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Introdução: As transfusões de sangue e as Reações transfusionais (RT) têm tido grande destaque nas discussões e estudos da hemoterapia atual, devido a necessidade e relevância para a prática transfusional e na busca em qualificar as transfusões e refinar a classificação das RT. As reações transfusionais febris não hemolíticas (RTFNH) apresentam um crescente no número de notificações e despertam a necessidade de mais estudos. Durante a estocagem dos hemocomponentes, ocorrem uma série de alterações morfológicas, aumento de potássio (K+) extracelular, hemólise e aumento de hemoglobina (Hb) sobrenadante. Analisar a qualidade e viabilidade do hemocomponente pode nos levar a verificar os fatores preditores de uma RT, procurando minimizar os riscos e selecionar um hemocomponente de melhor qualidade ao paciente. Objetivos: Avaliar potenciais fatores etiológicos na precipitação das RTFNH por meio da mensuração na concentração de sódio (Na+) e K+ no sobrenadante, a contagem leucocitária por mcL, o cultural e o Hematócrito (Ht) e Hb da bolsa de concentrado de hemácias (CH) envolvidas, comparando estes parâmetros em relação a um grupo controle de bolsas de CH. Analisar e comparar o perfil dos pacientes envolvidos com a RTFNH e do grupo controle e, estimar a frequência de culturais coletados positivos e os germes envolvidos. Metodologia: Estudo de caso-controle com seleção de amostras a partir de notificações de suspeita de RTFNH ao Serviço de Hemoterapia de um Hospital Universitário de Porto Alegre - RS, no período de setembro de 2015 a setembro de 2016. O grupo controle foi selecionado a partir da mesma população de bolsas, sendo pareadas por tipagem sanguínea e data de vencimento do hemocomponente, numa proporção de 1:2,1. Resultados: o total incluído foi de 124 bolsas, sendo 39(30,5%) do grupo RT e 85(69,5%) do grupo controle, onde uma série de variáveis foram avaliadas. A média de dias de estocagem das bolsas foi de 10,7(DP=6,7) dias, sendo que no grupo RT 12,1(DP=8,1), foi significativamente maior que no grupo controle 10(DP=5,8) com (P=0,037). Também quando avaliamos as dosagens de Ht as médias verificadas foram de 68,3(DP=7,27), sendo no grupo RT 71(DP=81) e 67(DP=6,5) no grupo controle e, na comparação dos grupos, observamos um P

Background Pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide. In vitro studies have demonstrated its effects on storage lesion, but little routine quality control data on blood banking outcomes have been reported. Materials and Methods Swirling of distributed products was monitored before and after implementation of Intercept pathogen inactivation. Metabolic parameters pH, glucose and lactic acid were determined in a random cohort of expired pathogen-inactivated products. Storage lesion indicators in apheresis concentrates with premature low swirling were compared to concentrates with normal swirling. Results During validation for implementing Intercept pathogen inactivation, pH and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pathogen-inactivated pooled buffy coat-derived products. In routine products, glucose exhaustion was more often found in apheresis compared to buffy coat-derived platelet concentrates despite 3–7% more plasma carryover in the former. Annual incidence of premature low swirling increased significantly by 50% following implementation of pathogen inactivation implementation for apheresis but not for pooled buffy coat platelet concentrates. In addition, apheresis concentrates with premature low swirling had a significantly higher median platelet count (5·0 × 1011) than unaffected products (3·5 × 1011). Conclusion The risk of increased storage lesion rates following Intercept pathogen inactivation is higher for apheresis than for buffy coat-derived platelet concentrates, especially when platelet contents are higher than 5·0 × 1011.

BACKGROUND The continued growth in the uses of umbilical cord blood (UCB) will require the development of meaningful post-thaw quality assays. This study examines both conventional and new measures for assessing UCB quality after long-term storage.

Red blood cells (RBCs) collected for transfusion deteriorate during storage. This deterioration is termed the "RBC storage lesion." There is increasing concern over the safety, therapeutic efficacy, and toxicity of transfusing longer-stored units of blood. The severity of the RBC storage lesion is dependent on storage time and varies markedly between individuals. Oxidative damage is considered a significant factor in the development of the RBC storage lesion. In this study, the variability during storage and heritability of antioxidants and metabolites central to RBC integrity and function were investigated. In a classic twin study, we determined the heritability of glutathione (GSH), glutathione disulfide (GSSG), the status of the GSSG,2H(+)/2GSH couple (Ehc), and total glutathione (tGSH) in donated RBCs over 56 days of storage. Intracellular GSH and GSSG concentrations both decrease during storage (median net loss of 0.52 ± 0.63 mM (median ± SD) and 0.032 ± 0.107 mM, respectively, over 42 days). Taking into account the decline in pH, Ehc became more positive (oxidized) during storage (median net increase of 35 ± 16 mV). In our study population heritability estimates for GSH, GSSG, tGSH, and Ehc measured over 56 days of storage are 79, 60, 67, and, 75%, respectively. We conclude that susceptibility of stored RBCs to oxidative injury due to variations in the GSH redox buffer is highly variable among individual donors and strongly heritable. Identifying the genes that regulate the storage-related changes in this redox buffer could lead to the development of new methods to minimize the RBC storage lesion.

