In this report, we present the results of a multicenter study to test analytic and diagnostic performance of soluble forms of amyloid precursor proteins α and β (sAPPα and sAPPβ) in the cerebrospinal fluid (CSF) of patients with different forms of dementing conditions. CSF samples were collected from 188 patients with early dementia (mini-mental state examination⩾20 in majority of cases) and mild cognitive impairment (MCI) in 12 gerontopsychiatric centers, and the clinical diagnoses were supported by neurochemical dementia diagnostic (NDD) tools: CSF amyloidβ peptides, Tau and phospho-Tau. sAPPα and sAPPβ were measured with multiplexing method based on electrochemiluminescence. sAPPα and sAPPβ CSF concentrations correlated with each other with very high correlation ratio (R=0.96, P<0.001). We observed highly significantly increased sAPPα and sAPPβ CSF concentrations in patients with NDD characteristic for Alzheimer's disease (AD) compared to those with NDD negative results. sAPPα and sAPPβ highly significantly separated patients with AD, whose diagnosis was supported by NDD findings (sAPPα: cutoff, 117.4 ng ml−1, sensitivity, 68%, specificity, 85%, P<0.001; sAPPβ: cutoff, 181.8 ng ml−1, sensitivity, 75%, specificity, 85%, P<0.001), from the patients clinically assessed as having other dementias and supported by NDD untypical for AD. We conclude sAPPα and sAPPβ might be regarded as novel promising biomarkers supporting the clinical diagnosis of AD.
With the growing number of elderly persons in the Western industrialized countries, the incidence of dementia disorders increases rapidly, resulting in an increasing burden on the health care system. In the case of Alzheimer's disease (AD), the increasing number of patients has not so far resulted in the achievement of accurate standards of durante vitam diagnosis. Although sensitivity of clinical diagnosis is relatively high (93%), specificity may be lower, being reported as 55% in a multicenter clinical-autopsy study.1 In expert hands the clinical diagnosis of AD is predictive of AD pathology in 80–90% of cases. Nevertheless, very early diagnosis of AD, and differential diagnosis of unusual presentations of patients with dementia remains difficult on clinical grounds.
With the introduction of potentially successful treatments for dementias that were previously considered to be irreversible (for a review, see Klafki2 and Hull3), the need for an early and differential diagnosis of dementia becomes even more urgent.4, 5, 6 As cerebrospinal fluid (CSF) is in direct contact with the central nervous system, it is obvious that any changes in biochemical composition of brain parenchyma should be predominantly reflected in CSF. Lumbar puncture is an easy procedure, with a low incidence of complications. In a large study by Andreasen et al.,7 only 4.1% of all patients experienced post-lumbar headache, and an even smaller incidence of 2% was reported in a study by Blennow et al.8 It is therefore reasonable to postulate that lumbar puncture is a feasible, moderately invasive procedure, and CSF analysis could possibly improve current clinical and neuroimaging-based approaches to diagnosis. Nevertheless, the low and limited amount of the material obtained with a lumbar puncture requires very precise planning of the laboratory tests to be performed. The growing number of possibly important biomarkers to be analyzed in a limited volume of the CSF makes techniques of a simultaneous analysis of several parameters in a single small-volume CSF sample (multiplexing) a method of choice in the future.9, 10
Materials and methods
Patients and lumbar punctures
The study was approved by the ethics committees of all the participating universities, and all patients and/or their relatives gave their informed consent.
A total of 12 German gerontopsychiatric university departments participated in the project (http://www.kompetenznetz-demenzen.de), recruiting a total of n=188 patients with early dementias (in a great majority of cases Mini Mental State Examination, MMSE⩾20) or mild cognitive impairment (MCI). Patients of this study were categorized according to (1) clinical and neuropsychological, and (2) neurochemical criteria.
