Effect of storage conditions on NSE, S100, and B2M stability in cerebrospinal fluid and serum

The reliability of cerebrospinal fluid (CSF) and serum biomarkers depends on sample integrity, which is strongly influenced by storage conditions. While -80 °C is considered the gold standard for long-term preservation, it requires specialized infrastructure, whereas -20 °C is more accessible but may compromise protein stability. This study aimed to evaluate the stability of three clinically relevant biomarkers - neuron-specific enolase (NSE), S100, and β-2 microglobulin (B2M), associated with neuronal injury and neuroinflammation - in CSF and serum samples stored at -20 °C and -80 °C over different time periods.

A total of 63 CSF and 56 serum samples from pediatric patients were analyzed. Samples were stored at -20 °C (24 h and 6 months) and -80 °C (6 and 12 months). NSE and S100 were measured by electrochemiluminescence immunoassays and B2M by turbidimetric immunoassay. Biomarker variability was expressed as relative percentage change from baseline. A maximum permissible error of ±15 % for CSF and ±10 % for serum was selected.

NSE and S100 were highly unstable at -20 °C. In CSF, after 6 months, NSE decreased by ∼74 % and S100 by ∼71 %; in serum, NSE declined by ∼54 % and S100 by ∼13 %. Remarkably, after only 24 h at -20 °C, CSF NSE dropped by 61 %. Storage at -80 °C largely preserved these two biomarkers, with declines below ∼15 % over 12 months. B2M levels remained stable under all conditions. The reduction in CSF NSE at -20 °C was strongly correlated with baseline concentrations.

CSF biomarkers NSE and S100 are highly susceptible to degradation at -20 °C, whereas B2M remains relatively stable. Strict adherence to -80 °C storage protocols is essential to ensure the reliability of biomarker-based diagnostics and research.