The Promise of Neurofilament Light (NfL) as a Biomarker
Neurofilament Light (NfL) is a structural component of axons that provides stability. When neurons are damaged or destroyed, NfL is released into the cerebrospinal fluid (CSF) and bloodstream. The more severe the damage, the higher the levels of NfL.(1) Elevated NfL levels indicate neuronal damage, regardless of the underlying cause. NfL has been linked to various conditions, including Alzheimer's Disease (AD), relapsing-remitting and progressive multiple Sclerosis (MS), Frontotemporal Dementia (FTD), atypical Parkinson's Disease (PD), stokes, Traumatic Brain Injury (TBI), and the neurotoxic effects of cancer treatments.(2)
Because NfL rises in response to various types of neuronal damage, it has limited use in differential diagnosis. However, it can differentiate between disease that involve neuron damage and those that do not, even when symptoms appear similar. For example, high NfL levels in behavioral variant FTD can distinguish it from psychiatric disorders, which do not cause neuronal damage and typically show normal NfL level. NfL concentrations can also help differentiate between PD and FTD; FTD, which involves more widespread neuronal damage, results in much higher NfL levels.(1)
As a biomarker of neurodegeneration, NfL may be useful for determining disease severity, tracking progression, and monitoring treatment effects.
NfL has predicative value: it can be detected years before symptoms appear, especially in individuals who are genetically predisposed to neurodegerenative diseases. For example, in sporadic AD, NfL levels begin to rise up to 10 years before clinical symptoms develop, and in familial AD, they can increase as early as 22 years before symptom onset. Tracking NfL levels allows researchers to monitor the early stages of neurodegenerative diseases.
Once symptoms appear, NfL levels can indicate disease severity. In AD, for example, NfL levels rise as cognitive function declines. NfL levels also correlate with neuroimaging and cognitive assessments of disease progression, making it a valuable metric for longitudinal studies.(1)
NfL has proven useful in monitoring clinical trials of treatments targeting neurodegenerative diseases. The FDA recently granted accelerated approval to tofersen for SOD1-associated amyotrophic lateral sclerosis (ALS) based on changes in blood NfL levels.(3) However, the FDA approved tofersen as an orphan drug for rare disease, thus allowing NfL to be used as a surrogate endpoint to expedite approval. The FDA does not formally recognize NfL as a prognostic, response, risk, susceptibility, or safety biomarker,(4) so it may take time before NfL becomes a standard basis for approval in more widely research diseases such as AD.
Meanwhile basic research on NfL continues to explore its role in health and diseases. Researchers are investigating:
- How NfL and other neurofilaments contribute to axonal transport and other functions in healthy neurons
- How NfL degrades and how injuries and diseases break down the neurofilaments, releasing NfL into body fluid
- How normal aging affects NfL levels and how these changes relate to cognitive decline or vulnerability to neurodegenerative diseases
In short, NfL is well-established in research as a biomarker of neurodegeneration, and is beginning to see use in clinical trials.
Challenges in Measuring Neurofilament Light
Despite widespread recognition as a biomarker of neurodegenerative disease, NfL measurement faces two major challenges: lack of standardization and the need for cut-off values to detect diseases accurately.
A lack of standardization makes it difficult to compare results across different assay platforms, biofluids, and research labs. As Mattson-Carlgen and colleagues explain. "reference measurement procedures ('gold standard' methods) or certified reference materials ('gold standard' samples) are not available for most neurodegeneration biomarkers, with the exception of CSF Ab42."(5) Ashrafzadeh-Kian and colleagues further note, "the lack of a certified reference material and reference method for NfL prevents the comparison of results between immunoassays from different manufacturers." Although measurements using different manufacturers' assays correlate well, they differ in absolute value, reducing their reliability and complicating the establishment of standard reference values and cut-off scores. Researchers need to develop conversion factors or chose a reference measurement procedure to standardize results.
Additionally, assays of serum versus plasma give correlated but different values.(3) The choice between these fluids appear to be dependent on the disease or clinical center rather than on empirical evidence. For example, studies on ALS and MS often use serum, while those on dementias favor plasma.
In short, researchers are trying to increase consistency of materials and procedures across platforms, biofluids, and research labs.
Need for Cut-off Values to Detect Neurodegenerative Diseases
Both researchers and clinicians need to interpret an individual's NfL levels as either healthy or indicative of disease. However, this judgement can be difficult without normative cut-off values that account for age and other confounding factors.
NfL levels naturally increase with age, typically by 2-4% per year. Thus, what is normal at age 60 might be abnormally high at age 30. The variability between individuals also increases with age. Thus, a 60 year old's NfL levels may need to diverge more from the average than a 30 year old's to be considered "abnormal". Several studies have reported a strong inverse correlation between Body Mass Index (BMI) and NfL levels. Individuals with higher BMI in general have a lower NfL levels.(5) Comorbid conditions, such as a history of stroke, cardiovascular lesions, or diabetes, also influence NfL levels, complicating interpretation. renal conditions indirectly affect NfL level because they influence blood concentration of substances. These conditions, becoming more common with age, further complicate the interpretation of NfL levels.(1)
As of July 15, 2024, reference intervals for newer assays are not available in the literature or from assay manufacturers, leaving researchers to establish them based on large, representative populations.(6)
Advances in Measuring Neurofilament Light
In the past year, researchers have introduced age-adjusted cut-off values for disease detection and found evidence supporting the use of plasma as a matrix. However, variability across assay platforms and laboratories remains a challenge.
