Cobas c502

Disease activity was evaluated using the SDAI (Simple Disease Activity Index) as measured using the arithmetic sum of tender and swollen 28-joint count, the patient’s and rheumatologist’s global assessment, and CRP in mg/dL as described in Aletaha et al [37 (link)]. For CRP analysis, fasting blood samples from RA patients were drawn during the visits, and serum was separated and examined within <2 h after collection using the cobas c 502 analyzer (Roche Diagnostics, Mannheim, Germany). All standardized procedures were conducted by clinical laboratory staff at Hescuro Clinic Bad Bocklet, Germany. Serum hs-CRP concentrations were determined with particle-enhanced immunoturbidimetric assay (CRP4, tina-quant C-Reactive Protein IV).

Inflammatory cytokines (TNF-α and IL-8) in plasma were measured by enzyme-linked immunosorbent assays (ELISA) (Invitrogen, Waltham, MA, USA). The levels of IL-6, hs-CRP and procalcitonin were quantified by an automated immunoturbidimetric assay (cobas c502, Roche Diagnostics). Neutrophil elastase (NE) level was measured by sandwich ELISA (Abcam270204, Cambridge, United Kingdom). Levels of Histone–DNA complexes were measured by the Cell Death Detection ELISA kit (Roche, Mannheim, Germany).
Levels of MPO–DNA complexes were measured using an adapted protocol from previous studies [47 (link),48 (link)]. Briefly, Costar 96 flat-well plates were coated with 1 µg/mL antihuman MPO antibody (Bio-Rad 0400-0002, Oxford, United Kingdom) in coating buffer from the Cell Death ELISA kit, overnight at 4 °C. After washing and blocking, sera or supernatants were added and incubated at room temperature for 90 min. The plate was washed and incubated with HRP-conjugated anti-DNA antibody (from the Cell Death kit), and color was developed with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Invitrogen, Waltham, MA, USA) followed by 2N H₂SO₄ to stop the reaction. The absorbance was measured at 450 nm.

WBC (lymphocytes, monocytes, and granulocytes) counts and the percentage of each component as well as platelet counts were measured using a HORIBA ABX diagnostic analyzer (HORIBA ABX SAS; Parc Euromedicine). Using these counts, the WBC to apolipoprotein A‐I ratio, monocyte to lymphocyte ratio (MLR), granulocyte to lymphocyte ratio (GLR), platelet to lymphocyte ratio (PLR), and monocyte to platelet ratio (MPR) were calculated.
High‐sensitivity C‐reactive protein (hs‐CRP), interleukin (IL)‐1β, IL‐2, IL‐6, IL‐12, tumor necrosis factor (TNF)‐α, and interferon (IFN)‐γ were measured as inflammatory markers. The hs‐CRP level was assessed with CRPHS reagent kits (Roche) and a Cobas C502 (Roche). IL‐1β, IL‐6, and TNF‐α levels were determined using Bio‐Plex Reagent kits (Bio‐Rad Laboratories) and a Luminex 200 (Luminex Corporation). IL‐2, IL‐12, and IFN‐γ levels were measured with Human IL‐2 ELISA kits (Cusabio Biotech), High Sensitivity Human IL‐12 (p70) ELISA kits (Genway Biotech Inc.), and IFN‐γ High Sensitivity Human ELISA kits (Abcam plc), respectively; the absorbance at 450 nm of the resulting reactions of the assays were evaluated using a Victor×5 2030 Multilabel Plate Reader (PerkinElmer).

Venous blood samples were collected with anticoagulant tubes after an overnight 12-h fast. The samples were centrifuged within an hour (30 min, RT, at 3000RPM; Centrifuge, DT5-4B, Beili Co., Beijing, China) and tested within two hours. The serum Hcy was measured with an assay kit (DiaSys Diagnostic Systems, Shanghai, China) based on an enzymatic cycling method. The reagent detection range was 1–50 umol/L. When the measured value of the sample exceeded the upper limit, it was diluted and remeasured. The sensitivity was 1 umol/L. The intra-assay and inter-assay coefficients of variation (CV%) were 2.2–4.8% and 3.2–5.5%, respectively. The measurement was performed on Roche COBAS c502 (Swiss). Quality control was conducted first every day. Specimen testing was conducted only after qualified quality control. Every month, statistical analysis of the quality control chart was conducted to ensure the stability of the quality control results. Every year, the laboratory participates in external quality assessment (EQA) conducted by the inspection center of the Ministry of Health to ensure the accuracy of the test results. The normal range of Hcy concentrations is between 5 and 15 umol/L and above 15 umol/L is considered to be hyperhomocysteinemia^{15 (link)}.

In the morning of the second day after admission, and after fasting for more than 8 h, 5 mL and 2 mL of venous blood were collected in vacuum tubes containing separation gel coagulant and EDTA-K2, respectively. The tubes were inverted eight times for mixing, and FPG, ALT, CREA and GA were assayed using an autoanalyzer (Roche Cobas C701) using the hexokinase, rate, sarcosine oxidase, and colorimetric methods, respectively. HGB and HbA1c% were assayed by immunoturbidimetry with an autoanalyzer (Cobas c502; Roche, Switzerland). The HbA1c% test was certified by the National Glycohemoglobin Standardization Program. After collecting fasting venous blood, 75 g anhydrous glucose was dissolved in 250 mL water and given orally. Venous blood was collected for glucose assays after 1, 2, and 3 h. After admission, bedside blood glucose meters (Yuyue 582) monitored blood glucose in peripheral capillaries using the oxidase method before and after three meals and before sleep. The SD of seven peripheral blood glucose values was calculated. The hypoglycemic treatment plan of the patient remained the same as before admission.