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Hello All,

There has been a lot of discussion about serological testing for celiac
disease recently, specifically regarding tTG (tissue Transglutaminase)
testing.  I will try to answer some of the many questions that have
appeared on this list about all of the tests.

First, and this applies to any of the blood tests, you must currently be
on a gluten containing diet for the tests to be accurate.  Antibodies
are produced by the immune system in response to substances that the
body perceives as threatening.  The immune response that your body
produces is it's response to being exposed to gluten in the diet and
it's subsequent effect on the intestinal mucosa.  If there is no gluten
in the diet, then there is no response that we can measure.  A brief
change in diet will not have a noticeable effect.  If you have been
gluten free for a week or so, it won't make any great difference.  The
response might be marginally less but the difference is insignificant
because the body has not had time to respond to the change.  Conversely,
if you have been gluten free for a protracted period of time and decide
to be tested, a brief challenge of a couple of weeks is not enough to
elicit a response and get an accurate test.  There are several steps
that take place to generate an immune response and it takes time both
for the positive reaction when gluten is present and to clear the
antibodies when gluten is eliminated.  There has been a great deal of
discussion about how much and how long a challenge should be and there
is no consensus. Talk with your Doctor.  My personal feeling is that the
minimum is 2 slices of bread per day for 6 weeks to get an accurate test
but I would not try to second guess the Doctor.

There are basically four tests that can be performed to aid in
diagnosing celiac disease.  Notice that I say they will "aid" in
diagnosing celiac disease.  Immunology is fairly accurate but it is far
from being an exact science.  All of the lab tests, regardless of the
type or source, are presented as  aids to diagnosis.  They should not be
used alone as a basis for diagnosis but rather are intended to be
considered in conjunction with the physical examination of the patient
as well as the reported symptoms, etc. by a trained physician.  There
has been a great deal of confusion about what the tests are and I hope
to alleviate some of the misunderstandings.

There are many terms that we hear.  tTG, IgA, IgG, ELISA, etc.  What are
all of these?  Some contributors to the list make reference to the "IgA"
or "IgG" test or to the "ELISA" test.  These labels are incomplete for
our purposes and could be referring to any number of different
tests.

We all have, within our bodies, a family of closely related although not
identical proteins that are capable of acting as antibodies.  These are
collectively referred to as "immunoglobulins".  Five major types of
immunoglobulins are normally present in the human adult.  They are IgG,
IgA, IgM, IgE and IgD.  Each of these is a shorthand way of writing
"immunoglobulin gamma G" (or A or M, etc.) and they each perform a
different function in our systems.  IgG is the principal immunoglobulin
in human serum.  It is important in providing immunity in a developing
fetus because it will pass across the placental barrier.  IgA is the
principal immunoglobulin in secretions from respiratory and intestinal
mucosa.  IgE is a gamma globulin produced by cells lining the intestinal
and respiratory tracts.  It produces the antibodies associated with most
hypersensitivity (allergic) responses.  It is associated with asthma,
hay fever, etc.  IgM is a globulin formed in almost every immune
response in the early part of the reaction.  IgD is a rare protein
present in normal sera in a tiny amount.  These designations refer to
the type of protein that is carrying the antibody in question.  Both IgG
and IgA subtypes of anti-gliadin antibody are produced, hence we refer
to them as "IgG gliadin" or "IgA gliadin".  Collectively they are
anti-gliadin antibodies.=20

ANTI-GLIADIN ANTIBODIES:

Both IgA and IgG anti-gliadin antibodies (AGA) are detected in sera of
patients with gluten sensitive enteropathy (celiac disease).  IgG
anti-gliadin antibodies are more sensitive but are less specific markers
for disease compared with IgA class antibodies.  IgA anti-gliadin
antibodies are less sensitive but are more specific.

In clinical trials, the IgA antibodies have a specificity of 97% but the
sensitivity is only 71%.  That means that, if a patient is IgA positive,
there is a 97% probability that they have CD.  Conversely, if the
patient is IgA negative, there is only a 71% probability that the
patient is truly negative for CD.   Therefore, a positive result is a
strong indication that the patient has the disease but a negative result
doesn't necessarily mean that they don't have it.  False positive
results are rather uncommon but false negative results can occur.

