Research

JAMA Neurology | Original Investigation

Concordance Between Different Amyloid Immunoassays and

Visual Amyloid Positron Emission Tomographic Assessment
Shorena Janelidze, PhD; Josef Pannee, PhD; Alvydas Mikulskis, PhD; Ping Chiao, PhD;
Henrik Zetterberg, MD, PhD; Kaj Blennow, MD, PhD; Oskar Hansson, MD, PhD

Supplemental content
IMPORTANCE Visual assessment of amyloid positron emission tomographic (PET) images has
been approved by regulatory authorities for clinical use. Several immunoassays have been
developed to measure -amyloid (A) 42 in cerebrospinal fluid (CSF). The agreement
between CSF A42 measures from different immunoassays and visual PET readings may
influence the use of CSF biomarkers and/or amyloid PET assessment in clinical practice and
trials.

OBJECTIVE To determine the concordance between CSF A42 levels measured using 5
different immunoassays and visual amyloid PET analysis.

DESIGN, SETTING, AND PARTICIPANTS The study included 262 patients with mild cognitive
impairment or subjective cognitive decline from the Swedish BioFINDER (Biomarkers for
Identifying Neurodegenerative Disorders Early and Reliably) cohort (recruited from
September 1, 2010, through December 31, 2014) who had undergone flutemetamol F 18
([18F]flutemetamol)labeled PET. Levels of CSF A42 were analyzed using the classic
INNOTEST and the newer modified INNOTEST, fully automated Lumipulse (FL), EUROIMMUN
(EI), and Meso Scale Discovery (MSD) assays. Concentrations of CSF A were assessed using
an antibody-independent mass spectrometrybased reference measurement procedure.

MAIN OUTCOMES AND MEASURES The concordance of CSF A42 levels and A42:A40 and
A42:tau ratios with visual [18F]flutemetamol PET status.

RESULTS Of 262 participants (mean [SD] age, 70.9 [5.5] years), 108 were women (41.2%) and
154 were men (58.8%). The mass spectrometryderived A42 values showed higher
correlations with the modified A42-INNOTEST (r = 0.97), A42-FL (r = 0.93), A42-EI
(r = 0.93), and A42-MSD (r = 0.95) assays compared with the classic A42-INNOTEST assay
(r = 0.88; P .01). The signal in the classic A42-INNOTEST assay was partly quenched by
recombinant A1-40 peptide. However, the classic A42-INNOTEST assay showed better
concordance with visual [18F]flutemetamol PET status (area under the receiver operating
characteristic curve [AUC], 0.92) compared with the newer assays (AUCs, 0.87-0.89;
P .01). The accuracies of the newer assays improved significantly when A42:A40 (AUCs,
0.93-0.95; P .01), A42 to total tau (T-tau) (AUCs, 0.94; P .05), or A42 to
phosphorylated tau (P-tau) (AUCs, 0.94-0.95; P .001) ratios were used. A combination of
the A42:A40 ratio and T-tau or P-tau level did not improve the accuracy compared with the
ratio alone.

CONCLUSIONS AND RELEVANCE Concentrations of CSF A42 derived from the new
immunoassays (modified INNOTEST, FL, EI, and MSD) may correlate better with the
antibody-independent mass spectrometrybased reference measurement procedure and
may show improved agreement with visual [18F]flutemetamol PET assessment when using
the A42:A40 or A42:tau ratios. These findings suggest the benefit of implementing the Author Affiliations: Author
CSF A42:A40 or A42:tau ratios as a biomarker of amyloid deposition in clinical practice affiliations are listed at the end of this
article.
and trials.
Corresponding Author: Shorena
Janelidze, PhD, Clinical Memory
Research Unit, Department of Clinical
Sciences Malm, Lund University,
Slvegatan 19, BMC Building B11,
JAMA Neurol. doi:10.1001/jamaneurol.2017.2814 221 84 Lund, Sweden
Published online November 6, 2017. (shorena.janelidze@med.lu.se).

(Reprinted) E1
2017 American Medical Association. All rights reserved.

