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Original Article |

Measurement of Phosphorylated Tau Epitopes in the Differential Diagnosisof Alzheimer Disease:  A Comparative Cerebrospinal Fluid Study FREE

Harald Hampel, MD; Katharina Buerger, MD; Raymond Zinkowski, PhD; Stefan J. Teipel, MD; Alexander Goernitz, MD; Niels Andreasen, MD, PhD; Magnus Sjoegren, MD; John DeBernardis, PhD; Daniel Kerkman, PhD; Koichi Ishiguro, PhD; Hideto Ohno, PhD; Eugeen Vanmechelen, PhD; Hugo Vanderstichele, PhD; Cheryl McCulloch, BS; Hans-Jürgen Möller, MD; Peter Davies, PhD; Kaj Blennow, MD, PhD
[+] Author Affiliations

From the Dementia Research Section and Memory Clinic, Alzheimer MemorialCenter and Geriatric Psychiatry Branch, Department of Psychiatry, Ludwig-MaximilianUniversity, Munich, Germany (Drs Hampel, Buerger, Teipel, Goernitz, and Möller);Applied NeuroSolutions Inc, Vernon Hills, Ill (Drs Zinkowski, DeBernardis,and Kerkman and Ms McCulloch); Karolinska Institute, Neurotec, Division ofGeriatric Medicine, Huddinge University Hospital, Stockholm, Sweden (Dr Andreasen);the Department of Clinical Neuroscience, Unit of Neurochemistry, Universityof Göteborg, Sahlgren's University Hospital, Mölndal, Sweden (DrsSjoegren and Blennow); Mitsubishi Kagaku Institute of Life Sciences, Tokyo,Japan (Dr Ishiguro); Mitsubishi Kagaku Medical, Tokyo (Dr Ohno); InnogeneticsNV, Gent, Belgium (Drs Vanmechelen and Vanderstichele); and the Departmentof Pathology, Albert Einstein College of Medicine, Bronx, NY (Dr Davies).Drs Hampel and Davies are consultants for Applied NeuroSolutions Inc. DrsZinkowski, DeBernardis, Kerkman, and Davies have stock and employee stockoptions and Ms McCulloch has employee stock options from Applied NeuroSolutionsInc.


Arch Gen Psychiatry. 2004;61(1):95-102. doi:10.1001/archpsyc.61.1.95.
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Background  Abnormal hyperphosphorylation of the microtubule-associated protein tau and its incorporation into neurofibrillary tangles are major hallmarks of the pathogenesis of Alzheimer disease (AD). Different tau phosphoepitopes can be sensitively detected in cerebrospinal fluid (CSF).

Objective  To compare the diagnostic accuracy of CSF concentrations of tau proteins phosphorylated at 3 pathophysiologically important epitopes (p-tau) to discriminate among patients with AD, nondemented control subjects, and patients with other dementias.

Design and Setting  Cross-sectional, bicenter, memory clinic–based studies.

Participants  One hundred sixty-one patients with a clinical diagnosis of AD, frontotemporal dementia, dementia with Lewy bodies, or vascular dementia and 45 nondemented controls (N = 206).

Main Outcome Measures  Levels of tau protein phosphorylated at threonine 231 (p-tau231), threonine 181 (p-tau181), and serine 199 (p-tau199). The CSF p-tau protein levels were measured using 3 different enzyme-linked immunosorbent assays.

Results  The mean CSF levels of the studied p-tau proteins were significantly elevated in patients with AD compared with the other groups. Applied as single markers, p-tau231and p-tau181 reached specificity levels greater than 75% between AD and the combined non-AD group when sensitivity was set at 85% or greater. Statistical differences between the assay performances are presented. Particularly, discrimination between AD and dementia with Lewy bodies was maximized using p-tau181at a sensitivity of 94% and a specificity of 64%, and p-tau231 maximized group separation between AD and frontotemporal dementia with a sensitivity of 88% and a specificity of 92%. Combinations of the 3 markers did not add discriminative power compared with the application as single markers.

Conclusions  The p-tau proteins in CSF come closest to fulfilling the criteria of a biological marker of AD. There is a tendency for p-tau proteins to perform differently in the discrimination of primary dementia disorders from AD.

Figures in this Article

Abnormal hyperphosphorylation of the microtubule-associated proteintau and its incorporation into neurofibrillary tangles are major componentsof the pathogenesis of Alzheimer disease (AD).

Using monoclonal antibodies specific for different phosphorylated epitopesof tau, enzyme-linked immunosorbent assays have been developed that sensitivelymeasure concentrations of phosphorylated tau protein (p-tau) in cerebrospinalfluid (CSF). Statistically significant increases in CSF p-tau concentrationsin patients with AD have recently been demonstrated in independent pilot studies,mainly using 3 different immunoassays specific for the phosphorylated epitopesthreonine 231 (p-tau231),1,2 threonine181 (p-tau181),3,4 andserine 199 (p-tau199).5 Evidencefrom these pilot studies indicates that quantification of tau phosphorylatedat these specific sites may improve early detection, differential diagnosis,and tracking of disease progression in AD (for a review see Blennow et al6,7).

In a recent study,1 CSF p-tau231 distinguished between patients with AD and those with other neurologicdisorders (ONDs) with a sensitivity of 85% and a specificity of 97%. Furthermore,p-tau231 significantly improved differential diagnosis betweenAD and other non-AD groups, particularly frontotemporal dementia (FTD).8 In AD vs FTD, p-tau231 correctly allocated91% of patients vs only 66% using total tau.8 Itohand colleagues5 showed that CSF p-tau199 discriminates between AD and the combined non-AD groups with a sensitivityand a specificity of 85%. The level of CSF p-tau181 was elevatedin patients with AD compared with patients with other dementias and controls,9 and p-tau181has been proposed as a potentialmarker for discriminating patients with AD from those with dementia with Lewybodies (DLB).10 Furthermore, CSF p-tau231 concentrations declined over time during the clinical progressionof AD and correlated with cognitive performance at baseline.11 Resultsof these first studies suggested that CSF p-tau proteins are promising biomarkercandidates for AD.

