The laboratory is challenged to provide guidance on celiac disease test ordering and interpretation while ensuring accurate performance and appropriate test utilization
Vol. 23 • Issue 9 • Page 38
Celiac Disease Testing
Celiac disease (CD) is an immune-mediated inflammatory process elicited by gluten that occurs in genetically susceptible individuals.1,2 The inflammatory response is triggered when partially digested gluten peptides (especially gliadin) reach the intestinal mucosa.2 These peptides are a substrate for tissue transglutaminase (TTG), which acts to deamidate gliadin, forming deamidated gliadin peptide (DGP).3 Both DGP and DGP-TTG complexes can be immunogenic, resulting in the production of antibodies against TTG and DGP, local inflammation, and damage to the intestinal mucosa causing malabsorption.3 This can lead to signs and symptoms such as anemia, failure to thrive, weight loss, diarrhea and bloating.1,3 HLA molecules encoded by the DQ2 and DQ8 haplotypes are associated with this immune process, forming part of the genetic basis of CD.4
Traditionally, CD diagnosis requires duodenal biopsy to characterize the degree of mucosal damage. Histological changes seen in CD include villous atrophy, crypt hyperplasia and increased intraepithelial lymphocytes. Classification of damage severity is outlined by the Marsh Criteria, which remains the gold standard for CD diagnosis.1,5-7 Histological analysis is not without its limitations, however (Table).8,9 Biopsy procedures are invasive, so other means of identifying CD patients have been sought. Serological tests, which detect antibodies produced during the mucosal immune reaction, provide a less invasive alternative.
Early indirect immunofluorescence methods detected CD-specific antibodies in a semi-quantitative fashion. The anti-endomysial antibody test (EMA) remains the most sensitive and specific of these.10,11 A positive test is defined by the presence of staining restricted to smooth muscle fibers, and titers can be reported for semi-quantitative results. This assay can be challenging due to scarcity of tissue substrates, subjective interpretation and method heterogeneity between labs.11-13 Other immunofluorescence-based markers such as reticulin and gliadin antibodies have been shown to have inferior sensitivity and specificity to EMA, and should not be used for CD testing.1,14,15
The antigenic target of EMAs was later discovered to be TTG;16 DGP was also found to be a target of antibodies in CD patients.17,18 This led to the production of quantitative immunoassays for both anti-TTG and anti-DGP antibodies. Solid-phase immunoassays using immobilized TTG or DGP are commonly used. Following incubation with a patient’s serum, anti-TTG or anti-DGP antibodies are detected using labeled anti-human, class-specific detection antibodies. Radiobinding assays have also been described.19 These assay formats allow for the use of a calibration curve and reporting in concentration units. A positive result is defined at a specific cut-off.1 However, because assay reagents vary by manufacturer, laboratories and clinicians need to be aware that cut-offs and results are not interchangeable.19-21
Other Testing Considerations
Several other important considerations must be made when using serological testing in CD. First, mucosal immune responses typically involve IgA-class antibodies; therefore, serological testing commonly targets IgA class antibodies. However, IgA deficiency occurs more frequently in CD patients compared to population estimates (2.5% versus 0.25%),22 and IgA-based serological tests may not provide informative results in these patients. IgA quantitation can help interpret CD serology and prompt the use of IgG-based
tests when necessary.23-25 The use of immunosuppressants can decrease immunoglobulin levels, also impairing result interpretation. Therefore, medication status of patients must be considered.1 Finally, some studies have suggested that not all serological markers perform equally in young children, so alternative testing strategies may be required in some patient groups.26,27
The nature of the immune response in CD should also be considered. Since antibodies are produced in response to gluten exposure, false-negative results may occur if a patient is on a gluten-free diet (GFD). However, since symptoms and antibodies remit upon removal of gluten, serological tests are useful for monitoring adherence to a GFD.1,28
A portion of CD risk is heritable and related to HLA molecules, specifically those encoded by DQ2 and DQ8 haplotypes.4,29,30 While these are thought to be required for the development of CD, their presence is not sufficient for diagnosis. Absence of HLA DQ2 or DQ8-associated alleles is thought to rule out CD, making HLA typing useful due to its high negative predictive value.1,4,28 In some situations, a positive HLA result may also aid in patient management decisions.1
