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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

Variations in reporting convention of data from the ex vivo LPS-stimulated WB cytokine release assay.
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Figure 2: Variations in reporting convention of data from the ex vivo LPS-stimulated WB cytokine release assay.

Mentions: Variations in sample handling prior to LPS stimulation caused discrepancies in assayed TNFα. Both time to LPS addition and storage temperature significantly effected supernatant TNFα concentration (both P < 0.001, 2-way RM ANOVA), increasing time leading to lower release and 20°C storage eliciting significantly greater release than either 4°C or 37°C (both P < 0.001, Tukey). No significant interaction was observed between the two variables (P = 0.2). Predilution of blood, for ease of technical performance and to enhance supernatant yield, as expected, leads to a fall in TNFα concentration per unit supernatant with an increased ratio of media to WB (Fig. 2A). This decrease was however not directly proportional, normalization by dilution factor failing to create equivalence between technical approaches (P < 0.01, one-way RM-ANOVA).


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)

Variations in reporting convention of data from the ex vivo LPS-stimulated WB cytokine release assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Variations in reporting convention of data from the ex vivo LPS-stimulated WB cytokine release assay.
Mentions: Variations in sample handling prior to LPS stimulation caused discrepancies in assayed TNFα. Both time to LPS addition and storage temperature significantly effected supernatant TNFα concentration (both P < 0.001, 2-way RM ANOVA), increasing time leading to lower release and 20°C storage eliciting significantly greater release than either 4°C or 37°C (both P < 0.001, Tukey). No significant interaction was observed between the two variables (P = 0.2). Predilution of blood, for ease of technical performance and to enhance supernatant yield, as expected, leads to a fall in TNFα concentration per unit supernatant with an increased ratio of media to WB (Fig. 2A). This decrease was however not directly proportional, normalization by dilution factor failing to create equivalence between technical approaches (P < 0.01, one-way RM-ANOVA).

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