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

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)

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

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

Traceability Chain

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 986310.

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.

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.

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

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

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

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

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

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 %

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.

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

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 (2013-09-16)

Characterisation of a calibrant Amino acid analysis

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

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

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

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