Chromosome specific DNA hybridization in suspension for flow cytometric detection of chimerism in bone marrow transplantation and leukemia

Flow cytometry was used to measure the fluorescence intensity of nuclei that were subjected to fluorescent in situ hybridization in suspension with chromosome specific DNA probes. Paraformaldehyde-fixed nuclei were protein digested with trypsin and hybridized simultaneously with a biotin-and DIG labeled chromosome specific centromere probe. A number of probes were tested in the suspension hybridizations. The method yielded fluorescent hybridization signals that allow discrimination between Y chromosome positive and negative nuclei when analyzed by flow cytometry. The method is especially suited for analysis of bone marrow cells derived from patients who have received a sex-mismatched allogeneic bone marrow transplantation. Male leukemia cells with a trisomy for chromosome 8 were mixed with normal female cells and simultaneously hybridized in suspension with a DIG labeled probe specific for chromosome 8 and the biotin labeled Y chromosome probe. Y chromosome positive or negative nuclei were s

The use of FISH with chromosome-specific repetitive DNA probes for the follow-up of leukemia patients

The use of fluorescence in situ hybridization (FISH) for the purpose of repeated follow-up examination of bone marrow samples from 38 leukemia patients was investigated. On the basis of conventional cytogenetic analysis, patients with acute leukemia whose leukemic cells carried numerical chromosomal aberrations were selected and followed with repetitive DNA probes that specifically hybridize to one chromosome type. Repeated cytogenetic metaphase analyses would have been laborious and not sensitive or quantitative enough to follow declining numbers of aberrant cells. FISH, as an interphase cytogenetic technique, provides a rapid and simple alternative with high sensitivity. Although FISH data before and after chemotherapy were in agreement with bone marrow cytology in 30 of 38 patients, discrepancies were noticed in specific cases. These could be explained by the presence of cytogenetically distinct subclones that behave differently during treatment, the presence of differentiated leukemic cells, changes in the chromosomal constitution caused by clonal relapse, or the fact that a numerical aberration is found by conventional chromosome banding analysis while the target region to which the probe is directed is still present in the nucleus as a diploid set