AgNOR staining and quantification

Nucleolar organiser regions (NORs) are defined as nucleolar components containing a set of argyrophilic proteins, which are selectively stained by silver methods. After silver-staining, the NORs can be easily identified as black dots exclusively localised throughout the nucleolar area, and are called "AgNORs". The NORs' argyrophilia is due to a group of nucleolar proteins, which have a high affinity for silver (AgNOR proteins). A number of studies carried out in different tumour types demonstrated that malignant cells frequently present a greater AgNOR protein amount than corresponding non-malignant cells. Moreover, in cancer tissues AgNOR protein expression was found to be strictly related to the cell duplication rate. Over the past 12 years, the "AgNOR method" has been applied in tumour pathology for both diagnostic and prognostic purposes. However, the lack of a standardised silver-staining protocol has led to much misinterpretation of actual structures evaluated in individual studies. Indeed, the absolute AgNOR scores reported by different authors for the same types of tumour are scarcely comparable and the results produced by these investigations sometimes seem to be conflicting. In order to achieve definitive standardisation of the AgNOR method and produce comparable data in all laboratories, the "International Committee on AgNOR Quantitation" was founded, and during the first Workshop "AgNORs in Oncology" held in Berlin in 1993 guidelines for AgNOR protein evaluation were first defined. The present paper discusses the main technical aspects of NOR silver-staining, and critically evaluates the methods commonly employed for AgNOR protein quantification in routine cyto-histopathology.