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  • Jefferson Adams
    Jefferson Adams

    Assessing Celiac Disease in Patients with Positive Tissue Transglutaminase Antibodies and Negative Endomysial Antibodies

    Caption: Photo: CC--Valerie_Everett

    Celiac.com 06/29/2012 - A group of researchers recently set out to study cases of positive tissue transglutaminase antibodies with negative endomysial antibodies to determine whether or not such cases amount to celiac disease.

    The team included Thomas Hornung; Pavel Gordins; Clare Parker; and Nicholas Thompson. They are variously affiliated with the departments of Gastroenterology, and Immunology at the Northern Deanery of Newcastle upon Tyne, and with the department of Gastroenterology at Freeman Hospital in Newcastle upon Tyne in the UK.

    Photo: CC--Valerie_EverettThe most sensitive and specific blood tests for diagnosing celiac disease are those that detect immunoglobulin A (IgA) antibodies against human tissue transglutaminase (tTGA) enzyme, and those that measure aspects of connective tissue covering individual smooth muscle fibers, endomysial antibodies (EMA).

    Because of the high sensitivity (up to 98%) and high specificity (around 96%) reported for the tTGA assay, detection of tTGA is currently the primary blood test used in screening for celiac disease.

    The tTGA test also has a high negative predictive value approaching 100%, which makes it an excellent test for excluding celiac disease in both high and low risk groups. In contrast, positive predictive value of the tTGA test is rather poor with values between 28.6% and 60.2% being reported in several studies.

    EMA, on the other hand, has extremely high specificity values close to 100% and positive predictive value values approaching 80%.[5 10] However, compared with tTGA, EMA has lower sensitivity, usually under 90%.

    This being the case, the present standard celiac disease screening strategy is to first use tTGA, and then confirm positive results using EMA. However, doing it this way, doctors often end up with a group of patients who show divergent test results.

    For their study, the researchers wanted to gauge the percentage of patients with positive tTGA and negative EMA, but who were confirmed with celiac disease upon biopsy, and to identify factors in these patients that may help to increase diagnostic accuracy in such patients.

    The research team identified 125 consecutive patients with positive tTGA and negative EMA, who subsequently underwent endoscopy with at least two biopsies from the second part of the duodenum.

    The team charted any tTGA result over 15 U/ml as positive. They excluded any patients with known celiac disease at the time of testing.

    They then reviewed patient notes to assess indications for celiac disease serological screening, including the presence of iron deficiency anaemia, and symptoms such as diarrhea or weight loss, and family history of celiac disease. They defined diarrhea as a bowel frequency of more than three times a day.

    They then assessed histological evidence of celiac disease based on subsequent duodenal biopsies, plus Marsh grading. In cases where patient histology was unclear, they relied on the clinical assessment of a consulting gastroenterologist. Unclear histology included minimal/mild increase in intraepithelial lymphocytes of not more than 30 per 100 enterocytes and without villous atrophy, plus mild villous blunting with no increase in intraepithelial lymphocytes.

    They then categorized patients as either celiac disease negative, or celiac disease positive. Patients with no histological evidence of celiac disease on duodenal biopsies or equivocal histology plus overall clinical impression of celiac disease absence were categorized as celiac disease negative. Patients with histological evidence of celiac disease on duodenal biopsies or equivocal histology plus overall clinical impression of celiac disease presence were categorized as celiac disease positive.

    To measure IgA anti-tTGA antibody the team used a commercially available enzyme linked immunosorbent assay called Aeskulisa, manufactured by Aesku Diagnostics GmbH in Wendelsheim, Germany.

    To detect IgA anti-EMA with the standard immunofluorescent method, they used commercial slides of monkey oesophagus sections (Euroimmun, Euroimmun AG, Lübeck, Germany). They used conjugated sheep antihuman IgA as a secondary antibody, relying on a test manufactured by Instrumentation Laboratory UK Ltd., in Warrington, UK.

    Overall, the team categorized 113 patients (90.4%) as celiac disease negative. Of these, 102 patients had no histological features of celiac disease, while 11 patients had unclear histology plus an overall clinical impression of not having celiac disease.

    They categorized twelve patients (9.6%) as celiac disease positive. Of these, 10 patients had positive histology, and two patients had unclear histology plus an overall clinical impression of having celiac disease.

    Of those with positive histology, 17% were Marsh grade I, 8% were Marsh grade II, 33% were Marsh grade IIIa, 17% were Marsh grade IIIb and 25% were Marsh grade IIIc. Those with celiac disease were more likely to be older and to have a higher tTGA level. The groups showed no difference in any clinical parameter.

