August 2007: Volume 33, Number 8
How Serological Tests Can Improve Diagnosis
By Robin G. Lorenz, MD, PhD
Historically diagnosed during childhood, celiac disease was once considered a relatively rare disease in which affected children failed to thrive after weaning and introduction of cereals into the diet (1). But as a result of new specific and sensitive serologic tests, it is now clear that this immune-mediated intestinal disease is under diagnosed. Population-based studies suggest that celiac disease, a type of gluten sensitive enteropathy (GSE), actually affects approximately 1% of the general U.S. population (2). More recent screening studies have shown that in Western countries, for each case of diagnosed disease, an average of 5–10 cases remain undiagnosed (3).
In fact, celiac disease is considered the most common genetic disease in Europe, with an estimated prevalence of 1 in 300 persons. In the U.S., however, diagnosis has been problematic. Many cases go undetected because the patient does not have a classic presentation. Instead, some patients present with symptoms related to chronic low-grade malabsorption, such as fatigue, anemia, neurologic dysfunction, bone changes, or skin rash.
Two forms of GSE are recognized today: celiac disease and dermatitis herpetiformis. Both are characterized by intolerance to the ethanol-soluble protein fraction of cereal grains in wheat known as gliadins. Affected individuals develop chronic inflammation of the small intestinal mucosa that may result in atrophy of intestinal villi, malabsorption, and a variety of clinical manifestations that can begin in either childhood or adult life.
The recent identification of the autoantigens involved in celiac disease has led to the development of new serologic diagnostic tests. In this article, I describe the clinical characteristics of celiac disease and the laboratory tests used to identify affected individuals. The criteria include the use of several serologic screening tests. These serological tests can also be used as markers of patient compliance with a gluten-free diet, as titers decline to zero after gluten withdrawal (4).
Because of the broad range of symptoms, celiac disease can be difficult to diagnose. The classic symptoms are diarrhea and weight loss; however, only a small percentage of the total celiac disease population presents with these classic symptoms. Other patients can be asymptomatic or have medical problems associated with malabsorption of oral nutrients (Table 1, below). These malabsorption syndromes can include iron deficiency, anemia due to the decreased absorption of iron, and osteoporosis secondary to malabsorption of calcium and vitamin D.
Clinical Features Encountered in GSE
|Typical Symptoms |
Failure to thrive
Anemic iron deficiency
Anemic folate deficiency
Nausea / vomiting
Recurrent abdominal pain
Recurrent apthous stomatitis
Pathologic fractures / osteoporosis
|Table adapted from references 1 and 2.|
Patients with dermatitis herpetiformis present with an itchy blistering skin eruption. These blisters usually show up in young adults, and they are more common in men and people originally from some areas of northern Europe. Biopsy of the skin lesions shows IgA deposited at the dermoepidermal junction of the skin.
Once a diagnosis of GSE has been established, the conventional treatment is a gluten-free diet. Gluten is the protein part of wheat, rye, barley, and other related grains, and affected individuals need to avoid products containing these grains.
Mechanism of Disease
A large body of work over the past decade has lead to a model for the pathogenesis of celiac disease (5). The majority of individuals who are susceptible to GSE express a particular major histocompatibility complex molecule, the HLA-DQ2 heterodimer encoded by the DQA1*0501 and DQB1*0201 genes, while a minority of patients express HLA-DQ8 (DQA1*03 and DQB1*0302). When individuals with these variants are exposed to dietary cereal grains, proteins such as gliadin cross the intestinal epithelial cell layer and initiate an immune response.
This immune response consists of intestinal T cells reacting against gliadin peptides deaminated by tissue transglutaminase. T-cell activation and cytokine production results in a cascade of downstream events that have yet to be described, but are known to damage the mucosa and result in the characteristic pathologic lesions of celiac disease: villous blunting, crypt hyperplasia, and lamina propria expansion (6).
Historical Diagnosis of GSE
In 1970, the European Society of Pediatric Gastroenterology and Nutrition (ESPGAN) published specific guidelines to identify patients with celiac disease. The diagnostic protocol consisted of three parts. The initial patient observation included the identification of gastrointestinal symptoms and histologic evidence of a flattened mucosa on an intestinal biopsy. Subsequently, after establishing a gluten-free diet, the patient had to show resolution of both clinical and histological disease. The final diagnostic step involved giving the patient a gluten challenge, with the actual diagnosis depending on the return of the symptoms and intestinal damage.
In the past 15 years, however, it has become clear that the majority of GSE does not present as the classical gastrointestinal form. Therefore, ESPGAN recently proposed a revised diagnostic protocol (Table 2, below) that includes serological screening tests (7). A brief overview of these tests is provided below. In addition, Table 3 presents the sensitivity and specificity of serum antibodies tests for identification of GSE.
Revised Criteria for the Diagnosis of GSE
- History and clinical presentation compatible with GSE
- Serological screening compatible with GSE
- Histological findings compatible with GSE
- Obvious clinical and serological response to a gluten free diet
- Subject >2 years old
- Rule out other clinical conditions mimicking GSE
Table adapted from reference 3.
Gliadin IgG and IgA Antibodies
The knowledge that celiac disease is characterized by a gluten intolerance advancing to chronic malabsorption led investigators in the early 1960s to recognize that high levels of anti-gliadin antibodies were present in serum of celiac patients. Serum gliadin antibodies are detected by a semi-quantitative ELISA and are predominantly of the IgG- and IgA- immunoglobulin classes.
To test for the antibodies, laboratorians incubate controls and diluted patient sera in microtiter wells precoated with purified gliadin antigen. The gliadin antibodies, if present, bind to the immobilized antigen. Washing steps remove unbound sample, and bound sample are detected with an enzyme-labeled, anti-human IgA, or IgG antibody. The sample value, in arbitrary units, is calculated based on a comparison with control samples run in every assay, and results are classified as negative, weakly positive, or moderate-to-strong positive.
