Axel Petzold

Neurofilament phosphoforms: surrogate markers for axonal injury, degeneration and loss.

This review on the role of neurofilaments as surrogate markers for axonal degeneration in neurological diseases provides a brief background to protein synthesis, assembly, function and degeneration. Methodological techniques for quantification are described and a protein nomenclature is proposed. The relevance for recognising anti-neurofilament autoantibodies is noted. Pathological implications are discussed in view of immunocytochemical, cell-culture and genetic findings. With reference to the present symposium on multiple sclerosis, the current literature on body fluid levels of neurofilaments in demyelinating disease is summarised.

Optical coherence tomography in multiple sclerosis: a systematic review and meta-analysis

Optical coherence tomography (OCT) is a new method that could aid analysis of neurodegeneration in multiple sclerosis (MS) by capturing thinning of the retinal nerve fibre layer (RNFL). Meta-analyses of data for time domain OCT show RNFL thinning of 20.38 microm (95% CI 17.91-22.86, n=2063, p<0.0001) after optic neuritis in MS, and of 7.08 microm (5.52-8.65, n=3154, p<0.0001) in MS without optic neuritis. The estimated RNFL thinning in patients with MS is greater than the extent expected in normal ageing, probably because of retrograde trans-synaptic degeneration and progressive loss of retinal ganglion cells, in addition to the more pronounced thinning caused by optic neuritis if present. RNFL thickness correlates with visual and neurological functioning as well as with paraclinical data. Developments that could improve understanding of the relation between structure and function in MS pathophysiology include spectral or Fourier domain OCT technology, polarisation-sensitive OCT, fluorescence labelling, structural assessment of action-potential propagation, and segmentation algorithms allowing quantitative assessment of retinal layers.

A consensus protocol for the standardization of cerebrospinal fluid collection and biobanking

There is a long history of research into body fluid biomarkers in neurodegenerative and neuroinflammatory diseases. However, only a few biomarkers in CSF are being used in clinical practice. One of the most critical factors in CSF biomarker research is the inadequate powering of studies because of the lack of sufficient samples that can be obtained in single-center studies. Therefore, collaboration between investigators is needed to establish large biobanks of well-defined samples. Standardized protocols for biobanking are a prerequisite to ensure that the statistical power gained by increasing the numbers of CSF samples is not compromised by preanalytical factors. Here, a consensus report on recommendations for CSF collection and biobanking is presented, formed by the BioMS-eu network for CSF biomarker research in multiple sclerosis. We focus on CSF collection procedures, preanalytical factors, and high-quality clinical and paraclinical information. The biobanking protocols are applicable for CSF biobanks for research targeting any neurologic disease.

Triple-H therapy in the management of aneurysmal subarachnoid haemorrhage

Cerebral vasospasm is a recognised but poorly understood complication for many patients who have aneurysmal subarachnoid haemorrhage and can lead to delayed ischaemic neurological deficit (stroke). Morbidity and mortality rates for vasospasm are high despite improvements in management. Since the middle of the 1970s, much has been written about the treatment of cerebral vasospasm. Hypervolaemia, hypertension, and haemodilution (triple-H) therapy in an intensive-care setting has been shown in some studies to improve outcome and is an accepted means of treatment, although a randomised controlled trial has never been undertaken. In this review, the rationale for this approach will be discussed, alongside new thoughts and future prospects for the management of this complex disorder.

Axonal damage markers in cerebrospinal fluid are increased in ALS

Objective: To test whether biomarkers for axonal degeneration correlated with clinical subtypes and were of use in predicting progression of ALS. Methods: Patients with ALS (n = 69), patients with Alzheimer disease (AD; n = 73), and age-matched controls (n = 33) were included in this prospective study. CSF levels of tau protein and neurofilaments (NfHSMI35) were measured using ELISA. In 49 patients with ALS, follow-up data were available (median follow-up 7 months). Results: CSF levels of NfHSMI35 were five times higher in patients with ALS (1.7 ng/mL) than in controls (0.3 ng/mL, p < 0.001) and 10 times higher than in patients with AD (0.14 ng/mL, p < 0.001). NfHSMI35 values were also higher in patients with upper motor neuron–dominant ALS than in patients with typical ALS (upper motor neuron + lower motor neuron) at p = 0.02. Values of NfHSMI35 were higher in ALS of more rapid progression. The values of NfH and tau did not correlate with CSF protein content. Conclusions: The authors propose that axonal damage markers in CSF may discriminate between subtypes of ALS and that they could be used as markers for therapeutic trials. CSF NfH was superior to tau in these discriminations.