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 Research Original Investigation
C
erebrospinal fluid (CSF) -amyloid (A) 42 and amy-
loid positron emission tomography (PET) have proved
to have high diagnostic accuracy for Alzheimer dis-
ease (AD) years before the onset of clinical symptoms and are
becoming an important part of the diagnostic workup.1 Both
biomarkers have shown correlations with postmortem plaque
measurements,2-5 and previous studies have indicated 80%
to 90% agreement between CSF A42 values and quantita-
tive amyloid PET data.6-8 For most immunoassays, the con-
cordance between the 2 biomarker modalities is further im-
proved with the CSF A42:A40 ratio (compared with CSF
A42 level alone), probably because the ratio corrects for
(1) interindividual variability in the overall A production;
(2) interindividual variability in the CSF turnover; (3) changes
in global levels of all A isoforms owing to non-ADrelated
abnormal findings, such as white matter lesions; and (4) vari-
ability owing to preanalytical factors.9-11
Several enzyme-linked immunosorbent assays (ELISAs) are
commonly used for measurements of A42 levels in CSF. In
general, the precision of an immunoassay may be compro-
mised by matrix interference when endogenous biological fac-
tors in the sample interact with the analyte of interest or non-
specifically bind to antibodies. Matrix interference affecting
A42 quantification has been reported for the initially devel-
oped INNOTEST A42 assay12 but seems to be minimized in the
newer A immunoassays from EUROIMMUN, Fujirebio, Meso
Scale Discovery, and Roche.13-15 A recent study suggested that
the diagnostic accuracy of CSF A biomarkers may vary de-
pending on the assays used.16 More specifically, CSF A42 mea-
sured with the classic INNOTEST (Fujirebio) kit17 performed bet-
ter in distinguishing patients with early-stage AD from
cognitively healthy control individuals than one of the newer
immunoassays. At same time, using the CSF A42:A40 ratio
improved the diagnostic accuracy of the newer EUROIMMUN
(EI; EUROIMMUN) A42 assay but not the classic A42
INNOTEST assay.16 Another report9 demonstrated higher con-
cordance between quantitative amyloid PET and the A42:
A40 ratio compared with CSF A42 level alone for the EI and
Meso Scale Discovery (MSD; Meso Scale Discovery) assays.
In most of the studies that have compared amyloid PET
with CSF A42 levels, the quantification of regional and global
amyloid PET ligand binding has used semiquantitative image
assessments of standardized uptake value ratio (SUVR). In clini-
cal practice, however, the standard approved by regulatory au-
thorities is visual classification of scans on a binary scale as
positive or negative for amyloid, with a negative finding indi-
cating that AD is unlikely. Visual readings of PET images agree
with PET SUVR values and with brain amyloid burden at
autopsy.18,19 Nevertheless, concordance between the visual
reading of PET and CSF A42 measures from different immu-
noassays has not been established and may influence the use
of amyloid PET and/or CSF analysis in clinical practice and
trials. In the present study, we investigated the agreement be-
tween visual amyloid PET analysis and CSF A42 measured
using 5 different A42 immunoassays or protocols
(INNOTEST A1-42 classical procedure, INNOTEST A1-42
modified procedure, fully automated Lumipulse [FL;
Fujirebio], EI A1-42, and MSD AN-42). For each of the 5 A42 standardized protocol.8,21
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Use of Different Amyloid Assays in Visual Assessment of Amyloid PET
Key Points
Question How well do the core cerebrospinal fluid biomarkers of
Alzheimer disease measured using different immunoassays agree
with visual amyloid positron emission tomographic assessment?
Findings In this study of 262 individuals with mild cognitive
complaints, the cerebrospinal fluid A42:A40 or A42:tau ratios
from several newer assays showed improved accuracy for
detection of cortical -amyloid fibrils as measured by positron
emission tomography.
Meaning These findings support implementation of the
cerebrospinal fluid A42:A40 and/or A42:tau ratios as
biomarkers of amyloid deposition in clinical practice and trials.
immunoassays, the performance of A42 and the A42:
A40, A42 to total tau (T-tau), and A42 to phosphorylated
tau (P-tau) ratios were determined using visual assessment
of flutemetamol F 18labeled PET images as the criterion
standard. Furthermore, an important advancement in
the CSF biomarker field was the recent development of a mass
spectrometrybased reference measurement procedure
(RMP),20 which allows antibody-independent quantification
of absolute CSF concentration of A42 with high accuracy and
is now considered to be the criterion standard method
for CSF A42 measurement. 1 However, implementation
of mass spectrometry in routine clinical laboratories is chal-
lenging owing to the complexity of the technology, the need
for in-house method development and validation and per-
sonal training, and high costs. Herein, we explored correla-
tion between CSF levels of amyloid biomarkers analyzed using
immunoassays and antibody-independent mass spectrometry
based RMP.
Methods
Study Participants
The study population included 262 patients with mild cogni-
tive impairment or subjective cognitive decline and no de-
mentia from the prospective and longitudinal Swedish
BioFINDER (Biomarkers for Identifying Neurodegenerative Dis-
orders Early and Reliably) cohort (http://www.biofinder.se) who
had undergone [ 18 F]flutemetamol PET evaluation from
September 1, 2010, through December 31, 2014. The diagnostic
criteria and cohort characteristics are described in the
eMethods and eTable 1 in the Supplement. The study was
approved by the Regional Ethical Review Board in Lund,
Sweden, and all study participants gave written informed
consent to participate in the study.
CSF Sampling and Analysis
The procedure and analysis of CSF samples were conducted
according to the Alzheimer Association Flowchart for CSF
biomarkers.21 Lumbar CSF samples were collected at 3 memory
clinics at the Skne University Hospital in Lund and Malm and
ngelholm Hospital in Sweden and analyzed according to a
jamaneurology.com
 Use of Different Amyloid Assays in Visual Assessment of Amyloid PET Original Investigation Research

