Biomarkers in neurology: Assessing diagnostic potential

Neurological disorders are responsible for significant long-term disabilities impacting an estimated one billion individuals and shortening the lives of approximately nine million others globally every year.¹

Early and accurate diagnosis is a priority in improving treatment response and in slowing down neurodegeneration and disease progression, regardless of outstanding advancements in therapeutics.

Recent advances in neurological research highlight the critical role of biomarkers. Biomarkers are indicators of normal biological activity, pathogenic activity, or a biological response to an exposure or intervention.² They are also emerging as vital tools in healthcare and clinical research.

Multiple studies have revealed their potential in diagnosis, prognosis, monitoring, drug development, and precision medicine. However, exhaustive corroboration is critical before these biomarkers can be adopted in a clinical setting.

The success of biomarker development is dependent on a process that starts with a discovery phase and concludes with clinical validation (Figure 1). Clinical validation determines the biomarker’s relationship to the clinical outcome of interest and discovers the statistical thresholds for decision-making within a given context of use.^3,4

This relationship is regularly described in terms of clinical sensitivity and specificity. The development process can include analytical validation that provides an assessment of a biomarker’s performance.

Characteristics that define the conditions that the candidate will reliably produce are the reproducibility, limit of detection, repeatability, limit of quantification and other accurate data.³ Candidate biomarkers are required to meet all conditions before being implemented as a research tool in clinical trials.

Figure 1. Overview of biomarker validation process.³ Image adapted from NINDS.NIH.gov, Focus on Biomarkers Research, available at: https://www.ninds.nih.gov/current-research/focus-tools-topics/focus-biomarkers-research

When biomarkers have been validated, they could be used to identify individuals at risk for developing neurological diseases. They could also be used to help monitor neurodegeneration and identify patients who could respond positively to a treatment.

Detecting variations in biomarker levels has to stringently match the clinical outcome of interest and support the proposed use. According to the Biomarkers, Endpoints, and other Tools (BEST) glossary, biomarkers are categorized into seven categories: diagnostic, monitoring, predictive, prognostic, response, safety, and risk (Figure 2)^.2

Clinical validation is typically linked with one of these categories and a specific context of use. Since a single biomarker can be associated with multiple categories and various applications across different diseases, clinical validation becomes essential in determining the optimal conditions for its utilization.

Figure 2. BEST Biomarker category and associated examples of the context of use.⁴ Image adapted from FDA.gov, Context of Use, available at: https://www.fda.gov/drugs/biomarker-qualification-program/context-use.

Neurological disorders are defined as diseases affecting the central and peripheral nervous system.5 More than 600 neuropathologies exist, including Alzheimer’s disease (AD), multiple sclerosis (MS), and Parkinson’s disease (PD).⁶

These conditions are challenging to differentiate and accurately identify because they share neuropathological hallmarks. Similar symptoms and a lack of precise diagnostic tools can result in inaccurate clinical assessment and delayed therapeutic management.

A collaborative effort is underway to pinpoint biomarker signatures for each neurological disorder. This initiative aims to streamline disease differentiation and enhance diagnostic accuracy.

Neurofilament light (NfL), phosphorylated tau protein (p-Tau), and glial fibrillary acidic protein (GFAP) have recently shown promise as biomarkers for a variety of neurological disorders.7 However, clinically validating biomarkers for every condition of use is a time-consuming and expensive procedure that can often pace further clinical implementation.

Advancements in high-throughput, ultrasensitive biomarker detection methods have recently unlocked new strategies for clinical validation.

With the ability to detect biomarkers alone or simultaneously, and even at very low levels, Quanterix’s Simoa® digital technology offers an innovative solution to perform fast and reliable clinical validation in an accessible, noninvasive, and cost-effective manner.

The bead-bead assays produce a higher sensitivity utilizing microwells that are 4.25 µm in diameter. Paramagnetic particles coupled with antibodies designed to bind the specific target, fluorescent detection antibodies, and an enzyme are added to the sample.

The specific target is captured on the paramagnetic beads, forming single antibody-antigen complexes. These complexes, along with a substrate that produces a fluorescent product in the presence of the enzyme, are dispersed into 216,000 microwells.

This innovative approach requires a significantly smaller number of target molecules to emit a detectable signal compared to previous technologies.

In the last few decades, NfL has become the gold standard in measuring neuronal damage and neurodegeneration. Under normal conditions, axons release NfL into the cerebrospinal fluid (CSF) and bloodstream at extremely low levels.

