Spurious tumor necrosis factor-α and interleukin-6 production by human monocytes from blood collected in endotoxin-contaminated vacutainer blood collection tubes [2]

Department of Psychology, University of California, Los Angeles, Los Ángeles, California, United States
Clinical Chemistry (Impact Factor: 7.91). 12/2004; 50(11):2215-6. DOI: 10.1373/clinchem.2004.040162
Source: PubMed
9 Reads
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Given the shortcomings in the measurement of pyrogenic contamination of pharmaceuticals and/or test substances by means of the rabbit pyrogen test and the Limulus amoebocyte lysate (LAL) test, several in vitro pyrogen tests have been developed based on the measurement of cytokine production by monocytes. In this study we measured cytokine production (IL-6, IL-8, IL-1beta, and TNF) in diluted whole blood (WB), mononuclear cells (MNC), and HEK cells stably transfected with CD14 and Toll-like Receptor-2 (TLR2) or TLR4, after stimulation with both standard pyrogens and contaminated substances. Our study demonstrated that in MNC, IL-6 production was more sensitive to pyrogen stimulation than IL-1beta and TNF production. The sensitivity of WB IL-8 production for pyrogens was comparable with that of MNC IL-6 production, but higher than WB IL-6 production. MNC IL-8 production as readout for pyrogenic stimulation was not useful due to high background IL-8 production. Surprisingly, contaminated culture media potently stimulated WB IL-8 production, but not MNC IL-6 production. Finally, the value of TLR-transfected HEK cells in the detection of pyrogenic contamination as well as the role of IL-10 in interindividual differences in cytokine production, is discussed. To summarize, the results presented herein together with literature data indicate that the measurement of WB IL-8 production may represent an advantageous alternative to the measurement of MNC IL-6 production, for the detection of pyrogenic contamination of pharmaceuticals.
    Journal of Immunological Methods 08/2008; 336(1):45-55. DOI:10.1016/j.jim.2008.03.010 · 1.82 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Biomarker assays are often conducted on whole blood samples in the course of drug development studies. Because bacterial lipopolysaccharide (LPS) (endotoxin) contamination is known to cause spontaneous cytokine production by monocytes, contamination of blood collection tubes may interfere with biomarker assay results. Whole blood from healthy donors was collected into plastic or glass sodium (Na(+))-heparin Vacutainer() blood collection tubes and heparinized syringes. Samples were analyzed for phosphoprotein response, cytokine production, and RNA expression. Tubes were tested for endotoxin contamination by use of the limulus amoebocyte lysate assay. Results of phospho-flow cytometry, branched DNA (bDNA), and ELISA assays indicated that a specific lot (#5339582) of plastic Na(+)-heparin Vacutainer tubes was highly contaminated with an endotoxinlike substance, and contamination was confirmed by the limulus amoebocyte lysate assay. Analysis of multiple-analyte panels revealed that analytes whose changed expression was predictive of LPS stimulation were increased when whole blood was incubated in contaminated tubes for 6 or 18 h. Two additional lots of plastic tubes tested had detectable amounts of endotoxin sufficient to strongly alter phospho-flow cytometry analyses, as determined by the fold change in phosphorylation of p38 mitogen-activated protein kinase in response to tumor necrosis factor alpha and LPS. In contrast, 3 lots of glass tubes had substantially lower levels of spontaneous blood activation. Endotoxin contamination associated with tubes from 3 lots of a particular type of plastic Na(+)-heparin Vacutainer tube dramatically affected biomarker assay measurements. Prescreening these tubes is suggested before their use in clinical sample analysis.
    Clinical Chemistry 09/2010; 56(9):1483-91. DOI:10.1373/clinchem.2006.144618 · 7.91 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Controlled, experimental studies on the effects of acute sleep loss in humans have shown that mediators of inflammation are altered by sleep loss. Elevations in these mediators have been found to occur in healthy, rigorously screened individuals undergoing experimental vigils of more than 24h, and have also been seen in response to various durations of sleep restricted to between 25 and 50% of a normal 8h sleep amount. While these altered profiles represent small changes, such sub-clinical shifts in basal inflammatory cytokines are known to be associated with the future development of metabolic syndrome disease in healthy, asymptomatic individuals. Although the mechanism of this altered inflammatory status in humans undergoing experimental sleep loss is unknown, it is likely that autonomic activation and metabolic changes play key roles.
    Best Practice & Research: Clinical Endocrinology & Metabolism 10/2010; 24(5):775-84. DOI:10.1016/j.beem.2010.08.014 · 4.60 Impact Factor
Show more

Similar Publications