Numerous assays are available to investigators for measuring cytokines, and a study by Parson and colleagues [1] shows that different assays yield different results. Which is correct? Why are there differences? The data from Dr. Mann and colleagues show that the presence of circulating soluble receptors to TNF may interfere with TNF immunoassays, depending on the assay kit. In the data presented, 500 ng/mL of the circulating soluble receptors resulted in the loss of detection of TNF by using the assay kit we used. However, the concentration of these receptors in their study was approximately 50 times higher than that in septic patients. Thus, the importance of their results is unclear.

Nevertheless, Dr. Mann and colleagues do raise important issues. Which is more critical to measure: total circulating TNF, TNF bound to its soluble receptor, or free TNF? Because TNF becomes inactive when bound to its receptor, and the infusion of the receptors improves outcome in septic animals, the quantitation of TNF that is bound to its soluble receptors may not be as important as free TNF. The free bioactive TNF that binds to TNF receptors on cells to induce cell activation may then contribute to the morbidity and mortality associated with sepsis.

In the study by Mann and colleagues, the assay kit from T Cell Science (vendor 1) did not detect TNF in the presence of its soluble receptor, whereas the kit from Genzyme Corp. (vendor 3) detected TNF even in the presence of high concentrations of the receptors. If you are using an in vitro cell culture system and you want to know the total amount of TNF produced by the cell, the assay kit from Genzyme Corp. might be better; if you are interested in the amount of free TNF, perhaps the kit from T Cell Science would be better. Even bioassays will not circumvent the limitations of immunoassays. Synergistic interactions exist between low levels of cytokines, resulting in an overestimation of the amount of cytokine present, and, in the case of interleukin-1, the endogenously produced interleukin-1-receptor antagonist interferes with the interleukin-1 bioassay. Interleukin-6 will still trigger a biological response, even if bound to its soluble receptor. Assays for cytokines are still evolving, and investigators should recognize that different assays may yield different results.