<<Disclaimer: Verify this information before applying it to your situation.>>
Tissue Transglutaminase Blood Tests
-----------------------------------
by Tom Ryan
[Tom Ryan is a Technical Service Specialist at INOVE Diagnostics, Inc.
This article is derived from two messages he recently sent to the
CELIAC e-mail list.<1><2>]
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 the CELIAC e-mail 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 its response to being exposed to gluten in the diet
and its 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 respon
se 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
long-lasting immunity. 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.
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.
[The specificity and sensitivity values will vary from lab to lab,
depending not only on technique but also on patient selection.--Dr.
Alexander, TCCSSG physician advisor.]
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 positiv e 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.
[Some cases never become negative.--Dr. Alexander] 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 clo se 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.
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.
|