In response to a neural injury, the circulating NfL concentrations increase in relation to the degree of underlying axonal damage. This occurs in many neurological disorders, highlighting the importance of NfL as a prominent biomarker for diagnosis and staging in neuropathology.⁸

Detection of CSF NfL reveals a substantial increase in concentrations for patients with neurological disorders, including MS, HIV-associated dementia (HAD), and mild traumatic brain injury.^8,9

In contrast, the rise in NfL levels in AD, PD, and PD dementia patients, while significant, is less abrupt.8 This difference highlights the importance of ultra-sensitive biomarker detection methods in distinguishing between healthy controls and various neuropathologies.

The inadequate sensitivity of detection is a long-standing limitation of NfL measurements to CSF. CSF samples require invasive lumbar punctures. It is not possible to perform frequent repeated lumbar punctures, presenting a limitation to the utilization of CSF biomarker measurements in clinical research and patient care settings.

The introduction of ultrasensitive Simoa® technology has facilitated a breakthrough in NfL detection in blood, achieving sensitivity down to the femto-level. This level of sensitivity was previously impossible to attain with traditional methods.¹⁰ The sensitivity of the Simoa® NfL immunoassay can provide reliable NfL blood detection in healthy controls and is 126 times higher than conventional ELISA for NfL quantification.¹⁰

With its unmatched sensitivity and accessibility, the Simoa® NfL blood test is used in a growing body of research covering many pathologies, especially neurodegenerative diseases.

CSF-based studies produced results with the Simoa® NfL blood-based assays exhibiting similar correlations between blood NfL levels and clinical outcomes.⁸ The capability of detecting NfL levels in blood allows Quanterix’s ultra-sensitive Simoa® technology to repeat high throughput analyses without invasive CSF collection.

NfLs are useful biomarkers of neuronal damage; missing specificity for one disease and the influence of other physiological factors, including age and body mass index, highlights how important quantitative measurements are for the adoption of biomarkers in neuropathologyUnderstanding NfL specificity of use and ensuring biomarker effectiveness in research and clinical settings requires the establishment of NfL threshold values specific to each neurological disorder.

Although NfL has proven to be a valuable biomarker of neuronal damage, its lack of specificity for individual diseases and susceptibility to influence from other physiological factors like age and body mass index highlight the significance of quantitative measurements for biomarker adoption in neuropathology.¹¹

Establishing specific NfL threshold values for each neurological disorder is crucial for understanding its specificity and ensuring the effectiveness of the biomarker as a tool in both research and clinical settings.

The lack of representative reference values to account for physiological influences on blood NfL could hinder its extensive utilization in research and clinical contexts. In 2022, Simoa® technology facilitated a significant international and comprehensive study aimed at establishing reference ranges of serum NfL in adults.¹²

The Simoa® NfL assay evaluated the abilities of serum NfL levels to determine disease activity and drug response in individual MS patients. The study utilized Simoa® digital technology and its capability of detecting serum NfL at ultra-low levels. NfL measurements were taken from a selection of normal, healthy adult individuals.

The study examined over 20,000 samples collected from a diverse group of donors, including different ages, a range of body mass indexes, and two large MS cohorts. The diverse patient set helped this study build a large statistical reference database and a new method that could support researchers and clinicians with the interpretation of serum NfL levels in blood.

This new method was validated and developed into an internet-based application.¹³ The potential of utilizing blood-based biomarkers to monitor the long-term effectiveness of disease-modifying therapies in a real-world setting is now becoming more of a reality.

In 2023, Simoa® technology participated in a second sizeable international study establishing serum NfL reference ranges to determine neurological disease activity in children and adolescents. This has expanded the future potential of the NfL blood test in clinical applications.¹⁴

The study examined 2,667 samples from donors aged 0 to 22 years of age, establishing a reference database that clinicians can utilize to interpret the results of serum NfL levels in pediatric patients.

The establishment of normative values in serum NfL for pediatric cases is a major improvement in the diagnosis and monitoring of neurological conditions in children and adolescents.

Reference values serve as a valuable tool for predicting neurological disease progression early on, facilitating the swift identification of high-risk patients and potentially expediting the application of disease-modifying therapies.

They also have the potential to streamline the diagnostic process for neurological disorders significantly and could speed up the clinical trial process by evaluating the growing array of targeted causal and disease-modifying treatments.

While biomarker detection and quantification offer valuable insights into neurological disorders like MS, it is essential to integrate biomarker measurements with other diagnostic methods to establish a definitive clinical diagnosis and ensure optimal therapeutic management.

