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BACKGROUND AND OBJECTIVES During the in vitro storage of red blood cells (RBCs), unfavourable changes (storage lesions) cause a rapid consumption of intracellular diphosphoglycerate. The latter deregulates the oxygen-haemoglobin binding potential, subsequently increasing oxygen saturation (SO2 ) and membrane degradation, transforming RBCs from biconcave discs to rigid spherical bodies (spheroechinocytes). Current laboratory techniques invasively extract RBC samples to assess the quality of red cell concentrate (RCC) units. Optical technologies could provide a means of assessing quality non-invasively. MATERIALS AND METHODS A photoacoustic (PA) imaging technique was developed for acquiring the SO2 of blood bags non-invasively. Seven RCC units were monitored every 3-5 days until expiry (6 weeks). Measurements were validated against a conventional blood gas analyzer (BGA). Using an image flow cytometry assay, morphological profile trends were compared against the SO2 trends during blood bag storage. RESULTS A strong correlation (r2 ≥ 0·95) was found when comparing temporal data between PA and BGA SO2 measurements. Inter-sample PA variability was found to be similar to that produced by BGA (±0·8%). A strong correlation was found to exist between the temporal changes in SO2 and relative spheroechinocyte population (0·79 ≤ r2 ≤ 0·97). CONCLUSION This study suggests that PA imaging can non-invasively track the SO2 of stored RBCs non-invasively. By longitudinally monitoring the change in SO2 , it is possible to infer the effects of the storage lesion on RBC morphology. This non-invasive monitoring technique allows for the assessment of blood bags, without compromising sterility pre-transfusion.

Red blood cell (RBC) storage in the blood bank is characterized by the progressive loss of metabolic regulation, a phenomenon that targets energy and antioxidant metabolism; While the progression of the storage lesion is inevitable, the rate at which this phenomenon occurs varies from donor to donor; Red blood cells from some donors at the end of storage are metabolically superior to RBCs from other donors at the beginning of storage, suggesting that the age of blood alone may not be a sufficiently accurate predictor of stored blood quality; and Animal studies and large-scale omics screening in blood donors have helped identify mechanistic contributors to the metabolic heterogeneity of stored blood units.

BACKGROUND Gamma irradiation of red blood cells (RBCs) is well recognized to exacerbate storage lesion formation, but the effect of storage after irradiation on in vivo oxygen delivery capacity of transfused RBCs is currently not known. STUDY DESIGN AND METHODS In 24 preterm infants with anemia receiving nonurgent transfusion of irradiated RBCs, we examined cerebral regional tissue oxygenation (crSO2 ) and time spent with peripheral arterial saturation (SpO2 ) less than 88%. Physiologic data were obtained immediately before, immediately after, and 5 days after transfusion. RESULTS We observed linear negative moderate correlations between time since irradiation and the magnitude of change in crSO2 (r = -0.60; 95% CI, -0.81 to -0.27; p = 0.0018) and time spent with SpO2 of less than 88% (r = -0.42; 95% CI, -0.71 to 0.003; p = 0.04) immediately after transfusion. In infants (n = 9) who received fresher RBCs (irradiated

With the development of several 'vein-to-vein' databases, which capture data on the entire donor-recipient continuum and link this data to health outcomes, there has been an increasing number of studies investigating the health effects of all aspects of the practice of transfusion medicine. The Scandinavian Donations and Transfusions (SCANDAT) database is one of several such databases, which includes all electronically available data on blood donors, donations and transfusions since the late 1960s in Sweden and the early 1980s in Denmark. The SCANDAT database has been used to characterise disease occurrence among blood donors and transfused patients, as well as to investigate possible health effects of blood donations, aspects of transfusion care and possible transfusion transmission of disease. Recent publications include studies on recipient mortality associated with the storage lesion, studies on the effects of donor demographics on patient mortality and health effects of frequent blood donation. Although this research approach is clearly very powerful, the appropriate analysis of such real-world data is complex and requires close methodological attention. The purpose of this review is to present some of the research conducted within the SCANDAT collaboration. We hope more international collaboration may help improve our understanding of the important remaining questions about donor and recipient health.

