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Background and Objectives: Donor factors influence the quality characteristics of red cell concentrates (RCCs) and the lesions that develop in these heterogeneous blood products during hypothermic storage. Teen male donors' RCCs contain elevated levels of biologically old red blood cells (RBCs). The aim of this study was to interrogate the quality of units of different donor ages and sexes to unravel the complex interplay between donor characteristics, long-term cold storage and, for the first time, RBC biological age., Materials and Methods: RCCs from teen males, teen females, senior males and senior females were density-separated into less-dense/young (Y-RBCs) and dense/old RBCs (O-RBCs) throughout hypothermic storage for testing. The unseparated and density-separated cells were tested for haematological parameters, stress (oxidative and osmotic) haemolysis and oxygen affinity (p50)., Results: The O-RBCs obtained from teen donor samples, particularly males, had smaller mean corpuscular volumes and higher mean corpuscular haemoglobin concentrations. While biological age did not significantly affect oxygen affinity, biologically aged O-RBCs from stored RCCs exhibited increased oxidative haemolysis and decreased osmotic fragility, with teenage male RCCs exhibiting the highest propensity to haemolyse., Conclusion: Previously, donor age and sex were shown to have an impact on the biological age distribution of RBCs within RCCs. Herein, we demonstrated that RBC biological age, particularly O-RBCs, which are found more prevalently in male teens, to be a driving factor of several aspects of poor blood product quality. This study emphasizes that donor factors should continue to be considered for their potential impacts on transfusion outcomes., (© 2024 The Authors. Vox Sanguinis published by John Wiley & Sons Ltd on behalf of International Society of Blood Transfusion.)

Background: We used laboratory indicators to evaluate the quality of pathogen-reduced red blood cell suspension (RBCS) compared with gamma-irradiated RBCS., Materials and Methods: To determine biochemical and metabolic parameters of RBCS, we obtained 50 whole blood units from healthy volunteers and randomized them into 2 groups: 25 were pathogen-reduced, and then, RBCS prepared from them. RBCS from the other 25 was gamma-irradiated. Sampling was carried out on day zero before and after treatment and at 7, 14, 21 and 28 days. To determine lymphocyte inactivation, we collected another 35 whole blood units. Each was sampled to form 3 study groups: untreated, gamma-irradiated and pathogen-reduced. Daily sampling was carried out during 3 days of storage., Results: The quality of RBCS from both groups was largely the same, except for haemolysis and red blood cell fragility, which were more pronounced in the pathogen-reduced group. This finding limited the shelf life of pathogen-reduced RBCS to 14 days. Lymphocyte viability was significantly reduced after both treatments. Proliferation of lymphocytes after pathogen reduction was reduced to the detection limit, while low-level proliferation was observed in gamma-irradiated samples., Conclusion: Pathogen-reduced red blood cells have acceptable quality and can be used for transfusion within 14 days. Results of inactivation of lymphocytes demonstrate that pathogen reduction technology, applied on WB, can serve as an alternative to irradiation., (© 2020 International Society of Blood Transfusion.)

Background and Objectives: In clinical practice, it has been shown that transfusion of packed red blood cells (pRBCs) with late shelf life increases the risk of post-transfusion complications., Objective: To study relationship of membrane stiffness, cytoskeleton structure and storage time of pRBCs., Materials and Methods: pRBCs were processed and stored according to blood bank procedure, for 42 days, at +4°C; pRBC samples were taken on days 3, 12, 19, 21, 24, 28, 35 and 42. Cytoskeleton images and membrane stiffness were studied using atomic force microscope., Results: In the course of the pRBC storage, the cytoskeleton network configuration underwent structural changes. Simultaneously, pRBC membrane stiffness was increasing, with the correlation coefficient 0·88. Until 19 days, the stiffness grew slowly, in 19-24 days there occurred a transition period, after which its growth rate was three times higher than the initial. A chain of pathological processes developed in pRBC during long storage: pH reduction (linked to increased oxidative stress), then cytoskeletal destruction and an associated increase in pRBC membrane stiffness., Conclusion: During prolonged storage of pRBCs and their acidification, there is a progression of pRBC cytoskeletal changes and associated increase of membrane stiffness, observed to increase in rate after days 19-24. Mutual measurements of cytoskeletal integrity and membrane stiffness may be useful quality assessment tool to study the molecular mechanisms of RBC structural degradation during storage., (© 2020 International Society of Blood Transfusion.)

Introduction: In vitro qualitative differences exist in red cell concentrates (RCCs) units processed from whole blood (WB) depending on the method of processing. Minimal literature exists on differences in processing and variability in quality data. Therefore, we collected information from blood manufacturers worldwide regarding (1) details of WB collection and processing used to produce RCCs and (2) quality parameters and testing as part of routine quality programmes., Methods: A secure web-based survey was developed, refined after pilot data collection and distributed to blood centres. Descriptive analyses were performed., Results: Data from ten blood centres in nine countries were collected. Six blood centres (60%) processed RCCs using the top-and-top (TAT) method which produces RCCs and plasma, and eight centres (80%) used the bottom-and-top (BAT) which additionally produces buffy coat platelets. Five of the centres used both processing methods; however, four favoured BAT processing. One centre utilized the Reveos automated system exclusively. All centres performed pre-storage leucoreduction. Other parameters demonstrated variability, including active cooling at collection, length of hold before processing, donor haemoglobin limits, acceptable collection weights, collection sets, time to leucoreduction, centrifugation speeds, extraction devices and maximum RCC shelf life. Quality marker testing also differed amongst blood centres. Trends towards higher RCC unit volume, haemolysis and residual leucoctyes were seen in the TAT compared with BAT processing across centres., Conclusion: Methods and parameters of WB processing and quality testing of RCCs differ amongst surveyed blood manufacturers. Further studies are needed to assess variations and to potentially improve methods and product quality., (© 2019 International Society of Blood Transfusion.)

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 (SO 2 ) 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 SO 2 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 SO 2 trends during blood bag storage., Results: A strong correlation (r 2  ≥ 0·95) was found when comparing temporal data between PA and BGA SO 2 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 SO 2 and relative spheroechinocyte population (0·79 ≤ r 2  ≤ 0·97)., Conclusion: This study suggests that PA imaging can non-invasively track the SO 2 of stored RBCs non-invasively. By longitudinally monitoring the change in SO 2 , 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., (© 2019 International Society of Blood Transfusion.)

Background and Objectives: The accumulation of microvesicles in erythrocyte concentrates during storage or irradiation may be responsible for clinical symptoms such as inflammation, coagulation and immunization. Our aim was to determine whether any of the cluster of differentiation (CD) molecules responsible for important functions are present on microvesicles, and if their expression level is dependent on the storage period of erythrocyte concentrates., Material and Methods: Erythrocyte microvesicles were isolated from 'fresh' (2 nd day) and 'old' (42 nd day) stored erythrocyte concentrates. Qualitative cytometric analysis of 0·5 µm, erythrocyte-derived, PS-exposing vesicles was performed using the annexin V-FITC, anti-CD235a-PE antibody and calibrated beads. The microvesicles were also visualized under a confocal microscope. The expression of the molecules CD235a, CD44, CD47, CD55, CD59 and of phosphatidylserine (PS) was compared using flow cytometry. Measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope., Results: The analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0·5 µm in the 'fresh' and 'old' samples. At day 2, the microvesicles had elevated expression levels of CD47, reduced expression levels of PS, CD55 and CD59. The phagocytosis index was higher for the microvesicles isolated from the 42-day-old erythrocyte concentrates., Conclusion: This research may bring us closer to understanding the factors responsible for erythrocyte ageing and to evaluate the quality of stored red blood concentrates intended for transfusion., (© 2019 International Society of Blood Transfusion.)

