Immunohistochemistry to Highlight Intraepithelial Lymphocytes in Formalin-Fixed and Frozen Duodenal Biopsies: Comparability of Results and Cut-Off Revision

Background and Aim: TcRγδ+ and CD3+ intraepithelial lymphocytes (IELs) counting is crucial for celiac disease (CD) diagnosis. TcRγδ+ staining by immunohistochemistry (IHC) is usually performed on frozen optimal-cutting-temperature-compound (OCT)-embedded sections that are not routinely stored in most hospitals. We implemented an IHC protocol for TcRγδ+ IELs on formalin-fixed paraffin-embedded (FFPE) duodenal sections, and performed it along with CD3 staining to estimate cut-offs useful for CD diagnosis for both markers. Methods: 40 untreated-CD and 60 No-CD controls for whom both FFPE and OCT-embedded duodenal samples were available have been included in the study. IHC for TcRγδ+ and CD3+ IELs was performed on all sections and stained cells were counted per mm of epithelium. Results: In FFPE sections, both TcR-γδ+ (20.6 ± 12.7) and CD3+ (60.3 ± 29.3) IELs were higher in CD than non-CD (1.9 ± 1.7; 23.9 ± 14.8, respectively; p < 0.0001). ROC curve analysis allowed to identify the following cut-offs to discriminate active CD from No-CD: 4 TcRγδ+ and 40 CD3+ IELs/mm of epithelium on FFPE, 4 TcRγδ+ IELs and 34 CD3+ IELs on OCT-embedded-sections. Cut-offs represented the 95th percentile of No-CD controls. Correlation analyses between FFPE and OCT showed a highly positive correlation for both markers (r-Pearson coefficient 0.7, p < 0.001). Conclusion: Upon validation of an IHC protocol to stain TcRγδ+ IELs on FFPE duodenal sections that shows high sensitivity and specificity to discriminate CD patients from controls, this study establishes cut-off values for TcRγδ+ and CD3+ IELs on IHC-stained OCT and FFPE sections, showing highly comparable results for both embedding methods. Notably, this will widen the use of TcRγδ+ IELs density assessment in clinical practice, allowing a more precise diagnostic definition of difficult CD cases.