Architect i2000 analyser

Serum samples were analysed by testing anti-HCV with a 3^rd generation assay using CMIA (enzymatic immunoassay with chemiluminescent detection) on the Architect i2000 analyser, by Abbott, USA [28 (link)]. The presence or absence of anti-HCV antibodies in the sample was determined based on comparing the value of the chemiluminescent signal in the reaction (RLU) with the cut-off value (CO). A sample was considered positive (reactive) when the sample had a chemiluminescent signal greater than or equal to the cut-off value based on the S/CO (sample/cut-off) formula. Samples with an S/CO < 1 were labelled negative (non-reactive), samples with S/CO values from 1–2 were defined as borderline reactive, and samples with S/CO > 2 were considered positive (reactive). All borderline reactive samples were tested using the confirmation immunoblot test INNO-LIA HCV Score (Fujirebio Europe N.V., Belgium) in the National Reference Laboratory. The test variant requiring a 3-hour incubation of the tested serum was performed.

Plasma levels of NTproBNP, hsCRP, CysC and MPO were measured at the University Medical Center Utrecht, the Netherlands, using a semi-automated ELISA robot (Freedom EVO, Tecan, Switzerland). Commercial antibody combinations were used to quantify NTproBNP (15 C4 and biotinylated 13G12, Hi-test Finland), hsCRP (Dy1707 duoset, R&D systems), CysC (Dy1196 duoset, R&D systems) and MPO (Dy3667 duoset, R&D systems). In brief, maxisorb plates were coated with mouse anti-human NTproBNP, hsCRP, CysC or MPO. Plates were blocked with 1 % bovine serum albumin, and incubated with supernatants. Plates were washed with phosphate buffered saline pH 7.4 with 0.05 % Tween 20. Bound factors were detected with biotin coupled detection antibodies. Biotin coupled antibodies were bound with streptavidin horseradish peroxidase (HRP), or goat-anti-human antibodies with rabbit-anti-goat HRP (DAKO, P0449). Detection was performed with SuperSignal West Pico Chemiluminescent substrate, and read with a luminometer. The intra-assay variation coefficient was 10 %. Levels of hsTnI were measured using the STAT hsTn1I assay on the clinically validated ARCHITECT i2000 analyser (Abbott Laboratories, Lisnamuck, Longford, Ireland).
All samples (Singaporean and Dutch) were randomly distributed across the plates for all analyses.

Routine blood examinations included leukocyte, neutrophil, and lymphocyte counts (cells^*10³/μL) and percentages. Serum biochemical parameters recorded were ferritin (ng/L) determined by chemiluminescence immunoassay in Architect i2000 analyser (Abbot), C-reactive protein (CRP) (mg/dL), and D-dimer (μg/L) quantified by immunoturbidimetry in Architect c16000 (Abbot) and ACL TOP 700 (Instrumentation Laboratory), respectively. We used a chemiluminescence assay (IMMULITE, Siemens, Germany) to determine serum soluble IL-2 receptor alpha (sIL-2rα or sCD25), and a human cytokine magnetic bead panel (Merck Millipore, Billerica, MA, USA) to measure levels of other cytokines associated with “cytokine storm”: IL-1β, IL-1 receptor antagonist (IL-1Ra), IL-6, IL-8, IL-17A, IL-18, IL-22, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-10.