Indeterminate biliary strictures (IBS) remain diagnostically challenging, as imaging, ERCP-based tissue sampling, and EUS-guided tissue acquisition (TA) often fail to differentiate malignant from benign lesions. Digital single-operator cholangioscopy (D-SOC) improves accuracy through direct visualization and targeted biopsies, although false-negative histology persists. This study evaluated whether biopsy orientation and cholangioscopic visual assessment using the Monaco classification improve diagnostic performance.

This retrospective single-center study included all consecutive patients undergoing D-SOC with direct biopsies for IBS at Infermi Hospital (Rimini, Italy) from 2017 to 2025. Two cohorts were analyzed: non-oriented biopsies (2017–2021) and oriented biopsies (2022–2025). Malignancy was defined according to a clinico-pathological standard (surgical histology or long-term follow-up). Primary outcomes were diagnostic yield, sensitivity, and specificity for malignancy. The Monaco classification was considered positive when ≥3 criteria were present. Diagnostic accuracy was further assessed using combined parallel (Monaco-positive or malignant histology) and serial (Monaco-positive and malignant histology) strategies.

A total of 103 patients were included (non-oriented 64.1%; oriented 35.9%). Overall biopsy diagnostic yield for malignancy was 62.1% (95% CI: 52.0–71.5), with sensitivity 57.1% (95% CI: 44.0–69.5) and specificity 100% (95% CI: 91.0–100). Diagnostic yield was comparable between groups: 61.5% (95% CI: 48.6–73.3) for non-oriented and 63.2% (95% CI: 46.0–78.2) for oriented biopsies. Sensitivity was 61.9% (95% CI: 45.6–76.4) and 47.6% (95% CI: 25.7–70.2), respectively; specificity remained 100% in both.Cholangioscopic visual assessment alone showed higher sensitivity (84.4%, 95% CI: 72.6–92.7) but lower specificity (65.9%, 95% CI: 50.1–79.5). Combined analysis improved diagnostic performance: the parallel model significantly increased sensitivity to 82.5% (95% CI: 70.9–90.9; p<0.05 vs biopsy alone), while the serial model maintained specificity at 100% (95% CI: 91.2–100), equivalent to histology.

D-SOC remains a valuable tool in evaluating indeterminate biliary strictures. Orientation of biopsy samples does not appear to provide an incremental diagnostic yield and sensitivity. Integrating visual cholangioscopic assessment through the Monaco classification substantially enhances the ability to identify malignant lesions, by reducing false negative results, while maintaining high specificity when used in a serial combination with biopsy. 

Further evidence is needed to better assess biopsy-handling techniques, optimize tissue acquisition strategies, and integrate artificial intelligence in the diagnostic pathway to increase the diagnostic performance for indeterminate biliary strictures.