Fresh Red Blood Cells Research Articles

Influence of Red Blood Cell Storage Time on Clinical Course and Cytokine Profile in Septic Shock Patients

Background: Previous investigations have suggested beneficial effects of fresh versus stored red blood cell transfusion in critically ill patients. The present study investigates the effects of red blood cell storage time on the clinical course and hemodynamic and laboratory parameters in patients with septic shock. Patients and Methods: 18 patients with septic shock received 2 erythrocyte units stored for ≤ 7 days (n = 8) or > 7 days (n = 10). The sequential organ failure assessment (SOFA) score was calculated for 7 days. Hemodynamic parameters (cardiac index, extravascular lung water) were determined using transpulmonary thermodilution. Laboratory parameters (lactate, base excess, C-reactive protein, procalcitonin, IL-1β, IL-6, TNF-α, sVCAM-1, sICAM-1) were monitored before and 1, 3, 6, 12, 24, and 48 h after transfusion. The Mann-Whitney-U test and Neumann test were used for group comparison and trend assessment, respectively. Results: We failed to observe significant differences with respect to SOFA scores between patients receiving fresh or stored erythrocytes. However, a significant trend towards an improvement in the SOFA score was found in the group receiving fresh erythrocytes (p < 0.01). No significant differences in hemodynamic or laboratory parameters were found between both groups. Conclusion: While the present findings do not provide clear-cut evidence supporting beneficial effects of fresh red blood cells in septic shock, they warrant larger randomized studies to confirm or refute such effects.

Pulmonary Hypertension During Autologous Transfusion of Stored Blood Is Prevented by Nitric Oxide Inhalation in Lambs

Abstract 39 Introduction:During extended storage, red blood cells (RBCs) undergo biochemical, mechanical, and functional changes. These changes reduce the viability of RBCs, resulting in elevated levels of the potent nitric oxide (NO) scavenger oxyhemoglobin in plasma. Recent studies have shown enhanced systemic vasoconstriction after challenge with tetrameric hemoglobin in murine endothelial dysfunction models. Based on human blood storage techniques, we developed and validated a new model for autologous transfusion of stored RBCs in lambs. We hypothesized that autologous transfusion of leukoreduced ovine RBCs stored for prolonged periods of time would increase plasma hemoglobin levels and induce pulmonary hypertension. We further hypothesized that inhalation of NO would prevent, and endothelial dysfunction would augment the pulmonary vasoconstriction induced by transfusing blood stored for prolonged periods. Methods:We studied three- to four-month-old Polypay lambs weighing 32±2 kg. Similar to current blood bank practices, leukoreduced ovine RBCs were stored in Adsol solution (additive solution-1, AS-1) for either 2 days (fresh red blood cells, FRBCs) or 40 days (stored red blood cells, SRBCs). Post-transfusion recovery of circulating biotinylated FRBCs (n=4) and SRBCs (n=4) was determined by flow cytometry.In separate experiments, 300 ml of autologous FRBCs (n=5) or SRBCs (n=6) were transfused over 30 min into awake lambs, which had been instrumented with carotid artery and pulmonary artery catheters under isoflurane anesthesia. Systemic and pulmonary hemodynamic parameters were measured continuously during and for 4 h after the transfusion. An additional group of animals receiving SRBCs concurrently inhaled 80 parts per million NO (n=4) at FiO2 0.25.We also studied the effects of transfusing FRBCs (n=4) or SRBCs (n=5) in lambs after acutely inducing endothelial dysfunction by IV injection of 25 mg·kg−1 of NG-nitro-L-arginine methyl-ester (L-NAME). An infusion of 5 mg·kg−1·h−1 L-NAME was continued throughout the experiment.Plasma hemoglobin and IL-6 levels were determined before and after transfusion. Tissue samples from the lung and liver were harvested 4 h after transfusion. Relative mRNA levels of inflammatory markers (IL-6, TNF-alpha, and myeloperoxidase) were measured by qPCR.All data are expressed as mean ± SEM. Results:Hemoglobin (41±6 vs. 148±8 mg/dl), potassium (3.7±0.4 vs. 7.9±0.9 mmol/l), and lactate levels (1.7±0.2 vs. 5.9±0.9 mmol/l) were higher in the supernatants of SRBCs than in those of FRBCs. Recovery of circulating biotinylated RBCs 24 h after autologous transfusion was 96±2% in FRBCs and 76±3% in SRBCs.Pulmonary arterial pressure (PAP) transiently increased from 13±0.3 to 18±1 mmHg (p<0.01) and pulmonary vascular resistance index (PVRI) from 108±8 to 156±14 dyne·sec·cm−5·m−2 (p<0.05) during the transfusion of SRBCs, but not FRBCs. This increase of PAP was temporally associated with an increase in plasma concentrations of hemoglobin. Transfusion of SRBCs did not produce systemic vasoconstriction. Concurrent inhalation of NO prevented the pulmonary vasoconstrictor effect induced by transfusing SRBCs, whereas the infusion of L-NAME potentiated the increase in PAP (16±0.3 to 26±2 mmHg, p<0.01) and PVRI (170±15 to 312±38 dyne·sec·cm−5·m−2, p<0.05) associated with transfusion of SRBCs.Plasma IL-6 levels did not change after transfusion of FRBCs or SRBCs. Lung and liver levels of mRNAs encoding inflammatory markers (IL-6, TNF-alpha, and myeloperoxidase) measured 4 h after transfusion did not differ in lambs receiving FRBCs or SRBCs. Conclusions:Ovine RBCs stored for 40 days have many in vitro storage properties and a post-transfusion recovery percentage similar to stored human RBCs. Autologous transfusion of leukoreduced SRBCs induces transient pulmonary hypertension associated with increased cell-free hemoglobin levels. This vasoconstrictor effect is increased in a model of L-NAME-induced endothelial dysfunction. Therefore, patients with disorders associated with pulmonary endothelial dysfunction might be more sensitive to pulmonary vasoconstriction associated with transfusion of SRBCs. Disclosures:Yu:Massachusetts General Hospital: Patents & Royalties. Bloch:MGH has received sponsored research grant funding from Ikaria LCC, the producer of NO gas in the US, in support of Dr. Bloch’s research program: Research Funding. Zapol:Dr. Warren Zapol receives royalties from patents on inhaled nitric oxide licensed by Massachusetts General Hospital to Linde Corp, Munich, Germany, and Ikaria Corp, Clinton, New Jersey. Dr. Zapol has applied for patents on inhaled nitric oxide and blood t: Patents & Royalties.

