Background and Objectives:Endoscopic ultrasonography (EUS)-guided needle-based confocal laser endomicroscopy (nCLE) characteristics of common types of pancreatic cystic lesions (PCLs) although identified lacks external validation and surgical histopathology was available in a minority of subjects. We sought to externally validate EUS-nCLE images for differentiating PCLs in a larger series of subjects with a definitive diagnosis.Methods:Six expert endosonographers, blinded to clinical data, reviewed nCLE images of PCLs from 29 subjects with surgical (n = 23) or clinical (n = 6) correlation. After 2 weeks, the assessors reviewed the same images in a different sequence. The performance characteristics of nCLE and the kappa-statistic for interobserver agreement (IOA, 95% confidence interval [CI]) and intraobserver reliability (IOR, mean, standard deviation [SD]) for the identification of nCLE image patterns were calculated. Landis and Koch interpretation of kappa values was used.Results:A total of 29 (16 mucinous PCLs, 13 nonmucinous PCLs) nCLE patient videos were reviewed. The overall sensitivity, specificity, and accuracy for the diagnosis of mucinous PCLs were 95%, 94%, and 95%, respectively. The IOA and IOR (mean, SD) were κ = 0.81 (almost-perfect), 95% CI 0.71–0.90 and κ = 0.86, 0.11 (almost-perfect), respectively. The overall specificity, sensitivity, and accuracy for the diagnosis of serous cystadenomas were 99%, 98%, and 98%, respectively. The IOA and IOR (mean, SD) for recognizing characteristic image pattern of serous cystadenomas were κ = 0.83 (almost-perfect), 95% CI 0.73–0.92 and κ = 0.85, 0.11 (almost-perfect), respectively.Conclusion:EUS-nCLE can provide virtual histology of PCLs with a high degree of accuracy and inter- and intra-observer agreement in differentiating mucinous versus nonmucinous PCLs. These preliminary results support larger multicenter studies to evaluate EUS-nCLE.