determination of igg and iga antibodies against native gliadin

8/13/2019 Determination of IgG and IgA Antibodies Against Native Gliadin

1/18Copyright ESPGHAN and NASPGHAN All rights reserved

Richter et al: Antibodies in celiac diagnosis in children aged up to two years

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JPGN Journal of Pediatric Gastroenterology and Nutrition Publish Ahead of Print

DOI: 10.1097/MPG.0b013e31824678fc

Determination of IgG and IgA antibodies against native gliadin

is not helpful for the diagnosis of coeliac disease

in children up to 2 years of age

Thomas Richter1, Xavier Bossuyt

2, Pieter Vermeersch

2, Holm H. Uhlig

3, Martin Stern

4, Al-

muthe Hauer5, Klaus-Peter Zimmer

6, Luisa Mearin

7, Johanna Hendrika Clementina de Roo

7,

Cornelia Dhnrich8, and Thomas Mothes

9

From the 1 Childrens Hospital of the Clinical Centre Sankt Georg Leipzig, Germany, 2

Dept. Laboratory Medicine of University Hospital Leuven, Belgium,3University Childrens

Hospital Leipzig, Germany,4University Childrens Hospital Tbingen, Germany,

5Univer-

sity Childrens Hospital Graz, Austria,6

University Childrens Hospital Gieen, Germany,7

Department of Paediatrics, Leiden University Medical Centre, The Netherlands, 8

EUROIMMUN Medizinische Labordiagnostika GmbH Lbeck, Germany,9Institute of Labo-

ratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig,

Germany

Address for correspondence: Professor Dr. Thomas Mothes, Department of Laboratory Medi-

cine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig, Liebigstrae 27,

D-04103 Leipzig, Germany (e-mail: [email protected])

T. Mothes, H. H. Uhlig, and C. Dhnrich submitted patents on methods for CD diagnosis (T.

Mothes, A. Osman, H. H. Uhlig, T. Gnnel, A. Dietl: Peptide und Verfahren zur

Diagnostik von Zliakie und Dermatitis herpetiformis and C. Probst, C. Dhnrich, W.

Schlumberger, W. Stcker, L. Komorowski, T. Mothes: Verfahren und Immunabsorbentien




2

zur spezifischen Detektion und Absorption Zliakie- und Dermatitis herpetiformis assoziierter

Antikrper). H. H. Uhlig was supported by the German Coeliac Society.

Key Words:antibodies, coeliac disease, deamidated gliadin, diagnosis, tissue transglutami-

nase

Abbreviations: AUC area under curve, CD coeliac disease, anti-dGli antibodies against

deamidated gliadin, anti-nGli antibodies against native gliadin, anti-tTG antibodies against

tissue transglutaminase, DOR diagnostic odds ratio, EmA endomysium antibodies, GFD glu-

ten-free diet; NLR negative likelihood ratio, PLR positive likelihood ratio, ROC receiver-

operating characteristics




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ABSTRACT

Objective:Assays for antibodies against native gliadin (anti-nGli) are still often assumed to

perform better in the diagnosis of coeliac disease (CD) in young children than tests for anti-

bodies to deamidated gliadin (anti-dGli), tissue transglutaminase (anti-tTG) and endomysium

(EmA). We compared the performance of assays for anti-nGli, anti-dGli, anti-tTG and EmA

in this age group.

Methods: We investigated retrospectively 184 children (42 coeliacs under normal diet and

142 controls) up to 2 years of age. IgA and IgG-anti-dGli, IgA and IgG-anti-nGli, IgA and

IgG-anti-tTG, and IgA-EmA were measured in serum. Areas under receiver-operating charac-

teristics curves (AUCs), sensitivities, specificities, positive (PPV) and negative predictive

values (NPV), positive and negative likelihood ratios (PLR and NLR), as well as diagnostic

odds ratios (DOR) were calculated.