Endothelial Deficiency Augments and Inhaled Nitric Oxide Prevents the Adverse Hemodynamic Effects of Transfusing Syngeneic Stored Blood in Mice

Abstract 38 Introduction:During storage, red blood cells (RBC) undergo progressive deleterious functional, biochemical and structural changes, producing a “storage lesion”. The storage lesion includes reversible and irreversible changes that begin within hours of storage, progress during storage, release free hemoglobin (Hb) and Hb-containing microvesicles, and impair RBC function and lifespan after transfusion. Our recent studies in mice with endothelial dysfunction revealed an enhanced systemic vasoconstrictor response to infusion of tetrameric Hb or Hb-based oxygen carriers. Therefore, we sought to test the hypothesis that endothelial dysfunction would predispose mice to the vasoconstrictor effects of infusion of fresh and stored murine blood. Methods:Murine leukoreduced RBC from C57BL/6 mice were prepared with 14% CPDA-1 anticoagulant and stored at 4°C for either ≤24 h (fresh red blood cells, FRBC) or 2 weeks (stored red blood cells, SRBC). RBC morphology, as well as ATP levels, 2,3-diphosphoglycerate (2,3-DPG) levels, and P50 were measured in FRBC and SRBC before transfusion. We also prepared murine RBC storage components, i.e. supernatant from FRBC or SRBC, oxidized supernatant from SRBC, and washed SRBC. We studied three groups of mice, awake wild-type mice (WT, C57BL/6) fed a standard diet, WT mice fed a high-fat diet (HFD) for 4–6 weeks (to induce endothelial dysfunction), and diabetic (db/db, C57BL/6 background) mice. Each group was transfused with FRBC, SRBC, and db/db mice also received RBC components (10% of total blood volume). Systolic blood pressure (SBP) was measured every 10 min in awake mice before and 2 h after transfusion with FRBC, SRBC or RBC components. A subgroup of mice transfused with SRBC also breathed air (FiO2=0.21) supplement with nitric oxide (NO, 80 parts per million (ppm)). Invasive hemodynamic measurements were performed in anesthetized mice in order to obtain mean arterial blood pressure, heart rate, and cardiac output. Blood and tissue samples were collected 2 h after FRBC or SRBC transfusion for determination of plasma Hb and iron levels, and measurement of lung and liver levels of mRNA encoding inflammatory cytokines. In addition, heme oxygenase-1 (HO-1) in lung and liver was measured 2 h after FRBC or SRBC transfusion. Results:SRBC were characterized by altered RBC morphology, decreased ATP and 2,3-DPG levels, and a reduced P50. Transfusion of SRBC into awake WT mice fed a standard diet or HFD produced no systemic hemodynamic changes. In contrast, transfusion of SRBC or supernatant from SRBC into db/db mice induced systemic hypertension that was prevented by concurrent inhalation of NO. Infusion of washed SRBC or oxidized SRBC supernatant into db/db mice did not induce systemic vasoconstriction or hypertension. Invasive hemodynamic studies confirmed that transfusion of SRBC and SRBC supernatant induced systemic vasoconstriction and hypertension, but transfusion of washed SRBC did not. Plasma Hb levels were greater in all mouse groups at 2 h after transfusion of SRBC but not after FRBC transfusion. Two hours after transfusion of SRBC, plasma interleukin-6 and iron levels, as well as hepatic HO-1 mRNA levels, were increased in all mouse groups. Conclusions:Syngeneic transfusion of SRBC or only the supernatant from SRBC but not washed SRBC produces systemic hypertension and vasoconstriction in db/db mice, which is prevented by oxidizing the supernatant of SRBC or breathing NO during SRBC transfusion. Infusion of SRBC induced a mild systemic inflammatory response in WT fed a standard diet or HFD, and db/db mice. Transfused cell-free oxyHb in the supernatant released from RBC during storage appears to be responsible for the vasoconstriction produced in db/db mice, since it is prevented by oxidizing the supernatant. Our current data support examining the link between the RBC storage lesion and cardiovascular and immunological perturbations in highly susceptible recipients with endothelial dysfunction. Disclosures:Yu:Massachusetts General Hospital: patents on inhaled nitric oxide and blood transfusion. Bloch:MGH has received sponsored research grant funding from Ikaria LLC, the maker of nitric oxide gas for inhalation in the US, in support of Dr. Bloch’s research program.: Research Funding. Zapol:Dr. Warren Zapol receives royalties from patents on inhaled nitric oxide licensed by Massachusetts General Hospital to Linde Corp, Munich, Germany, and Ikaria Corp, Clinton, New Jersey. Dr. Zapol has applied for patents on inhaled nitric oxide and blood t: Patents & Royalties.

