Detection of Y chromosome by in situ hybridization in combination with membrane antigens by two-color immunofluorescence.

Discrimination between donor and recipient peripheral blood cells through detection of the Y chromosome can be useful to document chimerism and engraftment after sex-mismatched bone marrow transplantation. Currently applied methods are hampered by the selection of cells (e.g., karyotyping of cells in metaphase) or by the fact that the detection of Y chromosome by in situ hybridization with specific probes does not allow further characterization of the cells. Although quinacrine staining of Y chromosomes can be performed on cells previously marked for membrane antigens, this staining is not fully discriminative between male and female cells. To circumvent this, a technique has been developed, in which mononuclear cells in suspension were stained for membrane antigens by the consecutive use of monoclonal antibodies and tetramethylrhodamine isothiocyanate conjugates. After the cells were spun down on slides and fixed with methanol/acetic acid and formaldehyde, in situ hybridization with a biotinylated Y-chromosome-specific DNA probe was performed. The probe was detected with avidin-fluorescein isothiocyanate and the signal was amplified by consecutive incubation with biotinylated anti-avidin and avidin-fluorescein isothiocyanate. The membrane staining for various antigens remained undisturbed during the hybridization procedure and the Y probe discriminated almost completely between male and female cells. Therefore, this approach allowed us to determine the chimerism within different subpopulations of unseparated mononuclear cells after sex-mismatched bone marrow transplantation with a sensitivity of 98% and a specificity of 100%.