Serologic analysis is invaluable in the diagnosis and management of gluten-sensitive enteropathy (GSE), a disease spectrum that may affect the bowel as in celiac disease (CD) or the skin as in dermatitis herpetiformis (DH) (1). Patients with CD develop autoantibodies and intestinal mucosal inflammation in response to ingestion of gluten (2). Patients with DH develop a papulovesicular cutaneous eruption most commonly on extensor elbows, knees, and buttocks also precipitated by ingestion of gluten (3). Autoantibodies in GSE can be detected using a variety of serologic assays; enzyme immunoassay (EIA) and indirect immunofluorescent antibody (IFA) are most commonly used. Detection of IgA antibodies is preferred, although screening for IgG isotype may be beneficial in IgA-deficient patients (4).
IgA antibodies against tissue transglutaminase (tTG) are present in most patients with GSE. tTG deamidates gliadin, the ethanol-soluble glycoprotein found in gluten (5). The humoral system targets both proteins as a consequence of a pathologic helper T-cell–mediated response (6). EIAs detecting IgA antibodies against guinea pig tTG were initially developed, but assays using native tTG purified from human red blood cells or human recombinant tTG are preferred for their increased sensitivity and specificity (7). In a recent study, semiquantitative units of IgA anti-tTG showed a strong correlation with degree of small bowel atrophy in pediatric CD (8). Pooled analysis suggests that sensitivities and specificities for tTG IgA EIAs exceed 95% (9). However, these values are likely lower in clinical practice, especially with partial villous atrophy (8,10). tTG, the cross-linking enzyme in endomysium, is recognized by serum autoantibodies in patients with GSE. IgA anti-tTG by EIA has a similar sensitivity and specificity to IgA anti-endomysium antibodies (EMA) detected by IFA. Compared with commercial EIA, IFA has the disadvantages of increased cost and observer variability (1).
Before tTG was isolated, patients with suspected CD were screened for anti-gliadin antibodies (AGA). However, the reported sensitivity and specificity for IgA anti-AGA in CD vary widely (9). In several studies, selective deamidation of gliadin increased performance for both IgA and IgG in adult and pediatric patients with CD (11,12). In fact, the sensitivity and specificity of EIA using synthetic deamidated gliadin peptides (DGP) is considerably superior to whole gliadin (11,13). Studies have shown IgA anti-DGP to have up to 97% concordance with IgA anti-tTG and EMA with sensitivities exceeding 92% and specificities more than or equal to 99% (12,14,15).
Clinicians may use serology to screen patients suspected of having GSE before small bowel mucosal or skin biopsy (the gold standards for CD and DH diagnosis, respectively) and to follow response to a gluten-free diet (GFD) (1). As serologic assays are further developed and deemed specific for CD or DH, their role may evolve from screening to definitively diagnostic (8,16). The tTG and DGP EIAs are commercially available and are specific and sensitive for CD. In this study, we evaluated the performance of a novel GSE screening assay in a cohort of pediatric CD and adult DH. Because screening for GSE is commonly performed in clinical practice, a combined screening assay with high specificity and sensitivity that eliminates the need for measuring total serum IgA would be beneficial.
PATIENTS AND METHODS
Sera from the following patient groups were analyzed: 111 retrospective pediatric patients suspected of having CD, all ingesting a normal diet undergoing duodenal biopsy (54 boys, 1.2–17.9 years, mean 7.7; 57 girls, 1.3–18.6 years, mean 8.5); 80 retrospective adult (>18 years) patients with DH on a normal diet at the time of their initial clinical evaluation; 50 prospective adult DH patients drawn postdiagnosis with some patients receiving treatment (dapsone) and/or on a GFD with variable compliance; and 49 adult and 77 pediatric age-matched normal controls. Sera from 10 patients submitted to ARUP Laboratories for GSE testing with IgA deficiency (<7.0 mg/dL) and IgG antibodies against 1 or more of the traditional serologic markers associated with GSE were also included. We used the recent North American Society for Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN) guidelines for assessment of a diagnosis of CD (1). Small bowel mucosal biopsy specimens were graded 0–3c according to the modified Marsh criteria as previously described (8,17–19). All of the patients with DH were confirmed by skin biopsy and demonstrated the characteristic IgA staining of dermal papillae by direct immunofluorescence (DIF) as previously described (20,21). Because none of the patients with DH had GI symptoms associated with CD, intestinal biopsy was not performed. These studies were approved by the University of Utah institutional review board and met the Health Information Portability and Accountability Act (HIPAA) patient confidentiality guidelines.
