Digesting all the options: laboratory testing for celiac disease.

http://informahealthcare.com/lab ISSN: 1040-8363 (print), 1549-781X (electronic) Crit Rev Clin Lab Sci, Early Online: 1–21 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10408363.2014.958813

REVIEW ARTICLE

Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by Ohio State University Libraries on 10/18/14 For personal use only.

Digesting all the options: Laboratory testing for celiac disease Vilte E. Barakauskas1,2, Grace Y. Lam3, and Mathew P. Estey1,2 1

DynaLIFEDx, Edmonton, Alberta, Canada and 2Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada, and 3Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada

Abstract

Keywords

Celiac disease is a complex immune-mediated disorder that is triggered by ingestion of gluten and related proteins in genetically susceptible individuals. Under conditions of increased intestinal permeability, gluten-derived peptides can travel across the intestinal epithelium and undergo deamidation catalyzed by the tissue transglutaminase (TTG) enzyme. This renders them immunogenic in individuals expressing specific human leukocyte antigen (HLA) DQ heterodimers. The resulting immune response is characterized by the production of antibodies against both deamidated gliadin peptides (DGP) and TTG, generation of pro-inflammatory cytokines and activation of cytotoxic T cells. This response damages the intestinal epithelium resulting in the wide range of gastrointestinal and systemic symptoms observed in those with celiac disease. Celiac disease diagnosis has traditionally been based on biopsy and histological examination of the small intestine. While this approach is still considered the gold standard, it is invasive and susceptible to both false-positive and false-negative results. Several laboratory tests have become available to aid in the diagnosis and monitoring of celiac disease, and are the focus of this review. These include serological tests for celiac disease-specific antibodies such as anti-endomysial antibodies, anti-TTG antibodies and anti-DGP antibodies of both the immunoglobulin A (IgA) and immunoglobulin G (IgG) class, genetic tests to elucidate HLA DQ status and ancillary tests such as total IgA. While some have suggested that laboratory tests may replace intestinal biopsy in specific circumstances, others maintain that this procedure remains a critical component of celiac disease diagnosis. We review the analytical methodology, strengths, weaknesses, diagnostic performance and clinical utility of the various laboratory tests for celiac disease. Potential future markers and tests that are now considered obsolete are also discussed. Current clinical practice guidelines for the diagnosis and management of celiac disease from the European Society for Pediatric Gastroenterology, Hepatology and Nutrition, the American College of Gastroenterology and the World Gastroenterology Organisation are summarized, and important differences between these guidelines are highlighted.

Biopsy, deamidated gliadin peptide, endomysial antibody, gluten, gliadin, human leukocyte antigen DQ2 and DQ8, serology, tissue transglutaminase History Received 8 January 2014 Revised 28 July 2014 Accepted 9 August 2014 Published online 19 September 2014

Abbreviations: ACG: American College of Gastroenterology; AGA: anti-gliadin antibody; CD: celiac disease; DGP: deamidated gliadin peptide; DH: dermatitis herpetiformis; ELISA: enzymelinked immunosorbent assay; EMA: endomysial antibodies; ESPGHAN: European Society for Pediatric Gastroenterology, Hepatology and Nutrition; HLA: human leukocyte antigen; IFA: indirect immunofluorescence; IgA: immunoglobulin A; IgG: immunoglobulin G; IgM: immunoglobulin M; GFD: gluten-free diet; MHC: major histocompatibility complex; MLPA: multiplex ligation-dependent probe amplification; NPV: negative predictive value; PCR: polymerase chain reaction; POC: point of care; ROC: receiver operating characteristic; SMA: smooth muscle antibody; SNP: single-nucleotide polymorphism; SSCP: single-strand conformation polymorphism; SSO: sequence-specific oligonucleotide; SSP: sequence-specific primer; TTG: tissue transglutaminase; ULN: upper limit of normal; WGO: World Gastroenterology Organisation

Introduction Referee: Dr. Julio Bai, Small Intestine Section, Department of Medicine, Hospital de Gastroenterologı´a ‘‘Dr. Carlos Bonorino Udaondo,’’ Buenos Aires; and School of Medicine, Universidad del Salvador, Buenos Aires, Argentina. Address for correspondence: Dr. Mathew Estey, DynaLIFEDx, #200, 10150-102 Street, Edmonton, Alberta, Canada, T5J 5E2. Tel: 780 4513702; ext. 8162. E-mail: [email protected]

Celiac disease (CD) is an immune-mediated disorder elicited by gluten in genetically susceptible individuals1. It is a relatively common condition, affecting roughly 1% of the population worldwide2,3, however, certain people are at higher risk of developing CD. These include individuals having an affected first-degree relative4, and those with

2

V. E. Barakauskas et al.

Down’s syndrome5,6, Turner’s syndrome7,8, selective immunoglobulin A (IgA) deficiency9, type I diabetes mellitus10–12 or other autoimmune conditions1,2.

fragments can enter the lamina propria of the intestine, where they have the potential to trigger an immune response if specific conditions are met.

Pathogenesis of CD

The role of tissue transglutaminase (TTG) and human leukocyte antigen (HLA) DQ2/DQ8

An overview of the pathogenesis of CD is depicted in Figure 1. This complex mechanism involves exposure to gluten-derived peptides, with a subsequent immune response resulting in inflammation and damage of the intestine. Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by Ohio State University Libraries on 10/18/14 For personal use only.

Crit Rev Clin Lab Sci, Early Online: 1–21

The environmental trigger The entire protein content of wheat is referred to as gluten, whereas gliadin is the proline-rich, alcohol soluble fraction of gluten implicated in CD3. Homologous proteins are found in other grains, including barley and rye. Dietary protein is normally broken down by digestive enzymes, generating fragments that are too small to elicit an immune response. However, the high proline content of gliadin renders it resistant to proteolysis, so large fragments remain intact13. Under conditions of increased intestinal permeability14, these Figure 1. The pathogenesis of celiac disease. Unlike other dietary proteins, gliadin is resistant to proteolysis by digestive enzymes. Consequently, large gliadin peptides remain intact in the small intestine. Under conditions of increased intestinal permeability, these peptides can cross the intestinal epithelium and undergo deamidation catalyzed by tissue transglutaminase (TTG). The resulting deamidated gliadin peptides have the capacity to trigger an immune response in genetically susceptible individuals (HLA DQ2 or DQ8 heterodimers). This response is characterized by the production of anti-DGP and anti-TTG antibodies, generation of pro-inflammatory cytokines and activation of cytotoxic T cells, and is responsible for the intestinal damage and symptoms observed in celiac disease. Refer text for details.

Gliadin peptides are highly enriched in proline-glutamine motifs, making them excellent substrates for the tissue transglutaminase (TTG) enzyme15,16. This enzyme, which is found in the lamina propria of the intestine, converts neutral glutamine residues to negatively charged glutamate residues17. In genetically susceptible individuals (see below), the resulting deamidated gliadin peptides (DGPs) can be highly immunogenic. Under normal conditions TTG is thought to be held in an inactive state, with tissue damage resulting in enzyme activation18. Human leukocyte antigen (HLA) DQ is a major histocompatibility complex (MHC) class II heterodimeric protein expressed on the surface of antigen-presenting cells, and is involved in the presentation of antigens to T cells. The genes

Laboratory testing for celiac disease


DOI: 10.3109/10408363.2014.958813

encoding the two HLA DQ subunits are highly polymorphic, yielding several different variants with unique antigen binding properties. In particular, HLA DQ2 and DQ8 have positively charged residues in their antigen-binding pockets, making them well suited to present negatively charged DGPs17,19,20. This triggers an immune response characterized by the production of pro-inflammatory cytokines and the activation of cytotoxic T cells, causing intestinal damage. At the same time, presentation of DGPs (which may be covalently bound to TTG as an intermediate in the deamidation reaction) also activates B cells which produce anti-DGP and anti-TTG antibodies3,21. Nearly all patients with CD express the HLA DQ2 or DQ8 haplotype22. However, since several other factors are required to trigger CD, including the ingestion of gluten, increased intestinal permeability and deamidation of gliadin by TTG, the presence of HLA DQ2 or DQ8 alone is not sufficient for the development of CD. Signs and symptoms of CD The immune response elicited by gluten damages the intestinal mucosa, impairing its function. Consequently, patients with CD may present with symptoms of malabsorption, including diarrhea, flatulence, weight loss, failure to thrive, abdominal distension, bloating, abdominal pain, anorexia and vomiting1–3,23. Systemic manifestations include iron deficiency, anemia, chronic fatigue, short stature, elevated liver enzymes, decreased bone mineral density, infertility, neurologic disorders and dermatitis herpetiformis (DH; a blistering skin rash). Patient presentation varies widely, both in the spectrum and magnitude of symptoms observed.

Intestinal biopsy: the gold standard for CD diagnosis Traditionally, CD diagnosis has relied on examining the state of the small intestine. Signs of villous atrophy (such as nodular or scalloped mucosa, reduced or lost duodenal folds and/or visible fissures) and/or inflammation may be observed during endoscopy24. However, data suggests that the degree of endoscopically visible mucosal pathology, which is estimated to span roughly 30–50% of the entire small-bowel mucosa, is less predictive of symptom severity than the extent of mucosal lesions as assessed by histology25. The histological hallmarks of CD include villous atrophy, crypt hyperplasia and increased intraepithelial lymphocytes (425 to 40 lymphocytes per 100 enterocytes)25–27. Current clinical practice guidelines endorse the use of the modified Marsh (Oberhuber) or Corazza classifications to determine the severity of intestinal pathology in CD1,23. These classification systems highlight the fact that patients with CD exhibit a varying spectrum of histological changes. Despite being regarded as the gold standard for CD diagnosis, intestinal biopsy has several limitations that can potentially lead to misdiagnosis. It is well documented that histological changes associated with CD can be patchy in nature, with variability occurring both between and within biopsy specimens28–33. It is therefore critical to examine an adequate number of biopsy specimens from different regions of the duodenum to avoid false-negative diagnoses. In addition, several studies have demonstrated that the

3

importance of performing duodenal bulb biopsies, as histological hallmarks of CD are restricted to this region in some patients28,33–35. Current clinical practice guidelines recommend that at least one specimen from the duodenal bulb and a minimum of four additional specimens from the remaining duodenum should be examined when performing biopsy for the investigation of CD1,23. Nevertheless, biopsy interpretation is confounded by the variable spectrum of histological features observed in CD, the patchy nature of these features, as well as the potential for artefacts related to suboptimal tissue processing and/or orientation36. A number of conditions may present with histological findings similar to those observed in CD. For example, villous distortion can be seen in other inflammatory conditions, including inflammatory bowel disease, autoimmune enteropathy, bacterial overgrowth and infections36,37. Lymphocytic infiltration without other histological features and a history of gastrointestinal symptoms is consistent with lymphocytic duodenosis38. In addition, medication injury may cause villous blunting and crypt hyperplasia36. Consequently, despite the fact that endoscopic biopsy is the gold standard for CD diagnosis, the above-described histological changes are by no means specific for CD. Traditionally, additional biopsies were performed on separate occasions to demonstrate histological resolution with a gluten-free diet (GFD) and lesion exacerbation following gluten challenge39. However, given the invasiveness and high cost associated with repeat biopsy, most diagnoses of CD are now confirmed with the resolution of symptoms upon implementation of a GFD, rather than histological changes. With the availability of highly sensitive and specific laboratory tests for the diagnosis of CD, the absolute requirement for intestinal biopsy has recently come under question. While some feel that biopsy is a crucial step in CD diagnosis23, others have suggested that it may not be required in all instances1. This issue is discussed further in the ‘‘Clinical Practice Guidelines’’ section.

Laboratory testing Several laboratory tests are available to support the diagnosis and management of CD. These can be broadly divided into two groups: serological and genetic testing. The analytical methodology, strengths and weaknesses of each of these tests are summarized in Table 1. Serological testing Anti-endomysial antibodies (EMA) Antibodies in the serum of patients with CD produce a staining pattern restricted to the connective fibers surrounding smooth muscle when detected by indirect immunofluoresence (IFA) on monkey esophageal tissue40. These antibodies were shown to be directed against the smooth muscle endomysium of the GI tract and therefore termed anti-endomysial antibodies (EMA). EMA production is induced by exposure to gliadin, and in vitro studies have identified specific peptides as particularly potent triggers41. Early studies showed EMA primarily consist of IgA class antibodies40. Use of human umbilical cord as substrate was subsequently

Endoscopy or Capsule biopsy

Not applicable.

Synthetic peptides

Biopsy

ELISA

DGP

Guinea pig liver, human TTG, or human recombinant TTG.

PCR combined with sequence-specific primers, sequence-specific oligonucleotide probes or sequencing.

ELISA

TTG

Monkey esophagus or human umbilical cord.

Reagent/Antigen

HLA-DQ2/DQ8 Molecular methods typing

Indirect immuno-fluoresence

Analytical principle

EMA

Test

Weaknesses

Other considerations

Produces a qualitative result. Allele resolution depends on method. Requires molecular testing equipment and reagents. Can be performed at any life or disease stage.

Allele frequency is relatively high in the Allele frequency is very high in CD general population. Presence of risk patients (high negative predictive alleles is not sufficient to prove disvalue). Can be detected even if patient ease. The same alleles have been is on a GFD. Novel assays allow for implicated in other diseases. increased throughput and decreased cost.

In the presence of intestinal pathology, Invasive and costly procedure. Expert gluten-dependence of symptoms or (pathologist) interpretation needed. lesions must still be demonstrated to Sensitivity is affected by patchy nature exclude other causes. of lesions, poor biopsy orientation, GFD. Other diseases produce similar biopsy findings.

Better accuracy than historical anti-gliadin tests. Accuracy decreases with GFD.

Useful in children less than 2 years of age. Accuracy is lower than that of TTG or EMA (except in children less than 2 May be detected earlier than TTG in years of age and IgG-DGP testing in some patients. IgG-DGP used in comthose with IgA deficiency). bination with IgA-TTG may increase testing accuracy. Easily automated. Produces a quantitative result.

Gold-standard for CD diagnosis. Villous atrophy is directly assessed and severity is graded.

Has become the first-line method for CD testing. Laboratory or assay-specific cut-offs needed if TTG results will be used to omit biopsy. Accuracy decreases with GFD.

IgA-based methods provide highly accur- Assays are not standardized. ate identification of CD, especially high sensitivity. IgG-based methods also available. Easily automated. POC testing available. Produces a quantitative result.

Produces semi-quantitative results (if titer IgA-based methods provide highly accur- Difficulty in obtaining reagents. Interis reported). Accuracy decreases with laboratory variation in cut-offs used. ate identification of CD, especially GFD. Experienced personnel needed for slide high specificity. IgG-based methods interpretation. Limited capacity for also available. automation.

Strengths

Table 1. Characteristics of laboratory tests for the diagnosis and management of CD. Biopsy is included for reference.