Results: From all tests investigated, only assays for IgG-anti-dGli, IgA-anti-tTG and IgA-

EmA had high specificity (0.96) connected with high sensitivity (0.86), with high positive

predictive values (0.52 and 0.69 at pre-test probabilities of 0.05 and 0.1, respectively), and

with high negative predictive values (0.99 and 0.98 at pre-test probabilities of 0.05 and

0.1, respectively). These assays also showed high PLR (24) at low NLR (0.15) and high

DOR (136).

Conclusions: Our results do not support the use of assays of anti-nGli to diagnose CD in

young children. IgA-anti-tTG, IgA-EmA and IgG-anti-dGli perform better than anti-nGli.




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Assays for IgA antibodies against tissue transglutaminase (anti-tTG), endomysium (EmA) as

well as for IgG antibodies against deamidated gliadin peptides (anti-dGli) in serum have a

high sensitivity and specificity for coeliac disease (CD) in children (1,2). However, in young

children (up to two years of age), antibodies against native gliadin (anti-nGli) are still often

assumed to have the best performance. At young age, IgA-EmA have been reported to have a

low sensitivity (3-8), with maximum values of 89 %. The sensitivity of IgA-anti-tTG ranged

between 83 % and 90 % (1,6,8,9). Specificity of IgG-anti-nGli was only 77 % at high sensi-

tivity (5). IgA-anti-nGli were claimed to be best in finding CD in young children (6) with sen-

sitivity between 82 and 97 % and specificity between 88 and 94 % (1,5-8). The diagnostic

performance of anti-dGli in very young children still needs to be established. Recently, it was

shown that in children younger than 2 years with signs of chronic enteropathy and high serum

levels of anti-nGli, but normal values of anti-tTG and of EmA, CD can be predicted by high

serum levels of anti-dGli (10).

The aim of our study was to compare the diagnostic performance of assays for IgA and IgG-

anti-dGli, for IgA and IgG-anti-nGli, for IgA and IgG-anti-tTG, and for IgA-EmA in the di-

agnosis of CD at young age.

MATERIALS AND METHODS

Patients

Sera of 184 children below two years of age with and without CD were retrospectively inves-

tigated. The patients were recruited from the Childrens Hospital of the Clinical Centre Sankt

Georg Leipzig, (Germany), University Hospital Leuven (Belgium), University Childrens

Hospitals of Leipzig, Tbingen, Mnchen and Gieen (Germany), Childrens University

Hospital Graz (Austria), and from the Department of Paediatrics of the University Medical

Centre Leiden (The Netherlands). The patients comprised 42 children with CD and 142 con-




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trols (100 females and 84 males, mean age 1.34 years, range 0.4 to 2.0 years). Sera were

withdrawn at the time of the diagnostic duodenal biopsies. All patients were biopsied under

normal diet due to suspicion of CD or other gastrointestinal disorders. Intestinal pathology of

all CD patients was in accordance with Marsh 2 or Marsh 3 criteria (11,12). All CD patients

improved under gluten-free diet (GFD). The study was approved by the Ethical committee of

the University of Leipzig as well as of the local Ethical committees of the participating cen-

tres.

Antibody assays

IgA and IgG antibodies against deamidated gliadin analogous fusion peptides (anti-dGli),

anti-nGli, anti-tTG, and IgA-EmA were measured (blinded to the histological diagnosis) with

test kits from EUROIMMUN Medizinische Labordiagnostika Lbeck, Germany. EmA were

estimated by indirect immunofluorescence analysis using a combination of primate oesopha-

gus, primate small intestine, and primate liver. The analyses were performed by

EUROIMMUN. Cut-offs were as suggested by the manufacturer (Table 1).

Statistics

The data were evaluated by receiver operating characteristic (ROC) analysis. The area under

the ROC-curves (AUC) was calculated. Differences between ROC-curves were evaluated by

pairwise comparison according to Chi-Square analysis. An error probability P of less than

0.05 was considered statistically significant. Non-inferiority testing was performed if there

was no statistically significant difference. For non-inferiority testing, the lower end of 90 per

cent confidence intervals of differences between AUCs was considered. Non-inferiority was

assumed if the lower end of this confidence interval was not below a zone of diagnostic indif-

ference of 0.01.