A worldwide multicentre comparison of assays for cerebrospinal fluid biomarkers in Alzheimer's disease

Background Different cerebrospinal fluid (CSF) amyloid-beta 1–42 (Aβ 1–42), total Tau (Tau) and Tau phosphorylated at threonine 181 (P-Tau) levels are reported, but currently there is a lack of quality control programmes. The aim of this study was to compare the measurements of these CSF biomarkers, between and within centres. Methods Three CSF-pool samples were distributed to 13 laboratories in 2004 and the same samples were again distributed to 18 laboratories in 2008. In 2004 six laboratories measured Aβ 1–42, Tau and P-Tau and seven laboratories measured one or two of these marker(s) by enzyme-linked immunosorbent assays (ELISAs). In 2008, 12 laboratories measured all three markers, three laboratories measured one or two marker(s) by ELISAs and three laboratories measured the markers by Luminex. Results In 2004, the ELISA intercentre coefficients of variance (interCV) were 31%, 21% and 13% for Aβ 1–42, Tau and P-Tau, respectively. These were 37%, 16% and 15%, respectively, in 2008. When we restricted the analysis to the Innotest® (N = 13) for Aβ 1–42, lower interCV were calculated (22%). The centres that participated in both years (N = 9) showed interCVs of 21%, 15% and 9% and intra-centre coefficients (intraCV) of variance of 25%,18% and 7% in 2008. Conclusions The highest variability was found for Aβ 1–42. The variabilities for Tau and P-Tau were lower in both years. The centres that participated in both years showed a high intraCV comparable to their interCV, indicating that there is not only a high variation between but also within centres. Besides a uniform standardization of (pre)analytical procedures, the same assay should be used to decrease the inter/intracentre variation.

The OSCAR-IB Consensus Criteria for Retinal OCT Quality Assessment

Background Retinal optical coherence tomography (OCT) is an imaging biomarker for neurodegeneration in multiple sclerosis (MS). In order to become validated as an outcome measure in multicenter studies, reliable quality control (QC) criteria with high inter-rater agreement are required. Methods/Principal Findings A prospective multicentre study on developing consensus QC criteria for retinal OCT in MS: (1) a literature review on OCT QC criteria; (2) application of these QC criteria to a training set of 101 retinal OCT scans from patients with MS; (3) kappa statistics for inter-rater agreement; (4) identification reasons for inter-rater disagreement; (5) development of new consensus QC criteria; (6) testing of the new QC criteria on the training set and (7) prospective validation on a new set of 159 OCT scans from patients with MS. The inter-rater agreement for acceptable scans among OCT readers (n = 3) was moderate (kappa 0·45) based on the non-validated QC criteria which were entirely based on the ophthalmological literature. A new set of QC criteria was developed based on recognition of: (O) obvious problems, (S) poor signal strength, (C) centration of scan, (A) algorithm failure, (R) retinal pathology other than MS related, (I) illumination and (B) beam placement. Adhering to these OSCAR-IB QC criteria increased the inter-rater agreement to kappa from moderate to substantial (0.61 training set and 0.61 prospective validation). Conclusions This study presents the first validated consensus QC criteria for retinal OCT reading in MS. The high inter-rater agreement suggests the OSCAR-IB QC criteria to be considered in the context of multicentre studies and trials in MS.

