Single-molecule Array Assay Research Articles

Neurofilament heavy chain in secondary progressive multiple sclerosis.

Biomarkers are needed to track progression in MS trials. Neurofilament heavy chain (NfH) has been underutilized due to assay limitations. To investigate the added value of cerebrospinal fluid (CSF) NfH in secondary progressive multiple sclerosis (SPMS) using contemporary immunoassays. This exploratory study was part of the MS-SMART trial. Clinical assessments (including expanded disability status scale, upper and lower limb function, visual acuity and symbol digit modalities test (SDMT)), CSF and serum sampling were acquired at baseline (n = 54), 48 and 96 weeks. Brain magnetic resonance imagings (MRIs) were obtained at baseline and 96 weeks. The NfL and NfH were measured using single-molecule array assay. Baseline CSF NfH and NfL correlated with information processing speed at 96 weeks, with CSF NfH showing stronger correlations (r = -0.49 for SDMT) than CSF NfL (r = -0.37 for SDMT). Baseline CSF NfL predicted poorer hand dexterity at baseline, 48 and 96 weeks. CSF NfH was the only predictor of cortical grey matter at baseline, while baseline CSF NfL was the only predictor of brain atrophy at 96 weeks. Serum neurofilaments showed limited associations. CSF neurofilaments are better outcomes than serum neurofilaments in small SPMS studies. CSF NfH and NfL variably predict worsening hand function, information processing speed and brain volume loss, possibly reflecting complementary aspects of neurodegeneration.

Comparison of Methods of Detecting IL-1β in the Blood of Alzheimer's Disease Subjects.

Interleukin (IL)-1β is a pro-inflammatory cytokine whose levels are increased in the brains of Alzheimer's disease (AD) patients. Despite the role of IL-1β in the pathology of AD, the fact that it is expressed at very low levels makes it a challenging cytokine to measure, hence limiting its potential use as a reliable biomarker. Moreover, being able to accurately and reliably measure the levels of IL-1 β in blood makes it possible to evaluate this cytokine as a potential biomarker of the inflammatory response in AD. In this study, we compared three quantification methodologies, Meso-Scale Discovery (MSD), both V-Plex and S-Plex versions, and Quanterix's SIMOA (Single-Molecule Array), to measure IL-1β in the serum of AD patients and age-matched controls. These assays are routinely used to measure IL-1β serum levels with high specificity and sensitivity in human AD patients, yet to the best of our knowledge, no study has compared all three techniques for their accuracy to measure IL-1β as biomarkers. Our findings indicate the two MSD assays can be used to measure IL-1β levels in AD and control serum, but the SIMOA assay showed the highest receiver operating characteristics (ROCs), with an area under the curve (AUC) of 0.9532, which can be compared to the AUC values for the V-Plex assay, 0.5660, and the S-Plex assay, 0.6632. Taken together, these data show that although all technologies are useful in the measurement of IL-1β in the blood, the SIMOA IL-1β 3.0 assay is more reliable and sensitive in measuring biomarkers of AD.

Association between glymphatic system function and cognitive impairment in elderly patients with late-onset epilepsy.

Recent studies have shown that late-onset epilepsy (LOE) is accompanied with cognitive decline and increased risk of dementia, particularly Alzheimer's disease (AD). However, the pathophysiological mechanism underlying the cognitive decline in LOE remains unclear. The aim of current study was to evaluate the relationship between glymphatic system (GS) function and cognitive decline in LOE patients using the diffusion tensor imaging (DTI) analysis along the perivascular space (DTI-ALPS). Medical records and neuro-imaging data were obtained from 21 LOE patients with cognitive decline, 14 LOE patients without cognitive decline, and 20 age- and sex-matched healthy controls (HCs). Plasma biomarkers including Aβ42 and Aβ40 were examined using single-molecule array (Simoa) assays. The DTI-ALPS parameter was calculated and correlated with the clinical characteristics of LOE, including age, seizure frequency, duration of epilepsy, Mini-Mental State Examination (MMSE), and Aβ42/40. Regression models were used to evaluate the influencing factors of DTI-ALPS index. LOE patients exhibited a decreased ALPS index and Aβ42/40 compared with the HCs. Post-hoc analysis indicated that the DTI-ALPS index and Aβ42/40 in LOE patients with cognitive decline was significantly lower in relative to LOE patients without cognitive decline and HCs. Spearman correlations showed a negative correlation between DTI-ALPS index and age, seizure frequency and disease duration while a positive correlations between the DTI-ALPS index and Aβ42/40 and MMSE scores in LOE patients. Linear regression analysis suggested that the DTI-ALPS index was independently related to age, Aβ42/40 and MMSE score after correcting for gender, education, and vascular risk factors. Our findings using DTI-ALPS method found a positive correlation between cognitive decline and GS dysfunction in LOE patients, and may indicate a potential internal link between age-related LOEU and dementia formation. Therefore, the DTI-ALPS index may serve as a potential imaging marker for diagnosing and monitoring the GS function in LOE patients.

