Evaluation of Novel Immunohistochemical Biomarkers for the Diagnosis of Celiac Disease Demonstrates the Utility of TCRδ Immunostaining

Background/Objectives: Celiac disease (CD) is a T-cell mediated autoimmune condition, triggered by gluten ingestion. Duodenal biopsy is the gold-standard diagnosis for CD, which is often limited by interobserver variability between pathologists. Immunohistochemistry (IHC) is a powerful technique for detecting biomarkers with potential diagnostic significance. This study aims to investigate five candidate biomarkers BTNL8, NKp46, TdT, THEMIS, and TCRδ that might improve the reproducibility of the diagnosis of CD. Methods: Formalin fixed paraffin-embedded material, surplus to diagnostic requirements was obtained from 46 subjects (untreated CD: n=21, CD treated with gluten-free diet: n=5; controls: n=20) and immunostained for BTNL8, NKp46, TdT, THEMIS and TCRδ. BTNL8 staining was scored on a 0-3 semi-quantitative scale. NKp46, TdT, THEMIS, and TCR delta-positive intra-epithelial lymphocytes (IELs) were quantified as mean counts per 100 epithelial cells (ECs). Results: TCRδ-positive IELs were markedly elevated in CD biopsies (median 9.4 IELs/100 ECs) compared to healthy controls (median 0.5 IELs/100 ECs; p<0.001), with a threshold of >2.1 TCRδ-positive IELs per 100 ECs yielding an AUC of 94% and interobserver agreement of 0.82. NKp46 expression was also increased in CD (median 13.8 IELs/100 ECs) versus controls (median 9.6; p<0.001), with >12.8 NKp46-positive IELs per 100 ECs achieving an AUC of 86% and interobserver agreement of 0.82. Immunostaining for the other biomarkers demonstrated less clear differences between CD and healthy controls. Conclusions: Corroborating several recent publications, TCRδ immunostaining provides high diagnostic accuracy and good interobserver agreement in the diagnosis of CD on duodenal biopsy, even for patients on a gluten-free diet.