Background Transfusion of long-stored red blood cells (RBCs) is associated with decreased in vivo RBC recovery, delivery of RBC breakdown products, and increased morbidity and mortality. Reducing the burden of this RBC “storage lesion” is a major challenge in transfusion medicine. Additive solution-7 (AS-7) is a new RBC storage solution designed to improve RBC metabolism by providing phosphate and increasing buffering capacity. Study Design and Methods Storage quality in AS-7 was measured in a prospective, randomized, three-center trial using units of whole blood from healthy human subjects whose RBCs were stored for up to 56 days in AS-7 (n = 120) or for 42 days in the control solution AS-1 (n = 60). Results Hemolysis and shedding of protein-containing microvesicles were significantly reduced in RBCs stored in AS-7 for 42 and 56 days compared with RBCs stored in AS-1. Autologous in vivo recoveries of RBCs stored in AS-7 was 88 ± 5% at 42 days (n = 27) and 82 ± 3% at 56 days (n = 27), exceeding recoveries of RBCs stored in currently used solutions. Conclusion Increasing the phosphate, pH range, and buffer capacity of a RBC storage system allowed RBCs to be stored better and longer than currently approved storage systems. AS-7 ameliorates the long-term storage lesion resulting in significantly increased viability in vitro and in vivo.

Background Platelet concentrates (PCs) can be prepared using three methods: platelet (PLT)-rich plasma, apheresis, and buffy coat. The aim of this study was to obtain a comprehensive data set that describes metabolism of buffy coat–derived PLTs during storage and to compare it with a previously published parallel data set obtained for apheresis-derived PLTs. Study Design and Methods During storage we measured more than 150 variables in 8 PLT units, prepared by the buffy coat method. Samples were collected at seven different time points resulting in a data set containing more than 8000 measurements. This data set was obtained by combining a series of standard quality control assays to monitor the quality of stored PLTs and a deep coverage metabolomics study using liquid chromatography coupled with mass spectrometry. Results Stored PLTs showed a distinct metabolic transition occurring 4 days after their collection. The transition was evident in PLT produced by both production methods. Apheresis-derived PLTs showed a clearer phenotype of PLT activation during early days of storage. The activated phenotype of apheresis PLTs was accompanied by a higher metabolic activity, especially related to glycolysis and the tricarboxylic acid cycle. Moreover, the extent of the activation differed between bags resulting in interbag variability in the storage lesion of apheresis-prepared PLTs. This may be related to donor-related polymorphism. Conclusion This study demonstrated two discrete metabolic phenotypes in stored PLTs prepared with both apheresis and buffy coat methods. PLT activation occurs during the first metabolic phenotype and might lead to a low reproducibility of the apheresis PCs.

Background In China apheresis platelets (PLTs) are stored in plasma for only 5 days, resulting in PLT inventory pressures. Anandamide (ANA) was reported to be a potential agent to inhibit PLT apoptosis. The aim of this study was to evaluate the characteristics of extended storage PLTs in plasma treated with ANA in vitro . Methods Apheresis PLTs ( n = 20) were prepared in plasma treated with ANA, and stored at 22 °C for up to 11 days. On day 1, 3, 5, 7, 9 and 11, PLTs were tested for PLT count, mean PLT volume (MPV), PLT distribution width (PDW), pH, pCO 2 , pO 2 , hypotonic shock response (HSR), phosphatidylserine (PS) exposure and soluble P-selectin content. Results PLTs stored in plasma with/without ANA didn't show significant differences during the first 5 days of storage. From the 7 th day on, PLTs stored in plasma with ANA displayed significantly lower PS expression, soluble P-selectin content and higher HSR scores than those stored in plasma without ANA ( P 0.05), respectively. Conclusion The extended storage of PLTs in plasma treated with 0.5 µmol/l ANA showed better characteristics of the PLTs, compared with the control group, which was suggested to potentially alleviate the PLT storage lesion.

Objective To evaluate the effect of leukoreduction (LR) as compared to standard nonleukoreduced (NLR) units on cytokine concentrations in canine erythrocyte concentrates during regular storage time. Design Randomized, experimental study. Setting University teaching hospital. Animals Ten random-source research dogs. Interventions One unit of whole blood was collected from each dog and randomized to standard processing (NLR, n = 5) or prestorage LR (n = 5). All units were stored at 4°C. Samples were aseptically collected from each unit weekly for 5 weeks. On day 35, 1 mL of blood was collected from each unit and submitted for aerobic culture. Measurement and Main Results An ELISA assay was used to analyze the concentrations of IL-1β, IL-8, TNF-α, and IL-10. There were no significant effects of either group or storage time for IL-1β, IL-10, or TNF-α. IL-8 concentration was significantly increased over the storage period in NLR units, and was significantly higher compared to LR units on days 28 and 35. Aerobic culture was negative on all units. Conclusions This study demonstrated a marked, storage time-dependent accumulation of IL-8 in canine erythrocyte concentrates. Prestorage LR attenuated the accumulation of IL-8. This chemokine may contribute to the proinflammatory effects of transfusion of stored erythrocyte concentrates.