4 V. E. Barakauskas et al. Crit Rev Clin Lab Sci, Early Online: 1–21


DOI: 10.3109/10408363.2014.958813

demonstrated to produce equivalent results, at times identifying more biopsy-confirmed celiac patients than monkey esophagus EMA assays42–45. Positive EMA results are seen in the majority of celiac patients. Meta-analysis of 34 studies which included biopsy results of untreated CD patients determined a pooled sensitivity of 93% and specificity of 99.7%, although antibody isotype was not considered in this analysis46. Individual studies using IgA-EMA report sensitivities ranging from approximately 60–100% and specificities of 80–100%40,45,47–51. Lower sensitivity estimates are usually derived from studies which attempt to reduce ascertainment bias, by measuring EMA in patients who are identified prospectively and irrespective of serological results46,48,52. For example, in a study of 2000 consecutive patients referred for endoscopy, 77 new cases of CD were identified based on histological criteria and supportive serology results, and EMA testing demonstrated a sensitivity of only 87% for CD (53). Similarly, Rostami et al. limited their analysis to CD patients diagnosed based on biopsy and response to GFD, with 69 patients meeting their inclusion criteria. In this study, EMA showed a sensitivity of only 60%, and this was attributed in part to heterogeneity in the degree of villous atrophy seen in patients53. Others report sensitivities of 78%51, 75%54, 87%55 and 100%56 when similar approaches to lessen ascertainment bias were undertaken. It must also be noted that patients with higher grade histological lesions are more likely to be positive for EMA57,53. Consequently, EMA sensitivity estimates also vary with the grade of villous atrophy. For example, higher sensitivity is noted in patients with total villous atrophy compared to those with partial villous atrophy. In addition to correlating with the severity of intestinal pathology, EMA titers change upon implementation of a gluten-free diet or gluten challenge40,47,58. In 79% of subjects who did not initially show villous atrophy, a positive EMA result (irrespective of titer) predicted subsequent intestinal pathology or symptom improvement on a GFD58. Patients with less severe intestinal pathology may show concordant EMA titers (low-positive or negative)59, although not all investigators agree48. A recent meta-analysis concluded that IgA-EMA was among the best performing tests in children (518 years of age), with most studies reporting sensitivities 90% and a pooled specificity reaching 98%60. Although most studies report 100% specificity, positive EMA results are known to occur in certain non-CD-patient populations. Positivity rates as high as 80% can be seen in the related gluten-sensitivity, DH40. However, EMA is not associated with other bowel disorders such as Crohn’s disease, ulcerative colitis or irritable bowel syndrome61. As with sensitivity estimates, EMA (and all serological marker) specificity estimates will vary depending on which biopsy findings are included in the patient and control groups. Some studies recognize Marsh 1 and 2 categories as indicative of potential CD, and therefore positive EMA results in these patients may increase the stated sensitivity, or at least not reduce specificity estimates62. Recognition of ‘‘potential’’ and ‘‘silent’’ subtypes of CD adds to the difficulty in assessing EMA specificity. Patients who are biopsy negative but EMA positive may therefore either be correctly identified


5

as one of these subtypes, or may have a false positive serology result46,52. IgG-EMA. Although IgA antibodies are most often associated with mucosal immunity, mucosal production of IgGEMA has been demonstrated in tissue culture supernatants of biopsy specimens63. Comparisons of IgG versus IgA-EMA performance are sparse; however, one report found that in IgA sufficient patients with positive IgA-EMA results, only 15% of subjects were IgG-EMA positive. Another study found this to be 12%, suggesting that IgG-EMA should not be used for general-purpose CD testing64,65. The utility of including IgGEMA in general population testing was also not obvious in a study of 512 samples tested for IgA-EMA at a reference laboratory. In this study, Prince et al. found that only 60% of IgA-EMA positive samples were also positive for IgG-EMA, although intestinal pathology was unknown66. Use of IgA and IgG-EMA to screen healthy Hungarian children identified six positive samples (1.5% of participants), five showed villous atrophy on biopsy, and the last showed only mild histological changes. Two of these samples were positive only for IgAEMA (this included the child with mild intestinal pathology); two were only IgG-EMA positive and subsequently shown to be IgA deficient (IgA50.1 g/L)67. The use of IgG-EMA, therefore, may be useful in IgA deficient patients. Case reports suggest that the sensitivity of IgG-EMA could be higher in this situation, with high IgG-EMA titers seen in a 12-year-old at presentation and following a four month gluten challenge, which decreased on a GFD64. In a study of IgAdeficient CD patients that included IgA-deficient controls, IgG-EMA was highly sensitive and specific for active disease, although only half of CD patients who adhered poorly to a GFD had positive IgG-EMA results. All IgA-sufficient, untreated CD patients were positive for both IgA- and IgGEMA, suggesting adequate performance of either EMA isotype in this population68. IgG-EMA may not be needed in all IgA-deficient cases, as there are reports of positive IgAEMA results in IgA-deficient CD patients69–71. Picarelli et al. (2001) have also reported a subset of IgA-sufficient CD patients who only tested positive for IgG-EMA (49 of 1399 total subjects)63. Thus, appropriate use of IgG-EMA remains somewhat unclear. Method considerations. EMA testing faces several chal-

lenges, one of which is the reliance on substrate sourced from endangered species43. Both monkey esophageal and monkey bladder tissue have been referenced in the literature40,70. Although human umbilical cord tissue was proposed as an equally performing alternative substrate, the staining pattern is different than observed in primate esophagus, so the transition to a new substrate is not seamless72. Rather than using umbilical cord tissue, a method using permeabilized umbilical vein cells has been proposed, and shown to be completely concordant with an EMA monkey esophagus method73. Although the incubation and staining stages of IFA methods can be automated, interpretation of the staining pattern remains a manual process, and is best performed by experienced personnel. A semi-quantitative result can be produced by reporting antibody titer. When reported in a


6


qualitative fashion, the titer at which a result is considered positive may not be standardized between laboratories. A comparison of eight US laboratories showed that a range of serum dilutions (1:20 to 1:2) were used for initial screening, possibly accounting for the range in EMA sensitivity observed among the labs74. Despite these differences, agreement between laboratories was high (kappa coefficient ¼ 0.74) and agreement of EMA results was higher than gliadin antibody results in the same sample set74. Difficulty in interpreting IFA results can arise due to background or nonspecific staining. Anti-smooth muscle antibodies (SMA) can stain muscle fibers, obscuring the endomysial staining pattern44. Sample dilution to remove SMA interference can unmask an EMA pattern75. SMA may be observed in various disorders including Crohn’s disease and autoimmune hepatitis43,44 but can also be found in healthy subjects75. IgG-EMA detection can be hindered by high background when using monkey esophagus. To help define positive IgG-EMA results, concomitant staining of jejunal tissue has been suggested. Lower background may be seen using human umbilical cord or appendix67. As with all IFA methods, potential method limitations arise due to fluorophore instability, photobleaching and tissue autofluorescence. Finally, as with all celiac serology, EMA performance is affected by the gluten status of the patient, and has been shown to be negative in CD patients anywhere from two to 12 months after cessation of gluten intake47. Thus, negative results need to be interpreted in the clinical context and related to biopsy results and gluten status of the patient. Anti-TTG antibodies In a landmark study, Dietrich et al. identified TTG as the unknown autoantigen targeted by EMA76. Pre-incubation of high titer celiac patient serum with commercial TTG almost completely abolished endomysial immunofluorescence, arguing that TTG is the major and possibly sole endomysial autoantigen. These authors also developed the first enzymelinked immunosorbent assay (ELISA) for measuring anti-TTG antibodies. TTG from guinea pig liver was coated on microtiter plates and used to capture anti-TTG antibodies from patient’s sera. These antibodies were subsequently detected by incubation with peroxidase-conjugated antihuman IgA antibodies with color development after addition of peroxidase substrate. Using this assay, the authors demonstrated that IgA-TTG antibodies were specifically elevated in patients with CD, with titers decreasing upon implementation of a GFD. In a follow-up study, this assay was shown to identify biopsy proven CD with a sensitivity of 98% and a specificity of 95%77. The anti-TTG assay had several major advantages over the anti-EMA assays including quantitative results, lack of inter-observer variability, ease of performance and capacity for automation. The work of Dieterich and colleagues76,77 led to the development of commercial IgA-TTG assays, which also used guinea pig liver TTG as the capture agent. Several studies demonstrated that these assays exhibited excellent diagnostic performance, achieving sensitivity and specificity comparable to that of IgA-EMA78–82. However, other work found that the specificity of these IgA-TTG assays was inferior to IgA-EMA,


particularly when the control group included patients with autoimmune diseases, such as autoimmune liver disease and type I diabetes mellitus83–86. Therefore, some authors recommended confirming positive IgA-TTG results with anti-EMA testing86. The high rate of false positive IgA-TTG results in these patients, however, was attributed to impurities in the guinea pig liver TTG capture agent83,84, and was greatly reduced when an ELISA using human TTG as the capture agent was employed. Several subsequent studies have confirmed the superior diagnostic performance of ELISAs based on human TTG compared to those employing guinea pig liver TTG87–92. The diagnostic performance of IgA-TTG assays using human TTG has been evaluated relative to IgA-EMA in many retrospective studies. In the vast majority of cases, both assays identified biopsy-proven CD with similar performance, routinely exhibiting sensitivities and specificities greater than 90%57,87,91,93–96. In many instances, IgA-TTG had slightly higher sensitivity57,87,91,93–96, whereas IgA anti-EMA showed slightly87,93,95,96 higher specificity. Van Meensel and colleagues evaluated the diagnostic accuracy of 10 different IgATTG assays (nine employing recombinant human TTG and one using purified human TTG as the capture agent) relative to IgA-EMA96. The sensitivity of the IgA-TTG assays ranged from 91% to 97%, compared to 90% for IgA-EMA, whereas the specificity ranged from 96% to 100% compared to 100% for IgA-EMA. The authors concluded that the diagnostic performance of IgA-TTG assays was superior to IgA-EMA, and positive IgA-TTG results do not need to be confirmed with IgA-EMA testing. While the overall diagnostic performance of all IgA-TTG assays was comparable, the numerical values obtained between assays were different. Consequently, the absolute values obtained using different IgA-TTG assays are not interchangeable. The diagnostic performance of IgA-TTG has been evaluated in several prospective studies, many of which have also shown diagnostic sensitivities and specificities greater than 90%57,88,97–100. However, work by Sugai and colleagues suggests that the diagnostic accuracy of IgA-TTG (and other serological tests for CD) depends on the pre-test probability of CD100, with performance declining in low-risk populations. For example, in a high-risk population (CD prevalence 39%), IgA-TTG exhibited 95% sensitivity, 98% specificity and an area under the receiver operating characteristic (ROC) curve of 0.997. In a low risk population (CD prevalence 3.3%), 77% sensitivity, 97% specificity and an area under the ROC curve of 0.921 were observed. Carroccio et al. compared the diagnostic performance of IgA-TTG and IgAEMA in a cohort of patients who underwent intestinal biopsy for suspected CD88. While both tests identified biopsyconfirmed CD with 100% sensitivity, the specificity of IgATTG was 97% compared to 100% for IgA-EMA. This small difference in specificity resulted in a significantly lower positive predictive value for IgA-TTG compared to IgA-EMA (80% versus 100%) in the population under study (which had a CD prevalence of 11.6%). Consequently, the authors suggested that positive IgA-TTG results should be confirmed with IgA-EMA testing. In a similar study by Hopper et al.57, IgA-TTG and IgA-EMA identified biopsy-confirmed CD with similar sensitivity (91% versus 87%, respectively), however,


DOI: 10.3109/10408363.2014.958813

IgA-EMA exhibited significantly higher specificity relative to IgA-TTG (98% versus 91%, respectively). As a result, the positive predictive values for IgA-TTG (29%) was significantly lower than that for IgA-EMA (64%) in the population under study (CD prevalence was 3.9%). Reeves and colleagues prospectively evaluated the diagnostic performance of several anti-TTG and anti-EMA assays in patients referred for gastroscopic assessment of possible CD70. While the diagnostic performance varied between manufacturers, the overall pattern was similar to that described in retrospective studies: IgA-TTG assays generally had higher sensitivity than anti-EMA assays (73–92% for IgA-TTG versus 62–68% for anti-EMA), whereas the specificity was generally higher for anti-EMA assays (80–99% for anti-EMA versus 81–95% for IgA-TTG). Several studies have demonstrated a strong link between very high IgA-TTG levels and histological findings consistent with CD101–108. Barker and colleagues retrospectively reviewed 103 patients who underwent both IgA-TTG testing and intestinal biopsy102. In this cohort, 58 patients (56%) had biopsy-confirmed CD. Fortynine patients had IgA-TTG levels greater than 100 U/mL [corresponding to five times the upper limit of normal (ULN)], with 48 of 49 (98%) showing histology consistent with CD. Thus, IgA-TTG greater than five times the ULN was 98% specific and 83% sensitive for CD, with a positive predicative value of 98%. The sole patient with very high IgA anti-TTG and normal biopsy was asymptomatic and underwent testing as part of routine screening. Based on these results, the authors suggest that biopsy is not necessary to diagnose CD in the setting of very high IgA-TTG levels. However, biopsy should still be performed in cases where the patient does not show symptomatic improvement on a GFD and in instances where IgA-TTG is only mildly or modestly elevated. Mubarak and colleagues performed a similar retrospective study of 283 pediatric patients105. In their cohort, 163 patients (58%) had biopsy findings diagnostic of CD. IgA-TTG levels greater than 100 U/mL (corresponding to 10 times the ULN) were found in 128 patients, with 124 of 128 (97%) showing histology diagnostic of CD. Therefore, IgA-TTG greater than 10 times the ULN was 97% specific and 76% sensitive for CD, with a positive predicative value of 97%. Of the four patients with very high IgA-TTG who did not exhibit biopsy lesions diagnostic of CD, one did not respond to a GFD. The remaining three had biopsy findings suggestive of an early stage of CD. Consequently, these authors also recommend that biopsy can be avoided in cases of very high IgA-TTG levels, as long as the patient shows symptomatic improvement on a GFD. Other retrospective studies have argued that an IgA antiTTG cut-off can be selected that yields 100% specificity and positive predictive value for biopsy-confirmed CD. In a group of patients positive for both IgA-TTG and anti-EMA, Alessio et al. found that all patients with IgA anti-TTG greater than seven times the ULN had duodenal damage consistent with CD101. Hill and colleagues examined patients with an elevated IgA-TTG (greater than 3.3 times the ULN) who underwent small bowel biopsy, and concluded that all patients

Markedly elevated IgA-TTG.


