Plasma p-tau231: a new biomarker for incipient Alzheimer's disease pathology.
Ashton Nicholas J,Pascoal Tharick A,Karikari Thomas K,Benedet Andréa L,Lantero-Rodriguez Juan,Brinkmalm Gunnar,Snellman Anniina,Schöll Michael,Troakes Claire,Hye Abdul,Gauthier Serge,Vanmechelen Eugeen,Zetterberg Henrik,Rosa-Neto Pedro,Blennow Kaj
The quantification of phosphorylated tau in biofluids, either cerebrospinal fluid (CSF) or plasma, has shown great promise in detecting Alzheimer's disease (AD) pathophysiology. Tau phosphorylated at threonine 231 (p-tau231) is one such biomarker in CSF but its usefulness as a blood biomarker is currently unknown. Here, we developed an ultrasensitive Single molecule array (Simoa) for the quantification of plasma p-tau231 which was validated in four independent cohorts (n = 588) in different settings, including the full AD continuum and non-AD neurodegenerative disorders. Plasma p-tau231 was able to identify patients with AD and differentiate them from amyloid-β negative cognitively unimpaired (CU) older adults with high accuracy (AUC = 0.92-0.94). Plasma p-tau231 also distinguished AD patients from patients with non-AD neurodegenerative disorders (AUC = 0.93), as well as from amyloid-β negative MCI patients (AUC = 0.89). In a neuropathology cohort, plasma p-tau231 in samples taken on avergae 4.2 years prior to post-mortem very accurately identified AD neuropathology in comparison to non-AD neurodegenerative disorders (AUC = 0.99), this is despite all patients being given an AD dementia diagnosis during life. Plasma p-tau231 was highly correlated with CSF p-tau231, tau pathology as assessed by [F]MK-6240 positron emission tomography (PET), and brain amyloidosis by [F]AZD469 PET. Remarkably, the inflection point of plasma p-tau231, increasing as a function of continuous [F]AZD469 amyloid-β PET standardized uptake value ratio, was shown to be earlier than standard thresholds of amyloid-β PET positivity and the increase of plasma p-tau181. Furthermore, plasma p-tau231 was significantly increased in amyloid-β PET quartiles 2-4, whereas CSF p-tau217 and plasma p-tau181 increased only at quartiles 3-4 and 4, respectively. Finally, plasma p-tau231 differentiated individuals across the entire Braak stage spectrum, including Braak staging from Braak 0 through Braak I-II, which was not observed for plasma p-tau181. To conclude, this novel plasma p-tau231 assay identifies the clinical stages of AD and neuropathology equally well as plasma p-tau181, but increases earlier, already with subtle amyloid-β deposition, prior to the threshold for amyloid-β PET positivity has been attained, and also in response to early brain tau deposition. Thus, plasma p-tau231 is a promising novel biomarker of emerging AD pathology with the potential to facilitate clinical trials to identify vulnerable populations below PET threshold of amyloid-β positivity or apparent entorhinal tau deposition.
Plasma neurofilament light chain levels in Alzheimer's disease.
Zhou Wenjun,Zhang Jie,Ye Fanlong,Xu Guangzheng,Su Hang,Su Yindan,Zhang Xiangyang,
Plasma neurofilament light (NFL) levels may be a marker of neuronal injury. We examined whether plasma NFL might be a potential biomarker for the prodromal and dementia stages of AD. Participants included 193 cognitively normal (CN), 198 amnestic mild cognitive impairment (aMCI) and 187 Alzheimer's disease (AD) individuals enrolled in the Alzheimer's Disease Neuroimaging Initiative (ADNI). Plasma NFL levels were examined by the Single Molecule array (Simoa) technique. Our results showed significantly increased plasma NFL levels in both AD (50.9pg/ml) and aMCI (43.0pg/ml) groups compared to CN (34.7pg/ml) group (both p<0.001), but with substantial overlap between the groups. Plasma NFL levels in AD group was also markedly increased, compared with aMCI group (p<0.001). Plasma NFL levels were positively associated with age (r=0.355, p<0.001) and negatively with global cognition (r=-0.355, p<0.001) in all subjects. Our results suggest that plasma NFL levels may not be a useful biomarker for the diagnosis of prodromal and dementia stages of AD.
Neurogranin and tau in cerebrospinal fluid and plasma of patients with acute ischemic stroke.
De Vos Ann,Bjerke Maria,Brouns Raf,De Roeck Naomi,Jacobs Dirk,Van den Abbeele Lien,Guldolf Kaat,Zetterberg Henrik,Blennow Kaj,Engelborghs Sebastiaan,Vanmechelen Eugeen
BACKGROUND:While neurogranin has no value as plasma biomarker for Alzheimer's disease, it may be a potential blood biomarker for traumatic brain injury. This evokes the question whether there are changes in neurogranin levels in blood in other conditions of brain injury, such as acute ischemic stroke (AIS). METHODS:We therefore explored neurogranin in paired cerebrospinal fluid (CSF)/plasma samples of AIS patients (n = 50) from a well-described prospective study. In parallel, we investigated another neuronal protein, i.e. tau, which has already been suggested as potential AIS biomarker in CSF and blood. ELISA as well as Single Molecule Array (Simoa) technology were used for the biochemical analyses. Statistical analyses included Shapiro-Wilk testing, Mann-Whitney analyses and Pearson's correlation analysis. RESULTS:In contrast to tau, of which high levels in both CSF and plasma were related to stroke characteristics like severity and long-term outcome, plasma neurogranin levels were only correlated with infarct volume. Likewise, CSF neurogranin levels were significantly higher in patients with an infarct volume > 5 mL than in patients with smaller infarct volumes. Finally, neurogranin and tau were significantly correlated in CSF, whereas a weaker relationship was observed in plasma. CONCLUSIONS:These findings indicate that although plasma and CSF neurogranin may reflect the volume of acute cerebral ischemia, this synaptic protein is less likely to be a potential AIS biomarker. Levels of tau correlated with severity and outcome of stroke in both plasma and CSF, in the present study as well as previous reports, confirming the potential of tau as an AIS biomarker.
Monitoring Disease Activity in Systemic Lupus Erythematosus With Single-Molecule Array Digital Enzyme-Linked Immunosorbent Assay Quantification of Serum Interferon-α.
Mathian Alexis,Mouries-Martin Suzanne,Dorgham Karim,Devilliers Hervé,Barnabei Laura,Ben Salah Elyès,Cohen-Aubart Fleur,Garrido Castillo Laura,Haroche Julien,Hie Miguel,Pineton de Chambrun Marc,Miyara Makoto,Sterlin Delphine,Pha Micheline,Lê Thi Huong Du,Rieux-Laucat Frédéric,Rozenberg Flore,Gorochov Guy,Amoura Zahir
Arthritis & rheumatology (Hoboken, N.J.)
OBJECTIVE:No simple or standardized assay is available to quantify interferon-α (IFNα) in routine clinical practice. Single-molecule array (Simoa) digital enzyme-linked immunosorbent assay (ELISA) technology enables direct IFNα quantification at attomolar (femtogram per milliliter [fg/ml]) concentrations. This study was undertaken to assess IFNα digital ELISA diagnostic performances to monitor systemic lupus erythematosus (SLE) activity. METHODS:IFNα concentrations in serum samples from 150 consecutive SLE patients in a cross-sectional study were determined with digital ELISA and a functional biologic activity assay (bioassay). According to their Safety of Estrogens in Lupus Erythematosus National Assessment version of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) flare composite scores, patients were divided into groups with inactive SLE (SLEDAI score of <4 or clinical SLEDAI score of 0) or active SLE (SLEDAI score of ≥4 or clinical SLEDAI score of >0), and into groups with no flare or mild/moderate flare or severe flare. RESULTS:Based on serum samples from healthy blood donors, the abnormal serum IFNα level threshold value was 136 fg/ml. Next, using receiver operating characteristic curves for an SLE patient series that was widely heterogeneous in terms of disease activity and organ involvement, the threshold IFNα value associated with active disease was determined to be 266 fg/ml. The digital ELISA-assessed serum IFNα level was a better biomarker of disease activity than the Farr assay because its specificity, likelihood ratio for positive results, and positive predictive value better discerned active SLE or flare from inactive disease. The digital ELISA was more sensitive than the bioassay for detecting low-abnormal serum IFNα concentrations and identifying patients with low disease activity. CONCLUSION:Direct serum IFNα determination with a highly sensitive assay might improve monitoring of clinical SLE activity and selection of the best candidates for anti-IFNα treatment.
Plasma IL-6 and IL-17A Correlate with Severity of Motor and Non-Motor Symptoms in Parkinson's Disease.
Green Holly F,Khosousi Shervin,Svenningsson Per
Journal of Parkinson's disease
The nature of the inflammatory response in Parkinson's disease (PD) remains to be better understood. Here, we used highly sensitive Single Molecule Array (SIMOA) technology to measure the levels of the inflammatory mediators Interleukin 6 (IL-6), Interleukin 17A (IL-17A), Tumour Necrosis Factor α (TNFα) and Transforming Growth Factor β (TGFβ) in plasma from PD patients and age- and gender-matched healthy controls. We report that IL-17A correlates with non-motor symptoms (NMS) scores, while IL-6 positively correlates with motor scores. We found no correlations between cytokines and disease duration suggesting that IL-6 and IL-17A are associated with disease severity rather than disease duration in this cohort, furthermore IL-17A may be involved in the underlying pathophysiology of NMS in PD.
Plasma neurofilaments correlate with disability in progressive multiple sclerosis patients.
Ferraro Diana,Guicciardi Claudio,De Biasi Sara,Pinti Marcello,Bedin Roberta,Camera Valentina,Vitetta Francesca,Nasi Milena,Meletti Stefano,Sola Patrizia
Acta neurologica Scandinavica
OBJECTIVES:Cerebrospinal fluid (CSF) and blood neurofilaments (NFLs) are markers of axonal damage and are being investigated, mostly in relapsing-remitting (RR) MS, as a marker of disease activity and of response to treatment, while there are less data in progressive MS patients. Primary aim was to measure NFL in plasma samples of untreated patients with primary (PP) and secondary (SP) progressive MS and to correlate them with disability, disease severity, and prior/subsequent disability progression. MATERIALS AND METHODS:Neurofilament concentrations were measured using SIMOA (Single Molecule Array, Simoa HD-1 Analyzer; Quanterix). RESULTS:Neurofilament concentrations were measured on plasma samples of 70 progressive (27 PP and 43 SP), 21 RRMS patients, and 10 HCs. Longitudinal plasma NFL (pNFL) concentrations (median interval between sampling: 25 months) were available for nine PP/SP patients. PNFL concentrations were significantly higher in PP/SP compared to RRMS patients. They correlated with EDSS and MS Severity Score values. There was no difference in pNFL levels between PP/SP patients with EDSS progression in the preceding year (14% of patients) or during a median follow-up of 27 months (41%). In the longitudinal sub-study, pNFL levels increased in all patients between sampling by a mean value of 23% while EDSS mostly remained stable (77% of cases). CONCLUSION:In PP/SP progressive MS patients, pNFL levels correlate with disability and increase over time, but are not associated with prior/subsequent disability progression, as measured by EDSS, which may not be a sufficiently sensitive tool in this context.
Serum neurofilament light chain (NFL) remains unchanged during electroconvulsive therapy.
Besse Matthias,Belz Michael,Folsche Thorsten,Vogelgsang Jonathan,Methfessel Isabel,Steinacker Petra,Otto Markus,Wiltfang Jens,Zilles David
The world journal of biological psychiatry : the official journal of the World Federation of Societies of Biological Psychiatry
Although there is consistent evidence that electroconvulsive therapy (ECT) is safe and well tolerated by the majority of patients, some authors still accuse ECT to inevitably cause brain damage and permanent memory loss, assertions that may increase patients' worries about a useful treatment. Recently, the measurement of neurofilament light chain (NFL) in peripheral blood was technically implemented, permitting longitudinal analysis of this biomarker for axonal damage. NFL is part of the axonal cytoskeleton and is released into the CSF and peripheral blood in the context of neuronal damage. In our study, blood from 15 patients with major depressive disorder receiving ECT was collected before the first ECT as well as 24 h and seven days after the last ECT, respectively. NFL concentrations were analysed using the ultrasensitive single molecule array (Simoa) technology. NFL concentrations did not differ between patients and healthy controls, and there was no significant change in NFL levels in the course of ECT. On the contrary, we even found a slight decrease in absolute NFL concentrations. Our study confirms the safety of ECT by using a most sensitive method for the detection of NFL in peripheral blood as a biomarker of neuronal damage.
Serum glial fibrillary acidic protein correlates with multiple sclerosis disease severity.
Högel Heidi,Rissanen Eero,Barro Christian,Matilainen Markus,Nylund Marjo,Kuhle Jens,Airas Laura
Multiple sclerosis (Houndmills, Basingstoke, England)
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.
Plasma amyloid is associated with the rate of cognitive decline in cognitively normal elderly: the SCIENCe project.
Verberk Inge M W,Hendriksen Heleen M A,van Harten Argonde C,Wesselman Linda M P,Verfaillie Sander C J,van den Bosch Karlijn A,Slot Rosalinde E R,Prins Niels D,Scheltens Philip,Teunissen Charlotte E,Van der Flier Wiesje M
Neurobiology of aging
Plasma biomarkers are promising prognostic tools in individuals with subjective cognitive decline (SCD). We aimed to investigate the relationships of baseline plasma amyloid beta (Aβ)42/Aβ40 and total Tau (tTau) with rate of cognitive decline, in comparison to relationships of baseline cerebrospinal fluid (CSF) Aβ42, tTau, and phosphorylated tau181 (pTau181) with rate of cognitive decline. We included 241 subjects with SCD (age = 61 ± 9, 40% female, Mini-Mental State Examination = 28 ± 2) with follow-up (average: 2 ± 2 years, median visits: 3 [range: 1-11]) for re-evaluation of neuropsychological test performance (attention, memory, language, and executive functioning domains). Using age, gender and education-adjusted linear mixed models, we found that lower plasma Aβ42/Aβ40 was associated with steeper rate of decline on tests for attention, memory, and executive functioning, but not language. Lower CSF Aβ42 was associated with steeper decline on tests covering all domains. Associations for plasma amyloid and cognitive decline mirror those of CSF amyloid. Plasma tTau was not associated with rate of cognitive decline, whereas CSF tTau and pTau181 were on multiple tests covering all domains.
Use of high-sensitivity digital ELISA improves the diagnostic performance of circulating brain-specific proteins for detection of traumatic brain injury during triage.
O'Connell Grant C,Alder Megan L,Smothers Christine G,Still Carolyn H,Webel Allison R,Moore Shirley M
: Historically, limited sensitivity associated with traditional immunoassay methods has prevented the use of brain-specific proteins as blood biomarkers of traumatic brain injury (TBI) during triage, as these proteins exhibit low circulating concentrations. Digital ELISA is a newly-developed technique that is up to 1000 times more sensitive than conventional ELISA methods. The purpose of this study was to determine whether the use of digital ELISA over conventional ELISA improves the performance of brain-specific proteins as blood biomarkers of TBI during triage.: Blood was sampled from TBI patients (n = 13) at emergency department admission, as well as from neurologically normal controls (n = 72). Serum levels of two brain-specific proteins, neurofilament light chain (NfL) and Tau, were measured via digital ELISA. Estimated conventional ELISA measures were generated by adjusting values according to the lower limits of detection achievable with commercially available conventional ELISA assays, and receiver operating characteristic (ROC) analysis was used to compare the diagnostic performance of digital ELISA measures to estimated conventional ELISA measures in terms of their ability to discriminate between TBI patients and controls.: Used in combination, digital ELISA measures of NfL and Tau could discriminate between groups with 100% sensitivity and 91.7% specificity. Estimated conventional ELISA measures could only discriminate between groups with 7.7% sensitivity and 94.4% specificity. This difference in diagnostic performance was statistically significant when comparing areas under ROC curves.: The use of digital ELISA over conventional ELISA methods improves the diagnostic performance of circulating brain-specific proteins for detection of TBI during triage.
Plasma β-amyloid in Alzheimer's disease and vascular disease.
Janelidze Shorena,Stomrud Erik,Palmqvist Sebastian,Zetterberg Henrik,van Westen Danielle,Jeromin Andreas,Song Linan,Hanlon David,Tan Hehir Cristina A,Baker David,Blennow Kaj,Hansson Oskar
Implementation of amyloid biomarkers in clinical practice would be accelerated if such biomarkers could be measured in blood. We analyzed plasma levels of Aβ42 and Aβ40 in a cohort of 719 individuals (the Swedish BioFINDER study), including patients with subjective cognitive decline (SCD), mild cognitive impairment (MCI), Alzheimer's disease (AD) dementia and cognitively healthy elderly, using a ultrasensitive immunoassay (Simoa platform). There were weak positive correlations between plasma and cerebrospinal fluid (CSF) levels for both Aβ42 and Aβ40, and negative correlations between plasma Aβ42 and neocortical amyloid deposition (measured with PET). Plasma levels of Aβ42 and Aβ40 were reduced in AD dementia compared with all other diagnostic groups. However, during the preclinical or prodromal AD stages (i.e. in amyloid positive controls, SCD and MCI) plasma concentration of Aβ42 was just moderately decreased whereas Aβ40 levels were unchanged. Higher plasma (but not CSF) levels of Aβ were associated with white matter lesions, cerebral microbleeds, hypertension, diabetes and ischemic heart disease. In summary, plasma Aβ is overtly decreased during the dementia stage of AD indicating that prominent changes in Aβ metabolism occur later in the periphery compared to the brain. Further, increased levels of Aβ in plasma are associated with vascular disease.
A digital enzyme-linked immunosorbent assay for ultrasensitive measurement of amyloid-β 1-42 peptide in human plasma with utility for studies of Alzheimer's disease therapeutics.
Song Linan,Lachno D Richard,Hanlon David,Shepro Adam,Jeromin Andreas,Gemani Dipika,Talbot Jayne A,Racke Margaret M,Dage Jeffrey L,Dean Robert A
Alzheimer's research & therapy
BACKGROUND:Amyloid-β 1-42 peptide (Aβ) is associated with plaque formation in the brain of patients with Alzheimer's disease (AD). Pharmacodynamic studies of AD therapeutics that lower the concentrations of Aβ in peripheral blood require highly sensitive assays for its measurement. A digital enzyme-linked immunosorbent assay (ELISA) using single molecule array (Simoa) technology has been developed that provides improved sensitivity compared with conventional ELISA methods using the same antibody reagents. METHODS:A sensitive digital ELISA for measurement of Aβ using antibodies 3D6 and 21F12 was developed. Assay performance was evaluated by repeated testing of pooled human plasma and buffer diluent quality control samples to determine relative accuracy, intra- and inter-assay precision, limit of detection (LOD), lower limit of quantification (LLOQ), dilutional linearity, and spike recovery. The optimized assay was used to quantify Aβ in clinical samples from patients treated with the β-site amyloid precursor protein cleaving enzyme 1 inhibitor LY2886721. RESULTS:The prototype assay measured Aβ with an LOD of 0.3 pg/ml and an LLOQ of 2.8 pg/ml in plasma, calibrated using an Aβ peptide standard from Fujirebio. Assay precision was acceptable with intra- and inter-assay coefficients of variation both being ≤10%. Dilutional linearity was demonstrated in sample diluent and immunodepleted human plasma. Analyte spike recovery ranged from 51% to 93% with a mean of 80%. This assay was able to quantify Aβ in all of the 84 clinical samples tested. A rapid reduction in levels of Aβ was detected within 1 h after drug treatment, and a dose-dependent decrease of Aβ levels was also observed over the time course of sample collection. CONCLUSIONS:This digital ELISA has potential utility in clinical applications for quantification of Aβ in plasma where high sensitivity and precision are required.
Blood-based NfL: A biomarker for differential diagnosis of parkinsonian disorder.
Hansson Oskar,Janelidze Shorena,Hall Sara,Magdalinou Nadia,Lees Andrew J,Andreasson Ulf,Norgren Niklas,Linder Jan,Forsgren Lars,Constantinescu Radu,Zetterberg Henrik,Blennow Kaj,
OBJECTIVE:To determine if blood neurofilament light chain (NfL) protein can discriminate between Parkinson disease (PD) and atypical parkinsonian disorders (APD) with equally high diagnostic accuracy as CSF NfL, and can therefore improve the diagnostic workup of parkinsonian disorders. METHODS:The study included 3 independent prospective cohorts: the Lund (n = 278) and London (n = 117) cohorts, comprising healthy controls and patients with PD, progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), and multiple system atrophy (MSA), as well as an early disease cohort (n = 109) of patients with PD, PSP, MSA, or CBS with disease duration ≤3 years. Blood NfL concentration was measured using an ultrasensitive single molecule array (Simoa) method, and the diagnostic accuracy to distinguish PD from APD was investigated. RESULTS:We found strong correlations between blood and CSF concentrations of NfL (ρ ≥ 0.73-0.84, ≤ 0.001). Blood NfL was increased in patients with MSA, PSP, and CBS (i.e., all APD groups) when compared to patients with PD as well as healthy controls in all cohorts ( < 0.001). Furthermore, in the Lund cohort, blood NfL could accurately distinguish PD from APD (area under the curve [AUC] 0.91) with similar results in both the London cohort (AUC 0.85) and the early disease cohort (AUC 0.81). CONCLUSIONS:Quantification of blood NfL concentration can be used to distinguish PD from APD. Blood-based NfL might consequently be included in the diagnostic workup of patients with parkinsonian symptoms in both primary care and specialized clinics. CLASSIFICATION OF EVIDENCE:This study provides Class III evidence that blood NfL levels discriminate between PD and APD.
Serum Neurofilament light: A biomarker of neuronal damage in multiple sclerosis.
