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Stimulated Whole Blood Cytokine Release as a Biomarker of Immunosuppression in the Critically Ill: The Need for a Standardized Methodology.

Segre E, Fullerton JN - Shock (2016)

Bottom Line: Normalization by monocyte count and pretreatment with a cyclooxygenase inhibitor (indomethacin 10  μM) reduced the coefficient of variation from 47.17% to 32.09%.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Clinical Pharmacology, Division of Medicine, University College London, London, UK.

ABSTRACT

Objective: 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.

No MeSH data available.


Related in: MedlinePlus

Effect of discrete variables on the ex vivo lipopolysaccharide (LPS)-stimulated whole blood (WB) cytokine release assay.
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Figure 1: Effect of discrete variables on the ex vivo lipopolysaccharide (LPS)-stimulated whole blood (WB) cytokine release assay.

Mentions: Stimulation with LPS (1 ng/mL) from different bacterial sources elicited significantly different concentrations of TNFα (P < 0.001, one-way RM-ANOVA, Fig. 1A), with “rough”-LPS derived from S. minnesota causing significantly greater TNFα release than the 3 “smooth”-LPS sources (all P < 0.01, Tukey). Cytokine release was further altered by the use of alternate anticoagulants (P < 0.001, one-way RM-ANOVA) being significantly lower when EDTA was employed than either LH or Na Cit (both P < 0.01, Tukey, Fig. 1C). While incubation of stimulated WB at 37°C was necessary—samples at 20°C releasing minimal TNFα (mean 89.27 pg/mL, Fig. 1E) —no significant difference was observed between agitated (mean 4,049 pg/mL, SD 1653) and nonagitated samples (4,459 pg/mL, SD 1967; P = 0.15, paired t test). Stimulation with either increasing doses of LPS (S. abortus equi), or for variable periods of time, predictably led to differential concentrations of TNFα in the supernatant when aspirated; 1 ng/mL and incubation time >4 h leading to maximal or equivalent responses (Fig. 1B and D). Spontaneous production of TNFα in un-stimulated blood was not observed.


Stimulated Whole Blood Cytokine Release as a Biomarker of Immunosuppression in the Critically Ill: The Need for a Standardized Methodology.

Segre E, Fullerton JN - Shock (2016)

Effect of discrete variables on the ex vivo lipopolysaccharide (LPS)-stimulated whole blood (WB) cytokine release assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836558&req=5

Figure 1: Effect of discrete variables on the ex vivo lipopolysaccharide (LPS)-stimulated whole blood (WB) cytokine release assay.
Mentions: Stimulation with LPS (1 ng/mL) from different bacterial sources elicited significantly different concentrations of TNFα (P < 0.001, one-way RM-ANOVA, Fig. 1A), with “rough”-LPS derived from S. minnesota causing significantly greater TNFα release than the 3 “smooth”-LPS sources (all P < 0.01, Tukey). Cytokine release was further altered by the use of alternate anticoagulants (P < 0.001, one-way RM-ANOVA) being significantly lower when EDTA was employed than either LH or Na Cit (both P < 0.01, Tukey, Fig. 1C). While incubation of stimulated WB at 37°C was necessary—samples at 20°C releasing minimal TNFα (mean 89.27 pg/mL, Fig. 1E) —no significant difference was observed between agitated (mean 4,049 pg/mL, SD 1653) and nonagitated samples (4,459 pg/mL, SD 1967; P = 0.15, paired t test). Stimulation with either increasing doses of LPS (S. abortus equi), or for variable periods of time, predictably led to differential concentrations of TNFα in the supernatant when aspirated; 1 ng/mL and incubation time >4 h leading to maximal or equivalent responses (Fig. 1B and D). Spontaneous production of TNFα in un-stimulated blood was not observed.

Bottom Line: Normalization by monocyte count and pretreatment with a cyclooxygenase inhibitor (indomethacin 10  μM) reduced the coefficient of variation from 47.17% to 32.09%.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Clinical Pharmacology, Division of Medicine, University College London, London, UK.

ABSTRACT

Objective: 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.

No MeSH data available.


Related in: MedlinePlus