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. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: Mult Scler Relat Disord. 2022 Feb 8;59:103676. doi: 10.1016/j.msard.2022.103676

Type of serum collection tube does not impact neurofilament light chain levels

Elias S Sotirchos 1, Kathryn C Fitzgerald 1, Matthew D Smith 1, Elena S Vasileiou 1, Yasmin Resto 1, Hannah-Noelle Lord 1, Ellen M Mowry 1, Peter A Calabresi 1
PMCID: PMC9249133  NIHMSID: NIHMS1814657  PMID: 35158190

SUMMARY:

Blood neurofilament light chain (NfL) has been reported to be a promising biomarker of neurological disease. NfL is predominantly measured in serum (sNfL), but there is a lack of reports regarding the effects of collection tubes on sNfL levels. We assessed sNfL levels using a novel immunoassay in 18 participants using 3 different types of serum collection tubes (no additive, with silica clot activator, and serum separator tubes). Variation observed in sNfL levels between samples from different collection tubes was similar to that observed in duplicate runs from the same tube. These findings support a lack of effect of type of serum collection tube on sNfL levels.

Keywords: neurofilament light chain, serum, collection tube, biomarker

INTRODUCTION

Neurofilaments are neuron-specific type IV intermediate filament heteropolymers composed of light, medium and heavy chains.[1] Neurofilaments are the dominant proteins of the neural cytoskeleton, are released into the extracellular space following neuro-axonal damage, and have thus been proposed as putative biomarkers of neuro-axonal injury in multiple neurological diseases.[1] Neurofilament light chain (NfL) especially, has been shown to be a promising biomarker because of its high solubility, and increased NfL levels have been found in the blood and cerebrospinal fluid (CSF) in several neurological disorders with underlying neuro-axonal degeneration.[2],[3]

Initial evaluation of NfL in neurological diseases focused on quantification in the CSF, given that the levels of NfL detected in the blood are considerably lower and thus more challenging to accurately quantify with conventional enzyme-linked immunosorbent assay (ELISA). However, CSF NfL clearly has limited potential for use as a biomarker given the necessity of an invasive procedure for CSF sampling. Recently, the application of newer immunoassays has enabled the measurement of the low concentrations of NfL in blood (usually serum) with high accuracy and reproducibility.[4],[5]

In addition to clinical validation in neurological diseases, understanding of pre-analytical factors that may impact measurements is critical for NfL to be used in clinical practice. However, various types of serum collection tubes are available, and factors such as additives (clot activator), tube material (plastic or glass) and presence of separator gel may be a potential source of pre-analytical variability in laboratory testing.[9] In this study, we aimed to assess whether the type of serum collection tube may affect serum NfL (sNfL) levels.

METHODS

Study Design and Participants

Johns Hopkins University Institutional Review Board approval was obtained for the study protocol, and written, informed consent was obtained from all participants. All blood samples were collected over a 10-day period in November 2020 and sNfL measurement was performed in December 2020.

Healthy controls (HC) were recruited for blood sampling from amongst Johns Hopkins University staff. Patients with a diagnosis of multiple sclerosis (MS) who were being seen for routine clinic visits were recruited from the Johns Hopkins MS Center.

Blood collection and sNfL measurement

Blood was collected by single venipuncture in three different types of serum collection tubes (in the following order):

  1. “No additive” tube: Glass serum tube, no additive, silicone-coated interior (BD catalog no. 366430)

  2. “Clot Activator” tube: Plastic serum tube, with silica act clot activator, silicone-coated interior (BD catalog no. 367820)

  3. “Serum separator” tube (SST): Plastic SST, with silica clot activator, polymer gel, silicone-coated interior (BD catalog no. 367988)

The blood was allowed to clot in an upright position for 30–45 min at room temperature and then centrifuged at 2000g (15min at room temperature for tubes without additive; 10min at 4° C for clot activator and SST tubes). Serum was divided into 1mL aliquots and stored at −80° C. Samples were not thawed until sNfL measurement was performed. Serum was examined for presence of hemolysis, and no significant hemolysis was observed in any of the collected samples.

Serum samples were shipped to Siemens Healthcare Laboratory, LLC (Berkeley, CA, USA) on dry ice and upon receipt, samples were stored at −80 °C. Prior to analysis, samples were thawed at room temperature and measurements were performed on the Atellica® Solution platform, using a novel, high-throughput acridinium-ester immunoassay, which has a range of 2 to 500pg/mL and excellent repeatability.[8] Serum from each tube was run in duplicate (from two separate aliquots) on the same day.