7

with IgA-TTG greater than 10 times the ULN had lesions characteristic of CD104. In a similar study, Zanini et al. found that IgA-TTG greater than five times the ULN exhibited 100% specificity and positive predictive value for duodenal atrophy106. While this was true for three different IgA-TTG assays, the sensitivity for duodenal atrophy varied greatly (10–59%) depending on the assay used. In addition, a cut-off of three times the ULN still gave a specificity and positive predictive value of 100% for one assay, resulting in a sensitivity of 70%. This study argues that the optimal IgA anti-TTG cut-off to identify patients who may not require biopsy will vary between assays. In a follow-up study, Mubarak and colleagues prospectively evaluated whether IgA-TTG greater than 100 U/mL (10 times the ULN) was sufficient for the diagnosis of CD107. Both IgA-TTG and IgA-EMA were measured in children suspected of having CD, and biopsy was performed on any patient with abnormal serology or a strong clinical suspicion. Of the 183 children included in the study, 120 had biopsy findings diagnostic of CD. Eighty-seven patients had an IgATTG greater than 10 times the ULN, all of whom had biopsyconfirmed CD. Therefore, IgA-TTG above 10 times the ULN had a specificity and positive predictive value of 100%, with a sensitivity of 73%. The authors concluded that biopsy is not necessary to confirm the diagnosis of CD in symptomatic patients with IgA-TTG greater than 100 U/mL. They emphasize that nearly all of the patients in their study were symptomatic, so biopsy should only be omitted in this group. Sandstrom et al. assessed the utility of IgA-TTG as a mass screening tool in over 7000 sixth graders108. Patients with abnormal serology (N ¼ 192) underwent intestinal biopsy (N ¼ 184), with 153 showing histological lesions consistent with CD. Forty-seven patients, all of whom were confirmed to have CD, had an IgA-TTG greater than 10 times the ULN. Therefore, even in a prospective study of asymptomatic patients, IgA-TTG greater than 10 times the ULN exhibited a specificity and positive predictive value of 100%. Perhaps not surprisingly, the proportion of CD cases with IgA-TTG greater than 10 times the ULN was much lower (sensitivity 31%) than that reported for a symptomatic population (sensitivity 73%)107. As part of their seminal work, Dieterich and colleagues also developed an ELISA for the detection of IgG-TTG antibodies76. The diagnostic performance of this test was inferior to that of IgA-TTG, likely due to the fact that it is IgA class antibodies that are prominently produced by the intestinal tract. Similar findings have been reported by others70, and it is widely accepted that IgA-TTG tests perform best in the general population. However, IgGTTG assays may have utility in the setting of selective IgA deficiency, since many such individuals are incapable of producing IgA-TTG antibodies. Villalta et al. assessed the diagnostic performance of several different IgG-TTG assays in identifying CD in patients with selective IgA deficiency109. It should be noted that only a fraction of patients in the control group had selective IgA deficiency. The sensitivity of these assays ranged from 75% to 95%, whereas the specificity varied from 94% to 100%. A limited number of studies have compared the performance of IgG-TTG and IgG-EMA in

Utility of IgG-TTG testing.

8



patients with selective IgA deficiency68,110,111, with most work demonstrating excellent concordance between the two tests110,111. In a small study of 20 CD patients and 10 healthy controls, all with selective IgA deficiency, Cataldo et al. found that both IgG-TTG and IgG-EMA were positive in all cases of CD68. While all control subjects were IgG-EMA negative, two had elevated titers of IgG-TTG. Given the small nature of this study, it is difficult to ascertain whether the specificity of IgG-TTG is inferior to that of IgG-EMA in the setting of IgA deficiency. Rapid point of care (POC) tests for antiTTG antibodies have been developed, with potential for use in a general physician’s office112,113. Retrospective studies suggest that these tests are highly accurate in identifying CD, with sensitivity and specificity comparable to laboratory based serological tests112–116. In a small prospective study, Raivio and colleagues measured POC IgA-TTG, laboratory IgA-TTG and IgA-EMA in patients suspected of having CD115. In this high risk population, the POC IgA-TTG test exhibited 97% concordance with both laboratory IgA-TTG and IgA-EMA. In addition, 94% of biopsy confirmed cases of CD were identified using the POC test. The performance of POC IgA-TTG tests as a screening tool in the general population has also been evaluated. Korponay-Szabo et al. measured POC IgA-TTG, laboratory IgA-TTG and IgA and IgG-EMA in nearly 2700 children117. Patients with positive serology were assessed by biopsy. While the concordance between the POC and laboratory results was over 99%, the POC test only identified 78% of biopsy confirmed cases of CD. Poor sensitivity as a screening tool has also been reported by others118, suggesting that POC IgA-TTG tests may not perform as efficiently as laboratory IgA-TTG tests when used in the general population.

Point of care testing.

Anti-deamidated gliadin peptide (DGP) antibodies In 2001, Aleanzi et al. demonstrated that deamidation of gliadin peptides led to enhanced recognition by circulating antibodies in patients with CD119. Schwertz and colleagues subsequently demonstrated that measuring antibodies directed against DGPs may be useful in the diagnosis of CD120. Since this time, several immunoassays have been developed to detect both IgA and IgG-DGP antibodies. These assays use synthetic DGPs to capture anti-DGP antibodies present in the patient’s serum or plasma, followed by incubation with labelled anti-human IgA or IgG antibodies (or both in some instances). Several studies have evaluated the diagnostic performance of these tests relative to anti-TTG assays. In all cases, both IgA and IgG-DGP were found to have either similar or inferior performance to IgA-TTG62,98,100,101,121–132. For example, prospective studies of high-risk populations98,100 have reported sensitivities 95% and specificities 93% for IgA-DGP, IgG-DGP and IgA-TTG (although lower diagnostic accuracy was observed in a low-risk population for all three serological tests100). In contrast, studies comparing IgG-DGP to IgG-TTG have suggested that the diagnostic performance of IgG-DGP is either similar or superior to that of


IgG-TTG101,121,124,129,130. Thus, IgG-DGP may be particularly useful in patients with IgA deficiency62,127. While anti-DGP antibody tests do not outperform IgA-TTG on an individual basis, there is evidence suggesting that anti-DGP assays are able to identify some individuals with CD who are IgA-TTG negative and do not have IgA deficiency123,124,133. In addition, some studies have argued that combining antiDGP tests with IgA-TTG leads to a small improvement in diagnostic performance. Brusca and colleagues showed that the combination of IgA-TTG and IgG-DGP improved the clinical sensitivity to 84% compared to 78% for IgA-TTG alone101. Importantly, the combination of these two tests retained a clinical specificity of 100%. It should be noted that none of the patients in this cohort had IgA deficiency, so the added value of IgG-DGP may have been underestimated. Niveloni et al. found that the combination of IgA-TTG and IgG-DGP improved clinical sensitivity to 100% compared to 95% for IgA-TTG alone98. Again, adding IgG-DGP did not alter the diagnostic specificity of 97.5%. In a cohort that included patients with IgA deficiency, Prause et al. found that IgG-DGP significantly improved the diagnostic performance of IgA-TTG alone when TTG cut-offs were set to maximize assay specificity127. At a specificity of 99.7%, IgA anti-TTG exhibited a sensitivity of 0%, whereas inclusion of IgG-DGP increased the sensitivity to 77.7% without altering specificity.

Combination testing to improve accuracy.

Several studies suggest that antiDGP antibodies may be particularly useful in young children. Mozo et al.134 found that in children 7 years of age or younger, IgG-DGP exhibited a diagnostic sensitivity and specificity of 100%. The performance of IgA-DGP was slightly inferior (sensitivity 95% and specificity 97%), although this difference was not statistically significant. It must be noted that all patients with CD in this study were selected for having elevated IgA-TTG antibodies. As a result, a head to head comparison of the diagnostic performance of anti-DGP antibodies relative to IgA-TTG was not possible. Amarri et al.135 measured IgG-DGP, IgA-TTG, and IgAEMA in 42 patients with malabsorption and 23 controls (with respiratory, neurological and/or infectious symptoms), all of whom were less than 2 years of age. All 23 controls were negative for the three serological markers, although biopsy was not performed to exclude CD. Thirty-three of the 42 patients with malabsorption were positive for IgG-DGP, IgATTG and IgA-EMA, and all had biopsy results consistent with CD. One patient was positive for IgG-DGP alone and displayed normal intestinal mucosa on biopsy. However, after 6 months on a gluten-containing diet, the IgG-DGP titer increased, IgA-TTG became positive and villous atrophy consistent with CD was observed on repeat biopsy. This is consistent with the work of Liu et al.136, who demonstrated that anti-DGP antibodies can precede the appearance of IgATTG in some patients. Interestingly, Amarri et al.135 described three additional patients with malabsorption who were positive for IgG-DGP alone. Although biopsies and IgA status were not obtained, all three were HLA-DQ2 positive. While this study suggests that IgG-DGP may be a useful marker for CD in patients less than 2 years of age, it is Utility in young children.



DOI: 10.3109/10408363.2014.958813

difficult to make any conclusions on its diagnostic performance relative to IgA-TTG and IgA-EMA. As part of a larger investigation, Basso et al.122 assessed the diagnostic performance of IgA-DGP, IgG-DPG and IgATTG in a group of children less than 2 years of age, in whom CD was either confirmed or ruled out histologically. IgADGP exhibited the poorest diagnostic performance of the three markers, with a sensitivity of 80% and a specificity of 94%. Both IgG-DPG and IgA-TTG showed a diagnostic specificity of 100%, whereas IgG-DPG had a slightly higher diagnostic sensitivity of 97% compared to 90% for IgA-TTG. However, two of the patients with CD had IgA deficiency. When these patients were excluded, both markers exhibited the same sensitivity (97%) and specificity (100%). The authors concluded that in the absence of IgA deficiency, IgG-DPG and IgA-TTG are equally reliable markers for identifying CD in patients less than 2 years of age. However, in cases of IgA deficiency, IgG-DPG (or IgG-TTG) should be measured. Similar results have been observed by others. In a subset of children aged 9–24 months in which CD was confirmed or ruled out by biopsy, Prause et al.127 found that IgG-DGP and IgA-TTG exhibited similar diagnostic performance (accuracy of approximately 90%). Only one patient in this cohort had IgA deficiency. Mubarak et al.137 measured IgA-DGP, IgGDGP, IgA-TTG and IgA-EMA in children suspected of having CD who had undergone small-bowel biopsy. None of the children were IgA deficient. In the subset of children less than 2 years of age, IgA-DGP, IgG-DGP and IgA-TTG all showed a diagnostic specificity of 100%. IgG-DPG had a slightly higher diagnostic sensitivity at 100% compared to 96% for IgA-DGP and IgA-TTG. The diagnostic performance of IgAEMA was slightly lower, with a specificity of 93% and sensitivity of 96%. However, none of these differences were statistically significant. Nevertheless, the authors concluded that IgG-DGP seems to be the best test in children less than 2 years of age, as it demonstrated 100% accuracy. Barbato et al.138 investigated a cohort of 40 children less than 2 years of age with signs and symptoms of chronic enteropathy. All patients had normal IgA-TTG and IgA-EMA results, with normal serum IgA concentrations. Anti-DGP antibodies were measured in all patients (and in 40 matched controls) and duodenal biopsy was performed. Eleven of the 40 patients had high concentrations of IgG-DGP, and nine of these patients also had elevated IgA-DGP. Interestingly, all of these patients had histological features consistent with CD, and showed improvement on a GFD. None of the remaining 29 patients had elevations in anti-DGP antibodies, and all showed improvement on a diet containing gluten, but free of cow’s milk and soy. In addition, none of the controls had elevated anti-DGP antibodies. This study raises the intriguing possibility that anti-DGP antibodies (particularly IgG-DGP) may have additional diagnostic value above IgA-TTG and IgA-EMA in young children, even when serum IgA concentrations are normal. A POC test detecting both IgA and IgG-DGP antibodies has recently been developed. This test exhibits high concordance with laboratory-based methods, at least in highrisk populations139,140; however, its performance has yet to be

POC testing.

9

evaluated at a population level, where the prevalence of CD is lower. There is some evidence suggesting that anti-DGP antibodies may be useful in monitoring patients diagnosed with CD. Initial work in a small number of children suggested that these antibodies may resolve sooner than IgA-TTG after starting a GFD136. Monzani and colleagues132 found that both IgA-DGP and a combined IgA + IgG-DGP method had a higher sensitivity than IgA-TTG in detecting strict compliance to a GFD in children (although the specificity of IgA-DGP was significantly lower than that of IgA-TTG). The authors concluded that IgA-TTG does not seem to be reliable in monitoring compliance to a GFD in children, and advocated for the use of anti-DGP antibodies. However, both IgA-DGP and IgA-TTG exhibited the best performance in monitoring long term compliance to a GFD in an adult population141. Thus, further studies will be required to establish the optimal means of monitoring patients with CD.

Monitoring patients with CD.

Total IgA Selective IgA deficiency, defined as total IgA50.07 g/L in the setting of normal IgG and immunoglobulin M (IgM)142, is much more common in patients with CD, occurring in 2.5% of patients as compared to 0.25% of the general population3. While IgA class serological tests exhibit the best overall performance in the diagnosis of CD, false-negative results could be obtained in those with selective IgA deficiency. Several approaches have been taken to avoid this pitfall. First, total IgA concentrations may be measured in all patients undergoing serological testing for CD69. IgG class serological tests could then be performed in those identified as having selective IgA deficiency109,111,129; however, there is variability in the definition of IgA deficiency among studies investigating serological assay performance. Some studies use the definition of50.06 to 0.07 g/L, others50.09 to 0.1 g/L and others define an insufficient state as two standard deviations below the population mean (the lower limit of the reference interval), or do not provide a clear definition63,66,67,69,70. In addition, not all total IgA assays have sufficient functional sensitivity to definitively identify selective IgA deficiency, which may influence the efficacy of this approach143. Second, several studies have suggested that very low concentrations of IgA anti-TTG may be indicative of selective IgA deficiency144–146. Consequently, an IgA antiTTG absorbance cutoff may be established, above which selective IgA deficiency is unlikely. Patients below the cutoff would undergo total IgA testing, and IgG class serological testing would be performed if selective IgA deficiency is confirmed. Finally, IgG class serological tests may be performed in addition to IgA class tests in all patients undergoing investigation for CD62,101,121,124. Interestingly, cost-effectiveness analyses of screening algorithms that have included models with and without total IgA testing do not encourage its use. This is primarily due to the high cost of measuring total IgA to exclude deficiency when CD prevalence is low; the effects of false negative IgA-based serological results need to be weighed against

10



Table 2. HLA alleles associated with CD and commonly targeted in CD testing. Allele Haplotype

DQA1

DQB1

DQ2 DR5 DR7 DQ8

*05:01 *05:01 *02:01 *03:01

*02:01 *03:01 *02:02 *03:02


DQ heterodimer subunits associated with each haplotype are indicated186,187.

this cost1,147. Although IgA deficiency occurs at a higher frequency in CD than the general population, it still occurs relatively rarely, and calls into question the need to test for IgA unless serology results do not match clinical suspicion148. There have also been reports of positive IgA-based serology in IgA-deficient patients71, such that a positive result without knowing IgA status may still be informative. Other serological tests for CD Reticulin antibodies. Anti-reticulin antibodies were first described in the serum of DH, celiac and some Crohn’s patients, and were shown to react with connective tissue in rat stomach, liver and kidney. The staining resembled the pattern produced by silver-impregnation methods which target reticulin149. Several reticulin staining patterns have been described149,150, but the ‘‘R1’’ pattern, characterized by peritubular staining in the kidney or liver periportal connective tissue, is most associated with CD. IgA-reticulin antibodies are most prevalent, although both IgA and IgG can be detected151. Early studies suggested that higher titers were associated with CD and DH, as opposed to Crohn’s disease, with highest titers seen in untreated children152. Antibodies in human serum that produce reticulin-like staining in rodent tissues are referred to as anti-reticulin antibodies, while those producing reticulin-like staining in primate substrates are referred to as EMA. Although IgAreticulin and EMA titers are highly correlated, they may not have identical antigen specificity153. Pre-absorption of patient serum with rodent liver homogenates abolishes reticulin but not EMA staining, while pre-absorption with human liver extracts abolishes both reticulin and EMA153,154. Detection of reticulin antibodies therefore uses similar methodology to that of EMA IFA, but uses rodent kidney as substrate, and produces a semi-quantitative result when titer is reported. Reviews of the literature have found reticulin antibodies to have specificities of 95–100%, but variable sensitivity ranging from 41% to 92% in adults and 29% to 100% in children with CD1,153,155. Reticulin antibody testing was superseded by EMA, which boasts higher sensitivity, and reticulin no longer appears in CD testing guidelines1,23,156.

