PRE-ANALYTICAL VARIABLES IN THE CONTEXT OF PUBLISHING/ESTABLISHING A STANDARDIZED PRE-ANALYTICAL PROTOCOL FOR ALZHEIMER’S DISEASE (AD) RESEARCH USE

DANNI LI, SILVIA FOSSATI, DOUGLAS GALASKO AND MICHELLE MIELKE,

ON BEHALF OF THE BBB PIA REFERENCE RANGES WORKING GROUP

GBSC AND SABB JOINT MEETING

10/30/18

BACKGROUND ON THE CHECKLIST COMPILED BY THE BIOFLUID BIOMARKERS PIA REFERENCE RANGES WORKING GROUP

• Reference ranges depend on many things: pre-analytical factors, assay methods, intended use, patient populations etc.

• In order to establish ref ranges, we need to have a comprehensive understanding of all these factors (including pre-analytical factors) and how they vary from studies to studies.

REFERENCE RANGES WORKING GROUP

• Chairperson/Facilitator: Dr. Michelle Mielke

• 22 members worldwide

• Initial goals/steps1. Identify and decide on a few promising biofluids based markers that may have future use at the

general physician level

2. Compare/contrast study sample collection; assays; populations (e.g., exclusion criteria)

3. While ultimate goal may be ‘normal ranges’, differences in study methods and clinical assays will be a factor

4. Decide on how best to move forward (e.g., standardize and re-assay measures?)

5. Identify studies interested in donating samples to this effort and participating

BBB PIA REFERENCE RANGES WORKING GROUP DEVELOPED AND TESTED A COMPREHENSIVE CHECKLIST

• Sample collection (pre-analytical variables)

• Assay information (analytical)

• Assay information (clinical) and statistical analysis

• Study population

Tested using 4 recently published manuscripts on blood amyloid peptides.

This check list was shared with the joint meeting members.

Checklist components:

Pre-analytical variablesThese 13 variables were selected based on literature, consensus, and the group’s clinical chemistry expertise.

Guidelines for the standardization of preanalytic variables for blood-based biomarker studies in Alzheimer's disease research.O'Bryant SE, et al.Alzheimers Dement. 2015 May;11(5):549-60. doi: 10.1016/j.jalz.2014.08.099. Epub2014 Oct 1. Review.

Sample collection (pre-analytical variables)

Teunissen et al 2018 JAD (Michelle)

Ovod et al 2018 AlzDem (Danni Li)

Lovhem et al 2017 AlzDem (Silvia)

Nakamura et al 2018 Nature Letter (Doug) NOTE: preanalyticalfactors may matter because data from Japanese subjects and AIBL subjects had different dynamic ranges.

Time of collection N/A Approximately hourly in 24 hours.

Early morning (80%) morning

Fasting status N/A Fasting over night (although blood samples were collected hourly for a 20 time points after IV bolus or oral intake of 800 mg labelled Leucine)

Yes. 4h fasting (20%) or overnight fasting (80%). The authors previously evaluated the effects of fasting with no significant variations in plasma ab40and ab42 (Hansson 2010)

unknown

If non fasting, time from last meal

N/A N/A Yes

Needle size and location of draw

N/A N/A N/A

Handling of tubes N/A Yes (samples were centrifuged immediately on collection).

N/A

Tube types and additives, e.g., EDTA, acid citrate, heparin

EDTA Polypropylene tubes (Axygen)

EDTA (however this is not clearly specified in the referenced paper (Hallmans 2003)

EDTA

Tube collection order N/A N/A N/A

Time of sample in collection tube

N/A I am not sure what this means. It is very likely this information is not provided.

N/A

Centrifugation parameters 1500-2500 g for 15 minutes

N/A N/A

Time from collection to freeze

N/A N/A N/A N/A

Temperature of freeze -80C negative 80 celsius negative 70 celsius -80C

Freeze-thaw cyles 1 No explicit information is provided (maybe never thawed samples?)

