<i>Ex vivo</i> fluorescence confocal microscopy for assessment of ureteroscopic biopsy adequacy in upper tract urothelial carcinoma

OBJECTIVES: To evaluate the feasibility of ex vivo fluorescence confocal microscopy (FCM) for real-time assessment of ureteroscopic biopsy specimen adequacy in patients with suspected upper tract urothelial carcinoma (UTUC). PATIENTS AND METHODS: In this prospective, single-centre feasibility study, four ureteroscopic biopsy specimens obtained from two patients undergoing evaluation for suspected UTUC were analysed using ex vivo FCM. Specimens were briefly stained with acridine orange, scanned within 50-60 s, and images were stored for deferred review. A board-certified genitourinary pathologist reviewed the FCM images in a blinded fashion and classified specimens as adequate or non-representative based on a pre-defined criterion of identifiable urothelial tissue. All specimens subsequently underwent routine haematoxylin and eosin (H&E) histopathological analysis for confirmation. RESULTS: Ex vivo FCM was successfully performed on all specimens without workflow interruption, with total preparation and image acquisition completed within approximately 5 min/specimen. Blinded FCM review identified urothelial tissue in three of four biopsy specimens, which were classified as adequate, while one specimen was composed predominantly of blood clot with only minimal, non-diagnostic cellular material and was therefore classified as non-representative. FCM-based adequacy assessments were concordant with corresponding H&E sections. Final histopathology demonstrated non-invasive papillary UC, high grade in one patient and low grade in the other; the non-representative specimen showed only focal atypical papillary urothelial proliferation admixed within the blood clot. CONCLUSION: Ex vivo FCM was feasible for immediate imaging of freshly obtained ureteroscopic biopsy specimens and allowed identification of urothelial-containing fragments as a marker of specimen adequacy while preserving tissue for standard histopathological evaluation. These preliminary findings support further investigation in larger cohorts to evaluate reproducibility and to clarify the potential role of this approach for intraoperative or real-time assessment of biopsy adequacy in UTUC, including correlation with final histopathological grade and stage.