The Indirect Antiglobulin (Coombs’) Test Is Specific but Less Sensitive Than the Direct Antiglobulin Test for Detecting Anti-Erythrocytic Autoantibodies and Thereby Immune-Mediated Hemolytic Anemia in Dogs

SIMPLE SUMMARY: In immune-mediated hemolytic anemia (IMHA), autoantibodies shorten the survival of red blood cells, thereby causing anemia. A life-threatening inflammatory and thrombotic complication may also develop in dogs with IMHA. Although pale or icteric mucous membranes and lethargy are common clinical signs, the diagnosis of IMHA is based on routine blood and immunological tests. The antiglobulin (Coombs’) test (AT) detects the presence of autoantibodies either bound to red blood cells (direct, DAT) or unbound in serum or plasma (indirect, IAT). While the DAT has been recommended for the diagnosis of canine IMHA, the value of the IAT is less clear. We tested five different IAT methods in 94 dogs suspected of having IMHA: half of which had DAT+ results. Slightly more than half of the DAT+ dogs were also IAT+ when assessed with the laboratory microtiter plate or in-clinic gel minitube kit methods. However, IAT+ results were seen in fewer than half of the DAT+ dogs when using the microcapillary, gel column, or immunochromatographic strip kit methods. Our results suggest that the DAT is superior for the diagnosis and monitoring of IMHA in dogs but that the IAT may be helpful for diagnosis when serum, but not fresh whole blood, is available from an anemic dog. ABSTRACT: The immunodiagnostic assessment of dogs suspected of having immune-mediated hemolytic anemia (IMHA) is based on persistent autoagglutination of erythrocytes (after three saline washes), marked spherocytosis, and a positive direct antiglobulin (Coombs’) test (DAT). However, the value of using the indirect antiglobulin test (IAT) for the detection of anti-erythrocytic autoantibodies in serum from dogs suspected of having IMHA is unclear. To evaluate the IAT, leftover serum samples from a large cohort of 94 dogs suspected of having IMHA and for which DAT results were known were incubated with DAT− erythrocytes, and five IAT techniques were performed (in part with different reagents and temperatures): microtiter plate (MICRO), microcapillary, laboratory gel column, gel minitube kit (GEL KIT), and immunochromatographic strip kit. Two IAT techniques (MICRO at 37 °C and GEL KIT with rabbit anti-dog polyvalent reagent) detected autoantibodies against erythrocytes in serum from 53% and 57% of DAT+ dogs, respectively, while other IATs performed less well. Moreover, while the analytic specificity of the IAT methods compared to the DAT ranged from 96–100%, the sensitivity range was only 9–57%. Thus, we still recommend DAT for diagnosis and monitoring of IMHA in dogs but conclude that a positive IAT result may aid diagnostically when serum is available, but fresh red blood cells are not.