Levels of CSF p-tau and the discriminative power of the 3 differentp-tau assays among patients with AD, nondemented controls, and patients withother dementias, however, have not yet been assessed in the same set of patients.Investigation of the 3 p-tau assays would allow for direct comparison of thediagnostic performance of the 3 markers to differentiate AD from other relevantdiseases. In the advent of large international multicenter trials (such asthe National Institute on Aging Initiative on Neuroimaging in Alzheimer'sDisease and the studies of the European Alzheimer's Disease Consortium) onthe value of biological markers and neuroimaging in the diagnosis of AD, thisis the first study to address the important clinical issue regarding the differentialdiagnostic performances of p-tau proteins as core biological marker candidates.

In particular, we tested the diagnostic accuracy of the 3 p-tau assaysaccording to the recommendations of a consensus group12 suggestinga sensitivity level of 85% or greater and a specificity level of at least75% for useful biomarkers of AD. In addition, we asked whether use of a combinationof the 3 markers might be superior to the application of single markers. Toour knowledge, this is the first comparative study applying 3 developed andpublished immunoassays detecting different p-tau epitopes on the same setof controls and patients to compare individual and combined diagnostic accuracy.Concentrations of p-tau231, p-tau181, and p-tau199 in the CSF were studied in the same group of patients with AD, DLB,FTD, or vascular dementia (VaD); patients with ONDs; and controls.

PATIENT SELECTION

A total of 206 individuals were studied. One hundred eight patientshad probable AD (National Institute of Neurological and Communicative Disordersand Stroke–Alzheimer's Disease and Related Disorders Association criteria)13 and 53 had other dementia disorders (24 patientswith FTD,14 22 with DLB,15 and7 with VaD).16 Structural and functional imagingresults were consistent with the diagnoses in these patients. Twenty-two patientswith ONDs were diagnosed as having mild psychiatric (eg, depressed mood) orneurologic (eg, dizziness) symptoms. One patient with OND experienced a bulbarsyndrome of unknown etiology. We also studied 23 controls. Study participantswere recruited at 2 academic expert centers: the Dementia Research Sectionand Memory Clinic, Alzheimer Memorial Center and Geriatric Psychiatry Branch,Department of Psychiatry, Ludwig-Maximilian University (22 patients with AD,6 with FTD, 7 with VaD, 9 with DLB, 1 with OND, and 13 controls), and theDepartment of Clinical Neuroscience, University of Göteborg (86 patientswith AD, 18 with FTD, 13 with DLB, 21 with OND, and 10 controls). Participantsfrom the former center had been studied previously with a different objective,and the results of this study have been published.8 Characteristicsof the patients and controls are given in Table 1. The protocol was approved by the local ethical committeesand the institutional review boards of the 2 participating medical centers.Informed consent was obtained from all participants.

Table Graphic Jump LocationTable 1. Characteristics of Patients and Controls and CSF p-Tau Values

Examination of the controls included medical history, physical examination,routine blood tests (blood cell count; international normalized ratio; partialthromboplastin time; and sodium, potassium, creatinine, urea, and blood glucoselevels), and a cognitive test using the Consortium to Establish a Registryfor Alzheimer's Disease battery.17 Ten of the23 controls were volunteers without any medical, neurologic, or psychiatricdisorders. Samples of CSF were collected from 13 controls while they underwentspinal anesthesia for surgery of the urinary tract or lower extremities. Theywere cognitively normal according to the Consortium to Establish a Registryfor Alzheimer's Disease battery (results within ±1 SD in all subtests).Three of these control subjects had diabetes mellitus as a somatic comorbidity.

CSF SAMPLING AND ANALYSES OF p-TAU PROTEINS

Samples of CSF were acquired via lumbar puncture between 9 and 11 AM according to a routine protocol (established by the 2 participatingmedical centers). Samples of CSF were collected in polypropylene tubes onice in 0.5-mL aliquots. For this study, a total of 1 mL was taken. Aliquotswere centrifuged at 4°C at 10 000g for 10minutes and stored at −80°C until analysis. The same procedureswere performed at the 2 sites involved in the study. There was no effect ofmedical centers on the variance of measured protein levels and no proteingradient in the CSF column for the markers (K.B., unpublished data, 2002).

Assay operators were masked to the diagnostic category of the samples.Levels of p-tau231 were measured using an enzyme-linked immunosorbentassay (Applied NeuroSolutions Inc).1 This assayuses a combination of CP27 (which recognizes amino acids 130-150 in normaltau and p-tau), Tau-1 (which recognizes amino acids 196-205 in nonphosphorylatedtau), and CP9 (which recognizes phosphothreonine 231). Experiments describingthe specificity of the detection antibody CP9 for phosphothreonine 231 havebeen reported previously.1 Full-length recombinanttau (441 amino acids) phosphorylated at threonine 231 was used to producea standard curve. Levels of p-tau231 in our patients were calculatedfrom the standard curve and expressed as CSF p-tau231 in picogramsper milliliter.

Levels of p-tau181 were measured using a sandwich enzyme-linkedimmunosorbent assay method (prototype version of Innotest Phospho-Tau [181p];Innogenetics), using a combination of monoclonal antibody HT7 (which recognizesamino acids 159-163 in normal tau and p-tau) and biotinylated monoclonal antibodyAT270 (which recognizes p-tau containing the phosphorylated threonine 181residue).3 A synthetic phosphopeptide was usedfor standardization.

Levels of p-tau199 were measured using a previously reportedsandwich enzyme-linked immunosorbent assay method (Mitsubishi Chemical Corp,Shinagawa, Japan), using a combination of monoclonal antibody HT7 and polyclonalantibody anti-PS199 (specific for tau phosphorylated at serine 199).5

STATISTICAL ANALYSES

Differences among groups regarding age were assessed using the Mann-Whitney(M-W) test and regarding sex distribution using the χ2 test.

Distributions of p-tau values differed statistically significantly fromnormal as revealed by the Kolmogorov-Smirnov test. Differences in mean CSFlevels of the 3 p-tau subtypes among all groups were assessed usingthe Kruskal-Wallis test. Pairwise comparisons between patients with AD andthe other groups were performed using the M-W test. Correlations between p-tausubtypes were assessed using the Spearman rank correlation.

Cutoff values for p-tau proteins were determined such that 85% of thepatients with AD were correctly identified according to the recommendationsof the consensus conference12 of 85% sensitivityfor an "excellent" biomarker. To determine differences in diagnostic accuracyamong markers, the specificity levels that correspond to the 85% sensitivitycutoff level for each marker in the comparison groups were compared betweenall possible pairs of markers using the McNemar test.