Gliadins comprise the alcohol-soluble component of gluten, and consist of heterogeneous polypeptide chains which produce four major sub-fractions upon electrophoresis, all of which can produce an immune response157. Anti-gliadin antibody (AGA) testing has been available since the 1980s, providing a more specific marker of

Anti-gliadin antibodies.

CD than the general class of wheat protein antibodies158. Early IFA methods were developed based on the observation that gliadin binds to reticulin fibers in mammalian and rodent tissue. Thus, patients with AGAs produce an R1 reticulin-like pattern in gliadin-treated rat tissue159. Initially AGA appeared more sensitive than anti-reticulin antibodies, but was proposed to be a marker of gluten sensitivity rather than CD because AGAs were detected in serum of children with transient gluten intolerance, cow’s milk protein sensitive enteropathy and Crohn’s disease159. Method advancements included the development of solidphase assays, first as a passive antiglobulin hemadsorption test, with antibodies detected by hemagglutination and later ELISA. ELISA AGA methods are still commercially available and can produce semi-quantitative or quantitative results158,160,161. Crude gliadin extracts and purified alphagliadin fractions have been used as the antigen source. Therefore, inter-assay variability can occur due to gliadin source and extract preparation, as well as differences in cutoffs and non-standardized reporting units157. Both IgA and IgG-gliadin antibodies can be detected. AGA performance is thought to be slightly better in children as compared with adults. IgG antibodies produce more false positive results as compared to IgA-AGA, as they are detected in other diseases as discussed above, and also in patients with Sjogren’s disease, sarcoidosis and rheumatoid arthritis, for example. IgA-AGA titers mirror gluten ingestion, while IgGAGA elevations persist longer following institution of a GFD157. Reviews of the literature have found the sensitivity and specificity of AGA (either IgA or IgG) to be between 40 and 100%. Studies that included IgA + IgG-AGA appeared to perform slightly better, with accuracy 470%70,157,162,163. The positive predictive value has been estimated at 30% in some populations162. Variable performance and the advent of more accurate markers has obviated the need for AGA testing, and AGA is no longer recommended for the screening or diagnosis of CD1,23,155,156. However, cases of biopsy-proven CD with positive AGA and negative TTG have been reported, and some suggest that there may still be a role for AGA in settings were EMA and DGP are not readily available164. Future serological and biochemical markers Research into more reliable, non-invasive markers for screening, earlier detection, diagnosis and monitoring remain a focus in CD. A number of potential markers with links to the pathogenesis of CD have recently been reviewed165, some of which are mentioned here. Knowledge of the in vivo interaction of TTG and gliadin underlies the rationale of serological assay modifications. One such assay detects not only TTG and DGP but also an epitope produced when DGP is cross-linked to TTG, termed the ‘‘neo-epitope’’166. Early investigation suggests that neoepitope detection may increase TTG assay performance70,166–168. Markers that reflect intestinal damage include Galectin-10, a lysophospholipase expressed in eosinophils and basophils, CTLA-4 (soluble cytotoxic T-lymphocyte antigen 4), assays of intestinal permeability and anti-actin antibodies165,169. Recent work suggests that intestinal permeability as assessed



DOI: 10.3109/10408363.2014.958813

using lactulose/mannitol ratios following oral administration of a carbohydrate solution may be an informative adjunct to TTG to monitor patient compliance with a GFD170. In the same study, zonulin, a protein involved in modulating intestinal permeability that is up-regulated in active CD, did not correlate well with histology and remained elevated in most patients irrespective of diet, suggesting it will not have a role in monitoring CD patients clinically170. IgA anti-actin antibodies can be assayed by IFA or ELISA169. Performance of the latter was prospectively assessed in high and low CDrisk cohorts, and was shown to be quite specific (490%) with suboptimal sensitivity (29–87%, depending on cohort and cutoff used)100. Although the sensitivity and specificity of these antibodies are not superior to IgA-TTG or IgA-EMA, and these antibodies were not recommended for use in CD diagnosis alone100,171, IgA anti-actin levels correlate with the severity of intestinal pathology and therefore, may be useful in reducing biopsies in patients with TTG levels less than 10 times the ULN172–174. Inflammatory cytokines, T-cell activation and dysregulation factors, and cytotoxic-T cell molecules have been studied for their potential to predict and monitor complicated CD states, such as refractory CD and enteropathy-associated Tcell lymphoma175. Elevations in pro-inflammatory cytokines are seen in those with active CD when compared with patients on prolonged GFD, and may represent markers of disease activity165,175,176. The future of serological and biochemical testing in CD will undoubtedly reflect the identification and validation of markers that reduce the number of invasive procedures needed for diagnosis and patient management and increase the accuracy of population-level screening. With improved understanding of disease pathophysiology comes the opportunity for more sensitive and specific markers, as reflected in the few examples illustrated above, and undoubtedly in those discovered in the future. Genetic testing HLA genes Human leukocyte antigen (HLA) molecules are involved in the presentation of antigens to the immune system. Six distinct genetic loci house more than 200 HLA genes which are found in the major histocompatibility complex (MHC) region on chromosome 6p177. MHC class II molecules are heterodimeric proteins expressed at the surface of antigen-presenting cells and are composed of alpha and beta subunits which form an antigen-binding cleft involved in antigen presentation to T cells. The HLA-class II region contains three distinct loci (DR, DQ and DP), each containing genes encoding alpha and beta HLA subunits (e.g. DQA1 and DQB1, respectively). These genes are highly polymorphic, and such variants are denoted by numbers preceded by an asterisk (i.e. DQA1 * 05). Further allelic variation may be distinguishable depending on the methods used to query HLA polymorphisms, and this variation is indicated by additional digits in the allele designation178. Strong linkage disequilibrium exists between DQ and DR genes, and haplotypes containing specific DQA1, DQB1 and DRB1 alleles are well-defined and sometimes enumerated and referred to as a ‘‘DQ/DR haplotype’’. Historically, HLA

11

molecules were identified serologically, but different combinations of DQA1, DQB1 and DRB1 alleles can exhibit similar serological specificity179, so allele-resolution HLA-typing cannot be determined solely based on serological specificity. HLA and CD Heritability of CD risk is demonstrated by the high degree of disease concordance between monozygotic twins (75%)180,181, lower concordance between dizygotic twins (11%)180 and increased risk of disease in first-degree relatives of CD patients (4–5%) when compared to the general population (1%)182,183. Linkage and association studies have implicated the MHC locus most consistently184,185. MHC class II molecules composed of DQ2 and DQ8 heterodimers, or alleles encoding the individual subunits (e.g. as found in the DR5 and DR7 haplotypes, Table 2) are associated with CD and this has been replicated in many populations. Examples from a few recent studies are described here, and illustrate that the same HLA DQA1 and DQB1 alleles, when present in the heterozygous or homozygous state, are associated with CD in populations across the globe. However, the exact frequency and enrichment relative to non-CD patients can vary. A Swedish screening study that used TTG serology to identify 153 biopsy-confirmed celiac cases (2% of total screened) in a population of sixth-grade children found all cases to carry DQ2 or DQ8 haplotypes. The frequency of either haplotype in the control population was 53%108. Vidales et al. characterized HLA-DQA1 and DQB1 alleles in 136 Spanish children with CD188. They found that DQA1*0501 and DQA1*0201 were the most frequent DQA1 alleles, carried by 85% of patients, and occurred at higher frequency in patients than in the reference population. DQB1 alleles encountered most frequently in patients were the *0201, *0202 and *0301 (85% of patients). The most frequent haplotypes encountered were DQ2, DR5 and DR7, with DQ2 occurring in 47% of the CD population, as compared to 7% of the reference population188. In a multi-center study including 1008 CD patients from France, Italy, Scandinavia and the UK, only 61 did not carry an HLA DQ2 or DQ8 heterodimer, and only 4 of these did not carry any DQ2 or DQ8 alleles22. Selfselecting Australian CD patients tested positive for DQ2, DR7 and/or DQ8 at a frequency of at least 99.7% after excluding four patients with biopsy results due to other conditions. Approximately 55% of the community cohort was positive for DQ2, DR7 or DQ8189. In a study of 72 Chilean CD patients, 96% of patients carried DQ2 or DQ8 haplotypes, while their frequency in the control group was 49%190. Association of DQ2 with CD in Asian children was demonstrated in 30 index cases, all of which were DQ2 positive183. A summary of DQ2 and DQ8 heterodimer frequencies in various CD populations can be found in recent guidelines1. DQ2/DQ8 typing methods HLA alleles differ from each other by multiple singlenucleotide polymorphisms (SNPs), thus differentiating between alleles involves identifying the combination of SNPs associated with a particular allele191. Molecular methods are therefore well-suited to HLA allele


12


identification, with several different methods in wide-spread use, including PCR-SSO (sequence-specific oligonucleotide primed PCR), PCR-SSP (sequence-specific primer based PCR) and sequencing. All methods include a PCR step, but differ in the way that an allele is detected. Given that only a few HLA alleles have been consistently associated with CD, extensive HLA typing is unnecessary when performed for risk assessment or screening purposes. In addition, commercially available kits do not always have sufficient resolution to identify the alleles of interest in CD192. As such, development and optimization of targeted typing approaches that increase sample throughput, decrease cost and utilize equipment routinely found in the clinical molecular laboratory have been the subject of recent research, and are also mentioned. Sequence-specific oligonucleotide probes (SSO) can be used to detect an allele after the HLA region of interest is amplified. The amplicon or, more commonly, the probes themselves, are immobilized and a different hybridization reaction is employed for each probe/allele combination193,194. Multiplex methods have recently been developed186. The ability to accurately identify unique HLA alleles by SSO depends on the specificity of the oligonucleotide probes used. Alleles that differ minimally from each other at the sequence level may not be distinguishable due to non-specific probe annealing, or may require the use of multiple probes to increase signal specificity for a given allele. In PCR-SSP, the HLA allele of interest is amplified using sequence-specific primers (SSP), complementary to the target polymorphism. Amplicon size represents the specific allele targeted by the primer. This can be determined by agarose gel or capillary electrophoresis191,195,196. Multiple primers/amplicons may be needed to identify haplotypes. Typing specificity depends on primer specificity and the use of additional primers and sequential amplification reactions increases resolution197. Method modifications to increase specificity and/or throughput have included restriction enzyme digestion198, use of real-time PCR and TaqMan probes199, doublestranded-DNA binding dyes192 and fluorescently-labelled locus-specific primers in addition to allele-specific primers200. Two related mutation scanning methods, singlestrand conformation polymorphism (SSCP) and heteroduplex analysis have also been used in DQ2/DQ8 identification. SSCP involves denaturation of the PCR product and electrophoresis in non-denaturing conditions, such that amplicons with different sequences will take on different secondary structure and possess different electrophoretic mobilities201. Heteroduplex analysis also exploits electrophoretic differences of DNA structure, but relies on reduced mobility of heteroduplexes (formed after a denaturation and re-annealing step) as compared to DNA homoduplexes, when a polymorphism is present201. Simultaneous amplification-detection of multiple CD-associated alleles by multiplex ligationdependent probe amplification (MLPA) was recently described202. MLPA-based methods have the added feature of being able to distinguish heterozygosity and homozygosity for a given allele based on the relative peak heights of the amplified probes202,203. Another approach described in the literature involves the detection of SNPs in linkage-disequilibrium with the CDassociated alleles, rather than the specific allele itself. This


approach tries to overcome difficulties encountered when targeting HLA alleles, which are surrounded by many sequence variants, making it difficult to design specific primers and probes204. The tag-SNP method is able to assess heterozygosity and homozygosity of the target DQ allele based on whether one or two SNP alleles were detected. This approach also requires fewer PCR reactions for correct allele identification when compared to conventional methods205. The highest resolution information about the polymorphisms/alleles of a given HLA gene is achieved by gene sequencing. It also allows for the discovery of novel polymorphisms, which will not be identified by SSO or SSP methods191. However, in-depth sequence information may not be necessary in the case of celiac-related testing, where only a few specific polymorphisms associated with disease need to be detected. Clinical utility of HLA-typing in CD Negative predictive value As illustrated above, studies in various populations indicate that the majority of patients carry DQ2 or DQ8 haplotypes. In most populations, the majority (85–95%) of patients carry DQ2 alleles, while 2–10% of patients carry DQ8187. Only a small percentage of patients do not express alleles of either haplotype and it has been suggested that this may be due to an incorrect diagnosis of CD189. Absence of DQ2 or DQ8 haplotypes appears greater in southern European celiac patients as compared to northern European populations, and most of these patients carried one half of the DQ2 heterodimer22. DQ2 and DQ8 heterodimer alleles are not rare in the general population, and occur much more frequently than does CD. This suggests that while DQ2/ DQ8 alleles are required for disease manifestation, on their own these alleles are not sufficient to cause disease, which gives DQ2/DQ8 typing a high negative predictive value (NPV)197. That is, the absence of DQ2 or DQ8 associated alleles makes the possibility of CD very unlikely, which would eliminate the need for serial serological monitoring of high-risk groups, individuals with unconvincing serological results or those who have begun a GFD prior to diagnostic evaluation156,206. Excluding biopsy Given the necessity of DQ2/DQ8 in developing CD, it has been suggested that a positive genetic test result may negate the need for intestinal biopsy in symptomatic patients with strongly positive serology1. However, increased diagnostic accuracy using this approach has been questioned by Husby et al.206, since serology and HLA-typing are likely correlated measures, so genetic testing may not provide orthogonal/ independent information to support a diagnosis of CD. Interestingly, cost analysis of different models of CD screening showed similar cost per case screened or case diagnosed when HLA-typing along with EMA was used to diagnose patients who screened positive with IgA-TTG, compared with biopsy of the same patients189. Thus, while biopsy is an expensive test, the utility of HLA-typing may

DOI: 10.3109/10408363.2014.958813

only be in reducing invasive procedures, rather than reducing cost of screening.


Risk stratification Recent studies have indicated that positive HLA-typing results may aid in determining risk of developing CD in atrisk populations. DQ2/DQ8 negative CD patients are very rare; however, it has been shown that in these cases, many patients carry one half of the DQ2 heterodimer, and this genotype may therefore be compatible with a CD diagnosis22. In a population enriched in high-risk patients (those with symptoms, a family history of CD or associated conditions), EMA positivity was related to the presence of DQ2 and DQ8 alleles in a dose-dependent manner. DQ2 homozygosity was seen most frequently, followed by DQ2 plus another high-risk allele. Only 1.5% of EMA positive patients did not carry any DQ2 or DQ8 alleles207. Analysis of a case-control data set estimated the risk of disease and demonstrated that the odds of developing disease varied with the number of copies of risk alleles. Risk of CD in subjects positive for DQ2 and DQ8 was 1:7. In those that were only DQ2 positive with two copies of the DQB1*02 allele, the risk was greater than in those carrying only one copy (1:10 versus 1:24). Interestingly, risk of disease in individuals that only carried one copy of the DQB1*02 allele was lower than the assumed disease prevalence of 1:100208,209. Similar risk stratification was seen in first-degree relatives, with all affected relatives possessing DQ2, DQ8 or DQB1*02/*02 genotypes208. Thus, HLA typing may be useful when determining which high-risk patients need to be monitored serologically and which patients do not need monitoring. By including genetic testing before repeat serological monitoring of celiac patient relatives, up to 60% of relatives would not need serological testing due to absence of DQ2/DQ8 alleles, which may result in cost containment210. Although screening for CD in the general population remains controversial, HLA genotype may represent a means of targeting population-level serological testing. Bjorck et al. describe a prospective cohort in Sweden who had HLA-DQ typing performed at birth, and were then followed-up at 3 years of age211. Those carrying the DQB1*02 and/or DQB1*03:02 alleles were asked to participate and screened for CD using IgA- and IgG-TTG. Those with positive results were tested again after 3 months and then offered intestinal biopsy. This approach identified 76 potential CD patients out of 1620 HLA-positive participants, and did not include 23 children that had already been diagnosed prior to the study. Seventy-one went on to biopsy and 56 met histological criteria for CD. Thus, 56 new cases were identified through this HLA-based targeting screening, while only 23 had been detected clinically up until this time211. This work suggests that risk stratification based on HLA typing may help direct serological testing resources in a clinically meaningful manner.