Loneliness and biomarkers of Alzheimer's disease, axonal damage, and astrogliosis: A coordinated analysis of two longitudinal cohorts.

Loneliness is associated with an elevated risk of dementia. There is mixed evidence from imaging studies on whether loneliness is associated with neuropathology in dementia-free adults. This study tests whether loneliness is associated with plasma neurobiomarkers of amyloid (Aβ42/Aβ40), phosphorylated tau 181 (pTau181), neurofilament light chain (NfL), and glial fibrillary acidic protein (GFAP) and imaging measures of amyloid and tau. Participants were cognitively unimpaired older adults from the Baltimore Longitudinal Study on Aging (BLSA; N=1,028 individuals and up to 2,277 neurobiomarker measurements; Baseline mean age=66, SD=15 years) and the UK Biobank (N=1,263 individuals and up to 2,526 neurobiomarker measurements; Baseline mean age=60, SD=7 years). Single-item measures of loneliness and the Quanterix Single Molecule Array assays were used in both samples. In a subset of BLSA participants, positron emission tomography (PET) was used to assess cerebral amyloid burden (n=220) and tau in the entorhinal cortex (n=102). In both samples and meta-analyses, loneliness was unrelated to plasma measures of Aβ42/Aβ40, pTau181, NfL, and GFAP. Changes in loneliness were also unrelated to changes in the plasma neurobiomarkers, and no consistent evidence of moderation by age, sex, or APOE ε4 allele was found. Loneliness was also unrelated to PET based measures of amyloid and tau. This study found no associations between loneliness and measures of Alzheimer's disease pathology, axonal damage, or astrogliosis.

Ultrasensitive detection of six sepsis-associated proteins in neonatal saliva

Sepsis is a life-threatening condition that affects millions of newborns every year. Current diagnostic practices require painful, often repetitive, blood collections to isolate and culture the causative microorganism. Definitive return of results can take up to 5 days, exposing newborns to potentially unnecessary antibiotics. Developing a more rapid, non-invasive assessment of infectious status based upon host immune response provides an alternative diagnostic approach to infection screening. However, additional blood collection for multiplex quantification of inflammatory biomarkers in neonates remains prohibitive. Alternatively, saliva may serve as an informative biofluid, though detection of biologically relevant proteins in saliva remains challenging, with analyte concentrations 100 to 1000 times lower compared to blood. To address this challenge, we developed a panel of six ultra-sensitive single-molecule array (Simoa) assays for inflammatory biomarkers known to be associated with neonatal sepsis: serum amyloid A1 (SAA1), lipopolysaccharide-binding protein (LBP), chemokines CCL20, CXCL6, CXCL12; and the adipokine resistin. When these assays were tested on 40 neonatal salivary samples, we found that CXCL6 and CCL20 were significantly elevated in infected and septic neonates compared to those uninfected. The small volumes of fluid used (~10 μL saliva) and limited concentrations of these analytes in saliva (pg/mL) underscore the importance of ultra-sensitive measurements in the development of non-invasive diagnostics and reinforces the value of neonatal saliva as a viable sample matrix for monitoring infection. We hypothesize these assays could be useful in future diagnostic tests to discriminate between infected and uninfected neonates.

Neurofilament Light Chain as a Discriminator of Disease Activity Status in MOG Antibody-Associated Disease.

In patients with myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD), acute disease activity is generally identified through medical history, neurologic examination, and imaging. However, these may be insufficient for detecting disease activity in specific conditions. This study aimed to investigate the dynamics of serum neurofilament light chain (sNfL) and serum glial fibrillary acidic protein (sGFAP) after clinical attacks and to assess their utility in discriminating attacks from remission in patients with MOGAD. We conducted a multicenter, retrospective, longitudinal study including 239 sera from 62 MOGAD patients assessed from 1995 to 2023 in a discovery and validation setup. Sera were measured for sNfL and sGFAP with a single-molecule array assay and for MOG-IgG with a live cell-based assay. sNfL and sGFAP Z scores and percentiles adjusted for age, body mass index, and sex (sGFAP) were calculated from a healthy control normative database. Mixed-effects regression models were used to characterize biomarkers' dynamics and to investigate associations between serum biomarkers, clinical variables, and disease activity status. Among the 62 study participants, 29 (46.8%) were female, with a median age at baseline of 40.0 years (interquartile range [IQR] 29.5-49.8) and a median duration of follow-up of 20.0 months (IQR 3.0-62.8). sNfL and sGFAP Z scores were nonlinearly associated with time from attack onset (p < 0.001 and = 0.002, respectively). During attacks, both biomarkers presented higher median values (sNfL Z score 2.9 [IQR 1.4-3.5], 99.8th; sGFAP Z score 0.4 [IQR -0.5 to 1.5], 65.5th) compared with remission (sNfL Z score 0.9 [IQR -0.1 to 1.6], 81.6th, p < 0.001; sGFAP Z score -0.2 [IQR -0.8 to 0.5], 42.1th; p < 0.001) across all clinical phenotypes. sNfL values consistently discriminated disease activity status in the discovery and validation cohorts, showing a 3.5-fold increase in the odds of attacks per Z score unit (odds ratio 3.5, 95% confidence interval 2.3-5.1; p < 0.001). Logistic models incorporating sNfL Z scores demonstrated favorable performance in discriminating disease activity status across both cohorts. sNfL Z scores may serve as a biomarker for monitoring disease activity in MOGAD.

