Measuring neurofilament light in human plasma and cerebrospinal fluid: a comparison of five analytical immunoassays
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Udit Sheth
,
Rebecca Harrison
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, usability, footprint, and intended use, should be considered.
Funding source: European Partnership on Metrology, co-financed from the European Union’™s Horizon Europe Research and Innovation Programme and by the Participating States
Award Identifier / Grant number: NEuroBioStand, #22HLT07
Funding source: ALS Association
Funding source: Association for Frontotemporal Degeneration
Funding source: Swedish State Support for Clinical Research
Award Identifier / Grant number: #ALFGBG-71320
Funding source: The Bluefield Project to Cure Frontotemporal Dementia
Funding source: National Institute of Neurological Disorders and Stroke
Award Identifier / Grant number: R01NS10547
Award Identifier / Grant number: U01NS107027
Award Identifier / Grant number: U54NS092091
Funding source: Alector, Inc.
Funding source: Diagnostics Accelerator at the Alzheimer’s Drug Discovery Foundation
Funding source: Foundation at the National Institute of Health Inc.
Funding source: Centrum for idrottsforskning
Award Identifier / Grant number: #2019-02397
Award Identifier / Grant number: #2022-01018
Award Identifier / Grant number: #2023-00356
Funding source: Biogen MA, Inc.
Funding source: Otsuka Pharmaceutical Development & Commercialization, Inc.
Funding source: Rainwater Charitable Foundation
Funding source: European Union’™s Horizon Europe Research and Innovation Programme
Award Identifier / Grant number: No 101053962
Funding source: Robert Packard Center for ALS Research, Johns Hopkins University
Acknowledgments
We thank Frank Shewmaker, Christine Swanson-Fischer and all other FNIH Biomarker Consortium members for their participation in this study.
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Research ethics: Not applicable.
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Informed consent: To compare assay performance, we purchased matching de-identified plasma and CSF samples from 20 participants with El Escorial defined probable or definite ALS and from 20 healthy controls from PrecisionMed’s inventory of samples collected under IRB-approved clinical protocols and eight pooled plasma and eight pooled CSF samples from University of Gothenburg collected under IRB-approved protocols.
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission. All authors contributed to the study design, analysis plan, data review, manuscript review, and approval of the final manuscript for submission. KF, RH and US also contributed to data analysis. TFG and US wrote the manuscript with input from all authors.
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Use of Large Language Models, AI and Machine Learning Tools: None declared.
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Conflict of interest: US, EGR, AB, RK, NB and TFG have no competing or conflicting interests. RH and EdD are employees of Astex Pharmaceuticals, which is an operationally independent wholly owned subsidiary of Otsuka Pharmaceutical Co., Ltd., which contributed to the funding of the work. KF and DG are employees of Biogen, for which they receive salary and company stock as compensation. MB reports consulting fees from Alaunos, Alector, Arrowhead, Biogen, Denali, Eli Lilly, Novartis, Roche, uniQure, and Woolsey. The University of Miami has licensed intellectual property to Biogen to support design of the ATLAS study. GAH is an employee of the Rainwater Charitable Foundation, which contributed funding to this work. OIK is an employee of Alector LLC and may have an equity interest in Alector, Inc. Alector LLC is a member of the consortium and has contributed funding for the work. YL is an employee of Otsuka Pharmaceutical Development & Commercialization (OPDC), and OPDC is part of the consortium and contributed funding to the work. HZ has served at scientific advisory boards and/or as a consultant for Abbvie, Acumen, Alector, Alzinova, ALZpath, Amylyx, Annexon, Apellis, Artery Therapeutics, AZTherapies, Cognito Therapeutics, CogRx, Denali, Eisai, Enigma, LabCorp, Merry Life, Nervgen, Novo Nordisk, Optoceutics, Passage Bio, Pinteon Therapeutics, Prothena, Quanterix, Red Abbey Labs, reMYND, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave, has given lectures sponsored by Alzecure, BioArctic, Biogen, Cellectricon, Fujirebio, Lilly, Novo Nordisk, Roche, and WebMD, and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (outside submitted work). LLM is an employee of the Bluefield Project to Cure Frontotemporal Dementia, which contributed funding to this work.
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Research funding: The following academic or private-sector partners provided financial or in-kind support for this study: Foundation at the National Institute of Health Inc., Robert Packard Center for ALS Research at Johns Hopkins, Alector, Inc., Biogen MA, Inc., Diagnostics Accelerator at the Alzheimer’s Drug Discovery Foundation, Otsuka Pharmaceutical Development & Commercialization, Inc., Rainwater Charitable Foundation, The ALS Association, The Association for Frontotemporal Degeneration, and The Bluefield Project to Cure Frontotemporal Dementia. MB is supported by U54NS092091, U01NS107027, and R01NS10547. HZ is a Wallenberg Scholar and a Distinguished Professor at the Swedish Research Council supported by grants from the Swedish Research Council (#2023-00356, #2022-01018 and #2019-02397), the European Union’s Horizon Europe Research and Innovation Programme under grant agreement No 101053962, Swedish State Support for Clinical Research (#ALFGBG-71320), and the European Partnership on Metrology, co-financed from the European Union’s Horizon Europe Research and Innovation Programme and by the Participating States (NEuroBioStand, #22HLT07).
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Data availability: De-identified NfL biomarker data are available upon request.
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Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2025-0610).
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