Challenges with laboratory testing Although laboratory testing has advanced the field of CD diagnosis and management and has the potential to reduce invasive biopsy procedures, it has not yet replaced biopsy completely. Several challenges are routinely encountered


13

when using serological markers of CD, the first of which is the gluten status of the patient. Unlike many other autoimmune conditions, CD symptoms, intestinal pathology, and associated antibodies can change, all of which mirror gluten exposure. While this means that serological testing has the potential to be used for monitoring patient compliance, reducing the need for repeat biopsy, it also alters the diagnostic performance of assays. The sensitivity of all serological markers is lower in patients who are not consuming gluten, making it difficult to assess those who present while on a self-imposed GFD. Another challenge arises due to assay heterogeneity. Substrates, antigen sources and calibrators vary between manufacturers. As a result, titers or antibody concentrations cannot be directly compared between assays, since a given patient’s serum may react differently depending on the reagents used. In addition, optimal cut-offs may differ between manufacturers, populations and laboratories106,212,213. For laboratories and physicians, the choice of which tests to offer and use poses a challenge as well. Although a wealth of literature exists, some of which has been reviewed here, interpretation of findings is not always straight-forward. Recruitment bias affects assay performance estimates reported in the literature48. Many patients are selected based on symptoms or serology results, with CD later confirmed by biopsy, an approach that can over-estimate the sensitivity of a given test. By the same token, choice of control groups can influence specificity estimates. Control groups have been selected based on negative serology results, symptoms, or referral for unrelated gastrointestinal disorders, for example. For ethical reasons control groups often do not have biopsy performed, or biopsy results may be restricted to those suspected of having gastrointestinal abnormalities. This itself may preclude application of results to the general public or population-wide screening approaches. When biopsy results are not available for control subjects, interpretation of study results is also limited because true CD status remains unknown46. Positive serological results in controls either reflect false-positive results or true-positive results in yet undiagnosed CD patients. This type of bias can be reduced with adequate study design – by performing biopsy on all subjects, irrespective of symptoms, serology or other characteristics100. However, this can be challenging because biopsy is an invasive procedure that is performed only if clinically indicated. Additional bias may occur due to inclusion of patients with differing biopsy lesion severity38,53; not all studies limit patient groups to those with severe grade lesions. It is therefore important to note how CD is defined in each published study to determine if findings are applicable to the local patient population. Evaluation of assay performance reported in the literature is made difficult by variability in terminology used. In particular, insufficient description of the methods employed, antibody isotypes detected and use of the term ‘‘gliadin antibody’’ with assays that detect DGP antibodies may prevent findings from being applied to the clinical laboratory. Although commercial kits for both antibody and genetic testing are available with potential for wide-spread use, CD testing is often relegated to the reference laboratory, and is


14



performed in separate sections of the lab (for instance, the genetics, tissue transplant, chemistry and/or immunology laboratories). Thus, clinical information and other lab findings are often not available and cannot be integrated into the laboratory investigation for a given sample, even though CD diagnosis requires synthesis of multiple signs, symptoms and measurements. Finally, now that both serological and genetic testing is available for clinical use, the disease entity can be subdivided into more categories. For example, latent and potential CD, by definition, can only be ascribed if HLA status of the patient is known1, illustrating how advances in laboratory testing have influenced the disease entity, with the potential to affect prevalence estimates as well.

Clinical practice guidelines Clinical practice guidelines for the diagnosis and management of CD were recently issued by several groups, including the European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN)1, the American College of Gastroenterology (ACG)23 and the World Gastroenterology Organisation (WGO)156. The recommended clinical use of laboratory testing for CD, according to these three clinical practice guidelines, is summarized in Table 3. ESPGHAN clinical practice guidelines The ESPGHAN guidelines are based on a systematic review of all literature on antibody testing for CD in pediatric patients from 2004 to 20091,60. These guidelines recommend IgA-TTG as the initial test for symptomatic patients. Total serum IgA should be assessed in individuals with unknown IgA status. Additional testing for IgG class antibodies (IgG-TTG, IgG-DGP or IgG-EMA) should be performed in

IgA deficient individuals. Given that EMA testing has the highest specificity for CD among all of the serological tests, it may be used to confirm low positive results of other serological markers. IgG and/or IgA-DGP may be used in cases where other serological tests are negative, but clinical suspicion of CD remains high. This is especially relevant in children less than 2 years of age. Given the extremely high NPV of HLA-DQ2/DQ8 typing, this test is recommended to exclude the diagnosis of CD. It should be offered when the diagnosis of CD is uncertain, such as cases where clinical suspicion remains high despite negative serology for CD-specific antibodies and equivocal biopsy findings. In addition, HLA-DQ2/DQ8 typing is recommended as the first line test for screening asymptomatic individuals who are at high risk for CD. These include first degree relatives of an individual with confirmed CD, and those with type I diabetes, Down’s syndrome or Turner’s syndrome. Based on the above-described recommendations, the ESPGHAN guidelines present two algorithms for CD testing in children and adolescents. The first algorithm1 should be applied to patients with otherwise unexplained symptoms of CD. In this approach, IgA-TTG is employed as the first-line test and total serum IgA is measured to exclude IgA deficiency (further testing may be required in those less than 2 years of age and those with IgA deficiency). If IgATTG is greater than 10 times the ULN, further laboratory testing can be performed to diagnose CD without performing biopsy, if the patient and guardians agree. The elevated IgATTG result should be confirmed by EMA testing on a separate blood sample. HLA-DQ2/DQ8 typing should also be performed. The diagnosis of CD is confirmed if the patient tests positive for both EMA and HLA-DQ2/DQ8. In instances of a positive IgA-TTG that is less than 10 times the ULN, biopsy

Table 3. Recommended clinical use of laboratory tests for CD according to 2012 and 2013 clinical practice guidelines1,23,156. Test IgA-TTG

IgG-TTG IgA-EMA

IgG-EMA IgA-DGP

IgG-DGP

Total IgA HLA-DQ2/DQ8

ESPGHAN

ACG

WGO

First-line test for symptomatic patients. Second-line test for high risk asymptomatic patients (firstline test if HLA typing is not available). Useful in patients with IgA deficiency. Confirmation of low positive IgATTG in asymptomatic patients. Diagnosis of celiac disease without biopsy. Useful in patients with IgA deficiency. Useful in children less than 2 years of age and in cases where other serological tests are negative but clinical suspicion is high. Useful in patients with IgA deficiency, children less than 2 years of age, and in cases where other serological tests are negative but clinical suspicion is high. Assessment of IgA status First-line test for high risk asymptomatic patients. Exclusion of celiac disease when the diagnosis is uncertain. Diagnosis of celiac disease without biopsy.

First-line test in patients over 2 years of age. Monitoring patients with celiac disease.

Diagnosis of celiac disease. POC test useful in low resource settings. Monitoring patients with celiac disease.

Useful in patients with IgA deficiency. No recommendation.

No recommendation. Diagnosis of celiac disease.

No recommendation. Should be combined with IgA-TTG in children less than 2 years of age. Monitoring patients with celiac disease. Useful in patients with IgA deficiency. Should be combined with IgA-TTG in children less than 2 years of age. Monitoring patients with celiac disease. Assessment of IgA status Exclusion of celiac disease in specific circumstances (see text).

No recommendation. Diagnosis of celiac disease. Useful in patients less than 3 years of age. Monitoring patients with celiac disease. Diagnosis of celiac disease. Useful in patients with IgA deficiency and children less than 3 years of age. No recommendation. Exclusion of celiac disease in equivocal cases.


DOI: 10.3109/10408363.2014.958813

should still be performed. It should be noted that some have questioned the approach of diagnosing CD in the absence of histological findings, and biopsy remains central to some guidelines23,214. While the performance of the ESPGHAN criteria for diagnosing CD in the absence of biopsy needs to be evaluated in a prospective manner1, recent retrospective studies have yielded encouraging results. Klapp and colleagues performed a retrospective review of 150 symptomatic patients who underwent small bowel biopsy for suspicion of CD215. All patients had IgA-TTG, IgA-EMA and HLA-DQ2/DQ8 testing performed at time of biopsy. One hundred and sixteen patients met the ESPGHAN criteria for the diagnosis of CD without biopsy (also referred to as the triple test: IgA-TTG greater than 10 times the ULN, positive EMA and HLA-DQ2/DQ8). One hundred and thirteen of these patients (97.4%) had histological findings consistent with CD, while the remaining three (2.6%) developed villous atrophy on follow-up. Thus, the triple test had a positive predictive value of 100%. Of the 34 patients who were negative for the triple test, intestinal biopsy confirmed CD in 25 and ruled it out in 9. Thus, the sensitivity of the triple test was 82% with a specificity of 100%. The authors concluded that all patients with CD would have been correctly diagnosed using the ESPGHAN criteria, and biopsy could have been avoided in 77% of patients in this cohort. However, the authors do stress that the results need to be validated in a large scale prospective study. It has been argued that omitting biopsy may result in additional diagnoses (such as gastritis, esophagitis and H. pylori infection) being missed in individuals with CD216. Thus, careful follow-up in these individuals is critical. A similar study was performed by Nevoral and colleagues217, who retrospectively reviewed symptomatic patients examined for suspicion of CD. Intestinal biopsy was performed in all patients, and IgA-TTG and IgA-EMA measured. HLA-DQ2/DQ8 typing was not performed. Of the 99 patients with IgA-TTG greater than 10 times the ULN and positive EMA results, 98 had biopsy findings consistent with CD, yielding a positive predictive value of 99%. Eighty-eight patients did not meet these serological criteria, with biopsy confirming CD in 41 and ruling it out in 47. Thus, the sensitivity of IgA-TTG greater than 10 times the ULN combined with positive EMA was 71%, with a specificity of 98%. This study raises the question of whether HLA typing is needed to diagnose CD without biopsy in symptomatic patients. Other combinations of serological tests may also have utility in this regard100,168,218,219. Thus, more work will be required to determine which combination of laboratory tests is best able to diagnose CD in the absence of biopsy. The second ESPGHAN algorithm1 for the diagnosis of CD should be used for patients who are at increased risk for developing CD, but do not currently have signs or symptoms suggestive of the disease. This algorithm differs in several ways from the symptomatic individuals’ algorithm. First, IgATTG is not recommended as the first-line test because the frequency of false positive results is higher in this population compared to symptomatic individuals220. Instead, HLA-DQ2/ DQ8 typing (if available) is recommended as the first line test. Due to its high NPV, absence of HLA-DQ2/DQ8 excludes patients from further CD testing. Patients who are positive for


15

HLA-DQ2/DQ8 should subsequently be tested for IgA-TTG and total IgA (with alternative testing in the setting of IgA deficiency). The second major difference compared to the algorithm for symptomatic patients, is that low positive IgA anti-TTG results (less than three times the ULN) should be confirmed by EMA testing. This aids in distinguishing false positive results from true positive low IgA-TTG titers. Finally, all asymptomatic patients with elevated IgA-TTG must undergo biopsy to confirm the diagnosis of CD (even if the result is greater than 10 times the ULN). ACG clinical practice guidelines The ACG makes many similar recommendations as the ESPGHAN with regards to the diagnosis of CD1,23. First, IgA-TTG is endorsed as the preferred first-line test due to its clinical sensitivity and specificity131. Second, total IgA should be assessed in individuals having a high pre-test probability for CD, especially in circumstances where IgATTG is negative. If IgA deficiency is identified, IgG-based serological testing for either IgG-TTG or IgG-DGP should be performed. Finally, IgA and/or IgG-DGP testing should be combined with IgA-TTG testing in patients less than 2 years of age. In contrast to the ESPGHAN, the ACG does not make any specific recommendations regarding EMA testing. In addition, they recommend that HLA-DQ2/DQ8 typing should not be used in the routine workup of CD, but should be reserved to rule out CD only in specific circumstances. These include patients with equivocal biopsy findings and negative serology, patients who commenced a GFD prior to any testing for CD, patients with discordant serology and biopsy findings, patients with suspected refractory CD in whom the diagnosis of CD is questionable, and patients with Down’s syndrome. Finally, the ACG guidelines do not endorse omission of intestinal biopsy in the setting of IgA-TTG greater than 10 times the ULN, positive anti-EMA, and positive HLADQ2/81,23. Instead, they emphasize that the diagnosis of CD requires histological findings consistent with the disease. The ACG also makes recommendations for the monitoring of patients who are diagnosed with CD. IgA-TTG or IgA (or IgG) -DGP antibodies should be measured to monitor adherence to a GFD. A decrease in baseline values should be evident within months of strict adherence141. While the optimal frequency of follow-up testing has not been established, annual testing is recommended until more evidence is available. WGO clinical practice guidelines The WGO has developed more general guidelines156 than those put forth by the ESPGHAN and ACG1,23. Different recommendations are provided based on different levels of resources that institutions or countries may have. According to these guidelines, the gold standard for CD diagnosis is positive intestinal biopsy findings with positive CD-specific antibodies; however, this requires the availability of both trained pathologists and a biochemistry laboratory. In settings where trained pathologists are not available, TTG, EMA or both may be measured depending on availability and experience. It is argued that IgG and IgA-DGP assays have similar


16


performance characteristics to IgA-TTG, and therefore are also feasible options. The guidelines note that the DGP assays are particularly useful for children under the age of three, whereas the IgG-DGP assay is especially useful in those with IgA deficiency. In settings where resources are scarce, POC testing for IgA-TTG may be considered. The WGO guidelines acknowledge that HLA-DQ2/DQ8 typing may be useful in excluding CD in equivocal cases, but do not provide more detailed indication for this testing. The WGO guidelines make similar recommendations as the ACG with regards to monitoring those with CD. They recommend follow-up serological testing (with IgA-TTG and/ or IgA-DGP being the preferred tests) every 3–6 months during the first-year post-diagnosis. Annual follow-up is recommended after the first year and once the patient is stable.

