1. Alzheimer’s Association CSF Pre-Analytical Consensus Protocol

2. Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Disclosure
James Hendrix, Ph.D. is a full-time employee of the Alzheimer’s Association and has no conflicts of interest with the content of this presentation.

4. Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Routine Clinical Practice
New platforms coming to measure CSF biomarkers in clinical practice
A simple protocol accepted and endorsed by diagnostic companies, academic KOL’s, physician groups and the Alzheimer’s Association is needed.
Goal: Minimize the systematic differences in the biomarker measurements
Focused on Ab1-42, Ab1-40, tTau, pTau
Research Use
Protocol not specific for research use but many of the same principles may apply. However, only Ab1-42, Ab1-40, tTau, and pTau have been evaluated in this protocol and it is unknown how other CSF biomarkers will behave.

5. Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Workgroup Members
ADx NeuroSciences
Hugo Vanderstichele, PhD
Roche Diagnostics
Richard Batrla, MD, MBA
Ekaterina Bauer, PhD, MBA
Nadja Baur-Kolarov, PhD
Veronika Corradini, MSc
Udo Eichenlaub, PhD
Valeria Lifke, PhD
Sandra Rutz, PhD
Simone Wahl, PhD
Alzheimer’s Association
Maria Carrillo, PhD
James Hendrix, PhD
April Ross, PhD, Project Manager
Euroimmun
Britta Brix, PhD
Fujirebio
Christina Hall, PhD
John Lawson, B.S., M.T.
Christopher Traynham, PhD
Manu Vandijck, MSc
Academic Experts
Kaj Blennow, MD, PhD, U. of Gothenburg
Robert Dean, MD, PhD, Indiana U.
Oskar Hansson, MD, PhD, Skane U. & Lund U
Jose Luis Molinuevo, MD, PhD, Barcelonabeta Brain Research Center
Saladax Biomedical
Salvatore Salamone, PhD
Meso Scale Discovery
Robert Umek, PhD

6. Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Overview of pre-analytical factors Impact factors on measured CSF biomarker concentrations
Transportation
Sample Collection
Tube filling volume
Tube transfer steps / pipetting
Mixing
Tube type
Temperature/ freeze-thaw
Measured biomarker concentration
Sample stability
Blood contamination

7. A new CSF tube for AD biomarkers was evaluated
Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
A new CSF tube for AD biomarkers was evaluated
Sarstedt has developed the following tube: Order Number
2.5 mL false bottom tube
protein low bind
sterile
mL scaling
75 x 13 mm, round
polypropylene
screw cap
suitable for short-term freezing
compatible with multiple systems

8. Impact of pre-analytical factors Mixing procedure and volume *
Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Impact of pre-analytical factors Mixing procedure and volume *
Mixing procedure and volume had a pronounced effect on Ab1-42 recovery; weaker impact on Ab1-40. No impact was observed on pTau or tTau.
Note: All samples in this and later experiments were assessed using new developed Protein Low Bind Tube from Sarstedt, 4-6h after LP („fresh“) if not indicated differently.
*Data were generated on the cobas® analyzer on behalf of Roche Diagnostics. The Elecsys® CSF Abeta42, tTau, and pTau immunoassays are not yet cleared or approved for clinical use in the USA.

9. Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Impact of pre-analytical factors * Volume (ml): Ab1-42 , Ab1-40 and tTau (no mixing)
volume had a pronounced effect on Ab1-42 recovery; weaker impact on Ab1-40. No impact was observed on pTau or tTau.
Ab1-42 recovery appeared stable when a volume of ml was used; accordingly, a conservative range ml is marked on the low-bind FBT and should be adhered for CSF collection.
*Data were generated on the cobas® analyzer on behalf of Roche Diagnostics. The Elecsys® CSF Abeta42, tTau, and pTau immunoassays are not yet cleared or approved for clinical use in the USA.

10. Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Impact of pre-analytical factors Blood contamination and storage: Ab1-42 , Ab1-40 and tTau (No mixing)
up to 1% blood contamination showed only weak impact on the Ab1-42 and Ab1-40 recovery and no effect on tTau and pTau. Evaluation of stability upon storage at RT and 4°C ongoing.
*Data were generated on the cobas® analyzer on behalf of Roche Diagnostics. The Elecsys® CSF Abeta42, tTau, and pTau immunoassays are not yet cleared or approved for clinical use in the USA.

11. Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Impact of pre-analytical factors Storage temperature: Ab1-42 , Ab1-40 and tTau*
preliminary experiments showed that Ab1-42 was stable for 48h at room temperature (RT) without mixing, and up to 15d at 4°C. Similar results were obtained for Ab1-40, pTau and tTau. Evaluation for longer times up to 7 days at RT ongoing.
*Data were generated on the cobas® analyzer on behalf of Roche Diagnostics. The Elecsys® CSF Abeta42, tTau, and pTau immunoassays are not yet cleared or approved for clinical use in the USA.

12. Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
CSF Stability*
4 oC RT
Mean SD
24 h
100
3.8 99
2.9
48 h
3.4 97
3.6
72 h
4.1
4.3
1 week 94
4.7
2 weeks 96
5.5 90
4 oC RT
Mean SD
24 h
104
4.3
101
5.1
48 h
100
4.7 98
3.8
72 h
5.3 95
5.0
1 week
5.6 96
6.7
2 weeks
103
4.5
2.5
4 oC RT
Mean SD
24 h 97
4.9 98
4.6
48 h 95
5.7 99
6.2
72 h
102
5.8
7.1
1 week
5.1
7.0
2 weeks 93
5.5 92
4.5
Results
Amyloid levels are stable on average for at least 1 week when stored at +4o C
Data normalized to the baseline biomarker concentrations measured on the day of CSF collection are shown as mean+sem (n=12). Changes in biomaker levels were tested using repeated measures ANOVA adjusted for multiple comparisons (with mean difference >5% and p<0.005 (0.05/10) considered significant).
*Data were generated by the Hansson lab (Lund University) with manual EUROIMMUN ELISA Kits.

13. Centrifugation Condition Temperature during Processing
Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Post Collection Volume, Centrifugation, and Temperature*
Condition
Fill Volume (mL)
Centrifugation Condition
Temperature during Processing A 8
2000 x g
(± 200 x g) for 10 min
5 ± 3°C B 5 C
No Centrifugation D
25 ± 3°C E
Donor concentrations ranged from ~950 – 2,400 pg/mL Aβ1-42
*Data were generated on the LUMIPULSE G1200 System on behalf of Fujirebio Diagnostics Inc.
Lumipulse G β-Amyloid 1-42 assay not currently cleared for use in USA
CSF was collected directly into the testing tubes
CSF collected via gravity drip
The first 2 mls were discarded if cloudy/bloody

14. Centrifugation Condition Temperature during Processing
Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Post Collection Volume, Centrifugation, and Temperature*
Condition B vs. A
Evaluation of 50% fill versus 80% fill with centrifugation and refrigerated condition during processing
No significant difference observed in 4 donors tested
Difference ranged from 0% to 6%.
Condition
Fill Volume (mL)
Centrifugation Condition
Temperature during Processing A 8
2000 x g
(± 200 x g) for 10 min
5 ± 3°C B 5 C
No Centrifugation D
25 ± 3°C E
*Data were generated on the LUMIPULSE G1200 System on behalf of Fujirebio Diagnostics Inc.
Lumipulse G β-Amyloid 1-42 assay not currently cleared for use in USA

15. Centrifugation Condition Temperature during Processing
Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Post Collection Volume, Centrifugation, and Temperature*
Condition B vs. A
Evaluation of 50% fill versus 80% fill with centrifugation and refrigerated condition during processing
No significant difference observed in 4 donors tested
Difference ranged from 0% to 6%.
Condition
Fill Volume (mL)
Centrifugation Condition
Temperature during Processing A 8
2000 x g
(± 200 x g) for 10 min
5 ± 3°C B 5 C
No Centrifugation D
25 ± 3°C E
Condition C vs. A
Evaluation of no centrifugation with refrigerated condition during processing
No significant difference observed in 4 donors tested
Difference ranged from -4% to 3%.
*Data were generated on the LUMIPULSE G1200 System on behalf of Fujirebio Diagnostics Inc.
Lumipulse G β-Amyloid 1-42 assay not currently cleared for use in USA