Disanto Giulio,Barro Christian,Benkert Pascal,Naegelin Yvonne,Schädelin Sabine,Giardiello Antonella,Zecca Chiara,Blennow Kaj,Zetterberg Henrik,Leppert David,Kappos Ludwig,Gobbi Claudio,Kuhle Jens,
Annals of neurology
OBJECTIVE:Neurofilament light chains (NfL) are unique to neuronal cells, are shed to the cerebrospinal fluid (CSF), and are detectable at low concentrations in peripheral blood. Various diseases causing neuronal damage have resulted in elevated CSF concentrations. We explored the value of an ultrasensitive single-molecule array (Simoa) serum NfL (sNfL) assay in multiple sclerosis (MS). METHODS:sNfL levels were measured in healthy controls (HC, n = 254) and two independent MS cohorts: (1) cross-sectional with paired serum and CSF samples (n = 142), and (2) longitudinal with repeated serum sampling (n = 246, median follow-up = 3.1 years, interquartile range [IQR] = 2.0-4.0). We assessed their relation to concurrent clinical, imaging, and treatment parameters and to future clinical outcomes. RESULTS:sNfL levels were higher in both MS cohorts than in HC (p < 0.001). We found a strong association between CSF NfL and sNfL (β = 0.589, p < 0.001). Patients with either brain or spinal (43.4pg/ml, IQR = 25.2-65.3) or both brain and spinal gadolinium-enhancing lesions (62.5pg/ml, IQR = 42.7-71.4) had higher sNfL than those without (29.6pg/ml, IQR = 20.9-41.8; β = 1.461, p = 0.005 and β = 1.902, p = 0.002, respectively). sNfL was independently associated with Expanded Disability Status Scale (EDSS) assessments (β = 1.105, p < 0.001) and presence of relapses (β = 1.430, p < 0.001). sNfL levels were lower under disease-modifying treatment (β = 0.818, p = 0.003). Patients with sNfL levels above the 80th, 90th, 95th, 97.5th, and 99th HC-based percentiles had higher risk of relapses (97.5th percentile: incidence rate ratio = 1.94, 95% confidence interval [CI] = 1.21-3.10, p = 0.006) and EDSS worsening (97.5th percentile: OR = 2.41, 95% CI = 1.07-5.42, p = 0.034). INTERPRETATION:These results support the value of sNfL as a sensitive and clinically meaningful blood biomarker to monitor tissue damage and the effects of therapies in MS. Ann Neurol 2017;81:857-870.
Serum neurofilament light is sensitive to active cerebral small vessel disease.
Gattringer Thomas,Pinter Daniela,Enzinger Christian,Seifert-Held Thomas,Kneihsl Markus,Fandler Simon,Pichler Alexander,Barro Christian,Gröbke Svenya,Voortman Margarete,Pirpamer Lukas,Hofer Edith,Ropele Stefan,Schmidt Reinhold,Kuhle Jens,Fazekas Franz,Khalil Michael
OBJECTIVE:To explore whether serum neurofilament light chain protein (NfL) levels are increased in patients with MRI-confirmed recent small subcortical infarcts (RSSI) compared to healthy controls and to determine the subsequent course and determinants of NfL levels in a longitudinal manner. METHODS:In a prospectively collected group of symptomatic patients with an RSSI (n = 79, mean age 61 ± 11 years, 67% male), we analyzed brain MRI and serum NfL using a Single Molecule Array (Simoa) assay at baseline and at 3 and 15 months after stroke. Community-dwelling healthy age- and sex-matched individuals with comparable severity of MRI white matter hyperintensities (WMH) (n = 53) served as controls. RESULTS:Patients with an RSSI had higher NfL baseline levels compared to controls (73.45 vs 34.59 pg/mL, < 0.0001), and they were increasingly higher with the time from stroke symptom onset to blood sampling (median 4 days, range 1-11 days, = 0.51, < 0.0001). NfL levels remained increased at the 3-month follow-up but returned to normal at 15 months after stroke. NfL levels were associated with RSSI size and baseline WMH severity and were especially high in patients with new, clinically silent cerebral small vessel disease (CSVD)-related lesions at follow-up. CONCLUSIONS:Serum NfL is increased in patients with an RSSI and the occurrence of new CSVD-related MRI lesions, even when clinically silent. This suggests NfL as a blood biomarker for active CSVD.
Long-Term Measurements of Human Inflammatory Cytokines Reveal Complex Baseline Variations between Individuals.
Wu Danlu,Dinh Trinh L,Bausk Bruce P,Walt David R
The American journal of pathology
Comprehensive characterization of the healthy human proteome baseline is essential for personalized medicine. Baseline data are necessary to understand the variation between individuals, as well as longitudinal variation within individuals. Many important protein biomarkers, such as cytokines, exist at extremely low or undetectable levels in the healthy state. This paper describes results from a 14-week study of healthy human subjects using ultrasensitive single-molecule array (Simoa) assays to measure both intra and intersubject variation of 15 cytokines. The results show a wide variation in the ranges of some cytokines between individuals and demonstrate that individual baseline values will be essential for predicting disease presence and progression. Although all of the studied cytokines demonstrated high temporal stability (or low intrasubject variation) over the entire study period, there were two distinct groups of cytokines that demonstrated either high (IL-8, IFN-γ, IL-2, IL-6, and IL-1β) or low (IL-15, TNF-α, IL-12 p70, IL-17A, GM-CSF, IL-12 p40, IL-10, IL-7, IL-1α, and IL-5) subject-to-subject variation. This work demonstrates that ultrasensitive assays are essential for characterizing human cytokines in healthy subjects. The results show that some cytokines vary by more than two orders of magnitude between individuals, making it an imperative to obtain individual baseline measurements if they are to play a role in health and disease diagnosis.
Single Molecule Arrays for ultra-sensitive detection of rat cytokines in serum.
Cohen Limor,Xie Liangxia,Xylas Mark E,Walt David R
Journal of immunological methods
Rats are used as animal models for many human diseases. Cytokines can serve as biomarkers indicative of these diseases or disease states. Techniques for measuring cytokine expression levels often do not provide the sensitivity needed to measure these biomarkers in biological fluids because the concentrations of many cytokines are below the detection limits of conventional methods. In this paper, we present ultra-sensitive digital immunoassays using Single Molecule Arrays (Simoa) for seven rat cytokines: TNF-α, IL-10, IL-17F, GM-CSF, IFN-γ, IL-4, and IL-1α. These ultra-sensitive immunoassays have limits of detection (LODs) in the femtomolar range and provide the ability to measure rat cytokines in serum below the LODs of conventional immunoassays. We also measured these cytokines in healthy rat serum to obtain baseline levels. The ability to measure cytokines present at low concentrations in rat serum will facilitate future studies of disease using rats as animal models.
Blood-based cerebral biomarkers in preeclampsia: Plasma concentrations of NfL, tau, S100B and NSE during pregnancy in women who later develop preeclampsia - A nested case control study.
Bergman Lina,Zetterberg Henrik,Kaihola Helena,Hagberg Henrik,Blennow Kaj,Åkerud Helena
OBJECTIVE:To evaluate if concentrations of the neuronal proteins neurofilament light chain and tau are changed in women developing preeclampsia and to evaluate the ability of a combination of neurofilament light chain, tau, S100B and neuron specific enolase in identifying neurologic impairment before diagnosis of preeclampsia. METHODS:A nested case-control study within a longitudinal study cohort was performed. 469 healthy pregnant women were enrolled between 2004-2007 and plasma samples were collected at gestational weeks 10, 25, 28, 33 and 37. Plasma concentrations of tau and neurofilament light chain were analyzed in 16 women who eventually developed preeclampsia and 36 controls throughout pregnancy with single molecule array (Simoa) method and compared within and between groups. S100B and NSE had been analyzed previously in the same study population. A statistical model with receiving characteristic operation curve was constructed with the four biomarkers combined. RESULTS:Plasma concentrations of neurofilament light chain were significantly increased in women who developed preeclampsia in gestational week 33 (11.85 ng/L, IQR 7.48-39.93 vs 6.80 ng/L, IQR 5.65-11.40) and 37 (22.15 ng/L, IQR 10.93-35.30 vs 8.40 ng/L, IQR 6.40-14.30) and for tau in gestational week 37 (4.33 ng/L, IQR 3.97-12.83 vs 3.77 ng/L, IQR 1.91-5.25) in contrast to healthy controls. A combined model for preeclampsia with tau, neurofilament light chain, S100B and neuron specific enolase in gestational week 25 displayed an area under the curve of 0.77, in week 28 it was 0.75, in week 33 it was 0.89 and in week 37 it was 0.83. Median week for diagnosis of preeclampsia was at 38 weeks of gestation. CONCLUSION:Concentrations of both tau and neurofilament light chain are increased in the end of pregnancy in women developing preeclampsia in contrast to healthy pregnancies. Cerebral biomarkers might reflect cerebral involvement before onset of disease.
Neurofilament as Neuronal Injury Blood Marker in Preeclampsia.
Evers Katrina Suzanne,Atkinson Andrew,Barro Christian,Fisch Urs,Pfister Marc,Huhn Evelyn A,Lapaire Olav,Kuhle Jens,Wellmann Sven
Hypertension (Dallas, Tex. : 1979)
Preeclampsia has been shown to be associated with changes in cerebral structure and cognitive function later in life. Nf (neurofilaments) are specific scaffolding proteins of neurons, and their quantification in serum has been proposed as a biomarker for neuroaxonal injury. We performed a prospective, longitudinal, single-center study at the University Hospital of Basel to determine serum Nf concentrations in pregnant women with singleton pregnancies and with high risk of preeclampsia or with early signs of preeclampsia. Enrollment started at 21 weeks of gestation, followed up with multiple visits until delivery. Sixty out of 197 women developed preeclampsia (30.5%). NfL (Nf light chain) was measured with a highly sensitive single molecule array (Simoa) assay, in addition to the established preeclampsia markers sFlt-1 (soluble fms-like tyrosine kinase-1) and PlGF (placental growth factor). The most important independent predictors of NfL were maternal age, number of pregnancies, and proteinuria. NfL levels increased during pregnancy and were significantly higher in women developing preeclampsia. The discriminatory accuracy of NfL, PlGF, and sFlt-1 in receiver operating characteristic curves analysis (area under the curve) of the overall group was 0.68, 0.81, and 0.84, respectively, and in women older than 36 years 0.7, 0.62, and 0.79, respectively. We conclude that increased axonal injury serum marker NfL predicts preeclampsia particularly in older women, with an accuracy similar to the established angiogenic factors. NfL may serve as an early indicator of preeclampsia-induced changes in cerebral structure and may help to stratify disease management.
Neurofilament levels, disease activity and brain volume during follow-up in multiple sclerosis.
Håkansson Irene,Tisell Anders,Cassel Petra,Blennow Kaj,Zetterberg Henrik,Lundberg Peter,Dahle Charlotte,Vrethem Magnus,Ernerudh Jan
Journal of neuroinflammation
BACKGROUND:There is a need for clinically useful biomarkers of disease activity in clinically isolated syndrome (CIS) and relapsing remitting MS (RRMS). The aim of this study was to assess the correlation between neurofilament light chain (NFL) in cerebrospinal fluid (CSF) and serum and the relationship between NFL and other biomarkers, subsequent disease activity, and brain volume loss in CIS and RRMS. METHODS:A panel of neurodegenerative and neuroinflammatory markers were analyzed in repeated CSF samples from 41 patients with CIS or RRMS in a prospective longitudinal cohort study and from 22 healthy controls. NFL in serum was analyzed using a single-molecule array (Simoa) method. "No evidence of disease activity-3" (NEDA-3) status and brain volume (brain parenchymal fraction calculated using SyMRI®) were recorded during 4 years of follow-up. RESULTS:NFL levels in CSF and serum correlated significantly (all samples, n = 63, r 0.74, p < 0.001), but CSF-NFL showed an overall stronger association profile with NEDA-3 status, new T2 lesions, and brain volume loss. CSF-NFL was associated with both new T2 lesions and brain volume loss during follow-up, whereas CSF-CHI3L1 was associated mainly with brain volume loss and CXCL1, CXCL10, CXCL13, CCL22, and MMP-9 were associated mainly with new T2 lesions. CONCLUSIONS:Serum and CSF levels of NFL correlate, but CSF-NFL predicts and reflects disease activity better than S-NFL. CSF-NFL levels are associated with both new T2 lesions and brain volume loss. Our findings further add to the accumulating evidence that CSF-NFL is a clinically useful biomarker in CIS and RRMS and should be considered in the expanding NEDA concept. CSF-CXCL10 and CSF-CSF-CHI3L1 are potential markers of disease activity and brain volume loss, respectively.
The role of microglia in processing and spreading of bioactive tau seeds in Alzheimer's disease.
Hopp Sarah C,Lin Yang,Oakley Derek,Roe Allyson D,DeVos Sarah L,Hanlon David,Hyman Bradley T
Journal of neuroinflammation
BACKGROUND:Misfolding of microtubule-associated protein tau (MAPT) within neurons into neurofibrillary tangles is an important pathological feature of Alzheimer's disease (AD). Tau pathology correlates with cognitive decline in AD and follows a stereotypical anatomical course; several recent studies indicate that tau pathology spreads inter-neuronally via misfolded tau "seeds." Previous research has focused on neurons as the source of these tau seeds. However, recent studies as well as the data contained herein suggest that microglia, the resident immune cells of the central nervous system, play a direct role in the spread of tau pathology. METHODS:Primary adult microglia were isolated from human AD cases and the rTg4510 tauopathy mouse model and used for analysis of gene expression, tau protein by Simoa technology, and quantification of tau seeding using a highly sensitive fluorescence resonance energy transfer (FRET) biosensing cell line for tau seeding and aggregation. RESULTS:Here, we show that microglia isolated from both human tauopathy and AD cases and the rTg4510 tauopathy mouse model stably contain tau seeds, despite not synthesizing any tau. Microglia releases these tau seeds in vitro into their conditioned media (CM). This suggests that microglia have taken up tau but are incapable of entirely neutralizing its seeding activity. Indeed, when in vitro microglia are given media containing tau seeds, they reduce (but do not eliminate) tau seeding. When microglia are treated with inflammagens such as lipopolysaccharide (LPS), interleukin-1β (IL1β), tumor necrosis factor α (TNFα), or amyloid-β, their ability to reduce tau seeding is unchanged and these factors do not induce seeding activity on their own. CONCLUSIONS:Overall, these data suggest that microglia have a complex role: they are capable of taking up and breaking down seed competent tau, but do so inefficiently and could therefore potentially play a role in the spread of tau pathology.
Neurofilament light chain serum levels correlate with 10-year MRI outcomes in multiple sclerosis.
Chitnis Tanuja,Gonzalez Cindy,Healy Brian C,Saxena Shrishti,Rosso Mattia,Barro Christian,Michalak Zuzanna,Paul Anu,Kivisakk Pia,Diaz-Cruz Camilo,Sattarnezhad Neda,Pierre Isabelle V,Glanz Bonnie I,Tomic Davorka,Kropshofer Harald,Häring Dieter,Leppert David,Kappos Ludwig,Bakshi Rohit,Weiner Howard L,Kuhle Jens
Annals of clinical and translational neurology
Objective:To assess the value of annual serum neurofilament light (NfL) measures in predicting 10-year clinical and MRI outcomes in multiple sclerosis (MS). Methods:We identified patients in our center's Comprehensive Longitudinal Investigations in MS at Brigham and Women's Hospital (CLIMB) study enrolled within 5 years of disease onset, and with annual blood samples up to 10 years ( = 122). Serum NfL was measured using a single molecule array (SIMOA) assay. An automated pipeline quantified brain T2 hyperintense lesion volume (T2LV) and brain parenchymal fraction (BPF) from year 10 high-resolution 3T MRI scans. Correlations between averaged annual NfL and 10-year clinical/MRI outcomes were assessed using Spearman's correlation, univariate, and multivariate linear regression models. Results:Averaged annual NfL values were negatively associated with year 10 BPF, which included averaged year 1-5 NfL values (unadjusted < 0.01; adjusted analysis < 0.01), and averaged values through year 10. Linear regression analyses of averaged annual NfL values showed multiple associations with T2LV, specifically averaged year 1-5 NfL (unadjusted < 0.01; adjusted analysis < 0.01). Approximately 15-20% of the BPF variance and T2LV could be predicted from early averaged annual NfL levels. Also, averaged annual NfL levels with fatigue score worsening between years 1 and 10 showed statistically significant associations. However, averaged NfL measurements were not associated with year 10 EDSS, SDMT or T25FW in this cohort. Interpretation:Serum NfL measured during the first few years after the clinical onset of MS contributed to the prediction of 10-year MRI brain lesion load and atrophy.
Serum GFAP as a biomarker for disease severity in multiple sclerosis.
Abdelhak A,Huss A,Kassubek J,Tumani H,Otto M
While neurofilament light chain (NfL) measurement in serum is a well-established marker of neuroaxonal damage in multiple sclerosis (MS), data on astroglial markers in serum are missing. In our study, glial fibrillary acid protein (GFAP) and NfL were measured in cerebrospinal fluid (CSF) and serum of MS patients and patients with other non-inflammatory neurological diseases (OND) using the Simoa technology. Clinical data like age, gender, expanded disability status scale (EDSS) and MRI findings were correlated to neurochemical markers. We included 80 MS patients: 42 relapsing-remitting MS (RRMS), 38 progressive MS (PMS), as well as 20 OND. Serum GFAP levels were higher in PMS compared to RRMS and OND (p < 0.001, p = 0.02 respectively). Serum GFAP levels correlated with disease severity in the whole MS group and PMS (Spearman-rho = 0.5, p < 0.001 in both groups). Serum GFAP correlated with serum NfL in PMS patients (Spearman-rho = 0.4, p = 0.01). Levels of serum GFAP were higher with increasing MRI-lesion count (p = 0.01). in summary, we report elevated levels of GFAP in the serum of MS patients. Since serum levels of GFAP correlate with the clinical severity scores and MRI lesion count, especially in PMS patients, it might be a suitable disease progression marker.
Plasma Amyloid as Prescreener for the Earliest Alzheimer Pathological Changes.
Verberk Inge M W,Slot Rosalinde E,Verfaillie Sander C J,Heijst Hans,Prins Niels D,van Berckel Bart N M,Scheltens Philip,Teunissen Charlotte E,van der Flier Wiesje M
Annals of neurology
OBJECTIVE:We investigated the association of plasma amyloid beta (Abeta)40, Abeta42, and total tau (tTau) with the presence of Alzheimer pathological changes in cognitively normal individuals with subjective cognitive decline (SCD). METHODS:We included 248 subjects with SCD (61 ± 9 years, 42% female, Mini-Mental State Examination = 28 ± 2) from the SCIENCe project and Amsterdam Dementia Cohort. Subjects were dichotomized as amyloid abnormal by cerebrospinal fluid (CSF) and positron emission tomography (PET). Baseline plasma Abeta40, Abeta42, and tTau were measured using Simoa technology. Associations between plasma levels and amyloid status were assessed using logistic regression analyses and receiver operating characteristic analyses. Association of plasma levels with risk of clinical progression to mild cognitive impairment (MCI) or dementia was assessed using Cox proportional hazard models. RESULTS:Fifty-seven (23%) subjects were CSF-amyloid abnormal. Plasma Abeta42/Abeta40 ratio and plasma Abeta42 alone, but not tTau, identified abnormal CSF-amyloid status (plasma ratio: area under the curve [AUC] = 77%, 95% confidence interval [CI] = 69-84%; plasma Abeta42: AUC = 66%, 95% CI: 58-74%). Combining plasma ratio with age and apolipoprotein E resulted in AUC = 83% (95% CI = 77-89%). The Youden cutoff of the plasma ratio gave a sensitivity of 76% and specificity of 75%, and applying this as a prescreener would reduce the number of lumbar punctures by 51%. Using PET as outcome, a comparable reduction in number of PET scans would be achieved when applying the plasma ratio as prescreener. In addition, low plasma ratio was associated with clinical progression to MCI or dementia (hazard ratio = 2.0, 95% CI = 1.4-2.3). INTERPRETATION:Plasma Abeta42/Abeta40 ratio has potential as a prescreener to identify Alzheimer pathological changes in cognitively normal individuals with SCD. Ann Neurol 2018;84:656-666.
Ultrasensitive Detection of Plasma Amyloid-β as a Biomarker for Cognitively Normal Elderly Individuals at Risk of Alzheimer's Disease.
Chatterjee Pratishtha,Elmi Mitra,Goozee Kathryn,Shah Tejal,Sohrabi Hamid R,Dias Cintia B,Pedrini Steve,Shen Kaikai,Asih Prita R,Dave Preeti,Taddei Kevin,Vanderstichele Hugo,Zetterberg Henrik,Blennow Kaj,Martins Ralph N
Journal of Alzheimer's disease : JAD
BACKGROUND:Aberrant amyloid-β (Aβ) deposition in the brain occurs two decades prior to the manifestation of Alzheimer's disease (AD) clinical symptoms and therefore brain Aβ load measured using PET serves as a gold standard biomarker for the early diagnosis of AD. However, the uneconomical nature of PET makes blood markers, that reflect brain Aβ deposition, attractive candidates for investigation as surrogate markers. OBJECTIVE:Investigation of plasma Aβ as a surrogate marker for brain Aβ deposition in cognitively normal elderly individuals. METHODS:Plasma Aβ40 and Aβ42 concentrations were measured using the ultrasensitive Single Molecule Array (Simoa) assay in 95 cognitively normal elderly individuals, who have all undergone PET to assess brain Aβ deposition. Based on the standard uptake value ratios (SUVR) obtained from PET imaging, using the tracer 18F-Florbetaben, plasma Aβ was compared between 32 participants assessed to have low brain Aβ load (Aβ-, SUVR <1.35) and 63 assessed to have high brain Aβ load (Aβ+, SUVR ≥1.35). RESULTS:Plasma Aβ42/Aβ40 ratios were lower in the Aβ+ group compared to the Aβ-group. Plasma Aβ40 and Aβ42 levels were not significantly different between Aβ-and Aβ+ groups, although a trend of higher plasma Aβ40 was observed in the Aβ+ group. Additionally, plasma Aβ42/Aβ40 ratios along with the known AD risk factors, age and APOEɛ4 status, resulted in Aβ+ participants being distinguished from Aβ-participants based on an area under the receiver operating characteristic curve shown to be 78%. CONCLUSION:Plasma Aβ ratios in this study are a potential biomarker for brain Aβ deposition and therefore, for preclinical AD. However, this method to measure plasma Aβ needs further development to increase the accuracy of this promising AD blood biomarker.
The prognostic value of the Tau protein serum level in metastatic breast cancer patients and its correlation with brain metastases.