We measured CSF A42 levels using the EI A1-42 and MSD

Table 1. Correlations Between As Measured
AN-42 according to the manufacturers instructions. We also Using MS and Different Immunoassaysa
measured CSF A42 (1-42) with the INNOTEST kit (Fujirebio).
MS-Derived MS-Derived
For the latter assay, the classic procedure17 as described in the Immunoassay A42 Level A42:A40 Ratio
products package insert and a modified procedure that mini- Classic A42-INNOTEST
mizes matrix interference effects were applied.15 The modifi- Pearson correlation r = 0.88 r = 0.87
cations included (1) dilution of conjugate 1 in conjugate dilu- Spearman correlation = 0.91 = 0.88
ent at 1:10 (classic procedure, 1:100 dilution), (2) addition of Modified A42-INNOTEST
only 10 L of samples or standards to the plate (classic proce- Pearson correlation r = 0.97 r = 0.70b
dure, 25 L of samples or standards), and (3) incubation of Spearman correlation = 0.96 b
= 0.74b
samples or standards for 3 hours (classic procedure, 1 hour in- A42-FL
cubation). In addition, CSF concentrations of A42 were de-
Pearson correlation r = 0.93c r = 0.64b
termined using the fully automated FL assay, which is based
Spearman correlation = 0.92 = 0.70b
on the modified INNOTEST procedure. We analyzed CSF A40
A42-EI
levels with kits from Fujirebio (INNOTEST A1-40), EI (A1-
Pearson correlation r = 0.93d r = 0.73b
40), and MSD (AN-40) according to the manufacturers in- d
Spearman correlation = 0.95 = 0.78b
structions, and the ratios with the corresponding CSF A42
A42-MSD
values (A42:A40) were calculated. We used the MSD elec-
Pearson correlation r = 0.95b r = 0.74b
torchemiluminescence assay (Vplex; MSD) for multiplex de-
b
Spearman correlation = 0.96 = 0.78b
tection of A42, A40, and A38 levels. Cerebrospinal fluid T-
tau and P-tau(181P) were quantified using EUROIMMUN and Abbreviations: A, amyloid ; EI, EUROIMMUN; FL, fully automated Lumipulse;
INNOTEST ELISA kits, respectively. These tau values and A42 MS, mass spectrometry; MSD, Meso Scale Discovery.
a
values from 5 different assays (classic INNOTEST, modified P < .001 for all comparisons except P = .02 for the correlation between
MS-derived A42 and A42-INC:A40 ratio. Differences between the
INNOTEST, FL, EI, and MSD) were used for the calculations of correlation coefficients were estimated using the Meng test.
the CSF A42:tau ratios. b
P < .001 compared with classic A42-INNOTEST.
Increasing concentrations of recombinant A40 peptide c
P = .01 compared with classic A42-INNOTEST.
(1-40 ng/mL) (rPeptide; Bogart) were added to 2 CSF samples d
P < .01 compared with classic A42-INNOTEST.
with low and high A42 concentrations. We measured A42
levels in spiked samples using the classic and modified
INNOTEST procedures and MSD assays. Finally, an antibody-
independent mass spectrometrybased method was used to
assess CSF concentrations of A4220 using an RMP (Joint Results
Committee for Traceability in Laboratory Medicine Database
identification number C11RMP9) and A4022 in a subgroup of CSF A Biomarkers: Associations Between ELISA
98 individuals. The [18F]flutemetamol PET imaging is and Mass Spectrometry Measurements
described in the eMethods in the Supplement. Of 262 participants (mean [SD] age, 70.9 [5.5] years), 108
were women (41.2%) and 154 were men (58.8%). To deter-
Statistical Analysis mine which of the immunoassays are more accurate in
We used SPSS (version 22; IBM), R (version 3.3.1), 23 and quantifying CSF levels of A42, we studied associations
MedCalc (version 16.8.4; MedCalc Software bvba) software for between CSF concentrations of A obtained with different
statistical analysis. Associations between ELISA and mass spec- immunoassays (classic INNOTEST and the newer modified
trometrybased biomarker concentrations were tested using INNOTEST, FL, EI, and MSD) and the antibody-independent
Pearson and Spearman correlation coefficient analyses. mass spectrometrybased RMP for A42 in a subgroup of 98
Differences between the correlation coefficients were esti- individuals. We found correlations between CSF levels of
mated using the Meng test for correlated correlation A42 measured using ELISAs and mass spectrometry
coefficients.24 Intermethod agreement between visual and (r>0.88; Table 1). However, the correlation coefficients
quantitative [18F]flutemetamol PET assessments was esti- between different ELISA and mass spectrometry measure-
mated with the Cohen statistic. The accuracies of CSF bio- ments varied signific antly. Correlations w ith mass
markers in detecting [18F]flutemetamol PET status were as- spectrometryderived A42 were greater for the new A42
sessed using receiver operating characteristic (ROC) curve assays (r = 0.97 for the modified INNOTEST, r = 0.93 for FL,
analysis and logistic regression models. Two areas under the r = 0.93 for EI, and r = 0.95 for MSD) than the classic
ROC curve (AUCs) were compared using the DeLong test.25 We INNOTEST assay (r = 0.88). In contrast, the correlation
dichotomized the study participants into groups with normal between the mass spectrometryderived A42:A40 ratio
and increased A deposition based on the previously estab- and the classic INNOTEST was significantly higher (r = 0.87)
lished [18F]flutemetamol SUVR cutoff of greater than 1.42.8 compared with the correlations between mass
Cutoffs for CSF biomarkers were determined using the Youden spectrometryderived A42:A40 and the A42 modified
J index and mixture modeling.26 P < .05 was considered to be INNOTEST (r = 0.70), A42-FL (r = 0.64), A42-EI (r = 0.73),
statistically significant. or A42-MSD (r = 0.74) assays (Table 1).

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 Research Original Investigation Use of Different Amyloid Assays in Visual Assessment of Amyloid PET

