122 Chapter 4 Burn Injury Is Associated With A Large, Continuous Surge of Immature Neutrophils, Classical and Non-Classical Monocytes To further explore the effect of severe burn injury on systemic granulocyte and monocyte subsets in time, we performed an unsupervised analysis using Flow Self-Organizing Map clustering (FlowSOM) (Figure 2). We used data from flow cytometry stainings of 7 patients from which samples of all time points were available. The FlowSOM cluster structure was determined based on all data from these patients and 10 healthy controls. We could define 5 main cell clusters: CD10dim neutrophils (nodes 8–13), CD10bright neutrophils (nodes 2–7), CD16¯ granulocytes (including eosinophils; node 14), classical CD14brightCD16¯ monocytes (node 16) and non-classical CD14dimCD16+ monocytes (node 1) (Figure 2A). Then, we analyzed the composition of these clusters in burn patients over time. CD10 was previously associated with the maturation stages of neutrophils [23–25]. In the first week post burn, the three mature (CD10bright) neutrophil populations (nodes 3–5) were hardly present and the majority of neutrophils was immature (CD10dim). From week 2 onward, mature CD10bright neutrophils reappeared, while immature CD10dim neutrophil numbers declined, but remained elevated for the remaining period of the study. The number of CD16¯ granulocytes slightly decreased in week 1 and returned to the level of healthy controls in week 2. Burn injury caused a shift toward more classical CD14brightCD16¯ monocytes and the elevated level of this subtype persisted for the whole study period.
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