Immune-mediated hemolytic anemia (Proceedings)

Disease conditions that lead to anemia can be divided into two general categories: regenerative and nonregenerative. Immune-mediated hemolytic anemia (IMHA) can fall under either category but is more commonly regenerative. Because the disease has a rapid onset and can quickly lead to severe illness or death, early diagnosis and treatment is necessary. The following discussion will review the causes, clinical signs, diagnosis, and current therapy of IMHA in dogs and cats. Preliminary results from a retrospective study will be included in the presentation.

Pathophysiology

The immune system recognizes or "tolerates" normal body tissues including red blood cells as harmless. During formation of lymphoid cells early in development, potentially self-reacting cells are destroyed or not allowed to mature. In some animals, a few of these cells escape and become "autoimmune", waiting for a trigger to start attacking normal tissue. Autoimmune responses can be divided into primary (idiopathic) and secondary (due to infectious disease, neoplasia, drugs, toxins, etc.). Other classifications take into account warm vs. cold antibody disease and various other factors (more relevant in human medicine).

In IMHA, IgG and IgM antibodies, which are normally produced in response to infectious organisms, are produced by plasma cells and directed against the membranes of red blood cells (rbcs). IgG-coated rbcs are detected as abnormal by macrophages, which primarily are in the spleen. As these rbcs circulate through the spleen, they are phagocytosed and removed from the bloodstream. This process results in extravascular hemolysis by the mononuclear phagocyte system (MPS) and leads to splenomegaly as well as loss of rbcs. As more rbcs are tagged with IgG, the liver begins participating in erythophagocytosis. When both IgG and IgM bind to rbcs, agglutination occurs (sticking together) which also leads to extravascular hemolysis in the spleen and liver.

Another mechanism in IMHA involves complement. Complement binds to Ig-coated rbcs, which activates the complement cascade. This arm of the innate immune system destroys its targets by forming membrane attack complexes, which "punch holes" in the rbc membranes and allow water and electrolytes to enter the cell, causing swelling and lysis. If this occurs in the circulation, it is called intravascular hemolysis. Complement-coated rbcs are also recognized in the spleen and liver and subject to phagocytosis (extravascular). Rbcs bound with IgM are more likely to be destroyed by complement-mediated destruction.

When the MPS removes only a portion of the rbc membrane instead of phagocytosing the entire cell, a spherocyte is formed. This is a damaged red cell with a spherical shape rather than concave disk, and it is likewise trapped in the spleen and destroyed. Spherocytes may be seen on blood smears which helps with diagnosis.

The trigger or underlying cause of immune-mediated hemolytic anemia (IMHA) is identified in 25-40% of canine cases and most of the feline cases. If a cause is found, it must be resolved before the immune-mediated rbc destruction stops. Therefore, a thorough diagnostic workup is necessary to rule in or rule out other diseases. Among the triggers are:

• Infections

     o Viral (FeLV, FIV, FIP)

     o Bacterial and fungal (leptospirosis, hemobartonellosis, histoplasmosis, endotoxemia, endocarditis, clostridial)

     o Parasitic (babesiosis, leishmaniasis, heartworm disease (caval syndrome), hookworms, ehrlichiosis, anaplasmosis, cytauxzoonosis)

• Neoplasia (leukemia, lymphoma, multiple myeloma, hemangiosarcoma, solid tumors)

• Other immune disorders (systemic lupus erythematosus, immunodeficiencies)

• Drugs (sulfas, cephalosporins, penicillins, vaccines, propylthiouracil, methimazole, procainamide, benzocaine, heparin, dipyrone, levamisole, phenylbutazone, quinidine, phenothiazines)

• Toxins (zinc, copper, propylene glycol, onion, garlic, rye grass, maple, castor bean, methylene blue, beestings)

• Intrinsic defects (methemoglobin reductase, pyruvate kinase, or phosphofructokinase deficiencies, idiopathic Heinz body anemia)

• Microangiopathic (disseminated intravascular coagulation, vasculitis, uremia)

Young to middle-aged dogs and cats are at higher risk. Breeds with a higher incidence include American Cocker Spaniels, Old English Sheepdogs, Doberman Pinschers, German Shepherds, Miniature Poodles, Miniature Schnauzers, Bichon Frises, Irish Setters, Collies, and English Springer Spaniels. This suggests that genetics play a role in the development of IMHA. Different breeds of dogs were found to have different major histocompatibility complexes (dog leukocyte antigen haplotypes) in one study of IMHA. While spayed females are suggested in some studies to be most susceptible, true gender differences or hormonal influences are difficult to determine. In cats, no particular breed or gender is associated.

