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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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    Thank you so much, this article is a God send! I just received my IGA test results today, it was 27 (a weak positive), but I had no idea what that meant. Your explanation told me everything I wanted to know.

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    Thanks for the article. I've been gluten-free for about 3 months and my doctor told me I did not need to be eating wheat to take the blood test. Think I'll have to be retested....

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    My son was tested for celiac disease after a year or so of feeling sick, having a rash on his elbows knees and legs, poor weight gain etc. The blood test came back Antigliadin IgA Antibody of 12 and Antigliadin IgG of 34. flagged as high. Then he had a stomach biopsy and it was inconclusive but remarked that it had to correlate with the Serologic findings. I don't know what that means. His pediatrician GI specialist said yes he had celiac...but I had to push to get the blood test and it came back to us in November 09 positive. She didn't say anything about it and I had to push for the endoscopy.

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    Excellent explanation. I recently tested positive by IgA (low but still positive). I have had many significant symptoms and chronic illnesses. I truly believe it is all due to gluten. My former doctor said the positive was weak and would not contribute to any of my health issues. I found a new doctor, went gluten free (1 month now) and am starting to feel the best I have ever felt in my life!

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    My daughter and son both tested negative on initial blood test. Because of horrific stomach pains, lack of focus and flu like symptoms she went gluten free anyways. Result: All symptoms gone. Her words after she got better "I felt like I was dying!" My son's excema, muscle pain and allergy symptoms--gone!

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    Please Help me to find out what is going on with me?

    I stopped eating gluten 1 year ago. I had a skin problem and It was suggested to me to try gluten free diet .I did that ,no milk and gluten for 2 mounts. After 2 mounts by accident some flour was on my plate .I was even surprise by the effect. My gyms and tong puff up and the tip of my tong felt burn I couldn't breathe normally for an about 20 min.

    Since then I tried food with gluten to see if it is going to be the same. Sure enough every time I had the same reaction. Sometimes I had swallow a little bit gluten without me knowing, then I get really sleepy and tired for the whole day with numbness on my feet and hands.The next day I am in abdomen pain and feel quite sick.Now I cook at home everything, only that way I feel safe .

    Lately or the past mount I am losing very quickly my energy ,I am getting lethargic and I feel pain in every muscle and bone in my body. I cannot concentrate easily.

    I am 45 years old and almost never got sick. I grow up on bread and filo dough, flour. It is true I work a lot , but I don think that this is the reason for me to feel always exhausted.

    I need to live my live somehow and not to be afraid to wake up every morning.

    I take some vitamins and minerals I have no insurance and I have not seen a Doctor for the problems .

    I read in Better Nutrition magazine that IgC gliadin antibody test is the best blood test to identify gluten sensitivity.

    I need to know what to do, to keep myself healthy and awake, and in good mind.

    I love the article ! I would be happy to know how to find out for sure about allergy I have.

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    Now I understand my family's lab results! My husband and I have 5 children. In a matter of 3 months, my husband, son, and daughter were diagnosed with celiac disease. First through blood work, then with biopsies. A little overwhelming :) My children go back in 2 months for more blood work to see if they are responding to their gluten free diet. Thank you for explaining this to us!! Now I understand what levels to look for.

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    I first started to suspect that I had celiac a few years ago, but when tested it came back normal. I noticed that when I ate certain foods or drank beer, I would bloat so bad it looked like I was pregnant. A few months ago, I started bloating with almost everything I ate. I saw a surgeon, who sent me to get an ultrasound, that sent me to get a CT scan, which sent me to a urologist, who said I had some bladder wall thickening, not the reason I was bloating, which sent me back to the doctor. I saw him on Monday, I told him I had been researching my symptoms and that I believed it was celiac. He's having me tested for it, but also a thyroid problem. I've bloated so much that I look like I'm 6 mos. pregnant, I've gained 8 lbs. since November. I have the floating stools, bloat, rashes on arms, legs, back etc.. I've got the worst gas anyone can imagine. At this point I don't care what tests/biopsies I have to endure if it means knowing what's wrong with me. I can't take much more.

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    I have had diarrhea for 6 months now. I had a baby 9 months ago...followed by a mystery rectal bleed a week later which was so severe I ended up needing surgery to stop the bleeding and a blood transfusion. About 3 months later, I developed diarrhea after I ate anything... sometimes even if I didn't eat anything. They have done tons of blood tests and stool cultures and tested for a bunch of stuff and still can't find anything. I've been through 3 doctors and 2 gastoenterologists. Had 2 colonoscopies (one for the bleed and one for the diarrhea) and they took biopsies and everything came back normal. My 2nd gasto tested for celiac and said I didn't have it after looking at the biopsies and blood tests... however my blood tests show my Gliadin IgA at 27 units, but my Gliadin IgG at 5 units. My tissue transglutamase AB IgG came back at 5 units and my tissue transglutamase AB IgA came back at 6 units. Some docs told me I have a gluten intolerance but I can still have limited amounts if I want. Gastro said I don't have it, and one doc told me she thinks I have celiac and to not eat gluten period. I've been gluten free for 2 months now, and I'm very careful... I don't eat out and I make sure any gluten items in my household stay far away from my food and my prep areas. I was feeling better gluten free with minimal diarrhea attacks, but still feel the bloating and gas every time I eat and within the last week or so the diarrhea has come back. Went to doc again and they did an ultrasound yesterday to check my gallbladder, pancreas ect. I should mention too.. over the last six months when this started I was down to 204 lbs (down from 260 at 39 weeks pregnant..204 was also my normal weight for about 2 years prior to pregnancy) and I am now 123 lbs as of my doctors appt yesterday. So I've been losing weight like crazy from this.... The most recent doctor thinks it's gluten intolerance and something else. Do the test results indicate gluten intolerance or celiac or neither? I'm getting so many different answers from doctors and even after reading this article am still not sure?!?! Please help!! I should also mention I am 26 years old....and a first time mom. Thanks.

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  • About Me

    Celiac.com's Founder and CEO, Scott was diagnosed with celiac disease  in 1994, and, due to the nearly total lack of information available at that time, was forced to become an expert on the disease in order to recover. Scott launched the site that later became Celiac.com in 1995 "To help as many people as possible with celiac disease get diagnosed so they can begin to live happy, healthy gluten-free lives."  In 1998 he founded The Gluten-Free Mall which he sold in 2014. He is co-author of the book Cereal Killers, and founder and publisher of Journal of Gluten Sensitivity.

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