Increased neurofilament light chain blood levels in neurodegenerative neurological diseases

Objective Neuronal damage is the morphological substrate of persisting neurological disability. Neurofilaments (Nf) are cytoskeletal proteins of neurons and their release into cerebrospinal fluid has shown encouraging results as a biomarker for neurodegeneration. This study aimed to validate the quantification of the Nf light chain (NfL) in blood samples, as a biofluid source easily accessible for longitudinal studies. Methods We developed and applied a highly sensitive electrochemiluminescence (ECL) based immunoassay for quantification of NfL in blood and CSF. Results Patients with Alzheimer’s disease (AD) (30.8 pg/ml, n=20), Guillain-Barré-syndrome (GBS) (79.4 pg/ml, n=19) or amyotrophic lateral sclerosis (ALS) (95.4 pg/ml, n=46) had higher serum NfL values than a control group of neurological patients without evidence of structural CNS damage (control patients, CP) (4.4 pg/ml, n=68, p<0.0001 for each comparison, p=0.002 for AD patients) and healthy controls (HC) (3.3 pg/ml, n=67, p<0.0001). Similar differences were seen in corresponding CSF samples. CSF and serum levels correlated in AD (r=0.48, p=0.033), GBS (r=0.79, p<0.0001) and ALS (r=0.70, p<0.0001), but not in CP (r=0.11, p=0.3739). The sensitivity and specificity of serum NfL for separating ALS from healthy controls was 91.3% and 91.0%. Conclusions We developed and validated a novel ECL based sandwich immunoassay for the NfL protein in serum (NfLUmea47:3); levels in ALS were more than 20-fold higher than in controls. Our data supports further longitudinal studies of serum NfL in neurodegenerative diseases as a potential biomarker of on-going disease progression, and as a potential surrogate to quantify effects of neuroprotective drugs in clinical trials.

A specific ELISA for measuring neurofilament heavy chain phosphoforms

Neurofilaments (Nf) are the major constitutents of the axoskeleton and body fluid Nf levels are an important tool for estimating axonal degeneration in vivo. This paper presents a new sandwich ELISA allowing quantification of the NfH(SMI35) phosphoform from CSF, brain tissue and cell culture homogenates. The sensitivity of the NfH(SMI35) ELISA is 0.2 ng/ml with a recovery of 119% and a mean within- and between-batch precision of 10.6% and 23%, respectively. CSF NfH(SMI35) was stable at 4 degrees C, is not influenced by freeze-thaw cycles, and proteolysis present at room temperature could be prevented by adding protease inhibitors. Aggregate formation was observed for HPLC-purified bovine NfH and could be resolved by sonication. The upper reference value for CSF NfH(SMI35) levels (0.73 ng/ml) was defined as the 95% cumulative frequency from 416 CSF samples. Based on this cutoff, a significantly higher proportion of patients with amyotrophic lateral sclerosis, space-occupying lesions, disc prolapse and subarachnoid haemorrhage had pathologically elevated NfH(SMI35) levels compared to patients with cluster headache or demyelinating disease.A new nomenclature is proposed to facilitate the comparison between ELISA, immunoblotting and immunocytochemistry.

Multiple sclerosis: Neurofilament light chain antibodies are correlated to cerebral atrophy.

Objective: To evaluate markers of axonal damage in CSF and serum of patients with different subtypes of MS in relation to measures of disease progression on MRI. Methods: In 51 patients with MS (21 relapsing-remitting, 20 secondary progressive, 10 primary progressive), levels of heavy and light neurofilaments (NfH and NfL) and antibodies to neurofilaments (anti-NfL and -NfH) as well as the total immunoglobulin G (IgG) were analyzed. MRI analysis included T2 hyperintense, T1 hypointense, and gadolinium enhancing lesions and markers of cerebral atrophy (ventricular and parenchymal fractions). Results: For the total group, correlations were found between the anti-NfL index and the parenchymal fraction (PF) (r = −0.51, p < 0.001), T2 lesion load (r = 0.41, p < 0.05), ventricular fraction (r = 0.37, p < 0.05), and T1 lesion load (r = 0.37, p < 0.05). For the anti-NfH index, a correlation was found with the PF (r = −0.39, p < 0.05). No correlations were found between the IgG index and MRI measures. Conclusions: Intrathecal production of anti-NfL antibodies may serve as a marker of tissue damage, particularly axonal loss, in MS.