Bernard F. Feldman, DVM, PHD
Alteration of the erythrocyte membrane is the final common pathway leading to hemolysis. The membrane alteration usually signals the reticuloendothelial macrophages to remove the damaged or altered red cell from the circulation. Immune-mediated hemolytic anemia (IHA) results from red cell membrane alteration due to immunoglobulin and/or complement and resultant intravascular hemolysis and/or extravascular mononuclear phagocyte (splenic essentially) attack.
Antibodies to red cells can arise as part of a normal immune response by a normal immune system or as a result of some abnormality of the control of the immune system. In the first case, antibodies may arise in response to infection or an allogeneic reaction to transfused blood or exposure to blood products; the antibodies formed may then cross react with the patient's own antigens, resulting in the destruction of the patient's cells. In some cases, the patient's antigens may be altered and a similar cross-reacting antibody may result. Abnormalities in the immune system may include the monoclonal proliferation of immunoglobulin producing cells. Most frequently this is due to the benign proliferation but may occur with malignant proliferation as well. In such instances the antibody is nearly always IgM and cold-reacting. The immune system may be stimulated by viral invasion, resulting in the production of antibodies to self antigens. Finally, there may be abnormalities of self control of the immune system (possibly diminished suppressor function) which results in the production of antibodies against self antigens; this may be confined to the red cells but often antibodies against other self antigens are also seen as in systemic lupus erythematosus (SLE).
DETECTION OF ANTIBODY
The ability to detect antibody in IHA will depend on the characteristics of the antibody. Agglutination occurs readily with IgM antibodies and poorly, if at all, with IgG antibodies. This is due to the fact that the larger IgM molecule can span the distance imposed by mutually repulsive forces that keep RBCs separated. When this happens IgM forms a lattice and agglutination occurs. Additionally a sufficient amount of antibody must be present for this phenomenon to occur. The direct antiglobulin test (DAT; Coombs' reaction) involves the species specific production of an antiantibody thus allowing agglutination to occur in vitro. The majority of autoimmune antibodies in human animals and nonhuman small animals are IgG1 and the remainder are predominantly IgG3. The use of antiIgG and anticomplement 3 antisera can aid in the determination the pattern of antibody or complement fixation in IHA. The antiglobulin test results are then of four types:
1. Antibody alone is fixed, generally antiIgG but no antiC3. In human animals this reaction is generally associated with specific blood groups and with specific drug-induced IHA.
2. Both IgG and C3 are found to be present on the RBC. This indicates that IgG antibody is capable of fixing complement. This pattern in human animals is seen in SLE and in idiopathic IHA. This never occurs when drugs are involved.
3. Complement only is fixed; antiIgG does not cause agglutination. This kind of DAT reaction has several causes. The antibody may not be IgG. The antibody may have low affinity due to temperature. Papainization of the RBCs may result in a positive immunoglobulin test because papain will increase the ability of the antibody to interact with the RBC surface antigen.
4. The test is negative. This results when the test is not performed in both cold and warm environs, when the affinity of the DAT antibodies is too low, when there are too few antibodies or complement molecules on the rbc surface, or when the DAT antibodies are too avid resulting in the prozone phenomenon that is, too many antiantibodies cover the antigens and lattice structures are prevented.
WHAT YOU SHOULD DO!
As the handling clinician you must ask your laboratory a series of questions. First is the DAT being uses species specific? Do they run positive and negative controls on each patient? Was the test run in both cold and warm environs? What dilutions were used to overcome potential prozone phenomena (generally dilutions out to 1:16 will do this)?
