continued efforts for standardization · ready-to-use calibrators (target conc. in pg/ml) id htau...
TRANSCRIPT
© 2014 Fujirebio Europe
Continued efforts for standardizationPresentation to GBSC meeting Copenhagen 2014
12 July 2014
Crowne Plaza Towers
© 2014 Fujirebio Europe
Outline
- Standardization of INNOTEST ® products
- Launch of the new INNOTEST ® β-AMYLOID (1-40)
- Resolving the INNOTEST ® β-AMYLOID (1-42) matrix effect
© 2014 Fujirebio Europe
For each of the INNOTEST® assays, Run Validation Control samples and Ready-to-Use Calibrators, and harmonize buffer composition are implemented.
Ready-to-Use Calibrators
• reduction of the variation during test performance
• increase ease of use (reduction hands-on time)
Run Validation Controls (RVC)
• validation of the test runs
• alignment with lab accreditation requirements (ISO15189)
Harmonized kit reagents
• improve ease of use
• facilitate automation
• step towards harmonized test procedures
Neuro INNOTEST ® improvements
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© 2014 Fujirebio Europe
All INNOTEST® assays have generic / harmonized kit components
� exchange of components over products and lots is possible!
• NO changes to the product concept and set-up were made !
Neuro INNOTEST ® improvementsHarmonization of buffer components
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INNOTEST® hTAU Ag INNOTEST® β-Amyloid(1-42) INNOTEST® β-Amyloid(1-40) INNOTEST® PHOSPHO-TAU(181P)
PLATE hTAU Ag PLATE β-Amyloid(1-42) PLATE β-Amyloid(1-40) PLATE PHOSPHO-TAU(181P)
Conjugate 1 Conjugate 1 Conjugate 1 Conjugate 1
Conjugate 2 Conjugate 2 Conjugate 2 Conjugate 2
Conjugate Diluent 1 = Conjugate Diluent 1 = Conjugate Diluent 1 ≠ Conjugate Diluent 1
Conjugate Diluent 2 = Conjugate Diluent 2 = Conjugate Diluent 2 = Conjugate Diluent 2
Sample Diluent = Sample Diluent = Sample Diluent = Sample Diluent
Substrate = Substrate = Substrate = Substrate
Substrate Buffer = Substrate Buffer = Substrate Buffer = Substrate Buffer
Stop Solution = Stop Solution = Stop Solution = Stop Solution
Wash Solution = Wash Solution = Wash Solution = Wash Solution
New composition
© 2014 Fujirebio Europe
Neuro INNOTEST ® improvementsOverview of the calibrators
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Storage: -25°C to -15°C
Single use only
each RTU CAL & RVC, 2 vials/ kit.
Previous Situation
NEW SITUATION
READY-TO-USE CALIBRATORS (target conc. in pg/ml)
ID hTau Ag β-AMYLOID (1-42)
P-Tau 181P
CAL 1 2500 4000 1000
CAL 2 900 1750 250
CAL 3 600 1000 125
CAL 4 300 500 62.5
CAL 5 150 125 31.3
CAL 6 75 62.5 15.6
RVC 1 800 1300 200
RVC 2 200 250 40
RUN VALIDATION CONTROL SAMPLES
(target conc. in pg/ml)
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© 2014 Fujirebio Europe
Impact of using RTU CALs compared to lab made dilutions series of concentrated standard was evaluated during round 1 of JPND WP A3 (lab defined SOP)
10 Experienced/ High-throughput labs evaluating 12 CSF samples with both calibration curves in parallel.
Neuro INNOTEST ® improvementsProduct validation: inter-site evaluation
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INNOTEST®
Inter-center variability
(%CV)% reduction
of
variabilitykit Standard RTU CAL
Aβ (1-42) 17,4% 12,2% 30%
hTau 13,3% 9,5% 29%
P-Tau 181P 12,0% 8,8% 26%
More detailed analysis and info � P4-049
© 2014 Fujirebio Europe
Test protocols for automated processing of the Neuro INNOTESTs are verified.