Darlix Amélie,Hirtz Christophe,Thezenas Simon,Maceski Aleksandra,Gabelle Audrey,Lopez-Crapez Evelyne,De Forges Hélène,Firmin Nelly,Guiu Séverine,Jacot William,Lehmann Sylvain
BACKGROUND:Metastatic breast cancer (MBC) prognosis is variable, depending on several clinical and biological factors. A better prediction of a patient's outcome could allow for a more accurate choice of treatments. The role of serum biomarkers in predicting outcome remains unclear in this setting. Tau, a microtubule-associated protein, is a neuronal marker that is also expressed in normal breast epithelial cells and cancer cells. Its tissue expression is associated with prognosis in MBC. However, the prognostic value of Tau serum levels in these patients is unknown. We aimed at evaluating the prognostic value of Tau (and other classical biomarkers) in MBC patients, and to assess its association with the presence of brain metastases (BM). METHODS:244 MBC patients treated at our institution (2007-2015) were retrospectively selected. The usual MBC clinical and pathological variables were collected, altogether with CA15-3, CEA and HER2 extra-cellular domain (ECD) serum levels. Tau serum levels were measured with a novel immunoassay (digital ELISA) using Single Molecule Array (Simoa) technology. Overall survival (OS) was estimated with the Kaplan-Meier method. To investigate prognostic factors, a multivariate analysis was performed. Cut-offs were set using the Youden index method associated with receiver-operating characteristics (ROC) curves to evaluate the accuracy of biomarkers to identify patients with BM. RESULTS:With a median follow-up of 40.8 months, median OS was 15.5 months (95%CI 12.4-20.2). Elevated serum levels of Tau were independently associated with a poor outcome in the whole population as well as in patients with (n = 86) and without BM (n = 158). Median serum Tau levels tended to be higher in patients with BM (p = 0.23). In univariate analysis, patients with BM had an increased risk of serum Tau > 3.17 pg/mL (OR = 2.2, p = 0.049). In multivariate analysis, high values of Tau (OR = 3.98, p = 0.034) accurately identified patients with BM in our cohort. CONCLUSIONS:Tau is a new biomarker of interest in MBC. Its serum level could represent an independent prognostic factor in these patients (both with and without BM). It also seems to be associated with the presence of BM. A validation of these results in an independent set of MBC patients is necessary to confirm these findings.
Comparison of plasma and cerebrospinal fluid neurofilament light in a multiple sclerosis trial.
de Flon Pierre,Laurell Katarina,Sundström Peter,Blennow Kaj,Söderström Lars,Zetterberg Henrik,Gunnarsson Martin,Svenningsson Anders
Acta neurologica Scandinavica
OBJECTIVE:The main objective of this study was to evaluate the axonal component neurofilament light protein (NFL) in plasma and cerebrospinal fluid (CSF) as an outcome measure in a clinical trial on disease-modifying treatments in multiple sclerosis. MATERIALS AND METHODS:Seventy-five patients with clinically stable relapsing-remitting multiple sclerosis (RRMS) participating in the clinical trial "Switch-To RItuXimab in MS" (STRIX-MS) were switched to rituximab from first-line injectable therapy and then followed up for 2 years. Thirty patients from the extension trial (STRIX-MS extension), accepting repeated lumbar punctures, were followed up for an additional 3 years. Plasma and CSF samples were collected yearly during the follow-up. NFL concentration in plasma was measured by an in-house NF-light assay on the Simoa platform with a Homebrew kit. NFL concentration in CSF was measured by sandwich ELISA. RESULTS:The mean levels of NFL, in both CSF and plasma, were low. The reduction of CSF-NFL was 25% during the first year of follow-up (from a mean of 471 [SD 393] to 354 [SD 174] pg/mL; P = 0.006) and was statistically significant. The corresponding reduction in plasma NFL was 18% (from 9.73 [SD 7.04] to 7.94 [SD 3.10] pg/mL; P = 0.055) and did not reach statistical significance. CONCLUSION:This study indicates that NFL in plasma is less sensitive as an endpoint in group comparisons than NFL in CSF. Given that plasma NFL is far easier to access, it is a promising and awaited method but further studies are needed to optimize the use in clinical trials.
Plasma neurofilament light chain and amyloid-β are associated with the kynurenine pathway metabolites in preclinical Alzheimer's disease.
Chatterjee Pratishtha,Zetterberg Henrik,Goozee Kathryn,Lim Chai K,Jacobs Kelly R,Ashton Nicholas J,Hye Abdul,Pedrini Steve,Sohrabi Hamid R,Shah Tejal,Asih Prita R,Dave Preeti,Shen Kaikai,Taddei Kevin,Lovejoy David B,Guillemin Gilles J,Blennow Kaj,Martins Ralph N
Journal of neuroinflammation
BACKGROUND:Blood markers indicative of neurodegeneration (neurofilament light chain; NFL), Alzheimer's disease amyloid pathology (amyloid-β; Aβ), and neuroinflammation (kynurenine pathway; KP metabolites) have been investigated independently in neurodegenerative diseases. However, the association of these markers of neurodegeneration and AD pathology with neuroinflammation has not been investigated previously. Therefore, the current study examined whether NFL and Aβ correlate with KP metabolites in elderly individuals to provide insight on the association between blood indicators of neurodegeneration and neuroinflammation. METHODS:Correlations between KP metabolites, measured using liquid chromatography and gas chromatography coupled with mass spectrometry, and plasma NFL and Aβ concentrations, measured using single molecule array (Simoa) assays, were investigated in elderly individuals aged 65-90 years, with normal global cognition (Mini-Mental State Examination Score ≥ 26) from the Kerr Anglican Retirement Village Initiative in Ageing Health cohort. RESULTS:A positive correlation between NFL and the kynurenine to tryptophan ratio (K/T) reflecting indoleamine 2,3-dioxygenase activity was observed (r = .451, p < .0001). Positive correlations were also observed between NFL and kynurenine (r = .364, p < .0005), kynurenic acid (r = .384, p < .0001), 3-hydroxykynurenine (r = .246, p = .014), anthranilic acid (r = .311, p = .002), and quinolinic acid (r = .296, p = .003). Further, significant associations were observed between plasma Aβ40 and the K/T (r = .375, p < .0005), kynurenine (r = .374, p < .0005), kynurenic acid (r = .352, p < .0005), anthranilic acid (r = .381, p < .0005), and quinolinic acid (r = .352, p < .0005). Significant associations were also observed between plasma Aβ42 and the K/T ratio (r = .215, p = .034), kynurenic acid (r = .214, p = .035), anthranilic acid (r = .278, p = .006), and quinolinic acid (r = .224, p = .027) in the cohort. On stratifying participants based on their neocortical Aβ load (NAL) status, NFL correlated with KP metabolites irrespective of NAL status; however, associations between plasma Aβ and KP metabolites were only pronounced in individuals with high NAL while associations in individuals with low NAL were nearly absent. CONCLUSIONS:The current study shows that KP metabolite changes are associated with biomarker evidence of neurodegeneration. Additionally, the association between KP metabolites and plasma Aβ seems to be NAL status dependent. Finally, the current study suggests that an association between neurodegeneration and neuroinflammation manifests in the periphery, suggesting that preventing cytoskeleton cytotoxicity by KP metabolites may have therapeutic potential.
Neurofilament light chain in serum of adolescent and adult SMA patients under treatment with nusinersen.
Wurster Claudia D,Steinacker Petra,Günther René,Koch Jan C,Lingor Paul,Uzelac Zeljko,Witzel Simon,Wollinsky Kurt,Winter Benedikt,Osmanovic Alma,Schreiber-Katz Olivia,Al Shweiki Rami,Ludolph Albert C,Petri Susanne,Hermann Andreas,Otto Markus,
Journal of neurology
OBJECTIVE:To determine the diagnostic and monitoring value of serum neurofilament light chain (NfL) in spinal muscular atrophy (SMA). METHODS:We measured serum NfL in 46 SMA patients at baseline and over 14 months of treatment with the antisense-oligonucleotide (ASO) nusinersen using the ultrasensitive single molecule array (Simoa) technology. Serum NfL levels of SMA patients were compared to controls and related to cerebrospinal fluid (CSF) NfL, blood-CSF barrier function quantified by the albumin blood/CSF ratio (Qalb) and motor scores (Hammersmith Functional Motor Scale Expanded, HFMSE; Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised, ALSFRS-R). RESULTS:Serum NfL levels of SMA patients were in the range of controls (p = 0.316) and did not correlate with CSF NfL (ρ = 0.302, p = 0.142) or Qalb (ρ = - 0.160, p = 0.293). During therapy, serum NfL levels were relatively stable with notable concentration changes in single SMA patients, however, within the control range. Higher NfL levels were associated with worse motor performance in SMA (baseline: HFMSE ρ = - 0.330, p = 0.025, ALSFRS-R ρ = - 0.403, p = 0.005; after 10 months: HFMSE ρ = - 0.525, p = 0.008, ALSFRS-R ρ = - 0.537, p = 0.007), but changes in motor scores did not correlate with changes in serum NfL. CONCLUSION:Diagnostic and monitoring performance of serum NfL measurement seems to differ between SMA subtypes. Unlike to SMA type 1, in adolescent and adult SMA type 2 and 3 patients, neurodegeneration is not reflected by increased NfL levels and short-term therapeutic effects cannot be observed. Long-term follow-up has to be performed to see if even low levels of NfL might be good prognostic markers.
Serum neurofilament light levels in normal aging and their association with morphologic brain changes.
Khalil Michael,Pirpamer Lukas,Hofer Edith,Voortman Margarete M,Barro Christian,Leppert David,Benkert Pascal,Ropele Stefan,Enzinger Christian,Fazekas Franz,Schmidt Reinhold,Kuhle Jens
Neurofilament light (NfL) protein is a marker of neuro-axonal damage and can be measured not only in cerebrospinal fluid but also in serum, which allows for repeated assessments. There is still limited knowledge regarding the association of serum NfL (sNfL) with age and subclinical morphologic brain changes and their dynamics in the normal population. We measured sNfL by a single molecule array (Simoa) assay in 335 individuals participating in a population-based cohort study and after a mean follow-up time of 5.9 years (n = 103). Detailed clinical examination, cognitive testing and 3T brain MRI were performed to assess subclinical brain damage. We show that rising and more variable sNfL in individuals >60 years indicate an acceleration of neuronal injury at higher age, which may be driven by subclinical comorbid pathologies. This is supported by a close association of sNfL with brain volume changes in a cross-sectional and especially longitudinal manner.
Neurotoxicity after hematopoietic stem cell transplant in multiple sclerosis.
Thebault Simon,Lee Hyunwoo,Bose Gauruv,Tessier Daniel,Abdoli Mohammad,Bowman Marjorie,Berard Jason,Walker Lisa,Rush Carolina A,MacLean Heather,Booth Ronald A,Narayanan Sridar,Arnold Douglas L,Tabard-Cossa Vincent,Atkins Harold L,Bar-Or Amit,Freedman Mark S
Annals of clinical and translational neurology
OBJECTIVE:Accelerated brain volume loss has been noted following immunoablative autologous hematopoietic stem cell transplantation (IAHSCT) for multiple sclerosis. As with other MS treatments, this is often interpreted as 'pseudoatrophy', related to reduced inflammation. Treatment-related neurotoxicity may be contributory. We sought objective evidence of post-IAHSCT toxicity by quantifying levels of Neurofilament Light Chain (sNfL) and Glial Fibrillary Acidic Protein (sGFAP) before and after treatment as markers of neuroaxonal and glial cell damage. METHODS:Sera were collected from 22 MS patients pre- and post-IAHSCT at 3, 6, 9, and 12 months along with 28 noninflammatory controls. sNfL and sGFAP quantification was performed using the SiMoA single-molecule assay. RESULTS:Pre-IAHSCT levels of sNfL and sGFAP were elevated in MS patients compared with controls (geometric mean sNfL 21.8 vs. 6.4 pg/mL, sGFAP 107.4 vs. 50.7 pg/mL, P = 0.0001 for both). Three months after IAHSCT, levels of sNfL and sGFAP increased from baseline by 32.1% and 74.8%, respectively (P = 0.0029 and 0.0004). sNfL increases correlated with total busulfan dose (P = 0.034), EDSS score worsening at 6 months (P = 0.041), and MRI grey matter volume loss at 6 months (P = 0.0023). Subsequent NfL levels reduced to less than baseline (12-month geometric mean 11.3 pg/mL P = 0.0001) but were still higher than controls (P = 0.0001). sGFAP levels reduced more slowly but at 12 months were approaching baseline levels (130.7 pg/mL). INTERPRETATION:There is direct evidence of transient CNS toxicity immediately after IAHSCT which may be chemotherapy mediated and contributes to transient increases in MRI atrophy.
Serum neurofilament light chain predicts long term clinical outcomes in multiple sclerosis.
Thebault Simon,Abdoli Mohammad,Fereshtehnejad Seyed-Mohammad,Tessier Daniel,Tabard-Cossa Vincent,Freedman Mark S
Serum neurofilament light chain (NfL) is emerging as an important biomarker in multiple sclerosis (MS). Our objective was to evaluate the prognostic value of serum NfL levels obtained close to the time of MS onset with long-term clinical outcomes. In this prospective cohort study, we identified patients with serum collected within 5 years of first MS symptom onset (baseline) with more than 15 years of routine clinical follow-up. Levels of serum NfL were quantified in patients and matched controls using digital immunoassay (SiMoA HD-1 Analyzer, Quanterix). Sixty-seven patients had a median follow-up of 18.9 years (range 15.0-27.0). The median serum NfL level in patient baseline samples was 10.1 pg/mL, 38.5% higher than median levels in 37 controls (7.26 pg/mL, p = 0.004). Baseline NfL level was most helpful as a sensitive predictive marker to rule out progression; patients with levels less 7.62 pg/mL were 4.3 times less likely to develop an EDSS score of ≥ 4 (p = 0.001) and 7.1 times less likely to develop progressive MS (p = 0.054). Patients with the highest NfL levels (3rd-tertile, > 13.2 pg/mL) progressed most rapidly with an EDSS annual rate of 0.16 (p = 0.004), remaining significant after adjustment for sex, age, and disease-modifying treatment (p = 0.022). This study demonstrates that baseline sNfL is associated with long term clinical disease progression. sNfL may be a sensitive marker of subsequent poor clinical outcomes.
Long Noncoding RNA POU3F3 and α-Synuclein in Plasma L1CAM Exosomes Combined with β-Glucocerebrosidase Activity: Potential Predictors of Parkinson's Disease.
Zou Jing,Guo Yue,Wei Lei,Yu Fang,Yu Bo,Xu Anding
Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics
Long noncoding RNAs (lncRNAs) are implicated in the autophagic-lysosomal pathway (ALP) and are closely linked to Parkinson's disease (PD) pathology. β-Glucocerebrosidase (GCase) has also been reported to be correlated with α-synuclein (α-syn) proteostasis. However, lncRNAs and α-syn in neural-derived L1CAM exosomes and GCase activity in the plasma of PD patients have not been studied. This study used an ultrasensitive methodology, fluorescence nanoparticle tracking analysis (NTA), to measure plasma L1CAM exosomes and Quanterix Simoa to measure α-syn concentrations in L1CAM exosomes. Eighty-five healthy controls and 93 PD patients were enrolled, and several scales were used to rate the severity of PD. Receiver operating characteristic (ROC) curves were applied to map the diagnostic accuracy of categorizing PD patients and healthy subjects. We found increased Linc-POU3F3 and α-syn concentrations in L1CAM exosomes and decreased GCase activity in PD patients compared with controls. The three biomarkers displayed obvious differences among PD patients based on gender, H-Y stage, and UPDRS-III distribution. Interestingly, Linc-POU3F3 was significantly positively correlated with α-syn in L1CAM exosomes and inversely correlated with GCase activity in PD patients. Significant correlations were observed among L1CAM exosomal Linc-POU3F3 levels, GCase activity, and PD severity, including motor/cognitive dysfunction. Additionally, the combination of Linc-POU3F3 and α-syn in L1CAM exosomes and GCase activity could discriminate PD patients from controls. These results suggest that L1CAM exosomal Linc-POU3F3, L1CAM exosomal α-syn, and GCase activity may shed light on the mechanism underlying the autophagic-lysosomal system in the pathogenesis of PD and could be used to assess the severity of PD.
Supplementary medication in multiple sclerosis: Real-world experience and potential interference with neurofilament light chain measurement.
Pape Katrin,Steffen Falk,Zipp Frauke,Bittner Stefan
Multiple sclerosis journal - experimental, translational and clinical
Background:As vitamins and dietary supplements are obtainable without prescription, treating physicians often ignore their intake by patients with multiple sclerosis (MS) and may therefore miss potential adverse effects and interactions. Objective:We aimed to assess the spectrum and intake frequency of supplementary medication in a cohort of MS patients and to analyse the effect of biotin intake on measurement of serum neurofilament light chain (sNfL), an emerging marker of disease activity. Methods:MS patients visiting our neurology outpatient clinic completed a questionnaire on their past or present use of vitamins or dietary supplements. In addition, the impact of two different doses of biotin (10 and 300 mg/day) on sNfL was studied in healthy volunteers. Results:Of 186 patients, 72.6% reported taking over-the-counter vitamins or dietary supplements currently or previously. Most frequently used was vitamin D (60.0%), followed by biotin. Female patients and patients with primary progressive MS tended to use supplements more frequently. Biotin intake did not interfere with sNfL measurement by single molecule array (Simoa). Conclusions:The use of vitamins and dietary supplements is frequent among patients with MS. Thus, treating physicians should be aware of the pitfalls of supplementary treatment and educate their patients accordingly.
Single Molecule Protein Detection with Attomolar Sensitivity Using Droplet Digital Enzyme-Linked Immunosorbent Assay.
Cohen Limor,Cui Naiwen,Cai Yamei,Garden Padric M,Li Xiang,Weitz David A,Walt David R
Many proteins are present at low concentrations in biological samples, and therefore, techniques for ultrasensitive protein detection are necessary. To overcome challenges with sensitivity, the digital enzyme-linked immunosorbent assay (ELISA) was developed, which is 1000× more sensitive than conventional ELISA and allows sub-femtomolar protein detection. However, this sensitivity is still not sufficient to measure many proteins in various biological samples, thereby limiting our ability to detect and discover biomarkers. To overcome this limitation, we developed droplet digital ELISA (ddELISA), a simple approach for detecting low protein levels using digital ELISA and droplet microfluidics. ddELISA achieves maximal sensitivity by improving the sampling efficiency and counting more target molecules. ddELISA can detect proteins in the low attomolar range and is up to 25-fold more sensitive than digital ELISA using Single Molecule Arrays (Simoa), the current gold standard tool for ultrasensitive protein detection. Using ddELISA, we measured the LINE1/ORF1 protein, a potential cancer biomarker that has not been previously measured in serum. Additionally, due to the simplicity of our device design, ddELISA is promising for point-of-care applications. Thus, ddELISA will facilitate the discovery of biomarkers that have never been measured before for various clinical applications.
Diagnostic and prognostic value of serum NfL and p-Tau in frontotemporal lobar degeneration.
Benussi Alberto,Karikari Thomas K,Ashton Nicholas,Gazzina Stefano,Premi Enrico,Benussi Luisa,Ghidoni Roberta,Rodriguez Juan Lantero,Emeršič Andreja,Simrén Joel,Binetti Giuliano,Fostinelli Silvia,Giunta Marcello,Gasparotti Roberto,Zetterberg Henrik,Blennow Kaj,Borroni Barbara
Journal of neurology, neurosurgery, and psychiatry
OBJECTIVE:To assess the diagnostic and prognostic value of serum neurofilament light (NfL) and serum phospho-Tau (p-Tau) in a large cohort of patients with frontotemporal lobar degeneration (FTLD). METHODS:In this retrospective study, performed on 417 participants, we analysed serum NfL and p-Tau concentrations with an ultrasensitive single molecule array (Simoa) approach. We assessed the diagnostic values of serum biomarkers in the differential diagnosis between FTLD, Alzheimer's disease (AD) and healthy ageing; their role as markers of disease severity assessing the correlation with clinical variables, cross-sectional brain imaging and neurophysiological data; their role as prognostic markers, considering their ability to predict survival probability in FTLD. RESULTS:We observed significantly higher levels of serum NfL in patients with FTLD syndromes, compared with healthy controls, and lower levels of p-Tau compared with patients with AD. Serum NfL concentrations showed a high accuracy in discriminating between FTLD and healthy controls (area under the curve (AUC): 0.86, p<0.001), while serum p-Tau showed high accuracy in differentiating FTLD from patients with AD (AUC: 0.93, p<0.001). In FTLD, serum NfL levels correlated with measures of cognitive function, disease severity and behavioural disturbances and were associated with frontotemporal atrophy and indirect measures of GABAergic deficit. Moreover, serum NfL concentrations were identified as the best predictors of survival probability. CONCLUSIONS:The assessment of serum NfL and p-Tau may provide a comprehensive view of FTLD, aiding in the differential diagnosis, in staging disease severity and in defining survival probability.
Cerebrospinal Fluid Biomarkers in Relation to MRZ Reaction Status in Primary Progressive Multiple Sclerosis.
Robinson Tilman,Abdelhak Ahmed,Bose Tanima,Meinl Edgar,Otto Markus,Zettl Uwe K,Dersch Rick,Tumani Hayrettin,Rauer Sebastian,Huss André
The MRZ reaction (MRZR) comprises the three antibody indices (AIs) against measles, rubella, and varicella zoster virus, reflecting an intrathecal polyspecific B cell response highly specific for multiple sclerosis (MS). Thus, MRZR can be used to confirm a diagnosis of primary progressive MS (PPMS) but its pathophysiological and wider clinical relevance is unclear. This study aimed to investigate whether PPMS patients with a positive MRZR (MRZR+) differ from those with a negative MRZR (MRZR-) according to cerebrospinal fluid (CSF) biomarkers of B cell activity, neuroaxonal damage or glial activity, and clinical features. (1) Methods: In a multicenter PPMS cohort ( = 81) with known MRZR status, we measured B cell-activating factor (BAFF), chemokine CXC ligand 13 (CXCL-13), soluble B cell maturation antigen (sBCMA), soluble transmembrane activator and CAML interactor (sTACI), and chitinase-3-like protein 1 (CHI3L1) in the CSF with enzyme-linked immunosorbent assays (ELISAs). Glial fibrillary acidic protein (GFAP) and neurofilament light chain (NfL) were detected in serum and CSF using single molecule array (SIMOA) technology. (2) Results: MRZR+ patients (45.7% of all PPMS patients) revealed higher levels of NfL in CSF compared to MRZR- patients (54.3%). There were positive correlations between each of sBCMA, sTACI, and intrathecal immunoglobin G (IgG) synthesis. Additionally, NfL concentrations in serum positively correlated with those in CSF and those of GFAP in serum. However, MRZR+ and MRZR- patients did not differ concerning clinical features (e.g., age, disease duration, Expanded Disability Status Scale (EDSS) at diagnosis and follow-up); CSF routine parameters; CSF concentrations of BAFF, CXCL-13, sBCMA, sTACI, CHI3L1, and GFAP; or serum concentrations of GFAP and NfL. (3) Conclusions: In PPMS patients, MRZR positivity might indicate a more pronounced axonal damage. Higher levels of the soluble B cell receptors BCMA and transmembrane activator and CAML interactor (TACI) in CSF are associated with a stronger intrathecal IgG synthesis in PPMS.
Serum neurofilament light chain level associations with clinical and cognitive performance in multiple sclerosis: A longitudinal retrospective 5-year study.
