Reverse chromosome painting for the identification of marker chromosomes and complex translocations in leukemia

Background: Chromosome banding techniques and in situ hybridization reveal the majority of chromosomal aberrations. However, difficulties remain in ca ses of highly contracted chromosomes, poor quality of the metaphases or the presence of markers with the involvement of several chromosomes. Here, it is demonstrated that reverse painting can be applied successfully starting with bone marrow cells from primary acute myelocytic leukemias (AML). Methods: This was accomplished by culturing the leukemic cells with a cockt ail of various growth factors, which yielded sufficient numbers of cells in cycle to harvest chromosomes for sorting. Aberrant chromosomes were flow-s orted and amplified by degenerate oligonucleotide-primed PCR. The resulting products were labeled by nick-translation and hybridized on normal metapha se spreads. Results: Two patients with marker chromosomes in their leukemia tells were analyzed in detail. The hybridization pattern displayed the composition of the aberrant sorted chromosome. Results were compared with conventional cyt ogenetic analyses that were performed on material obtained from the same as pirate. The reverse-painting technique enabled identification of aberration s that were not detected by conventional cytogenetic analysis. Conclusions: Primary AML cells can be cultured in vitro, using optimal cult ure conditions, facilitating the production of high quality now karyotypes, suitable for sorting of marker chromosomes to produce DOP-PCR derived chro mosome painting probes for reverse painting. Valuable additional cytogeneti c information can thus be obtained about complex chromosomal rearrangements or structural aberrations that could not be completely resolved by convent ional cytogenetic analysis. (C) 1999 Wiley-Liss, Inc.