16. Centrifugation Condition Temperature during Processing
Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Post Collection Volume, Centrifugation, and Temperature*
Condition B vs. A
Evaluation of 50% fill versus 80% fill with centrifugation and refrigerated condition during processing
No significant difference observed in 4 donors tested
Difference ranged from 0% to 6%.
Condition
Fill Volume (mL)
Centrifugation Condition
Temperature during Processing A 8
2000 x g
(± 200 x g) for 10 min
5 ± 3°C B 5 C
No Centrifugation D
25 ± 3°C E
Condition C vs. A
Evaluation of no centrifugation with refrigerated condition during processing
No significant difference observed in 4 donors tested
Difference ranged from -4% to 3%.
Condition D vs. A
Evaluation of no centrifugation with room temperature condition during processing
No significant difference observed in 5 donors tested
Difference ranged from -3% to 1%.
*Data were generated on the LUMIPULSE G1200 System on behalf of Fujirebio Diagnostics Inc.
Lumipulse G β-Amyloid 1-42 assay not currently cleared for use in USA

17. Centrifugation Condition Temperature during Processing
Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Post Collection Volume, Centrifugation, and Temperature*
Condition B vs. A
Evaluation of 50% fill versus 80% fill with centrifugation and refrigerated condition during processing
No significant difference observed in 4 donors tested
Difference ranged from 0% to 6%.
Condition
Fill Volume (mL)
Centrifugation Condition
Temperature during Processing A 8
2000 x g
(± 200 x g) for 10 min
5 ± 3°C B 5 C
No Centrifugation D
25 ± 3°C E
Condition C vs. A
Evaluation of no centrifugation with refrigerated condition during processing
No significant difference observed in 4 donors tested
Difference ranged from -4% to 3%.
Condition D vs. A
Evaluation of no centrifugation with room temperature condition during processing
No significant difference observed in 5 donors tested
Difference ranged from -3% to 1%.
Condition E vs. A
Evaluation of centrifugation with room temperature condition during processing
No significant difference observed in 5 donors tested
Difference ranged from -2% to 2%.
*Data were generated on the LUMIPULSE G1200 System on behalf of Fujirebio Diagnostics Inc.
Lumipulse G β-Amyloid 1-42 assay not currently cleared for use in USA

18. Collection Tube Sarstedt Cat#
Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Ultra low bind tube evaluation*
A to B
A to C
Donor (n=19) conc. ranged from ~400 – 3,000 pg/mL Aβ 1-42
No significant differences observed:
B vs. A  differences between -9% to 8%
C vs. A  differences between -12% to 2%
 More investigations needed on alternative tubes
Tube
Collection Tube Sarstedt Cat#
Fill Volume (mL) A 8 B
PR001 2 C
1.2
*Data were generated on the LUMIPULSE G1200 System on behalf of Fujirebio Diagnostics Inc., Lumipulse G β-Amyloid 1-42 assay not currently cleared for use in USA

19. No tube transfer or further handling before analysis
Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Proposed Routine-use Pre-Analytical Protocol
Collection Site
Testing Site
Sample Collection
Collection
Storage*
Sample Handling
Measurement
Fresh samples (Routine )
Discard first 2 mL, then directly collect mL by drip method directly into a Low Bind (i.e. Sarstedt) tube.
No centrifugation, freezing, mixing/inverting or tube transfers
Storage up to 15 days at 2-8 oC
No mixing;
immediate measurement
Measure: Ab1-42, Ab1-40, tTau, pTau
No tube transfer or further handling before analysis
*The Storage step assumes the transport of samples however the group does not yet have data on sample stability during transport .

20. Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Additional Issues
Collection Method
Drip method recommended over syringe pull. However, syringe pull could be used after the mL routine sample is collected if additional research sample is required.
Needle
Atraumatic needle recommended
LP Tray
Recommended LP tray(s) needed for US Market that are dedicated to CSF biomarkers, under discussion. ViveBio tray under consideration.
Low Bind Tubes
Collection tubes constructed of Sarstedt Low Bind Polypropylene material is the consortium’s current recommended best practice in routine use.
Sarstedt 2.5 mL false-bottom low bind tube ( ) or 1.5 mL low bind tube ( ) have been evaluated but more studies are planned to confirm that they are equivalent.
Mixing / transport with the Sarstedt tubes needs to be evaluated as the caps for these tubes are not made of the Low Bind PP material.
The Workgroup is encouraging other manufacturers to produce similar tubes that could also be tested.
Sample Storage
Long term storage data is not available at this time. Mid-term and long term storage studies at -20°C with other tube types need to be performed. A storage recommendation will be made at a later date.

21. Alzheimer’s Association CSF Pre-Analytical Consensus Protocol
Next Steps
Further Research
Alternative tubes
Transportation
Long Term Storage
Communication of Consensus Protocol
Future publication planned
Engagement with future conferences and societies

22. Alzheimer’s Association CSF Pre-Analytical Consensus Protocol