The QUANTA Lite h-tTG/DGP Screen (product number 704575) and individual IgA and IgG EIAs for tTG and DGP were donated by INOVA Diagnostics (San Diego, CA). The QUANTA Lite h-tTG/DGP Screen uses purified synthetic DGP (gliadin II) and native human tissue transglutaminase (h-tTG) bound to polystyrene microtiter wells under conditions that preserve the antigens in their native state. After incubation with diluted patient serum and washing away the unbound sample, enzyme-labeled antihuman IgA and IgG conjugate (divalent) was added to each well. After washing the wells, a chromogenic substrate was added and optical density was measured at 450 nm using a spectrophotometer (Molecular Devices, Sunnyvale, CA).
These assays have been approved by the Food and Drug Administration for in vitro diagnostic use, and cutoff values recommended by the manufacturer (<20 U = negative; all assays) were used for statistical analysis. IgA anti-EMA IFA results for our pediatric patients with CD and their correlation to tTG IgA and Marsh grade were previously reported (8).
Sensitivity, specificity, positive and negative predictive values, and area under the receiver operating characteristic curve (AUC) were calculated for each assay. Ninety-five percent confidence intervals were also computed for sensitivity, specificity, and positive and negative predictive values. All of the statistics were generated using SAS software, version 9.1 of the SAS system for Windows (SAS Institute Inc, Cary, NC).
Fifty-four of the 111 pediatric patients evaluated for GSE were diagnosed as having CD. Forty-nine of these 54 (90.7%) patients had a Marsh grade of 3a-c and the remaining 5 had Marsh 0–1 pathology. In this cohort of pediatric patients with CD, the tTG/DGP screen achieved slightly higher sensitivity than traditional IgA anti-tTG (92.6% vs 90.7%, respectively) by detecting 1 additional patient (Table 1). This patient, a 6-year-old girl with Marsh 3c histopathology, was found to be positive only for IgG anti-DGP (46 U) when assessed by the individual assays. Specificity for the tTG/DGP screen was slightly less than that of IgA anti-tTG (96.1% vs 100%) in pediatric normal controls (Table 1). Sera from 3 pediatric normal controls produced low positive values (22, 23, and 24 U) by the tTG/DGP screen. Each of these 3 pediatric controls produced values (15–19 U) slightly under the cutoff for 2 of the 4 individual markers tested (data not shown). One additional pediatric normal control gave positive results (35 U) for DGP IgA only; results for all other assays were well below the cutoff. This sample was repeated in which the original results were confirmed. Duodenal biopsy was not performed on the pediatric normal controls.
The tTG/DGP screen detected all 10 IgA-deficient (<7 mg/dL) sera sent to ARUP Laboratories for GSE testing that were IgG positive against tTG and/or DGP (data not shown); clinical information was not available for these patients. Two of the 54 pediatric patients diagnosed as having CD demonstrated depressed levels (7–68 mg/dL) of serum IgA. One patient (Marsh 3c; serum IgA 38.7 mg/dL) produced positive results for the tTG/DGP screen (106 U), IgA anti-tTG (44 U), IgA anti-DGP (34 U), and IgG anti-DGP (110 U), whereas the second patient (Marsh 3a; serum IgA 39.1 mg/dL) was negative (<20 U) by all of the assays; this patient improved on a GFD.