Jakimovski Dejan,Zivadinov Robert,Ramanthan Murali,Hagemeier Jesper,Weinstock-Guttman Bianca,Tomic Davorka,Kropshofer Harald,Fuchs Tom A,Barro Christian,Leppert David,Yaldizli Özgür,Kuhle Jens,Benedict Ralph Hb
Multiple sclerosis (Houndmills, Basingstoke, England)
BACKGROUND:A limited number of studies investigated associations between serum neurofilament light chain (sNfL) and cognition in persons with multiple sclerosis (PwMS). OBJECTIVE:To assess cross-sectional and longitudinal associations between sNfL levels, clinical, and cognitive performance in PwMS and age-matched healthy controls (HCs). MATERIALS:One hundred twenty-seven PwMS (85 relapsing-remitting MS/42 progressive MS), 20 clinically isolated syndrome patients, and 52 HCs were followed for 5 years. sNfL levels were measured using the single-molecule array (Simoa) assay and quantified in picograms per milliliter. Expanded Disability Status Scale (EDSS), walking, and manual dexterity tests were obtained. At follow-up, Brief International Cognitive Assessment for MS (BICAMS) was utilized. Cognitively impaired (CI) status was derived using HC-based -scores. Age-, sex-, and education-adjusted analysis of covariance (ANCOVA) and regression models were used. Multiple comparison-adjusted values of < 0.05 were considered significant. RESULTS:In PwMS, sNfL levels were cross-sectionally associated with walking speed ( = 0.235, = 0.036), manual dexterity ( = 0.337, = 0.002), and cognitive processing speed (CPS; =-0.265, = 0.012). Baseline sNfL levels predicted 5-year EDSS scores ( = 0.25, = 0.012), dexterity ( = 0.224, = 0.033), and CPS ( =-0.205, = 0.049). CI patients had higher sNfL levels (27.2 vs. 20.6, = 0.016) and greater absolute longitudinal sNfL increase when compared with non-CI patients (4.8 vs. 0.7, = 0.04). CONCLUSION:Higher sNfL levels are associated with poorer current and future clinical and cognitive performance.
Evaluation of a novel immunoassay to detect p-tau Thr217 in the CSF to distinguish Alzheimer disease from other dementias.
Hanes Jozef,Kovac Andrej,Kvartsberg Hlin,Kontsekova Eva,Fialova Lubica,Katina Stanislav,Kovacech Branislav,Stevens Eva,Hort Jakub,Vyhnalek Martin,Boonkamp Lynn,Novak Michal,Zetterberg Henrik,Hansson Oskar,Scheltens Philip,Blennow Kaj,Teunissen Charlotte E,Zilka Norbert
OBJECTIVE:To investigate whether tau phosphorylated at Thr217 (p-tau T217) assay in CSF can distinguish patients with Alzheimer disease (AD) from patients with other dementias and healthy controls. METHODS:We developed and validated a novel Simoa immunoassay to detect p-tau T217 in CSF. There was a total of 190 participants from 3 cohorts with AD (n = 77) and other neurodegenerative diseases (n = 69) as well as healthy participants (n = 44). RESULTS:The p-tau T217 assay (cutoff 242 pg/mL) identified patients with AD with accuracy of 90%, with 78% positive predictive value (PPV), 97% negative predictive value (NPV), 93% sensitivity, and 88% specificity, compared favorably with p-tau T181 ELISA (52 pg/mL), showing 78% accuracy, 58% PPV, 98% NPV, 71% specificity, and 97% sensitivity. The assay distinguished patients with AD from age-matched healthy controls (cutoff 163 pg/mL, 98% sensitivity, 93% specificity), similarly to p-tau T181 ELISA (cutoff 60 pg/mL, 96% sensitivity, 86% specificity). In patients with AD, we found a strong correlation between p-tau T217 and p-tau T181, total tau and β-amyloid 40, but not β-amyloid 42. CONCLUSIONS:This study demonstrates that p-tau T217 displayed better diagnostic accuracy than p-tau T181. The data suggest that the new p-tau T217 assay has potential as an AD diagnostic test in clinical evaluation. CLASSIFICATION OF EVIDENCE:This study provides Class III evidence that a CSF immunoassay for p-tau T217 distinguishes patients with AD from patients with other dementias and healthy controls.
Amyloid-β misfolding as a plasma biomarker indicates risk for future clinical Alzheimer's disease in individuals with subjective cognitive decline.
Stockmann Julia,Verberk Inge M W,Timmesfeld Nina,Denz Robin,Budde Brian,Lange-Leifhelm Julia,Scheltens Philip,van der Flier Wiesje M,Nabers Andreas,Teunissen Charlotte E,Gerwert Klaus
Alzheimer's research & therapy
BACKGROUND:We evaluated Aβ misfolding in combination with Aβ ratio as a prognostic tool for future clinical progression to mild cognitive impairment (MCI) or dementia due to Alzheimer's disease (AD) in individuals with subjective cognitive decline (SCD). METHODS:Baseline plasma samples (n = 203) from SCD subjects in the SCIENCe project and Amsterdam Dementia Cohort (age 61 ± 9 years; 57% male, mean follow-up time 2.7 years) were analyzed using immuno-infrared-sensor technology. Within 6 years of follow-up, 22 (11%) individuals progressed to MCI or dementia due to AD. Sensor readout values > 1646 cm reflected normal Aβ folding; readouts at ≤ 1646 cm reflected low and at < 1644 cm high misfolding. We used Cox proportional hazard models to quantify Aβ misfolding as a prognostic biomarker for progression to MCI and dementia due to AD. The accuracy of the predicted development of MCI/AD was determined by time-dependent receiver operating characteristic (t-ROC) curve analyses that take individual follow-up and conversion times into account. Statistical models were adjusted for age, sex, and APOEε4 status. Additionally, plasma Aβ data measured by SIMOA were statistically analyzed and compared. RESULTS:All 22 patients who converted to MCI or AD-dementia within 6 years exhibited Aβ misfolding at baseline. Cox analyses revealed a hazard ratio (HR) of 19 (95% confidence interval [CI] 2.2-157.8) for future conversion of SCD subjects with high misfolding and of 11 (95% CI 1.0-110.1) for those with low misfolding. T-ROC curve analyses yielded an area under the curve (AUC) of 0.94 (95% CI 0.86-1.00; 6-year follow-up) for Aβ misfolding in an age, sex, and APOEε4 model. A similar model with plasma Aβ ratio yielded an AUC of 0.92 (95% CI, 0.82-1.00). The AUC increased to 0.99 (95% CI, 0.99-1.00) after inclusion of both Aβ misfolding and the Aβ ratio. CONCLUSIONS:A panel of structure- and concentration-based plasma amyloid biomarkers may predict conversion to clinical MCI and dementia due to AD in cognitively unimpaired subjects. These plasma biomarkers provide a noninvasive and cost-effective alternative for screening early AD pathological changes. Follow-up studies and external validation in larger cohorts are in progress for further validation of our findings.
Neurofilament Light Chain Levels Are Associated with Disease Activity Determined by No Evident Disease Activity in Multiple Sclerosis Patients.
Szilasiová Jarmila,Rosenberger Jaroslav,Fedičová Miriam,Mikula Pavol,Urban Peter,Gdovinová Zuzana,Vitková Marianna,Hanes Jozef,Stevens Eva
INTRODUCTION:There is a need for blood biomarkers of disease activity in multiple sclerosis (MS). The aim of the study was to assess the relationship between plasma neurofilament light chain (pNfL) and disease activity as defined by the concept three-domain no evident disease activity (NEDA-3). METHODS:Levels of pNfL (SIMOA) were examined in 159 MS patients and analyzed in relationship to NEDA-3 status (absence of relapse, disability score worsening, and brain magnetic resonance activity) during the last 12 months. The accuracy of the proposed model was evaluated by calculating the area under the receiver operating characteristics (ROC) curve. From the pNfL cutoff, we evaluated the NEDA-NfL status (no relapse, no Expanded Disability Status Scale [EDSS] worsening, and pNfL below the cutoff value). RESULTS:Levels of pNfL were significantly higher in MS patients than in healthy controls (p < 0.001). From a total of 159 patients, 80 (50.3%) achieved NEDA-3 status, while 79 (49.7%) patients showed evident disease activity (EDA) status. pNfL were significantly lower in the NEDA-3 group than in the EDA group (pNfL mean 7.06 pg/mL [standard deviation (SD) 2.37] vs. pNfL mean 13.04 pg/mL [SD 7.07]) (p < 0.001). ROC analysis showed that pNfL predicts NEDA-3 status (sensitivity and specificity were 80.5 and 72.7%, respectively, p < 0.001), and NEDA-NfL predicts NEDA-3 status (sensitivity and specificity were 97.1 and 82.9%, respectively, p < 0.001). CONCLUSION:The results show that pNfL levels are a useful biomarker of disease activity determined by NEDA status in patients with MS and could be an alternative to brain magnetic resonance investigation.
Cerebrospinal fluid N-224 tau helps discriminate Alzheimer's disease from subjective cognitive decline and other dementias.
Cicognola Claudia,Hansson Oskar,Scheltens Philip,Kvartsberg Hlin,Zetterberg Henrik,Teunissen Charlotte E,Blennow Kaj
Alzheimer's research & therapy
BACKGROUND:Elevated cerebrospinal fluid (CSF) concentrations of total tau (T-tau) and phosphorylated tau at Thr181 (P-tau181) protein are typical of Alzheimer's disease (AD). However, the T-tau assay measures only the mid-region of the protein, while tau in CSF is instead composed of a series of fragments. One fragment species in particular, N-224, shows increased levels in AD compared to controls. In this multicentre study, we performed a clinical validation of the N-224 assay in cohorts including patients with subjective cognitive decline (SCD), mild cognitive impairment (MCI), AD, non-AD dementias and controls. METHODS:Cohorts consisted of 30 SCD and 30 probable AD from the Amsterdam Dementia Cohort (cohort 1) and 539 controls, 195 SCD, 232 MCI, 137 AD and 253 non-AD from the Swedish BioFINDER study (cohort 2). All samples had AD core biomarkers (Aβ42, T-tau, P-tau181) measurements. N-224 was measured with an in-house ultrasensitive Simoa assay. RESULTS:N-224 levels were significantly higher in AD compared to SCD (cohort 1: p = 0.003) and in AD compared to all other diagnostic groups in cohort 2 (control, SCD, MCI and non-AD, p < 0.0001). Within the non-AD group, N-224 showed significantly lower concentrations compared to AD in Parkinson's disease (PD, p < 0.0001), Parkinson's disease dementia (PDD, p = 0.004), progressive supranuclear palsy (PSP, < 0.0001), multiple system atrophy (MSA, p = 0.002) and parkinsonisms not otherwise specified (NOS, p = 0.007). In cohort 1, higher concentrations of N-224 were associated to lower Mini-Mental State Examination (MMSE) scores (R = 0.318, β = 0.564, p ≤ 0.0001) and could accurately identify a pathological (< 24) MMSE score (p < 0.0001, AUC = 0.824). CONCLUSIONS:N-224 tau can distinguish AD subjects from SCD and can discriminate subgroups of non-AD dementias from AD. Therefore, N-224 may be a useful addition to the tau biomarker toolbox for the study of tau species in CSF and for better understanding disease pathogenesis.
Neurofilament Light Chain as A Biomarker for Brain Metastases.
Winther-Larsen Anne,Hviid Claus Vinter Bødker,Meldgaard Peter,Sorensen Boe Sandahl,Sandfeld-Paulsen Birgitte
BACKGROUND:Brain metastases are feared complications in cancer. Treatment by neurosurgical resection and stereotactic radiosurgery are only available when metastatic lesions are limited and early detection is warranted. The neurofilament light chain (NfL) is a sensitive neuron-specific biomarker released following neuronal decay. We explored serum NfL as a biomarker of brain metastases. METHODS:Serum was collected from 43 stage IV lung cancer patients with brain metastases and 25 stage I lung cancer patients. Serum was collected at time of cancer diagnosis and at time of brain metastasis diagnosis. In nine patients with brain metastases, additional samples were available between the two time points. NfL was quantified by Single Molecule Array (Simoa)™. RESULTS:The median NfL level was significantly higher in patients with brain metastases than in patients without (35 versus 16 pg/mL, = 0.001) and separated patients with an area under the curve of 0.77 (0.66-0.89). An increase in NfL could be measured median 3 months (range: 1-5) before the brain metastasis diagnosis. Further, a high level of NfL at time of brain metastasis diagnosis correlated with an inferior survival (hazard ratio: 2.10 (95% confidence interval: 1.11-3.98)). CONCLUSIONS:This study implies that NfL could be a potential biomarker of brain metastases.
Stage-specific links between plasma neurofilament light and imaging biomarkers of Alzheimer's disease.
Benedet Andréa L,Leuzy Antoine,Pascoal Tharick A,Ashton Nicholas J,Mathotaarachchi Sulantha,Savard Melissa,Therriault Joseph,Kang Min Su,Chamoun Mira,Schöll Michael,Zimmer Eduardo R,Gauthier Serge,Labbe Aurélie,Zetterberg Henrik,Rosa-Neto Pedro,Blennow Kaj,
Brain : a journal of neurology
Neurofilament light (NfL) is a marker of neuroaxonal injury, a prominent feature of Alzheimer's disease. It remains uncertain, however, how it relates to amyloid and tau pathology or neurodegeneration across the Alzheimer's disease continuum. The aim of this study was to investigate how plasma NfL relates to amyloid and tau PET and MRI measures of brain atrophy in participants with and without cognitive impairment. We retrospectively examined the association between plasma NfL and MRI measures of grey/white matter volumes in the Alzheimer's Disease Neuroimaging Initiative [ADNI: n = 1149; 382 cognitively unimpaired control subjects and 767 cognitively impaired participants (mild cognitive impairment n = 420, Alzheimer's disease dementia n = 347)]. Longitudinal plasma NfL was measured using single molecule array (Simoa) technology. Cross-sectional associations between plasma NfL and PET amyloid and tau measures were independently assessed in two cohorts: ADNI [n = 198; 110 cognitively unimpaired, 88 cognitively impaired (MCI n = 67, Alzheimer's disease dementia n = 21), data accessed October 2018]; and Translational Biomarkers in Aging and Dementia [TRIAD, n = 116; 74 cognitively unimpaired, 42 cognitively impaired (MCI n = 16, Alzheimer's disease dementia n = 26), data obtained November 2017 to January 2019]. Associations between plasma NfL and imaging-derived measures were examined voxel-wise using linear regression (cross-sectional) and linear mixed effect models (longitudinal). Cross-sectional analyses in both cohorts showed that plasma NfL was associated with PET findings in brain regions typically affected by Alzheimer's disease; associations were specific to amyloid PET in cognitively unimpaired and tau PET in cognitively impaired (P < 0.05). Longitudinal analyses showed that NfL levels were associated with grey/white matter volume loss; grey matter atrophy in cognitively unimpaired was specific to APOE ε4 carriers (P < 0.05). These findings suggest that plasma NfL increases in response to amyloid-related neuronal injury in preclinical stages of Alzheimer's disease, but is related to tau-mediated neurodegeneration in symptomatic patients. As such, plasma NfL may a useful measure to monitor effects in disease-modifying drug trials.
The diagnostic and prognostic capabilities of plasma biomarkers in Alzheimer's disease.
Simrén Joel,Leuzy Antoine,Karikari Thomas K,Hye Abdul,Benedet Andréa Lessa,Lantero-Rodriguez Juan,Mattsson-Carlgren Niklas,Schöll Michael,Mecocci Patrizia,Vellas Bruno,Tsolaki Magda,Kloszewska Iwona,Soininen Hilkka,Lovestone Simon,Aarsland Dag, ,Hansson Oskar,Rosa-Neto Pedro,Westman Eric,Blennow Kaj,Zetterberg Henrik,Ashton Nicholas J
Alzheimer's & dementia : the journal of the Alzheimer's Association
INTRODUCTION:This study investigated the diagnostic and disease-monitoring potential of plasma biomarkers in mild cognitive impairment (MCI) and Alzheimer's disease (AD) dementia and cognitively unimpaired (CU) individuals. METHODS:Plasma was analyzed using Simoa assays from 99 CU, 107 MCI, and 103 AD dementia participants. RESULTS:Phosphorylated-tau181 (P-tau181), neurofilament light, amyloid-β (Aβ42/40), Total-tau and Glial fibrillary acidic protein were altered in AD dementia but P-tau181 significantly outperformed all biomarkers in differentiating AD dementia from CU (area under the curve [AUC] = 0.91). P-tau181 was increased in MCI converters compared to non-converters. Higher P-tau181 was associated with steeper cognitive decline and gray matter loss in temporal regions. Longitudinal change of P-tau181 was strongly associated with gray matter loss in the full sample and with Aβ measures in CU individuals. DISCUSSION:P-tau181 detected AD at MCI and dementia stages and was strongly associated with cognitive decline and gray matter loss. These findings highlight the potential value of plasma P-tau181 as a non-invasive and cost-effective diagnostic and prognostic biomarker in AD.
Plasma glial fibrillary acidic protein is elevated in cognitively normal older adults at risk of Alzheimer's disease.
Chatterjee Pratishtha,Pedrini Steve,Stoops Erik,Goozee Kathryn,Villemagne Victor L,Asih Prita R,Verberk Inge M W,Dave Preeti,Taddei Kevin,Sohrabi Hamid R,Zetterberg Henrik,Blennow Kaj,Teunissen Charlotte E,Vanderstichele Hugo M,Martins Ralph N
Glial fibrillary acidic protein (GFAP), an astrocytic cytoskeletal protein, can be measured in blood samples, and has been associated with Alzheimer's disease (AD). However, plasma GFAP has not been investigated in cognitively normal older adults at risk of AD, based on brain amyloid-β (Aβ) load. Cross-sectional analyses were carried out for plasma GFAP and plasma Aβ1-42/Aβ1-40 ratio, a blood-based marker associated with brain Aβ load, in participants (65-90 years) categorised into low (Aβ-, n = 63) and high (Aβ+, n = 33) brain Aβ load groups via Aβ positron emission tomography. Plasma GFAP, Aβ1-42, and Aβ1-40 were measured using the Single molecule array (Simoa) platform. Plasma GFAP levels were significantly higher (p < 0.00001), and plasma Aβ1-42/Aβ1-40 ratios were significantly lower (p < 0.005), in Aβ+ participants compared to Aβ- participants, adjusted for covariates age, sex, and apolipoprotein E-ε4 carriage. A receiver operating characteristic curve based on a logistic regression of the same covariates, the base model, distinguished Aβ+ from Aβ- (area under the curve, AUC = 0.78), but was outperformed when plasma GFAP was added to the base model (AUC = 0.91) and further improved with plasma Aβ1-42/Aβ1-40 ratio (AUC = 0.92). The current findings demonstrate that plasma GFAP levels are elevated in cognitively normal older adults at risk of AD. These observations suggest that astrocytic damage or activation begins from the pre-symptomatic stage of AD and is associated with brain Aβ load. Observations from the present study highlight the potential of plasma GFAP to contribute to a diagnostic blood biomarker panel (along with plasma Aβ1-42/Aβ1-40 ratios) for cognitively normal older adults at risk of AD.
Neurofilament light is a novel biomarker for mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes.
Zheng Yong-Sheng,Sun Chong,Wang Rong,Chen Ne,Luo Su-Shan,Xi Jian-Ying,Lu Jia-Hong,Zhao Chong-Bo,Li Yu-Xin,Zhou Lei,Lin Jie
Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a complicated maternally inherited disorder lacking of sensitive and specific biomarkers. The objective of this study was to investigate the serum neurofilament light chain (NfL) as a novel biomarker of neurological dysfunction in MELAS. Patients with different status of MELAS were enrolled in this study. The Mini-Mental State Examination (MMSE) was given to the participants to evaluate cognition status. Multiple functional MRI was performed on the participants. Blood samples were collected and the serum NfL concentrations were determined by the single-molecule array technology (Simoa). This study enrolled 23 patients with MELAS, 15 people in the acute attack phase of MELAS and 10 people in the remission phase, including 2 patients in both acute attack and remission phase. Sixteen healthy controls (HCs) were also enrolled. Serum NfL level increased significantly in patients with MELAS. Serum NfL level in the acute attack group (146.73 [120.91-411.31] pg/ml, median [IQR]) was higher than in the remission group (40.31 [19.54-151.05] pg/ml, median [IQR]) and HCs group (7.70 [6.13-9.78] pg/ml, median [IQR]) (p < 0.05). The level of NfL in the remission phase group was higher than in HCs group (p < 0.05). A negative correlation was found between the serum NfL level and MMSE (p = 0.006, r = -0.650). The NfL concentration correlated positively with stroke-like lesion volume in the brain (r = 0.740, p < 0.001). Serum NfL may serve as a novel biomarker for the neurological dysfunction in MELAS patients.
Brain-derived Neurotrophic Factor in the Aqueous Humor of Glaucoma Patients.
Igarashi Tsutomu,Nakamoto Kenji,Kobayashi Maika,Suzuki Hisaharu,Arima Takeshi,Tobita Yutaro,Takao Kazuhiro,Igarashi Toru,Okuda Takahisa,Okada Takashi,Takahashi Hiroshi
Journal of Nippon Medical School = Nippon Ika Daigaku zasshi
BACKGROUND:Brain-derived neurotrophic factor (BDNF) may be involved in the pathogenesis of glaucoma. BDNF concentrations reported in previous studies have varied widely, and the concentration of BDNF in aqueous humor is unknown. In this study, BDNF concentrations in the aqueous humor of glaucoma patients and control patients were measured with ELISA kits. METHODS:This prospective, observational study examined BDNF levels in aqueous humor in 62 eyes of 43 patients who underwent cataract surgery or trabeculectomy (11 glaucoma patients and 32 non-glaucoma cataract patients as controls). BDNF concentrations were examined by 4 different enzyme-linked immunosorbent assay (ELISA) techniques. RESULTS:The mean ± SD patient age was 72.0 ± 10.1 (range 35 to 87) years. Two of the techniques detected no BDNF in aqueous humor in any samples (n=3 and n=9, respectively); the average value was less than zero. An ultrasensitive ELISA kit did not yield reliable measurements. Finally, in an even more sensitive ELISA (Simoa-HD1), performed by an outside contractor, 25 (54.3%) eyes were below the detection limit, including 20 (55.6%) control and 5 (50%) glaucoma cases. For eyes with detectable BDNF, the overall BDNF concentration was 0.158 pg/mL (n=21): 0.196 pg/mL (n=16) in controls and 0.034 pg/mL (n=5) in glaucoma cases. CONCLUSIONS:BDNF level in aqueous humor varies widely.
