• Autoantibodies;
  • Direct antiglobulin test;
  • Immune-mediated hemolytic anemia;
  • Osmotic fragility


Difficulties with the direct antiglobulin test (DAT) and its apparent lack of sensitivity and specificity for immune-mediated hemolytic anemia (IMHA) in dogs have raised skepticism regarding its diagnostic value.


To compare different DATs and other hematologic parameters in dogs.


Anticoagulated blood samples from 59 nonanemic and 46 anemic dogs (± IMHA) from a research colony and veterinary clinics.


Prospective observational study: Immunochromatographic strip, gel microcolumn, and capillary techniques were compared with standard microtiter DAT using 2 polyvalent antiglobulins. Spherocytosis, autoagglutination, osmotic fragility, and clinical data were assessed.


Blood samples from all 59 nonanemic dogs were DAT-. Among 46 anemic dogs, 33 were suspected of IMHA, but only 20 were DAT+. Old and new DAT methods yielded comparable and consistent results even after storage of chilled blood samples for 1 week. Spherocytosis and autoagglutination (that did not persist after washing) were noted in 15 and 16 DAT+ dogs, respectively. The other 26 anemic dogs, including 21 previously transfused dogs and 4 with autoagglutination, tested DAT- by the other methods. Osmotic fragility was increased in 70% (19/27) of anemic and all 15 DAT+ dogs tested. Limited follow-up testing revealed DAT+ results for 3–70 days.

Conclusions and Clinical Importance

The novel strip and capillary DAT methods are promising adjunct in-clinic tools. Despite prior immunosuppressive treatment and presence of autoagglutination, the DAT was positive in anemic dogs with IMHA. Transfusion did not cause false DAT+ results. Our results support DAT as a cornerstone in the diagnosis of canine IMHA.