Ex vivo induction of cytokine mRNA expression in human blood samples

The interest in the quantitative analysis of cytokine mRNA profiles has inc reased 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 kno wn to what extent differences in blood collection and preparation technique s may cause ex vivo alteration of quantitative cytokine mRNA levels. We the refore evaluated the effect of blood sampling and the impact of cell separa tion on interleukin (IL)-2, IL-4, interferon (IFN)-gamma and tumour necrosi s factor (TNF)-alpha mRNA expression in an intraindividual study design (n= 8). Two different blood sampling procedures were applied. A whole blood sam ple 1 was collected by constant moderate blood flow into a blood collection tube containing lithium-heparin. Moreover, a second sample from the same d onor was collected by a 5-fold acceleration of blood flow. Furthermore, per ipheral blood mononuclear cell (PBMC) were isolated from the first whole bl ood sample by density separation over Ficoll-Hypaque. The quantification of cytokine mRNA expression was performed by real-time PCR in native whole bl ood/PBMC samples or unstimulated cultures. We found a significant increase of IL-2, IL-4 and TNF-cu mRNA expression (P=0.018, P=0.038, P=0.018) in who le blood samples collected by rapid sampling. The isolation of PBMC by dens ity 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 b lood sample preparation. Our data clearly demonstrate that differences in t he blood sampling technique or cell separation should be considered as impo rtant factors for non-physiological ex vivo induction of cytokine mRNA expr ession. The current data emphasize the need for data on the impact of ex vi vo variation in order to extract reliable and consistent information, parti cularly when cytokine mRNA expression data from healthy blood donors are in cluded in clinical studies. (C) 2001 Elsevier Science B.V. All rights reser ved.