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Updated: Aug 22, 2026
FISH for Pre-implantation Genetic Diagnosis
Published on: February 23, 2011
Technical report. Radiation sensitivity testing by fluorescence in-situ hybridization: how many metaphases have to be
U Keller1, G Grabenbauer, A Kuechler
1Department of Radiation Oncology, Friedrich-Alexander-University Erlangen-Nuremberg, Universitaetsstr. 27, D-91054 Erlangen, Germany.
Purpose:
The technique of three-colour fluorescence in-situ hybridization (FISH) is generally regarded as 'gold standard' for detecting chromosomal aberrations. The question was: how many metaphases should be counted to get reliable results?
Material And Methods:
Peripheral blood lymphocytes were irradiated in vitro (2.0 Gy). Metaphase chromosomes (1, 2, 4) were labelled by means of three-colour FISH and chromosomal aberrations (breaks per metaphase [B/M], complex chromosomal rearrangements per metaphase [CCR/M]) were analysed. To evaluate the correlation between the number of metaphases counted and the reliable detection of the rate of break events, B/M and CCR/M were scored using 250-1,000 metaphases in steps of 50 unirradiated cells, and from 50 to 200 metaphases in steps of 10 after 2 Gy.
Results:
Analysing spontaneously occurring aberrations, B/M values based on 500 and 750 counted metaphases agreed well with those B/M values from 1,000 scored metaphases. After counting 150 metaphases after 2 Gy, the confidence interval of B/M values was about 44% smaller and the confidence interval of CCR/M values was about 41% smaller compared with values obtained after counting 100 metaphases.
Conclusions:
Scoring the number of spontaneous aberrations, reliable results can be obtained after counting 500 metaphases. After 2 Gy, a minimum of 150 metaphases should be analysed.
