Evaluation of nucleolus organizer regions (NORs) by automatic image analysis: A contribution to standardization

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Abstract
This study summarizes our experiences with the silver staining of nucleolus organizer regions (AgNORs) in a total of 580 tumours from ten different tissues. In contrast to other investigators, we made use of automatic image analysis for the evaluation of AgNORs. This provided good reproducibility as determined by the standard cumulative means technique and intra-observer (r1) and inter-observer (r2) agreement in 30 benign (r1 = 0.83-0.95, r2= 0.76–0.92) and 50 malignant tissue samples (r1=0.72–0.85, r2 = 0.51–0.78). By using a series of staining times on sections from 30 tissue blocks taken from the ten types of tissue investigated, considerable variation in the argyrophilic staining of NORs in different tissues and in different blocks from one tumour was shown. The mean AgNOR area of resting lymphocytes or connective tissue cells within tissue blocks of the same organ system varied up to four-fold, even though identical staining times had been used. The most suitable silver reaction time which rendered a good diagnostic difference in the AgNOR content of benign and malignant tissue ranged, for example, in the breast cancer specimens, from 23 to 35 min. We therefore conclude that the staining time has to be adjusted to the individual silver-binding characteristics of each tissue block or even each section. The use of internal staining standards like lymphocytes or connective tissue cells in the same tissue section is mandatory. This, in turn, is most precisely controlled by morphometry.