Artificial microvascular network: a new tool for measuring rheologic properties of stored red blood cells

The progressive deterioration of red blood cell (RBC) rheologic properties during refrigerated storage may reduce the clinical efficacy of transfusion of older units. This article describes the development of a microfluidic device designed to test the rheologic properties of stored RBCs by measuring their ability to perfuse an artificial microvascular network (AMVN) comprised of capillary-size microchannels arranged in a pattern inspired by the real microvasculature. In the AMVN device, the properties of RBCs are evaluated by passing a 40% hematocrit suspension of RBCs through the network and measuring the overall perfusion rate. The sensitivity of the AMVN device to the storage-induced change in rheologic properties of RBCs was tested using five prestorage leukoreduced RBC units stored in AS-1 for 41 days. The AMVN perfusion rate for stored RBCs was 26 ± 4% (19%-30%) lower than for fresh RBCs. Washing these stored RBCs in saline improved their performance by 41 ± 6% (the AMVN perfusion rate for washed stored RBCs was still 15 ± 2% lower than for fresh RBCs). The measurements performed using the AMVN device confirm a significant decline in the rheologic properties of RBCs in units nearing expiration and demonstrate the sensitivity of the device to these storage-induced changes. The AMVN device may be useful for testing the effect of new storage conditions, additive solutions, and rejuvenation strategies on the rheologic properties of stored RBCs in vitro.

Impaired adenosine-5′-triphosphate release from red blood cells promotes their adhesion to endothelial cells: A mechanism of hypoxemia after transfusion*

Transfusion of red blood cells has been linked to disappointing clinical outcomes in the critically ill, but specific mechanisms of organ dysfunction after transfusion remain poorly understood. We tested the hypothesis that red blood cell storage impairs the ability of red blood cells to release adenosine-5'-triphosphate and that impaired adenosine-5'-triphosphate release was injurious in vivo, in part through increased red blood cell adhesion. Prospective, controlled, mechanistic study. University research laboratory. Human and mouse blood donors; nude mouse transfusion recipients. Manipulation of adenosine-5'-triphosphate release, supplemental adenosine-5'-triphosphate, and antibodies to red blood cell and endothelial adhesion receptors were used in vitro and in vivo to probe the roles of released adenosine-5'-triphosphate and adhesion in responses to (transfused) red blood cells. The ability of stored red blood cells to release adenosine-5'-triphosphate declined markedly within 14 days after collection despite relatively stable levels of adenosine-5'-triphosphate within the red blood cells. Inhibiting adenosine-5'-triphosphate release promoted the adhesion of stored red blood cells to endothelial cells in vitro and red blood cell sequestration in the lungs of transfused mice in vivo. Unlike transfusion of fresh human red blood cells, stored red blood cell transfusion in mice decreased blood oxygenation and increased extravasation of red blood cells into the lung's alveolar air spaces. Similar findings were seen with transfusion of fresh red blood cells treated with the adenosine-5'-triphosphate release inhibitors glibenclamide and carbenoxolone. These findings were prevented by either coinfusion of an adenosine-5'-triphosphate analog or pretransfusion incubation of the red blood cells with an antibody against the erythrocyte adhesion receptor Landsteiner-Wiener (intercellular adhesion molecule-4). The normal flow of red blood cells in pulmonary microvessels depends in part on the release of antiadhesive adenosine-5'-triphosphate from red blood cells, and storage-induced deficiency in adenosine-5'-triphosphate release from transfused red blood cells may promote or exacerbate microvascular pathophysiology in the lung, in part through increased red blood cell adhesion.

Plasma levels of sphingosine 1-phosphate are strongly correlated with haematocrit, but variably restored by red blood cell transfusions

Anaemia and RBC (red blood cell) transfusion may be associated with worse clinical outcomes, especially with longer blood storage duration prior to transfusion. The mechanisms underlying these harmful effects are unknown. RBCs have been proposed to buffer plasma S1P (sphingosine 1-phosphate), a lysophospholipid essential for the maintenance of endothelial integrity and important in the regulation of haematopoietic cell trafficking. The present study examined the effect of anaemia, RBC transfusion and RBC storage duration on plasma S1P levels. Plasma S1P from 30 individuals demonstrated a linear correlation with Hct (haematocrit; R2=0.51, P<0.001) with no evidence for a plateau at Hct values as low as 19%. RBC transfusion in 23 anaemic patients with baseline mean Hct of 22.2±0.34% (value is the mean±S.D.) increased Hct to 28.3±0.6% at 72 h. Despite an Hct increase, RBC transfusion failed to elevate plasma S1P consistently. A trend towards an inverse correlation was observed between RBC storage duration and the post-transfusion increase in plasma S1P. After 30 days of storage, RBC S1P decreased to 19% of that observed in fresh (3–7-day-old) RBC segments. RBC membranes contain low levels of both S1P phosphatase and S1P lyase activities that may account for the decline in S1P levels with storage. Our results support a role for RBCs in buffering plasma S1P and identify a disturbance in the capacity after transfusion. Changes in S1P content may contribute to an RBC storage lesion. Further studies should investigate the clinical significance of alterations in circulating S1P levels and the potential value of enriching stored RBCs with S1P.