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    My tissue transglutaminase test was positive and all other blood tests and the endoscopy were negative. My doctor said because the tissue transglutaminase was the most accurate that I definitely had celiac disease. Are you saying that I might not actually have it?! After going gluten-free I never had a point where I felt way better like other people with celiac disease I have talked to when they went gluten-free.

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    My tissue transglutaminase test was positive and all other blood tests and the endoscopy were negative. My doctor said because the tissue transglutaminase was the most accurate that I definitely had celiac disease. Are you saying that I might not actually have it?! After going gluten-free I never had a point where I felt way better like other people with celiac disease I have talked to when they went gluten-free.

    Hi Kevin, did you ever find out anything more about your situation? I got a low positive tTGA test and a negative EMA but was also diagnosed as celiac. Did you have a biopsy done to confirm? Would be interested in hearing your experience and how you are doing now.

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  • About Me

    Jefferson Adams earned his B.A. and M.F.A. at Arizona State University, and has authored more than 2,000 articles on celiac disease. His coursework includes studies in biology, anatomy, medicine, science, and advanced research, and scientific methods. He previously served as Health News Examiner for Examiner.com, and devised health and medical content for Sharecare.com. Jefferson has spoken about celiac disease to the media, including an appearance on the KQED radio show Forum, and is the editor of the book "Cereal Killers" by Scott Adams and Ron Hoggan, Ed.D.

  • Related Articles

    Scott Adams
    The following abstract was submitted to celiac.com directly by William Dickey, Ph.D., a leading celiac disease researcher and gastroenterologist who practices at Altnagelvin Hospital, Londonderry, Northern Ireland.
    Scandinavian Journal of Gastroenterology 2005; 40: 1240-3.
    Dickey W, Hughes DF, McMillan SA.
    Celiac.com 09/27/2005 - What does a positive endomysial antibody (EmA) test mean if the biopsy does not show villous atrophy? The authors studied 35 patients where this was the case. In the authors practice, these patients account for 10% of all EmA positives.
    Firstly, the lack of villous atrophy did not necessarily mean a normal biopsy: 14 patients had excess inflammatory cells (lymphocytes) consistent with a mild abnormality of gluten sensitivity.
    Secondly, many of these patients had typical celiac features: twelve had a family history of celiac, five had dermatitis herpetiformis and thirteen had osteopenia or osteoporosis on DEXA scan.
    After discussion, 27 patients opted to take a gluten-free diet from the first biopsy: 26 of these had clinical improvement. Seven of eight patients who persisted with a normal diet developed villous atrophy on follow-up biopsies.
    The authors conclude that a positive EmA result indicates gluten sensitivity even if biopsies do not show villous atrophy. While a biopsy remains important as a baseline reference, these patients should be offered a gluten-free diet to allow clinical improvement and prevent the development of villous atrophy. There may be no such thing as a "false positive" EmA, although the authors emphasise that the same conclusion cannot yet be applied to tissue transglutaminase antibody results.

    Jefferson Adams
    More and more people with celiac disease present atypical symptoms that are clinically indistinguishable from other gastrointestinal disorders. A new study shows that upwards of 4% of people with generalized gastrointestinal complaints  show elevated celiac disease antibodies when screened.
    A team of researchers recently set out to assess rates of celiac disease in patients with gastrointestinal symptoms, and to catalog the common manifestations of atypical expressions of celiac disease. The research team was made up of Mohammad Rostami Nejad, Kamran Rostami, Mohamad Amin Pourhoseingholi, Ehsan Nazemalhosseini Mojarad, Manijeh Habibi, Hossein Dabiri, and Mohammad Reza Zali.
    The team designed and executed a cross sectional study that included 5,176 individuals chosen randomly from self-referred patients within a primary care setting in Tehran province from 2006-2007.
    In all, 670 of the 5176, or 13% of patients self-referred to a general practitioner suffered from gastrointestinal complaints.  All 670 subjects with gastrointestinal symptoms underwent celiac blood tests, including total immunoglobulin A (IgA) and anti-tissue transglutaminase (tTG) antibodies. Individuals showing IgA deficiency underwent screening for IgG tTG.
    Of the 670 investigated for gastrointestinal complaints, a total of 22 patients, 17 women and 5 men, showed positive anti-tTG results (95% CI: 1.70-4.30). Another 8/670 showed IgA deficiency, with 3 of those 8 subjects showing positive IgG tTG.  Dyspepsia (indigestion) was the chief complaint in 25 patients withpositive blood tests and cases that were analogous to the rest of thesubjects.
    In all, 3.3% of serologically screened samples excluding IgA-deficient showed celiac disease antibodies, compared to 3.7% of those IgA-deficient subjects with positive tTG-IgG.
    Generalized gastrointestinal complaints are a common indication of atypical celiac disease. This study points to high rates of celiac disease antibodies among patients with generalized gastrointestinal symptoms (3.7%).
    Clinicians and patients will benefit from greater vigilance regarding atypical presentation of celiac disease and its association with generalized gastrointestinal symptoms.
    Source:
    Journal of Gastrointestinal Liver Disease - September 2009 Vol.18 No 3, 285-291