Objectives This study describes the biochemical changes – also known as the storage lesion – that occur in canine packed red blood cells during ex vivo storage. Materials and Methods Ten 125-mL units of non-leuco-reduced packed red blood cells in citrate phosphate dextrose adenine were obtained from a commercial blood bank within 24 hours of donation. Samples were aseptically collected on days 1, 4, 7, 14, 28, 35 and 42 for measurement of sodium, potassium, chloride, lactate, glucose, pH and ammonia concentrations. All units were cultured on day 42. Friedman's repeated measures test with Dunn's multiple comparison test was used for non-parametric data. A repeated-measures analysis of variance with Tukey's multiple comparison test was used for parametric data. Alpha was set to 0·05. Results All analytes changed significantly during storage. The mean ammonia on day 1 (58·14 g/dL) was significantly lower (P

We present a novel automated system for morphology analysis of red blood cells (RBC) under flow. RBC concentrates collected by blood banks for transfusions are stored for periods of up to several weeks, during which time a number of changes occur, collectively termed the storage lesion. Typically the extent of hemolysis is the defining criterion to determine the acceptability of the RBCs for transfusions. Morphological changes are related with biochemical alteration during the storage of RBCs. The typical blood smear procedure for determining such changes is a labor-intensive and potentially biased manual process. The advantage of the flow morphometry system presented here is that it provides fully automated morphological classification of RBCs with large sample numbers in a short time. Our system uses a commercially available flow cell and flow conditions that prevent adhesion of RBCs, thus eliminating the need for blocking agents such as albumin that affect the distribution of cell shapes. Our morphometry results are validated by comparison with standard biochemical assays (hemolysis, ATP) for blood from 17 donors stored under blood bank conditions for 13 weeks. We show that the percentage of spherocytes present can be used to estimate the status of RBC concentrates. © 2017 International Society for Advancement of Cytometry.

Background Storage lesion may explain the rapid clearance of up to 25% of transfused red blood cells (RBCs) in recipients. Several alterations affect stored RBC but a quantitative, whole cell-based predictor of transfusion yield is lacking. Because RBCs with reduced surface area are retained by the spleen, we quantified changes in RBC dimensions during storage. Study design and methods Using imaging flow cytometry we observed the dimension and morphology of RBCs upon storage, along with that of conventional biochemical and mechanical markers of storage lesion. We then validated these findings using differential interference contrast (DIC) microscopy and quantified the accumulation of microparticles (MPs). Results Mean projected surface area of the whole RBC population decreased from 72.4 to 68.4 µm2 , a change resulting from the appearance of a well-demarcated subpopulation of RBCs with reduced mean projected surface (58 µm2 , 15.2%-19.9% reduction). These "small RBCs" accounted for 4.9 and 23.6% of all RBCs on Days 3 and 42 of storage, respectively. DIC microscopy confirmed that small RBCs had shifted upon storage from discocytes to echinocytes III, spheroechinocytes, and spherocytes. Glycophorin A-positive MPs and small RBCs appeared after similar kinetics. Conclusion The reduction in surface area of small RBCs is expected to induce their retention by the spleen. We propose that small RBCs generated by MP-induced membrane loss are preferentially cleared from the circulation shortly after transfusion of long-stored blood. Their operator-independent quantification using imaging flow cytometry may provide a marker of storage lesion potentially predictive of transfusion yield.

