1. Alz. Assoc. CSF Reference Method Sub-group
Notes of 21nov2011
Alz. Assoc. CSF Reference Method Sub-group

2. Goal
Quantify concentration of analyte as accurately as possible in the reference material to then be used to compare other methods and platforms available…

3. Methods to evaluate (for pros/cons)
MRM published by Waters et al… (need cite)
Absorbance based methods
PPD (Randy Jenkins)- Assay from ICAD poster
Proteome Sciences - “validated” ab42 assay
C2N – quant ab42 and “total ab” assay
Immunoassay based approaches
MSD, Myriad RBM, INNX/Fujirebio
Manu Vandijeck had a new methodogy from Ab42 std working group…
Note: need request validation reports from each.

4. Reference Methods Issues (2)
Issue: are we considering what is available today or what may need to get developed
Consideration of pre-analytic methods and their impact on the analytical methods
Oligomer vs monomer of Ab42
What is the minimum validation required? What is the quality standard?
What is overall target goal? FDA clearance or approval, operate in research space only?
Resources to achieve the goals?
Suggest pull EMA opinions based on BMS submission regarding CSF biomarkers
MSD-RUO initially, require more than MSD alone if tied to drug or indication, working on how take to next level, what are clinical study design and samples for a PMA . . .
Invite DX companies: OCD, Roche to the “table”, CRO have done “validated” Pfizer/ICON, BARC, and Covance
Issue: is method/assay validated and available? Where should it be done?

5. TC Notes 17jan2012 3 methods for 3 analytes: ab42, tau, pTau
Possible methods: MS, immunoassay methods, HPLC, spectrophotemetry basis
Method have or method to develop? May differ depending on the analyte.
If method reference is available, who should conduct the method? Commercial company or international organization? (NIST, etc)
Case Example of Testosterone/Hormone: done by special laboratory in academia (Holland) as a reference method for the world … (??)
MS is one of the candidate methods (e.g. PPD or others), needs to be data driven decisions to assess the possible reference methods. Need to push to develop data itself.
Q: Which technique are reference methods? 3 options … (KB)
Suggestion: to gather the available data on each possible reference method on an analyte by analyte basis in priority order (AS)
Suggestion (HZ): could collaborate on Kaj’s reference method paper with deadline of April to review candidate reference methods
Create 3 by 4 table of analyte by method (MS, immunoassay methods, HPLC, spectrophotemetry basis… (MC)
Note CAMD meeting with FDA to discuss suitability of CSF markers (MRI and CSF), Feb. 28/29th, , AA would like to push to get some progress before (MC).
Document the progress or state of methodology would be worthwhile… (LS) longer term is reference materials and reference methods
Rec’d to focus on Ab42 and tau, then circle back later to pTau which appears more difficult with multiple isoforms (HV, Charlotte ?), pTau offers clinical differential value
Suggest: stepwise order, Ab42, then Tau, then pTau in this order of focus (?)
For CAMD FDA mtg, would like to show FDA there are approaches to standardization (this groups work and plan), we are moving forward (MC)
Longer term goal is harmonization, but this is longer term and uncertain as to when it will occur (LS), should be transparent about this gap of harmonization
Look in other fields, go for serum proteins or hormones as examples, assays in field for long time and still not harmonized across assays… (KB)
Next steps: table with 3 analytes, 3 or 4 methods
Suggest start with Ab42 (KB)
MS, immuno assay, HPLC, possible for spectro … should look at data directly (RB)
Promising paper to include Lei Wang (Biomarker … Theresa will forward for inclusion).

