Comparison of circulating MV levels in burns patients on day 0 and day 2.

<p>Plasma from burns patients on admission day 0 and post-admission day 2 was analyzed by flow cytometry to quantify MVs of different cellular origin: CD45+ leukocyte-derived (<b>A</b>), CD105+ endothelia-derived (<b>B</b>), CD66b+/CD11b+ granulocyte-derived (<b>C</b>), and CD45+/CD14+ monocyte-derived (<b>D</b>). Data from individual patients are log-transformed and analyzed by paired t test (<b>A</b>, <b>C</b> and <b>D</b>) or Wilcoxon signed rank test (<b>B</b>). *p < 0.05.</p

Circulating MV levels were elevated in burns patients.

<p>Plasma from healthy volunteers (HV), burns patients on day of admission, and sepsis patients was analyzed by flow cytometry to quantify MVs of different cellular origin: CD45+ leukocyte-derived (<b>A</b>), CD105+ endothelial-derived (<b>B</b>), CD66b+/CD11b+ granulocyte-derived (<b>C</b>), and CD45+/CD14+ monocyte-derived (<b>D</b>). Data are log-transformed and analyzed by one-way ANOVA with Tukey’s tests (<b>A</b>, <b>C</b> and <b>D;</b> mean ± SD) or Kruskal-Wallis with Dunn’s tests (<b>B;</b> median ± interquartile range). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

Evaluation of flow cytometry gating strategy for MVs in plasma.

<p>Suspensions of neutrophil-derived MVs and MV-depleted healthy volunteer plasma were stained with antibodies, individually or after addition of MVs to plasma (volume ratio: 1:4). Larger events were excluded using 1.3 μm beads (not shown) and a gate (<b>R1</b>) for neutrophil-derived MVs was drawn based on CD66b+, CD11b+ double-staining and a cut-off boundary defined by detergent-mediated selective lysis of MVs. Isotype-matched control staining provided confirmation of the detergent-lysis method for identification of non-MV background events.</p

Receiver operator characteristic curves for circulating leukocyte MVs in burns patients.

<p>Receiver operating characteristic (ROC) curves and corresponding area under the curve values for: leukocyte-derived MVs, neutrophil-derived MVs and CRP (<b>A</b>), and for BOBI, ABSI and TBSA (<b>B</b>). *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

Comparison of circulating MV levels in burns and sepsis patient survivors and non-survivors.

<p>Levels of MV subtypes were compared between burns patients that recovered (survivors, n = 10) or died in burns ICU (non-survivors, n = 5) (<b>A</b>) and sepsis patients that recovered (n = 11) or died (n = 4) in the general ICU (<b>B</b>). Data are log-transformed and analyzed by t tests (for burns: leukocyte-, granulocyte- and monocyte-derived MVs; sepsis: leukocyte- and granulocyte-derived MVs; mean ± SD) or Mann-Whitney U tests (for the remainder; median ± interquartile range). Levels of total leukocyte- and granulocyte-derived MVs were higher in burns non-survivors than survivors, **<i>p</i> < 0.01.</p

Burns patient demographics, assessment scores and outcome, with survivor and non-survivor comparisons.

<p>Burns patient demographics, assessment scores and outcome, with survivor and non-survivor comparisons.</p

Flow cytometric identification of MV sub-types.

<p>Dots plots of burns patient plasma stained for MVs using fluorophore-conjugated monoclonal antibodies to cell-surface markers (<b>A</b>). Plots show events that have been gated on forward light scatter using 1.3 μm calibration beads to define the upper size limit. For CD45/CD14 MV analysis, total CD45+ events are shown in the larger region 1 (<b>R1</b>) and CD45+/CD14+ events in the smaller sub-region 2 (<b>R2</b>). MV identification is confirmed by sensitivity to detergent lysis (Triton X-100, 0.1%)(<b>B</b>). Matched antibody isotype and fluorophore control plots are shown for comparison (<b>C</b>).</p

Spearman rank correlation coefficients for MV subpopulations in burns patients.

<p>Spearman rank correlation coefficients for MV subpopulations in burns patients.</p