Improved laboratory diagnostics for celiac disease.

Gluten intake is associated with irritable bowel disease and a gluten free diet
(GFD) is increasingly used without prior diagnosis as it is thought to reduce
abdominal complaints. While a GFD is essential for celiac disease (CD)
treatment, it may be detrimental in the general population as it changes intake
of fiber and vitamins. CD patients need additional diagnostics for other
auto-immune diseases and specific follow-up. Identification of CD patients and
exclude CD diagnosis in individuals on a self-initiated GFD is therefore
important. Standard diagnostic tests fail as they become negative in
individuals on a GFD. Gluten challenge (GC) may induce reappearance of
(auto)antibodies and duodenal abnormalities but in a substantial proportion of
CD patients a GC is insufficiënt. Furthermore, in patients with atypical
presentation of CD and/or patients with antibody deficiencies (Common Variable
Immune Deficiency) standard diagnostic tests may be inconclusive. For this
group of patients there is a medical need for additional tests that will help
the clinician to either confirm or exclude the diagnosis CD.
Gliadin specific T-cells are detectable in blood of CD patients, even after
prolonged GFD, but undetectable in gluten consuming controls. Furthermore, the
γδ T-cell proportion among duodenal intraepithelial lymphocytes (IEL) is high
in CD and remains high after recovery on a GFD.

Develop cellular blood tests applicable in routine specialized diagnostic
settings for CD in patients on a GFD and patients with idiopathic villous
atrophy and atypical presentation of CD. **

IEL and LPL will be isolated from duodenal biopsies. Dextramer/Tetramer
technology will be used to identify gliadin specific T-cells in peripheral
blood, using the additional differentiation, activation and homing markers
(CD45RA, CD62L, CD38, integrin β7). We will use the methodology used to detect
rare malignant cells in minimal residual disease in whole peripheral blood of
patients in remission of hematological malignancies. This will be applicable in
a diagnostic routine setting as it does not require enrichment of cells prior
to analysis but involves analysis of small populations in large numbers of
cells after bulk-lysis of erythrocytes.

Extensive phenotyping of γδT-cells in IEL, LPL and peripheral blood will be
done by multi (up to 24) parameter analysis using AURORA technology
The AURORA advanced flow-cytometry technology (3-laser system) enables a
sensitive 24 color assay due to its special technology analyzing the full
spectra of the fluorochromes. This approach overcomes limitations of spectral
overlap in standard flow cytometry.

The methodology to detect gliadin specific T-cells and γδT-cell phenotyping
panels using the markers listed above, will first be established. For this we
will use both peripheral blood and duodenal biopsies from active CD patients,
CD patients on a gluten free diet and non-celiac disease controls.

The phenotype of the cells of active CD patients and patients responding to a
GFD will be compared to γδT-cells in IEL and LPL of patients with abdominal
complaints without evidence of CD. Subsequently we will investigate whether
these cells are detectable in peripheral blood and in potential CD patients on
a self-initiated GFD. We will initially focus on cell surface markers. The
multidimensional data will be analyzed with specialized software such as FCS
express and tSNE analysis to establish the most discriminating clinical
relevant phenotype in order to limit the marker panel to a routinely applicable
flow-cytometry based diagnostic test. If this panel does not lead to a *CD
related phenotype*, we will explore additional markers which will also include
intracellular functional markers such as IL-21, IL-17A, IFNγ, Granzyme B and
CTLA4.

We will subsequently use the established optimized panels to determine
presence of gliadin specific T-cells and γδT-cells with specific phenotypes in
IEL (γδT-cells only), LPL and peripheral blood of active CD patients, patients
on a GFD and controls to gather statistical support for the detection of
gliadin specific T-cells and/or γδT-cells with a *CD-related phenotype* in
peripheral blood of CD patients and CD patients on a gluten free diet.

There are no direct benefits for the patients in this study. The risk of
participation is considered to be very low. In adults, there is a limited risk
to taking extra biopsies during a planned endoscopy. Taking biopsies during
endoscopy can cause intra-intestinal or intramural haemorrhage, or even
perforation. The risk is estimated to be < 1 : 10000. A maximum total of 6
extra biopsies will be taken during endoscopy.

Celiac disease patients taking a gluten challenge can experience celiac
diesease related complaints. There are no risks related to a short term (max 4
weeks) gluten use in CD patients. Blood will be drawn solely for the purpose of
this study in the group of healthy volunteers and the group of celiac disease
patients that take a glutenchallenge, risks of venapuncture are negligable.
Healthy controls need to vistit the hospitla once, the CD patients will need to
visist the hospital at least two times and maximal 5 times.