Manu Vandijck

Longitudinal Cerebrospinal Fluid Biomarker Changes in Preclinical Alzheimer Disease During Middle Age.

IMPORTANCE Individuals in the presymptomatic stage of Alzheimer disease (AD) are increasingly being targeted for AD secondary prevention trials. How early during the normal life span underlying AD pathologies begin to develop, their patterns of change over time, and their relationship with future cognitive decline remain to be determined. OBJECTIVE To characterize the within-person trajectories of cerebrospinal fluid (CSF) biomarkers of AD over time and their association with changes in brain amyloid deposition and cognitive decline in cognitively normal middle-aged individuals. DESIGN, SETTING, AND PARTICIPANTS As part of a cohort study, cognitively normal (Clinical Dementia Rating [CDR] of 0) middle-aged research volunteers (n = 169) enrolled in the Adult Children Study at Washington University, St Louis, Missouri, had undergone serial CSF collection and longitudinal clinical assessment (mean, 6 years; range, 0.91-11.3 years) at 3-year intervals at the time of analysis, between January 2003 and November 2013. A subset (n = 74) had also undergone longitudinal amyloid positron emission tomographic imaging with Pittsburgh compound B (PiB) in the same period. Serial CSF samples were analyzed for β-amyloid 40 (Aβ40), Aβ42, total tau, tau phosphorylated at threonine 181 (P-tau181), visinin-like protein 1 (VILIP-1), and chitinase-3-like protein 1 (YKL-40). Within-person measures were plotted according to age and AD risk defined by APOE genotype (ε4 carriers vs noncarriers). Linear mixed models were used to compare estimated biomarker slopes among middle-age bins at baseline (early, 45-54 years; mid, 55-64 years; late, 65-74 years) and between risk groups. Within-person changes in CSF biomarkers were also compared with changes in cortical PiB binding and progression to a CDR higher than 0 at follow-up. MAIN OUTCOMES AND MEASURES Changes in Aβ40, Aβ42, total tau, P-tau181, VILIP-1, and YKL-40 and, in a subset of participants, changes in cortical PiB binding. RESULTS While there were no consistent longitudinal patterns in Aβ40 (P = .001-.97), longitudinal reductions in Aβ42 were observed in some individuals as early as early middle age (P ≤ .05) and low Aβ42 levels were associated with the development of cortical PiB-positive amyloid plaques (area under receiver operating characteristic curve = 0.9352; 95% CI, 0.8895-0.9808), especially in mid middle age (P < .001). Markers of neuronal injury (total tau, P-tau181, and VILIP-1) dramatically increased in some individuals in mid and late middle age (P ≤ .02), whereas the neuroinflammation marker YKL-40 increased consistently throughout middle age (P ≤ .003). These patterns were more apparent in at-risk ε4 carriers (Aβ42 in an allele dose-dependent manner) and appeared to be associated with future cognitive deficits as determined by CDR. CONCLUSIONS AND RELEVANCE Longitudinal CSF biomarker patterns consistent with AD are first detectable during early middle age and are associated with later amyloid positivity and cognitive decline. Such measures may be useful for targeting middle-aged, asymptomatic individuals for therapeutic trials designed to prevent cognitive decline.

Global standardization measurement of cerebral spinal fluid for Alzheimer's disease: An update from the Alzheimer's Association Global Biomarkers Consortium

Recognizing that international collaboration is critical for the acceleration of biomarker standardization efforts and the efficient development of improved diagnosis and therapy, the Alzheimers Association created the Global Biomarkers Standardization Consortium (GBSC) in 2010. The consortium brings together representatives of academic centers, industry, and the regulatory community with the common goal of developing internationally accepted common reference standards and reference methods for the assessment of cerebrospinal fluid (CSF) amyloid β42 (Aβ42) and tau biomarkers. Such standards are essential to ensure that analytical measurements are reproducible and consistent across multiple laboratories and across multiple kit manufacturers. Analytical harmonization for CSF Aβ42 and tau will help reduce confusion in the AD community regarding the absolute values associated with the clinical interpretation of CSF biomarker results and enable worldwide comparison of CSF biomarker results across AD clinical studies.

Changes in plasma amyloid beta in a longitudinal study of aging and Alzheimer's disease

A practical biomarker is required to facilitate the preclinical diagnosis of Alzheimers disease (AD).

