FLUORESCENT IN-SITU HYBRIDIZATION ON FLOW-SORTED CELLS AS A TOOL FOR EVALUATING MINIMAL RESIDUAL DISEASE OR CHIMERISM AFTER ALLOGENEIC BONE-MARROW TRANSPLANTATION

We studied the feasibility and the sensitivity of fluorescent in situ hybridization (FISH) using leukemic or host/donor-specific probes on f low-sorted cells to assess minimal residual disease (MRD) or chimerism in transplanted patients in complete remission. We first performed ex perimental models of MRD and chimerism by mixing HL60 cells and normal lymphocytes in different proportions. Over 80% HL60 cells were obtain ed from mixtures of 5% HL60 cells in peripheral blood mononuclear cell s (PBMC). We then evaluated MRD and mixed chimerism in a chronic myelo genous leukemia patient in relapse after allogeneic sex-mismatched bon e marrow transplantation (BMT), who had received a donor lymphocyte in fusion (DLI). Three months after DLI, mixed chimerism was observed in each bone marrow (BM)-sorted lineage (CD13+, CD14+, CD20+, and CD3+), with the highest level of recipient cells in the granulocytic lineage (CD13+). Five months after DLI, host cells were at a low level but rem ained detectable in the granulocytic lineage. In the same sample, the bcr-abl gene was detected in the granulocytic lineage and not in the l ymphocytes. We also studied chimerism in an aplastic anemia sex-mismat ched transplanted female patient. We determined the proportion of reci pient total lymphocytes, CD4+ and CD8+ lymphocytes, and CD14+ monocyte s under cyclosporin A therapy on five peripheral blood samples and one BM sample over 5 months. Results showed a regular decrease in recipie nt total lymphocytes (26.6% to 10.6%) and monocytes (20.7% to 8%). CD8 +-recipient cells decreased rapidly, while CD4+ remained stable (17%), This work demonstrates the feasibility of FISH after cell sorting, co mbining the sensitivities of both flow cytometry and FISH and the spec ificities of both immunophenotyping and genotype analysis. (C) 1998 Wi ley-Liss, Inc.