Although originally thought to be a disease occurring predominantly in individuals of European descent, Celiac Disease (CD) is present worldwide. Large epidemiologic studies report a global prevalence of 0.8-2.0%, with rates as high as 3.0% reported in a Swedish cohort. CD is an autoimmune disease triggered primarily by gliadin, the protein component of gluten, which is found in wheat, barley, and rye. In persons with CD, gluten consumption results in intestinal inflammation, villous atrophy, and destruction of the gastrointestinal mucosa. Although it may present at any age, CD is most commonly diagnosed in young adults exhibiting diarrhea, steatorrhea, bloating, abdominal pain, flatulence, and weight loss. Depending on disease severity and diet, symptoms may be intermittent or chronic; individuals who do not monitor gluten intake are more likely to experience flares. Given the non-specific and variable clinical presentations associated with disease, parallel laboratory testing can aid in the diagnosis of CD. The majority of CD testing is performed via assessment of autoantibody production, either via immunoassay or immunofluorescent imaging.

CD serologic tests can be categorized based on the antigen detected and induces autoantibodies against gliadin (AGA), deaminated gliadin peptides (DGP), and tissue transglutaminase (TTG) (Table 1). Assessment of AGA was one of the first tests used to evaluate patients with suspected CD; the sensitivity of AGA is variable, ranging from 46-87% (1). Detection of autoantibodies to deaminated gliadin peptides (DGP) improves upon the performance of AGA assays and is utilized in specific populations today. However, it was a research discovery that revolutionized CD testing. In the 1980s, a research team reported that sera from individuals with CD binds specifically to the endomysium of esophageal tissue sections, which may be visualized with immunofluorescence. Subsequent work led to the development of the endomysial antibody (EMA) assay. Similar to the original report, the EMA assay is an immunofluorescent technique requiring tissue sections, imaging, and skilled manual interpretation. Although still in use, EMA testing is more time and resource intensive than other CD testing methods, namely immunoassays. For nearly 20 years following the initial discovery, the mechanism of CD sera reactivity remained unknown. However, diligent research efforts revealed autoantibodies in CD sera were binding to the tissue transglutaminase (TTG), which is highly abundant in the endomysium. Autoantibody TTG-IgA testing is currently the gold standard method and is recommended as the single preferred test for diagnostic assessment of CD by American and European professional guidelines (2).

Table 1. Performance Characteristics of Tests for the Evaluations of Celiac Disease.

Test Antigen Sensitivity Specificity
TTG-IgA Tissue transglutaminase 81-100 97-99
TTG-IgG Tissue transglutaminase 27-100 77-95
EMA Tissue transglutaminase 74-100 99-100
DGP-IgA Deaminated gliadin peptides 75-78 95-100
DGP-IgG Deaminated gliadin peptides 65-71 95-98
AGA-IgA Gliadin 46-87 70-98
AGA-IgG Gliadin 42-93 84-97

TTG is an intracellular enzyme released following tissue destruction to coordinate repair of the extracellular matrix in response to injury. TTG levels increase in response to intestinal inflammation and damage, and as the injury persists, antibodies against TTG are produced. TTG antibodies are measured via ELISA, utilizing recombinant TTG to capture autoantibodies in sera. Results are semi-quantitative and reported as units per milliliter accompanied by a positive or negative interpretation. Of note, TTG assays are not standardized and there is variability in interpretation across laboratories. Generally, the threshold for positivity is between 10-20 U/mL, with some labs reporting “weak positivity” for results within this range. In the U.S., TTG-IgA is the first test used in the assessment of CD, and if positive, CD would be confirmed with duodenal biopsy. European guidelines allow for a CD diagnosis in symptomatic children if TTG-IgA is five times above the upper reference limit, thereby eliminating the need for biopsy. Clinical trials are ongoing to assess if this diagnostic algorithm is appropriate in adult populations.

Although TTG-IgA has excellent performance characteristics in most populations, there are specific clinical scenarios where TTG-IgA testing is not recommended. Selective IgA deficiency is a common immunodeficiency; its prevalence is 10-15 times higher in patients with CD. Examination of TTG-IgA in an IgA deficient individual may result in a false negative result; thus, it is critical to measure total IgA with TTG-IgA. For IgA deficient individuals, professional guidelines recommend ordering TTG-IgG and DGP-IgG to increase specificity, as TTG-IgG has lower sensitivity than TTG-IgA. There has been significant debate surrounding the role of TTG-IgA testing in patients less than 2 years of age. Several small clinical trials reported decreased sensitivity of TTG-IgA in young children. However, in 2020 a large-scale multi-center clinical trial demonstrated a sensitivity of 98% for TTG-IgA in patients less than two years old (3). In 2022, the American College of Gastroenterology revised its recommendations, moving away from a combinatorial approach, stating TTG-IgA testing alone is more accurate than combinatorial testing across pediatric populations.

All autoantibody testing requires patients to adhere to a gluten containing diet for 6 to 8 weeks prior to testing. Testing while already on a gluten-free diet, or evaluation of a person who has maintained a gluten containing diet for less than 6 weeks, may result in a false negative result. The only testing available for those already on a gluten-free diet or those unable to tolerate a gluten-containing diet is genetic testing. Although rare, false positives may occur in patients with other autoimmune conditions, such as type 1 diabetes or lupus, presumably due to cross-reactivity with high levels of autoantibody production.

Clinical research has led to significant breakthroughs in the treatment and management of individuals with CD. Evaluation of test performance is essential to proper diagnostic assessment and providing patients timely access to treatment. It is essential to critically examine the performance characteristics of clinical assays to understand if a combinatorial testing approach is best for a specific patient. For the general population TTG-IgA exhibits sensitivity and specificity approaching 100%, so the addition of other concurrent testing would lower the overall sensitivity and specificity. However, in instances where TTG-IgA is not possible (IgA deficiency) and the alternative approaches have less robust performance characteristics, the addition of another test improves sensitivity, resulting in a more accurate diagnosis. The evolution of CD testing highlights the interdisciplinary nature of diagnostic medicine and exemplifies how the expertise of clinical laboratorians may improve evidence-based medicine and support excellent patient care.


  1. Armstrong D, Don-Wauchope AC, Verdu EF. Testing for gluten-related disorders in clinical practice: The role of serology in managing the spectrum of gluten sensitivity. Canadian Journal of Gastroenterology. 2011.
  2. Rubio-Tapia A, Hill ID, Semrad C, Kelly CP, Lebwohl B. American College of Gastroenterology Guidelines Update: Diagnosis and Management of Celiac Disease. American Journal of Gastroenterology. 2023;118.
  3. Khan MR, Silvester JA, Sparks B, Hintze Z, Ediger T, Larson JJ, et al. The Utility of IgA-Based Serologic Markers in Diagnosing Celiac Disease in Children 24 Months of Age or Younger. Journal of Pediatrics. 2020;224.