Pre-analytical long-term stability of neopterin and neurofilament light in stored cerebrospinal fluid samples

Introduction

Biomarkers are considered “A defined characteristic that is measured as an indicator of normal biological processes, pathogenic processes or responses to an exposure or intervention” [1]. Several markers of inflammation and neuronal damage have been proposed as diagnostic or prognostic markers of neurological disease. These biomarkers can be measured in different bodily fluids, such as plasma, serum, urine and cerebrospinal fluid (CSF). Among the markers of neuroinflammation, the quantity of neopterin in CSF has long been used as a biomarker of different infectious and non-infectious neurological conditions, such as multiple sclerosis, hypoxic-ischemic encephalopathy (HIE), human T cell lymphotropic virus type-1 (HTLV-1) -associated myelopathy (HAM), human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) and cerebral malaria [2], [3], [4], [5], [6]. Interest in neurofilament light (Nfl), a structural axonal protein that is released into CSF following neuronal damage [7] is increasing. Like neopterin, Nfl CSF concentrations are increased in several neurological diseases [8, 9]. Although the quantification of these markers is valuable, pre-analytical variables may impact results [10, 11]. For example, it is known that there is a risk of sample degradation during storage. This is particularly important, when performing retrospective studies that rely on the analysis of stored samples. Therefore, the present research aimed to address whether long-term storage of CSF samples at −80 °C would impact on the concentrations of neopterin and neurofilament light, markers of neuroinflammation and neuronal damage that are widely used in research and increasingly adopted into routine follow-up of patients with neurological conditions.

Materials and methods

Twenty-one anonymised CSF samples, obtained from patients with HAM during their clinical follow-up, were randomly selected and included in the study. These patients attended the National Centre for Human Retrovirology (NCHR), at St Mary`s Hospital, London and had donated samples to the Communicable Diseases Research Tissue Bank following written informed consent (National Research Ethics Reference 20/SC/026). Samples were collected by lumbar puncture and centrifugated (16,000 rcf for 1 min) within 24 h of collection and transported at room temperature. CSF supernatant was stored at −80 °C for 0.5–11 years (Median 3.5 years) before testing for neopterin and for 1–8 years (Median 3) before testing the first time for Nfl. Stored samples were retested, with a three-year interval, after one freeze and thaw cycle, for neopterin (RE59321, IBL International, Hamburg, Germany) (n=10) and neurofilament light (10–7,001, UmanDiagnostics, Umea, Sweden) (n=11) by ELISA (Figure 1). Previous data indicate stability of these markers when submitted to one freeze-thaw cycle. Samples were tested undiluted for neopterin and diluted 1:2 for Nfl. Assays were performed according to the manufacturer’s instructions. Briefly, Nfl ELISA uses two non-competing monoclonal antibodies to Nfl protein. One is immobilized on a solid phase and binds to the Nfl in the sample. The second antibody acts as a detector that interacts with the bound target protein and signals through biotin – horseradish peroxidase avidin binding. The signal obtained corresponds directly to the amount of Nfl which is extrapolated from the standard curve. Neopterin ELISA is a competitive ELISA. In this assay, an unknown amount of antigen in the sample and a fixed amount of enzyme labelled antigen compete for binding at rabbit-anti-neopterin. Both antigen-antibody complexes bind to the goat-anti-rabbit antibody coating the microtiter wells. All unbound antigen is removed by washing and the intensity of the colour developed after the substrate incubation is inversely proportional to the amount of antigen in the sample. The quantity is extrapolated from the manufacturer’s standard curve. All samples were tested in duplicate and the average concentration used for analysis. Mean, standard deviation (SD) and coefficient of variation between the two repeat tests were calculated for neopterin and Nfl for each sample. The concentrations of neopterin and Nfl were compared between the first and second test using paired t test. Correlations were evaluated with the Spearman test, and p<0.05 was considered to reflect statistically significance. Patients with HAM were classified according to disease activity based on the levels of neopterin in CSF, as proposed by Sato and colleagues, as low <6 nmol/L, moderate 6–43 nmol/L and high >43 nmol/L [3]. The concordance between the two results was assessed. Reference change values (RCV) were calculated using the formula RCV=2^1/2 · Z · (CV_(A)² + CV_(I)²)^1/2, as previously described [12]. Z is the number of standard deviations appropriate to the probability and set as 1.96 (p<0.05). CV biological (CV_(I)) was 0 as there were no resampling.

Figure 1:

Graphic summary of methods.

Results

Data are shown in the Table 1. Results for both neopterin and Nfl concentration were consistent when comparing the two time-points. Neopterin concentrations ranged from 12.4 to 64 nmol/L in 2018 and from 11.5 to 64.4 nmol/L, when retested in 2021 (Mean [SD]: 2018=30 [18.4]; 2021=33 [19.1], p=0.03). Neurofilament light concentrations ranged from 79.9 to 3,733 pg/mL in 2018 and from 86.3 to 3,332 pg/mL, when retested in 2022 (Mean [SD]: 2019=1,138 [1,272]; 2022=1,009 [1,114], p=0.002). There was a strong correlation between the results obtained at the two time-points for both neopterin and Nfl (Figure 2). The coefficient of variation ranged between 0.4 and 25.7% for neopterin and 5.1–13.3% for Nfl with a mean (SD) for CV of 9 (9.5) and 8 (2.6), respectively and was not impacted by the duration of storage to first test. There was no correlation between duration of storage and the absolute concentration of either biomarker. According to neopterin concentration, three patients had high disease activity, while the remaining seven had moderate disease activity. Most importantly, all results were concordant when retested.

