1. IRMM/IFCC Project Overview
Henrik Zetterberg, MD, PhD
Professor of Neurochemistry
University of Gothenburg and University College London
Ingrid Zegers, PhD
Joint Research Centre, the European Commission's science and knowledge service

2. Reference Materials for AD IVDs
The EC adopted the Communication from the Commission COM(2009) 380/4 to the European Parliament and the Council on a European initiative on AD and other dementias
4 key areas are identified → support early diagnosis
Reliable biomarker measurements will improve early diagnosis
Alzheimer biomarkers have been added to the criteria for diagnosis
→ early diagnoses
→ enable clinical studies on treatments for the early stages of the disease
Clinical reference materials developed in support of the implementation of the EU Directive on In Vitro Diagnostics Medical Devices (IVD-MD) (Directive 98/79/EC)

3. Collaborations and International Context
GBSC!
IFCC Working Group for CSF Proteins Develop reference materials for CSF proteins, main targets are A1-42, T-tau and P-tau.
The Alzheimer’s Association Funding a quality control program for CSF biomarkers, actively supports the standardisation of biomarkers
The European Commission funds the project BIOMARKAPD in the context of the EU Joint Programme - Neurodegenerative Disease Research
Industry SMEs and multinationals are developing assays for Alzheimer’s biomarkers; many are keen to collaborate in the standardisation process
SME = Small and medium-sized enterprises

4. Biomarkers of Interest
Amyloid 1-42 (A1-42)
Healthy: >500 ng/L
Decrease in Alzheimer patients by about 50 %
Reflects plaques
Total tau protein (T-tau)
Healthy: <300 ng/L
Increased in AD, Creutzfeld-Jacob (CJD), and stroke (by about 300 %)
Reflects the intensity of neuronal degeneration
6 isoforms
Phosphorylated tau protein (P-tau)
Increased in AD, but not CJD and stroke
Many phosphorylation sites, but Thr181 is main one
Reflects neurofibrillary tangles

5. Traceability Chain

6. First Steps in CRM Production Process
Essential steps:
Development of Reference Measurement Procedure
Preparation & characterization of calibrant
Feasibility studies:
Collection
Storage
Processing
Commutability
Characterization
643 ± 48 ng/L (CV = 7.5 %)
M. Bjerke et al., International Journal of Alzheimer’s Disease 2010; 2010: pii

7. Commutability Study I 5 immunoassays & 1 LC-MS method
Tested 16 candidate CRM formats & addition of detergent
M. Bjerke et al., CCLM 2016, ePub 10/2015.

8. Commutability Study II
8 immunoassays & 1 LC-MS method
Tested 1 CSF pool & 3 Aβ1-42 spike levels
Only non-spiked CSF pool commutable for all tested method combinations
→ Decision to produce 3 CRMs
M. Bjerke et al., CCLM 2016, ePub 10/2015.

9. Candidate CRMs at three concentration levels (ran in quadruplicates)
Commutability Study III
Candidate CRMs at three concentration levels (ran in quadruplicates)
Thirty-four individual CSF samples (ran in duplicates)
Six different methods plus the RMP
Samples analyzed by the manufacturers
Analyzed using Passing-Bablok regression and Spearman’s rho 9

10. Commutability III- Result example
Individual CSF
Candidate CRM
Passing-Bablok
95% CI
y=x 10

11. Commutability III- Slopes and correlations
Correlations (upper right) and slopes (lower left) 11

12. Processing Feasibility Studies
Manual vs. machine filling
Stirring vs. no stirring
Flash vs. slow freezing
Aβ1-42 concentration over time while stirring

13. Conditions chosen: - Machine filling - Stirring - Slow freezing
Statistical evaluation of data:
Soverall = 3.8 %
Srepeatability = 3.2 %
(method variability)
Sbetween vial = 1.9 %
(between vial variability) 13

14. Candidate CRMs Processing
Matrix materials: 3 CSF pools (6-7 different patients per pool), not spiked
Filled 0.5 mL/vial
3 levels: low, medium and high Aβ1-42 concentration
Stored at -70 °C
Homogeneity
Abeta1-42:
ERM-DA480/IFCC: Roche = 538 ng/L; ADx = 310 ng/L
ERM-DA481/IFCC: Roche = 826 ng/L; ADx = 533 ng/L
ERM-DA482/IFCC: Roche = 1603 ng/L; ADx = 858 ng/L
Abeta1-40:
ERM-DA480/IFCC: 6474 ng/L; Swb = 4.63 %; Sbb = 0.23%; u*bb = 0.92%
ERM-DA481/IFCC: 5994 ng/L; Swb = 5.95 %; Sbb = N/A; u*bb = 1.41 %
ERM-DA482/IFCC: 7668 ng/L; Swb = 4.90 %; Sbb = 2.65 %; u*bb = 1.16 %

15. LC-MS Ring Trial I
4 labs  4 SRM methods, same sample preparation method
12 CSF samples
J. Pannee et al., Alzheimer's & Dementia, Manuscript accepted.

16. LC-MS Ring Trial II
5 labs  common Aβ1-42 calibrant & dilution protocol
20 CSF samples
9 %

17. Aβ1-42 Calibrator
Calibration of LC-MS measurements for the value assignment of the CRM
Recombinant Aβ1-42 in 20% ACN, 1% NH4OH
Characterisation: Amino acid analysis (AAA) and purity assessment
Problem: Repeatability and between AA variation of AAA method
AAA data from INTERNAL TEST REPORT #2835 ( )

18. Characterisation of a calibrant
Amino acid analysis

19.       Traceability Chain Two MS methods formally certified
by JCTLM:
-C12RMP1
-C11RMP9    

20. CRM Production Process
Feasibility studies (collection, processing, characterisation, commutability)
Material selection & collection
Planning
Homogeneity
study
Interim transport
and storage
Storage
Processing
Short-term
stability study
Long-term
stability study
Commutability
study
Value assignment
Follow-up stability
monitoring of CRM
Assessment
by experts
Documentation
CRM Distribution and Sales

21. Next Steps Finish characterisation of calibrant Value assignment
Validated LC-MS methods
6 laboratories or methods
3 samples
At least 2 days
Common protocol for calibration
Look into other CSF AD biomarker
T-tau
P-tau
Aβ1-40

22. Acknowledgements Julia Kuhlmann
Sébastien Boulo Jean Charoud-Got Hendrik Emons Amalia Muñoz-Pineiro Heinz Schimmel Stefanie Trapmann Virginie Tregoat Ingrid Zegers Ulf Andreasson Maria Bjerke Kaj Blennow Josef Pannee Eric Portelius Henrik Zetterberg Magda Korecka Les Shaw Tobias Bittner Andreas Leinenbach Hugo Vanderstichele Erik Stoops Erin E. Chambers Rand G. Jenkins