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Diagnostic biomarkers of Alzheimer disease International Congress on Quality in Laboratory Medicine Helsinki, 8 Feb 2018 Maryline Simon, Clinical Science Leader
Agenda
1. What is Alzheimer’s disease?
2. Technical performance of the Elecsys ß-Amyloid (1-42) CSF assay
3. Technical performance of the Elecsys Total-Tau CSF and Elecsys Phospho-Tau (181P) CSF assay
4. How Biomarkers relate to Alzheimer’s disease diagnosis
WHAT IS ALZHEIMER?
Dementia One of the most growing diseases with limited treatment and diagnosis
2013 → XXmillion people 2030 → 76 million people 2050 → XX million people The field is desperate for advancements to alleviate the heavy burden on patients, caregivers, and healthcare systems.
* WHO: 10 facts of dementia. www.who.int/features/factfiles/dementia/en/ Source: Alzheimer’s Society: www.alzheimers.org.uk/site/scripts/documents_info.php?documentID=412
2013 → 48 million people 2030 → 76 million people 2050 → 130 million people The field is desperate for advancements to alleviate the heavy burden on patients, caregivers, and healthcare systems.
Biomarkers are the earliest signs of Alzheimer’s “Jack curves”
Amyloid-beta accumulation Tau accumulation Brain structure alterations Memory impairment Functional impairment
Time Normal
Abnormal
Magn
itude
Preclinical AD Prodromal AD (=MCI)
AD dementia
>10 years ~5-7 years ~7-10 years
• Subjective symptoms can occur much later than pathology starts • Biomarkers become detectable many years before clinical onset
Clinical function
Memory
Normal
First lesions
Major lesions Bra
in
struct
ure
Jack CR Jr, et al. Lancet Neurol 2010;9:119–28
TECHNICAL PERFORMANCE OF THE BIOMARKERS
Elecsys β-Amyloid (1-42) CSF, Elecsys Total-Tau CSF and Elecsys Phospho-Tau (181P) CSF
ElectroChemiLuminescence ImmunoAssay (ECLIA) for use with Elecsys and cobas e fully automated analyzers
Easy calibration
Faster result reporting
High precision
Low sample volumes
Wide measuring range
Less hands-on time Fewer redraws/retests Right results, first time
Efficient workflow with Elecsys assays Reagent concept and advanced assay design
Easy to handle
No preparation
Long onboard stability
ELECSYS ß-AMYLOID (1-42) CSF
ElectroChemiLuminescence ImmunoAssay (ECLIA) for use with Elecsys and cobas e fully automated analyzers
Design of the Elecsys ß-Amyloid (1-42) CSF assay to measure the ß-amyloid peptide 1-42 with monoclonal antibodies in a sandwich format
Biotinylated monoclonal ß –amyloid (1-42) specific antibody (21F12)
Ruthenylated monoclonal ß-amyloid (1-42) specific antibody (3D6)
9 min
Streptavidin microparticle added
ß-amyloid
Measurement
ß-amyloid Ru 9 min
18 min. turn around time
200 – 1700 pg/mL measuring range
50 L Sample volume
<200 pg/mL LeMR (LOQ)
Ru
Stability Performance data
Analyte 8 hours @ 15-25 °C 24 hours @ 2-8 °C 8 weeks @ -20 °C >1.5 years @ -70 °C (real time) 3 freeze/thaw cycles
Reagent after opening @ 2-8 °C: 8 weeks on the analyzer: 4 weeks
Calibration after 4 weeks when using the same regent lot after 7 days when using the same lot on the analyzer
Robust reagent and calibration stability for weeks
Linearity across a broad range facilitates accurate results
All results > 1700 pg/mL are above cutoff = patients are negative to report higher values is clinically not relevant (but may be interesting scientifically)
LoQ <11.28 pg/mL
Normal measuring range 200 -1,700 pg/mL
Source: Bittner T, et al. Alzheimer’s & Dementia. 2016; 517-526 NB: NOT TO SCALE
No ‘Hook’ Effect up to ~12,000 pg/mL pg/mL
Internal interference studies showed no effects
Compound Concentration tested Interference?