N/A

Aliquot size 0.5 ml N/A 1.5mL unknown

Assays (including analytical performance) and biochemical testing processMethodology (e.g., antibody based immunoassay, mass spectrometry based) and platform if relevant (e.g., MSD)If ELISA or similar: chemiluminescent/ fluorescent/ PCR readoutValidated for multiplexing or notCommericially available or home brewedQuantitative (e.g., calibration curve established to derive quantitative results)

Quality control samples used Calibration standards used e.g., recombinant peptides (including source/vendor)Compatible additives (EDTA, heparin etc) Compatible biofluid (plasma or serum) Between day precisionsAssay sensitivity Assay specificityVolume of sample neededIs sample diluted? What diluent?LLODULODLOQLinear rangeHeterophile Abs cause interference?Intralab CV for assay (intra-assay)Intralab CV for assay (inter-assay)Inter lab CV for assaySamples tested randomized?Samples tested blinded?Samples tested in multiple batches?If in batches, did each batch contain equal N for each study group?Reagent lot changed during the biochemical testing process?Dilution experiments performed during assay validationSpike-in experiments performedDetergent, protease inhibitor or other reagent addedImmunoprecipitation protocol

Assay Methodology and biochemical process variables

Assays (including clinical performance) and statistical analysis

Intended use or context of use

Dependent variable

Independent variable: AD related outcomes (e.g.,clinical diagnosis, neuroimaging data, or other fluid biomakers)

Clinical sensitivity

Clinical specificity

Positive predictive value

Negative predictive value

Effect size

Brief discrption of the statistical method used

Has the results been validated in other independent samples? If so, were the assay conducted by a different lab?

Assay clinical performance and Statistical analysisVariables

Study populationSample size AgeSexEthinicityRaceAPOE e4 Smoking status (current/former/never)GestationDietMedications (e.g., lipid lowering, antihypertensive)No-AD comorbidiites: cardiovascular diseases, diabetes, cerebrovascular disease (e.g., stroke), hypertension, psychiatric disorders, TBIAlchohol use: current/former/neverPhysical activity levelExclusion criteria usedBMIRenal functionDiabetes

Study Population Variables

#1: ORIGINAL RESEARCH STUDIES ARE NOT DESIGNED TO STUDY PRE-ANALYTICAL/ANALYTICAL VARIABLES

• Comprehensive pre-analytical variables information are lacking from original research studies (e.g., 4 references included in our checklist exercise).

• Purpose of these original research studies are not to evaluate pre-analytical or analytical variables.

• Studies of pre-analytical/analytical variables require a different study design.

Insights gained from this exercise that may be helpful for a pre-analytical protocol discussion:

EXAMPLES OF STUDIES DESIGNED TO EVALUATE PRE-ANALYTICAL VARIABLES

#1B. STUDY RESULTS ARE DIFFERENT DUE TO MANY FACTORS, NOT JUST IN HOW SAMPLES ARE COLLECTED (EG. PRE-ANALYTICAL VARIABLES), BUT ASSAY METHODS, PATIENT POPULATIONS ETC.

• If interested in assay method and performance difference, do a round robin study as Kaj/Henrik suggested using the same set of samples collected under the most pristine condition possible (eliminate pre-analytical variables) to be measured by different methods

• If interested in pre-analytical variables, do a preanalytical variable study (responsibility of research group, a few groups or assay manufacturer) since these preanalytical variables may be method specific.

• If interested in comparing clinical performance of specific context of use, do a round robin study with the same set of samples collected under the most pristine condition possible with clinical information.

#2: PRE-ANALYTICAL VARIABLES CAN BE METHOD (OR TARGET) SPECIFIC.

• This does not mean that a generic pre-analytical protocol for all AD biomarkers is not possible.

• However, studies of pre-analytical variables should include all intended methods/biomarkers.

• A clarifying question could be: will the standardized protocol be applicable to all promising AD blood biomarkers/methods?

• Amyloid peptides (Simoa, IMR, IP-MS, and etc)

• NFL (Simoa, IMR and etc)

• Total Tau (Simoa, IMR and etc)

• P-tau (Simoa, IMR and etc)

TOP BIOMARKER CANDIDATES (FOR RR WORKING GROUP)

We received a total of 29 votes (up to 3 candidates per member)

• NfL (9)• Screening/diagnosis, prognosis, progression, and treatment efficacy

• p-tau 181 (5), total tau (4), p-tau 231 (1), p-tau 396 (1)• Screening/diagnosis, prognosis, progression, and treatment efficacy

• Amyloid beta 42, 40, 38 or ratio (4)• Screening/diagnosis and treatment efficacy

QUESTIONS

•Should the standardized protocols be analyte and/or method specific?

• If so, what blood analytes and methods should these standardized protocols include?

•Can we establish best pre-analytical protocols from the literature or do we need to test them in new studies?