Abstract INTRODUCTION Blood‐based biomarkers (BBMs) have revolutionized Alzheimer's disease diagnosis and monitoring. Their pre‐analytical stability requires scrutiny. This study assessed pre‐analytical effects to inform a standardized sample handling protocol. METHODS Assessed pre‐analytical variations included collection tube type, hemolysis, centrifugation settings, centrifugation/storage delays, tube transfers, and freeze‐thawing ( n = 15/experiment). Phosphorylated tau (pTau) isoforms were measured with Simoa, Lumipulse, MesoScale Discovery, and immunoprecipitation‐mass spectrometry. Amyloid‐beta (Aβ42, Aβ40), glial fibrillary acidic protein (GFAP), and neurofilament light (NfL) protein were measured with Simoa. RESULTS All assessed BBM levels varied by over 10% by collection tube type. Aβ peptides were the most sensitive, and their levels declined >by more than 10% under storage and centrifugation delays, more steeply at room temperature (RT) compared with 2°C to 8°C. NfL and GFAP levels increased by more than 10% upon RT/−20°C storage. pTau isoforms demonstrated stability across most pre‐analytical variations. DISCUSSION We established an evidence‐based handling protocol to ensure reliable sample handling for neurological BBMs upon adoption in clinics, trials, and research. Highlights Sample handling protocols can mitigate pre‐analytical effects on BBM results. We developed an evidence‐based, expert‐consensus plasma sample handling protocol. Primary collection tube and delays to centrifuging or freezing impact AD BBMs. Plasma pTau217 is highly resistant to pre‐analytical sample handling variations. Plasma Aβ42 and Aβ40 were most sensitive to pre‐analytical variations.
Verberk et al. (2025) studied this question.