Hemoglobin ≤ 11 mg/dL no
Bilirubin ≤ 0.3 mg/dL no
Lipemia (Intralipid®) ≤ 3 mg/dL no
Biotin ≤ 30 ng/mL no
Serum Albumin ≤ 0.17 g/dL no
Human IgG ≤ 0.02g/dL no
Human IgA ≤ 0.003 g/dL no
Human IgM ≤ 0.002 g/dL no
Rheumatoid Factor 14 IU/mL no
Interference studies: Endogenous interferences study
Interference studies: Common drug and special pharmaceutical interferences:
Common Pharmaceuticals Drug conc [mg/L]
Interference? Special Pharmaceuticals Drug conc [mg/L]
Interference?
N-Acetylcysteine 150 no Atorvastatin 6 no Ampicillin-Na 1000 no Clopidogrel 0.3 no Ascorbic acid 300 no Digoxin 5 no Calcium Dobesilate 200 no Donepezil 30 no Cyclosporine 5 no Escitalopram 15 no Cefoxitine 250 no Esomeprazole 170 no Heparin 5000 U/L no Furosemide 90 no Levodopa 20 no Galantamine 250 no Methyldopa +1.5 20 no Hydrochlorothiazide 120 no Metronidazole 200 no Lisinopril 500 no Phenylbutazone 100 no Memantine 250 no Doxycycline 10 no Metformin 4000 no Acetylsalicylic Acid 1000 no Metoprolol 900 no Rifampicin 60 no Rivastigmine 45 no Acetaminophen 200 no Simvastatin 3 no Ibuprofen 500 no Theophylline 100 no
Internal interference studies showed no effects
Aβ1–42 concentration correlation LC-MS vs Elecsys® assay
Assay standardized to a listed IFCC reference procedure
* International Federation of Clinical Chemistry (IFCC) Certified Reference Material (CRM) and Reference Measurement Procedure (RMP) Source: Shaw LM, et al. Alzheimer’s & Dementia 2016; 12:668
CV
<3.5%
GOAL: Evaluate external reproducibility of the Elecsys® β-Amyloid (1–42) assay under varying conditions
2 sites used all 3 lots and 2 sites
used 2 lots
Within-run variability
Between-run variability
Between-day variability
Between-laboratory variability
Lot-to-lot variability
University of Gothenburg
VU University Medical Center
Amsterdam
Covance Indianapolis
Washington University at St. Louis
external sites
different lots 5 days 2 aliquots
per run 2 runs per day 4 3
* Roche Diagnostics: this product is not yet available in the USA.
Multicenter evaluation study of the fully automated Elecsys® β-Amyloid (1–42) assay*: methods
CV
<3.5%
Parameter variability as %CV
Sample (mean Aβ(1–42) concentration)
Control 1 485pg/mL
Control 2 869pg/mL
CSF 1 342pg/mL
CSF 2 662pg/mL
CSF 3 755pg/mL
CSF 4 935pg/mL
CSF 5 1472pg/mL
Range CSF samples
Within-run (repeatability) 1.02 0.90 1.56 1.35 2.66 1.50 2.04 1.35–2.66
Between-run 0.54 0.47 0.17 0.75 2.46 0.71 0.23 0.17–2.46
Between-day 1.35 1.41 1.75 1.44 0 0.95 1.58 0–1.75
Between-laboratory 0.66 0.76 0 0.46 3.49 0.75 1.75 0–3.49
Lot-to-lot 0.59 1.03 2.25 1.46 0.61 0.89 0.78 0.61–2.25
Total reproducibility†
(95% CI) 1.99
(1.65; 2.49)
2.16
(1.67; 3.05)
3.25
(2.22; 6.04)
2.61
(1.96; 3.88)
5.07
(3.64; 8.33)
2.24
