Adaptation of the agyrophil technique for nucleolar organizer regions to canine peripheral blood smears

Nucleolar organizer regions (NORs) are chromosomal segments which are selectively stained by silver methods and evaluated as agyrophilic nucleolar organizer regions (AgNORs). The evaluation of AgNORs provides an insight into the level of cellular proliferation. This technique has extensively and sparsely been used in human and veterinary histopathology respectively. However, two major drawbacks have been irreproducible results and excessive staining precipitates. This study seeks to adapt the technique to canine peripheral blood smears in order to establish a routine staining protocol. Standardized volumes and concentrations of silver nitrate, gelatin, and formic acid were applied to smears at different temperatures for varying lengths of time. The technique was applied to unfixed and fixed smears. In some cases, a reducing agent (1% potassium iodide) was applied. It was shown that the optimum staining protocol was achieved by applying standardized solutions to a fixed smear at 46°C for 50 minutes. It is concluded that the staining protocol outlined in this study is practicable, and produces excellent and reproducible results that would enhance evaluation of AgNORs in canine peripheral blood cells.