Conclusions Laboratory testing for CD has made steady progress over the years and reflects improved knowledge of disease pathophysiology. Clear links between molecular mechanisms and serological and genetic markers are evident, supporting their use in the clinic. However, it has also led to heterogeneity in both our definition CD and the manner in which test performance is evaluated. In characterizing test accuracy, limitations of biopsy as the gold standard and the use of other serological tests as the reference method need to be kept in mind, as do the populations studied. Regardless of this heterogeneity, however, improvements in laboratory testing for CD are evident, and demonstrate that IgA-TTG has taken the appropriate position as the first-line test in recently updated clinical guidelines. Inclusion of anti-DGP testing in the newest guidelines provides an opportunity for further study of the utility of these markers in patient diagnosis and management, as data is accumulated through clinical use. It seems likely that use of anti-TTG and anti-DGP will become more wide-spread owing to ease of use of the ELISA and/or POC testing formats. Similarly, modifications to increase throughput and decrease cost of HLA-typing may increase use of this testing. Demand for testing is not likely to subside, given that many patients remain undiagnosed, as well as heighted public interest in diet-modification. Among the controversies that require additional study, remain the omission of biopsy for diagnosis and the most effective combination of testing for screening and diagnosis purposes. Prospective and laboratory-specific data is needed to address these issues, given the current lack of standardization between assays and laboratories. Future challenges for laboratories may also include elimination of tests now rendered obsolete (such as gliadin and reticulin antibodies), optimization of CD test menu and reflexive algorithms, educating ordering physicians and determining the clinical efficacy of these changes. Given the gains in test sensitivity and specificity made in recent years, further improvements in test accuracy may prove difficult. The novel biochemical markers being evaluated in research settings are likely to add to the performance of existing assays, rather than serve as stand-alone tests. There may, however, be an opportunity for novel testing in sub-sets of patients who are only now being


identified, for example, through non-classical means such as DQ2/DQ8 typing, in whom CD risk will need to be determined and appropriate follow-up prescribed.

Declaration of interest Vilte E. Barakauskas and Mathew P. Estey are employees of DynaLIFEDx.

References 1. Husby S, Koletzko S, Korponay-Szabo IR, et al. European Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of coeliac disease. J Pediatr Gastroenterol Nutr 2012;54:136–60. 2. Fasano A, Catassi C. Clinical practice. Celiac disease. N Engl J Med 2012;367:2419–26. 3. Green PH, Cellier C. Celiac disease. N Engl J Med 2007;357: 1731–43. 4. Rubio-Tapia A, Van Dyke CT, Lahr BD, et al. Predictors of family risk for celiac disease: a population-based study. Clin Gastroenterol Hepatol 2008;6:983–7. 5. Goldacre MJ, Wotton CJ, Seagroatt V, Yeates D. Cancers and immune related diseases associated with Down’s syndrome: a record linkage study. Arch Dis Child 2004;89:1014–17. 6. Wouters J, Weijerman ME, van Furth AM, et al. Prospective human leukocyte antigen, endomysium immunoglobulin A antibodies, and transglutaminase antibodies testing for celiac disease in children with Down syndrome. J Pediatr 2009;154: 239–42. 7. Bonamico M, Pasquino AM, Mariani P, et al. Prevalence and clinical picture of celiac disease in Turner syndrome. J Clin Endocrinol Metab 2002;87:5495–8. 8. Frost AR, Band MM, Conway GS. Serological screening for coeliac disease in adults with Turner’s syndrome: prevalence and clinical significance of endomysium antibody positivity. Eur J Endocrinol 2009;160:675–9. 9. Lenhardt A, Plebani A, Marchetti F, et al. Role of human-tissue transglutaminase IgG and anti-gliadin IgG antibodies in the diagnosis of coeliac disease in patients with selective immunoglobulin A deficiency. Dig Liver Dis 2004;36:730–4. 10. Hansen D, Brock-Jacobsen B, Lund E, et al. Clinical benefit of a gluten-free diet in type 1 diabetic children with screening-detected celiac disease: a population-based screening study with 2 years’ follow-up. Diabetes Care 2006;29:2452–6. 11. Salardi S, Volta U, Zucchini S, et al. Prevalence of celiac disease in children with type 1 diabetes mellitus increased in the mid-1990 s: an 18-year longitudinal study based on anti-endomysial antibodies. J Pediatr Gastroenterol Nutr 2008;46:612–14. 12. Volta U, Tovoli F, Caio G. Clinical and immunological features of celiac disease in patients with Type 1 diabetes mellitus. Expert Rev Gastroenterol Hepatol 2011;5:479–87. 13. Shan L, Molberg O, Parrot I, et al. Structural basis for gluten intolerance in celiac sprue. Science 2002;297:2275–9. 14. Fasano A, Shea-Donohue T. Mechanisms of disease: the role of intestinal barrier function in the pathogenesis of gastrointestinal autoimmune diseases. Nat Clin Pract Gastroenterol Hepatol 2005; 2:416–22. 15. Dorum S, Arntzen MO, Qiao SW, et al. The preferred substrates for transglutaminase 2 in a complex wheat gluten digest are Peptide fragments harboring celiac disease T-cell epitopes. PLoS One 2010;5:e14056. 16. Dorum S, Qiao SW, Sollid LM, Fleckenstein B. A quantitative analysis of transglutaminase 2-mediated deamidation of gluten peptides: implications for the T-cell response in celiac disease. J Proteome Res 2009;8:1748–55. 17. Sollid LM, Jabri B. Celiac disease and transglutaminase 2: a model for posttranslational modification of antigens and HLA association in the pathogenesis of autoimmune disorders. Curr Opin Immunol 2011;23:732–8. 18. Siegel M, Strnad P, Watts RE, et al. Extracellular transglutaminase 2 is catalytically inactive, but is transiently activated upon tissue injury. PLoS One 2008;3:e1861.


DOI: 10.3109/10408363.2014.958813

19. van de Wal Y, Kooy Y, van Veelen P, et al. Selective deamidation by tissue transglutaminase strongly enhances gliadin-specific T cell reactivity. J Immunol 1998;161:1585–8. 20. Qiao SW, Sollid LM, Blumberg RS. Antigen presentation in celiac disease. Curr Opin Immunol 2009;21:111–17. 21. Sollid LM, Molberg O, McAdam S, Lundin KE. Autoantibodies in coeliac disease: tissue transglutaminase–guilt by association? Gut 1997;41:851–2. 22. Karell K, Louka AS, Moodie SJ, et al. HLA types in celiac disease patients not carrying the DQA1*05-DQB1*02 (DQ2) heterodimer: results from the European Genetics Cluster on Celiac Disease. Hum Immunol 2003;64:469–77. 23. Rubio-Tapia A, Hill ID, Kelly CP, et al. ACG clinical guidelines: diagnosis and management of celiac disease. Am J Gastroenterol 2013;108:656–76; quiz 77. 24. Oxentenko AS, Grisolano SW, Murray JA, et al. The insensitivity of endoscopic markers in celiac disease. Am J Gastroenterol 2002; 97:933–8. 25. Marsh MN, Crowe PT. Morphology of the mucosal lesion in gluten sensitivity. Bailliere’s Clin Gastroenterol 1995;9:273–93. 26. Fry L, Seah PP, McMinn RM, Hoffbrand AV. Lymphocytic infiltration of epithelium in diagnosis of gluten-sensitive enteropathy. Br Med J 1972;3:371–4. 27. 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;102:330–54. 28. Gonzalez S, Gupta A, Cheng J, et al. Prospective study of the role of duodenal bulb biopsies in the diagnosis of celiac disease. Gastrointest Endosc 2010;72:758–65. 29. Lebwohl B, Kapel RC, Neugut AI, et al. Adherence to biopsy guidelines increases celiac disease diagnosis. Gastrointest Endosc 2011;74:103–9. 30. Pais WP, Duerksen DR, Pettigrew NM, Bernstein CN. How many duodenal biopsy specimens are required to make a diagnosis of celiac disease? Gastrointest Endosc 2008;67:1082–7. 31. Ravelli A, Villanacci V, Monfredini C, et al. How patchy is patchy villous atrophy?: distribution pattern of histological lesions in the duodenum of children with celiac disease. Am J Gastroenterol 2010;105:2103–10. 32. Valitutti F, Di Nardo G, Barbato M, et al. Mapping histologic patchiness of celiac disease by push enteroscopy. Gastrointest Endosc 2014;79:95–100. 33. Weir DC, Glickman JN, Roiff T, et al. Variability of histopathological changes in childhood celiac disease. Am J Gastroenterol 2010;105:207–12. 34. Bonamico M, Thanasi E, Mariani P, et al. Duodenal bulb biopsies in celiac disease: a multicenter study. J Pediatr Gastroenterol Nutr 2008;47:618–22. 35. Evans KE, Aziz I, Cross SS, et al. A prospective study of duodenal bulb biopsy in newly diagnosed and established adult celiac disease. Am J Gastroenterol 2011;106:1837–42. 36. Pai RK. A practical approach to small bowel biopsy interpretation: celiac disease and its mimics. Semin Diagn Pathol 2014;31: 124–36. 37. Shah VH, Rotterdam H, Kotler DP, et al. All that scallops is not celiac disease. Gastrointest Endosc 2000;51:717–20. 38. Walker MM, Murray JA, Ronkainen J, et al. Detection of celiac disease and lymphocytic enteropathy by parallel serology and histopathology in a population-based study. Gastroenterology 2010;139:112–19. 39. McNeish AS, Harms HK, Rey J, et al. The diagnosis of coeliac disease. A commentary on the current practices of members of the European Society for Paediatric Gastroenterology and Nutrition (ESPGAN). Arch Dis Child 1979;54:783–6. 40. Chorzelski TP, Beutner EH, Sulej J, et al. IgA anti-endomysium antibody. A new immunological marker of dermatitis herpetiformis and coeliac disease. Br J Dermatol 1984;111:395–402. 41. Picarelli A, Di Tola M, Sabbatella L, et al. 31–43 amino acid sequence of the alpha-gliadin induces anti-endomysial antibody production during in vitro challenge. Scand J Gastroenterol 1999; 34:1099–102. 42. Ladinser B, Rossipal E, Pittschieler K. Endomysium antibodies in coeliac disease: an improved method. Gut 1994;35:776–8.


17

43. Volta U, Molinaro N, de Franceschi L, et al. IgA anti-endomysial antibodies on human umbilical cord tissue for celiac disease screening. Save both money and monkeys. Dig Dis Sci 1995;40: 1902–5. 44. Amara W, Husebekk A. Improved method for serological testing in celiac disease–IgA anti-endomysium antibody test: a comparison between monkey oesophagus and human umbilical cord as substrate in indirect immunofluorescence test. Scand J Clin Lab Invest 1998;58:547–54. 45. Carroccio A, Cavataio F, Iacono G, et al. IgA antiendomysial antibodies on the umbilical cord in diagnosing celiac disease. Sensitivity, specificity, and comparative evaluation with the traditional kit. Scand J Gastroenterol 1996;31:759–63. 46. Lewis NR, Scott BB. Systematic review: the use of serology to exclude or diagnose coeliac disease (a comparison of the endomysial and tissue transglutaminase antibody tests). Aliment Pharmacol Ther 2006;24:47–54. 47. Kapuscinska A, Zalewski T, Chorzelski TP, et al. Disease specificity and dynamics of changes in IgA class anti-endomysial antibodies in celiac disease. J Pediatr Gastroenterol Nutr 1987;6: 529–34. 48. Cummins A, Thompson F. Sensitivity of anti-endomysial antibody in detecting celiac disease. Gastroenterology 2002;122: 246–7. 49. Ozgenc F, Aksu G, Aydogdu S, et al. Association between antiendomysial antibody and total intestinal villous atrophy in children with coeliac disease. J Postgrad Med 2003;49:21–4; discussion 4. 50. Levine A, Bujanover Y, Reif S, et al. Comparison of assays for anti-endomysial and anti-transglutaminase antibodies for diagnosis of pediatric celiac disease. Isr Med Assoc J 2000;2:122–5. 51. 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: 181–3. 52. Collin P, Maki M, Kaukinen K. Revival of gliadin antibodies in the diagnostic work-up of celiac disease. J Clin Gastroenterol 2010;44:159–60. 53. Rostami K, Kerckhaert J, Tiemessen R, et al. Sensitivity of antiendomysium and antigliadin antibodies in untreated celiac disease: disappointing in clinical practice. Am J Gastroenterol 1999;94:888–94. 54. Dahele A, Kingstone K, Bode J, et al. Anti-endomysial antibody negative celiac disease: does additional serological testing help? Dig Dis Sci 2001;46:214–21. 55. Feighery C, Weir DG, Whelan A, et al. Diagnosis of glutensensitive enteropathy: is exclusive reliance on histology appropriate? Eur J Gastroenterol Hepatol 1998;10:919–25. 56. Biagi F, Bianchi PI, Campanella J, et al. The prevalence and the causes of minimal intestinal lesions in patients complaining of symptoms suggestive of enteropathy: a follow-up study. J Clin Pathol 2008;61:1116–18. 57. Hopper AD, Hadjivassiliou M, Hurlstone DP, et al. What is the role of serologic testing in celiac disease? A prospective, biopsyconfirmed study with economic analysis. Clin Gastroenterol Hepatol 2008;6:314–20. 58. Kurppa K, Rasanen T, Collin P, et al. Endomysial antibodies predict celiac disease irrespective of the titers or clinical presentation. World J Gastroenterol 2012;18:2511–16. 59. Rostami K, Kerckhaert JP, Tiemessen R, et al. The relationship between anti-endomysium antibodies and villous atrophy in coeliac disease using both monkey and human substrate. Eur J Gastroenterol Hepatol 1999;11:439–42. 60. Giersiepen K, Lelgemann M, Stuhldreher N, et al. Accuracy of diagnostic antibody tests for coeliac disease in children: summary of an evidence report. J Pediatr Gastroenterol Nutr 2012;54: 229–41. 61. Di Tola M, Sabbatella L, Anania MC, et al. Anti-tissue transglutaminase antibodies in inflammatory bowel disease: new evidence. Clin Chem Lab Med 2004;42:1092–7. 62. Volta U, Granito A, Parisi C, et al. Deamidated gliadin peptide antibodies as a routine test for celiac disease: a prospective analysis. J Clin Gastroenterol 2010;44:186–90. 63. Picarelli A, di Tola M, Sabbatella L, et al. Identification of a new coeliac disease subgroup: antiendomysial and anti-

18

64. 65. 66.


67.

68.

69. 70. 71. 72. 73.

74. 75. 76. 77. 78.

79. 80.

81. 82. 83.

84. 85.

