Detection of intracellular tumor necrosis factor alpha in stimulated fetalcells

Background. It is unknown if immature fetal cells produce tumor necrosis fa ctor (TNF) alpha in the same manner that adult cells do. The aim of this st udy was to determine the feasibility of early detection of intracellular TN F produced by circulating human monocytes (Mo) and lymphocytes (Ly) using h ow cytometry and to compare the stimulation profiles of mature and fetal ce lls. Material and methods. Fetal umbilical cord blood (n = 10) and adult volunte er blood (n = 10) were obtained. In vitro stimulation with endotoxin (LPS) and ionomycin-PMA was performed. Brefeldin A was added to prevent extracell ular transport of TNF. Cell type was determined by using CD-14 marker separ ating monocyte and lymphocyte populations. Antihuman TNF monoclonal antibod y was used to detect intracellular TNF by flow cytometry analysis. Results. Thirty to sixty thousand cells were analyzed per sample. Average T NF expression of stimulated fetal Mo was 28.2%, and that of fetal Ly was on ly 1.1%. Adult stimulated Ly had an average TNF expression of 31.9% and adu lt Mo, 29.6% (P < 0.05 for adult Ly vs fetal Ly). Conclusion. TNF flow cytometry analysis allows assessment of individual cel l types and their ability to produce that cytokine. Fetal cells are able to produce TNF when stimulated, but the stimulation profile of Ly differs fro m that of adult samples. This observation may be of clinical importance in evaluating the response of immature cells to a septic stimulus. Flow cytome try is reliable, reproducible, quick, and easily obtained from a small samp le of peripheral blood. Clinical use will be applicable once appropriate co ntrols are developed, as reported in this study. (C) 1999 Academic Press.