Levels of plasma cytokines, their receptors or immune-related peptides, are used in experimental and clinical medicine. However, no standards are available regarding the use of anticoagulants or blood processing including the technique of blood collection or time delay between blood sampling and centrifugation. Blood was collected from 10 patients in order to assay interleukin 6, soluble interleukin 6 receptor, soluble interleukin 2 receptor and soluble transferrin receptor by enzyme-linked immunosorbent assay (ELISA). At the time of sampling, five different collecting tubes were used: allowing coagulation, in the presence of EDTA, with EDTA being administered to the serum after its separation, in citrated and in heparinized blood. Blood was centrifuged 10 min after collection and separated serum or plasma was immediately frozen at -20 degrees C. To study the effect of time delay between blood sampling and processing, blood from 11 other patients was sampled, allowing coagulation; or in EDTA tubes and stored at 4 degrees C. The blood was centrifuged 10 min, 8 h and 24 h later, and the separated serum or EDTA plasma was stored at -20 degrees C until thawed for protein determination. Plasma interleukin 6 and soluble interleukin 6 receptor concentrations did not depend on the type of anticoagulant used or the time delay between sampling and processing. Soluble interleukin 2 receptor concentrations were not influenced by time delay before centrifugation, but concentrations were increased 10-fold in EDTA plasma exclusively when EDTA had contact with blood cells. Soluble transferrin receptor concentrations rose progressively with storage time before centrifugation. Contact of EDTA to whole blood or serum resulted in the same increase of soluble transferrin receptor concentration. The results suggest that standardization of the use of anticoagulant and time delay before centrifugation and serum or plasma separation is necessary for soluble interleukin 2 receptor and soluble transferrin receptor.