Measuring neurofilament light in human plasma and cerebrospinal fluid: a comparison of five analytical immunoassays

Abstract

Objectives: Neurofilament light (NfL) is an established biofluid marker of neuroaxonal injury for neurological diseases. Several high-throughput and sensitive immunoassays have been developed to quantify NfL in blood and cerebrospinal fluid (CSF), facilitating the use of NfL as a biomarker in research and clinical practice. However, because of the lack of rigorous comparisons of assays, it has been difficult to determine whether data are comparable and whether assay performance differs. Here, we compared the performance of five NfL immunoassays.

Methods: To assess the five NfL immunoassays (Fujirebio, ProteinSimple, Quanterix, Roche and Siemens), we used pooled plasma or pooled CSF, as well as unique samples from 20 healthy controls and 20 individuals with El Escorial defined probable or definite amyotrophic lateral sclerosis (ALS), to evaluate precision, parallelism and/or bias. We also examined correlations between plasma and CSF NfL concentrations within and across assays and evaluated their ability to differentiate healthy controls from individuals with ALS.

Results: Four of the five assays demonstrated exemplary performance based on our analyses of precision and parallelism. Across the five assays, NfL concentrations were lower in plasma than in CSF, although they displayed a high degree of correlation. We noted bias across assays; plasma NfL concentrations were lowest for the Roche assay and highest for the ProteinSimple assay. In addition, all assays reliably distinguished healthy controls from individuals with ALS using plasma or CSF NfL.

Conclusions: Four NfL assays demonstrated similar analytic performance. Alongside performance, other factors such as costs, accessibility, useability, footprint, and intended use, should be considered.

Keywords: amyotrophic lateral sclerosis; biomarker; cerebrospinal fluid; immunoassays; neurofilament light chain; plasma.

Figure 1:. Parallelism measures for plasma and CSF NfL concentrations.

NfL measurements across a series of manufacturer-recommended dilutions using pooled plasma (A) or CSF (B) samples. Data are shown on the Log10 scale for ease of visualization. For data shown on the linear scale, please refer to Figure S1.

Figure 2:. Correlations of plasma or CSF NfL concentrations between assays.

Correlations of plasma NfL (A) or CSF NfL (B) concentrations across all five assays. Assay names at the top of each graph serve as the reference assay, depicted by a black line, to which the other assays are compared. Fujirebio=gold, ProteinSimple=pink, Quanterix=green, Roche=purple, Siemens=lavender, reference assay=black.

Figure 3:. Plasma and CSF NfL concentrations in controls and patients with ALS.

For each assay, plasma NfL (A) and CSF NfL (B) concentrations in 20 controls and 20 patients with ALS were compared. Maximum and minimum NfL concentrations are represented by the top and bottom whiskers, respectively. The 75% and 25% quartiles are represented by the upper and lower bounds of the box, respectively, and the median is represented by the mid-line of the box. Data are shown using a Log10 scale for ease of visualization. P-values from two-sided, unpaired t-tests are shown. For data visualized on the linear scale, please refer to Figure S4.