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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.