There remain disparities regarding the routinely used diagnostic techniques and interpretations of specific test results for diagnosis of IMHA in veterinary clinics as well as in laboratories, as recently illustrated in a small survey by the Veterinary and Comparative Clinical Immunology Society. In 2019, the American College of Veterinary Internal Medicine (ACVIM) consensus statement offered similar guidelines, but the data substantiating these recommendations were sparse. For many years, we have recommended that IMHA be diagnosed by evidence of in vivo hemolysis and one of three specific immunohematological test results: persistent autoagglutination after three saline washes, marked spherocytosis, and/or a positive DAT result. Approaches to the diagnosis of IMHA in dogs remain controversial, with some investigators and practicing clinicians applying strict hematological and immunological parameters, while others use a combination of RBC agglutination, spherocytosis or responsiveness to immunosuppressive therapy as diagnostic criteria. Immune-mediated hemolytic anemia (IMHA) is driven by accelerated destruction of antibody-coated red blood cells (RBCs). They confirm marked spherocytosis and a positive DAT result as useful clinical parameters for diagnosing and potentially monitoring this disease. These concordant results underscore the diagnostic value of both laboratory and in-clinic DAT methods for IMHA in dogs. Furthermore, dogs with DAT positive results were more severely anemic and more likely to have active bone marrow responses compared to dogs with negative DAT results. Marked spherocytosis (small spherical RBCs) and to a lesser extend autoagglutination (clumping of RBCs) also correlated with the DAT results. The results of four laboratory and two in-clinic DAT kits were comparable and consistent. Using a variety of direct antiglobulin (Coombs’) test (DAT) methods, antibody-coated RBCs were detected in approximately half of the samples from dogs with suspected IMHA. In addition, 28 samples from healthy non-anemic dogs served as negative controls. We compared routine and specific blood test methods in samples from 126 dogs suspected of having IMHA. The clinical diagnosis of IMHA requires blood tests and can be challenging due to the lack of established reference methods and reagents. In addition, use of these tests for monitoring response of IMHA dogs to treatment might be valuable. Conclusionsīased upon this comparative prospective survey, all in-clinic and laboratory DAT techniques produced similar results when performed by trained personnel and can therefore be recommended for detection of antibody-coated erythrocytes and immunohematological diagnosis. Of the sample from 12 DAT+ dogs collected during treatment, 10 remained DAT+ when tested 1–24 weeks after initial assessment. Clinical follow-up was available for 42 dogs. There was good correlation between spherocytosis and DAT results from the six DAT techniques, but the correlation with autoagglutination was only fair. Among the DAT+ samples, 57% had agglutination, 87% had spherocytosis, and 45% had both. Macroscopic agglutination in tubes or on slides was observed in 48 samples after 1:1 and 1:4 blood to saline dilution, but only persisted in four samples after washing. DAT+ dogs were more severely anemic and more likely to have erythroid regeneration compared to DAT- dogs. Notably, DAT results were comparable and consistent across all evaluated methods regardless of antiglobulin and temperature used. Among the 126 samples submitted for DAT 67 were positive by a DAT utilizing microtiter plates with goat anti-dog antiglobulin DAT at 22☌. Samples from healthy dogs yielded negative results with all immunodiagnostic tests. Samples were also subjected to different DATs: a gel minitube and an immunochromatographic strip kit used in clinics neutral gel column cards, microtiter plates (at 4°, 22°, and 37☌), capillary tubes, and flow cytometry used in laboratories. Material and methodsĪnticoagulated blood samples from 126 dogs suspected of having IMHA submitted to a diagnostic veterinary laboratory for a routine direct antiglobulin test (DAT) and from 28 healthy control dogs were evaluated for spherocytosis and autoagglutination before and after three saline washes. As data on the performance of immunohematological tests was lacking, we undertook a comparative analysis. A 2019 ACVIM consensus statement on diagnostics for immune-mediated hemolytic anemia (IMHA) in dogs made testing recommendations.
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