Abstract
Objectives
Neurofilament light chain (NfL) has emerged as a promising biomarker for detecting and monitoring axonal injury. Until recently, NfL could only be reliably measured in cerebrospinal fluid, but digital single molecule array (Simoa) technology has enabled its precise measurement in blood samples where it is typically 50–100 times less abundant. We report development and multi-center validation of a novel fully automated digital immunoassay for NfL in serum for informing axonal injury status.
Methods
A 45-min immunoassay for serum NfL was developed for use on an automated digital analyzer based on Simoa technology. The analytical performance (sensitivity, precision, reproducibility, linearity, sample type) was characterized and then cross validated across 17 laboratories in 10 countries. Analytical performance for clinical NfL measurement was examined in individual patients with relapsing remitting multiple sclerosis (RRMS) after 3 months of disease modifying treatment (DMT) with fingolimod.
Results
The assay exhibited a lower limit of detection (LLoD) of 0.05 ng/L, a lower limit of quantification (LLoQ) of 0.8 ng/L, and between-laboratory imprecision <10 % across 17 validation sites. All tested samples had measurable NfL concentrations well above the LLoQ. In matched pre–post treatment samples, decreases in NfL were observed in 26/29 RRMS patients three months after DMT start, with significant decreases detected in a majority of patients.
Conclusions
The sensitivity characteristics and reproducible performance across laboratories combined with full automation make this assay suitable for clinical use for NfL assessment, monitoring in individual patients, and cross-comparisons of results across multiple sites.
-
Research ethics: The local Institutional Review Board deemed the study exempt from review.
-
Informed consent: Informed consent was obtained from all individuals included in this study.
-
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
-
Competing interests: David Wilson is an employee at Quanterix. Dandan Chan was an employee at Quanterix when the work was completed. Lei Chang was an employee at Quanterix when the work was completed. Robert Mathis was an employee at Quanterix when the work was completed. Inge Verberk states no conflict of interest. Xavier Montalban states no conflict of interest. Manuel Comabella states no conflict of interest. Nicolas Fissolo states no conflict of interest. Bibi Bielekova states no conflict of interest. Ruturaj Masvekar states no conflict of interest. Tanuja Chitnis states no conflict of interest. Tjalf Ziemssen states no conflict of interest. Katja Akgün states no conflict of interest. Kaj Blennow states no conflict of interest. Henrik Zetterberg states no conflict of interest. Wolfgang Brück states no conflict of interest. Gavin Giovannoni states no conflict of interest. Sharmilee Gnanapavan states no conflict of interest. Stefan Bittner states no conflict of interest. Frauke Zipp states no conflict of interest. Giancarlo Comi states no conflict of interest. Roberto Furlan states no conflict of interest. Sylvain Lehmann states no conflict of interest. Simon Thebault states no conflict of interest. Mark Freedman has consulted for Quanterix and received consultant fees. Amit Bar-Or states no conflict of interest. Marty Kramer states no conflict of interest. Markus Otto states no conflict of interest. Steffen Halbgebauer states no conflict of interest. Kevin Hrusovsky was an employee at Quanterix when the work was completed. Tatiana Plavina states no conflict of interest. Michael Khalil states no conflict of interest. Fredrik Piehl states no conflict of interest. Heinz Wiendl states no conflict of interest. Ludwig Kappos states no conflict of interest. Aleksandra Maceski states no conflict of interest. Eline Willemse states no conflict of interest. David Leppert states no conflict of interest. Charlotte Teunissen has a collaboration contract with Quanterix. Jens Kuhle has consulted for Quanterix and received consultant fees.
-
Research funding: None declared.
-
Data availability: The data that support the findings of this study are available from the corresponding author, JK, upon reasonable request.
