Comparison between plasma and cerebrospinal fluid biomarkers for the early diagnosis and association with survival in prion disease

ObjectiveTo compare the diagnostic accuracy and the prognostic value of blood and cerebrospinal fluid (CSF) tests across prion disease subtypes.MethodsWe used a single-molecule immunoassay to measure tau and neurofilament light chain (NfL) protein levels in the plasma and assessed CSF total(t)-tau, NfL and protein 14-3-3 levels in patients with prion disease (n=336), non-prion rapidly progressive dementias (n=106) and non-neurodegenerative controls (n=37). We then evaluated each plasma and CSF marker for diagnosis and their association with survival, taking into account the disease subtype, which is a strong independent prognostic factor in prion disease.ResultsPlasma tau and NfL concentrations were higher in patients with prion disease than in non-neurodegenerative controls and non-prion rapidly progressive dementias. Plasma tau showed higher diagnostic value than plasma NfL, but a lower accuracy than the CSF proteins t-tau and 14-3-3. In the whole prion cohort, both plasma (tau and NfL) and CSF (t-tau, 14-3-3 and NfL) markers were significantly associated with survival and showed similar prognostic values. However, the intrasubtype analysis revealed that only CSF t-tau in sporadic Creutzfeldt-Jakob disease (sCJD) MM(V)1, plasma tau and CSF t-tau in sCJD VV2, and plasma NfL in slowly progressive prion diseases were significantly associated with survival after accounting for covariates.ConclusionsPlasma markers have lower diagnostic accuracy than CSF biomarkers. Plasma tau and NfL and CSF t-tau are significantly associated with survival in prion disease in a subtype-specific manner and can be used to improve clinical trial stratification and clinical care.

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Evaluation of neurofilament light chain in the cerebrospinal fluid and blood as a biomarker for neuronal damage in experimental pneumococcal meningitis

Journal of Neuroinflammation ◽

10.1186/s12974-020-01966-3 ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Ngoc Dung Le ◽

Lukas Muri ◽

Denis Grandgirard ◽

Jens Kuhle ◽

David Leppert ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Single Molecule ◽

Light Chain ◽

Pneumococcal Meningitis ◽

Neuronal Damage ◽

Neuronal Injury ◽

Health Concern ◽

Neurofilament Light Chain ◽

Neurofilament Light ◽

Inflammatory Phase

Abstract Background Pneumococcal meningitis (PM) remains a global public health concern and affects all age groups. If acquired during infancy or childhood, permanent neurofunctional deficits including cognitive impairment, cerebral palsy, and secondary epilepsy are typical sequelae of neuronal injury. Determination of patients at risk for the development of brain injury and subsequent neurofunctional sequelae could help to identify patients for focused management. Neurofilament light chain (NfL) is an axonal cytoskeletal protein released upon neuronal injury into the cerebrospinal fluid (CSF) and blood. As little is known about the course of neurofilament release in the course of PM, we measured CSF and serum NfL levels longitudinally in experimental PM (ePM). Methods Eleven-day-old infant Wistar rats were infected intracisternally with Streptococcus pneumoniae and treated with ceftriaxone. At 18 and 42 h post-infection (hpi), the blood and CSF were sampled for NfL measurements by a single molecule array technology. Inflammatory cytokines and MMP-9 in CSF were quantified by magnetic bead multiplex assay (Luminex®) and by gel zymography, respectively. Results In ePM, CSF and serum NfL levels started to increase at 18 hpi and were 26- and 3.5-fold increased, respectively, compared to mock-infected animals at 42 hpi (p < 0.0001). CSF and serum NfL correlated at 18 hpi (p < 0.05, r = 0.4716) and 42 hpi (p < 0.0001, r = 0.8179). Both CSF and serum NfL at 42 hpi strongly correlated with CSF levels of IL-1β, TNF-α, and IL-6 and of MMP-9 depending on their individual kinetics. Conclusion Current results demonstrate that during the peak inflammatory phase of ePM, NfL levels in CSF and serum are the highest among CNS disease models studied so far. Given the strong correlation of CSF versus serum NfL, and its CNS-specific signal character, longitudinal measurements to monitor the course of PM could be performed based on blood sample tests, i.e., without the need of repetitive spinal taps. We conclude that NfL in the serum should be evaluated as a biomarker in PM.