Clinical signs

The effects of hemolytic anemia and tissue hypoxia lead to signs that may or may not be recognized by owners. Lethargy, weakness, and anorexia may be the only presenting complaints. Physical exam findings may include fever, pale or icteric mucous membranes, hepatosplenomegaly on palpation, tachypnea, weak rapid pulses, systolic heart murmur, and lymphadenopathy. A few patients with concurrent thrombocytopenia may have petechiae, ecchymoses, epistaxis, or other signs of coagulopathy. The history should include questions about any recent illnesses or drugs, toxins, vaccinations, estrus, ticks, along with suspected onset of clinical signs (rapid progression of anemia typically leads to more severe disease). If IMHA is on the ruleout list, the owners should be informed at this time about the extensive diagnostics and treatments needed as well as the poor prognosis (30-70% survival rates are found in the literature).

Diagnostic testing

A complete blood count, biochemistry profile, and urinalysis should be performed first to assess the anemia and possibly rule out other diseases. The blood smear needs careful analysis, so relying only an in-house CBC analyzer will miss important findings.

Approximately 2/3 of animals have a moderate to severe regenerative anemia, characterized by reticulocytosis, polychromasia, anisocytosis, and nucleated rbcs. Up to 1/3 have nonregenerative anemia for several reasons: in acute disease regeneration may not have occurred, or the antibodies are directed against both rbcs and rbc precursors in the bone marrow, or pathologic changes in the bone marrow lead to ineffective erythropoiesis. A neutrophilia (mild to marked) with or without a left shift suggests tissue necrosis (centrilobular hepatic is common). Many patients have mild thrombocytopenia, which if severe (<50,000/μL) suggests Evan's Syndrome, a combination of IMHA and immune-mediated thrombocytopenia. Spherocytes may be seen on a routine blood smear, and appear as small round rbcs with no central pallor. Approximately 90% of dogs with IMHA have spherocytosis. Blood smears should also be carefully screened for rbc and wbc parasites (e.g. Ehrlichia, Babesia).

The profile reflects organ damage due to hypoxia or hemoglobin-induced injury. Hemolysis (hemoglobinemia) or icteric serum (bilirubinemia) may be seen. Azotemia and increased liver enzymes may occur, with increased bilirubin a common finding. The urine may be discolored due to hemoglobinuria or bilirubinuria. Elevations in C-reactive protein has been reported.

Diagnostic imaging such as abdominal radiographs and ultrasound helps detect underlying disorders such as metallic foreign bodies (zinc) and neoplasia. Thoracic radiographs are indicated in animals with tachypnea or respiratory distress.

To help support a diagnosis of IMHA, additional tests are needed. A drop of fresh blood is placed on a slide and observed macro-and microscopically for autoagglutination, with or without a drop of saline mixed in. Approximately 40-90% of dogs and most cats with IMHA test positive for autoagglutination. Coomb's testing (direct antiglobulin test) is the next step but is not necessary with a positive agglutination test. A positive Coomb's is not diagnostic of IMHA as secondary causes of hemolytic anemia also result in positive results and false negatives are possible (especially if medications have been started). The test method is not standardized, and results from different labs cannot be directly compared. Complete Coomb's testing includes both monovalent and polyvalent antiserum at 4°C and 37°C, which helps differentiate primary from secondary IMHA. Coagulation testing to identify DIC and possible risk of thromboembolic disease is indicated, as the prognosis worsens with coagulopathies. Bone marrow aspiration (cytology) and histopathology (core biopsy) are helpful in working up cases of nonregenerative anemia, neutropenia, and/or thrombocytopenia.

Therapy

The initial treatment can be directed against secondary or underlying causes of the hemolytic anemia, such as antirickettsial, antibiotic, or antiprotozoal medication. Supportive measures such as intravenous fluids for rehydration and oxygen for respiratory compromise may be necessary. Care should be taken when placing IV catheters (small gauge, sterile prep) as thromboembolism is an ongoing risk, and gloves should be worn when handling the animal to minimize risk of spreading infection.