We prospectively studied the dose-dependent effect of transfused stored red blood cells (RBCs) on recipient RBC indices, deformability and cell density in 10 patients administered stored RBCs for blood transfusion during general surgery. There were dose-dependent decreases in mean corpuscular volume and increases in mean corpuscular haemoglobin concentration after completion of 4- and 6-unit stored RBC transfusions. The amount of dense populations increased proportionately with the amount of stored RBCs transfused. The maximal deformability index value was significantly and dose-dependently decreased, suggesting that hemodynamic blood flow, especially the microcirculation may be impaired in patients who receive large amounts of stored RBCs., (© 2018 International Society of Blood Transfusion.)

Background and Objectives: Infusion of by-products of red blood cell (RBC) storage-induced degradation as well as of the residual plasma proteins and the anticoagulant-preservative solution contained in units of stored blood serve no therapeutic purpose and may be harmful to some patients. Here, we describe a prototype of a gravity-driven system for bedside washing of stored RBCs., Materials and Methods: Stored RBCs were diluted to 10% haematocrit (Hct) with normal saline, matching the conventional washing procedure. The dilute RBC suspensions were passed through a column of coiled tubing to allow RBC sedimentation in normal gravity, thus separating them from the washing solution. Washed RBCs were collected using bifurcations located along the tubing. Washing efficiency was quantified by measuring Hct, morphology, deformability, free haemoglobin and total-free protein., Results: The gravity-driven washing system operating at 0·5 ml/min produced washed RBCs with final Hct of 36·7 ± 3·4% (32·3-41·2%, n = 10) and waste Hct of 3·4 ± 0·7% (2·4-4·3%, n = 10), while removing 80% of free haemoglobin and 90% of total-free protein. Washing improved the ability of stored RBCs to perfuse an artificial microvascular network by 20%. The efficiency of washing performed using the gravity-driven system was not significantly different than that of conventional centrifugation., Conclusions: This proof-of-concept study demonstrates the feasibility of washing stored RBCs using a simple, disposable system with efficiency comparable to that of conventional centrifugation, and thus represents a significant first step towards enabling low-cost washing of stored blood at bedside., (© 2017 International Society of Blood Transfusion.)

Background and Objectives: Enumeration of residual white blood cells in leucoreduced blood components is essential part of quality control. Digital PCR has substantially facilitated quantitative PCR and was thus evaluated for measurements of leucocytes., Materials and Methods: Target for quantification of leucocytes by digital droplet PCR was the blood group gene RHCE. The SPEF1 gene was added as internal control for the entire assay starting with automated DNA extraction. The sensitivity of the method was determined by serial dilutions of standard samples. Quality control samples were analysed within 24 h, 7 days and 6 months after collection. Routine samples from leucodepleted red blood cell concentrates (n = 150) were evaluated in parallel by flow-cytometry (LeucoCount) and by digital PCR., Results: Digital PCR reliably detected at least 0·4 leucocytes per assay. The mean difference between PCR and flow-cytometric results from 150 units was -0·01 (±1·0). DNA samples were stable for up to at least six months. PCR measurement of leucocytes in samples from plasma and platelet concentrates also provided valid results in a pilot study., Conclusion: Droplet digital PCR to enumerate leucocytes offers an alternative for quality control of leucoreduced blood products. Sensitivity, specificity and reproducibility are comparable to flow-cytometry. The option to collect samples over an extended period of time and the automatization introduce attractive features for routine quality control., (© 2017 International Society of Blood Transfusion.)

Background and Objectives: Pathogen reduction technology using amustaline (S-303) was developed to reduce the risk of transfusion-transmitted infection and adverse effects of residual leucocytes. In this study, the viability of red blood cells (RBCs) prepared with a second-generation process and stored for 35 days was evaluated in two different blood centres., Materials and Methods: In a single-blind, randomized, controlled, two-period crossover study (n = 42 healthy subjects), amustaline-treated (Test) or Control RBCs were prepared in random sequence and stored for 35 days. On day 35, an aliquot of 51 Cr/ 99m Tc radiolabeled RBCs was transfused. In a subgroup of 26 evaluable subjects, 24-h RBC post-transfusion recovery, mean life span, median life span (T 50 ) and life span area under the curve (AUC) were analysed., Results: The mean 24-h post-transfusion recovery of Test and Control RBCs was comparable (83·2 ± 5·2 and 84·9 ± 5·9%, respectively; P = 0·06) and consistent with the US Food and Drug Administration (FDA) criteria for acceptable RBC viability. There were differences in the T 50 between Test and Control RBCs (33·5 and 39·7 days, respectively; P < 0·001), however, these were within published reference ranges of 28-35 days. The AUC (per cent surviving × days) for Test and Control RBCs was similar (22·6 and 23·1 per cent surviving cells × days, respectively; P > 0·05). Following infusion of Test RBCs, there were no clinically relevant abnormal laboratory values or adverse events., Conclusion: RBCs prepared using amustaline pathogen reduction meet the FDA criteria for post-transfusion recovery and are metabolically and physiologically appropriate for transfusion following 35 days of storage., (© 2017 The Authors. Vox Sanguinis published by John Wiley & Sons Ltd on behalf of International Society of Blood Transfusion.)

Background: Although most studies have shown that little haemolysis is induced by infusion pumps, there are some notable exceptions. Only limited data are available on the actual infusion pumps that are most used in hospitals in Quebec and elsewhere, namely, the Infusomat ® Space (peristaltic), Plum A+™ (piston) and Colleague ® CXE (shuttle) pumps., Methods: Haemolysis and potassium levels were compared before and after the use of the three different infusion pumps. Using 135 units of packed red blood cells (RBCs) aged from 10 to 28 days, 27 measurements were taken for each pump at various flow rates (30, 60, 150, 300 and 450 ml/h) and were compared with measurements taken before using the pumps. The range of flow rates was chosen to cover those of paediatric and adult transfusions., Results: The shuttle- and piston-type pumps resulted in low haemolysis levels. The peristaltic-type pump produced significantly more haemolysis, which worsened at low flow rates, but the absolute value of haemolysis remained within the range recommended by the regulatory agencies in North America and Europe. Approximately two-thirds of the haemolysis produced by the peristaltic-type pump seemed to be secondary to the use of an antisiphon valve (ASV) on the transfusion line recommended by the manufacturer. Potassium levels did not increase with the use of the pumps., Conclusion: Modern infusion pumps widely used in hospitals in Quebec and elsewhere produce non-threatening levels of haemolysis during the transfusion of packed RBCs aged from 10 to 28 days. ASVs appear to induce additional haemolysis, and we do not recommend using them for blood transfusion., (© 2017 International Society of Blood Transfusion.)

Background: Among transfused patients, the effect of the duration of red blood cell storage on mortality remains unclear. This study aims to compare the mortality of patients who were transfused with fresher versus older red blood cells., Methods: We performed an updated systematic search in the CENTRAL, MEDLINE, EMBASE and CINAHL databases, from January 2015 to October 2016. RCTs of hospitalized patients of any age comparing transfusion of fresher versus older red blood cells were eligible. We used a random-effects model to calculate pooled risk ratios (RRs) with corresponding 95% confidence interval (CI)., Results: We identified 14 randomized trials that enrolled 26 374 participants. All-cause mortality occurred in 1219 of 9531 (12·8%) patients who received a transfusion of fresher red blood cells and 1810 of 16 843 (10·7%) in those who received older red blood cells (RR: 1·04, 95% CI: 0·98-1·12, P = 0·90, I 2 = 0%, high certainty for ruling out benefit of fresh blood, moderate certainty for ruling out harm of fresh blood). In six studies, in-hospital death occurred in 691 of 7479 (9·2%) patients receiving fresher red cells and 1291 of 14 757 (8·8%) receiving older red cells (RR: 1·06, 95% CI: 0·97-1·15, P = 0·81, I 2 = 0%, high certainty for ruling out benefit of fresh blood, moderate certainty for ruling out harm of fresh blood)., Conclusion: Transfusion of fresher red blood cells does not reduce overall or in-hospital mortality when compared with older red blood cells. Our results support the practice of transfusing patients with the oldest red blood cells available in the blood bank., (© 2017 International Society of Blood Transfusion.)

