Study participants
Our study only included cognitively normal individuals from the Chinese Alzheimer’s Biomarker and LifestyLE (CABLE) study. Since 2017, CABLE is a large-scale cohort study mainly focusing on Alzheimer’s risk factors and biomarkers in Chinese Han population [9]. CABLE aims to identify genetic, environmental, and lifestyle risk factors as well as AD biomarkers in Chinese Han population. All participants in the CABLE were enrolled at Qingdao Municipal Hospital, Shandong Province, China.
All participants were Han Chinese aged between 50 to 90 years. The exclusion criteria were (1) central nervous system infection, head trauma, epilepsy, multiple sclerosis, or other major neurological disorders; (2) major psychological disorders (e.g., depression); (3) severe systemic diseases (e.g., malignant tumours, circulatory diseases such as severe heart failure and kidney failure) that may affect CSF or blood levels of AD biomarkers including Aβ and tau; (4) family history of genetic disease. All participants underwent clinical and neuropsychological assessments, and blood and CSF sample collection for the next day. Participants were required to fast for at least 8 h prior. Demographic information, AD risk factor profile, and medical history were also collected by a comprehensive questionnaire and an electronic medical record system. Participants in the study were outpatients and inpatients. We excluded participants with serious medical conditions under the guidance of a professional and experienced physician.
Haemoglobin measurements and definition of anaemia
Haemoglobin was measured from blood samples using an automated haematology analyser in the laboratory at Qingdao Municipal Hospital. Anaemia was defined as haemoglobin concentrations < 13 g/dl for men and < 12 g/dl for women. The severity of anaemia was classified as mild (11–12.9 g/dl for men and 11–11.9 g/dl for women), moderate (8–10.9 g/dl), or severe (< 8 g/dl) according to World Health Organization criteria [10].
CSF AD biomarker measurements
The collection and management of CSF samples conform with the international consensus on standardization of CSF research [11]. CSF was collected in 10 ml polypropylene tubes by lumbar puncture in the morning after overnight fasting and was gently mixed to avoid gradient effects. Polypropylene tubes that were used for sample collection do not absorb protein. In the case of traumatic lumbar puncture, the first 1–2 mL of CSF was discarded and sample was collected after transparency [12]. CSF sample was sent to the laboratory at room temperature within 2 h. These specimens were centrifuged at 2000 g for 10 min. CSF samples were separated and stored in an enzyme-free EP (Eppendorf) tube (AXYGEN; PCR-02-C) at − 80 °C until assay. The thaw/freezing cycle was limited not to surpass 2 times. According to the manufacturer’s recommendations, CSF concentrations of Aβ42, Aβ40, t-tau, and p-tau were determined with the ELISA kit (Innotest β-AMYLOID (1–42),β-AMYLOID (1–40), hTAU-Ag, and PHOSPHO-TAU (181p); Fujirebio, Ghent, Belgium) on the microplate reader (Thermo Scientific™ Multiskan™ MK3). All ELISA measurements were performed by experienced technicians in strict accordance with the manufacturer’s instructions. They were blinded to the clinical information. Moreover, the within-batch CV was < 5% and the inter-batch CV was < 15%.
Covariates and cognitive assessment
Fundamental covariates included age, gender, education years, APOE ε4 alleles. Participants were classified as APOE ε4 noncarriers (participants with no copies of the APOE ε4 gene), and APOE ε4 carriers (individuals with at least one copy of the APOE ε4 gene). DNA was extracted from the blood samples using QIAamp DNA Blood Mini Kit (250). Restriction fragment length polymorphism (RFLP) technology was used to genotype rs7421 and rs429358 to define APOE ε4 alleles. Hypertension, diabetes mellitus and dyslipidemia were determined using self-report, medication use, and verification of hospital records by an adjudicator according to prescribed algorithms. History of stroke and coronary heart disease were based on self-report, clinic data, and medication use. Glomerular filtration rate was estimated using the Cockcroft-Gault equation. Cognitive function was assessed using the adopted China-Modifed Mini-Mental State Examination (CM-MMSE).
Statistical analysis
Wilcoxon tests and chi-squared tests were used to examine continuous variables (age, educational level, cognitive scores, and glomerular filtration rate, etc.) and categorical variables (gender, APOE ε4 alleles, history of coronary heart disease, history of stroke, history of hypertension,history of diabetes mellitus, and history of dyslipidaemia, etc) between groups respectively. Multiple linear regression models were used to investigate the associations of anaemia with CSF AD biomarkers. Age, gender, educational levels, APOE ε4 alleles comorbidities (history of coronary heart disease, history of stroke, history of hypertension, history of diabetes mellitus, history of dyslipidaemia), and glomerular filtration rate were included as covariates. P < 0.05 was considered statistically significant. All statistical analyses were performed with R software (version 3.6.1).