On the other hand, the IgG anti-gliadin antibodies are 91% specific and
have an 87% sensitivity.  This means that they will show positive
results more readily but there isn't as strong a correlation with CD.
It is less specific.  Patients with other conditions but not afflicted
with CD will occasionally show positive results.  IgG anti-gliadin
antibodies are detectable in approximately 21% of patients with other
gastrointestinal disorders. This test might yield false positive results
but is less likely to yield false negative results.

A sensitive testing protocol includes testing for both IgA and IgG
anti-gliadin antibodies since a significant portion of celiac patients
(approx. 2-5%) are IgA deficient.  This combined IgA and IgG
anti-gliadin antibody assay has an overall sensitivity of 95% with a
specificity of 90%.

The type of test used to detect the anti-gliadin antibodies is called an
ELISA.  This is an acronym and it stands for Enzyme Linked
Immuno-Sorbent Assay.  "ELISA" is not a test in itself.  It is a method
of testing and it is a relatively simple test to perform.  It involves
putting a measured amount of diluted patient serum into the wells of a
specially constructed and prepared plate and incubating it for a period
of time with various chemicals.  The end result is a color change, the
intensity of which is dependent upon the concentration of anti-gliadin
antibody (or other protein being measured) in the patient serum.  The
ability of this colored solution to absorb light at a particular
wavelength can be measured on a laboratory instrument and mathematically
compared with solutions that contain a known amount of
anti-gliadin antibody to arrive at a number for the amount of antibody
present.  The sample can then be classified as negative, (0-20 units);
weak positive, (21-30 units); or moderate to strong positive if greater
than 30 units.

The purpose of testing for anti-gliadin antibodies includes, in addition
to diagnosis of gluten sensitive enteropathy, monitoring for compliance
to a gluten free diet.  IgA gliadin antibodies increase rapidly in
response to gluten in the diet and decrease rapidly when gluten is
absent from the diet.  The IgA anti-gliadin antibodies can totally
disappear in 2-6 months on a gluten free diet, so they are useful as a
diet control.  By contrast, IgG anti-gliadin antibodies need a long
time, sometimes more than a year, to become negative.  The reverse is
also true.  That is, a patient with CD who has been on a gluten free
diet and tests negative for IgA anti-gliadin antibodies, will show a
rapid increase in antibody production when challenged by gluten in the
diet.  Approximately 90% of challenged patients will yield a positive
IgA anti-gliadin result within 14-35 days after being challenged.  The
IgG antibodies are somewhat slower.

ENDOMYSIAL ANTIBODIES:

IgA class anti-endomysial antibodies (AEA) are very specific, occurring
only in CD and DH.  These antibodies are found in approximately 80% of
patients with DH and in essentially 100% of patients with active CD.
IgA endomysial antibodies are more sensitive and specific than gliadin
antibodies for diagnosis of CD.  Antibody titers (dilutions) are found
to parallel morphological changes in the jejunum and can also be used to
reflect compliance with gluten-free diets.  Titers decrease or become
negative in patients on gluten free diets and reappear upon gluten
challenge.

The test for anti-endomysial antibodies is more subjective and more
complicated for the lab to perform than the anti-gliadin assays.  It
involves serially diluting some of the patients serum, that is, diluting
it by 1/2 then 1/4, 1/8, 1/16, etc. and putting these dilutions on a
glass slide that has some sort of tissue affixed to it.  The slide is
then processed with various solutions and examined under a fluorescent
microscope to determine if any of that serum binds to any of the
proteins in the tissue.  If so, then that patient is confirmed as having
antibodies to that particular protein.  This method of testing is called
an IFA or sometimes IIFA.  It stands for Indirect Immuno-Fluorescent
Assay.

The selection of which tissue slide to use is determined by what
specific protein, hence which antibody, you are specifically looking
for.  Endomysial antibodies react with the endomysium, which is a sheath
of reticular fibrils that surround each muscle fiber.  Therefore, to
detect endomysial antibodies, you would want to use a tissue substrate
that contains a lot of muscle tissue.  The substrate used most often for
this assay is distal sections of the esophagus.  These are very thinly
sliced and fixed to the slide.  They contain muscle fibers and not much
else so there is a lot of endomysium available to react with the
anti-endomysial antibodies.