Background and Objectives: According to European guidelines, the temperature of whole blood (WB) has to be maintained at 20-24°C until processing within 24 h, but in blood bank practice, WB is frequently held at temperatures between 18-25°C. We aimed to assess the impact of these small temperature deviations on the quality of the blood components., Materials and Methods: After rapid cooling, 7 WB units were held overnight at 18°C and 8 units at 25°C, reflecting worst case holding conditions, and separated into a red cell concentrate (RCC), plasma and buffy coat (BC). RCCs were filtered at test temperature and stored for 42 days at 2-6°C. BCs were processed to single-BC platelet concentrates (sPC) and stored up to Day 8 at 20-24°C., Results: After overnight hold at 18°C, 2,3-DPG in WB decreased by 34 ± 9%, while at 25°C the decrease was 82 ± 6%. Accordingly, the 2,3-DPG levels in the RCCs in the 25°C group were significantly lower than in the 18°C group (2·2 ± 1·4 vs. 10·4 ± 2·9 μmol/g Hb). RCCs and sPCs in the 25°C group showed higher initial lactate levels and lower pH compared to the 18°C group, but these differences levelled off at the end of storage. RCCs showed small differences in ATP levels and haemolysis. Plasma in both groups showed comparable Factor VIII:C levels., Conclusion: The temperature of WB during overnight hold strongly affects initial 2,3-DPG levels of RCCs and supports the maintenance of temperature limits between 20 and 24°C. Other in vitro effects of the temperature deviations were small and of no practical relevance., (© 2016 International Society of Blood Transfusion.)

The use of di-ethylhexyl-phthalate (DEHP) in blood bags is under discussion due to toxicity concerns and possible restrictions. A questionnaire among 15 blood centres in nine countries showed that none so far have fully switched to non-DEHP blood bags. If centres had to change, sites with a 42-day outdate would choose for a shorter outdating period, while others would allow a higher haemolysis rate (but within current specifications). To improve red cell quality, about half of the centres are willing to move to an alternative red cell storage solution, while the other half would not change for various reasons., (© 2017 International Society of Blood Transfusion.)

Background and Objectives: The accumulation of non-polar lipids arachidonic acid, 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE and 15-HETE during storage of transfusion products may play a role in the onset of transfusion-related acute lung injury (TRALI), a syndrome of respiratory distress after transfusion., Materials and Methods: We investigated non-polar lipid accumulation in red blood cells (RBCs) stored for 42 days, plasma stored for 7 days at either 4 or 20°C and platelet (PLT) transfusion products stored for 7 days. Furthermore, we investigated whether transfusion of RBCs with increased levels of non-polar lipids induces TRALI in a 'two-hit' human volunteer model. All products were produced following Dutch Blood Bank protocols and are according to European standards. Non-polar lipids were measured with high-performance liquid chromotography followed by mass spectrometry., Results: All non-polar lipids increased in RBCs after 21 days of storage compared to baseline. The non-polar lipid concentration in plasma increased significantly, and the increase was even more pronounced in products stored at 20°C. In platelets, baseline levels of 5-HETE and 15-HETE were higher than in RBCs or plasma. However, the non-polar lipids did not change significantly during storage of PLT products. Infusion of RBCs with increased levels of non-polar lipids did not induce TRALI in LPS-primed human volunteers., Conclusion: We conclude that non-polar lipids accumulate in RBC and plasma transfusion products and that accumulation is temperature dependent. Accumulation of non-polar lipids does not appear to explain the onset of TRALI (Dutch Trial Register - NTR4455)., (© 2016 International Society of Blood Transfusion.)

Background and Objectives: The present general plasticizer di-2-ethylhexyl-phthalate in polyvinylchloride (PVC) blood bags is only physically dispersed in PVC and will therefore leach into blood components. The objective of this study was to perform a first preliminary red blood cell (RBC) storage evaluation in a new blood bag manufactured of polyolefin without any inclusion of potentially migrating substances., Study Design and Methods: This is a RBC storage study for 42 days. Blood collection was performed in a polyolefin-based PVC-free blood bag. RBCs were prepared within 8 h. Two different RBC additive solutions were used, either PAGGS-M or PAGGG-M. We weekly measured pH, K + , glucose, lactate, haemolysis, red cell ATP and 2,3-DPG., Results: RBC storage in PAGGS-M resulted in high haemolysis levels already after 21 days, exceeding the European maximum limit of 0·8%, and low ATP levels by the end of the storage period. With PAGGG-M, haemolysis exceeded 0·8% after 28 days of storage. For additional parameters, the results were comparable to those of previous studies in conventional blood bags., Conclusion: This is a first preliminary study of RBC storage in a new type of blood bags. PAGGG-M gave encouraging results except for its inability to prevent increased haemolysis. There will be room for further development of RBC additive solutions to address the haemolysis problems. Plasma should also be tested regarding the stability of coagulation and activation pathway variables. There may also be a potential for future use of the bag for preparation of pooled buffy-coat-derived platelets., (© 2016 International Society of Blood Transfusion.)

Background and Objectives: To determine the value of erythropoietin in reducing allogeneic transfusions, it is important to assess the effects, safety and costs for individual indications. Previous studies neither compared the effects of erythropoietin between total hip and total knee arthroplasty, nor evaluated the safety or costs. We performed a meta-analysis to assess the effects of erythropoietin in total hip and knee arthroplasty separately. Safety and costs were evaluated as secondary outcomes., Materials and Methods: A systematic literature search was performed to identify randomized controlled trials evaluating the effect of erythropoietin in total hip and knee arthroplasty until April 2014. Study data were extracted using standardized forms and pooled using a random-effects model. Strength of the evidence was evaluated using Cochrane's Collaboration's tool for risk of bias assessment., Results: Seven studies were included (2439 patients). Erythropoietin significantly reduced exposure to allogeneic transfusion in both hip (RR 0·45; 95%CI 0·33-0·61) and knee (RR 0·38; 95%CI 0·27-0·53) arthroplasty, without differences between indications (P = 0·44). Mean number of transfused red blood cell units was significantly decreased in erythropoietin-treated patients (mean difference -0·57; 95%CI -0·86 to -0·29)(unable to split). No differences in thromboembolic or adverse events were found. Only one study evaluated costs, so that no pooled cost-effectiveness estimates could be given., Conclusion: Erythropoietin is effective in both hip and knee arthroplasty and can be considered as safe. However, the decision to use erythropoietin on a routine base should be balanced against its costs, which may be relatively high., (© 2016 The Authors. Vox Sanguinis published by John Wiley & Sons Ltd on behalf of International Society of Blood Transfusion.)

A retrospective analysis was conducted on 20 D(-) liver transplant (LT) recipients transfused with D(+) RBCs perioperatively and screened for RBC antibodies between 2 and 6 months later. None developed anti-D detectable by the indirect antiglobulin test. Two patients produced weak anti-D that reacted only with papain-treated RBCs at 10 and 11 days without any sign of immune haemolysis. Antibodies became quickly undetectable. These data suggest an unusual pattern of alloimmunization in LT recipients with rapid, weak and transient antibody response and support the safety of transfusing D(+) RBCs in most of D(-) patients during LT surgery., (© 2016 International Society of Blood Transfusion.)

Background and Objectives: Preclinical studies generated the hypothesis that older stored red blood cells (RBCs) can increase transfusion risks. To examine the most updated and complete clinical evidence and compare results between two trial designs, we assessed both observational studies and randomized controlled trials (RCTs) studying the effect of RBC storage age on mortality., Materials and Methods: Five databases were searched through December 2014 for studies comparing mortality using transfused RBCs having longer and shorter storage times., Results: Analysis of six RCTs found no significant differences in survival comparing current practice (average storage age of 2 to 3 weeks) to transfusion of 1- to 10-day-old RBCs (OR 0·91, 95% CI 0·77-1·07). RBC storage age was lower in RCTs vs. observational studies (P = 0·01). The 31 observational studies found an increased risk of death (OR 1·13, 95% CI 1·03-1·24) (P = 0·01) with increasing age of RBCs, a different mortality effect than RCTs (P = 0·02)., Conclusion: RCTs established that transfusion of 1- to 10-day-old stored RBCs is not superior to current practice. The apparent discrepancy in mortality between analyses of RCTs and observational studies may in part relate to differences in hypotheses tested and ages of stored RBCs studied. Further trials investigating 1- to 10-day-old stored RBC benefits would seem of lower priority than studies to determine whether 4- to 6-week stored units have safety and efficacy equivalent to the 2- to 3-week-old stored RBCs commonly transfused today., (© 2016 International Society of Blood Transfusion.)

