Production of human tumor necrosis factor from whole blood ex vivo

Since monocytes are the major source of tumor necrosis factor-alpha (TNF) in whole blood, we have developed a new method for stimulating TNF production using heparinized human whole blood instead of isolated monocytes. Test agents were dissolved in endotoxin-free buffer and 25 microliters aliquots were added directly to 225 microliters of whole blood. Following incubation at 37 degrees C, TNF levels were measured directly from diluted plasma (10%) by enzyme-linked immunoassay or L929 bioassay. Unstimulated whole blood released no detectable TNF (less than 150 pg/ml; less than 40 U/ml) over a 24 hour incubation period, but significant TNF release could be detected following a 6 hour incubation with 10 ng/ml of LPS (2163 pg/ml; 390 +/- 240 U/ml). In contrast to methods using isolated monocytes, the measurement of TNF production in whole blood ex vivo avoids monocyte activation by an adherence step, reduces the risk of contamination by endotoxin during isolation, and eliminates potentially confounding exogenous serum factors. More importantly, this method examines monocyte TNF release in response to stimuli in the relevant physiologic milieu.