with azathioprine also failed to show real benefits. Anecdotal reports suggest that cyclosporine, mycophenylate, leflunomide and human intravenous immunoglobulin may have some efficacy and may be associated with fewer side effects. Liposomal clodronate (dichloromethylene diphosphonate) has been used to deplete macrophages and block clearance of opsonized cells in mouse models of autoimmune disease and may be a promising option in dogs; studies are in progress.
Splenectomy has also been rarely considered as a secondary treatment option for IMHA. Indeed the spleen may be enlarged in dogs with IMHA, harbor a trigger (histopathology) and be the main site of red cell destruction. Animals undergoing a splenectomy should not receive any other immunosuppressive agents than glucocorticosteroids at least a week prior to surgery and thereafter, receive prophylactic antibiotics, and be carefully monitored for the development of infection postoperatively.
It should be noted that an apparent therapeutic response to immunosuppressive therapy is insufficient evidence for the diagnosis of IMHA. Response to therapy may be indicated by a hematocrit that rises or stabilizes, an appropriate reticulocytosis, diminished autoagglutination, and fewer spherocytes and ultimately a negative Coombs'test result. As glucocorticosteroid therapy is associated with well-known side effects, the initial dose will be tapered by reducing the amount by one-third every 7–14 days and moving toward an every other day therapy. In secondary IMHA with appropriate control of the underlying disease, the tapering can be accomplished more rapidly. Because of the potential of gastrointestinal ulceration by glucocorticosteroids, gastrointestinal protectants such as sucralfate may be considered. Because dogs with IMHA suffer from an immune deregulation, which may have been triggered by an infection and are treated with immunosuppressive agents, these patients are prone to experience infections. It is, therefore, prudent to administer preventative as well as therapeutic antibiotics to these dogs with IMHA on immunosuppressive therapy.
How to Recognize and Treat or Prevent Thromboembolic Complications?
Thromboemboli and DIC are unique serious complications that greatly contribute to the morbidity and mortality of dogs with IMHA. Although the pathogenesis remains unknown, venipuncture, catheters, confinement, and glucocorticosteroids as well as other immunosuppressive agents may be contributing factors. Thus far, only one limited heparin trial (utilizing high doses and close dose adjustments) has shown successful prevention of these life-threatening hemostatic problems in canine IMHA. Predisposing factors should, whenever possible, be limited, and adequate perfusion and tissue oxygenation should be provided with fluids and transfusions. Generally, anticoagulation therapy is instituted after there is some evidence or suspicion of thromboemboli. Unfractionated Heparin (dose of 50–300 U/kg subcutaneously every 6 hours or by continuous intravenous infusion) or Low Molecular Weight Heparin (LMWH; Dalteperin 150 IU/kg sc every 12 hours) are the most commonly used drugs. The replacement of coagulation factors and antithrombin III has not been proven to be beneficial. Antiplatelet agents may also be used and for instance an ultralow dose of aspirin (0.5 mg kg once daily) has been proposed, but other studies question its efficacy. Other antithrombotic agents such as modern antithrombotic agents have been used occasionally, but their efficacy and safety remain also unproven. Dogs being treated with anti-thrombotic agents should be carefully monitored by thromboelastography and other parameters.
Because the severity of IMHA ranges from indolent to life-threatening disease, therapy has to be tailored for each patient and depends in part on whether the IMHA is primary or secondary in nature. Removal of the triggering agent or treatment of the underlying condition can bring the IMHA rapidly under control. Prednisolone or dexamethasone are the first-line immunosuppressive drugs. Alternatives in case of resistance or unacceptable side effects may include cyclosporine and human intravenous immunoglobulin. The mortality remains high despite aggressive therapy and transfusion support. The major complications caused by thromboembolism are difficult to predict, treat and prevent.
Additional references are available from author. Author's studies were supported in part by grants from the National Institutes of Health (RR002512) and the AKC Canine Health and other foundations.
Contact: Dr. Urs Giger, School of Veterinary Medicine, University of Pennsylvania, 3900 Delancey Street, Philadelphia, Pennsylvania, USA. firstname.lastname@example.org; http://research.vet.upenn.edu/penngen.