BACKGROUND Pulsed xenon (Xe) flash without any photoreactive compounds has been shown to inactivate a type of bacteria spiked into platelet (PLT) suspension in plasma, but enhanced the PLT storage lesion (PSL). Predicting reduction of PSL with increasing bactericidal ability, pulsed Xe flash was filtered through a band-stop filter, which excluded ultraviolet (UV)A, UVB, and visible light. STUDY DESIGN AND METHODS Apheresis PLT concentrates (PCs) inoculated with bacteria were irradiated with filtered Xe flash (fXe treatment). For in vitro functional quality assessment, PLT aggregation and thrombin generation together with other assays that monitor the PSL were investigated. RESULTS Staphylococcus aureus and Streptococcus dysgalactiae could be inactivated without regrowth during 6 days of storage. PC variables, such as PLT count, concentrations of soluble CD40 ligand, and ratio of aggregated PLTs, were not significantly different between fXe-treated and untreated PCs after 6 days of storage, while PAC-1 binding increased in the fXe-treated PLTs. Responsiveness of fXe-treated PLTs to ADP was maintained over a 6-day storage period as shown by the up regulation of P-selectin expression and induction of both integrin αIIbβ3 conformational change and PLT aggregation. The fXe-treated PLTs showed a sustained aggregation curve in response to ADP, whereas untreated PLTs transiently aggregated and then subsequently dissociated. Thrombin-generating kinetics of fXe-treated PLTs via PLT membrane surface were equivalent to those of untreated PLTs. CONCLUSIONS The fXe treatment inactivated bacteria in apheresis PCs in plasma without additional chemical compounds. The fXe-treated PCs retained acceptable in vitro properties of PC quality and PLT functionality.

The introduction of omics technologies, in particular proteomics and metabolomics in the field of transfusion medicine, has unveiled the complexity of the storage lesion. Even though it is now universally accepted that the storage lesion affects red cell survival upon transfusion and thus, theoretically, the safety and effectiveness of the transfusion therapy, no significant association between storage duration and transfusion outcomes has been observed in any of the large-scale randomized clinical trials performed so far. This disconnection between laboratory and clinical evidence may be in part reconciled by the appreciation of the underlying biological variability among donors and recipients, utterly affecting the severity of the storage lesion and the clinical effects of the transfusion therapy. While in the last few years the transfusion community has been split between those seeing the glass half full or half empty, considerations on the influence of biological variability and prestorage variables (e.g. handling and processing) on the final quality of packed red blood cells have prompted a re-assessment of whether we do really know what is in the glass (the final blood product). Controversy persists between the supporters of the clinical irrelevance of the storage lesion and those questioning the design and statistical power of the evidence-based studies looking for a correlation between storage duration and untoward consequences of transfusion. In the meantime, alternative storage strategies and solutions have been designed that promise to deliver better quality blood products for both factions.

BACKGROUND Transfusion of platelets (PLTs) is a common therapy in a number of clinical settings. However, it is well understood that there is substantial donor-to-donor variation in how well PLTs store and thus the quality of the products that are transfused. The basis of such variation is poorly understood, and there are limited metrics by which units of PLTs can be assessed for their posttransfusion performance. It has repeatedly been demonstrated that myriad biologic changes take place during PLT storage; however, which of the changes correlate with quality of the stored PLTs and/or are mechanistically involved in PLT function remains undetermined. STUDY DESIGN AND METHODS The current study tested stored PLTs from 21 normal subjects, combining high-resolution metabolomics of stored PLTs with in vivo PLT recoveries and survivals. Both individual analytes and metabolic pathways that correlate with posttransfusion PLT viability were identified. RESULTS Caffeine metabolites were associated with poor PLT recovery; caffeine metabolism was not ongoing in the PLT bag and remained at prestorage levels. Acylcarnitines, particular fatty acid metabolites, and oxidized fatty acids were associated with poor PLT survivals. Of the myriad metabolic changes during PLT storage, these are the first reported metabolic findings to begin distinguishing which changes are of functional importance regarding posttransfusion PLT performance. CONCLUSIONS Together, these findings provide novel mechanistic insights into the functional biology of the PLT storage lesion as well as identifying potential targets for modifying donor environment (e.g., caffeine consumption) and also metrics of quality assessment for stored human PLTs.

