Analysis of fluid phase mediators

Abstract

Sputum cellular indices are valid, reliable and responsive to change 1–7. Increasingly, numerous inflammatory mediators are being measured in the fluid phase of sputum; these include cytokines, chemokines, granulocyte proteins, markers of vascular leakage, eicosanoids, proteases and others. Many of these mediators are included in table 1⇓, which gives details of methods used by investigators to process sputum and measure mediator levels. The table also provides the median/mean levels measured in studied subject groups to give an indication of the expected levels of these mediators in sputum. However, the reproducibility, precision and validity of many of these measurements in sputum have not been investigated and, therefore, their utility as a research and clinical tool remains uncertain and requires confirmation. The following issues are important in the analysis of mediators: 1) choice of methods for measuring fluid­phase mediators; 2) points to consider when planning an immunoassay; and 3) evaluation of the measurement of a soluble mediator in sputum, i.e. validation of the measurement method. The three main types of method used for the measurement of sputum fluid­phase mediators are bioassays, enzyme assays and immunoassays. ### Bioassays Bioassays rely on the retention of biological activity and the ability to exert a measurable effect, such as proliferation of cells, bone marrow colony formation or chemotaxis 3, 11, 58. Sputum processing with mucolytics such as dithiothreitol (DTT) or dithioerythritol, which are strong reducing agents, may decrease the biological activity of cytokines, many of which rely on disulphide bonds to provide a stable structure for bioactivity. Bioassays also have the disadvantage of being inconvenient, time­consuming and lacking in specificity 59. In addition, the presence of commonly occurring endogenous cytokine inhibitors, although allowing an estimate of net activity, may result in significant underestimation of total cytokine levels 58. ### Enzyme assays Many …