Alloimmunization to transfused HOD red blood cells is not increased in mice with sickle cell disease

Increased rates of red blood cell (RBC) alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease. Transgenic sickle mice, which express human α and β(S) globin, were transfused with fresh or 14-day-stored RBCs containing the HOD (hen egg lysozyme, ovalbumin, and human Duffy(b) ) antigen; some recipients were inflamed with poly(I : C) before transfusion. Anti-HOD alloantibody responses were subsequently measured by enzyme-linked immunosorbent assay and flow crossmatch; a cohort of recipients had posttransfusion serum cytokines measured by bead array. Both Berkeley and Townes homozygous (SS) and heterozygous (AS) mice had similar rates and magnitude of anti-HOD RBC alloimmunization after fresh HOD RBC transfusion compared with control animals; under no tested condition did homozygous SS recipients make higher levels of alloantibodies than control animals. Unexpectedly, homozygous SS recipients had blunted cytokine responses and lower levels of anti-HOD alloantibodies after transfusion of 14-day stored RBCs, compared with control animals. In sum, homozygous β(S) expression and the ensuing disease state are not alone sufficient to enhance RBC alloimmunization to transfused HOD RBCs in two distinct humanized murine models of sickle cell disease under the conditions examined. These data suggest that other factors may contribute to the high rates of RBC alloimmunization observed in humans with sickle cell disease.

Transfusion of fresh murine red blood cells reverses adverse effects of older stored red blood cells

Although a subset of recent studies have suggested that red blood cell (RBC) storage length is associated with adverse patient outcomes, others have shown no such relationship. Adults may be transfused with RBC units of different storage lengths, and existing studies do not take into consideration that fresh RBCs may alter responses to concurrently transfused stored RBCs. To test this possibility, we utilized a murine model and investigated transfusion outcomes of fresh, stored, or fresh-plus-stored RBCs. Fresh, 14-day-stored or fresh plus 14-day-stored leukoreduced RBCs from HOD-transgenic donors (with RBC-specific expression of hen egg lysozyme, ovalbumin, and human Duffy(b)) were transfused into naïve C57BL/6 recipients. Serum cytokines and anti-HOD alloimmunization were evaluated after transfusion. In six of six experiments (n = 90 mice total), a proinflammatory serum cytokine storm of interleukin-6, keratinocyte-derived chemokine/CXCL1, and monocyte chemoattractant protein-1 was observed in transfusion recipients of stored but not fresh RBCs, along with high degrees of anti-HOD alloimmunization. However, concurrent transfusion of fresh HOD RBCs along with stored HOD RBCs significantly decreased these adverse outcomes (p < 0.05). These results are consistent with fresh murine HOD RBCs losing protective properties during storage, and introduce a previously unrecognized variable in RBC storage studies. If translatable to humans, uniform "old blood" groups may be needed in future clinical studies to more accurately investigate the biologic effects of older RBC units.

Rapid clearance of transfused murine red blood cells is associated with recipient cytokine storm and enhanced alloimmunogenicity

Fourteen-day stored red blood cells (RBCs) containing an RBC-specific transgenic antigen (HOD) induce a recipient proinflammatory cytokine storm and are significantly more immunogenic compared to fresh RBCs. Given that recipient mice clear transfused stored RBCs more rapidly than fresh RBCs, we hypothesized that rapid RBC clearance was associated with adverse transfusion outcomes. HOD RBCs were treated by two distinct methods known to lead to rapid posttransfusion RBC clearance: phenylhydrazine or heat. HOD antigen expression was analyzed on the treated cells before transfusion, and RBC recovery, recipient cytokine response, and recipient anti-HOD alloimmunization response were measured after transfusion. Phenylhydrazine and heat treatment each led to near complete RBC clearance in recipients by 24 hours posttransfusion, without significantly altering HOD antigen expression on the transfused RBCs. Recipients of phenylhydrazine- or heat-treated RBCs had elevated circulating levels of keratinocyte-derived chemokine/CXCL-1, monocyte chemoattractant protein-1, and interleukin-6 after transfusion. Furthermore, phenylhydrazine- or heat-treated RBCs were significantly more immunogenic than control RBCs, with a mean 25.1- and 10.3-fold enhancement, respectively, of anti-HOD alloimmunization magnitude by flow cytometric crossmatch. Three separate insults to RBCs (storage, phenylhydrazine, or heat treatment) result in rapid posttransfusion clearance, with a recipient proinflammatory cytokine storm and enhanced alloimmunogenicity. These data are consistent with the hypothesis that rapid clearance of RBCs is causally involved in these outcomes and suggest that human donor RBCs with favorable posttransfusion clearance profiles may be less immunogenic.

Can fructose 1–6 diphosphate (FDP) be an additive to ECC during cardiac surgery?

Extracorporeal circulation (ECC) is the basic support system for patients undergoing cardiac surgery, during which oxygen free radicals (OFR) are generated and involved in ischemic reperfusion injury. This study was done to investigate the protective function of fructose 1–6 diphosphate (FDP) with fresh human red blood cells (RBCs) as a model in vitro and presumably during ECC and cardiac surgery. RBCs were exposed to OFR and mechanical trauma, and then FDP was incubated to RBCs alone and then to RBCs plus OFR. Malondialdehyde (MDA) and red cell filtration rate (RFR) were measured. There was a significant decrease of MDA in RBCs exposed to FDP plus OFR (p<0.01,p<0.001) and an increase of RFR (p<0.05,p<0.001) when compared with RBCs alone or with OFR. A negative correlation was found between MDA and RFR. These results suggest that the protective action of FDP is promising as an additive during ECC in man so as to improve the blood cell rheology and the microcirculation in cardiac surgery with ECC.