Table 2. ROC Analysis of CSF As for Distinguishing Abnormal From Normal Visual Reading Assessments of Flutemetamol F 18Labeled PET
Cutoff (Cutoff for Mixture Youden J
Immunoassaya AUC (95% CI) Modeling Analysis)b Index Sensitivity Specificity
Classic A42-INNOTEST 0.92 (0.89-0.95) 548.00 (524.00) 0.78 0.96 0.82
Classic INNOTEST A42:A40 ratio 0.92 (0.88-0.95) 0.06 (0.05) 0.73 0.91 0.82
Classic INNOTEST A42:T-tau ratio 0.94 (0.91-0.97)c 1.79 (1.23) 0.84 0.96 0.87
Classic INNOTEST A42:P-tau ratio 0.95 (0.92-0.98)c,d 10.30 (8.11) 0.83 0.95 0.89
Modified A42-INNOTEST 0.87 (0.83-0.91)e 1091.00 (1129.00) 0.67 0.92 0.74
Modified INNOTEST A42:A40 ratio 0.93 (0.90-0.96)f 0.12 (0.10) 0.79 0.92 0.87
Modified INNOTEST A42:T-tau ratio 0.94 (0.91-0.97)g 3.30 (2.38) 0.83 0.96 0.88
Modified INNOTEST A42:P-tau ratio 0.95 (0.92-0.97)g 19.60 (16.50) 0.82 0.95 0.87
A42-EI 0.88 (0.84-0.92)e 449.00 (479.00) 0.63 0.82 0.80
A42-EI:A40 ratio 0.93 (0.90-0.96)h 0.10 (0.10) 0.81 0.93 0.88
A42-EI:T-tau ratio 0.94 (0.90-0.97)h 1.44 (1.11) 0.81 0.96 0.86
A42-EI:P-tau ratio 0.94 (0.91-0.96)i 9.59 (7.26) 0.81 0.95 0.87
A42-MSD 0.89 (0.85-0.93)j 506.00 (500.00) 0.70 0.94 0.76
A42-MSD:A40 ratio 0.95 (0.93-0.98)k 0.08 (0.09) 0.86 0.96 0.89
A42-MSD:T-tau ratio 0.94 (0.91-0.97)l 1.39 (1.12) 0.85 0.96 0.89
A42-MSD:P-tau ratio 0.95 (0.92-0.98)k 9.89 (7.84) 0.83 0.96 0.87
Abbreviations: A, amyloid ; AUC, area under the curve; CSF, cerebrospinal d
P .05 compared with classic A42-INNOTEST level.
fluid; EI, EUROIMMUN; MSD, Meso Scale Discovery; PET, positron emission e
P .001 compared with classic A42-INNOTEST level.
tomography; P-tau, phosphorylated tau; ROC, receiver operating characteristic; f
P .01 compared with modified A42-INNOTEST level.
T-tau, total tau.
a
g
P .001 compared with modified A42-INNOTEST level.
Cerebrospinal fluid levels of T-tau and P-tau were quantified with
EUROIMMUN and INNOTEST enzyme-linked immunosorbent assay kits,
h
P .01 compared with A42-EI level.
respectively. These tau values and A42 values from the 5 different assays i
P .001 compared with A42-EI level.
(classic and modified INNOTESTs, fully automated Lumipulse, EI, and MSD) j
P .01 compared with classic A42-INNOTEST level.
were used for the calculations of the CSF A42:tau ratios.
b
k
P .001 compared with A42-MSD level.
Cutoffs for A42 are given in pg/mL. l
P .05 compared with A42-MSD level.
c
P .01 compared with classic INNOTEST A42:A40 ratio.

Concordance Between Visual and Quantitative
[18F]flutemetamol PET Assessments
With use of visual readings of [18F]flutemetamol PET, 113 pa-
tients (43.1%) were classified as A positive and 149 patients
(56.9%) as A negative. When applying the SUVR cutoff, 132
patients (50.4%) showed abnormal amyloid deposition,
whereas composite SUVR values were within the reference
range in 130 patients (49.6%). The Cohen was 0.81, suggest-
ing very good agreement between the visual and quantitative
[18F]flutemetamol PET assessment methods.27 Visual and
quantitative [18F]flutemetamol PET data disagreed in 25 cases
(9.5%), of which 22 (8.4%) were A positive based on the SUVR
cutoff but A negative based on visual analysis (Figure 1A).
Visual A PET vs CSF AD Biomarker Assessments
We investigated how accurately CSF A42 levels could pre-
dict visual [18F]flutemetamol PET assessment when using the
commercially available classic A42-INNOTEST assay or the
newer modified A42-INNOTEST, A42-EI, and A42-MSD ass-
says. The results of ROC curve analysis are shown in Table 2
and eTable 2 in the Supplement. We found that classic A42-
INNOTEST was more accurate than the newer assays (modi-
fied A42-INNOTEST, A42-EI, and A42-MSD) in distinguish-
ing individuals with normal and abnormal visual readings
(Figure 1B). Next, we studied whether the A42:A40, A42:
T-tau, or A42:P-tau ratios improved the accuracy of the 4 as-
says. For the classic A42-INNOTEST, only the A42:P-tau but
not the A42:A40 or the A42:T-tau ratios exhibited a sig-
nificantly higher AUC than A42 alone in predicting visual clas-
sification of [18F]flutemetamol PET (Table 2, eTable 2 in the
Supplement, and Figure 1C). For all the newer assays (modi-
fied A42-INNOTEST, A42-EI, and A42-MSD), all 3 ratios (ie,
A42:A40, A42:T-tau, and A42:P-tau) were comparable and
significantly more accurate than A42 levels alone (Table 2,
eTable 2 in the Supplement, and Figure 1D-F). The results for
the A42-FL assay were comparable to those of the modified
A42-INNOTEST assay and are shown in eFigure 1 in the
Supplement. Furthermore, using logistic regression analysis,
we found that a combination of the A42:A40 ratio and
T-tau (AUCs, 0.92-0.95) or P-tau (AUCs, 0.92-0.93) for any of
the 4 A42 assays did not have better concordance with vi-
sual [18F]flutemetamol PET classification than the A42:
A40 ratio alone (AUCs, 0.92-0.95; P > .14 when comparing the
AUC of 2 ROC curves). The performance of T-tau and P-tau was
similar in all the statistical tests and, therefore, only data for
P-tau are presented hereinafter.
Cutoff for the Different Immunoassays
The A42 levels in CSF are most often bimodally distributed,28
and more reliable cutoffs likely can be derived when bio-
marker levels show 2 clearly distinct populations.29 Fre-
quency plots revealed improved bimodal separation of
[18F]flutemetamol PETpositive and negative populations
based on the A42:A40 and A42:P-tau ratios compared with

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 Use of Different Amyloid Assays in Visual Assessment of Amyloid PET Original Investigation Research

Figure 1. Cerebrospinal Fluid (CSF) Alzheimer Disease (AD) Biomarkers and Flutemetamol F 18Labeled Positron Emission Tomography (PET)

