Towards quantifying axonal injury in blood samples of patients affected by multiple sclerosis

Background. Neuro-axonal injury is a hallmark of the underlying pathological processes in neurodegenerative disorders. Reliable quantification and longitudinal follow-up of such damage via a biofluid marker would be a highly relevant adjunctive tool in the treatment workup for patients with multiple sclerosis (MS). The neurofilament proteins have emerged as the first biomarker bearing promise for a clinical application beyond a research tool. For the first time a biomarker specifically indicative of neuronal damage can be quantified in an easily accessible fluid source, i.e. in serum or plasma. Second (Enzyme-linked immunosorbent assays (ELISA)) and third generation (electrochemiluminescence based (ECL) assays) measuring systems lacked sufficient sensitivity to reliably measure neurofilaments throughout the range of concentrations found in blood samples, and specifically failed to define normal levels. The single molecule array system (SIMOA) marks a qualitative technological advancement as it provides the sensitivity to quantify physiologic neurofilament levels. This has paved the way to investigate neurofilaments in a range of neurological disorders, and specifically in diseases with smoldering course of neurodegeneration. Objective. We aimed to develop and validate a highly sensitive SIMOA assay for the neurofilament light chain (NfL). Using this assay, we investigated blood-based neurofilament light chain (NfL) as fluid biomarker of disease activity, treatment response, and as a predictor of the long-term course of disability and morphological features of neurodegeneration in MS. Further, we are evaluating in a third work stream the validity of NfL as a tool to detect suboptimal treatment with current standard MS therapies. Methods. In the first study, we quantified serum NfL (sNfL) in two independent MS patient cohorts: (i) in a cross-sectional cohort (142 patients) NfL in serum and CSF was correlated with magnetic resonance imaging (MRI) data, ii) in a longitudinal cohort (246 patients) from the Swiss MS Cohort study (SMSC) NfL levels in two samples post-switch to a new disease modifying treatment were compared to pre-switch levels and with those from 254 healthy controls from the Genome-Wide Association Study of Multiple Sclerosis (GeneMSA). In the second study, we quantified yearly serum sNfL in 259 MS patients followed up in the GeneMSA study for up to 10 years and 259 healthy controls who had also a one year follow up blood sampling. Results and interpretation. NfL levels in CSF and blood were highly correlated, thus supporting the concept that serum is a valid biofluid source to determine accurately neuronal damage within the central nervous system compartment. sNfL levels were higher in relapsing and progressive forms of MS, compared to healthy controls and were associated with current clinical and MRI disease activity. Finally, sNfL levels independently predicted future disability worsening, and cranial and spinal cord volume loss. Conclusion. Our data demonstrate that NfL can be reliably quantified in peripheral blood and CSF. Levels are associated with a) concurrent clinical and MRI measures of acute and chronic disease activity, b) response to DMT and c) long-term course of disability. This supports the potential of sNfL to become the first precision medicine tool to monitor subclinical disease activity and suboptimal treatment response

Serum neurofilament light chain is a biomarker of acute and chronic neuronal damage in early multiple sclerosis

Background Monitoring neuronal injury remains one key challenge in early relapsing-remitting multiple sclerosis (RRMS) patients. Upon axonal damage, neurofilament – a major component of the neuro-axonal cytoskeleton – is released into the cerebrospinal fluid (CSF) and subsequently peripheral blood. Objective To investigate the relevance of serum neurofilament light chain (sNfL) for acute and chronic axonal damage in early RRMS. Methods sNfL levels were determined in 74 patients (63 therapy-naive) with recently diagnosed clinically isolated syndrome (CIS) or RRMS using Single Molecule Array technology. Standardized 3 T magnetic resonance imaging (MRI) was performed at baseline and 1–3 consecutive follow-ups (42 patients; range: 6–37 months). Results Baseline sNfL correlated significantly with T2 lesion volume (r = 0.555, p < 0.0001). There was no correlation between baseline sNfL and age, Expanded Disability Status Scale (EDSS) score or other calculated MRI measures. However, T2 lesion volume increased (r = 0.67, p < 0.0001) and brain parenchymal volume decreased more rapidly in patients with higher baseline sNfL (r = −0.623, p = 0.0004). Gd-enhancing lesions correlated positively with sNfL levels. Initiation of disease-modifying treatment led to a significant decrease in sNfL levels. Conclusion sNfL indicates acute inflammation as demonstrated by correlation with Gd+ lesions. It is a promising biomarker for neuro-axonal damage in early multiple sclerosis (MS) patients, since higher baseline sNfL levels predicted future brain atrophy within 2 years

