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Diagnostic tools and
technology transfer
MediCel Workshop
In vitro diagnosis of coeliac disease
• The serologic in vitro diagnosis of coeliac disease is
based on the following tests:
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tTG
DPG
AEA
AGA
• In addition, recently, genetic tests have also become
available on the market.
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Technologies
• Available technologies can be adapted to any lab
size and needs, both in terms of financial and
operative resources
• ELISA
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tTG, AGA, DPG
• IFI
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AEA
• Dot-Blot
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tTG, AGA
• PCR
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HLA
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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
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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
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tTG Workshop 2009
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tTG - Rapid test
Sample addition
Reading window
Red Latex-tTG complex
Blue Latex
tTG
Anti-tTG
Other antibodies
Positive results
Negative
results
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Rapid tests - Literature
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Deamidated Gliadin Peptides
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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
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EB 10
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Antiendomysium test
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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
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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.
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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.
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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.
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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.
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What are the requirements ?
• Complete HLA status:
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DQ
DR
• Identification of patients at risk.
• Possibility to exclude some patients before
proceeding to a further testing for risk definition.
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What we look for
Most common haplotypes associated to Coeliac Disease
Haplotype
Coded by alleles
In linkage disequilibrium with
DQA1*05/DQB1*02
DR3
DQA1*0201/DQB1*02
DR7
DQ8
DQA1*03/DQB1*0302
DR4
DQ7
DQA1*05/DQB1*0301
DR11
DQ2
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Two level-approach
• First Level: screening (e.g. Eu-Gen)
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DQ 2 and DQ8
Selection of patients requiring additional tests
• Second level: definition of predisposition
(e.g. Eu-Gen Risk)
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DR (3, 4, 7, 11) and DQ (2, 8) genotypes
Complete haplotypes
DQB1*02 status
Relative risk to develop Coeliac Disease
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First level approach
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DQA1*05
• Mix
Inclusion/Exclusion
kit DQB1*0302
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DQ2
2 mixes containing specific primers for:
Mix 6
DQB1*02
DR4
DQA1*05, DQB1*02, DRB1*04, DQB1*0302.
Mix 4
DQA1*05
DQB1*0302
DQ2/DQ8
Positive response to any allele: indicates need of further
Mix 6testingDQB1*02
DR4
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Mix 4
DQA1*05
DQB1*0302
Negative response:
Mix 6low orDQB1*02
DR4
minimal risk. Further
tests are not required
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Mix 4
DQA1*05
DQB1*0302
Mix 6
DQB1*02
DR4
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DQ8
Non DQ2/8
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Second level approach
• 8 mixes with specific primers providing a combined
results allowing:
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Identification of DQ and DR genotypes
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Definition of complete haplotypes
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Definition of DQB1*02 status (homo / heterozygosis) if
present
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Definition of relative risk for any tested patient.
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Eu-Gen Risk
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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.
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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.
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Techniques Applied
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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 glutenfree 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.
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Features of the system
• Standardisation of calibrators
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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.
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CD MEDICS instrument connectivity
Hospital
Information
System
Demographics
Requests/ Replies
Laboratory
Workstation
CD MEDICS
Instrument
Laboratory Orders /
Observations
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Thank you for your attention