Recruitment of subjects
All patients aged 50 years or older were recruited from neurological clinics at National Yang Ming Chiao Tung University Hospital (NYCUH)( (former National Yang-Ming University Hospital). All participants received cognitive assessments. Dementia was diagnosed according to the National Institute on Aging and Alzheimer’s Association (NIA-AA) criteria [27]. Trained research assistants also administered the Chinese version of the MMSE[28], which features a total score of 30. The Clinical Dementia Rating (CDR) was also used to determine the severity of dementia after separate semi-structured interviews with the patient and a knowledgeable informant were conducted by a neurologist or psychologist. The CDR scores are categorized as follows: 0 for normal, 0.5 for MCI or very mild dementia (VMD) [29, 30], 1 for mild dementia, 2 for moderate dementia, and 3 for severe dementia [31]. Individuals with VMD were classified according to the presentation of mild impairment in two or more cognitive domains and a slight decline in daily function; cognitive deficits sufficient to interfere with independence in daily life, community affairs, or at-home hobbies; or based on the outcome of the CDR. MCI was diagnosed according to the NIA-AA-recommended criteria, defined as a change in cognition with impairment in one or more cognitive domains but no evidence of impairment in social or occupational functions as assessed by the CDR, activities of daily living (ADL) [32], and instrumental activities of daily living (IADL) [33]. The final diagnosis was reviewed after one year of follow-up.
The research was approved by the Institutional Review Board (IRB) of NYCUH (IRB No.2017A033 and No. 2018A004). Written informed consent was obtained from all participants in the study. All patients with dementia and their proxies provided written informed consent. Informed consent could only be signed directly by patients with MCI because the IRB agreed that patients with MCI, who suffered from cognitive impairments in one or more cognitive domains but presented with no evidence of impairment in social or occupational functions, were capable of understanding the study procedures and non-invasive assessments being performed in this study. We also explained this informed consent requirement to any proxies who accompanied MCI patients.
All participants received medical, neurological, neuropsychological, and psychiatric assessments and blood examinations. The neurological assessments performed for each participant included a cerebral computed tomography scan to exclude intracranial pathologies (i.e., brain tumors or stroke) that may have contributed to cognitive decline.
Plasma preparation
A 9-ml EDTA tube (455036, Greiner) was used to draw blood. No fasting was required for the blood draw. The tube was gently inverted 10 times immediately after the blood draw. A swing-out (bucket) rotor was used to centrifuge the blood at 15–25°C at 1500–2500 × g for 15 minutes. Then, 1 ml plasma (supernatant) was transferred to a fresh 1.5-ml Eppendorf tube. All aliquoted plasma samples were stored at − 80°C within 4.5 hours after the blood draw and prior to performing biomarker assays.
Assays of plasma biomarkers
The frozen human plasma sample was moved from − 80°C to wet ice and room temperature. To assaying Aβ1 − 40, T-Tau, and p-Tau181 (Tau phosphorylated at threonine 181), 40 µl plasma was mixed with 80 µl of the respective reagent (MF-AB0-0060, MF-TAU-0060, and MF-PT1-0060; MagQu). For assaying Aβ1 − 42, 60 µl plasma was mixed with 60 µl reagent (MF-AB2-0060, MagQu). For each batch of measurements, calibrators (CA-DEX-0060, CA-DEX-0080, MagQu) and control solutions were used. An immunomagnetic reduction (XacPro-S361, MagQu) analyzer was utilized. For each sample, duplicate measurements were performed for each biomarker. The averaged concentration of the duplicate measurements was reported.
Statistical analysis
Continuous variables for each measurement are presented as the mean ± standard deviation. SPSS (version 22.0) for Windows (SPSS Inc., Chicago, IL, USA) was used to perform statistical analyses. Baseline demographic characteristics, including age, MMSE score, and CDR-Sum of Boxes (SOB) scores, were coded as continuous variables and compared using a T-test to determine p-values. Receiver operating characteristic curves were analyzed for each plasma biomarker to explore cutoff values, sensitivity, specificity, and area under the curve (AUC) for the differentiation of various types of dementia. All statistical tests were two-tailed, and significance levels were established at p-values of less than 0.05.