Abstract
Background/Objectives: Accurate intraoperative margin assessment is essential for ensuring complete tumour excision in breast-conserving surgery, minimising local recurrence, and avoiding reoperations. Ex vivo fusion confocal microscopy (EVFCM) provides real-time, high-resolution imaging of fresh, unfixed tissues that closely resembles conventional histological imaging. This study aimed to validate the diagnostic performance and clinical feasibility of EVFCM for real-time intraoperative margin assessment during breast cancer surgery. Methods: A prospective observational diagnostic validation study was conducted using 144 breast tissue specimens. The samples were stained with acridine orange and fast green and scanned using a VivaScope 2500M-G4 system. Two breast pathologists independently evaluated the EVFCM images, blinded to the conventional histology results, which served as the reference standard. The diagnostic accuracy, sensitivity, specificity, and interobserver agreement were calculated using Cohen's κ. Results: Interobserver agreement was almost perfect for neoplasia detection (97.3%, κ = 0.942) and tumour type classification (93.8%, κ = 0.883). The EVFCM achieved 93.7% sensitivity and specificity, with 94.0% accuracy for tumour detection (κ = 0.929, p < 0.001); 95.8% accuracy for tumour type classification (κ = 0.925, p < 0.001); and 95.1% accuracy for invasive subtype identification (κ = 0.907, p < 0.001). For margin assessment, EVFCM achieved 80% sensitivity, 100% specificity, and 99.3% accuracy (κ = 0.857, p < 0.001), whereas margin distance evaluation (<2 mm vs. ≥2 mm) yielded 75% sensitivity, 100% specificity, and 98.6% accuracy (κ = 0.854, p < 0.001). Conclusions: EVFCM enables rapid, high-resolution imaging of fresh breast tissue, facilitating real-time intraoperative margin evaluation with excellent diagnostic concordance and workflow efficiency. Its integration into surgical practice could reduce re-excisions, enhance oncological safety, and improve patient outcomes in breast-conserving surgeries.