Phosphorylated tau (p-tau) 217 is a promising blood biomarker for Alzheimer's disease (AD). However, most p-tau217 assays have been validated solely in ethylenediaminetetraacetic acid (EDTA) plasma, leaving the clinical applicability of serum p-tau217 largely unexplored despite serum being a preferred matrix in many clinical laboratories. To address this gap, we compared p-tau217 concentrations and diagnostic performances in matched plasma and serum samples using four research-use-only assays, including three from commercial sources i.e., Lumipulse, ALZpath, NULISA, and one from University of Pittsburgh. Paired plasma and serum samples were processed from the same venipuncture collection and assessed with the four p-tau217 assays following manufacturer-recommended procedures in two research cohorts (N=84). Plasma and serum p-tau217 levels varied across assays; the ALZpath, Pittsburgh, and NULISA methods showed significantly lower p-tau217 levels in serum compared with plasma (p<0.0001), while Lumipulse showed higher or non-significant differences in serum. Yet, strong correlations (rho >0.8) were observed between plasma and serum p-tau217 pairs. Both plasma and serum p-tau217 demonstrated strong classification accuracies to differentiate clinical AD from normal controls, with high AUC (up to 0.963) for all methods. The exception was the Pittsburgh assay, where plasma p-tau217 had superior AUC than serum p-tau217 (plasma: 0.912, serum: 0.844). The rest of the assays had equivalent accuracies in both matrices. Serum p-tau217 performs equivalently as plasma p-tau217 for most assessed assays. Serum can therefore be used in place of plasma for p-tau217 assessment for research and clinical purposes.
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