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Virchows ArchivThe European Journal of Pathology ISSN 0945-6317 Virchows ArchDOI 10.1007/s00428-013-1448-7

Anti-TCR gamma antibody in celiacdisease: the value of count on formalin-fixed paraffin-embedded biopsies.

Silvia Lonardi, Vincenzo Villanacci,Luisa Lorenzi, Alberto Lanzini,Francesco Lanzarotto, Nice Carabellese,Umberto Volta, et al.

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ORIGINAL ARTICLE

Anti-TCR gamma antibody in celiac disease: the valueof count on formalin-fixed paraffin-embedded biopsies.

Silvia Lonardi & Vincenzo Villanacci & Luisa Lorenzi &Alberto Lanzini & Francesco Lanzarotto &

Nice Carabellese & Umberto Volta & Fabio Facchetti

Received: 24 April 2013 /Accepted: 2 July 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Small bowel intraepithelial lymphocytosis (IL)may depend from different causes, including celiac disease(CD). Demonstration of increased number of duodenal T cellreceptor gamma-delta (TCRγδ) positive intraepithelial lym-phocytes (IELs) has been used to support CD diagnosis onfrozen material. This work evaluates a new commerciallyavailable anti-TCRγ antibody on formalin-fixed paraffinembedded (FFPE) small bowel biopsies. Anti-CD3 andanti-TCR CγM1 (clone γ3.20) from Thermo Scientific wereapplied by immunohistochemistry on 59 FFPE biopsies from18 cases of CD with mild/severe atrophy, 19 cases of IL inCD patients on gluten-free diet (IL-GFD), 14 cases of IL(6/14 with positive CD-related serology), and 8 controls(CTR) with mild duodenitis and negative CD serology andgenotyping. IELs/100 epithelial cells were counted in at leastsix high power fields. CD3+ and TCRγ+ IELs were signif-icantly higher in CD, IL-GFD, and IL compared with CTR,but in contrast to CD3+ IELs, TCRγ+ IELs were significant-ly increased in CD and IL-GFD compared with IL.Furthermore, TCRγ+ IELs discriminated between IL withnegative and positive CD-related serology (p=0.02). TCRγ+IELs can be identified on FFPE samples and their evaluation

adds useful information for the work-up of small bowelbiopsies in CD diagnosis. In fact, TCRγ staining coupledwith CD3, may represent an additional tool to recognizecases of latent/potential CD when serology and clinical dataare not conclusive or when the histological diagnosis re-mains equivocal.

Keywords TCRγδ . Celiac disease . Intraepitheliallymphocytosis . Latent celiac disease . TCRCγΜ1

Introduction

Intraepithelial lymphocytes (IELs) that phenotypically ex-press CD3 on T cell surface are normal residents of thesmall intestinal mucosa where they modulate and activateimmune surveillance against endoluminal pathogens orfood antigens [1]. The upper normal limit of CD3+IELs in the proximal small intestine is 25 per 100epithelial cells [2, 3]; values greater than this cutoff valuein the presence of normal villous structure define acondition of intraepithelial lymphocytosis (IL). Many fac-tors have been identified in association with IL includingHelicobacter pylori infection, use of nonsteroidal anti-inflammatory drugs, immune dysregulations [4], and alsogluten sensitivity, a condition characterized by adverse reac-tion to gluten ingestion in patients in whom celiac disease(CD) has been ruled out.

In normal conditions, the large majority of IELs expressthe alpha/beta T cell receptor, and only a minor fractionexpresses the gamma/delta T cell receptor (TCRγδ) on thesurface. Many studies have reported that TCRγδ+ T cellpopulation is expanded in gluten-related enteropathy [5–8].Notably, Järvinen et al. reported that TCRγδ+ IELs had apositive predictive value (PP) of 0.95 and a negative PP of0.85 in the detection of CD [9]; additionally, TCRγδ+ IELs,differently from the total number of CD3+ IELs, remain

S. Lonardi (*) :V. Villanacci : L. Lorenzi : F. FacchettiDepartment of Molecular and Translational Medicine, Section ofPathology, University of Brescia, Brescia, Italye-mail: silvia.lona@gmail.com

A. Lanzini : F. LanzarottoGastroenterology Unit, Spedali Civili-University of Brescia,Brescia, Italy

N. CarabelleseImmunology Unit, Spedali Civili, Brescia, Italy

U. VoltaDepartment of Medical and Surgical Sciences, St. Orsola-MalpighiHospital, Bologna, Italy

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elevated also during a gluten-free diet [10–12], configuringTCRγδ as a good marker of latent/potential CD [5, 13].

