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Inter-individual and intra-individual variability in TNF-alpha production by human peripheral blood cells in vitro
L T Bakiyeva1, R A Brooks, N Rushton
1Orthopaedic Research Unit, Box 180, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK. l.t.bakiyeva.01@cantab.net
Insights
Reliability of TNF-alpha assays is questionable. Both isolated peripheral blood mononuclear cell (PBMC) and whole blood assays (WBA) show high intra-individual variability, making them unsuitable for assessing inter-individual differences in cytokine production.
Area of Science:
- Immunology
- Cellular Biology
Background:
- Tumor Necrosis Factor-alpha (TNF-alpha) production assays are used to differentiate high and low cytokine producers.
- Commonly employed methods include isolated peripheral blood mononuclear cells (PBMC) and whole blood assays (WBA).
Purpose of the Study:
- To assess the reliability and reproducibility of PBMC and WBA for studying TNF-alpha production.
- To determine if these assays can accurately distinguish between high and low cytokine producers.
Main Methods:
- PBMC assays (n=5) were conducted weekly for 6 weeks; WBAs (n=4) were conducted weekly for 4 weeks.
- Stimulatory agents included polymethylmethacrylate particles for PBMC and endotoxin for WBA.
- TNF-alpha levels were quantified using ELISA.
Main Results:
- Significant intra- and inter-individual variability in TNF-alpha secretion was observed.
- Unpredictable fluctuations in cytokine production from the same donor were noted.
- Correcting for cell numbers did not reduce this variability.
Conclusions:
- PBMC and WBA are unreliable for evaluating inter-individual variability in TNF-alpha production due to high intra-individual variability.
- Differences in cell confluency may contribute to variability in PBMC assays.
- These models are not suitable for assessing inter-individual differences in human peripheral blood cytokine secretion.
Background:
Two assays--isolated peripheral blood mononuclear cells (PBMC) and a whole blood assay (WBA)--are commonly used to study TNF-alpha production by an individual in order to distinguish between high and low cytokine producers. We assessed the reliability and reproducibility of these assays.
Methods:
The PBMC assays (n=5) were performed weekly over a period of 6 weeks and the WBAs (n=4) weekly over a 4-week period. Polymethylmethacrylate particles (approx. 6 x 10(2) particles/cell) and optimal concentrations of endotoxin (6.25 and 12.5 ng/ml) were used as the stimulatory agents in PBMC and WBAs, respectively. TNF-alpha production was measured by ELISA.
Results:
There was a high degree of both intra- and inter-individual variability of TNF-alpha secretion, with unpredictable changes in the amount of the cytokine produced by cells from the same donor. This variability could not be eliminated by correcting for cell numbers.
Conclusion:
The PBMC and WBA models of TNF-alpha production by human peripheral blood cells cannot be used for the evaluation of inter-individual variability in cytokine secretion due to the high intra-individual variability observed. In the case of PBMC this is partly due to differences in the confluency of the cells between individuals.
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