Development and multi-center validation of a fully automated digital immunoassay for neurofilament light chain: toward a clinical blood test for neuronal injury

Research output: Contribution to journalResearch articleContributedpeer-review


  • David Wilson - , Quanterix Corp (Author)
  • Dandan Chan - , Quanterix Corp (Author)
  • Lei Chang - , Quanterix Corp (Author)
  • Robert Mathis - , Quanterix Corp (Author)
  • Inge Verberk - , Amsterdam University Medical Centers (UMC) (Author)
  • Xavier Montalban - , Vall d'Hebron Research Institute (VHIR) (Author)
  • Manuel Comabella - , Vall d'Hebron Research Institute (VHIR) (Author)
  • Nicolas Fissolo - , Vall d'Hebron Research Institute (VHIR) (Author)
  • Bibi Bielekova - , National Institutes of Health (NIH) (Author)
  • Ruturaj Masvekar - , National Institutes of Health (NIH) (Author)
  • Tanuja Chitnis - , Harvard Medical School (HMS) (Author)
  • Tjalf Ziemssen - , Department of Neurology (Author)
  • Katja Akgün - , Department of Neurology (Author)
  • Kaj Blennow - , University of Gothenburg (Author)
  • Henrik Zetterberg - , University of Gothenburg (Author)
  • Wolfgang Brück - , University Medical Center Göttingen (Author)
  • Gavin Giovannoni - , Barts Health NHS Trust (Author)
  • Sharmilee Gnanapavan - , Barts Health NHS Trust (Author)
  • Stefan Bittner - , University Medical Center Mainz (Author)
  • Frauke Zipp - , University Medical Center Mainz (Author)
  • Giancarlo Comi - , Vita-Salute San Raffaele University, IRCCS Hospital San Raffaele (Author)
  • Roberto Furlan - , Vita-Salute San Raffaele University (Author)
  • Sylvain Lehmann - , CHU Montpellier (Author)
  • Simon Thebault - , Ottawa Hospital Research Institute (Author)
  • Mark Freedman - , Ottawa Hospital Research Institute (Author)
  • Amit Bar-Or - , University of Pennsylvania (Author)
  • Marty Kramer - , Sanofi-Aventis (Author)
  • Markus Otto - , Ulm University Medical Center (Author)
  • Steffen Halbgebauer - , Ulm University Medical Center (Author)
  • Kevin Hrusovsky - , Quanterix Corp (Author)
  • Tatiana Plavina - , Quanterix Corp (Author)
  • Michael Khalil - , Medical University of Graz (Author)
  • Fredrik Piehl - , Karolinska Institutet (Author)
  • Heinz Wiendl - , University of Münster (Author)
  • Ludwig Kappos - , University of Basel (Author)
  • Aleksandra Maceski - , University of Basel (Author)
  • Eline Willemse - , University of Basel (Author)
  • David Leppert - , University of Basel (Author)
  • Charlotte Teunissen - , Amsterdam University Medical Centers (UMC) (Author)
  • Jens Kuhle - , University of Basel (Author)


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 NfLmeasurement 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 afterDMT 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 crosscomparisons of results across multiple sites.


Original languageEnglish
Pages (from-to)322-331
Number of pages10
JournalClinical chemistry and laboratory medicine
Issue number2
Early online date13 Sept 2023
Publication statusPublished - 26 Jan 2024

External IDs

Scopus 85172480921



  • Biomarkers, Hematologic Tests, Humans, Immunoassay, Intermediate Filaments, Neurofilament Proteins, Neurons, Reproducibility of Results