Blood neurofilament light as a potential endpoint in Phase 2 studies in MS

Objectives To assess whether neurofilament light chain (NfL) could serve as an informative endpoint in Phase 2 studies in patients with relapsing-remitting multiple sclerosis (RRMS) and estimate the sample size requirements with NfL as the primary endpoint. Methods Using data from the Phase 3 FREEDOMS study, we evaluated correlation of NfL at Month 6 with 2-year outcomes: relapses, confirmed disability worsening (CDW), new or enlarging T2 lesions (active lesions), and brain volume loss (BVL). We compared the proportion of treatment effect (PTE) on 2-year relapses and BVL explained by 6-month log-transformed NfL levels with the PTE explained by the number of active lesions over 6 months. We estimated sample size requirements for different treatment effects. Results At Month 6, blood NfL levels (pg/mL, median [range]) were lower in the fingolimod arm (fingolimod (n = 132) 18 [8-247]; placebo (n = 114) 26 [8-159], P &lt; 0.001). NfL at 6 months correlated with number of relapses (r = 0.25, P &lt; 0.001), 6-month CDW (hazard ratio 1.83, P = 0.012), active lesions (r = 0.46, P &lt; 0.001), and BVL (r = -0.41, P &lt; 0.001) at Month-24. The PTE (95% CI) on 24-month relapses and BVL explained by 6-month NfL was 25% (8-60%) and 60% (32-132%), and by 6-month active lesions was 28% (11-66%) and 45% (18-115%), respectively. Assuming a 20-40% treatment-related reduction in NfL levels, 143-28 patients per arm will be required. Conclusions Blood NfL may qualify as an informative and easy-to-measure endpoint for future Phase 2 clinical studies that captures both inflammatory- and noninflammatory-driven neuroaxonal injury in RRMS

Serum glial fibrillary acidic protein correlates with multiple sclerosis disease severity

BACKGROUND:: Cerebrospinal fluid (CSF) levels of two soluble biomarkers, glial fibrillary acidic protein (GFAP) and neurofilament light chain (NfL), have been shown to associate with multiple sclerosis (MS) disease progression. Now, both biomarkers can be detected reliably in serum, and importantly, their serum levels correlate well with their CSF levels.OBJECTIVE:: To evaluate the usability of serum GFAP measurement as a biomarker of progressive disease and disease severity in MS.METHODS:: Clinical course, Expanded Disability Status Scale (EDSS), disease duration, patient age and magnetic resonance imaging (MRI) parameters were reviewed in 79 MS patients in this cross-sectional hospital-based study. Serum samples were collected for measurement of GFAP and NfL concentrations using single molecule array (Simoa) assay. A cohort of healthy controls was evaluated for comparison.RESULTS:: Higher serum concentrations of both GFAP and NfL were associated with higher EDSS, older age, longer disease duration, progressive disease course and MRI pathology.CONCLUSION:: Earlier studies have demonstrated that GFAP, unlike NfL, is not increased in association with acute focal inflammation-related nervous system damage. Our work suggests that GFAP serum level associates with disease progression in MS and could potentially serve as an easily measurable biomarker of central nervous system (CNS) pathology related to disease progression in MS.</p

