The Alzheimer s Association QC program for CSF biomarkers

The Alzheimer’s Association QC program for CSF biomarkers • Ongoing ( 2009 -) project to monitor CSF biomarker lab and assay performance • Led by Gothenburg University, funded by the Alzheimer’s Association Principle for the QC program: For each round, 3 QC samples (pooled CSF) are sent out 2 unique samples - for comparisons between labs 1 identical sample - for comparisons over time Frequency: 3 times per year > 90 labs At present 17 rounds completed Newly added assay developments and formats: - Innotest ELISAs with ready-to-use (RTU) calibrators - MSD 6 E 10 Vplex assays - EUROIMMUNE/ADx assays - Roche cobas Elecsys fully automated assay

First paper in 2011 – Rounds 1 -2 Between laboratory CVs of 13 -36%

Results from Rounds 7 -17 - Innotest è Between laboratory CVs for the Innotest assays tend to go down No clear differences between Aβ 42 and T-tau, but P-tau show somewhat lower variability Aliquots of pooled CSF are stable over >3. 5 years !

Results from Rounds 7 -17 – Alz. Bio 3 è Between laboratory CVs for Alz. Bio 3 show no clear trend for reduced variability Few labs make results more uncertain

Results from Rounds 7 -17 – Meso Scale (MSD) è Between laboratory CVs for MSD(6 E 10) Aβ assays show a tendency for reduced variability

Results from Rounds 7 -17 – Euro. Immune / ADx results on the A and B samples 16 A 16 B 17 A 17 B Aβ 42 57 % 19. 4 % 6. 5 % 8. 2 % Aβ 40 45. 5 % 22. 7 % 9. 1% 8. 4 % T-tau 6% 21 % 17. 4% - è The Euro. Immune / ADx asays have just been enrolled in the QC program Few labs and few rounds make results uncertain – last round shows good performance for the Aβ assays

Sources of variability for CSF biomarker measurements SOURCE Pre-analytical factors CAUSE • E. g. type of test tubes - Not applicable in the QC program • Laboratory standardization (e. g. pipette calibration) Analytical factors • Analytical procedures (e. g. time, temperature, manual or pipetting robot) • Training / certification of technicians (e. g. pipetting variability) • Data handling (e. g. calibration curve) • Reagent (e. g. antibody) purity and variability Assay manufacturing • Kit production (e. g. plate coating) • Calibrator stability and format • Lot-to-lot consistency (batch bridging procedure) Analytical factors and assay quality and stability have a large impact on outcome

Sources of variability for CSF biomarker in the QC program Between lot INNOTEST ELISA Contribution to variation % Between laboratory Overall variability CV % 23% 18% 19% Analytical factors (laboratory and technician procedures) and assay quality and stability have a large impact on variability Mattson N et al. Alzheimer’s & Dementia 2013; 9: 251 -261

Impact of automation of ELISA methods Data from the Clinical Neurochemistry Lab, Sahlgrenska University Hospital, Sweden Internal quality control (QC) samples (pooled CSF) run on every ELISA plate (Innotest) All runs during 1 year Manual ELISA 134 QC samples – CV 14. 2% Pipetting robot - Tecan 230 QC samples – CV 11. 0% èAutomation of ELISAs for CSF Aβ(1– 42) provides some improvement in variability

CSF biomarker assays on fully automated clinical analyzers • Fully automated lab analyzer – no variation due to differences in laboratory procedures or between technicians – will give reduced between-run, between-batch and between-lab variations Paves the way for uniform cut-off levels Single sample analysis no need to ”wait” for samples to fill up an ELISA plate clinician Roche Diagnostics – Cobas Elecsys fast results (< 30 min) to the

Roche Diagnostics Elecsys assay – first Round 2016 Cobas Elecsys results on the A and B samples 14 A Aβ 42 14 B 15 A 15 B 16 A 16 B 17 A 17 B 2. 9 % 4. 4 % 4. 6 % 3. 4 % 3. 0 % 2. 5 % 1. 9 % 3. 2 % The Cobas Elecsys assay show minimal variability

