This was an exploratory sub-study of a previously performed study registered in the international trial register with ID number: ISRCTN79036545. All study participants provided written consent for exploratory analyses of material obtained during study execution.

The main study was approved by the ethics committee of the Leiden University Medical Center (LUMC), the Netherlands. The study was conducted according to the Dutch act on Medical Research Involving Human Subjects (WMO) and in compliance with Good Clinical Practice (ICH-GCP) and the Declaration of Helsinki.

Samples of 100 healthy male and female participants of 65 years of age and older were selected from the main study in health elderly [21]. All subjects were healthy volunteers without cognitive complaints who registered for participation voluntarily. Of these 100 subjects, 50 subjects were selected with a CSF Aβ1-42 profile consistent with Alzheimer’s disease and were classified as subjects with probable brain amyloidosis, referred to as preclinical AD. A healthy control group of 50 subjects was selected based on subjects having high levels of CSF Aβ1-42. Aβ1-42 was measured in CSF using the fully automated Elecsys platform [22] at the Neurochemistry Lab Amsterdam UMC, using in-house confirmed cutoffs [23]. Lowered Aβ levels classified as amyloid abnormal and consistent with the presence of Alzheimer pathology were dichotomized by creating a group of “Aβ positive subjects” (Aβ+ = < 1000 pg/ml) and “Aβ negative subjects” (Aβ− = > 1000 pg/ml). All the subjects visited Centre for Human Drug Research (CHDR) between October 2017 and November 2018. Main exclusion criteria were a diagnosis of a cognitive disorder (including but not limited to MCI, AD, Lewy Body dementia [LBD], frontotemporal dementia [FTD]), history of psychiatric disease in the past 3 years, Mini-Mental State Examination (MMSE) ≤ 24, Geriatric Depression Scale (GDS) ≥ 6, presence of drug or alcohol abuse (<2 standard drinks per day for female and <3 standard drinks per day for male), use of any medication that was expected to influence central nervous system function or is contraindicative of the performance of a lumbar puncture.

All subjects visited the clinical research unit once and underwent blood sampling at predefined time points (0, 2, and 4 h]). A single lumbar puncture was performed for the collection of CSF (at 4 h, either in the morning or afternoon). Furthermore, an automated CNS test battery was performed to collect data related to different domains of CNS functioning. The Clinical Dementia Rating scale (CDR) was assessed during the study day.

In the context of a post hoc analysis, subjects were also dichotomized based on the Ptau/Aβ1-42 ratio. Previous studies have shown that the use of ratio scores may be superior to the use of a single biomarker [24, 25]. Ptau information was known from the main study and determined by measuring Ptau in CSF using the fully automated Elecsys platform [22] at the Neurochemistry Lab Amsterdam UMC, using the Ptau/ Aβ1-42 ratio >0.02 cutoff for preclinical AD definition. Subjects with a score <0.02 were classified as healthy subjects.

Approximately 10mL of blood was collected via an i.v. catheter placed in an antecubital vein in the arm in appropriate K2EDTA tubes (BD, USA) at the predefined time points mentioned above. Following blood centrifugation within 1 h at 2000g for 10 min at 4°C, the plasma aliquots were divided into 0.5mL aliquots in Sarstedt polypropylene tubes and stored at −80°C. All blood samples for analyses of YKL-40, GFAP, MCP-1, and eotaxin-1 are collected in a non-fasted state within 1 h of collection of the CSF sample

Lumbar punctures were performed by a trained physician with a 25G atraumatic lumbar puncture needle (Braun, 25G). The needle was placed at the L3–L4 or L4–L5 interspace with the subject in supine or sitting position. 4 ml CSF was collected in a 15 mL polypropylene tube (Corning, USA). CSF was centrifuged within 1 h, at 2000g for 10 min at 4°C, and stored at −80°C [26].

Apolipoprotein E (APOE) genotyping was performed after isolating DNA from EDTA blood by the laboratory of human genetics (department of human genetics and endocrinology, Leiden University Medical Center LUMC). DNA was isolated using a QIAamp DNA Blood MINI kit after which a polymerase chain reaction (PCR) technique was applied on the clean DNA. A sequential analysis (according to the Sanger method) then determined the APOE genotype. One or 2 APOE ε4 alleles classified subjects as APOE ε4 carriers, when no APOE ε4 alleles were present a subject was classified as noncarrier.

YKL-40 (Chitinase 3-like 1 [CHI3L1]) was measured in the plasma samples using the CHI3L1 Human ELISA Kit (Thermo Fisher) according to the manufacturer’s instructions. YKL-40 was measured previously in a larger sample and not for the sole purpose of this study [21]. Results of the 100 subjects selected for this study have been used in the analyses.

Plasma GFAP concentrations were measured at Amsterdam University Medical Centers (Amsterdam UMC) using the Simoa GFAP Discovery kit on the Single molecule array (Simoa) platform (Quanterix, Billerica, USA). MCP-1 and Eotaxin-1 were also measured at the Amsterdam UMC using Meso scale discovery (MSD, Rockville, MD, USA) assays according to the kit instructions.

Visual checks on the ranges of biomarker and clinical characteristic test scores for each group based on CSF amyloid beta status were done using scatter plots, as well as Tukey boxplots. Independent T-test, Pearson chi-square test, and Mann-Whitney tests were applied as appropriate.

To establish differences between subject groups in biomarkers, data is analyzed using an ANCOVA, where age, sex, and E4 status are added to the model as covariates. After including all covariates, the analysis was repeated with only the significant covariates added to the model. Variables were Log transformed where applicable. Least square means were calculated for all 4 inflammatory biomarkers using both the Aβ+/Aβ− cutoff and Ptau/Aβ1-42 ratio. All analyses were carried out using SAS for Windows V9.4 (SAS Institute, Inc., Cary, NC, USA). A p-value of <0.05 was considered significant.