Clinical guidelines from a number of organizations have been recently updated to reflect advancements in our understanding of CD pathophysiology, biology and genetics. The lab needs to be aware of these updates to provide guideline-based services, optimize testing algorithms, and understand changes in ordering patterns. The reader is referred to the full guideline documents for complete information.1,5,14,28,31
The recent European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) guideline update includes novel approaches to CD testing in children, with separate algorithms proposed for symptomatic patients and asymptomatic patients at high risk of CD (for example, those with affected first-degree relatives, other autoimmune conditions, or certain chromosomal and endocrine abnormalities).1 The former advocates for IgA-TTG testing first, along with ascertainment of IgA sufficiency. Very high TTG results may not need confirmation by biopsy, but can be substantiated by EMA and HLA testing.1 While prospective data is still needed, a few studies have yielded promising results for this approach.32,33 In asymptomatic high-risk children, HLA testing is suggested as the first-line test due to its high negative predictive value. TTG testing is then reserved for those who are HLA-positive, or have not had HLA typing performed. Confirmation of low-positive TTG results by EMA is recommended to reduce the number of false-positive results and unnecessary biopsies.1
Guidelines from other agencies are applicable to a broader age group and may not distinguish between low and high-risk individuals. These include documents from the American College of Gasteroenterology (ACG), North American Society for Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHN), the World Gastroenterology Organization (WGO) and the UK National Institute for Health and Care Excellence (NICE).5,14,28,31 Most of these organizations recommend IgA-TTG as the first-line test and outline its use in symptomatic or high-risk individuals. The WGO suggests TTG and/or DGP, with the addition of EMA to increase specificity if screening in a general population.28 Biopsy remains the gold standard for diagnosis and is not omitted in any case by these organizations, with possible exceptions in resource-limited settings.28
- Husby S, Koletzko S, Korponay-Szabo IR, et al. European Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of coeliac disease. J Pediatr Gastroenterol Nutr 2012;54:136-60.
- Fasano A, Shea-Donohue T. Mechanisms of disease: the role of intestinal barrier function in the pathogenesis of gastrointestinal autoimmune diseases. Nat Clin Pract Gastroenterol Hepatol 2005;2:416-22.
- Alaedini A, Green PH. Narrative review: celiac disease: understanding a complex autoimmune disorder. Annals of internal medicine 2005;142:289-98.
- Sollid LM. Molecular basis of celiac disease. Annu Rev Immunol 2000;18:53-81.
- Rubio-Tapia A, Hill ID, Kelly CP, Calderwood AH, Murray JA. ACG clinical guidelines: diagnosis and management of celiac disease. Am J Gastroenterol 2013;108:656-76.
- Marsh MN. Grains of truth: evolutionary changes in small intestinal mucosa in response to environmental antigen challenge. Gut 1990;31:111-4.
- Oberhuber G, Granditsch G, Vogelsang H. The histopathology of coeliac disease: time for a standardized report scheme for pathologists. European journal of gastroenterology & hepatology 1999;11:1185-94.
- Husby S, Murray JA. New aspects of the diagnosis of celiac disease in children, adolescents, and adults. Mayo Clin Proc 2013;88:540-3.
- Pai RK. A practical approach to small bowel biopsy interpretation: celiac disease and its mimics. Semin Diagn Pathol 2014;31:124-36.
- Chorzelski TP, Beutner EH, Sulej J, et al. IgA anti-endomysium antibody. A new immunological marker of dermatitis herpetiformis and coeliac disease. Br J Dermatol 1984;111:395-402.
- Volta U, Molinaro N, de Franceschi L, Fratangelo D, Bianchi FB. IgA anti-endomysial antibodies on human umbilical cord tissue for celiac disease screening. Save both money and monkeys. Dig Dis Sci 1995;40:1902-5.
- Amara W, Husebekk A. Improved method for serological testing in celiac disease–IgA anti-endomysium antibody test: a comparison between monkey oesophagus and human umbilical cord as substrate in indirect immunofluorescence test. Scand J Clin Lab Invest 1998;58:547-54.
- Murray JA, Herlein J, Mitros F, Goeken JA. Serologic testing for celiac disease in the United States: results of a multilaboratory comparison study. Clin Diagn Lab Immunol 2000;7:584-7.
