Laboratory diagnostics for celiac disease

In recent years, advances in laboratory diagnostic tests for celiac disease (CD) have transformed the way the disease is diagnosed and monitored, reducing the number of small bowel biopsies performed significantly. Key laboratory investigations comprise the determination of autoantibodies against tissue transglutaminase (anti-tTG) or endomysium (EmA) antibodies against deamidated gliadin peptides and the CD-associated human leukocyte antigens (HLA) DQ2 and DQ8. Newly published guidelines confirm the pivotal role of serological and genetic testing in CD diagnostics.

Celiac disease—a multifaceted entity

CD (also known as gluten-sensitive enteropathy or non-tropical sprue) is an autoimmune disease caused in genetically predisposed individuals by consumption of gluten-containing cereals. It is characterized by inflammation of the small intestine, resulting in atrophy of the small intestinal villi and reduced resorption of nutrients. Clinical symptoms are fatigue, abdominal pain, diarrhea, effects of malabsorption such as weight loss, anemia and growth retardation in children, vomiting, constipation, and bone pains. Some CD patients additionally suffer from Duhring’s dermatitis herpetiformis, a recurrent skin disease characterized by subepidermal blisters. CD patients may also manifest with non-typical symptoms such as osteoporosis, neuropathies, carditis, pregnancy problems, or lymphoma. The only effective treatment for CD is adherence to a gluten-free diet.

Recently, it has become increasingly evident that CD exists not only in its classical form, but also more frequently in asymptomatic, silent, latent, and potential forms. The different forms have been defined more precisely.¹ Individuals with silent CD do not show any symptoms, but nevertheless exhibit specific antibodies, relevant HLA alleles, and villous atrophy. Those with latent CD do not currently have an enteropathy, but have had a gluten-dependent enteropathy in the past and exhibit compatible HLA; they may be antibody-positive or -negative and may or may not show symptoms. Potential CD refers to persons with positive antibodies and compatible HLA but as yet no symptoms, who may or may not go on to develop a gluten-dependent enteropathy.

While the prevalence of symptomatic CD is around 0.1%, the total prevalence of the disease in all its forms is estimated to be as high as 1%. Thus, the classical variant appears to represent only the tip of the celiac iceberg. What was once believed to be a rare enteropathy of childhood is now considered to be a much more common multi-organ disease.

Disease pathogenesis

The triggers of the pathological process leading to CD are prolamins, which are protein components of gluten found in many types of cereal. Gliadin is the most common prolamin, occurring, for example, in wheat. Prolamins are only partly digested in the small intestine. The resulting fragments (peptides) are taken up into the intestinal wall and chemically modified (deamidated) by the enzyme tTG. At this point they become pathologically relevant in CD patients. The immune system of affected individuals is genetically predisposed to develop immune reactions against both the deamidated peptides and transglutaminase. These reactions cause chronic inflammation of the small-intestinal mucosa via an as yet unknown mechanism.

New ESPGHAN diagnostic criteria

Early this year, the European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) released a revised set of guidelines for the diagnosis of CD.¹ These guidelines were put together by a group of 17 international experts in the field. Since the publication of the previous ESPGHAN guidelines in 1990, significant progress has been made in the development of specific antibody tests and the understanding of the role genetics play in CD. These advances have now been incorporated into the new diagnostic criteria, which are based on two algorithms: one for symptomatic and one for asymptomatic individuals.

In individuals with symptoms suggestive of CD, the first-line approach to diagnosis is the determination of specific anti-tTG antibodies of class IgA in patient serum. In order to exclude the possibility of an IgA deficiency, either total IgA or specific IgG (e.g., deamidated gliadin) should be investigated in parallel. If the anti-tTG antibody titer is very high (>10 times upper normal limit), and if this result is reinforced by positive EmA and compatible HLA, it is no longer considered necessary to perform a biopsy. If serological/genetic findings are inconclusive, results must be confirmed by histological examination of duodenal biopsy tissue to demonstrate villous atrophy and crypt hyperplasia. Diagnostic tests should be carried out in individuals on a gluten-containing diet. A gluten challenge is now only performed under exceptional circumstances.

In asymptomatic persons with a high risk factor—for example, those with type 1 diabetes mellitus, Down’s syndrome, autoimmune thyroid or liver disease, Turner’s syndrome, Williams’ syndrome, or selective IgA deficiency—and in first-degree relatives of CD patients—HLA-DQ2/DQ8 determination is the first-line analysis, followed up by specific antibody tests. In this group duodenal biopsy is a prerequisite for a definite diagnosis of CD.

The individual serological and genetic markers for CD, their roles in diagnosis, and the technologies used for their detection are described in detail in the following sections. All of the assays described here are FDA cleared.

Antibodies against tissue transglutaminase

Autoantibodies against tTG are the most important serological markers for CD. They are alternatively known as EmA, depending on the test method used: EmA are determined using indirect immunofluorescence (Figure 1), while anti-tTG are detected using monospecific test systems such as ELISA (Figure 2).

EmA and anti-tTG antibodies of immunoglobulin class IgA possess a very high sensitivity and specificity for CD. They virtually never occur in healthy individuals or patients with other intestinal diseases, whereas in untreated CD their prevalence is near 100%.

