SIMULTANEOUS DETECTION OF X-CHROMOSOMES AND Y-CHROMOSOMES BY 2-COLOR FLUORESCENCE IN-SITU HYBRIDIZATION IN COMBINATION WITH IMMUNOPHENOTYPING OF SINGLE CELLS TO DOCUMENT CHIMERISM AFTER SEX-MISMATCHED BONE-MARROW TRANSPLANTATION

A powerful approach to documenting engraftment after allogeneic BMT is the quantification of the degree of chimaerism in distinct haematopoi etic cell lineages, This cannot be achieved by the recently developed, quantitative, modifications of PCR amplification of highly polymorphi c DNA markers, unless this technique is applied to separated cell popu lations, Here, we report the development of a new method, in which cel ls are simultaneously characterized by enzymatic immunophenotyping and identified for their origin by two-colour fluorescence in situ hybrid ization with X and Y chromosome-specific DNA probes (XY-FISH/immunosta ining), The method enables the rapid, reliable and quantitative analys is of chimaerism within distinct cell lineages after sex-mismatched BM T, without the requirement for cell separation techniques, This is ill ustrated by investigation of the pattern of chimaerism in patients rec eiving a sex-mismatched BMT for the treatment of primary immunodeficie ncies. The results obtained with the quantitative XY-FISH/immunostaini ng method show a good correlation with the data generated by the semi- quantitative analysis of PCR amplified minisatellites in FAGS-sorted t ell fractions, In addition, XY-FISH/immunostaining was successfully ap plied to detect materno-fetal engraftment of T cells in a SCID patient .