Alzheimer's disease cerebrospinal fluid biomarker in cognitively normal subjects

In a large multicentre sample of cognitively normal subjects, as a function of age, gender and APOE genotype, we studied the frequency of abnormal cerebrospinal fluid levels of Alzheimers disease biomarkers including: total tau, phosphorylated tau and amyloid-β1-42. Fifteen cohorts from 12 different centres with either enzyme-linked immunosorbent assays or Luminex® measurements were selected for this study. Each centre sent nine new cerebrospinal fluid aliquots that were used to measure total tau, phosphorylated tau and amyloid-β1-42 in the Gothenburg laboratory. Seven centres showed a high correlation with the new Gothenburg measurements; therefore, 10 cohorts from these centres are included in the analyses here (1233 healthy control subjects, 40-84 years old). Amyloid-β amyloid status (negative or positive) and neurodegeneration status (negative or positive) was established based on the pathological cerebrospinal fluid Alzheimers disease cut-off values for cerebrospinal fluid amyloid-β1-42 and total tau, respectively. While gender did not affect these biomarker values, APOE genotype modified the age-associated changes in cerebrospinal fluid biomarkers such that APOE ε4 carriers showed stronger age-related changes in cerebrospinal fluid phosphorylated tau, total tau and amyloid-β1-42 values and APOE ε2 carriers showed the opposite effect. At 40 years of age, 76% of the subjects were classified as amyloid negative, neurodegeneration negative and their frequency decreased to 32% at 85 years. The amyloid-positive neurodegeneration-negative group remained stable. The amyloid-negative neurodegeneration-positive group frequency increased slowly from 1% at 44 years to 16% at 85 years, but its frequency was not affected by APOE genotype. The amyloid-positive neurodegeneration-positive frequency increased from 1% at 53 years to 28% at 85 years. Abnormally low cerebrospinal fluid amyloid-β1-42 levels were already frequent in midlife and APOE genotype strongly affects the levels of cerebrospinal fluid amyloid-β1-42, phosphorylated tau and total tau across the lifespan without influencing the frequency of subjects with suspected non-amyloid pathology.

Assessing the commutability of reference material formats for the harmonization of amyloid-β measurements.

Abstract Background: The cerebrospinal fluid (CSF) amyloid-β (Aβ42) peptide is an important biomarker for Alzheimer’s disease (AD). Variability in measured Aβ42 concentrations at different laboratories may be overcome by standardization and establishing traceability to a reference system. Candidate certified reference materials (CRMs) are validated herein for this purpose. Methods: Commutability of 16 candidate CRM formats was assessed across five CSF Aβ42 immunoassays and one mass spectrometry (MS) method in a set of 48 individual clinical CSF samples. Promising candidate CRM formats (neat CSF and CSF spiked with Aβ42) were identified and subjected to validation across eight (Elecsys, EUROIMMUN, IBL, INNO-BIA AlzBio3, INNOTEST, MSD, Simoa, and Saladax) immunoassays and the MS method in 32 individual CSF samples. Commutability was evaluated by Passing-Bablok regression and the candidate CRM termed commutable when found within the prediction interval (PI). The relative distance to the regression line was assessed. Results: The neat CSF candidate CRM format was commutable for almost all method comparisons, except for the Simoa/MSD, Simoa/MS and MS/IBL where it was found just outside the 95% PI. However, the neat CSF was found within 5% relative distance to the regression line for MS/IBL, between 5% and 10% for Simoa/MS and between 10% and 15% for Simoa/MSD comparisons. Conclusions: The neat CSF candidate CRM format was commutable for 33 of 36 method comparisons, only one comparison more than expected given the 95% PI acceptance limit. We conclude that the neat CSF candidate CRM can be used for value assignment of the kit calibrators for the different Aβ42 methods.

Increased CSF α-synuclein levels in Alzheimer's disease: Correlation with tau levels

Given the difficult clinical differential diagnosis between Alzheimers disease (AD) and dementia with Lewy bodies (DLB), growing interest resulted in research on α‐synuclein as a potential cerebrospinal fluid biomarker (CSF) for synucleinopathies.

Alzheimer Disease Biomarker Testing in Cerebrospinal Fluid: A Method to Harmonize Assay Platforms in the Absence of an Absolute Reference Standard

To the Editor: The acceptance of the clinical utility of protein biomarkers for Alzheimer disease diagnosis and their integration into routine clinical testing require extensive standardization at different levels (preanalytical, analytical, postanalytical) (1). The approval of such biomarkers by regulatory authorities is hampered in part by ( a ) uncertainty regarding the accuracy of values produced with different measurement technologies, ( b ) the absence of international reference standards, ( c ) the absence of validated reference methods for measuring absolute analyte concentrations, and ( d ) a lack of well-defined recommendations for immunoassay validation (2). The present letter provides a solution for harmonization of cutoff concentration values for different technology platforms by performing immunoassays that use calibrators consisting of undiluted cerebrospinal fluid (CSF).1 For this exploratory study, 197 CSF samples had been analyzed previously at the same time (on 5 different immunoplates) by ELISA [INNOTEST® β-Amyloid(1–42); Innogenetics, single analyte, CE version] and Luminexs xMAP assay system with the INNO-BIA AlzBio3 assay (Innogenetics; multiplexed, research use only assay) (3). Differences in the design of the assays have previously been described (4). The plate layouts for CSF testing for the 2 assay formats were identical. No new test runs were performed for the current study. Kit calibrators consisted of synthetic β-amyloid(1–42) dissolved in a phosphate-buffered solution. After run validation (criteria: out of range, precision, bead count), 31 CSF …

