ALZHEIMERS ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL ALZHEIMERS ASSOCIATION

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ALZHEIMER’S ASSOCIATION CSF PRE-ANALYTICAL CONSENSUS PROTOCOL

ALZHEIMER’S ASSOCIATION CSF PRE-ANALYTICAL CONSENSUS PROTOCOL

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Disclosure James Hendrix, Ph. D. is a full-time

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL 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.

PHOTO, VIDEO AND AUDIO POLICY Photography is welcome in this presentation. Video and audio

PHOTO, VIDEO AND AUDIO POLICY Photography is welcome in this presentation. Video and audio recording are prohibited.

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Routine Clinical Practice • • New platforms coming

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL 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 Ab 1 -42, Ab 1 -40, t. Tau, p. Tau Research Use • Protocol not specific for research use but many of the same principles may apply. However, only Ab 1 -42, Ab 1 -40, t. Tau, and p. Tau have been evaluated in this protocol and it is unknown how other CSF biomarkers will behave.

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Workgroup Members ADx Neuro. Sciences Roche Diagnostics Hugo

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

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Overview of pre-analytical factors Impact factors on measured

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Overview of pre-analytical factors Impact factors on measured CSF biomarker concentrations Tube type Sample Collection Tube filling volume Tube transfer steps / pipetting Measured biomarker concentratio n Temperatur e/ freezethaw Blood contaminat ion Mixing Transportat ion Sample stability

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL A new CSF tube for AD biomarkers was

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL A new CSF tube for AD biomarkers was evaluated Sarstedt has developed the following tube: Order Number 63. 614. 699 • 2. 5 m. L false bottom tube • polypropylene • protein low bind • screw cap • sterile • suitable for short-term freezing • 1. 5 -2 m. L scaling • 75 x 13 mm, round • compatible with multiple systems

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Impact of pre-analytical factors Mixing procedure and volume

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Impact of pre-analytical factors Mixing procedure and volume * • Mixing procedure and volume had a pronounced effect on Ab 1 -42 recovery; weaker impact on Ab 1 -40. No impact was observed on p. Tau or t. Tau. Note: All samples in this and later experiments were assessed using new developed Protein Low Bind Tube from Sarstedt, 4 -6 h after LP („fresh“) if not indicated differently. *Data were generated on the cobas® analyzer on behalf of Roche Diagnostics. The Elecsys® CSF Abeta 42, t. Tau, and p. Tau immunoassays are not yet cleared or approved for clinical use in the USA.

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Impact of pre-analytical factors * Volume (ml): Ab

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Impact of pre-analytical factors * Volume (ml): Ab 1 -42 , Ab 1 -40 and t. Tau (no mixing) • • volume had a pronounced effect on Ab 1 -42 recovery; weaker impact on Ab 1 -40. No impact was observed on p. Tau or t. Tau. Ab 1 -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 Abeta 42, t. Tau, and p. Tau immunoassays are not yet cleared or approved for clinical use in the USA.

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Impact of pre-analytical factors Blood contamination and storage:

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Impact of pre-analytical factors Blood contamination and storage: Ab 1 -42 , Ab 1 -40 and t. Tau (No mixing) • up to 1% blood contamination showed only weak impact on the Ab 1 -42 and Ab 1 -40 recovery and no effect on t. Tau and p. Tau. 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 Abeta 42, t. Tau, and p. Tau immunoassays are not yet cleared or approved for clinical use in the USA.

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Impact of pre-analytical factors Storage temperature: Ab 1

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Impact of pre-analytical factors Storage temperature: Ab 1 -42 , Ab 1 -40 and t. Tau* • preliminary experiments showed that Ab 1 -42 was stable for 48 h at room temperature (RT) without mixing, and up to 15 d at 4°C. Similar results were obtained for Ab 1 -40, p. Tau and t. Tau. 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 Abeta 42, t. Tau, and p. Tau immunoassays are not yet cleared or approved for clinical use in the USA.

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL CSF Stability* 4 o. C Mean 24 h

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL CSF Stability* 4 o. C Mean 24 h 100 48 h 99 72 h 100 1 week 2 weeks RT SD Mean Results 4 o. C SD 4 o. C RT Mean SD 3. 8 99 2. 9 24 h 104 4. 3 101 5. 1 24 h 97 4. 9 98 4. 6 3. 4 97 3. 6 48 h 100 4. 7 98 3. 8 48 h 95 5. 7 99 6. 2 4. 1 97 4. 3 72 h 98 5. 3 95 5. 0 72 h 102 5. 8 102 7. 1 99 4. 1 94 4. 7 1 week 100 5. 6 96 6. 7 1 week 99 5. 1 98 7. 0 96 5. 5 90 4. 3 2. 5 2 weeks 93 5. 5 92 4. 5 Amyloid levels are stable on average for at least 1 week when stored at +4 o C 2 weeks 103 4. 5 98 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.

