Preanalytical approaches to improve recovery of amyloid-β peptides from CSF as measured by immunological or mass spectrometry-based assays.

BACKGROUND

Amyloid-β 1-42 (Aβ) peptide is a well-established cerebrospinal fluid (CSF) biomarker for Alzheimer's disease (AD). Reduced levels of Aβ are indicative of AD, but significant variation in the absolute concentrations of this analyte has been described for both healthy and diseased populations. Preanalytical factors such as storage tube type are reported to impact Aβ recovery and quantification accuracy. Using complementary immunological and mass spectrometry-based approaches, we identified and characterized preanalytical factors that influence measured concentrations of CSF Aβ peptides in stored samples.

METHODS

CSF from healthy control subjects and patients with AD was aliquoted into polypropylene tubes at volumes of 0.1 ml and 0.5 ml. CSF Aβ concentrations were initially measured by immunoassay; subsequent determinations of CSF Aβ, Aβ, Aβ, Aβ, and Aβ concentrations were made with an absolute quantitative mass spectrometry assay. In a second study, CSF from healthy control subjects and patients with dementia was denatured with guanidine hydrochloride (GuHCl) at different stages of the CSF collection and aliquoting process and then measured with the mass spectrometry assay.

RESULTS

Two distinct immunoassays demonstrated that CSF Aβ concentrations measured from 0.5-ml aliquots were higher than those from 0.1-ml aliquots. Tween-20 surfactant supplementation increased Aβ recovery but did not effectively resolve measured concentration differences associated with aliquot size. A CSF Aβ peptide mass spectrometry assay confirmed that Aβ peptide recovery was linked to sample volume. Unlike the immunoassay experiments, measured differences were consistently eliminated when aliquots were denatured in the original sample tube. Recovery from a panel of low-retention polypropylene tubes was assessed, and 1.5-ml Eppendorf LoBind® tubes were determined to be the least absorptive for Aβ. A comparison of CSF collection and processing methods suggested that Aβ peptide recovery was improved by denaturing CSF earlier in the collection/aliquoting process and that the Aβ/Aβ ratio was a useful method to reduce variability.

CONCLUSIONS

Analyte loss due to nonspecific sample tube adsorption is a significant preanalytical factor that can compromise the accuracy of CSF Aβ measurements. Sample denaturation during aliquoting increases recovery of Aβ peptides and improves measurement accuracy. The Aβ/Aβ ratio can overcome some of the quantitative variability precipitated by preanalytical factors affecting recovery.