Population-based blood screening for preclinical Alzheimer’s disease in a British birth cohort at age 70

Abstract Alzheimer’s disease has a preclinical stage when cerebral amyloid-β deposition occurs before symptoms emerge, and when amyloid-β-targeted therapies may have maximum benefits. Existing amyloid-β status measurement techniques, including amyloid PET and CSF testing, are difficult to deploy at scale, so blood biomarkers are increasingly considered for screening. We compared three different blood-based techniques—liquid chromatography-mass spectrometry measures of plasma amyloid-β, and single molecule array (Simoa) measures of plasma amyloid-β and phospho-tau181—to detect cortical 18F-florbetapir amyloid PET positivity (defined as a standardized uptake value ratio of >0.61 between a predefined cortical region of interest and eroded subcortical white matter) in dementia-free members of Insight 46, a substudy of the population-based British 1946 birth cohort. We used logistic regression models with blood biomarkers as predictors of amyloid PET status, with or without age, sex and APOE ε4 carrier status as covariates. We generated receiver operating characteristics curves and quantified areas under the curves to compare the concordance of the different blood tests with amyloid PET. We determined blood test cut-off points using Youden’s index, then estimated numbers needed to screen to obtain 100 amyloid PET-positive individuals. Of the 502 individuals assessed, 441 dementia-free individuals with complete data were included; 82 (18.6%) were amyloid PET-positive. The area under the curve for amyloid PET status using a base model comprising age, sex and APOE ε4 carrier status was 0.695 (95% confidence interval: 0.628–0.762). The two best-performing Simoa plasma biomarkers were amyloid-β42/40 (0.620; 0.548–0.691) and phospho-tau181 (0.707; 0.646–0.768), but neither outperformed the base model. Mass spectrometry plasma measures performed significantly better than any other measure (amyloid-β1-42/1-40: 0.817; 0.770–0.864 and amyloid-β composite: 0.820; 0.775–0.866). At a cut-off point of 0.095, mass spectrometry measures of amyloid-β1-42/1-40 detected amyloid PET positivity with 86.6% sensitivity and 71.9% specificity. Without screening, to obtain 100 PET-positive individuals from a population with similar amyloid PET positivity prevalence to Insight 46, 543 PET scans would need to be performed. Screening using age, sex and APOE ε4 status would require 940 individuals, of whom 266 would proceed to scan. Using mass spectrometry amyloid-β1-42/1-40 alone would reduce these numbers to 623 individuals and 243 individuals, respectively. Across a theoretical range of amyloid PET positivity prevalence of 10–50%, mass spectrometry measures of amyloid-β1-42/1-40 would consistently reduce the numbers proceeding to scans, with greater cost savings demonstrated at lower prevalence.

Purified anti-β-Amyloid 17-24 (4G8) and anti-β-Amyloid 1-16 antibodies (6E10, both Biolegend, San Diego, CA) were added to the magnetic beads at a final concentration of 137 µg/mL, each in a separate tube and mixed for one hour on a roller at +20 °C. The beads were washed three times in twice the original volume and then suspended in the original volume with PBS prior to combining the content of the two tubes.
Plasma samples were centrifuged at 2500 RCF for 10 minutes at +4 °C directly after thawing at room temperature. 250 µL of each sample and calibrator was transferred to a KingFisher deep-well 96 plate (Thermo Scientific,#95040450). 20 µL of the 2.7 ng/mL IS solution was added to all samples (including calibrators) giving a final concentration of 200 pg/mL in samples. The samples were diluted with 660 µL PBS and the sample plate was placed in a KingFisher™ Flex Purification System (Thermo Fisher Scientific, #5400630), where the samples were mixed for 20 minutes. After removing the plate, 20 µL of 10 % Triton X-100 and 50 µL of magnetic beads with bound antibodies were added to each well and the plate was placed in the KingFisher™ Flex. After the samples and beads were mixed for 1.5 hours, the beads from each well were washed in 1 mL 0.2 % Triton x100 in PBS followed by 1 mL PBS and finally 1 mL 50 mM ammonium bicarbonate for 10 seconds in each solution.
Finally. the Aβ peptides were eluted from the antibodies by mixing the beads from each sample in 0.1 mL 0.5 % formic acid in water (in a KingFisher 96 KF microplate 200μL, Thermo Fisher Scientific, #97002540) for four minutes. The eluates were dried using vacuum centrifugation using a Savant SC210A SpeedVac Concentrator (Thermo Fisher Scientific, #SC210A-230) without applying heat.

