Flow cytometric evaluation of bound IgG on erythrocytes of anaemic dogs

The detection of red blood cell (RBC)-bound immunoglobulins in case of anaemia with the direct agglutination test (DAT or Coombs test) has been reported to be of low sensitivity. We therefore tested the applicability of flow cytometry for the detection of canine IgG on RBC using two different IgG-specific secondary reagents: goat-anti-dog IgG (GαD-IgG) and rabbit-anti-dog IgG (RαD-IgG). Membrane staining RBC samples were performed at 4 °C. Comparisons of agglutination test at 37 °C and 4 °C showed, that binding of the secondary antibodies at 4 °C was more sensitive compared to agglutination at 37 °C and the two antisera differed considerable in their agglutination activity. Binding of GαD-IgG and RαD-IgG to RBC of healthy dogs (n=15) was low and mean fluorescence intensities were taken to calculate thresholds above which RBC of patients were judged positive. As in agglutination tests, both secondary antisera displayed considerable differences (concentration-dependent binding and histogram profiles) after flow cytometric analysis. Using flow cytometry, with GαD-IgG 8 of 17 agglutination-negative patients were positive and RαD-IgG was positive with 3 of 3 agglutination-negative RBC samples. Thus, flow cytometric analysis of proved to be a sensitive technique, detecting RBC-bound canine IgG of DAT-negative patients. The results of both techniques, however, are significantly influenced by the used IgG-specific polyclonal reagents.