134 Chapter 4 (for subject details see Supplementary Table 1A). The study protocol with number “NL54823.094.15” was approved by the METc of the VU Medical Center (Amsterdam, the Netherlands). Patients were eligible from 18 years of age with a burned total body surface area (TBSA) of ≥15%. Subjects were included between April 2018 and April 2020. Venous blood samples were collected on a daily basis when present on ICU or twice per week when transferred to the infirmary. Blood samples taken from 20 healthy volunteers served as controls (METc approved under protocol number “NL54823.094.15”). Blood levels of C-reactive protein (CRP), albumin and thrombocytes were determined according to standard diagnostic laboratory procedures as part of standard burn care. Blood samples for flow cytometry were collected in ethylenediaminetetraacetic acid (EDTA) tubes and were stored at 4°C until analysis (<3 h). Only blood samples from working days were used for flow cytometric analysis. The frequency of sampling of each individual patient is presented in Supplementary Table 1B. The patients were treated according to standard burn care, including fluid resuscitation. All patients received analgesics (including paracetamol, NSAIDs and opiates) and antibiotics One of the included patients died two days after the trauma. Three patients contracted pneumonia, one patient had an infected hematoma and none of the patients had sepsis or full-blown infection due to their burn injuries. Colonization of burn wounds was noted, and the predominant bacterial species were Staphylococcus aureus (10/20), Enterococcus cloaca (10/20), Pseudomonas aeruginosa (7/20) and Escherichia coli (5/20). Flow Cytometry Plasma was separated from blood cells by centrifugation for 10 min at 450 × g and stored at -80 °C. Erythrocyte lysis buffer (1.5 mM NH4Cl, 0.1 mM NaHCO3 and 0.01 mM EDTA (Life Technologies, Paisley, UK) in demineralized water) was used to remove erythrocytes from the blood cells. Blood immune cells were resuspended in Dulbecco’s phosphate buffered saline (Gibco, ThermoFisher, Paisley, UK) containing 0.2 mM bovine serum albumin (Fisher Scientific, Pittsburgh, PA) and 0.01 mM EDTA. Cell concentrations were determined by a flow cytometer (MACS Quant Analyzer 10, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Cell suspensions of 2.5 × 105 cells each were stained with different antibody combinations (see panels in Supplementary Table 2) and were analyzed by flow cytometry (MACS Quant Analyzer 10). Samples with more than 40% dead cells (based on 7-AAD staining (Miltenyi)) were excluded from the analysis. Singlet events were gated based on FSC. Viable CD45+ cells were gated and subtyped based on expression of the markers in the 3 staining panels: innate panel (CD10, CD11b, CD14, CD15, and CD16), eosinophil panel (CD9, CD15, and CD16), and lymphocyte panel (CD3, CD4, CD25, CD127, CCR4/CD194, and CCR6/CD196). Manual data analysis was performed using the FlowLogic software (Inivai Technologies, Victoria, Australia).
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