Objectives: Amyloid-β (Aβ) peptides in cerebrospinal fluid (CSF), including Aβ42 (residues 1-42) and Aβ40 (residues 1-40), are utilized as biomarkers in the diagnostic workup of Alzheimer's disease. Careful consideration has been given to the pre-analytical and analytical factors associated with measurement of these peptides via immunoassays; however, far less information is available for mass spectrometric methods. As such, we performed a comprehensive evaluation of pre-analytical and analytical factors specific to Aβ quantification using mass spectrometry.
Methods: Using our quantitative mass spectrometry assay for Aβ42 and Aβ40 in CSF, we investigated the potential for interference from hemolysate, bilirubin, lipids, and anti-Aβ-antibodies. We also optimized the composition of the calibrator surrogate matrix and Aβ recovery during and after solid phase extraction (SPE).
Results: There was no interreference observed with total protein up to 12 g/L, hemolysate up to 10% (v/v), bilirubin up to 0.5% (v/v), intralipid up to 1% (v/v), or anti-Aβ-antibodies at expected therapeutic concentrations. For hemolysate, bilirubin and lipids, visual CSF contamination thresholds were established. In the analytical phase, Aβ recovery was increased by ∼50% via SPE solvent modifications and by over 150% via modification of the SPE collection plate, which also extended analyte stability in the autosampler.
Conclusions: Attention to mass spectrometric-specific pre-analytical and analytical considerations improved analytical sensitivity and reproducibility, as well as, established CSF specimen acceptance and rejection criteria for use by the clinical laboratory.
Keywords: Alzheimer’s disease; amyloid-β peptides; biomarkers; mass spectrometry; pre-analytical phase; therapeutics.
© 2021 Lauren M. Forgrave et al., published by De Gruyter, Berlin/Boston.