In Vitro Gliadin Challenge:
Diagnostic Accuracy and Utility for
the Difficult Diagnosis of Celiac
Disease
Am J Gastroenterol 2012; 107:111–117;
Raffaella Tortora MD et al.
Dr Mohammad Sadrkabir
INTRODUCTION
• Celiac disease is a digestive disorder that damages the
small intestine. Patients with celiac disease cannot
tolerate gluten-rich cereals and, in particular gliadin,
which is the toxic fraction of the gluten of wheat.
• Diagnosis of celiac disease is based on the evidence of
high levels of serum auto-antibodies, including antitissue transglutaminase antibodies (a-tTG) or antiendomysium antibodies (EMA) and on the evidence of
mucosal damage in the duodenal biopsy .
• Diagnosis of celiac disease can be difficult in patients
who begin treatment with a gluten-free diet (GFD) after
non-standardized food intolerance tests and in the
absence of adequate diagnostic work-up.
• Once this treatment is initiated, diagnosis of celiac
disease requires the reintroduction of gluten into the
patient's diet for an appropriate period.
• Diagnosis of celiac disease can be difficult when the
search of serum antibodies and the duodenal biopsy
results are not concordant.
• Genetic markers as the HLA DQ2–DQ8 genes are of
help only in excluding celiac disease as their
presence is common also in non-celiac subjects .
• In the presence of gliadin, the duodenal mucosa of
celiac patients modifications that, in part, can be
reproduced in vitro.
• The short-term in vitro exposure of the duodenal
mucosa of celiac patients to gliadin induces crypt
hyperplasia, villus atrophy, and T lymphocyte
recruitment in the lamina propria, increasing the
number of intraepithelial T lymphocytes.
• The in vitro response of the duodenal mucosa to gliadin
is generally defined as the gliadin challenge, a test which
could represent a tool for objective assessment of celiac
disease when diagnosis is difficult.
• The present study aimed to evaluate the diagnostic
accuracy of the in vitro gliadin challenge in patients
without celiac disease, with celiac disease, and in
patients with difficult diagnosis.
METHODS
The study
included three
groups of
patients: negative
controls, positive
controls, and
difficult
diagnoses.
• The study enrolled patients undergoing upper endoscopy
at a tertiary center for food intolerance and celiac
disease.
• The inclusion criteria were adult age (≥18 years) and
informed written consent.
• Participants underwent, a dietary interview for
evaluation of treatment with GFD, upper endoscopy for
duodenal biopsies, and assessment of HLA status, serum
a-tTG levels , and EMA .
• Negative controls were patients without celiac disease as
assessed by negative serology and histology selected to
match sex and age of celiac patients of the positive
control group. The negative control group included
patients with peptic disease (n=21), gastroesophageal
reflux disease (n=22), irritable bowel syndrome (n=7),
and inflammatory enteropathies (n=2 with autoimmune
enteropathy, three with Crohn's disease, one with acute
gastroenteritis, one with Giardia L. infestation).
• Positive controls included celiac disease patients under
treated and untreated conditions. Treated celiac patients
were on GFD since at least 1 year after complete
diagnostic work-up including serology and histology.
Untreated celiac patients were cases with positive
serology who underwent upper endoscopy for
completion of diagnostic work-up.
• Difficult diagnosis group was made of patients with
suspected celiac disease in whom diagnosis could not be
made because the diagnostic work-up was initiated while
on treatment with GFD and/or of non-concordant
diagnostic tests. The list of combination of nonconcordant tests included the presence of EMA
antibodies with the absence of a-tTG or vice versa and
the presence of antibodies with the absence of
histological abnormalities in the duodenal mucosa.
Follow-up studies
• After completion of the gliadin challenge,
participants of the difficult diagnosis group were
prescribed a gluten-containing diet and were
followed up for up 1 year with assessments of serum
a-tTG and EMA every 3 months. Celiac disease
diagnosis was made during follow-up if celiac
disease-specific serology was consistently positive
for both a-tTG and EMA. If the serology was
constantly negative during the follow-up, celiac
disease was excluded.
