## 4 Discussion  
Ex vivo Fluorescence Confocal Microscopy (eFuCM) is a technique that provides high-resolution and real-time digital imaging of native tissues, resembling Hematoxylin-Eosin (H&E)-stained slides. This technology has been increasingly utilized in surgical settings, particularly in dermatology and urology [6; 9; 13; 22]. This prospective observational study is the first to specifically address the use of eFuCM for the diagnosis of fresh non-tumoral liver biopsies, while also examining the influence of pathologist experience with eFuCM, a factor that has not been previously explored. Figure 4: **(a,b)**: Case 14. Steatosis vacuoles (*) visible on H&E slides, 40\(\times\) (**b**), which could not be correctly identified in eFuCM digital images (**a**); (**c,d**): Case 4. Stablished cirrhosis on eFuCM images (**c**) and in the H&E 40\(\times\) (**d**); (**e,f**): Case 20. eFuCM image (**e**) and H&E 100\(\times\) (**f**) of the colorectal carcinoma metastasis. Our findings demonstrate that eFuCM is well-suited for identifying fundamental liver structures and for detecting key non-neoplastic parenchymal changes such as cirrhosis, steatosis and portal inflammation, with generally acceptable levels of agreement compared to conventional histology. However, as we have shown, the accuracy of eFuCM interpretation is closely related to the pathologist's familiarity with the technology. The significant discrepancy between conventional microscopy and eFuCM, observed in cases where the pathologist lacked prior experience, highlights the existence of a learning curve. This learning curve must be taken into account in future research and when implementing this technology in pathology departments for the liver as well as other tissues. Future research should involve multiple pathologists with varying levels of expertise in both conventional microscopy and eFuCM to achieve more reliable conclusions. There were certain limitations in the agreement between eFuCM and the conventional liver pathology, particularly in assessing lobular inflammation, grading inflammation and staging fibrosis. Similar challenges have been observed in conventional H&E liver biopsy evaluations, with previous studies showing significant variability both between and within observers [22; 23; 24; 25]. In our study, difficulties also arose in identifying the acute hepatitis pattern, and in two cases, it was impossible to distinguish between confluent necrosis and bridging fibrosis. This underscores the limitations of eFuCM in fully replacing conventional histochemical stains like Reticulin and Masson's Trichrome, which are critical in liver pathology for fibrosis staging. Only two previous studies have specifically addressed the usefulness of eFuCM for liver biopsies. Our findings align with those of [PERSON] et al. [23] who reported an excellent suitability for tumor diagnosis (\(\kappa=1.00\)) and a moderate-to-good suitability for assessing inflammation severity and localization (\(\kappa=0.4\)-\(0.6\)), with similar limitations, such as challenges in evaluating cytoplasmic features. However, unlike our study, [PERSON] et al. used various types of liver samples, not just liver core biopsies. The other study focused on intraoperative consultations for donor liver biopsies, demonstrating better correlations with eFuCM than conventional H&E staining [19]. eFuCM is emerging as an attractive alternative to using frozen sections for evaluating donor liver specimens, and it may also hold potential for intraoperative consultations, such as margin evaluation. A significant strength of eFuCM is its ability to perform real-time histopathological diagnosis without losing tissue in the process, as occurs with conventional or frozen diagnosis procedures. As demonstrated in other pathologies where samples are scarce, such as renal biopsies [13; 22], prostate biopsies [24; 25] or small colorectal polyps [26], this technology can be useful for the rapid evaluation of liver core biopsies, where material is precious for additional IHC or molecular analysis.