The immune-mediated hemolytic anemia (IMHA) is the most common diagnosed immune disorder in dogs. The direct antiglobulin test (DAT) is realized to confirm the IMHA. The flow cytometric technique has been used to detect erythrocyte-bound antibodies in human and most recently in dogs.

A direct flow cytometric erythrocyte immunofluorescence assay (FC) was developed and compared with direct antiglobulin test (DAT) for detection of erythrocyte-bound immunoglobulin (IgG and IgM) and complement (C3) in three dogs with IMHA attended at the Veterinarian Hospital of São Paulo State University-Campus of Jaboticabal-Brazil. The three dogs (all female) were 2-4 year-old. One dog was mongrel (D1) and two were English Cocker Spaniel (D2 and D3). The tests were performed during the diagnostic and repeated weekly after the beginning of treatment with prednisone (1mg/kg q12h/PO). Healthy dogs were used as a negative control.

The FC and DAT were negative for erythrocyte-bound immunoglobulin in all healthy dogs. During the IMHA diagnostic, the three dogs were positives for IgG and IgM on FC, whereas all dogs were positives for IgG and just one dog for IgM on DAT. C3 was positive for one dog on FC. After seven days of treatment, all dogs showed a improved clinical aspect, where D1 showed an increase of IgG level, D2 showed a decrease of IgG level and D3 showed a negative DAT. The D2 and D3 animals did not show important variation on FC, whereas D1 showed a IgG increase. After 14 days of treatment, D1 showed an aggravation of clinical aspect, low decrease of IgG and IgM on FC and negative DAT. D1 dead after 15 days of treatment. D3 showed a high decrease of IgM, IgG and C3 on FC after 14 days of treatment, however, the treatment was discontinued and it was observed an increase of immunoglobulins on FC when the fourth clinical consultation was performed, where the DAT remained negative. After the therapy recovery, the values decreased back. D2 showed negative DAT and high decrease of immunoglobulins after 21 days of treatment, however, as the prednisone decreased (0.5/kg q24/PO), there was an increase of IgG and C3 on FC, and positive IgG on DAT.

The FC showed to be a more sensitive method for erythrocyte-bound immunoglobulin and complement detection than DAT, mainly with the use of prednisone. In this work, a correlation between the interruption or reduction of treatment with the increase of immunoglobulin amount was detected on FC.