155 Local Immune Response in Burn Patients Figure 2. Local neutrophil response to burn injury. (A) Myeloperoxidase (MPO) immunohistochemical DAB staining of a representative section of healthy skin and burn tissue (from 15 days post burn) (black scale bar = 100 μm). (B) MPO+ area of tissue sections. Flow cytometrybased quantification of: (C) Absolute number of neutrophils (CD15+CD16+ granulocytes) per mg tissue; (D) Percentage of CD10+ (mature) neutrophils (CD15+CD16+ granulocytes); (E) MFI of CD11b on neutrophils (CD15+CD16+ granulocytes) in tissue; (F) MFI of CD66b on neutrophils (CD15+CD16+ granulocytes) in tissue. (G) Unsupervised clustering of neutrophil (CD15+CD16+ granulocytes) flow data from healthy skin and burn tissue, 5 clusters are highlighted. Node size represents relative size of population and node diagram shows expression level of markers. (H) Percentage of neutrophils within each cluster. Error bars in H show boxplot, p values were calculated using Mann-Whitney U statistical test, significant differences are indicated by black asterisks: *p < 0.05; **p < 0.01; ***p < 0.001. Burn injury increases macrophage numbers and affects differentiation Macrophage differentiation was assessed by analyzing the CD14 and CD16 expression of the monocytic cell population (among which are monocytes and macrophages) using flow cytometry. In both healthy skin and burn tissue the majority of these cells expressed a classical phenotype (CD14highCD16¯) (Figures 3A,B). In burn tissue from PBW 3, the proportion of classical monocytic cells decreased while the proportion of intermediate 5
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