    Jefferson Adams
    Celiac.com 06/17/2010 - In a recent letter to the editors of Clinical Chemistry, Carolina Arguelles-Grande, Gary L. Norman, Govind Bhagat, and Peter H. R. Green describe how hemolysis interferes with the detection of anti–tissue transglutaminase antibodies in celiac disease.
    They are variously affiliated with the Departments of Medicine and Pathology at Columbia University's College of Physicians and Surgeons in New York, and with INOVA Diagnostics, Inc., in San Diego, CA.
    Using human recombinant or erythrocyte tTG-IgA–based ELISA assays to measure anti–tissue transglutaminase (tTG) antibodies is one of the favored methods for diagnosing celiac disease.
    However, assessments of various tTG kits have shown variations in sensitivity, which has raised some alarms among clinicians. Many clinicians suspect that hemolysis plays a role in these variations.
    To assess the effect of hemolysis on tTG-IgA titers, the team looked at blood samples from 9 patients with biopsy-confirmed, active celiac disease who chose to participate in the study.
    They split the samples into 3 groups, with three samples in each group. They divided the samples according to tTG-IgA concentration after thawing. They categorized the samples as high titer (>185 U), intermediate titer (100–140 U), and borderline titer (20–50 U).
    The team hemolyzed a whole-blood sample taken from 1 tTG/DGP-seronegative patient. They measured hemoglobin in the sample at 149 g/L of hemoglobin. They repeatedly froze and thawed the sample until 90% of cells hemolyzed. They then serially diluted in ratios of 1:2, 1:5, 1:10, 1:50, 1:100, 1:500 in PBS to obtain hemoglobin concentrations of 67.1, 26.8, 13.4, 2.7, 1.3, and 0.27 g/L, respectively. They then added to each sample at a 1:1 ratio.
    For the tTG sequestration assessment, the team added human recombinant tTG from Diarect AG for final concentrations of 0.04, 0.02, 0.01, and 0.002 g/L. The team used undiluted serum as the baseline titer reference, and serum diluted 1:2 in PBS as a control.
    To measure antibody titers, they used 2 ELISA test kits: QUANTA LiteTM h-tTG IgA (human erythrocyte tTG-IgA based) and Gliadin II (DGP-IgA based) from INOVA Diagnostics, Inc.  The team conducted blinded screens per manufacturer instructions, and compared the results for each group using the Mann–Whitney U-test, with P values <0.05 considered significant.
    They discovered that adding hemolyzed blood (HB) to sera of patients with active celiac disease lowered levels of anti-tTG, with intermediate- and borderline-titer groups seeing the largest reduction. Anti-DGP antibodies remained unchanged.
    Total average titer loss of anti-tTG vs anti-DGP antibodies was 36% vs 13% in the high-titer groups (P 0.026), 45% vs 3% (P = 0.026) in the intermediate titer groups, and 51% vs 2% in the borderline-titer groups (P = 0.0022)
    The team also found that adding ever higher concentrations of hemoglobin lowered the titers of anti-tTG, but not of anti-DGP, causing negative anti-tTG results in samples with low tTG antibody concentrations.
    The anti-tTG titer decreased 2%–65% in the high-titer groups, 1%–81% in the intermediate-titer groups, and 16%–74% in the borderline-titer group at hemoglobin concentrations of 0.3– 67.1 g/L.
    This compares with a decrease in anti-DGP titers of 10%–16% for high-titer groups, 4%–8% for intermediate-titer groups, and 7%–3% for the borderline-titer groups at hemoglobin concentrations of 0.3– 67.1 g/L.
    In all groups, tTG titer reduction was greater at higher concentrations of HB/HGB and gradually recovered as the red tint started to vanish at about 13 g/L of HGB, until complete visual disappearance at about 0.3g/L HGB).
    In the intermediate- and borderline-titer groups, titer reduction induced false-negative results at 20 U, with the anti-tTG, but not anti-DGP assays for HGB concentrations  ≥13 or ≥0.3 g/L, respectively.
    They also found that raising concentrations of exogenous tTG (recombinant human tTG) to intermediate-titer blood samples triggered a significant reduction in anti-tTG assay titers similar to that seen with hemoglobin (range, 32%–82%; mean, 69%), as compared with that of anti-DGP titers (mean, 18%; range, 1%–38%; P = 0.0159).
    Hemolysis is clearly indicated by a red tint in serum plasma, and is one of the most common reasons for labs to reject specimens. Visible hemolysis starts at about 0.5 g/L of hemoglobin and is obvious above 1.3 g/L of hemoglobin.
    The results show that that hemolysis does interfere with the detection of anti-tTG antibodies, and that visibly hemolyzed blood samples generate false-negative anti–tTG-IgA results.
    These findings may explain false-negative tests for celiac disease that arise when clinicians use tTG-IgA assays. They encourage clinicians and laboratories to take measures to avoid hemolysis. If they notice hemolyzed blood samples, they should alert physicians so new blood samples can be taken. If redrawing samples is not possible, hemolyzed samples should be measured for anti-DGP antibodies.
    Clinicians who suspect hemolysis should consider using anti-DGP serological tests, which are not influenced by hemolysis.
    Source:

     Clinical Chemistry. 2010;56:1034-1036. DOI: 10.1373/clinchem.2010.143263

    Jefferson Adams
    Celiac.com 04/22/2014 - Blood tests are highly valuable for diagnosing celiac disease. However, their role in gauging mucosal healing in celiac children who have adopted gluten-free diets is unclear.
    A team of researchers recently set out to compare the performance of antibody tests in predicting small-intestinal mucosal status in diagnosis and follow-up of pediatric celiac disease.
    The research team included Edith Vécsei, Stephanie Steinwendner, Hubert Kogler, Albina Innerhofer, Karin Hammer, Oskar A Haas, Gabriele Amann, Andreas Chott, Harald Vogelsang, Regine Schoenlechner, Wolfgang Huf, and Andreas Vécsei.
    They are variously affiliated with the Clinical Department of Pathology and the Department of Internal Medicine III of the Division for Gastroenterology and Hepatology, the Center for Medical Physics and Biomedical Engineering, the Department of Pediatrics and Pediatric Gastroenterology of St. Anna Children's Hospital, all at Medical University Vienna, and with the Institute of Pathology and Microbiology, Wilhelminenspital in Vienna, and with the Department of Food Science and Technology, Institute of Food Technology, University of Natural Resources and Life Sciences in Vienna, Austria.
    The team conducted a prospective cohort study at a tertiary-care center, where 148 children received biopsies either for symptoms ± positive celiac disease antibodies (group A; n = 95) or following up celiac disease diagnosed ≥ 1 year before study enrollment (group B; n = 53).
    Using biopsy (Marsh ≥ 2) as the criterion standard, they calculated areas under ROC curves (AUCs) and likelihood-ratios to gauge the performance of antibody tests against tissue transglutaminase (TG2), deamidated gliadin peptide (DGP) and endomysium (EMA).
    They found that AUC values were higher when tests were used for celiac disease diagnosis compared with follow-up: 1 vs. 0.86 (P = 0.100) for TG2-IgA, 0.85 vs. 0.74 (P = 0.421) for TG2-IgG, 0.97 vs. 0.61 (P = 0.004) for DPG-IgA, and 0.99 vs. 0.88 (P = 0.053) for DPG-IgG, respectively.
    Empirical power was 85% for the DPG-IgA comparison, and on average 33% (range 13–43) for the non-significant comparisons. A total of 88.7% of group B children showed mucosal healing, at an average of 2.2 years after primary diagnosis.
    Only the negative likelihood-ratio of EMA was low enough (0.097) to effectively rule out persistent mucosal injury. However, out of 12 EMA-positive children with mucosal healing, 9 subsequently tested EMA-negative.
    Among the celiac disease antibodies examined, negative EMA most reliably predict mucosal healing. In general, however, antibody tests, especially DPG-IgA, are of limited value in predicting the mucosal status in the early years after celiac diagnosis, though they may do better over a longer time.
    Source:
    BMC Gastroenterology 2014, 14:28. doi:10.1186/1471-230X-14-28

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    And he needs to be super strict in his gluten free diet! SUPER strict, not just low gluten. No cross contamination, NONE.  I am so sorry, there are no short cuts with the testing. It flat out sucks but there you have it.  Welcome to the forum!
    Hi TDZ, My understanding is the same, a full gluten challenge is needed for the DH diagnosis.  The method the use for DH is to take a skin biopsy from next to a lesion, not on it.  They check the biopsy for IgA antibodies. I don't know of any way to shortcut the process and avoid eating gluten to get tested.  There may be a test some  day that doesn't require it, but for now I don't think there are any out there. One thing he might not have tried is avoiding iodine.  Some of the m
    Hello, new here and new to the whole thing! My husband has been battling this rash and assorted digestive issues for years. He was diagnosed with contact dermatitis by the dermatologist, had some steroid injections and various creams over the last couple of years, and then in November he went to the ER and they said eczema and gave him steroid pills. This was after a huge bloom that pretty much hit him from head to toe, where it had been mostly arms and legs before. He finally concluded he
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