- Hill ID, Dirks MH, Liptak GS, et al. Guideline for the diagnosis and treatment of celiac disease in children: recommendations of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition. J Pediatr Gastroenterol Nutr 2005;40:1-19.
- Nandiwada SL, Tebo AE. Testing for antireticulin antibodies in patients with celiac disease is obsolete: a review of recommendations for serologic screening and the literature. Clin Vaccine Immunol 2013;20:447-51.
- Dieterich W, Ehnis T, Bauer M, et al. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997;3:797-801.
- Aleanzi M, Demonte AM, Esper C, Garcilazo S, Waggener M. Celiac disease: antibody recognition against native and selectively deamidated gliadin peptides. Clin Chem 2001;47:2023-8.
- Schwertz E, Kahlenberg F, Sack U, et al. Serologic assay based on gliadin-related nonapeptides as a highly sensitive and specific diagnostic aid in celiac disease. Clin Chem 2004;50:2370-5.
- Li M, Yu L, Tiberti C, et al. A report on the International Transglutaminase Autoantibody Workshop for Celiac Disease. Am J Gastroenterol 2009;104:154-63.
- Beltran L, Koenig M, Egner W, et al. High-titre circulating tissue transglutaminase-2 antibodies predict small bowel villous atrophy, but decision cut-off limits must be locally validated. Clinical and experimental immunology 2014;176:190-8.
- Vermeersch P, Geboes K, Marien G, Hoffman I, Hiele M, Bossuyt X. Defining thresholds of antibody levels improves diagnosis of celiac disease. Clin Gastroenterol Hepatol 2013;11:398-403.
- Green PH, Cellier C. Celiac disease. N Engl J Med 2007;357:1731-43.
- McGowan KE, Lyon ME, Butzner JD. Celiac disease and IgA deficiency: complications of serological testing approaches encountered in the clinic. Clin Chem 2008;54:1203-9.
- Korponay-Szabo IR, Dahlbom I, Laurila K, et al. Elevation of IgG antibodies against tissue transglutaminase as a diagnostic tool for coeliac disease in selective IgA deficiency. Gut 2003;52:1567-71.
- Villalta D, Tonutti E, Prause C, et al. IgG antibodies against deamidated gliadin peptides for diagnosis of celiac disease in patients with IgA deficiency. Clin Chem 2010;56:464-8.
- Lagerqvist C, Dahlbom I, Hansson T, et al. Antigliadin immunoglobulin A best in finding celiac disease in children younger than 18 months of age. J Pediatr Gastroenterol Nutr 2008;47:428-35.
- Barbato M, Maiella G, Di Camillo C, et al. The anti-deamidated gliadin peptide antibodies unmask celiac disease in small children with chronic diarrhoea. Dig Liver Dis 2011;43:465-9.
- Bai JC, Fried M, Corazza GR, et al. World Gastroenterology Organisation global guidelines on celiac disease. J Clin Gastroenterol 2013;47:121-6.
- Greco L, Romino R, Coto I, et al. The first large population based twin study of coeliac disease. Gut 2002;50:624-8.
- Fasano A, Berti I, Gerarduzzi T, et al. Prevalence of celiac disease in at-risk and not-at-risk groups in the United States: a large multicenter study. Arch Intern Med 2003;163:286-92.
- Recognition and assessment of coeliac disease. NHS, 2009. (Accessed July 19, 2014, at http://www.nice.org.uk/CG86.)
- Klapp G, Masip E, Bolonio M, et al. Celiac disease: the new proposed ESPGHAN diagnostic criteria do work well in a selected population. J Pediatr Gastroenterol Nutr 2013;56:251-6.
- Guandalini S, Newland C. Can we really skip the biopsy in diagnosing symptomatic children with celiac disease. J Pediatr Gastroenterol Nutr 2013;57:e24.
Sidebar: Case for the Neo-epitope By Dr. Torsten Matthias
The so called neo-epitope is the complex of tissue transglutaminase (tTg) and gliadin peptides proven to form in vitro and in vivo.
