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  • Scott Adams
    Scott Adams

    Interpretation of Celiac Disease Blood Test Results

    Reviewed and edited by a celiac disease expert.

    Caption: Images: CC--Scott Robinson

    The following detailed explanation of serological tests for celiac disease was written by Tom Ryan, Technical Service Specialist, INOVA Diagnostics, Inc.

    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 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 will not 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 which 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.

    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 celiac disease. Conversely, if the patient is IgA negative, there is only a 71% probability that the patient is truly negative for celiac disease. Therefore, a positive result is a strong indication that the patient has the disease but a negative result does not necessarily mean that they don not 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 is not as strong a correlation with celiac disease. It is less specific. Patients with other conditions but not afflicted with celiac disease 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 celiac disease 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 celiac disease and DH. These antibodies are found in approximately 80% of patients with DH and in essentially 100% of patients with active celiac disease. IgA endomysial antibodies are more sensitive and specific than gliadin antibodies for diagnosis of celiac disease. 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 ½ then ¼, 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 judgment 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 judgment 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 celiac disease 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 celiac disease 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:

    • An initial positive gut biopsy;
    • 6 months on a gluten free diet;
    • A second, negative gut biopsy;
    • A gluten challenge for 6 months and;
    • 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.

    Edited by Scott Adams


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    I just had a positive small intestinal biopsy 7/2012 and am now having labs done. I had TTG IgA done and it was >3 and negative Endomyisal Ab in 2010. Do you think I just wasn't fully reacting to gluten? Has anyone else had similar issues? I'm Fibromyagia, Chronic Iron Deficiency Anemia, irritable bowel syndrome, migraines, sleep problems, depression, anxiety, hair loss, burning/sore tongue, & vision problems.

    Danelia and Everyone else,

    I have a ton of symptoms. You know when something is not right with your body and hope your finding it. There seems to be a root cause to you being tired and having pain everywhere. with lethargy and being afraid to wake up in the morning.

    I have the same this for almost four years now, plus heart flip flops, arythmias, numb body sections, MEMORY LOSS, can't focus. Can't make decisions. I can get very anxious for no reason. My hair is falling out more and more. I had low basal body temperature for months with a minor sore throught and achiness. Im freezingEasy And I'm just putting the two and two together. I am so exhausted and heavy feeling 24/7 and this never lets up. the symptoms just kept coming. Slowly and slowly...more and more. I went through...thinking it was Candida, then I got the ELISA test for lyme disease. Came back equivicol. What a joke. It was the most inaccurate way to do this test...whether I have lyme or not. It just took me till now to know that many blood tests, especially ones we don't look up....Can cost you the quality of your life. I had to get on ssi disability at 25. Im now 29. I lost everything. Im 99 percent sure I have lyme. medicare and Medicaid I hear don't pay for the appropritate tests or treatments. I have to pay out of pocket to see a dr. that will listen to you and give you accurate results. So insurance...in my situation, seems, many of these doctors...don't listen, don't care, and make you think your crazy.

    The pattern of this is so obvious. Also Dental amalgams that contain 50 percent mercury can do a great job on the fatigue so can getting a root canal. Root canals are dead teeth. Get them out if you have them. These alone can make you so tired and lethargic. They create so much havoc to every body system, they make bacteria and the mercury corrodes teeth and jaw bone which can have almost more profound effects than all the silver mercury amalgams you have in your teeth. then you get more terrible bacteria. NEUROTOXIC bacteria.

     

    Anything with gluten or suger or a carb will make any type of invfestation that you have worse. I'm too sick most of the time to care, but I have to.

     

    Theres many things I believe that can make blood results not so sufficient, especially in people who have multiple symtoms going on. I am so sick...but labs come back normal? Really? Mercury predisposes you to autoimmune disorders. People are genetically predisposed to be sensitive to it and all the other metals. that are on an amalgam. and it's parterns in crime (bacteria, parasites, candida,...)go undetected that they rob you of your nutrients, as they attack you kidney, and bind to cells like hemoglobin giving you less oxygen as a result making you more tired. Your results from the blood work for everything says and looks normal.