Progress in Standardization: Improving Lab Consistency
Several international groups are working to develop an Rn RMP for measuring NfL level in blood. They have already increased standardization of cerebrospinal fluid (CSF) measurements through pre-analytical protocols and automated, standardized lab tests, and hope to extend these techniques to blood.(7)
In November 2021, the Standardization of Alzheimer's Blood Biomarkers (SABB) workgroup of the Global Biomarker Standardization Consortium (GBSC) of the Alzheimer's Association developed a standardized operating procedure (SOP) for handling plasma. However, this SOP predates some of the empirical evidence and may need updating. Additionally, SOPs still need to be developed for the analytic and post-analytic phases of research.(8)
Some organizations working on standardizing NfL measurement:
Multi-Omics Interdisciplinary Research Integration to accelerate Dementia Biomarker Development (MIRIADE): validates biomarkers from multiple platforms and created a roadmap for developing and disseminating biomarkers
Alzheimer's Association Global Biomarker Standardization consortium (GBSC): Develops reference methods and materials. Its quality Control Program brings together labs worldwide to synchronize procedures and compare their results using the same assay.
International Federation of Clinical Chemistry's Biomarkers of Neurodegenerative Diseases Working Group: Seeks to standardize measurements across assay platforms globally.
These efforts may be reducing variability between labs and over time. For example, a January 2024 multicenter trial by Linnemann and colleagues of blood NfL levels in large FTD cohort using SIMOA showed excellent consistency and high reliability between labs, even when assessed at different tine points by partly different kits. This supports an earlier 2020 multicenter study that showed good cross-lab reliability of blood NfL levels in ALS.
Consensus on Biofluids
Recent evidence favors serum over plasma as the preferred biofluid for NfL assays. In January 2023, Andreassen and colleagues compared neat and variously spiked serum and plasma as matrices for NfL assays, using a difference in bias approach. They argued that serum makes a better choice for a CRM matrix than plasma due to serum being more commutable than plasma. Khalil and colleagues also recommend using serum due to its simpler and more standardizable production process.(3)
Progress Toward Normative Cut-Off Values
Age-based cut-off values are becoming more common. In January 2024, Beltran proposed a set age-based cutoffs based on a U.S. cohort. By April 2024, large reference databases had been established for serum levels in adults and children, along with "growth curve" models, which enable more precise interpretation at the individual level.
The US FDA is supporting faster development of assays for measuring NfL, at least in Multiple Sclerosis (MS). Quanterix's SIMOA platform was granted FDA Breakthrough Device Status 4/22/22 for MS, as was Roche's Elecsys on 11/9/23. Although driven by clinical concerns, this support contributes expertise to improving the measurement tools basic researchers use. It remain to be seen on how the FDA designation affect the research community's effort to develop standardization and cut-off values.
Conclusion
NfL makes an excellent biomarker for assessing neurodegenerative disease severity, predicting progression, and evaluating treatments. The next challenges for researchers is to standardize the process of measuring NfL levels and establish reference values for research and eventually, clinical use.
References:
- Coppens, S. et al., "Neurofilament-Light, a Promising Biomarker: Analytical, Metrological and Clinical Challenges", Int. J. Mol Sci., 24(14):11624, (2023), DOI: 10.3390/ijms241411624 (link)
- Johnson, K. "Neurofilament Light Chain Detects Early Chemotherapy-Related Neurotoxicity", Medscape, June 24 (2024) (link)
- Khalil, M. et al., "Neurofilaments as biomarkers in neurological disorders-towards clinical application", Nat Rev Neurol., 20:269, (2024), DOI: 10.1038/s41582-024-00955-x (link)
- Benatar, M. et al., "Biomarker Qualification for Neurofilament Light Chain in Amyotrophic Lateral Sclerosis: Theory and Practice", Ann Neurol., 95(2):211, (2024), DOI: 10.1002/ana.26860 (link)
- Mattsson-Carlgren, N. et al., "Increasing the reproducibility of fluid biomarker studies in neurodegenerative studies", Nat Commun., 11:6252, (2024), DOI: 10.1038/s41467-020-19957-6 (link)
- Ashrafzadeh-Kian, S. et al., "Head-tohead comparison of four plasma neurofilament light chain (NfL) immunoassays", Clin Chim Acta., 561:119817, (2024), DOI: 10.1016/j.cca.2024.119817 (link)
- Willemse, E.A.J. et al., "Comparing CSF amyloid-beta biomarker ratios for two automated immunoassays, Elecsys and Lumipulse, with amyloid PET status", Alzheimers Dement., 13(1):e12182, (2021), SOI: 10.1002/dad2.12182 (link)
- Verberk, I.M.W. 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 Bllod Biomarkers (SABB) working group", Alhzeimers Dement., 18(8): 1484, (2022), DOI: 10.1002/alz.12510 (link)