Signs of neuroaxonal injury in preeclampsia-A case control study.
Andersson Malin,Oras Jonatan,Thörn Sven Egron,Karlsson Ove,Kälebo Peter,Zetterberg Henrik,Blennow Kaj,Bergman Lina
BACKGROUND:Cerebral injury is a common cause of maternal mortality due to preeclampsia and is challenging to predict and diagnose. In addition, there are associations between previous preeclampsia and stroke, dementia and epilepsy later in life. The cerebral biomarkers S100B, neuron specific enolase, (NSE), tau protein and neurofilament light chain (NfL) have proven useful as predictors and diagnostic tools in other neurological disorders. This case-control study sought to determine whether cerebral biomarkers were increased in cerebrospinal fluid (CSF) as a marker of cerebral origin and potential cerebral injury in preeclampsia and if concentrations in CSF correlated to concentrations in plasma. METHODS:CSF and blood at delivery from 15 women with preeclampsia and 15 women with normal pregnancies were analysed for the cerebral biomarkers S100B, NSE, tau protein and NfL by Simoa and ELISA based methods. MRI brain was performed after delivery and for women with preeclampsia also at six months postpartum. RESULTS:Women with preeclampsia demonstrated increased CSF- and plasma concentrations of NfL and these concentrations correlated to each other. CSF concentrations of NSE and tau were decreased in preeclampsia and there were no differences in plasma concentrations of NSE and tau between groups. For S100B, serum concentrations in preeclampsia were increased but there was no difference in CSF concentrations of S100B between women with preeclampsia and normal pregnancy. CONCLUSION:NfL emerges as a promising circulating cerebral biomarker in preeclampsia and increased CSF concentrations point to a neuroaxonal injury in preeclampsia, even in the absence of clinically evident neurological complications.
Plasma Tau and Neurofilament Light in Frontotemporal Lobar Degeneration and Alzheimer Disease.
Illán-Gala Ignacio,Lleo Alberto,Karydas Anna,Staffaroni Adam M,Zetterberg Henrik,Sivasankaran Rajeev,Grinberg Lea T,Spina Salvatore,Kramer Joel H,Ramos Eliana M,Coppola Giovanni,La Joie Renaud,Rabinovici Gil D,Perry David C,Gorno-Tempini Maria Luisa,Seeley William W,Miller Bruce L,Rosen Howard J,Blennow Kaj,Boxer Adam L,Rojas Julio C
OBJECTIVE:To test the hypothesis that plasma total tau (t-tau) and neurofilament light chain (NfL) concentrations may have a differential role in the study of frontotemporal lobar degeneration syndromes (FTLD-S) and clinically diagnosed Alzheimer disease syndromes (AD-S), we determined their diagnostic and prognostic value in FTLD-S and AD-S and their sensitivity to pathologic diagnoses. METHODS:We measured plasma t-tau and NfL with the Simoa platform in 265 participants: 167 FTLD-S, 43 AD-S, and 55 healthy controls (HC), including 82 pathology-proven cases (50 FTLD-tau, 18 FTLD-TDP, 2 FTLD-FUS, and 12 AD) and 98 participants with amyloid PET. We compared cross-sectional and longitudinal biomarker concentrations between groups, their correlation with clinical measures of disease severity, progression, and survival, and cortical thickness. RESULTS:Plasma NfL, but not plasma t-tau, discriminated FTLD-S from HC and AD-S from HC. Both plasma NfL and t-tau were poor discriminators between FLTD-S and AD-S. In pathology-confirmed cases, plasma NfL was higher in FTLD than AD and in FTLD-TDP compared to FTLD-tau, after accounting for age and disease severity. Plasma NfL, but not plasma t-tau, predicted clinical decline and survival and correlated with regional cortical thickness in both FTLD-S and AD-S. The combination of plasma NfL with plasma t-tau did not outperform plasma NfL alone. CONCLUSION:Plasma NfL is superior to plasma t-tau for the diagnosis and prediction of clinical progression of FTLD-S and AD-S. CLASSIFICATION OF EVIDENCE:This study provides Class III evidence that plasma NfL has superior diagnostic and prognostic performance vs plasma t-tau in FTLD and AD.
Modulation of Cardiometabolic Disease Markers by Type I Interferon Inhibition in Systemic Lupus Erythematosus.
Casey Kerry A,Smith Michael A,Sinibaldi Dominic,Seto Nickie L,Playford Martin P,Wang Xinghao,Carlucci Philip M,Wang Liangwei,Illei Gabor,Yu Binbing,Wang Shiliang,Remaley Alan T,Mehta Nehal N,Kaplan Mariana J,White Wendy I
Arthritis & rheumatology (Hoboken, N.J.)
OBJECTIVE:Neutrophil dysregulation and the type I interferon (IFN) axis have been proposed to contribute to premature cardiovascular disease, a leading cause of mortality in patients with systemic lupus erythematosus (SLE). In the present study, we evaluated the ability of anifrolumab, a type I IFN receptor-blocking antibody, to reduce neutrophil extracellular trap (NET) formation and modulate cardiometabolic disease markers in comparison to placebo. METHODS:Study subjects comprised patients with moderate-to-severe SLE who were enrolled in phase IIb of the MUSE trial (A Phase II, Randomized Study to Evaluate the Efficacy and Safety of MEDI-546 in Subjects with Systemic Lupus Erythematosus), with healthy individuals as controls. Blood samples were collected from SLE patients (n = 305) and healthy controls (n = 10-20) before the initiation of treatment (baseline) and from SLE patients after they had been treated with 300 mg of anifrolumab (n = 99) or placebo (n = 102). Baseline IFN gene signature test status was determined, and the IFN gene signature (21-gene panel) was monitored over time. Serum proteins were measured by multiplex immunoassay or ultrasensitive Simoa assay. NET complexes, cholesterol efflux capacity (CEC), and glycoprotein acetylation (GlycA) and other lipid parameters were assessed in plasma. RESULTS:Formation of NET complexes and levels of tumor necrosis factor (TNF) and interleukin-10 (IL-10) were correlated with extent of type I IFN pathway activity. NET complexes and IL-10 levels were up-regulated in SLE patients compared to healthy controls (P < 0.008). The cardiometabolic disease markers CEC and GlycA were also found to be dysregulated in patients with SLE (P < 0.001 versus healthy controls). Type I IFN receptor inhibition with anifrolumab significantly reduced NET complexes and GlycA and improved CEC compared to baseline (P < 0.05) whereas no improvements were seen with placebo. Levels of TNF and IL-10 were reduced with anifrolumab compared to placebo (P < 0.05). CONCLUSION:These data support a key role for type I IFNs in modulating factors contributing to SLE vasculopathy and suggest that inhibition of this pathway could decrease cardiovascular risk in individuals with SLE.
Plasma P-tau181 to Aβ42 ratio is associated with brain amyloid burden and hippocampal atrophy in an Asian cohort of Alzheimer's disease patients with concomitant cerebrovascular disease.
Chong Joyce R,Ashton Nicholas J,Karikari Thomas K,Tanaka Tomotaka,Saridin Francis N,Reilhac Anthonin,Robins Edward G,Nai Ying-Hwey,Vrooman Henri,Hilal Saima,Zetterberg Henrik,Blennow Kaj,Lai Mitchell K P,Chen Christopher P
Alzheimer's & dementia : the journal of the Alzheimer's Association
INTRODUCTION:There is increasing evidence that phosphorylated tau (P-tau181) is a specific biomarker for Alzheimer's disease (AD) pathology, but its potential utility in non-White patient cohorts and patients with concomitant cerebrovascular disease (CeVD) is unknown. METHODS:Single molecule array (Simoa) measurements of plasma P-tau181, total tau, amyloid beta (Aβ)40 and Aβ42, as well as derived ratios were correlated with neuroimaging modalities indicating brain amyloid (Aβ+), hippocampal atrophy, and CeVD in a Singapore-based cohort of non-cognitively impaired (NCI; n = 43), cognitively impaired no dementia (CIND; n = 91), AD (n = 44), and vascular dementia (VaD; n = 22) subjects. RESULTS:P-tau181/Aβ42 ratio showed the highest area under the curve (AUC) for Aβ+ (AUC = 0.889) and for discriminating between AD Aβ+ and VaD Aβ- subjects (AUC = 0.903). In addition, P-tau181/Aβ42 ratio was associated with hippocampal atrophy. None of the biomarkers was associated with CeVD. DISCUSSION:Plasma P-tau181/Aβ42 ratio may be a noninvasive means of identifying AD with elevated brain amyloid in populations with concomitant CeVD.
[Clinical chemistry laboratory assay of circulating oncology biomarkers : limitations and perspectives].
Sqalli G,Cavalier E,Ladang A
Revue medicale de Liege
In current practice, the use of circulating oncological biomarkers by clinicians is almost inseparable from cancer patients management. However, the interpretation of the results is not always easy because it is more specific to laboratory medicine and involves notions of peri-analytical orders as well as analytical sensitivity and specificity. In the past, the development of new analytical techniques improved the analytical sensitivity or allowed the implementation of new biomarkers; this observation would still be true today. Mass spectrometry, microRNA assay, or Single Molecule Array (SiMoA) are recent analytical developments with very good analytical performances that could contribute to the improvement of cancer patient management.
Ultra-sensitive protein detection via Single Molecule Arrays towards early stage cancer monitoring.
Schubert Stephanie M,Arendt Lisa M,Zhou Wenhui,Baig Shazia,Walter Stephanie R,Buchsbaum Rachel J,Kuperwasser Charlotte,Walt David R
The early diagnosis of cancers and continued monitoring of tumor growth would be greatly facilitated by the development of a blood-based, non-invasive, screening technique for early cancer detection. Current technologies for cancer screening and detection typically rely on imaging techniques or blood tests that are not accurate or sensitive enough to definitively diagnose cancer at its earliest stages or predict biologic outcomes. By utilizing Single Molecule Arrays (SiMoA), an ultra-sensitive enzyme-linked immunosorbent assay (ELISA) technique, we were able to measure increasing levels of prostate specific antigen (PSA) within murine serum over time, which we attribute to tumor development. The measured concentrations of PSA were well below the detectable limits of both a leading clinical diagnostic PSA ELISA assay as well as a commercial ultra-sensitive PSA assay. Our work benchmarks the role of SiMoA as a vital tool in monitoring previously non-detectable protein biomarkers in serum for early cancer detection and offers significant potential as a non-invasive platform for the monitoring of early stage cancer.
Quantification of plasma phosphorylated tau to use as a biomarker for brain Alzheimer pathology: pilot case-control studies including patients with Alzheimer's disease and down syndrome.
Tatebe Harutsugu,Kasai Takashi,Ohmichi Takuma,Kishi Yusuke,Kakeya Tomoshi,Waragai Masaaki,Kondo Masaki,Allsop David,Tokuda Takahiko
BACKGROUND:There is still a substantial unmet need for less invasive and lower-cost blood-based biomarkers to detect brain Alzheimer's disease (AD) pathology. This study is aimed to determine whether quantification of plasma tau phosphorylated at threonine 181 (p-tau181) is informative in the diagnosis of AD. METHODS:We have developed a novel ultrasensitive immunoassay to quantify plasma p-tau181, and measured the levels of plasma p-tau181 in three cohorts. RESULTS:In the first cohort composed of 20 AD patients and 15 age-matched controls, the plasma levels of p-tau181 were significantly higher in the AD patients than those in the controls (0.171 ± 0.166 pg/ml in AD versus 0.0405 ± 0.0756 pg/ml in controls, p = 0.0039). The percentage of the subjects whose levels of plasma p-tau181 exceeded the cut-off value (0.0921 pg/ml) was significantly higher in the AD group compared with the control group (60% in AD versus 16.7% in controls, p = 0.0090). In the second cohort composed of 20 patients with Down syndrome (DS) and 22 age-matched controls, the plasma concentrations of p-tau181 were significantly higher in the DS group (0.767 ± 1.26 pg/ml in DS versus 0.0415 ± 0.0710 pg/ml in controls, p = 0.0313). There was a significant correlation between the plasma levels of p-tau181 and age in the DS group (R = 0.4451, p = 0.0013). All of the DS individuals showing an extremely high concentration of plasma p-tau181 (> 1.0 pg/ml) were older than the age of 40. In the third cohort composed of 8 AD patients and 3 patients with other neurological diseases, the levels of plasma p-tau181 significantly correlated with those of CSF p-tau181 (R = 0.4525, p = 0.023). CONCLUSIONS:We report for the first time quantitative data on the plasma levels of p-tau181 in controls and patients with AD and DS, and these data suggest that the plasma p-tau181 is a promising blood biomarker for brain AD pathology. This exploratory pilot study warrants further large-scale and well-controlled studies to validate the usefulness of plasma p-tau181 as an urgently needed surrogate marker for the diagnosis and disease progression of AD.
Digital direct detection of microRNAs using single molecule arrays.
Cohen Limor,Hartman Mark R,Amardey-Wellington Aaron,Walt David R
Nucleic acids research
MicroRNAs (miRNAs) are involved in many biological pathways, and detecting miRNAs accurately is critical for diagnosing a variety of diseases including cancer. However, most current methods for miRNA detection require lengthy sample preparation and amplification steps that can bias the results. In addition, lack of specificity and reproducibility give rise to various challenges in detection of circulating miRNAs in biological samples. In this work, we applied the Single Molecule Array (Simoa) technique to develop an ultra-sensitive sandwich assay for direct detection of multiple miRNAs without pre-amplification. We successfully detected miRNAs at femtomolar concentrations (with limits of detection [LODs] ranging from 1 to 30 fM) and high specificity (distinguishing miRNAs with a single nucleotide mismatch). This method was effective against a range of diverse target sequences, suggesting a general approach for miRNA detection. To demonstrate the practical application of this technique, we detected miRNAs in a variety of sample types including human serum and total RNA. The high sensitivity and simple workflow of the Simoa method represent excellent advantages for miRNA-based diagnostics of human diseases.
Neuro biomarker levels measured with high-sensitivity digital ELISA differ between serum and plasma.
O'Connell Grant C,Alder Megan L,Webel Allison R,Moore Shirley M
Digital ELISA-based assays for blood biomarkers of neurological disease are on the verge of clinical use. Here, we aimed to determine whether different preanalytical blood processing techniques influence results. Concentrations of neurofilament light chain (NfL), Tau and amyloid beta (Aβ) were measured in human plasma and serum specimens using digital ELISA and compared between blood products. Measured levels of NfL were highly equivalant between serum and plasma in all analyses, however, measured levels of Tau and Aβ were consistently lower in serum relative to plasma. Tau and Aβ are likely lost during clotting in serum preparations, and should be assayed in plasma to get an accurate measure of circulating levels.
Diagnosis of ischemic stroke using circulating levels of brain-specific proteins measured via high-sensitivity digital ELISA.
O'Connell Grant C,Alder Megan L,Smothers Christine G,Still Carolyn H,Webel Allison R,Moore Shirley M
Limited lower detection ranges associated with traditional immunoassay techniques have prevented the use of brain-specific proteins as blood biomarkers of stroke in the acute phase of care, as these proteins are often only present in circulation at low concentrations. Digital ELISA is a newly developed technique with allows for quantification of proteins in biofluids with up to 1000 times greater sensitivity than conventional ELISA techniques. The purpose of this study was to determine whether the extended lower limits of detection associated with digital ELISA could enable the use of brain-specific proteins as blood biomarkers of ischemic stroke during triage. Blood was sampled from ischemic stroke patients (n = 14) at emergency department admission, as well as from neurologically normal controls matched in terms of risk factors for cardiovascular disease (n = 33). Plasma levels of two brain-specific axonal proteins, neurofilament light chain (NfL) and tau, were measured via digital ELISA, and receiver-operating characteristic analysis was used to determine their ability to discriminate between groups. Plasma levels of NfL and tau were both significantly elevated in stroke patients versus controls, and could respectively discriminate between groups with 92.9% sensitivity / 84.9% specificity, and 85.7% sensitivity / 54.6% specificity. Furthermore, adjustment of measured NfL and Tau levels according to the lower-limits of detection associated with commercially-available conventional ELISA assays resulted in a dramatic and statistically significant decrease in diagnostic performance. Collectively, our results suggest that the increased analytical sensitivity of digital ELISA could enable the use of brain-specific proteins as blood biomarkers of ischemic stroke during triage.
Performance Evaluation of a Multiplex Assay for Simultaneous Detection of Four Clinically Relevant Traumatic Brain Injury Biomarkers.
Korley Frederick K,Yue John K,Wilson David H,Hrusovsky Kevin,Diaz-Arrastia Ramon,Ferguson Adam R,Yuh Esther L,Mukherjee Pratik,Wang Kevin K W,Valadka Alex B,Puccio Ava M,Okonkwo David O,Manley Geoffrey T
Journal of neurotrauma
Traumatic brain injury (TBI) results in heterogeneous pathology affecting multiple cells and tissue types in the brain. It is likely that assessment of such complexity will require simultaneous measurement of multiple molecular biomarkers in a single sample of biological fluid. We measured glial fibrillary acidic protein (GFAP), ubiquitin c-terminal hydrolase L1 (UCH-L1), neurofilament light chain (NF-L) and total tau in plasma samples obtained from 107 subjects enrolled in the Transforming Research and Clinical Knowledge in Traumatic Brain Injury Pilot (TRACK-TBI Pilot) Study using the Quanterix Simoa 4-Plex assay. We also measured NF-L using the Simoa singleplex assay. We computed the correlation between the different biomarkers and calculated the discriminative value of each biomarker for distinguishing between subjects with abnormal versus normal head computed tomography (CT). We found a strong correlation between NF-L values derived from the multiplex and singleplex assays (correlation coefficient = 0.997). Among biomarker values derived from the multiplex assay, the strongest correlation was between the axonal and neuronal markers, NF-L and UCH-L1 (coefficient = 0.71). The weakest correlation was between the glial marker GFAP and the axonal marker tau (coefficient = 0.06). The areas under the curves for distinguishing between subjects with/without abnormal head CT for multiplex GFAP, UCH-L1, NF-L, and total tau were: 0.88 (95% confidence interval 0.81-0.95), 0.86 (0.79-0.93), 0.84 (0.77-0.92), and 0.77 0.67-0.86), respectively. We conclude that the multiplex assay provides simultaneous quantification of GFAP, UCH-L1, NF-L, and tau, and may be clinically useful in the diagnosis of TBI as well as identifying different types of cellular injury.
Detection of amyloid β oligomers toward early diagnosis of Alzheimer's disease.
Hwang Soyoon Sarah,Chan Hon,Sorci Mirco,Van Deventer James,Wittrup Dane,Belfort Georges,Walt David
Amyloid β (Aβ) peptide accumulation in the brain is considered to be one of the hallmarks of Alzheimer's disease. Here, we compare two analytical techniques for detecting neurotoxic Aβ oligomers - Quartz Crystal Microbalance with Dissipation (QCM-D) and Single Molecule Array (Simoa). Both detection methods exploit a feature of the monoclonal antibody bapineuzumab, which targets N-terminal residues 1-5 of Aβ with high affinity and use it as both a capture and detection reagent. Assays developed with the two methods allow us to specifically recognize neurotoxic Aβ oligomers and higher aggregates such as fibrils but discriminate against Aβ monomer species. We find that for detection of Aβ oligomers, Simoa was roughly 500 times more sensitive than the QCM-D technique with limits of detection of 0.22 nM and 125 nM, respectively.
Comparison of Different Platform Immunoassays for the Measurement of Plasma Alpha-Synuclein in Parkinson's Disease Patients.
Youssef Priscilla,Kim Woojin S,Halliday Glenda M,Lewis Simon J G,Dzamko Nicolas
Journal of Parkinson's disease
BACKGROUND:The identification of reliable biomarkers in Parkinson's disease (PD) would provide much needed diagnostic accuracy, a means of monitoring progression, objectively measuring treatment response, and potentially allowing patient stratification within clinical trials. Whilst the assessment of total alpha-synuclein in biofluids has been identified as a promising biomarker, conflicting trends in these levels across patient plasma samples relative to controls has limited its use. Different commercially available assay platforms that have been used to measure alpha-synuclein may contribute to different study outcomes. OBJECTIVE:To compare different platform immunoassays for the measurement of total alpha-synuclein using the same plasma samples from 49 PD patients and 47 controls. METHODS:Total plasma alpha-synuclein concentrations were assessed using the BioLegend, MesoScale Discovery, and Quanterix platform in plasma samples from PD patients and matched controls. RESULTS:A significant increase in total plasma alpha-synuclein was observed in PD patients using the Biolegend (10%), Mesoscale Discovery (13%) and Quanterix (39%) assays. The Mesoscale Discovery and Quanterix assays showed the strongest correlations (r = 0.78, p < 0.0001) with each other, whilst the Quanterix platform demonstrated the lowest variation and highest effect size. Inclusion of age, sex and hemoglobin levels as covariates in the analysis of total alpha-synuclein improved the ability of all three immunoassays to detect a significant difference between patients and controls. CONCLUSION:All three immunoassays were sensitive enough to detect group level differences between PD patients and controls, with the largest effect size observed with the Quanterix assay. These results may help inform assay choices in ongoing clinical trials.
Serum neurofilament light chain is a discriminative biomarker between frontotemporal lobar degeneration and primary psychiatric disorders.
Katisko Kasper,Cajanus Antti,Jääskeläinen Olli,Kontkanen Aleksi,Hartikainen Päivi,Korhonen Ville E,Helisalmi Seppo,Haapasalo Annakaisa,Koivumaa-Honkanen Heli,Herukka Sanna-Kaisa,Remes Anne M,Solje Eino
Journal of neurology
Due to the significant clinical overlap between frontotemporal lobar degeneration (FTLD) spectrum disorders and late-onset primary psychiatric disorders (PPD), diagnostic biomarkers reflecting the different underlying pathophysiologies are urgently needed. Thus far, elevated cerebrospinal fluid (CSF) levels of neurofilament light chain (NfL) have been reported in various neurological conditions. Furthermore, recent advancements in ultrasensitive analytical methods (e.g., single molecule array, Simoa) have enabled sensitive and less invasive NfL detection also from blood samples. In this study, we evaluated the potential of serum NfL (sNfL) as a diagnostic tool between FTLD and PPD. We analyzed sNfL levels with Simoa from 125 participants including patients from FTLD (n = 91) and PPD (n = 34) spectra. Our results show that sNfL levels are higher in the FTLD group compared to the PPD group as well as in separate clinical subtypes of FTLD compared to different psychiatric manifestations (i.e., mood or psychotic disorders). At single-subject level, discrimination between FTLD and PPD was possible with 80% sensitivity and 85% specificity (AUC = 0.850, 95% CI 0.776-0.923), and between behavioral variant frontotemporal dementia (bvFTD) and PPD with 79% sensitivity and 85% specificity (AUC = 0.830, 95% CI 0.732-0.908). These findings highlight the potential of sNfL as a discriminating biomarker for FTLD over PPD in patients with wide-ranging behavioral, psychiatric and cognitive symptoms.