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Post Collection Volume, Centrifugation, and Temperature* Condition Fill

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Post Collection Volume, Centrifugation, and Temperature* Condition Fill Volume (m. L) A 8 B 5 C 8 2000 x g (± 200 x g) for 10 min No Centrifugation D 8 No Centrifugation 25 ± 3°C E 8 2000 x g (± 200 x g) for 10 min 25 ± 3°C Centrifugation Condition Temperature during Processing 5 ± 3°C Donor concentrations ranged from ~950 – 2, 400 pg/m. L Aβ 1 -42 *Data were generated on the LUMIPULSE G 1200 System on behalf of Fujirebio Diagnostics Inc. Lumipulse G β-Amyloid 1 -42 assay not currently cleared for use in USA

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Post Collection Volume, Centrifugation, and Temperature* Condition Fill

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Post Collection Volume, Centrifugation, and Temperature* Condition Fill Volume (m. L) A 8 B 5 C 8 2000 x g (± 200 x g) for 10 min No Centrifugation D 8 No Centrifugation 25 ± 3°C E 8 2000 x g (± 200 x g) for 10 min 25 ± 3°C Centrifugation Condition Temperature during Processing 5 ± 3°C *Data were generated on the LUMIPULSE G 1200 System on behalf of Fujirebio Diagnostics Inc. Lumipulse G β-Amyloid 1 -42 assay not currently cleared for use in USA 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%.

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Post Collection Volume, Centrifugation, and Temperature* Condition Fill

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Post Collection Volume, Centrifugation, and Temperature* Condition Fill Volume (m. L) A 8 B 5 C 8 2000 x g (± 200 x g) for 10 min No Centrifugation D 8 No Centrifugation 25 ± 3°C E 8 2000 x g (± 200 x g) for 10 min 25 ± 3°C Centrifugation Condition Temperature during Processing 5 ± 3°C *Data were generated on the LUMIPULSE G 1200 System on behalf of Fujirebio Diagnostics Inc. Lumipulse G β-Amyloid 1 -42 assay not currently cleared for use in USA 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 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%.

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Post Collection Volume, Centrifugation, and Temperature* Condition Fill

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Post Collection Volume, Centrifugation, and Temperature* Condition Fill Volume (m. L) A 8 B 5 C 8 2000 x g (± 200 x g) for 10 min No Centrifugation D 8 No Centrifugation 25 ± 3°C E 8 2000 x g (± 200 x g) for 10 min 25 ± 3°C Centrifugation Condition Temperature during Processing 5 ± 3°C *Data were generated on the LUMIPULSE G 1200 System on behalf of Fujirebio Diagnostics Inc. Lumipulse G β-Amyloid 1 -42 assay not currently cleared for use in USA 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 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%.

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Post Collection Volume, Centrifugation, and Temperature* Condition Fill

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Post Collection Volume, Centrifugation, and Temperature* Condition Fill Volume (m. L) A 8 B 5 C 8 2000 x g (± 200 x g) for 10 min No Centrifugation D 8 No Centrifugation 25 ± 3°C E 8 2000 x g (± 200 x g) for 10 min 25 ± 3°C Centrifugation Condition Temperature during Processing 5 ± 3°C 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 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 *Data were generated on the LUMIPULSE G 1200 System on behalf of during processing Fujirebio Diagnostics Inc. • No significant difference observed in 5 donors tested Lumipulse G β-Amyloid 1 -42 assay not currently cleared for use in USA • Difference ranged from -2% to 2%.

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Ultra low bind tube evaluation* A to B

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Ultra low bind tube evaluation* A to B A to C Donor (n=19) conc. ranged from ~400 – 3, 000 pg/m. L Aβ 1 -42 Collection Tube Fill Volume • • No significant differences observed: Sarstedt Cat# (m. L) Ø B vs. A differences between -9% to 8% A 62. 610. 018 8 Ø C vs. A differences between -12% to 2% B 60. 614. PR 001 2 More investigations needed on alternative tubes C 72. 703. 600 1. 2 *Data were generated on the LUMIPULSE G 1200 System on behalf of Fujirebio Diagnostics Inc. , Lumipulse G β-Amyloid 1 -42 assay not currently cleared for use in USA Tube

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Proposed Routine-use Pre-Analytical Protocol Collection Site Sample Collection

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Proposed Routine-use Pre-Analytical Protocol Collection Site Sample Collection Testing Site Collection Storage* Sample Handling Measurement Fresh samples (Routine ) Discard first 2 m. L, then directly collect 1. 5 -2 m. L by drip method directly into a Low Bind (i. e. Sarstedt) tube. No centrifugation, freezing, mixing/inverting or tube transfers No tube transfer or further handling before analysis Storage up to 15 days at 2 -8 o. C No mixing; immediate measurement *The Storage step assumes the transport of samples however the group does not yet have data on sample stability during transport. Measure: Ab 1 -42, Ab 1 -40, t. Tau, p. Tau

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Collection Method • Additional Issues Drip method recommended

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL Collection Method • Additional Issues Drip method recommended over syringe pull. However, syringe pull could be used after the 1. 5 -2 m. L 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. Vive. Bio 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 m. L false-bottom low bind tube (63. 614. 699) or 1. 5 m. L 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 PREANALYTICAL CONSENSUS PROTOCOL Next Steps Further Research • • • Alternative

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL 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 PREANALYTICAL CONSENSUS PROTOCOL alz. org

ALZHEIMER’S ASSOCIATION CSF PREANALYTICAL CONSENSUS PROTOCOL alz. org