LC-MS/MS
Mobile phases for the liquid chromatography (LC) were prepared using ultrapure (type 1) water from a Merck Synergy UV water purification system (Merck, #SYNSVHFWW) set to a water resistivity of 18.2 MΩ.cm at 25 °C, Acetonitrile (ACN), Far UV HPLC Gradient grade (Fisher Scientific, #A/0627/17X) and Ammonium hydroxide solution, puriss. p.a., reag. ISO, reag. Ph. Eur., ~25% NH3 basis (Sigma-Aldrich, #30501-1L-D). Mobile phase A consisted of 5 % ACN and 0.3 % concentrated ammonia solution (v/v) in water and mobile phase B consisted of 4 % water and 0.1 % (v/v) concentrated ammonia solution in ACN. Wash solution consisted of 50 % ACN and 4 % concentrated ammonia solution (volume/volume) in water. LC was performed using an UltiMate™ 3000 system (Thermo Fisher Scientific). The analytical columns were a Proswift RP-4H 1×250 mm monolithic column (Thermo Fisher Scientific, #066640) maintained at 50 °C. A dual pump and column approach was used to increase throughput of the method, where one pump eluted the analyte from one column as the other pump equilibrated the second column before switching columns using a 10-port valve.
The dried samples were dissolved in 50 µL 20 % ACN and 4 % concentrated ammonia solution (volume/volume) in water and placed on shaker for 15 minutes at 600 rpm. The samples were then placed in an autosampler. After injection of 40 µL of the dissolved sample, gradient elution was performed with one pump at a flow rate of 0.3 mL/min with the following gradient steps: 0 min, 5 % B; 3 min, 20 % B; 3.5 min, 95 % B; 3.9 min, 95 % B; 4 min, 5 % B. The second pump simultaneously equilibrated the second column at a flow rate of 0.3 mL/min with the following gradient steps: 0 min, 95 % B; 2 min, 95 % B; 3 min, 5 % B; 5 min, 5 % B. The auto sampler injector needle and tubing were washed with wash solution after each sample injection.
Mass spectrometric analysis was performed using a quadrupole-Orbitrap hybrid mass spectrometer (Q-Exactive) equipped with a heated electrospray ionization source (HESI-II) (Thermo Fisher Scientific, Bremen, Germany) using the following ion source parameters: probe position D, sheath gas 35, auxiliary gas 15, spray voltage 4.40 kV, S-lens RF 55, heater +350 °C, and capillary temperature +320 °C. The precursors were isolated with isolation windows of 2.5 m/z followed by fragmentation using a normalized collision energy of 19.0. Parallel reaction monitoring (PRM) was performed by recording fragment spectra at a resolution of 17.500 using an automatic gain control (AGC) target of 5×10 5 charges and a maximum injection time of 250 ms. Ion chromatograms were constructed by summing the peak areas of selected ions fragment of the 4+ charge states for the respective Aβ peptides and their corresponding internal standards (Supplementary table 1). The PRM method was scheduled to scan for one Aβ peptide and its internal standard at a time to maximize sensitivity. Data processing and quantification was performed using Thermo Xcalibur 4.1.

List of supplementary tables
Supplementary  The ion types and charge states are identical for each native peptide and its corresponding internal standard ( 15 N-labelled).
Abbreviations: Aβ, amyloid-β; m/z, mass to charge ratio.  The Pearson correlation coefficient, r, and the Bonferroni-corrected P value for each correlation are shown, with P < 0.05 in bold.

Precursor ion Product ions m/z (ion type and charge state)
Abbreviations: Aβ, amyloid-β; LC-MS, liquid chromatography-mass spectrometry; p-tau181, phospho-tau181.     Log-fold change is shown for biomarkers that were log-transformed prior to linear regression (all except the LC-MS plasma composite, for which a linear regression coefficient is shown), with their p values and the overall R2 of each multivariate model.