Blood-Based Biomarkers for Identifying Disease Activity in AQP4-IgG–Positive Neuromyelitis Optica Spectrum Disorder

The temporal dynamics of serum glial fibrillary acidic protein (sGFAP) and serum neurofilament light chain (sNfL) as biomarkers of disease activity for neuromyelitis optica spectrum disorder (NMOSD) remain underexplored. To determine optimal timing for assessing sGFAP and sNfL, establish cutoff values differentiating between attacks and remissions in NMOSD, and evaluate these findings across independent cohorts. This retrospective, longitudinal, multicenter cohort study was conducted among patients with aquaporin-4 antibody (AQP4-IgG)-positive NMOSD. Patients with available stored serum samples were included, totaling 181 patients with 625 samples. Discovery cohort samples were collected from February 2008 to October 2023 and validation cohort samples were collected from January 2013 to October 2023. A combined analysis of both cohorts was conducted from November 2023 to March 2024. sNfL and sGFAP concentrations, measured by a single-molecule array assay. The primary outcomes were the optimal timing of assessing sGFAP and sNfL and the adjusted cutoff values for evaluating disease activity in NMOSD. The discovery cohort consisted of 366 samples from 78 Korean patients (median [IQR] age, 35 [30-42] years; 73 female patients [95%]), while the validation cohort included 190 samples from 34 German patients (median [IQR] age, 54 [39-61] years; 32 female patients [94%]) and 69 samples from 69 Brazilian patients (median [IQR] age, 46 [35-55] years; 62 female patients [90%]). Six-month postattack temporal biomarker dynamics were analyzed in 202 samples from 74 patients in the discovery cohort: sGFAP levels peaked within the first week and sNfL levels peaked at 5 weeks postattack. The optimal time frames for evaluating attacks were within 1 week for sGFAP and from 1 to 8 weeks for sNfL, with remission defined as at least 6 months postattack. z Score cutoffs of 3.0 for sGFAP and 2.1 for sNfL effectively distinguished between attack and remission phases, indicated by area under the curve values of 0.95 (95% CI, 0.88-1.02) and 0.87 (95% CI, 0.82-0.91), respectively. The discovery cohort time frames and cutoff values were applied to the validation cohort, achieving 71% sensitivity and 94% specificity for sNfL and 100% sensitivity and specificity for sGFAP in the German and Brazilian cohorts. This longitudinal cohort study established optimal timing and thresholds for sGFAP and sNfL, which were consistent in independent cohorts, supporting these biomarkers' effectiveness in distinguishing NMOSD attacks from remission.

Plasma p-tau217 in Alzheimer's disease: Lumipulse and ALZpath SIMOA head-to-head comparison.

Plasma phosphorylated-tau217 (p-tau217) has been shown to be one of the most accurate diagnostic markers for Alzheimer's disease. No studies have compared the clinical performance of p-tau217 as assessed by the fully automated Lumipulse and single molecule array (SIMOA) AlZpath p-tau217. The study included 392 participants, 162 with Alzheimer's disease, 70 with other neurodegenerative diseases with CSF biomarkers and 160 healthy controls. Plasma p-tau217 levels were measured using the Lumipulse and ALZpath SIMOA assays. The ability of p-tau217 assessed by both techniques to discriminate Alzheimer's disease from other neurodegenerative diseases and controls was investigated using receiver operating characteristic analyses. The p-tau217 levels measured by the two techniques demonstrated a strong correlation, showing a consistent relationship with CSF p-tau181 levels. In head-to-head comparison, Lumipulse and SIMOA showed similar diagnostic accuracy for differentiating Alzheimer's disease from other neurodegenerative diseases [area under the curve (AUC) 0.952, 95% confidence interval (CI) 0.927-0.978 versus 0.955, 95% CI 0.928-0.982, respectively] and healthy controls (AUC 0.938, 95% CI 0.910-0.966 and 0.937, 95% CI 0.907-0.967 for both assays). This study demonstrated the high precision and diagnostic accuracy of p-tau217 for the clinical diagnosis of Alzheimer's disease using fully automated or semi-automated techniques.

