Richard O. Francis

Transfusion of red blood cells after prolonged storage produces harmful effects that are mediated by iron and inflammation

Although red blood cell (RBC) transfusions can be lifesaving, they are not without risk. In critically ill patients, RBC transfusions are associated with increased morbidity and mortality, which may increase with prolonged RBC storage before transfusion. The mechanisms responsible remain unknown. We hypothesized that acute clearance of a subset of damaged, stored RBCs delivers large amounts of iron to the monocyte/macrophage system, inducing inflammation. To test this in a well-controlled setting, we used a murine RBC storage and transfusion model to show that the transfusion of stored RBCs, or washed stored RBCs, increases plasma nontransferrin bound iron (NTBI), produces acute tissue iron deposition, and initiates inflammation. In contrast, the transfusion of fresh RBCs, or the infusion of stored RBC-derived supernatant, ghosts, or stroma-free lysate, does not produce these effects. Furthermore, the insult induced by transfusion of stored RBC synergizes with subclinical endotoxinemia producing clinically overt signs and symptoms. The increased plasma NTBI also enhances bacterial growth in vitro. Taken together, these results suggest that, in a mouse model, the cellular component of leukoreduced, stored RBC units contributes to the harmful effects of RBC transfusion that occur after prolonged storage. Nonetheless, these findings must be confirmed by prospective human studies.

Prolonged red cell storage before transfusion increases extravascular hemolysis

BACKGROUND. Some countries have limited the maximum allowable storage duration for red cells to 5 weeks before transfusion. In the US, red blood cells can be stored for up to 6 weeks, but randomized trials have not assessed the effects of this final week of storage on clinical outcomes. METHODS. Sixty healthy adult volunteers were randomized to a single standard, autologous, leukoreduced, packed red cell transfusion after 1, 2, 3, 4, 5, or 6 weeks of storage (n = 10 per group). 51-Chromium posttransfusion red cell recovery studies were performed and laboratory parameters measured before and at defined times after transfusion. RESULTS. Extravascular hemolysis after transfusion progressively increased with increasing storage time (P < 0.001 for linear trend in the AUC of serum indirect bilirubin and iron levels). Longer storage duration was associated with decreasing posttransfusion red cell recovery (P = 0.002), decreasing elevations in hematocrit (P = 0.02), and increasing serum ferritin (P < 0.0001). After 6 weeks of refrigerated storage, transfusion was followed by increases in AUC for serum iron (P < 0.01), transferrin saturation (P < 0.001), and nontransferrin-bound iron (P < 0.001) as compared with transfusion after 1 to 5 weeks of storage. CONCLUSIONS. After 6 weeks of refrigerated storage, transfusion of autologous red cells to healthy human volunteers increased extravascular hemolysis, saturated serum transferrin, and produced circulating nontransferrin-bound iron. These outcomes, associated with increased risks of harm, provide evidence that the maximal allowable red cell storage duration should be reduced to the minimum sustainable by the blood supply, with 35 days as an attainable goal. REGISTRATION. ClinicalTrials.gov NCT02087514. FUNDING. NIH grant HL115557 and UL1 TR000040.

Strain-specific red blood cell storage, metabolism, and eicosanoid generation in a mouse model

Red blood cell (RBC) transfusion is a lifesaving therapy, the logistic implementation of which requires RBC storage. However, stored RBCs exhibit substantial donor variability in multiple characteristics, including hemolysis in vitro and RBC recovery in vivo. The basis of donor variability is poorly understood.

A comparison of adverse reaction rates for PAS C versus plasma platelet units.

Plasma constituents have been implicated in some types of platelet (PLT) transfusion reactions. Leukoreduced apheresis PLTs stored in InterSol have 65% less plasma than apheresis PLTs stored in 100% plasma (PPs). This study compared transfusion reaction rates in InterSol PLTs (PLT additive solution [PAS] C) versus PPs.

Transfusion of stored blood impairs host defenses against Gram-negative pathogens in mice.

Although human red blood cell (RBC) units may be refrigerator stored for up to 42 days, transfusion of older RBCs acutely delivers a large bolus of iron to mononuclear phagocytes. Similarly, iron dextran circulates in plasma for hours to days and is progressively cleared by mononuclear phagocytes, which return iron to plasma. Finally, malaria infection continuously delivers iron to macrophages by intra‐ and extravascular hemolysis. Studies suggest that iron administration increases infectious risk.

