Astrocytes are fundamental in neural functioning and homeostasis in the central nervous system. These cells respond to injuries and pathological conditions through astrogliosis, a reactive process associated with neurodegenerative diseases such as Alzheimer's disease. This process is thought to begin in the early stages of these conditions. Glial fibrillary acidic protein (GFAP), a type III intermediate filament protein predominantly expressed in astrocytes, has emerged as a key biomarker for monitoring this response. During astrogliosis, GFAP is released into biofluids, making it a candidate for non-invasive diagnosis and tracking of neurodegenerative diseases. Growing evidence positions GFAP as a biomarker for Alzheimer's disease with specificity and disease-correlation characteristics comparable to established clinical markers, such as Aβ peptides and phosphorylated tau protein. To improve diagnostic accuracy, particularly in the presence of confounders and comorbidities, incorporating a panel of biomarkers may be advantageous. This review will explore the potential of GFAP within such a panel, examining its role in early diagnosis, disease progression monitoring and its integration into clinical practice for Alzheimer's disease management.

Neurodegenerative dementias are progressive diseases that cause neuronal network breakdown in different brain regions often because of accumulation of misfolded proteins in the brain extracellular matrix, such as amyloids or inside neurons or other cell types of the brain. Several diagnostic protein biomarkers in body fluids are being used and implemented, such as for Alzheimer's disease. However, there is still a lack of biomarkers for co-pathologies and other causes of dementia. Such biofluid-based biomarkers enable precision medicine approaches for diagnosis and treatment, allow to learn more about underlying disease processes, and facilitate the development of patient inclusion and evaluation tools in clinical trials. When designing studies to discover novel biofluid-based biomarkers, choice of technology is an important starting point. But there are so many technologies to choose among. To address this, we here review the technologies that are currently available in research settings and, in some cases, in clinical laboratory practice. This presents a form of lexicon on each technology addressing its use in research and clinics, its strengths and limitations, and a future perspective.

Neurofilament-light chain (Nf-L) is a non-specific early-stage biomarker widely studied in the context of neurodegenerative diseases (NDD) and traumatic brain injuries (TBI), which can be measured in biofluids after axonal damage. Originally measured by enzyme-linked immunosorbent assay (ELISA) in cerebrospinal fluid (CSF), Nf-L can now be quantified in blood with the emergence of ultrasensitive assays. However, to ensure successful clinical implementation, reliable clinical thresholds and reference measurement procedures (RMP) should be developed. This includes establishing and distributing certified reference materials (CRM). As a result of the complexity of Nf-L and the number of circulating forms, a clear definition of what is measured when immunoassays are used is also critical to achieving standardization to ensure the long-term success of those assays. The use of powerful tools such as mass spectrometry for developing RMP and defining the measurand is ongoing. Here, we summarize the current methods in use for quantification of Nf-L in biofluid showing potential for clinical implementation. The progress and challenges in developing RMP and defining the measurand for Nf-L standardization of diagnostic tests are addressed. Finally, we discuss the impact of pathophysiological factors on Nf-L levels and the establishment of a clinical cut-off.

Neurofilament-light chain (Nf-L) is an early marker monitoring axonal damage which occur in many forms of dementia. During neurodegeneration, Nf-L is released into biological fluid and is quantifiable in cerebrospinal fluid (CSF) and blood. Currently, its quantification is performed by immunoassays. While those ones are sensitive and precise, the development of a reference measurement procedure (RMP) using mass spectrometry (MS) approaches traceable to the International System of Units (SI) is essential to achieve standardisation. Following the characterisation of three commercially available proteins as potential primary calibrator, one has been chosen and used for the method development. Here, we present the purity assessment and quantification traceable to the SI of the primary calibrator and preliminary data on the development of Nf-L quantification in CSF by LC-MS. A recombinant Nf-L protein from Promise was characterized, SI traceably quantified via amino acid analysis and used as primary calibrator for the development of a LC-MS method for Nf-L. A triple quadrupole (QQQ) mass spectrometer coupled to a capillary liquid chromatography (LC) was used to set up a multiple reaction monitoring (MRM) method to monitor Nf-L tryptic peptides and assess the potential of developing a RMP for Nf-L. The homogeneity of Nf-L from Promise was assessed on 3 aliquots prepared at 1.7 nmol/g. The mass fraction content in each vial was estimated at 1nmol/g ± 0.02 nmol/g. A LC-MRM method was developed to monitor Nf-L tryptic peptides and assess their suitability for the development of a RMP. Fifteen peptides were selected. The limit of detection in solution is 1 ng/g for 6 peptides. The sample clean-up was developed using Nf-L spiked in artificial and real CSF. Several solid-phase extraction cartridges were assessed in term of recovery and volume load. The use of protein precipitation was also assessed to increase the sensitivity. Preliminary experiments show a sensitivity of 100ng/g. Immunocapture will be used to achieve the required sensitivity. A LC-MRM based method and a sample clean-up procedure were developed for the quantification of Nf-L in CSF. A first assessment of sensitivity was carried out and the utilisation of immunocapture is on-going to reach the required sensitivity.