When comparing serology to histology, there were 13 patients with discordant results (Table 2). Of the 6 patients having Marsh 0–1/serology+ results, 2 were diagnosed with latent CD (because there was a strong family history of CD), 1 with eosinophilic esophagitis, 1 with CD (improved on GFD), and 2 were unknown because there was no follow-up visit or a GFD was refused (Table 2). These last 2 patients likely had CD based on gastrointestinal symptoms and having multiple positive serologic results; both patients were also IgA anti-EMA+ by IFA with a titer of 1:80 (Table 2). Of the 7 patients with Marsh 3a–c histopathology and negative serology, 4 were diagnosed with CD (improved on GFD), 2 with inflammatory bowel disease (IBD), and 1 with eosinophilic gastroenteritis (Table 2).
Performance of the tTG/DGP screen in DH is shown in Tables 3 and 4. Percent sensitivity and specificity were 65 and 100 for retrospective DH, and 62 and 100 in prospective DH. Although superior to tTG IgA alone, the low sensitivity of the tTG/DGP assay makes it unsuitable as a screening test for this form of GSE. In addition, the median tTG/DGP screen value for DH was much lower than in pediatric CD (Fig. 1).
According to published guidelines, serologic testing is of primary importance in screening for GSE. We found a commercially available anti-tTG/DGP EIA screen (IgA + IgG) to be more sensitive than IgA anti-tTG alone for pediatric CD and DH. The tTG/DGP screen identified 1 pediatric patient with CD (6.1 years/girl; Marsh 3c) that was IgA anti-tTG negative. The serum from this patient exhibited only IgG against DGP, demonstrating the utility of screening for both IgA and IgG autoantibodies in patients suspected of having GSE. This would also be useful in cases of IgA deficiency, in which patients with CD are thought to produce IgG-class autoantibodies to GSE antigens (22,23).
Guidelines indicate serum total IgA should be performed in all patients screened for IgA-class celiac serologies (1). In our pediatric population at risk for CD, 2 patients with histologic changes had depressed levels (7–68 mg/dL) of total IgA and none were found to be completely IgA deficient (<7 mg/dL). The first, graded as a Marsh 3c on biopsy, had a positive tTG/DGP screen (106 U) and specific increases in IgA anti-tTG (44 U), IgA anti-DGP (34 U), and IgG anti-DGP (110 U). The second had a Marsh 3a lesion and was seronegative by all assays. Repeat or follow-up intestinal biopsy was not available in this patient to confirm the histologic diagnosis of CD. The patient improved, however, on a GFD. The tTG/DGP screen uniformly identified 10 IgA-deficient patient sera (found positive for IgG anti-tTG and/or DGP) submitted to ARUP Laboratories for GSE testing, but it is unknown whether these patients were diagnosed as having CD. Consequently, measuring total serum IgA may be unnecessary if using this novel screening assay.
Cost analysis based on reagent/kit list prices and the testing of 1 specimen showed a cost savings of 6% by substituting total serum IgA and anti-tTG (IgA or IgG) for the IgA/IgG anti-tTG/DGP screen. In contrast, the estimated cost of performing the 4 celiac-associated assays (IgA anti-tTG, IgG anti-tTG, IgA anti-DGP, IgG anti-DGP) separately compared with that of the tTG/DGP screen showed a cost savings of 72%.
Despite the sensitivity of serologic testing, the diagnosis of CD requires clinical judgment. As seen in Table 2, serology and histology can be discordant, making the diagnosis of CD unclear in some patients. Negative serologies occurred in approximately 8% of our pediatric patients with CD with Marsh 3 lesions. In 1 study, one fifth of IgA anti-EMA–negative patients had Marsh 3a or greater lesions (24). Histopathologic discordance with serology may be attributable to sampling error, poorly oriented biopsy specimens, or erroneous biopsy interpretation. All 7 discordant Marsh 3 cases were considered seronegative CD in our statistical analysis. However, 3 of these patients were found to have villous atrophy from non-GSE disease. The 4 remaining cases responded to a GFD and likely represent true seronegative CD. Biopsy after rechallenge with gluten can be useful to establish true seronegative CD. Screening for predisposing HLA markers DQ2 or DQ8 can also provide supporting evidence. Unfortunately, these data were not available in these patients. If these unusual cases are all misdiagnosed and excluded from analysis, then sensitivity and negative predictive value for the tTG/DGP screen increase to 100%.
