Continued efforts for standardization · READY-TO-USE CALIBRATORS (target conc. in pg/ml) ID hTau...

© 2014 Fujirebio Europe

Continued efforts for standardizationPresentation to GBSC meeting Copenhagen 2014

12 July 2014

Crowne Plaza Towers

[email protected]


Outline

- Standardization of INNOTEST ® products

- Launch of the new INNOTEST ® β-AMYLOID (1-40)

- Resolving the INNOTEST ® β-AMYLOID (1-42) matrix effect


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

3


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

4

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


Neuro INNOTEST ® improvementsOverview of the calibrators

5

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)

5


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

6

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


Test protocols for automated processing of the Neuro INNOTESTs are verified.

Dynex Technologies DS2® plate processing instrument

Update of INNOTEST ® assaysAutomation

7

INNOTEST® update info:

P1-158

P1-159

P4-037


Steps towards accuracy based assaysBringing contexts of use together in one product

8

clinical focus

reliable cut-off values

patient diagnosis / stratification

AD pathology severity

dynamic focus

change in biomarker

patient follow-up/

treatment monitoring


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


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

10

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


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

11

mean

At a MRD of 1 / 4, the average variability over back calculated CSF concentrations = 5%


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

12

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


INNOTEST® β-AMYLOID (1-42) feasibilityEvaluation spiking with A β peptides

13

Spike recovery with Aβ1-42 peptide

Addition of increasing amounts of Aβ1-40 peptide


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

14

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%


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

15


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

Continued efforts for standardization · READY-TO-USE CALIBRATORS (target conc. in pg/ml) ID hTau...

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