Oxidative stress-dependent oligomeric status of erythrocyte peroxiredoxin II (PrxII) during storage under standard blood banking conditions

Although biochemical properties of 2-Cys peroxiredoxins have been extensively studied in various cell lines and organisms, redox-induced structural transitions of peroxiredoxin II (PrxII) in human erythrocytes certainly warrant further investigation. In this work, cytosol and membrane ghosts of both fresh erythrocytes (cells obtained just after blood collection) and 28-day stored erythrocytes were analyzed by proteomics tools. We demonstrated that in fresh red blood cells PrxII exhibits four different oligomeric states in cytosol, whereas no PrxII complexes are in the membrane. The highest molecular weight PrxII protein complex (440 kDa) was proven to derive from the association between tetrameric catalase (CAT, 232 kDa) and decameric PrxII, whereas oligomers at 140, 100 and 67 kDa resulted to be homo-polymeric complexes composed of variable copies of PrxII monomeric subunits. Interestingly, the 440 kDa complex contained both reduced and oxidized (disulphide-linked dimers) PrxII decamers. Upon oxidative stress (28-day storage), the PrxII oligomers at 100 kDa in the cytosol disappeared and the CAT–PrxII hetero-oligomeric complex at 440 kDa is converted to a higher molecular weight structure (480 kDa) due to the presence therein of cross-linked species of PrxII and hemoglobin. More interestingly, oxidized red cell membranes contained the CAT–PrxII complex detected in 0-day cytosol as a consequence of protein recruitments induced by oxidative stress, however it showed a greater percentage of PrxII dimers. Finally, since the adoption of distinct PrxII structures is known to be closely related to different functions, peroxidase activity assays were performed demonstrating a positive reaction for oligomers at 440 kDa (both in cytosol and membrane compartment) and at 140 kDa. Our results contribute to clarify structural and functional switching of peroxiredoxin II in erythrocytes, thus possibly opening new scenarios in the biological roles played by this protein in defense mechanisms against oxidative stress, especially with the reference to red cell storage lesions.

The effects of long‐term storage of human red blood cells on the glutathione synthesis rate and steady‐state concentration

Banked red blood cells (RBCs) undergo changes that reduce their viability after transfusion. Dysfunction of the glutathione (GSH) antioxidant system may be implicated. We measured the rate of GSH synthesis in stored RBCs and applied a model of GSH metabolism to identify storage-dependent changes that may affect GSH production. RBC units (n = 6) in saline-adenine-glucose-mannitol (SAGM) solution were each divided into four transfusion bags and separate treatments were applied: 1) SAGM (control), 2) GSH precursor amino acids, 3) aminoguanidine, and 4) glyoxal. RBCs were sampled during 6 weeks of storage. Rejuvenated RBCs were also analyzed. After 6 weeks, the ATP concentration declined to 50 ± 5.5% (p < 0.05) of that in the fresh RBCs. For control RBCs, the GSH concentration decreased by 27 ± 6.5% (p < 0.05) and the rate of GSH synthesis by 45 ± 8% (p < 0.05). The rate of GSH synthesis in rejuvenated and amino acid-treated RBCs was unchanged after 6 weeks. Modeling identified that the decline in GSH synthesis was due to decreased intracellular substrate concentrations and reduced amino acid transport, secondary to decreased ATP concentration. This study has uniquely shown that the glutathione synthesis rate decreased significantly after 6 weeks in stored RBCs. Our results have identified potential opportunities for improvement of banked blood storage.

The antimicrobial activity of inert oligonuclear polypyridylruthenium(ii) complexes against pathogenic bacteria, including MRSA

The minimum inhibitory concentrations (MIC) of a series of synthetic inert polypyridylruthenium(II) complexes against four strains of bacteria--Gram positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and Gram negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa)--have been determined. The results demonstrate that for the dinuclear ruthenium(II) complexes ΔΔ/ΛΛ-[{Ru(phen)(2)}(2){μ-bb(n)}](4+) {where phen = 1,10-phenanthroline; bb(n) = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n = 2, 5, 7, 10, 12 or 16)} the complexes linked by the bb(12), bb(14) and bb(16) ligands are highly active, with MIC values of 1 μg mL(-1) against both S. aureus and MRSA, and 2-4 and 8-16 μg mL(-1) against E. coli and P. aeruginosa, respectively. The mononuclear complex [Ru(Me(4)phen)(3)](2+) showed equal activity (on a mole basis) against S. aureus compared with the Rubb(12), Rubb(14) and Rubb(16), but was considerably less active against MRSA and the two Gram negative bacteria. For the dinuclear Rubb(n) family of complexes, the antimicrobial activity was related to the octanol-water partition coefficient (logP). However, the highly lipophilic mononuclear complex Δ-[Ru(phen)(2)(bb(16))](2+) was significantly less active than Rubb(16), highlighting the importance of the dinuclear structure. Preliminary toxicity assays were also carried out for the ΔΔ isomers of Rubb(7), Rubb(10), Rubb(12) and Rubb(16) against two human cells lines, fresh red blood cells and THP-1 cells. The results showed that the dinuclear ruthenium complexes are significantly less toxic to human cells compared to bacterial cells, with the HC(50) and IC(50) values 100-fold higher than the MIC for the complex that showed the best potential--ΔΔ-Rubb(12).

