Trauma represents a main cause for morbidity and mortality in the young [1]. Since improved resuscitation strategies and surgical techniques have led to a decreased mortality due to exsanguination, the relative importance of inflammatory and infectious complications has increased as more patients survive the initial phase of trauma [2]. Neutrophils are believed to play a crucial role in the development of inflammatory complications, such as multiple organ dysfunction (MODS), acute respiratory distress syndrome (ARDS) and sepsis [3,4,5]. Several markers on neutrophils have been linked with impaired outcome after trauma. Especially the parameters FcgRIII/CD16, Mac-1/CD11b, L-Selectin/CD62L, neutral endopeptidase/CD10 and FcgRI/CD64 have come into focus in recent studies [6,7,8]. Novel advances in fully automated point-of-care (PoC) technologies allow for rapid near patient flow analyses including PoC studies on neutrophil characterization. A standardized protocol for fully automated PoC analysis of neutrophils has been established at the trauma bay and validated in a trauma center in the Netherlands [7, 9].

This study aimed to validate the standardized protocol at another international centerand assess the feasibility of transferring both the protocol and associated logistics from The Netherlands to a second center in Switzerland.

This study has been performed at two European level one trauma centers in Zurich (Switzerland) and Utrecht (the Netherlands). In both study centers ethical approval (Zurich: BASEC 2017 − 01380, Utrecht: PREDICT 22U-0385 and AQUIval 25U-0305), as well as patient informed consent were obtained. A group of eight healthy volunteers in Zurich was sampled in parallel with the severely injured trauma patients. In Utrecht blood samples from eight healthy volunteers were obtained from the institute Mini Donor Service (Protocol-No 07-125/C).

Blood samples were collected and immediately (< 0.5 h) analyzed using the fully automated AQUIOS CL “load & go” flow cytometer (Beckman Coulter Life Sciences, Miami, FL, USA). The flow cytometer in Zurich was installed and calibrated in accordance with the standardized protocol settings from Utrecht [7, 9]. Needle positioning, laser settings, and compensation parameters in Zurich were adapted to predefined values from Utrecht. The device automatically pipettes blood from a tube into a 96-well plate, followed by staining with an 18 µL customized antibody panel containing CD16-FITC (clone 3G8), CD11b-PE (clone Bear1), CD62L-ECD (clone DREG56), CD10-PC5 (clone ALB1), and CD64-PC7 (clone 22). After 15 min, red blood cells were lysed with 335 µL AQUIOS Lysing Reagent A and stopped with 100 µL AQUIOS Lysing Reagent B, before analysis in the flow cell. The results were exported as FCS 3.1 High Res Listmode Files (.lmd) and imported to FlowJo (De Novo Software, Glendale, CA, USA, Version 10.10.0). Granulocytes were manually gated based on forward and sideward scatter. Statistical analysis was performed using R-Studio (R Foundation for Statistical Computing, Vienna, Austria, Version 2024.12.1 + 563). A two-sided Mann-Whitney-tests with a significance level of 0.05 was used. Diagrams were designed using GraphPad Prism (GraphPad Software, Boston, United States, Version10).