Patients with early AD (D-AD; n=69) were diagnosed according to the criteria of International Classification of Diseases-10 (ICD-10), and the National Institute of Neurological and Communicative Disorders and the Stroke-Alzheimer's Disease and Related Disorders Association.11 Patients with other early dementias (D-O; n=27) fulfilled the criteria of corresponding disorders (vascular dementia, n=3; frontotemporal degeneration, n=10; dementia with Lewy bodies, n=4; Parkinson's disease, n=1; corticobasal degeneration, n=1; progressive supranuclear palsy, n=1; other and unclassified, n=7). Cognitive dysfunction was assessed with Consortium to Establish a Registry for AD (CERAD) neuropsychological test battery,12 WMS-R logical memory,13 trail making test14 and clock drawing test.15 Functional decline was assessed using informant questionnaires The Bayer Activities of Daily Living (B-ADL),16 and Informant Questionnaire on Cognitive Decline (IQCODE) in the elderly.17 Distinction of MCI and dementia was based on Clinical Dementia Rating (CDR) score.18 A diagnosis of MCI corresponded to a CDR score of 0.5, a diagnosis of mild dementia corresponded to a CDR score of 1.0. Severity of dementia was graded according to the MMSE,19 with the score being available for 185 patients.
Patients with MCI fulfilled the modified criteria of Petersen et al.,20 and were subdivided into two groups: those with clinical, neuropsychological and neuroradiological signs suggesting development of AD (MCI-AD; n=54), and those with the signs suggesting development of other dementias (MCI-O; n=38).
In the second categorization, patients were divided according to the neurochemical findings in the CSF: the group with pathologic results of neurochemical dementia diagnosis (NDD+; n=103) characterized by decreased CSF amyloidβ (Aβ) x-42/x-40 concentration ratio (Aβ ratio; cutoff, 0.1121) accompanied by increased CSF concentration of phospho-Tau181 (pTau181; cutoff 70 pg ml−1), and the group with normal results of neurochemical dementia diagnosis (NDD−; n=48) characterizing with normal amyloidβ ratio (that is higher than 0.11) accompanied by normal pTau181 (below 50 pg ml−1). Note that n=37 patients with ‘borderline’ CSF pTau181 concentration (50–70 pg ml−1) were not considered for this categorization.
Patients of the study were further categorized according to combined clinical and neurochemical data, that is D-AD, D-O, MCI-AD and MCI-O patients were selected fulfilling corresponding NDD criteria: positive or negative values of Aβ ratio and pTau181: D-AD/NDD+, n=54; D-O/NDD−, n=7; MCI-AD/NDD+, n=30; and MCI-O/NDD−, n=20. The demographic data of the patients are shown in Table 1.
Lumbar punctures were performed with the patients in the sitting position according to the protocol described elsewhere.22 Briefly, after collection of the CSF for routine diagnosis (2–5 ml), additionally 4.5 ml of the CSF for this study was sampled into the same polypropylene test tube. The tube was gently shaken, and the CSF was centrifuged immediately after collection (1600 g, room temperature, 15 min) and then aliquoted into 16 polypropylene test tubes (250 μl each), and frozen within 30–40 min after the puncture. The CSF was at no time thawed/refrozen. The analysis was performed in one center (University of Erlangen) to which all the aliquots of frozen CSF were shipped on dry ice.
In all the subjects of this study, routine neurochemical dementia diagnostics was performed in the CSF, including: Aβ1–42, Aβx-42, Aβx-40, Aβ ratio, total Tau and pTau181, according to the protocols described elsewhere.21, 23 Briefly, with the assay of the Genetics Co. (Zürich, Switzerland) Aβx-42 peptide was measured, as the capturing antibody of this assay is not N terminus specific, and with the assay of Innogenetics (Ghent, Belgium), Aβ1–42 peptide was measured, as the capturing antibody of this assay shows N terminus specificity. In both assays, the detecting antibodies are specific for the position 42.
sAPPα and sAPPβ CSF concentrations were measured with a multiplexing assay of Meso Scale Discovery (Gaithersburg, MD, USA). The assay uses 6E10 (Signet Covance, Dedham, MA, USA), having an epitope of amino acids 3–8 of the human Aβ sequence, as capturing antibody for sAPPα, and ANGU, raised in rabbit against the peptide sequence of amino acids 591–596 of APP695,24 as capturing antibody for sAPPβ. As detecting antibody, P2-1 is used for recognizing an epitope in the N-terminal domain of APP.25 Briefly, 150 μl of the blocking buffer was added to all wells of the plate, and after 1 h of incubation the wells were washed with the washing buffer. Next, 25 μl of standards or samples was applied, and the plate was incubated for 1 h at room temperature on a shaker. After the next washing step, the incubation was followed with 25 μl of the detection antibody per well at the final concentration of 1 nM. After the next washing, the reading buffer was added to all wells, and was followed by 10 min of incubation the plate was read with the Sector Imager (Meso Scale Discovery). All measurements were performed in duplicate.