Fleckenstein et al characterized the molecular structure of the covalent complexes between tissue transglutaminase and gliadin peptides.1 Ciccocioppo et al demonstrated that the deamidation and crosslinking of gliadin peptides by transglutaminases formed supramolecular complexes in normal and diseased duodenal mucosa and that the level of both molecules was increased in active celiac disease.2
Several external studies proved the superior performance of ELISA assays using the neo-epitope as antigen to capture autoantibodies linked to celiac disease (CD) and dermatitis herpetiformis (DH). In 2006, Reeves published an Australian multicenter study in which the neo-epitope screening test performed best compared to 11 competitor assays.3 In 2008, Marcos demonstrated that the neo-epitope ELISA assay was a much better screening tool for CD in pediatric and adult samples compared with assays using pure tTg.4
McMillan revealed in his study that high positive results for neo-epitope antibodies correlate with the severity of mucosal damage.5 Therefore, he stated that confirmatory small bowl biopsy might not be needed in patients with high levels of CD antibodies.
Jaskowski showed that neo-epitope IgA assays exhibited good sensitivity and specificity in pediatric population confirmed by biopsy.6 Moreover, this study proved its very good correlation with MARSH criteria also in pediatrics.
A year later, Tozzoli stated that the neo-epitope ELISA assay was able to identify CD patients who have been tested negative with conventional antibody assays.7 In the same year, Tonutti demonstrated that the neo-epitope IgA test became positive earlier than anti-tTg assays.8
Two years later, Bizzaro confirmed that the neo-epitope ELISA assay had a very high sensitivity and that this method for measuring anti-complex antibodies could be used as a reliable test for screening in the general population or in at-risk groups.9 He also stated that one could hypothesize that these antibodies were present early in the natural course of CD and, therefore, had a predictive value for clinical and subclinical disease patients. In 2013, Lytton stated that the neo-epitope IgA autoantibodies represented a new and sensitive marker for DH.10
These and further studies (comprising more than 50,000 samples) demonstrated the superior performance of the neo-epitope antigen as a highly sensitive and specific screening assay for both adults and pediatrics. Due to its very good correlation with MARSH criteria, biopsy could be potentially avoided in cases of high levels of neo-epitope antibodies. Studies also demonstrated that the neo-epitope IgA antibodies represented a sensitive marker for both CD and DH.
More importantly, it showed that antibodies to the neo-epitope could be positive up to 12 months earlier than anti-tTg antibodies. This shortens the time to diagnosis, which has an important impact on the health improvement status of the patients.
Dr. Matthias has nearly two decades of experience with R&D in the autoimmune field. AESKU, based in Wendelsheim, Germany, is a research-focused supplier of products and services for early detection diagnosis.
- Fleckenstein B. et al. Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides. J.Biol.Chem. 279 (2004) 17607-17616
- Ciccocioppo R. et al. Gliadin and tissue transglutaminase complexes in normal and celiac duodenal mucosa. Clin.Exp.Immunol. 134 (2003) 516-524
- Reeves G.E. Diagnostic accuracy of coeliac serological tests: a prospective study. Eur.J.Gastroenterol. Hepatol. 18(5) (2006) 493-501
- Marcos M.G. et al. tTg neo-epitopes: a complex of deamidated gliadin peptides crosslinked with tissue transglutaminase as an ideal screening tool for celiac disease in comparison to tissue transglutaminase. Presented at the 6th International Congress on Autoimmunity 2008
- McMillan S.A. et al. Relationship between coeliac serology and small bowel biopsy findings in clinical practice. Presented at the International Celiac Disease Symposium 2009
- Jaskowski T.D. et al. Tissue transglutaminase (tTg) neo-epitope ELISA: a useful tool for the detection of celiac disease in pediatric samples. Presented at the International Celiac Disease Symposium 2009
- Tozzoli R. et al. Clinical relevance and diagnostic accuracy of a new ELISA method for the detection of autoantibodies to the gliadin-transglutaminase complex. Presented at the 7th International Congress on Autoimmunity in 2010
- Tonutti E. et al. Antibodies to the transglutaminase-deamidated gliadin peptides complex: a new serological approach to the diagnosis of celiac disease. Presented at the 7th International Congress on Autoimmunity in 2010
- Bizzaro N. et al. Cutting-Edge issues in celiac disease and in gluten intolerance. Clinic. Rev. Allerg. Immunol. 42(3) (2012) 279-287
- Lytton SD et al. Neo-epitope tissue transglutaminase autoantibodies as a biomarker of the gluten sensitive skin disease–dermatitis herpetiformis. Clin. Chim. Acta. 415 (2013) 346-349