To develop predictive cutoff values that optimized the combined useof the different p-tau markers, we used classification tree analysis withSYSTAT 7.0 (SPSS Inc, Chicago, Ill). Classification tree analysis uses recursivepartitioning to consider all possible binary splits of the data in pursuitof optimal classification.18 The analysis consideredall 3 p-tau markers simultaneously to create cutoff values that maximize separationamong groups, resulting in a decision tree. The number of branches dependson the separation that has been achieved at the first split. To avoid overfittingof the data, generation of further branches was interrupted if sensitivityor specificity declined below 80%. A similar technique has been presentedin earlier CSF studies in AD.19

To show sensitivity and specificity levels over the entire range ofcutoff levels, we determined receiver operating characteristic curves.

CSF p-TAU LEVELS

As illustrated in Figure 1 and Table 1, levels of all p-tau subtypeswere significantly increased in patients with AD compared with the other groupsstudied. Because the controls were significantly younger than the patientswith AD, we investigated correlations between p-tau subtypes and age. In patientswith AD, there was no correlation between the 3 p-tau subtypes and age (ρ= –0.050 to 0.002; P = .61-.93). In controls,p-tau181 (ρ = 0.61; P = .002) andp-tau231 (ρ = 0.41; P = .05) correlatedwith age, but p-tau199 did not (ρ = 0.05; P = .80). Therefore, we repeated the analyses in a group of 23 patientswith AD and 23 controls who were matched for age (M-W1 = 208; P = .21) and sex (χ21<0.001; P>.99). Differences between the AD group and the controlgroup remained unchanged and statistically significant for all3 p-tau subtypes (p-tau231: M-W1 = 32; P<.001; p-tau181: M-W1 = 52.5; P<.001; p-tau199: M-W1 = 46; P<.001). Consequently, we included all of the patients with AD inour analyses. Correlations among the 3 p-tau subtypes are given in Table 2.

Place holder to copy figure label and caption
Figure 1

Levels of cerebrospinal fluid(CSF) phosphorylated tau protein (p-tau)231 (A), p-tau181 (B), and p-tau199 (C) in patients and controls. Dashed linesrepresent the cutoff level when sensitivity was set at 85% or higher. Asteriskindicates differences from AD at P<.001; dagger,differences from AD atP<.05; AD, Alzheimer disease;ONDs, other neurologic disorders; DLB, dementia with Lewy bodies; FTD, frontotemporaldementia; and VaD, vascular dementia.

Graphic Jump Location
Table Graphic Jump LocationTable 2. Correlations Between p-Tau Epitopes*
SENSITIVITY AND SPECIFICITY OF THE SINGLE MARKERS

Specificity levels of single markers using p-tau proteins when sensitivitywas set at 85% or higher, as recommended by a consensus report, are givenin Table 3.12 Inthe case of p-tau199, several individuals in each comparisongroup had a value equal to the 85% sensitivity cutoff value. Therefore, forp-tau199, a lower and an upper limit is given for the specificity,corresponding to 2 alternatives: (1) all patients with this specific valuewere allocated to the AD group and (2) all patients with this specific valuewere allocated to the comparison group. In the differentiation of patientswith AD from those in the non-AD group, p-tau231 (85%) and p-tau181 (81%) reached specificity levels of 75% or higher, but p-tau199 did not (61%-72%). All 3 p-tau proteins showed excellent specificitylevels when patients with AD were compared with those with ONDs and controls.

Table Graphic Jump LocationTable 3. Specificity of Single Markers With Sensitivity Set at 85%or Higher and Differences in Diagnostic Accuracy Between the Single Markers*

Comparing AD to FTD, p-tau231and p-tau181 showedgood discriminative power, with specificity levels of 92% and 79%, respectively.For p-tau199, specificity ranged from 42% to 54% in the discriminationof AD from FTD. When AD was compared with DLB, p-tau181 showeda specificity of 68%, whereas for p-tau231 the specificity was64%. For p-tau199, specificity ranged between 50% and 64%. Valuesfor AD vs VaD are not separately given owing to the small sample size of patientswith VaD (n = 7).

We tested for differences in diagnostic accuracy among p-tau proteins(Table 3). Discriminative powerbetween AD and the combined non-AD groups was significantly higher for p-tau231 (McNemar test P = .004 to P<.001) and p-tau181 (McNemar test P = .10 to P<.001) compared with p-tau199. Diagnostic accuracy was also significantly higher using p-tau231 and p-tau181 compared with p-tau199 in differentiatingAD from FTD (p-tau231 vs p-tau199: McNemar test P = .004 to P<.001; p-tau181 vs p-tau199: McNemar test P =.07 to P = .01). There was no statistically significantdifference in diagnostic accuracy among p-tau proteins for discriminationbetween patients with AD vs ONDs and DLB. Receiver operating characteristiccurves for pairwise comparisons of marker levels between patients with ADand comparison groups are shown in Figure2.

Place holder to copy figure label and caption
Figure 2

Receiver operating characteristiccurves for cerebrospinal fluid phosphorylated tau protein (p-tau)231, CSF p-tau181, and CSF p-tau199 when patientswith Alzheimer disease were compared with the combined non–Alzheimerdisease group (A), the control group (B), patients with other neurologic disorders(C), patients with dementia with Lewy bodies (D), and patients with frontotemporaldementia (E). Diagonal lines indicate an area of 50%, indicating no differencein marker levels between groups.

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COMBINATION OF THE MARKERS

We used classification tree analysis to investigate whether a combinationof p-tau markers would improve group discrimination (Table 4). For AD vs the combined non-AD groups, AD vs controls,AD vs ONDs, and AD vs FTD, we found that p-tau231 accounted formaximal group discrimination, whereas the other p-tau subtypes added no additionaldiscriminatory power. Similarly, p-tau181 alone maximized groupseparation between patients with AD and those with DLB and between the ADand VaD groups (data not shown). For discrimination between AD and non-ADdementias, a combination of p-tau231 and p-tau181resultedin a slight increase in sensitivity from 86% to 94% at the cost of decreasedspecificity from 75% to 66%, resulting in only a slight increase in correctclassification accuracy (from 83% to 85%) (Figure 3).