Validation of serum neurofilaments as prognostic and potential pharmacodynamic biomarkers for ALS.
Benatar Michael,Zhang Lanyu,Wang Lily,Granit Volkan,Statland Jeffrey,Barohn Richard,Swenson Andrea,Ravits John,Jackson Carlayne,Burns Ted M,Trivedi Jaya,Pioro Erik P,Caress James,Katz Jonathan,McCauley Jacob L,Rademakers Rosa,Malaspina Andrea,Ostrow Lyle W,Wuu Joanne,
OBJECTIVE:To identify preferred neurofilament assays and clinically validate serum neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) as prognostic and potential pharmacodynamic biomarkers relevant to amyotrophic lateral sclerosis (ALS) therapy development. METHODS:In this prospective, multicenter, longitudinal observational study of patients with ALS (n = 229), primary lateral sclerosis (n = 20), and progressive muscular atrophy (n = 11), biological specimens were collected, processed, and stored according to strict standard operating procedures (SOPs). Neurofilament assays were performed in a blinded manner by independent contract research organizations. RESULTS:For serum NfL and pNfH measured using the Simoa assay, there were no missing data (i.e., technical replicates below the lower limit of detection were not encountered). For the Iron Horse and Euroimmun pNfH assays, such missingness was encountered in ∼4% and ∼10% of serum samples, respectively. Mean coefficients of variation for NfL in serum and CSF were both ∼3%. Mean coefficients of variation for pNfH in serum and CSF were ∼4%-5% and ∼2%-3%, respectively, in all assays. Baseline serum NfL concentration, but not pNfH, predicted the future Revised ALS Functional Rating Scale (ALSFRS-R) slope and survival. Incorporation of baseline serum NfL into mixed effects models of ALSFRS-R slopes yields an estimated sample size saving of ∼8%. Depending on the method used to estimate effect size, use of serum NfL (and perhaps pNfH) as pharmacodynamic biomarkers, instead of the ALSFRS-R slope, yields significantly larger sample size savings. CONCLUSIONS:Serum NfL may be considered a clinically validated prognostic biomarker for ALS. Serum NfL (and perhaps pNfH), quantified using the Simoa assay, has potential utility as a pharmacodynamic biomarker of treatment effect.
Comparison of Simoa and Ella to assess serum neurofilament-light chain in multiple sclerosis.
Gauthier Audrey,Viel Sébastien,Perret Magali,Brocard Guillaume,Casey Romain,Lombard Christine,Laurent-Chabalier Sabine,Debouverie Marc,Edan Gilles,Vukusic Sandra,Lebrun-Frénay Christine,De Sèze Jérôme,Laplaud David Axel,Castelnovo Giovanni,Gout Olivier,Ruet Aurélie,Moreau Thibault,Casez Olivier,Clavelou Pierre,Berger Eric,Zephir Hélène,Trouillet-Assant Sophie,Thouvenot Eric,
Annals of clinical and translational neurology
We compared Simoa and Ella immunoassays to assess serum neurofilament-light chain levels in 203 multiple sclerosis patients from the OFSEP HD study. There was a strong correlation (ρ = 0.86, p < 0.0001) between both platforms. The Ella instrument overestimated values by 17%, but as the data were linear (p = 0.57), it was possible to apply a correction factor to Ella results. As for Simoa , serum neurofilament-light chain levels measured by Ella were correlated with age and EDSS and were significantly higher in active multiple sclerosis, suggesting that these assays are equivalent and can be used in routine clinical practice.
Highly specific and ultrasensitive plasma test detects Abeta(1-42) and Abeta(1-40) in Alzheimer's disease.
Thijssen Elisabeth H,Verberk Inge M W,Vanbrabant Jeroen,Koelewijn Anne,Heijst Hans,Scheltens Philip,van der Flier Wiesje,Vanderstichele Hugo,Stoops Erik,Teunissen Charlotte E
Plasma biomarkers that reflect specific amyloid beta (Abeta) proteoforms provide an insight in the treatment effects of Alzheimer's disease (AD) therapies. Our aim was to develop and validate ready-to-use Simoa 'Amyblood' assays that measure full length Abeta and Abeta and compare their performance with two commercial assays. Linearity, intra- and inter-assay %CV were compared between Amyblood, Quanterix Simoa triplex, and Euroimmun ELISA. Sensitivity and selectivity were assessed for Amyblood and the Quanterix triplex. Clinical performance was assessed in CSF biomarker confirmed AD (n = 43, 68 ± 6 years) and controls (n = 42, 62 ± 5 years). Prototype and Amyblood showed similar calibrator curves and differentiation (20 AD vs 20 controls, p < 0.001). Amyblood, Quanterix triplex, and ELISA showed similar linearity (96%-122%) and intra-assay %CVs (≤ 3.1%). A minor non-specific signal was measured with Amyblood of + 2.4 pg/mL Abeta when incubated with 60 pg/mL Abeta. A substantial non-specific signal of + 24.7 pg/mL Abeta was obtained when 40 pg/mL Abeta was measured with the Quanterix triplex. Selectivity for Abeta at physiological Abeta and Abeta concentrations was 125% for Amyblood and 163% for Quanterix. Amyblood and Quanterix ratios (p < 0.001) and ELISA Abeta concentration (p = 0.025) could differentiate AD from controls. We successfully developed and upscaled a prototype to the Amyblood assays with similar technical and clinical performance as the Quanterix triplex and ELISA, but better specificity and selectivity than the Quanterix triplex assay. These results suggest leverage of this specific assay for monitoring treatment response in trials.
Glial Activation Markers in CSF and Serum From Patients With Primary Progressive Multiple Sclerosis: Potential of Serum GFAP as Disease Severity Marker?
Abdelhak Ahmed,Hottenrott Tilman,Morenas-Rodríguez Estrella,Suárez-Calvet Marc,Zettl Uwe K,Haass Christian,Meuth Sven G,Rauer Sebastian,Otto Markus,Tumani Hayrettin,Huss André
Frontiers in neurology
In progressive multiple sclerosis (MS), glial activation is thought to be a relevant mechanism of disability progression. Therefore, assessment of the glial cell activity is, in the emerging treatment era of primary progressive MS (PPMS), more important than ever. To test the association of cerebrospinal fluid (CSF) and serum markers of glial activation in PPMS patients; including glial fibrillary acidic protein (GFAP), chitinase-3-like protein 1 (CHI3L1), soluble variant of triggering receptor expressed on myeloid cells 2 (sTREM2), and marker of neuroaxonal damage (Neurofilament light chain, NfL) as well as clinical severity. CSF and serum samples from PPMS patients were collected in the MS-centers at Universities of Freiburg ( = 49), Ulm ( = 27), Muenster ( = 11), and Rostock ( = 6). sTREM2 and CHI3L1 levels were measured using the previously reported ELISA assays, while NfL and GFAP were measured using SIMOA assays. Clinical data included age, gender, disease duration, treatment status, and Expanded Disability Status Scale (EDSS). 93 CSF samples and 71 matching serum samples were analyzed. The median age of patients was 49 years and disease duration 4.5 years. GFAP correlated with EDSS after correction for age (β = 0.3, = 0.001). Furthermore, EDSS was higher in patients with a GFAP level ≥ 151.7 pg/ml compared to patients with GFAP below this cut-off (5.5 vs. 4.0, = 0.009). Other markers did not correlate with the clinical severity. Moreover, we found a correlation between NfL and GFAP, sTREM2 and CHI3L1 (ρ = 0.4 for GFAP and sTREM2, ρ = 0.3 for CHI3L1, < 0.01 for sTREM2 and CHI3L1 and <0.001 for GFAP). CHI3L1 did not correlate with GFAP but with sTREM2 (ρ = 0.4, < 0.01). The correlation between the glial activation markers in CSF with the markers of neuroaxonal demise supports the notion of the glial involvement in PPMS. The positive correlation between GFAP with disease duration and GFAP with the clinical severity of the disease may highlight a particular role of the astrocytes in PPMS and mark the potential of GFAP as a disease severity marker.
Neurofilament light chain as a blood biomarker to differentiate psychiatric disorders from behavioural variant frontotemporal dementia.
Al Shweiki Mhd Rami,Steinacker Petra,Oeckl Patrick,Hengerer Bastian,Danek Adrian,Fassbender Klaus,Diehl-Schmid Janine,Jahn Holger,Anderl-Straub Sarah,Ludolph Albert C,Schönfeldt-Lecuona Carlos,Otto Markus
Journal of psychiatric research
The overlapping symptoms of behavioural variant frontotemporal dementia (bvFTD) and primary psychiatric disorders (such as depressive disorder, schizophrenia spectrum, and bipolar disorder) present a challenge for the differential diagnosis of bvFTD in middle and older-aged people. Neurofilaments are cytoskeletal proteins in the neurons, and several studies have reported elevated levels of neurofilament light chain (NfL) in cerebrospinal fluid of neurodegenerative as well as psychiatric disorders. The study aims to determine the utility of serum NfL levels as a biomarker to differentiate between bvFTD and psychiatric disorder. In our study, we investigated the levels of NfL in the serum of schizophrenia (n = 11), depression (n = 28), bipolar (n = 11), bvFTD (n = 20) patients and controls (n = 27) by single molecule array (Simoa) technology. The schizophrenia, depression and bipolar patients did not show significant changes in serum NfL levels in comparison to the control group (p > 0.99). The serum NfL levels were significantly elevated in bvFTD patients in comparison to the control cohort (p < 0.0001), depression (p < 0.0001), schizophrenia (p < 0.0002) and bipolar patients (p < 0.0083). We propose serum NfL as a biomarker to differentiate bvFTD from psychiatric disorders and to rule out neurodegeneration in the course of psychiatric disorders.
Alpha-Synuclein Protofibrils in Cerebrospinal Fluid: A Potential Biomarker for Parkinson's Disease.
von Euler Chelpin Marianne,Söderberg Linda,Fälting Johanna,Möller Christer,Giorgetti Marco,Constantinescu Radu,Blennow Kaj,Zetterberg Henrik,Höglund Kina
Journal of Parkinson's disease
BACKGROUND:Currently, there is no established biomarker for Parkinson's disease (PD) and easily accessible biomarkers are crucial for developing disease-modifying treatments. OBJECTIVE:To develop a novel method to quantify cerebrospinal fluid (CSF) levels of α-synuclein protofibrils (α-syn PF) and apply it to clinical cohorts of patients with PD and atypical parkinsonian disorders. METHODS:A cohort composed of 49 patients with PD, 12 with corticobasal degeneration (CBD), 22 with progressive supranuclear palsy, and 33 controls, that visited the memory clinic but had no biomarker signs of Alzheimer's disease (AD, tau<350 pg/mL, amyloid-beta 42 (Aβ42)>530 pg/mL, and phosphorylated tau (p-tau)<60 pg/mL) was used in this study. The CSF samples were analyzed with the Single molecule array (Simoa) technology. Total α-synuclein (α-syn) levels were analyzed with a commercial ELISA-kit. RESULTS:The assay is specific to α-syn PF, with no cross-reactivity to monomeric α-syn, or the β- and γ-synuclein variants. CSF α-syn PF levels were increased in PD compared with controls (62.1 and 40.4 pg/mL, respectively, p = 0.03), and CBD (62.1 and 34.2 pg/mL, respectively, p = 0.02). The accuracy of predicting PD using α-syn PF is significantly different from controls (area under the curve 0.68, p = 0.0097) with a sensitivity of 62.8% and specificity of 67.7%. Levels of total α-syn were significantly different between the PD and CBD groups (p = 0.04). CONCLUSION:The developed method specifically quantifies α-syn PF in human CSF with increased concentrations in PD, but with an overlap with asymptomatic elderly controls.
Dynamics of plasma biomarkers in Down syndrome: the relative levels of Aβ42 decrease with age, whereas NT1 tau and NfL increase.
Mengel David,Liu Wen,Glynn Robert J,Selkoe Dennis J,Strydom Andre,Lai Florence,Rosas H Diana,Torres Amy,Patsiogiannis Vasiliki,Skotko Brian,Walsh Dominic M
Alzheimer's research & therapy
BACKGROUND:Down syndrome (DS) is the most common genetic cause of Alzheimer's disease (AD), but diagnosis of AD in DS is challenging due to the intellectual disability which accompanies DS. When disease-modifying agents for AD are approved, reliable biomarkers will be required to identify when and how long people with DS should undergo treatment. Three cardinal neuropathological features characterize AD, and AD in DS-Aβ amyloid plaques, tau neurofibrillary tangles, and neuronal loss. Here, we quantified plasma biomarkers of all 3 neuropathological features in a large cohort of people with DS aged from 3 months to 68 years. Our primary aims were (1) to assess changes in the selected plasma biomarkers in DS across age, and (2) to compare biomarkers measured in DS plasma versus age- and sex-matched controls. METHODS:Using ultra-sensitive single molecule array (Simoa) assays, we measured 3 analytes (Aβ42, NfL, and tau) in plasmas of 100 individuals with DS and 100 age- and sex-matched controls. Tau was measured using an assay (NT1) which detects forms of tau containing at least residues 6-198. The stability of the 3 analytes was established using plasma from ten healthy volunteers collected at 6 intervals over a 5-day period. RESULTS:High Aβ42 and NT1 tau and low NfL were observed in infants. Across all ages, Aβ42 levels were higher in DS than controls. Levels of Aβ42 decreased with age in both DS and controls, but this decrease was greater in DS than controls and became prominent in the third decade of life. NT1 tau fell in adolescents and young adults, but increased in older individuals with DS. NfL levels were low in infants, children, adolescents, and young adults, but thereafter increased in DS compared to controls. CONCLUSIONS:High levels of Aβ42 and tau in both young controls and DS suggest these proteins are produced by normal physiological processes, whereas the changes seen in later life are consistent with emergence of pathological alterations. These plasma biomarker results are in good agreement with prior neuropathology studies and indicate that the third and fourth decades (i.e., 20 to 40 years of age) of life are pivotal periods during which AD processes manifest in DS. Application of the assays used here to longitudinal studies of individuals with DS aged 20 to 50 years of age should further validate the use of these biomarkers, and in time may allow identification and monitoring of people with DS best suited for treatment with AD therapies.
Intraindividual Neurofilament Dynamics in Serum Mark the Conversion to Sporadic Parkinson's Disease.
Wilke Carlo,Dos Santos Marcia Cristina Teixeira,Schulte Claudia,Deuschle Christian,Scheller Dieter,Verbelen Moira,Brockmann Kathrin,von Thaler Anna-Katharina,Sünkel Ulrike,Roeben Benjamin,Bujac Sarah,Metzger Florian G,Maetzler Walter,da Costa Andre Nogueira,Synofzik Matthis,Berg Daniela
Movement disorders : official journal of the Movement Disorder Society
BACKGROUND AND OBJECTIVES:With disease-modifying treatment strategies on the horizon, stratification of individual patients at the earliest stages of Parkinson's disease (PD) is key-ideally already at clinical disease onset. Blood levels of neurofilament light chain (NfL) provide an easily accessible fluid biomarker that might allow capturing the conversion from prodromal to manifest PD. METHODS:We assessed longitudinal serum NfL levels in subjects converting from prodromal to manifest sporadic PD (converters), at-risk subjects, and matched controls (72 participants with ≈4 visits), using single-molecule array (Simoa) technique. RESULTS:While NfL levels were not increased at the prodromal stage, subjects converting to the manifest motor stage showed a significant intraindividual acceleration of the age-dependent increase of NfL levels. CONCLUSIONS:The temporal dynamics of intraindividual NfL blood levels might mark the conversion to clinically manifest PD, providing a potential stratification biomarker for individual disease onset in the advent of precision medicine for PD. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.
Tau, S100B and NSE as Blood Biomarkers in Acute Cerebrovascular Events.
Onatsu Juha,Vanninen Ritva,JÄkÄlÄ Pekka,Mustonen Pirjo,Pulkki Kari,Korhonen Miika,Hedman Marja,HÖglund Kina,Blennow Kaj,Zetterberg Henrik,Herukka Sanna-Kaisa,Taina Mikko
In vivo (Athens, Greece)
BACKGROUND/AIM:We aimed to analyze the diagnostic value of total tau (T-tau), S-100 calcium-binding protein B (S100B) and neuron-specific enolase (NSE) as blood-based biomarkers in acute ischemic stroke (AIS) or transient ischemic attack (TIA), and their correlation with symptom severity, infarct size, etiology and outcome. PATIENTS AND METHODS:A total of 102 patients with stroke and 35 with TIA were analyzed. Subacute (63.8±50.1 h) plasma T-tau was measured with the single-molecule array (Simoa) method and NSE and S100B were evaluated for comparison. We evaluated biomarkers associations with: (i) diagnosis of AIS or TIA, (ii) cerebral infarction volume in the brain computed tomography, (iii) stroke etiology, (iv) clinical stroke severity and (iv) functional outcome after three months. RESULTS:T-tau was higher in patients with stroke [1.0 pg/ml (IQR=0.3-2.2)] than with TIA [0.5 pg/ml (IQR=0.2-1.0), p=0.02]. The levels of S100B were also increased in stroke [0.082 μg/l (IQR=0.049-0.157)] patients compared to TIA patients [0.045 μg/l (IQR=0.03-0.073), p<0.001]. However, when the results were adjusted for confounders, significance was lost. Serum levels of NSE among patients with AIS [11.85 μg/l (IQR=9.30-16.14)] compared to those with TIA [10.96 μg/l (IQR=7.98-15.33), p=0.30] were equal. T-tau and S100B concentrations significantly correlated with cerebral infarction volume (r=0.412, p<0.001) and (r=0.597, p<0.001), also after corrections (p<0.001). mRS scores at three-month follow-up correlated with T-tau (r=0.248, p=0.016) and S100B concentrations (r=0.205, p=0.045). CONCLUSION:For the diagnosis of TIA vs. AIS, blood T-tau and S100B concentrations discriminated only modestly. Additionally, groups were not separable after measuring of T-tau and S100B levels in the blood. T-tau and S100B concentrations correlated with the infarct size, but were not alone predictive for functional outcome at 3 months.
Optimization and qualification of the single molecule array digital immunoassay for IL-12p70 in plasma of cancer patients.
Gupta Vinita,Kalia Navdeep,Yadav Mahesh,Noyes Amy,Good Jeremy,Hendricks Robert,Xu Wenfeng
AIM:Cytokine/chemokine levels can reflect the pharmacodynamics of checkpoint inhibitors. The single molecule array (Simoa) HD-1 is a sensitive next-generation immunoassay platform for quantification of low abundance proteins, with potential for cancer immunotherapy mechanism of action studies. RESULTS:The Simoa IL-12p70 reagents, standard curve and test conditions were optimized for improved precision and linearity of dilution in plasma of cancer patients. The assay achieved a lower limit of quantification of 0.08 pg/ml, with 27/29 samples recording above lower limit of quantification, precision ≤20% CV and accuracy within 80-120%. CONCLUSION:Simoa enabled quantification of IL-12p70 at sub-pg/ml levels in cancer patients and was superior to Simple Plex™ and Aushon in overall performance. This study qualifies the user-modified IL-12p70 immunoassay to measure pharmacodynamic changes in plasma during cancer immunotherapy.
Assessment of an ultra-sensitive IFNγ immunoassay prototype for latent tuberculosis diagnosis.
Ben Salah Elyes,Dorgham Karim,Lesénéchal Mylène,Pease Camille,Allard Laure,Dragonetti Céline,Gorochov Guy,Guihot Amélie,Sterlin Delphine
European cytokine network
Worldwide there are about 1.7 billion individuals with latent tuberculosis infection (LTBI) and only 5% to 15% will develop active tuberculosis (TB). It is recommended to treat only those most at risk of developing active TB to avoid problems of drug resistance. LTBI diagnosis involves reviewing the individual's medical history, physical examination, and biological tests. Interferon gamma release assays (IGRA) can yield "undeterminate" or "uncertain" results, which makes clinical management decisions difficult. We assessed an ultra-sensitive immunoassay prototype based on single molecule array (SiMoA) technology to evaluate its overall performance, and in particular, its performance for indeterminate and uncertain positive or negative samples, as classified by the results from the current ELISA technique used for IFNγ quantification. We analyzed samples from hospitalized or consulting patients and healthcare workers from three hospitals in Paris, previously classified as negative (n = 30), positive (n = 35), uncertain negative (n = 25), uncertain positive (n = 31), or indeterminate (n = 30). We observed that with the SiMoA assay 83.3% of the indeterminate samples became interpretable and could be classified as negative, whereas 74% of uncertain positive samples were classified as positive. Most uncertain negative samples (72%) were reclassified as uncertain positive (68%) or positive (4%). The results suggest that the ultra-sensitive SiMoA IFNγ assay could represent a useful tool for the identification of true positive and negative samples among those giving indeterminate or uncertain results with the TB IGRA assay currently used.
Plasma biomarker profiles and the correlation with cognitive function across the clinical spectrum of Alzheimer's disease.
Xiao Zhenxu,Wu Xue,Wu Wanqing,Yi Jingwei,Liang Xiaoniu,Ding Saineng,Zheng Li,Luo Jianfeng,Gu Hongchen,Zhao Qianhua,Xu Hong,Ding Ding
Alzheimer's research & therapy
BACKGROUND:Plasma biomarkers showed a promising value in the disease diagnosis and management of Alzheimer's disease (AD). However, profiles of the biomarkers and the associations with cognition across a spectrum of cognitive stages have seldom been reported. METHODS:We recruited 320 individuals with cognitive impairment and 131 cognitively normal participants from a memory clinic and a community cohort. Participants were classified into 6 groups based on their Clinical Dementia Rating (CDR) scores and clinical diagnosis, including AD, amnestic mild cognitive impairment (aMCI), and normal cognition (NC). A battery of neuropsychological tests was used to assess the global and domain-specific cognition. Plasma Aβ, Aβ, Aβ/Aβ, total tau (t-tau), neurofilament protein light chain (NfL), and phosphorylated tau at threonine 181 (p-tau181) were quantified using the single-molecule array (Simoa) platform. RESULTS:All the plasma markers (Aβ, Aβ, Aβ/Aβ, t-tau, NfL, p-tau181) showed certain discrepancies among NC, aMCI, and AD groups. The p-tau181 level showed a continuous escalating trend as the CDR scores increased from 0 (NC group) to 3 (severe AD). Compared with other biomarkers, p-tau181 had correlations with broader cognitive domains, covering global cognition (r = -0.536, P < 0.0001), memory (r = -0.481, P < 0.0001), attention (r = -0.437, P < 0.0001), visuospatial function (r = -0.385, P < 0.0001), and language (r = -0.177, P = 0.0003). Among participants with CDR ≥ 1, higher p-tau181 was correlated with worse global cognition (r = -0.301, P < 0.001). CONCLUSIONS:Plasma p-tau181 had correlations with broader cognitive domains, suggesting its potential as a promising clinical-relevant blood-based biomarker.
