Biomarker validation: Methods and matrix matter

We read with interest the research article from Casanova et al. for an upcoming edition of Alzheimer’s & Dementia (2016, article in press). We appreciate the efforts that these investigators have expended in attempting to replicate our plasma lipidomic findings in preclinical Alzheimer’s disease [1]. We enthusiastically endorse independent validation of new findings in science but assert that valid replication requires identical methods including those related to clinical characterization and biospecimen collection, processing, and analysis. The methodology used by Casanova et al., in particular, metabolomic analysis of serum rather than plasma makes their study distinct but renders direct comparison with our study invalid. A brief delineation of these critical differences is important for the reader as they effectively preclude comparison of results. Casanova et al. attempted to replicate our findings using samples of convenience from two well-established longitudinal studies, the Baltimore Longitudinal Study on Aging (BLSA) and the Age, Gene/Environment Susceptibility— Reykjavik Study (AGES-RS). These studies do not use many of the methods we used in our study that was specifically designed to discover blood-based biomarkers for preclinical Alzheimer’s disease. As a result, Casanova et al. were restricted by the methodologies used by these extant studies rather than replicating the methodology used in our study. In these terms, their study cannot be considered a replication of ours. Several of the methodological differences are significant. First and foremost, we used plasma. Casanova et al. used serum. Whether this was a conscious decision or a matter of sample availability is not clear, but it is clear that partitioning of metabolites is nonuniform and the matrix can have a profound effect on the findings. Recent studies have shown that levels of certain metabolites can differ greatly between serum and plasma in the same individuals [2], and these differences may be even more significant when measuring glycerophospholipids [3]. Of note, two metabolites in our panel shown in Table 4 of Casanova et al. were excluded from the original study by Yu et al. [4] because of low-measurement stability.