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Diagnostic Testing Algorithms for Celiac Disease

Click CC box for captions; full transcript is below.

Published: June 2010Print Record of Viewing
Updated: June 2011

Dr. Snyder will discuss the new celiac disease panels available from Mayo Medical Laboratories and the algorithmic approach of each.

Presenter: Melissa Snyder, PhD

Hot Topic Q&A

Hot Topic Q&A is a 30-minute conference call during which the Hot Topic presenter answers questions submitted by email. Hot Topic Q&A for Celiac was held on June 22, 2010 at 3:00pm CST. Read the transcript.

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Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Our presenter for this program is Melissa Snyder, PhD, Director of the Antibody Immunology Laboratory, in the Division of Clinical Biochemistry and Immunology at Mayo Clinic. Dr. Snyder will discuss the new celiac disease panels available from Mayo Medical Laboratories and the algorithmic approach of each.

Introduction to Celiac Disease

Celiac disease is a chronic inflammatory condition that primarily affects the small intestine.

It is caused by an inflammatory response mounted by the patient’s own immune system against dietary gluten.

This inflammatory response ultimately results in damage and atrophy of the villae within the small intestine. In the upper figure, you see a biopsy of a normal small intestine, with intact villae. In the bottom 2 figures, you see the partial and total villous atrophy that occurs in celiac disease as a result of the inflammatory response.

Clinical Features of Celiac Disease

The clinical symptoms generally associated with celiac disease are a result of the intestinal inflammation and villous atrophy.

Patients may present with abdominal pain, diarrhea, and/or vomiting. Adult patients with longer-standing disease may show symptoms of malnutrition, including iron-deficient anemia and osteoporosis. Children may present with a failure to thrive.

The point to stress here is that the symptoms of celiac disease are generally nonspecific, sometimes making for a challenging diagnosis.

Patients with celiac disease may have evidence of immunologic abnormalities, specifically a selective IgA deficiency. This type of IgA deficiency occurs approximately 10-fold more frequently in patients with celiac disease as compared to the general population. IgA deficiency is important to recognize because, as we will discuss later, many specific antibody tests used to diagnose celiac disease detect antibodies of the IgA isotype. Celiac disease is also associated with other several autoimmune endocrine and liver disorders. In addition, dermatitis herpetiformis occurs frequently in patients with celiac disease and is associated with one of the same autoantibodies implicated in the pathogenesis of celiac disease.

Development of Celiac Disease

For celiac disease to develop, the proper environmental exposure must occur in an individual with genetic susceptibility.

The environmental component is exposure to protein from wheat, barley, or rye.

Collectively, the protein agent from these cereal grains is known as gluten.

The genetic component of celiac disease had been inferred from observations that the disease occurred in families, with family members of individuals with a confirmed diagnosis of celiac disease being at a greater risk of being affected themselves. Ultimately, specific alleles of the human leukocyte antigen complex, abbreviated as HLA, were demonstrated to be responsible for much of the genetic susceptibility. These two specific alleles are HLA-DQ2 and HLA-DQ8. The HLA-DQ2 allele is found in approximately 90 to 95% of individuals with celiac disease; the remaining 5 to 10% possess the HLA-DQ8. Since HLA-DQ2 or HLA-DQ8 are found in virtually all patients with celiac disease, the absence of these alleles virtually excludes celiac disease as a diagnosis. However, the presence of either allele is not diagnostic for the disease. In other words, the HLA alleles are necessary, but not sufficient, for celiac disease to present in a given individual.

Diagnosis of Celiac Disease

The diagnosis of celiac disease relies significantly on laboratory and biopsy evaluation.

A presumptive diagnosis can be established if a patient has positive serology, which I will expand on in a moment, and an intestinal biopsy that demonstrates villous atrophy. Once a presumptive diagnosis has been established, the patient will be started on a gluten-free diet. The goal of this treatment is to remove the initiating source of the inflammatory response. Once gluten has been successfully abolished from the diet, the patient should begin to see resolution of their clinical symptoms, which is often accompanied by conversion to a negative serology and reconstitution of the villae in the small intestine. Using these criteria, a definitive diagnosis of celiac disease can be established.

As I stated in the last slide, laboratory serology plays a key role in establishing a presumptive diagnosis of celiac disease.

