Reference proteins to improve Core 1 and Core 2 Alzheimer's disease CSF and plasma biomarkers.

Concentration-based fluid biomarkers represent an informative and cost-effective way to detect and monitor Alzheimer's disease (AD) pathology. However, non-AD-related inter-individual variation in biofluids can also affect biomarker concentrations. Here, we investigated whether normalization of cerebrospinal fluid (CSF) and plasma biomarkers to reference proteins, such as amyloid-β40 (Aβ40) and non-phosphorylated mid-region tau (np-tau), improves their robustness and reliability of representing AD pathology load. Using the Swedish BioFINDER-2 cohort (n=1702, 50.7% male, mean [SD] age 68.4 [12.2] years), we compared the associations between tau/Aβ-PET load and fluid biomarkers alone versus in a ratio with a reference protein (Aβ40 or np-tau) in univariate linear regression models. Fluid biomarkers included CSF and plasma measures of p-tau217, p-tau181, p-tau205, np-tau181-190, np-tau195-210, np-tau212-221, Aβ42, Aβ40, and CSF MTBR-tau243, SNAP-25, neurogranin, YKL-40, sTREM2, and plasma eMTBR-tau243. Biomarkers were measured with mass spectrometry assays and/or immunoassays. In addition, we performed validation and extended analyses, comparing for example group-level diagnostic differences and longitudinal biomarker trajectories, in three independent prospective cohorts: BioFINDER-1, Knight Alzheimer Disease Research Center (ADRC), and Translational Biomarkers in Aging and Dementia (TRIAD), as well as in an Italian multiple sclerosis cohort. CSF Aβ40 normalization significantly strengthened the associations of several core CSF AD biomarkers, including CSF MTBR-tau243, p-tau isoforms and synaptic biomarkers, with tau-PET (ΔR²=0.064-0.24) and Aβ-PET (ΔR² = 0.016-0.28). Normalization to CSF np-tau mainly improved concordance with Aβ-PET (ΔR² = -0.0059-0.19). The strongest association with tau-PET was observed for MTBR-tau243/Aβ40 (R² = 0.78, compared to 0.65 for non-normalized MTBR-tau243), and with Aβ-PET for p-tau217/np-tau (R² = 0.65, compared to 0.46 for non-normalized p-tau217). Plasma biomarker associations with tau-PET improved when using normalization to plasma Aβ40 or np-tau (ΔR² = 0.004-0.14), with the strongest effect for eMTBR-tau243/np-tau (R² = 0.72 versus 0.60). Associations with Aβ-PET were enhanced with np-tau normalization (ΔR² = 0.018-0.16, strongest for p-tau217/np-tau: R² = 0.62 versus 0.53). The results were replicated in Knight ADRC and TRIAD. Furthermore, longitudinal analyses showed that Aβ40 normalization typically reduced inter-individual rather than intra-individual variability over time. Normalization did not enhance group-level differences in inflammatory CSF biomarkers in AD, nor did it improve biomarker associations in the multiple sclerosis cohort. In conclusion, normalization of CSF and plasma biomarkers to reference proteins, such as Aβ40 or np-tau, enhances their association with brain tau and Aβ pathology, making already high-performing AD fluid biomarkers even more accurate.