Using methods previously described [30-32], the ability of the PLT and PRBC supernatants to prime N-formylmethionyl-leucyl-phenylalanine (fMLP) induced respiratory burst function of human neutrophils was measured in vitro and compared to that of buffer, fresh autologous selleck chemicals llc plasma and platelet-activating factor (PAF).Statistical analysesThe clinical incidence of TRALI was compared using two-way contingency tables and the Fisher’s exact test. Physiological differences between groups were made using a two-way ANOVA with Bonferroni’s multiple comparisons adjustment. Physiological data were subsequently modelled using a mixed model with a random intercept for each sheep in the R statistical package [33]. These models were used to examine differences between groups over time whilst accounting for repeated data [34].

Data from LPS-treated sheep that developed TRALI due to either “stored PRBC” or “stored PLT” transfusion were also compared using mixed modelling. Neutrophil priming ability of the different supernatants and controls was compared using a one-way ANOVA with Bonferroni’s multiple comparisons adjustment. In all cases statistical significance was determined at P < 0.05.ResultsTRALI did not develop in healthy sheepThe absence of ALI in the sham group demonstrated that the anaesthetic, surgical and experimental protocols did not induce lung injury (Table (Table1).1). Similarly, sheep that were infused with saline as a control first event and then transfused with either "fresh PRBC" or "stored PRBC," (that is, saline-fresh and saline-stored groups) did not develop TRALI.

A single sheep displayed evidence of hypoxaemia that worsened following transfusion of “stored PRBC.”Transfusion of “stored PRBC” caused TRALI in LPS-primed sheepThe dose of LPS, infused as a first event resulted in profound neutropenia (mean �� SD: 2.03 �� 0.86 neutrophils �� 109/L at baseline vs. 0.14 �� 0.03 neutrophils �� 109/L post-LPS; P < 0.0001 by paired t-test). The LPS-control group of sheep confirmed that this dose of LPS was insufficient to induce ALI (Table (Table1).1). One of the five LPS-fresh sheep and four of the five sheep in the LPS-stored group were diagnosed with TRALI (Table (Table1).1). This demonstrated that TRALI predominantly developed in sheep that received LPS-infusion followed by "stored PRBC" transfusion (P = 0.01 LPS-stored group vs. saline-fresh, saline-stored and LPS-fresh groups). Analysis of the lung section wet/dry weights (mean �� SD: sham = 5.33 �� 0.21; saline-fresh = 5.53 �� 0.37; saline-stored = 5.89 �� 0.52; LPS-control = 5.84 �� 0.96; LPS-fresh = 5.38 �� 0.73; LPS-stored = 6.99 �� 1.2) confirmed this finding (P = 0.0038 LPS-stored group vs. saline-fresh, saline-stored Entinostat and LPS-fresh groups by unpaired t-test).