Background and Objectives: Quality control (QC) data collected by blood services are used to monitor production and to ensure compliance with regulatory standards. We demonstrate how analysis of quality control data can be used to highlight the sources of variability within red cell concentrates (RCCs)., Materials and Methods: We merged Canadian Blood Services QC data with manufacturing and donor records for 28 227 RCC between June 2011 and October 2014. Units were categorized based on processing method, bag manufacturer, donor age and donor sex, then assessed based on product characteristics: haemolysis and haemoglobin levels, unit volume, leucocyte count and haematocrit., Results: Buffy-coat method (top/bottom)-processed units exhibited lower haemolysis than units processed using the whole-blood filtration method (top/top). Units from female donors exhibited lower haemolysis than male donations. Processing method influenced unit volume and the ratio of additive solution to residual plasma., Conclusions: Stored red blood cell characteristics are influenced by prestorage processing and donor factors. Understanding the relationship between processing, donors and RCC quality will help blood services to ensure the safety of transfused products., (© 2016 International Society of Blood Transfusion.)

Background and Objectives: Damage-associated molecular patterns (DAMPs) are found in transfusion products, but their potential impacts are not fully understood. We examined the influence of manufacturing method on levels of mitochondrial (mt) DNA and extracellular vesicle (EV) DAMPs in red cell concentrates (RCCs)., Materials and Methods: Eighty-seven RCCs were prepared using nine different methods (6-15 units/method), including three apheresis, five whole blood (WB)-derived leucoreduced (LR) and one WB-derived non-LR method. On storage days 5 and 42, levels of mtDNA (by PCR) and number and cell of origin of EVs (by flow cytometry) were assessed in RCC supernatants., Results: There was a 100-fold difference in mtDNA levels among methods, with highest levels in non-LR, followed by MCS+ and Trima apheresis RCCs. There was a 10-fold difference in EV levels among methods. RBC-derived CD235a+ EVs were found in fresh RCCs and increased in most during storage. Platelet-derived CD41a+ EVs were highest in non-LR and Trima RCCs and did not change during storage. WBC-derived EVs were low in most RCCs; CD14+ EVs increased in several RCCs during storage., Conclusion: DAMPs in RCCs vary by manufacturing method. MtDNA and EV could be informative quality markers that may be relevant to RCC immunomodulatory potential., (© 2016 International Society of Blood Transfusion.)

Background: Although recent randomized controlled trials have not found increased risk of morbidity/mortality with older red blood cells (RBCs), several large trials will be completed soon providing power to detect smaller risks if indeed they exist. Hence, there may still be a need for inventory management policies that could reduce the age of transfused RBCs without compromising availability or resulting in excessive outdates., Materials and Methods: We developed a computer simulation model based on data from an acute care hospital in Hamilton, Ontario. We evaluated and compared the performance of certain practical ordering and allocation policies in terms of outdate rate, shortage rate and the distribution of the age of issued RBCs., Results: During the 1-year period for which we analysed the data, 10349 RBC units were transfused with an average issue age of 20·7 days and six units were outdated (outdate rate: 0·06%). Adopting a strict first in, first out (FIFO) allocation policy and an order-up-to ordering policy with target levels set to five times the estimated daily demand for each blood type, reduced the average issue age by 29·4% (to 14·6 days), without an increase in the outdate rate (0·05%) or resulting in any unmet demand. Further reduction of issue age without a significant increase in outdate rate was observed when adopting non-FIFO threshold-based allocation policies and appropriately adjusting the order-up-to levels., Conclusion: A significant reduction of issue age could be possible, without compromising availability or resulting in excessive outdates, by properly adjusting the ordering and allocation policies at the hospital level., (© 2016 International Society of Blood Transfusion.)

Background: The only accepted way to avoid transfusion-associated graft-versus-host disease is irradiation of blood components. With respect to irradiation and associated storage time, different recommendations exist. We examined early irradiated (day +3) leucoreduced red blood cell units for irradiation-associated damages during storage., Study Design and Methods: We studied 80 leucoreduced units from two manufacturers. All RBCs were stored in the additive solution saline-adenine-glucose-mannitol (SAG-M) and leucoreduced on collection day. Forty components were irradiated on day +3 with 30 Gy, 40 served as non-irradiated controls. Samples were drawn and analysed from these 80 units on days +3, +7, +14, +21, +28, +35 and +42., Results: From day +14, there was a significant difference in the in vitro haemolysis rate between the non-irradiated and the irradiated components. Two of the irradiated units showed a haemolysis rate over the recommended limit of 0·8% on day +42. Potassium and lactate dehydrogenase activity increased faster in irradiated groups during storage., Conclusions: Our findings show that leucoreduced RBCs which are irradiated early after collection show an obvious radiation damage over storage period. Interestingly, on days +28, +35 and +42 the quality of RBCs which were irradiated on day +3 was nearly identical to the quality of RBCs which were irradiated on day +14 in a former series. Early irradiation does not cause more damage of RBCs during subsequent storage than irradiation on day +14. The maximum storage period of irradiated RBCs should remain restricted to 28 days from collection, however independently from the day of irradiation within this period., (© 2015 International Society of Blood Transfusion.)

Background and Objectives: The influence that blood component separation methods have on changes to the red blood cell membrane during storage is not well understood. In Canada, red cell concentrates (RCCs) are produced using the buffy coat (BC, top/bottom) and the whole-blood filtration (WBF, top/top) methods, and this study aimed at comparing their influence on the characteristics of the extracellular vesicles (EV) which accumulated in the respective products during storage., Materials and Methods: Using flow cytometry, dynamic light scattering and mass spectrometry, we assessed RCC EVs for concentration, size, lipid composition and correlation with supernatant haemoglobin (Hb)., Results: Accumulation of RBC EVs (CD235a(+) ) with storage time was similar in WBF and BC RCCs. The size of the EVs changed from <100 nm at d5 to near 200 nm by d42, with the EVs from WBF being smaller (P < 0·001) than BC RCCs at all storage times. The amount of EV-bound Hb in the WBF and BC units was similar (about 10% of total supernatant Hb). WBF EVs and BC EVs displayed similar lipid composition., Conclusion: Haemolysis and EVs increase in BC and WBF RCCs during storage. Differences in the size characteristics of the EVs in WBF and BC RCCs suggest that non-RBC EVs are more prevalent in WBF products. Understanding the impact that manufacturing has on the characteristics of the different populations of EVs in RCCs will aid quality improvement efforts., (© 2015 International Society of Blood Transfusion.)

Background and Objectives: The morphology of red blood cells (RBCs) deteriorates progressively during hypothermic storage. The degree of deterioration varies between individual cells, resulting in a highly heterogeneous population of cells contained within each RBC unit. Current techniques capable of categorizing the morphology of individual stored RBCs are manual, laborious and error-prone procedures that limit the number of cells that can be studied. Our objective was to create a simple, automated system for high-throughput RBC morphology classification., Materials and Methods: A simple microfluidic device, designed to enable rapid, consistent acquisition of images of optimally oriented RBCs, was fabricated using soft lithography. A custom image analysis algorithm was developed to categorize the morphology of each individual RBC in the acquired images. The system was used to determine morphology of individual RBCs in several RBC units stored hypothermically for 6-8 weeks., Results: The system was used to automatically determine the distribution of cell diameter within each morphological class for >1 000 000 individual stored RBCs (speed: >10 000 cells/h; accuracy: 91·9% low resolution, 75·3% high resolution). Diameter mean and standard deviation by morphology class were as follows: discocyte 7·80 ± 0·49 μm, echinocyte 1 7·61 ± 0·63 μm, echinocyte 2 7·02 ± 0·61 μm, echinocyte 3 6·47 ± 0·42 μm, sphero-echinocyte 6·01 ± 0·26 μm, spherocyte 6·02 ± 0·27 μm, stomatocyte 1 6·95 ± 0·61 μm and stomatocyte 2 7·32 ± 0·47 μm., Conclusions: The automated morphology classification procedure described in this study is significantly simpler, faster and less subjective than conventional manual procedures. The ability to evaluate the morphology of individual RBCs automatically, rapidly and in statistically significant numbers enabled us to perform the most extensive study of stored RBC morphology to date., (© 2015 International Society of Blood Transfusion.)