References
1. Yuan, A, Rao, MV, Veeranna, Nixon, RA. Neurofilaments at a glance. J Cell Sci 2012;125:3257–63. https://doi.org/10.1242/jcs.104729.Search in Google Scholar PubMed PubMed Central
2. Bridel, C, van Wieringen, WN, Zetterberg, H, Tijms, BM, Teunissen, CE, Alvarez-Cermeño, JC, et al.. Diagnostic value of cerebrospinal fluid neurofilament light protein in neurology: a systematic review and meta-analysis. JAMA Neurol 2019;76:1035–48. https://doi.org/10.1001/jamaneurol.2019.1534.Search in Google Scholar PubMed PubMed Central
3. Hansson, O, Janelidze, S, Hall, S, Magdalinou, N, Lees, AJ, Andreasson, U, et al.. Blood-based NfL: a biomarker for differential diagnosis of parkinsonian disorder. Neurology 2017;88:930–7. https://doi.org/10.1212/wnl.0000000000003680.Search in Google Scholar
4. Gaiottino, J, Norgren, N, Dobson, R, Topping, J, Nissim, A, Malaspina, A, et al.. Increased neurofilament light chain blood levels in neurodegenerative neurological diseases. PLoS One 2013;8:e75091. https://doi.org/10.1371/journal.pone.0075091.Search in Google Scholar PubMed PubMed Central
5. Bergman, J, Dring, A, Zetterberg, H, Blennow, K, Norgren, N, Gilthorpe, J, et al.. Neurofilament light in CSF and serum is a sensitive marker for axonal white matter injury in MS. Neurol Neuroimmunol NeuroInflammation 2016;3:e271. https://doi.org/10.1212/nxi.0000000000000271.Search in Google Scholar
6. Rosengren, LE, Karlsson, JE, Karlsson, JO, Persson, LI, Wikkelsø, C. Patients with amyotrophic lateral sclerosis and other neurodegenerative diseases have increased levels of neurofilament protein in CSF. J Neurochem 1996;67:2013–8. https://doi.org/10.1046/j.1471-4159.1996.67052013.x.Search in Google Scholar PubMed
7. Rissin, DM, Kan, CW, Campbell, TG, Howes, SC, Fournier, DR, Song, L, et al.. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat Biotechnol 2010;28:595–9. https://doi.org/10.1038/nbt.1641.Search in Google Scholar PubMed PubMed Central
8. Barro, C, Chitnis, T, Weiner, HL. Blood neurofilament light: a critical review of its application to neurologic disease. Ann Clin Transl Neurol 2020;7:2508–23. https://doi.org/10.1002/acn3.51234.Search in Google Scholar PubMed PubMed Central
9. Novakova, L, Zetterberg, H, Sundström, P, Axelsson, M, Khademi, M, Gunnarsson, M, et al.. Monitoring disease activity in multiple sclerosis using serum neurofilament light protein. Neurology 2017;89:2230–7. https://doi.org/10.1212/wnl.0000000000004683.Search in Google Scholar PubMed PubMed Central
10. Disanto, G, Barro, C, Benkert, P, Naegelin, Y, Schädelin, S, Giardiello, A, et al.. Serum Neurofilament light: a biomarker of neuronal damage in multiple sclerosis. Ann Neurol 2017;81:857–70. https://doi.org/10.1002/ana.24954.Search in Google Scholar PubMed PubMed Central
11. Calabresi, P. Serum neurofilament light (NfL) for disease prognosis and treatment monitoring in multiple sclerosis patients: is it ready for implementation into clinical care? ECTRIMS Online Library. Calabresi P, 231907; 2018:158 p. https://learn.ectrims.eu/ectrims/2018/ectrims-2018/231907/peter.calabresi.serum.neurofilament.light.28nfl29.for.disease.prognosis.and.html?f=listing%3D0%2Abrowseby%3D8%2Asortby%3D1%2Asearch%3Dcalabresi [Accessed March 2023].Search in Google Scholar
12. Kapoor, R, Sellebjerg, F, Hartung, H-P, Arnold, D, Freedman, M, Jeffery, D, et al.. Natalizumab reduces serum concentrations of neurofilament light chain in secondary progressive multiple sclerosis patients from the phase 3 ASCEND study. Neurology 2019;92(15 Suppl).10.1212/WNL.92.15_supplement.S12.008Search in Google Scholar
13. Calabresi, P, Kuhle, J, Arnold, D, Kinkel, R, Kappos, L, Singh, C, et al.. Serum neurofilament light (NfL) for disease prognosis and treatment monitoring in multiple sclerosis patients: toward implementation in clinical care. Neurology 2019;92(15 Suppl). https://doi.org/10.1212/WNL.0000000000007295.Search in Google Scholar PubMed
14. Kuhle, J, Kropshofer, H, Haering, DA, Kundu, U, Meinert, R, Barro, C, et al.. Blood neurofilament light chain as a biomarker of MS disease activity and treatment response. Neurology 2019;92:e1007–5. https://doi.org/10.1212/wnl.0000000000007032.Search in Google Scholar
15. Thebault, S, Abdoli, M, Fereshtehnejad, SM, Tessier, D, Tabard-Cossa, V, Freedman, MS. Serum neurofilament light chain predicts long term clinical outcomes in multiple sclerosis. Sci Rep 2020;10:10381. https://doi.org/10.1038/s41598-020-67504-6.Search in Google Scholar PubMed PubMed Central
