Value of the silver-stained nucleolar organizer regions technique in the differentiation between benign and malignant lesions in urine cytology.

U bladder cancer is a common disease. In Iraq, it represents 12.3% of cancer cases among men, and 4.9% of cancer cases among women.1 Cytological examination of urinary specimens is a relatively simple diagnostic procedure that can aid in the diagnosis of disease anywhere in the urinary tract; and nowadays, examination of urine cytology is regarded as part of the routine investigation of patients with hematuria, prostatism, and suspected urinary tract neoplasia. It is most useful in detecting high-grade bladder cancers; however, its limitation lies in the diagnosis of low-grade urothelial carcinoma since the cells usually do not exhibit the typical features of malignancy.2 This problem has motivated the development of new techniques to augment routine methods, and to improve the accuracy, and reproducibility of prognostication. One of these ancillary techniques is the study of proliferative markers. Ncleolar organizer regions [NORs] are loops of ribosomal DNA (rDNA) occurring in the nucleoli of cells. Its evaluation is regarded as an indicator of cell proliferation. So, the aim of this study is to evaluate the application of the argyrophilic nucleolar organizer region (AgNOR) technique on urine cytology in the diagnosis of neoplastic, and non-neoplastic lesions of the bladder, using AgNOR count, size, and dispersion as parameters for evaluation and to study the correlation of these parameters with tumor grade. The study was performed on 64 urinary specimens. Thirty of these were positive of malignant cells (22 males and 8 females with a male to female ratio of 2.75:1) and the selection criteria included patients complaining of gross hematuria and/or prostatism with radiological investigations suggestive of bladder tumor and confirmed by histopathological study of biopsy specimens taken cystoscopically. The other set represents 34 urine samples that were obtained from patients complaining of other urological diseases (such as vesical stone and urinary tract infection) and they were confirmed to be negative for malignancy both by pylogram and cytological study of urine samples and their stained slides were adequately cellular so they were considered as a control. Urine samples were collected from Iraqi patients attending Medical City and AlKadhimiya Teaching Hospitals, Baghdad, Iraq between May 2006 and December 2006. From each patient, 30 ml of mid stream freshly voided urine samples was collected. The samples were kept in an ice box, and processed within 2 hours from collection. Urine samples were centrifuged at 3000 rpm for 15 minutes. The supernatants were decanted, and the sediments were smeared on pre-albumenized slides using the micropipette method for obtaining the sediments. Four slides were prepared for each sample, and stained by routine Hematoxylin and Eosin stain. From these slides, the most representative ones were chosen to be re-stained with silver stain without prior de-staining. Following the standard procedure adopted by Khan et al,3 solution A was prepared by dissolving 500 mg gelatin in 25 ml de-ionized water along with 250 μl of formic acid (2% gelatin and 1% formic acid), and freshly prepared solution B was prepared by dissolving silver nitrate in distilled de-ionized water to make a 50% solution. Then, the working solution was prepared just before the staining by mixing one volume of solution A and 2 volumes of solution B. The nuclei were stained light yellow, and AgNORs were visualized as brown black discrete dots of variable size within the nuclei. Using oil immersion (× 1000), all distinguishable black to brown dots within the nucleus were identified, and treated as one AgNOR, and fine focusing was carried out to reduce the effect of dust particles, and deposit debris that interfered with the AgNOR dots. The mean AgNOR (mAgNOR) per nucleus was calculated by counting the AgNOR dots in 50 nuclei, and the average was counted. In addition to the AgNOR counts, variations in AgNOR size, and distribution were also recorded using the criteria of Ahsan et al.4 The final diagnosis (gold standard) was determined by the histopathological report of subsequent bladder biopsies. There were no cases with confirmed diagnosis of grade 1 urothelial carcinoma (papillary urothelial neoplasms of low malignant potential according to the World Health Organization/International Society of Urological Pathology classification system.)5 So, the diagnoses were grouped in 3 categories: (I) nonneoplastic lesions; (II) low grade carcinoma; and (III) high grade carcinoma. The AgNOR results of all nuclei were calculated, and expressed as the mean (± standard deviation). Student’s t test was used to compare mean Brief Communication