To test possible cross-reactivity, a standard of sAPPα at the concentration of 200 ng ml−1 was applied into two wells of the plate, and a standard of sAPPβ at the concentration of 200 ng ml−1 was applied into two different wells of the plate. Moreover, Aβ42 synthetic peptide at the concentration of 4 ng ml−1, and Aβ40 synthetic peptide at the concentration of 4 ng ml−1 were tested for cross-reactivity of sAPPα and sAPPβ assays.
To analyze intraassay imprecision, n=8 CSF samples were tested 10 times on 1 plate, and to analyze interassay imprecision, 1 CSF sample was tested on 5 different plates during 5 different experiments.
Furthermore, to test the antibodies for potential cross-reactivity with other CSF proteins, SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting were performed. Briefly, three CSF samples were dissolved in SDS sample buffer, heated to 95 °C for 5 min and separated on 7.5% T/2.67% C SDS–PAGE. The separated proteins were blotted onto polyvinylidene difluoride membranes (Millipore Immobilon-P 0.45 μM) by semi-dry transfer (Hoefer Semiphor, San Francisco, CA, USA) at 1 mA cm−2 for 60 min with 25 mM Tris, 192 mM glycine, 20% (v/v) methanol. After blotting, the membranes were rinsed briefly with deionized H2O and blocked with 2% (w/v) ECL Advance blocking reagent (GE-Healthcare, Munich, Germany) in phosphate-buffered saline (PBS)/0.075% Tween-20 (PBS-T) for 60 min at room temperature. The blots were then incubated overnight at 4 °C with primary antibodies (1 μg ml−1) in blocking buffer. To control for proteins that could cross-react with the secondary antibodies, parallel blots were incubated overnight in blocking solution in the absence of primary antibodies. The membranes were washed with PBS-T (two short rinses plus 3 × 10 min) and incubated with horseradish peroxidase-conjugated secondary antibodies for 45 min at room temperature. After washing, the blots were developed with ECL-Plus chemiluminescence substrate (GE Healthcare) according to the manufacturer's instructions.
Storage stability of sAPPα and sAPPβ CSF concentrations was tested with n=5 CSF samples divided after lumbar puncture into four aliquots (A–D): A, frozen and stored at −80 °C for 3 days; B, stored at room temperature (ca. +20 °C) for 3 days; C, stored at +4 °C for 3 days; and D, frozen at −80 °C and exposed to three freeze/thaw cycles during 3 days of storage. Following 3 days of samples’ storage, sAPPα and sAPPβ in all aliquots of all five samples were measured on one plate.
Apolipoprotein E genotyping (APOE) has been performed after obtaining additional patient's informed consent. Leukocyte DNA was isolated with the Qiagen blood isolation kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). The APOE genotype was studied as described by Hixson and Vernier.26
If not stated otherwise, the results are presented as medians and interquartile ranges. Statistical differences between groups were analyzed with analysis of variance (ANOVA) followed by Scheffe's post hoc test. Correlations between measured values were analyzed with Pearson's correlation factor. Differences were considered significant if P<0.05. Analyses were performed with Statistica 7.0 (Statsoft, Tulsa, OK, USA).
Analytical performance of the assay
Intraassay imprecision, expressed as coefficients of variation for three (out of eight) samples representing the highest, middle and the lowest sAPPα and sAPPβ concentrations is presented in Table 2. In the remaining five samples, imprecision turned out to be below 5% for both biomarkers (with one exception: 7.1% in case of sAPPβ in one CSF sample).
