Cobas c702 module

ProGN and proUGN concentrations in plasma and urine were quantified by using the Human Proguanylin ELISA and the Prouroguanylin Human ELISA kits (Cat. Nos. RD191046100R and RD191069200R, respectively; BioVendor a.s., Brno, Czech Republic). The ELISAs were performed according to the manufacturer’s instructions. The ELISA plate optical readings were done by using a SPECTRAmax microplate reader (Molecular Devices, Sunnyvale, CA, USA), and concentrations were calculated by using SoftMaxPro Software version 7.1 (Molecular Devices, Sunnyvale, CA, USA) using a curve fit based on its 4-parameter logistic nonlinear regression model.
Urine chloride, sodium, potassium, and protein levels were measured using the Cobas^® c 702 module (Roche, Basel, Switzerland); urine creatinine was measured using a AU680 Clinical Chemistry Analyzer (Beckman Coulter, Brea, CA, USA); and urine zinc was measured using inductively coupled plasma mass spectrometry (ICP-MS) on an ELAN DRC-e (PerkinElmer Inc., Waltham, MA, USA), all performed by the Laboratory Clinic at HUS.

Morning blood samples were drawn after an overnight fast. The PAC and PRA were measured by radioimmunoassay (SPAC-S aldosterone and PRA kits, respectively; TFB Inc., Tokyo, Japan) using a Cobra II Gamma Counter (Packard Instrument Co., Meriden, CT). For the PAC assay, the lower limit of detection was >1.53 ng/dL, and the intra-assay and inter-assay coefficients of variation (CVs) were <3.2 and <6.7%, respectively. For the PRA assay, the lower limit of detection was >0.09 ng/mL/h and the intra-assay and inter-assay CVs were <8.3 and <9.7%, respectively.
Serum potassium levels were measured using a Roche ISE Standard Low/High (Roche Diagnostics, Mannheim, Germany) ion selective electrode (ISE) and a Cobas 8000 ISE analyzer (Roche Diagnostics). The intra-assay and inter-assay CVs were 0.5 and 1.6%, respectively. Serum creatinine was measured in a kinetic colorimetric assay using the Roche CREAJ2 kit (Roche Diagnostics) and a Cobas c702 module (Roche Diagnostics). The intra-assay and inter-assay CVs were <2.3 and <2.7%, respectively. Glomerular filtration rate (GFR) was estimated using the Cockcroft–Gault equation (18 (link)).

The patient’s age, gender and race, medical history, HbA1c, current medications, details of meals, beverage and blood sampling were documented in a short questionnaire. Time from the end of the last meal to blood taking was computed for each patient and rounded down to the nearest hour.
The blood samples were sent from the polyclinic laboratories to the central Pathological Laboratory in the Singapore General Hospital twice daily. The specimens were processed using the Roche Cobas c702 module, which leveraged on spectrophotometry to measure the TC, HDL-C, and TG. The LDL-C was calculated based on the Friedewald formula, so no value would be obtained if the TG was > 4.5 mmol/l. The Friedewald formula is: $$LDL\text{-}C=TotalCholesterollevels(\mathit{TC})-HDL\text{-}C-\left[TG/2.2\right]\text{mmol/l}\left(or\left[TG/5\text{mg/dl}\right]\right)$$ and only when TG is < 4.5 mmol/l (TG < 400 mg/dl), beyond which the formula is deemed inaccurate^{23 (link)}. The results were reported daily and automatically channelled to the EMR, which were then retrieved by the research coordinators and recorded in the study documents. The Systeme International (SI) units “mmol/l” was used to define each component of the lipid profile (1 mmol/l cholesterol = 38.7 mg/dl).

Serum IFX and ADL serum concentrations were measured with an ELISA developed by Sanquin (Sanquin, Amsterdam, The Netherlands) [14 (link),15 (link)]. The lower and upper limits of the IFX quantification ranged from 0.002 µg/mL to 120 µg/mL. The lower limit of the ADL quantification was 0.01 µg/mL and there was no upper limit. A photometric lab assay (Cobas c702 module, Roche, Basel, Switzerland) was used to measure plasma CRP concentrations (referred to as ‘CRP lab assay’). The lower limit of the CRP assay was 0.3 mg/L with no upper limit. FCP concentrations were assessed with an automated enzyme fluoroimmunoassay: Elia Phadia (Phadia 250, Thermo Fisher Scientific, Uppsala, Sweden) with a range between 3.8–6000 mg/kg.

Glycaemia during the fasting test was determined by the Accu-Chek^® Inform II system (Roche Diagnostics), 3–7 times a day. The ketonuria during the fasting test was determined twice a day by semi-quantitative testing using diagnostic strips (Diaphan, Erba Lachema, Brno, Czech Republic). Blood glucose was measured in mmol/L (1 mmol/L = 18.0182 mg/dL). The HbA1c values were measured chromatographically (high-performance liquid chromatography using the Cobas system, Cobas 8000 Analyzer, Cobas c702 module, Roche Diagnostics, Basel, Switzerland), set in mmol/mol.

Participants’ venous blood and first morning spot urine samples for the clinical chemistry tests were obtained after a minimum of 8 hr overnight fasting. Serum aliquots were centrifuged; and both serum and urine samples were stored at −80 °C before being assayed. Serum levels of inflammation-related cytokines (pro-inflammatory markers: IL-1β, IL-6, IL-8, and TNF-α; and an anti-inflammatory marker: IL-10) and a urinary oxidative stress marker (8-OHdG) were quantified by commercially available enzyme-linked immunosorbent assay kits according to the manufacturers’ protocols. The inter- and intra-assay coefficients of variation (CVs) were, respectively, 7.88 and 3.56 for IL-1β, 6.45 and 3.94 for IL-6, 9.12 and 6.99 for IL-8, 7.27 and 4.55 for IL-10, and 8.97 and 4.46 for TNF-α; and the minimum detection limits for the corresponding biomarkers were, respectively, 0.08, 0.14, 0.04, 0.21, and 0.29 pg/mL. The inter- and intra-assay CV values of urine 8-OHdG levels were 16.42 and 2.27, respectively, and the detection limit was 0.01 ng/mL. Urine creatinine concentrations were measured using Jaffe and enzymatic assays (Roche Diagnostics, Cobas c702 module). Urinary 8-OHdG levels were expressed as a ratio of creatinine levels to correct for the effect of urine dilution.

After the face-to-face interviews and anthropometric measurements, participants were appointed to the next morning (8:30 am–9:30 am) at our assigned hospitals to collect fasting blood. Around 5 ml of blood sample was collected after overnight fasting to determine the contents of total cholesterol (TC), triglyceride (TG), apolipoprotein A1 (Apo A1), apolipoprotein B (Apo B), high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C). After clotting for 30 minutes, the blood sample was centrifuged for 10 minutes at 3500 rpm. Around 2.0 ml of pure serum sample was transferred to the barcoded storage tubes and stored at 4 °C. The sample was measured within 2 hours by Cobas c 702 module (Roche Diagnostics) for clinical chemistry analysis.