Proteomics studies have shown differential expression of numerous proteins in dementias but have rarely led to novel biomarker tests for clinical use. The Marie Curie MIRIADE project is designed to experimentally evaluate development strategies to accelerate the validation and ultimate implementation of novel biomarkers in clinical practice, using proteomics-based biomarker development for main dementias as experimental case studies. We address several knowledge gaps that have been identified in the field. First, there is the technology-translation gap of different technologies for the discovery (e.g., mass spectrometry) and the large-scale validation (e.g., immunoassays) of biomarkers. In addition, there is a limited understanding of conformational states of biomarker proteins in different matrices, which affect the selection of reagents for assay development. In this review, we aim to understand the decisions taken in the initial steps of biomarker development, which is done via an interim narrative update of the work of each ESR subproject. The results describe the decision process to shortlist biomarkers from a proteomics to develop immunoassays or mass spectrometry assays for Alzheimer's disease, Lewy body dementia, and frontotemporal dementia. In addition, we explain the approach to prepare the market implementation of novel biomarkers and assays. Moreover, we describe the development of computational protein state and interaction prediction models to support biomarker development, such as the prediction of epitopes. Lastly, we reflect upon activities involved in the biomarker development process to deduce a best-practice roadmap for biomarker development.

Neurofilament light chain (Nf-L) is a well-known biomarker for axonal damage; however, the corresponding circulating Nf-L analyte in cerebrospinal fluid (CSF) is poorly characterized. We therefore isolated new monoclonal antibodies against synthetic peptides, and these monoclonals were characterized for their specificity on brain-specific intermediate filament proteins. Two highly specific antibodies, ADx206 and ADx209, were analytically validated for CSF applications according to well-established criteria. Interestingly, using three different sources of purified Nf-L proteins, a significant impact on interpolated concentrations was observed. With a lower limit of analytical sensitivity of 100 pg/mL using bovine Nf-L as the calibrator, we were able to quantify the Nf-L analyte in each sample, and these Nf-L concentrations were highly correlated to the Uman diagnostics assay (Spearman rho = 0.97, p < 0.001). In the clinical diagnostic groups, the new Nf-L ELISA could discriminate patients with Alzheimer’s disease (AD, n = 20) from those with frontotemporal lobe dementia (FTD, n = 20) and control samples with subjective cognitive decline (SCD, n = 20). Henceforth, this novel Nf-L ELISA with well-defined specificity and epitopes can be used to enhance our understanding of harmonizing the use of Nf-L as a clinically relevant marker for neurodegeneration in CSF.

Neurofilament‐light chain (NF‐L) is a known early marker for neurodegeneration. This marker which is essential for the axonal structure, is quantifiable in biological fluids after neuronal damage but is not specific to a given pathology. Currently, the methods for detecting/quantifying this biomarker are mainly based on immunoassays. While the performance of immunoassay‐based assays is good in term of sensitivity and precision, mass spectrometry (MS) approaches are required to achieve standardisation. Those aiding to improved characterisation/quantification of the calibrator and potential for developing a reference measurement procedure traceable to the International System of Units. Furthermore MS has the potential of multiplexing NF‐L with other specific biomarkers for dementia. The characterization and quantification of a number of primary calibrators and their suitability for harmonization of immunoassay based results and development of a reference measurement procedure is here assessed.