(1.84; 2.86)
3.22
(2.52; 4.46)
2.24–5.07
* Roche Diagnostics: this product is not yet available in the USA. †Total reproducibility includes all factors (estimated from a variance components model): sites, lots, days, runs, within-run
MCE study (Reproducibility) showed high precision
ELECSYS TOTAL-TAU CSF AND ELECSYS PHOSPHO-TAU (181P) CSF
ElectroChemiLuminescence ImmunoAssay (ECLIA) for use with Elecsys and cobas e fully automated analyzers
pTau
Biotinylated monoclonal Tau specific antibody/ies
Ruthenylated monoclonal Tau specific antibody (PC1C6)
9 min
Streptavidin microparticle added Measurement
Tau Tau
Bi-pTau (181P) mAb (11H5V1)
Ru
Design of the Elecsys Total-Tau CSF and Elecsys Phospho-Tau (181P) CSF assays …to measure the total Tau /or phosphorylated (181P)
Tau with monoclonal antibodies in a sandwich format
Ru
Bi-Tau mAbs (5.28.461 and 4.35.411)
tTau
9 min
Stability Performance data
Analyte
5 days @ 15-25°C 14 days @ 2-8°C 3 months @-20°C 6 months @-80 °C 3 freeze/thaw cycles
Reagent after opening @ 2-8 °C: 8 weeks on the analyzer: 4 weeks
Callibration after 4 weeks when using the same regent lot after 7 days when using the same lot on the analyzer
…Stability shown for weeks
Robust reagent and calibration stability
Linearity across a broad range facilitates accurate results …Enough for Clinical routine use
No ‘Hook’ Effect up tTau ~3,000 pg/mL pTau ~300 pg/mL
Normal measuring range
tTau: 80 -1300 pg/mL pTau: 8 -120 pg/mL
MR of Tau assays cover almost 100% of values from IU population
LoQ tTau <62.6 pg/ml pTau < 3.90 pg/mL
Internal interference studies
Common drug and special pharmaceutical interferences:
Compound Concentration tested tTau/(pTau*) Interference?
Rheumatoid factors ≤ 4 IU/mL/ (12 IU/mL*) no
Bilirubin ≤ 6.6 mg/dL /(3 mg/dL*) no
Lipemia (Intralipid®) ≤ 200 mg/dL no
Biotin ≤ 50 ng/mL no
Serum Albumin ≤ 0.7 g/dL no
Human IgG ≤ 0.09 g/dL no
Human IgA ≤ 0.016 g/dL no
Human IgM ≤ 0.004 g/dL no
Hemoglobin ≤ 100 mg/dL no
No effects pTau
Albumin , 0.7g/dL Bilirubin, 6.6mg/dL
Biotin, 60ng/L Haemoglobin, 100mg/dL
IgA, 0.16g/dL IgG, 0.09g/dL
IgM, 0.004 g/dL Rheumatoid factor, 4.2lU/mL
Albumin , 0.7 g/dL Bilirubin, 3 mg/dL
Biotin, 54ng/L Haemoglobin, 100mg/dL
IgA, 0.16g/dL IgG, 0.09g/dL
IgM, 0.004 g/dL Rheumatoid factor, 12lU/mL
tTau
90 95 100 105 110 90 95 100 105 110
A B
Figure 2. Interference of the Elecsys® tTau and pTau Assays by (A) endogenous compounds, (B) common drugs and (C) special drugs
Recovery (%) Recovery (%)
tTau pTau tTau pTau
Internal interference studies No effects
Common drug and special pharmaceutical interferences:
Common Pharmaceuticals Drug conc [mg/L] tTau/(pTau*) Interference? Special Pharmaceuticals Drug conc
[mg/L] tTau/(pTau*) Interference?