V. E. Barakauskas et al. transglutaminase antibodies of IgG class in the absence of selective IgA deficiency. J Intern Med 2001;249:181–8. Beutner EH, Kumar V, Chorzelski TP, Szaflarska-Czerwionka M. IgG endomysial antibodies in IgA-deficient patient with coeliac disease. Lancet 1989;1:1261–2. Sulkanen S, Collin P, Laurila K, Maki M. IgA- and IgG-class antihuman umbilical cord antibody tests in adult coeliac disease. Scand J Gastroenterol 1998;33:251–4. Prince HE, Norman GL, Binder WL. Immunoglobulin A (IgA) deficiency and alternative celiac disease-associated antibodies in sera submitted to a reference laboratory for endomysial IgA testing. Clin Diagn Lab Immunol 2000;7:192–6. Korponay-Szabo IR, Kovacs JB, Czinner A, et al. High prevalence of silent celiac disease in preschool children screened with IgA/ IgG antiendomysium antibodies. J Pediatr Gastroenterol Nutr 1999;28:26–30. Cataldo F, Lio D, Marino V, et al. IgG(1) antiendomysium and IgG antitissue transglutaminase (anti-tTG) antibodies in coeliac patients with selective IgA deficiency. Working Groups on Celiac Disease of SIGEP and Club del Tenue. Gut 2000;47:366–9. McGowan KE, Lyon ME, Butzner JD. Celiac disease and IgA deficiency: complications of serological testing approaches encountered in the clinic. Clin Chem 2008;54:1203–9. Reeves GE, Squance ML, Duggan AE, et al. Diagnostic accuracy of coeliac serological tests: a prospective study. Eur J Gastroenterol Hepatol 2006;18:493–501. Valletta E, Fornaro M, Pecori S, Zanoni G. Selective immunoglobulin A deficiency and celiac disease: let’s give serology a chance. J Investig Allergol Clin Immunol 2011;21:242–4. Izzi L. Anti-endomysium antibodies detection in the celiac disease screening: indirect immunofluorescence pattern using umbilical cord sections as substrate. ALTEX 1998;15:141–3. Castellino F, Scaglione N, Grosso SB, Sategna-Guidetti C. A novel method for detecting IgA endomysial antibodies by using human umbilical vein endothelial cells. Eur J Gastroenterol Hepatol 2000;12:45–9. Murray JA, Herlein J, Mitros F, Goeken JA. Serologic testing for celiac disease in the United States: results of a multilaboratory comparison study. Clin Diagn Lab Immunol 2000;7:584–7. Lasagni D, Ferrari R, Lapini M. Unmasking anti-endomysial antibodies in coeliac subjects positive for anti-smooth muscle antibodies. Acta Paediatr 1999;88:462–4. Dieterich W, Ehnis T, Bauer M, et al. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997;3:797–801. Dieterich W, Laag E, Schopper H, et al. Autoantibodies to tissue transglutaminase as predictors of celiac disease. Gastroenterology 1998;115:1317–21. Chan AW, Butzner JD, McKenna R, Fritzler MJ. Tissue transglutaminase enzyme-linked immunosorbent assay as a screening test for celiac disease in pediatric patients. Pediatrics 2001;107:1–4. Gillett HR, Freeman HJ. Comparison of IgA endomysium antibody and IgA tissue transglutaminase antibody in celiac disease. Can J Gastroenterol 2000;14:668–71. Poddar U, Thapa BR, Nain CK, Singh K. Is tissue transglutaminase autoantibody the best for diagnosing celiac disease in children of developing countries? J Clin Gastroenterol 2008;42: 147–51. Troncone R, Maurano F, Rossi M, et al. IgA antibodies to tissue transglutaminase: an effective diagnostic test for celiac disease. J Pediatr 1999;134:166–71. Vitoria JC, Arrieta A, Arranz C, et al. Antibodies to gliadin, endomysium, and tissue transglutaminase for the diagnosis of celiac disease. J Pediatr Gastroenterol Nutr 1999;29:571–4. Carroccio A, Giannitrapani L, Soresi M, et al. Guinea pig transglutaminase immunolinked assay does not predict coeliac disease in patients with chronic liver disease. Gut 2001;49: 506–11. Clemente MG, Musu MP, Frau F, et al. Antitissue transglutaminase antibodies outside celiac disease. J Pediatr Gastroenterol Nutr 2002;34:31–4. Koop I, Ilchmann R, Izzi L, et al. Detection of autoantibodies against tissue transglutaminase in patients with celiac disease and dermatitis herpetiformis. Am J Gastroenterol 2000;95:2009–14.


86. Weiss B, Bujanover Y, Avidan B, et al. Positive tissue transglutaminase antibodies with negative endomysial antibodies: low rate of celiac disease. Isr Med Assoc J 2004;6:9–12. 87. Burgin-Wolff A, Dahlbom I, Hadziselimovic F, Petersson CJ. Antibodies against human tissue transglutaminase and endomysium in diagnosing and monitoring coeliac disease. Scand J Gastroenterol 2002;37:685–91. 88. Carroccio A, Vitale G, Di Prima L, et al. Comparison of antitransglutaminase ELISAs and an anti-endomysial antibody assay in the diagnosis of celiac disease: a prospective study. Clin Chem 2002;48:1546–50. 89. Fabiani E, Peruzzi E, Mandolesi A, et al. Anti-human versus antiguinea pig tissue transglutaminase antibodies as the first-level serological screening test for coeliac disease in the general population. Dig Liver Dis 2004;36:671–6. 90. Tonutti E, Visentini D, Bizzaro N, et al. The role of antitissue transglutaminase assay for the diagnosis and monitoring of coeliac disease: a French-Italian multicentre study. J Clin Pathol 2003;56: 389–93. 91. Wolters V, Vooijs-Moulaert AF, Burger H, et al. Human tissue transglutaminase enzyme linked immunosorbent assay outperforms both the guinea pig based tissue transglutaminase assay and anti-endomysium antibodies when screening for coeliac disease. Eur J Pediatr 2002;161:284–7. 92. Wong RC, Wilson RJ, Steele RH, et al. A comparison of 13 guinea pig and human anti-tissue transglutaminase antibody ELISA kits. J Clin Pathol 2002;55:488–94. 93. Baudon JJ, Johanet C, Absalon YB, et al. Diagnosing celiac disease: a comparison of human tissue transglutaminase antibodies with antigliadin and antiendomysium antibodies. Arch Pediatr Adolesc Med 2004;158:584–8. 94. Collin P, Kaukinen K, Vogelsang H, et al. Antiendomysial and antihuman recombinant tissue transglutaminase antibodies in the diagnosis of coeliac disease: a biopsy-proven European multicentre study. Eur J Gastroenterol Hepatol 2005;17:85–91. 95. Tesei N, Sugai E, Vazquez H, et al. Antibodies to human recombinant tissue transglutaminase may detect coeliac disease patients undiagnosed by endomysial antibodies. Aliment Pharmacol Ther 2003;17:1415–23. 96. Van Meensel B, Hiele M, Hoffman I, et al. Diagnostic accuracy of ten second-generation (human) tissue transglutaminase antibody assays in celiac disease. Clin Chem 2004;50:2125–35. 97. Hopper AD, Cross SS, Hurlstone DP, et al. Pre-endoscopy serological testing for coeliac disease: evaluation of a clinical decision tool. BMJ 2007;334:729. 98. Niveloni S, Sugai E, Cabanne A, et al. Antibodies against synthetic deamidated gliadin peptides as predictors of celiac disease: prospective assessment in an adult population with a high pretest probability of disease. Clin Chem 2007;53:2186–92. 99. Parizade M, Bujanover Y, Weiss B, et al. Performance of serology assays for diagnosing celiac disease in a clinical setting. Clin Vaccine Immunol 2009;16:1576–82. 100. Sugai E, Moreno ML, Hwang HJ, et al. Celiac disease serology in patients with different pretest probabilities: is biopsy avoidable? World J Gastroenterol 2010;16:3144–52. 101. Alessio MG, Tonutti E, Brusca I, et al. Correlation between IgA tissue transglutaminase antibody ratio and histological finding in celiac disease. J Pediatr Gastroenterol Nutr 2012;55:44–9. 102. Barker CC, Mitton C, Jevon G, Mock T. Can tissue transglutaminase antibody titers replace small-bowel biopsy to diagnose celiac disease in select pediatric populations? Pediatrics 2005; 115:1341–6. 103. Donaldson MR, Book LS, Leiferman KM, et al. Strongly positive tissue transglutaminase antibodies are associated with Marsh 3 histopathology in adult and pediatric celiac disease. J Clin Gastroenterol 2008;42:256–60. 104. Hill PG, Holmes GK. Coeliac disease: a biopsy is not always necessary for diagnosis. Aliment Pharmacol Ther 2008;27:572–7. 105. Mubarak A, Wolters VM, Gerritsen SA, et al. A biopsy is not always necessary to diagnose celiac disease. J Pediatr Gastroenterol Nutr 2011;52:554–7. 106. Zanini B, Magni A, Caselani F, et al. High tissue-transglutaminase antibody level predicts small intestinal villous atrophy in adult patients at high risk of celiac disease. Dig Liver Dis 2012;44: 280–5.


DOI: 10.3109/10408363.2014.958813

107. Mubarak A, Wolters VM, Gmelig-Meyling FH, et al. Tissue transglutaminase levels above 100 U/mL and celiac disease: a prospective study. World J Gastroenterol 2012;18:4399–403. 108. Sandstrom O, Rosen A, Lagerqvist C, et al. Transglutaminase IgA antibodies in a celiac disease mass screening and the role of HLADQ genotyping and endomysial antibodies in sequential testing. J Pediatr Gastroenterol Nutr 2013;57:472–6. 109. Villalta D, Alessio MG, Tampoia M, et al. Diagnostic accuracy of IgA anti-tissue transglutaminase antibody assays in celiac disease patients with selective IgA deficiency. Ann N Y Acad Sci 2007; 1109:212–20. 110. Dahlbom I, Olsson M, Forooz NK, et al. Immunoglobulin G (IgG) anti-tissue transglutaminase antibodies used as markers for IgAdeficient celiac disease patients. Clin Diagn Lab Immunol 2005; 12:254–8. 111. 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;52:1567–71. 112. Baldas V, Tommasini A, Trevisiol C, et al. Development of a novel rapid non-invasive screening test for coeliac disease. Gut 2000;47:628–31. 113. Sorell L, Garrote JA, Acevedo B, Arranz E. One-step immunochromatographic assay for screening of coeliac disease. Lancet 2002;359:945–6. 114. Nemec G, Ventura A, Stefano M, et al. Looking for celiac disease: diagnostic accuracy of two rapid commercial assays. Am J Gastroenterol 2006;101:1597–600. 115. Raivio T, Kaukinen K, Nemes E, et al. Self transglutaminasebased rapid coeliac disease antibody detection by a lateral flow method. Aliment Pharmacol Ther 2006;24:147–54. 116. Raivio T, Korponay-Szabo I, Collin P, et al. Performance of a new rapid whole blood coeliac test in adult patients with low prevalence of endomysial antibodies. Dig Liver Dis 2007;39: 1057–63. 117. Korponay-Szabo IR, Szabados K, Pusztai J, et al. Population screening for coeliac disease in primary care by district nurses using a rapid antibody test: diagnostic accuracy and feasibility study. BMJ 2007;335:1244–7. 118. Alarida K, Harown J, Ahmaida A, et al. Coeliac disease in Libyan children: a screening study based on the rapid determination of anti-transglutaminase antibodies. Dig Liver Dis 2011;43:688–91. 119. Aleanzi M, Demonte AM, Esper C, et al. Celiac disease: antibody recognition against native and selectively deamidated gliadin peptides. Clin Chem 2001;47:2023–8. 120. 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;50:2370–5. 121. Agardh D. Antibodies against synthetic deamidated gliadin peptides and tissue transglutaminase for the identification of childhood celiac disease. Clin Gastroenterol Hepatol 2007;5: 1276–81. 122. Basso D, Guariso G, Fogar P, et al. Antibodies against synthetic deamidated gliadin peptides for celiac disease diagnosis and follow-up in children. Clin Chem 2009;55:150–7. 123. Dahle C, Hagman A, Ignatova S, Strom M. Antibodies against deamidated gliadin peptides identify adult coeliac disease patients negative for antibodies against endomysium and tissue transglutaminase. Aliment Pharmacol Ther 2010;32:254–60. 124. Jaskowski TD, Donaldson MR, Hull CM, et al. Novel screening assay performance in pediatric celiac disease and adult dermatitis herpetiformis. J Pediatr Gastroenterol Nutr 2010;51:19–23. 125. Naiyer AJ, Hernandez L, Ciaccio EJ, et al. Comparison of commercially available serologic kits for the detection of celiac disease. J Clin Gastroenterol 2009;43:225–32. 126. Olen O, Gudjonsdottir AH, Browaldh L, et al. Antibodies against deamidated gliadin peptides and tissue transglutaminase for diagnosis of pediatric celiac disease. J Pediatr Gastroenterol Nutr 2012;55:695–700. 127. Prause C, Richter T, Koletzko S, et al. New developments in serodiagnosis of childhood celiac disease: assay of antibodies against deamidated gliadin. Ann N Y Acad Sci 2009;1173:28–35. 128. Sakly W, Mankai A, Ghdess A, et al. Performance of antideamidated gliadin peptides antibodies in celiac disease diagnosis. Clin Res Hepatol Gastroenterol 2012;36:598–603.


19

129. Villalta D, Tonutti E, Prause C, et al. IgG antibodies against deamidated gliadin peptides for diagnosis of celiac disease in patients with IgA deficiency. Clin Chem 2010;56:464–8. 130. Health Quality Ontario. Clinical utility of serologic testing for celiac disease in Ontario: an evidence-based analysis. Ont Health Technol Assess Ser 2010;10:1–111. 131. Lewis NR, Scott BB. Meta-analysis: deamidated gliadin peptide antibody and tissue transglutaminase antibody compared as screening tests for coeliac disease. Aliment Pharmacol Ther 2010;31:73–81. 132. Monzani A, Rapa A, Fonio P, et al. Use of deamidated gliadin peptide antibodies to monitor diet compliance in childhood celiac disease. J Pediatr Gastroenterol Nutr 2011;53:55–60. 133. Sugai E, Hwang HJ, Vazquez H, et al. New serology assays can detect gluten sensitivity among enteropathy patients seronegative for anti-tissue transglutaminase. Clin Chem 2010;56:661–5. 134. Mozo L, Gomez J, Escanlar E, et al. Diagnostic value of antideamidated gliadin peptide IgG antibodies for celiac disease in children and IgA-deficient patients. J Pediatr Gastroenterol Nutr 2012;55:50–5. 135. Amarri S, Alvisi P, De Giorgio R, et al. Antibodies to deamidated gliadin peptides: an accurate predictor of coeliac disease in infancy. J Clin Immunol 2013;33:1027–30. 136. Liu E, Li M, Emery L, et al. Natural history of antibodies to deamidated gliadin peptides and transglutaminase in early childhood celiac disease. J Pediatr Gastroenterol Nutr 2007;45: 293–300. 137. Mubarak A, Gmelig-Meyling FH, Wolters VM, et al. Immunoglobulin G antibodies against deamidated-gliadin-peptides outperform anti-endomysium and tissue transglutaminase antibodies in children 52 years age. Apmis 2011;119:894–900. 138. Barbato M, Maiella G, Di Camillo C, et al. The anti-deamidated gliadin peptide antibodies unmask celiac disease in small children with chronic diarrhoea. Dig Liver Dis 2011;43:465–9. 139. Benkebil F, Combescure C, Anghel SI, et al. Diagnostic accuracy of a new point-of-care screening assay for celiac disease. World J Gastroenterol 2013;19:5111–17. 140. Bienvenu F, Besson Duvanel C, Seignovert C, et al. Evaluation of a point-of-care test based on deamidated gliadin peptides for celiac disease screening in a large pediatric population. Eur J Gastroenterol Hepatol 2012;24:1418–23. 141. Nachman F, Sugai E, Vazquez H, et al. Serological tests for celiac disease as indicators of long-term compliance with the gluten-free diet. Eur J Gastroenterol Hepatol 2011;23:473–80. 142. Conley ME, Notarangelo LD, Etzioni A. Diagnostic criteria for primary immunodeficiencies. Representing PAGID (PanAmerican Group for Immunodeficiency) and ESID (European Society for Immunodeficiencies). Clin Immunol 1999;93:190–7. 143. Estey MP, Cohen AH, Colantonio DA, et al. CLSI-based transference of the CALIPER database of pediatric reference intervals from Abbott to Beckman, Ortho, Roche and Siemens Clinical Chemistry Assays: direct validation using reference samples from the CALIPER cohort. Clin Biochem 2013;46: 1197–219. 144. Fernandez E, Blanco C, Garcia S, et al. Use of low concentrations of human IgA anti-tissue transglutaminase to rule out selective IgA deficiency in patients with suspected celiac disease. Clin Chem 2005;51:1014–16. 145. Lowbeer C, Wallinder H. Undetectable anti-tissue transglutaminase IgA antibody measured with EliA Celikey indicates selective IgA deficiency. Clin Chim Acta 2010;411:612. 146. Sinclair D, Saas M, Turk A, et al. Do we need to measure total serum IgA to exclude IgA deficiency in coeliac disease? J Clin Pathol 2006;59:736–9. 147. Dorn SD, Matchar DB. Cost-effectiveness analysis of strategies for diagnosing celiac disease. Dig Dis Sci 2008;53:680–8. 148. Lock RJ, Pitcher MC, Unsworth DJ. IgA anti-tissue transglutaminase as a diagnostic marker of gluten sensitive enteropathy. J Clin Pathol 1999;52:274–7. 149. Seah PP, Fry L, Hoffbrand AV, Holborow EJ. Tissue antibodies in dermatitis herpetiformis and adult coeliac disease. Lancet 1971;1: 834–6. 150. Rizzetto M, Doniach D. Types of ‘reticulin’ antibodies detected in human sera by immunofluorescence. J Clin Pathol 1973;26: 841–51.


