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

Sheth, U.; Harrison, R.; Ferber, K.; Rosenbaugh, E. G.; Bevis, A.; Khillan, R.; Benatar, M.; Bjorklund, N. L.; Di Daniel, E.; Harris, G. A.; Kahn, O. I.; Liu, Y.; Zetterberg, H.; Mitic, L. L.; Graham, D.; Gendron, T. F.

doi:10.1101/2025.05.05.652212

bioRxiv · 10.1101/2025.05.05.652212

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

Sheth, U.·Harrison, R.·Ferber, K.·Rosenbaugh, E. G.·Bevis, A.·Khillan, R.·Benatar, M.·Bjorklund, N. L.·Di Daniel, E.·Harris, G. A.·Kahn, O. I.·Liu, Y.·Zetterberg, H.·Mitic, L. L.·Graham, D.·Gendron, T. F.

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Abstract

ObjectivesNeurofilament 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. MethodsTo 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. ResultsFour 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. ConclusionsFour NfL assays demonstrated similar analytic performance. Alongside performance, other factors such as costs, accessibility, useability, footprint, and intended use, should be considered.

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Sheth, U., Harrison, R., Ferber, K., Rosenbaugh, E. G., Bevis, A., Khillan, R., Benatar, M., Bjorklund, N. L., Di Daniel, E., Harris, G. A., Kahn, O. I., Liu, Y., Zetterberg, H., Mitic, L. L., Graham, D., Gendron, T. F.. 2025-05-09. Measuring neurofilament light in human plasma and cerebrospinal fluid: a comparison of five analytical immunoassays. https://doi.org/10.1101/2025.05.05.652212

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