For the cut-offs suggested by the manufacturer, diagnostic accuracies, sensitivities, and speci-

ficities were calculated. Significance of differences (P < 0.05) was evaluated applying




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McNemars test. For non-inferiority testing (see above), the proportions of children being

false-negative or false-positive were compared between two tests. The z-test was applied for

calculation of the 90 per cent confidence intervals of differences in proportions and for com-

parison of sensitivities and specificities in the different age groups.

RESULTS

From all tests investigated, only assays for IgG anti-dGli, IgA anti-tTG and IgA EmA had

high specificity (0.96) connected with high sensitivity (0.86), with high positive predic-

tive values (0.52 and 0.69 at pre-test probabilities of 0.05 and 0.1, respectively), and with

high negative predictive values (0.99 and 0.98 at pre-test probabilities of 0.05 and 0.1,

respectively, Table 1).

The IgG-anti-tTG assay showed the highest specificity (0.993). The specificity of the two

anti-dGli tests (0.972 and 0.965) was comparable with that of the assays for IgA-anti-tTG

(0.972) and IgA-EmA (0.958). The specificity of the IgG-anti-nGli test was significantly

lower than that of all other tests. The IgG-anti-tTG-test had a significantly higher specificity

than the IgA-anti-nGli assay. The 2 anti-dGli assays were non-inferior to the IgG-anti-nGli

test.

The IgG-anti-nGli test exhibited the highest sensitivity (0.929). The sensitivity of the IgG-

anti-dGli test (0.857) was as high as that of the IgA-anti-tTG and the IgA-EmA assay, and

higher than that of IgA-anti-dGli (0.810), IgA-anti-nGli (0.785), and IgG-anti-tTG (0.476)

assays. The low sensitivity of the latter test differed significantly from that of all other tests.

Positive likelihood ratios (PLR) were lowest for IgG-anti-nGli. These antibodies, however,

showed the also lowest negative likelihood ratio (NLR).

The diagnostic odds ratios (DOR) as an integrated measure of sensitivity and specificity were

lowest for IgA and IgG-anti-nGli (48.4 and 26.3, respectively) and highest for IgA-anti-tTG




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and IgG-anti-dGli (207 and 164, respectively).Similarly, ROC analysis revealed that from all

antibody tests examined, the AUCs of the IgA-anti-tTG and of the IgG-anti-dGli assay were

highest (0.951 and 0.950, respectively). Statistical evaluation of the ROC analysis showed

that the IgG-anti-dGli assay was non-inferior to that of IgA-anti-nGli, IgG-anti-nGli, IgG-

anti-tTG and IgA-EmA. The IgA-tTG assay was noninferior to the IgA-anti-dGli, the IgA-

anti-nGli and the IgA-EmA assay.

Mean concentrations of IgA and IgG-anti-dGli, of IgA-anti-tTG and of IgA-EmA were 50

fold higher in CD patients than in controls. Median concentrations were even increased at

least 200 fold. For IgA and IgG-anti-nGli and for IgG-anti-tTG, the factor of increase was

lower (Table 1).

Positive and negative predictive values of the tests in the children up to two years of age are

shown in Figure 1 as a function of pre-test probability. Positive predictive values of the anti-

dGli, anti-tTG, and EmA tests were higher (0.52 at a pre-test-probability of 0.05 and 0.69

at a pre-test probability of 0.1) than that of anti-nGli assays (0.37 at a pre-test probability of

0.05 and

0.55 at a pre-test probability of 0.1). The negative predictive value of the IgG-anti-

tTG test was lowest (0.97 at a pre-test probability of 0.05 and 0.94 at a pre-test probability of

0.1). Only IgA and IgG-anti-dGli, IgA-anti-tTG and IgA-EmA showed a high positive predic-

tive value in combination with a high negative predictive value.