Detection of EmA using indirect immunofluorescence is considered the reference standard for CD-specific antibodies due to its unsurpassed sensitivity and specificity. However, the microscopic evaluation required is demanding and dependent on the proficiency of laboratory staff. Enzyme immunoassays for detection of anti-tTG antibodies are often preferred due to their simplicity, cost-effectiveness, and automatability, combined with their high sensitivity and specificity. Modern ELISAs for determination of anti-tTG antibodies are based on recombinant human tTG. A multitude of clinical studies have confirmed the efficacy of this method, with high-quality tests yielding a sensitivity of 90% to 100% and a specificity of 95% to 100% for active CD.

Antibodies against deamidated gliadin peptides

Antibodies against gliadin also occur in patients with CD, and their determination can strengthen a diagnosis. However, the choice of test system for detecting these antibodies is critical. Conventional test systems based on native full-length gliadin frequently yield positive reactions with sera from healthy individuals, limiting the significance of the analysis. These conventional assays, however, are now obsolete.

Recent scientific knowledge has revealed that only a tenth of the epitopes of the gliadin molecule are diagnostically relevant, and these must be present in deamidated form.² Based on these observations, a novel recombinant gliadin-analogous fusion peptide (GAF), consisting of two nonapeptide components expressed in trimeric form (3X), was created (Figure 3). The remaining 90% of the molecule was omitted, as it serves predominantly as a target for unspecific reactions. This designer fusion protein is now used as the target antigen in the anti-gliadin (GAF-3X) ELISA, providing significantly higher sensitivity and specificity than conventional anti-gliadin ELISAs.^3,4 In a multicenter study using a total of more than 900 sera, the new test yielded a sensitivity (at 95% specificity) of 83%/94% (IgA/IgG) compared to 54%/31% for a conventional anti-gliadin ELISA. This represents an increase of 29% for IgA and 63% for IgG, significantly enhancing the relevance of the analysis.

Use of the anti-gliadin (GAF-3X) ELISA in combination with the Anti-tTG ELISA significantly increases the serological detection rate for CD and dermatitis herpetiformis.⁵ The IgG version of the ELISA is particularly valuable for identifying CD patients with an IgA deficiency,⁶ which is frequently associated with CD. Determination of antibodies against deamidated gliadin is also suitable for assessing disease activity and for monitoring a gluten-free diet or a gluten-load test.

HLA-DQ2 and DQ8

HLA-DQ2 and -DQ8 are associated with CD and occur in almost all patients. Around 95% exhibit the DQ2 genotype, while the remainder have the DQ8 genotype. Although these parameters are not particularly specific, since they occur in around a third of the healthy population, they have a high negative predictive value. Hence, DQ2/DQ8 determination is an extremely valuable analysis for exclusion diagnostics. If neither DQ2 nor DQ8 is detected in a patient, then CD can be virtually excluded.

With the publication of the new ESPGHAN guidelines, HLA analysis has gained a new significance in the diagnosis of CD. DQ2/DQ8 is now recommended as the first tool for screening persons at risk of CD, as defined by the presence of an associated disease or family history. Notably, the prevalence of CD in first-degree relatives of patients is around 10%, in identical twins 70%, and in non-identical twins 11%. Individuals who are negative for DQ2/DQ8 can be reassured that their risk of having or developing CD is virtually zero. HLA analysis is also important for excluding CD as a cause of disease in cases where diagnosis is inconclusive due to ambiguous serological/biopsy results, especially in infants or in patients who are already on a gluten-free diet, and for differentiation of CD from other intestinal diseases.

HLA-DQ2/DQ8 alleles can be determined using microarray test systems such as the EUROArray system. This analysis is simple to perform. Disease-associated gene sections are amplified from purified genomic patient DNA samples by the polymerase chain reaction (PCR) (Figure 4Figure 5) and documentation of results is fully automated using specially developed software. In clinical studies employing precharacterized samples, this microarray yielded a sensitivity of 100% and a specificity also of 100%,⁷ demonstrating its ability to deliver accurate and reliable results in HLA analysis.

Conclusions

Serological and genetic tests are an integral component of CD diagnostics and diet monitoring. They not only facilitate the diagnosis of symptomatic CD, sparing many patients the discomfort and stress of a biopsy, but they also enable cases of asymptomatic, atypical, silent, and latent CD to be more easily recognized.

In pediatrics, all cases of unexplained growth disorders, retarded development, and chronic diarrhea should be considered for the possibility of CD. Likewise, patients with autoimmune diseases such as type 1 diabetes mellitus, certain chromosomal aberration diseases, osteoporosis, pregnancy problems, or lymphoma, which are known to be associated with CD, should also be screened. The importance of identifying individuals with non-classical forms of the disease is underscored by the risk of long-term health consequences such as the development of tumors. Further studies will help to establish the exact burden of CD on the population.

Jacqueline Gosink, PhD, is a Product Manager at EUROIMMUN AG in Luebeck. Germany. She started her career in the laboratory, gaining research experience in biochemistry before moving into the diagnostics industry. As part of an international team, she is responsible for promoting new innovations in medical diagnostics to laboratories worldwide.

References

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4. Prause C, et al. New developments in serodiagnosis of childhood celiac disease. Ann. N.Y.Acad.Sci. 2009;1173:28-35.
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