REVIEW

Progress Update: Fluid and Imaging Biomarkers

in Alzheimer’s Disease
Courtney L. Sutphen, Anne M. Fagan, and David M. Holtzman
Alzheimer’s disease (AD) is a growing health crisis around the world. Although significant progress has been made in our understanding
of AD pathogenesis, there is currently no effective treatment to delay onset or prevent the disease. The focus has now shifted to the
identification and treatment of AD in the early clinical stages as well as before cognitive symptoms emerge—during the long preclinical
stage. It is possible that diagnosis of individuals with AD will be more accurate when clinical symptoms and signs are combined with
biomarkers, which can improve both the diagnostic and prognostic accuracy of AD and its differentiation from the other
neurodegenerative diseases. This review discusses fluid and imaging biomarkers that have shown promise in such areas, as well as
some of the current challenges that face the field.

based on guidelines established by the National Institute of

Key Words: Alzheimer’s disease, biomarker, cerebrospinal fluid, Neurological Disorders and Stroke–Alzheimer’s Disease and Related
neuroimaging, diagnosis, prognosis Disorders Association, although the sensitivity and specificity of
such a diagnosis is lower than desirable (4). It is now widely
accepted that AD starts with a “preclinical” stage beginning 10 to 20

A
lzheimer’s disease (AD) is a chronic, progressive neuro- years before the onset of cognitive decline. The addition of
degenerative disease that slowly strips individuals of their biomarkers to the diagnostic criteria for AD may increase the
memories and other cognitive functions. The World Health sensitivity and specificity of both the diagnostic and prognostic
Organization estimated that as many as 35.6 million people capabilities currently available through clinical and cognitive assess-
worldwide were living with dementia in 2012, the majority of ment. One study suggests that it may be possible for biomarkers to
whom had AD. It is estimated that those numbers will approx- be used reliably as indicators of disease stage (5). One goal of
imately double every 20 years without the aid of treatment or studying biomarkers is to identify those with AD pathologic
prevention (1). Clinical trials of AD therapeutics to date have been changes before clinical signs emerge, as well as predict the odds
unsuccessful in reversing, halting, or slowing cognitive decline. that such individuals will clinically progress and at what rate. A
A widely held belief is that some of this failure is due to the recent workgroup cautions that biomarkers are not yet ready for
exclusive enrollment of individuals who already exhibit mild or clinical use (6); however, a small number of biomarkers for AD are
moderate dementia, stages of AD that are accompanied by robust currently used in clinical practice (see Current CSF Biomarker
neuronal cell death. At even earlier stages of the disease (very Utilization and Challenges later in the article).
mild dementia and mild cognitive impairment due to AD), neuron Current AD therapies are only available after a clinical
loss in certain critical brain regions is already significant (2). Thus, diagnosis is made and are limited to symptomatic drugs that
it is important to diagnose individuals at early disease stages and offer a modest, temporary improvement in or stabilization of
enroll them in clinical trials to identify and apply therapies that cognitive decline in some patients. However, these drugs do not
have the best chance of preserving normal cognitive function target the underlying pathology of the disease. Recent clinical
(Figure 1). This does not imply that there is no merit in treating trials have focused on anti-Aβ antibodies that bind to either
symptomatic individuals. Indeed, because neurodegeneration aggregated or soluble forms of Aβ and encourage the removal or
continues to progress through advancing clinical stages, develop- neutralization of these species from the brain. Phase III trials of
ment of therapies that could block or slow this degeneration is the anti-Aβ antibodies bapineuzumab and solanezumab in mild
also highly desirable. Given the prospect of long-term treatment, to moderate dementia believed to be due to AD ended in late
especially in asymptomatic cases and in elderly individuals who 2012. Neither drug achieved its primary cognitive endpoint, and
potentially exhibit other age-related comorbid conditions, rigor- an increased risk of vasogenic edema was seen with bapineuzu-
ous safety testing, as was performed in initial studies of mab (7,8). However, in the solanezumab trial, prespecified
antihypertensive and cholesterol-lowering medications, will be analyses of individuals with mild dementia (Mini-Mental State
required. Examination score 20–26) revealed an approximately 30% slow-
Currently, a definitive diagnosis of AD requires postmortem ing of cognitive decline as measured by the Alzheimer’s Disease
identification of the pathologic hallmarks of the disease: extrac- Assessment Scale—Cognitive Subscale and significant improve-
ellular amyloid plaques composed mainly of aggregated amyloid-β ment over placebo on some measures of activities of daily living
(Aβ) peptides and neurofibrillary tangles composed mainly of (9). An additional Phase III trial of this compound in mild
hyperphosphorylated tau (p-tau) (3). Clinical diagnosis of AD is dementia is planned.
As the AD community moves toward a treatment, it seems
From the Department of Neurology (CLS, AMF, DMH), Charles F. and
certain that biomarkers will play a key role in the process.
Joanne Knight Alzheimer’s Disease Research Center (CLS, AMF, DMH), A biomarker is defined as any measurable characteristic that
Hope Center for Neurological Disorders (CLS, AMF, DMH), and can be used as an indicator of a particular disease, and with such
Department of Development Biology (DMH), Washington University a broad definition, it is certain that many options will exist for any
School of Medicine, St. Louis, Missouri. given disease. It is the goal of investigators to determine the most
Address correspondence to David Holtzman, M.D., 660 S Euclid Avenue, effective combination of biomarkers to enable identification,
Box 8111, St. Louis, MO 63110; E-mail: [email protected]. differentiation, and treatment of the disease in question. Cogni-
Received May 2, 2013; revised Jul 1, 2013; accepted Jul 21, 2013. tive measures can serve as a biomarker of disease; however, fluid