Background Hypothermic storage of red blood cell (RBC) concentrates for up to 42 days is associated with biochemical, molecular, morphological, and mechanical modifications. This "storage lesion" increases with storage duration and is associated with increased clearance of transfused storage-damaged RBCs from the recipient's circulation in the first few hours post-transfusion. This rapid clearance reduces transfusion efficacy, but how it occurs is not fully elucidated. RBCs with reduced surface area called "storage-induced micro-erythrocytes" (SMEs) were recently described. Their proportion increases from 2% to 23% during storage. Their reduced surface-to-volume ratio is expected to induce rapid mechanical clearance by the spleen. We aimed to evaluate whether SMEs can be used as a marker of transfusion efficacy, if this subpopulation of RBCs is preferentially cleared by the spleen after transfusion, and if so, by which mechanisms. Methods We evaluated the proportion of SMEs in stored RBC concentrates in vitro using ImageStream and correlated it to the 51Chromium-labeled 24h post-transfusion recovery (24hPTR) in vivo in 31 healthy human volunteers. We then investigated the fate of SMEs during 8 ex vivo perfusions of human spleens (16 RBC concentrates stored for 35-42 days). Finally, we developed a mouse transfusion model to assess the fate of SMEs in vivo and determine their main mechanisms of clearance. Results The proportion of SMEs in RBC concentrates at day 42 of storage correlated negatively with 24hPTR in healthy volunteers (r=-0.42, P5 passages through the spleen). The percentage of SMEs correlated with splenic retention rate ex vivo (r=0.46, p In our mouse transfusion model, SMEs accumulated during RBC storage. The 24hPTR also decreased with storage duration (64% on Day 14 vs. 95% on Day 1). The decrease in 24hPTR of long-stored RBCs was mostly due to clearance of the SME subpopulation. SME and morphologically normal long-stored RBC subpopulations displayed clearances of 83% and 13%, respectively. Stored RBCs accumulated predominantly in the spleen post-transfusion, and were mainly ingested by macrophages. In macrophage-depleted mice, 24hPTR improved (from 64% to 79%), splenic accumulation and clearance of SMEs were delayed, and the proportion of inflammatory monocytes increased and mediated clearance. In splenectomized mice, clearance of SMEs was not delayed, but increased accumulation was observed in the liver and bone marrow, and increased erythrophagocytosis by inflammatory monocytes was also observed. Conclusions We show that the proportion of SMEs correlates with 24hPTR in healthy human volunteers and with retention in human spleens perfused ex vivo. In vivo mouse data confirms these findings, showing that SMEs are cleared from the recipient circulation during the 24h following transfusion. Clearance of SMEs is delayed in macrophage-depleted mice, suggesting a central role of macrophages in this process. The human spleen is also likely to clear SMEs from the recipient's circulation, as suggested by experiments with human spleens perfused ex vivo. However, the spleen is not required, because SME clearance is not affected in splenectomized mice. This suggests that other organs may compensate to remove SMEs and highlights the importance of eliminating these morphologically-altered RBCs. Finally, quantification of SMEs is an operator-independent, reproducible marker of transfusion efficacy. It can be used to assess the potential of new processes to prepare and store RBC concentrates. Pre-transfusion quantification of SMEs could benefit chronically transfused patients, for whom improved transfusion efficacy is expected to reduce transfusion-induced iron overload. Disclosures Roussel: Zimmer Biomet: Research Funding. MARIN:Zimmer Biomet: Research Funding. Spitalnik:Hemanext: Membership on an entity's Board of Directors or advisory committees; Tioma, Inc.: Consultancy. Hermine:AB science: Consultancy, Equity Ownership, Honoraria, Research Funding; Celgene: Research Funding; Novartis: Research Funding. Buffet:Zimmer Biomet: Research Funding. Amireault:Zimmer Biomet: Research Funding.

The medical effects of transfusing stored RBCs is an area of significant concern that has received substantial attention in recent years. Retrospective trials show all possible outcomes, including sequelae from transfusing older RBCs, no difference between older and fresher RBCs, and a benefit to older RBCs. Several prospective clinical trials are under way to further investigate potential untoward effects of stored RBCs. Thus far, the issue of potential sequelae from transfusing stored RBCs remains a highly controversial issue. However, what is not controversial is that RBC storage is an unnatural state during which a series of substantial changes take place to the stored RBCs. These changes result in the formation of cellular and chemical entities known to have biological activities in other settings, giving rise to several distinct hypotheses by which stored RBCs may alter recipient biology. Herein, the clinical background and basic science of RBC storage are reviewed, with a particular focus on factors that may complicate hypothesis testing and obfuscate underlying biologies. The complexity of the RBC storage lesion, donor-to-donor variation, and the diversity of recipient pathophysiologies remain a challenge to prospective trials assessing the safety of stored RBCs.

Blood transfusion saves many lives but carries significant risk of injury. Currently, red blood cell (RBC) concentrates can be stored up to 42 days. Concerns have recently been raised about the safety and efficacy of transfusing stored RBCs. Refrigerated storage results in a 'storage lesion' that is reflected by metabolic derangements, RBC shape modification, rheological changes, oxidative injury to lipids and proteins, alterations in oxygen affinity and delivery, increased adhesion of RBCs to endothelial cells, and accumulation of bioactive substances in storage media. In animal models, transfusion of aged, but not fresh, RBCs induces organ injury, inflammation, coagulopathy, and impaired oxygen delivery. A number of clinical studies, mostly observational or retrospective and from a single centre, have reported an association between transfusion of older RBCs and increased clinically significant outcomes, such as increased morbidity and mortality in certain patient populations, including trauma, critical care, and cardiac surgery. Others, however, have failed to indicate an influence of RBC age on outcome. The quality of evidence is currently too poor to make recommendations to change current transfusion practice; however, the transfusion community looks forward to the results of randomized trials currently addressing the long-standing question regarding the effects of RBC storage on clinically significant outcomes.