Background and Objectives: Red cells frozen with glycerol may require gamma-irradiation after thawing and deglycerolization for transfusion to at-risk patients. Both freezing and irradiation are known to cause red cell damage. However, the effect of irradiation on the quality of deglycerolized red cells and the optimal shelf life of such a component is currently unknown., Materials and Methods: Red cells (<7 days) were pooled, split and glycerolized using an ACP-215 automated cell washer (n = 12 pairs) and frozen at -80°C. Red cells were thawed, deglycerolized and resuspended in SAG-M. One of each pair was gamma-irradiated, while the other served as a control. Products were stored at 2-6°C and sampled for in vitro testing immediately after irradiation, and at 24 and 48 h postirradiation., Results: Irradiation of deglycerolized red cells led to a >1·5-fold increase in extracellular potassium, compared to control units at 24 and 48 h postirradiation. Other parameters, including haemolysis, were not significantly affected by irradiation postdeglycerolization., Conclusion: Deglycerolized, irradiated red cells had increased supernatant potassium, but remained of acceptable quality for 24 h postirradiation., (© 2015 International Society of Blood Transfusion.)

Background: AS-7 is a new alkaline hypotonic red cell additive solution (AS) shown to improve red cell quality during storage compared with AS-1. We sought to compare red cells stored in AS-7 with those stored in SAGM using RCC that were either untreated, or washed or irradiated on day 14 of storage., Study Design and Methods: A pooled and split study design was used to produce seven identical RCC (four in SAGM and three in AS-7). At day 14 following donation, two RCC (one in SAGM and one in AS-7) were gamma irradiated and three RCC (two in SAGM and one in AS-7) were washed and resuspended in either SAGM or AS-7. RCC were sampled for analysis throughout storage and at end of shelf life: day 28 for washed or irradiated and day 35 for untreated RCC., Results: For untreated, washed or irradiated RCC, those stored in AS-7 had lower haemolysis, red cell microvesicles and supernatant potassium content than RCC in SAGM. In addition, ATP levels and pH were better maintained in AS-7 RCC than in SAGM RCC., Conclusion: These data suggest that the quality of these components may be improved by storage in AS-7 compared with SAGM., (© 2015 International Society of Blood Transfusion.)

Background and Objectives: Transfusion of red blood cells (RBC) is beneficial for the patient but can also be harmful, as randomized trials have demonstrated increased infection rates, bleeding and mortality. The study aim was to investigate the response of the vascular system (the haemostatic function and the endothelium) to RBC transfusion., Materials and Methods: Blood was sampled from patients with various transfusion-dependent haematologic diseases before 1 and 24 h after RBC transfusion. Primary and secondary haemostasis was evaluated by whole-blood impedance aggregometry (Multiplate) and by thromboelastography (TEG). Samples were analysed by ELISA for biomarkers reflecting endothelial activation and damage (sICAM-1, syndecan-1, sThrombomodulin (sTM), sVE-Cadherin), platelet activation (sCD40L) and inflammation (hsCRP)., Results: A total of 58 patients were enrolled in the study. Median age was 71 years. Compared to before transfusion, patients had slightly reduced coagulability 1 h after RBC transfusion, assessed by TEG. However, transfusion of older RBC products (>14 days) was associated with increased coagulability (all P < 0·05). The level of syndecan-1 increased slightly 24 h after transfusion (median 12·4 (IQR 9-23) vs. 13·2 (9-25) ng/ml, P < 0·01), indicating increased glycocalyx degradation., Conclusion: Overall, RBC transfusion was associated with reduced coagulability and endothelial glycocalyx degradation. Transfusion of older RBCs was however associated with increased coagulability. The changes observed were small to moderate and the clinical relevance of these findings should be investigated in larger studies., (© 2015 International Society of Blood Transfusion.)

Background and Objectives: Cytomegalovirus poses a risk to transfusion safety as its transmission to an immunocompromised recipient may lead to significant clinical sequelae. Once infection is established, it is lifelong and generally asymptomatic. Strategies to reduce the risk of transfusion-transmitted CMV (TT-CMV) include donor serological testing and blood component leucodepletion to deplete the transmissible reservoir. We estimate the residual risk for non-CMV antibody screened, leucodepleted (LD-only) fresh blood components., Materials and Methods: We established an approach to estimate the risk of TT-CMV under various scenarios. We estimated the probability of an infectious component, for both red cells and platelets, as a function of the observed WBC filter failure rate and the probability that such a unit was also contaminated with infectious virus., Results: Using this model, the estimated combined residual risk of LD-only red cell and platelet units was very low, 1 in 13 575 000 (95%CI:1 in 1 344 167 000-1 in 1 730 000) as was the individual residual risk estimate for LD-only red cells, 1 in 7 790 000 (95%CI: 1 in 771 307 000-1 in 993 000) and LD-only platelets, where a zero risk was estimated (95%CI: 0-1 in 1 074 000)., Conclusion: We describe a novel approach to assess the residual risk of LD-only components. This can be applied generally using local data. Our risk estimate for LD-only blood components in Australia is below the threshold of 1 in 1 million, generally considered negligible. This provides a useful indicator of the relative safety of LD-only components to assist clinical decisions when serologically screened inventory is unavailable., (© 2015 International Society of Blood Transfusion.)

Cryopreserved (frozen) red blood cells have been used in transfusion medicine since the Vietnam war. The main method to freeze the red blood cells is by usage of glycerol. Although the usage of cryopreserved red blood cells was promising due to the prolonged storage time and the limited cellular deterioration at subzero temperatures, its usage have been hampered due to the more complex and labour intensive procedure and the limited shelf life of thawed products. Since the FDA approval of a closed (de) glycerolization procedure in 2002, allowing a prolonged postthaw storage of red blood cells up to 21 days at 2-6°C, cryopreserved red blood cells have become a more utilized blood product. Currently, cryopreserved red blood cells are mainly used in military operations and to stock red blood cells with rare phenotypes. Yet, cryopreserved red blood cells could also be useful to replenish temporary blood shortages, to prolong storage time before autologous transfusion and for IgA-deficient patients. This review describes the main methods to cryopreserve red blood cells, explores the quality of this blood product and highlights clinical settings in which cryopreserved red blood cells are or could be utilized., (© 2014 International Society of Blood Transfusion.)

Background and Objectives: Semi-automatic separation devices can be used for the separation of centrifuged whole blood into leucoreduced red cell concentrate (LR-RCC), plasma and buffy coat (BC) and to make platelet concentrates (PC) from pooled BCs. To improve and to obtain a more uniform and standardized process, the CompoMat G5 (Fresenius) was implemented, a new-generation semi-automated device., Materials and Methods: Uniform programs for WB separation and preparation of PCs were validated, using collection and pooling systems with CompoFlow (CF) closures, which can be automatically opened by the G5. Cell counts were performed and compared with historic data of blood components obtained with the formerly used Compomat G4 and Optipress II. After implementation, different adjustments were made to improve product quality., Results: Leucoreduced red cell concentrates (280 ± 15 ml, 53 ± 5 g haemoglobin) and plasma (317 ± 16 ml) met European guidelines. BCs (48 ± 2 ml, 0·42 ± 0·05 l/l, 93 ± 25 × 10(9) platelets) contained a similar platelet (PLT) content as BC prepared before with the Compomat G4. A relatively high percentage (4-6%) of PCs (330 ± 17 ml, 330 ± 50 × 10(9) PLT, 0·12 ± 0·21 × 10(6) leucocytes) contained <250 × 10(9) PLT which was the subject of improvement studies. After implementation, RCC and BC discard decreased and workload was less. Operator complaints were also less frequent., Conclusion: The same high-quality blood components can be prepared by using the CompoMat G5 as previously with other semi-automated devices. Improvement was realized by automation of the opening process by the use of collection systems with CF closures, which led to a decrease in discarded units and workload., (© 2014 International Society of Blood Transfusion.)