Reading this test involves viewing the reacted slides with a fluorescent
microscope to make the determination.  This requires a highly skilled
and trained eye and, of necessity, is somewhat subjective.  You are
looking for a green fluorescence in the endomysium covering the muscle
fibers.  The test is reported as the "titer" or final dilution in which
the fluorescence can still clearly be seen.  As you can imagine, this is
very subjective.  There are no standardized values and it is up to the
judgement of the particular technician what the endpoint titer is.
Recently, (1998) the endomysial antigen targeted by the anti-endomysial
antibodies was identified as the protein cross-linking enzyme known as
tissue transglutaminase (tTG).  This has enabled the production of an
antigen specific ELISA assay incorporating tTG as a reliable and
objective alternative to the traditional and subjective
Immunofluorescence based assays.  In clinical trials, the correlation
with the endomysial IFA assay has been shown to be close to 100%.  This
is a test that has been very well received in the professional
community.  It is an ELISA, like the anti-gliadin antibody test and, as
such, is not subject to interpretation like the IFA.  That is the
greatest advantage to this new test!  With this or any ELISA, the
response is measured on an instrument that calculates the amount of
light of a particular wavelength that is absorbed by the solution and
prints out a numerical result.  There is no chance of human error
skewing the results because there is no judgement call involved.  The
ELISA plate, regardless of what you are testing for, is processed with
at least three control sera (sometimes as many as eight) in addition to
the unknown sample being tested.  There is a negative serum and at least
two positive sera containing different levels of the antibody being
tested.  There are specific requirements for the absorption levels of
these three controls.  That is, each of them has a minimum or maximum
(or both) number that must be seen by the instrument in order for it to
be a valid test.  If there is any variance from these expected numbers,
it is an indication that something went wrong and the test results are
discarded and the test repeated.  There is therefore no way the
technician could report inaccurate results, (assuming they diluted the
sample correctly).  Either the test was valid, and you can rely upon the
accuracy of the result, or the test is invalid, and the entire result
discarded.  If any error was made during the processing of the ELISA
plate, it would result in the control sera numbers being out of range
and the entire test result would be thrown out.

In summary, the tTG ELISA is measuring the same thing that the
endomysial IFA is measuring but with a method that is more sensitive and
specific and not subject to interpretation.

IgA class Reticulin antibodies are found only in Celiac disease and
dermatitis herpetiformis.  These antibodies are found in approximately
60% of CD patients and 25% of DH patients.  This test is falling into
disuse because of the limited utility and the availability of better
tests.  It is an IFA performed on a tissue substrate with all the
attendant problems that go along with it.

The development of all of these serum assays has tremendously simplified
the diagnosis of CD and improved the accuracy as well.  The original
criteria for diagnosis according to the European Society for Pediatric
Gastroenterology and Nutrition, (ESPGAN), involved a year of arduous
studies with:  a) an initial positive gut biopsy,  b) 6 months on a
gluten free diet,  c) a second, negative gut biopsy,  d) a gluten
challenge for 6 months and e) a third, positive gut biopsy.  The revised

ESPGAN criteria call for positive results in two of the serological
tests confirmed by a single positive biopsy.  In practice, many
gastroenterologists are utilizing the serologies in conjunction with a
controlled diet and the clinical presentation to form a basis for
diagnosis without the need for the invasive procedure.

Through the auspices of the Celiac Disease Foundation and others, a
professional symposium and workshop was organized earlier this year in
Marina Del Rey, California with participants from Europe as well as the
U.S. to establish standards for reporting test results.  This should
improve testing and diagnosis even more.  At the conclusion of this
conference a Celiac Disease Standardization Committee was formed to
investigate and make recommendations on a standardized method of
reporting results.

Due to the numerous requests I have received, I am publishing a list of
several labs throughout the country that offer tTG testing.  It is part
two of this posting.

Tom Ryan
Technical Service Specialist
INOVA Diagnostics, Inc.