Detection of mycobacterial lipoarabinomannan in serum for diagnosis of active tuberculosis.
Brock Mary,Hanlon David,Zhao Mingwei,Pollock Nira R
Diagnostic microbiology and infectious disease
Urinary detection of Mycobacterium tuberculosis lipoarabinomannan (LAM) for tuberculosis (TB) diagnosis is well characterized, but the utility of serum LAM detection remains unclear. We developed an assay for serum LAM detection using single-molecule array (Simoa), purified M. tuberculosis LAM, and anti-LAM monoclonal antibodies and evaluated performance on diluted/heat-treated serum samples from patients with and without active TB and/or HIV. The Simoa assay had a limit of detection of 0.35 pg/mL and lower limit of quantification of 0.942 pg/mL. Corrected serum LAM concentrations ranged from 0 to 132.0 pg/mL [median 1.71, interquartile range (IQR) 0.94-6.80] in 90 TB+ patients and from 0 to 2.29 pg/mL (median 1.03, IQR 0.47-1.69) in 55 TB- patients. Using a cutoff of 2.3 pg/mL for 100% specificity, assay sensitivity was 37% in all TB+ subjects (33/90; 95% CI 0.27-0.48), 47% in TB+/HIV+ subjects (26/55; 0.34-0.61), and 60% in TB+/HIV+/smear+ subjects (21/35; 0.42-0.76). Mycobacterial LAM is detectable in serum with high specificity and reasonable sensitivity using Simoa.
Simultaneous detection of small molecules, proteins and microRNAs using single molecule arrays.
Wang Xu,Walt David R
Biological samples such as blood, urine, cerebrospinal fluid and saliva contain a large variety of proteins, nucleic acids, and small molecules. These molecules can serve as potential biomarkers of disease and therefore, it is desirable to simultaneously detect multiple biomarkers in one sample. Current detection techniques suffer from various limitations including low analytical sensitivity and complex sample processing. In this work, we present an ultrasensitive method for simultaneous detection of small molecules, proteins and microRNAs using single molecule arrays (Simoa). Dye-encoded beads modified with specific capture probes were used to quantify each analyte. Multiplex competitive Simoa assays were established for simultaneous detection of cortisol and prostaglandin E2. In addition, competitive and sandwich immunoassays were combined with a direct nucleic acid hybridization assay for simultaneous detection of cortisol, interleukin 6 and microRNA 141. The multi-analyte Simoa assay shows high sensitivity and specificity, which provides a powerful tool for the analysis of many different samples.
Development of immunoprecipitation - two-dimensional liquid chromatography - mass spectrometry methodology as biomarker read-out to quantify phosphorylated tau in cerebrospinal fluid from Alzheimer disease patients.
Bijttebier Sebastiaan,Theunis Clara,Jahouh Farid,Martins Dina Rodrigues,Verhemeldonck Marc,Grauwen Karolien,Dillen Lieve,Mercken Marc
Journal of chromatography. A
In Alzheimer's disease (AD) brain, one of the histopathological hallmarks is the neurofibrillary tangles consisting of aggregated and hyperphosphorylated tau. Currently many tau binding antibodies are under development to target the extracellular species responsible for the spreading of the disease in the brain. As such, an in-house developed antibody JNJ-63733657 with picomolar affinity towards tau phosphorylated at both T212 and T217 (further named p217+tau) was recently tested in phase I clinical trial NCT03375697. Following multiple dose administration in healthy subjects and subjects with AD, there were dose dependant reductions in free p217+tau fragments in cerebrospinal fluid (CSF) following antibody administration, as measured with a novel single molecule ELISA assay (Simoa PT3 x PT82 assay), demonstrating epitope engagement of the therapeutic antibody [Galpern, Haeverans, Janssens, Triana-Baltzer, Kolb, Li, Nandy, Mercken, Van Kolen, Sun, Van Nueten, 2020]. Total p217+tau levels also were reduced in CSF as measured with the Simoa PT3 x PT82 assay. In this study we developed an orthogonal immunoprecipitation - liquid chromatography - triple quadrupole mass spectrometry (IP-LC-TQMS) assay to verify the observed reductions in total p217+ tau levels. In this assay, an excess of JNJ-63733657 is added to the clinical CSF to ensure all p217+tau is bound by the antibody instead of having a pool of bound and unbound antigen and to immunoprecipitate all p217+tau, which is followed by on-bead digestion with trypsin to release surrogate peptides. Tryptic peptides with missed cleavages were monitored when phosphorylation occurred close to the cleavage site as this induced miscleavages. Compared with acidified mobile phases typically used for peptide analysis, reversed phase LC with mobile phase at basic pH resulted in sharper peaks and improved selectivity and sensitivity for the target peptides. With this setup a diphospho-tau tryptic peptide SRTPSLPTPPTREPK*2 could be measured with pT217 accounting for at least one of the phospho-sites. This is the first time that the presence of a diphopsho-tau peptide is reported to be present in human CSF. A two-dimensional LC-TQMS method was developed to remove matrix interferences. Selective trapping of diphospho-peptides via a metal oxide chromatography mechanism was achieved in a first dimension with a conventional reversed phase stationary phase and acidified mobile phase. Subsequent elution at basic pH enabled detection of low picomolar p217+tau levels in human CSF (lower limit of quantification: 2 pM), resulting in an approximate 5-fold increase in sensitivity. This enabled the quantification of total p217+tau in CSF leading to the confirmation that in addition to reductions in free p217+tau levels total p217+tau levels were also reduced following administration of the tau mAb JNJ-63733657, correlating with the previous measurement with the PT3 x PT82 Simoa assay. An orthogonal sample clean-up using offline TiO/ZrO combined with 1DLC-TQMS was developed to confirm the presence of mono-ptau (pT217) tryptic peptides in CSF.
Evaluation of highly sensitive immunoassay technologies for quantitative measurements of sub-pg/mL levels of cytokines in human serum.
Yeung David,Ciotti Shawn,Purushothama Shobha,Gharakhani Elham,Kuesters Geoffrey,Schlain Brian,Shen Chase,Donaldson Douglas,Mikulskis Alvydas
Journal of immunological methods
A comprehensive cross-platform and cross-assay evaluation using nine technology platforms and four cytokine immunoassays (IL-6, TNFα, IL-17a, IL-2) was performed by comparing assay precision, sensitivity, parallelism and data correlation between platforms. The precision was acceptable for most evaluated assays. In addition to comparing the analytical assay sensitivity using a spiked recombinant analyte in buffer, forty serum samples from both normal controls and multiple sclerosis patients were used to measure the frequency of endogenous analyte detection (FEAD) as a parameter of each assay's ability to detect the endogenous analyte. The highest FEAD measurements were observed on the Simoa™, Erenna®, Milliplex® and Imperacer® platforms. However, only Simoa and Erenna results showed a high correlation across all evaluated cytokine assays, followed by a more moderate correlation of results across platforms for the V-plex™, high sensitivity ELISA and the Ella™ IL-6 and TNFα assays. In contrast, results from the evaluated cytokine assays on the Milliplex, AMMP™ ViBE® and Imperacer platforms did not correlate to each other nor to other evaluated assays. Acceptable parallelism was observed for the Simoa, Erenna, V-plex and Ella assays but not for the Milliplex, AMMP ViBE and Imperacer assays. In conclusion, the Simoa, Erenna,V-plex and Ella platforms performed well in one or more evaluated cytokine assays. Among those, the Simoa and Erenna assays had the highest sensitivity for detection of cytokines present at sub-pg/mL levels in human serum. In addition, the cross-platform and cross-assay comparisons demonstrated that different immunoassays may yield different results, which underscores the importance of performing such comparative evaluations, especially in the absence of reliable reference standards for the quantitative assessments of biomarkers in immunoassays.
Development and evaluation of an ultrasensitive free VEGF-A immunoassay for analysis of human aqueous humor.
Göpfert Jens C,Reiser Astrid,Carcamo Yañez Viviana A,Pohle Annelie,Wessels Uwe,Heine Anne,Joos Thomas O,Petit-Frère Corinne,Nogoceke Everson,Stubenrauch Kay-Gunnar
Novel bifunctional VEGF-A neutralizing therapies are being developed for the treatment of retinal vascular diseases such as age-related macular degeneration and diabetic retinopathy. In developing new therapeutic drugs, only small aqueous humor sample volumes are available for analyzing several parameters. Highly sensitive detection methods must be applied in analyzing VEGF-A levels in ocular fluids in order to demonstrate VEGF-A suppression following drug administration. A highly sensitive immunoassay for VEGF-A was developed on the single molecule array (Simoa) platform, and validated before being used for the analysis of clinical aqueous humor samples from patients treated with anti-VEGF-A therapeutics. This highly sensitive immunoassay allows the detection of baseline VEGF-A levels and suppression effects after drug administration, even in sample volumes as low as 12 μl. The Simoa VEGF-A assay is a valuable tool for the reliable monitoring of VEGF-A suppression after intravitreal administration of anti-VEGF-A drugs.
Plasma amyloid β 40/42 ratio predicts cerebral amyloidosis in cognitively normal individuals at risk for Alzheimer's disease.
Vergallo Andrea,Mégret Lucile,Lista Simone,Cavedo Enrica,Zetterberg Henrik,Blennow Kaj,Vanmechelen Eugeen,De Vos Ann,Habert Marie-Odile,Potier Marie-Claude,Dubois Bruno,Neri Christian,Hampel Harald, ,
Alzheimer's & dementia : the journal of the Alzheimer's Association
INTRODUCTION:Blood-based biomarkers of pathophysiological brain amyloid β (Aβ) accumulation, particularly for preclinical target and large-scale interventions, are warranted to effectively enrich Alzheimer's disease clinical trials and management. METHODS:We investigated whether plasma concentrations of the Aβ/Aβ ratio, assessed using the single-molecule array (Simoa) immunoassay, may predict brain Aβ positron emission tomography status in a large-scale longitudinal monocentric cohort (N = 276) of older individuals with subjective memory complaints. We performed a hypothesis-driven investigation followed by a no-a-priori hypothesis study using machine learning. RESULTS:The receiver operating characteristic curve and machine learning showed a balanced accuracy of 76.5% and 81%, respectively, for the plasma Aβ/Aβ ratio. The accuracy is not affected by the apolipoprotein E (APOE) ε4 allele, sex, or age. DISCUSSION:Our results encourage an independent validation cohort study to confirm the indication that the plasma Aβ/Aβ ratio, assessed via Simoa, may improve future standard of care and clinical trial design.
Plasma extracellular vesicles detected by Single Molecule array technology as a liquid biopsy for colorectal cancer.
Wei Ping,Wu Fei,Kang Bin,Sun Xiaohua,Heskia Fabienne,Pachot Alexandre,Liang Ji,Li Dawei
Journal of extracellular vesicles
Circulating extracellular vesicles (EVs) were recognized as a promising source of diagnostic biomarker. However, there are limited studies published in this area, partly due to the limited number of detection platforms capable of detecting extracellular vesicles. In this study, extracellular vesicle immunoassays were developed using the Single Molecule array technology (SiMoa) and their clinical applications to cancer diagnosis were evaluated. Two extracellular vesicle detection assays, CD9-CD63 and Epcam-CD63, were designed to detect universal extracellular vesicles and tumour-derived extracellular vesicles, respectively. Our results show that CD9-CD63 and Epcam-CD63 SiMoa assays specifically detect extracellular vesicles but not free proteins with high sensitivities. The Epcam-CD63 levels detected in cancer cell culture media were consistent with levels of Epcam-expressing EVs isolated from the same cancer cell lines and detected by Western blot. Furthermore, the assays distinguish cancerous from non-cancerous plasma samples. The highest CD9-CD63 and Epcam-CD63 signals were observed in colorectal cancer patients comparing to healthy and benign controls. Both assays showed superior diagnostic performance for colorectal cancer. In addition, our results show that CD9-CD63 detection is an independent prognosis factor for both progression free survival and overall survival, while Epcam-CD63 detectionis an independent prognosis factor for OS.
Single-Molecule Arrays for Ultrasensitive Detection of Blood-Based Biomarkers for Immunotherapy.
Cohen Limor,Keegan Alissa,Walt David R
Methods in molecular biology (Clifton, N.J.)
Single-molecule array (Simoa) technology enables ultrasensitive protein detection that is suited to the development of peripheral blood-based assays for assessing immuno-oncology responses. We adapted a panel of Simoa assays to measure systemic cytokine levels from plasma and characterized physiologic variation in healthy individuals and preanalytic variation arising from processing and handling of patient samples. Insights from these preclinical studies led us to a well-defined set of Simoa assay conditions, a specimen processing protocol, and a data processing approach that we describe here. Simoa enables accurate quantitation of soluble immune signaling molecules in an unprecedented femtomolar range, opening up the potential for liquid biopsy-type approaches in immuno-oncology. We are using the method described here to distinguish PD-1 inhibitor nonresponders as early as after one dose after therapy and envision applications in characterizing PD-1 inhibitor resistance and detection of immune-related adverse effects.
A fully-automated, six-plex single molecule immunoassay for measuring cytokines in blood.
Rivnak Andrew J,Rissin David M,Kan Cheuk W,Song Linan,Fishburn Matthew W,Piech Tomasz,Campbell Todd G,DuPont Derek R,Gardel Melissa,Sullivan Sean,Pink Brian A,Cabrera Carlos G,Fournier David R,Duffy David C
Journal of immunological methods
We report a system and assay for performing fully-automated measurement of 6 proteins simultaneously with single molecule sensitivity. The system combines handling of samples, reagents, and consumables, with a module for imaging single molecule arrays (Simoa) to enable immunoassays that have high sensitivity (~fg/mL), are multiplexed, and are fully-automated. A 6-plex cytokine Simoa assay for IL-6, TNF-α, GM-CSF, IL-10, IL-1β, and IL-1α was developed on the system. The assays had limits of detection in the range 0.01-0.03pg/mL, and the average imprecision (CV) of the Simoa signal was 4.2%. This assay was used to measure the concentrations of these cytokines in the plasma of patients with Crohn's Disease (CD), before and after treatment with anti-TNF-α antibody drugs, and in the serum of Type 1 diabetics. Concentrations of TNF-α and IL-6 in the CD samples determined using the fully-automated, multiplex Simoa assay had good correlation with the manual, single-plex assays previously reported. Drug treatment caused reductions in the mean concentration of all 6 cytokines in the plasma of CD patients. The concentrations of 4 cytokines were significantly higher in diabetics compared to healthy controls. The system could enable the widespread, multiplexed measurement of protein biomarkers with low abundance.
Simplified Digital Enzyme-Linked Immunosorbent Assay Using Tyramide Signal Amplification and Fibrin Hydrogels.
Maley Adam M,Garden Padric M,Walt David R
Many protein biomarkers occur at very low concentrations in biofluids like blood and saliva, and ultrasensitive detection methods are required in order to measure them. Approaches such as digital enzyme-linked immunosorbent assays (ELISA) and single molecule arrays (Simoa) have been developed to accurately quantitate protein concentrations as low as attomolar levels. Although these techniques are being implemented in research and clinical laboratories to develop ultrasensitive clinical diagnostic assays, the size and cost of the instruments required to run these digital assays have precluded them from being implemented into point-of-care diagnostic formats. Here, we report the development of a simplified digital ELISA format that is more amenable to point-of-care technologies, referred to as catalyzed reporter deposition digital ELISA (CARD-dELISA). On-bead signal generation using the CARD tyramide signal amplification technique is combined with bead immobilization in fibrin hydrogels for single molecule counting in a simplified workflow format. CARD-dELISA allows for ultrasensitive protein detection (IL-6: ∼1 fM) with a dynamic range similar to the conventional Simoa assay. We use CARD-dELISA to measure IL-6 in saliva samples and show good agreement with conventional Simoa.
Head-to-head comparison of amplified plasmonic exosome Aβ42 platform and single-molecule array immunoassay in a memory clinic cohort.
Tanaka Tomotaka,Ruifen Joyce Chong,Nai Ying-Hwey,Tan Chin Hong,Lim Carine Z J,Zhang Yan,Stephenson Mary C,Hilal Saima,Saridin Francis N,Gyanwali Bibek,Villaraza Steven,Robins Edward G,Ihara Masafumi,Schöll Michael,Zetterberg Henrik,Blennow Kaj,Ashton Nicholas J,Shao Huilin,Reilhac Anthonin,Chen Christopher
European journal of neurology
BACKGROUND AND PURPOSE:Various blood biomarkers reflecting brain amyloid-β (Aβ) load have recently been proposed with promising results. However, to date, no comparative study amongst blood biomarkers has been reported. Our objective was to examine the diagnostic performance and cost effectiveness of three blood biomarkers on the same cohort. METHODS:Using the same cohort (n = 68), the performances of the single-molecule array (Simoa) Aβ40, Aβ42, Aβ42/Aβ40 and the amplified plasmonic exosome (APEX) Aβ42 blood biomarkers were compared using amyloid positron emission tomography (PET) as the reference standard. The extent to which these blood tests can reduce the recruitment cost of clinical trials was also determined by identifying amyloid positive (Aβ+) participants. RESULTS:Compared to Simoa biomarkers, APEX-Aβ42 showed significantly higher correlations with amyloid PET retention values and excellent diagnostic performance (sensitivity 100%, specificity 93.3%, area under the curve 0.995). When utilized for clinical trial recruitment, our simulation showed that pre-screening with blood biomarkers followed by a confirmatory amyloid PET imaging would roughly half the cost (56.8% reduction for APEX-Aβ42 and 48.6% for Simoa-Aβ42/Aβ40) compared to the situation where only PET imaging is used. Moreover, with 100% sensitivity, APEX-Aβ42 pre-screening does not increase the required number of initial participants. CONCLUSIONS:With its high diagnostic performance, APEX is an ideal candidate for Aβ+ subject identification, monitoring and primary care screening, and could efficiently enrich clinical trials with Aβ+ participants whilst halving recruitment costs.
Correlation between CSF and blood neurofilament light chain protein: a systematic review and meta-analysis.
Alagaratnam Jasmini,von Widekind Sophia,De Francesco Davide,Underwood Jonathan,Edison Paul,Winston Alan,Zetterberg Henrik,Fidler Sarah
BMJ neurology open
Objective:To assess the overall pooled correlation coefficient estimate between cerebrospinal fluid (CSF) and blood neurofilament light (NfL) protein. Methods:We searched Medline, Embase and Web of Science for published articles, from their inception to 9 July 2019, according to Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. Studies reporting the correlation between CSF and blood NfL in humans were included. We conducted a random-effects meta-analysis to calculate the overall pooled correlation coefficient estimate, accounting for correlation technique and assay used. Heterogeneity was assessed using the I statistic test. In sensitivity analyses, we calculated the pooled correlation coefficient estimate according to blood NfL assay: single-molecule array digital immunoassay (Simoa), electrochemiluminescence (ECL) assay or ELISA. Results:Data were extracted from 36 articles, including 3961 paired CSF and blood NfL samples. Overall, 26/36 studies measured blood NfL using Simoa, 8/36 ECL, 1/36 ELISA and 1 study reported all three assay results. The overall meta-analysis demonstrated that the pooled correlation coefficient estimate for CSF and blood NfL was r=0.72. Heterogeneity was significant: I=83%, p<0.01. In sensitivity analyses, the pooled correlation coefficient was similar for studies measuring blood NfL using Simoa and ECL (r=0.69 and r=0.68, respectively) but weaker for ELISA (r=0.35). Conclusion:Moderate correlations are demonstrated between CSF and blood NfL, especially when blood NfL was measured using Simoa and ECL. Given its high analytical sensitivity, Simoa is the preferred assay for measuring NfL, especially at low or physiological concentrations, and this meta-analysis supports its use as the current most advanced surrogate measure of CSF NfL. PROSPERO registration number:CRD42019140469.
Sensitivity of Single-Molecule Array Assays for Detection of Clostridium difficile Toxins in Comparison to Conventional Laboratory Testing Algorithms.
Banz Alice,Lantz Aude,Riou Brigitte,Foussadier Agnès,Miller Mark,Davies Kerrie,Wilcox Mark
Journal of clinical microbiology
Guidelines recommend the use of an algorithm for the laboratory diagnosis of infection (CDI). Enzyme immunoassays (EIAs) detecting toxins cannot be used as standalone tests due to suboptimal sensitivity, and molecular tests suffer from nonspecificity by detecting colonization. Sensitive immunoassays have recently been developed to improve and simplify CDI diagnosis. Assays detecting CD toxins have been developed using single-molecule array (SIMOA) technology. SIMOA performance was assessed relative to a laboratory case definition of CDI defined by positive glutamate dehydrogenase (GDH) screen and cell cytotoxicity neutralizing assay (CCNA). Samples were tested with SIMOA assays and a commercial toxin EIA to compare performance, with discrepancy resolution using a commercial nucleic acid-based test and a second cell cytotoxicity assay. The SIMOA toxin A and toxin B assays showed limits of detection of 0.6 and 2.9 pg/ml, respectively, and intra-assay coefficients of variation of less than 10%. The optimal clinical thresholds for the toxin A and toxin B assays were determined to be 22.1 and 18.8 pg/ml, respectively, with resultant sensitivities of 84.8 and 95.5%. In contrast, a high-performing EIA toxin test had a sensitivity of 71.2%. Thus, the SIMOA assays detected toxins in 24% more samples with laboratory-defined CDI than the high performing toxin EIA (95% [63/66] versus 71% [47/66]). This study shows that SIMOA toxin assays have a higher sensitivity than currently available toxin EIA and have the potential to improve CDI diagnosis.
Sequential Protein Capture in Multiplex Single Molecule Arrays: A Strategy for Eliminating Assay Cross-Reactivity.
