A New Method for Improving Metaphase Chromosome Spreading

Abstract

Background: The success of complex molecular cytogenetic studies depends on having properly spread chromosomes. However, inconsistency of optimum chromosome spreading remains a major problem in cytogenetic studies. Methods: The metaphase spreading process was carefully timed to identify the most critical phase of chromosome spreading. The effects of dropping height of cell suspension, slide condition, drying time, fixative ratio, and relative humidity on the quality of metaphase spreads were studied by quantitative examination of metaphase chromosome spreads. Normal and immortalized human epithelial ovarian cells, neuroblastoma cells, and normal lymphocytes were tested. Results: Humidity over the slide was the most important variable affecting the quality of chromosome spreads. Consistent improvement in chromosome spreading (larger metaphase area, less chromosome overlaps, or lower frequencies of broken metaphases) was obtained for all cell types if dynamic cell rehydration, occurring as fixative absorbs moisture from air, was made to coincide with the prompt fixation of spread chromosomes to the slide. This was achieved by dropping cells on dry glass slides placed in a shallow metal tray and then quickly lowering the tray into a covered 50°C water bath for slide drying. Conclusions: A new and simple method for improving metaphase chromosome spreading was developed based on our study on the characteristics of chromosome spreading. © 2002 Wiley-Liss, Inc.