Assessing the feasibility of dried blood spot (DBS) collection for blood‐based biomarkers of Alzheimer’s Disease in rural South Africa

AbstractBackgroundThe characterization of Alzheimer’s disease (AD) and AD related dementias (ADRD) pathophysiology has been revolutionized by the development of highly sensitive blood‐based biomarkers. Although blood‐based biomarkers allow for greater access, cost effectiveness, and scalability, there are limitations for their implementation in resource‐constrained low‐ and middle‐income countries (LMICs) and rural settings, where access to equipment, freezers, and assays is often limited. Dried blood spot (DBS) collection emerges as a promising, convenient, and cost‐effective method for acquiring blood samples in these contexts, but it is unclear whether highly sensitive assays typically applied to cerebrospinal fluid (CSF), plasma, or serum can detect biomarker concentrations accurately. This pilot study assessed the feasibility of using DBS to measure blood‐based AD biomarkers by examining the agreement between ADRD biomarker concentrations derived from DBS and from plasma in older adults in rural South Africa.MethodData were collected from the Health and Aging in Africa: A Longitudinal Study of an INDEPTH Community in South Africa (HAALSI), an ongoing population cohort of 5,059 adults aged ≥40 years living in Agincourt, South Africa. This study focuses on a sub‐sample of 95 participants, whose venous blood and DBS were collected during the HAALSI visit and later tested for blood‐based ADRD biomarkers, including neurofilament light chain (NfL), phosphorylated‐tau181 (p‐tau181), and glial fibrillary acidic protein (GFAP). Plasma and DBS markers were analyzed using commercially available Single molecule array assays (Quanterix) at the University of Gothenburg. We ran pairwise correlations that examined the association between the plasma and DBS measures of p‐tau181, NfL, and GFAP.ResultThe sample consisted of 70.5% women, with an average age of 67.53 years (SD=9.69). We observed a positive correlation between plasma and DBS GFAP concentrations (r=0.78, p‐value&lt;0.001). However, DBS and plasma concentrations were not correlated for p‐tau181 (r=0.05, p‐value=0.62) or for NfL (r=‐0.04, p‐value=0.68).ConclusionThese preliminary analyses suggest that GFAP concentration, a marker of astrogliosis, can be measured consistently in DBS. These findings have important implications for the selection and analysis of AD blood‐based biomarkers in low‐resource and LMIC settings, where venous blood draws may not be feasible.

Neurodegeneration is highly associated with comorbidity burden in HFrEF patients

Abstract Background Heart failure (HF) is associated with cognitive decline and risk for dementia. Blood levels of amyloid beta 40 and 42 (Aβ40, Aβ42), tau protein (tau), and neurofilament light chain (NfL) have been found to be altered in neurodegenerative disease. Previous research has established a clear association between these neuromarkers and the risk of developing cognitive dysfunction and mortality in the general population. Objective The aim of this study was to investigate the association between the respective neuromarkers and comorbidity burden, as well as to explore whether comorbidity burden impacts the relationship between neuromarkers and outcome in HF patients. Methods Plasma levels of Aβ40, Aβ42, tau, and NfL were quantified using a single-molecule array assay. The primary outcome parameter was all-cause mortality. Results A total of 470 HFrEF patients were enrolled in the study, with a median age of 62 years (IQR: 52–72); 77% were male. Median NT-proBNP concentration was 1812 pg/ml (IQR: 718–3881). Elevated levels of all biomarkers were closely associated with comorbidity burden (Figure 1). All neuromarkers showed increased levels in individuals with chronic kidney disease (CKD) and known carotid artery stenosis (CAS), except for Aβ40, which did not show significant elevation in CAS (NfL: p&amp;lt;0.001 for both; t-tau: p&amp;lt;0.001 and p=0.041; Aβ40: p&amp;lt;0.001 and p=0.050; Aβ42: p&amp;lt;0.001 and p=0.033, respectively). In patients with type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD), only NfL levels were significantly elevated (p&amp;lt;0.001 for both), whereas atrial fibrillation (AF) was associated with higher levels of NfL and t-tau (p&amp;lt;0.001 for both). In individuals with iron deficiency, only Aβ42 levels were significantly higher (p=0.025). All biomarkers showed significant associations with all-cause mortality [crude HR for an increase in 1-log unit (95%CI): NfL 4.44 (3.02-6.53), t-tau 5.04 (2.97-8.58), Aβ40 3.90 (2.27-6.72), Aβ42 5.14 (2.84-9.32); p&amp;lt;0.001 for all] (Figure 2). These associations remained statistically significant after adjusting for comorbidity burden including AF, T2DM, CAD, CKD, CAS and ID [adjusted HR for an increase in 1-log unit (95%CI): NfL 3.28 (1.93-5.57), t-tau 3.96 (2.07-7.57), Aβ40 2.42 (1.34-4.38), Aβ42 3.05 (1.56-5.97); p&amp;lt;0.01 for all]. Conclusion The findings suggest a close connection between elevated levels of markers of neurodegeneration and increased comorbidity burden in HFrEF patients. While this association may indicate the cumulative risk leading to neuronal injury and HFrEF progression, a causal relationship cannot be excluded. The observed accelerated neurodegeneration in HFrEF underscores the need for further investigation into this complex interplay.Figure 1Figure 2

