T and B cell crossmatching using three-colour flow cytometry

We have developed a highly sensitive method for the simultaneous crossmatching of T and B cells using three-colour flow cytometry. This method identifies T and B cell populations in a lymphocyte sample and analyses each for the presence of bound patient IgG alloantibodies. A fluorescein isothiocyanate conjugated rabbit anti-human IgG antibody was used to detect bound IgG following incubation of donor lymphocytes with patient serum. R-phycoerythrin conjugated anti-human CD19, and Quantum Red conjugated anti-human CD3 monoclonal antibodies were used to detect T and B cell populations, respectively. A comparison of results between conventional and three-colour flow cytometry crossmatches showed that there was no difference in detection of bound IgG between the two methods for ten test sera, doubling dilutions of a positive control serum and 72 donor-recipient pairs. Three-colour flow cytometry has proved particularly helpful in determining whether an antibody is directed against donor T and/or B cells and was shown to be more sensitive than complement-dependent cytotoxicity assay in eight out of 72 crossmatches.