Blood banking underpins modern medical care, but blood storage, necessary for testing and inventory management, reduces the safety and efficacy of individual units of red blood cells (RBCs). Stored RBCs are damaged by the accumulation of their own waste products, by enzymatic and oxidative injury, and by metabolically programmed cell death. These chemical activities lead to a complex RBC storage lesion that includes haemolysis, reduced in vivo recovery, energy and membrane loss, altered oxygen release, reduced adenosine tri-phosphate and nitric oxide secretion, and shedding of toxic products. These toxic products include lysophospholipids that can cause transfusion-related acute lung injury, free iron that can potentiate infections and cause inflammation, and shed microvesicles that can scavenge nitric oxide and potentiate inflammation and thrombosis. However, most of the obvious negative outcomes of RBC storage are uncommon and appear to be related to exceptionally bad units. Generally, the quality of stored RBCs is highly related to the conditions of storage, so refrigerator temperature, intact bags, residual leucocyte counts and visible haemolysis remain excellent general measures. Specific biochemical measures, such as adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) concentrations, calcium and potassium content or lipid breakdown products, require specialized measures that are not widely available, involve destructive testing and generally reflect only a part of the storage lesion. This review describes a number of components of the storage lesion and their measurement and attempts to access the utility of the measures., (© 2014 International Society of Blood Transfusion.)

Background: Red blood cells (RBCs) are routinely stored in liquid state at temperatures below 6°C, and RBC unit core temperature should not exceed 10°C during transport. Since the critical temperature of 10°C was chosen mostly arbitrarily, this study investigated the effect of both constant temperature settings as well as multiple rewarming cycles on stored RBCs with respect to morphology, biochemical parameters and haemolysis., Materials and Methods: Buffy coat-depleted filtered RBCs were used as standard products. RBCs were stored at 1-6°C (reference group, n = 12), 13 and 22°C (test groups, n = 12 each) or stored at 1-6°C and warmed up five times to 10, 13, or 22°C for a period of 24 h each. Various biochemical parameters were measured weekly. RBCs were further investigated using electron microscopy., Results: Red blood cells stored constantly at 13 or 22°C showed stable haemolysis rates until day 28 and day 14, respectively. RBCs stored at 1-6°C with five warming-up periods to 10, 13 or 22°C each lasting 24 h (total 120 h) did not exceed the limit of the haemolysis rate at the end of storage. Differently shaped erythrocytes were found in all samples, but more crenate erythrocytes appeared after 42 days of storage independent of temperature profiles., Conclusion: Red cells can be kept at constant temperatures above 6°C without apparent harmful effects at least until day 14, whereas multiple warming cycles for no longer than 24 h at 10, 13 or 22°C with subsequent cooling do not cause quality loss as assessed using the in vitro assays employed in this study., (© 2013 International Society of Blood Transfusion.)

Background and Objective: The Reveos automated blood processing system has been developed to combine primary and secondary processing of whole-blood units, resulting in a plasma unit, a red-blood-cell concentrate and an interim platelet unit per input. The aim of this study was to determine product specifications and in vitro quality of components produced by the Reveos system., Materials and Methods: Whole blood was processed using the Reveos system and compared with historical Reference units produced using semi-automated methods. Reveos red cells were leucoreduced and stored in SAGM at 4°C. Reveos plasma was frozen at -30°C and factor activity was assessed after thawing. Reference red cell, plasma and buffy coats were produced by top and bottom processing. Leucoreduced Reveos and Reference platelet concentrates were prepared by pooling four interim platelet units or four buffy coats, respectively, with SSP+., Results: Processing with the Reveos system was faster (76 min) than semi-automated separation (92 min). The red cell and platelet yields were higher in the units prepared by the Reveos system. The Reference and Reveos red cell and plasma units had very similar in vitro quality parameters. The platelet concentrates were also similar in many in vitro parameters, including pH, glucose and lactate metabolism, hypotonic shock response and phosphatidylserine expression, although platelet activation markers (CD62P and cytokine levels) were higher in the Reveos units., Conclusion: The Reveos system can improve blood component efficiencies through reductions in processing time, whilst maintaining similar component quality., (Vox Sanguinis © 2013 International Society of Blood Transfusion.)

Background and Objectives: There are manual and automated methods to separate whole blood (WB) available. The Atreus whole blood processing system is an automated method, which combines centrifugation and expression of components into a single device. A major difference to conventional methods is that centrifugation temperature is not controlled at 22°C. The aim of this study was to examine the influence of increased centrifugation temperatures on the quality of red-blood-cell concentrates (RCC) after active cooling of WB prior to processing., Materials and Methods: A total of 28 WB were processed: 16 at centrifugation temperatures of up to 28°C (1st protocol) and 12 at 34°C (2nd protocol). RCC quality parameters were tested weekly for 42 days., Results: Red-blood-cell concentrates (RCC) quality complied with the European and German guidelines. Haemolysis was not significantly different throughout storage. Significant statistical differences were detected between both protocols in potassium concentration at the end of storage and in ATP levels at the day of processing., Conclusion: Centrifugation temperatures of up to 34°C are well tolerated by the red blood cells with minimal interference with the RCC quality parameters., (© 2013 International Society of Blood Transfusion.)

Haemoglobin A1c (HbA1c ) represents a key biomarker in diabetes diagnosis and management, as it is indicative of recent blood glucose concentrations. Glycation of haemoglobin is a non-enzymatic irreversible process that is promoted by the prolonged exposure of erythrocytes to high glucose concentrations, a condition that is known to occur under blood banking conditions. However, controversial data indicate no clear hint as to whether and to which extent HbA1c accumulates during red blood cell storage. Hereby, we propose the application of a validated MALDI-TOF mass-spectrometry-based method to this issue and report the observation about HbA1c levels apparently increasing over storage progression., (© 2013 International Society of Blood Transfusion.)

Background: During storage, red blood cells (RBCs) lose their membrane stability, leading to haemolysis and microparticle (MP) formation. The use of RBCs stored for more than 28 days has been associated with an increased incidence of deep vein thrombosis. However, the exact mechanism by which coagulation activation is enhanced in stored RBCs is still unknown., Objectives: To investigate the relevant potential procoagulant activities of MPs and study the relative procoagulant factors for initiating the coagulation on MPs in stored RBCs., Study Design and Methods: MPs were isolated from the plasma of RBC units stored in citrate-phosphate-dextrose-adenine. At seven storage time-points (d0, d7, d14, d21, d28, d35 and d42), MPs were morphologically observed, quantified and analysed for tissue factor, factor XI (FXI) and their thrombin-generating potential., Results: MPs were observed using electron microscopy. The size of the MPs ranged from 0·272 μm to 0·973 μm in diameter. During the storage of RBCs in plastic bags, the MP concentration increased from 3389 ± 218/μl at day 0 to 61 586 ± 2237/μl at d42. Thrombin generation was dependent on the total number of MPs (r = 0·987). Anti-human FXI antibody inhibited thrombin concentrations by 50·3% compared with control plasma, whereas antitissue factor and antitissue factor pathway inhibitor failed to reduce thrombin concentrations., Conclusions: Our study provides evidence that MP formation due to RBC storage might propagate coagulation not only by exposing phosphatidylserine, but also by initiating thrombin generation independently of tissue factor in a FXI -dependent manner., (© 2013 International Society of Blood Transfusion.)

