Cerebrospinal Fluid Biomarkers in Alzheimer's Disease

The ADNI is a large, multicenter, longitudinal neuroimaging study, launched in 2004 by the National Institute on Aging, the National Institute of Biomedical Imaging and Bioengineering, the Food and Drug Administration, private pharmaceutical companies, and nonprofit organizations. ADNI includes 819 adult subjects, 55 to 90 years old, who meet entry criteria for a clinical diagnosis of amnestic MCI (n = 397), probable AD (n = 193), or normal cognition (n = 229). Participants receive baseline and periodic physical and neurological examinations and standardized neuropsychological assessments, and provide biological samples (blood, urine, and in a subset, CSF) throughout the study. Imaging (magnetic resonance imaging and for a subset, F-fluorodeoxyglucose positron emission tomography and Pittsburgh compound B positron emission tomography) is performed at baseline and at regular intervals thereafter (for reviews and more details, see Shaw and colleagues,7 Mueller and coauthors,11 (link) and http://www.adni-info.org/). All AD subjects met National Institute of Neurological and Communication Disorders/Alzheimer’s Disease and Related Disorders Association criteria for probable AD with a Mini-Mental State Examination score between 20 and 26, a global Clinical Dementia Rating of 0.5 or 1, a sum-of-boxes Clinical Dementia Rating of 1.0 to 9.0, and, therefore, are only mildly impaired. Entry criteria for amnestic MCI subjects include a Mini-Mental State Examination score of 24 to 30 and a Memory Box score of at least 0.5, whereas other details on the ADNI cohort can be found online at: http://www.nia.nih.gov/Alzheimers/ResearchInformation/ClinicalTrials/ADNI.htm. In brief, exclusion criteria included any serious neurological disease other than possible AD, any history of brain lesions or head trauma, or psychoactive medication use (including antidepressants, neuroleptics, chronic anxiolytics, or sedative hypnotics).
Baseline CSF samples were obtained in the morning after an overnight fast from 416 ADNI subjects (AD = 102, MCI = 200, NC = 114 with average [± standard deviation] ages of 75 ± 8, 75 ± 7, and 76 ± 5 years, respectively; Table 1) from individuals enrolled at 56 participating centers at the time the subjects entered ADNI (ie, baseline). Their demographic, clinical, and APOε genotyping results are comparable with that in the full ADNI patient population (http://www.adni-info.org/index). Lumbar puncture was performed with a 20- or 24-gauge spinal needle as described in the ADNI procedures manual (http://www.adni-info.org/). In brief, CSF was collected into collection tubes provided to each site, then transferred into polypropylene transfer tubes followed by freezing on dry ice within 1 hour after collection, and shipped overnight to the ADNI Biomarker Core laboratory at the University of Pennsylvania Medical Center on dry ice. Aliquots (0.5ml) were prepared from these samples after thawing (1 hour) at room temperature and gentle mixing. The aliquots were stored in bar code–labeled polypropylene vials at −80°C. Written informed consent was obtained for participation in these studies, as approved by the institutional review board at each participating center.
An independent set of premortem CSF samples from 56 autopsy-confirmed AD cases and 52 cognitively normal elderly subjects followed by the University of Pennsylvania Alzheimer’s Disease Clinical Core provided an independent analysis sample set that was matched with the ADNI samples with respect to age (mean ± standard deviation [95% confidence interval]: 71 ± 10 [69–74] and 70 ± 11 [67–73] years, respectively) at the time of their lumbar puncture. The cases and control subjects were evaluated and followed as described previously,12 (link)–14 and all of these CSF samples were collected at University of Pennsylvania Alzheimer’s Disease Clinical Core using standardized methodology including storage of aliquots in polypropylene vials maintained in the repository at −80°C.12 (link)–14 Written informed consent was obtained for participation in these studies, which was approved by the University of Pennsylvania Institutional Review Board.
Aβ_1-42, t-tau, and p-tau_181p were measured in each of the 416 CSF ADNI baseline aliquots using the multiplex xMAP Luminex platform (Luminex Corp, Austin, TX) with Innogenetics (INNO-BIA AlzBio3; Ghent, Belgium; for research use–only reagents) immunoassay kit–based reagents. Full details of this combination of immunoassay reagents and analytical platform are provided elsewhere.15 (link),16 In brief, Innogenetics kit reagents included well-characterized capture monoclonal antibodies specific for Aβ_1-42(4D7A3), t-tau(AT120), and p-tau_181p (AT270), each chemically bonded to unique sets of color-coded beads, and analyte-specific detector antibodies (HT7, 3D6). Calibration curves were produced for each biomarker using aqueous buffered solutions that contained the combination of three biomarkers at concentrations ranging from 56 to 1,948pg/ml for recombinant tau, 27 to 1,574pg/ml for synthetic Aβ_1-42 peptide, and 8 to 230pg/ml for a tau synthetic peptide phosphorylated at the threonine 181 position (ie, the p-tau_181p standard). Before performing these analyses of the ADNI and the independent autopsy-based CSF samples in the ADNI University of Pennsylvania ADNI Biomarker Core laboratory, an interlaboratory study was conducted to qualify the performance conditions, including all major variables that can affect the test results, for the immunoassay reagents and analytical platform. These studies were conducted using strategies and procedures to standardize the assay similar to those that Vanderstichele and colleagues16 described. This investigation (Shaw and colleagues, manuscript in preparation, but see summary of these data online at: http://www.adni-info.org/) provided the basis for achieving day-to-day reproducibility for the three biomarkers of less than 10% variation for CSF pool samples and less than 7% for aqueous quality controls. The ADNI baseline CSF samples were analyzed over a 14-day period and included test–retest analyses of 29 of the samples that further substantiated the analytical performance (r² values for comparison of initial test result with retest result of 0.98, 0.90, and 0.85 for t-tau, Aβ_1-42, and p-tau_181p, respectively for 29 randomly selected samples). Only subjects with a valid test result for all 3 biomarkers are included in this study, that is, 114 NC, 196 MCI, and 100 AD subjects.
APOε genotyping was done for all ADNI study candidates using EDTA blood samples collected at the screening visit (see Table 1). TaqMan quantitative polymerase chain reaction assays were used for genotyping APOε nucleotides 334 T/C and 472 CT with an ABI 7900 real-time thermo-cycler (Applied Biosystems, Foster City, CA) using DNA freshly prepared from EDTA whole blood. A total of 96 samples randomly selected from the total of 1,131 subjects screened before inclusion (or exclusion) into the ADNI study were retested by sequencing using an ABI 3130 sequencer (Applied Biosystems). Except for the 5 samples that failed to sequence, the remaining 91 were concordant with the Taq-Man genotyping results.
Receiver operating characteristic curve (ROC) and logistic regression (LR) analyses were done using SAS v 9.1.3 (SAS Institute, Cary, NC) and R v 2.7.1. Between-group differences for each biomarker were assessed by the Mann–Whitney U test using GraphPad Prism, v 5.