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

2. 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)

3. 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

4. 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

5. Longitudinal data from the QC program
Aβ1-42 ELISA %
RTU ELISA %
T-tau ELISA % RTU ELISA %
P-tau ELISA % RTU ELISA %
• Change to RTU calibrators for the Innotest ELISAs gave a reduction in CVs

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

7. 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

8. 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

9. 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

10. 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

11. CSF Aβ42 on the fully automated Lumipulse instruments
Lumipulse G 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

12. 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

13. 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 %

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

15. 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

16. Thanks for listening !