N-Acetylcysteine 1660 no Atorvastatin 2 (6) no Ampicillin-Na 1000 no Clopidogrel 0.1 (0.3) no Ascorbic acid 300 no Digoxin 5 (3) no Cyclosporine 5 no Donepezil 10 (30) no Cefoxitine 2500 (2000) no Escitalopram 10 no Heparin 500 U/L (5000 U/L) no Esomeprazole 56 (170) no Levodopa 3 (20) no Furosemide 50 no Methyldopa +1.5 20 no Galantamine 83 no Metronidazole 200 no Hydrochlorothiazide 40 no Phenylbutazone 400 no Lisinopril 500 no Doxycycline 7.5 (50) no Memantine 83 no Acetylsalicylic Acid 1000 no Metformin 3000 (2000) no Rifampicin 60 no Metoprolol 500 (900) no Acetaminophen 200 no Rivastigmine 15 no Ibuprofen 500 no Simvastatin 1 (3) no Theophylline 100 no
Parameter variability as %CV
Sample (mean tTau concentration)
Control 1 172.4 pg/mL
Control 2 449.7 pg/mL
CSF 1 211.2 pg/mL
CSF 2 289.2 pg/mL
CSF 3 413.5 pg/mL
CSF 4 638 pg/mL
CSF 5 1084 pg/mL
Range CSF samples
Within-run (repeatability)
1.20 1.15 1.53 1.63 1.31 1.74 1.81 1.31–1.81
Between-run 1.98 1.90 2.25 2.19 2.02 2.14 2.04 2.02–2.25
Between-day 2.31 2.22 2.73 2.73 2.41 2.76 2.73 2.41–2.76
Between-laboratory 2.67 2.15 0.883 1.09 1.02 1.13 1.55 0.883–1.55
Lot-to-lot 5.02 4.28 5.76 5.57 5.35 5.61 5.92 5.35–5.92
Total reproducibility† 6.14 5.28 6.43 6.30 5.95 6.35 6.70 5.95–6.43
MCE Study (Reproducibility Study) Elecsys tTau shows high precision
CV
<3.5%
Parameter variability as %CV
Sample (mean pTau concentration)
Control 1 14.20 pg/mL
Control 2 33.15 pg/mL
CSF 1 19.79 pg/mL
CSF 2 28.82 pg/mL
CSF 3 40.69 pg/mL
CSF 4 61.93 pg/mL
CSF 5 102.4 pg/mL
CSF 6 108.9 pg/mL
Range CSF samples
Within-run (repeatability)
1.57 1.32 1.40 1.33 1.43 1.75 1.49 1.53 1.33–1.75
Between-run 1.31 1.45 1.87 1.99 2.19 1.59 2.11 2.31 1.59–2.31
Between-day 2.04 1.96 2.34 2.39 2.62 2.36 2.59 2.77 2.34–2.77
Between-laboratory 2.23 2.38 1.05 1.37 2.47 2.39 2.50 2.62 1.05–2.62
Lot-to-lot 3.33 2.06 1.76 1.98 2.78 3.05 2.97 2.78 1.76–3.05
Total reproducibility† 4.50 3.70 3.11 3.39 4.55 4.54 4.67 4.72 3.11–4.72
MCE Study (Reproducibility Study)
Elecsys pTau shows high precision
Enhanced QC Concept
Ensuring high precision and accuracy
To ensure high precision and accuracy of Abeta42, tTau and pTau measurements in all laboratories world-wide, we have elaborated an „Enhanced Quality Control Concept“ (eQC Concept), where each laboratory establishes it‘s own measurement bias and CV.
The eQC Concept is based on Total Error Requirements, which were established through clinical considerations.
The maximal allowable Total Error (TEa) for tTau and pTau assays is: TEa (%) = relative bias (%) + 1.65 x CV (%) = 26.5% with: relative bias ≤ 10% and CV ≤ 10%
Key featuresw tTau pTau Abeta42
Measuring range 80 - 1300 pg/mL 8 - 120 pg/mL 200 - 1700 pg/mL
Limit of Quantification (LoQ) 63 pg/mL 4 pg/mL 11 pg/ml
Within-run precision CV 0.7 – 1.2 % CV 1.5 – 2.7 % CV 0.8 – 1.6 %
Within-lab precision CV 1.4 – 2.0 % CV 1.9 – 3.5 % CV 1.6 – 2.9 %
External reproducibility (3 sites) CV 5.9 - 6.4 % CV 3.1 - 4.7 % CV 2.2 - 5.1 %
Lot-to-lot consistency (3 lots) r=0.998 (Pearson) r=0.999 (Pearson) r=0.996 (Pearson)
Platform consistency (e411 vs. e601) r=1.00 (Pearson) r=1.00 (Pearson) n.a.
