Inter-individual and intra-individual variability in TNF-alpha production by human peripheral blood cells in vitro.
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.


CONCLUSIONS
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.
Bakiyeva
L T
LT
Orthopaedic Research Unit, Box 180, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK. l.t.bakiyeva.01@cantab.net
Brooks
R A
RA
Rushton
N
N
D000328:Adult; D016207:Cytokines / Q000378:metabolism; D004731:Endotoxins / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000378:metabolism; D019904:Polymethyl Methacrylate / Q000737:chemistry; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000378:metabolism
D016207:Cytokines; D004731:Endotoxins; D014409:Tumor Necrosis Factor-alpha; D019904:Polymethyl Methacrylate