An improved system for quantifying AgNOR and PCNA in canine tumors

Background: Quantifying silver stained nucleolar organizer regions (AgNORs) and proliferation cell nuclear antigens (PCNA) are useful techniques to me asure proliferative activity of tumor cells; however, the nonspecific depos ition of stains and overlappings of AgNOR and PCNA counts between grades of tumors hamper their applications. Materials and methods: Fifty-two surgica l specimens from dogs, including mast cell tumors, perianal gland tumors an d hyperplasias, fibromas, fibrosarcomas, and normal tissues were studied. T he 3 mum dewaxed sections of formalin-fixed tissues were stained to detect AgNORs by a modified inverted incubation technique in a newly developed sil ver staining device. Data were collected and analyzed using a high-resoluti on digital microscope camera and image analysis software. Sequential sectio ns were also stained for PCNA using an immunohistochemical method. Results: The improved system for quantifying AgNOR provided more accurate and non-o ver lapping mean AgNOR counts, which enable us to distinguish benign states from malignant changes. The mean AgNOR cut-off points that discriminated g rade II or III mast cell tumors from grade I, perianal gland carcinomas fro m adenomas (ol hyperplasia), fibrosarcomas fi om non-fibrosarcoma tissues, were 60, 14.1, 9.4, and 8.8 respectively. The mean AgNOR areas, relative Ag NOR areas, and PCNA positive rates of some malignant and non-malignant tiss ues (benign tumor and normal tissues) were significantly different (P < 0.0 5). Conclusions: This improved system is a sensitive and rather precise met hod for quantifying the AgNOR and PCNA. It provides a valuable objective me asurement for differentiating benign and malignant tumors.