Blood transfusions are indicated when the anemia is of rapid onset or severe, although there are no specific PCV numbers or "triggers". Packed red cell transfusions are useful if available, but whole blood has the advantage of providing plasma, which contains other potentially beneficial compounds such as antithrombin. Synthetic hemoglobin solution (Oxyglobin, Biopure) is indicated if canine blood products are not readily available.

Immunosuppression remains the main treatment for IMHA. As soon as the disorder is diagnosed, corticosteroids are administered to decrease Ig- and complement-binding to rbcs and to decrease production of antibody and cytokines. If the animal tolerates oral medication, then that route is preferred to injectables. Prednisone, prednisolone, or methylprednisolone at high doses (1-2 mg/kg PO twice a day) are started and continued long-term. A common mistake is to taper or reduce this dose too quickly. At least two weeks of high-dose therapy are necessary, and the hematocrit should be stabilized (not dropping or still increasing) before dose reduction is considered. The PCV is rechecked every other day or every third day at first, then weekly after the animal has responded. If the oral route is not possible, injectable dexamethasone at 0.5 mg/kg once a day is appropriate but should be switched to oral prednisone as soon as possible.

Tapering steroid therapy is done slowly over several months. Approximately every 2-4 weeks, a 25% dose reduction is attempted. In some animals, the PCV will begin to drop and the previous dose will have to be resumed. Side effects associated with high doses of prednisone are common (PU/PD, polyphagia, incontinence, weight gain) but in most cases are tolerated. Gastroprotectant therapy may be considered but is not routinely indicated.

If additional immunosuppression is required to increase or stabilize the hematocrit, or if corticosteroid side effects are not tolerated, the next drug to add is azathioprine. Some clinicians start this concurrently with corticosteroids instead of waiting. The usual dose is 2 mg/kg once a day to start (may be tapered to every other day after 1-2 weeks). The onset of action may be delayed for 1-3 weeks, as it inhibits lymphocyte function. A CBC is checked weekly as azathioprine may cause myelosuppression. Other side effects may include pancreatitis, increased liver enzymes, and GI distress. Cats do not tolerate azathioprine well and it should only be used in rare cases at a lower dose (0.3 mg/kg).

Cyclophosphamide was recommended in the past but recent studies have indicated little or no benefit for IMHA. Danazol has been suggested but is unlikely to improve survival.

Cyclosporine (Atopica, Novartis) suppresses cell-mediated immunity but is expensive and has more side effects (vomiting, diarrhea, anorexia). The suggested dose is 10 mg/kg daily (or 5 mg/kg twice a day). This is higher than the label dose for atopic dermatitis, and serum levels should be monitored every 2-4 weeks. Concurrent ketoconazole is sometimes used to decrease the dose and expense in larger dogs. Cyclosporine is not myelosuppressive like azathioprine and may be useful in nonregenerative IMHA.

Human intravenous immunoglobulin may be useful but is expensive and clinical experience is limited. Mycophenolate mofetil (CellCept, Myfortic) is a newer human immunosuppressant drug that is expensive but may be considered for resistant cases. Leflunomide (Arava, Aventis) has anecdotally been reported to be effective. Other therapeutic measures include splenectomy and plasmapheresis, but neither shows a consistent benefit.

The prognosis for dogs and cats affected with IMHA should be guarded, as relapses are common and complications such as thromboembolic disease and DIC can occur. Low-molecular-weight heparin (dalteparin, enoxaparin) may be useful although plain (unfractionated) heparin has not been shown to have significant anticoagulant effects even with frequent dosing (300 IU SC q6h). Ultra-low-dose aspirin (0.5 mg/kg) may increase survival. At this low dose, it is safe to use along with immunosuppressive doses of corticosteroids, but close monitoring for GI effects (especially diarrhea) is necessary. The most rewarding cases are those in which there is a rapid response to corticosteroid therapy and no complications or relapses when tapering the drugs. As stated above, secondary or triggering causes of hemolytic anemia need to be specifically treated along with immunosuppressive therapy.

Retrospective study

Medical records from all dogs seen at the University of Missouri Veterinary Medical Teaching Hospital 2006-2009 were searched for cases of IMHA as a diagnosis. 54 cases were identified – most were seen on referral while a small number presented on an emergency basis. Historical and clinical signs were tabulated along with treatments administered as a percent of the total.

Outcomes to date (2009) were as follows: 14/52 alive (27%), 35/52 dead (67%). 18 dogs were euthanized for IMHA or complications, 4 dogs were euthanized for unrelated causes, and 3 dogs died of unrelated causes (10 were lost to followup).