0006-3223/$36.00 BIOL PSYCHIATRY 2014;75:520–526

http://dx.doi.org/10.1016/j.biopsych.2013.07.031 & 2014 Society of Biological Psychiatry
C.L. Sutphen et al. BIOL PSYCHIATRY 2014;75:520–526 521

Established Core CSF Biomarkers

Three proteins are typically considered the gold standards for
AD CSF biomarkers—Aβ42, total-tau (tau), and p-tau. These
proteins are the most abundant components of amyloid plaques
and neurofibrillary tangles, respectively. Each of these analytes
has been extensively studied and validated in a variety of cohorts
worldwide and, although absolute concentrations of each marker
may vary, similar results have been reported in multiple studies.
Aβ42. Aβ42 is a 42 amino acid peptide created from the
processing of the amyloid precursor protein. There are multiple
lengths of Aβ peptides; the 42 amino acid form is the most
abundant in amyloid plaques. Significantly reduced levels of CSF
Aβ42 in individuals diagnosed with AD, compared with cognitively
Figure 1. Proposed biomarker trajectories in Alzheimer’s disease (AD). normal, age-matched individuals, is one signature of amyloid
The clinical stages of Alzheimer’s disease are classified as “very mild AD/
mild cognitive impairment (MCI),” “mild,” “moderate,” and “severe.” These
plaques (12). Studies have shown that CSF Aβ42 levels correlate
stages correspond to scores of .5, 1, 2, and 3, respectively, on the Clinical inversely with amyloid plaque load in the brain as determined by
Dementia Rating (CDR) scale (represented in the bar below the plot). All postmortem histology (13) and concomitant in vivo plaque
clinical stages are associated with the presence of amyloid plaques, but measurement using amyloid imaging (14–18). CSF Aβ42 is likely
the plaque buildup begins in the preclinical stage, approximately 15 years low in the presence of amyloid deposition due to its sequestra-
before the onset of cognitive symptoms, and can be measured reliably tion in plaques (19). Low CSF Aβ42 is useful as a marker that
using cerebrospinal fluid (CSF) amyloid-β (Aβ)42 and positron emission
tomography (PET) imaging with Pittsburgh Compound B (PiB; red line).
predicts future clinical disease progression and rate of cognitive
Neurofibrillary tangles seem to develop gradually later in the preclinical decline, especially in the early clinical stages of the disease (20–
stage, the severity of which correlates well with CSF tau and p-tau181 22). However, Aβ42 alone is not a sufficient biomarker for AD
levels (blue line). Synaptic and neuronal loss occurs robustly in association diagnosis and prognosis (23,24), nor does it mark the presence of
with the onset of dementia and may be represented by CSF tau and/or other AD pathology.
visinin-like protein-1 (VILIP-1) and magnetic resonance imaging (MRI), Total-Tau and p-Tau. Tau is a microtubule-associated protein
functional MRI (fMRI), and 18fluorodeoxyglucose (FDG)-PET (green line).
The trajectories of inflammatory markers such as CSF YKL-40 are unknown
that is considered to be a biomarker of neurodegeneration in AD.
at this time. The goal of AD biomarker research is to identify those High levels of tau in CSF may reflect neuronal damage, as is
preclinical individuals who will go on to develop dementia due to AD. suggested by increases in tau after acute neuronal injury such as
Reprinted by permission from Macmillan Publishers Ltd: Perrin et al.; stroke and traumatic brain injuries (25,26). Phospho-tau levels
Nature 461:916-922, copyright (2009). correlate with total tau and also correlate closely with neuro-
fibrillary tangle load (27). Tau is normally released by neurons in
(cerebrospinal fluid [CSF], plasma, serum) and imaging (magnetic the absence of cell death. Evidence from wild type and P301S tau
resonance imaging [MRI] and positron emission tomography transgenic mice suggests tau is continuously secreted from
[PET]) biomarkers as measures of underlying pathology and healthy neurons into the brain interstitial fluid space (28). In
disease progression are the focus of this review. Each modality addition, both soluble and aggregated forms of tau have been
presents its own challenges for identifying or developing viable shown to be secreted by cultured cells (29). CSF tau and p-tau181
markers, test validation, and intra- and interlab consistency in are significantly increased in AD and mild cognitive impairment
measurement. (MCI) (21,27,30). Levels of p-tau231 in CSF are also associated with
disease progression in AD cases (31). Tau and p-tau are similar to
CSF Biomarkers Aβ42 in diagnostic and prognostic performance but are not
sufficient biomarkers on their own.
CSF is considered a prime source for AD biomarkers because
many proteins and metabolites in CSF directly reflect the internal Current CSF Biomarker Utilization and Challenges
milieu of the brain. A lumbar puncture (LP) is necessary for the In current clinical practice, in the absence of a disease
collection of CSF, which makes it somewhat more invasive than a modifying therapy for AD, CSF is only obtained for the assess-
blood draw. However, complications stemming from LP are not ment of Aβ42, tau or p-tau181 by most clinicians under specific
frequent (10,11), and when the procedure is performed by circumstances. For example, in mildly symptomatic individuals,
experienced clinicians, it is usually not painful. especially in individuals younger than 60 years, CSF assessment

