Ex vivo induction of cytokine mRNA expression in human blood samples

The interest in the quantitative analysis of cytokine mRNA profiles has increased substantially in recent years. This is based on the potential use of basal cytokine mRNA expression as sensitive markers for in vivo lymphocyte activation in a variety of clinical settings. However, it is less well known to what extent differences in blood collection and preparation techniques may cause ex vivo alteration of quantitative cytokine mRNA levels. We therefore evaluated the effect of blood sampling and the impact of cell separation on interleukin (IL)-2, IL-4, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha mRNA expression in an intraindividual study design (n=8). Two different blood sampling procedures were applied. A whole blood sample 1 was collected by constant moderate blood flow into a blood collection tube containing lithium-heparin. Moreover, a second sample from the same donor was collected by a 5-fold acceleration of blood flow. Furthermore, peripheral blood mononuclear cell (PBMC) were isolated from the first whole blood sample by density separation over Ficoll-Hypaque. The quantification of cytokine mRNA expression was performed by real-time PCR in native whole blood/PBMC samples or unstimulated cultures. We found a significant increase of IL-2, IL-4 and TNF-alpha mRNA expression (P=0.018, P=0.028, P=0.018) in whole blood samples collected by rapid sampling. The isolation of PBMC by density gradient separation prompted on upregulation of the mRNA levels of IL-2, IL-4 and TNF-alpha 5-9-fold (P=0.018, P=0.018, P=0.018). In contrast, IFN-gamma mRNA expression was not significantly influenced by differences in blood sample preparation. Our data clearly demonstrate that differences in the blood sampling technique or cell separation should be considered as important factors for non-physiological ex vivo induction of cytokine mRNA expression. The current data emphasize the need for data on the impact of ex vivo variation in order to extract reliable and consistent information, particularly when cytokine mRNA expression data from healthy blood donors are included in clinical studies.