Combined detection of phenotype and Y chromosome by immunoenzyme labelling and in situ hybridisation on peripheral lymphocytes

Abstract A novel staining method for simultaneously determining the immunophenotype and sex of peripheral lymphocytes is described. Cell surface markers on lymphocytes are identified using specific monoclonal antibodies located by a peroxidase anti-peroxidase staining technique (PAP). Lymphocytes are subsequently hybridised with a biotinylated Y chromosome-specific sequence probe and this is located by avidin-biotin-alkaline phosphatase staining. Following the two staining steps the preparation is examined and in the same field lymphocytes show brown peroxidase staining of cell surface markers and red staining of the Y chromosome. The application of this combined staining technique provides a convinient method for studying the development of different cell lineages following sex-mismatched bone-marrow transplantation and for the identification of chimeric situations. The method has been shown to be sensitive and reproducible.