Identification and count of TCRγδ+ IELs are convention-ally performed on cryosectioned snap-frozen biopsies [14].In the clinical setting, however, formalin-fixed paraffin-embedded (FFPE) samples have now largely substitutedfrozen samples, leading to a better morphological qualityand sample orientation and solving the problem of handlingfrozen samples. Because of these technical aspects, the countof TCRγδ+ IELs is mainly limited to the research setting andhas rarely been adopted in common practice for routinarydiagnosis of the small bowel diseases [15, 16]. In fact, incommon practice, IELs are counted using anti-CD3 and anti-CD8 antibodies that work on FFPE, and counting of TCRγδis not routinely carried out due to the lack of an antibodyworking on FFPE samples [15].

In this study, we evaluated a new anti-TCRγ antibody,suitable on FFPE tissues as recently reported [17], on smallbowel biopsies and propose it as a routine staining coupledwith CD3+ to better identify initial cases of CD with normalmucosa architecture and pathological number of IELs.

Material and methods

Fifty-nine cases were selected retrospectively among patientsundergoing small intestinal biopsy in the Gastroenterology Unitof Spedali Civili di Brescia between years 2010 and 2011. Thepatients included 20 males and 39 females, ranging from 2 to80 years of age with diagnosis of celiac disease with mild/severeatrophy (CD, 18 cases), IL in CD patients on gluten-free diet(IL-GFD, 19 cases), and IL (14 cases, 6 with positive tTG-IgAserology). Eight cases characterized by mild duodenitis withnormal IELs, negative for CD-related serology and for DQ2-DQ8 genotyping, were considered as controls (CTR).

Diagnosis was made by the combination of histological,serological, and genetic criteria (when available). Histologicalclassification was based on Marsh–Oberhuber criteria; serolo-gy involved detection of anti-tissue tranglutaminase (tTG)and/or anti-endomysial (EMA) IgA antibodies, and genotypinginvolved human leukocyte antigen (HLA) identification ofDQ2-DQ8 haplotypes that are characteristic of CD. CD wasdiagnosed on Marsh III lesion on histology and positivetTG/EMA serology in all cases. HLA typing was performedin a minority of cases. IL was diagnosed on IEL count >25/100epithelial cells (EC). The absolute count of IELs per 100 ECcorrelates well with IEL density expressed as cells per milli-meters of epithelium (7, 9).

Serial-cut sections (4 μm thick) of FFPE biopsies were usedfor immunohistochemical staining. CD3+ IELs were stainedusing rabbit monoclonal (SP7) anti-CD3, diluted 1:100 andTCRγ+ IELs using mouse monoclonal (γ3.20) anti-TCRCγM1, diluted 1:400, both from Thermo Scientific (Fremont,

CA). After the appropriate antigen retrieval, the reactions wererevealed using EnVision (Dako, Glostrup, Denmark) andNovolink Polymer (Novocastra Laboratories, Newcastle uponTyne, UK), respectively. Diaminobenzidine was used as chro-mogen and Meyer's hematoxylin as counterstaining.

Sections were digitalized with Aperio Scanscope CS(Nikon), and IELs/100 epithelial cells (EC) were countedin at least six high power fields (×60) per case. A mean of thecount values was obtained. Scanned slides were visualizedusing ImageScope software.

In order to better define IL, serological values of anti-tTGIgA antibodies were considered. The cutoff value is >7 AU/mland divides IL into two groups defined as ILwith negative andIL with positive serology.

Results are expressed as mean ± SD, and differences weretested for statistical significance using unpaired t test. For statis-tic analysis, theMann–Whitney test was applied (p value<0.05).Graphs were created using GraphPad Prism 5 Software.

Results

Immunohistochemical staining for anti-CD3 and anti-TCRγwas carried out on small bowel specimens of 18 CD, 19 IL-GFD, 14 IL, and 8 CTR patients. Examples of immunohis-tochemistry for CD3+ and TCRγ+ IELs are shown inFig. 1a, b for CD and in Fig. 1c–f for IL samples.

Results for CD3+ IEL count are shown in Fig. 2a. CD3+ IELcount was significantly higher in CD (52.0±15.3, p=0.0004),IL-GFD (43.6±18.3 p=0.0019), and IL (46.3±16.6, p=0.0006)compared with that of the CTR group (24.1±7.6). There was nostatistically significant difference between CD3+ IEL count inCD, IL-GFD and IL patients.

Results for TCRγ+ IEL count are shown in Fig. 2b. TCRγ+IEL count was significantly higher in CD (21.4±11.7,p<0.0001), IL-GFD (16.2±8.7, p=0.0004), and IL (9.9±6.6,p=0.0046) compared to CTR group (3.4±1.0). By contrast toCD3+ IEL count, TCRγ+ IEL count was significantly higherin CD (21.4±11.7, p=0.0010) and IL-GFD (16.2±8.7,p=0.0126) than in IL (9.9±6.6, p=0.0004) (not shown).Furthermore, TCRγ+ IEL count was significantly higher(14.2±8, p=0.02) in IL patients with positive CD-related se-rology (representative staining in Fig. 1d) than in those with ILand negative CD-related serology (6.7±2.9) (representativestaining in Fig. 1f and data in Fig. 2c).

