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.
Robson
A
A
Tissue Typing Laboratory, St Mary's Hospital, Manchester, UK.
Martin
S
S
D001402:B-Lymphocytes / Q000166:cytology*; D005189:False Positive Reactions; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes; D006650:Histocompatibility Testing; D006801:Humans; D007518:Isoantibodies / Q000032:analysis; D013154:Spleen / Q000166:cytology; D013601:T-Lymphocytes / Q000166:cytology*
D005456:Fluorescent Dyes; D007518:Isoantibodies