BACKGROUND:
Clinical translation of blood-based biomarkers for dementia diagnosis requires a robust understanding of their stability under real-world pre-analytical handling conditions. We investigated the effects of varied pre-analytical handling conditions on quantification of 124 brain-derived proteins relevant to dementia research in human plasma.
METHODS:
Plasma samples from symptomatic individuals and asymptomatic relatives attending two specialist cognitive clinics were subjected to a range of pre-analytical conditions (n=10 each), including: pre-centrifugation delay at room temperature (RT) for 30 minutes, 1 or 3 hours, or at 2-8°C for 3 or 24 hours; post-centrifugation delay prior to -80°C storage for 0, 4 or 24 hours at RT; 4, 24 hours or 2 weeks at 2-8°C; or 2 weeks at -20°C; 1-4 freeze/thaw cycles; and post-centrifugation storage for up to 2 weeks at -20°C and transport in a Bio-Freeze device or storage at -80°C and transport on dry ice. Protein abundance was quantified using the NULISAseq CNS Disease 120 Panel (ARGO immunoassay platform; Alamar Biosciences). Non-parametric Friedman or Wilcoxon tests (p<0.05) assessed statistical significance, and a ±10% median relative change threshold defined likely clinical significance.
RESULTS:
ANXA5 values reduced by a median of 10% with 3-hour pre-centrifugation delay at RT. Across post-centrifugation delays, concentrations of six proteins (ANXA5, ARSA, NRGN, oligo-SNCA, RUVBL2, YWHAZ) showed increases of>10% with intermediate storage at -20°C for 2 weeks, five (ANXA5, APOE4, ARSA, ENO2, HBA1) increased and one (Aβ42) reduced with storage at 2-8°C for 2 weeks and 2 (ENO2, HBA1) increased with storage at RT for 24 hours. No analyte showed clinically significant differences for either pre-centrifugation delays at 2-8°C, or for up to four freeze-thaw cycles, or between the two storage and transport conditions.
CONCLUSION:
Plasma measurements on the NULISAseq CNS panel are mostly robust to pre-centrifugation delays of up to 3 hours at RT and 24 hours at 2-8°C, but some proteins quantifications are significantly altered with post-centrifugation intermediate storage at either 2-8 °C or -20°C for 2 weeks. Recently developed standardised sample handling protocols may need to be updated to ensure the reliability and reproducibility of blood-based biomarker measurements on this multiplexed panel.