Radiation sensitivity testing by fluorescence in-situ hybridization:: how many metaphases have to be analysed?

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 250-1000 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 1000 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.