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Objective Salivary glands of primary Sjögren’s syndrome (pSS) patients characteristically harbour periductal infiltrates in which lymphoepithelial lesions (LELs) can develop. LELs are composed of hyperplastic ductal epithelium with infiltrating lymphocytes and may assist in the challenging diagnostic process of pSS. As manual identification of LELs remains difficult, we aimed to identify LELs by using an objective digital image analysis (DIA) algorithm that detects intraepithelial lymphocytes. Methods A virtual triple staining technique developed for this study was used to count intraepithelial lymphocytes in consecutive slides stained for CD3 (T-lymphocytes), high molecular weight cytokeratin (hmwCK) (striated ducts) and CD20 (B-lymphocytes) in labial and parotid gland biopsies in a diagnostic cohort of 109 sicca patients. Patients were classified as pSS or non-SS according to the ACR-EULAR classification criteria. Results T-lymphocytes were detected in almost all analyzed ducts of pSS and non-SS patients, whereas intraepithelial B-lymphocytes were present in 59–68% of labial and parotid gland biopsies of pSS patients, against only 2–3% of patients classified as non-SS. Intraepithelial B-lymphocytes were found in almost all striated ducts with hyperplasia (LELs). Remarkably, around 25% of analyzed striated ducts without hyperplasia of pSS patients also contained B-lymphocytes (precursor-LELs). Furthermore, presence of intraepithelial B-lymphocytes was associated with clinical parameters of pSS (i.e. serology). Conclusion Intraepithelial B-lymphocytes are a specific finding in salivary gland biopsies of pSS patients and can be used as an objective alternative to LEL scoring. Therefore, identification of B-lymphocyte containing ducts should be added to the diagnostic histopathological work-up of patients suspected of pSS.

Digital image analysis of intraepithelial B-lymphocytes to assess lymphoepithelial lesions in salivary glands of Sjögren’s syndrome patients