NO production from healthy RBCs (type O, Rh negative) measured by flow cytometry after incubation with sera from trauma patients or healthy donors by using DAF-FM diacetate. NO production from RBCs was expressed as mean fluorescence intensity of DAF-FM diacetate fluorescence. A, we used 104 samples from trauma patients (n, 0 hour =104, 6 hour =18, 24 hour =31 and 72 hour =14) and 38 samples from healthy donors (HD). The mean fluorescence intensity [Mean ± SD (min - max)] of nitric oxide production was [1568 ± 730 (578 – 3455)] for healthy donors, [2542 ± 934 (1392– 8093)] for 0 hour, [2809 ± 1189 (1488 – 6137)] for 6 hour, [2084 ± 594 (1318–4134)] for 24 hour and [1469 ± 430 (935 – 2437)] for 72 hour patients. B, Heat inactivation of complement in sera from trauma patients eliminates their ability to trigger NO production. To inactivate complement, sera were incubated 55°C for 30 min. RBCs were incubated with sera from trauma patients before or after inactivation of complement. Individual sera with/without inactivation of complement are shown. Straight lines are used to connect individual points to help visualize how different they are in each sample.