• Washing older blood before transfusion reduces plasma iron, improving outcomes from established infection in canines.• In contrast, washing fresh blood before transfusion increases in vivo plasma CFH release, worsening outcomes.In a randomized controlled blinded trial, 2-year-old purpose-bred beagles (n 5 24), with Staphylococcus aureus pneumonia, were exchanged-transfused with either 7-or 42-dayold washed or unwashed canine universal donor blood (80 mL/kg in 4 divided doses). Washing red cells (RBC) before transfusion had a significantly different effect on canine survival, multiple organ injury, plasma iron, and cell-free hemoglobin (CFH) levels depending on the age of stored blood (all, P < .05 for interactions). Washing older units of blood improved survival rates, shock score, lung injury, cardiac performance and liver function, and reduced levels of non-transferrin bound iron and plasma labile iron. In contrast, washing fresh blood worsened all these same clinical parameters and increased CFH levels. Our data indicate that transfusion of fresh blood, which results in less hemolysis, CFH, and iron release, is less toxic than transfusion of older blood in critically ill infected subjects. However, washing older blood prevented elevations in plasma circulating iron and improved survival and multiple organ injury in animals with an established pulmonary infection. Our data suggest that fresh blood should not be washed routinely because, in a setting of established infection, washed RBC are prone to release CFH and result in worsened clinical outcomes. (Blood. 2014;123(9):1403-1411 IntroductionTransfusion of older stored canine universal donor blood in a canine model of experimental Staphylococcus aureus pneumonia results in markedly increased lung injury and mortality rates.1 Transfusion with older blood is also associated with increased levels of cell-free hemoglobin (CFH), transferrin bound iron (TBI), non-TBI (NTBI) and plasma labile iron (PLI). NTBI represents iron excess bound to proteins that do not normally handle circulating iron, and PLI is the toxic iron moiety in plasma. Whereas increased nitric oxide scavenging by CFH causing vasoconstriction and vascular injury and increased available iron promoting bacterial growth represent 2 candidate mechanisms of injury, multiple other biological changes have been documented with increasing blood storage interval.2,3 Some of these changes involve the release into the plasma of biologically active proteins, microvesicles, potassium, acid, and plasticizer, all of which can be reduced by means of standard red cell (RBC) washing procedures. [4][5][6][7][8][9][10] The clinical effect(s) of washing on the RBC storage lesion has not been studied.RBC washing has long been performed to reduce potassium levels in stored blood transfused to neonates, debris from RBCs recovered during surgery, cryoprotectant glycerol from cryopreserved RBCs, and plasma proteins from blood intended for patients who have been sensitized to those proteins.11-13 Automated cell washers cap...

BACKGROUND In experimental pneumonia we found transfused older blood increased mortality and lung injury that was associated with increased in vivo hemolysis and elevated plasma cell-free Hb (CFH), non-transferrin bound (NTBI), and plasma labile (PLI) iron levels. In the present study, we additionally analyze identically treated animals that received lower or higher bacterial doses. STUDY DESIGN AND METHODS Two-year-old purpose-bred beagles (n=48) challenged intrabronchially with Staphylococcus aureus [0 (n=8), 1.0 (n=8), 1.25 (n=24) and ≥1.5 (n=8) × 109 CFU/kg) were exchange transfused with either 7- or 42-day-old canine universal donor blood (80 ml/kg in 4 divided doses). RESULTS The greater increases in cell-free Hb (CFH) with older blood over days following exchange proved relatively independent of bacterial dose. The lesser increases in CFH observed with fresher blood were bacterial dose-dependent potentially related to bacterial hemolysins. Without bacterial challenge, levels of CFH, NTBI, and PLI were significantly higher with older vs. fresher blood transfusion but there was no significant measurable injury. With higher dose bacterial challenge, the elevated NTBI and PLI levels declined more rapidly and to a greater extent after transfusion with older vs. fresher blood, and older blood was associated with significantly worse shock, lung injury, and mortality. CONCLUSION The augmented in vivo hemolysis of transfused older red cells appears to result in excess plasma cell-free Hb and iron release, which requires the presence of established infection to worsen outcome. These data suggest that transfused older red cells increase the risks from infection in septic subjects.

While S-nitrosothiols are regarded as important elements of many NO-dependent signal transduction pathways, the physiological mechanism of their formation remains elusive. Here, we demonstrate a novel mechanism by which cytochrome c may represent an efficient catalyst of S-nitrosation in vivo. In this mechanism, initial binding of GSH to ferric cytochrome c is followed by reaction of NO with this complex, yielding ferrous cytochrome c and GSNO. We show that when submitochondrial particles or cell lysates are exposed to NO in the presence of cytochrome c, there is a robust formation of protein S-nitrosothiols. In the case of submitochondrial particles protein S-nitrosation is paralleled with an inhibition of mitochondrial complex I. These observations raise the possibility that cytochrome c is a mediator of S-nitrosation in biological systems, particularly during hypoxia, and that release of cytochrome c in to the cytosol during apoptosis potentially releases a GSNO synthase activity which could modulate apoptotic signaling.

Background No studies have been performed comparing intravenous iron with transfused red cells (RBC) for treating anemia during infection. In a previous report, transfused older RBC increased free iron release and mortality in infected animals when compared to fresher cells. We hypothesized that treating anemia during infection with transfused fresh RBC, with minimal free iron release, would prove superior to intravenous iron therapy. Methods Purpose-bred-beagles (n=42) with experimental S. aureus pneumonia rendered anemic were randomized to be transfused RBCs stored for 7-days or one of two intravenous iron preparations (7mg/kg), iron sucrose, a widely-used preparation, or ferumoxytol, a newer formulation that blunts circulating iron levels. Results Both irons increased the alveolar-arterial-oxygen gradient at 24–48-hours (p=0.02–0.001), worsened shock at 16-hours (p=0.02– p=0.003 respectively), and reduced survival (transfusion 56%; iron sucrose 8%, p=0.01; ferumoxytol 9%, p=0.04). Compared to fresh RBC transfusion, plasma iron measured by non-transferrin-bound iron (NTBI) levels increased with iron sucrose at 7, 10, 13, 16, 24 and 48-hours, (p=0.04 to p<0.0001) and ferumoxytol at 7, 24 and 48-hours (p=0.04 to p=0.004). No significant differences in cardiac filling pressures or performance, hemoglobin or cell free hemoglobin were observed. Conclusions During canine experimental bacterial pneumonia, treatment of mild anemia with intravenous iron significantly increased free iron levels, shock, lung injury, and mortality compared to transfusion of fresh RBC. This was true for iron preparations that do or do not blunt circulating free iron level elevations. These findings suggest that treatment of anemia with intravenous iron during infection should be undertaken with caution.

Macrophages are main effectors of heme metabolism, increasing transiently in the liver during heightened disposal of damaged or senescent red blood cells (sRBC). Macrophages are also essential in defense against microbial threats, but pathologic states of heme excess may be immunosuppressive. Herein, we uncovered a mechanism whereby an acute rise in sRBC disposal by macrophages led to an immunosuppressive phenotype following intrapulmonary Klebsiella pneumoniae infection characterized by increased extrapulmonary bacterial proliferation and reduced survival from sepsis in mice. The impaired immunity to K. pneumoniae during heightened sRBC disposal was independent of iron acquisition by bacterial siderophores, as K. pneumoniae mutant lacking siderophore function recapitulated findings observed with wildtype strain. Rather, sRBC disposal induced a liver transcriptomic profile notable for suppression of Stat1 and interferon-related responses during K. pneumoniae sepsis. Excess heme handling by macrophages recapitulated STAT1 suppression during infection that required synergistic NRF1 and NRF2 activation but was independent of heme oxygenase-1 induction. Whereas iron was dispensable, the porphyrin moiety of heme was sufficient to mediate suppression of STAT1dependent responses in human and mouse macrophages and promoted liver dissemination of K. pneumoniae in vivo. Thus, cellular heme metabolism dysfunction negatively regulates the STAT1 pathway with implications in severe infection.