6. TC Notes 17jan2012 (2)
Agree focus on Ab42 3 methods for 3 analytes: ab42, tau, pTau
MS papers on:
Waters published in Analytical Biochem (Erin ? At Waters , presented at ASM last week and published)
PPD poster at ICAD, based on Waters method with important modifications
ASM meeting abstracts
PPD works in GLP environment, subjected to some validation, should include them in the process (OL), deeply involved in method for over 2 yrs now
Penn has spoken extensively with Rand Jenkins at PPD (LS) and now has the PPD methods installed in the Penn lab…
Action items: collect papers, validation reports, etc on Ab42 methods available . . .
Next call on Jan 31. Must be able to get materials to Maria in advance of next call
Ab42 MS : ? Kaj and Henrik collate works, Les to join
Ab42 Immunoassay candidate methods: ? Amsterdam to take lead (RB and Charlotte, Hugo?)
Ab42 HPLC: ? Method for preparation of sample, not detection … total vs denatured Ab42?
Ab42 SDS/Page and Western blot : ? Piotr etal working on this …
Ab42 Spectrophotometery : ?
Possible need for two independent (but linked) steps in the reference method:
Sample prep / handling / extraction – e.g. HPLC
Quantitation / detection method – MS, immunoassays, put spectrophotometry on hold
Or do we just focus on a single method ? For review of what exists for Ab42, this is only what is available, be pragmatic on what exists without need for further development (OL)
Clearly need Analytical performance characterized and ironed out before any clinical performance evaluation or assessment is considered (0L)
Step 1 of AB42: focus on MS assays as they exist today?
Suggest review published criteria for a reference method: AACC, WHO etal; look at criteria/characteristics (come to agreement) and then evaluate putative reference methods against them (RB)

7. TC Notes 31jan2012 (1)
Review of past actions since last meeting-data / materials shared
Abstract from PPD method, Waters paper out from Pfizer group (suggest present to the group), PPD additional information – Bill comfortable with distribution
What the plan is for analytical validation, # analytes, grouping, etc… ?
U Penn able to replicate PPD work flow nicely. Focus on Ab42 important, at sacrifice of other analyte performance and 2-42 are also at reasonable concentrations.
U Penn focusing on AB1-38, AB1-40, AB1-42 via MS in an adapted form of the PPD based work flow.
INNX assays: yes, they are specific to the n-term aa1, rec’d AB1-42.
Q re K Blennow data on Abx-40/Abx-42 as similar to 1-40, 1-42? A: maybe due to CSF samples, could need to compare on the same technology platform.

8. TC Notes 31jan2012 (2) PPD Poster discussion follows:
Lot 1,2,3 are individual human lots of CSF
Authentic CSF materials with spiked material and look at recovery to determine Bias
Q: what was spiked, in what liquid were the spiked analyte prepared in?
DMSO stock solution, then dilute into aCSF solution for nominal solution, to then spike this nominal solution into aCSF and the method of standard additions in hu CSF.
Note: purchase peptides AnaSpec but 1-40, 1-42 from R-peptide, non amyloid from Phoenix pharma.
Extraction recovery is from previous expts is from 70-80%, calculated into via non-radio internal standard adjusts for differential extraction recovery.
Stable isotope labeled internal standards to adjust for extraction differences: 5 standards are IS labeled, repeater pipettes within 3%.
Omar clarifies, use of internal standards via repeaters are well established methods
Challenge in LC and MS to get broad coverage, about to break in class 14/15/16/17 for one method, another method 34/37/38/40/42/43/39 would be another MS class … assay direction
PPD doing 34-43, then back for 14-17, perhaps consider zero in on ab1-42 only
Rat serum for non-specific blocker, now prefer BSA, can BSA be in tube ahead of time
Pre-analytical variables should be considered
Les U Penn to try the SUH AA QC samples program , PPD as well..
Possible Next steps:
Organize what we know amongst the 3 methods, Les/Omar to generate slide(s)
Draft validation plan to review at next meeting (Holly/Adam)
Need specify: the peptide vendors, extractions, spikes, solvents, how many analytes to be measured in the MS reference method
then go thru an extensive validation exercise
then talk about next steps to get IFCC to recognize a reference method; are we measuring the same thing, correlations between MS and immunoassay results