Table Graphic Jump LocationTable 4. Sensitivity, Specificity, and Correctly Allocated Cases (CACs)for Group Comparisons Derived From Tree Analysis*
Place holder to copy figure label and caption
Figure 3

Group discrimination for Alzheimerdisease (AD) vs non-AD using phosphorylated tau protein (p-tau)231 andp-tau181derived from tree analysis. The vertical and horizontallines indicate the cutoffs for cerebrospinal fluid p-tau231 andp-tau181, respectively, derived from tree analysis.

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EFFECT OF AGE, SEX, MINI-MENTAL STATE EXAMINATION SCORE, AND CENTERON CSF p-TAU LEVELS IN THE AD GROUP

Levels of CSF p-tau231 (ρ = −0.18; P = .06) and p-tau181 (ρ = 0.002; P = .99) did not correlate with the Mini-Mental State Examination scorein patients with AD. Only for p-tau199 did we found a correlationwith the Mini-Mental State Examination score (ρ = −0.25; P = .01). There was no significant effect of sex (p-tau231: M-W107 = 1302; P = .55; p-tau181: M-W107 = 1375; P = .89; p-tau199: M-W107 = 1203; P = .22) or age (p-tau231: ρ = 0.002; P = .98; p-tau181: ρ= −0.05; P = .61; p-tau199: ρ= −0.013; P = .89) on levels of p-tau proteins.Levels of p-tau did not differ significantly between participating centers(p-tau231: M-W107 = 903; P =.74; p-tau181: M-W107 = 822; P =.34; p-tau199: M-W107 = 813; P =.31).

In the present study, we investigated the diagnostic performance of3 different pathophysiologically important CSF tau phosphorylation epitopes(p-tau231, p-tau181, and p-tau199)2022 to discriminate patientswith AD from those with other clinically important causes of dementia, thosewith ONDs, and nondemented controls. All 3 phosphorylation sites were studiedusing 3 recently developed and reported immunoassays.1,3,5 Sofar, independent early studies of the single phosphorylation sites have indicatedstatistically significant discriminative power between the AD and non-AD studygroups. Subsequently, CSF p-tau proteins in general were suggested as promisingbiological marker candidates for AD. In none of these pilot studies, however,were all 3 p-tau protein assays applied in the same set of subjects and patientsto look at differential diagnostic assay accuracy. With the advent of internationallarge-scale multicenter trials on putative biomarkers and neuroimaging inAD, the important issue has not yet been addressed of how p-tau assays performin general and whether there are potential relevant differences in assay performance,particularly in diagnostic sensitivity and specificity. Moreover, it is notyet known whether a combination of different p-tau epitopes or assays mightimprove diagnostic accuracy.

Our group showed that concentrations of all 3 p-tau proteins were equallysignificantly increased in patients with AD compared with the other groupsstudied. This finding is in strong agreement with all previously reportedresults.1,3,5,810

In a next step, discriminative power of the individual p-tau proteinswas studied. To interpret the clinical significance of the determined diagnosticaccuracy, we followed the recommendations of a consensus report12 foruseful (ideal) biomarkers of AD, determining specificity levels after thesensitivity level is 85% or higher. According to a consensus report on molecularand biochemical markers of AD, a useful (ideal) biomarker should yield a specificitylevel of at least 75% to 85%. In the differentiation of AD from non-AD, p-tau231 and p-tau181, but not p-tau199, reached therecommended specificity level. All 3 p-tau proteins, however, showed excellentspecificity levels when patients with AD were compared with nondemented controls.

For the clinically relevant differential diagnosis of AD, it is essentialto have a marker or a set of markers that discriminates AD from other clinicallyrelevant dementias. Therefore, we included patients with FTD, DLB, and VaDas well. In the discrimination between AD and FTD, p-tau231andp-tau181 fulfilled the proposed biomarker criteria for sensitivityand specificity. Comparison of the markers revealed that p-tau231andp-tau181 discriminated better than p-tau199 betweenthe AD group and the combined non-AD group and between AD and FTD.

The high level of discrimination between AD and FTD may originate indistinct differences in the biochemical and molecular signatures of tau-relatedpathophysiological changes between the 2 diseases.23 Therewas an increase in the p-tau protein level in some patients with DLB and VaD.Concomitant AD-type neuropathological changes in the brain, including neurofibrillarytangles, has been described for many patients with VaD24 andDLB25 who are clinically indistinguishablefrom those with "pure" VaD and DLB, respectively. In a clinical setting, ithas to be assumed that patients with VaD and DLB are heterogeneous regardingunderlying AD characteristic neuropathological changes in the brain, resultingin an increase in the p-tau protein level in at least some patients with VaDand DLB.

Using marker combinations did not add discriminative power comparedwith applying single markers. This might be a consequence of the high intercorrelationof the markers and the accurate discrimination between groups applied as singlemarkers. Group separation was maximized between AD and FTD using p-tau231and between AD and DLB using p-tau181.

In addition, we considered the effect of potentially confounding factorson CSF p-tau levels to assess the clinical applicability of p-tau proteins.Mini-Mental State Examination score accounted for approximately 5% of thevariance in p-tau levels, being significant only for p-tau199.This effect was not reported in previous studies,5,8 andit should be followed in independent samples. There was no effect of age andsex on levels of p-tau. In addition, different diagnostic centers did notaffect variance of p-tau levels. These findings indicate that p-tau proteinsmay be valuable markers for the clinical diagnosis of AD irrespective of age,sex, and diagnostic center.

To our knowledge, this is the first comparative study applying 3 developedand published immunoassays detecting different p-tau epitopes on the sameset of subjects and patients to compare the individual and combined diagnosticaccuracy. The results of this study indicate that all 3 p-tau assays performnearly equally well in discriminating patients with AD from nondemented controls.Both p-tau231 and p-tau181 fulfill the proposed criteriafor useful biomarkers in the differentiation of AD and non-AD and particularlyof AD and FTD.

