Cytokine assays in human sera and tissues

The use of accurate and sensitive methods for the measurement of cytokines in body fluids is an absolute prerequisite for the proper use of these mediators in clinical practice. Many factors contribute to the complexity of cytokines quantitation: these molecules circulate at very low levels (e.g. pg/ml) under various molecular forms, the existence of circadian rhythms has been described, and the presence of inhibitors (binding proteins, soluble receptors, autoantibodies) can potentially interfere in the assays. Blood collection for cytokines needs particular attention to prevent possible contamination by endotoxins, which can trigger cytokines cellular production after sampling. Bioassays historically preceded immunoassays; the latter techniques are now very popular, but there is an urgent need for standardisation between the different kits commercially available. Nevertheless, due to the essentially local effects of cytokines, the study of their circulating levels only represents the 'tip of the iceberg' and is of limited value for a global understanding of the pathophysiology of these mediators. This explains the development of other approaches to assess the ability of cells to produce cytokines. These include the ELISPOT assay, the measurement of cell-associated cytokines by flow cytometry, and the study of cytokine secretion by isolated peripheral blood mononuclear cells or by whole blood test. All these techniques, associated with a local detection of cytokines by immunohistochemistry or in situ hybridization and reverse transcriptase polymerase chain reaction (RT-PCR), appear to be complementary tools for a better understanding of the multiple aspects of the biology of cytokines.