Gilboa Tal,Maley Adam M,Ogata Alana F,Wu Connie,Walt David R
Advanced healthcare materials
Measurements of multiple biomolecules within the same biological sample are important for many clinical applications to enable accurate disease diagnosis or classification. These disease-related biomarkers often exist at very low levels in biological fluids, necessitating ultrasensitive measurement methods. Single-molecule arrays (Simoa), a bead-based digital enzyme-linked immunosorbent assay, is the current state of the art for ultrasensitive protein detection and can detect sub-femtomolar protein concentrations, but its ability to achieve high-order multiplexing without cross-reactivity remains a challenge. Here, a sequential protein capture approach for multiplex Simoa assays is implemented to eliminate cross-reactivity between binding reagents by sequentially capturing each protein analyte and then incubating each capture bead with only its corresponding detection antibody. This strategy not only reduces cross-reactivity to background levels and significantly improves measurement accuracies, but also enables higher-order multiplexing. As a proof of concept, the sequential multiplex Simoa assay is used to measure five different cytokines in plasma samples from Coronavirus Disease 2019 (COVID-19) patients. The ultrasensitive sequential multiplex Simoa assays will enable the simultaneous measurements of multiple low-abundance analytes in a time- and cost-effective manner and will prove especially critical in many cases where sample volumes are limited.
Using Antigen-antibody Binding Kinetic Parameters to Understand Single-Molecule Array Immunoassay Performance.
Dinh Trinh L,Ngan Kevin C,Shoemaker Charles B,Walt David R
This paper provides insights into the performance of single-molecule array (Simoa) immunoassays by examining the effects of various capture and detector antibody-antigen binding kinetic parameters. Simoa is similar to other immunoassays in that the overall Simoa performance is heavily dependent on the choice of antibodies; however, little is known about how the different properties of the antibodies result in the wide variations in assay performance. Here, we focus on antibody-antigen binding kinetics and demonstrate how the association (k) and dissociation (k) rate constants of the capture and detection antibodies affect Simoa performance. We compared six different antibodies with over a four-log range of equilibrium dissociation constants (K) and found that Simoa assay performance had an inverse relationship to the k value of the detection antibody. The Simoa fluorescent signals were highest when the k of the detection antibody was less than 10 s. The capture antibody k did not have as significant an effect, but a k of less than 10 s was preferred. We also found that the k values of the capture and detection antibodies were not important factors for Simoa performance. Therefore, the assay optimization process could be accelerated by choosing detection antibodies with the slowest k values.
Ultrasensitive immunoassay allows measurement of serum neurofilament heavy in multiple sclerosis.
Verberk Inge M W,Koel-Simmelink Marleen,Twaalfhoven Harry,Vrenken Hugo,Korth Carsten,Killestein Joep,Teunissen Charlotte E,Bridel Claire
Multiple sclerosis and related disorders
BACKGROUND:Neurofilament heavy (NfH) is a promising biomarker for neuro-axonal damage in Multiple Sclerosis (MS). We compared the performance of high-sensitivity serum-NfH immunoassays, with as aim to investigate the value of serum-NfH as biomarker for MS. METHODS:We measured serum-NfH in 76 MS patients with Simoa (one commercial, one in-house) or Luminex assays. Serum-NfH measured by the immunoassay with greatest sensitivity was related to clinical and radiological outcomes with age and sex-adjusted linear regression analysis, and to biological outcomes cerebrospinal fluid (CSF)-NfH, serum neurofilament light (NfL) and CSF-NfL with Spearman's correlation analysis. RESULTS:With the commercial Simoa assay, we obtained 100% serum-NfH detectability (in-house Simoa: 70%, Luminex: 61%), with lowest coefficient of variation (CV) between duplicates of 11%CV (in-house Simoa: 22%CV, Luminex: 30%CV). Serum-NfH quantified with the commercial Simoa assay was associated with disease duration (standardized beta (sβ) = 0.28, p = 0.034), T2 lesion volume (sβ = 0.23, p = 0.041), and tended to associate with black hole count (sβ = 0.21, p = 0.084) but not with Expanded Disease Disability Score (EDSS) or normalized brain volume (all: p>0.10). Furthermore, serum-NfH showed correlations with CSF-NfH (rho = 0.27, p = 0.018) and serum-NfL (rho=0.44, p < 0.001), but not with CSF-NfL. CONCLUSIONS:Serum-NfH can be quantified with high-sensitivity technology. Cross-sectionally, we observed some weak correlations of serum-NfH with MS disease burden parameters, suggesting there might be some utility for serum-NfH as biomarker for MS disease burden.
Correlations between serum and CSF pNfH levels in ALS, FTD and controls: a comparison of three analytical approaches.
Wilke Carlo,Pujol-Calderón Fani,Barro Christian,Stransky Elke,Blennow Kaj,Michalak Zuzanna,Deuschle Christian,Jeromin Andreas,Zetterberg Henrik,Schüle Rebecca,Höglund Kina,Kuhle Jens,Synofzik Matthis
Clinical chemistry and laboratory medicine
Background Phosphorylated neurofilament heavy (pNfH), a neuronal cytoskeleton protein, might provide a promising blood biomarker of neuronal damage in neurodegenerative diseases (NDDs). The best analytical approaches to measure pNfH levels and whether serum levels correlate with cerebrospinal fluid (CSF) levels in NDDs remain to be determined. Methods We here compared analytical sensitivity and reliability of three novel analytical approaches (homebrew Simoa, commercial Simoa and ELISA) for quantifying pNfH in both CSF and serum in samples of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and control subjects. Results While all three assays showed highly correlated CSF measurements, Simoa assays also yielded high between-assay correlations for serum measurements (ϱ = 0.95). Serum levels also correlated strongly with CSF levels for Simoa-based measurements (both ϱ = 0.62). All three assays allowed distinguishing ALS from controls by increased CSF pNfH levels, and Simoa assays also by increased serum pNfH levels. pNfH levels were also increased in FTD. Conclusions pNfH concentrations in CSF and, if measured by Simoa assays, in blood might provide a sensitive and reliable biomarker of neuronal damage, with good between-assay correlations. Serum pNfH levels measured by Simoa assays closely reflect CSF levels, rendering serum pNfH an easily accessible blood biomarker of neuronal damage in NDDs.
Single molecule array (Simoa) assay with optimal antibody pairs for cytokine detection in human serum samples.
Wu Danlu,Milutinovic Milena Dumont,Walt David R
Concentrations of cytokines in bodily fluids reflect the physiological or pathological state of the patient and can be used for prognosis, disease diagnosis or for monitoring therapeutic efficacy. However, in the bodily fluids of healthy or sub-healthy individuals, many cytokines are present at concentrations that are near or below the detection limits of current methods. Here we selected antibody pairs to be employed in the single molecule array (Simoa) assay for ten cytokines including GM-CSF, TNF-α, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, and IL-10. The limits of detection (LODs) obtained were as low as 90 aM-6 fM. These assays allow detection of cytokines in healthy human serum samples at levels significantly below the detection limits of conventional ELISA assays. We provide detailed antibody pair information as well as the concentration profiles of ten cytokines in healthy human serum to serve as reference data for further ultrasensitive immunoassay development and future clinical applications.
Rhodopsin in plasma from patients with diabetic retinopathy - development and validation of digital ELISA by Single Molecule Array (Simoa) technology.
Petersen Eva Rabing Brix,Olsen Dorte Aalund,Christensen Henry,Hansen Søren Berndt,Christensen Cramer,Brandslund Ivan
Journal of immunological methods
BACKGROUND:Diabetic retinopathy (DR) is the most frequent cause of blindness among younger adults in the western world. No blood biomarkers exist to detect DR. Hypothetically, Rhodopsin concentrations in blood has been suggested as an early marker for retinal damage. The aim of this study was therefore to develop and validate a Rhodopsin assay by employing digital ELISA technology, and to investigate whether Rhodopsin concentrations in diabetes patients with DR are elevated compared with diabetes patients without DR. METHODS:A digital ELISA assay using a Simoa HD-1 Analyzer (Quanterix©, Lexington, MA 02421, USA) was developed and validated and applied on a cohort of diabetes patients characterised with (n=466) and without (n=144) DR. RESULTS:The Rhodopsin assay demonstrated a LOD of 0.26ng/l, a LLOQ of 3ng/l and a linear measuring range from 3 to 2500ng/l. Total CV% was 32%, 23%, 19% and 17% respectively at the following Rhodopsin concentrations: 1, 3, 5 and 13ng/l. Recovery was 17%, 34%, 51% and 55% respectively at Rhodopsin concentrations of 2, 10, 50 and 250ng/l. There was no statistically significant difference in the plasma concentration of Rhodopsin between the diabetes patients with or without DR, but significantly increased number of DR patients having concentrations above the LOD. CONCLUSION:We developed and validated a digital ELISA method for quantification of Rhodopsin in plasma but found no statistically significant difference in the plasma concentration of Rhodopsin between diabetes patients with DR compared to diabetes patients without DR, though significantly more DR patients had values above the LOD.
A Multi-site In-depth Evaluation of the Quanterix Simoa from a User's Perspective.
Chunyk Allison Given,Joyce Alison,Fischer Saloumeh K,Dysinger Mark,Mikulskis Alvydas,Jeromin Andreas,Lawrence-Henderson Rosemary,Baker Dana,Yeung David
The AAPS journal
An in-depth evaluation of the Quanterix© Simoa™ platform was undertaken by scientists from the AAPS Emerging Technologies Focus Group to determine the overall performance of the technology as well as provide guidance to future users. In order to test the platform in a non-GLP bioanalytical setting, a cross-site evaluation of the Quanterix IL-6 biomarker kit was performed. Parameters tested during this evaluation included sensitivity, accuracy and precision, and parallelism in human serum from normal individuals. The results demonstrated improved sensitivity compared to the claimed sensitivity of other commercially available IL-6 kits and showed excellent site-to-site reproducibility. Observed issues included difficulties with system reliability and a lack of parallelism and specificity in a subset of samples. Overall, these results demonstrate that while there are challenges to the Simoa platform this technology offers automation capabilities and excellent sensitivity that enhance bioanalysis especially of low-abundance analytes.
Development of an ultra-sensitive Simoa assay to enable GDF11 detection: a comparison across bioanalytical platforms.
Myzithras Maria,Li Hua,Bigwarfe Tammy,Waltz Erica,Gupta Priyanka,Low Sarah,Hayes David B,MacDonnell Scott,Ahlberg Jennifer,Franti Michael,Roberts Simon
BACKGROUND:Four bioanalytical platforms were evaluated to optimize sensitivity and enable detection of recombinant human GDF11 in biological matrices; ELISA, Meso Scale Discovery, Gyrolab xP Workstation and Simoa HD-1. Results & methodology: After completion of custom assay development, the single-molecule ELISA (Simoa) achieved the greatest sensitivity with a lower limit of quantitation of 0.1 ng/ml, an improvement of 100-fold over the next sensitive platform (MSD). DISCUSSION & CONCLUSION:This improvement was essential to enable detection of GDF11 in biological samples, and without the technology the sensitivity achieved on the other platforms would not have been sufficient. Other factors such as ease of use, cost, assay time and automation capability can also be considered when developing custom immunoassays, based on the requirements of the bioanalyst.
Establishment of neurofilament light chain Simoa assay in cerebrospinal fluid and blood.
Hendricks Robert,Baker Dana,Brumm Jochen,Davancaze Teresa,Harp Chris,Herman Ann,Büdingen H-Christian von,Townsend Michael,Fischer Saloumeh K
Neurofilament light (NfL) chain is an established cerebrospinal fluid (CSF) biomarker for neuroaxonal injury. The highly sensitive Quanterix Simoa™ platform is evaluated for NfL measurement in both CSF and blood. There is a need to link historical ELISA data that use bovine NfL to that of Simoa using a recombinant human (rhuman) NfL standard. The Simoa NF-light Advantage Kit was validated for CSF and qualified for serum and plasma, using both rhuman and bovine NfL calibrators. Matched CSF, serum and plasma samples from 112 multiple sclerosis patients were analyzed using both calibrators. In multiple sclerosis, there is a good correlation between blood and CSF NfL levels. A conversion factor of approximately 5:1 was established between bovine and rhuman NfL calibrators.
The Simoa HD-1 Analyzer: A Novel Fully Automated Digital Immunoassay Analyzer with Single-Molecule Sensitivity and Multiplexing.
Wilson David H,Rissin David M,Kan Cheuk W,Fournier David R,Piech Tomasz,Campbell Todd G,Meyer Raymond E,Fishburn Matthew W,Cabrera Carlos,Patel Purvish P,Frew Erica,Chen Yao,Chang Lei,Ferrell Evan P,von Einem Volker,McGuigan William,Reinhardt Marcus,Sayer Heiko,Vielsack Claus,Duffy David C
Journal of laboratory automation
Disease detection at the molecular level is driving the emerging revolution of early diagnosis and treatment. A challenge facing the field is that protein biomarkers for early diagnosis can be present in very low abundance. The lower limit of detection with conventional immunoassay technology is the upper femtomolar range (10(-13) M). Digital immunoassay technology has improved detection sensitivity three logs, to the attomolar range (10(-16) M). This capability has the potential to open new advances in diagnostics and therapeutics, but such technologies have been relegated to manual procedures that are not well suited for efficient routine use. We describe a new laboratory instrument that provides full automation of single-molecule array (Simoa) technology for digital immunoassays. The instrument is capable of single-molecule sensitivity and multiplexing with short turnaround times and a throughput of 66 samples/h. Singleplex and multiplexed digital immunoassays were developed for 16 proteins of interest in cardiovascular, cancer, infectious disease, neurology, and inflammation research. The average sensitivity improvement of the Simoa immunoassays versus conventional ELISA was >1200-fold, with coefficients of variation of <10%. The potential of digital immunoassays to advance human diagnostics was illustrated in two clinical areas: traumatic brain injury and early detection of infectious disease.
Correlational Analysis of ALS Progression and Serum NfL Measured by Simoa Assay in Chinese Patients.
Sugimoto Kazuo,Han Yi,Song Yuebo,Gao Ying
Frontiers in neurology
Neurofilament light chain (NFL) was believed to be a promising biomarker for the diagnosis of Amyotrophic lateral sclerosis (ALS) and disease burden evaluation. To determine the serum NFL level and its clinical relevance, including its association with disease severity [evaluated by the ALS Functional Rating Scale-revised (ALSFRS-r) score and King's College staging system] and progression (evaluated by the disease progression rate (DPR) and diagnostic delay), in ALS patients in China. Serum NFL levels were detected using the Single Molecule Array (Simoa) technology in 30 ALS patients and 20 healthy controls (HCs). There were significantly elevated levels of serum NFL in patients with ALS than in the HCs < 0.001). The serum NFL levels were significantly higher in rapidly progressive ALS and patients in Stage 3 than in slowly progressive ALS and patients in Stage 2 ( < 0.001, = 0.019; = 0.033). Furthermore, the serum NFL levels negatively correlated with the diagnostic delay ( = 0.23, = 0.016), the ALSFRS-r score ( = 0.15, = 0.047) and disease duration ( = 0.15, = 0.034), and positively correlated with the DPR ( = 0.42, < 0.001). The present study preliminarily investigated the diagnostic value of serum NFL and its clinical relevance in the Chinese ALS population using the ultrasensitive Simoa technology. The results demonstrated that the level of serum NFL may become a potential biomarker for ALS diagnosis and indicate disease severity and progression.
The kinetic profile and clinical implication of SCC-Ag in squamous cervical cancer patients undergoing radical hysterectomy using the Simoa assay: a prospective observational study.
Ye Shuang,Sun Xiaohua,Kang Bin,Wu Fei,Zheng Zhong,Xiang Libing,Lesénéchal Mylène,Heskia Fabienne,Liang Ji,Yang Huijuan
BACKGROUND:To study the kinetic profile and clinicopathological implications of squamous cell carcinoma antigen (SCC-Ag) in cervical cancer patients who underwent surgery by a self-developed SCC-Ag single molecule assay (Simoa) prototype immunoassay. METHODS:Participants were prospectively enrolled between 04/2016 and 06/2017. Consecutive serum samples were collected at five points: day 0 (the day before surgery), postoperative day 4, weeks 2-4, months 2-4 and months 5-7. In total, 92 patients and 352 samples were included. The kinetic change in SCC-Ag levels and their associations with clinicopathological characteristics were studied. RESULTS:Simoa SCC-Ag was validated by comparison with the Architect assay. SCC-Ag levels measured by the Simoa assay were highly correlated with the Architect assay's levels (Pearson's correlation coefficient = 0.979, Passing-Bablok regression slope 0.894 (0.847 to 0.949), intercept - 0.009 (- 0.047 to 0.027)). The median values for each time-point detected by the Simoa assay were 2.49, 0.66, 0.61, 0.72, and 0.71 ng/mL, respectively. The SCC-Ag levels decreased dramatically after surgery and then stabilized and fluctuated to some extent within 6 months. Patients with certain risk factors had significantly higher SCC-Ag values than their negative counterparts before surgery and at earlier time points after surgery, while no difference existed at the end of observation. Furthermore, although patients with positive lymph nodes had sustained higher SCC-Ag levels compared to those with negative lymph nodes, similar kinetic patterns of SCC-Ag levels were observed after surgery. Patients who received postoperative treatment had significantly higher SCC-Ag values than those with surgery only at diagnosis, while no difference existed after treatment. CONCLUSIONS:The Simoa SCC-Ag prototype was established for clinical settings. The SCC-Ag levels were higher in patients with risk factors, whereas the kinetic trend of SCC-Ag might be mainly affected by postoperative adjuvant therapy. These data indicate that the SCC-Ag level might be a good predictor for the status of cervical cancer, including disease aggressiveness and treatment response.
Measurement of IL-21 in human serum and plasma using ultrasensitive MSD S-PLEX® and Quanterix SiMoA methodologies.
Poorbaugh Josh,Samanta Tanushree,Bright Stuart W,Sissons Sean E,Chang Ching-Yun,Oberoi Pankaj,MacDonald Angus J,Martin Andrea P,Cox Karen L,Benschop Robert J
Journal of immunological methods
IL-21 is a pleiotropic cytokine that plays a key role in modulating inflammatory responses, including the promotion of autoimmune diseases. Several groups have quantitated circulating levels of IL-21 in plasma and serum samples using various commercial ELISAs. We determined, however, that the most commonly used commercial assays in published literature were not specific or sensitive enough to detect levels of IL-21 in heparin plasma or serum from healthy human individuals. This finding prompted an effort to develop more specific and sensitive methods to quantitate IL-21 in complex biological matrices using proprietary anti-IL-21 antibodies with the Quanterix SiMoA platform and the Meso Scale Discovery (MSD) S-PLEX® format. Assays developed on both technology platforms were characterized in heparin plasma and serum using spike recoveries across a range of concentrations. Each method was able to detect sub-pg/mL levels of IL-21 (predicted Limit of Detection [LOD] of approximately 1.0 fg/mL for both the Quanterix SiMoA and MSD S-PLEX® platforms) which is 200-500 times lower than current commercial assays. Additionally we demonstrated that rheumatoid factor did not interfere with measuring IL-21 in the Quanterix SiMoA assay. Results obtained with the two new ultrasensitive assays showed a strong correlation (r = 0.9428; p < .0001). Additionally, IL-21 levels were significantly increased in samples from patients with Systemic Lupus Erythematosus (mean+/- SD: n = 14, 202.64 +/- 111.47 fg/mL, p = .0001 for Quanterix SiMoA and 275.4 +/- 174.66 fg/mL p = .0001 for MSD S-PLEX®) as well as in samples from patients with Sjögren's Syndrome (mean+/- SD: n = 11, 122.18 +/- 84.50 fg/mL, p = .0029 for Quanterix SiMoA and 183.64 +/- 153.00 fg/mL, p = .0082 for MSD S-PLEX®) when compared to healthy donors (mean+/- SD: n = 11, 38.1 +/- 27.8 fg/mL for Quanterix SiMoA and 58.1 +/- 30.7 fg/mL for MSD S-PLEX®). These ultrasensitive assays, for the first time, allow for the accurate quantitation of human IL-21 in heparin plasma and serum. In addition, these experiments also provide a direct comparison of the MSD S-PLEX® format and Quanterix SiMoA platform technologies, which may have broader implications to future application of these methods to evaluate low abundance proteins in complex biological matrices.
Comparison of ELISA- and SIMOA-based quantification of plasma Aβ ratios for early detection of cerebral amyloidosis.
De Meyer Steffi,Schaeverbeke Jolien M,Verberk Inge M W,Gille Benjamin,De Schaepdryver Maxim,Luckett Emma S,Gabel Silvy,Bruffaerts Rose,Mauroo Kimberley,Thijssen Elisabeth H,Stoops Erik,Vanderstichele Hugo M,Teunissen Charlotte E,Vandenberghe Rik,Poesen Koen
Alzheimer's research & therapy
BACKGROUND:Blood-based amyloid biomarkers may provide a non-invasive, cost-effective and scalable manner for detecting cerebral amyloidosis in early disease stages. METHODS:In this prospective cross-sectional study, we quantified plasma Aβ/Aβ ratios with both routinely available ELISAs and novel SIMOA Amyblood assays, and provided a head-to-head comparison of their performances to detect cerebral amyloidosis in a nondemented elderly cohort (n = 199). Participants were stratified according to amyloid-PET status, and the performance of plasma Aβ/Aβ to detect cerebral amyloidosis was assessed using receiver operating characteristic analysis. We additionally investigated the correlations of plasma Aβ ratios with amyloid-PET and CSF Alzheimer's disease biomarkers, as well as platform agreement using Passing-Bablok regression and Bland-Altman analysis for both Aβ isoforms. RESULTS:ELISA and SIMOA plasma Aβ/Aβ detected cerebral amyloidosis with identical accuracy (ELISA: area under curve (AUC) 0.78, 95% CI 0.72-0.84; SIMOA: AUC 0.79, 95% CI 0.73-0.85), and both increased the performance of a basic demographic model including only age and APOE-ε4 genotype (p ≤ 0.02). ELISA and SIMOA had positive predictive values of respectively 41% and 36% in cognitively normal elderly and negative predictive values all exceeding 88%. Plasma Aβ/Aβ correlated similarly with amyloid-PET for both platforms (Spearman ρ = - 0.32, p < 0.0001), yet correlations with CSF Aβ/t-tau were stronger for ELISA (ρ = 0.41, p = 0.002) than for SIMOA (ρ = 0.29, p = 0.03). Plasma Aβ levels demonstrated poor agreement between ELISA and SIMOA with concentrations of both Aβ and Aβ measured by SIMOA consistently underestimating those measured by ELISA. CONCLUSIONS:ELISA and SIMOA demonstrated equivalent performances in detecting cerebral amyloidosis through plasma Aβ/Aβ, both with high negative predictive values, making them equally suitable non-invasive prescreening tools for clinical trials by reducing the number of necessary PET scans for clinical trial recruitment. TRIAL REGISTRATION:EudraCT 2009-014475-45 (registered on 23 Sept 2009) and EudraCT 2013-004671-12 (registered on 20 May 2014, https://www.clinicaltrialsregister.eu/ctr-search/trial/2013-004671-12/BE ).