Serum neurofilament light chain in response to probiotics in bi-center, double-blind, randomized, placebo-controlled clinical trial (CleverAge Biota).

Neurodegenerative disorders affecting the brain and spinal cord are caused by a large number of factors. More recently, imbalances in gut microbiota are found to be one factor linked directly to neurological dysfunction. Probiotics prevent cognitive decline. For the first time, the effect of probiotics was assessed by monitoring the concentrations of the neurodegeneration biomarker neurofilament light chains (NfL) in a well-defined group of community-dwelling individuals. The aim of this study was to determine whether administration of our new probiotics could reduce NfL concentrations. The serum NfL concentrations were measured in total of 190 serum samples of 85 older community-dwelling individuals. The participants were randomly divided into two groups: the PROPLA group and the PLAPRO group. Individuals in the PROPLA group started with a three-month use of probiotics and continued with a three-month use of placebo while the order was reversed in the PLAPRO group. The participants underwent detailed examinations at three time points: at baseline, in three and six months. The serum NfL concentrations were determined using ultrasensitive single-molecule array (SIMOA) assay. Longitudinal comparisons of NfL concentrations between samplings at different time points in the PROPLA and PLAPRO groups showed no statistically significant differences. Baseline NfL concentrations at the beginning of the study and in the succeeding samplings were not significantly different for the two groups in cross-sectional comparisons. Serum NfL concentrations were not influenced by the three-month use of probiotics.

Blood biomarkers of neuronal injury and astrocytic reactivity in electroconvulsive therapy.

Despite electroconvulsive therapy (ECT) being recognized as an effective treatment for major depressive episodes (MDE), its application is subject to controversy due to concerns over cognitive side effects. The pathophysiology of these side effects is not well understood. Here, we examined the effects of ECT on blood-based biomarkers of neuronal injury and astrocytic reactivity. Participants with a major depressive episode (N = 99) underwent acute ECT. Blood was sampled just before (T0) and 30 min after (T1) the first ECT session, as well as just before the sixth session (T2; 48-72 h after the fifth session). Age- and sex-matched controls (N = 99) were recruited from the general population. Serum concentrations of neurofilament light chain (NfL), total tau protein, and glial fibrillary acidic protein (GFAP) were measured with ultrasensitive single-molecule array assays. Utilizing generalized least squares regression, we compared baseline (T0) biomarker concentrations against those of our control group, and calculated the shifts in serum biomarker concentrations from baseline to immediately post-first ECT session (T1), and prior to the sixth session (T2). Baseline analysis revealed that serum levels of NfL (p < 0.001) and tau (p = 0.036) were significantly elevated in ECT recipients compared with controls, whereas GFAP levels showed no significant difference. Relative to T0, serum NfL concentration neither changed at T1 (mean change 3.1%, 95%CI -0.5% to 6.7%, p = 0.088) nor at T2 (mean change -3.2%, 95%CI -7.6%to1.5%, p = 0.18). Similarly, no change in total tau was observed (mean change 3.7%, 95%CI -11.6% to 21.7%, p = 0.65). GFAP increased from T0 to T1 (mean change 20.3%, 95%CI 14.6to26.3%, p < 0.001), but not from T0 to T2 (mean change -0.7%, 95%CI -5.8% to 4.8%, p = 0.82). In conclusion, our findings suggest that ECT induces a temporary increase in serum GFAP, possibly reflecting transient astrocytic activation. Importantly, we observed no indicators of neuronal damage or long-term elevation in any assessed biomarker.

Plasma pTau181 and pTau217 predict asymptomatic amyloid accumulation equally well as amyloid PET.

