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.

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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Background: Canine histiocytic sarcoma (HS) is an aggressive malignancy. Hyperferritinemia has been documented in dogs with HS and could serve as a tumor marker aiding in diagnosis and treatment. In people, hyperferritinemia is found in inflammatory diseases, liver disease, and hemolysis, and thus may occur in dogs with these conditions.Objective: To determine if serum ferritin concentration is a tumor marker for canine HS. Animals: Dogs with HS (18), inflammatory diseases (20), liver disease (24), immune-mediated hemolytic anemia (IMHA) (15), and lymphoma (23).Methods: Prospective, observational, cohort study: Serum ferritin concentration was measured at initial diagnosis. Parametric methods were used to compare mean log ferritin concentrations among disease categories. Receiver-operating characteristic curves and likelihood ratios were used to evaluate serum ferritin concentration as a tumor marker.Results: Varying proportions of dogs with IMHA (94%), HS (89%), liver disease (79%), lymphoma (65%), and inflammatory diseases (40%) had hyperferritinemia. Dogs with IMHA had significantly higher mean ferritin concentration than dogs in all other categories. Dogs with HS had significantly higher mean ferritin concentration than those in the inflammatory disease and lymphoma categories. Mean serum ferritin concentration was not significantly different between dogs with HS and those with liver disease. Decision thresholds were determined to distinguish IMHA and HS from the other diseases associated with hyperferritinemia.Conclusion: Hyperferritinemia is common in dogs with HS and, after IMHA is ruled out, the degree of hyperferritinemia may be useful in differentiating dogs with HS from dogs with inflammatory diseases, liver disease, and lymphoma.

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