title: Fluorescent in situ hybridization on flow-sorted cells as a tool for evaluating minimal residual disease or chimerism after allogenic bone marrow transplantation.
abstract: We studied the feasibility and the sensitivity of fluorescent in situ hybridization (FISH) using leukemic or host/donor-specific probes on flow-sorted cells to assess minimal residual disease (MRD) or chimerism in transplanted patients in complete remission. We first performed experimental models of MRD and chimerism by mixing HL60 cells and normal lymphocytes in different proportions. Over 80% HL60 cells were obtained from mixtures of 5% HL60 cells in peripheral blood mononuclear cells (PBMC). We then evaluated MRD and mixed chimerism in a chronic myelogenous leukemia patient in relapse after allogeneic sex-mismatched bone marrow transplantation (BMT), who had received a donor lymphocyte infusion (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 remained detectable in the granulocytic lineage. In the same sample, the bcr-abl gene was detected in the granulocytic lineage and not in the lymphocytes. We also studied chimerism in an aplastic anemia sex-mismatched transplanted female patient. We determined the proportion of recipient total lymphocytes, CD4+ and CD8+ lymphocytes, and CD14+ monocytes under cyclosporin A therapy on five peripheral blood samples and one BM sample over 5 months. Results showed a regular decrease in recipient 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, combining the sensitivities of both flow cytometry and FISH and the specificities of both immunophenotyping and genotype analysis.
authors.affiliation: Laboratoire d'Hematologie, Université de Bordeaux II, France.
authors.forename: S
authors.initials: S
authors.lastname: Cotteret
authors.forename: F
authors.initials: F
authors.lastname: Belloc
authors.forename: J M
authors.initials: JM
authors.lastname: Boiron
authors.forename: C
authors.initials: C
authors.lastname: Bilhou-Nabera
authors.forename: P
authors.initials: P
authors.lastname: Dumain
authors.forename: C
authors.initials: C
authors.lastname: Boyer
authors.forename: F
authors.initials: F
authors.lastname: Lacombe
authors.forename: J
authors.initials: J
authors.lastname: Reiffers
authors.forename: P
authors.initials: P
authors.lastname: Bernard
mesh_terms: D000741:Anemia, Aplastic / Q000235:genetics / Q000628:therapy; D016026:Bone Marrow Transplantation; D002648:Child; D002678:Chimera; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005838:Genotype; D018922:HL-60 Cells; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000628:therapy; D018365:Neoplasm, Residual / Q000175:diagnosis* / Q000235:genetics; D010641:Phenotype