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Opioids, including fentanyl, are often administered to patients who may be at risk for the consequences of impaired immune function. We performed a clinical study to test the effects of the synthetic opioid fentanyl on human immune function. Participants received an IV fentanyl initial dose of 3 μg/kg followed by a 2-h IV infusion of 1.2 μg · kg−1 · h−1. Peripheral blood was drawn before and after fentanyl administration to test for neutrophil phagocytic function, neutrophil antibody-dependent cell cytotoxicity, natural killer cell cytotoxicity, percentage of lymphocyte populations, T-lymphocyte proliferative response, and in vivo antibody response to a pneumococcal vaccine inoculation given at the end of the fentanyl infusion. Fentanyl exposure under the conditions of this study caused a rapid and significant increase in natural killer cell cytotoxicity, which was coincident with an increase in the percentage of CD16+ and CD8+ cells in peripheral blood. Fentanyl did not significantly affect any of the other immune measurements.