A PET analysis B ROC curve for A42

3.5 1.0

3.0
0.8
[18F]flutemetamol SUVR

2.5
0.6

Sensitivity
2.0

0.4
1.5 A42-INC, AUC = 0.92
A42-INM, AUC = 0.87
0.2 A42-EI, AUC = 0.88
1.0
A42-MSD, AUC = 0.89

0.5 0
Negative Positive 0 0.2 0.4 0.6 0.8 1.0
Visual PET Analysis 1Specificity

C ROC curve for A42-INC D ROC curve for A42:A40 ratio

1.0 1.0

0.8 0.8

0.6 0.6
Sensitivity

Sensitivity

A42-INM, AUC = 0.87

0.4 0.4 A42-EI, AUC = 0.88
A42-INC, AUC = 0.92 A42-MSD, AUC = 0.89
A42-INC:A40, AUC = 0.92 A42-INM:A40, AUC = 0.93
0.2 A42-INC:T-tau, AUC = 0.94 0.2 A42-EI:A40, AUC = 0.93
A42-INC:P-tau, AUC = 0.95 A42-MSD:A40, AUC = 0.95

0 0
0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0
1Specificity 1Specificity

E ROC curve for A42:T-tau ratio F ROC curve for A42:P-tau ratio
1.0 1.0

0.8 0.8

0.6 0.6
Sensitivity

Sensitivity

A42-INM, AUC = 0.87 A42-INM, AUC = 0.87

0.4 A42-EI, AUC = 0.88 0.4 A42-EI, AUC = 0.88
A42-MSD, AUC = 0.89 A42-MSD, AUC = 0.89
A42-INM:T-tau, AUC = 0.94 A42-INM:P-tau, AUC = 0.95
0.2 A42-EI:T-tau, AUC = 0.94 0.2 A42-EI:P-tau, AUC = 0.94
A42-MSD:T-tau, AUC = 0.94 A42-MSD:P-tau, AUC = 0.95

0 0
0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0
1Specificity 1Specificity

A, Concordance between visual and quantitative amyloid PET analysis.
Composite standardized uptake value ratio (SUVR) of [18F]flutemetamol data in
patients who were classified as negative (n = 149) or positive (n = 113) for
amyloid findings with visual PET assessment are shown. The dotted line
represents a cutoff of greater than 1.42 SUVR. Data points indicate individuals.
B-F, Correlation of CSF AD biomarkers with [18F]flutemetamol PET status
according to visual analysis. Levels of CSF -amyloid (A) 42 were analyzed with
the classic INNOTEST (INC), modified INNOTEST (INM), EUROIMMUN (EI) and
Meso Scale Discovery (MSD) assays. To calculate the CSF A42:A40 ratio, we
used A40 kits from the assay vendors Fujirebio (INC and INM), EUROIMMUN
(EI), and Meso Scale Discovery (MSD). Levels of CSF total tau (T-tau) and
phosphorylated tau (P-tau) were measured with the EI and INNOTEST assays,
respectively. Receiver operating characteristic (ROC) curves were generated for
A42 level (B-F), the A42:A40 ratio (C [INC] and D [INM, EI, and MSD]), the
A42:T-tau ratio (C [INC] and E [INM, EI, and MSD]), and the A42:P-tau ratio
(C [INC] and F [INM, EI, and MSD]) to determine their accuracy in differentiating
A-negative (n = 149) and A-positive (n = 113) visual readings. AUC indicates
area under the ROC curve.

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 Research Original Investigation Use of Different Amyloid Assays in Visual Assessment of Amyloid PET

Figure 2. Frequency Plots of Cerebrospinal Fluid (CSF) -Amyloid (A) 42 Levels and A42:A40 and A42 to Phosphorylated Tau (P-tau) Ratios

A A42-INC B A42-INC:A40 ratio C A42-INC:P-tau ratio

20 25 30
A positive
20 A negative
15

No. of Values
No. of Values

No. of Values
20
15
10
10
10
5
5

0 0 0
0 500 1000 1500 0 0.05 0.10 0.15 0.20 0 10 20 30 40
A42-INC, pg/mL A42-INC:A40 A42-INC:P-tau

D A42-INM E A42-INM:A40 ratio F A42-INM:P-tau ratio

30 20 20

15 15

No. of Values
No. of Values
No. of Values

20

10 10

10
5 5

0 0 0
0 1000 2000 3000 4000 0 0.1 0.2 0.3 0 20 40 60
A42-INM, pg/mL A42-INM:A40 A42-INM:P tau

G A42-EI H A42-EI:A40 ratio I A42-EI:P-tau ratio

30 20 30

15
No. of Values
No. of Values

No. of Values
20 20

10

10 10
5

0 0 0
0 500 1000 1500 2000 0 0.05 0.10 0.15 0.20 0.25 0 5 10 15 20 25 30 35
A42-EI, pg/mL A42-EI:A40 A42-EI:P-tau

J A42-MSD K A42-MSD:A40 ratio L A42-MSD:P-tau ratio

25 30 30

20
No. of Values

No. of Values
No. of Values

20 20
15

10
10 10
5

0 0 0
0 500 1000 1500 0 0.05 0.10 0.15 0.20 0 10 20 30 40
A42-MSD, pg/mL A42-MSD:A40 A42-MSD:P tau

Histograms of frequency distribution for CSF A42 levels and the A42:A40 with the Youden J index. EI indicates EUROIMMUN; INC, classic INNOTEST;
and A42:P-tau ratios across groups with A-positive (n = 113) and A-negative INM, modified INNOTEST; and MSD, Meso Scale Discovery.
(n = 149) visual ratings. Veritical dashed lines indicate cutoff points associated

the A42 measurement alone when using the modified A42-
INNOTEST, A42-EI, and A42-MSD assays (Figure 2D-F, G-I,
and J-L). This effect was less pronounced for the classic A42-
INNOTEST assay (Figure 2A-C). The cutoffs for each bio-
marker were determined based on the Youden J index in which
visual read of [18F]flutemetamol PET was used as the out-
come. Mixture modeling, which is independent of the
standard of truth (ie, PET imaging results), produced similar
cutoff values (Table 2). Analytical imprecision and within-
participant variability may increase the risk for misclassifica-
tion, especially when sensitivity and/or specificity markedly
decrease around cutoff points. In our study, the trends for sen-