Table 1. Comparison of the Core Alzheimer’s Disease (AD) Cerebrospinal Fluid Biomarkers in Other Neurodegenerative Diseases
Diagnosis Aβ42 tau p-tau181 Other

AD ↓↓ (20,21,86,95,96) ↑ (20,21,86,95,96) ↑ (20,86,95) p-tau231: ↑ (97)

FTD — (21) — (21) — (21)
DLB ↓ (21,86,95,98) ↑ (86,95,98) — (21,86,95)
PDD — (86,95) —/↓ (86,95) — (86,95)
CJD ↓ (99) ↑↑ (12,100) ↑ (12)
Depression ↑ (96) — (96) — (96) p-tau231: — (97)
Comparisons are reported as difference from a normal cerebrospinal fluid biomarker profile. ↑: increase, ↑↑: large increase, ↓: decrease, ↓↓: large
decrease, —: no difference. Numbers in parentheses indicate relevant references.
Aβ, amyloid-β; AD, Alzheimer’s disease; CJD, Creutzfeldt-Jakob disease; FTD, frontotemporal dementia; DLB, dementia with Lewy bodies; PDD,
Parkinson’s disease with dementia.

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can be useful in differential diagnosis between other neuro- sensor protein. An increase in CSF VILIP-1 have been observed
degenerative diseases such as frontotemporal dementia and even in AD and is a strong predictor of future cognitive decline in
certain psychiatric disorders such as depression. It is worth noting individuals with MCI/mild dementia and in cognitively normal
that in addition to CSF, some clinicians obtain 18fluorodeoxyglu- control subjects (36–38). Another candidate with a similar degree
cose (FDG)-PET scans to aid in distinguishing possible frontotem- of validation in CSF is chitinase-3-like protein-1 (YKL-40), an
poral dementia from AD because there is usually a different astrocytic protein that is upregulated in neuroinflammatory
pattern of abnormalities between these disorders. The CSF conditions (39). In two reports, YKL-40 performed nearly as well
profiles for common biomarkers in a selection of common as the core biomarkers for both diagnosis and prognosis in AD
disorders and neurodegenerative diseases are shown in Table 1. versus control subjects (5,40). However these results were not
A current challenge in the fluid biomarker field is protocol replicated in a subsequent study (41). There are countless addi-
standardization. A worldwide quality control study has reported tional novel CSF biomarkers for AD, although few have been
significant inter- and intralab variability in the measurement of validated to the extent of VILIP-1 or YKL-40 (42).
the three core CSF markers, even when using the same assay kits It is possible that antibody-free assays, such as mass spec-
and lot numbers (32,33). Generally, intralab is lower than interlab trometry, will provide more reliable means to measure current
variability. One goal of the ongoing quality control initiative is to and novel biomarkers for AD. Some studies have shown promis-
determine accurate “cutoff” values that can identify an individual ing differences between preclinical AD or MCI and control groups
as biomarker-positive or -negative and has utility for both clinical using mass spectrometry to measure nonprotein metabolites
trial design/evaluation and disease diagnosis. However, current such as F2-isoprostanes (43,44) and lipids such as sulfatides
CSF biomarkers are continuous variables, and clinicians should be (45). Such data have yet to be reported in large cohorts
aware that treating them as such may be helpful in day-to-day comparable to other promising biomarkers exploring CSF, plasma,
practice—for instance, if a patient presents with a clinical or imaging markers.
syndrome consistent with early AD but with biomarker values
that are borderline, strict cutoff values are not likely to be as
useful as clinical judgment. Blood Biomarkers
Animal Models of AD Identification of blood biomarkers (plasma and serum) for AD
The discovery of human genetic mutations in certain families has been disappointing. Possible contributing factors include low
