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

J Immunol Methods. 2014 Jan 15;402(1-2):43-9. doi: 10.1016/j.jim.2013.11.008. Epub 2013 Nov 22.


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 (6.6pg/mL), recovery in cerebrospinal fluid (CSF) (between 90 and 104%), linearity (from 6.6-1250pg/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 24hours) 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 21days 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.

Keywords: AD; ALS; Alzheimer disease; CIS; CSF; Cerebrospinal fluid; ELISA; Enzyme Linked Immuno Sorbent Assay; FTD; HQC; LBD; LOD; LOL; LP; LQC; Lewy bodies disease; Limit of linearity; MS; Neurofilament heavy (NfH) protein; Neurofilament light (NfL) protein; Nf; NfH; NfH SinglePlex Luminex assay; NfL; Pre-analytical variables; SD; Stability; amyotrophic lateral sclerosis; cerebrospinal fluid; clinically isolated syndrome; frontotemporal dementia; high quality control; limit of detection; low quality control; lumbar puncture; multiple sclerosis; neurofilament; neurofilament heavy; neurofilament light; standard deviation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Biomarkers / cerebrospinal fluid
  • Enzyme-Linked Immunosorbent Assay*
  • Freezing
  • Humans
  • Neurofilament Proteins / blood
  • Neurofilament Proteins / cerebrospinal fluid*
  • Observer Variation
  • Protein Stability
  • Reproducibility of Results
  • Temperature
  • Time Factors


  • Biomarkers
  • Neurofilament Proteins
  • neurofilament protein L
  • neurofilament protein H