Chromosome spreading techniques for primary gastrointestinal tumors

Abstract

The requirement for well spread out chromosomes for the cytogenetic analysis of primary gastrointestinal tumors led us to develop new techniques. These techniques involved two main procedures: (1) preliminary incubation with culture medium in the presence of collagenase, Dispase, and colcemid, for 3 h, and (2) treatment with an extremely hypotonic solution (0.044M KCl) for 30 min. The techniques were applied to 11 gastrointestinal malignancies (including 1 early gastric cancer and 1 metastatic liver lesion of colon cancer) and significant increases (P < 0.01) in the number of metaphases of analyzable karyotypes were obtained, compared with a previous method in which the standard hypotonic molarity of KCL (0.075 M) was employed. The mean value for metaphase numbers of the analyzable karyotypes was 37.0 +/- 3.7% in the 5 gastric cancers and 44.7 +/- 4.8% in the 5 colon cancers and 1 metastatic lesion. These values were three times and more than twice, respectively, the values obtained by the previous method. A fluorescence in situ hybridization (FISH) study was carried out on one cologenic tumor, the alpha-satellite centromere-specific probe 17 being used. Deletion of the long arm of chromosome 17 was demonstrated. The method proposed here could yield a sufficient number of metaphases without the use of tissue culture that might cause alteration of karyotype. It can be employed with small biopsy specimens and in studies utilizing the FISH technique.

A new method for improving metaphase chromosome spreading

“…Reports based on quantitative investigation on improving the quality of the metaphase spreads are scarce. Even in a few quantitative studies (2-4) and a number of qualitative studies (5)(6)(7)(8)(9), there are apparently conflicting points regarding the speculated mechanisms and applied techniques. Spurbeck et al (4) emphasized that chromosome spreading depends on the natural drying time of the fixative and that approximately 90 s of drying time is needed for best spreading under the ambient condition of 20°C and relative humidity of 55%.…”

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

“…Cytogenetic slide preparation is essential for both classical banding procedures and in situ protocols, such as fluorescence in situ hybridization (FISH). Slide preparation techniques were described in numerous publications (1)(2)(3)(4)(5)(6)(7). Almost every cytogenetic laboratory inherited a procedure, which was then refined to yield good results.…”

Microtechnologies Enable Cytogenetics

“…In recent years, there has been considerable interest in fathoming the underlying process of chromosome spreading (Chattopadhyay et al, 1992;Gibas et al, 1987;Sasai et al, 1996). These explorations have also resulted in a number of devices aimed at automating the chromosome spreading protocol (Henegariu et al, 2001;Qu et al 2008;Yamada et al, 2008).…”