A New Intraepithelial γδ T-Lymphocyte Marker for Celiac Disease Classification in Formalin-Fixed Paraffin-Embedded (FFPE) Duodenal Biopsies

Abstract

Background: The histopathologic diagnosis of celiac disease (CD) may be challenging when the duodenal biopsies mucosal injury is limited. Intraepithelial T-lymphocytes (IELs) can be useful to characterize the degree of mucosal inflammation. A small fraction of IELs expresses the γδ T-cell receptor (named γδ-IELs), whose density, determined by flow cytometry or frozen section immunohistochemistry (IHC), is a specific marker for CD.

Aim: To establish a new IHC assay for γδ-IELs applicable to formalin-fixed paraffin-embedded (FFPE) duodenal biopsies.

Methods: We analyzed γδ-IELs using IHC in 138 duodenal biopsies using a standard IHC staining protocol with a new monoclonal antibody H-41. IELs were quantitated with digital image analysis.

Results: Compared to those in non-celiac controls (n = 51), γδ-IEL density was significantly increased in newly diagnosed celiac disease patients (n = 22, p < 0.0001). In ROC-curve analysis, the cutoff of 6.5 γδ-IELs/100 enterocytes distinguished optimally active CD patients from non-celiac controls (sensitivity 96%, specificity 95%). γδ-IEL density in CD patients on a gluten-free diet (n = 53) were also higher than in controls (p < 0.0001), but lower than those in newly diagnosed CD (p < 0.0001). The diagnostic value of γδ-IELs outperformed that of CD3 + IELs in both patient groups. γδ-IELs were better than CD3 + IELs distinguishing between celiac disease and conditions histologically mimicking celiac disease (n = 12).

Conclusions: Intraepithelial γδ T-lymphocytes can be stained and quantitated reliably in FFPE duodenal biopsies. The results showed excellent specificity and sensitivity for celiac disease. The new IHC method of detection of γδ-IELs is a promising addition to the routine histopathologic assessment methodology of celiac disease.

Keywords: Autoimmune; Celiac disease; Diagnostics; Histopathology; Inflammation; Lymphocyte; Small bowel.

Fig. 1

Examples of immunohistochemistry of TCR γδ intraepithelial lymphocytes on formalin-fixed paraffin-embedded duodenal sections using monoclonal antibody H-41. Panel A represents non-celiac control, panel B represents a celiac patient on a gluten-free diet, and Panel C represents active celiac disease. Hematoxylin counterstain. Magnification × 200

Fig. 2

Distribution of duodenal CD3 + and TCR γδ + intraepithelial lymphocyte densities in non-celiac controls, celiac mimics, celiac disease patients on a GFD, and celiac disease patients with active celiac disease. Median and 95% confidence intervals are shown with horizontal lines. Dashed horizontal lines show the diagnostic cutoffs (6.5 and 25, respectively)

Fig. 3

Receiver-operator-curve (ROC curve) analysis of discrimination between cases with active celiac disease and non-celiac controls. The ROC curve corresponds to TCR γδ-IELs/100 enterocytes

Fig. 4

Distribution CD3 + and γδ IEL densities in celiac patients according to the Marsh classification. Medians are shown with horizontal lines and 95% confidence intervals with whiskers. Dashed horizontal lines show the diagnostic cutoffs (6.5 and 25, respectively)

Fig. 5

Comparison of CD3 and γδ IHC on serial formalin-fixed paraffin-embedded sections of duodenal biopsies representing active celiac disease (A, B), and drug-related duodenitis (C, D). The lack of intraepithelial TCR-γδ labeling suggests a diagnosis other than celiac disease. Hematoxylin counterstain. Magnification × 200