BACKGROUND Previous studies have shown that baseline hematologic characteristics concerning or influencing red blood cell (RBC) properties might affect storage lesion development in individual donors. This study was conducted to evaluate whether variation in hemolysis, microparticle accumulation, phosphatidylserine (PS) exposure, and other storage lesion–associated variables might be a function of the prestorage hematologic and biologic profiles of the donor. STUDY DESIGN AND METHODS Ten eligible, regular blood donors were paired and studied before donation (fresh blood) and during storage of RBCs in standard blood banking conditions. Plasma and cellular characteristics and modifications were evaluated by standard laboratory and biochemical or biologic analyses as well as by statistical and network analysis tools. RESULTS Nitrate/nitrite and other bioactive factors exhibited high interdonor variability, which further increased during storage in a donor-specific manner. Storage lesion evaluators, including RBC fragility and PS exposure, fluctuated throughout the storage period in proportion to their values in fresh blood. Donors’ levels of phosphatidylserine exposure and hemoglobin F correlated with stored cells’ mean cell (RBC) Hb concentration, oxidative stress markers, and cellular fragility. DISCUSSION Storage lesion indicators change in an orderly fashion, namely, by following donor-related prestorage attributes. These correlations are illustrated for the first time in “prestorage versus storage” biologic networks, which might help determine the best candidates for in vivo biomarkers of storage quality and provide deeper insight into the apparently complex donor variation effect on the RBC storage lesion.

The storage of red blood cells under standard blood-banking conditions leads to a decline in red cell quality and function known as the red cell storage lesion. These changes are linked to alterations in cellular biochemical processes, biomechanical effects and damage from oxidation. Although storage-related alterations in red cells can be readily measured in the laboratory, the clinical impact of the storage lesion is not as easy to demonstrate. While some immediate differences between fresh and stored cells can be measured, red cells recover in the circulation to some degree upon transfusion with a concomitant reduction in the effect of the storage lesion. The clinical literature is inconsistent with studies arguing that long-term stored red cells have a clinical impact and other studies including recent randomized controlled trials have failed to show any effect of the storage lesion on patient outcomes. Amelioration of the storage lesion will require further development of novel storage processes as well as an increased understanding of the role that donor-specific factors play in product quality.

Background Controversy exists regarding the optimal duration of storage of units of canine packed red blood cells (PRBC) prior to transfusion, resulting in different blood usage strategies between hospitals. Two hospitals were studied: At North Carolina State University (NCSU), usage protocol dictates that the oldest units of PRBC (stored the longest) are prioritized for transfusion; whereas, at Ontario Veterinary College (OVC), units of PRBC are sorted according to age (days after collection) and PRBC are selected for transfusion according to clinician or technician discretion, such that PRBC could be administered after any storage duration, with no preference given to older units. Objective To evaluate differences in the age of PRBC administered to dogs at 2 veterinary hospitals with different age-related PRBC usage protocols during a 1-year period (2010–2011). Key Findings The average age of units of PRBC administered at NCSU was older than that at OVC (P < 0.001). Additionally, a higher number of PRBC units older than 14 days old were transfused at NCSU (P < 0.001), and the age of the oldest administered PRBC unit was also older at NCSU (P < 0.001). NCSU discarded approximately 15 expired PRBC units, whereas OVC discarded 96 expired PRBC units during the study period. Significance A standardized protocol for the priority administration of the oldest stored PRBC resulted in the administration of significantly older PRBC, but fewer expired units of PRBC were discarded.

BACKGROUND Providers commonly transfuse sickle cell disease (SCD) patients with fresh red blood cells (RBCs) as treatment for acute chest syndrome (ACS). The objective of this study was to determine if there is an association between the storage duration of RBCs and length of hospitalization and oxygen requirement after transfusion in pediatric SCD patients with ACS. STUDY DESIGN AND METHODS This is a retrospective cohort study of pediatric SCD patients with ACS treated with a simple RBC transfusion over 8.5 years at a single institution. Multivariate generalized estimation equation analysis was used to identify associations between storage duration of RBCs and outcome measures. RESULTS A total of 234 ACS episodes in 131 subjects were included. The median storage duration of the oldest unit of transfused RBCs was 17 days (interquartile range, 11-26). The majority of ACS episodes, 77.4%, were treated with 1 unit of transfused RBCs; 20.9% received 2 units; and 1.7% received 3 or more units of RBCs. There was no association between the storage duration of the oldest unit of transfused RBCs and either duration of hospitalization or supplemental oxygen requirement after transfusion in multivariate analyses. CONCLUSION This retrospective study is one of the first to investigate the role of the storage lesion in children with SCD and does not support the preferential transfusion of fresh RBCs for ACS. Ultimately, a randomized controlled trial is necessary to determine whether the storage age of RBCs affects outcomes for patients with SCD and ACS.

