Plasma Phosphorylated Tau 217 Levels Are Highly Stable under Common Pre‐analytical Sample Handling Procedures

AbstractBackgroundThe Standardization of Alzheimer’s Blood Biomarkers working group issued a standardized operating procedure (SOP) to mitigate effects of common pre‐analytical sample handling procedures on the Alzheimer’s blood‐based biomarkers. This SOP was based mainly on results obtained with blood‐based amyloid‐beta40 and amyloid‐beta42 assays. Now that various blood‐based phosphorylated (p)Tau assays enter the fore, we need to re‐assess the validity of the proposed blood handling SOP. We aimed to understand the effect of common pre‐analytical sample handling procedures on blood pTau217 levels.MethodBlood was obtained from 62 volunteers at the polyclinic phlebotomy department (n = 31) and the Alzheimer Center Amsterdam (n = 31) of Amsterdam University Medical Centers. Blood samples from volunteers were handled according to one of the following experiments: variation in primary collection tube (K2EDTA versus serum, sodium‐citrate, lithium‐heparin), delayed centrifugation of K2EDTA tubes while kept at room temperature (RT; 20‐25°C) or in the fridge (2‐8°C; 30min versus 4h, 7h, 24h, 72h), hemolysis of K2EDTA plasma (no hemolysis versus low (100mg/dL hemoglobin) and high (500 mg/dL hemoglobin)) and repeated freeze‐thawing (FT) of K2EDTA plasma (no additional FT versus +1, +2, +4FT). Sample sets were measured with the pTau217 AlzPath prototype assay on the Simoa HD‐X. Recovery% was calculated as the concentration measured in the experimental condition divided over the concentration measured in the relevant reference condition. Median recovery change >10% was considered relevant.ResultEffects of the pre‐analytical sample handling variations on plasma pTau217 levels are presented in figure 1. Blood‐based pTau217 levels were highest in K2EDTA plasma (median (interquartile range (IQR)) %Recovery compared to K2EDTA, in serum: 34% (29‐42%), in sodium‐citrate: 21% (15‐27%), in lithium‐heparin: 7% (6‐8%)). Delayed centrifugation up to 72h either when tubes were kept at RT or when kept cold did not affect pTau217 levels, neither did hemolysis and repeated freeze‐thawing.ConclusionUsing a primary collection tube other than K2EDTA results in substantially lower pTau217 levels. Otherwise, pTau217 level seems highly stable in plasma under diverse pre‐analytical sample handling conditions. According to the results, the consensus pre‐analytical blood handling SOP as published by the Standardization of Alzheimer’s Blood Biomarkers working group is still valid.