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
First paper in 2011 – Rounds 1-2
 Between laboratory CVs of 13-36%
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 !
Results from Rounds 7-17 – AlzBio3
 Between laboratory CVs for AlzBio3 show no clear trend for reduced variability
 Few labs make results more uncertain
Results from Rounds 7-17 – Meso Scale (MSD)
 Between laboratory CVs for MSD(6E10) Aβ assays show a tendency for reduced variability
Results from Rounds 7-17 – EuroImmune / ADx
EuroImmune / ADx results on the A and B samples
16A
16B
17A
17B
Aβ42
57 %
19.4 %
6.5 %
8.2 %
Aβ40
45.5 %
22.7 %
9.1%
8.4 %
T-tau
6%
21 %
17.4%
-
 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
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%
Automation 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
Aβ42
14A
14B
15A
15B
16A
16B
17A
17B
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 Aβ42 and T-tau on the fully automated Lumipulse instruments
Characteristics:
Lumipulse G1200
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
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
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 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
CSF +
internal Aβ standards
Guanidine HCl
 denaturation
SPE antibody-free
purification
Elute + dry
+ redissolve in
ACN and NH4OH
Aβ1-40
Endogenous Aβ
Aβ standards
Aβ1-38
Vantage Triple Quad mass spectrometry
5.5
6.0
Aβ1-42
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 Aβ42:
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 Aβ42 published
 SRM mass spec suitable as a Reference
Measurement Procedure (RMP) for CSF Aβ42
Mean of all labs
How do amyloid PET and CSF Aβ42 compare ?
Study design: 118 patients with cognitive complaint
examined for both CSF biomarkers - as part of clinical routine – 2 years
and amyloid 18F-flutemetamol PET
Cut-offs: CSF Aβ42
< 647 pg/mL
18F-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 Aβ42 vs. amyloid PET
Study design: 103 patients with cognitive complaint
examined for
and
Aβ42
CSF Aβ using SRM mass spec
amyloid 18F-flutemetamol PET
Aβ42/40 ratio
 High concordance between amyloid PET and SRM mass spec, especially for the CSF Aβ42/40 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 Aβ42” vs. “amyloid PET positive/negative”
Low
Positive
Cut-off >1.11 SUVR
Negative
B
Cut-off <192 pg/mL
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