Gliadin challenge
• The in vitro gliadin challenge was performed by
expert biologists who were blind to the
characteristics of the patients.
• Eight duodenal fragments were collected from each
patient.
• Two fragments were obtained for routine histology.
• The remaining six fragments were used for gliadin
challenge and placed in ice-cold tissue-culture
medium within 20 min and cultured as follows.
• The gliadin challenge was performed adding a
gliadin digest (1 mg/ml) to four samples.
• Two fragments were cultured for 3 h for evaluation of
early markers of inflammation namely: PY99 (antiphospho-tyrosine-mAb), HLA-DR , and ICAM-1
(intercellular cell adhesion molecule).
• Two fragments were cultured for up to 24 h for
evaluation of the delayed markers of inflammation,
namely CD3 (a marker of mature T lymphocyte), CD25
(interleukin-2 receptor in both lymphoid and myeloid
cells), and CD69 (a marker of T-cell activation).
• The remaining two fragments served as controls and
were cultured for 3 or 24 h in the same way without the
addition of gliadin (blank samples).
• All samples underwent also the search for the
transglutaminase 2 IgA (TG2–IgA) deposits before and
after 3 and 24 h.
Assessment of immunofluorescence intensity
• Intensity of immunofluorescence was scored from 0 to 3
for each one of the seven markers .
• Intensity score of PY99 was based on the percent of
stained cells per 100 epithelial cells for (0: <25%; 1: 25–
49%; 2: 50–74%; 3: ≥75%).
• Intensity score of HLA-DR was based on the cellular
localization of expression (0: no expression; 1:
expression in basolateral membrane; 2: expression in
basolateral membrane and cytoplasmic compartment; 3:
expression in basolateral membrane, cytoplasmic
compartment, and apical membrane).
• The evaluation of HLA-DR expression focused on villous
enterocytes at time point 3 h and on crypt enterocytes at
time point 24 h, respectively.
• Intensity score was based on the absolute count of
positive cells per mm2 of mucosa for ICAM-1, CD69,
CD3, and CD25 (0: <5 cells, 1: 5–29 cells, 2: 30–59 cells,
3: ≥60 cells).
• Intensity score of TG2 and IgA was based on the coexpression of both markers below the basement
membrane .
• For each one of the markers, the challenge was
considered positive when the intensity score in gliadinexposed samples was at least one unit higher than the
intensity score in blank samples.
Statistical analysis
• Analysis of variance was used for comparisons of
continuous variables among the groups.
• For each marker, the rate of positive cases in
negative controls and of negative cases in positive
controls was used to calculate the area under the
receptor-operated curve (ROC).
RESULTS
• Of the 347 participants enrolled in the study, 10 were
excluded (n=4 with superficial biopsy samples, n=3 with
necrotic samples, and n=3 with contaminated cultures).
Thus, the study cohort consisted of 337 patients.
Challenge in negative controls and
positive controls
• Table 1 reports for each marker the prevalence of
positive cases (i.e., false-positive cases) in negative
controls and of negative cases positive controls (i.e.,
false-negative cases).
• PY99 had the second highest rate of false-positive cases.
• HLA-DR had no false-positive case at both times of
incubation and only one false-negative case.
• ICAM-1 and CD69 had the highest rate of false-positive
cases and no false-negative case.
• CD3 and CD25 had high rates of false-positive cases and
of false-negative cases.
• TG2–IgA deposits had the highest rate of false-negative
cases.
The highest value
of the area under
the ROC was found
for HLA-DR at
both incubation
times. The area
under the ROC of
HLA-DR did not
significantly
increase when data
of this marker were
combined with
data of anyone of
the other markers
(not shown).
Examples of
immunostaining in samples
of negative and positive
controls after incubation
with gliadin-free medium
(blank) and with gliadinrich medium (PT-gliadin).