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 Use of Different Amyloid Assays in Visual Assessment of Amyloid PET
sitivity and/or specificity near the cutoff points appeared to
be less steep for the A42:A40 and A42:P-tau ratios than for
A42 levels alone when A42 was measured using the newer
assays (modified A42-INNOTEST, A42-EI, or A42-MSD)
(eFigure 2B-D, F-H, and J-L in the Supplement), but this was
not the case for the classic A42-INNOTEST assay (eFigure 2A,
E, and I in the Supplement).
To further assess the concordance between CSF biomark-
ers and visual PET assessment in terms of classification per-
formance, we dichotomized CSF A variables into abnormal
(CSF A-positive) and normal (CSF A-negative) values based
on the optimal cutoff points corresponding to the highest
Youden J indices. The number of cases with discordant CSF
A status compared with visual PET assessments was higher
for the newer assays (48 [18.3%] for modified A42-
INNITEST; 49 [18.7%] for A42-EI; and 42 [16.0%] for A42-
MSD) than for the classic A42-INNOTEST (31 [11.8%]) and con-
sisted of mainly CSF A42-positive and visual PET-negative
cases (eTable 3 in the Supplement). However, the number of
discordant cases for the newer assays (modified A42-
INNOTEST, A42-EI, and A42-MSD) was reduced signifi-
cantly when using the A42:A40 or A42:P-tau ratios (eTable
3 in the Supplement). As shown in Figure 3, using cutoffs ob-
tained from the 2 ratios (A42:A40 or A42:P-tau) resulted
in better separation of visual PET-positive and -negative cases
than CSF A42 levels alone, and the ratios mainly reduced the
number of cases in the discordant group that were positive for
CSF A42 and negative for visual PET findings (eTable 3 in the
Supplement). The results were similar for CSF biomarkers
measured using antibody-independent mass spectrometry
based RMP and quantitative PET assessment (eResults and
eFigure 3 in the Supplement).
Spiking of CSF Samples With A40
Addition of recombinant A1-40 peptide to CSF samples caused
a spike leveldependent decrease in measured CSF A42 con-
centrations by as much as 62% when measured using the clas-
sic INNOTEST assay (eFigure 4A and D in the Supplement). In
contrast, no differences in A42 concentrations were ob-
served when the samples were analyzed using the newer modi-
fied INNOTEST procedure (eFigure 4B and E in the Supple-
ment) and MSD assay (eFigure 4C and F in the Supplement).
Discussion
In this study, we showed that the new modified A42-
INNOTEST, A42-FL, A42-EI, and A42-MSD assays corre-
lated better with A42 values obtained with antibody-
independent mass spectrometrybased RMP compared with
the classic A42-INNOTEST assay. The classic A42-
INNOTEST instead correlated better with the A42:A40 ra-
tio obtained with mass spectrometrybased RMP compared
with the other A42 assays, a phenomenon that might be
explained by the fact that the signal in the classic A42-
INNOTEST assay is somewhat quenched by A40 levels. Of
interest, the classic A42-INNOTEST assay had a higher accu-
racy to predict visual PET assessment outcome compared with
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Original Investigation Research
the newer modified A42-INNOTEST, A42-FL, A42-EI, and
A42-MSD immunoassays. However, the CSF A42:A40
ratios from these assays performed better than the correspond-
ing A42 levels alone and had comparable performance to the
classic A42-INNOTEST level. The accuracy of the classic A42-
INNOTEST assay did not improve by using the A42:A40 ra-
tio. For all the A42 assays, the addition of T-tau and P-tau to
A42:A40 did not improve the ability to predict cortical A
accumulation compared with the A42:A40 ratios alone.
The INNOTEST ELISA is one of the most extensively used
assays for assessment of A42 levels in CSF. However, this
assay is limited by the matrix interference that may lead to in-
accurate estimates of A42 concentrations.12 The matrix ef-
fects are reduced in the newer assays (modified INNOTEST, FL,
EI, and MSD), which are based on the analysis of diluted CSF
samples.13-15 Accordingly, we found that CSF concentrations
of A42 derived from antibody-independent mass spectro-
metry procedure matched better with the data from the newer
assays than the classic INNOTEST assay.
Visual rating is the only approved procedure for assess-
ment of amyloid PET scans in the clinic and when selecting AD
cases for clinical trials.1 Although quantitative approaches pro-
vide more informative data that might be critical for investi-
gating longitudinal changes in amyloid burden and treat-
ment effects, the concordance between visual and quantitative
methods for establishing amyloid status is very high. Similar
to previous studies,19,30 we found that only 25 cases (9.5%)
were discordant according to visual and quantitative PET analy-
sis with very good intermethod agreement (Cohen = 0.81).
Most discordant cases were found in individuals who had been
classified as amyloid negative by the visual analysis but as amy-
loid positive by SUVR analysis, demonstrating that visual analy-
sis is more conservative than SUVR analysis; this finding is con-
sistent with those of previous studies.19
Cerebrospinal fluid A42 is a biomarker of amyloid dep-
osition that is often used interchangeably or in combination
with PET imaging in the diagnostic workup in the clinic or in
clinical trials.1 The agreement between CSF A42 and quan-
titative amyloid PET data has ranged from 80% to 90%,6-8 and
this agreement might be improved by using the A42:A40 or
A42:tau ratios.9,10,31 However, for implementation of CSF AD
biomarkers (A42 level, A42:A40 ratio, and/or A42:tau ra-
tio) in routine clinical practice, establishing the concordance
with the clinically approved visual amyloid PET ratings, and
studying whether the concordance rate is affected by the choice
of the immunoassay for CSF A42 measurements are impor-
tant. Herein, we showed that newer A42-EI and A42-MSD
assays have similar performance with respect to association
of visual read outcome and exhibit acceptable accuracy with
visual [18F]flutemetamol PET status when using the A42:
A40 ratio. With the classic INNOTEST assay, the AUC for A42
alone was close to the A42:A40 ratios from the EI and MSD
assays and did not increase any further when the classic
INNOTEST A42:A40 ratio was used. The modification of the
classic INNOTEST assay, which improved the correlation with
mass spectrometrybased RMP A42 values, resulted in de-
creased accuracy of A42 levels alone but improved perfor-
mance for the A42:A40 ratio, as was the case for other A42
(Reprinted) JAMA Neurology Published online November 6, 2017 E7
 Research Original Investigation Use of Different Amyloid Assays in Visual Assessment of Amyloid PET