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.

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.

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 (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

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.

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

BACKGROUND In recent times there has been debate around whether longer storage time of blood is associated with increased rates of adverse outcomes following transfusion. It is unclear whether results focused on cardiac or critically ill patients apply to a maternity population. This study investigates whether older blood is associated with increased morbidity and readmission in women undergoing obstetric transfusion. STUDY DESIGN AND METHODS Women giving birth in hospitals in New South Wales, Australia between July 2006‐December 2010 were included in the study population if they had received between 1‐4 red cell units during the birth admission. Information on women’s characteristics, transfusions and outcomes were obtained from 5 routinely collected datasets including blood collection, birth and hospitalisation data. Generalised propensity score methods were used to determine the effect of age of blood on rates of severe morbidity and readmission, independent of confounding factors. RESULTS Transfusion data were available for 2990 women, with a median age of blood transfused of 20 days (interquartile range 14,27 days). There were no differences in the age of blood transfused between women with and without severe morbidity (21 (14,28) vs 22 (15,30) days), and in women readmitted or not (22 (14,28) vs 22 (16,30) days). After considering potential confounding factors, no relationship was found between the age of blood transfused and rates of severe morbidity and readmission. CONCLUSION Among women receiving low volume transfusions during a birth admission, there was no evidence of increased rates of adverse outcomes following transfusion with older blood. NHMRC, Australian Red Cross, NSW Clinical Excellence Commission, ARC

The purpose of this review is to draw the attention of general readers to the importance of cellular exocytic vesiculation as a normal mechanism of development and subsequent adjustment to changing conditions, focusing on red cell (RBC) vesiculation. Recent studies have emphasized the possible role of these microparticles as diagnostic and investigative tools. RBCs lose membrane, both in vivo and during ex vivo storage, by the blebbing of microvesicles from the tips of echinocytic spicules. Microvesicles shed by RBCs in vivo are rapidly removed by the reticuloendothelial system. During storage, this loss of membrane contributes to the storage lesion and the accumulation of the microvesicles are believed to be thrombogenic and thus to be clinically important.

The demand for blood components is constantly increasing, while the exclusion criteria for donors are strengthened in order to reach maximal safety for donors and patients. To counterbalance reduced availability of volunteers, multicomponent collections (MCC) is an attractive approach to produce more than one component during a single apheresis procedure from one donor, such as packed red blood cells (PRBCs) and platelet concentrates (PCs). Further, the exposures of patients to a limited number of donors reduces the possibility of alloimmunization and transfusion-related diseases. We measured the quality of PRBCs and PCs obtained by MCC, using the MCS+ device with the LDPRBC program, Revision B, and compared them with the quality of manually collected PRBCs and PCs collected with the Revision C2 of the MCS+. We found higher pH levels and lower hemolysis assessed by means of fHb and K+ in the supernatant of PRBCs over the whole storage period of 42 days in MCC-derived PRBCs. The functional metabolism assessed by intracellular ATP was higher in PRBCs collected by MCC than in manually collected units. Furthermore, PCs obtained during MCC showed an increase in p-selectin expression on day 5 of storage compared to PCs collected with the Revision C2 of the MCS+. The p-selectin expression on MCC platelets was within the range of p-selectin expression found in PCs obtained by other apheresis devices. These results indicate less storage lesion in MCC-derived PRBCs compared to manually collected units and no compromise in the quality of MCC PCs obtained in the same apheresis procedure. J. Clin. Apheresis 18:21–25, 2003. © 2003 Wiley-Liss, Inc.