Discussion

In clinical practice, a high TCRγδ+ IEL count may help inidentifying patients with latent or so-called potential celiacdisease defined as a clinical condition presenting with posi-tive or borderline serology for CD without villous atrophy on

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Fig 1 Serial sections from FFPEsmall bowel biopsies stained forCD3 (a, c, and e) and TCRγ(clone γ3.20) (b, d, and f). In aand b, a representative case ofceliac disease (CD) displays amarked increase of CD3+lymphocytes in the atrophicepithelium and in lamina propria(a) and a significant increase ofTCRγ+ intraepitheliallymphocytes (IELs) (b). In c, d,e, and f, two representative casesof intraepithelial lymphocytosis(IL) with positive (c and d) andnegative (e and f) tTG serology.CD3 staining illustrates a clearincrease of IELs inarchitecturally preserved villiwhereas TCRγ stainings show asignificant increase of TCRγ+IELs only in the case withpositive CD-related serology (d).Sections are counterstained withMayer's hematoxylin.Magnification is ×200. Scale baris 100 μm

Fig. 2 Graphs of counts of CD3 (a) and TCRγ+ IELs/100 EC (b) inCD, IL-GFD, IL, and CTR cases. Both CD3 and TCRγ were signifi-cantly different for each group compared to CTR. P value in a: singleasterisk (*) CD vs CTR p=0.0004, double asterisk (**) IL-GFD vsCTR p=0.0019, triple asterisk (***) IL vs CTR p=0.0006. P value in

b: single asterisk (*) CD vs CTR p<0.0001, double asterisk (**) IL-GFD vs CTR p=0.0004, triple asterisk (***) IL vs CTR p=0.0046. Inc, IL group (14 cases) was divided based on tTG serology; eight caseswere negative, and six cases were positive. TCRγ+ IELs/100 EC countwas significantly different between the groups (p=0.02).

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biopsy. tTG is recommended in clinical practice for CDserological screening, but false positive results may occurin conditions such as primary biliary cirrhosis, rheumatoidarthritis, and inflammatory bowel disease [18, 19]. EMArepresents the gold standard of CD-related serology but isdifficult to standardize [20] because it requires skill andexperience in immunofluorescent assay [21].

The observations reported indicate that there are caseswhere IL cannot be confidently attributed to latent/potentialCD but to other causes unrelated to gluten exposure. In thesecases, detection of increased TCRγδ+ IEL count may beuseful for a correct diagnosis. Studies carried out on frozentissue have shown a direct correlation between an increasednumber of TCRγδ+ IELs and latent/potential celiac disease[22–24]. Based on this finding, lymphogram on IEL isolatedby flow cytometry has been proposed as an initial screeningfor CD [25] given the distinctive feature of increased numberof total IELs and TCRγδ+ IELs in CD [26]. Despite the highpositive predictive value of increased TCRγδ+ IELs fordiagnosing latent or potential CD, the count of TCRγδ+IELs has been of limited diagnostic utility for the practicingpathologist due to the lack of an anti-TCRγδ antibody suit-able for FFPE tissue [15, 16].

In this work, we tested a new commercially available anti-TCRγ antibody suitable for FFPE tissue and evaluated itsusefulness for the work-up of small bowel biopsies in thediagnosis of CD in clinical practice. As already demonstratedon frozen biopsies [9], on FFPE TCRg+ cells were veryabundant in CD, IL-GFD, and IL compared to those of theCTR. TCRγ+ IEL count was also significantly higher in CDand IL-GFD groups, conditions within the spectrum ofgluten-related disease, than in that of the IL, a heterogeneouscondition associated with a variety of causes. It is of partic-ular importance the finding that TCRγ+ IELs increased incases associated with positive CD-related serology that arelikely to represent early CD stages.

In this study, we did not test the anti-TCRγ antibody onenteropathies—except for CD—that occasionally have beenreported to be characterized by increased number ofTCRγδ+ IELs. Kokkonen et al. reported a significantlyhigher density of TCRγδ+ cells in children suffering of foodallergy similar to those observed in CD, but differently fromCD, IL was not present in children with food allergy [27].Spencer et al. reported 4/18 cases (22 %) of cow milk-sensitive enteropathy and post-enteritis syndrome with highnumbers of TCRγδ+ IELs but normal IELs. These findingshighlight that a diagnosis of CD requires the presence of IL,because high TCRγδ+ IELs alone can be misleading for thediagnosis of CD [28].

In conclusion, our work shows the feasibility to performanti-TCRγ immunohistochemistry together with anti-CD3 onFFPE small intestinal biopsies in routine clinical practice, andwe propose this as a tool to recognize cases of latent or

potential CD when serology and clinical data are not conclu-sive or when the histological diagnosis remains equivocal.

Acknowledgments SL is supported by “Borsa di studio Prof RobertoTosoni” (Garda Vita, BCC del Garda). All authors approved the finalversion of the article, including the authorship list.

Conflict of interest The authors declare no competing financial interests.

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