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Hi Ron and all list members.

I have received a couple of questions about test results lately, so I think
it's time once again to answer them for the entire list.

Both IgA and IgG gliadin antibodies (AGA or anti-gliadin antibodies) 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 very 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.  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 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.

IgA class anti-endomysial antibodies (AEA) are very specific, occuring 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 hence more
expensive.  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.

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 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 operator what the endpoint titer is.  That is the reason the lab
you mentioned lets the pathologist make the final call.  It is also the
reason they don't perform this assay unless it is indicated by a positive
test for anti-gliadin antibodies.

Recently, the endomysial antigen targeted by the anti-endomysial antibodies
has been 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 assay has
been shown to be close to 100%.  This is a test that we will all be hearing
a great deal about in the near future.

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 is being organized early next year 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.

As I mentioned earlier, the level of the IgA antibodies responds fairly
quickly to changes in the diet while the IgG tends to lag behind.  Although
I am not certain of any hard and fast rule, I believe it is generally
considered that a minimum of 6 weeks challenge is required before testing
to assure accurate results.  If this is correct, your original results
might be suspect and I believe you are wise to continue the challenge as
you said.  I hope it isn't too miserable for you.

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.

Now, with apologies to all, (It is not my intent to offend anyone), I'd
like to offer my own opinion on a subject I see on this list frequently.
That is with those who are self diagnosed.  Although in some, perhaps many,
cases the self diagnosis is accurate but, in my humble opinion, you are
doing yourself a great disservice by not getting a professional diagnosis.
There is no way you can be certain that the diagnosis is correct until and
unless the diagnosis is confirmed by an examination by a trained physician
experienced with this disease with or without serological testing.
Starting a Gluten Free diet with subsequent abatement of symptoms does not
prove CD conclusively and it makes a firm diagnosis very difficult for the
physician should you chose to consult one in the future.  It could be the
result of an IgE mediated allergy rather than an IgA/IgG mediated
malabsorption which is easier to deal with on a less stringent basis or it
could be the result of a placebo effect.  An allergy might feel just as
miserable but it doesn't do the same damage that CD does and it can be
managed.

Don't be too hard on your General Practitioner for not being fully
conversant with these test results.  Until recently, the awareness of
celiac disease in the U.S. has been lagging behind the rest of the world.
I have a great deal of literature available.  If he (or she) would like to
have copies, give them my EMail address and ask them to contact me.  I'll
be glad to provide copies.

I apologize to all for the length of this.  I hope I didn't bore everyone
too badly.  I must also point out that I am not a physician.  I am not
qualified to offer medical advice and it is not my intention to do so but I
work extensively with the serological tests we have been discussing
including performing them, training technicians to perform them and
assisting with interpretation of results.  I am a Technical Service
Specialist for a company that produces the tests.

Best regards,

Tom