Figure 3. Separation of Populations by Visual Positron Emission Tomography (PET) Findings

A A42-INC vs A40 B A42-INC vs P-tau

25 000 A42 level cutoff 200 A42 level cutoff
175
20 000 Ratio

P-tau Level, pg/mL

cutoff 150
A40 Level, pg/mL

Ratio
15 000 125
cutoff
100
10 000
75

50
5000
25

0 0
0 250 500 750 1000 1250 0 250 500 750 1000 1250
A42-INC Level, pg/mL A42-INC Level, pg/mL
C A42-INM vs A40 D A42-INM vs P-tau
30 000 200
A42 level cutoff Ratio A42 level cutoff
cutoff 175 Ratio
25 000 cutoff
150
P-tau Level, pg/mL
A40 Level, pg/mL

20 000
125

15 000 100

75
10 000
50
5000
25

0 0
0 500 1000 1500 2000 2500 3000 3500 0 500 1000 1500 2000 2500 3000 3500
A42-INM Level, pg/mL A42-INM Level, pg/mL
E A42-EI vs A40 F A42-EI vs P-tau

15 000 200
Ratio A42 level cutoff
A42 level cutoff cutoff 175 Ratio
12 500 cutoff
150
P-tau Level, pg/mL
A40 Level, pg/mL

10 000
125

7500 100

75
5000
50
2500
25

0 0
0 500 1000 1500 2000 0 500 1000 1500 2000
A42-EI Level, pg/mL A42-EI Level, pg/mL
G A42-MSD vs A40 H A42-MSD vs P-tau

15 000 200 Separation of populations with

A42 level cutoff A42 level
Ratio cutoff
Positive PET finding positive and negative PET findings
cutoff 175 Negative PET finding used cutoffs for cerebrospinal fluid
12 500
(CSF) levels of -amyloid (A) 42 and
150
P-tau Level, pg/mL

Ratio the A42:A40 and A42 to

A40 Level, pg/mL

10 000
125 cutoff phosphorylated tau (P-tau) ratios.
Scatterplots of CSF A42 levels
7500 100 against A40 and P-tau levels; A42
75 was measured using the classic
5000 INNOTEST (INC), modified INNOTEST
50 (INM), EUROIMMUN (EI), and Meso
2500 Scale Discovery (MSD) assays. Dotted
25
lines indicate Youden J index cutoffs
0 0 for CSF A42 levels. Solid lines
0 250 500 750 1000 1250 1500 0 250 500 750 1000 1250 1500
indicated Youden J index cutoffs
A42-MSD Level, pg/mL A42-MSD Level, pg/mL for the CSF A42:A40 and
A42:P-tau ratios.

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 Use of Different Amyloid Assays in Visual Assessment of Amyloid PET Original Investigation Research