Figure 2:

Stability of neopterin and neurofilament light in CSF samples stored at −80 °C. Comparison of neopterin and neurofilament light concentration in cerebrospinal fluid samples (CSF) retested with a 3-year interval. (A, D) Values were compared using paired t test and p-values are shown. (C, E) Spearman test was used to assess correlation between the results when retested. Each dot represents one sample.

Discussion

In the present study we assessed the effect of long-term storage on the quantification of the CSF biomarkers neopterin and neurofilament light by ELISA. Historical cohorts and stored samples are needed to establish appropriate cut-off values and for the feasibility and reliability of studies focusing on rare diseases, diseases with long period of incubation or with slow progression, as for example, HTLV-1-associated myelopathy (HAM). In this scenario, to understand if and how long-term storage influences analyte stability is key. Here, the two biomarkers assessed, neopterin and Nfl, were stable during storage at −80 °C, when retested 3-years later. Although there was a statistically significant difference between the means, this difference (3 nmol/L for neopterin, 127 pmol/mL for neurofilament light) was not clinically relevant given the absolute differences in the ranges associated with the described disease states. This is supported by the small coefficient of variation observed for most samples. Only two samples (samples 3 and 8) had a coefficient of variation higher than 20% when retested for neopterin. In both cases the quantity of neopterin had increased when retested, pointing against analyte degradation, and being probably related to inter-assay variability. Although sublimation/evaporation cannot be disregarded, all samples were stored in the same condition. It is important to note that the variation has not impacted clinical classification according to disease activity [3]. This reinforces the importance of monitoring differences in reagents batches to identify potential impact of inter-assay variability in results. The inclusion of inter-assay controls is strongly advised as well as inter-laboratory comparison.

Our results add to the literature about the impact of pre-analytical factors on the reliability of biomarkers concentration. Previous studies have assessed the impact of a range of variables on the concentration of different biomarkers, but data on CSF samples is limited. In addition, long-term stability is usually limited to up to 21 days with only one study showing stability of these biomarkers in serum samples stored for up to 20 years [13]. Here we have reassessed CSF samples with a 3-year interval.

One study evaluated the impact of blood collection tube type, and of ethylene diamine tetraacetic acid (EDTA), plasma delayed centrifugation, centrifugation temperature, aliquot volume, delayed storage, and freeze-thawing cycles on the concentration of different biomarkers such as Nfl, amyloid beta (Aβ)42 and 40, Aβ oligomerization-tendency (OAβ), amyloid precursor protein (APP)699-711, glial fibrillary acidic protein (GFAP), total tau (t-tau), and phosphorylated tau 181. This study found that while the collection tube type resulted in different values of all assessed markers, delayed plasma centrifugation and storage affected Aβ and t-tau only. In addition, centrifugation temperature affected t-tau measurement. All the other markers, including Nfl, were resistant to all handling variations tested [11]. Based on these findings the authors proposed a standardized operating procedure for plasma handling, for the quantification of blood-based biomarkers to be used on research and clinical settings [11]. Others have shown that Nfl is also resistant to delayed freezing of serum and plasma (up to 7 days) [14] and repeated freeze-thaw cycles [10, 15], [16], [17]. There is also data showing that Nfl in serum is not affected by collection tube type, centrifugation temperature, delayed storage after centrifugation [10] nor delayed centrifugation [18]. In addition, a recent study confirmed that NfL is stable in serum and plasma samples stored at −80 °C for up to 20 and 16 years, respectively [13], which accords with our findings for CSF. CSF Nfl is also resistant to up to four freeze-thaw cycles and to blood contamination which may accidently happen during sampling [19]. Interestingly, NfL concentration was stable in CSF stored at −20 °C and 37 °C compared to samples stored at −80 °C, for 21 days. In contrast, Neurofilament heavy (Nfh) concentration in CSF had a significant linear decline when stored at the same conditions. A reduction of 18% was observed at day 21 for samples at −20 °C, 26% when kept at 4 °C and 20 °C, and up to 71% reduction on Nfh was detected when samples were stored at 37 °C [19].

Regarding neopterin, data on the impact of pre-analytical variables are extremely limited. One study showed that neopterin in plasma is resistant to up to 10 freeze-thaw cycles and when kept at temperatures varying from −70 °C to 24 °C for up to 20 days [20]. The same study obtained different concentrations of neopterin in plasma, when using kits from different manufactures [20]. Our study has some limitations, including the small sample size and the lack of baseline data on fresh samples. However, our data confirms the stability of neopterin in stored samples and shows, for the first time, the stability of this marker in CSF stored long-term at −80 °C and we conclude that results from retrospective analyses will be reliable.