Exclusion of chromosomal mosaicism in amniotic fluid cultures: determination of number of colonies needed for accurate analysis.

Most laboratories use the in situ or flask culture method and a two-stage approach to mosaicism detection. Determination of the optimum number of metaphases to be counted depends largely on whether or not the colonies that grow from the cells in the amniotic fluid can be considered independent. Previous statistical analysis of data from mixed male and female amniocyte cultures indicated that for mosaicism detection these colonies can be considered independent (Cheung et al., 1990). This analysis was repeated with a set of mosaic cases from two independent prenatal diagnosis programmes. The same degree of colony independence was found with this set of data. This result was used to calculate the level of mosaicism that is excluded at a particular confidence level when set numbers of colonies are analysed at each stage. The tables generated apply directly to the in situ method and with modification they can be used with the flask method. The conclusions are (1) analysis of cells from multiple colonies enhances the likelihood of excluding true mosaicism; (2) analysis of more than one metaphase per colony offers little advantage in excluding mosaicism; and (3) the two-stage approach is the most efficient. These conclusions should be used together with the expected clinical outcome of the actual cytogenetic abnormality, as discussed by Hsu et al. (1992).