The CSF biomarkers Aβ 42 and T-tau on the fully automated Lumipulse instruments Characteristics: Lumipulse G 1200 Lumipulse G 600 II (benchtop model) Reaction time 25 minutes Throughput 120 tests per hour 60 tests per hour Analytical performance (in-house data) CSF Aβ 42 CVs of 2 -5% within-run, between instrument and between-day CSF T-tau CVs of 1 -3% within-run, between instrument and between-day The Lumipulse assays are not yet in the QC program

The IFCC Work Group for CSF proteins and The Global Biomarker Standardization Consortium Reference Measurement Procedure (RMP) High precision Mass spectrometry-based technique Certified calibrator (amino acid analysis) Tested in Round Robin studies Intended use Golden standard method for absolute quantification Set level in the Certified Reference Material (CRM) Large aliquoted CSF pool Exact level set using RMPs Golden standard CSF with exact levels Commutability between assays tested Tested for long-term stability etc. Intended use Distribution to kit vendors and large labs Harmonize CSF levels between assay formats Assure stability between production lots

Reference method for CSF Aβ 42 - Validated ”Golden standard” method Antibody-free Single Reaction Monitoring (SRM) Triple Quad mass spec method for CSF Aβ isoforms CSF + internal Aβ standards Separation of Aβ by HPLC Guanidine HCl denaturation SPE antibody-free purification Elute + dry + redissolve in ACN and NH 4 OH Aβ 1 -40 Endogenous Aβ Aβ standards Aβ 1 -38 Vantage Triple Quad mass spectrometry 5. 5 6. 0 Aβ 1 -42 6. 5 Time (min) 7. 0 Quantification of Aβ isoforms • Isotope labelled Aβ calibrator added to the CSF sample (and thus processed identically) • No antibodies involved absolute quantification without interference (matrix effects) 7. 5

• Four laboratories with SRM methods for CSF Aβ 42: University of Gothenburg University of Pennsylvania PPD, USA Waters, USA • 12 CSF samples analyzed • One CSF served as a Candidate Reference Material Value for Individual lab Candidate reference methods for CSF Aβ 42 published SRM mass spec suitable as a Reference Measurement Procedure (RMP) for CSF Aβ 42 Mean of all labs

How do amyloid PET and CSF Aβ 42 compare ? Study design: 118 patients with cognitive complaint examined for both CSF biomarkers - as part of clinical routine – 2 years and amyloid 18 F-flutemetamol PET Cut-offs: CSF Aβ 42 < 647 pg/m. L 18 F-flutemetamol PET > 1. 42 Validation cohort n= 38 Original cohort n= 118 Positive PET+CSF or Negative PET+CSF 92 % Positive PET+CSF or Negative PET+CSF 97 % Palmquist S, et al, JAMA Neurol 2014

Mass spec method for CSF Aβ 42 vs. amyloid PET Study design: 103 patients with cognitive complaint examined for and Aβ 42 CSF Aβ using SRM mass spec amyloid 18 F-flutemetamol PET Aβ 42/40 ratio High concordance between amyloid PET and SRM mass spec, especially for the CSF Aβ 42/40 ratio Shahim P, et al, unpublished

How can we apply absolute biomarker cut-off levels for AD ? Younger AD patients showed extensive pathological changes (cortical plaques and tangles) when compared with people of similar age, but in older age, pathological changes in AD patients approached that of old age alone. Marked overlap in pathology (plaques and tangles) between AD and aging Elderly demented patients have many different types of pathology (not only plaques and tangles) Even if we have exact biomarkers – how do we apply a cut-off ?

“Absolute levels of CSF Aβ 42” vs. “amyloid PET positive/negative” Low Positive Cut-off >1. 11 SUVR Negative B Cut-off <192 pg/m. L Normal A Continuum in pathology between AD and aging gives a continuum in CSF Aβ levels and amyloid PET SUVs Is it better to report actual values to the clinician instead of “positive” or “negative” ?

We are doing great progress in the GBSC and IFCC WG-CSF - Standardization may be difficult