Whether “older” red cells (RBCs) are as efficacious as “younger” RBCs is currently unresolved and awaiting the outcome of several large prospective clinical trials in the US and Canada. In an effort to proactively advance our understanding of the RBC storage lesion prior to the completion of these trials, NHLBI established a targeted research program in 2009 entitled, “Immunomodulatory, Inflammatory and Vasoregulatory Properties of Transfused RBC Units as a Function of Preparation and Storage.” The investigative teams funded by this initiative and their research topics have been previously reported.1 These teams, as well as principle investigators from two of the large prospective clinical RBC storage trials, and other Transfusion Medicine experts gathered on August 28th and 29th, 2012 in Bethesda, MD for an NHLBI sponsored Working Group exploring “Strategies to Optimize Red Blood Cell (RBC) Products.” The first day consisted of progress reports from the eight investigative teams, a presentation about a canine sepsis model to investigate the effect of older blood on survival, and updates from two of the ongoing prospective RBC storage clinical trials. On the second day, researchers provided presentations on a planned study to identify genetic determinants that affect RBC storage, the influence of manufacturing method and irradiation on RBC in vitro quality during storage, development of a new additive solution, the effects of storage containers composition on RBC quality, post collection manipulations, and the possible impact of a reduced RBC shelf life on inventory. This article provides a summary of many of the presentations given during the two-day workshop.

The von Willebrand factor (vWf) activity, as measured by the ristocetin co-factor (vWf:RCo) and collagen binding (vWf:CBA) assays, declined progressively in standard blood units stored at 4 degrees C after a 2-day storage period. This loss of activity was accompanied by a loss and degradation of high molecular weight (HMW) vWf multimers. In studies using a paired design, filtration of blood with a high efficiency leucocyte-removal filter, prior to storage at 4 degrees C, led to significantly improved maintenance of vWf:RCo and vWf:CBA compared with unfiltered units (P < 0.01 after 8 days). Loss and degradation of HMW vWf decreased when blood was filtered prior to 4 degrees C storage. Filtration had no effect on vWf-associated activities when blood was stored at 22 degrees C for 10 days. These results indicate that part of the storage lesion of vWf in banked blood is due to leucocyte-mediated removal and degradation of HMW vWf. This has implications when specifying plasma for the production of vWF concentrates and may also play a role in the haemostatic lesion associated with massive transfusion of stored blood.

Functional and metabolic characteristics of fresh and three-week stored erythrocytes from patients with sickle cell anemia were compared. The storage-related changes in ATP, 2,3-DPG, and P50 in sickle erythrocytes were similar to those in control (HbA) red blood cells. After storage in CPD, sickle erythrocytes maintained significantly higher levels of 2,3-DPG (mean 2.20 +/- 0.73 mM/ml RBC) than did control cells (mean 0.36 +/- 0.13 mM/ml RBC). The posttransfusion recovery and survival of stored SS erythrocytes in autologous recipients and in an animal test system were at least as good as those before storage. Tolerance of the storage lesion by sickle erythrocytes is probably related to their young mean cell age. These results also suggest that the option of autotransfusion should be explored for selected patients with sickle cell disease in special clinical settings.

Newly prepared platelet concentrates (PC) and platelet poor plasma (PPP) stored at either room temperature (RT) or 4 C were analyzed for ammonia content initially, and after storage at 24, 48, and 72 hours. These studies demonstrate that ammonia rapidly accumulates in PC stored at RT. This accumulation appears to be temperature dependent, as evidenced by the observed differences in ammonia accumulation in PC stored at 4 C and RT. Whether ammonia acts as a toxic metabolite in the inhibition of platelet function and the development of the "storage lesion," which occurs in PC stored at RT, needs further clarification. Additional studies are also necessary to establish the clinical efficacy of transfusing ammonia-containing platelet concentrates into premature neonates with immature functioning livers, and perhaps into adults with severe liver disease.