Statistical methods

Statistical analyses were performed with R Version 4.1.0 (https://www.r-project.org/). For comparisons between tube types, duplicate measurements from each tube type were averaged. The within-subject coefficient variation (CV) was calculated for each pair of measurements using the logarithmic method, as previously described.[10] We also calculated intraclass correlation coefficients (ICC) for each pair of measurements using a two-way random effects model for consistency.[11] Furthermore, we constructed Bland-Altman plots to demonstrate the data for pairs of measurements.[12]

RESULTS

A total of 18 participants were recruited (7 HC, 11 MS). Age (mean±SD) was 39.8±10.1 years for HC and 41.3±14.4 years for MS participants. There were 2 female participants in the HC group (28.6%) and 7 female participants in the MS group (63.6%).

The CV for duplicate measurements from the same serum collection tube (different aliquots) was 6.9% (95% CI: 4.6–9.3%), 7.5% (95% CI: 5.0–10.1%) and 11.3% (95% CI: 7.5–15.2%) for the no additive, clot activator only, and SST tubes, respectively. The corresponding ICC were 0.98 (95% CI: 0.96–0.99), 0.97 (95% CI: 0.93–0.99), and 0.96 (95% CI: 0.90–0.99). Bland-Altman plots for the duplicate measurements from each collection tube are shown in Figure 1.

Figure 1.

Figure 1.

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Bland-Altman plots demonstrating the difference in sNfL levels between duplicate runs (y-axis) and the mean of sNfL levels in duplicate runs (x-axis) for each type of serum collection tube. The solid line corresponds to the mean difference and the dashed lines correspond to the 95% limits of agreement.

When comparing the measurement across tube types, no systematic difference was observed, as shown in the Bland-Altman plots in Figure 2. The CV for no additive vs clot activator was 6.7% (95% CI: 4.5–9.0%), no additive vs SST was 9.8% (95% CI: 6.5–13.2%), and for clot activator vs SST was 8.1% (5.4–10.9%), which were overall similar to the variability observed for duplicate measurements from the same serum collection tube. The corresponding ICC were 0.98 (95% CI 0.95–0.99), 0.98 (95% CI 0.93–0.99) and 0.97 (0.93–0.99).

Figure 2.

Figure 2.

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Bland-Altman plots comparing sNfL (in pg/mL) across pairs of serum collection tube types. The y-axis corresponds to the difference between sNfL measurements obtained for a given pair of tubes and the x-axis demonstrates the mean of these measurements. The solid line corresponds to the mean difference and the dashed lines correspond to the 95% limits of agreement.

DISCUSSION

We assessed the potential effect of type of serum collection tubes on NfL measurement, across three different commonly used tube types, and we found no evidence for any systematic effect on sNfL levels. These results suggest that sNfL measurements using serum collected in different tubes may be pooled in analyses (e.g. when utilizing biobanked samples from studies utilizing different serum collection tubes). Limitations of this study include the relatively low sample size and lack of participants with markedly elevated sNfL levels; however the results did not demonstrate any evidence for presence of a systematic difference across the range of values examined.

Notably, NfL levels measured using Single Molecule Array (SIMOA) in ethylenediaminetetraacetic acid (EDTA) anti-coagulated plasma samples have been reported to be lower compared to serum samples, although equivalent NfL concentrations were found in serum, heparin-treated plasma and EDTA-treated plasma in a study using the acridinium ester immunoassay that we also used.[6]–[8] This highlights the fact that, with the availability of multiple assays for sNfL measurement, one must consider that the effect of pre-analytical factors may vary by assay, and results of studies examining these factors on a given assay may not necessarily generalize to others.

ACKNOWLEDGMENTS

This study was funded by the National Institutes of Health (K23NS117883 to E.S.S and U01NS111678 to P.A.C.) and National Multiple Sclerosis Society (RG 1904–33800 to P.A.C.).

Footnotes

Disclosures:

Elias Sotirchos has received speaker honoraria from Viela Bio, Alexion and Biogen and consulting fees from Viela Bio, Horizon Therapeutics, Alexion and Genentech.

Kathryn Fitzgerald, Elena Vasileiou and Matthew Smith report no disclosures.

Ellen Mowry has grants from Biogen, Genzyme and Genentech, is site PI for studies sponsored by Biogen and Genentech, has received free medication for a clinical trial from Teva and receives royalties for editorial duties from UpToDate.

Peter Calabresi has received consulting fees from Biogen, Nervgen, Avidea, and Disarm Therapeutics and is PI on grants from Principia, and Genentech.

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