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.

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The value of automatic image analysis in the investigation of nucleolus regions (AgNOR) has been examined in tissue sections of 52 malignant and 30 benign breast lesions. Determination of the AgNOR number per cell alone revealed a considerable overlap between benign (range 1.2-3.8) and malignant specimens (range 1.5-16.2). They differed however, highly significantly (P less than 0.001) in their AgNOR sizes. In benign breast disorders the mean AgNOR area per tumour ranged from 0.22 microns2 to 1.07 microns2 (mean 0.39 microns2), whereas in carcinomas AgNOR sites ranged from 0.05 microns2 to 0.22 microns2 (mean 0.09 microns2). AgNOR counts showed a good correlation with histopathological grade (P less than 0.05), aneuploidy (P less than 0.01), proliferation rate as determined by Ki67 immunostaining (P less than 0.01), as well as oestrogen and progesterone receptor content (P less than 0.01). Image analysis proved to be advantageous over AgNOR counting alone as it facilitated the standardization of the AgNOR technique itself and thus, significantly improved its diagnostic specifity.

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The comparison of the diagnostic and prognostic significance of histology, immunohistochemical parameters (PSA, PSP), and silver-stained nucleolar organizer regions (AgNORs) was estimated in paraffin sections taken of 63 prostatic carcinomas prior to therapy. AgNORs were visualized with a one-step silver staining technique with the appropiate staining time determined by preliminary staining-time series. The mean AgNOR number per cell (n) and the mean AgNOR area per silver-stained dot (A) were determined by means of an automatic image analysis system. Thereby prostatic carcinomas exhibited multiple small AgNORs within their nuclei (n = 4.7, A = 0.09 µm2), whereas benign prostatic epithelium showed few but large silver-stained particles (n = 1.8, A = 0.27 µm2; p < 0.001). This relationship was then calculated as a quotient of AgNOR number and area (NQ = n/A) which provided additional information for the diagnosis of malignancy as well as survival. Univariate survival analysis disclosed a set of four variables predicting death from prostatic cancer: cribriform growth pattern, AgNOR quotient, histological grade, and PSA immunoreactivity. Of these parameters, immunoreactivity of PSA failed to prove its prognostic significance in multivariate survival analysis (Cox model). No relation to prognosis was found for the number as well as the area of AgNORs alone. Therefore, image analysis proved to be a prerequisit for the feasibility of this promising technique by providing objective and reproducible results.