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Inflammation-related plasma and CSF biomarkers for multiple sclerosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1912839117 ◽

2020 ◽

Vol 117 (23) ◽

pp. 12952-12960 ◽

Cited By ~ 6

Author(s):

Jesse Huang ◽

Mohsen Khademi ◽

Lars Fugger ◽

Örjan Lindhe ◽

Lenka Novakova ◽

...

Keyword(s):

Multiple Sclerosis ◽

Cerebrospinal Fluid ◽

Neurological Diseases ◽

Area Under The Curve ◽

Csf Biomarkers ◽

Healthy Controls ◽

Protein Biomarkers ◽

Neurofilament Light Chain ◽

Diagnostic Efficacy ◽

Neurofilament Light

Effective biomarkers for multiple sclerosis diagnosis, assessment of prognosis, and treatment responses, in particular those measurable in blood, are largely lacking. We have investigated a broad set of protein biomarkers in cerebrospinal fluid (CSF) and plasma using a highly sensitive proteomic immunoassay. Cases from two independent cohorts were compared with healthy controls and patients with other neurological diseases. We identified and replicated 10 cerebrospinal fluid proteins including IL-12B, CD5, MIP-1a, and CXCL9 which had a combined diagnostic efficacy similar to immunoglobulin G (IgG) index and neurofilament light chain (area under the curve [AUC] = 0.95). Two plasma proteins, OSM and HGF, were also associated with multiple sclerosis in comparison to healthy controls. Sensitivity and specificity of combined CSF and plasma markers for multiple sclerosis were 85.7% and 73.5%, respectively. In the discovery cohort, eotaxin-1 (CCL11) was associated with disease duration particularly in patients who had secondary progressive disease (PCSF< 4 × 10−5,Pplasma< 4 × 10−5), and plasma CCL20 was associated with disease severity (P= 4 × 10−5), although both require further validation. Treatment with natalizumab and fingolimod showed different compartmental changes in protein levels of CSF and peripheral blood, respectively, including many disease-associated markers (e.g., IL12B, CD5) showing potential application for both diagnosing disease and monitoring treatment efficacy. We report a number of multiple sclerosis biomarkers in CSF and plasma for early disease detection and potential indicators for disease activity. Of particular importance is the set of markers discovered in blood, where validated biomarkers are lacking.

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Cerebrospinal fluid levels of chitinase 3-like 1 and neurofilament light chain predict multiple sclerosis development and disability after optic neuritis

Multiple Sclerosis Journal ◽

10.1177/1352458515574148 ◽

2015 ◽

Vol 21 (14) ◽

pp. 1761-1770 ◽

Cited By ~ 60

Author(s):

S Modvig ◽

M Degn ◽

H Roed ◽

TL Sørensen ◽

HBW Larsson ◽

...

Keyword(s):

