Cobas 601

The eight TSH immunoassays used in this study were as follows: the ADVIA CentaurXP (Siemens Healthineers, Tarrytown, NY, USA), the Immulite 2000 (Siemens Healthineers, Gwynedd, UK), the DXI800 (Beckman Coulter, Brea, CA, USA), the Autolumo A2000plus (Autobio Diagnostics, Zhengzhou, China), the Maglumi2000plus (Snibe, Shenzhen, China), the Cobas 601 (Roche Diagnostics, Mannheim, Germany), the Architect i2000sr (Abbott Diagnostics, Abbott Park, IL, USA), and the Liaison XL (DiaSorin S.p.A, Saluggia, Italy). Each of these assays was performed using the respective manufacturer’s reagents and calibrators. Detailed information on these assays is provided in S1 Table.

The CA125 and HE4 markers serum level was determined in all patients. The Roche Elecsys^® CA125 II assay is a tumor marker test for use with blood samples to support monitoring and surveillance of ovarian cancer patients, and together with HE4, aids in risk assessment of patients with pelvic mass with the Risk of Ovarian Malignancy Algorithm (ROMA).
Carbohydrate antigen 125 (CA125) and Human Epididymis Protein (HE4) were detected using the full automatic chemiluminescence analyzer Cobas601 along with the corresponding kit and were used according to laboratory protocol (Abbott Laboratories, Roche Diagnostics). Serum HE4 and CA125 levels were calculated for ROMA index value using Roche ROMA index of ovarian cancer risk assessment software. The serum CA125 and HE4 reference ranges were <35 U/mL and <140 pmol/L, respectively.
The ROMA index was calculated according to the levels of CA125, HE4, and menopausal status. Marker values were input to the ovarian cancer risk assessment software, followed by automatic calculation of the corresponding ROMA index. When Roche Elecsys specificity was 75%, premenopausal women with a ROMA value ≥11.4% and post-menopausal women with ROMA value ≥29.9% had a higher risk of ovarian cancer (https://diagnostics.roche.com/global/en/article-listing/roma-calculator.html; last accessed on 16 November 2020).

Blood samples were collected to analyze HIV related parameters (current CD4 cell count and current HIV viral load). As a general rule, blood samples were obtained within one month of DXA scanning. Glomerular filtration rate (GFR) was estimated by using the chronic kidney disease (CKD)-epidemiology collaboration equation. Also, individuals were screened for thyroid function (thyroid-stimulating hormone, TSH), hepatitis C (HCV-RNA), diabetes mellitus, gonadal function (total testosterone), vitamin D status (serum determination of 25-dihydroxyvitamin D by standardized electrochemiluminescence method (Cobas 601 Roche) and parathyroid function (serum parathyroid hormone, PTH) by chemiluminescence (Immulite 2000 Siemens).
The BMD was measured by dual X-ray absorptiometry (DXA) using Hologic densitometer (Hologic 4500, Bedford, USA) and included the values for bone mineral density (g/cm²), T-score or Z-score at level of L1-L4 lumbar spine and right or left femoral neck. The same DXA scanning was used throughout the study.

CSF samples were obtained by lumbar puncture, immediately centrifugated at 1000× g at 4 °C for 10 min and aliquoted in polypropylene tubes of 1.5 mL and stored at −80 °C until further analysis, according to international guidelines [47 (link)]. AD CSF biomarker measurements were performed in the biochemistry unit of Lariboisière Hospital [36 (link)]. Aβ42, Aβ40, t-Tau and p-Tau were first measured by ELISA, using available kits (INNOTEST^®; Fujirebio, Ghent, Belgium) until July 2018, according to the manufacturer’s instruction. Then, the concentration of these biomarkers was measured by the analyzer Cobas 601 with the electrochemiluminescent reagents Elecsys^® (Roche Diagnostics GmbH, Germany). The CSF profiles of patients were classified in A+T+N+/A-T-N-/or intermediate according to cut-offs determined by the laboratory.
CSF levels of α-syn were measured using an ELISA kit (ADx Neuroscience ELISA Alphasynuclein (Ghent, Belgium) available via EUROIMMUN, Lübeck, Germany), validated for research for the quantification of human total α-syn in CSF [48 (link)].

Detection kits were provided by Beijing Rejing Biotechnology Co., Ltd.; micro spin column method was used for AFP-L3 separation, and analyzed with Roche Cobas 601 automatic biochemical immunoassay analyzer.