Storage of human erythrocytes in CPD produced a lesion of the erythrocyte membrane manifested by abnormal endocytosis in resealed ghosts and in intact erythrocytes. Endocytosis produced by resealing Ca, Mg, and ATP into ghosts was impaired by five weeks of storage and this defect was promptly reversed by the prior regeneration of ATP in the stored erythrocytes. Drug-induced endocytosis was studied in intact stored erythrocytes. Vinblastine endocytosis was not affected by storage. Chlorpromazine endocytosis was not affected by storage. Chlorpromazine endocytosis was variably but never completely inhibited by storage. Chlorpromazine endocytosis was variably but never completely inhibited by storage, and restoration of ATP occasionally resulted in complete restoration of chlorpromazine endocytosis to base line values. However, primaquine endocytosis was usually totally inhibited after three to four weeks of storage at a time when residual ATP levels were 30 to 50 per cent of base line values. Restoration of ATP levels to at least base line values did not completely primaquine endocytosis to control values. Study of primaquine endocytosis provides an opportunity for defining an erythrocyte membrane storage lesion.

Fetal red cells are well suited for intrauterine life; however, little is known about their response to postnatal environments. The purpose of this work was to investigate the metabolic and membrane changes affecting newborn red cells during their exposure to storage in citrate- phosphate-dextrose (CPD) and citrate-phosphate-dextrose-adenine (CPDA- 1). The findings suggest that newborn red cells are affected more by storage than are adult cells. These accelerated storage changes in the red cells of newborns may be related indirectly to the rapid adenosine triphosphate (ATP) decline. As is the case with adult red cells, fetal cells withstand storage in CPDA-1 better than in CPD. The storage lesion in these cells was partly reversible, as in adult cells, by incubation with adenosine. It was therefore concluded that newborn red cells obtained from placentas and stored for several weeks in CPD or CPDA-1 media or other media that improve the metabolic profile of these cells may be acceptable for transfusion.

The effect of phosphate buffer on the course of pH, ATP, and 2,3-PDG of CPD red blood cells stored at three temperatures was observed. Basic phosphate at an equilibrated level of 10 mM (as iP) maintained pH above 7.00 and ATP and 2,3-DPG above 70 per cent of initial value in cells stored at 37 C for 24 hours. In contrast however, at 25 and 4 C no buffering was obtained with basic phosphate concentrations up to 50 mM, but values for both ATP and 2,3-DPG were higher in phosphate treated aliquots than in controls throughout storage. When the pH of blood stored at 4 C was adjusted into the range 7.15 to 7.25 with tromethamine and the level of iP raised to 10 mM by addition of Na2HPO4 on day seven, it was found that ATP and 2,3-DPG levels were maintained at 90 and 120 per cent, while control levels fell to 60 and 12 per cent, respectively at 21 days. The process described parallels the normal repair of damaged red blood cells of bank blood that occurs in vivo following transfusion.

Aggregation and adhesion of stored platelets were studied by a modified Wright technique. Comparisons of platelet rich plasma collected in ACD solution were made with and without aggregating agents, and at storage temperatures of 5°C, 24°C and 37°C. Platelets showed a loss of response to 0.5 μg ADP/ml, collagen and agar after storage for 4–6 h at the three temperatures with the greatest loss at 37°C and the least change at 5°C. The response to 5μg ADP/ml also was best maintained at 5°C. After 6–8 h of storage at 5°C. there was some decrease in free (single) platelets after rotation in the absence of aggregating agents, with a marked decrease after 48 hours storage. With storage at 24°C and 37°C, decrease in single platelets after rotation was not demonstrated during the first 24 h. A response to 5 μg ADP/ml was maintained for more than a week with the 5°C preparations. Acidification diminished the response to ADP for the first few days of storage and also depressed platelet aggregation and adhesion in the absence of aggregating agents on prolonged storage. The results of this in vitro study would appear to correlate with the loss of hemostatic effect of stored platelets on transfusion. The sustained response to 5 μg ADP/ml at 5°C suggests that this may be the best temperature for short term storage, although there is less spontaneous aggregation of platelets at the end of 24 h with 24°C storage. Also the ‘storage lesion’ appears to include loss of ADP or inability to release this substance from platelets.