Maglio Mariantonia, Marano Antonella, Mandile Roberta, Angelino Pasqualina, D'Ambrosio Angelo, Mirra Anna, Auricchio Renata, Greco Luigi, Troncone Riccardo, Discepolo Valentina
European Laboratory for the Investigation of Food Induced Diseases (ELFID), University Federico II, Via Pansini 5, 80131, Naples, Italy.
Department of Translational Medical Science, Section of Pediatrics, University Federico II, Via Pansini 5, 80131, Naples, Italy.
Dig Dis Sci. 2025 Jul 16. doi: 10.1007/s10620-025-09168-8.
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.
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.
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).
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.
T细胞受体γδ（TcRγδ）⁺和CD3⁺上皮内淋巴细胞（IELs）计数对乳糜泻（CD）诊断至关重要。通过免疫组织化学（IHC）对TcRγδ⁺进行染色通常在冷冻的最佳切片温度化合物（OCT）包埋切片上进行，而大多数医院并不常规保存此类切片。我们在福尔马林固定石蜡包埋（FFPE）的十二指肠切片上实施了一种针对TcRγδ⁺ IELs的IHC方案，并将其与CD3染色一起进行，以确定对两种标志物均有助于CD诊断的临界值。
本研究纳入了40例未经治疗的CD患者和60例非CD对照，这些患者均有FFPE和OCT包埋的十二指肠样本。对所有切片进行TcRγδ⁺和CD3⁺ IELs的IHC检测，并对每毫米上皮内的染色细胞进行计数。
在FFPE切片中，CD患者的TcR - γδ⁺（20.6 ± 12.7）和CD3⁺（60.3 ± 29.3）IELs均高于非CD患者（分别为1.9 ± 1.7；23.9 ± 14.8；p < 0.0001）。ROC曲线分析确定了以下用于区分活动性CD与非CD的临界值：FFPE切片上每毫米上皮4个TcRγδ⁺和40个CD3⁺ IELs，OCT包埋切片上4个TcRγδ⁺ IELs和34个CD3⁺ IELs。临界值代表非CD对照的第95百分位数。FFPE与OCT之间的相关性分析显示两种标志物均呈高度正相关（Pearson相关系数r = 0.7，p < 0.001）。
在验证了一种在FFPE十二指肠切片上对TcRγδ⁺ IELs进行染色且对区分CD患者与对照具有高敏感性和特异性的IHC方案后，本研究确定了IHC染色的OCT和FFPE切片上TcRγδ⁺和CD3⁺ IELs的临界值，显示两种包埋方法的结果具有高度可比性。值得注意的是，这将扩大TcRγδ⁺ IELs密度评估在临床实践中的应用，从而对疑难CD病例进行更精确的诊断定义。
