MediCel Workshop Diagnostic tools and technology transfer.

MediCel Workshop

Diagnostic tools and technology transfer

• 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.

In vitro diagnosis of coeliac disease

Elfid 2001-2010 2

• 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

Technologies

Elfid 2001-2010 3

• 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

Tissue transglutaminase

Elfid 2001-2010 4

• 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

tTG - Elisa kits

Elfid 2001-2010 5

tTG Workshop 2009

6

Elfid 2001-2010

tTG - Rapid test

Elfid 2001-2010 7

Reading windowSample addition

Red Latex-tTG complex

Blue Latex

tTG

Anti-tTG

Other antibodies

Positive resultsNegative results

Rapid tests - Literature

8

Elfid 2001-2010

• 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

Deamidated Gliadin Peptides

Elfid 2001-2010 9

EB 10

Elfid 2001-2010

• 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

Antiendomysium test

Elfid 2001-2010 11

• 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.

Conclusions

Elfid 2001-2010 12

• 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.

Genetic tests and Coeliac Disease

Elfid 2001-2010 13

• 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.

Present Situation

Elfid 2001-2010 14

What are the requirements ?

Elfid 2001-2010 15

What we look for

16

Most common haplotypes associated to Coeliac Disease

Haplotype Coded by alleles In linkage disequilibrium with

DQ2DQA1*05/DQB1*02 DR3

DQA1*0201/DQB1*02 DR7

DQ8 DQA1*03/DQB1*0302 DR4

DQ7 DQA1*05/DQB1*0301 DR11

Elfid 2001-2010

• 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

Two level-approach

Elfid 2001-2010 17

• 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

First level approach

Elfid 2001-2010 18

• 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.

Second level approach

Elfid 2001-2010 19

Eu-Gen Risk

Elfid 2001-2010 20

• Technology available• Complete information about the patient’s status• Easy interpretation of results

• Experienced technicians are required for proper test performance.

Genetic test for Coeliac Disease

Elfid 2001-2010 21

22

Elfid 2001-2010

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 2001-2010 23

2

Techniques Applied

Elfid 2001-2010 24

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 2001-2010 25

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 2001-2010 26

CD MEDICS instrument connectivity

Elfid 2001-2010 27

Hospital Information System

Hospital Information System

Laboratory Workstation

Laboratory Workstation

CD MEDICS InstrumentCD MEDICS Instrument

Demographics Requests/ Replies

Laboratory Orders / Observations

Thank you for your attention