In 19 (45 %) of our CD patients up to two years of age, all 7 antibody tests were positive (Ta-

ble 2). In 12 further CD patients (29 %) all but the IgG-anti-tTG antibodies were above the

cut-offs. In 3 of the CD patients antibody positivity was restricted only to IgG-anti-nGli anti-

bodies. In further 2 patients none of the tests was positive. There were 6 children (14 %) with

negative IgA-anti-tTG among the CD patients. Details of these patients are shown in Table 3.

In 89 (63 %) of the controls up to 2 years of age, none of the tests was positive (Table 2). In

further 35 controls (25 %) only the concentration of IgG-anti-nGli was elevated. In 3 controls




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(2 %), only IgA- and IgG-anti-nGli were positive. In 2 controls (1 %) IgA-anti-tTG together

with IgG-anti-nGli were elevated. In 2 further controls (1 %), all but IgA-anti-tTG and IgG-

anti-tTG were positive. In the remaining 11 controls, different combinations of antibody posi-

tivity were observed, which occurred only once. Altogether, there were 4 controls with in-

creased IgA-anti-tTG. In all of them, concentration of IgA-anti-tTG was higher than twofold

the cut-off. In 2 of them, also an elevated concentration of IgG-anti-dGli was found. In one of

these 2 controls, the antibody concentration could be controlled after 19 months (both anti-

bodies positive) and after 23 months (both antibodies negative).

DISCUSSION

Except for IgG-anti-nGli, the specificity of all antibody tests was high for CD for patients up

to 2 years. However, the assays with high specificity (IgA- and IgG-anti-dGli and anti-tTG,

IgA-anti-nGli and EmA) exhibited a low sensitivity, whereas the low specificity of the IgG-

anti-nGli test was associated with a high sensitivity. Using paired indicators of diagnostic

performance such as sensitivity and specificity or PLR and NLR can be a disadvantage in

comparing the performance of competing tests, especially if one test does not outperform the

other on both indicators. The DOR can be used as a single indicator of diagnostic

performance and combines the strengths of sensitivity and specificity as well as of PLR and

NLR and is independent on prevalence (13). Regarding the DOR, the performance of the IgA

and IgG anti-nGli was worst.

For further evaluation of the tests, calculation of predictive values at defined pre-test probabil-

ity is useful. In contrast to screening conditions with a prevalence of about 1 %, in paediatric

gastroenterology centres a higher pre-test probability has to be assumed. At a pre-test prob-

ability of CD of about 0.05, all tests except the assays for IgA- and IgG-anti-nGli result in




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post-test probabilities of at least 0.52 (Figure 1). The post-test probability of IgA- and IgG-

anti-nGli tests was below 0.4. A high positive predictive value reduces the number of patients

who need to undergo endoscopy. The negative predictive value is very high for all tests (

0.99) except for IgG-anti-tTG (0.97). A high negative predictive value assures that the num-

ber of CD patients among the children with negative test results is minimum (at most 1 %).

Thus, from all tests investigated, only assays for IgG anti-dGli, IgA anti-tTG and IgA EmA

had high specificity (0.96) connected with high sensitivity (0.86), with high positive pre-

dictive values (0.52 and 0.69 at pre-test probabilities of 0.05 and 0.1, respectively), and

with high negative predictive values (0.99 and 0.98 at pre-test probabilities of 0.05 and

0.1, respectively). Therefore, these assays should be preferred over tests measuring anti-nGli.

A selection bias in our study cannot be excluded. First of all, biopsies are more likely to be

performed in symptomatic patients with positive results for CD specific antibodies such as

IgA-EmA and IgA-anti-tTG, which favours these tests. Furthermore, most children in this

study had not been re-challenged with gluten in order to confirm the diagnosis of CD, which

wasrequired according to thepreviousESPGHAN criteria on the diagnosis of CD (14). Re-

cently, it was suggested that routine gluten challenge in patients below two years of age is not

necessary when patients have villous atrophy in combination with positive EmA (7). Accord-

ing to forthcoming ESPGHAN guidelines, a later gluten challenge should only be performed

if villous atrophy was found in children below to years of age, who are negative for CD spe-

cific antibodies in order to confirm CD as a cause of the enteropathy (15). Six of our patients

were classified as coeliacs in the absence of increased IgA-anti-tTG (however, one of them

positive for IgA-EmA and this patient and another positive for routine assay of IgA-anti-tTG

outside of this study). The latter finding shows that occasionally diverse results may be ob-

tained when tests of different suppliers are compared (16). In fact, none of the remaining four

patients was re-challenged with gluten until now, mainly due to their youngage. According to


