Correlation between in vivo hemolysis and the amount of red cell-bound IgG measured by flow cytometry

A flow cytometry method was used to compare the amount of red cell (RBC)-bound IgG in 73 patients with and without immune hemolytic anemia (IHA). The positive results in 10 of the direct antiglobulin tests (DATs) were idiopathic, and those in 25 were due to methyldopa therapy; 38 of the 73 DAT-positive patients were babies born to women with IgG alloantibodies of potential clinical significance. Normal blood donors with (n = 30) and without (n = 121) positive DATs were also tested. RBCs that had been strongly sensitized (4+ indirect antiglobulin test) in vitro with different quantities of IgG anti-D, but that had similar antiglobulin test (AGT) titration scores, could easily be differentiated by flow cytometry. The mean percent fluorescence of RBCs, incubated with fluorescein-labeled anti-IgG, from neonatal patients with IHA was higher than that of RBCs from those without IHA, but there was no statistical difference in the other groups. There was considerable overlap in the respective ranges of percent fluorescence of RBCs from patients with and without IHA in all groups. It was not possible to define a clear quantitative threshold differentiating patients with IHA from those without. Although flow cytometry was more precise and reproducible than standard serology (e.g., AGT titration scores), correlations of the amount of RBC-bound IgG and in vivo hemolysis were similar.