Although there is no doubt that tau phosphorylation differs in AD, itis hard to speculate why. There have been few studies of phosphoserine 199and phosphothreonine 181 in the human brain. Except for one study,22 all we really know about these sites is that theyare phosphorylated in advanced AD neuropathological changes. The antibodiesused in the 181 and 199 assays have not been investigated to the same extentas the antibodies to phosphothreonine 231. It is well established that phosphorylationof threonine 231 is a very early event in AD, occurring before the formationof paired helical filaments in neurons of the hippocampus.21 Accordingto Augustinack and collegues,22 phosphorylationat threonine 181 and serine 199 occurs later, and these are only found toany appreciable extent in intracellular tangles. Reactivity to TG3, an antibodythat recognizes phosphothreonine 231, is found in pretangles, intracellulartangles, and extracellular tangles and so is present at all stages of thedisease. Augustinack and colleagues also suggest that several kinases canphosphorylate 199 and 231, but only extracellular regulated protein kinase2 phosphorylates 181. We suggest further investigations of the temporal sequenceof phosphorylations of the 3 sites, using the same antibodies as used in theCSF assays. Moreover, other potentially pathophysiologically relevant p-tauepitopes, such as serine 396 and serine 404, need to be further explored intheir ability to differentiate between relevant dementia disorders.26

Another relevant issue is to distinguish patients with mild cognitiveimpairment (MCI) from controls and particularly to predict AD in MCI. We showedthat CSF p-tau231 levels are elevated in patients with MCI comparedwith controls.27 In this longitudinal study,high p-tau231 levels at baseline correlated with the rate of cognitivedecline in Mini-Mental State Examination scores in patients with MCI. A subgroupof patients with MCI converted to AD. In agreement with the analysis of ratesof cognitive decline, increased levels of p-tau231 correlated withconversion to AD. De Leon and colleagues28 showeda longitudinal increase in p-tau231 levels in patients with MCI.Elevated levels of CSF p-tau181in patients with MCI compared withcontrols have also been shown.29 Future studiesare warranted to further explore CSF p-tau proteins in MCI and to comparetheir diagnostic and prognostic value.

This study was conducted in an academic clinical setting. Diagnoseswere performed by experienced dementia experts according to National Instituteof Neurological and Communicative Disorders and Stroke–Alzheimer's Diseaseand Related Disorders Association criteria,13 withan estimated positive predictive value of 89% to 100%.30 Partof our sample is enrolled in an ongoing neuropathological program designedto provide autopsy-confirmed diagnoses. In addition to autopsy-confirmed determinationof assay performance, population-based studies are warranted to establishCSF p-tau proteins as potential biomarkers for routine diagnostic use. Thesestudies are currently under way in international large-scale multicenter approaches.One large network, the National Institute on Aging Initiative on Neuroimagingin Alzheimer's Disease, will potentially start to evaluate neuroimaging thisyear, as well as an array of potential biomarkers in a 5-year longitudinalapproach in 650 individuals (patients with MCI, patients with AD, and controls).In their recent proceedings, a subconsortium, the Biological Marker WorkingGroup, has determined that measurement of CSF p-tau levels is a "feasiblecore marker" within the National Institute on Aging initiative.31

Reprints: Raymond Zinkowski, PhD, Applied NeuroSolutions Inc, 50Lakeview Pkwy, Vernon Hills, IL 60061 (e-mail: zinkowski@moleculargeriatrics.com).

Submitted for publication September 11, 2002; final revision receivedJune 24, 2003; accepted July 3, 2003.

This study was supported by grants from the Volkswagen-Foundation, Hannover,Germany (Dr Hampel); the Hirnliga e.V., Nürmbrecht, Germany (Drs Hampeland Buerger); a grant from the Medical Faculty, Ludwig-Maximilian University(Drs Buerger, Teipel, and Hampel); grants 11560 and 14002 from the SwedishMedical Research Council, Stockholm (Dr Blennow); and a grant from the Stiftelsenför Gamla Tjänarinnor, Stockholm (Dr Blennow).

We thank Felician Jancu, Bea Riemenschneider, Jenny Wagner, and OliverPogarell, MD, for clinical support; Thomas Nolde, PhD, and Heike Gluba fortechnical assistance; and Arun Lawrence Warren Bokde, PhD, and Jens Prüssner,PhD, for helpful discussion of the manuscript.

This study was presented in part at the 8th International Conferenceon Alzheimer's Disease and Related Disorders 2002, Alzheimer's Association;July 23, 2002; Stockholm, Sweden.

Corresponding authors: Katharina Buerger, MD, and Harald Hampel, MD,Dementia Research Section and Memory Clinic, Alzheimer Memorial Center andGeriatric Psychiatry Branch, Department of Psychiatry, Ludwig-Maximilian University,Nussbaumstrasse 7, 80336 Munich, Germany (e-mail: katharina.buerger@psy.med.uni-muenchen.de; hampel@psy.med.uni-muenchen.de).

