Flow Cytometric Analyses of the Subclasses of Red Cell IgG Antibodies

Abstract

We report on flow cytometric IgG subclass determinations of red cell antibodies using polyclonal FITC-labeled antibodies. The limit of detection of this method was 1 ng anti-D per 1 x 10(7) red cells. The inter- and intra-assay coefficients of variance were 8.2 and 2.3%, respectively. In 8 newborns with a positive direct antiglobulin test (DAT) in the gel centrifugation test (GCT), due to ABO antibodies, IgG1 was detected in all and IgG2 additionally in 4 of these cases. In 5 severe cases of hemolytic disease of the newborn (HDN) due to anti-D, large amounts of IgG1 were found, and in 3 of these 5, IgG3 in combination with IgG1. In 8 mild or moderate HDN cases (4 anti-D, 2 anti-E, 1 anti-Fya, 1 anti-Jka), phototherapy sufficed, and IgG1 was the only antibody. In 7 adult patients with malignant lymphoma and a positive DAT (GCT), only small amounts of IgG1 red cell autoantibodies could be demonstrated by flow cytometry. In 5 further patients with malignant lymphoma, a positive DAT, and severe hemolytic anemia, large amounts of IgG1 autoantibodies were found and IgG3 was also present in 3 of these cases. Flow-cytometric determination of IgG subclasses may be a useful tool in immunohematology, since subclass determinations were possible in all of these cases. This method is suited for clinical routine and offers the possibility of sufficient standardization.

A newly developed gel centrifugation test for quantification of RBC‐bound IgG antibodies and their subclasses IgG1 and IgG3: comparison with flow cytometry

“…Sonneborn, PhD, Biotest, Dreieich, Germany) for positive controls and for calibrations. Anti‐D sera were tested after incubation with O, D.ccEe RBCs by using the IAT as described 3 …”

A dispermic chimerism in a 2-year-old Caucasian boy

“…Following subsequent incubation with FITC-conjugated sheep anti-humanIgG, cells were analyzed on a FACScalibur [5] (Becton Dickinson, Heidelberg, Germany).…”

Efficacy of D– red blood cell transfusion and rituximab therapy in autoimmune hemolytic anemia with anti‐D and panreactive autoantibodies arising after hematopoietic stem cell transplant

“…IgG subclasses (IgG 1 , IgG 2 , IgG 3 , and IgG 4 ) of autoantibodies were determined using plasma samples taken on Days 2, 7, and 10, as previously described . Type O DccEE and type A ddccEE RBCs were sensitized by the patient's plasma for 60 minutes at 37°C.…”