Rapid Human Chromosome Aberration Analysis Using Fluorescencein SituHybridization

Abstract

We have used in situ hybridization of repeat-sequence DNA probes, specific to the paracentromric locus 1q12 and the telomeric locus 1p36, to fluorescently stain regions that flank human chromosome 1p. This procedure was used for fast detection of structural aberrations involving human chromosome 1p in two separate experiments. In one, human lymphocytes were irradiated with 0, 0.8, 1.6, 2.4 and 3.2 Gy of 137Cs gamma-rays. In the other, human lymphocytes were irradiated with 0, 0.09, 0.18, 2.0, 3.1 and 4.1 Gy of 60Co gamma-rays. The frequencies (per cell) of translocations and dicentrics with one breakpoint in 1p and one elsewhere in the genome were determined for cells irradiated at each dose point. These frequencies both increased with dose, D, in a linear-quadratic manner. The delta, alpha, and beta coefficients resulting from a fit of the equation f(D)=delta + alphaD + betaD2 to the translocation frequency dose-response data were 0.0025, 0.0027 and 0.0037 for 137Cs gamma-rays, and 0.0010, 0.0041, and 0.0057 for 60Co gamma-rays. The delta, alpha, and beta coefficients resulting from a fit to the dicentric frequency dose-response data were 0.0005, 0.0010 and 0.0028 for 137Cs gamma-rays and 0.0001, 0.0002 and 0.0035, for 60Co gamma-rays. Approximately 32,000 metaphase spreads were scored in this study. The average analysis rate was over two metaphase spreads per minute. However, an experienced analyst was able to find and score one metaphase spread every 10s. The importance of this new cytogenetic analysis technique for biological dosimetry and in vivo risk assessment is discussed.

The use of fluorescence in situ hybridisation combined with premature chromosome condensation for the identification of chromosome damage

“…This technique, often termed 'chromosome painting', has two obvious advantages: it identifies specific chromosomes, and chromosome damage is easily visualised and scored. Fluorescent in situ hybridisation has been used, not only for routine cytogenetic analysis, such as detecting trisomy 21, but also for the detection of translocation in metaphase cells from individuals exposed to ionising radiation (Lucas et al, 1989;Pinkel et al, 1988).…”

Is FISH painting an appropriate biological marker for dose estimates of suspected accidental radiation overexposure? A review of cases investigated in France from 1995 to 1996.

“…Cells containing visible EC were scored separately and not included in the total data. To compare the yields of stable chromosome aberrations with those of the unstable aberrations, all observed frequencies were genomic estimated using the formula of Lucas et al (6,7). So a full genome equivalent cell number (cell equivalent) was calculated and the corresponding genomic estimated translocation number was termed (TR+ TT)eq.…”

“…On the other hand, it appears that translocations persist for many years after exposure and that their scoring may be an indication of past overexposure. FISH painting using whole human chromosome-specific DNA probes has opened new possibilities for detecting some interchromosomal exchanges (i.e., translocations, insertions) using a cocktail of composite DNA probes specific to some chromosomes (5,6). The data obtained by the analysis of only a few chromosomes (the painted ones) generally are scaled up to full genomic frequency by assuming a random distribution of break points.…”

“…The data obtained by the analysis of only a few chromosomes (the painted ones) generally are scaled up to full genomic frequency by assuming a random distribution of break points. FISH painting, therefore, provides easy identification and classification of radiation-induced chromosome aberrations (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16).…”

Dicentric chromosome frequency analysis using slit‐scan flow cytometry

“…The dose rate was 4.05 Gylmin measured at the sample location using a Victoreen ion chamber calibrated to a source traceable to the National Bureau of Standards. Absorbed doses to the cells were obtained from ionchamber readings as previously described (17). …”