Correlation to Innotest r=0.98 (Spearman) r=0.96 (Spearman) r=0.95 (Spearman)
Reagent stability 4°C > 25 months > 25 months > 25 months
Onboard stability 20°C > 4 weeks > 4 weeks > 4 weeks
No cross reactivity for n.a. non-p181P-Tau Abeta(1-38) & (1-40)
Reference Method/Material Weighted tTau Roche Ref. Material
Weighted pTau Roche Ref. Material LC-MS (Leinenbach et al. 2014)
…to ensure minimal batch variation
Analytical Performance tTau, pTau and Abeta42 assays Summary
Alzheimer’s Association Quality Control (AAQC) Program for CSF biomarkers
CSF=cerebrospinal fluid; CV=coefficient of variation Source: CPS MSA after Roche Dx – Roche Rx update medical (Nov. 2016)
Aim: to standardize biomarker measurements between laboratories (research and clinical)
Design: - Aliquots of pooled CSF samples sent to participating laboratories - 2 unique samples per round for comparisons
between laboratories - PLUS QC sample maintained over multiple rounds
for longitudinal comparisons - 3 rounds per year - ~90 laboratories participate - Final report for each round includes:
- Biomarker concentrations for individual laboratories - Mean biomarker concentrations and %CV
for all laboratories - Longitudinal stability (percentage deviation over time)
CSF=cerebrospinal fluid; CV=coefficient of variation Source: CPS MSA after Roche Dx – Roche Rx update medical (Nov. 2016)
Between laboratory CV (percent): Between laboratory CV (percent):
INNOTEST@
β-Amyloid (1-42) Fujirebio
Euroimmune / ADx
β-Amyloid (1-42)
AlzBio3
β-Amyloid (1-42) Fujirebio
Meso-Scale Human Aβ42
IBL Aβ 1-42
Elecsys@
β-Amyloid (1-42) Roche Diagnostics
Lumipulse@
β-Amyloid (1-42) Fujirebio
Round ELISA ELISA Luminex ECL V-PLEX ELISA Fully automated Fully automated
2014-14A 18 16 low n 2,9
2014-14B 21 18 low n 4,4
2014-15A 15 7,1 12 4,6
2014-15B 17 14 12 3,4
2014-16A 27 57 40 13 3
2014-16B 17 19 30 11 2,5
2015-17A 19 6,5 17 21 1,9
2015-17B 14 8,2 15 20 3,2
2015-18A 13 22 25 10 7,2
2015-18B 13 16 13 9,4 4,7
2015-19A 13 13 15 10 3
2015-19B 13 13 14 13 1,5
2016-20A 17 18 ND 11 2
2016-20B 21 15 ND 14 Level out of range
2016-21A 15 18 22 8,1 10 6,3
2016-21B 11 15 29 7,4 5,2 4
2016-22A 14 12 28 39 5,9 7,5
2016-22B 19 13 24 37 6,7 9,4
2017-23A 15 7,3 12 16 1,6 21
2017-23B 16 13 18 19 3 8,6
2017-24A 15 8,9 too few 29 13 3 13
2017-24B 13 9,9 too few 25 3,1 5,6 14
2017-25A 13 9,0 23 20 6 11 10
2017-25B 14 9,5 24 21 8,7 12 14
Mean 16,0 15,2 21,6 17,2 7,7 4,7 10,8
The Alzheimer’s Association QC program – results 2014-2018
Between laboratory CV (percent): Between laboratory CV (percent):
INNOTEST@
Total tau Fujirebio
Euro Immune Total tau
AlzBio3
Total tau Fujirebio
Elecsys@
Total tau Roche Diagnostics
Lumipulse@
Total tau Fujirebio
INNOTEST@
Phospho tau Fujirebio
AlzBio3
Phospho tau Fujirebio
Elecsys@
Phospho tau Roche Diagnostics
Round ELISA ELISA Luminex Fully
automated
Fully
automated Round ELISA Luminex
Fully
automated
2014-16A 19 6 16 2014-16A 16 25
2014-16B 20 21 18 2014-16B 17 22
2015-17A 28 17 20 2015-17A 16 34
2015-17B 20 <LLOQ 19 2015-17B 13 6
2015-18A 14 10 13 2015-18A 10 32
2015-18B 12 7,2 13 2015-18B 10 7
2015-19A 15 23 18 2015-19A 12 58
