Cobas 6000 system

This study is based on a cohort of 412 NSCLC patients described in previous studies [11, (link)13] (link). Patients were treated with either nivolumab or pembrolizumab at the Netherlands Cancer Institute between March 2013 and September 2019. Retrospective collection of data was approved by the local institutional review board and ethics committee (PTC NKI-AvL, NL45524.031.13). Blood samples were provided prior to treatment initiation and bi-weekly thereafter as part of regular care. CYFRA, CEA, CA125 and NSE were measured on a Cobas 6000 system (Roche) and SCC on a Kryptor system (Thermo Fisher).

Venous blood was collected into 0.3 mL citrate buffer tubes (Monovettes, Sarstedt, Nuremberg, Germany). Plasma concentrations of fibrinogen (lower limit of detection, LOD: 30 mg/dL) and VWF antigen (lower LOD: 270 mg/L) were determined with a multiplexed particle-based flow cytometric assay (Cytolab, Regensdorf, Switzerland). Plasma D-dimer concentration (lower LOD: 0 ng/mL) was determined with an enzyme-linked immunosorbent assay (Technozym D-dimer, Technoclone, Vienna, Austria). Inter-and intra-assay coefficients of variation were <15% for all analyses. CRP plasma levels were determined with the cobas 6000 system (Roche Diagnostics, Switzerland).

Blood was collected from the antecubital vein for analyses of several blood variables. Plasma concentrations of free fatty acids (NEFA-HR set; Wako Chemicals), free glycerol (Glycerol kit; R-Biopharm), total glycerol (ABX Triglyceriden CP; Horiba ABX), and glucose (ABX Glucose HK CP; Horiba ABX) were measured on a COBAS PENTRA centrifugal spectrophotometer (Horiba ABX). Plasma triglyceride concentrations were calculated by subtracting free glycerol from the total glycerol concentrations, and serum insulin was analyzed on a Gamma Counter (2470 Automatic Gamma Counter Wizard2 Wallac; Perkin-Elmer) with a Human Insulin specific RIA kit (Millipore). Serum TSH was measured by an Electrochemiluminescence Immunoassay Kit on a COBAS 6000 system (Roche Diagnostica), and free thyroxine (FT4) was analyzed by a solid-phase time-resolved fluoroimmunoassay kit on an AutoDELFIA system (PerkinElmer). Plasma inflammatory marker C-reactive protein (CRP) was measured with a particle-enhanced immunoturbidimetric assay on a COBAS PENTRA system (Horiba ABX). BA concentrations were determined in plasma by phase liquid chromatography associated to tandem mass spectrometry (LC-MS/MS, in Multiple Reaction Monitoring [MRM] mode) after extraction by protein precipitation. The use of internal deutered standards allowed the quantification.