Use of mouse models to study the mechanisms and consequences of RBC clearance

Mice provide tractable animal models for studying the pathophysiology of various human disorders. This review discusses the use of mouse models for understanding red‐blood‐cell (RBC) clearance. These models provide important insights into the pathophysiology of various clinically relevant entities, such as autoimmune haemolytic anaemia, haemolytic transfusion reactions, other complications of RBC transfusions and immunomodulation by Rh immune globulin therapy. Mouse models of both antibody‐ and non‐antibody‐mediated RBC clearance are reviewed. Approaches for exploring unanswered questions in transfusion medicine using these models are also discussed.

Frequency of glucose-6-phosphate dehydrogenase-deficient red blood cell units in a metropolitan transfusion service.

BACKGROUND: Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency is characterized by red blood cell (RBC) destruction in response to oxidative stress. Although blood donors are not routinely screened for G6PD deficiency, the transfusion of stored G6PD‐deficient RBCs may have serious adverse outcomes. By measuring G6PD enzyme activity of RBC units from a large metropolitan hospital transfusion service, we sought to determine 1) the prevalence of G6PD‐deficient RBC units, 2) if G6PD activity changes during storage, and 3) if G6PD activity in segments correlates with its activity in the bags.

Red blood cell transfusion is associated with increased hemolysis and an acute phase response in a subset of critically ill children.

In healthy adults, transfusion of older stored red blood cells (RBCs) produces extravascular hemolysis and circulating non–transferrin‐bound iron. In a prospective, observational study of critically ill children, we examined the effect of RBC storage duration on the extent of hemolysis by comparing laboratory measurements obtained before, and 4 hr after, RBC transfusion (N = 100) or saline/albumin infusion (N = 20). Transfusion of RBCs stored for longer than 4 weeks significantly increased plasma free hemoglobin (P < 0.05), indirect bilirubin (P < 0.05), serum iron (P < 0.001), and non‐transferrin‐bound iron (P < 0.01). However, days of storage duration poorly correlated (R2<0.10) with all measured indicators of hemolysis and inflammation. These results suggest that, in critically ill children, most effects of RBC storage duration on post‐transfusion hemolysis are overwhelmed by recipient and/or donor factors. Nonetheless, we identified a subset of patients (N = 21) with evidence of considerable extravascular hemolysis (i.e., increased indirect bilirubin ≥0.4 mg/dL). In these patients, transfusion‐associated hemolysis was accompanied by increases in circulating non‐transferrin‐bound iron and free hemoglobin and by an acute phase response, as assessed by an increase in median C‐reactive protein levels of 21.2 mg/L (P < 0.05). In summary, RBC transfusions were associated with an acute phase response and both extravascular and intravascular hemolysis, which were independent of RBC storage duration. The 21% of transfusions that were associated with substantial hemolysis conferred an increased risk of inducing an acute phase response. Am. J. Hematol. 90:915–920, 2015.

Rapid Detection of Klebsiella pneumoniae Carbapenemase Genes in Enterobacteriaceae Directly From Blood Culture Bottles by Real-Time PCR

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae are endemic in New York City hospitals and have been associated with serious infections globally. A real-time polymerase chain reaction (RT-PCR) assay was developed to detect carbapenem resistance attributable to KPC from blood culture bottles positive for gram-negative bacilli. Culture confirmation of carbapenemase production included automated imipenem and meropenem susceptibility testing and ertapenem susceptibility testing by disk-diffusion. A total of 323 Enterobacteriaceae isolates were tested, of which 8.7% (n = 28) demonstrated carbapenem-resistance by automated and manual susceptibility testing methods or by RT-PCR. The sensitivity, specificity, and positive and negative predictive values of the RT-PCR assay when compared with the automated method were 92.9%, 99.3%, 92.9%, and 99.3%, respectively, and 96.4%, 99.7%, 96.4%, and 99.7%, respectively, when compared with the ertapenem disk-diffusion method. RT-PCR is a rapid and reliable means of detecting carbapenem resistance due to KPC-plasmids in Enterobacteriaceae directly from blood culture bottles.

Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1–7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13C1-aspartate or 13C5-adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and – preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo. Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates.