Diagnostic tools and technology transfer

1. Diagnostic tools and technology transfer MediCel Workshop

2. In vitro diagnosis of coeliac disease • The serologic in vitro diagnosis of coeliac disease is based on the following tests: • tTG • DPG • AEA • AGA • In addition, recently, genetic tests have also become available on the market.

3. Technologies • Available technologies can be adapted to any lab size and needs, both in terms of financial and operative resources • ELISA • tTG, AGA, DPG • IFI • AEA • Dot-Blot • tTG, AGA • PCR • HLA

4. Tissue transglutaminase • Tissue transglutaminase is the auto-antigen responsible for AEA positivity in coeliac disease. • The introduction of tTG is a milestone in the knowledge and in vitro diagnosis of CD. • tTG based kits are the reference method for the in vitro diagnosis of coeliac disease • Both ELISA and Rapid tests are available

5. tTG - Elisa kits ELISA tTG IgA is the reference method/test for in vitro diagnosis of coeliac disease Higher sensitivity than AEA IgG should be used in case of Total IgA deficiency mainly Kits on the market show different sensitivity and specificity values depending on the chosen antigen and/or diagnostic approach Need of instrumentation can be limited to an Elisa reader

6. tTG Workshop 2009

7. tTG - Rapid test Sample addition Reading window Positive results Negative results

8. Rapid tests - Literature

9. Deamidated Gliadin Peptides Newly introduced in the market More sensitive and specific than AGA Useful in suspected CD patients Limited use in patients with other gastrointestinal disorders Might be a complement to tTG assays in young children Useful in GFD follow-up

11. Antiendomysium test • Highest specificity • Sensitivity lower than tTG • IFA technology normally available in all labs • Raising cost of and limited availability of animal substrate • HUC is an alternative

12. Conclusions • tTG autoantibodies is still the best test for CD. • tTG autoantibody assays vary greatly qualitatively. • IgG tTG are not as reliable as IgA in screening procedures. • The introduction of DPG IgG might be helpful in some cases. • Children younger than 2 yrs of age • GFD follow up • Total Serum IgA deficiency • Both ELISA and Rapid test are available. • Limited need of instrumentation.

13. Genetic tests and Coeliac Disease During the last years, many papers have been published about genetics of CD. Both HLA and non-HLA have been extensively studied. An increased number of patients is today subjected to genetic tests.

14. Present Situation • Request of genetic tests for CD is growing up rapidly. • Detection of DQ2 a/o DQ8 heterodimers only does no longer meet customers' needs. • Knowledge of DR status is now a MUST and is required in medium/small centres dealing with Coeliac Disease, not only reference centres.

15. What are the requirements ? • Complete HLA status: • DQ • DR • Identification of patients at risk. • Possibility to exclude some patients before proceeding to a further testing for risk definition.

16. What we look for

17. Two level-approach • First Level: screening (e.g. Eu-Gen) • DQ 2 and DQ8 • Selection of patients requiring additional tests • Second level: definition of predisposition (e.g. Eu-Gen Risk) • DR (3, 4, 7, 11) and DQ (2, 8) genotypes • Complete haplotypes • DQB1*02 status • Relative risk to develop Coeliac Disease

18. First level approach Mix 4 DQA1*05 DQB1*0302 Mix 6 DQB1*02 DR4 DQ2 • Inclusion/Exclusion kit • 2 mixes containing specific primers for: • DQA1*05, DQB1*02, DRB1*04, DQB1*0302. • Positive response to any allele: indicates need of further testing • Negative response: low or minimal risk. Further tests are not required DQ2/DQ8 Mix 4 DQA1*05 DQB1*0302 Mix 6 DQB1*02 DR4 Mix 4 DQA1*05 DQB1*0302 Mix 6 DQB1*02 DR4 DQ8 Mix 4 DQA1*05 DQB1*0302 Mix 6 DQB1*02 DR4 NonDQ2/8

19. Second level approach • 8 mixes with specific primers providing a combined results allowing: • Identification of DQ and DR genotypes • Definition of complete haplotypes • Definition of DQB1*02 status (homo / heterozygosis) if present • Definition of relative risk for any tested patient.

20. Eu-Gen Risk

21. Genetic test for Coeliac Disease • Technology available • Complete information about the patient’s status • Easy interpretation of results • Experienced technicians are required for proper test performance.

23. CD-MEDICS Overall Objectives • Development of disposable microchip for screening of coeliac disease in a portable/hand-held device carrying out multi-analyte tests • Simultaneous detection of coeliac disease associated autoantibodies (DPG and tissue transglutaminase) and HLA-DQ2 and DQ8 genes. • The device will have embedded communication abilities for result storage and easy follow-up, management and monitoring of the patient’s response to withdrawal of gluten from the diet.

24. Techniques Applied 2

25. Clinically Relevant Outcome • Population based screening, and/or screening of high-risk groups and based on the data regarding HLA status, design individually tailored dietary options. • Monitoring for adherence to the appropriate gluten-free diet either at their GP’s office, or from their own home, with data being communicated directly to their electronic medical record. • Neonatal screening in risk groups to identify newborns who may develop coeliac disease.

26. Features of the system • Standardisation of calibrators • Levels of IgA and IgG anti-tTG antibodies measured in many patient samples. • To establish absolute concentrations in terms of ng/mL rather than arbitrary units. • IgA and IgG anti-tTG sera of coeliac disease patients to be used as standard calibrator/controls. • Clinical evaluation to see if specificity and sensitivity can be improved.

27. CD MEDICS instrument connectivity Hospital Information System CD MEDICS Instrument Demographics Requests/ Replies Laboratory Workstation Laboratory Orders / Observations

28. Thank you for your attention