title: Chromosome specific DNA hybridization in suspension for flow cytometric detection of chimerism in bone marrow transplantation and leukemia.
abstract: 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 sorted onto microscope slides and subsequently classified as being leukemic or not by fluorescence microscopy, on the basis of the presence of a trisomy for chromosome 8. A 120-fold enrichment could be achieved when 300 Y positive nuclei were sorted from a mixture originally containing 0.5% leukemia cells. Given the specificity of the flow cytometry and FISH procedure, the combination of the two methods can reach a lower detection level of 1 per 250,000.
authors.affiliation: Institute of Hematology, Erasmus University, Rotterdam, The Netherlands.
authors.forename: G J
authors.initials: GJ
authors.lastname: Arkesteijn
authors.forename: S L
authors.initials: SL
authors.lastname: Erpelinck
authors.forename: A C
authors.initials: AC
authors.lastname: Martens
authors.forename: A
authors.initials: A
authors.lastname: Hagenbeek
mesh_terms: D016026:Bone Marrow Transplantation; D002678:Chimera / Q000235:genetics*; D002869:Chromosome Aberrations; D004247:DNA / Q000235:genetics*; D015342:DNA Probes; D005260:Female; D005434:Flow Cytometry / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods* / Q000706:statistics & numerical data; D007938:Leukemia / Q000235:genetics; D008297:Male; D012680:Sensitivity and Specificity; D014960:X Chromosome; D014998:Y Chromosome
chemical_list: D015342:DNA Probes; D004247:DNA