Background and Objectives: Previous data that showed maintenance of reticulocyte percentage in whole blood stored in CPDA-1 have led to the assumption that reticulocyte maturation becomes arrested during refrigerated storage. However, reticulocyte behaviour in red-blood-cell units stored in additive solutions has not yet been studied. This study was thus aimed at determining reticulocyte count and reticulocyte subtypes in red-blood-cells units stored in AS-1., Materials and Methods: Reticulocyte percentage and subtypes were determined by flow cytometry with thiazole orange in six red-blood-cells units stored in AS-1., Results: Reticulocyte count was 26.8 ± 4.6 × 10(9) /l at week 0.5 and 8.2 ± 2.9 × 10(9) /l at week 6. Total haemolysis during storage was 0.19 ± 0.08%. High-fluorescence reticulocytes were 2.0 ± 3.2 × 10(9) /l at week 0.5 and decreased by weeks 2, 4 and 6. Low-fluorescence reticulocytes were 22.1 ± 3.1 × 10(9) /l at week 0.5 and decreased by weeks 4 and 6., Conclusion: A significant decrease in reticulocytes occurred during red-blood-cells units' storage in AS-1. Even if it were assumed that all of haemolysed cells during storage were reticulocytes, there are a number of them whose disappearance cannot be explained by this mechanism. Changes observed in reticulocyte subtypes suggest that they mature during storage., (© 2013 The Author(s) Vox Sanguinis © 2013 International Society of Blood Transfusion.)

Introduction: We investigated whether haemolysis in red cells suspended in plasma was affected by the lipid content and/or methylene blue (MB) treatment of fresh-frozen plasma (FFP). We also investigated whether haemolysis was affected by the conditions under which lipaemic plasma was stored., Methods: Study 1: Visibly lipaemic (n = 22) or nonlipaemic FFP (n = 24) units were thawed, pooled and split into identical pairs, one of which was MB treated. These units were used to resuspend red cell concentrates (RCC) and tested for haemolysis immediately and after 24 and 48 h of storage at 2-6°C. Study 2: Fresh plasma was aliquoted into 15-ml tubes and stored in one of four ways as follows: room temperature; 2-6°C; frozen and thawed; or twice frozen and thawed. A sample of RCC was resuspended in each of these plasmas and haemolysis measured after 2 h. Study 3: Plasma was divided into 15-ml tubes and stored as in study 2 followed by storage left standing upright in a refrigerator (2-6°C) for 24 h (with the exception of the room temperature sample). Plasma was separated into top, middle and bottom fractions and used to resuspend RCC that were assessed for haemolysis after 2 h., Results: The levels of haemolysis in RCC were immediately greater when suspended in lipaemic plasma (0·70 ± 0·53% v 0·05 ± 0·06% for nonlipaemic plasma), which increased further on subsequent storage for 48 h (1·22 ± 0·40% v 0·15 ± 0·14% for nonlipaemic plasma). This was irrespective of whether plasma was MB treated. Lipaemic plasma stored frozen and then thawed resulted in the greatest haemolysis. In lipaemic plasma stored at 2-6°C, the chylomicron-rich top fraction caused the highest level of haemolysis., Conclusion: Haemolysis in red cells is increased in those suspended in lipaemic plasma and is dependent upon the storage conditions of that plasma prior to suspension. These data are relevant to the choice of plasma used to suspend red cells for neonatal exchange transfusion., (© 2012 The Author(s). Vox Sanguinis © 2012 International Society of Blood Transfusion.)

Background and Objectives: There is no automated, accurate assay for the enumeration of residual red blood cells (rRBCs) in non-RBC components for transfusion, despite the potential risk of allo-immunization when mismatched components are transfused., Materials and Methods: The automated ADVIA 120 cerebrospinal fluid (CSF) assay, which is approved to count RBCs and WBCs in CSF samples, was optimized and tested to measure rRBC in platelet concentrate (PC) and plasma components., Results: Sample dilution, incubation time and reagent volume were optimized for use with non-RBC blood products. The assay was linear (R(2) = 0·99), even at low rRBCs counts. Intra- and inter-assay variation gave coefficients of variance (CV) between 2·2 and 9·4% and 2·6 and 14·9%, respectively, depending on rRBC levels. Good correlation (r = 0·995) was found between the automated assay and manual counting, which is considered the gold standard. Using the automated assay, the range of rRBCs (count/unit) in buffy-coat platelet concentrate (PCs) was 27-5505 × 10(6) and in apheresis PCs was 1-361 × 10(6)., Conclusion: The ADVIA CSF assay is a sensitive, precise and accurate means to assess rRBC counts in non-RBC components., (© 2012 The Author(s). Vox Sanguinis © 2012 International Society of Blood Transfusion.)

Background and Objectives: Blood components must be stored under controlled temperature conditions, for reasons of component quality and safety. However, there are occasions when components may be exposed to conditions outwith the defined limits. This study aimed to generate prospective data on the effect of red cell exposure to extremes of temperature., Materials and Methods: Study 1: red cell concentrates (RCC) in saline, adenine, glucose and mannitol (SAGM), made after ambient overnight hold of whole blood, were exposed to either +22°C or -2°C for up to three periods of 3 h on days 3, 8 and 15 of storage, followed by a 5 h exposure on day 29. Study 2: RCC in SAGM were exposed to 25°C for 24 or 48 h from day 2. In vitro markers of cell quality were measured during storage to 43 days, and compared with control units that had been stored at 2-6°C., Results: Multiple short-term exposures to +22°C or -2°C did not cause any significant changes to pH, haemolysis, supernatant potassium, cellular ATP, 2,3-DPG, or deformability, when compared to control units. Exposure of RCC to 25°C for 24 or 48 h caused a significant fall in pH, ATP, and deformability., Conclusion: Red cells may be damaged by prolonged exposure to warm temperatures, but repeated short-term exposure to 22°C or -2°C does not appear to affect the in vitro quality of RCC. It is important to note that no bacterial growth studies were performed during this study., (© 2012 The Author(s). Vox Sanguinis © 2012 International Society of Blood Transfusion.)

Most methods for quality control of white blood cell (WBC) depletion in blood products are based on flow cytometric techniques. Nearly all commercial kits are based on propidium iodide staining of the DNA and subsequently counting those DNA based events as residual WBC. Here, we could show that a substantial proportion of those events are derived from nucleated red blood cells and therefore not specific for WBCs (e.g. in erythrocyte products 30%). We developed a flow cytometric method for residual WBC counting applying simultaneous DNA- and WBC-specific surface staining to enable this., (© 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.)

Background and Objectives: The Fc function assessment of immunoglobulin products is performed to confirm that the biological activity of immunoglobulin products is not affected by manufacturing and storage conditions. The European Pharmacopoeia (EP) test is cumbersome and time-consuming especially with respect to the derivatization of red blood cells (RBC) with rubella antigen via tanninization. We investigated the KODE biosurface engineering technology for inserting specific antigens into red blood cell membranes to create kodecytes and evaluated their suitability for use in the Fc function assay., Materials and Methods: Human RBC were derivatized with function-spacer-lipid (FSL) constructs comprising of a peptide epitope of a cytomegalovirus (CMV) surface protein, conjugated to a spacer and lipid tail. These kodecytes were used in an Fc function assay based on the monitoring of complement-mediated haemolysis of immunoglobulin-coated red cells. Optimization of FSL construct, immunoglobulin and complement concentration was undertaken. Fc values obtained for various immunoglobulin batches were compared with those from the EP-based method. Carbohydrate-bearing FSLs Galili (Galα1-3Galβ1-4GlcNAcβ), Galα1-4GlcNAcβ and GalNAcα1-3Galβ were also evaluated., Results: Fc function indices determined with CMV peptide construct-coated red cells were comparable to those obtained with the EP-based method with respect to specificity and precision. Carbohydrate-bearing FSLs revealed Fc indices lower than expected., Conclusion: The use of CMV kodecytes was shown to be a convenient means of generating red cells for the determination of Fc function of immunoglobulin products and offers the possibility of significantly reducing the time required to perform this assay., (© 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.)

