Participants
Patients (n = 100: AD, 20; VaD, 20; FTD, 20; PD, 20; MCI, 20) in the study were recruited from the Huashan Hospital of Fudan University (Shanghai, China). Matched healthy controls (n = 40) were recruited from the Chinese Alzheimer’s Biomarker and LifestylE (CABLE; Qingdao, China) study previously described in detail [20,21,22]. The participants capable of communicating, cooperating with physical and cognitive examinations, and giving consent to the blood draw as well as other necessary ancillary diagnostic tests were included. We excluded participants with severe systemic illness or central nervous system diseases attributed to non-NDDs, such as central nervous system infectious diseases and epilepsy. All procedures conformed to the tenets set forth by the Helsinki Declaration. All participants or legal guardians gave their written informed consent. Ethics approval was received from the institutional review boards of each participating center.
Diagnostic procedures
General cognitive tests including Mini-Mental Status Examination (MMSE), Montreal Cognitive Assessment (MoCA) were performed by neuropsychological professionals blinded to the study design and subsequent procedures. Participants without formal education completed the Montreal Cognitive Assessment-Basic (MoCA-B) questionnaire instead of the original MoCA. Some other tests critical for diagnosis like Clinical Dementia Rating scale (CDR), Activities of Daily Living (ADL), the Unified Parkinson’s Disease Rating Scale (UPDRS), and Frontal Behavioral Inventory (FBI) were also performed. Small portions of the translated international questionnaires were appropriately adjusted to ensure applicability to the Chinese Han population.
Diagnoses were made by experienced neurologists who were unaware of the research procedures. AD dementia was diagnosed according to the National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) criteria [23], as well as the National Institute on Aging-Alzheimer’s Association (NIA-AA) research framework [24]. All patients with AD dementia had strong PET evidence of the disease or CSF-confirmed Aβ-positive pathology. VaD was diagnosed according to the National Institute of Neurological Disorders and Stroke and the Association Internationale pour la Recherche et l’Enseignement en Neurosciences (NINDS-AIREN) criteria [25]. FTD was diagnosed according to the revised criteria set forth by the International Behavioral Variant of FTD Criteria Consortium [26]. PD was diagnosed according to the clinical criteria set forth by the International Movement Disorders Society (MDS) in 2015 [27]. MCI was diagnosed when there was objective evidence of cognitive decline while no significant evidence of impaired social or self-care ability. Healthy individuals recruited in the study were required to be matched to the patients with AD in terms of age and sex. In addition, all controls had an MMSE score > 24 at the screening visit and no signs of cognitive decline, thus not meeting the criteria for MCI or any NDD.
Blood sample collection and analyses
All laboratory personnel were blinded to clinical information. To obtain plasma, venous blood samples collected in EDTA-containing tubes were centrifuged at 1800 rpm for 15 min at 4 °C. The supernatant was immediately removed, frozen in 200 μL aliquots, and stored at
-80 °C until further processing.
Plasma Aβ42, Aβ40, p-tau181, GFAP, and NfL levels were quantified using single molecule arrays (SiMoA), an ultra-sensitive enzyme-linked immunosorbent assay (ELISA) technique on an automated SiMoA HD-X platform (Quanterix, Billerica, MA, USA). The SiMoA Human Neurology 4-Plex E assay was used to measure GFAP, Aβ40, Aβ42, and NfL levels, whereas p-tau181 was quantified by the SiMoA p-tau181 Advantage V2 assay.
Quantification of isoAsp in blood HSA and IgGs against deamidated HSA
As described before [18], the plasma samples were sonicated, centrifuged and their protein concentrations were measured. All plasma samples and HSA standards were diluted to 5 μg/mL and analyzed in triplicates by an indirect Enzyme-Linked Immunosorbent Assay (ELISA). The isoAsp levels in each blood sample were calculated according to the standard curve of HSA constructed from standards on each plate. The results were normalized by the average value in the control group.
The IgG antibodies were purified from plasma using the Melon Gel IgG Purification Kit (Thermo Fisher Scientific, San Diego, CA, USA). To determine in each sample the amount of IgG against aged HSA (aHSA, ≈60% isoAsp), the purified IgG antibodies were used as the primary antibody in the indirect ELISA [17], while the secondary antibody was Goat anti-Human IgG (H + L) Secondary Antibody conjugated with HRP (Thermo Fisher Scientific, San Diego, CA, USA). To eliminate the effect of sample location on the plate, the data were normalized by the average reading in each row and column.
Statistical analysis
The data analysis was performed using GraphPad Prism (version 8.0.2) and coding using R software (version 3.4.4). The parameters distributed normally were expressed as mean ± standard deviation (SD), and group differences were analyzed using two-tailed Student’s t-test. Otherwise, parameters were stated as medians with interquartile ranges, and a Mann-Whitney U test or a Kruskal-Wallis test was applied. The receiver operating characteristic (ROC) curves of Cohort 1 were adjusted for age, sex, education years and APOE ε4 carrier status according to the reported method [28, 29], while the ROC curves of Cohort 2 were adjusted for age, sex, and education years.