Laboratory assays

Blood samples were transported fresh to a single central laboratory (journey time less than 1 hour from each ICU) for analysis by staff proficient in all of the techniques described below. Patients’ neutrophils were isolated from citrated whole blood by dextran sedimentation and discontinuous Percoll gradient.²⁰ Neutrophil phagocytosis was quantified as the percentage of neutrophils ingesting ≥2 zymosan particles (derived from the cell wall of the yeast Saccharomcyes cerevisiae), which were opsonised using serum from the same patient whose neutrophils were being assessed.¹¹ Neutrophils were incubated with serum-opsonised zymosan particles for 30 min and the proportion ingesting two or more particles was determined at light microscopy.

Neutrophil chemotaxis in response to formyl-methionine-leucine-phenylalanine was estimated using the subagarose method,²¹ and superoxide generation using a cytochrome c reduction assay.²² Neutrophil apoptosis was measured by flow cytometric analysis of annexin V and propidium iodide in whole blood.²³ Monocyte HLA-DR expression in whole blood was estimated by flow cytometry using a Quantibrite kit (BD Biosciences, Oxford, UK).²⁴ Concentrations of interleukin (IL) 1β, IL-6, IL-8, IL-10, IL-12p70, TNFα and GM-CSF were determined by cytometric bead array (Becton Dickinson). All flow cytometry was carried out using a FACSCanto II flow cytometer (Becton Dickinson).