In comparison to IgA-class antibodies, this data supports the conclusion that IgG anti-gliadin antibodies are more sensitive, but less specific markers for disease. On the other hand, IgA anti-gliadin antibodies are less sensitive but more specific. These observations can be partially explained by the fact that investigators have reported elevated levels of gliadin IgG antibodies in patients with atopic eczema, pemphigus, pemphigoid, Sjögren’s syndrome, rheumatoid arthritis, and sarcoid. Approximately 5% of the normal population will be positive for gliadin IgG antibodies; however, antigliadin antibody tests are no longer routinely recommended because of their lower sensitivity and specificity (8).
Serum Endomysial IgA Antibodies
Endomysial IgA antibody is detected in 70–76% of patients with dermatitis herpetiformis and >90% of patients with celiac disease who are on a gluten-containing diet, but the occurrence of this antibody decreases significantly on gluten-free diets.
The assay for endomysial antibodies employs an indirect immunofluorescence technique that uses monkey esophagus frozen sections as a substrate. Trained laboratory technologists evaluate the slide for presence of bright apple-green staining of the reticulin-like fibers in connective tissue around smooth muscle fibers in the monkey esophagus. All patient samples determined to be positive for the correct fluorescent pattern are endpoint titered by 1:2 dilutions to determine the strength of the endomysial IgA antibodies.
These endomysial IgA antibody titers have been shown to parallel the intestinal histopathological pattern seen on biopsy, with more severe intestinal atrophy correlating with a higher titer. Although this indirect immunofluorescent assay relies on the use of scarce materials, such as monkey tissues, and is observer-dependent and time-consuming, it boasts a reported predictive value close to 100% in most clinical settings (Table 3, below).
Sensitivity and Specificity of Serum Antibodies in GSE
| || |
|Anti-gliadin IgA || |
|Anti-gliadin IgG || |
|Anti-endomysial IgA || |
|Anti-guinea pig tTG || |
|Anti-human tTG || |
|Table adapted from references 1, 3, and 9. |
Endomysial IgG antibodies have been found in IgA-deficient patients with celiac disease, although it has a much lower specificity than endomysial IgA antibodies.
Transglutaminase IgA Autoantibodies
Recently, investigators identified the endomysial autoantigen as tissue transglutaminase (tTG). This enzyme belongs to a family of calcium-dependent enzymes that cross-link glutamine and lysine residues in substrate proteins. The first commercially available ELISAs for this enzyme used guinea pig tTG and were reproducible, sensitive, and specific tests to measure autoantibodies to tTG. However, ELISAs based on human recombinant or erythrocyte tTG have recently become available. In several studies, these human based ELISAs have been shown to be more sensitive than the guinea pig substrate (Table 3, above). These ELISAs are semi-quantitative and correlate well with semi-quantitative endomysial IgA antibodies by indirect immunofluorescence, with increasing ELISA units of transglutaminase IgA antibodies showing a positive correlation with endomysial IgA titers.
Total Serum IgA
IgA deficiency is approximately 10 times more common in people with celiac disease than in the general population. The diagnostic criteria for GSE relies on IgA antibodies to endomysium, gliadin, and transglutaminase to establish the serologic diagnosis; therefore, labs should consider determining serum IgA levels on any patient who has symptoms consistent with celiac disease but who is negative for endomysial IgA and transglutaminase IgA. Recently published data indicate that 94–100% of IgA-deficient patients with celiac disease have high-titer serum IgG gliadin antibodies (9). The adult reference range for serum IgA is 70–370 mg/dL, with IgA deficiency usually defined as a serum IgA level below 5 mg/dL.
Who Should be Screened for GSE?
While current data do not support screening the general population for celiac disease, the availability of automated assays with high diagnostic sensitivity and specificity for GSE provides clinicians with the opportunity to screen patients more easily. In addition to screening patients with symptoms such as malabsorption, fatigue, anemia, neurologic dysfunction, bone changes, or skin rash, clinicians can also rely upon the new serological tests to readily screen children with failure to thrive or with persistent diarrhea.
The spectrum of patients for whom celiac disease testing should be considered also includes those with Type 1 diabetes, where the prevalence of GSE is reported to be 5–10%. In addition, investigators have recently proposed that patients with osteoporosis may also have a higher incidence of GSE.
Toward Improved Diagnosis
Although awareness of celiac disease is increasing, the disease can still prove hard to recognize. Not all patients present with the classical symptoms of diarrhea and failure to thrive. Many have atypical and silent presentations, and the only treatment of the disease continues to be strict adherence to a gluten-free diet.
The high reliability, sensitivity, and specificity of the anti-tTG ELISA make it the screening test of choice in high-risk populations, and laboratories should consider including the test as part of their menu. As part of the laboratory’s protocol, patients should be asked to maintain a gluten-containing diet for the test. The test is easy to perform and is considered the initial diagnostic test of choice because of its high sensitivity and specificity.
For most people with celiac disease, following a gluten-free diet will stop symptoms, heal existing intestinal damage, and prevent further damage. Improvements begin quickly, within days of starting the diet. In order to stay well, people with celiac disease must avoid gluten for the rest of their lives. REFERENCES
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Robin G. Lorenz, MD, PhD is an Associate Professor of Medicine, Associate Director of the Pathology Residency Program, and Director of the Medical Scientist Training Program at the University of Alabama at Birmingham. Dr. Lorenz also is Vice Chairholder for the Clinical Laboratory Standards Institutes Area Committee on Immunology and Ligand Assay.