The primary antibodies associated with celiac disease are tissue transglutaminase, or TTG, antibodies; deamidated gliadin antibodies; and endomysial antibodies, or EMA. Testing for TTG and deamidated gliadin antibodies involve testing for IgA and IgG isotypes, while the EMA assay detects only the IgA isotype. The testing methodology is also different. TTG and deamidated gliadin antibodies are detected using plate-based enzyme immunoassays, while EMAs are detected by an immunofluorescent assay using a monkey esophagus substrate. These tests, although different in their methodologies, are related. The EMA assay is so-named because it detects an antigen in the endomysium, which is the connective tissue that surrounds smooth muscle fibers. It was subsequently determined that the antigen target of the EMA was tissue transglutaminase. It was this discovery that led to development of immunoassays specific for TTG. The antibodies against gliadin are related to the dietary gluten that initiates the inflammation in celiac disease. When the protein gluten is ingested, it is digested into smaller peptides. The ethanol-soluble fraction of gluten is referred to as gliadin. The first enzyme immunoassays developed tested for antibodies against gliadin. However, these assays were inferior to the TTG antibody and EMA assays and generally were not recommended. The newest generation of gliadin antibody assays uses a novel form of this antigen, specifically deamidated gliadin. These assays detect antibodies against gliadin that has undergone enzymatic deamidation by tissue transglutaminase. These newer assays specific for deamidated gliadin are similar to the TTG antibody assays in both sensitivity and specificity.

When testing for celiac disease using antibody serology, it is also important to remember that these antibody levels generally will decrease with proper adherence to a gluten-free diet.

HLA Typing For Celiac Disease

The other laboratory test available for celiac disease is HLA typing specifically for the DQ2 and DQ8 alleles. The HLA-DQ molecules are composed of an alpha and a beta chain. Identification of both chains is required in order to determine if either the DQ2 or DQ8 are present. This type of testing is performed using PCR amplification of the alpha and beta chains, followed by allele-specific identification on a bead-based multiplex platform. For the HLA typing, the results are reported out as "permissive genes present," "permissive genes absent," or "equivocal."

For HLA-DQ2, DQA1*05xx with DQB1*0201 or *0202 are considered permissive for celiac disease, even if an individual is heterozygous for either allele. In contrast, DQA1*0201 with DQB1*0202 must be present as a homozygote in ordered to be considered permissive for celiac disease. If only one copy of DQA1*0201 with DQB1*0202 is found, the results are considered to be equivocal.

For HLA-DQ8, a single copy of DQA1*03xx with DQB1*0302 would be considered to be permissive for celiac disease. Again, although this testing is not required to establish a diagnosis of celiac disease, it can be useful for ruling out the disease in cases where neither allele is detected.

Diagnosis of Celiac Disease

Given the variety of tests that are available for the diagnosis of celiac disease, choosing the tests that are most appropriate for a given patient, not to mention interpreting the results, can be a challenge. The clinical labs at Mayo, working closely with our GI colleagues, have established several algorithms to aid in the diagnosis of celiac disease. This first diagnostic algorithm is designed as a guide to help clinicians better order and interpret test results related to celiac disease. The algorithm itself is not available as an orderable test, but every test outlined within the algorithm is available clinically.

The remaining algorithms, which I will show you in a moment, are laboratory algorithms and are available as orderable clinical tests.


Here is the diagnostic algorithm in its entirety. When looking at it for the first time, it can seem very confusing. In the next series of slides, I will take you through the algorithm step-by-step to clarify the pathways.

Because of the prevalence of IgA deficiency in patients with celiac disease, the algorithm begins with assessment of total IgA levels.

If the IgA levels are within the age-adjusted reference range, the patient is identified as not having an IgA deficiency; the most appropriate second level of testing in the context of celiac disease would be for TTG antibodies of the IgA isotype.

If this test result is positive, a diagnosis of celiac disease is possible, and the recommendation is to proceed to a biopsy of the small intestine.

In a case like this, no other serologic testing or HLA typing is necessary.

On the other hand, if the TTG IgA antibody test is negative, a diagnosis of celiac disease is very unlikely. However, it is important to remember that a certain percentage of patients with celiac disease may be seronegative. If the testing for TTG IgA antibody is negative, but celiac disease is highly suspected based on clinical presentation or perhaps a family history, the most appropriate test with which to proceed would the HLA-DQ2 and DQ8 typing. I will expand upon the interpretation of these test results in a moment.

