This prospective cohort study analyzed neutrophil functionality within the first 2 weeks after trauma in severely injured trauma patients. Patients were only included after written informed consent of the patient or his/her legal representative, in accordance with the Declaration of Helsinki. All experiments were performed in accordance with the relevant guidelines and regulations. The study was approved by the University Medical Centre Utrecht ethical review committee (protocol no. 13/325). The trial was registered online on the website of the Central Committee on Research Involving Human Subjects before participant enrollment started (NL43279.041.13). The process and storage of data were in accordance with privacy and ethics regulations.
Severely injured patients ≥ 18 years of age with an expected ICU stay of ≥ 48 h were included between November 2018 and July 2019. Patients were excluded if they recently (< 3 months before hospital admission) used immunosuppressive medication, had an immunosuppressive disorder, or were admitted to ICU because of isolated neurologic injury. Blood was drawn in sodium heparine tubes as soon as possible after trauma (< 12 h), after 3 days, 6 days, 10 days, and 15 days. Control blood samples were provided by anonymous, sex- and age-matched, healthy volunteers. Data concerning patient characteristics, trauma mechanism, injuries, resuscitation, and treatment were obtained from the electronic medical record system. The injury severity score (ISS)  based on the abbreviated injury scale 2008 (AIS08)  was obtained from the National Trauma Registration database that collects data of all trauma patients admitted to the emergency department [35, 36].
Fluorescent double-labeling of bioparticles
pHrodo® Green Staphylococcus aureus (S. aureus) BioParticlesTM (Thermo Fisher Scientific, Waltham, MA, USA) were labeled with PromoFluor 520 LSS NHS ester (PF520) (PromoCell, Heidelberg, Germany) following the instructions of the manufacturer. In short, PF520 was dissolved in DMSO at a concentration of 2.5 mg/ml and the bioparticles were suspended in a 0.1-M NaHCO3 pH 9 buffer at room temperature at a concentration of 10 mg/ml (3 × 109 particles/ml). Bioparticles were sonicated to prevent clumping. Then, PF520 was added drop-wise while the bioparticle suspension was vortexed. The suspension was mixed for 1 h in the dark at room temperature, after which the double-labeled bioparticles were washed 3 times. Bioparticles were suspended in a pH 7.4 buffer containing 20 mM HEPES, 132 mM NaCl, 6 mM KCl, 1 mM MgSO4, 1.2 mM KH2PO4, 1.0 mM CaCl2, 5 mM glucose, and 5 mg/ml human serum albumin (Albuman 200 g/l, Sanquin, Amsterdam, the Netherlands).
Flow cytometry analysis
All experiments were performed with whole blood using the fully automated AQUIOS CL® “Load & Go” flow cytometer (Beckman Coulter, Brea, CA, USA) at 30 °C . Firstly, the AQUIOS CL® automatically incubated whole blood with the double-labeled S. aureus bioparticles (end concentration of 10 × 106/ml) and with antibody-fluorochrome conjugates for the neutrophil receptors CD16 (clone 3G8, PE labeled; Beckman Coulter) and CD62L (clone DREG56, ECD labeled; Beckman Coulter). Then, after 10, 20, 40, and 60 min of incubation, the AQUIOS CL® was programmed to aspirate part of the sample, to lyse red blood cells (RBCs), and to perform flow cytometric analysis of the leukocytes. Lysing is performed by the addition of 335 μl of lysing reagent A (Beckman Coulter) followed by 100 μl of lysing reagent B (Beckman Coulter). Lysing reagent A is a cyanide-free lytic reagent that lyses red blood cells, and lysing reagent B slows the reaction caused by reagent A and preserves the white blood cells for measurement in the flow cell. The .LMD files were exported and analyzed using Kaluza Analysis Software v2.1 (Beckman Coulter).
Analysis of neutrophil subsets and functionality
The gating strategy is shown in Supplementary Figures 1 and 2. Granulocytes were identified based on their specific forward/side scatter pattern (Supplementary Figure 1a). Neutrophils were identified by selecting granulocytes with CD16 expression (thereby excluding eosinophils) (Supplementary Figure 1b). Percentages of CD16dim/CD62Lbright neutrophils, CD16bright/CD62Lbright neutrophils, and CD16bright/CD62Ldim neutrophils were analyzed as previously described (Supplementary Figure 2) . The acidification of neutrophil phagolysomes was investigated by analyzing both pHrodo® Green fluorescence and PF520 fluorescence. The fluorescence of pHrodo® Green increases when the pH in the phagolysosome decreases [27, 38], while the fluorescence of PF520 is not sensitive for pH changes. Combined analysis of these fluorochromes allows for assessment of phagocytosis, expressed as percentage of PF520-positive neutrophils, and neutrophil phagosomal acidification, expressed as the ratio pHrodo® Green fluorescence divided by PF520 fluorescence (Supplementary Figure 1c-d). This ratio was measured per PF520-positive neutrophil to correct for the number of phagocytosed bioparticles within the cell. The mean ratio of all neutrophils was used as an indicator of acidification. Additionally, mean fluorescence intensities (MFIs) of PF520 and pHrodo® were calculated to gain insight into changes in MFI over time and how this influences the ratio pHrodo® Green fluorescence divided by PF520 fluorescence.
Data were analyzed with IBM SPSS version 23 (IBM Corporation, NY, USA) and GraphPad Prism version 8 (GraphPad, La Jolla, CA, USA). The distribution of continuous variables was assessed with the use of the Shapiro-Wilk test and through visual inspection. Clinical outcomes and demographics were presented as median with interquartile range (IQR) and compared between outcome groups using a Fisher’s exact test or a Mann-Whitney U test, as indicated. Generalized estimating equations (GEE) were used to compare neutrophil subset percentages, neutrophil functionality, PF520 MFI, and pHrodo® MFI over time between patients who later develop an infection and patients who do not, and to correct for within-subject correlation. Outcome data of GEE analysis were presented as the beta coefficient (β) with p value. Additionally, to investigate the differences between these groups for every time point, a Mann-Whitney U test was used because data were not normally distributed. Furthermore, neutrophil phagosomal acidification after 60 min was compared between the 3 neutrophil subsets using a one-way ANOVA with a follow-up comparison of the means using Tukey’s correction for multiple comparisons, since data were normally distributed. Statistical significance was defined as a p value < 0.05.