Rejuvenation improves roller pump–induced physical stress resistance of fresh and stored red blood cells

Retrospective studies on transfusion recipients suggested that transfusion of older red blood cells (RBCs) was associated with higher morbidity. Similar studies were also done on cardiac surgery patients who were placed on cardiac bypass pumps. It is possible that stored RBCs are more fragile and could be more easily damaged by these pumps, thus leading to additional morbidity. Fresh and stored (42 days) RBCs, rejuvenated and nonrejuvenated, were compared in resistance to physical stress, induced by a roller pump, and osmotic fragility changes during physical stress to model RBCs going through cardiac bypass instruments. In addition, posttransfusion in vivo recovery was evaluated in an immunodeficient mouse model to minimize species differences between transfusion product and recipient. Fresh RBCs were more resistant to both osmotic and physical stress than stored cells. After 2 hours of physical stress, the osmotic stress resistance of fresh cells declined and was the same as for stored cells. Rejuvenated fresh cells did not demonstrate a decline in osmotic resistance during the stress test and both fresh and stored cells had the same improved resistance to osmotic stress before and after the physical stress. Rejuvenation slightly improved recovery of fresh RBCs but almost doubled the recovery of stored cells in the mouse model. Our studies suggest that rejuvenation improves roller pump-induced physical and osmotic stress resistance of stored RBCs.

Duration of Red Cell Storage Influences Mortality After Trauma

Although previous studies have identified an association between the transfusion of relatively older red blood cells (RBCs) (storage ≥ 14 days) and adverse outcomes, they are difficult to interpret because the majority of patients received a combination of old and fresh RBC units. To overcome this limitation, we compared in-hospital mortality among patients who received exclusively old versus fresh RBC units during the first 24 hours of hospitalization. Patients admitted to a Level I trauma center between January 2000 and May 2009 who received ≥ 1 unit of exclusively old (≥ 14 days) vs. fresh (< 14 days) RBCs during the first 24 hours of hospitalization were identified. Risk ratios (RRs) and 95% confidence intervals (CIs) were calculated for the association between mortality and RBC age, adjusted for patient age, Injury Severity Score, gender, receipt of fresh frozen plasma or platelets, RBC volume, brain injury, and injury mechanism (blunt or penetrating). One thousand six hundred forty-seven patients met the study inclusion criteria. Among patients who were transfused 1 or 2 RBC units, no difference in mortality with respect to RBC age was identified (adjusted RR, 0.97; 95% CI, 0.72-1.32). Among patients who were transfused 3 or more RBC units, receipt of old versus fresh RBCs was associated with a significantly increased risk of mortality, with an adjusted RR of 1.57 (95% CI, 1.14-2.15). No difference was observed concerning the mean number of old versus fresh units transfused to patients who received 3 or more units (6.05 vs. 5.47, respectively; p = 0.11). In trauma patients undergoing transfusion of 3 or more RBC units within 24 hour of hospital arrival, receipt of relatively older blood was associated with a significantly increased mortality risk. Reservation of relatively fresh RBC units for the acutely injured may be advisable.

Hemolysis of red blood cells after cell washing with different automated technologies: clinical implications in a neonatal cardiac surgery population

In subsets of pediatric cardiac surgery patients, red blood cells (RBCs) are often washed to reduce extracellular potassium (K) to avoid hyperkalemia, but mechanical manipulation and time delay in issuing washed products may increase hemolysis and K. This study's purpose was to evaluate the quality of washed RBCs with regard to hemolysis and extracellular K using different cell washers as a function of postprocessing time. Fresh (<4 days old) RBCs were washed on COBE 2991 blood cell processors (Model 1 and Model 2) or the Fresenius Continuous AutoTransfusion System (CATS), and K and hemolysis index (HI) were analyzed. Academic pediatric hospitals were surveyed to ascertain practice trends regarding indications for washing, washing device, and expiration time for washed RBCs. K concentration at 24 hours for units washed with the COBE devices met or exceeded prewash values. At 12 hours, there was a significant difference (p < 0.001) in K concentration between all devices, with the CATS maintaining the lowest K concentration. HI increased immediately after wash on all devices and showed a significant difference between the COBE devices and CATS at times of more than 6 hours (p < 0.01). At storage times beyond 4 hours, hemoglobin exceeded 100 mg/dL on the COBE Model 1. Survey of pediatric hospitals indicated that COBE devices are commonly used, and storage time after washing was 12 hours or more in blood banks queried. Hemolysis levels vary among different cell washers. Decreasing the expiration time of units after washing may be warranted.

Fresh Murine Red Blood Cells Abrogate the Enhanced Alloimmunogenicity of Stored Murine Red Blood Cells