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Conflicts of interest: T. Mothes, H. H. Uhlig, and C. Dhnrich submitted patents on methods

for CD diagnosis (T. Mothes, A. Osman, H. H. Uhlig, T. Gnnel, A. Dietl: Peptide und

Verfahren zur Diagnostik von Zliakie und Dermatitis herpetiformis and C. Probst, C.

Dhnrich, W. Schlumberger, W. Stcker, L. Komorowski, T. Mothes: Verfahren und

Immunabsorbentien zur spezifischen Detektion und Absorption Zliakie- und Dermatitis

herpetiformis assoziierter Antikrper).

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2. Prause C, Ritter M, Probst C, et al. Antibodies against deamidated gliadin as new and accu-

rate biomarkers of childhood coeliac disease.J Pediatr GastroenterolNutr2009b;49:52-8.

3. Brgin-Wolff A, Gaze H, Hadziselimovic F, et al. Antigliadin and antiendomysium anti-

body determination for coeliac disease.Arch Dis Child1991;66:941-7.

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5. Tonutti E, Visentini D, Bizzaro N, et al. The role of antitissue transglutaminase assay for

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Pathol 2003;56;389-93.




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6. Lagerqvist C, Dahlbom I, Hansson T, et al. Antigliadin immunoglobulin A best in finding

celiac disease in children younger than 18 months of age. J Pediatr Gastroenterol Nutr

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7. Wolters VM, van de Nadort C, Gerritsen SAM, et al. Is gluten challenge really necessary

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Gastroenterol Nutr2009;48:56670.

8. Maglio M, Tosco A, Paparo F, et al. Serum and intestinal celiac disease-associated antibod-

ies in children with celiac disease younger than 2 years of age. J Pediatr Gastroenterol Nutr

2010;50:43-8.

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tides for celiac disease diagnosis and follow-up in children. Clin Chem2009;55:150-7.

10. Barbato M, Maiella, G, Di Cam illo C, Guida S, Valitutti F, Lastrucci G, Mainiero F,

Cucchiara S. The anti-deamidated gliadin peptide antibodies unmask celiac disease in small

children with chronic diarrhoea.Dig Liver Dis 2011;43:465-469

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troenterology 1992;102:33054.

12. Oberhuber G, Granditsch G, Vogelsang H. The histopathology of coeliac disease: time for

a standardized report scheme for pathologists. Eur J Gastroenterol Hepatol 1999;11:1185

94.

13. Glas AS, Lijmer JG, Prins MH, Bonsel GJ, Bossuyt PMM. The diagnostic odds ratio: a

single indicator of test performance.J Clin Epidemiol2003;56:11291135.




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14. Walker-Smith JA, Guandalini S, Schmitz J, et al. Revised criteria for diagnosis of coeliac

disease. Report of Working Group of European Society of Paediatric Gastroenterology and

Nutrition.Arch Dis Child 1990;65:909-11.

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FIGURE LEGENDS

FIGURE 1. Predictive values of the different antibody assays in the diagnosis of CD in children up to

two years of age in dependence on pre-test probability.The thick grey line in the lower diagram collec-

tively indicates negative predictive values of IgG-anti-dGli, IgA-anti-tTG as well as IgA-EmA, which

are very similar. The vertical dotted line indicates a pre-test probability of 0.05.