ICAM, intercellular cell
adhesion molecule;
TG2/IgA, transglutaminase
2/IgA.
• Analyses were repeated after exclusion of treated celiac
patients to investigate the possible confounding of
treatment with GFD. Area under the ROC for HLA-DR
was the highest one also in this set of analyses (area
under ROC=0.99).
• Findings for other markers were similar to data of table
2 with exception of TG2–IgA. The area under the ROC of
TG2–IgA increased up to 0.90 after exclusion of treated
celiac patients.
• Analyses were done also focusing on blank samples to
investigate the accuracy of the expression of these
markers without the incubation with gliadin. For all
markers, the area under the ROC was much lower than
values found with the use of the gliadin challenge. The
highest value was found for the area under the ROC of
HLA-DR 24 h (0.71), the lowest value for the area under
the ROC of TG2–IgA (0.64).
• The inaccuracy of immunofluorescence assessment in
blank samples mainly reflected a high number of falsenegative cases.
Gliadin challenge results in difficult diagnosis
group
• Thirty-nine patients of the difficult diagnosis group
accepted to stop GFD after the gliadin challenge to
undergo a re-assessment of the celiac disease-specific
serology.
• Table 3 reports data about accuracy of markers of the
gliadin challenge in these 39 patients divided by
presence/absence of celiac disease-specific antibodies in
the analyses repeated under untreated conditions.
• The comparison between these two subgroups tended to
give results, which were similar to the results of the
comparison between negative controls and positive
controls. HLA-DR had the highest area under the ROC
also in this set of analyses.
DISCUSSION
• The present study shows that in the gliadin challenge
test, HLA-DR has high accuracy for celiac disease
diagnosis because of high specificity and high sensitivity.
• Findings for this marker were consistent at both
incubation times, in the absence and in the presence of
treatment with GFD, and in patients with difficult
diagnosis because of non-concordant tests and/or the
confounding of GFD initiated before an appropriate
diagnosis of celiac disease.
• Findings for the other markers of in vitro mucosal
response to gliadin were less accurate and did not
increase the accuracy of information derived by the
evaluation of HLA-DR alone.
• The data from this study indicate the diagnostic
accuracy of the in vitro gliadin challenge in the
identification of celiac disease in the event of a
difficult diagnosis.
• In the present study, we observed a clear
modification in the expression of markers of the Tcell immune response following in vitro gliadin
challenge almost exclusively in celiac disease.
• We experienced a 3% failure rate in the ability to
cultivate biopsies for the in vitro challenge with
gliadin; however, standardized procedures may
lower the percentage of these technical problems
(such as obtaining superficial tissue samples).
• Several patients in the difficult diagnosis group were on
GFD without any evidence of celiac disease ,
demonstrating that a number of patients who receive a
diagnosis of celiac disease on the basis of negative
specific serum markers and minimal alteration of
mucosa at histology are unlikely to have celiac disease.
• In our hands, TG2–IgA deposits had high accuracy only
when analyses were limited to patients who were not on
GFD
• According to previous studies, full appraisal of the
immunological response to gliadin should be conducted
and analyzed for a number of markers for up to 24 h . On
the basis of our findings, we suggest a gliadin challenge
with testing of HLA-DR alone and only for 3 h of culture
with gliadin.
• This simplified in vitro gliadin challenge might be less an
expensive procedure than repeated endoscopies and
biopsies. Although in this simplified form the gliadin
challenge should be performed strictly limited to cases in
which a GFD or non-concordant tests interfere with a
correct diagnosis of celiac disease.
• Further investigation is needed on the in vitro gliadin
challenge in non-celiac gluten-sensitive individuals.
Conclusion
• The in vitro gliadin challenge is not only a model for
studying the mechanisms of the mucosal immune
response in celiac disease but also a useful tool for
reaching a final diagnosis in “difficult” cases of celiac
disease, in which sensitivity to gluten is suspected
but undemonstrated by standard diagnostic tests.
Thank you for
your attention