20


151. Eade OE, Lloyd RS, Lang C, Wright R. IgA and IgG reticulin antibodies in coeliac and non-coeliac patients. Gut 1977;18: 991–3. 152. Seah PP, Fry L, Holborow EJ, et al. Antireticulin antibody: incidence and diagnostic significance. Gut 1973;14:311–15. 153. Maki M. The humoral immune system in coeliac disease. Bailliere’s Clin Gastroenterol 1995;9:231–49. 154. Hallstrom O. Comparison of IgA-class reticulin and endomysium antibodies in coeliac disease and dermatitis herpetiformis. Gut 1989;30:1225–32. 155. Nandiwada SL, Tebo AE. Testing for antireticulin antibodies in patients with celiac disease is obsolete: a review of recommendations for serologic screening and the literature. Clin Vaccine Immunol 2013;20:447–51. 156. Bai JC, Fried M, Corazza GR, et al. World Gastroenterology Organisation global guidelines on celiac disease. J Clin Gastroenterol 2013;47:121–6. 157. Troncone R, Ferguson A. Anti-gliadin antibodies. J Pediatr Gastroenterol Nutr 1991;12:150–8. 158. Unsworth DJ, Kieffer M, Holborow EJ, et al. IgA anti-gliadin antibodies in coeliac disease. Clin Exp Immunol 1981;46:286–93. 159. Unsworth DJ, Manuel PD, Walker-Smith JA, et al. New immunofluorescent blood test for gluten sensitivity. Arch Dis Child 1981;56:864–8. 160. Ascher H, Lanner A, Kristiansson B. A new laboratory kit for antigliadin IgA at diagnosis and follow-up of childhood celiac disease. J Pediatr Gastroenterol Nutr 1990;10:443–50. 161. Gonczi J, Skerritt JH, Mitchell JD. A reliable screening test for coeliac disease: enzyme-linked immunosorbent assay to detect anti-gliadin antibodies in serum. Aust N Z J Med 1991; 21:723–31. 162. Leffler DA, Schuppan D. Update on serologic testing in celiac disease. Am J Gastroenterol 2010;105:2520–4. 163. van der Windt DA, Jellema P, Mulder CJ, et al. Diagnostic testing for celiac disease among patients with abdominal symptoms: a systematic review. JAMA 2010;303:1738–46. 164. Benson BC, Mulder CJ, Laczek JT. Anti-gliadin antibodies identify celiac patients overlooked by tissue transglutaminase antibodies. Hawaii J Med Public Health 2013;72:14–17. 165. Moore JK, West SR, Robins G. Advances in celiac disease. Curr Opin Gastroenterol 2011;27:112–18. 166. Matthias T, Pfeiffer S, Selmi C, Eric Gershwin M. Diagnostic challenges in celiac disease and the role of the tissue transglutaminase-neo-epitope. Clin Rev Allergy Immunol 2010;38: 298–301. 167. Lytton SD, Antiga E, Pfeiffer S, et al. Neo-epitope tissue transglutaminase autoantibodies as a biomarker of the gluten sensitive skin disease–dermatitis herpetiformis. Clin Chim Acta 2013;415:346–9. 168. Barak M, Rozenberg O, Froom P, et al. Challenging our serological algorithm for celiac disease (CD) diagnosis by the ESPGHAN guidelines. Clin Chem Lab Med 2013;51:e257–9. 169. Carroccio A, Brusca I, Iacono G, et al. Anti-actin antibodies in celiac disease: correlation with intestinal mucosa damage and comparison of ELISA with the immunofluorescence assay. Clin Chem 2005;51:917–20. 170. Duerksen DR, Wilhelm-Boyles C, Veitch R, et al. A comparison of antibody testing, permeability testing, and zonulin levels with small-bowel biopsy in celiac disease patients on a gluten-free diet. Dig Dis Sci 2010;55:1026–31. 171. Fabbro E, Rubert L, Quaglia S, et al. Uselessness of anti-actin antibody in celiac disease screening. Clin Chim Acta 2008;390: 134–7. 172. Carroccio A, Brusca I, Iacono G, et al. IgA anti-actin antibodies ELISA in coeliac disease: a multicentre study. Dig Liver Dis 2007; 39:818–23. 173. Achour A, Thabet Y, Sakly W, et al. IgA anti-actin antibodies in celiac disease. Gastroenterol Clin Biol 2010;34:483–7. 174. Schirru E, Danjou F, Cicotto L, et al. Anti-actin IgA antibodies identify celiac disease patients with a Marsh 3 Intestinal damage among subjects with moderate anti-TG2 levels. Biomed Res Int 2013;2013:630463. 175. Tack GJ, van Wanrooij RL, Von Blomberg BM, et al. Serum parameters in the spectrum of coeliac disease: beyond standard antibody testing–a cohort study. BMC Gastroenterol 2012;12:159.


176. Manavalan JS, Hernandez L, Shah JG, et al. Serum cytokine elevations in celiac disease: association with disease presentation. Hum Immunol 2010;71:50–7. 177. McCluskey J, Kanaan C, Diviney M. Nomenclature and serology of HLA class I and class II alleles. Curr Protoc Immunol 2003;52:1S:A.1S.1–A.1S.8. 178. Nunes E, Heslop H, Fernandez-Vina M, et al. Definitions of histocompatibility typing terms. Blood 2011;118:e180–3. 179. Nepom GT, Erlich H. MHC class-II molecules and autoimmunity. Annu Rev Immunol 1991;9:493–525. 180. Greco L, Romino R, Coto I, et al. The first large population based twin study of coeliac disease. Gut 2002;50:624–8. 181. Hervonen K, Karell K, Holopainen P, et al. Concordance of dermatitis herpetiformis and celiac disease in monozygous twins. J Invest Dermatol 2000;115:990–3. 182. Fasano A, Berti I, Gerarduzzi T, et al. Prevalence of celiac disease in at-risk and not-at-risk groups in the United States: a large multicenter study. Arch Intern Med 2003;163:286–92. 183. Srivastava A, Yachha SK, Mathias A, et al. Prevalence, human leukocyte antigen typing and strategy for screening among Asian first-degree relatives of children with celiac disease. J Gastroenterol Hepatol 2010;25:319–24. 184. Babron MC, Nilsson S, Adamovic S, et al. Meta and pooled analysis of European coeliac disease data. Eur J Hum Genet 2003; 11:828–34. 185. Garner CP, Ding YC, Steele L, et al. Genome-wide linkage analysis of 160 North American families with celiac disease. Genes Immun 2007;8:108–14. 186. Beni V, Zewdu T, Joda H, et al. Gold nanoparticle fluorescent molecular beacon for low-resolution DQ2 gene HLA typing. Anal Bioanal Chem 2012;402:1001–9. 187. Sollid LM. Molecular basis of celiac disease. Annu Rev Immunol 2000;18:53–81. 188. Vidales MC, Zubillaga P, Zubillaga I, Alfonso-Sanchez MA. Allele and haplotype frequencies for HLA class II (DQA1 and DQB1) loci in patients with celiac disease from Spain. Hum Immunol 2004;65:352–8. 189. Anderson RP, Henry MJ, Taylor R, et al. A novel serogenetic approach determines the community prevalence of celiac disease and informs improved diagnostic pathways. BMC Med 2013;11: 188. 190. Parada A, Araya M, Perez-Bravo F, et al. Amerindian mtDNA haplogroups and celiac disease risk HLA haplotypes in mixedblood Latin American patients. J Pediatr Gastroenterol Nutr 2011;53:429–34. 191. Little AM. An overview of HLA typing for hematopoietic stem cell transplantation. Methods Mol Med 2007;134:35–49. 192. Profaizer T, Eckels D, Delgado JC. Celiac disease and HLA typing using real-time PCR with melting curve analysis. Tissue Antigens 2011;78:31–7. 193. Buyse I, Decorte R, Baens M, et al. Rapid DNA typing of class II HLA antigens using the polymerase chain reaction and reverse dot blot hybridization. Tissue Antigens 1993;41:1–14. 194. Joda H, Beni V, Curnane D, et al. Low-medium resolution HLADQ2/DQ8 typing for coeliac disease predisposition analysis by colorimetric assay. Anal Bioanal Chem 2012;403:807–19. 195. Olerup O, Zetterquist H. HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation. Tissue Antigens 1992;39:225–35. 196. Megiorni F, Mora B, Bonamico M, et al. A rapid and sensitive method to detect specific human lymphocyte antigen (HLA) class II alleles associated with celiac disease. Clin Chem Lab Med 2008; 46:193–6. 197. Olerup O, Aldener A, Fogdell A. HLA-DQB1 and -DQA1 typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours. Tissue Antigens 1993;41:119–34. 198. Lavant EH, Carlson JA. A new automated human leukocyte antigen genotyping strategy to identify DR-DQ risk alleles for celiac disease and type 1 diabetes mellitus. Clin Chem Lab Med 2009;47:1489–95. 199. Reinton N, Helgheim A, Shegarfi H, Moghaddam A. A one-step real-time PCR assay for detection of DQA1*05, DQB1*02 and

DOI: 10.3109/10408363.2014.958813

200. 201. 202.


203. 204. 205. 206. 207.

208. 209. 210.

DQB1*0302 to aid diagnosis of celiac disease. J Immunol Methods 2006;316:125–32. Lavant EH, Agardh DJ, Nilsson A, Carlson JA. A new PCR-SSP method for HLA DR-DQ risk assessment for celiac disease. Clin Chim Acta 2011;412:782–4. Wallace AJ. SSCP/heteroduplex analysis. Methods Mol Biol 2002; 187:151–63. van Beek EM, Roelandse-Koop EA, Vijzelaar R, et al. A multiplex assay to rapidly exclude HLA-DQ2.5 and HLA-DQ8 expression in patients at risk for celiac disease. Clin Chem Lab Med 2013;51: 1191–8. Best DH, Lyon E, Roberts KA, et al. Quick Guide to Molecular Diagnostics. Washington, DC: American Association for Clinical Chemistry; 2013. de Bakker PI, McVean G, Sabeti PC, et al. A high-resolution HLA and SNP haplotype map for disease association studies in the extended human MHC. Nat Genet 2006;38:1166–72. Monsuur AJ, de Bakker PI, Zhernakova A, et al. Effective detection of human leukocyte antigen risk alleles in celiac disease using tag single nucleotide polymorphisms. PLoS One 2008;3:e2270. Husby S, Murray JA. New aspects of the diagnosis of celiac disease in children, adolescents, and adults. Mayo Clin Proc 2013; 88:540–3. Pietzak MM, Schofield TC, McGinniss MJ, Nakamura RM. Stratifying risk for celiac disease in a large at-risk United States population by using HLA alleles. Clin Gastroenterol Hepatol 2009;7:966–71. Megiorni F, Mora B, Bonamico M, et al. HLA-DQ and risk gradient for celiac disease. Hum Immunol 2009;70:55–9. Piccini B, Vascotto M, Serracca L, et al. HLA-DQ typing in the diagnostic algorithm of celiac disease. Rev Esp Enferm Dig 2012; 104:248–54. Chang M, Green PH. Genetic testing before serologic screening in relatives of patients with celiac disease as a cost containment method. J Clin Gastroenterol 2009;43:43–50.


21

211. Bjorck S, Brundin C, Lorinc E, et al. Screening detects a high proportion of celiac disease in young HLA-genotyped children. J Pediatr Gastroenterol Nutr 2010;50:49–53. 212. Beltran L, Koenig M, Egner W, et al. High-titre circulating TTG2 antibodies predict small bowel villous atrophy, but decision cut-off limits must be locally validated. Clin Exp Immunol 2014;176: 190–8. 213. Vermeersch P, Geboes K, Marien G, et al. Defining thresholds of antibody levels improves diagnosis of celiac disease. Clin Gastroenterol Hepatol 2013;11:398–403; quiz e32. 214. Hogberg L, Stenhammar L. Celiac disease: pediatric celiac disease–is a diagnostic biopsy necessary? Nat Rev Gastroenterol Hepatol 2012;9:127–8. 215. Klapp G, Masip E, Bolonio M, et al. Celiac disease: the new proposed ESPGHAN diagnostic criteria do work well in a selected population. J Pediatr Gastroenterol Nutr 2013;56:251–6. 216. Guandalini S, Newland C. Can we really skip the biopsy in diagnosing symptomatic children with celiac disease. J Pediatr Gastroenterol Nutr 2013;57:e24. 217. Nevoral J, Kotalova R, Hradsky O, et al. Symptom positivity is essential for omitting biopsy in children with suspected celiac disease according to the new ESPGHAN guidelines. Eur J Pediatr 2013 Nov 15. 218. Burgin-Wolff A, Mauro B, Faruk H. Intestinal biopsy is not always required to diagnose celiac disease: a retrospective analysis of combined antibody tests. BMC Gastroenterol 2013;13:19. 219. Clouzeau-Girard H, Rebouissoux L, Taupin JL, et al. HLA-DQ genotyping combined with serological markers for the diagnosis of celiac disease: is intestinal biopsy still mandatory? J Pediatr Gastroenterol Nutr 2011;52:729–33. 220. Vecsei A, Arenz T, Heilig G, et al. Influence of age and genetic risk on anti-tissue transglutaminase IgA titers. J Pediatr Gastroenterol Nutr 2009;48:544–9.

Serological testing for celiac disease in adults.

Serologic testing in celiac disease: Practical guide for clinicians.

Timely but above all useful diagnosis of celiac disease.

Gluten, Dysbiosis, and Genetics in Celiac Disease: All Are Important.

All of the above: When multiple correct response options enhance the testing effect.

Clinical utility of celiac disease-associated HLA testing.

Laboratory testing for iron status.

Laboratory testing for factor inhibitors.

Options for prenatal testing for Huntington's disease using linked DNA probes.

Laboratory aspects of von Willebrand disease: test repertoire and options for activity assays and genetic analysis.

Therapeutic approaches for celiac disease.

Emerging drugs for celiac disease.

Point-of-care testing for celiac disease has a low sensitivity in endoscopy.

Laboratory testing in the porphyrias.

Digesting GWAS.

Current options for providing sustained analgesia to laboratory animals.

Celiac disease.

Celiac disease.

Application of Bayesian decision-making to laboratory testing for Lyme disease and comparison with testing for HIV.

TNFA Haplotype Genetic Testing Improves HLA in Estimating the Risk of Celiac Disease in Children.

Laboratory testing in neurorheumatology.

Laboratory testing and standardisation.

Preoperative laboratory testing for the oral and maxillofacial surgery patient.

Technology: testing all possibilities.