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Table 1

TABLE 1. Performance of different antibody tests in children aged up to 2 years

IgA-anti-dGli

IgG-anti-dGli

IgA-anti-nGli

IgG-anti-nGli

IgA-anti-tTG

IgG-anti-tTG

IgA-EmA*

Test-No. 1 2 3 4 5 6 7

Cut-off (U/L) 25.0 25.0 25.0 25.0 20.0 1.0 10

AUC 0.936 0.9503,4,6,7 0.920 0.931 0.9511,3,7 0.912 0.925

Specificity 0.97234 0.9653

4 0.930

4,6 0.669

1,2,3,5,6,70.972

4 0.993

3,45 0.958

4

Sensitivity 0.8106 0.857

6 0.786

4,6 0.9291

3,6 0.857

6 0.476

1,2,3,4,5,7 0.857

3,6

PLR 28.7 24.3 11.2 2.81 30.4 67.6 20.3

NLR 0.196 0.148 0.231 0.107 0.147 0.528 0.149

DOR 147 164 48.4 26.3 207 128 136

95 % CI 41.7 to 515 47.5 to 569 18.2 to 129 7.72 to 89.5 55.5 to 773 16.4 to 1004 41.4 to 447

AbC CD (U/L)

Mean 437 296 252 241 427 1.13 773

Median; range 580; 0.4-711 286; 0.8-700 200; 1.0-551 200; 3.3-520 457; 0.5-700 0.0-5.5 320; 0-10000

AbC Co (U/L)

Mean 3.46 5.62 9.01 35.73 3.40 0.09 1.06

Median; range 1.0; 0.0-94.3 1.4; 0.2-173 1.5; 0.3-191 10.0; 0.6-220 0.74; 0.0-98.5 0.04; 0.00-1.37 0; 0-100

* For EmA the reciprocal values of the titres are presented. CI: Confidence interval; AbC CD: Antibody concentration in CD

patients; AbC Co: Antibody concentration in control patients. Superscripts denote significant differences to tests with therespective numbers. Subscripts denote non-inferiority to the tests with the respective numbers. Results of non-inferiority tests

only shown if there was no statistically significant difference.




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Table 2

TABLE 2.Antibody profiles in CD patients and controls aged up to 2 years

IgA-anti-dGli

IgG-anti-

dGli

IgA-anti-

nGli

IgG-anti-

nGli

IgA-anti-tTG

IgG-anti-tTG

IgA-EmA

Number ofpatients

CD + + + + + + + 19

+ + + + + - + 12

- - - + - - - 3

- - - - - - - 2

Con-

trols- - - - - - - 89

- - - + - - - 35

- - + + - - - 3

- - - + + - - 2

+ + + + - - + 2

For sake of clarity, from the total of 184 patients, profiles are only shown if occurring more than in one patient. Six CD pa-

tients and 11 control patients had unique antibody profiles.


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Table 3

TABLE 3. Patients with negative IgA-anti-tTG classified as CD

No Total IgA HLA Histology IEL Other positive

antibodies

Further findings

1 sIgAD DQ8 Marsh 3A >40 IgG-anti-nGli Routine test of IgAanti-tTG negative

2 Normal n.d. Marsh 2 >40 None IgA-anti-tTG, IgA-EmA, IgA- and IgG-anti-dGli

positive 64 days before endoscopy. Preterm GFD?

3 Normal n.d. Marsh 3C >40 None Routine test of IgA-anti-tTG negative

4 Normal n.d. Marsh 3C >40 IgG-anti-nGli Routine tests of IgA-anti-tTG and IgA-EmA

positive. Progressive decrease in total IgA during

follow up

5 Normal DQ2 Marsh 3B >40 IgG-anti-nGli Routine test of IgA-anti-tTG negative

6 Unknown n.d. Marsh 3A >40 IgA-anti-nGli

IgG-anti-nGli

IgG-anti-dGli

IgA-EmA

Routine tests of IgA-anti-tTGand IgA-EmA positive

None of the patients was re-challenged with gluten. sIgAD: secretory IgA-deficiency. n.d.: not determined.Routine tests: performed in clinical routine at the time of endoscopy outside of this study.Lack of IgA improbable since IgA-anti-nGli and IgA-EmA increased.

determination of igg and iga antibodies against native gliadin

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