Methods

Overview of assessments and examinations throughout the study

A.   Clinical Evaluation and Cognition

Cognitive testing

The cognitive battery is detailed in the table provided. The paper-and-pencil tests, standard in clinical practice for decades, are currently used in our ongoing BioFINDER-2 study (NCT03174938) and are performed annually. Global cognition is assessed using the Mini-Mental State Examination (MMSE) and, during the screening phase, the telephone version of the Montreal Cognitive Assessment (T-MoCA). Attention and executive function are assessed through the Trail Making Test A and B (TMT), the Symbol Digit Modalities Test (SDMT), and the Digit Symbol Substitution Test (DSST). Memory is evaluated using Logical Memory, the Free and Cued Selective Reminding Test (FCSRT), and the 10-word delayed recall test from ADAS-cog, which includes a recognition component. Verbal ability is gauged through category (animals, fruits, and vegetables) and letter (FAS) fluency tests, along with the 15-item short version of the Boston Naming Test. The FCSRT, Logical Memory, DSST, MMSE, and category fluency are utilized to compile the AD-sensitive Preclinical Alzheimer Cognitive Composite (PACC) score, employed in several clinical trials involving pre-symptomatic (preclinical) AD patients.

Cognitive symptoms

All participants rate their memory and attention/executive function using the Cognitive Function Instrument (CFI). This is also administered to an informant.

Functional ability

Activities of Daily Living are evaluated using the informant-based Functional Activities Questionnaire (FAQ).

Neuropsychiatric symptoms

Neuropsychiatric symptoms and Behavioral and Psychological Symptoms of Dementia (BPSD) are assessed using the Mild Behavioral Impairment Checklist (MBI-C) and the Apathy Evaluation Scale (AES), with AES featuring both self-rated and informant-rated versions. Mood and anxiety are further assessed using the Hospital Anxiety and Depression Scale (HADS).

Quality of Life (QoL)

Overall health status is rated by the participants using the EQ-5D from Euro-QoL.

Sleep

The presence of REM sleep behavior disorder is evaluated using a validated composite question from the Mayo Sleep Questionnaire. Sleep quality is assessed with the Sleep Scale from the Medical Outcome Study (MOS).

Cognitive reserve

Premorbid cognition and cognitive reserve are approximated using the Cognitive Reserve Index questionnaire (CRI-q), focusing on the sub-items “Education” and “Working activity” but excluding “Leisure time”.

B.   Cerebrospinal fluid (CSF) and blood sampling and analyzes

Blood is drawn at the pre-screening visit into an EDTA tube (10 mL). After centrifugation (2000g, +4°C, 10 min), plasma samples are aliquoted into LoBind tubes (Sarstedt) and transported for immediate analysis of plasma p-tau217 and APOE genotype. Lumbar CSF samples are collected at the inclusion visit following a standardized protocol in line with the Alzheimer’s Association Flow Chart for CSF biomarkers. Lumbar punctures are performed between 8-12 am, collecting 25 mL of CSF in LoBind tubes (Eppendorf), which are then stored on ice for 5-20 minutes before centrifugation (2000g, +4°C, 10 min). Subsequently, the CSF is aliquoted in approximately 1 mL portions into LoBind tubes (Sarstedt) and stored at -80°C until batch analyses are conducted. Approximately 0.5 mL of CSF from each individual is analyzed directly at baseline for AD biomarkers (Aβ42/Aβ40 ratio and Aβ42/P-tau ratio) according to the clinical routine at Memory Clinic at Skåne University Hospital. The main plasma collection occurs during the same visit as the lumbar puncture. Blood is drawn into tubes containing either EDTA or Lithium heparin as an anticoagulant. Following centrifugation, plasma samples are aliquoted in approximately 1 mL portions into LoBind tubes (Sarstedt) and stored at -80°C pending biochemical analyses.

C.  Amyloid PET

Amyloid PET is performed in the study using PET/CT cameras (GE Discovery MI) with the ligand 18F-flutemetamol to detect and quantify amyloid deposits in the brain. GE Healthcare provides the precursor for 18F-flutemetamol. A 20-minute scan is conducted 90-110 minutes after the injection of 18F-flutemetamol, allowing for detailed imaging of amyloid accumulation. This setup ensures that the process aligns with established protocols for amyloid PET imaging, facilitating reliable and consistent data collection across study participants.

D.  Tau PET

Tau PET is conducted using PET/CT cameras (GE Discovery MI) with the ligand 18F-RO948, specifically designed to detect and quantify tau aggregates in the brain. A 20-minute scan is performed approximately 70 minutes after the injection of the tracer, ensuring precise timing for optimal imaging results. This method provides critical insights into the distribution and density of tau proteins.

E.    Magnetic Resonance Imaging (MRI)

Magnetic Resonance Imaging (MRI) in the study is conducted using a 3 Tesla MRI system (Siemens Prisma), utilized consistently across all participants and visits. This MRI suite employs a variety of techniques to thoroughly investigate different aspects of brain structure and function. These include three-dimensional magnetization-prepared rapid acquisition with gradient echo (3D MPRAGE) for studying regional brain volume, MR spectroscopy (MRS) for metabolism, diffusion tensor imaging (DTI) and functional MRI (fMRI) for structural and functional connectivity, arterial spin labeling (ASL) for regional blood flow, and susceptibility-weighted imaging (SWI) as well as fluid-attenuated inversion recovery (FLAIR) for detecting iron deposition and small vessel disease, respectively. The complete protocol requires approximately 60 minutes and is performed without the use of any contrast-enhancing agents.