Standardization of interphase Ag‐NOR measurement by means of an automated image analysis system using lymphocytes as an internal control

Using an automated image analysis system, we have developed a procedure for standardizing the measurement of silver-stained proteins of the nucleolar organizer regions (NORs) in cancer cells, irrespective of the fixative employed and the time of the staining reaction. We observed that the area of Ag-NOR proteins from the same tumour was smaller in samples fixed with formalin-containing solutions, buffered formalin, and Bouin liquid, compared with those fixed with absolute ethanol and 'methcarn' solution (1.77 +/- 0.26micron 2 and 2.36 +/- 0.35 micron2 versus 3.34 +/- 0.54 micron2 and 3.72 +/- 0.61 micron2). Increased values of the Ag-NOR area were also observed after lengthening the silver staining reaction. However, in both cases no difference was observed in the ratio between Ag-NOR area of cancer cells and that of the lymphocytes infiltrating the stroma. The value of the ratio, which is called the 'Ag-NOR index', was in fact very similar, for the same cancer, after employing different fixatives or staining times. The use of lymphocyte Ag-NOR area as an internal control for the standardization of Ag-NOR evaluation in cancer tissues was made possible by the fact that lymphocyte Ag-NOR area is almost constant in human tumours independent of sex and age.