1. 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 6E10 Vplex assays
- EUROIMMUNE/ADx assays
- Roche cobas Elecsys fully automated assay

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

3. Results from Rounds 7-17 - Innotest
Between laboratory CVs for the Innotest assays tend to go down
No clear differences between Aβ42 and T-tau, but P-tau show somewhat lower variability
 Aliquots of pooled CSF are stable over >3.5 years !

4. Results from Rounds 7-17 – AlzBio3
Between laboratory CVs for AlzBio3 show no clear trend for reduced variability
 Few labs make results more uncertain

5. Results from Rounds 7-17 – Meso Scale (MSD)
Between laboratory CVs for MSD(6E10) Aβ assays show a tendency for reduced variability

6. Results from Rounds 7-17 – EuroImmune / ADx
16A 16B 17A 17B
Aβ42 57 % 19.4 % 6.5 % 8.2 %
Aβ % 22.7 % 9.1% 8.4 %
T-tau 6% 21 % 17.4% -
EuroImmune / ADx results on the A and B samples
The EuroImmune / 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

7. Sources of variability for CSF biomarker measurements
CAUSE
Pre-analytical factors
• E.g. type of test tubes - Not applicable in the QC program
Analytical factors
• Laboratory standardization (e.g. pipette calibration)
• Analytical procedures (e.g. time, temperature, manual or pipetting robot)
• Training / certification of technicians (e.g. pipetting variability)
• Data handling (e.g. calibration curve)
Assay manufacturing
• Reagent (e.g. antibody) purity and variability
• 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

8. Sources of variability for CSF biomarker in the QC program
INNOTEST ELISA
Between lot
Between laboratory
Contribution to variation %
Overall variability CV % % % %
 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:

9. 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 some improvement in variability

10. 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
 fast results (< 30 min) to the clinician
Roche Diagnostics – Cobas Elecsys

11. Roche Diagnostics Elecsys assay – first Round 2016
14A 14B 15A 15B 16A 16B 17A 17B
Aβ % 4.4 % 4.6 % 3.4 % 3.0 % 2.5 % 1.9 % 3.2 %
Cobas Elecsys results on the A and B samples
 The Cobas Elecsys assay show minimal variability

12. The CSF biomarkers Aβ42 and T-tau on the fully automated Lumipulse instruments
Characteristics: Lumipulse G Lumipulse G600 II
(benchtop model)
Reaction time 25 minutes 25 minutes
Throughput 120 tests per hour 60 tests per hour
Analytical performance (in-house data)
CSF Aβ42  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

13. The IFCC Work Group for CSF proteins and
The Global Biomarker Standardization Consortium
High precision
Mass spectrometry-based technique
Certified calibrator (amino acid analysis)
Tested in Round Robin studies
Intended use
Set level in the Certified Reference Material
Reference Measurement Procedure (RMP)
Golden standard method
for absolute quantification
Large aliquoted CSF pool
Exact level set using RMPs
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
Certified Reference Material (CRM)
Golden standard CSF
with exact levels

14. Reference method for CSF Aβ42 - Validated ”Golden standard” method
Antibody-free Single Reaction Monitoring (SRM) Triple Quad mass spec method for CSF Aβ isoforms
Separation of Aβ by HPLC
Vantage Triple Quad mass spectrometry
CSF +
internal Aβ standards
Guanidine HCl
 denaturation
SPE antibody-free
purification
Elute + dry
+ redissolve in
ACN and NH4OH
Quantification of Aβ isoforms
5.5
6.0
6.5
7.0
7.5
Time (min)
Aβ1-38
Aβ1-42
Aβ1-40
Endogenous Aβ
Aβ standards
• Isotope labelled Aβ calibrator added to the CSF sample (and thus processed identically)
• No antibodies involved
 absolute quantification without interference (matrix effects)

15. Candidate reference methods for CSF Aβ42 published
Mean of all labs
Value for Individual lab
• Four laboratories with SRM methods for CSF Aβ42:
University of Gothenburg
University of Pennsylvania
PPD, USA
Waters, USA
• 12 CSF samples analyzed
• One CSF served as a Candidate Reference Material
 SRM mass spec suitable as a Reference
Measurement Procedure (RMP) for CSF Aβ42

16. How do amyloid PET and CSF Aβ42 compare ?
Study design: patients with cognitive complaint
examined for both CSF biomarkers - as part of clinical routine – 2 years
and amyloid 18F-flutemetamol PET
Cut-offs: CSF Aβ < 647 pg/mL
18F-flutemetamol PET > 1.42
Original cohort n= 118
Positive PET+CSF or Negative PET+CSF %
Validation cohort n= 38
Positive PET+CSF or Negative PET+CSF %
Palmquist S, et al, JAMA Neurol 2014

17. Mass spec method for CSF Aβ42 vs. amyloid PET
Study design: patients with cognitive complaint
examined for CSF Aβ using SRM mass spec
and amyloid 18F-flutemetamol PET
Aβ42/40 ratio
Aβ42
 High concordance between amyloid PET and SRM mass spec, especially for the CSF Aβ42/40 ratio
Shahim P, et al, unpublished

18. 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 ?

19. “Absolute levels of CSF Aβ42” vs. “amyloid PET positive/negative”
Cut-off <192 pg/mL A
Normal
Low
Cut-off >1.11 SUVR B
Positive
Negative
 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” ?

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