If the result for the TTG IgA antibody test falls in the weakly positive range, further serologic testing may be of use, specifically the deamidated gliadin IgA antibody and the EMA test.

If either the EMA or deamidated gliadin IgA tests are positive, then a biopsy of the small intestine would be warranted.

If both the EMA and deamidated gliadin IgA tests are negative, then it is possible that the result from the TTG IgA antibody test was a false positive, and it is unlikely that the patient has celiac disease. However, as stated before, some patients with celiac disease may be seronegative. If celiac disease is highly suspected, then HLA-DQ2 and DQ8 typing would be appropriate.

The test for celiac disease HLA typing identifies the DQ alpha and beta chains that correspond to the DQ2 and DQ8 alleles.

If neither DQ2 nor DQ8 is identified in the patient, celiac disease is almost conclusively excluded as a diagnosis.

On the other hand, if either DQ2 or DQ8 are present, then a diagnosis of celiac disease is possible.

In this case, if the clinical symptoms are strongly suggestive, then proceeding with a biopsy would be suggested.

That covers the pathways in the algorithm that apply to patients with normal concentrations of total IgA. Let’s move on now to individuals with low total IgA concentrations. There are 2 possible scenarios.

Individuals may have a detectable amount of total IgA, which is greater than 1 mg/dL, but below the age-adjusted reference range. For these individuals, the recommended testing is TTG and deamidated gliadin antibodies, both IgA and IgG isotypes.

If the patient has completely undetectable IgA, this would be defined as a selective IgA deficiency.

For these patients, testing for celiac disease should include TTG and deamidated gliadin antibodies, but only the IgG isotypes.

In addition, for patients with selective IgA deficiency who have a history of recurrent infections, further testing for a possible immunodeficiency is warranted. As an initial evaluation, IgG subclasses and total IgG and IgM, in addition to the total IgA, should be assessed.

Coming back to the celiac disease algorithm, if all the TTG and deamidated gliadin antibody tests are negative in these individuals, then celiac disease becomes an unlikely diagnosis. For individuals in whom the disease is highly suspected, HLA typing may be informative.

On the other hand, if any of the specific antibody test results are equivocal or positive, the patient should undergo a small intestinal biopsy. Following the biopsy, it is important to consider the pathology results in the context of the serology lab data.

If the biopsy of the patient is positive, meaning it shows evidence of villous atrophy, and the serology for more one or more specific antibodies is positive, then a presumptive diagnosis of celiac disease is established.

However, if the biopsy was performed based on positive serology and the pathology comes out to be negative, then we have a situation where the results are not conclusive.

For these cases, HLA typing is appropriate if it had not been previously performed.

If the HLA typing is negative for both DQ2 and DQ8, then both the biopsy and HLA result indicate that celiac disease is not a probable diagnosis. This likely means that the serology result was a false positive.

If the patient is positive for DQ2 or DQ8, then celiac disease remains a possibility. In this case, both the serology and HLA results are positive, but the biopsy is negative. If the patient is symptomatic, the biopsy may represent a false negative and the patient may, in fact, have a sensitivity to gluten. If the patient is asymptomatic, the negative biopsy may accurately reflect the fact that the patient’s small intestinal villae have not been exposed to an inflammatory response. However, given the positive serology and compatible HLA type, these patients should be followed for possible future development of active celiac disease.


To summarize the diagnostic algorithm, testing for total IgA is useful in identifying individuals with IgA deficiency. For this sub-group of individuals, testing for specific antibodies of the IgA isotype is of little use.

The purpose of the TTG and deamidated gliadin antibody tests is to identify individuals with suspected celiac disease. These are the individuals in whom a small intestinal biopsy is most warranted. One caveat to this testing strategy is that TTG and deamidated gliadin antibody titers generally decrease when a patient has been following a gluten-free diet.

For the HLA typing, although a positive result does not confirm a diagnosis of celiac disease, a negative result, meaning that both DQ2 and DQ8 alleles are absent, essentially rules out the diagnosis. This may be of use in individuals with equivocal serologic and/or biopsy results.

Laboratory Algorithms

To simplify test ordering, our laboratories have now implemented 3 new testing algorithms. No one algorithm is applicable to all patients being evaluated for celiac disease. However, we believe that these various testing strategies will be useful for many patients in a variety of situations.

The first cascade is the Celiac Disease Comprehensive Cascade, which includes both serologic and genetic testing.

