The impact of pre-analytical variables on the stability of neurofilament proteins in CSF, determined by a novel validated SinglePlex Luminex assay and ELISA

doi:10.1016/j.jim.2013.11.008

Journal of Immunological Methods

Volume 402, Issues 1–2, 15 January 2014, Pages 43-49

https://doi.org/10.1016/j.jim.2013.11.008 Get rights and content

Abstract

Background

Neurofilament (Nf) proteins have been shown to be promising biomarkers for monitoring and predicting disease progression for various neurological diseases. The aim of this study was to evaluate the effects of pre-analytical variables on the concentration of neurofilament heavy (NfH) and neurofilament light (NfL) proteins.

Methods

For NfH an in-house newly-developed and validated SinglePlex Luminex assay was used; ELISA was used to analyze NfL.

Results

For the NfL ELISA assay, the intra- and inter-assay variation was respectively, 1.5% and 16.7%. Analytical performance of the NfH SinglePlex Luminex assay in terms of sensitivity (6.6 pg/mL), recovery in cerebrospinal fluid (CSF) (between 90 and 104%), linearity (from 6.6–1250 pg/mL), and inter- and intra-assay variation (< 8%) were good. Concentrations of both NfL and NfH appeared not negatively affected by blood contamination, repeated freeze–thaw cycles (up to 4), delayed processing (up to 24 hours) and during long-term storage at − 20 °C, 4 °C, and room temperature. A decrease in concentration was observed during storage of both neurofilament proteins up to 21 days at 37 °C, which was significant by day 5.

Conclusions

The newly developed NfH SinglePlex Luminex assay has a good sensitivity and is robust. Moreover, both NfH and NfL are stable under the most prevalent pre-analytical variations.

Introduction

The Nf protein is a heteropolymer composed of three subunits, Nf light (NfL), medium (NfM), and heavy (NfH) (Petzold et al., 2003). The NfL is the most abundant form (ratio: 4:2:1, NfL:NfM:NfH) and contains four sites for phosphorylation. NfH contains more than 100 potential phosphorylation sites. Nf proteins have been shown to be promising biomarkers for monitoring and predicting disease progression for various neurological diseases (Deisenhammer et al., 2006). Nf heavy (NfH) and light (NfL) chain proteins are elevated early in the disease course of MS (multiple sclerosis) (Kuhle et al., 2010, Norgren et al., 2004) and NfL chain has prognostic value for conversion of CIS (clinically isolated syndrome) patients to MS (Teunissen et al., 2009). Moreover, NfH protein in brain interstitial fluid has prognostic value in patients with traumatic brain injury (Petzold et al., 2011), NfH and NfL were related to negative outcomes in subarachnoid hemorrhage (Lewis et al., 2008, Zanier et al., 2011) and both NfH and NfL were elevated and related to disability in amyotrophic lateral sclerosis (Reijn et al., 2009, Steinacker et al., 2011). Moreover, NfL concentrations were reduced after Natalizumab treatment of MS patients (Gunnarsson et al., 2011) and NfH protein levels in blood were increased after aggressive chemotherapy (Petzold et al., 2010a).

We recently showed by western blot analysis and ELISA that NfL protein is not stable in neat CSF during one day at room temperature (Koel-Simmelink et al., 2011). Variability in pre-analytical CSF sampling and handling can significantly impact accuracy of results in large cohort studies. Therefore, we studied the effects of pre-analytical variables on NfH and NfL as measured with our newly developed and validated NfH SinglePlex Luminex assay and by ELISA for NfL used in recently published studies (Kuhle et al., 2013, Petzold et al., 2010b). First, we thoroughly validated the newly developed NfH SinglePlex Luminex assay. We next focussed on effects of delayed processing, different storage temperatures, freeze–thaw cycles and effect of blood contamination.

Section snippets

CSF samples and materials

Donor CSF samples were obtained by informed consent and processed anonymously. Two pools of surplus CSF, with low (LQC) and high (HQC) NfH concentrations served as internal quality references. These were prepared in Sarstedt polypropylene tubes and aliquots were stored at − 80 °C.

Purified bovine spinal cord NfH (200 kD, #62210, > 98% purity as determined by SDS gel electrophoresis) and NfL (68kD, #62208, > 98% purity as determined by SDS gel electrophoresis) was obtained from Progen Biotechnik GmbH,

Analytical performance of NfL assay

The CV of duplicates (n = 149) was 1.5 ± 1.1%. In two internal quality control CSF samples the between-assay variation was 17.4% at 999 pg/mL and 15.9% at 50,789 pg/mL (sample diluted 50x in the assay) averaging at: 16.7% analyzed over 5 independent runs.

Reproducibility of the standard curve

The mean standard curve for NfH as determined over eight independent assays is shown in Fig. 1. Limit of linearity (LOL) of the standard curve was determined by linear regression analysis. Individual standard curves showed a high degree of linearity

Discussion

The results of this study show that our SinglePlex Luminex assay for detection of NfH is specific for NfH protein, is free of the artifacts of non-parallelism, and is tolerant to significant blood contamination of CSF. We checked for cross reactivity with NfM (19.8%) and NfL (0%). It is known that neurofilament proteins have 30–50% homology with vimentin, desmin, and GFAP (Blumenberg, 1989). However we loaded CSF on a SDS-gel and after separation and blotting, immobilized proteins were probed

Conflict of interest

None of the authors have a conflict of interest to declare.

Acknowledgements

We would like to acknowledge the technical assistance of W. van Geel and M. Verbeek. We would like to thank A.F.S. Petzold for useful discussions. This study was financially supported by EDAR and the Koningin Wilhelmina Fonds.

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The impact of pre-analytical variables on the stability of neurofilament proteins in CSF, determined by a novel validated SinglePlex Luminex assay and ELISA

Abstract

Background

Methods

Results

Conclusions

Introduction

Section snippets

CSF samples and materials

Analytical performance of NfL assay

Reproducibility of the standard curve

Discussion

Conflict of interest

Acknowledgements

J. Neuroimmunol.

Brain Res.

J. Neurol. Sci.

J. Immunol. Methods

J. Immunol. Methods

Biochem. Biophys. Res. Commun.

Evolution of homologous domains of cytoplasmic intermediate filament proteins and lamins

Mol. Biol. Evol.

Guidelines on routine cerebrospinal fluid analysis report from an EFNS task force

Eur. J. Neurol.

EFNS guidelines on disease-specific CSF investigations

Eur. J. Neurol.

Axonal damage in relapsing multiple sclerosis is markedly reduced by natalizumab

Ann. Neurol.

Predicting the stability of biological standards and products

Biom. J.

The neurofilament light chain is not stable in vitro

Ann. Neurol.

A comparative study of CSF neurofilament light and heavy chain protein in MS

Mult. Scler.