Reduced ex vivo lipopolysaccharide (LPS) stimulated whole blood pro-inflammatory cytokine release is a hallmark of immunosuppression in the critically ill and predicts adverse clinical outcomes. No standard technique for performing the assay currently exists. The impact of methodological heterogeneity was determined.Design, Setting, Subjects, and Interventions:
Clinical experimental study set in a research laboratory. Venous blood from 5 to 10 healthy volunteers/experiment (total participant group: 18 subjects, 72% men, mean age 32) was stimulated ex vivo to evaluate the effect of variables identified via literature review on tumor necrosis factor-α (TNFα) release. These included sample handling, stimulation technique, and incubation conditions. Reporting convention was additionally assessed.Main Results:
Measured TNFα release was significantly altered by source of LPS, concentration of LPS employed, duration and temperature of incubation prior to supernatant aspiration, and predilution of blood (repeated measures ANOVA, all P < 0.01). Sample handling prior to stimulation (anticoagulant employed, time to LPS addition, and storage temperature) also caused significant alterations in TNFα release. Considerable interindividual variation was observed (range 1,024–4,649 pg/mL, mean 2,339 pg/mL). Normalization by monocyte count and pretreatment with a cyclooxygenase inhibitor (indomethacin 10 μM) reduced the coefficient of variation from 47.17% to 32.09%.Conclusions:
Inconsistency in interlaboratory methodology and reporting impairs interpretation, comparability, and reproducibility of the ex vivo LPS-stimulated whole blood cytokine release assay. A standardized validated technique is required. The advent of trials of immunoadjuvant agents renders this a clinical imperative.