Marsh 2 changes were uncommon in our pediatric population, occurring in only 3 of 111 individuals. All 3 had uniformly negative serologies and were not assigned a diagnosis of CD. Our findings are consistent with NASPGHAN guidelines indicating that Marsh 2 changes are less specific for CD, and such patients require positive serology and clinical judgment to make the diagnosis (1). Positive serologic tests with Marsh 0 and 1 changes are an important population and need to be followed expectantly for development of CD (Table 2). Nearly 10% of our pediatric patients diagnosed as having CD were in this category (biopsy negative/serology positive). Two patients could be classified as having latent CD because of strong family histories. Only 1 Marsh 1 patient with positive serology convincingly responded to a GFD and was considered to have CD. Of particular interest was our patient having positive serology, Marsh 1 histopathology, and eosinophilic esophagitis because this disorder has recently been recognized as comorbid with CD (25). This patient responded to elimination of wheat and other food antigens. Our identification of 4 symptomatic, serologic negative subjects with CD supports the consensus that duodenal biopsy is the gold standard for diagnosis (1). Seropositive patients with only minor histological changes should be followed longitudinally serologically and for response to a GFD. Patient results such as these may be a result of sampling error (26,27).
The tTG/DGP screen was slightly less specific for GSE than IgA anti-tTG alone. Three of the 77 (3.9%) sera from pediatric normal controls produced low positive results. These 3 patients also produced values slightly under the cutoff for 2 of the 4 individual markers, and the combined effect of the 2 different antibodies is the likely cause of the low positive results observed in the tTG/DGP screen. We are not aware whether these patients ever had small bowel biopsy or clinical/family history of CD because these subjects were unidentified controls. For at-risk or general population screening, low positive values should certainly be interpreted with caution with confirmation via biopsy.
The new tTG/DGP screen performed slightly better than IgA anti-tTG when detecting pediatric patients diagnosed with CD. The sensitivity of the tTG/DGP screen in DH was low (62%–65%) and should not be used as a screening tool for this form of GSE. Although further evaluation in other patient populations is needed, this dual antigen/isotype assay is highly sensitive for CD and may make serum IgA testing unnecessary. Further comparison of the combined tTG/DGP assay versus serum IgA and IgA anti-tTG is necessary in patients with relative and absolute IgA deficiency to determine whether the combined assay obviates the need for serum IgA testing.
The authors thank INOVA Diagnostics (San Diego, CA) for donating the IgA/IgG anti-tTG/DGP EIA screen and the individual tTG and DGP EIA kits for this study.
1. 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.
2. Green PH, Jabri B. Celiac Disease
. Annu Rev Med 2006; 57:207–221.
3. Humbert P, Pelletier F, Dreno B, et al
. Gluten intolerance and skin diseases. Eur J Dermatol 2006; 16:4–11.
4. Kumar V, Jarzabek-Chorzelska M, Sulej J, et al
. Celiac disease
and immunoglobulin a deficiency: how effective are the serological methods of diagnosis? Clin Diagn Lab Immunol 2002; 9:1295–1300.
5. Rostom A, Murray JA, Kagnoff MF. American Gastroenterological Association (AGA) Institute technical review on the diagnosis and management of celiac disease
. Gastroenterology 2006; 131:1981–2002.
6. Kagnoff MF. Overview and pathogenesis of celiac disease
. Gastroenterology 2005; 128:S10–S18.
7. Hill ID. What are the sensitivity and specificity of serologic tests for celiac disease
? Do sensitivity and specificity vary in different populations? Gastroenterology 2005; 128:S25–S32.
