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 25-28 September, 2018 | Singapore
If erythrocytes are only partially phagocytized or lysed
by complement in circulation, erythrocytes with reduced surface area to volume ratio, known as spherocytes, are formed. They appear spherical and microcytic with no central pallor and are considered fragile. Note proper areas on the blood smear needs to be reviewed to find spherocytes in between single regular discoid red cells. Large numbers of spherocytes (>20/microscopic high power field) are nearly diagnostic for IMHA, whereas small numbers may be seen with other conditions including DIC, endotoxemia and zinc intoxication. In our experience all dogs with marked spherocytosis and suspected to have IMHA also had a positive Coombs’ test. However, only 60-80% of dogs with a positive Coombs’ test or clinically diagnosed with IMHA had marked spherocytosis. Hereditary spherocytosis due to genetic membrane defects has rarely been seen in dogs, but should be considered as a differential diagnosis in dogs with negative Coombs’ test results.
Because of the difficulties with the Coombs’ test
(see below), Slappendale had proposed to use the erythrocytic osmotic fragility test at specific saline concentrations as a mean to diagnose IMHA and this test is currently used in various clinics in Europe. However, there are many other reasons for increased fragility of erythrocytes beside IMHA including hereditary red cell defects. This test is not used in human medicine and
has not been shown to be superior to determination
of marked spherocytosis and a positive Coombs’ test
in dogs with IMHA. The osmotic fragility test is also a cumbersome and not well standardized technique.
Positive Direct Coombs’ Test Result
The direct Coombs’ test is also known as direct antiglobulin test (DAT) 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 (subagglutinating titer). Separate canine-specific IgG, IgM, and C3b antibodies as well as polyvalent antiglobulin reagents are available. They are added at various concentrations after washing the patient’s erythrocytes free of plasma (3x as shown above) and mixtures are generally incubated at room temperature or 37°C (cold agglutinins appear to be rarely of clinical importance and rarely cause hemolysis). The strength of the Coombs’ reaction does not necessarily predict the severity of hemolysis, but reaction changes are useful in monitoring the disease.
Typically tube or microtiter methods have been used exclusively in the reference or teaching laboratory setting, but a flow cytometric method has also been introduced in a couple of places. A standardized, sensitive, and simple gel column method was available
by DiaMed (Switzerland), but unfortunately the company was sold to another company which decided to not pursue the veterinary market. A novel standardized antiglobulin test method has just been developed by Alvedia (France) similar to the immunochromatographic strip technique for blood typing of dogs and cats (see updates on blood typing and crossmathing). Although many commercial laboratories offer Coombs’ testing for dogs, clinicians have questioned the tests sensitivity and specificity and often forgo the test and/or use response to therapy as a diagnostic. However, negative Coombs’ test results may be seen because of technical reasons, insufficient quantities of bound antibodies, the presence of weakly bound antibodies, or the disease in remission. The Coombs’ test stays positive for days to months after initiating treatment. A few days of immunosuppressive therapy will likely not reverse the Coombs’ test result, as unlikely a transfusion would cause a positive Coombs’ test result. Thus, dogs with negative Coombs’ test results should be reevaluated for other causes of hemolytic anemia.
In a recent prospective study of anemic and non- anemic dogs we compared various direct Coombs’ test methods including microtiter plate assays, gel column, capillary, and immunochromatographic techniques using polyvalent antiglobulins in a laboratory setting and found excellent correlations between tests and with spherocytosis and without noticeable interference by immunosuppressive or transfusion therapy in anemic dogs.
In conclusion, a diagnosis of IMHA requires the documentation of red blood cell destruction and an immune process. While regenerative anemia, icterus, and hyperbilirubinuria are suggesting a hemolytic anemia, evidence of true autoagglutination, spherocytosis, and/or a positive direct Coombs’ test are required to document immune destruction. The authors also recommend monitoring IMHA patients for the disappearance of these immunological parameters to adjust and taper therapy.
Therapeutic Considerations
A diagnosis of IMHA requires the documentation of red blood cell destruction and an immune process. While regenerative anemia, icterus, and hyperbilirubinuria are indicating the presence of a hemolytic anemia, evidence of (1) true autoagglutination after washing, (2) marked spherocytosis, and/or (3) a positive direct Coombs’
test are required to document immune destruction.
The prognostic factors for IMHA are poorly defined unless IMHA is secondary to an underlying disease. Severe anemia, icterus, leukocytosis, hypoalbuminemia and thrombotic evidence are unfavorable findings. Because the severity of IMHA ranges from indolent to life-threatening disease and serious complications seen with IMHA, therapy has to be tailored for each patient and depends in part on whether the IMHA is primary or

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