Comparative study of immunohematological tests with canine blood samples submitted for a direct antiglobulin (Coombs’) test

BACKGROUND: A 2019 ACVIM consensus statement on diagnostics for immune-mediated hemolytic anemia (IMHA) in dogs made testing recommendations. As data on the performance of immunohematological tests was lacking, we undertook a comparative analysis. MATERIAL AND METHODS: Anticoagulated 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. 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°C), capillary tubes, and flow cytometry used in laboratories. RESULTS: Samples from healthy dogs yielded negative results with all immunodiagnostic tests. Among the 126 samples submitted for DAT 67 were positive by a DAT utilizing microtiter plates with goat anti-dog antiglobulin DAT at 22°C. Notably, DAT results were comparable and consistent across all evaluated methods regardless of antiglobulin and temperature used. DAT+ dogs were more severely anemic and more likely to have erythroid regeneration compared to DAT- dogs. 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. Among the DAT+ samples, 57% had agglutination, 87% had spherocytosis, and 45% had both. There was good correlation between spherocytosis and DAT results from the six DAT techniques, but the correlation with autoagglutination was only fair. Clinical follow-up was available for 42 dogs. Of the sample from 12 DAT+ dogs collected during treatment, 10 remained DAT+ when tested 1–24 weeks after initial assessment. CONCLUSIONS: Based 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. In addition, use of these tests for monitoring response of IMHA dogs to treatment might be valuable. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40575-021-00107-0.