Update on the Alzheimer’s Association Quality Control (QC) program for CSF biomarkers Kaj Blennow, Gothenburg University, Sweden

Background for starting the Alzheimer’s Association CSF program Large variability for CSF Aβ42 across laboratories Need of standardization efforts Variability due to: Pre-analytical factors e.g. type of test tube, CSF transfer, freeze-thaw effects, Analytical factors e.g. analytical procedures, technician training, run acceptance Assay manufacturing e.g. reagent purity, plate coating variability, calibrator stability, lot-to-lot consistency (batch bridging procedure)

The Alzheimer’s Association QC program for CSF biomarkers • Ongoing project since 2009 • Led by Gothenburg University, funded by the Alzheimer’s Association (private sponsor) 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 Goal for the QC program Assess analytical and assay variability (NOT pre-analytical factors) monitor CSF biomarker levels between clinical clinical laboratories  harmonize levels between labs  monitor assay performance and batch-to-batch variation for assays  stimulate kit vendors to produce high quality assays

Assays in the Alzheimer’s Association QC program • Innotest Aβ1-42 ELISA (Innogenetics) / Fujirebio Since Round 1 (2009) Aβ1-42, T-tau, P-tau RTU (ready-to-use) calibrator version Since Round 15 (2014) • Alzbio3 xMAP (Innogenetics) / Fujirebio Since Round 1 (2009) Aβ1-42, T-tau, P-tau Luminex • MSD ECL MesoScale Since Round 1 (2009) Aβ42, Aβ40, Aβ38 V-Plex (validated assays) Since Round 12 (2013) • Euroimmune ELISA ADx / Euroimmune Since Round 16 (2014) Aβ1-42, Aβ1-40, T-tau • Elecsys Aβ1-42 ECL-Cobas Roche Diagnostics Since Round 16 (2014) Fully automated • Lumipulse Aβ1-42 ECL Fujirebio Since Round 21 (2016) Fully automated

Longitudinal data from the QC program Aβ1-42 ELISA 21.6 % RTU ELISA 18.7% T-tau ELISA 22.1 % RTU ELISA 16.1% P-tau ELISA 18.6% RTU ELISA 13.0% • Change to RTU calibrators for the Innotest ELISAs gave a reduction in CVs

New MesoScale V-Plex assays in the QC program • Change to V-Plex validated assays gives an apparent reduction in CVs

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% Automation of ELISAs for CSF Aβ(1–42) provides minor improvement in variability

The CSF biomarker assays on fully automated clinical analyzers • Fully automated - minimize variations due to differences in laboratory procedures - reduced between-run, between-batch and between-lab variations  Will allow uniform cut-off levels Single sample analysis  fast results (< 30 min) to the clinician Roche Diagnostics – Cobas Fujirebio - Lumipulse

standardized to the mass spectrometry RMP for CSF Aβ42 (r= 0.93) MP03 vs. P02 r = 0.998 MP03 vs. P03 r = 0.996 P02 vs. P03 r = 0.997 Minimal variations between lots of reagents

The Cobas Elecsys fully automated assay - first round in the Alzheimer’s Association QC program Innotest ELISA Cobas Elecsys  fully automated instruments give a marked reduction in between-lab variability

CSF Aβ42 on the fully automated Lumipulse instruments Lumipulse G1200 Lumipulse G600 II (benchtop model) Analytical performance (in-house data) CSF Aβ42  CVs of 2-5% within-run, between instrument and between-day IFCC-WG CSF Commutability study III 37 individual CSFs 3 candidate Aβ1-42 CRMs (low, medium, high) Analyzed for Aβ1-42 by Lumipulse G1200 and the SRM mass spec Certified Reference Method r= 0.98 The Lumipulse assay is standardized to the mass spectrometry RMP Aβ42

The Lumpulse fully automated assay - first round in the Alzheimer’s Association QC program Round 21 – 2016 8 different results for Lumipulse Aβ1-42 on QC samples 21A and 21B 2 results from different lots of reagents  fully automated instruments give a marked reduction in between-lab variability

Data from the latest round - Round 21 (2016) for CSF Aβ42 6 different Aβ42 assays: 2 ELISAs, 2 assays on fully automated instruments, Luminex xMAP and MesoScale (MSD) V-Plex Innotest ELISA ADx/Euroimmune ELISA CV 11 % CV 15 % Alzbio3 xMAP CV 29 % MSD ECL CV 7.4 % Cobas Elecsys CV 5.2 % Lumipulse CV 4.0 %

The Alzheimer’s Association QC program – results 2014-2016

The Alzheimer’s Association QC program – summary of status New assay versions show improved performance, with lower between-lab CVs, both for: - Innotest RTU calibrator ELISAs - MSD V-Plex ECL methods New assays on fully automated analyzers show dramatic improvement in performance The QC program will:  Continue to monitor the performance of the AD CSF biomarker assays - between lab CVs - longitudinally between batches of reagents  Continue to serve as an aid to individual labs - monitoring accuracy of measurements - basis for accreditation of AD CSF biomarker assays

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