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Buerger  KZinkowski  RTeipel  SJTapiola  TArai  HBlennow  KAndreasen  NHofmann-Kiefer  KDeBernardis  JKerkman  DMcCulloch  CKohnken  RPadberg  FPirttila  TSchapiro  MBRapoport  SIMoeller  HJDavies  PHampel  H Differential diagnosis of Alzheimer's disease with cerebrospinal fluidlevels of tau protein phosphorylated at threonine 231. Arch Neurol. 2002;591267- 1272
PubMed Link to Article
Sjoegren  MDavidsson  PTullberg  MMinthon  LWallin  AWikkelso  CGranerus  AKVanderstichele  HVanmechelen  EBlennow  K Both total and phosphorylated tau are increased in Alzheimer's disease. J Neurol Neurosurg Psychiatry. 2001;70624- 630
PubMed Link to Article
Vanmechelen  EVan Kerschaver  EBlennow  KDe Deyn  PPGalasko  DParnett  LSindic  CJMArai  HRiemenschneider  MHampel  HPottel  HValgaeren  AHulstaert  FVanderstichele  H CSF-phospho-tau (181P) as a promising marker for discriminating Alzheimer'sdisease from dementia with Lewy bodies. Iqbal  KSisodia  SWinblad  Beds.Alzheimer'sDisease: Advances in Etiology, Pathogenesis and Therapeutics. New York,NY John Wiley & Sons Inc2001;285- 293
Hampel  HBuerger  KKohnken  RTeipel  SJZinkowski  RMoeller  HJRapoport  SIDavies  P Tracking of Alzheimer's disease progression with CSF tau protein phosphorylatedat threonine 231. Ann Neurol. 2001;49545- 546
PubMed Link to Article
 Consensus report of the Working Group on: "Molecular and BiochemicalMarkers of Alzheimer's Disease": the Ronald and Nancy Reagan Research Instituteof the Alzheimer's Association and the National Institute on Aging WorkingGroup. Neurobiol Aging. 1998;19109- 116
PubMed Link to Article
McKhann  GDrachman  DFolstein  MKatzman  RPrice  DStadlan  EM Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDAWork Group under the auspices of the Department of Health and Human ServicesTask Force on Alzheimer's disease. Neurology. 1984;34939- 944
PubMed Link to Article
Lund and Manchester Groups, Clinical and neuropathological criteria for frontotemporal dementia. J Neurol Neurosurg Psychiatry. 1994;57416- 418
PubMed Link to Article
McKeith  IGGalasko  DKosaka  KPerry  EKDickson  DWHansen  LASalmon  DPLowe  JMirra  SSByrne  EJLennox  GQuinn  NPEdwardson  JAInce  PGBergeron  CBurns  AMiller  BLLovestone  SCollerton  DJansen  ENBallard  Cde Vos  RAWilcock  GKJellinger  KAPerry  RH Consensus guidelines for the clinical and pathologic diagnosis of dementiawith Lewy bodies (DLB). Neurology. 1996;471113- 1124
PubMed Link to Article
Roman  GCTatemichi  TKErkinjuntti  TCummings  JLMasdeu  JCGarcia  JHAmaducci  LOrgogozo  JMBrun  AHofman  A  et al.  Vascular dementia: diagnostic criteria for research studies: reportof the NINDS-AIREN International Workshop. Neurology. 1993;43250- 260
PubMed Link to Article
Morris  JCHeyman  AMohs  RCMoody  DMO'Brien  MDYamaguchi  TGrafman  JDrayer  BPBennett  DAFisher  MOgata  JKokmen  EBermejo  FWolf  PAGorelick  PBBick  KLPajeau  AKBell  MADeCarli  CCulebras  AKorczyn  ADBogousslavsky  JHartmann  AScheinberg  P The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Neurology. 1989;391159- 1165
PubMed Link to Article
Breiman  LFriedman  JHOlshen  RAStone  CJ Classification and Regression Trees.  Belmont, Calif Wadsworth International Group1984;
Galasko  DChang  LMotter  RClark  CMKaye  JKnopman  DThomas  RKholodenko  DSchenk  DLieberburg  IMiller  BGreen  RBasherad  RKertiles  LBoss  MASeubert  P High cerebrospinal fluid tau and low amyloid-beta-42 levels in theclinical diagnosis of Alzheimer disease and relation to apolipoprotein E genotype. Arch Neurol. 1998;55937- 945
PubMed Link to Article
Goedert  MJakes  RCrowther  RACohen  PVanmechelen  EVandermeeren  MCras  P Epitope mapping of monoclonal antibodies to the paired helical filamentsof Alzheimer's disease: identification of phosphorylation sites in tau protein. Biochem J. 1994;301 (pt 3) 871- 877
PubMed
Vincent  IZheng  JHDickson  DWKress  YDavies  P Mitotic phosphoepitopes precede paired helical filaments in Alzheimer'sdisease. Neurobiol Aging. 1998;19287- 296
PubMed Link to Article
Augustinack  JCSchneider  AMandelkow  EMHyman  BT Specific tau phosphorylation sites correlate with severity of neuronalcytopathology in Alzheimer's disease. Acta Neuropathol. 2002;10326- 35
PubMed Link to Article
Delacourte  A Biochemical and molecular characterization of neurofibrillary degenerationin frontotemporal dementias. Dement Geriatr Cogn Disord. 1999;10 (suppl 1) 75- 79
PubMed Link to Article
Kosunen  OSoininen  HPaljarvi  LHeinonen  OTalasniemi  SRiekkinen  PJ  Sr Diagnostic accuracy of Alzheimer's disease: a neuropathological study. Acta Neuropathol. 1996;91185- 193
PubMed Link to Article
Gomez-Isla  TGrowdon  WBMcNamara  MNewell  KGomez-Tortosa  EHedley-Whyte  ETHyman  BT Clinicopathologic correlates in temporal cortex in dementia with Lewybodies. Neurology. 1999;532003- 2009
PubMed Link to Article
Hu  YYHe  SSWang  XGrundke-Iqbal  IIqbal  KWang  J Levels of nonphosphorylated and phosphorylated tau in cerebrospinalfluid of Alzheimer's disease patients: an ultrasensitive bienzyme-substrate-recycleenzyme-linked immunosorbent assay. Am J Pathol. 2002;1601269- 1278
PubMed Link to Article
Buerger  KTeipel  SJZinkowski  RBlennow  KArai  HEngel  RHofmann-Kiefer  KMcCulloch  CPtok  UHeun  RAndreasen  NDeBernardis  JKerkman  DMoeller  HDavies  PHampel  H CSF tau protein phosphorylated at threonine 231 correlates with cognitivedecline in MCI subjects. Neurology. 2002;59627- 629
PubMed Link to Article
De Leon  MJSegal  STarshish  CYDeSanti  SZinkowski  RMehta  PDConvit  ACaraos  CRusinek  HTsui  WSaint Louis  LADeBernardis  JKerkman  DQadri  FGary  ALesbre  PWisniewski  TPoirier  JDavies  P Longitudinal cerebrospinal fluid tau load increases in mild cognitiveimpairment. Neurosci Lett. 2002;333183- 186
PubMed Link to Article
Andreasen  NVanmechelen  EVanderstichele  HDavidsson  PBlennow  K Cerebrospinal fluid levels of total-tau, phospho-tau and A beta 42predicts development of Alzheimer's disease in patients with mild cognitiveimpairment. Acta Neurol Scand Suppl. 2003;17947- 51
PubMed Link to Article
Nagy  ZEsiri  MMHindley  NJJoachim  CMorris  JHKing  EMMcDonald  BLitchfield  SBarnetson  LJobst  KASmith  AD Accuracy of clinical operational diagnostic criteria for Alzheimer'sdisease in relation to different pathological diagnostic protocols. Dement Geriatr Cogn Disord. 1998;9219- 226
PubMed Link to Article
Frank  RAGalasko  DHampel  HHardy  Jde Leon  MJMehta  PDRogers  JSiemers  ETrojanowski  JQ Biological markers for therapeutic trials in Alzheimer's disease: proceedingsof a working group: NIA initiative on neuroimaging in Alzheimer's disease. Neurobiol Aging. 2003;24521- 536
PubMed Link to Article

