Hi, I’m Matt Binnicker, the Director of Clinical Virology and Vice Chair of Practice
in the Department of Laboratory Medicine and Pathology at Mayo Clinic.
Celiac disease is an inherited, autoimmune disorder that affects the digestive process
of the small intestine.
And did you know that it’s estimated that 1% of the U.S. population, or about 3 million
people, are living with this disease?
Well, because of its significance, it’s important that we understand the clinical
manifestations of Celiac disease, as well as how to test for it.
In this month’s Hot Topic, my colleague, Dr. Melissa Snyder, will review the diagnostic
testing algorithms for celiac disease, and highlight the advantages and limitations of
certain tests, including serology and genetic assays.
I hope you enjoy this month’s Hot Topic, and I want to personally thank you for allowing
Mayo Clinic the opportunity to be a partner in your patients’ healthcare.
Thank you, Dr. Binnicker.
Hello, Everyone.
My name is Melissa Snyder, and I am the Co-Director of the Antibody Immunology Laboratory at Mayo
Clinic in Rochester, Minnesota.
I’m so glad you are able to join me for a brief discussion about celiac disease and
the role of diagnostic testing algorithms.
Before beginning the presentation, I have one disclosure to make.
I have served as a member of the Strategic Advisory Committee for Inova Diagnostics,
which is an in vitro diagnostic company that manufactures and sells kits for celiac serology
testing.
For many of us in the health care field, utilization management, and how most efficiently to use
laboratory testing in patient care is a critical issue.
While viewing this presentation, take some time to consider a few points, including how
this testing might be used in your practice, when the tests should be ordered, and how
the results from this testing might impact patient management.
Celiac disease is a chronic inflammatory condition that primarily affects the small intestine.
It is caused by an inflammatory response mounted by the patient’s own immune system, which
ultimately results in damage and atrophy of the villae within the small intestine.
In the figure to the left on this slide, you see a biopsy of a normal small intestine,
with intact villae.
In the middle and right-most figures, you see the partial and total villous atrophy
that can occur in celiac disease as a result of the inflammatory response.
The clinical manifestations associated with celiac disease can be classified as gastrointestinal,
malabsorptive, or extra-gastrointestinal.
In the context of the gastrointestinal symptoms, patients may present with diarrhea, weight
loss, steatorrhea (fatty stool that can cause fecal incontinence), or abdominal pain, just
to name a few.
Because of the villous atrophy, patients with celiac disease may not be able to absorb nutrients
from their food.
As a result, patients may show symptoms of malabsorption, including iron-deficient anemia,
various vitamin deficiencies, hypoproteinemia (low protein levels in blood), or hypocalcemia
(low calcium levels in blood).
Young children may even present with a failure to thrive.
Lastly, patients with celiac disease may show manifestations that appear to have little
to do with the gastrointestinal system.
Reports in the literature have demonstrated associations between celiac disease and ataxia
(mobility issues), infertility, arthralgia (joint pain), dermatitis herpetiformis (chronic
blistering), hyposplenism (abnormal spleen function), and a variety of autoimmune conditions.
The point to stress here is that the symptoms of celiac disease may be nonspecific, sometimes
making for a challenging diagnosis.
For celiac disease to develop, an individual must have the both the genetic susceptibility
and the proper environmental exposure.
The genetic component of celiac disease had been inferred from observations that the disease
occurred in families.
Overall, in the United States, celiac disease occurs in approximately 1% of the total population.
In first-degree relatives of individuals with celiac disease, the prevalence increases to
10%.
Ultimately, specific alleles of the human leukocyte antigen complex, namely HLA-DQ2
and HLA-DQ8, were demonstrated to be responsible for much of the genetic susceptibility for
celiac disease.
The environmental component that causes celiac disease is dietary exposure to protein from
the cereal grains, namely wheat, barley, and rye.
Collectively, the protein agent from these cereal grains known to be associated with
celiac disease is gluten.
An initial diagnosis of celiac disease can be established if a patient has positive serology,
which I will expand on in a moment, and an intestinal biopsy that demonstrates villous
atrophy.
Once this initial diagnosis has been established, the patient will be started on a gluten-free
diet.
The goal of this treatment is to remove the environmental exposure of dietary gluten,
which is the trigger for the inflammatory response in the small intestine.
A definitive diagnosis of celiac disease can be established after gluten has been successfully
abolished from the diet.
The patient should begin to see resolution of their clinical symptoms, which is often
accompanied by conversion to a negative serology and reconstitution of the intestinal villae.
As I stated in the last slide, laboratory serology plays a key role in establishing
a presumptive diagnosis of celiac disease.
The primary antibodies associated with celiac disease are endomysial antibodies (or EMA);
tissue transglutaminase (or TTG) antibodies; and gliadin antibodies.
The antibodies against gliadin are related to the dietary gluten that initiates the inflammation
in celiac disease.