Dynex Technologies DS2® plate processing instrument
Update of INNOTEST ® assaysAutomation
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INNOTEST® update info:
P1-158
P1-159
P4-037
© 2014 Fujirebio Europe
Steps towards accuracy based assaysBringing contexts of use together in one product
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clinical focus
reliable cut-off values
patient diagnosis / stratification
AD pathology severity
dynamic focus
change in biomarker
patient follow-up/
treatment monitoring
© 2014 Fujirebio Europe
Steps towards accuracy based assaysBringing contexts of use together in one product
9
clinical focus
reliable cut-off values
patient diagnosis / stratification
AD pathology severity
dynamic focus
change in biomarker
patient follow-up/
treatment monitoring
2008, Neurobiol Aging
© 2014 Fujirebio Europe
Evaluation of the assay “variables”:
• kit components used
• test procedure (volumes and incubation times)
• assay set-up: simultaneous vs. sequential incubation of sample and detection conjugate
INNOTEST® β-AMYLOID (1-42) feasibilityParameter review
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Variable Conjugate 1 incubation time,Fixed sample incubation time (1h)
Aβ(1-42) pg/mlO
D45
0n
m101 102 103 1040
1
2
3
1h 30'1h 1h1h 2h1h 3h1h o/n
Incubation TimeSample - Conjugate 1:
Variable Sample incubation time,Fixed conjugate 1 time (1h)
Aβ(1-42) pg/ml
OD
450n
m
101 102 103 1040
1
2
3
30' 1h1h 1h2h 1h3h 1h
Incubation TimeSample - Conjugate 1:
o/n 1h
© 2014 Fujirebio Europe
Optimal assay procedure was defined
Tested with a set of 16 CSF samples for determination of MRD � dilution in kit Sample Diluent
INNOTEST® β-AMYLOID (1-42) feasibilityDetermination of minimal required dilution
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mean
At a MRD of 1 / 4, the average variability over back calculated CSF concentrations = 5%
© 2014 Fujirebio Europe
A set of 8 CSF samples was pre-treated with a low volume of Tween solution, afterwards the neat and pre-treated CSF were directly diluted with kit Sample Diluent in a pre-plate.
Evaluation over 3 runs
INNOTEST® β-AMYLOID (1-42) feasibilityNo need for Tween at test set-up
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Mean conc. (pg/ml) % CV Mean conc. (pg/ml) % CV
CSF1 1324 6% 1241 5% 7%
CSF2 909 8% 831 9% 9%
CSF3 1131 9% 1064 10% 6%
CSF4 375 5% 356 8% 5%
CSF5 853 4% 892 4% -4%
CSF6 1898 5% 1818 4% 4%
CSF7 1079 3% 993 5% 9%
CSF8 812 1% 755 7% 8%
Mean 5% 6% 6%
pre-treatment with Tween (30 min)
+ 1/4 dilution in SD
only 1/4 dilution in
Sample DiluentIDConc. diff.
induced by Tween
© 2014 Fujirebio Europe
INNOTEST® β-AMYLOID (1-42) feasibilityEvaluation spiking with A β peptides
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Spike recovery with Aβ1-42 peptide
Addition of increasing amounts of Aβ1-40 peptide
© 2014 Fujirebio Europe
40 individual CSF samples + 2 CSF pools in each run
3 independent runs for each method
INNOTEST® β-AMYLOID (1-42) feasibilityPreliminary comparison PI to improved procedure
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Standard curves over runs
Aβ (1-42) pg/ml
OD
(450
nm)
100 101 102 103 1040
1
2
3
run 1_PI
run1_Impr
run2_PI
run2_Impr
Run3_PI
Run3_Impr
inter-run %CV = ± 7%
© 2014 Fujirebio Europe
The ease of use for the Neuro INNOTEST® assays was increased
RTU CALs
included RVC samples
automation
Implementation of RTU Calibrators for the Neuro INNOTEST® assays reduced inter-site variability significantly. This result should become visible in the AA ww QC program.
Launch of the INNOTEST® β-AMYLOID(1-40) will help in CSF AD BM evaluation for “high producers”
Possible improvements to the INNOTEST® β-AMYLOID(1-42) assay eliminated the matrix effect, further evaluation and re-assessment of the clinical cut-off are required
The addition of Tween at assay set-up had little impact of the outcome, early introduction of Tween in the pre-analytical trajectory needs to be investigated further.
Continued efforts for standardizationConclusion
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© 2014 Fujirebio Europe
Acknowledgment
Vesna Kostanjevecki
Hilde Decraemer
Roger Moonen
Bart De Decker
Caroline Byl
Martine Dauwe
Katrien Allcock
Marjan De La Court
Nathalie Le Bastard
Katsumi Aoyagi
Peggy Ruymbeeck
John Lawson
Sandra Pereson
Annelies Vandersteen
Laura Vernoux
Yasmine Djellas
Heather Darby
and many more