Red blood cells (RBCs) undergo biochemical and structural changes during storage, commonly referred to as the "storage lesion." Evidence suggests that the longer the RBC product is stored, the less effective is the transfused blood. Many studies linking morbidity to transfusion have not considered duration of RBC storage as a variable that may modulate the effect. In addition, the effects of supply and demand and RBC inventory management strategies have been incompletely investigated. It is possible to envision a blood management system based on modern inventory management strategies that could greatly reduce storage duration.

Multilayer preventative strategies have been instituted to enhance transfusion safety for patients in need of critical blood components. Presently blood safety is at its highest levels, with the implementation of precautionary/preventative measures against vCJD, bacterial and viral contamination of the blood supply. The implementation of these strategies together with advances in automation and computerization led to significant improvements in standardisation for transfusion practices. These include validation, verification, adherence to GLP and GMP and other regulatory requirements. In most European countries, universal prestorage leukodepletion is routine practice. In France proactive pathogen inactivation treatments [PITs] have been implemented emphasizing patient safety. This at least conceptually reduces the risk of transfusing viable WBCs, emerging bacteria and viruses, all with potential transfusion complications. In the UK, prion removal filters for red cell products are used selectively for special groups of patients. Some research establishments are exploring the potential impact of pathogen inactivation of whole blood or red cell components, using the new generation of S-303 PIT and the prion removal filters in combination. It needs to be determined whether such a combined strategy, applied synergistically, enhances red cell transfusion safety without compromising the overall criteria of acceptability. It is necessary to critically examine the impact of a new generation of PIT technologies, which may exacerbate the red cell storage lesion and cause the development of undesirable antibodies in the recipient. The development of innovative laboratory tools is vital to study impacts of these measures on the quality of stored blood and their clinical outcome. The ultimate aim of red cell transfusion is to provide oxygen enriched red blood cells to the microcirculations and tissues. Definitive studies are needed to establish the potential unforeseen negative long term toxicity, overall efficacy/quality and the clinical outcome of treated stored blood.

Background Hypothermic storage (HS) of red blood cells (RBCs) leads to a progressive deterioration of cell quality. Mitigating these deleterious changes could allow maintenance or even an improvement of RBC in vitro quality. The aim was to determine the effect of a cold rejuvenation treatment of RBCs and in particular to assess the connection between ATP levels, RBC deformability, and morphology during RBC storage. Study Design and Methods A pool-and-split design of leukoreduced CPD–saline-adenine-glucose-mannitol–packed RBC units was used to generate three groups: untreated controls, sham-treated units, and units treated with a cold (1-6°C) rejuvenation solution on Day 28, 35, or 42 of cold storage. Units were followed until Day 49 of storage and assessed for ATP concentration, morphology, deformability, and other in vitro quality variables including hemolysis, pH, and supernatant potassium levels. Results At every treatment time, rejuvenation was associated with a significant increase in intracellular ATP (p

Red blood cell (RBC) transfusions are a vital part of the medical management for several conditions and can be a life saving intervention. RBCs are refrigerator stored in blood banks to allow for a sufficient inventory to be readily available for patient care. These RBCs are stored in additive solutions at 1–6 °C. The extended storage of RBCs results in the RBC storage lesion, which is a series of biochemical and biomechanical changes that occur in the RBC and storage media during ex vivo preservation, reducing RBC survival and function once transfused. During storage, there are changes in RBC morphology and deformability, metabolomics, membrane consistency, and oxidation status. Furthermore, older, stored RBCs are more likely to be hemolyzed, both by intravascular and extravascular hemolysis. Several observational studies suggest that the RBC storage lesion is clinically relevant with better outcomes when fresher blood is transfused. However, recent randomized controlled trials suggest no significant differences between transfusion of fresh blood and the current standard of practice. However, these studies did not test whether transfusion of the oldest RBCs (i.e., between 35 and 42 days old) is harmful. Thus, despite these trials, controversy still remains regarding how significant the RBC storage lesion is clinically and whether RBCs transfused after prolonged storage cause harm.

During storage, erythrocytes suffered from biomechanical alterations called the "storage lesion”, which may caused hemolysis. The hemolysis released LDH into the plasma. The LDH that was released during hemolysis made it an adequate instrument to assess the quality of in vitro blood products. The aims of this study were to analyse the alteration of LDH level at day 1, 3, 7, 14, and 28 in the WB and PRC, to analyse the correlation between LDH level with storage duration, and also to analyse enhancement differences of LDH level between WB and PRC.This research was an observational study with a cross-sectional design. As the samples there were 11 bags of WB and 10 bags of PRC. Blood products were kept in bloodbank with the temperature range of 2–6° C. The LDH level was measured with the Beckman Chemistry Analyzer. There were statistically significant alterations of LDH level started from day 7 of storage in both blood products (p

BACKGROUND AND OBJECTIVES Blood operators routinely monitor the pH of apheresis platelets as a marker of the so-called storage lesion, which can result from manufacturing problems. It is also suspected that some donor characteristics can increase the risk of poor platelet storage. To explore this hypothesis, we analysed a large, multinational data set of quality control (QC) pH test results on apheresis platelets. MATERIALS AND METHODS For the period between September 2011 and August 2014, seven blood operators in Canada, the USA, the Netherlands, the United Kingdom, France and Australia provided pH QC test results and donor characteristics on a total of 21,671 apheresis platelets. RESULTS Some variations in pH distribution between blood operators were in part explained by differences in collection, processing and testing methods. Younger age and female gender were significantly associated with a pH value below the 10th percentile. Among donors who had two or more pH measurements (n = 3672), there was a strong correlation between pH results (r = 0·726; P < 0·0001). CONCLUSION The strong intradonor correlation of pH measurements and the association between donor characteristics and pH results suggest that donor factors play a role in the quality of platelets.

