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.
Elfid

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
Elfid

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
Elfid

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
Elfid

6. tTG Workshop 2009
Elfid

7. tTG - Rapid test Negative results Positive results
Sample addition
Reading window
Red Latex-tTG complex
Blue Latex
tTG
Anti-tTG
Other antibodies
Negative results
Positive results
Elfid

8. Rapid tests - Literature
Elfid

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
Elfid

10. Elfid

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
Elfid

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.
Elfid

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.
Elfid

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.
Elfid

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.
Elfid

16. What we look for Most common haplotypes associated to Coeliac Disease
Coded by alleles
In linkage disequilibrium with
DQ2
DQA1*05/DQB1*02
DR3
DQA1*0201/DQB1*02
DR7
DQ8
DQA1*03/DQB1*0302
DR4
DQ7
DQA1*05/DQB1*0301
DR11
Elfid

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
Elfid

18. First level approach DQ2 DQ2/DQ8 DQ8 Non DQ2/8 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
Mix 4 DQA1*05 DQB1*0302
Mix 6 DQB1*02 DR4
Non DQ2/8
DQ2
DQ8
DQ2/DQ8
Elfid

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.
Elfid

20. Eu-Gen Risk 
Elfid

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.
Elfid

22. Elfid

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.
Elfid

24. Techniques Applied
Elfid 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.
Elfid

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.
Elfid

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

28. Thank you for your attention