NfLs potential as a biomarker of neuronal injury is vast. Thorough validation is required before any benefit can be achieved in clinical applications. A landmark study was recently published that validated the analytical performance of the Simoa® NfL assay using 17 testing centers in 10 different countries located in Europe and North America.¹⁵

The study is a comprehensive validation completed in real-world laboratory settings that highlights the Simoa® NfL blood test’s reliability and versatility. Combined with established reference datasets, these studies position the Simoa® NfL assay as the gold standard in blood measurement of the important Nfl biomarker.

The Simoa® NfL assay has the potential to revolutionize the treatment and management of neurology patients.

In 2022, the Simoa® NfL assay was granted Breakthrough Device designation by the US Food and Drug Administration (FDA) as a prognostic aid in assessing the risk of disease activity in patients diagnosed with relapsing-remitting MS (RRMS).¹⁶

NfL testing holds a prominent place in therapeutic trials and drug efficacy monitoring, providing researchers, clinicians and patients with more effective treatments and interventions for neurology conditions.

Quanterix continues to make important advancements in ultra-sensitive biomarker detection, propelling scientific and healthcare breakthroughs to new heights.

Image Credit: Quanterix

1. Feigin VL, Vos T, Nichols E, et al. The global burden of neurological disorders: translating evidence into policy. Lancet Neurol. 2020;19(3):255-265. doi:10.1016/S1474-4422(19)30411-9
2. FDA-NIH Biomarker Working Group. BEST (Biomarkers, EndpointS, and other Tools) Resource [Internet]. Silver Spring (MD): Food and Drug Administration (US); 2016-. Glossary. 2016 Jan 28 [Updated 2021 Nov 29]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK338448/
3. National Institute of Neurological Disorders and Stroke. Focus On Biomarkers Research. Accessed September 15, 2023. https://www.ninds.nih. gov/current-research/focus-tools-topics/focus-biomarkers-research
4. FDA. Context of Use. Published online July 7, 2021. Accessed September 15, 2023. https://www.fda.gov/drugs/biomarker-qualification-program/context-use
5. World Health Organization. Mental health: neurological disorders. Accessed September 15, 2023. https://www.who.int/news-room/questions-and-answers/item/mental-health-neurological-disorders#:~:text=These%20disorders%20include%20epilepsy%2C%20Alzheimer,trauma%2C%20and%20neurological%20disorders%20as
6. Matilla-Dueñas A, Corral-Juan M, Rodríguez-Palmero Seuma A, et al. Rare Neurodegenerative Diseases: Clinical and Genetic Update. In: Posada de la Paz M, Taruscio D, Groft SC, eds. Rare Diseases Epidemiology: Update and Overview. Advances in Experimental Medicine and Biology. Springer International Publishing; 2017:443-496. doi:10.1007/978-3-319-67144-4_25
7. Baiardi S, Quadalti C, Mammana A, et al. Diagnostic value of plasma p-tau181, NfL, and GFAP in a clinical setting cohort of prevalent neurodegenerative dementias. Alzheimer’s Res Ther. 2022;14(1):153. doi:10.1186/s13195-022-01093-6
8. Gaetani L, Blennow K, Calabresi P, Di Filippo M, Parnetti L, Zetterberg H. Neurofilament light chain as a biomarker in neurological disorders. J Neurol Neurosurgery Psychiatry. 2019;90(8):870-881. doi:10.1136/jnnp-2018-320106
9. Bridel C, van Wieringen WN, Zetterberg H, et al. Diagnostic Value of Cerebrospinal Fluid Neurofilament Light Protein in Neurology. JAMA Neurol. 2019;76(9):1035-1048. doi:10.1001/jamaneurol.2019.1534
10. Kuhle J, Barro C, Andreasson U, et al. Comparison of three analytical platforms for quantification of the neurofilament light chain in blood samples: ELISA,

About Quanterix

From discovery to diagnostics, Quanterix’s ultrasensitive biomarker detection is fueling breakthroughs only made possible through its unparalleled sensitivity and flexibility. Quanterix’s Simoa® technology has delivered the gold standard for earlier biomarker detection in blood, serum or plasma, with the ability to quantify proteins that are far lower than the Limit of Quantification (LoQ) of conventional analog methods.

Its industry-leading precision instruments, digital immunoassay technology and CLIA-certified Accelerator laboratory have supported research that advances disease understanding and management in neurology, oncology, immunology, cardiology and infectious disease. Quanterix has been a trusted partner of the scientific community for nearly two decades, powering research published in more than 2,500 peer-reviewed journals.

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