1. Institute for Reference Materials and Measurements (IRMM)
Joint Research Centre
Institute for Reference Materials and Measurements (IRMM)

2. Abeta42 standardisation
Definition of the SI unit
e.g. AAA,
refractive index
“Pure” peptide calibrator
Reference procedure, immunoassay ring trial
CRM
matrix material
Value transfer 1:
LC-MS, immunoassay
Manufacturer’s working calibrator
Value transfer 2: immunoassay
Manufacturer’s product calibrator
Immunoassay
Routine sample
Aim: Setting up a reliable traceability chain linking results on routine samples to a common reference, the international system of units (SI) 2

3. ✔ ✔ Definition of the SI unit Purity assessment calibrant
Purity assessment amino acids ✔
Pure amino acids
Amino acid analysis validation ✔
Amino acid analysis
Abeta42 calibrant
Commutability studies
Correlation studies
LC-MS Reference procedure
CRM
matrix material
Processing
LC-MS ring trial
Feasibility studies
processing

4. Calibrator batch: - Intended use: Calibration of LC-MS measurements for the value assignment of the CRM - Format: recombinant Abeta42 in 20 % acetonitrile, 1 % ammoniumhydroxide - Concentration: determined by Amino acid analysis and purity assessment

5. Calibrator batch: Amino Acid analysis: - Problem in-house with repeatability and between AA variation, indicating a problem.

6. Possible explanations: - Purity: very high purity (Q-TOF, ion-trap, sequencing) - Digestion conditions optimised - Different sequences present? Unlikely as recombinant, confirmed by MS sequencing - Problematic interaction between solvent and HCl → freezedrying of samples before measurements: good conditions found on a different test batch To be repeated on actual calibrant (ongoing)

7. ✔ ✔ ✔ ✔ ✔ ✔ Definition of the SI unit Purity assessment calibrant
Purity assessment amino acids ✔
Pure amino acids
Amino acid analysis validation ✔ ✔
Amino acid analysis ✔
Abeta42 calibrant ✔
Commutability studies
Correlation studies
LC-MS Reference procedure
CRM
matrix material
Processing
LC-MS ring trial
Feasibility studies
processing

8. LC-MS reference method
Must give results that correlate with routine assays → correlation studies: OK
Pannee et al (2013) J Alzheim Dis
Leinenbach et al (2014) Clin Chem
Korecka et al (2014) J Alzheim Dis
Bjerke et al (2015) CCLM submitted

9. Ring trial with common protocol for calibration
9 %
6 datasets:
Uncertainty for value assignment:
Acceptable for value assignment of the CRM !

10. ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ Definition of the SI unit
Purity assessment calibrant ✔
Purity assessment amino acids ✔
Pure amino acids
Amino acid analysis validation ✔ ✔
Amino acid analysis ✔
Abeta42 calibrant ✔
Commutability studies
Correlation studies ✔
LC-MS Reference procedure ✔
CRM
matrix material
Processing
LC-MS ring trial ✔
Feasibility studies
processing

11. Commutability: a reference material must behave in the same way as a patient sample (2 studies, 10 methods)
Bjerke et al, CCLM submitted

12. Processing tests: - Manual filling/ machine filling - Stirring/ not stirring (no difference) - Abeta42 concentration over time while stirring (no slope) - Flash freezing versus slow freezing (better slow freezing)
Drift according to filling sequence (aCSF)
problem at beginning and end of filling (aCSF)

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)
Results from last test (CSF)

14. → Decision on format of the matrix reference material: - CSF pool
- No spiking
- 0.5 mL/vial
- 3 levels: low, medium and high
- Stored at -70°C

15. 1st level filled

16. ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ Definition of the SI unit
Purity assessment calibrant ✔
Purity assessment amino acids ✔
Pure amino acids
Amino acid analysis validation ✔ ✔
Amino acid analysis ✔
Abeta42 calibrant ✔
Commutability studies ✔
Correlation studies ✔
LC-MS Reference procedure ✔ ✔
CRM
matrix material
Processing ✔
LC-MS ring trial ✔
Feasibility studies
processing ✔

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

18. → Next step 1: homogeneity study first material
→ Next step 2: processing 2 other levels
→ Next step 3: Value assignment:
- LC-MS methods validated
- 6 laboratories or methods
- 3 levels
- At least 2 days
- Common protocol for calibration

19. Thanks to a large collaboratve network:
GBSC
IFCC
Participating laboratories
Manufacturers
Some people devoting a majority of their time:
Josef Pannee, Julia Kuhlmann, Maria Bjerke, Magdalena Korecka, Andreas Leinenbach…