The effect of preanalytical factors on cerebrospinal fluid and plasma proteomics: a systematic experimental study

Preanalytical variations (PAVs) are a major source of irreproducibility in biomarker studies. While their effects on blood have been widely examined, their impact on cerebrospinal fluid (CSF) proteomics remains less well characterized. We systematically evaluated common PAVs to inform biobanking and biomarker research.

CSF and plasma samples were collected at an ISO 20387-accredited biobank. For each condition, three or four independent paired samples were analyzed. PAVs included storage temperature, processing delay, centrifugation speed, storage tube type, freeze-thaw cycles, and blood contamination. Protein abundance was quantified using SomaScan. Selected analytes were further evaluated using orthogonal immunoassays. Fold changes were calculated as linear RFU ratios relative to baseline. Paired t-tests with false discovery rate (FDR) correction were performed. Analytes showing detectable or larger changes were defined based on predefined fold-change thresholds with nominal p < 0.05.

Plasma proteomes were highly sensitive to delays and temperature, with 969 analytes showing large changes after 24 h at 25 °C (counts refer to large changes), compared to 26 analytes in CSF. At 4 °C, CSF remained stable for 24 h (4 changes), whereas plasma showed extensive changes after 24 h (465) and 72 h (813). Storage at - 20 °C caused substantial shifts in both matrices (CSF: 144; plasma: 1,164), whereas no analytes differed in plasma stored at - 50 °C relative to - 80 °C. Freeze-thaw cycles up to three resulted in few altered analytes, whereas 11 cycles led to pronounced changes (CSF: 198; plasma: 21). Hemolysis in plasma induced 28 changes, whereas CSF blood contamination ≤ 500 cells/mm³ was associated with few changes. After FDR correction, no analytes remained significant in CSF, whereas multiple analytes remained significant in plasma under selected conditions.

Storage temperature and processing delay were the most influential PAVs, with plasma more susceptible than CSF. For optimal quality, CSF should be processed within 4 h or kept at 4 °C, plasma within 2 h, and both stored at ≤ - 80 °C. Freeze-thaw cycles should be limited to three.