The dynamic phase of preclinical Alzheimer's disease, as characterized by accumulating cortical amyloid-β, is a window of opportunity for amyloid-β-lowering therapies to have greater efficacy. Biomarkers that accurately predict amyloid-β accumulation may be of critical importance for participant inclusion in secondary prevention trials and thus enhance development of early Alzheimer's disease therapies. We compared the abilities of baseline plasma pTau181, pTau217 and amyloid-β PET load to predict future amyloid-β accumulation in asymptomatic elderly. In this longitudinal cohort study, baseline plasma pTau181 and pTau217 were quantified using single molecule array assays in cognitively unimpaired elderly selected from the community-recruited F-PACK cohort based on the availability of baseline plasma samples and longitudinal amyloid-β PET data (median time interval = 5 years, range 2-10 years). The predictive abilities of pTau181, pTau217 and PET-based amyloid-β measures for PET-based amyloid-β accumulation were investigated using receiver operating characteristic analyses, correlations and stepwise regression analyses. We included 75 F-PACK subjects (mean age = 70 years, 48% female), of which 16 were classified as amyloid-β accumulators [median (interquartile range) Centiloid rate of change = 3.42 (1.60) Centiloids/year). Plasma pTau181 [area under the curve (95% confidence interval) = 0.72 (0.59-0.86)] distinguished amyloid-β accumulators from non-accumulators with similar accuracy as pTau217 [area under the curve (95% confidence interval) = 0.75 (0.62-0.88) and amyloid-β PET [area under the curve (95% confidence interval) = 0.72 (0.56-0.87)]. Plasma pTau181 and pTau217 strongly correlated with each other (r = 0.93, Pfalse discovery rate < 0.001) and, together with amyloid-β PET, similarly correlated with amyloid-β rate of change (r pTau181 = 0.33, r pTau217 = 0.36, r amyloid-β PET = 0.35, all Pfalse discovery rate ≤ 0.01). Addition of plasma pTau181, plasma pTau217 or amyloid-β PET to a linear demographic model including age, sex and APOE-ε4 carriership similarly improved the prediction of amyloid-β accumulation (ΔAkaike information criterion ≤ 4.1). In a multimodal biomarker model including all three biomarkers, each biomarker lost their individual predictive ability. These findings indicate that plasma pTau181, plasma pTau217 and amyloid-β PET convey overlapping information and therefore predict the dynamic phase of asymptomatic amyloid-β accumulation with comparable performances. In clinical trial recruitment, confirmatory PET scans following blood-based prescreening might thus not provide additional value for detecting participants in these early disease stages who are destined to accumulate cortical amyloid-β. Given the moderate performances, future studies should investigate whether integrating plasma pTau species with other factors can improve performance and thus enhance clinical and research utility.

Quantification of neurofilament light and glial fibrillary acidic protein in finger-prick blood.

An accurate diagnosis of neurodegenerative disease and traumatic brain injury is important for prognostication and treatment. Neurofilament light and glial fibrillary acidic protein (GFAP) are leading biomarkers for neurodegeneration and glial activation that are detectable in blood. Yet, current recommendations require rapid centrifugation and ultra-low temperature storage post-venepuncture. Here, we investigated if these markers can be accurately measured in finger-prick blood using dried plasma spot cards. Fifty patients (46 with dementia; 4 with traumatic brain injury) and 19 healthy volunteers underwent finger-prick and venous sampling using dried plasma spot cards and aligned plasma sampling. Neurofilament light and GFAP were quantified using a Single molecule array assay and correlations between plasma and dried plasma spot cards assessed. Biomarker concentrations in plasma and finger-prick dried plasma spot samples were significantly positively correlated (neurofilament light ρ = 0.57; GFAP ρ = 0.58, P < 0.001). Finger-prick neurofilament light and GFAP were significantly elevated after acute traumatic brain injury with non-significant group-level increases in dementia (91% having Alzheimer's disease dementia). In conclusion, we present preliminary evidence that quantifying GFAP and neurofilament light using finger-prick blood collection is viable, with samples stored at room temperature using dried plasma spot cards. This has potential to expand and promote equitable testing access, including in settings where trained personnel are unavailable to perform venepuncture.

Neurofilament Light Chain Concentration in Cerebrospinal Fluid in Children with Acute Nontraumatic Neurological Disorders.

(1) Introduction: This pilot study aimed to analyze neurofilament light chain levels in cerebrospinal fluid (cNfL) in a cohort of children with different acute nontraumatic neurological conditions. (2) Methods: This prospective observational cohort study consisted of 35 children aged 3 months to 17 years and was performed from November 2017 to December 2019. Patients' clinical data were reviewed, and patients were assigned to the following groups: n = 10 (28.6%) meningitis, 5 (14.3%) Bell's palsy, 7 (20.0%) febrile non-CNS infection, 3 (8.6%) complex febrile seizure, 4 (11.4%) idiopathic intracranial hypertension, and 6 (17.1%) others. cNfL levels were measured using a sensitive single-molecule array assay. (3) Results: The cNfL levels [median (range)] in children with meningitis were 120.5 pg/mL (58.1-205.4), in Bell's palsy 88.6 pg/mL (48.8-144.5), in febrile non-CNS infection 103.9 pg/mL (60.1-210.8), in complex febrile seizure 56 pg/mL (53.2-58.3), and in idiopathic intracranial hypertension 97.1 pg/mL (60.1-124.6). Within the meningitis group, children with Lyme neuroborreliosis (LNB) had significantly higher cNfL concentrations (median 147.9 pg/mL; range 87.8-205.4 pg/mL) than children with enterovirus meningitis (72.5 pg/mL; 58.1-95.6 pg/mL; p = 0.048) and non-significantly higher cNfL levels when compared to Bell's palsy (88.6 pg/mL; 48.8-144.5 pg/mL; p = 0.082). There was no correlation between cNfL levels and age. (4) Conclusions: Although the number of patients in this pilot study cohort is limited, higher cNfL levels in children with LNB compared to those with viral meningitis (significant) and Bell's palsy (trend) may indicate the potential of cNfL as a biomarker in the differential diagnosis of pediatric meningitis and facial palsy.

