Diagnosing AIHA

Animals with AIHA may exhibit lethargy or weakness, exercise intolerance, difficulty breathing, rapid breathing, vomiting, diarrhea, excessive thirst and urination, depression and lack of appetite.

Further examinations may physically reveal pale mucous membranes, brownish-red urine, rapid heartbeat, rapid breathing, enlargement of the spleen or liver, jaundice, fever, a heart murmur and inflammation of the lymph nodes.

The diagnosis of AIHA can be made by demonstrating the presence of antibodies on red blood cell membranes and by ruling out other causes. Study of the red blood cell morphology may reveal the presence of a large number of spherocytes (abnormally shaped, damaged red blood cells), which suggests AIHA but may also form as a result of some other type of red blood cell damage.

Therefore, to confirm a diagnosis of AIHA, in saline autoagglutination needs to be observed or a positive DAT test should be performed.

• Autoagglutination, the automatic clumping together of red blood cells, is tested by placing a large drop of blood on a slide. Autoagglutination is distinguished by diluting the blood with saline solution to disperse cells aggregated by rouleaux. The persistent presence of autoagglutination is diagnostic for AIHA since it is secondary to antibodies overcoming antigens on red blood cell membranes, so finding in saline autoagglutination makes the DAT unnecessary. The DAT would only need to be done in these circumstances to find the specific antibody class responsible for the agglutination.
• Only a minority of patients with AIHA exhibit true autoagglutination.
• In patients whose cells don't autoagglutinate, a Direct Antiglobulin Test, aka DAT, aka Coomb's Test, should be done to confirm the presence of antibodies on red blood cell membranes. This tests for IgG, IgM and complement. A positive test result in the presence of anemia is consistent with the diagnosis for AIHA. False negative results do occur and shouldn't rule out AIHA.

Your vet should consider performing additional clinical and laboratory tests with cases of AIHA:

• Packed Cell Volume, aka PCV, to determine the red blood cell count, extent of the anemia and need for a transfusion (usually PCV<15%).
• Examination of blood smear under a microscope to determine presence of reticulocytosis and spherocytosis. Reticulocytosis, a large number of baby red blood cells prematurely released by the bone marrow, confirms red blood cell regeneration. Spherocytosis, a large number of abnormally shaped, damaged red blood cells, confirms red blood cell destruction. Polychromasia (many colors in the blood), anisocytosis (irregular dissolution of cells), and leukocytosis (large number of white blood cells) may also be present.
• Reticulocyte Count to check if bone marrow is compensating for anemia.
• A Complete Blood Count, aka CBC, should be done to confirm severe anemia through lab work. Actual numbers and percentages can be determined for red blood cells and white blood cells. Red blood cell morphology can be reviewed and a platelet count can also be performed.
• Biochemistry: hyperbilirubinemia (elevated bilirubin levels in the blood), hyperproteinemia (elevated protein levels in the blood), hemoglobinemia (presence of free hemoglobin in blood plasma), increased liver enzymes.
• Platelet Count to check for thrombocytopenia in case antibodies are there for both red blood cells and platelets.
• Urinalysis: hemoglobinuria and bilirubinuria (hemoglobin or bilirubin in urine).
• Check feces for a change in color which could indicate hemolysis.
• Anti Nuclear Antibody (ANA) titer if Systemic Lupus Erythemytosis is the suspected primary disorder.
• Positive Serotologic Titer for Infectious Diseases. Babesia, Ehrlichia, Leishmania, Hemobartonella and Dirofilaria should all be tested.
• Thoracic and Abdominal Radiographs and Ultrasonographic Imaging are used to check the internal organs, to rule out foreign bodies or tumors, to detect blood clots, accumulation of fluids in the abdomen and size of the liver and spleen.

Regenerative & Nonregenerative Anemia

A Complete Blood Count (CBC) and reticulocyte count most commonly show a regenerative, macrocytic, hypochromic anemia, meaning many abnormally large, lightly pigmented red blood cell precursors are present in the blood. A significant portion of AIHA cases first appear to be nonregenerative due to peracute disease, meaning a sudden onset in <3 days, which hasn't given the bone marrow enough time to try to compensate for the shortage by firing out baby red blood cells.

If the bone marrow is working reticulocytes (baby red blood cells, precursors to red blood cells) will be present. Sometimes it can take almost up to a week for signs of regeneration to appear.

A lack or low count of reticulocytes at presentation can be due to peracute or acute onset or nonregenerative anemia. A persistent lack of reticulocytes or suspicion of nonregenerative anemia warrants a bone marrow aspirate to see if the bone marrow even has the ability to make new red blood cells.

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