Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays.
Author(s): de Jager W, Bourcier K, Rijkers GT, Prakken BJ, Seyfert-Margolis V
Publication: BMC Immunol, 2009, Vol. 10, Page 52
PubMed ID: 19785746 PubMed Review Paper? No
Suggested by: ISBER
Purpose of Paper
The purpose of this paper was to determine the effects of anticoagulant, storage, and freeze-thaw cycling on cytokine levels in serum and plasma.
Conclusion of Paper
Most cytokines measured in this study were expressed at too low levels to allow for comparisons between anticoagulants, but interleukin (IL)-18 was expressed highly in all specimens and was not affected by the type of anticoagulant used. However, levels of IL-6 were much higher in citrated plasma than in heparinated plasma, EDTA-plasma or serum. When plasma and serum specimens were spiked with cytokines, sodium heparin allowed for the most consistent cytokine recovery. When cytokine production was stimulated in sodium heparin-plasma with lipopolysaccharides (LPS), levels of IL-13, IL-15, IL-17 and IL-8 (CXCL8) declined within 1 year of storage at -80 degrees C, but IL-2, IL-4, IL-12 and IL-18 were stable at -80 degrees C for up to 3 years. In stimulated plasma, IL-6 and IL-10 were stable through five freeze-thaw cycles, but IL-4 and tumor necrosis factor alpha (TNFa) increased, while IL-1a, IL-5, IL-13, IL-15, IL-17, interferon (IFN) gamma and CXCL8 levels decreased with freeze-thaw cycling.
Studies
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Study Purpose
The purpose of this study was to determine the effects of anticoagulant, storage, and freeze-thaw cycling on cytokine levels in normal, spiked, and/or stimulated blood.
Summary of Findings:
Most cytokines measured in this study were expressed at too low levels to allow for comparisons between anticoagulants, but interleukin (IL)-18 was expressed highly in all specimens and was not affected by the type of anticoagulant used. However, levels of IL-6 were much higher in citrated plasma than in heparinated plasma, EDTA-plasma or serum. When plasma and serum specimens were spiked with cytokines, sodium heparin allowed for the most consistent cytokine recovery. When cytokine production was stimulated in sodium heparin-plasma with lipopolysaccharides (LPS), levels of IL-13, IL-15, IL-17 and IL-8 (CXCL8) declined within 1 year of storage at -80 degrees C, but IL-2, IL-4, IL-12 and IL-18 were stable at -80 degrees C for up to 3 years. In stimulated plasma, IL-6 and IL-10 were stable through five freeze-thaw cycles, but IL-4 and tumor necrosis factor alpha (TNFa) increased, while IL-1a, IL-5, IL-13, IL-15, IL-17, interferon (IFN) gamma and CXCL8 levels decreased with freeze-thaw cycling.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Immunoassay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature -80 degrees C
Storage Storage duration 0 years
1 year
2 years
3 years
4 years
Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles
3 cycles
4 cycles
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Biospecimen Aliquots and Components Biospecimen components LPS
No LPS or added cytokines
Added cytokines
Biospecimen Acquisition Anticoagulant EDTA
None
Sodium citrate
Sodium heparin