Longitudinal analysis of serum neurofilament light chain levels as marker for neuronal damage in hereditary transthyretin amyloidosis

Objective To evaluate serum neurofilament light chain (sNfL) as biomarker of disease onset, progression and treatment effect in hereditary transthyretin (ATTRv) amyloidosis patients and TTR variant (TTRv) carriers. Methods sNfL levels were assessed longitudinally in persistently asymptomatic TTRv carriers (N = 12), persistently asymptomatic ATTRv amyloidosis patients (defined as asymptomatic patients but with amyloid detectable in subcutaneous abdominal fat tissue) (N = 8), in TTRv carriers who developed polyneuropathy (N = 7) and in ATTRv amyloidosis patients with polyneuropathy on treatment (TTR-stabiliser (N = 20) or TTR-silencer (N = 18)). Polyneuropathy was confirmed by nerve conduction studies or quantitative sensory testing. sNfL was analysed using a single-molecule array assay. Results sNfL increased over 2 years in persistently asymptomatic ATTRv amyloidosis patients, but did not change in persistently asymptomatic TTRv carriers. In all TTRv carriers who developed polyneuropathy, sNfL increased from 8.4 to 49.8 pg/mL before the onset of symptoms and before polyneuropathy could be confirmed neurophysiologically. In symptomatic ATTRv amyloidosis patients on a TTR-stabiliser, sNfL remained stable over 2 years. In patients on a TTR-silencer, sNfL decreased after 1 year of treatment. Conclusion sNfL is a biomarker of early neuronal damage in ATTRv amyloidosis already before the onset of polyneuropathy. Current data support the use of sNfL in screening asymptomatic TTRv carriers and in monitoring of disease progression and treatment effect.

Serum neurofilament light chain associates with symptom burden in Lyme neuroborreliosis patients: a longitudinal cohort study from Norway

ObjectivesSerum neurofilament light chain (sNfL), an indicator of neuronal damage, is increasingly recognized as a potential biomarker for disease activity in neurodegenerative disorders. In this study, we wanted to investigate sNfL as a prognostic marker in a large, well-defined population of 90 patients with Lyme neuroborreliosis (LNB). In addition, we sought to explore associations between symptoms and sNfL levels during the acute phase of LNB.Materials and methodsPatients diagnosed with definite or possible LNB were recruited from a double-blinded, placebo-controlled, multi-center trial, in which the participants were randomly assigned to 2 or 6 weeks of oral doxycycline treatment. The sNfL levels were measured using a single molecule array assay at both diagnosis and 6-month follow-up, and analysed against clinical parameters, variations in symptom burden and long-term complaints as assessed by a composite clinical score.ResultsAt the time of diagnosis, approximately 60% of the patients had elevated sNfL levels adjusted for age. Notably, mean sNfL levels were significantly higher at diagnosis (52 pg/ml) compared to 6 months after treatment (12 pg/ml, p < 0.001), when sNfL levels had normalized in the majority of patients. Patients with objective signs of spinal radiculitis had significantly higher baseline sNfL levels compared to patients without spinal radiculitis (p = 0.033).ConclusionOur findings suggest that sNfL can serve as a biomarker for peripheral nerve tissue involvement in the acute phase of LNB. As found in an earlier study, we confirm normalization of sNfL levels in blood after treatment. We found no prognostic value of acute-phase sNfL levels on patient outcome.

