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Giemsa-staining method

Depertment of Genetics

Biodosimetry of human exposure to radiation:
A manual for detecting stable chromosome aberrations by the conventional Giemsa staining method

Akio A. Awa
Former Chief of Genetics Department, Radiation Effects Research Foundation, Hiroshima
Contents
1.
Introduction
2.
Preparation of metaphases
3.
Selection of metaphases
4.
Nomenclatures for describing aberrations
5.
Characteristics of chromosome groups (karyotyping)
6.
Normal variation of chromosomes
7.
Stable aberrations - introduction
8.
Stable aberrations - deletion
9.
Stable aberrations - inversion
10.
Stable aberrations - reciprocal translocation
11.
Example of misinterpretation
12.
Conclusions

1. Introduction

The frequency of chromosome aberrations in blood lymphocytes has been used to estimate radiation doses received by individuals. There are two kinds of aberrations, stable and unstable.

Unstable aberrations, represented by dicentrics and centric rings, are easily detected, but they are lost with time following cell divisions. Thus, as years pass following radiation exposure, the frequency declines, and these aberrations are not useful for dose estimation. In contrast, stable aberrations, represented by reciprocal translocations and pericentric inversions (i.e., inversions that include centromeres), are not affected by cell division and hence do not disappear with time. However, these aberrations have their own problem: they are difficult to identify by the conventional method and hence the detection efficiency has remained low.

We started to examine chromosome aberration frequencies of atomic-bomb survivors in the late 1960s, but unstable aberrations had mostly disappeared by that time. Hence, detection of stable aberrations was the only choice. Only the conventional Giemsa staining method was available in those days, so we worked to establish a laboratory manual and continued the work for nearly 30 years.

Recently, a new chromosome painting technique (FISH) has become available to replace conventional Giemsa staining. This new technique uses painting of specific chromosomes with fluorescent dyes through in situ hybridization and enables chromosomal exchanges between the painted and non-painted chromosomes to be detected objectively. When we compared the results of the two methods for the same 200 survivors, we found that the conventional method detected translocations with nearly 70% of that by FISH (Nakano M et al. Int J Radiat Biol 77:971-7, 2001).

We felt recently that too much emphasis is given to FISH, as if no other methods can detect stable aberrations, which is not true for us. For this reason we decided to open this manual to the public so that scientists working in this field may understand that there are different methods to detect stable aberrations.

Although FISH has many technical advantages, the costs of performing FISH and installing the ultraviolet microscope are high. Thus, we sought to provide information that the conventional Giemsa staining can also detect a fairly good fraction of stable aberrations if one is well familiar with human karyotypes.

2. Preparation of metaphases

This manual has been prepared for those who experienced in cytogenetics, so I thus omitted technical procedures for preparing metaphases. Please refer to published manuals for this purpose. For example, refer to

Verma RS and Babu A: Human chromosomes. Manual of basic techniques. New York, Pergamonn Press, 1989.