Macroagglutination and blood smear evaluations
Preparing two blood slides is imperative in diagnosing IMHA and should be performed at the outset in every suspected case.
On the first slide, mix a drop of blood with a drop of saline solution, gently agitate the mixture, and then visually inspect
it for macroagglutination. On the second slide, you can evaluate a blood smear in-house and also send it to a clinical pathologist
for evaluation of spherocytes (Figure 1), Heinz bodies, blood parasites, and microagglutination (Figure 2). Spherocytes and erythrocyte macroagglutination and microagglutination are commonly found on blood smears in patients with
IMHA. As many as 89% of dogs with IMHA have spherocytes; however, dogs with secondary hemolysis from hypophosphatemia, zinc
intoxication, or splenic disease can also have them in small numbers.1,14
1. A peripheral blood smear from an anemic dog reveals that the majority of the RBCs are small, dark, and lack central pallor,
which indicates they are spherocytes. Several large polychromatophilic cells are also present, which indicate a regenerative
response to the anemia (Wright's-Giemsa stain; 1000X)
Direct Coombs test
The direct Coombs test, also called the direct antiglobulin test, can be used when IMHA is suspected but autoagglutination is not seen. This test is run on an EDTA blood sample and will
identify antibodies or complement on a patient's RBC surface. A positive result on a direct Coombs test does not distinguish
between primary and secondary causes of IMHA.1 False positive results are seen in both dogs and cats and can be a result of concurrent diseases such as neoplasia, infections,
inflammatory conditions, or recent drug administration.15 False negative results are also common and can occur in up to 42% of all dogs with IMHA.16 Thus, this test can be prone to errors and is not sensitive enough to detect low levels of clinically relevant antibodies.9 Flow cytometry also detects antierythrocyte antibodies and is more sensitive for detecting IgG than is the Coombs test,
but it is not yet widely available to general practitioners.15,17
2. A peripheral blood smear from an anemic dog reveals that the RBCs are prominently clumped together, which indicates agglutination.
A saline dispersion test should be performed for confirmation. Additional findings in this smear include moderate polychromasia
and rare nucleated RBCs, which indicate a regenerative response, small round cells lacking central pallor suggestive of spherocytes,
and a neutrophilic leukocytosis, which indicates inflammation (Wright's-Giemsa stain; 500X).
Obtain abdominal radiographs to investigate triggers of hemolytic anemia, such as ingestion of zinc-containing foreign objects
or abdominal neoplasia. If abdominal radiographs reveal suspicious findings, an abdominal ultrasonographic examination is
warranted. In middle-aged to older dogs, it is recommended that routine thoracic radiographs also be obtained to investigate
whether primary or metastatic neoplasia may be a cause of IMHA.
Other diagnostic tests
A thorough search for underlying infection is warranted in cases of IMHA. Chronic infections, such as pyometra, abscesses,
urinary tract infections, and discospondylitis, have all been associated with triggering IMHA.1 Transmissible causes of IMHA, such as infection with the hemoprotozoan Babesia gibsoni, are increasingly frequent in the United States.18 A recent study described a breed predisposition to B. gibsoni infection in American pit bull terriers.18 Studies have also described transmission associated with blood transfusions and dog bites.19-21 Other infectious organisms, such as Ehrlichia and Dirofilaria species, have also been associated with anemia in dogs.22,23 Mycoplasmosis does not cause a clinically relevant anemia in dogs that have not undergone a splenectomy, but it can cause
a mild to severe anemia in cats.1