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.

Bivariate, Chimerism, Chromosome, DNA-probe, Dual laser, Fluorescence microscopy, Human, Hybridization, Sex difference, Suspension
dx.doi.org/10.1002/cyto.990190410, hdl.handle.net/1765/67600
Cytometry
Department of Hematology

Arkesteijn, G.J.A, Erpelinck, S.L.A, Martens, A.C.M, & Hagenbeek, A. (1995). Chromosome specific DNA hybridization in suspension for flow cytometric detection of chimerism in bone marrow transplantation and leukemia. Cytometry, 19(4), 353–360. doi:10.1002/cyto.990190410