Elecsys assay

Serum was stored at −80°C until measurements were performed in a single batch for each measure at the end of the study. All biochemical markers were assessed at 0 and 6 months, except for serum 25(OH)D which was assessed every 2 months. Serum 25(OH)D was analysed by an automated chemiluminescent immunoassay (DiaSorin, USA, CV 6.6%). Parathyroid hormone was assessed by electrochemiluminescence immunoassay (Roche e602) and high-sensitive C-reactive protein (hs-CRP) by immunoturbidimetric immunoassay (Roche c701). Lipid profiles were measured using standard methods (Roche c701). Serum IL-6 and TNF-α were measured by a chemiluminescent enzyme immunoassay (Immulite, Siemens, USA). Glycated haemoglobin (HbA_1c) was assessed by a DCCT-aligned ion exchange HPLC method (Bio-Rad Variant II Turbo 2.0 analysers, CV 1.6%). Adiponectin was measured by ELISA (R&D Systems, USA) and total osteocalcin by the automated Elecsys assay (Roche, Australia). Undercarboxylated osteocalcin was measured using the same automated assay described for total osteocalcin. However, measurement was preceded by sample pre-treatment with a 100 mg/mL hydroxyapatite slurry, following the method described by Gundberg [27] (link).

Three separate subgroups were analyzed here from the overall cohort based on amyloid status as determined by 18F‐florbetapir PET (or CSF Aβ42 if PET was not available) and cognition as measured by the CDR Global cognitive scale. The first group (“controls”) comprised individuals with no cognitive impairment (CDR = 0) who were amyloid‐negative (Aβ‐). The second group (“preclinical AD”) comprised individuals who have no cognitive impairment (CDR = 0) but were amyloid‐positive (Aβ+). The final group (“mild AD”) comprised individuals with cognitive impairment (CDR = 0.5 or 1, representing “very mild” or “mild” dementia) who were also amyloid‐positive (Aβ+). A sensitivity analysis was performed with a requirement that mild AD individuals also showed altered levels of CSF P‐tau181 (measured by an Elecsys assay [Roche Diagnostics GmbH, Penzberg, Germany]; P‐tau was defined as positive when CSF P‐tau181> 27 using a previously published cutoff²² in addition to being Aβ‐positive. These groups are relevant for current AD trials which have a heightened focus on early disease stages and acknowledge an increased willingness of regulatory agencies to recognize early staging of AD through the coupling of cognition and biomarkers of Aβ pathology.¹⁶

The tumour biomarkers CA125, CA15‐3, CA19‐9, CEA, CYFRA 21‐1, AFP, alongside with the HF marker (NT‐proBNP) were assessed in venous blood samples. Blood samples were centrifuged for 15 min at 2500 g at 4°C, and afterwards, plasma was collected and stored at −80°C until further analysis. All six tumour biomarkers were measured by the Roche Elecsys^® assay on a cobas e 411 analyzer using standard methods (Roche Diagnostics GmbH, Mannheim, Germany). This platform allows to quantitatively measure human CA125, CA15‐3, CA19‐9, CEA, CYFRA 21‐1, and AFP levels in plasma with high sensitivity.

The Siemens SARS-CoV-2 IgG (SCOVG) assay was performed on an automated ADVIA Centaur XPT System (Siemens Healthineers) according to the manufacturer’s instructions. The immunoassay detects anti-SCOVG antibodies directed against the S1 domain of the viral spike protein (including the immunologically relevant receptor binding domain). The Elecsys assay from Roche detecting high-affinity antibodies (including IgG) directed against the nucleocapsid protein of SARS-CoV-2 was performed according to the manufacturer’s instructions for samples collected at the University Hospital Tübingen. Results are reported in index values for the Roche assay and the SCOVG assay. For the latter, an index value of 1 corresponds to 1 U/ml; 1 U/ml can be converted to 21.80 binding antibody units/ml according to the manufacturer. The final interpretation of positivity is determined by an antibody titer of ≥1.0 U/ml given by the manufacturer. Values of <0.1 were set to 0.1. One hundred was the highest measurable index value with the SCOVG assay. Quality control was performed following the manufacturer’s instructions on each day of testing.