Improvements in cytogenetic slide preparation: Controlled chromosome spreading, chemical aging and gradual denaturing

Background: Metaphase spreading is an essential technique for clinical and molecular cytogenetics. Results of classical banding techniques as well as complex fluorescent in situ hybridization (FISH) applications, such as comp arative genomic hybridization (CGH) or multiplex FISH (M-FISH), are greatly influenced by the quality of chromosome spreading and pretreatment of the slide prior to hybridization. Materials and Methods: Using hot steam and a metal plate with a temperature gradient across its surface, a reproducible protocol for slide preparation . aging, and hybridization was developed. Results: This protocol yields good chromosome spreads from even the most di fficult cell suspensions and is unaffected by the environmental conditions. Chromosome spreads were suitable for both banding and FISH techniques comm on to the cytogenetic laboratory. Chemical aging is a rapid slide pretreatm ent procedure for FISH applications, which allows freshly prepared cytogene tic slides to be used for in situ hybridization within 30 min, thus increas ing analytical throughput and reducing benchwork. Furthermore, the graduall y denaturing process described allows the use of fresh biologic material wi th optimal FISH results while protecting chromosomal integrity during denat uring. Conclusion: The slide preparation and slide pretreatment protocols can be p erformed in any laboratory, do not require specialized equipment, and provi de robust results. (C) 2001 Wiley-Liss. Inc.