Coeliac Disease Study Group SELF TRANSGLUTAMINASE-BASED ONE-MINUTE ONSITE BLOOD TEST FOR CELIAC DISEASE Ilma Korponay-Szabó, Tiina Raivio, Katri Kaukinen, Judit B.Kovács, Pekka Collin, Kaija Laurila, Judit B.Kovács, Pekka Collin, Kaija Laurila, Jukka Partanen, Markku Mäki Pediatric Research Centre and Dept. of Internal Medicine, University of Tampere, Finland, University of Tampere, Finland, University of Debrecen, Debrecen, Hungary Heim Pál Children’s Hospital, Budapest, Hungary

Coeliac Disease Study Group Circulating antibodies against type-2(tissue) tansglutaminase (TG) are good non-invasive markers of gluten-sensitive enteropathy.Circulating antibodies against type-2(tissue) tansglutaminase (TG) are good non-invasive markers of gluten-sensitive enteropathy. Antibodies against TG are currently measured in specialized laboratories using ELISA or immunofluorescent method (endomysial antibody test, EMA).Antibodies against TG are currently measured in specialized laboratories using ELISA or immunofluorescent method (endomysial antibody test, EMA). The testing is expensive and the tournaround time of results is considerably long.The testing is expensive and the tournaround time of results is considerably long. → Q uick, easy to perform, economical → Q uick, easy to perform, economical and widely accessible celiac tests and widely accessible celiac tests are needed are needed BACKGROUND AND AIMS

Coeliac Disease Study Group PRINCIPLE OF THE TESTING Whole blood samples contain endogeneous TG antigen inside normal red blood cells. TG Hemolysis Hemolysis TG RBC self-TG can be used as antigen to detect anti-TG antibodies present in the plasma of the same sample. of the same sample. TG Formation of self-TG/anti-TGcomplexes TG Label(anti-IgA) Capture of the labelled complexes by TG-binding protein

Coeliac Disease Study Group PRINCIPLE OF THE TESTING Whole blood samples contain endogeneous TG antigen inside normal red blood cells. TG Hemolysis Hemolysis TG RBC self-TG can be used as antigen to detect anti-TG antibodies present in the plasma of the same sample. of the same sample. TG Formation of self-TG/anti-TGcomplexes TG Label(anti-IgA) Nunc Immunstick™ surface (Maxisorp) Sensitive and specific for CD Color reaction entirely dependent on the presence of RBC-TG (no color when using TG-/- mouse RBC+serum TG+/+ TG-/- RBC

Coeliac Disease Study Group PRINCIPLE OF THE TESTING Whole blood samples contain endogeneous TG antigen inside normal red blood cells. TG Hemolysis Hemolysis TG RBC self-TG can be used as antigen to detect anti-TG antibodies present in the plasma of the same sample. of the same sample. TG Formation of self-TG/anti-TGcomplexes TG Label(anti-IgA) Capture of the labelled complexes by TG-binding protein line Lateral flow

Coeliac Disease Study Group The lateral flow system Biocard™ Celiac Disease Test AniBiotech, Vantaa, Finland

Coeliac Disease Study Group A/ Retrospective cohort (n=224) GI patients undergoing endoscopy Gold standard: histology Evaluation on stored whole blood by blinded observers untreated celiac patients  12 also after 1y on diet non-celiac controls age: 1,8-72 years, median, 14 Comparison with serum antibody measurements EMA (indirect immunofluorescence on monkey esophagus) TG-Ab ELISA (Celikey™, Pharmacia) PATIENTS & METHODS Clinical diagnosis in the controls Dyspepsia or diarrhea61 GOR1515 IBD14 Cong. sucrase deficiency5 FAP4 Recurrent abdominal pain, IBS 4 Pancreatic disease (CF, Swachman-Diamond sy) 2 Nonspecific rash1 All107

Coeliac Disease Study Group B/ Prospective cohort (n=168) - Symptomatic patients (n=69) (GI, iron deficiency, short stature, dental enamel defects, chronic fatigue) - Relatives of CD subjects (n=72) - Healthy volunteers (n=27) Testing in the outpatient office or at home Gold standard: EMA/TG-Ab (kappa-statistics) Subjects with positive results were referred for small-bowel biopsy PATIENTS & METHODS