When the protein gluten is ingested, it is digested into smaller peptides.
The ethanol-soluble fraction of gluten is referred to as gliadin.
The first immunoassays developed tested for antibodies against unmodified gliadin.
However, these assays were inferior to the TTG antibody and EMA assays, and generally,
they were not recommended.
The newest generation of gliadin antibody assays uses a novel form of this antigen,
specifically deamidated gliadin.
These newer assays specific for deamidated gliadin offer improved diagnostic utility
and are preferred over assays using unmodified gliadin.
Testing for each of these antibodies can involve either assessing for IgA and IgG isotypes.
A variety of methodologies are available for these serology tests.
TTG and deamidated gliadin antibodies are generally detected using plate-based enzyme
immunoassays, although bead-based multiplex assays are becoming more common.
EMAs, on the other hand, are detected by immunofluorescent assays using some source of smooth muscle
tissue, such as a monkey esophagus substrate.
In addition to serology tests, genetic testing can be useful in the evaluation of a patient
with suspected celiac disease.
In this context, testing will focus on assessing for the presence of the HLA-DQ2 and HLA-DQ8
alleles.
Both the serologic and genetic testing can be important in establishing a diagnosis of
celiac disease.
However, it is important to appreciate the limitations of each type of testing.
For the serologic testing, we must deal with the issue of selective IgA deficiency.
Selective IgA deficiency is generally defined as the absence of detectable IgA immunoglobulin
in the presence of normal IgG and IgM production.
Although relatively rare, it is clearly more common in patients with celiac disease compared
with the general population.
In celiac diagnostic testing, the IgA isotype for the celiac-specific serologies is more
sensitive and specific compared to the IgG isotypes.
It is for this reason that the IgA isotype antibodies are preferred as a diagnostic test.
However, for patients with selective IgA deficiency, testing for the IgA isotype antibodies is
not useful, and testing for the IgG isotype antibodies is necessary.
The other issue that can impact the utility of the serology testing is the effect of a
gluten-free diet.
In a patient with celiac disease, removal of gluten from the diet leads to “down regulation”
of the inflammatory immune response, ultimately leading to reduced autoantibody production.
This is useful when monitoring patients with celiac disease, as decreasing concentrations
of the celiac-specific autoantibodies is interpreted as having a favorable response to the gluten-free
diet.
However, if a patient is already following a gluten-free diet before the diagnosis of
celiac disease has been established, there is a risk of a false-negative diagnostic serology
test.
Genetic testing for the celiac-associated HLA alleles also has some important caveats.
HLA-DQ2 is present in 90% to 95% of patients with celiac disease, while HLA-DQ8 is found
in the remaining 5% to 10%.
Because HLA-DQ2 and HLA-DQ8 are detected in virtually all patients with celiac disease,
it might appear that genetic testing would be the preferred diagnostic test for this
disorder.
Unfortunately, this is not the case because 30% to 40% of the general population of the
United States is positive for HLA-DQ2 and/or HLA-DQ8, yet, we must remember that only 1%
of the population has celiac disease.
So, what does this mean for the utility of HLA typing for celiac disease?
The power of the HLA testing lies in a negative result.
If a patient is negative for both HLA-DQ2 and HLA-DQ8, we can exclude celiac disease
as a diagnosis, since the patient does not have the genetic component, which is required
to develop the disease.
In contrast, if the patient is positive for either HLA-DQ2 or HLA-DQ8, we can only say
that the patient has the genetic susceptibility for celiac disease, although he or she may
never develop the disease in his/her lifetime.
At this point, I think it is important to summarize the test performance and clinical
utility of the various serologic and genetic tests used for the diagnosis of celiac disease.
The IgA isotypes for TTG and deamidated gliadin consistently have shown the best combination
of sensitivity and specificity.
EMA IgA generally demonstrates excellent specificity.
However, because EMA is performed by indirect immunofluorescence, this testing can have
some analytical challenges for the laboratory.
If we consider the IgG isotypes for TTG and deamidated gliadin, we find that they are
probably most appropriate for patients with a selective IgA deficiency.
And lastly, for HLA-DQ2 and HLA-DQ8, we find these to be most useful as a “rule-out test”
to exclude celiac disease as a diagnosis.
Given the variety of tests that are available for the diagnosis of celiac disease, choosing
the tests that are most appropriate for a given patient—not to mention interpreting
the results—can be a challenge.
The clinical labs at Mayo, working closely with our GI (gastrointestinal) colleagues,
have established several algorithms to aid in the diagnosis of celiac disease.
These algorithms involve reflexing of tests within the laboratory and are available as
orderable clinical tests.
No one algorithm is applicable to all patients being evaluated for celiac disease.
However, we believe that these various testing strategies will be useful for many patients
in a variety of situations.
The first cascade is the Celiac Disease Serology Cascade, or CDSP.
This algorithm is applicable to most patients and incorporates a reflex approach to serologic
testing.
