[Establishment of reference intervals for plasma p-tau181, Aβ1-42, Aβ1-40, and Aβ1-42/40 ratio and a preliminary...

Objective: To establish reference intervals for Alzheimer's disease (AD)-related biomarkers in plasma and cerebrospinal fluid (CSF) of adults in Shanghai using single molecule immune detection. Methods: In this cross-sectional study, which included residual plasma samples from 454 healthy individuals and CSF samples from 106 non-AD patients were collected from the Department of Laboratory Medicine, Huashan Hospital, Fudan University, between March and July 2024. The concentrations of phosphorylated tau protein-181 (p-tau181), amyloid-beta peptide 1-42 (Aβ1-42), and amyloid-beta peptide 1-40 (Aβ1-40) were detected using the AST-Dx90 fully automated fluorescence immunoassay analyzer. After normality testing and outlier removal, reference intervals were determined by the non-parametric method and validated using plasma samples from 20 healthy individuals. Results: Spearman correlation analysis revealed no significant correlations with age for plasma Aβ1-42 (R=0.047, P=0.344), the Aβ1-42/40 ratio (R=-0.050, P=0.326), or for CSF p-tau181 (R=-0.078, P=0.438), Aβ1-42 (R=0.002, P=0.980), Aβ1-40 (R=-0.084, P=0.400), and the Aβ1-42/40 ratio (R=0.173, P=0.083) (|R|<0.2, P>0.05). Although plasma p-tau181 (R=-0.102, P=0.032) and Aβ1-40 (R=0.150, P=0.002) showed statistically significant associations with age (P<0.05), the strength of these correlations was very weak (|R|<0.2).Z-tests indicated that there were no statistically significant differences (Z<Z*) in plasma p-tau181 (Z=0.81, Z*=4.08), Aβ1-42 (Z=1.90, Z*=3.94), Aβ1-40 (Z=1.03, Z*=3.96), or the Aβ1-42/40 ratio (Z=0.22, Z*=3.84) between sex subgroups. Similarly, no significant differences were observed for plasma p-tau181 (Z=1.21, Z*=4.08) and Aβ1-40 (Z=3.00, Z*=3.96) between subgroups stratified by the median age (58 years). Therefore, there is no need to establish age-and sex-stratified reference intervals. Consequently, the reference intervals established in this study were as follows: plasma p-tau181≤5.90 pg/ml, Aβ1-42: 26.79-160.30 pg/ml, Aβ1-40: 147.77-501.10 pg/ml, Aβ1-42/40≥0.08; CSF p-tau181≤27.95 pg/ml, Aβ1-42≥179.07 pg/ml, Aβ1-40≤14 466.00 pg/ml, Aβ1-42/40≥0.05. The validation compliance rate for the plasma reference intervals was≥90%. Conclusion: This study successfully established reference intervals for p-tau181, Aβ1-42, Aβ1-40, and Aβ1-42/40 ratio in both plasma and CSF of adults in Shanghai based on single molecule immune detection.