Multiple Sclerosis ◽

Cerebrospinal Fluid ◽

Optic Neuritis ◽

Light Chain ◽

Oligoclonal Bands ◽

Enzyme Linked Immunosorbent Assay ◽

Csf Biomarkers ◽

Neurofilament Light Chain ◽

Neurofilament Light

Background: Cerebrospinal fluid (CSF) biomarkers have been suggested to predict multiple sclerosis (MS) after clinically isolated syndromes, but studies investigating long-term prognosis are needed. Objective: To assess the predictive ability of CSF biomarkers with regard to MS development and long-term disability after optic neuritis (ON). Methods: Eighty-six patients with ON as a first demyelinating event were included retrospectively. Magnetic resonance imaging (MRI), CSF leukocytes, immunoglobulin G index and oligoclonal bands were registered. CSF levels of chitinase-3-like-1, osteopontin, neurofilament light-chain, myelin basic protein, CCL2, CXCL10, CXCL13 and matrix metalloproteinase-9 were measured by enzyme-linked immunosorbent assay. Patients were followed up after 13.6 (range 9.6–19.4) years and 81.4% were examined, including Expanded Disability Status Scale and MS functional composite evaluation. 18.6% were interviewed by phone. Cox regression, multiple regression and Spearman correlation analyses were used. Results: Forty-six (53.5%) developed clinically definite MS (CDMS) during follow-up. In a multivariate model MRI ( p=0.0001), chitinase 3-like 1 ( p=0.0033) and age ( p=0.0194) combined predicted CDMS best. Neurofilament light-chain predicted long-term disability by the multiple sclerosis severity scale ( p=0.0111) and nine-hole-peg-test ( p=0.0202). Chitinase-3-like-1 predicted long-term cognitive impairment by the paced auditory serial addition test ( p=0.0150). Conclusion: Neurofilament light-chain and chitinase-3-like-1 were significant predictors of long-term physical and cognitive disability. Furthermore, chitinase-3-like-1 predicted CDMS development. Thus, these molecules hold promise as clinically valuable biomarkers after ON as a first demyelinating event.

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Blood-based NfL

Neurology ◽

10.1212/wnl.0000000000003680 ◽

2017 ◽

Vol 88 (10) ◽

pp. 930-937 ◽

Cited By ~ 156

Author(s):

Oskar Hansson ◽

Shorena Janelidze ◽

Sara Hall ◽

Nadia Magdalinou ◽

Andrew J. Lees ◽

...

Keyword(s):

Diagnostic Accuracy ◽

Single Molecule ◽

Area Under The Curve ◽

Diagnostic Workup ◽

Healthy Controls ◽

Class Iii ◽

Early Disease ◽

Neurofilament Light Chain ◽

Neurofilament Light ◽

Parkinsonian Disorders

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, p ≤ 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 (p < 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.

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CSF neurofilament light concentration is increased in presymptomatic CHMP2B mutation carriers

Neurology ◽

10.1212/wnl.0000000000004799 ◽

2017 ◽

Vol 90 (2) ◽

pp. e157-e163 ◽

Cited By ~ 6

Author(s):

Nina Rostgaard ◽

Peter Roos ◽

Erik Portelius ◽

Kaj Blennow ◽

Henrik Zetterberg ◽

...

Keyword(s):

Continuous Process ◽

Csf Biomarkers ◽

Neurofilament Light Chain ◽

Cross Sectional ◽

Neurofilament Light ◽

Preclinical Phase ◽

Mutation Carriers ◽

Amyloid Aβ ◽

Multiplexed Immunoassay ◽

T Tau

ObjectiveA rare cause of familial frontotemporal dementia (FTD) is a mutation in the CHMP2B gene on chromosome 3 (FTD-3), described in a Danish family. Here we examine whether CSF biomarkers change in the preclinical phase of the disease.MethodsIn this cross-sectional explorative study, we analyzed CSF samples from 16 mutation carriers and 14 noncarriers from the Danish FTD-3 family. CSF biomarkers included total tau (t-tau) and neurofilament light chain (NfL) as a marker for neurodegeneration, phosphorylated tau (p-tau) as a marker for tau pathology, β-amyloid (Aβ) 38, 40, and 42 (Aβ38, Aβ40, and Aβ42) to monitor Aβ metabolism, and YKL-40 as a marker of neuroinflammation. Aβ isoform concentrations were measured using a multiplexed immunoassay; t-tau, p-tau, NfL, and YKL-40 concentrations were measured using sandwich ELISAs.ResultsCSF NfL concentration was significantly increased in mutation carriers vs noncarriers. Further, CSF NfL concentration was significantly higher in symptomatic mutation carriers compared to presymptomatic carriers, and also significantly higher in presymptomatic carriers compared to noncarriers. No differences in t-tau and p-tau and YKL-40 concentrations between controls and mutation carriers were observed. CSF concentrations of the Aβ peptides Aβ38 and Aβ40 but not Aβ42 were significantly lower in mutation carriers compared to noncarriers.ConclusionsIncreased NfL levels in presymptomatic individuals and in symptomatic patients with FTD-3 indicate a continuous process of neurodegeneration from the presymptomatic to symptomatic state. Although not specific for FTD-3 pathology, our data suggest that CSF NfL could serve as a valuable biomarker to detect onset of neurodegeneration in FTD-3 mutation carriers.