     

    But blood work only can tell the levels available! They don't measure performance and functioning!

     

    Hope this helps, I've been walking around with vapors from the boat load of mercury I had in my mouth for 20 years. And been really really sick for the last 4 without thinking of it. I also too know Im dying. But just have questions about my IGG labs.

    Ref range says <20 Units.

    Gliadin IGA is 5

    Gliadin IGG is 4

    TTG AB Iga is4

    and TTG AB IGG is 8.

    Does this sound right?

    I can't get any antibodies for ElISA (the lyme) test, But results came back equivicol? What else can't I make antibodies for?

    Just know that you have to do your own research, Ask for different tests. Even the great natropathic Drs. don't know how your symptoms connect.

    Try colloidal silver, it's amazing. It stopped my heart from beating out of its chest for a week and a half straight. I took a teaspoon of that and within mintues I could catch my breath and get my appetite back. DEF a bacteria issue. Magnesium for anxiety and GABA. Lots of Vitamin C. achidolpolus, B-complex. Melatonin for sleep. Vitamin D.

     

    Hope this helps.

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    So... IgA of 219 means I have celiac disease?

    My test result 8-5-13. 152

    19 units or less : negative

    20-30 units: weak positive

    31 units or greater: moderate to strong positive.

     

    I am gluten-free since 8-6-13. feeling better everyday.

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    Finally an explanation! I was low on my IgA but high positive on IgG and so sick and my doc said, 'at some point in the past, gluten made you very sick, but you're fine now.'.. He was totally ignoring my symptoms! That was two years ago and he still doesn't believe I have it, even though since then he diagnosed me with osteopenia.. I've been off gluten for about 3 months now, and my chronic migraines and stomach problems are gone! my doc still doesn't believe I have it, but IBS instead! give me a break! And these are Seattle gastroenterologists!

    Me too! In Australia - "dying" for a proper diagnosis.

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    Oh wow. I hadn't had gluten for months when I had the blood test, and my doctor wouldn't do an endoscopy. He also wouldn't show me the results. Just said, "I told you that you didn't have it."

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    Interesting article. One note: the definitions of sensitivity and specificity are not correct: in this context, sensitivity is the fraction of people with celiac that would have a positive test result. Likewise, specificity is the fraction of people without celiac that would have a negative test result. The terms you describe are positive and negative predictive value, which are usually harder to measure because they depend on the fraction of a population that is being treated. The difference between these can be quite large: if you assume 1% of a population has a disease a specificity of 97% means that most people (>74%) that test positive actually don't have the disease. Which is presumably why additional confirmation tests are necessary. For more information, see the Wikipedia article for sensitivity and specificity.

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    In the hopes this will help someone, I'm posting my before and after Celiac panel test results.

     

    On 4/10/09 (diagnosed)

    Deamidated Gliad IgAH 127

    Deaminated Gliad IgGH 104

    Tranglut IgA 43

     

    On 6/12/09 (gluten free diet)

    127 -> 58 (goal under 10)

    104-> 48 (goal under 10)

    43 -> 10 (goal under 3)

     

    So after being on gluten free diet from 4/10/09-6/12/09, 8 weeks, already seeing dramatic reductions in antibody levels. Not done yet, but are headed in the right direction. I didn't see this kind of information posted anywhere, so hoping this information is helpful.

    I was diagnosed yesterday. She said something about my results being 57.6 and they are suppose to be under 15. I have no idea what this means but she has told me absolutely no gluten.

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    My test results are extremely confusing and I do not have an official diagnosis of Celiac. My blood test (Igg/Iga) was 210, well above normal on both. My biopsy was negative but I was positive for one of two genetic markers. My gastroenterologist did not diagnose me as Celiac but the nutritionist they sent me to said "oh absolutely" and put me on a gluten free diet. How does one get a real answer?

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