Heparin and EDTA as anticoagulant differentially affect cytokine mRNA level of cultured porcine blood cells

Abstract

Cytokine mRNA expression profiles serve to characterize immune cell activation in different test systems. Both, diluted whole blood and isolated PBMC are widely applied for these studies. Comprehensive data regarding the suitability of different anticoagulants for profiling cytokine expression are not available for the pig. Therefore the aim of this study was to compare the effect of two commonly used anticoagulants (heparin and EDTA) on the cytokine expression pattern of porcine blood cells. IL-1alpha, IL-2, IL-4, IL-6, IL-10 and IFN-gamma mRNA levels were detected ex-vivo and upon in-vitro stimulation in diluted porcine whole blood and isolated PBMC by real-time PCR. The cells were stimulated with ConA or LPS, known to act on different target cells and implying different signalling pathways. Additionally the integrity of the isolated RNA was investigated. Ex-vivo cytokine expression pattern of fresh whole blood were not affected by the investigated anticoagulants. In contrast, stimulation of cultured diluted whole blood or PBMC resulted in significant differences depending on the applied anticoagulant. Using EDTA we found a significantly decreased capacity of whole blood to express cytokines. However, isolated PBMC from EDTA anticoagulated blood showed a higher cytokine expression capacity than PBMC from heparinized blood. Comparing diluted whole blood and PBMC we found that cultured porcine whole blood responded better to bacterial products than isolated PBMC, probably because sufficient auxiliary plasma derived factors such as LPS-binding protein, are present. However, isolated PBMC showed a higher T-cell response than diluted whole blood. In conclusion, our findings underline that each application demands a specific assay system.