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

Octavian Henegariu, Nyla A. Heerema, Lisa Lowe Wright, Patricia Bray-Ward, David C. Ward, Gail H. Vance

Research output: Contribution to journalArticle

151 Scopus citations

Abstract

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 comparative 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 difficult cell suspensions and is unaffected by the environmental conditions. Chromosome spreads were suitable for both banding and FISH techniques common to the cytogenetic laboratory. Chemical aging is a rapid slide pretreatment procedure for FISH applications, which allows freshly prepared cytogenetic slides to be used for in situ hybridization within 30 min, thus increasing analytical throughput and reducing bench-work. Furthermore, the gradually denaturing process described allows the use of fresh biologic material with optimal FISH results while protecting chromosomal integrity during denaturing. Conclusion: The slide preparation and slide pretreatment protocols can be performed in any laboratory, do not require specialized equipment, and provide robust results.

Original languageEnglish (US)
Pages (from-to)101-109
Number of pages9
JournalCytometry
Volume43
Issue number2
DOIs
StatePublished - Feb 1 2001
Externally publishedYes

Keywords

  • Alcohol
  • CGH
  • Cytogenetics
  • FISH technique
  • G-banding
  • Hybridization
  • M-FISH
  • Metaphase spreading
  • Protocol
  • Slide aging

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Biophysics
  • Hematology
  • Endocrinology
  • Cell Biology

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