16. Ning, L, Wang, B. Neurofilament light chain in blood as a diagnostic and predictive biomarker for multiple sclerosis: a systematic review and meta-analysis. PLoS One 2022;17:e0274565. https://doi.org/10.1371/journal.pone.0274565.Search in Google Scholar PubMed PubMed Central
17. Wilson, DH, Rissin, DM, Kan, CW, Fournier, DR, Piech, T, Campbell, TG, et al.. The Simoa HD-1 analyzer: a novel fully automated digital immunoassay analyzer with single-molecule sensitivity and multiplexing. J Lab Autom 2016;21:533–47. https://doi.org/10.1177/2211068215589580.Search in Google Scholar PubMed
18. Kan, CW, Rivnak, AJ, Campbell, TG, Piech, T, Rissin, DM, Mösl, M, et al.. Isolation and detection of single molecules on paramagnetic beads using sequential fluid flows in microfabricated polymer array assemblies. Lab Chip 2012;12:977–85. https://doi.org/10.1039/c2lc20744c.Search in Google Scholar PubMed
19. CLSI. Evaluation of the linearity of quantitative measurement procedure: a statistical approach; approved guideline. In: CLSI Document EP6-A, 2nd ed. Wayne (PA): CLSI; 2003.Search in Google Scholar
20. Lee, JW, Devanarayan, V, Barrett, YC, Weiner, R, Allinson, J, Fountain, S, et al.. Fit-for-purpose method development and validation for successful biomarker measurement. Pharm Res 2006;23:312–28. https://doi.org/10.1007/s11095-005-9045-3.Search in Google Scholar PubMed
21. Wayne, PA. CLSI protocols for determination of limits of detection and limits of quantitation. In: Approved Guideline CLSI Document EP17-A. Wayne, PA: CLSI; 2004.Search in Google Scholar
22. Wayne, PA, CLSI. Evaluation of precision performance of quantitative measurement methods; approved guideline. In: CLSI Document EP5–A2, 2nd ed. Wayne, PA: CLSI; 2004.Search in Google Scholar
23. Benkert, P, Meier, S, Schaedelin, S, Manouchehrinia, A, Yaldizli, Ö, Maceski, A, et al.. Serum neurofilament light chain for individual prognostication of disease activity in people with multiple sclerosis: a retrospective modelling and validation study. Lancet Neurol 2022;21:246–57. https://doi.org/10.1016/s1474-4422(22)00009-6.Search in Google Scholar PubMed
24. Hviid, CVB, Madsen, AT, Winther-Larsen, A. Biological variation of serum neurofilament light chain. Clin Chem Lab Med 2021;60:569–75. https://doi.org/10.1515/cclm-2020-1276.Search in Google Scholar PubMed
25. Gauthier, A, Viel, S, Perret, M, Brocard, G, Casey, R, Lombard, C, et al.. Comparison of SimoaTM and EllaTM to assess serum neurofilament-light chain in multiple sclerosis. Ann Clin Transl Neurol 2021;8:1141–50. https://doi.org/10.1002/acn3.51355.Search in Google Scholar PubMed PubMed Central
26. Verberk, IMW, Misdorp, EO, Koelewijn, J, Ball, AJ, Blennow, K, Dage, JL, et al.. Characterization of pre-analytical sample handling effects on a panel of Alzheimer’s disease-related blood-based biomarkers: results from the Standardization of Alzheimer’s Blood Biomarkers (SABB) working group. Alzheimers Dement 2022;18:1484–97. https://doi.org/10.1002/alz.12510.Search in Google Scholar PubMed PubMed Central
27. van Lierop, ZYGJ, Verberk, IMW, van Uffelen, KWJ, Koel-Simmelink, MJ, in ’t Veld, L, Killestein, J, et al.. Pre-analytical stability of serum biomarkers for neurological disease: neurofilament-light, glial fibrillary acidic protein and contactin-1. Clin Chem Lab Med 2022;60:842–50. https://doi.org/10.1515/cclm-2022-0007.Search in Google Scholar PubMed
28. Altmann, P, Leutmezer, F, Zach, H, Wurm, R, Stattmann, M, Ponleitner, M, et al.. Serum neurofilament light chain withstands delayed freezing and repeated thawing. Sci Rep 2020;10:19982. https://doi.org/10.1038/s41598-020-77098-8.Search in Google Scholar PubMed PubMed Central
29. Altmann, P, Ponleitner, M, Rommer, PS, Haslacher, H, Mucher, P, Leutmezer, F, et al.. Seven day pre-analytical stability of serum and plasma neurofilament light chain. Sci Rep 2021;11:11034. https://doi.org/10.1038/s41598-021-90639-z.Search in Google Scholar PubMed PubMed Central
30. Akgün, K, Kretschmann, N, Haase, R, Proschmann, U, Kitzler, HH, Reichmann, H, et al.. Profiling individual clinical responses by high-frequency serum neurofilament assessment in MS. Neurol Neuroimmunol NeuroInflammation 2019;6:e555. https://doi.org/10.1212/nxi.0000000000000555.Search in Google Scholar
31. Freedman, M, Gnanapavan, S. Consortium of multiple sclerosis centers (CMSC) expert panel. CMSC consensus statement on neurofilament proteins in multiple sclerosis. Int J MS Care 2021;22:294. https://doi.org/10.7224/1537-2073.2020-140.Search in Google Scholar PubMed PubMed Central
Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2023-0518).
© 2023 Walter de Gruyter GmbH, Berlin/Boston
