Qualification of the analytical and clinical performance of CSF biomarker analyses in ADNI

Abstract

The close correlation between abnormally low pre-mortem cerebrospinal fluid (CSF) concentrations of amyloid-β1-42 (Aβ(1-42)) and plaque burden measured by amyloid imaging as well as between pathologically increased levels of CSF tau and the extent of neurodegeneration measured by MRI has led to growing interest in using these biomarkers to predict the presence of AD plaque and tangle pathology. A challenge for the widespread use of these CSF biomarkers is the high variability in the assays used to measure these analytes which has been ascribed to multiple pre-analytical and analytical test performance factors. To address this challenge, we conducted a seven-center inter-laboratory standardization study for CSF total tau (t-tau), phospho-tau (p-tau(181)) and Aβ(1-42) as part of the Alzheimer's Disease Neuroimaging Initiative (ADNI). Aliquots prepared from five CSF pools assembled from multiple elderly controls (n = 3) and AD patients (n = 2) were the primary test samples analyzed in each of three analytical runs by the participating laboratories using a common batch of research use only immunoassay reagents (INNO-BIA AlzBio3, xMAP technology, from Innogenetics) on the Luminex analytical platform. To account for the combined effects on overall precision of CSF samples (fixed effect), different laboratories and analytical runs (random effects), these data were analyzed by mixed-effects modeling with the following results: within center %CV 95% CI values (mean) of 4.0-6.0% (5.3%) for CSF Aβ(1-42); 6.4-6.8% (6.7%) for t-tau and 5.5-18.0% (10.8%) for p-tau(181) and inter-center %CV 95% CI range of 15.9-19.8% (17.9%) for Aβ(1-42), 9.6-15.2% (13.1%) for t-tau and 11.3-18.2% (14.6%) for p-tau(181). Long-term experience by the ADNI biomarker core laboratory replicated this degree of within-center precision. Diagnostic threshold CSF concentrations for Aβ(1-42) and for the ratio t-tau/Aβ(1-42) were determined in an ADNI independent, autopsy-confirmed AD cohort from whom ante-mortem CSF was obtained, and a clinically defined group of cognitively normal controls (NCs) provides statistically significant separation of those who progressed from MCI to AD in the ADNI study. These data suggest that interrogation of ante-mortem CSF in cognitively impaired individuals to determine levels of t-tau, p-tau(181) and Aβ(1-42), together with MRI and amyloid imaging biomarkers, could replace autopsy confirmation of AD plaque and tangle pathology as the "gold standard" for the diagnosis of definite AD in the near future.

Fig. 1

Box plots of the within-center %CV for each of seven participating centers for a Aβ_1–42, b t-tau and c p-tau₁₈₁ measured in two aqueous kit quality control samples, three aqueous run validation samples and five CSF pools in three separate analytical runs using INNO-BIA AlzBio3 reagents and the Luminex platform

Fig. 2

Plots of average % of grand mean values for each center and for each of five CSF pools for a Aβ_1–42, b t-tau and c p-tau₁₈₁. The concentrations of Aβ_1–42, t-tau and p-tau₁₈₁ were determined as described using pristine aliquots of each of five CSF pools in three analytical runs, and the grand mean values for each biomarker were determined. For each of the seven participating centers the % of the grand mean value was determined for each of the five CSF pools by dividing the average value over the three analytical runs by the grand average value and plotting the % of the grand mean value for each pool and each center

Fig. 3

Bar chart plots for the total, between-center and within-center %CV values derived for each CSF pool from the Mixed Effects statistical model for the CSF pools data set for a Aβ_1–42, b t-tau and c p-tau₁₈₁

Fig. 4

Longitudinal plots of Aβ_1–42, t-tau and p-tau₁₈₁ concentrations measured in never previously thawed aliquots of a CSF pool #52 (n = 51) and b CSF pool #45 (n = 36)

Fig. 5

Linear regression plots of concentrations, measured in never previously thawed CSF aliquots from 118 ADNI subjects, utilizing 2–3 subjects randomly selected from each of 38 analytical runs. For each randomly selected subject, a second never previously thawed aliquot was included in the run following analysis of the first never previously thawed aliquot. In plots a–c, the Aβ_1–42, t-tau and p-tau₁₈₁ concentration values obtained in the second aliquot (retest) are plotted against the biomarker concentration value obtained in the original analytical run (test) and linear regression analyses performed. In plots d–f, the % difference between the test and retest values are plotted versus the average value for each test/retest pair of concentrations. The shaded area around each linear regression line is the 95% confidence interval for the regression line. In plots d–f, the dotted lines are the 95% confidence intervals for the mean difference lines (solid lines)

Fig. 6

Kaplan–Meier time to conversion to AD survival curves for ADNI subjects who had a diagnosis of mild cognitive impairment at their baseline visit. The small vertical lines are censored data, and the number of subjects at risk is noted at the bottom of the plot. In a, the survival curves are shown for MCI subjects with CSF Aβ_1–42 concentrations above or below the threshold value of 192 pg/mL at their baseline. In b, the survival curves are shown for MCI subjects with CSF t-tau/Aβ_1–42 ratio values above or below the threshold value of 0.39