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

Screening for Urological Malignancies Using a Urine Exfoliated Cell Fluorescence Assay in a Hematuria Population: A Prospective Clinical Study

This is a prospective, single-center diagnostic clinical study designed to evaluate the performance of a urine-based immunofluorescence cytology test for the detection of urological malignancies in patients presenting with hematuria. The study will enroll 170 eligible participants who are scheduled for cystoscopy and/or ureteroscopy. Urine samples will be collected prior to endoscopy and subjected to both the index immunofluorescence assay and DNA ploidy analysis, with the results compared against the reference standard of endoscopic biopsy and histopathology. The primary objective is to estimate the sensitivity, specificity, and negative predictive value of the immunofluorescence test. Secondary objectives include evaluating the diagnostic accuracy of DNA ploidy analysis, comparing the two index tests using paired statistical methods (e.g., McNemar's test), and assessing their agreement with the reference standard via Kappa statistics. Sample size calculation, based on an assumed malignancy rate of 15% and a target of at least 23 pathology-confirmed malignant cases, ensures adequate power for the primary comparison. The study will follow standardized operating procedures, with independent, blinded interpretation by trained cytopathologists. Statistical analyses will be performed using R or SPSS, with a two-sided significance level of 0.05. The trial is scheduled to commence in April 2026 and is expected to conclude by June 2027, with the aim of providing evidence for a non-invasive, rapid, and cost-effective screening tool in hematuria populations.

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.

Inclusion Criteria:

• Presenting with hematuria requiring urological malignancy workup

Age ≥ 18 years

Planned to undergo cystoscopy and/or ureteroscopy

Able to provide written informed consent

Exclusion Criteria:

• Prior confirmed diagnosis of urological malignancy

Participation in another clinical trial within 3 months prior to enrollment

Any clinical condition that, per investigator judgment, may interfere with study execution

Inadequate urine sample quality or volume