Screening for Urological Malignancies Using a Urine Exfoliated Cell Fluorescence Assay in a Hematuria Population

Hematuria is a critical early warning sign of urological malignancies, including bladder cancer, renal pelvic cancer, and ureteral cancer, with approximately 10%-20% of patients with gross hematuria ultimately diagnosed with malignancy. Although cystoscopy and ureteroscopy remain the diagnostic gold standard, their invasive nature and high cost limit their widespread use in initial screening. Conventional urinary cytology, while non-invasive, has suboptimal sensitivity (approximately 30%-50%) for low-grade tumors, failing to meet the demands of early screening.

This study utilizes a urine-based immunofluorescence cytology test employing acridine orange compound fluorescent dye, which specifically binds to nucleic acids: emitting green fluorescence with double-stranded DNA and orange-red fluorescence with single-stranded RNA. Previous basic research has demonstrated significantly increased RNA content in the nucleolar regions of malignant cells, providing a biological rationale for this technique to non-invasively detect tumor cells by identifying abnormal nuclear morphology and nucleic acid metabolism.

This is a single-center, prospective diagnostic accuracy study. A total of 170 participants presenting with hematuria and scheduled for cystoscopy and/or ureteroscopy to rule out urological malignancies will be enrolled. Prior to endoscopic examination, fresh urine samples will be collected from each participant. The same specimen will undergo two independent tests simultaneously: (1) the urine-based immunofluorescence cytology test (manual interpretation under fluorescence microscopy) and (2) DNA ploidy analysis. Histopathological biopsy results from suspicious lesions detected during endoscopy will serve as the reference standard. The operators performing the immunofluorescence and DNA ploidy tests, as well as the endoscopists and pathologists, will be mutually blinded to ensure objective results.

Sample size calculation was based on a paired diagnostic test design using McNemar's test. With a two-sided alpha of 0.05 and power (1-beta) of 0.80, and based on literature and our center's preliminary data, we assume a sensitivity of 90% for the immunofluorescence test and 52% for DNA ploidy analysis. Considering a 15% malignancy detection rate in our hematuria population and a 10% sample inadequacy rate, the final target sample size is 170 participants.

For primary analysis, the Clopper-Pearson exact method will be used to calculate the sensitivity, specificity, and negative predictive value of the immunofluorescence test with their 95% confidence intervals. Secondary analyses include: Kappa statistics to evaluate agreement between each test and the reference standard; McNemar's test to compare the sensitivity and specificity between the two tests; receiver operating characteristic (ROC) curve analysis with area under the curve (AUC) comparison; and exploratory subgroup analyses stratified by tumor type (e.g., bladder cancer, upper tract urothelial carcinoma) and tumor grade (low-grade vs. high-grade). All statistical tests will be two-sided, and a P-value < 0.05 will be considered statistically significant.