Introduction: The irradiation of blood components is used to inactivate T-lymphocytes to prevent transfusion-associated graft-versus-host (GVH) disease in susceptible recipients. X-irradiation can be used as an alternative to gamma irradiation and does not involve the use of a radioactive source. This study investigated the effect of X-irradiation on the quality of various red cell concentrates (RCC)., Methods: Whole blood units were processed into RCC in additive solution (SAG-M) or RCC in plasma suitable for intra-uterine (IUT) or neonatal exchange transfusion. RCC-SAG-M were irradiated at day 14 and sampled before and 0, 7 and 14 days following irradiation. RCC-IUT and RCC-Exchange were irradiated on day 4 and sampled before and 0 and 24 h following irradiation. Extracellular potassium levels and free plasma haemoglobin (haemolysis) were compared in X or gamma-irradiated units during storage., Results: X-irradiation of RCC in SAG-M, or RCC for IUT or Exchange resulted in haemolysis comparable with gamma irradiation and levels were below the current UK/CE limit of 0.8% at the end of shelf life. X-irradiation of RCC-SAG-M or RCC-IUT with haematocrit of 75% and 85% resulted in potassium leakage comparable with gamma-irradiation. However, X-irradiation of RCC for neonatal exchange transfusion showed statistically significantly higher supernatant potassium levels after 24 h than gamma irradiation, although this difference is considered to be clinically insignificant., Conclusion: X-irradiation of RCC in SAG-M or IUT or RCC in plasma suitable for neonatal exchange transfusion resulted in acceptable levels of haemolysis and potassium leakage compared with the current process of gamma irradiation., (© 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.)

Background: Whole blood is stored at room temperature (RT) until processing into components. After separation and filtration, the RCC has to be cooled from RT to +2 - 6°C. Different start times of the cooling process and different cooling rates can be encountered in daily routine. The effect of these parameters of the initial cooling of leucoreduced red cell concentrates (LR-RCC) on in vitro quality is not known., Methods: In paired experiments (n=12), LR-RCCs in SAGM were cooled immediately after preparation from RT to +2 to +6°C either 'fast' (within 2½ h) or 'slow' (within 10-24 h) or 'slow' after a holding period of 6, 12, 18 or 24 h. Units were then stored at +2-6°C for 42 days and sampled at regular intervals for in vitro analysis., Results: Irrespective of the start time and cooling rate during the initial cooling process, all units maintained good in vitro quality up to Day 42 with haemolysis <0·8%. Adenosine triphosphate (ATP) levels remained >2·7 μmol/g Hb in 99% of all units up to Day 35. Differences in pH, ATP content and 2,3-DPG content between the groups were largest at Day 2 or 3 but generally disappeared during storage., Conclusion: Start time and cooling rate of the initial cooling process had minor effects on in vitro quality of red cells. LR-RCCs can be stored up to 24 h before cooling down to +2-6°C without deleterious effects on in vitro parameters during 42-day storage., (© 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.)

Background and Objectives: Neonates undergoing exchange transfusion require <5-day-old red cells suspended in plasma. This study assesses the effect of replacing the saline, adenine, glucose and mannitol (SAGM) of prion reduced (P-Capt) red cells with either methylene blue-treated plasma (MBTFFP) or OctaplasLG to reduce the risk of variant Creutzfelt-Jakob disease transmission., Materials and Methods: Twenty leucoreduced red cell units in SAGM were prion reduced on day 1. The SAGM was replaced by MBTFFP (n=10) or OctaplasLG (n=10). The units were irradiated and stored at 4°C for 24 h. A further 20 units were stored for 5 days before being processed as above. Haemolysis (%), potassium, ATP, 2,3-DPG and plasma proteins were measured., Results: Haemolysis remained low (≤0·16%). Following irradiation and storage, red cells in both types of plasma showed similar changes in potassium and ATP concentrations. The 2,3-DPG concentrations were well maintained although lower in red cells in OctaplasLG compared with those in MBTFFP (4·79 vs. 6·83 μmoles/g Hb on day 6). MBTFFP contained lower concentrations of fibrinogen, FV and FVIII. In OctaplasLG, alpha-2-antiplasmin was approximately 0·4 U/ml lower than in MBTFFP. After 24 h at 4°C, free protein S in OctaplasLG fell from 0·82 to 0·57 IU/ml. Other plasma proteins, in both types of plasma, were stable., Conclusions: Red cells in both types of plasma demonstrated similar storage characteristics. The plasma proteins, except protein S in OctaplasLG, were stable over 24 h at 4°C in both types of plasma, and low FVIII concentrations were noted in the MBTFFP (group O) units used., (© 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.)

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., (© 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.)

Background and Objectives: The DiaMed Impact R tests platelet function under close to physiological flow conditions. The machine is designed to use whole blood but by adding back compatible red cells, it can be used to study stored platelet concentrates. To date, red cells ≤14 days old have been used. In this study, the effect on the assay of using red cells stored for up to 60 days was examined., Material and Methods: This study looked at buffy coat-derived platelet concentrates on day 2 of storage along with various stored red-blood-cells (RBC). To determine whether the age of the RBC is a factor in supporting adhesion and aggregation, platelets were assayed with either RBC stored between 2 and 60 days or with separated 'young' and 'old' red cell populations obtained using a centrifugation method and confirmed by percoll gradient analysis., Results: A statistically significant difference was observed between red-blood-cells stored for ≤20 days compared with those which have been stored for 21-60 days in respect of their ability to support platelet adhesion (SC) and aggregation (AS) (P<0·01). Separating red cells by centrifugation into top (young population) and bottom (old population) showed that the effect of storage was much greater than was any difference between young and old at the individual time-points e.g. 'young' red cells from stored units were poorer at supporting platelet adhesion and aggregation than 'young' red cells from fresh units., Conclusion:   Results suggest that the red cells should be stored for less than 21 days when using this assay. This assay may also allow assessment of red cell functionality., (© 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.)

Background: The haemolysis level at the end of storage is a performance parameter for RBC preparations. In the evaluation of new devices or new processes for processing blood, it is relevant to evaluate whether the haemolysis is linked to (1) specific characteristics of the blood donor, or (2) the nature of the blood-processing methodologies., Materials and Methods: As part of the validation of a new automated whole blood processing system compared to the current manual methods, randomized, paired crossover studies were conducted evaluating measures of blood component quality, including RBC haemolysis over 42 days of storage., Results: The association between haemolysis and the individual subject was evaluated by modelling haemolysis with independent predictors of treatment (control and test processing) and leucocyte reduction as fixed factors with donor and laboratory as random effects in a mixed-effects ANOVA model. It was found that the day 42 haemolysis values were strongly dependent on the donor subject, with an intraclass correlation coefficient of 0.81., Conclusions: The data reported in this study suggest a link between the specific whole blood donor and the haemolysis levels observed in red-blood-cell units stored refrigerated for 42 days. Additional research to identify possible donor characteristics associated with haemolysis during storage is warranted.

Background and Objectives: Radio frequency identification (RFID) can be a key enabler for enhancing productivity and safety of the blood product supply chain. This article describes a systematic approach developed by the RFID Blood Consortium for a comprehensive feasibility and impact assessment of RFID application in blood centre operations., Materials and Methods: Our comprehensive assessment approach incorporates process-orientated and technological perspectives as well as impact analysis. Assessment of RFID-enabled process redesign is based on generic core processes derived from the three participating blood centres. The technological assessment includes RFID tag readability and performance evaluation, testing of temperature and biological effects of RF energy on blood products, and RFID system architecture design and standards. The scope of this article is limited to blood centre processes (from donation to manufacturing/distribution) for selected mainstream blood products (red blood cells and platelets)., Results: Radio frequency identification can help overcome a number of common challenges and process inefficiencies associated with identification and tracking of blood products. High frequency-based RFID technology performs adequately and safely for red blood cell and platelet products. Productivity and quality improvements in RFID-enabled blood centre processes can recoup investment cost in a 4-year payback period., Conclusion: Radio frequency identification application has significant process-orientated and technological implications. It is feasible and economically justifiable to incorporate RFID into blood centre processes.