Luminex performance assay

The release of cytokines by PMNs was quantified in 6 h supernatants of C. albicans infection. PMNs (1 × 10⁵ cells/well) were infected in 24 wells with C. albicans (multiplicity of infection [MOI] of 1) for 6 h. IL-8 release was determined using human IL-8 magnetic Luminex performance assay (R&D Systems) and read using a multiplex array reader Bio-Plex 200 system (Bio-Rad Laboratories). The cytokine concentration was based on the standard curve supplied by the manufacturer.

Upon laboratory reception, blood samples were centrifuged at 6°C at a speed of 1008xg for 15 minutes. The supernatants were collected in sterile falcon tubes and centrifuged again at 1500xg for 10 minutes at 6°C. Resulting platelet poor plasma per sample was separated into two vials and stored at -80°C for later use to perform the immunoassays (Fig 1).
Stored plasma samples were thawed at 4–8°C. Immunoassays were performed in duplicates using VEGF and PlGF Magnetic Luminex Performance Assay (Human Angiogenesis Premixed KitA; R&D Systems, a bio-techne brand), and Human VEGF R1/Flt-1 Quantikine ELISA Kit (R&D Systems), following manufacturer’s instructions. Plasma levels of angiogenic factors were measured in pg/ml, and validated commercial control samples for each analyte were assayed in parallel as a quality control measure. Commercial control samples used include; Luminex Performance Assay, Human Angiogenesis Panel A Kit Controls (R&D Systems, a bio-techne brand) for VEGF and PlGF luminex assays, and Quantikine Immunoassay Control Set 935 for Human VEGF R1 (R&D Systems, a bio-techne brand) for sFlt1 ELISA assays. Inter-assay coefficients of variation for VEGF and PlGF was 10.03% and 9.72% respectively whereas that of sFlt1 was 8.08%.

Routine laboratory testing was carried out for all patients, including serum vitamin D levels. Within the first 72 h after admission, blood samples were taken at 8.00 a.m. in fasting conditions and were immediately frozen at −80 °C. The following parameters were determined by Luminex^® Performance Assay (R&D Systems, Minneapolis, MN, USA): tumor necrosis factor alpha (TNF-α), with a sensitivity of 0.29 pg/mL; interleukin 8 (IL-8), with a sensitivity of 1.97 pg/mL; and interleukin 6 (IL-6), with a sensitivity of 0.36 pg/mL (data provided by the manufacturer).

Stored AF samples were analyzed for cytokines, matrix metalloproteinases and chemokines. Cytokines including interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IFN-γ, and TNF-α were measured with Bio-Plex Pro assays (BIO-RAD Laboratories, Inc., Hercules, CA, USA). Matrix metalloproteinases (MMPs; MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-12) and chemokines (Complement Factor D/Adipsin, Serpin E1/PAI-1, Adiponectin/Acrp30, C-Reactive Protein, CCL2/MCP-1, Leptin, Resistin) were measured with Luminex Performance Assay (R&D Systems, Inc., Minneapolis, MN, USA). In these multiplex assays, the antibodies are covalently coupled to microspheres with a unique fluorescent dye, thereby enabling the determination of concentrations of each analyte using a Bio-Plex 200 analyzer (BIO-RAD). For the purpose of analysis, values below the lower limit of detection for each analyte were recorded as the lower limit of quantification (LLOQ).