with an autosomal-dominant pattern of AD phenotypic expres- expression of target biomarker proteins in the periphery that
sion facilitated the critical development of transgenic mouse could make quantification of central nervous system-derived
models that are widely used in AD research today. Significant analytes difficult, as well as the relatively higher levels of total
effort has been dedicated to investigation of the pathologic protein in plasma and serum compared with CSF that could
similarities between such models and the actual human disease. interfere with analyte detection.
For example, in the Tg2576 APP transgenic mouse model, levels
of CSF Aβ42 decrease as plaques begin to deposit in the brain (34). Plasma Amyloid-β42
This mirrors what is seen in the humans carrying such disease- Findings from the many published studies of plasma Aβ
causing mutations, where decreases in CSF Aβ42 were seen in species have been contradictory (46). Some groups report slightly
mutation carriers at least 15 years before their estimated age of higher plasma levels of either Aβ42 or Aβ40 in AD, although with
symptom onset (35). broad overlap between AD and control groups, whereas most
studies find no change. Some studies report that a high level of
plasma Aβ42, or a high Aβ42/Aβ40 ratio, is an indicator of increased
Novel CSF Biomarkers risk for future AD; however, other studies have reported the
opposite. Although levels of CSF Aβ42 are negatively correlated
Because it is likely that no single biomarker will perform with plaque load, as evidenced by amyloid imaging, levels of
satisfactorily on its own, identification and development of plasma Aβ species are not (47).
additional CSF biomarkers that do not directly reflect AD
pathology (plaques and tangles) but instead reflect more general Plasma Tau
processes such as neurodegeneration and inflammation might be There are conflicting reports as to the behavior of tau in the
useful. Proteomics and multianalyte profiling (MAP) are two periphery; some report that plasma tau decreases in AD (48),
common techniques used to identify novel fluid biomarkers. whereas another study found tau increases in AD (49). These
These nonbiased approaches can be used to identify proteins and differences are likely due to variability created by extremely low
lipids in human fluids that have differential expression between levels of tau in plasma. Considering the behavior of tau in CSF, it
groups of individuals. MAP analysis relies on bead-based immu- seems plausible that, as the disease progresses, tau would
noassay techniques or microscopy/flow cytometry to quantify a become more abundant in plasma. Ultrasensitive and other
large number of analytes from a relatively small volume of assays are being developed to address the usefulness of measur-
sample. This method is useful for a fast assessment of novel ing tau in the blood (49).
biomarkers, although it is often limited to panels of analytes
chosen by the manufacturers of MAP kits. A challenge presented Novel Blood Biomarkers
by unbiased approaches to biomarker identification has been in There are few well-characterized biomarkers in blood com-
identifying biomarkers that have high enough sensitivity and pared with CSF and imaging. Novel serum candidates include
specificity to warrant investigation in large cohorts. C-reactive protein (50) and the presence of antibodies in serum
Of the novel CSF biomarkers proposed to date, few have been that recognize selective peptoid ligands in AD versus control
validated in large, independent cohorts. One candidate is visinin- samples (51). Novel plasma biomarkers, at least recently, have
like protein-1 (VILIP-1), a neuron-specific intracellular calcium been presented as panels rather than individual biomarkers (see

