Rapid detection of IgG subclasses on DAT positive RBC membranes by flow cytometry (FC)

Red blood cell (RBC)-bound IgG can cause hemolysis resulting, e.g. in severe cases of autoimmune hemolytic anemias (AIHA) or in hemolytic disease of the newborn (HDN). Serologic detection and differentiation of these antibodies are often difficult in cases of low antibody titers. We investigated 36 cases of poly- and monospecific IgG-positive DATs by flow cytometry. The RBC samples were washed, diluted, and incubated with monoclonal antibodies directed against IgG1, IgG2, IgG3, and IgG4, respectively. Analysis was performed on a flow cytometer. Nine cases were negative for all 4 IgG subclasses, 8 cases were positive for IgG2, 5 for IgG1, 5 for IgG3, and 3 for all four subclasses. In 6 patients we found combinations of 2 or 3 subclasses (2 for IgG1 and IgG2, 1 for IgG1 and IgG3, 1 for IgG3 and IgG4, 1 for IgG1, IgG2 and IgG4, 1 for IgG1, IgG3 and IgG4). Serological differentiation revealed specific anti-bodies only in 3 cases (anti-Lea, anti-Leb, anti-P1). The type of IgG subclass and the amount of RBC-bound IgG is relevant for the degree of in vivo RBC destruction. Flow cytometry provides a rapid, highly sensitive, cost efficient, and specific tool for IgG detection, including subgroups.

We therefore recommend flow cytometric analysis to be integrated into the serological decision process as an additional method for serological problem cases.