DETECTION OF MIXED CHIMERISM AND LEUKEMIC RELAPSE AFTER ALLOGENEIC BONE-MARROW TRANSPLANTATION IN SUBPOPULATIONS OF LEUKOCYTES BY FLUORESCENT IN-SITU HYBRIDIZATION IN COMBINATION WITH THE SIMULTANEOUS IMMUNOPHENOTYPIC ANALYSIS OF INTERPHASE CELLS

Serial blood and marrow specimens from eight adult recipients of sex-m ismatched transplants (BMT) for chronic myeloid leukemia (CML, n = 3), Ewing sarcoma (n = 1), acute myeloid leukemia (AML) in second remissi on (n = 1), acute lymphatic leukemia (ALL, n = 1) and multiple myeloma (n = 2) were analyzed by the simultaneous immunophenotypic CD3, CD4, CD8, CD20, CD34, CD10 and genotypic analysis (for X and Y chromosomes) . This combined technique of moAb/APAAP staining for cell surface and cytoplasmic antigens and fluorescence in situ hybridization (FISH) for the detection of sex chromosomes allowed the qualitative and quantita tive evaluation of mixed chimerism and/or relapse. Using the same slid es for moAb/APAAP and FISH allowed the simultaneous identification of the cell lineage, the lymphocyte subpopulation and the genotype (XX or YX) in every blood or BM specimen analyzed. A mixed chimerism in the T cell (CD4, CD8(+): median 26% host cells, range 5-44%) and in the my elomonocytic cell population (CD14(+) median 16% host cells, range 5-5 0%) was observed at day +7 after BMT. By days +14 to +18 this mixed ch imerism was reduced to 18% host T cells (range 5-50%) and 7% host myel omonocytic cells (range 0-20%). Beyond days +21 to +28 a stable donor chimerism for T cells, myelomonocytic cells and granulocytes was obser ved in seven of eight patients. Still 0.5-1% host cells of different l ineages were detectable in five from the eight patients at later time points (>day + 100). In three patients with CML these cells were CD13 or CD13, CD34 positive and in one was CD4, CD8 positive. In two patien ts with common-ALL and multiple myeloma respectively, the host cell le ukemic relapse expressing the original phenotype was detected at days +176 and +294, respectively. Thus, this method provides new possibilit ies for investigating the kinetics of mixed chimerism as well as early detection of leukemic relapse after BMT.