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The Potential of Biomarker Panels later in the article). More work such as the posterior cingulate and medial temporal lobe early in
needs to be done before blood biomarkers can be considered the disease process and gradually expand to areas including the
useful for AD diagnosis and prognosis. frontal association cortices, providing both diagnostic and prog-
nostic capabilities (67,68). However, these changes are not
typically seen until the symptomatic phase of AD (69).
Imaging Biomarkers Use of amyloid imaging PET biomarkers began with develop-
ment of the first radioligand specific to fibrillar Aβ, Pittsburgh
Imaging biomarkers capture a broad range of AD-associated Compound B (PiB), in the mid-2000s (70). Other amyloid imaging
processes, from brain size and structure to the presence of radioligands have since become available that utilize 18F instead
protein aggregation. Imaging biomarkers can be noninvasive or of 11C (e.g., florbetapir, flutemetamol, florbetaben, and AZD4694)
moderately invasive based on the modality used (i.e., MRI vs. PET (71), allowing for studies to be carried out in locations that do not
with radioactivity, respectively). A particular advantage for imag- have access to a cyclotron (72,73). In AD, levels of fibrillar Aβ are
ing biomarkers is the continual improvement of equipment/ significantly increased when measured by amyloid PET (16). These
software and discovery of new radioimaging ligands that either increases correlate inversely with CSF Aβ42 levels and offer some
improve on or add to the pool of available proteins that can be value for diagnosis and prognosis in early stages of the disease,
imaged in vivo. As with fluid biomarkers, interlab standardization including the preclinical period (18,74–76). Florbetapir (also
is paramount but can be difficult because of the use of different known as Amyvid) was recently approved by the Food and Drug
makes and models of scanners, each with their own idiosyncrasies Administration for use in patients being evaluated for AD and
(52). In studies such as the Alzheimer’s Disease Neuroimaging other causes of cognitive decline (77). The widespread use of
Initiative, use of software to analyze scans and implementation of these agents outside of clinical trials will probably await approval
a phantom—a small, standardized object that contains filling of the first effective disease modifying treatment. Molecular
readable by MRI—has helped reduce some variability. imaging of other disease-related aggregated proteins, such as
tau and synuclein, is a critical need in the field and is currently
MRI Biomarkers under development.
Volumetric MRI is one of the most studied imaging bio-
markers. The measurement of the size of a brain region at a
single time point and within-individuals longitudinally allows for Potential of Biomarker Panels
detection of atrophy in either whole brain or targeted areas (53–
55). In many studies, a marked decrease in volume is observed in An ideal biomarker panel will 1) have clear cutoff values for
AD; this is seen both in normalized whole brain volume and biomarker positivity, 2) identify AD preclinically and reflect the
in specific areas such as the hippocampus and entorhinal cortex different stages of the disease throughout its course, 3) offer
(56,57). Volumetric MRI performs as well as the CSF gold standard differentiation between AD and other dementias, and 4) be
biomarkers for diagnosis and prognosis, especially in the more simple and not costly, such as might be afforded by a blood
advanced clinical stages of the disease (58,59). Currently, how- test. Such a panel will likely need to include markers of AD
ever, few diagnostic software programs for volumetric MRI have pathology as well as supplementary markers of more general
Food and Drug Administration approval, and the process of neuroinflammatory and neurodegenerative processes.
analyzing such scans is labor-intensive and time-consuming.
Both task-based and resting-state functional MRI (fMRI) are Statistical Methods in Biomarker Analysis
promising imaging biomarkers for AD (60). The difference in Depending on the specific scientific objectives of analyzing
magnetization between oxygen-rich and oxygen-poor blood can biomarker data, a wide range of analytic tools can be used. One
be measured using fMRI to detect changes in connectivity approach is to compare biomarkers across well-established
between areas of the brain while an individual is performing a clinical stages and identify biomarker cutoffs that best discrim-
task or resting. Some studies have shown fMRI differences inate clinical stages by optimizing well-established measures of
between individuals with MCI versus control subjects on task- diagnostic accuracy (e.g., Youden index or the distance of receiver
based assessments (61). Of particular interest in resting-state fMRI operating characteristic curve/surface) to the ideal diagnosis (78).
assessments is the default mode network, a network of brain These cutoffs can then be applied to individuals who are
regions that is most active when a person is not engaged in a cognitively normal to define those with preclinical AD. Statistical
specific cognitive task and is deactivated when an individual is methods that combine multiple biomarkers to optimize the
externally stimulated or engaged in a specific task. In AD, default diagnostic accuracy in both a parametric (79) and nonparametric
mode network resting connectivity is weakened, whereas in some setting (80) can be used to improve the diagnostic accuracy.
cases, other networks have increased activity (62,63). This change Another potential use of biomarkers is the prediction of the
in activity has been shown to have diagnostic and prognostic longitudinal course of the disease. Standard “survival” analysis
ability in small cohorts (64). Standardization and validation in such as those based on Cox proportional hazards models (81) and
large AD cohorts is needed (65), although a recent large study of generalized illness-death models (82) can be implemented.
500 individuals showed progressive decline in resting-state func-
tional connectivity across multiple networks with disease pro- Current Biomarker Panels
gression (66). Whether fMRI will prove useful in differential Of the fluid biomarkers, small panels that assess CSF Aβ42 with
diagnosis, prognosis, or in clinical trials awaits further studies. tau and/or p-tau181 are good diagnostic tools for predicting
cognitive decline in individuals with cognitive impairment (22,83),
PET Biomarkers as well as in cognitively normal individuals who have preclinical
FDG-PET is a highly studied radioligand that acts as an AD (20,22,83,84). The sensitivity and specificity for Aβ42, tau, and
indicator of glucose metabolism and, by proxy, neuronal activity. p-tau181 alone range from 80% to 90% (12). Some studies have
Reductions in FDG-PET are observed in AD in vulnerable areas shown that the tau(s)/Aβ42 ratios have outstanding sensitivity (at