8. Donaldson MR, Firth SD, Wimpee H, et al
. Correlation of duodenal histology with tissue transglutaminase
and endomysial antibody levels in pediatric celiac disease
. Clin Gastroenterol Hepatol 2007; 5:567–573.
9. Rostom A, Dube C, Cranney A, et al
. The diagnostic accuracy of serologic tests for celiac disease
: a systematic review. Gastroenterology 2005; 128:S38–S46.
10. Abrams JA, Diamond B, Rotterdam H, et al
. Seronegative celiac disease
: increased prevalence with lesser degrees of villous atrophy. Dig Dis Sci 2004; 49:546–550.
11. Aleanzi M, Demonte AM, Esper C, et al
. Celiac disease
: antibody recognition against native and selectively deamidated gliadin peptides
. Clin Chem 2001; 47:2023–2028.
12. 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; 50:2370–2375.
13. Skovbjerg H, Koch C, Anthonsen D, et al
. Deamidation and cross-linking of gliadin peptides by transglutaminases and the relation to celiac disease
. Biochim Biophys Acta 2004; 1690:220–230.
14. Prince HE. Evaluation of the INOVA diagnostics enzyme-linked immunosorbent assay kits for measuring serum immunoglobulin G (IgG) and IgA to deamidated gliadin peptides
. Clin Vaccine Immunol 2006; 13:150–151.
15. Sugai E, Vazquez H, Nachman F, et al
. Accuracy of testing for antibodies to synthetic gliadin-related peptides in celiac disease
. Clin Gastroenterol Hepatol 2006; 4:1112–1117.
16. Barker CC, Mitton C, Jevon G, et al
. Can tissue transglutaminase
antibody titers replace small-bowel biopsy to diagnose celiac disease
in select pediatric populations? Pediatrics 2005; 115:1341–1346.
17. Dandalides SM, Carey WD, Petras R, et al
. Endoscopic small bowel mucosal biopsy: a controlled trial evaluating forceps size and biopsy location in the diagnosis of normal and abnormal mucosal architecture. Gastrointest Endosc 1989; 35:197–200.
18. Oberhuber G, Granditsch G, Vogelsang H. The histopathology of coeliac disease: time for a standardized report scheme for pathologists. Eur J Gastroenterol Hepatol 1999; 11:1185–1194.
19. Marsh MN. Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity (‘celiac sprue'). Gastroenterology 1992; 102:330–354.
20. Hull CM, Liddle M, Hansen N, et al
. Elevation of IgA anti-epidermal transglutaminase antibodies in dermatitis herpetiformis
. Br J Dermatol 2008; 159:120–124.
21. Nicolas ME, Krause PK, Gibson LE, et al
. Dermatitis herpetiformis
. Int J Dermatol 2003; 42:588–600.
22. Dahlbom I, Olsson M, Forooz NK, et al
. Immunoglobulin G (IgG) anti-tissue transglutaminase
antibodies used as markers for IgA-deficient celiac disease
patients. Clin Diagn Lab Immunol 2005; 12:254–258.
23. 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; 52:1567–1571.
24. Dickey W, Hughes DF, McMillan SA. Reliance on serum endomysial antibody testing underestimates the true prevalence of coeliac disease by one fifth. Scand J Gastroenterol 2000; 35:181–183.
25. Ooi CY, Day AS, Jackson R, et al
. Eosinophilic esophagitis in children with celiac disease
. J Gastroenterol Hepatol 2008; 23:1144–1148.
26. Bonamico M, Mariani P, Thanasi E, et al
. Patchy villous atrophy of the duodenum in childhood celiac disease
. J Pediatr Gastroenterol Nutr 2004; 38:204–207.
27. Ravelli A, Bolognini S, Gambarotti M, et al
. Variability of histologic lesions in relation to biopsy site in gluten-sensitive enteropathy
. Am J Gastroenterol 2005; 100:177–185.