Detection of chimerism in subpopulations of cells by fluorescence in situ hybridization and immunofluorescent staining of cell surface antigens

Fluoresence in situ hybridization (FISH) enables the visualization of the X and Y chromosomes in nuclei of interphase cells using probes which recognize repetitive sequences on the X and Y chromosomes. The process of cell preparation, hybridization and visualization of the probes within the nuclei lasts between 16 and 24 h. Because of these features the method is ideal for monitoring engraftment after sex-mismatched bone marrow transplant (BMT). So far, the FISH technique has been applied to assess the degree of chimerism in either unseparated peripheral blood cells or unseparated bone marrow cells. However, for early detection of rejection or relapse, the degree of chimerism in sub-populations of cells is probably more informative. A new FISH protocol was developed with the aim of simultaneously visualizing the surface antigens by fluorescent antibodies and X or Y chromosomes with fluorescent probes. After development of the new FISH protocol, it was applied to patient samples after sex-mismatched BMT. The degree of chimerism was assessed comparing the results of the new FISH protocol with a 'standard' FISH protocol which is employed both in our and other labatories to detect chromosomes in interphase nuclei. Both methods gave similar results. The new FISH protocol was applicable for the detection of Y and X chromosomes. The clinical relevance of this new FISH protocol is illustrated by the results obtained on peripheral blood samples of five BMT patients who were mixed chimeras after sex-mismatched BMT. A clear distinction between engraftment of T and B cells was noted. In some patients the T cells were of donor origin while the B cells were of host origin.