Serial plasma cell–free hemoglobin levels (CFH), nitric oxide consumption capability, and metHb levels. (A) Serial values of total plasma cell–free hemoglobin (CFH) in 42-day-old (top plot) compared with 7-day-old (bottom plot) transfused stored blood. Mean values for CFH by treatment group over time are represented by a thick gray line. The various forms of dashed lines represent individual animals CFH values over time. The 42-day-old blood was similar to 7-day-old blood before exchange transfusion (4 hours; P = NS) and not elevated compared with baseline. However, CFH was increased in 42- versus 7-day-old blood during transfusion (7, 10, and 13 hours; P = .002 to P < .0001) and remained elevated to 48 hours (24 and 48 hours; both P < .0001). The accuracy of measuring CFH using Drabkins was confirmed by correlating at 24 hours (a time of a wide range of values) with measures using the Winterbourne³⁸  and deconvolution methods (R² > 0.995; see supplemental Figure 2). The slopes are not significantly different from 1 (0.987 ± 0.012, 0.980 ± 0.015), the intercepts are not significantly different from 0 (2.03 ± 1.68, −2.07 ± 2.20), and R² are at least 0.995, confirming the accuracy of our methodology measuring CFH. (B) Similar to the increases in total CFH levels, NO consumption capability of plasma was increased in 42- (closed circle, solid line) versus 7-day-old (open circle, dashed line) from 7 to 48 hours (P = .02 to P < .0001). (C) To confirm the CFH was in the reduced species form, we measured the proportion of metHb in total plasma Hb using spectral de-convolution of absorption spectra. Less than 10% of CFH was in the metHb species form indicating approximately 90% of the CFH was in the reduced oxyHb species form consistent with the plasma NO consumption capability findings. Although low on a percentage basis, 42-day-old blood had significantly more metHb by the end of the first infusion (7 hours, P < .0001) that remained higher until 24 hours (P = .04) compared with 7-day-old blood. Data presented (B-C) as mean ± SE.