Combination protein biomarkers predict multiple sclerosis diagnosis and outcomes

Establishing biomarkers to predict multiple sclerosis diagnosis and prognosis has been challenging using a single biomarker approach. We hypothesised that a combination of biomarkers would increase the accuracy of prediction models to differentiate multiple sclerosis from other neurological disorders and enhance prognostication for people with multiple sclerosis. We measured 24 fluid biomarkers in the blood and cerebrospinal fluid of 77 people with multiple sclerosis and 80 people with other neurological disorders, using ELISA or Single Molecule Array assays. Primary outcomes were multiple sclerosis versus any other diagnosis, time to first relapse, and time to disability milestone (Expanded Disability Status Scale 6), adjusted for age and sex. Multivariate prediction models were calculated using the area under the curve value for diagnostic prediction, and concordance statistics (the percentage of each pair of events that are correctly ordered in time for each of the Cox regression models) for prognostic predictions. Predictions using combinations of biomarkers were considerably better than single biomarker predictions. The combination of cerebrospinal fluid [chitinase-3-like-1 + TNF-receptor-1 + CD27] and serum [osteopontin + MCP-1] had an area under the curve of 0.97 for diagnosis of multiple sclerosis, compared to the best discriminative single marker in blood (osteopontin: area under the curve 0.84) and in cerebrospinal fluid (chitinase-3-like-1 area under the curve 0.84). Prediction for time to next relapse was optimal with a combination of cerebrospinal fluid[vitamin D binding protein + Factor I + C1inhibitor] + serum[Factor B + Interleukin-4 + C1inhibitor] (concordance 0.80), and time to Expanded Disability Status Scale 6 with cerebrospinal fluid [C9 + Neurofilament-light] + serum[chitinase-3-like-1 + CCL27 + vitamin D binding protein + C1inhibitor] (concordance 0.98). A combination of fluid biomarkers has a higher accuracy to differentiate multiple sclerosis from other neurological disorders and significantly improved the prediction of the development of sustained disability in multiple sclerosis. Serum models rivalled those of cerebrospinal fluid, holding promise for a non-invasive approach. The utility of our biomarker models can only be established by robust validation in different and varied cohorts.

Emerging biomarkers for improving pregnancy planning in multiple sclerosis.

Patient disability, relapse rate, and age are used for family planning in multiple sclerosis (MS). However, the need for more accurate biomarkers is widely recognized. We aimed to explore the influence of age on neurofilament light chain (sNfL), which reflects acute inflammation; glial fibrillary acidic protein (GFAP), associated with disability progression independent of relapses; and anti-Müllerian hormone (AMH), reflecting ovarian reserve, to provide a tailored family planning strategy. This case-control study included 95 MS patients and 61 healthy control women (HCW). sNfL and GFAP levels were measured using a sensitive single-molecule array assay. AMH levels were measured by the automated Elecsys® Anti-Müllerian Hormone Assay. We observed no significant differences in AMH values between MS patients and the control group within any of the age-matched categories. Age exhibited a negative correlation with AMH values in both groups, as expected. Nevertheless, our findings suggest a slight tendency toward reduced ovarian reserve in MS patients (rho MS patients = -0.67, p < 0.0001; rho HCW = -0.43, p = 0.0006). Interestingly, among the 76 MS participants under 40 years old, we identified ten individuals (13.1%) with AMH levels below 0.7 ng/ml, indicative of a low ovarian reserve, and an additional six individuals (7.8%) with AMH levels between 0.7 ng/ml and 0.9 ng/ml, suggesting a potential risk of premature ovarian failure. Conversely, sNfL and GFAP levels in the MS group exhibited high variability but showed no significant association with age intervals. We found no significant differences in AMH, sNfL or GFAP values between MS patients and the control group within any of the age-matched categories. The assessment of AMH, sNFL and GFAP levels at MS onset facilitates personalized therapeutic and family planning strategies for childbearing-age women.

Analysis of Clinical Samples of Pancreatic Cyst's Lesions with A Multi-Analyte Bioelectronic Simot Array Benchmarked Against Ultrasensitive Chemiluminescent Immunoassay.

Pancreatic cancer, ranking as the third factor in cancer-related deaths, necessitates enhanced diagnostic measures through early detection. In response, SiMoT-Single-molecule with a large Transistor multiplexing array, achieving a Technology Readiness Level of 5, is proposed for a timely identification of pancreatic cancer precursor cysts and is benchmarked against the commercially available chemiluminescent immunoassay SIMOA (Single molecule array) SP-X System. A cohort of 39 samples, comprising 33 cyst fluids and 6 blood plasma specimens, undergoes detailed examination with both technologies. The SiMoT array targets oncoproteins MUC1 and CD55, and oncogene KRAS, while the SIMOA SP-X planar technology exclusively focuses on MUC1 and CD55. Employing Principal Component Analysis (PCA) for multivariate data processing, the SiMoT array demonstrates effective discrimination of malignant/pre-invasive high-grade or potentially malignant low-grade pancreatic cysts from benign non-mucinous cysts. Conversely, PCA analysis applied to SIMOA assay reveals less effective differentiation ability among the three cyst classes. Notably, SiMoT unique capability of concurrently analyzing protein and genetic markers with the threshold of one single molecule in 0.1mL positions it as a comprehensive and reliable diagnostic tool. The electronic response generated by the SiMoT array facilitates direct digital data communication, suggesting potential applications in the development of field-deployable liquid biopsy.