2015-19B 14 29 11 2015-19B 11 35
2016-20A 14 12 7 2016-20A 14 54
2016-20B 15 9,4 13 2016-20B 17 14
2016-21A 17 20 18 2016-21A 12 48
2016-21B 20 5,1 14 2016-21B 11 20
2016-22A 15 6,6 15 2016-22A 7,0 60
2016-22B 19 4,4 23 2016-22B 14 52
2017-23A 14 14 11 2017-23A 10 44
2017-23B 12 18 8,5 2017-23B 9,9 33
2017-24A 13 5,7 too few 3,8 2017-24A 12 too few 20
2017-24B 13 19 too few 3,6 2017-24B 13 too few 1,7
2018-25A 14 18 5,2 1,3 4 2017-24A 9,1 28 1,3
2018-25B 13 22 36 2,2 6,5 2017-24B 11 36 2,2
Mean 16,0 14,1 15,5 2,7 5,3 Mean 12,3 33,7 1,8
The Alzheimer’s Association QC program – results 2014-2018
CLINICAL VALIDATION & UTILITY
Elecsys β-Amyloid (1-42) CSF, Elecsys Total-Tau CSF and Elecsys Phospho-Tau (181P) CSF
Patients with cognitive decline
or
For the detection of amyloid
Patients with cognitive decline
…adapt therapy for personalized healthcare CSF biomarkers can be used to
Identify patients at higher risk of progression
3.0
2.5
2.0
1.5
0 6 12 24 Visit
CDR-
SB
…Patient identification
Biomarker status Abeta42, tTau and pTau PET concordance and progression claim (CE)
CSF biomarkers concordance with visual read amyloid-PET
Alternative to amyloid-PET (PET concordance) Identification of subject likely to progress (progression)
Aβ421000 pg/mL Aβ421000 pg/mL
tTau/Aβ420.28 tTau/Aβ420.28
pTau/Aβ420.024 pTau/Aβ420.024
tTau300 pg/mL* pTau27 pg/mL*
pTau
pg/m
L
Bio
FIN
DE
R
pTau vs Abeta42
N = 277
25
50
75
ADNI
tTau
pg/m
L
tTau vs Abeta42
N = 277
250
500
750
pTau
pg/m
L
N = 646
25
50
75
tTau
pg/m
L
N = 646
600
800
200
400
1000 2000 3000 1000 2000 3000
1000 2000 3000 1000 2000 3000
Abeta42 pg/mL Abeta42 pg/mL
100 1000
100
PET visual read
Negative
Positive
Cut-off
Abeta42=β-Amyloid(1–42); ADNI=Alzheimer's Disease Neuroimaging Initiative; CI=confidence interval; CSF=cerebrospinal fluid; NPA=negative percentage agreement; OPA=overall percentage agreement; PET=positron emission tomography; PPA=positive percentage agreement; pTau=phosphorylated Tau; tTau=total Tau.A
* tTau and pTau have no values as alternatives to amyloid-PET as they are not relevant.
CSF Biomarkers identify MCI-patients at higher risk of progression
Cut-off: 0.028 N: 300 (BM-) N: 319 (BM+)
0 6 12 24
1.5
2.0
2.5
3.0
Figure 2. Change in CDR-SB over time according to pTau/Abeta42 status (ADNI progression cohort, n=619).
Poster presentation CTAD 2017
Single assay or in Ratio
pTau/ Albeta 42, tTau/
Abeta 42
Patients assessed for AD
Patient (SCD, MCI and AD)
No plaques Cognitive issue
due to other non AD cause
Presence of amyloid plaques, Informs HCP’s on likelihood that
symptoms are caused by an underlying AD pathological process.
Abeta42 Positive
No or low amyloid plaques
Cognitive impairment probably not due to AD
Positive Negative Negative
Patients with confirmed MCI
Single assay or in Ratio
Lower risk of progression within next 2 years
higher risk of progression within next 2 years
implications for patients, their relatives & and their carers
Patient (MCI)
Negative
Positive
Biomarkers Abeta42, tTau and pTau, ratio recommended Summary utility
Doing now what patients need next
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