Interassay imprecision was 7% for sAPPα, and 9% for sAPPβ. We did not observe significant cross-reactivity between sAPPα and sAPPβ assays, and similarly we did not observe cross-reactivity of any of the two assays with either Aβ42 or Aβ40 (data not shown). Western blotting analysis of the sAPPα and sAPPβ-capturing antibodies is presented in Figure 1. We did not observe significant cross-reactivity of the two antibodies with CSF proteins. Bands observed in the molecular mass of approximately 50 kDa in Figures 1a and c represent the most probable unspecific binding of the secondary antibodies, as they were present on the control blots (developed without primary sAPPα or sAPPβ antibodies, Figures 1b and d, respectively), as well. Furthermore, western blot analysis confirmed the absence of cross-reactivity between sAPPα- and sAPPβ-capturing antibodies.
Stability of sAPP concentrations under different storage conditions
The results of storage experiments are presented in Figure 2 (normalized for the results of the samples stored at −80 °C (aliquot A) representing 100%).
Statistical analysis of the results (ANOVA for repeated measurements with post hoc Scheffe's test) revealed no significant differences between the concentrations in the samples stored under these four conditions regarding sAPPα, but the concentrations of sAPPβ in the samples stored at −80 °C turned out to be slightly lower than those stored at room temperature (P<0.01) and at +4 °C (P<0.05), and the concentrations in the samples stored at room temperature were slightly higher than those of the samples exposed for repeated freeze/thaw cycles (P<0.05). Three repetitions of refreezing did not influence the results of samples stored at −80 °C.
Correlation between sAPPα and sAPPβ CSF concentrations
We observed very high correlation (R=0.96, P<0.001) between the CSF concentrations of both sAPP forms, as presented in Figure 3.
sAPPα and sAPPβ weakly correlated with Aβx-40 (R=0.46 and R=0.48, respectively, P<0.001 in both cases). Correlation with other biomarkers, if at all, was only marginal (R<0.35, data not shown).
sAPPα and sAPPβ as potential biomarkers of Alzheimer's disease
The results of the sAPPα and sAPPβ CSF concentrations in NDD+ and NDD− patients are presented in Figure 4.
The concentration of both biomarkers turned out to be highly significantly (P<0.001) higher in NDD+ compared to NDD−.
Interestingly, we did not observe any difference between NDD+ subjects carrying the APOE ɛ4 allele versus noncarriers of this allele, and similarly there were no difference between NDD− subjects with and without the APOE ɛ4 allele (data not shown).
The results of sAPPα and sAPPβ CSF concentrations in patients whose clinical diagnoses were supported by neurochemical findings (that is Aβ ratio and pTau181 in agreement with the clinical diagnoses) are presented in Figure 5.
We observed significant differences of the concentrations of both biomarkers between D-AD/NDD+ and D-O/NDD−, as well as MCI-AD/NDD+ and MCI-O/NDD−. There were no statistical differences concerning sAPPα or sAPPβ between D-AD/NDD+ and MCI-AD/NDD+, and between D-O/NDD− and MCI-O/NDD−, respectively. We did not see statistically significant correlation between either sAPPα or sAPPβ and age of patients (data not shown). Overall, the female patients had higher sAPPα and sAPPβ concentrations compared to the male patients (data not shown).
Cutoff values, sensitivities and specificities
Cutoff values, sensitivities and specificities of the biomarkers (sAPPα and sAPPβ) are presented in Table 3. For the calculation of cutoffs, D-AD and MCI-AD groups were combined and the cutoff values were calculated optimally separating this group from D-O and MCI-O according to the Youden's index.
In this report, we present the results of a multicenter study on soluble forms of amyloid precursor proteins α and β in CSF of patients with different types of early dementia and MCI.
One of the important issues in clinical neurochemistry is the management of human CSF samples that are very precious material obtained by lumbar puncture, a procedure regarded as invasive. The volume of a sample is sometimes, due to for example technical problems, extremely small (in critical cases less than 1 ml) and the number of biomarkers to study or to measure routinely grows rapidly. To solve this conflict, different multiplexing platforms have been developed and established in laboratories enabling simultaneous measurements of more than one biomarker in one sample volume, during one analytical run, saving precious material, time and effort.27
When establishing a new assay in a laboratory, a critical question must be addressed of reliability of the results, and as a matter of fact assays with high imprecision cannot be considered for routine use even if they promise help in diagnosis of patients. Therefore, before starting analysis of patients’ samples, a set of experiments was performed to control the assay's performance, which turned out to be well acceptable (intraassay imprecision less than 5% in low-, middle-, and high-concentration range, and interassay imprecision less than 10%).