Release of vesicles from cells is a universal biological system, an adaptive cellular response to endogenous or external physiological or stressful stimuli and a genius means for intercellular, inter-organ and even inter-organism communication. These secreted vesicles that are collectively designated extracellular vesicles (EVs) have increasingly attracted the interest of cell biologists due to their imaginable interactions with every piece of the known biological systems in both health and disease states. Although EVs isolation and characterization are challenges, owing to their particular physicochemical features and complex biology, recent technological innovation has offered better understanding and inevitably, driven the revision of previously established theories on them. However, a crucial question remains unsolved: the physiological relevance of EVs in vivo. Since membrane vesiculation is an integral part of red blood cell (RBC) aging and homeostatic machinery and a prominent feature of RBC storage lesion, the characterization of storage EVs and their probable clinical relevance with the therapeutic or adverse effects of transfusions are extremely important targets in the research fields of transfusion biology and medicine. The scientists involved should transfer nascent knowledge and state-of-the-art technological tools in the packed RBC unit in order to: (i) update the inventory of biochemical and biophysical features of storage EVs; (ii) gain insight into the molecular pathways/signals underlying their generation; and (iii) clarify their dependence on blood donor, storage strategies and analytical variations, in order to step forward on understanding their interactions with stored or recipient target cells.

Background and objectives Metabolomics studies have revealed transition points in metabolic signatures of red cells during storage in SAGM, whose clinical significance is unclear. We set out to investigate whether these transition points occur independent of storage media and define differences in the metabolism of red cells in additive solutions. Materials and methods Red cell concentrates were stored in SAGM, AS-1, AS-3 or PAGGSM, and sampled fourteen times spanning Day 1-46. Following quality control, the samples were split into extracellular and intracellular aliquots. These were analysed with ultra-high-performance liquid chromatography coupled to mass spectrometry analysis affording quantitative metabolic profiles of both intra- and extracellular red cell metabolites. Results Differences were observed in glycolysis, purine salvage, glutathione synthesis and citrate metabolism on account of the storage solutions. Donor variability however hindered the accurate characterization of metabolic transition time-points. Intracellular citrate concentrations were increased in red cells stored in AS-3 and PAGGSM media. The metabolism of citrate in red cells in SAGM was subsequently confirmed using 13 C citrate isotope labelling and shown to originate from citrate anticoagulant. Conclusion Metabolic signatures that discriminate between 'fresh' and 'old' stored red cells are dependent upon additive solutions. Specifically, the incorporation and metabolism of citrate in additive solutions with lower chloride ion concentration is altered and impacts glycolysis.

Background/Aims: During storage, units of human red blood cells (pRBCs) experience membrane destabilization and hemolysis which may cause harm to transfusion recipients. This study investigates whether inhibition of acid sphingomyelinase could stabilize erythrocyte membranes and prevent hemolysis during storage. Methods: Human and murine pRBCs were stored under standard blood banking conditions with and without the addition of amitriptyline, a known acid sphingomyelinase inhibitor. Hemoglobin was measured with an electronic hematology analyzer and flow cytometry was used to measure erythrocyte size, complexity, phosphatidylserine externalization, and band 3 protein expression. Results: Cell-free hemoglobin, a marker of hemolysis, increased during pRBC storage. Amitriptyline treatment decreased hemolysis in a dose-dependent manner. Standard pRBC storage led to loss of erythrocyte size and membrane complexity, increased phosphatidylserine externalization, and decreased band 3 protein integrity as determined by flow cytometry. Each of these changes was reduced by treatment with amitriptyline. Transfusion of amitriptyline-treated pRBCs resulted in decreased circulating free hemoglobin. Conclusion: Erythrocyte storage is associated with changes in cell size, complexity, membrane molecular composition, and increased hemolysis. Acid sphingomyelinase inhibition reduced these changes in a dose-dependent manner. Our data suggest a novel mechanism to attenuate the harmful effects after transfusion of aged blood products.

Background The RBCs storage lesion is most carefully viewed as the sum of all the changes in RBCs occurring during the course of storage and that limit their survival. Materials and methods Erythrocytes were isolated from stored blood at regular intervals. Oxidative stress markers were analyzed to determine the changes during the storage. Results Antioxidant enzymes – (SOD and CAT), and SH showed insignificant variation whereas hemolysis, MDA and AOPP showed significant variations. Conclusion The oxidative stress has not successfully overridden the protection offered by the endogenous antioxidant system. Prolonged storage may result in the onset of erythrocyte deterioration. This clearly indicates that the erythrocytes are capable of attenuating ROS with 2 weeks of storage.

