CSF biomarker variability in the Alzheimer's Association quality control program

Abstract

Background: The cerebrospinal fluid (CSF) biomarkers amyloid beta 1-42, total tau, and phosphorylated tau are used increasingly for Alzheimer's disease (AD) research and patient management. However, there are large variations in biomarker measurements among and within laboratories.

Methods: Data from the first nine rounds of the Alzheimer's Association quality control program was used to define the extent and sources of analytical variability. In each round, three CSF samples prepared at the Clinical Neurochemistry Laboratory (Mölndal, Sweden) were analyzed by single-analyte enzyme-linked immunosorbent assay (ELISA), a multiplexing xMAP assay, or an immunoassay with electrochemoluminescence detection.

Results: A total of 84 laboratories participated. Coefficients of variation (CVs) between laboratories were around 20% to 30%; within-run CVs, less than 5% to 10%; and longitudinal within-laboratory CVs, 5% to 19%. Interestingly, longitudinal within-laboratory CV differed between biomarkers at individual laboratories, suggesting that a component of it was assay dependent. Variability between kit lots and between laboratories both had a major influence on amyloid beta 1-42 measurements, but for total tau and phosphorylated tau, between-kit lot effects were much less than between-laboratory effects. Despite the measurement variability, the between-laboratory consistency in classification of samples (using prehoc-derived cutoffs for AD) was high (>90% in 15 of 18 samples for ELISA and in 12 of 18 samples for xMAP).

Conclusions: The overall variability remains too high to allow assignment of universal biomarker cutoff values for a specific intended use. Each laboratory must ensure longitudinal stability in its measurements and use internally qualified cutoff levels. Further standardization of laboratory procedures and improvement of kit performance will likely increase the usefulness of CSF AD biomarkers for researchers and clinicians.

Fig. 1

Measurements of blinded quality control test samples. Dots with error bars show mean measured concentrations and standard deviation from all participating sites (the samples were made from different pools of cerebrospinal fluid [CSF], so constant concentrations were not expected). Connected lines show the coefficient of variation (CV; right-hand y-axes). (A–C) INNOTEST Enzyme-linked immunosorbent assay (ELISA). (D–F) INNO-BIA xMAP. (G–I) Meso Scale Discovery (MSD) amyloid beta (Aβ) triplex. The CV for xMAP total tau (T-tau) sample 7B (E) was very high (64%) because of a single extreme outlier (the CV was 22% after removal of this outlier). P-tau, phosphorylated tau.

Fig. 2

(A, B) Within-laboratory longitudinal coefficients of variation (CVs) were calculated by repeated measurements at reference laboratories (Refx; using six measurements per sample, varying number of samples per laboratory) and in the whole program (using the quality control longitudinal sample at laboratories measuring the sample at least three times during rounds 1 through 7). Data are means of CV (error bars are standard deviations) for each biomarker, ordered by laboratory (x-axes). Enzyme-linked immunosorbent assay (ELISA) reference laboratory 4 (Ref 4) only used two lots of analytical kits for each analyte, which limits the influence of lot-dependent variability. Meso Scale Discovery (MSD) data are not included in the figure, since only one reference laboratory reported data for MSD within-laboratory longitudinal CV (mean CV at that laboratory was 11% to 17% (standard deviation, 4%–8%) for all amyloid beta (Aβ) triplex measurements using either 6E10 or 4G8 as the detection antibody. T-tau, total tau; P-tau, phosphorylated tau.

Fig. 3

Measurements of longitudinal quality control samples. Bars show mean measured concentrations (error bars are standard deviations) for the quality control longitudinal sample (constant concentrations expected). Connected lines show the coefficient of variation CV (right-hand y-axes) for all laboratories (blue) and reference laboratories (red). ELISA, enzyme-linked immunosorbent assay; Aβ, amyloid beta; T-tau, total tau; P-tau, phosphorylated tau.

Fig. 4

Between-kit lot and between-laboratory contributions to variability. Bars show the contribution from the different components to the overall variability according to variance component analysis for enzyme-linked immunosorbent assay (ELISA) (A) and xMAP (B). The bars are stacked. Note that the components do not sum to 100% because there is also an influence of within-laboratory and within-kit lot variability that contributes to the total. Aβ, amyloid beta; T-tau, total tau; P-tau, phosphorylated tau.

Fig. 5

Bias and imprecision plots. Bias (systematic variation) at a particular laboratory was estimated by comparing the measurement at that laboratory with the mean of all reported measurements for that particular sample. Imprecision (random variation) was estimated by calculating the variance of the results. Laboratories with high bias (defined as > 30% or ≤ 30% from the mean) or high imprecision (defined as > 20%) for at least two analytes are indicated by special symbols. Three of these laboratories (■, ◆, and ✖) only provided data for one to two rounds, which make their estimates uncertain. Laboratory provided data for three rounds, but on review it was found that they were all analyzed at the same time point, which may contribute to a strong bias. The remaining laboratories, indicated by special symbols, provided data for five to nine rounds. ELISA, enzyme-linked immunosorbent assay; Aβ, amyloid beta; T-tau, total tau; P-tau, phosphorylated tau.