A critical comparison of frequently used methods for the analysis of tumornecrosis factor-alpha expression by human immune cells

A variety of methods have been developed for the measurement of tumor necro sis factor (TNF)-alpha synthesis by immune cells. Here we have compared the results of the most common used methods, including in vitro stimulation of whole blood or peripheral blood mononuclear cell (PBMC) cultures with phyt ohaemagglutinin (PHA) or lipopolysaccharide (LPS) and RT-PCR analysis of TN F-alpha transcription in unstimulated PBMC. When we used EDTA treated blood samples we observed a significant correlation between the PHA and LPS stim ulated TNF-alpha responses in whole blood or PBMC cultures. In contrast, TN F-alpha concentrations obtained from PHA and LPS stimulated whole blood cul tures from citrate-treated blood did not show a correlation We also found t hat the PHA stimulated TNF-alpha response was significantly higher in PBMC than in whole blood cultures, whereas the highest LPS stimulated TNF-alpha response was observed in citrate-treated blood. Moreover, the TNF-alpha res ponse in both, citrate and EDTA treated whole blood cultures was significan tly higher after LPS than after PHA stimulation. In contrast, in PBMC cultu res the PHA stimulated TNF-alpha response was significantly higher than the LPS stimulated response. The results of RT-PCR analysis revealed a signifi cant correlation with the PHA stimulated TNF-alpha response, both in whole blood assays and in PBMC cultures. In addition our results demonstrate that these different methods can only be compared when the influence of externa l factors such as the immediate processing of blood samples or the use of a n appropriate anticoagulant and stimulant is considered. (C) 2000 Elsevier Science B.V. All rights reserved.