The second cascade is the Celiac Disease Comprehensive Cascade, and has the test ID “CDCOM.”
This algorithm includes both serologic and genetic testing.
Lastly, we have the Celiac Disease Comprehensive Cascade for Patients on a gluten-free diet,
or CDGF.
This cascade only performs serology in the context of a positive genetic test.
Now, I will go through each algorithm, beginning with the Serologic Cascade.
The Serologic Cascade begins with total IgA quantitation.
All further testing reflexes automatically within the lab, based on the IgA result.
The IgA result is classified as normal, or within the age-adjusted reference rage, as
low, being still detectable but below the reference range, or as deficient, or undetectable
by our nephelometric assay.
All samples with a normal IgA result would automatically reflex to a TTG-IgA antibody.
For all samples testing positive or negative, no further testing would be required.
The final report would include the total IgA and TTG-IgA results, along with an interpretive
comment.
However, if the TTG-IgA result falls into the equivocal range, then EMA and deamidated
gliadin-IgA testing is performed.
These results, along with the total IgA and TTG-IgA results would be included in the final
report.
On the other side of the cascade, those individuals who have no detectable IgA or have a selective
IgA deficiency would have TTG and deamidated gliadin testing performed but only the IgG
isotypes.
These results would be released as part of the final report, along with the total IgA
quantitation.
Finally, for those individuals with low but detectable IgA, TTG, and deamidated gliadin,
both IgA and IgG isotypes would be performed.
This cascade is designed to perform all testing necessary to identify patients who may have
celiac disease and in whom a biopsy would be suggested.
It is not applicable to patients who have been following a gluten-free diet, due to
the possibility of a false-negative serology test result.
The Comprehensive Cascade is identical to the Serologic Cascade except that HLA typing
is also performed.
The Comprehensive Cascade begins with both total IgA and HLA-DQ typing.
All further testing reflexes automatically within the lab based on the total IgA result
and occurs independent of the HLA result.
As far as the serology reflexing is concerned, the same pathways are followed as in the Serologic
Cascade.
The IgA results are classified as normal, low, or deficient.
For normal IgA, a TTG-IgA is performed.
For positive and negative results, no further testing is required.
If the TTG-IgA is weakly positive, EMA and deamidated gliadin-IgA are performed, the
results of which are included in the final report.
For individuals with selective IgA deficiency, testing for the IgG isotype for TTG and deamidated
gliadin antibodies is performed followed by the release of the final report.
For a low IgA result, both isotypes for TTG and deamidated gliadin are performed followed
by the interpretive report.
For a patient who has instituted a gluten-free diet in whom the diagnosis of celiac disease
has not been confirmed, the Comprehensive Cascade for Patients on a Gluten-Free Diet
may be appropriate.
In this algorithm, only the HLA-DQ typing is performed initially.
For those individuals who have neither the DQ2 nor DQ8 alleles, celiac disease is virtually
excluded as a diagnosis.
At this point, testing for celiac disease should stop, and other potential diagnoses
related to the patient’s clinical presentation should be evaluated.
On the other hand, a positive result for DQ2 or DQ8 does not establish a diagnosis of celiac
disease—it means only that celiac disease is a possible diagnosis.
At this point, further testing should be performed, specifically all of the serologic tests.
Depending upon how long the patient has been following the gluten-free diet, and how strict
the diet is, some of these serologic tests may provide a positive result.
In that case, the interpretation would be that the results of all laboratory testing
are consistent with celiac disease and that a biopsy should be performed.
If all results are negative, celiac disease has not been completely ruled out, since this
could simply be a reflection of a successful gluten-free diet.
At this point, the clinician must determine how likely the diagnosis of celiac disease
is and if further evaluation, such as a gluten challenge, should be considered.
To summarize, Mayo Medical Laboratories offers three laboratory reflex algorithms for celiac
disease, which targets the most appropriate testing for the individual patient.
Each cascade has a specific utility.
The Celiac Disease Serology Cascade is the most widely applicable algorithm and uses
targeted serology testing for identification of patients in whom celiac disease is a possible
diagnosis and for whom a biopsy may be indicated.
The Celiac Disease Comprehensive Cascade includes serologic and genetic testing and is designed
for the small sub-set of patients in whom HLA-DQ2 and HLA-DQ8 typing is desired.
The Celiac Disease Comprehensive Cascade for Patients on a gluten-free diet relies on genetic
testing to exclude celiac disease in patients who have initiated a gluten-free diet prior
to a confirmed diagnosis of celiac disease.
One final point to mention is that all celiac testing offered by Mayo Medical Laboratories
is also available as individually orderable tests.
The testing algorithms are most useful for diagnostic evaluation, while the individual
tests are most appropriate for monitoring patients and their response to gluten-free
diets.
I hope this presentation has provided you with useful information regarding laboratory
testing for celiac disease and has helped to clarify the many options available for
diagnostic testing.
Thank you for your participation.