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A panel of nine cerebrospinal fluid biomarkers may identify patients with atypical parkinsonian syndromes

Journal of Neurology Neurosurgery & Psychiatry ◽

10.1136/jnnp-2014-309562 ◽

2015 ◽

Vol 86 (11) ◽

pp. 1240-1247 ◽

Cited By ~ 116

Author(s):

N K Magdalinou ◽

R W Paterson ◽

J M Schott ◽

N C Fox ◽

C Mummery ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Amyloid Precursor Protein ◽

Area Under The Curve ◽

Precursor Protein ◽

Csf Biomarkers ◽

Neurofilament Light Chain ◽

Neurofilament Light ◽

Monocyte Chemoattractant Protein 1 ◽

Parkinsonian Syndromes ◽

Healthy Elderly

BackgroundPatients presenting with parkinsonian syndromes share many clinical features, which can make diagnosis difficult. This is important as atypical parkinsonian syndromes (APSs) such as progressive supranuclear palsy (PSP), multiple system atrophy (MSA) and corticobasal syndrome (CBS) carry a poor prognosis, compared with patients with Parkinson's disease (PD). In addition, there is overlap between APS and dementia diseases, such as Alzheimer's disease (AD) and frontotemporal dementia (FTD).ObjectiveTo use a panel of cerebrospinal fluid (CSF) biomarkers to differentiate patients with APS from PD and dementia.MethodsA prospective cohort of 160 patients and 30 control participants were recruited from a single specialist centre. Patients were clinically diagnosed according to current consensus criteria. CSF samples were obtained from patients with clinical diagnoses of PD (n=31), PSP (n=33), CBS (n=14), MSA (n=31), AD (n=26) and FTD (n=16). Healthy, elderly participants (n=30) were included as controls. Total τ (t-τ), phosphorylated τ (p-τ), β-amyloid 1–42 (Aβ42), neurofilament light chain (NFL), α-synuclein (α-syn), amyloid precursor protein soluble metabolites α and β (soluble amyloid precursor protein (sAPP)α, sAPPβ) and two neuroinflammatory markers (monocyte chemoattractant protein-1 and YKL-40) were measured in CSF. A reverse stepwise regression analysis and the false discovery rate procedure were used.ResultsCSF NFL (p<0.001), sAPPα (p<0.001) and a-syn (p=0.003) independently predicted diagnosis of PD versus APS. Together, these nine biomarkers could differentiate patients with PD from APS with an area under the curve of 0.95 and subtypes of APS from one another. There was good discriminatory power between parkinsonian groups, dementia disorders and healthy controls.ConclusionsA panel of nine CSF biomarkers was able to differentiate APS from patients with PD and dementia. This may have important clinical utility in improving diagnostic accuracy, allowing better prognostication and earlier access to potential disease-modifying therapies.

