Optimisation of interphase fluorescence in situ hybridisation for detection of common aneuploidies
The optimisation of a simple, reliable and practical method of interphase FISH which allows prenatal diagnosis of major chromosome aneuploidies using a minimum volume of amniotic fluid sample was the overall objective of this study. When all the probes required were available, the study continued by developing the technique of ratio-mixing FISH for simultaneous detection of the five major chromosome aneuploidies. The technique of five-colour ratio mixing FISH which has been presented here is simple and straightforward, since only two haptenisation and detection systems have been employed to visualise simultaneously five different targets in five distinguishable colours. The steps of denaturation, hybridisation and detection are the same as those used in a uni-colour FISH experiment. The results obtained from hybridisation of an unselected series of 20 uncultured lymphocytes and 27 uncultured amniocytes indicate that the technique is reliable and can be used for simultaneous detection of major chromosome aneuploidies. In order to provide a practical strategy for clinical diagnostic purposes, the use of a three colour ratio-mixing FISH and a dual colour was investigated to visualise the five probe sets on two slides from the same sample. A total unselected series of 45 uncultured lymphocytes and 60 uncultured aminocytes were hybridised with different probe combinations using three colour ratio-mixing FISH. The results indicate that the major chromosome aneuploidies can be simply and reliably identified on two slides from the same sample, using a three colour ratio-mixing FISH to detect the chromosomes X, Y and 21 and a dual colour to detect chromosomes 13 and 18. The failure rate was reduced to 4 per cent using this approach.