BACKGROUND: The handling of cerebrospinal fluid (CSF) affects the biomarker quantification used to diagnose Alzheimer's disease (AD). Only specialized centers can test for AD markers. The precise timing and freezing is required to correctly measure these biomarkers. Therefore, the effects of CSF storage temperature and repeated freeze/thaw cycles on CSF stability were investigated. METHODS: Drop coating deposition Raman spectroscopy in combination with principal component analysis was used to analyze CSF and its dialyzed form (ELISA confirmed the removal of up to 80% of the AD markers). The advantage of this approach is that no prior knowledge of the biomarkers is necessary and that both the concentration and the protein structure of intact CSF are analyzed. RESULTS: Dialyzed CSF was stable for up to 5 h after its collection, while native CSF started to denature nearly immediately. Most of the unstable proteins were denatured within 24 h. The dialyzed CSF was not affected by freeze/thaw cycles, but the native CSF exhibited significant progressive changes, even after the first freezing. The mechanism as well as the resulting structures of the freeze-denatured proteins differed from those of the temporally denatured proteins, although both protein sets began with the same initial proteins. CONCLUSIONS: CSF must be processed immediately, within 5 h of collection. Flash cooling is recommended for freezing CSF, but any freeze/thaw cycle will affect the protein component of CSF.

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