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80% specificity) in identifying groups of individuals with amyloid diagnosis of preclinical and symptomatic AD, to the exclusion of
deposition in the brain, ranging from 92% to 98%, and perform other forms of dementia. Should current or future clinical trials of
better than Aβ42 alone (sensitivity 85%–89%) (85). Other small AD drugs provide evidence that symptoms of the disease can be
panels of novel biomarkers have been studied by groups around halted or delayed, such a diagnosis will be critical in giving the
the world, some showing differential diagnostic sensitivity and right patients the right therapies at the right time.
specificity as high as 90% and 80%, respectively, for AD versus
dementia with Lewy bodies and Parkinson’s disease with demen- This work was supported by the National Institutes of Health
tia (86). The threshold for biomarker sensitivity and specificity (Grant Nos. P50-AG05681, P01-AG03991, and P01-26276).
considered acceptable for individual diagnosis remains unclear. We thank Drs. Tammie Benzinger and Chengjie Xiong for helpful
A panel of 18 plasma biomarkers was found suitable for comments.
diagnosis of AD both in the preclinical and clinical stages (87); Courtney L. Sutphen reports no biomedical financial interests or
however, these results were not replicated in a subsequent study potential conflicts of interest. Anne M. Fagan reports being a
(88). Other studies have reported panels of plasma biomarkers, member of the advisory board for Roche and Eli Lilly. David M.
including apolipoprotein E, B-type natriuretic peptide, C-reactive Holtzman cofounded C2N Diagnostics, LLC, and is on the scientific
protein, and pancreatic polypeptide, that identify individuals with advisory board. He consults for AstraZeneca, Genentech, and Bristol
AD dementia from normal control subjects or accurately reflect a Myers Squibb. His laboratory receives research grant support from
diagnosis of AD in a discovery cohort (87,89,90). This indicates the AstraZeneca, Eli Lilly, and Biogen.
need for replication and the use of large, independent sample
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