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      Frequently Asked Questions About Celiac Disease   04/07/2018

      This Celiac.com FAQ on celiac disease will guide you to all of the basic information you will need to know about the disease, its diagnosis, testing methods, a gluten-free diet, etc.   Subscribe to Celiac.com's FREE weekly eNewsletter   What are the major symptoms of celiac disease? Celiac Disease Symptoms What testing is available for celiac disease?  Celiac Disease Screening Interpretation of Celiac Disease Blood Test Results Can I be tested even though I am eating gluten free? How long must gluten be taken for the serological tests to be meaningful? The Gluten-Free Diet 101 - A Beginner's Guide to Going Gluten-Free Is celiac inherited? Should my children be tested? Ten Facts About Celiac Disease Genetic Testing Is there a link between celiac and other autoimmune diseases? Celiac Disease Research: Associated Diseases and Disorders Is there a list of gluten foods to avoid? Unsafe Gluten-Free Food List (Unsafe Ingredients) Is there a list of gluten free foods? Safe Gluten-Free Food List (Safe Ingredients) Gluten-Free Alcoholic Beverages Distilled Spirits (Grain Alcohols) and Vinegar: Are they Gluten-Free? Where does gluten hide? Additional Things to Beware of to Maintain a 100% Gluten-Free Diet What if my doctor won't listen to me? An Open Letter to Skeptical Health Care Practitioners Gluten-Free recipes: Gluten-Free Recipes
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    HEMOLYSIS INTERFERES WITH THE DETECTION OF ANTI-TISSUE TRANSGLUTAMINASE ANTIBODIES IN CELIAC DISEASE


    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.


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    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:


    Image Caption: New research on hemolysis and celiac disease.
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  • Related Articles

    Jefferson Adams

    Celiac.com 07/10/2007 - A study published recently in the American Journal of Gastroenterology tracks the appearance and disappearance of antibodies associated with childhood risk celiac disease, and suggests that key antibodies often disappear even when gluten is still present in the diet.
    A team of Finnish doctors set out to evaluate the natural history of antibodies versus tissue transglutaminase (TGA), endomysium (EMA), reticulin (ARA), and gliadin (AGA-IgG and AGA-IgA). They looked at data for children genetically at risk for celiac disease, specifically, children who carried HLA-conferred risk of celiac disease who had been monitored frequently since birth. The research team was made up of S. Simell, S. Hoppu, A. Hekkala, T. Simell, M.R. Ståhlberg, M. Viander, H. Yrjänäinen, J. Grönlund, P. Markula, V. Simell, M. Knip, J. Ilonen, H. Hyöty, O. Simell.
    The team looked at serum samples from 1,320 children who were genetically at risk for celiac disease. Serum samples taken between 2000 and 2003 were assessed for TGA. Samples testing positive for TGA were evaluated for all five antibodies. Also, all future samples for the given patient were similarly evaluated. Also, positive TGA patients were encouraged to have a duodenal biopsy.
    The assessment was completed in August 2004. At that time, the test subjects ranged in age from 1 year to 9.5 years, with a mean age of 4.1 years. In all, 49 children (3.7%) were TGA positive. 26 of these TGA positive children submitted to biopsy. Celiac disease was diagnosed by biopsy in 20 of the 26. Of the 49 children who tested TGA positive, AGA-IgA surfaced at an average age of 2 years (+/- 1.5 over a range of 0.5 to 6.6 years for subjects). Compared to AGA-IgA, TGA, EMA, and ARA all surfaced together about 1 year later (TGA at 3.2 +/- 1.5, 1.0-7.0 yr, P < 0.001).
    Key Antibodies Can Vanish Early in Childhood Celiac Disease
    Even with ongoing gluten consumption, positive TGA values disappeared in 49%, EMA values disappeared in 49%, ARA values disappeared in 43%, AGA-IgA values disappeared in 41%, and AGA-IgG in 32%.
    The research team concluded that there are likely potential triggers for celiac disease that are active before AGA-IgA surfaces, or about 3 months earlier on average than when the TGA-associated antibodies appear.
    In a significant number of children, antibodies vanish spontaneously. This indicates that in many cases, conditions allow the regulatory immune phenomena to eliminate incipient celiac disease in genetically at-risk children even when gluten is still significant part of the diet.
    Am J Gastroenterol 2007;102:1–10
     

    Jefferson Adams
    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.
    The 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.
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    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.
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    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.
    Source:
    Frontline Gastroenterol. 2012;3(2):81-83.

    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.
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    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.
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    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.
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    Source:
    BMC Gastroenterology 2014, 14:28. doi:10.1186/1471-230X-14-28

    Jefferson Adams
    Celiac.com 06/06/2014 - Celiac disease guidelines suggest that some patients with high anti-tTG ab levels might be diagnosed without biopsy.
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    The team reviewed data on 366 anti-tTG ab-positive individuals who received duodenal biopsies. The team conducted anti-tTG ab screens before patients began a gluten-free diet, and they expressed anti-tTG ab results in terms of fold-rise by calculating ratio of observed values with cutoff value. Celiac disease was diagnosed only in patients with positive serology, villous atrophy greater than Marsh grade 2, and clear response to gluten-free diet.
    Average anti-tTG fold-rise in groups with Marsh grade ≤2 was 2.6 (±2.5), grade 3a was 4.0 (±3.9), 3b was 5.7 (±5.1), and 3c was 11.8 (±8.0). Overall positive likelihood ratio for diagnosing celiac disease was 15.4 and 27.4 at 12- and 14-fold-rise of anti-tTG ab titer, respectively.
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    Contrary to emerging wisdom, even patients with anti-tTG ab levels less than 2-times baseline should receive mucosal biopsies, because many patients with celiac disease have such low levels.
    Source:
     J Clin Gastroenterol. 2014 Feb 27.

  • Recent Articles

    Jefferson Adams
    Celiac.com 04/23/2018 - A team of researchers recently set out to learn whether celiac disease patients commonly suffer cognitive impairment at the time they are diagnosed, and to compare their cognitive performance with non-celiac subjects with similar chronic symptoms and to a group of healthy control subjects.
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    J Clin Gastroenterol. 2018 Mar 1. doi: 10.1097/MCG.0000000000001018.

    Connie Sarros
    Celiac.com 04/21/2018 - Dear Friends and Readers,
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    Jefferson Adams
    Celiac.com 04/20/2018 - A digital media company and a label data company are teaming up to help major manufacturers target, reach and convert their desired shoppers based on dietary needs, such as gluten-free diet. The deal could bring synergy in emerging markets such as the gluten-free and allergen-free markets, which represent major growth sectors in the global food industry. 
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    fdfworld.com

    Jefferson Adams
    Celiac.com 04/19/2018 - Previous genome and linkage studies indicate the existence of a new disease triggering mechanism that involves amino acid metabolism and nutrient sensing signaling pathways. In an effort to determine if amino acids might play a role in the development of celiac disease, a team of researchers recently set out to investigate if plasma amino acid levels differed among children with celiac disease compared with a control group.
     
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    Source:
    PLoS One. 2018; 13(3): e0193764. doi: & 10.1371/journal.pone.0193764

    Jefferson Adams
    Celiac.com 04/18/2018 - To the relief of many bewildered passengers and crew, no more comfort turkeys, geese, possums or other questionable pets will be flying on Delta or United without meeting the airlines' strict new requirements for service animals.
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    Source:
    cnbc.com