AbstractAbstract 663 Introduction:The potential deleterious effects of “older” stored red blood cells (RBCs) are currently an area of intense interest. There are clinical data both to support and refute the concept that transfusing older units of RBCs leads to negative sequelae. Recently, we demonstrated in a murine model that RBCs collected in CPDA-1 and stored at 4 degrees C for 14 days prior to transfusion enhance the alloantibody response as compared to freshly collected and transfused RBCs. Although the most likely explanation is that stored RBCs acquire damage that leads to increased immunogenicity, the data are equally consistent with the hypothesis that fresh RBCs have some immunoregulatory properties that are lost with storage. Materials and Method:The HOD mouse expresses an RBC specific transgene consisting of a triple fusion protein: hen egg lysozyme fused in frame to a portion of ovalbumin fused in frame to human Fyb (HEL-OVA-DUFFY (HOD)). RBCs from the HOD mouse were collected in CPDA-1, leukoreduced (LR) over a Pall neonatal leukoreduction filter, and stored at an Hct of 75% at 4 degrees C for 14 days. C57BL/6 recipients were transfused with either 1 “unit” (i.e. 100 microliters) of fresh LR HOD RBCs, 1 unit of 14 d stored LR HOD RBCs, or a combination of 1 unit of each. Additional control animals received 2 units of fresh LR HOD RBCs or 2 units of 14 d stored LR HOD RBCs. HEL antigen expression on fresh and stored RBCs was evaluated prior to transfusion by flow cytometry. Alloimmunization was assessed 2 weeks post-transfusion using a flow cytometric crossmatch with HOD targets and adjusting background to wild-type FVB controls. Statistical analysis completed using Prism Graphpad software (one-way ANOVA). Results:In 3 of 3 experiments (n=65 recipients), 14 d stored murine RBCs were significantly more immunogenic than fresh RBCs. HEL expression was not significantly altered as a result of storage. However, in each experiment, the rate of immunization in response to 1 unit of stored plus 1 unit of fresh RBCs was lower than alloimmunization to 1 unit of stored RBCs alone. This trend was seen in all 3 experiments, and reached statistical significance in 2 of 3 experiments (p=0.015, p=0.11, p=0.017). Overall, 14 of 15 recipients of stored RBCs responded, 3 of 15 recipients of fresh RBCs responded, and 7 of 15 recipients of 1 unit of fresh and 1 unit of stored RBCs responded by making a detectable alloantibody 2 weeks post-transfusion. The decrease in alloimmunization rate derived from mixing fresh with stored RBCs was not due to dose effect alone, as recipients of 2 units of fresh RBCs responded in a manner similar to that of recipients of 1 unit, and recipients of 2 units of stored RBCs had an enhanced alloantibody magnitude compared to recipients of 1 unit of stored RBCs. Conclusions:These data appear to reject the hypothesis that the increased alloimmune response to stored RBCs is only due to a gain in immunogenicity and support a hypothesis in which fresh RBCs have an immunoregulatory activity that is lost upon storage. In addition to suggesting that RBC immunogenicity changes with storage, these results may be critically important to current and planned human studies. Several randomized clinical studies in humans are proposed or underway to investigate the outcomes resulting from transfusing older vs. younger RBCs, and the study of blood immunogenicity has been proposed to be included. In general, the design of these studies compares “fresh” RBCs to “standard issue” RBCs. Although the average age of “standard issue” RBCs varies from region to region, it often consists of a mixture of older and younger units. Our results suggest that fresh RBCs in the standard issue arm may conceal whatever potential increase in immunogenicity may occur upon storage of RBCs. Moreover, to the extent that alloimmunization results can be extrapolated to other medical outcomes, the same issue may result in false rejection of the hypothesis that older RBCs have a worse outcome in the context of standard issue groups that receive a mixture of fresh and old RBCs. While the current findings are in a murine model, they raise an important issue that has not been studied in humans, and suggest that future studies will require the inclusion of randomized arms receiving solely fresh RBCs or solely stored RBCs. Disclosures:No relevant conflicts of interest to declare.

Transfusions of Red Blood Cells Stored for 40–42 Days Induce Circulating Non-Transferrin-Bound Iron (NTBI) In Healthy Adults

AbstractAbstract 662To compare the effects on iron metabolism of transfusion of packed red blood cells (RBCs) after prolonged (40-42 days) and short-term (3–7 days) storage, we prospectively studied 9 healthy adults. Each volunteer donated a standard, leukoreduced, double RBC unit by automated apheresis and was then transfused with the first autologous RBC unit after 3–7 days of storage and the second after 40–42 days of storage. Timed blood samples were obtained just before each transfusion and immediately, 1, 2, 4, 24, and 72-hours after transfusion. Plasma non-transferrin bound iron (NTBI) was quantified by an ultrafiltration assay, serum hepcidin determined by an immunoassay, and complete blood counts and other laboratory measures obtained by standard methods. For each analyte, differences after transfusion of the older and fresher RBCs were evaluated by 2-way ANOVA with a Bonferroni post-test. At 24 hours after transfusion, similar increases in mean hemoglobin concentration were found with the older (0.83 ± 0.54 (SD) g/dL) and fresher (0.74 ± 0.58 (SD) g/dL) units (p=0.30). At 4 hours after transfusion of older RBCs there was a significant, ~3-fold increase over baseline in mean serum iron (by 160 ± 99 mcg/dL with older vs. −7 ± 25 mcg/dL with fresher RBCs, p<0.001), along with ~2-fold rise in mean total serum bilirubin (by 0.52 ± 0.37 mg/dL with older vs. −0.09 ± 0.18 mg/dL with fresher RBCs, p<0.001). There were no significant differences in circulating lactate dehydrogenase or haptoglobin at any time. The significant increases in mean transferrin saturation (p<0.001) and mean change in plasma NTBI (p<0.001) seen after transfusions of older RBCs are shown in the accompanying Figure. [Display omitted] After transfusion of fresher and older blood, mean serum hepcidin concentrations were not significantly different at any time and no significant differences were found in mean serum C-reactive protein (CRP) or interleukin (IL)-6 levels. Current FDA standards allow refrigerated RBC units to be stored for up to 42 days before transfusion based on the criteria that, on average, less than 25% of the transfused RBCs will be cleared from the circulation in the first 24 hours. In critically ill patients, observational studies identified increases in serious infections, multi-organ failure, and mortality associated with transfusion of older stored RBCs, but the underlying mechanisms have yet to be determined. In healthy volunteers, our results indicate that transfusions of RBCs stored for 3–7 days did not significantly increase serum iron or induce the appearance of plasma NTBI. In contrast, transfusion of RBCs stored for 40–42 days significantly increased serum iron and transferrin saturation and produced substantial amounts of plasma NTBI, in association with the apparently extravascular clearance of a subpopulation of the transfused RBCs. Plasma NTBI can promote bacterial growth and induce oxidative injury. The observed increases in plasma NTBI in healthy volunteers after transfusion of older stored RBCs produced no clinically-evident adverse effects. Nonetheless, in critically ill patients, increases in plasma NTBI may enhance the risk of sepsis, multi-organ failure, and death. Disclosures:Olbina:Intrinsic LifeSciences LLC: Employment. Westerman:Intrinsic Life Sciences: Employment, Membership on an entity's Board of Directors or advisory committees.

Quality of Life Outcomes of the Age of Blood In Highly Transfusion Dependent Patients with Myelodysplasia: Prospective Randomized Controlled N-of-1 Studies

AbstractAbstract 4411 Introduction:Anemia is the most common objective manifestation of the myelodysplastic syndromes (MDS), and excessive fatigue is experienced in over 80% of patients. Transfusion is essential for the management of anemia in these patients. Past studies have investigated the age of transfused blood and its effect on outcome in various acute clinical settings such as critically ill, post-cardiac surgery, trauma, and brain injury patients. However, few studies have investigated the age of transfused blood in chronically transfused patients such as MDS and its impact on quality of life (QoL) and hemoglobin (Hb) levels in these populations. Methods:The aim of our study was to compare QoL outcomes between fresh (transfused within 7 days of donation) and standard issue (transfused between 8 to 42 days, mean 20.5 days locally) blood in highly transfusion dependent patients including those with MDS. The Functional Assessment Cancer Therapy Anemia subscale (FACT-An) was utilized for QoL assessment in a series of prospective “N-of-1” trials design. An “N-of-1” study was performed for each of the participants. Each participant was randomized to receive a total of four transfusions of fresh blood and four transfusions of standard issue blood. Randomization tables were created for each participant “N-of-1” study a priori. The participants, nurses and investigators were blinded to the age of blood administered. Prior to each transfusion, and again after 24 hours, participants completed the FACT-An survey. Post transfusion Hb levels were measured prior to a subsequent transfusion. For each FACT-An parameter and Hb level, mean results were derived for the four fresh blood transfusions and for the four standard blood transfusions. These were analyzed using an unpaired Student's t-test. Results:Twenty patients were enrolled into the study at the London Health Sciences Centre (LHSC), London, Ontario, Canada. Participants included 8 males and 12 females with a mean age of 64.5 years. There were a total of 13 MDS participants (5 males, 8 females) and seven participants (3 males, 4 females) with other diseases that required them to be transfusion dependent. At the time of abstract submission, 1 participant has completed his “N-of-1” study. For this one individual, analysis comparing the pooled results of the four fresh blood transfusions versus the pooled results of the four standard issue blood transfusions was done. No significant difference was seen between fresh blood transfusions versus standard blood transfusions for all of the FACT-An parameters and post-transfusion Hb levels. Conclusions:Improving QoL is an essential component in the management of chronic diseases such as the myelodysplastic syndromes. We have designed a series of “N-of-1” studies to detect a potential difference between fresh versus standard issue red blood cells. The preliminary data from a single participant who has completed this “N-of-1” study suggest that there is no significant difference in QoL measures and Hb levels. The study has fully enrolled and demonstrated the feasibility of this novel approach to evaluating the clinical benefits of transfusion. To our knowledge this is the first reported “N-of-1” study in transfusion. Disclosures:No relevant conflicts of interest to declare.

The effects of cryopreservation on red blood cell rheologic properties

In transfusion medicine, frozen red blood cells (RBCs) are an alternative for liquid-stored RBCs. Little is known about the rheologic properties (i.e., aggregability and deformability) of thawed RBCs. In this study the rheologic properties of high-glycerol frozen RBCs and postthaw stored in saline-adenine-glucose-mannitol medium were compared to those of conventionally liquid-stored and fresh RBCs. Fresh RBCs were obtained from healthy volunteers. Leukoreduced liquid-stored and thawed deglycerolized RBC units were obtained from the Sanquin Blood Bank. RBCs were tested for aggregability (aggregation index [AI]), deformability (elongation index [EI]), and various hematologic variables. The AI of thawed RBCs was reduced, compared to fresh and liquid-stored RBCs (p<0.05). The EI of stored RBCs was significantly enhanced over a shear stress range of 2.0 to 50Pa compared to fresh RBCs (p<0.05). No significant differences in EI between thawed and 21- or 35-day liquid-stored RBCs were observed. The osmotic fragility, hemolysis, mean cell volume, and mean cell hemoglobin concentration of thawed RBCs were markedly altered, compared to fresh and liquid-stored RBCs (p< 0.05). The adenosine triphosphate (ATP) content of thawed RBCs was similar to 3- or 21-day liquid-stored and fresh RBCs. Thawed RBCs are more fragile than conventionally liquid-stored and fresh RBC. The freeze-thaw-wash process, however, did not adversely affect the aggregability and deformability or the ATP content of thawed RBCs. Based on the rheologic properties, cryopreserved RBCs are a valuable alternative to liquid-stored RBCs.