Background The storage lesion of packed red blood cells (PRBCs) consists of biochemical changes associated with increased inflammatory mediators and decreased oxygen-carrying capacity. The effect of storage on the coagulation system is less well studied. The purpose of this study is to determine how PRBC storage time affects the activated coagulation time (ACT), using an in vitro model. Methods Type-matched PRBCs and fresh-frozen plasma were mixed, warmed to 37°C, and recalcified. The microsample ACT was repeatedly determined by 2 methods (ACT+ and ACT-LR) at 1 week, 3 weeks, and 5 weeks after date of donation. Hematocrit, pH, and ionized calcium were measured to assess how the model compared to physiologic circumstances. Statistical analysis was performed using the Student 2-tailed t-test with unequal variance. Results Hematocrit, pH, and ionized calcium were successfully maintained within narrow ranges. The model was notably acidotic, consistent with circumstances of shock and massive transfusion. There was no significant difference in the ACT+ between week 1 and week 3 ( P = .183), but there was a significant difference between week 3 and week 5 ( P = .029) and between week 1 and week 5 ( P = .007). The ACT-LR showed a significant difference between week 1 and week 3 ( P = .001), but not between week 3 and week 5 ( P = .286). There was again a strong difference between week 1 and week 5 results ( P = .011). Conclusion In an isolated in vitro model, the storage lesion of PRBCs is associated with decreased coagulation. This may have relevance for transfusion practices in coagulation-sensitive circumstances such as trauma.

The potential adverse effects of transfusion of red blood cells after prolonged storage have been hotly debated. During refrigerated storage, red blood cells are damaged, a process known as the red blood cell "storage lesion." We hypothesized that the delivery of a bolus of iron derived from these rapidly cleared, damaged, red blood cells is responsible for some of the adverse effects of transfusion. Iron may play a role in producing a pro-inflammatory response to transfused red blood cells, potentially through the effects of reactive oxygen species on stress pathways and inflammasome activation. Furthermore, the excess iron may impair the host's ability to combat infection by its innate iron-withholding pathways. This symposium paper summarizes the background for the "iron hypothesis" as it relates to transfusion of red blood cells after prolonged refrigerated storage. It also includes a summary of the data from recent murine and human studies, and concludes with a discussion of several unresolved questions arising from these published studies.

BACKGROUND: The temporal pattern of the biologic mechanism linking red blood cell (RBC) storage duration with clinical outcomes is yet unknown. This study investigates how such a temporal pattern can affect the power of randomized controlled trials (RCT) to detect a relevant clinical outcome mediated by the transfusion of stored RBCs. STUDY DESIGN AND METHODS: This study was a computer simulation of four RCTs, each using a specific categorization of the RBC storage time. The trial's endpoint was evaluated assuming five hypothetical temporal patterns for the biologic mechanism linking RBC storage duration with clinical outcomes. RESULTS: Power of RCTs to unveil a significant association between RBC storage duration and clinical outcomes was critically dependent on a complex interaction among three factors: 1) the way the RBC storage time is categorized in the trial design, 2) the temporal pattern assumed for the RBC storage lesion, and 3) the age distribution of RBCs in the inventory from which they are picked up for transfusion. For most combinations of these factors, the power of RCTs to detect a significant treatment effect was below 80%. All the four simulated RCTs had a very low power to disclose a harmful clinical effect confined to last week of the maximum 42-day shelf life of stored RBCs. CONCLUSIONS: Ongoing RCTs may lack enough power to settle the issue of whether or not the transfusion of stored blood has a negative clinical impact. A precautionary reduction of the maximum storage time to 35 days is advisable.

BACKGROUND: For the past 30 years, red blood cell (RBC) storage systems have been licensed in the United States based on the demonstration that 24-hour in vivo recovery was greater than 75% and hemolysis was less than 1%. Now additional requirements for storage system licensure have being added. The meaning and value of these new requirements have been questioned. STUDY DESIGN AND METHODS: The literature regarding the performance of present and suggested new tests for RBC licensure was reviewed. RESULTS: 51Cr 24-hr in vivo recovery has an intrinsic 4% error of measurement whereas the error in measures of hemolysis is less than 0.1%. Both measures have large donor-dependent end-of-storage variability; nevertheless, they have successfully guided RBC storage system development for six decades. Adenosine 5′-triphosphate and 2,3-diphosphoglycerate are difficult to measure accurately and international shared-sample studies suggest 6 and 11% coefficients of variation across laboratories. There is no readily available way to measure the oxygen equilibrium curve accurately. The new failure criteria provide no useful information and randomly fail good products. CONCLUSIONS: Attempts to expand the useful regulatory requirements for RBC storage system licensure are limited by poor understanding of the storage lesion and its effect of RBC performance. Measures of 51Cr 24-hour in vivo recovery remain critical and resources for this measure are limiting. The interaction between limited testing resources and large donor variability remains a major limit on RBC storage system development. It is important that new required tests contribute meaningful information and not make development and licensure of better products more difficult.

A primary function of blood transfusion services is to ensure the safety of the community's blood supply. Multilayer strategies of safety need to be incorporated into the processing of blood components in order to minimize untoward transfusion events related to infections or the blood storage lesion. While there have been considerable technical advances over the past few decades to advance blood component safety, there is a need for continued improvement. One significant problem area is transfusion transmitted bacterial infections. In infectious disease, blood borne bacteria are the major cause of morbidity in transfusion medicine. Proactive implementation of pathogen inactivation technologies, PIT can help to eliminate bacterial contamination of blood. This is accomplished by inactivation of various known and emerging bacterial pathogens, which have been identified through their nucleic acid sequences. The current PIT has additional positive impacts on transfusion safety by reducing adverse clinical events associated with viable residual leukocytes in the blood. Given the valuable benefits of pathogen inactivation, there is a practicable need to advance this technology. In order for a particular pathogen inactivation strategy to gain widespread acceptance, it must satisfy a number of important criteria. The technology shall be: (1) Effective to eliminate pathogens. (2) Uncomplicated and cost-effective to implement in the preparation of blood components. (3) Minimally toxic while maintaining the quality of blood storage products for transfusion purposes. (4) Safe. Currently, PIT is not suitable for all blood components. Future advances in PIT for whole blood and red cell components should expand the application of this technology across a broader range of blood products. Based on available laboratory and clinical data, current pathogen inactivation procedures appear to leave the cell components sufficiently viable to achieve an effective transfusion. Novel technologies continue to evolve for the inactivation of pathogens in the blood supply. We need to ensure these procedures do not worsen the storage lesion causing potential harm to patients. Quality research and development supporting multi-centre clinical trials are necessary. One purpose of such trials is to determine whether this new generation of procedures for processing blood yields bioequivalency regarding the quality of the products. Validated haematological assays should be utilized to test the extent of the blood storage lesion. Post-marketing surveillance involving active haemovigilance is necessary regarding possible adverse outcomes of transfusion.