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Identification of cerebrospinal fluid biomarkers for parkinsonism using a proteomics approach

npj Parkinson s Disease ◽

10.1038/s41531-021-00249-9 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Tainá M. Marques ◽

Anouke van Rumund ◽

Iris Kersten ◽

Ilona B. Bruinsma ◽

Hans J.C.T. Wessels ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Current Knowledge ◽

Shotgun Proteomics ◽

Selected Reaction Monitoring ◽

Csf Biomarkers ◽

Forest Modelling ◽

Neurofilament Light Chain ◽

Tryptic Peptides ◽

Neurofilament Light ◽

Peptide Profiles

AbstractThe aim of our study was to investigate cerebrospinal fluid (CSF) tryptic peptide profiles as potential diagnostic biomarkers for the discrimination of parkinsonian disorders. CSF samples were collected from individuals with parkinsonism, who had an uncertain diagnosis at the time of inclusion and who were followed for up to 12 years in a longitudinal study. We performed shotgun proteomics to identify tryptic peptides in CSF of Parkinson’s disease (PD, n = 10), multiple system atrophy patients (MSA, n = 5) and non-neurological controls (n = 10). We validated tryptic peptides with differential levels between PD and MSA using a newly developed selected reaction monitoring (SRM) assay in CSF of PD (n = 46), atypical parkinsonism patients (AP; MSA, n = 17; Progressive supranuclear palsy; n = 8) and non-neurological controls (n = 39). We identified 191 tryptic peptides that differed significantly between PD and MSA, of which 34 met our criteria for SRM development. For 14/34 peptides we confirmed differences between PD and AP. These tryptic peptides discriminated PD from AP with moderate-to-high accuracy. Random forest modelling including tryptic peptides plus either clinical assessments or other CSF parameters (neurofilament light chain, phosphorylated tau protein) and age improved the discrimination of PD vs. AP. Our results show that the discovery of tryptic peptides by untargeted and subsequent validation by targeted proteomics is a suitable strategy to identify potential CSF biomarkers for PD versus AP. Furthermore, the tryptic peptides, and corresponding proteins, that we identified as differential biomarkers may increase our current knowledge about the disease-specific pathophysiological mechanisms of parkinsonism.

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Neuronal and Glia-Related Biomarkers in Cerebrospinal Fluid of Patients with Acute Ischemic Stroke

Journal of Central Nervous System Disease ◽

10.4137/jcnsd.s13821 ◽

2014 ◽

Vol 6 ◽

pp. JCNSD.S13821 ◽

Cited By ~ 48

Author(s):

Clara Hjalmarsson ◽

Maria Bjerke ◽

Björn Andersson ◽

Kaj Blennow ◽

Henrik Zetterberg ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Ischemic Stroke ◽

White Matter ◽

Acute Ischemic Stroke ◽

Experimental Studies ◽

Csf Biomarkers ◽

Stroke Severity ◽

Neurofilament Light ◽

Age Related ◽

T Tau

Background Cerebral ischemia promotes morphological reactions of the neurons, astrocytes, oligodendrocytes, and microglia in experimental studies. Our aim was to examine the profile of CSF (cerebrospinal fluid) biomarkers and their relation to stroke severity and degree of white matter lesions (WML). Methods A total of 20 patients (mean age 76 years) were included within 5–10 days after acute ischemic stroke (AIS) onset. Stroke severity was assessed using NIHSS (National Institute of Health stroke scale). The age-related white matter changes (ARWMC) scale was used to evaluate the extent of WML on CT-scans. The concentrations of specific CSF biomarkers were analyzed. Results Patients with AIS had significantly higher levels of NFL (neurofilament, light), T-tau, myelin basic protein (MBP), YKL-40, and glial fibrillary acidic protein (GFAP) compared with controls; T-Tau, MBP, GFAP, and YKL-40 correlated with clinical stroke severity, whereas NFL correlated with severity of WML (tested by Mann–Whitney test). Conclusions Several CSF biomarkers increase in AIS, and they correlate to clinical stroke severity. However, only NFL was found to be a marker of degree of WML.

