Quantitative evaluation of AgNOR in liver cells by high-resolution image cytometry

This study is aimed at determining the relationship between polyploidy and the amount of nucleolar organiser region (AgNOR) positive dots or aggregates using high resolution image analysis. Liver imprints from mice in which hepatocyte polyploidy is very well documented and related to variation in nuclear area were used as a model for this investigation. A technical variant of the AgNOR method using a Triton X-100 treatment was developed for removal of some proteins from the cytoplasm, which produced a clearer and cleaner background. Feulgen-stained preparations were used to detect the association of various ploidy degrees with their respective values of nuclear area and to subsequently furnish a basis for the association of the nuclear areas in AgNOR-stained cell preparations with different ploidy levels. A high correlation between nuclear and AgNOR-stained areas was revealed and was demonstrated to be much higher than the correlation between nuclear area and the number of AgNOR positive dots or aggregates. The use of two different thresholds for segmentation of the grey levels of the AgNOR-stained material demonstrated the importance of the appropriate decision to obtain the best results to be associated with polyploidy in terms of the real biological event involved, the nuclear area being in this case correlated to Feulgen-DNA values.