Validation and Clinical Utility of ELISA Methods for Quantification of Amyloid-β Peptides in Cerebrospinal Fluid Specimens from Alzheimer's Disease Studies

The aim of this study was to validate assays for measurement of amyloid-β (Aβ) peptides in cerebrospinal fluid (CSF)specimens according to regulatory guidance and demonstrate their utility with measurements in specimens from Alzheimer’s disease (AD) studies. Methods based on INNOTEST(®)β-AMYLOID(1-42) and prototype INNOTEST(®)β-AMYLOID(1-40) ELISAkits were developed involving pre-analytical sample treatment with Tween-20 for reliable analyte recovery.Validation parameters were evaluated by repeated testing of CSF pools collected and stored in the same manner as clinical specimens. Intra- and interassay coefficients of variation were ≤11% and relative accuracy was within ± 10% for both analytes. Dilutional linearity was demonstrated for both analytes from a spiked CSF pool, but not from a non-spiked native CSF pool. Recovery of standard Aβ peptide spikes standard ranged from 77% to 93%. No interference was observed from the investigational drugs LY2811376, LY2886721, LY3002813, or semagacestat. Aβ(1-40) and Aβ(1-42) were stable in CSF for up to 8 hours at room temperature and during 5 f reeze-thaw cycles from ≤−20◦C and ≤−70◦C. In frozen native CSF specimens, Aβ(1-40) was mostly stable up to 3 years at ≤−70◦C, whereas stability of Aβ(1-42) was limited to 221 days. Dose-dependent changes in measured CSF Aβ were observed in healthy volunteers up to 36 hours after treatment with the-site cleavage enzyme inhibitor LY2886721. In conclusion, rigorous validation tests have successfully demonstrated the strengths and operational limitations of these INNOTEST(®)-based assays.They have proved to be robust and reliable tools for pharmacodynamic evaluations of investigational AD therapeutics in clinical trials.

IMPROVEMENTS TO INNOTEST® PHOSPHO-TAU 181P

replicates and technical replicates were analyzed for 3 AD and 3 non-AD over ten consecutive days. For the vast majority of the peptides CVs were <5%. The second, 6 AD and 6 non-AD, indicated slightly increased levels of cystatin C, beta-2-microglobulin, and APP, as well as clearly increased levels of chromogranin A, secretogranin-2, and neurosecretory protein VGF for AD. Moreover, for complex samples the advantage of high resolution instrumentation became evident because of the superior possiblity to avoid signal interferences. Conclusions: The assay is reproducible and consumes little amount per analysis. Preliminary results should be interpreted with caution due to the limited sample material. Data from a larger study, using clinically well characterized samples, is under way and will be presented.

IMPROVEMENTS TO INNOTEST® HTAU AG

properties of these proteins and the interference of matrix components make the establishment of referencematerials very challenging. As a consequence up to now, the trust in the in commercial assays is limited. Since CSF quality control samples were not broadly available. Methods: A large number of individual CSF samples were pooled to establish 5 samples with different A b 1-42 and Ab 1-40 concentrations. Different compounds were added to the pools before lyophilization. The pools underwent one freeze-thaw cycle before aliquotation (250 ml, polypropylene screw-cap tubes). All aliquots were lyophilized and stored at -20 C. The consistency and homogeneity of the prepared panel was evaluated using three different lot numbers of the assays and analysis in 3 different laboratories by different users. In addition, the performance of the proficiency panel was integrated into a multicenter study and compared to performances of neat CSF or run-validation samples (1⁄4 analyte in buffer). Results: Addition of components do not affect analytical behavior of the CSF sample. Lyophilisation of the samples was successful. Stability was considerably increased by that procedure as analytes showed only 5% CV after one week at 37 C. Neat CSF, with and without the additives, as well as samples from the proficiency panel, showed a very good parallelism (1⁄4 absence of matrix interference in the assays) upon dilution for the three analytes evaluated in the study. The production process did not change the intrinsic characteristics of the CSF samples. Overall variability in a multicenter study for the proficiency panel did not exceed > 10%, including the sum of % CVof intra-run, inter-run, inter-operator, and inter-lab. Conclusions: A panel of 5 stabilized and lyophilized CSF samples with target values covering the whole range of the calibration curve of the A b 1-42 , Ab 1-40 and Total-Tau ELISA. The excellent precision data obtained in a multicenter study make this panel an excellent new tool to verify the quality of the biomarker measurements in research settings, but also for routine labs. In the absence of an international reference standard, this commercially available proficiency panel may significantly increase the trust in the values measured by ELISA of CSF diagnostics.