Effects of Certain Pre-analytical Factors on the Performance of Plasma Phospho-tau217

Introduction: Pre-analytical factors can cause substantial variability in the measurements of cerebrospinal fluid (CSF) and plasma biomarkers of Alzheimer's disease (AD). However, their effects on the performance of one of the most promising plasma AD biomarkers, phosphorylated tau (p-tau)217, are not known.

Methods: We included 50 amyloid-β positive (Aβ) and 50 Aβ participants from the Swedish BioFINDER-1 study. Plasma and CSF p-tau217 were measured using an immunoassay developed by Lilly Research Laboratories. We examined the effect of four plasma handling conditions, i.e., (1) thawing at room temperature (RT) with no centrifugation, (2) thawing at RT followed by centrifugation, (3) thawing on ice with no centrifugation, and (4) thawing on ice followed by centrifugation. In addition, we also tested the effects of up to 3 freeze-thaw cycles on the associations of plasma p-tau217 with AD-related pathologies measured with CSF p-tau217 and CSF Aβ42/Aβ40.

Results: In the whole cohort (combining Aβ and Aβ participants), we found significant correlations between plasma p-tau217 and both CSF p-tau217 (R 0.614-0.717, p < 0.001) and CSF Aβ42/Aβ40 (Spearman R, - 0.515 to - 0.652, p < 0.001) for each of the four tested conditions. Correlations between plasma and CSF p-tau217 were also significant for all conditions in the Aβ group (R, 0.506-0.579, p < 0.001). However, in this Aβ subgroup, correlations with CSF Aβ42/Aβ40 were only significant for centrifuged samples (thawed at RT, R =  - 0.394, p = 0.010; thawed on ice, R =  - 0.406; p = 0.007). In Aβ participants, correlations between plasma and CSF p-tau217 were again significant only for centrifuged samples (thawed at RT, R = 0.394, p = 0.007; thawed on ice, R = 0.334; p = 0.022), with no correlations seen between plasma p-tau217 and CSF Aβ42/Aβ40 for any of the conditions. While the accuracy of plasma p-tau217 to identify individuals with abnormal CSF Aβ42/Aβ40 or CSF p-tau217 status was high, the AUCs for samples thawed at RT and analyzed without centrifugation were numerically lower than the AUCs of other conditions (CSF Aβ42/Aβ40 = 0.845 vs 0.872-0.884; CSF p-tau217 = 0.866 vs 0.908-0.924, p > 0.11). P-tau217 concentration was consistently higher in non-centrifuged samples than in centrifuged samples (p ≤ 0.021). There were no differences between samples freeze-thawed once, twice, or three times.

Conclusion: Centrifugation improved the performance of plasma p-tau217, but thawing temperatures and up to three freeze-thaw cycles did not have a significant impact. These results may inform the future development of standardized sample-handling protocols for AD biomarkers.