assays evaluated herein. These results could in part be ex-
plained by our spiking data, which suggested that A42 con-
centrations obtained using the classic INNOTEST were influ-
enced by A1-40 and that in individuals with higher CSF levels
of A1-40, these effects might be more pronounced, resulting
in a lower signal. Further analysis revealed that the CSF A42:
A40 ratios from the newer immunoassays (modified
INNOTEST, EI, and MSD) had a more pronounced bimodal dis-
tribution allowing improved separation of [18F]flutemetamol
PET-positive and -negative populations compared with CSF
A42 levels alone. Moreover, compared with A42 levels alone,
the rate of decrease in sensitivity and specificity values around
the cutoff points was less steep when using the A42:A40 ra-
tios. However, we did not observe differences in the distribu-
tions and behavior of sensitivity and specificity values near cut-
off between A42 level and A42:A40 ratio for the classic
INNOTEST assay. These results indicate that the A42:A40
ratio cutoffs for the new assays (modified INNOTEST, EI, and
MSD) may be more reliable in terms of performance that is more
robust with respect to small changes in cutoff values. In agree-
ment with our findings, the CSF A42:A40 ratio has also been
shown to provide a better prediction of prodromal AD com-
pared with A42 level alone when using the A42-EI and A42-
MSD assays but not the classic A42-INNOTEST or xMAP
AlzBio3 (Fujirebio) assays.16,32
In the present study, the A42:tau ratios were better pre-
dictors of visual [18F]flutemetamol PET classification than were
A42 levels alone. However, we did not observe improved ac-
curacy when the A42:A40 ratios were combined with T-tau
or P-tau compared with the A42:A40 ratios alone. Biochemi-
cal mechanisms underlying the better performance of the A42:
A40 and A42:tau ratios are not known. Interindividual vari-
ability in the production of CSF, the production and secretions
of proteins by neurons such as A and tau, and preanalytical fac-
tors may affect the accuracy of CSF A42 levels in predicting
amyloid status. Although all these effects could be partly com-
pensated by using the ratios to A40 or tau, at present, which
of the mechanisms are mainly responsible for the better per-
formance of the ratios and whether these mechanisms differ for
ratios to A40 or tau remain unclear. For example, some data
suggest that CSF levels of tau are less affected by preanalytical
factors than A peptides.33-35 Consequently, variations in pre-
analytical handling should in theory be better compensated for
by using A42:A40 ratios11 than A42:tau ratios, suggesting
that the A42:A40 ratios might be more useful in clinical prac-
tice as well as for tracking the emergence of amyloid deposi-
tion in longitudinal studies.
Limitations
One limitation that could have possibly influenced the
result of the present study is that PET images were assessed
by a single rater. However, this is unlikely considering that
the findings were the same for quantitative measures
(SUVR) of amyloid PET.
Conclusions
We showed that the newer A42 assays (modified INNOTEST,
EI, and MSD) correlated better with the antibody-free RMP
than did the classic INNOTEST assay, possibly because the
signal in the classic INNOTEST assay is partly quenched by
A1-40. However, the accuracy to correlate with visual
[18F]flutemetamol PET status was decreased in the newer A42
assays, a limitation that is overcome by using a A42:A40 or
A42:tau ratios. The CSF A42:A40 or A42:tau ratios from
the newer assays showed improved accuracy for detection of
cortical A fibrils as measured by PET. Moreover, the sensi-
tivities and specificities of these newer assays were less influ-
enced by moderate changes in the cutoffs when A42:A40
or A42:tau ratios were used, a finding that is important
when samples will be analyzed consecutively over time. The
precision of the A42:A40 ratios in differentiating
[18F]flutemetamol PETpositive and negative visual reads did
not improve further when combined with T-tau or P-tau
values. Thus, our study provides a comprehensive overview
of the correlation of the performance of CSF biomarkers across
different immunoassays with amyloid PET status, which may
influence the selection of immunoassays and biomarkers
in future studies. Furthermore, our findings support imple-
mentation of the CSF A42:A40 and/or the A42:tau ratios
as biomarkers of amyloid deposition in clinical practice.

ARTICLE INFORMATION
Accepted for Publication: July 24, 2017.
Published Online: November 6, 2017.
doi:10.1001/jamaneurol.2017.2814
Open Access: This article is published under the
JN-OA license and is free to read on the day of
publication.
Author Affiliations: Clinical Memory Research
Unit, Department of Clinical Sciences Malm, Lund
University, Lund, Sweden (Janelidze, Hansson);
Institute of Neuroscience and Physiology,
Department of Psychiatry and Neurochemistry, the
Sahlgrenska Academy at the University of
Gothenburg, Gothenburg, Sweden (Pannee,
Zetterberg, Blennow); Biogen, Cambridge,
Massachusetts (Mikulskis, Chiao); Clinical
Neurochemistry Laboratory, Sahlgrenska University
Hospital, Gothenburg, Sweden (Zetterberg,
Blennow); Department of Molecular Neuroscience,
University College London Institute of Neurology,
Queen Square, London, England (Zetterberg);
Memory Clinic, Skne University Hospital, Malm,
Sweden (Hansson).
Author Contributions: Drs Janelidze and Hansson
had full access to all the data in the study and take
responsibility for the integrity of the data and the
accuracy of the data analysis.
Study concept and design: Mikulskis, Chiao,
Blennow, Hansson.
Acquisition, analysis, or interpretation of data: All
authors.
Drafting of the manuscript: Janelidze, Chiao,
Hansson.
Critical revision of the manuscript for important
intellectual content: Pannee, Mikulskis, Zetterberg,
Blennow, Hansson.
Statistical analysis: Janelidze.
Obtained funding: Zetterberg, Blennow, Hansson.
Administrative, technical, or material support:
Mikulskis, Chiao, Zetterberg, Hansson.
Study supervision: Hansson.
Conflict of Interest Disclosures: Drs Mikulskis and
Chiao report employment by Biogen. Dr Zetterberg
reports serving on advisory boards for Eli Lilly,
Roche Diagnostics, and Pharmasum Therapeutics.
Drs Zetterberg and Blennow report serving as
cofounders of Brain Biomarker Solutions in
Gothenburg AB, a GU Venturesbased platform
company at the University of Gothenburg. Dr
Blennow reports serving on advisory boards or as a
consultant (unrelated to the work presented in the
present study) for Eli Lilly and Company, Fujirebio
Europe, IBL International, Novartis, and Roche
Diagnostics. Dr Hansson reports serving on
advisory boards or as a consultant for Eli Lilly and
Company and receiving research support from

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 Research Original Investigation
Roche, GE Healthcare, and AVID
Radiopharmaceuticals. No other disclosures were
reported.
Funding/Support: This study was supported by the
European Research Council, the Swedish Research
Council, the Swedish Alzheimer Foundation, the
Swedish Brain Foundation, the Marianne and
Marcus Wallenberg Foundation, and the Swedish
Federal Government under the ALF Agreement.
Doses of [18]F-flutemetamol injection were
sponsored by GE Healthcare. EUROIMMUN A42,
A40, and T-tau kits were provided by
EUROIMMUN. Analysis of cerebrospinal fluid
samples using INNOTEST and Lumipulse kits were
performed at Fujirebio Europe.
Role of the Funder/Sponsor: The sponsors had no
role in the design and conduct of the study;
collection, management, analysis, and
interpretation of the data; and preparation, review,
or approval of the manuscript; and decision to
submit the manuscript for publication.
Additional Contributions: The authors thank the
study participants and the research nurses involved
in the study for their invaluable contributions.
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