Alzheimer’s Association CSF Pre-Analytical Consensus Protocol

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.

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.

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

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

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 63.614.699 2.5 mL false bottom tube protein low bind sterile 1.5 -2 mL scaling 75 x 13 mm, round polypropylene screw cap suitable for short-term freezing compatible with multiple systems

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.

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 1.25-2.25 ml was used; accordingly, a conservative range 1.5 -2 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.

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.

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.

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.

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

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

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

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

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

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 62.610.018 8 B 60.614.PR001 2 C 72.703.600 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

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 1.5-2 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 .

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 1.5-2 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 (63.614.699) or 1.5 mL low bind tube (72.703.600) 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.

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

Alzheimer’s Association CSF Pre-Analytical Consensus Protocol