Blood transfusion is an integral component of life-saving fluid resuscitation for anemic, surgical, oncological, and severely injured patients. However, there have been well-publicized studies suggesting that blood may have harmful side-effects. One possible factor contributing to adverse effects of transfusion is the duration of red blood cell storage prior to transfusion. Our group is exploring possible mechanisms of a putative “storage lesion”, including measuring modulation of the immune system or changes in adhesion properties between endothelial cells and transfused red blood cells. The following review will explore changes that occur during red blood cell storage and how these might impact pathways that are important in transfusion recipients’ clinical outcome, as well as provide a broad overview of the research our group is performing on the topic.

During storage of red blood cells (RBC), these cells develop storage lesions. The clinical relevance of these storage lesions is heavily discussed in literature. In this review, different aspects of the storage lesion are shown and how these potentially affect posttransfusion performance of the RBC. An overview of the conflicting literature on the clinical relevance of prolonged storage is given, summarizing the evidence on associations with mortality, length of stay, (postoperative) infections and organ failure. Subsequently, possible explanations are given for the conflicting results in the clinical studies and suggestions on how to proceed.

The mechanical fragility index (MFI) is an in vitro measure of sublethal injury to RBCs. In our previous experiments, we demonstrated that an increase in sublethal injury (increasing MFI) was a component of the RBC storage lesion, and that the MFI was significantly higher amongst the RBC units from male donors compared to pre-menopausal female donors during storage. It was hypothesized that hormonal or menstrual factors contributed to this difference. In this study, we found that RBC units donated by post-menopausal women demonstrated an MFI that was significantly higher than those donated by pre-menopausal women throughout storage.

Background The mechanical fragility index (MFI) is an in vitro measurement of the extent of RBC sublethal injury. Sublethal injury might constitute a component of the RBC storage lesion, thus the MFI was determined serially during routine RBC storage. Methods Leucoreduced AS-5- and SAGM-preserved RBCs were stored under routine blood bank conditions. The mechanical fragility (MF) of each unit was serially measured during storage. Results For both AS-5 and SAGM units, male and female RBCs demonstrated statistically significant increases in the MFI during storage. The MFI was significantly lower in AS-5 units compared to SAGM units throughout storage. Female RBCs had significantly lower MFI vs. male RBCs in both AS-5 and SAGM units at all times. No significant differences in MFI were observed between ABO groups for both genders for AS-5 RBCs. Conclusions The MF of RBCs increases during storage. Both gender and preservation solution influenced the MFI; however, the male:female MFI ratios were similar at all time-points and remained stable, suggesting that gender-based biological differences exist independent of storage solution. The MF could be a useful test for evaluating the effect of novel interventions intended to mitigate the susceptibility of RBCs to sublethal injury during storage.

BACKGROUND: Proteomics methods may be used to analyze changes occurring in stored blood products. These data sets can identify processes leading to storage-associated losses of blood component quality such as the platelet (PLT) storage lesion (PSL). The optimal strategy to perform such analyses to obtain the most informative data sets, including which proteomics methods, is undefined. This study addresses relative differences among proteomics approaches to the analysis of the PLT storage lesion. STUDY DESIGN AND METHODS: Changes to the PLT proteome between Days 1 and 7 of storage were analyzed with three complementary proteomic approaches with final mass spectrometry analysis: two-dimensional (2D) gel electrophoresis/differential gel electrophoresis (DIGE), isotope tagging for relative and absolute quantitation (iTRAQ), and isotope-coded affinity tagging (ICAT). Observed changes in concentration during storage of selected proteins were confirmed by immunoblotting. RESULTS: In total, 503 individual proteins changed concentration over a 7-day storage period. By method, a total of 93 proteins were identified by 2D gel/DIGE, 355 by iTRAQ, and 139 by ICAT. Less than 16 percent of the 503 proteins, however, were identified by not more than at least two proteomic approaches. Only 5 proteins were identified by all approaches. Membrane protein changes were not reliably detected with 2D gel/DIGE methods. CONCLUSION: Although proteomics analyses identified many storage-associated protein changes, these varied significantly by method suggesting that a combination of protein-centric (2D gel or DIGE) and peptide-centric (iTRAQ or ICAT) approaches are essential to acquire adequate data. The use of one proteomics method to study changes in stored blood products may give insufficient information.