Thinking in terms of routine application of the potential biomarkers, it is important to address a practical question of sample delivery and storage.28, 29, 30 In case of both candidate biomarkers in this study, no difference was observed between the results from samples stored at +4 °C and room temperature, and between samples constantly kept frozen (at −80 °C) or exposed for refreezing, respectively. This could mean that CSF samples may be sent between laboratories via regular mail without necessity of cooling, and that precious aliquots of experimental study samples can be thawed and refrozen. However, at least for sAPPβ, cutoffs and ranges must not be mixed for frozen and nonfrozen samples. The limitation of this conclusion is, however, a small number of samples studied.
Perhaps one of the most intriguing outcomes of this study is the surprisingly high correlation between the CSF concentrations of the two forms of soluble APP. In such a case, an artifact has to be considered, and the explanation could be for example cross-reactivity between antibodies for sAPPα and sAPPβ, and/or cross-reactivity of the assay with some other CSF molecules. However, as we could exclude such unspecificity of the antibodies, we may carefully conclude our observation corresponds to a real physiological phenomenon. In this case, perhaps it is justified to postulate the presence of some kind of hypothetical factor regulating metabolism of APP, for example by controlling activities of α and β secretases. Certainly more studies are necessary to prove or reject this hypothesis.
We observed highly significantly increased sAPPα and sAPPβ CSF concentration in patients with AD compared to patients with other dementias if clinical diagnosis was supported by findings of routine neurochemical dementia diagnosis (amyloidβ peptides, Tau and pTau181). This raises a question of reliability of differential diagnosis in early dementia. It is known that clinical diagnosis in later stages of dementias might be incorrect in a considerable percentage of patients, and the diagnostic uncertainty in early cases, as these included into our study (MMSE⩾20), is probably even larger. Therefore, it is justified to postulate that CSF biomarkers should be routinely measured to increase the accuracy of differential diagnosis. In a recently published report, Engelborghs et al. tested diagnostic performance of the CSF biomarkers: Aβ1–42, total Tau and pTau181 on the ground of autopsy-controlled cases, and concluded that dementia could be discriminated from controls with sensitivity of 86% and specificity of 89%, obviously showing the value of biomarkers in differential dementia diagnosis.31
Our results show no differences between sAPPα or sAPPβ concentrations of patients in MCI stage and those already having early dementias. This fully supports the observations of Blennow and co-workers,32, 33 as well as our recently published data27 showing alterations of CSF Aβ1–42, total Tau and pTau181 in cases of MCI subjects with increased risk to develop AD compared to those without such risk. Considering all these reports, strong evidence indicates that the AD-indicative alterations of the CSF biomarkers can be observed years before the onset of dementia. This hypothesis certainly requires further studies, and longitudinal observations of AD/other dementia patients as well as subjects with pre-MCI stage should be involved. If it could be proven, however, it would certainly have high impact on prophylactics and preventive treatment of AD in the future.
Concluding, we feel sAPPα and sAPPβ may represent novel promising markers of AD especially if combined with markers already routinely applied. More studies are necessary to further characterize analytical and diagnostic performance of the assay.
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This study was supported by the following grants from the German Federal Ministry of Education and Research (BMBF): Kompetenznetz Demenzen (01 GI 0420), HBPP-NGFN2 (01 GR 0447), and the Forschungsnetz der Früh- und Differenzialdiagnose der Creutzfeldt-Jakob-Krankheit und der neuen Variante der CJK (01 GI 0301), and by the EU grants cNEUPRO (contract no. LSHM-CT-2007-037950), and neuroTAS (contract no. LSHB-CT-2006-037953). We appreciate aliquots of 6E10 and ANGU antibodies provided by Dr R Umek (Meso-Scale Discovery) for western blot experiments.
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