OTHER MEANS OF DIAGNOSIS
There does not seem to be a major age or sex predisposition to IHA in nonhuman animals. There does seem to be a breed predilection with Cocker Spaniels, Old English Sheepdogs, Poodles, German Shepherds and Doberman Pinschers predominating. The presence of spherocytes helps but there is a caveat here. Unless a trained technologist or cytologist is reviewing the smears, spherocytes often are incorrectly identified. Conversely without training spherocytes are often missed. With feline and equine RBCs spherocytosis is often missed because of the small cell size. Spherocytosis is suggested when the MCV is below normal or at the low end of the reference interval despite the large numbers of reticulocytes which often accompany IHA and when the MCHC is above the reference interval. This results when smaller cells (spherocytes) are present (MCV) and have only lost membrane without loss of intracellular content (MCHC). Slappendel from the State University of Utrecht suggests that performing a standard osmotic fragility test at half strength will reveal increased osmotic fragility in IHA patients. Attempting to cause or disperse agglutination has been singularly unrewarding in this author's hands.
Increased reticulocyte production indices without clinical evidence of bleeding and without hypoproteinemia suggest hemolysis. The caveat here is that nonimmune hemolysis can occur and...spherocytosis can occur without IHA (hypophosphatemia often causes spherocytosis).
IHA in the acute fulminant form is most often complicated by disseminated intravascular coagulation (DIC). Decreasing fibrinogen concentrations and platelet counts while the activated partial thromboplastin time (APTT) and the prothrombin time (PT) prolong and the finding of decreased concentrations of fibrinogen (Thrombin Time; fibrinogen), antithrombin III (ATIII) and protein C (PC) in concert with increased fibrin(ogen) degradation products (FDPs) all indicate DIC. This constellation of test abnormalities associated with an anemic patient suggests IHA-induced DIC.
Therapy should be rational and should be based on a large body of evidence rather than a single test. The DAT for example may be positive in some septic nonanemic patients and, as discussed earlier, can be negative in some IHA patients.
Cold reacting antibodies are best treated by minimizing exposure to cold and that may even include cold water. Often cold reacting antibodies are associated with paraneoplastic or neoplastic disease so a clinical search for those entities must be initiated. In cold reacting IHA the spleen is seldom the source of antibody production and splenectomy is not usually recommended.
Low dose prednisone (1 mg/kg/day) is recommended by this author as there has been no apparent correlation between glucocorticoid dosage and success or lack thereof. There is no therapeutic advantage in using other forms of glucocorticoids. If remission is not easily achieved then this regime is not successful and further immunomodulation is indicated. This author prefers to begin with azathioprine and prednisone and if unsuccessful after several weeks of therapy uses prednisone and cyclophosphamide. The use of anabolic steroids has been singularly unsuccessful in the treatment of IHA.
If ATIII and/or PC concentrations are decreased, heparinized plasma therapy is initiated. Fresh or thawed fresh frozen plasma is incubated with heparin (100 IU/patient kg) at 37°C for 30 minutes to one hour before delivery to the patient. Amounts of thusly treated plasma may be delivered at the rate of 6-10 mls/kg q8h. Heparin must always be tapered through the next 48 hours. Subcutaneous heparin always follows heparinized plasma infusions. Movement of ATIII or PC towards the reference interval suggests success. Normalization of APTT, PT, fibrinogen concentrations, and platelet count suggests success.
Transfusion with dog erythrocyte antigen 1.1, 1.2, and 7 negative washed red blood cells, without major and minor crossmatch reactions can be used effectively. Volume overload generally does not occur unless more than 20 percent of blood volume is delivered too quickly (dog blood volume is approximately 90 mls/kg; cat blood volume is approximately 80 mls/kg).
Treatments with human gamma globulin concentrates and with cyclosporine have been suggested. As the author has not seen sufficient data to recommend these approaches, the author cannot, at this time, recommend these forms of therapy.
Splenectomy may be considered when other forms of therapy have been deemed unsuccessful. Many clinicians report success subsequent to splenectomy in that other drugs may be removed or dosages lowered, and when relapse occurs, remission is more quickly achieved.
Treatment for chronic cases of IHA (those that do not respond immediately to modest therapeutic intervention) most be extended for a minimum of 3 to 6 months. Many cases require continuous or intermittent lifetime therapy.
References are available on request.