Figures

Place holder to copy figure label and caption
Figure 1

Levels of cerebrospinal fluid(CSF) phosphorylated tau protein (p-tau)231 (A), p-tau181 (B), and p-tau199 (C) in patients and controls. Dashed linesrepresent the cutoff level when sensitivity was set at 85% or higher. Asteriskindicates differences from AD at P<.001; dagger,differences from AD atP<.05; AD, Alzheimer disease;ONDs, other neurologic disorders; DLB, dementia with Lewy bodies; FTD, frontotemporaldementia; and VaD, vascular dementia.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2

Receiver operating characteristiccurves for cerebrospinal fluid phosphorylated tau protein (p-tau)231, CSF p-tau181, and CSF p-tau199 when patientswith Alzheimer disease were compared with the combined non–Alzheimerdisease group (A), the control group (B), patients with other neurologic disorders(C), patients with dementia with Lewy bodies (D), and patients with frontotemporaldementia (E). Diagonal lines indicate an area of 50%, indicating no differencein marker levels between groups.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3

Group discrimination for Alzheimerdisease (AD) vs non-AD using phosphorylated tau protein (p-tau)231 andp-tau181derived from tree analysis. The vertical and horizontallines indicate the cutoffs for cerebrospinal fluid p-tau231 andp-tau181, respectively, derived from tree analysis.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable 1. Characteristics of Patients and Controls and CSF p-Tau Values
Table Graphic Jump LocationTable 2. Correlations Between p-Tau Epitopes*
Table Graphic Jump LocationTable 3. Specificity of Single Markers With Sensitivity Set at 85%or Higher and Differences in Diagnostic Accuracy Between the Single Markers*
Table Graphic Jump LocationTable 4. Sensitivity, Specificity, and Correctly Allocated Cases (CACs)for Group Comparisons Derived From Tree Analysis*