BACKGROUND: Current FDA regulations allow the prestorage pooling of whole blood–derived platelet concentrates (PCs) of identical ABO type in a recently cleared platelet (PLT) pooling bag (Acrodose, Pall Medical). It is unclear how ABO-mixed PC pools would store if pooled before storage. STUDY DESIGN AND METHODS: Pools consisting of ABO-identical PLTs and mixed A and O PLTs in varying proportions were evaluated on Days 1, 5, and 7 of storage with measures of the PLT storage lesion, lymphocyte activation, and activation of the complement and coagulation system. Data were analyzed by analysis of variance. RESULTS: Pools did not differ on Day 7 in pH (p = 0.63), hypotonic shock response (p = 0.25), extent of shape change (p = 0.26), morphology score (p = 0.18), or white cell count (p = 0.79), but surface P-selectin expression was more evident in the ABO-mixed pools (p = 0.02). Small microscopic clumps of PLTs were observed in all pools, but were more prominent in the ABO-mixed pools (p

This study describes a new method for comparison of fluid motion and associated shear forces with various modes of platelet agitation. Fluid motion generated by flatbed, tumbler-type and circular platelet agitators was investigated. Trajectory tracings of polyethylene beads suspended inside platelet storage bags were used to calculate motion parameters. Mean particle speeds generated by flatbed agitation were approximately 25-fold greater than those generated by 3-rpm tumbler-type agitation, corresponding to a wall shear stress increase in approximately sevenfold for flatbed agitation. As such, flatbed agitation is more likely to contribute to shear-associated changes in platelets than other modes of agitation.

OBJECTIVE To assess changes in biochemical and biophysical properties of canine RBCs during cold (1° to 6°C) storage in a licensed RBC additive solution (the RBC preservation solution designated AS-1) supplemented with ascorbic acid. SAMPLE Blood samples from 7 neutered male Greyhounds; all dogs had negative results when tested for dog erythrocyte antigen 1.1. PROCEDURES Blood was collected into citrate-phosphate-dextrose and stored in AS-1. Stored RBCs were supplemented with 7.1mM ascorbic acid or with saline (0.9% NaCl) solution (control samples). Several biochemical and biophysical properties of RBCs were measured, including percentage hemolysis, oxygen-hemoglobin equilibrium, and the kinetic rate constants for O2 dissociation, carbon monoxide association, and nitric oxide dioxygenation. RESULTS Greyhound RBCs stored in AS-1 supplemented with ascorbic acid did not have significantly decreased hemolysis, compared with results for the control samples, during the storage period. CONCLUSIONS AND CLINICAL RELEVANCE In this study, ascorbic acid did not reduce hemolysis during storage. Several changes in stored canine RBCs were identified as part of the hypothermic storage lesion.

To determine the safety and efficacy of cryopreserved packed red blood cell (CPRBC) transfusion in trauma patients.Liquid packed red blood cells (LPRBCs) have an abbreviated shelf-life and worsening storage lesion with age. CPRBCs are frozen 2 to 6 days after donation, stored up to 10 years, and are available for 14 days after thawing and washing. CPRBCs can be utilized in diverse settings, but the effect on clinical outcomes is unknown.We performed a prospective, randomized, double-blind study at 5 level 1 trauma centers. Stable trauma patients requiring transfusion were randomized to young LPRBCs (≤14 storage days), old LPRBCs (14 storage days), or CPRBCs. Tissue oxygenation (StO2), biochemical and inflammatory mediators were measured, and clinical outcomes were determined.Two hundred fifty-six patients with well-matched injury severity and demographics (P0.2) were randomized (84 young, 86 old, and 86 CPRBCs). Pretransfusion and final hematocrits were similar (P0.68). Patients in all groups received the same number of units postrandomization (2 [1-4]; P0.05). There was no difference in the change in tissue oxygenation between groups. CPRBCs contained less α2-macrogobulin, haptoglobin, C-reactive protein, and serum amyloid P (P0.001). Organ failure, infection rate, and mortality did not differ between groups (P0.2).Transfusion of CPRBCs is as safe and effective as transfusion of young and old LPRBCs and provides a mechanism to deliver PRBCs in a wide variety of settings.

Background and Objectives Apheresis platelet concentrates sometimes contain persistent aggregates (PA). Because apheresis involves extracorporeal circulation, we hypothesized that interactions between GPIbα and von Willebrand factor (VWF) underlie their origin. Materials and Methods Platelets in donations with PA were compared to aggregate-free (AF) controls. Flow cytometry was used to determine platelet bound VWF. Degranulation was measured using P-selectin expression in flow cytometry and cytokine release using immunosorbent assays. Platelet adhesion to VWF was assessed in hydrodynamic flow and real-time video microscopy. Results Platelets in PA concentrates had significantly more (P = 0·009, n ≥ 8) bound VWF compared to AF platelets, but differences in VWF concentration, VWF collagen binding, activated VWF or GPIbα expression were not found. Degranulation was higher (P = 0·030, n = 7) in PA than AF concentrates on day 1 of storage, but adhesion to immobilized VWF under hydrodynamic flow conditions was normal at that moment. On day 6, however, significantly less VWF adhesion (P = 0·009, n ≥ 6) was found for PA platelets compared to AF, indicating accelerated storage lesion in PA products. In a model that mimicks PA formation by chemically induced binding of VWF to platelets, we found that degranulation, phosphatidylserine expression and metabolism did not differ with paired controls at any time during subsequent storage. Conclusion Accelerated storage lesion is found in concentrates with PA, but this cannot be explained solely by increased platelet bound VWF following apheresis. Therefore, additional stressors are probably responsible for the increases observed in platelet degranulation and storage lesion in products with PA.