The second cascade is the Celiac Disease Serology Cascade. This algorithm is similar to the Comprehensive Cascade, except that genetic testing is not performed.

Lastly, we have the Celiac Disease Comprehensive Cascade for Patients on a Gluten-Free Diet. This cascade only performs serology in the context of a positive genetic test. Now, I will go through each algorithm, beginning with the Comprehensive Cascade.

Algorithm Breakdown

The Comprehensive Cascade begins with both total IgA and HLA-DQ typing. All further testing reflexes automatically within the lab based on the IgA result. This reflexing occurs regardless of the HLA result.

The IgA result is classified as normal, or within the age-adjusted reference range, as low, being still detectable but below the reference range, or as deficient, or undetectable by our nephelometric assay.

All samples with a normal IgA result would automatically reflex to a TTG-IgA antibody.

For all samples testing positive or negative, no further testing would be required. The final report would include the total IgA, TTG-IgA antibody, and HLA results, along with an interpretive comment. However if the TTG-IgA result falls into the equivocal range, then EMA and deamidated gliadin-IgA testing is performed. These results, along with the total IgA, TTG-IgA, and HLA typing results, would be included in the final report.

On the other side of the cascade, those individuals who have no detectable IgA or have a selective IgA deficiency would have TTG and deamidated gliadin testing performed, but only the IgG isotypes. These results would be released as part of the final report.

Finally, for those individuals with low but detectable IgA TTG and deamidated gliadin, both IgA and IgG isotypes, would be performed. Again, all those serologic results, along with the HLA typing would be included in the final report.

This cascade is designed to perform all testing necessary to identify patients who may have celiac disease and in whom a biopsy would be suggested. It is not applicable to patients who have been following a gluten-free diet or who have been previously typed for the celiac-associated HLA-DQ alleles. For individuals who are already known to be DQ2 or DQ8 positive, or for individuals who do not want the HLA typing performed, the serologic cascade may be an option.

The Serology Cascade is identical to the Comprehensive Cascade except that the HLA typing is not performed. As far as the serology reflexing is concerned, the same pathways are followed as in the Comprehensive Cascade.

The IgA results are classified as normal, low, or deficient. For normal IgA, a TTG-IgA is performed. For positive and negative results, no further testing is required. If the TTG-IgA is equivocal, EMA and deamidated gliadin-IgA are performed, the results of which are included in the final report.

For individuals with selective IgA deficiency, testing for the IgG isotype for TTG and deamidated gliadin antibodies is performed followed by the release of the final report. For a low IgA results, both isotypes for TTG and deamidated gliadin are performed followed by the interpretive report.

The main drawback of serology testing is the gluten-free diet, which can result in depressed antibody titers. For this particular situation, we have developed the Gluten-Free Cascade.

For a patient who has instituted a gluten-free diet in whom the diagnosis of celiac disease has not been confirmed the Comprehensive Cascade for Patients on a Gluten-Free Diet may be appropriate. In this algorithm, only the HLA-DQ typing is performed initially. For those individuals who have neither the DQ2 nor DQ8 alleles, celiac disease is virtually excluded as a diagnosis.

At this point, testing for celiac disease should stop and other potential diagnoses related to the patient’s clinical presentation should be evaluated.

On the other hand, a positive result for DQ2 or DQ8 does not establish a diagnosis of celiac disease – it means only that celiac disease is a possible diagnosis. At this point, further testing should be performed, specifically all of the serologic tests.

Depending upon how long the patient has been following the gluten-free diet, and how strict the diet is, some of these serologic tests may provide a positive result. In that case, the interpretation would be that the results of all laboratory testing are consistent with celiac disease and that a biopsy should be performed.

If all results are negative, celiac disease has not been completely ruled out, since this could simply be a reflection of a successful gluten-free diet. At this point, the clinician must determine how likely the diagnosis of celiac disease is and if further evaluation, such as a gluten challenge, should be considered.


To summarize all of the potential strategies we have the diagnostic testing algorithm which may be useful for some patients. This algorithm provides flexibility, but requires that each test be ordered individually.

The laboratory algorithms, on the other hand, use automatic reflexing to perform the necessary tests. Each cascade has a specific utility. Knowledge about the patient’s clinical presentation, past treatment, and previous lab testing is critical to selecting the most appropriate lab algorithm.


I hope this presentation has provided you with useful information regarding laboratory testing for celiac disease and has helped to clarify the many options available for diagnostic testing.