Coeliac Disease Study Group TEST RESULT Positive Negative Control line Celiac antibody detection (at 5 minutes) Biocard™ Celiac Disease Test AniBiotech, Vantaa, Finland

Coeliac Disease Study Group RESULTS Celiac disease (severe villous atrophy) Non-celiac (normal villous architecture) Rapid test+ 1147* Rapid test Total Sensitivity (%) Specificity (%) Rapid test 97.4 (CI: ) 93.5 (CI: ) EMA 99.1 (CI: ) 100 TG-Ab ELISA 99.1 (CI: ) 100 *Anti-TG antibodies deposited in the gut mucosa: 3 Partial villous atrophy in the past: 1 Retrospectively studied patients / Correlation with histology:

Coeliac Disease Study Group Agreement of lateral flow test results with standard laboratory antibody tests from serum poor good excellent 0.89 Biocard / EMA 0.88 Biocard / TG-ELISA Kappa statistics Inter-observer agreement on 28 samples: same result

Coeliac Disease Study Group % Prospectively studied patients: (fresh blood samples) EMA+ EMA- Rapid test+ 301 Rapid test Total31137 TG-Ab ELISA+ TG-Ab ELISA- Rapid test+ 301 Rapid test Total30138 Concordant: 98.8% Concordant: 99.4%

Coeliac Disease Study Group Positive CD by biopsy Negative CD by biopsy Rapid test 3025/261381/9 EMA3126/271370/8 TG-Ab3025/261381/9 Prospectively studied patients: 35 /168 had a jejunal biopsy performed 26 new celiac cases were diagnosed 68 g/l = lowest hemoglobin level among CD patients Self TG-based rapid test result : +

Coeliac Disease Study Group Ten of the 12 rapid test+ coeliac patients reevaluated after one year on diet had negative lateral flow test result. The two patients still positive had TG- antibody ELISA levels near the cut-off (4.6 and 4.2 U/ml). Lateral flow test results before and after a gluten-free diet for one year positive lateral flow negative lateral flow Cut-off TG-Ab ELISA

Coeliac Disease Study Group Results are available within 5 minutes onsite.Results are available within 5 minutes onsite. The testing is simple and easy to perform from capillary blood.The testing is simple and easy to perform from capillary blood. No recombinant / purified TG2 antigen is needed.No recombinant / purified TG2 antigen is needed. No laboratory equipment is needed.No laboratory equipment is needed. No blood centrifugation is needed.No blood centrifugation is needed. Suitable for home useSuitable for home use ADVANTAGES

Coeliac Disease Study Group Not meant for final diagnosis (biopsy is still required)Not meant for final diagnosis (biopsy is still required) May be negative if the subject had adopted a gluten-free diet before testingMay be negative if the subject had adopted a gluten-free diet before testing Not suitable to show decrease or increase in antibody concentrations (plus/minus test)Not suitable to show decrease or increase in antibody concentrations (plus/minus test) Immunoglobulin deficiency (IgA)Immunoglobulin deficiency (IgA) LIMITATIONS

Coeliac Disease Study Group The self TG-based rapid celiac antibody test was highly accurate in detecting new celiac cases.The self TG-based rapid celiac antibody test was highly accurate in detecting new celiac cases. Its sensitivity was comparable to that of EMA testing and TG-antibody detection by ELISA.Its sensitivity was comparable to that of EMA testing and TG-antibody detection by ELISA. The rapid test was able to show the clearence of antibodies after a prolonged gluten-free diet.The rapid test was able to show the clearence of antibodies after a prolonged gluten-free diet. Immediate availability of results may help physicians to speed up the diagnostic process and to evaluate dietary compliance.Immediate availability of results may help physicians to speed up the diagnostic process and to evaluate dietary compliance. CONCLUSIONS

Coeliac Disease Study Group  Case finding among symptomatic patients in the office or ward in the office or ward  Screening of subjects at-risk for celiac disease family members, autoimmune patients, pregnant women family members, autoimmune patients, pregnant women  Population screening studies  Diet monitoring in the office or by home testing (longterm surveillance) of celiac antibody detection at the point of care POTENTIAL APPLICATIONS