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


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.
Keller
U
U
Department of Radiation Oncology, Friedrich-Alexander-University Erlangen-Nuremberg, Universitaetsstr. 27, D-91054 Erlangen, Germany.
Grabenbauer
G
G
Kuechler
A
A
Sauer
R
R
Distel
L
L
D002869:Chromosome Aberrations; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008677:Metaphase; D011836:Radiation Tolerance