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Neuronal and glial CSF biomarkers in multiple sclerosis: a systematic review and meta-analysis

Reviews in the Neurosciences ◽

10.1515/revneuro-2020-0145 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Sara Momtazmanesh ◽

Parnian Shobeiri ◽

Amene Saghazadeh ◽

Charlotte E. Teunissen ◽

Joachim Burman ◽

...

Keyword(s):

Multiple Sclerosis ◽

Meta Analysis ◽

Csf Biomarkers ◽

Neurofilament Light Chain ◽

Neurofilament Light ◽

Meta Regression ◽

Meta Analyses ◽

Astroglial Activation ◽

In Cis ◽

T Tau

Abstract Multiple sclerosis (MS) is a neurodegenerative disease associated with inflammatory demyelination and astroglial activation, with neuronal and axonal damage as the leading factors of disability. We aimed to perform a meta-analysis to determine changes in CSF levels of neuronal and glial biomarkers, including neurofilament light chain (NFL), total tau (t-tau), chitinase-3-like protein 1 (CHI3L1), glial fibrillary acidic protein (GFAP), and S100B in various groups of MS (MS versus controls, clinically isolated syndrome (CIS) versus controls, CIS versus MS, relapsing-remitting MS (RRMS) versus progressive MS (PMS), and MS in relapse versus remission. According to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses, we included 64 articles in the meta-analysis, including 4071 subjects. For investigation of sources of heterogeneity, subgroup analysis, meta-regression, and sensitivity analysis were conducted. Meta-analyses were performed for comparisons including at least three individual datasets. NFL, GFAP, t-tau, CHI3L1, and S100B were higher in MS and NFL, t-tau, and CHI3L1 were also elevated in CIS patients than controls. CHI3L1 was the only marker with higher levels in MS than CIS. GFAP levels were higher in PMS versus RRMS, and NFL, t-tau, and CHI3L1 did not differ between different subtypes. Only levels of NFL were higher in patients in relapse than remission. Meta-regression showed influence of sex and disease severity on NFL and t-tau levels, respectively and disease duration on both. Added to the role of these biomarkers in determining prognosis and treatment response, to conclude, they may serve in diagnosis of MS and distinguishing different subtypes.

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Detection of Cerebrospinal Fluid Neurofilament Light Chain as a Marker for Alpha-Synucleinopathies

Frontiers in Aging Neuroscience ◽

10.3389/fnagi.2021.717930 ◽

2021 ◽

Vol 13 ◽

Author(s):

Sezgi Canaslan ◽

Matthias Schmitz ◽

Anna Villar-Piqué ◽

Fabian Maass ◽

Karin Gmitterová ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Single Molecule ◽

Light Chain ◽

Clinical Symptoms ◽

Alpha Synuclein ◽

Test System ◽

Marker Protein ◽

Neurofilament Light Chain ◽

Neurofilament Light ◽

Different Types

Alpha-synucleinopathies, such as Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), are a class of neurodegenerative diseases. A diagnosis may be challenging because clinical symptoms partially overlap, and there is currently no reliable diagnostic test available. Therefore, we aimed to identify a suitable marker protein in cerebrospinal fluid (CSF) to distinguish either between different types of alpha-synucleinopathies or between alpha-synucleinopathies and controls. In this study, the regulation of different marker protein candidates, such as alpha-synuclein (a-Syn), neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and total tau (tau) in different types of alpha-synucleinopathies, had been analyzed by using an ultrasensitive test system called single-molecule array (SIMOA). Interestingly, we observed that CSF-NfL was significantly elevated in patients with DLB and MSA compared to patients with PD or control donors. To differentiate between groups, receiver operating characteristic (ROC) curve analysis resulted in a very good diagnostic accuracy as indicated by the area under the curve (AUC) values of 0.87–0.92 for CSF-NfL. Furthermore, we observed that GFAP and tau were slightly increased either in DLB or MSA, while a-Syn levels remained unregulated. Our study suggests NfL as a promising marker to discriminate between different types of alpha-synucleinopathies or between DLB/MSA and controls.

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