References

Kohnken  RBuerger  KZinkowski  RMiller  CKerkman  DDeBernardis  JShen  JMoeller  HJDavies  PHampel  H Detection of tau phosphorylated at threonine 231 in cerebrospinal fluidof Alzheimer's disease patients. Neurosci Lett. 2000;287187- 190
PubMed Link to Article
Buerger  KZinkowski  RTeipel  SJArai  HDeBernardis  JKerkman  DMcCulloch  CPadberg  FFaltraco  FGoernitz  ATapiola  TRapoport  SIPirttila  TMoeller  HJHampel  H Differentiation of geriatric major depression from Alzheimer's diseasewith CSF tau protein phosphorylated at threonine 231. Am J Psychiatry. 2003;160376- 379
PubMed Link to Article
Vanmechelen  EVanderstichele  HDavidsson  PVan Kerschaver  EVan Der Perre  BSjogren  MAndreasen  NBlennow  K Quantification of tau phosphorylated at threonine 181 in human cerebrospinalfluid: a sandwich ELISA with a synthetic phosphopeptide for standardization. Neurosci Lett. 2000;28549- 52
PubMed Link to Article
Schoenknecht  PPantel  JHunt  AVolkmann  MBuerger  KHampel  HSchroeder  J Levels of total tau and tau protein phosphorylated at threonine 181in patients with incipient and manifest Alzheimer's diesase. Neurosci Lett. 2003;339172- 174
PubMed Link to Article
Itoh  NArai  HUrakami  KIshiguro  KOhno  HHampel  HBuerger  KWiltfang  JOtto  MKretzschmar  HMoeller  HJImagawa  MKohno  HNakashima  KKuzuhara  SSasaki  HImahori  K Large-scale, multicenter study of cerebrospinal fluid tau protein phosphorylatedat serine 199 for the antemortem diagnosis of Alzheimer's disease. Ann Neurol. 2001;50150- 156
PubMed Link to Article
Blennow  KVanmechelen  EHampel  H CSF total tau, Ab42 and phosphorylated tau protein as biomarkers forAlzheimer's disease. Mol Neurobiol. 2001;2487- 97
PubMed Link to Article
Blennow  KHampel  H CSF markers for incipient Alzheimer's disease. Lancet Neurol. 2003;2605- 613
PubMed Link to Article
Buerger  KZinkowski  RTeipel  SJTapiola  TArai  HBlennow  KAndreasen  NHofmann-Kiefer  KDeBernardis  JKerkman  DMcCulloch  CKohnken  RPadberg  FPirttila  TSchapiro  MBRapoport  SIMoeller  HJDavies  PHampel  H Differential diagnosis of Alzheimer's disease with cerebrospinal fluidlevels of tau protein phosphorylated at threonine 231. Arch Neurol. 2002;591267- 1272
PubMed Link to Article
Sjoegren  MDavidsson  PTullberg  MMinthon  LWallin  AWikkelso  CGranerus  AKVanderstichele  HVanmechelen  EBlennow  K Both total and phosphorylated tau are increased in Alzheimer's disease. J Neurol Neurosurg Psychiatry. 2001;70624- 630
PubMed Link to Article
Vanmechelen  EVan Kerschaver  EBlennow  KDe Deyn  PPGalasko  DParnett  LSindic  CJMArai  HRiemenschneider  MHampel  HPottel  HValgaeren  AHulstaert  FVanderstichele  H CSF-phospho-tau (181P) as a promising marker for discriminating Alzheimer'sdisease from dementia with Lewy bodies. Iqbal  KSisodia  SWinblad  Beds.Alzheimer'sDisease: Advances in Etiology, Pathogenesis and Therapeutics. New York,NY John Wiley & Sons Inc2001;285- 293
Hampel  HBuerger  KKohnken  RTeipel  SJZinkowski  RMoeller  HJRapoport  SIDavies  P Tracking of Alzheimer's disease progression with CSF tau protein phosphorylatedat threonine 231. Ann Neurol. 2001;49545- 546
PubMed Link to Article
 Consensus report of the Working Group on: "Molecular and BiochemicalMarkers of Alzheimer's Disease": the Ronald and Nancy Reagan Research Instituteof the Alzheimer's Association and the National Institute on Aging WorkingGroup. Neurobiol Aging. 1998;19109- 116
PubMed Link to Article
McKhann  GDrachman  DFolstein  MKatzman  RPrice  DStadlan  EM Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDAWork Group under the auspices of the Department of Health and Human ServicesTask Force on Alzheimer's disease. Neurology. 1984;34939- 944
PubMed Link to Article
Lund and Manchester Groups, Clinical and neuropathological criteria for frontotemporal dementia. J Neurol Neurosurg Psychiatry. 1994;57416- 418
PubMed Link to Article
McKeith  IGGalasko  DKosaka  KPerry  EKDickson  DWHansen  LASalmon  DPLowe  JMirra  SSByrne  EJLennox  GQuinn  NPEdwardson  JAInce  PGBergeron  CBurns  AMiller  BLLovestone  SCollerton  DJansen  ENBallard  Cde Vos  RAWilcock  GKJellinger  KAPerry  RH Consensus guidelines for the clinical and pathologic diagnosis of dementiawith Lewy bodies (DLB). Neurology. 1996;471113- 1124
PubMed Link to Article
Roman  GCTatemichi  TKErkinjuntti  TCummings  JLMasdeu  JCGarcia  JHAmaducci  LOrgogozo  JMBrun  AHofman  A  et al.  Vascular dementia: diagnostic criteria for research studies: reportof the NINDS-AIREN International Workshop. Neurology. 1993;43250- 260
PubMed Link to Article
Morris  JCHeyman  AMohs  RCMoody  DMO'Brien  MDYamaguchi  TGrafman  JDrayer  BPBennett  DAFisher  MOgata  JKokmen  EBermejo  FWolf  PAGorelick  PBBick  KLPajeau  AKBell  MADeCarli  CCulebras  AKorczyn  ADBogousslavsky  JHartmann  AScheinberg  P The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Neurology. 1989;391159- 1165
PubMed Link to Article
Breiman  LFriedman  JHOlshen  RAStone  CJ Classification and Regression Trees.  Belmont, Calif Wadsworth International Group1984;
Galasko  DChang  LMotter  RClark  CMKaye  JKnopman  DThomas  RKholodenko  DSchenk  DLieberburg  IMiller  BGreen  RBasherad  RKertiles  LBoss  MASeubert  P High cerebrospinal fluid tau and low amyloid-beta-42 levels in theclinical diagnosis of Alzheimer disease and relation to apolipoprotein E genotype. Arch Neurol. 1998;55937- 945
PubMed Link to Article
Goedert  MJakes  RCrowther  RACohen  PVanmechelen  EVandermeeren  MCras  P Epitope mapping of monoclonal antibodies to the paired helical filamentsof Alzheimer's disease: identification of phosphorylation sites in tau protein. Biochem J. 1994;301 (pt 3) 871- 877
PubMed
Vincent  IZheng  JHDickson  DWKress  YDavies  P Mitotic phosphoepitopes precede paired helical filaments in Alzheimer'sdisease. Neurobiol Aging. 1998;19287- 296
PubMed Link to Article
Augustinack  JCSchneider  AMandelkow  EMHyman  BT Specific tau phosphorylation sites correlate with severity of neuronalcytopathology in Alzheimer's disease. Acta Neuropathol. 2002;10326- 35
PubMed Link to Article
Delacourte  A Biochemical and molecular characterization of neurofibrillary degenerationin frontotemporal dementias. Dement Geriatr Cogn Disord. 1999;10 (suppl 1) 75- 79
PubMed Link to Article
Kosunen  OSoininen  HPaljarvi  LHeinonen  OTalasniemi  SRiekkinen  PJ  Sr Diagnostic accuracy of Alzheimer's disease: a neuropathological study. Acta Neuropathol. 1996;91185- 193
PubMed Link to Article
Gomez-Isla  TGrowdon  WBMcNamara  MNewell  KGomez-Tortosa  EHedley-Whyte  ETHyman  BT Clinicopathologic correlates in temporal cortex in dementia with Lewybodies. Neurology. 1999;532003- 2009
PubMed Link to Article
Hu  YYHe  SSWang  XGrundke-Iqbal  IIqbal  KWang  J Levels of nonphosphorylated and phosphorylated tau in cerebrospinalfluid of Alzheimer's disease patients: an ultrasensitive bienzyme-substrate-recycleenzyme-linked immunosorbent assay. Am J Pathol. 2002;1601269- 1278
PubMed Link to Article
Buerger  KTeipel  SJZinkowski  RBlennow  KArai  HEngel  RHofmann-Kiefer  KMcCulloch  CPtok  UHeun  RAndreasen  NDeBernardis  JKerkman  DMoeller  HDavies  PHampel  H CSF tau protein phosphorylated at threonine 231 correlates with cognitivedecline in MCI subjects. Neurology. 2002;59627- 629
PubMed Link to Article
De Leon  MJSegal  STarshish  CYDeSanti  SZinkowski  RMehta  PDConvit  ACaraos  CRusinek  HTsui  WSaint Louis  LADeBernardis  JKerkman  DQadri  FGary  ALesbre  PWisniewski  TPoirier  JDavies  P Longitudinal cerebrospinal fluid tau load increases in mild cognitiveimpairment. Neurosci Lett. 2002;333183- 186
PubMed Link to Article
Andreasen  NVanmechelen  EVanderstichele  HDavidsson  PBlennow  K Cerebrospinal fluid levels of total-tau, phospho-tau and A beta 42predicts development of Alzheimer's disease in patients with mild cognitiveimpairment. Acta Neurol Scand Suppl. 2003;17947- 51
PubMed Link to Article
Nagy  ZEsiri  MMHindley  NJJoachim  CMorris  JHKing  EMMcDonald  BLitchfield  SBarnetson  LJobst  KASmith  AD Accuracy of clinical operational diagnostic criteria for Alzheimer'sdisease in relation to different pathological diagnostic protocols. Dement Geriatr Cogn Disord. 1998;9219- 226
PubMed Link to Article
Frank  RAGalasko  DHampel  HHardy  Jde Leon  MJMehta  PDRogers  JSiemers  ETrojanowski  JQ Biological markers for therapeutic trials in Alzheimer's disease: proceedingsof a working group: NIA initiative on neuroimaging in Alzheimer's disease. Neurobiol Aging. 2003;24521- 536
PubMed Link to Article

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