Neurofilament Light Chain: Blood Biomarker of Neonatal Neuronal Injury

Background: Neurofilament light chain (NfL) is a highly promising biomarker of neuroaxonal injury that has mainly been studied in adult neurodegenerative disease. Its involvement in neonatal disease remains largely unknown. Our aim was to establish NfL plasma concentrations in preterm and term infants in the first week of life.Methods: Plasma NfL was measured by single molecule array immunoassay in two neonatal cohorts: cohort 1 contained 203 term and preterm infants, median gestational age (GA) 37.9 weeks (interquartile range [IQR] 31.9–39.4), in whom venous and arterial umbilical cord blood was sampled at birth and venous blood at day of life (DOL) 3; cohort 2 contained 98 preterm infants, median GA 29.3 weeks (IQR 26.9–30.6), in whom venous blood was sampled at DOL 7.Results: Median NfL concentrations in venous blood increased significantly from birth (18.2 pg/mL [IQR 12.8–30.8, cohort 1]) to DOL 3 (50.9 pg/mL [41.3–100, cohort 1]) and DOL 7 (126 pg/mL [78.8–225, cohort 2]) (p &lt; 0.001). In both cohorts NfL correlated inversely with birth weight (BW, Spearman's rho −0.403, p &lt; 0.001, cohort 1; R −0.525, p &lt; 0.001, cohort 2) and GA (R −0.271, p &lt; 0.001, cohort 1; R −0.487, p &lt; 0.001, cohort 2). Additional significant correlations were found for maternal age at delivery, preeclampsia, delivery mode, 5-min Apgar, duration of oxygen supplementation, sepsis, and brain damage (intraventricular hemorrhage or periventricular leukomalacia). Multivariable logistic regression analysis identified the independent predictors of NfL in cohort 1 as BW (beta = −0.297, p = 0.003), delivery mode (beta = 0.237, p = 0.001) and preeclampsia (beta = 0.183, p = 0.022) and in cohort 2 as BW (beta = −0.385, p = 0.001) and brain damage (beta = 0.222, p = 0.015).Conclusion: Neonatal NfL levels correlate inversely with maturity and BW, increase during the first days of life, and relate to brain injury factors such as intraventricular hemorrhage and periventricular leukomalacia, and also to vaginal delivery

Comparison of three analytical platforms for quantification of the neurofilament light chain in blood samples: ELISA, electrochemiluminescence immunoassay and Simoa

AbstractBackground: Neuronal damage is the morphological substrate of persisting neurological disability. Neurofilaments (Nf) are specific cytoskeletal proteins of neurons and their quantification has shown encouraging results as a biomarker for axonal injury. Methods: We aimed at comparing a widely used conventional ELISA for Nf light chain (NfL) with an electrochemiluminescence-based method (ECL assay) and a newly developed single-molecule array (Simoa) method in clinically relevant cerebrospinal fluid (CSF) and serum samples. Results: Analytical sensitivity was 0.62 pg/mL for Simoa, 15.6 pg/mL for the ECL assay, and 78.0 pg/mL for the ELISA. Correlations between paired CSF and serum samples were strongest for Simoa (r=0.88, p<0.001) and the ECL assay (r=0.78, p<0.001) and weaker for ELISA measurements (r=0.38, p=0.030). CSF NfL measurements between the platforms were highly correlated (r=1.0, p<0.001). Serum NfL levels were highly related between ECL assay and Simoa (r=0.86, p<0.001), and this was less visible between ELISA-ECL assay (r=0.41, p=0.018) and ELISA-Simoa (r=0.43, p=0.013). Multiple sclerosis (MS) patients had significantly higher serum NfL levels than controls when measured with Simoa (p=0.001) but not with the other platforms. Conclusions: We found Simoa to be more sensitive than ELISA or the ECL assay. Our results support the feasibility of quantifying NfL in serum; the results correlate with the more-established CSF NfL test. The highly sensitive Simoa technology deserves further studies in larger patient cohorts to clarify whether serum NfL could be used in the future to measure disease severity and determine prognosis or response to treatment interventions in neurological diseases

Accurate and versatile 3D segmentation of plant tissues at cellular resolution

Quantitative analysis of plant and animal morphogenesis requires accurate segmentation of individual cells in volumetric images of growing organs. In the last years, deep learning has provided robust automated algorithms that approach human performance, with applications to bio-image analysis now starting to emerge. Here, we present PlantSeg, a pipeline for volumetric segmentation of plant tissues into cells. PlantSeg employs a convolutional neural network to predict cell boundaries and graph partitioning to segment cells based on the neural network predictions. PlantSeg was trained on fixed and live plant organs imaged with confocal and light sheet microscopes. PlantSeg delivers accurate results and generalizes well across different tissues, scales, acquisition settings even on non plant samples. We present results of PlantSeg applications in diverse developmental contexts. PlantSeg is free and open-source, with both a command line and a user-friendly graphical interface