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Additive solutions are used to limit changes that red blood cells (RBCs) undergo during storage. Several studies have shown better preservation of glucose and redox metabolism using the alkaline additive solution PAGGGM (phosphate-adenine-glucose-guanosine-gluconate-mannitol). In this randomized open-label intervention trial in 20 healthy volunteers, the effect of storage, PAGGGM vs SAGM (saline-adenine-glucose-mannitol), on posttransfusion recovery (PTR) and metabolic restoration after transfusion was assessed. Subjects received an autologous biotinylated RBC concentrate stored for 35 days in SAGM or PAGGGM. As a reference for the PTR, a 2-day stored autologous biotinylated RBC concentrate stored in SAGM was simultaneously transfused. RBC phenotype and PTR were assessed after transfusion. Biotinylated RBCs were isolated from the circulation for metabolomics analysis up to 24 hours after transfusion. The PTR was significantly higher in the 2-day stored RBCs than in 35-day stored RBCs 2 and 7 days after transfusion: 96% (90 to 99) vs 72% (66 to 89) and 96% (90 to 99) vs 72% (66 to 89), respectively. PTR of SAGM- and PAGGGM-stored RBCs did not differ significantly. Glucose and redox metabolism were better preserved in PAGGGM-stored RBCs. The differences measured in the blood bag remained present only until 1 day after transfusion. No differences in RBC phenotype were found besides an increased complement C3 deposition on 35-day RBCs stored in PAGGGM. Our data indicate that despite better metabolic preservation, PAGGGM is not a suitable alternative for SAGM because storage in PAGGGM did not result in an increased PTR. Finally, RBCs recovered from circulation after transfusion showed reversal of the metabolic storage lesion in vivo within a day. This study is registered in the Dutch trial register (NTR6492).

Platelet components are commonly transfused to patients for a variety of indications, including patients with low platelet counts or patients with platelet dysfunction who are bleeding or at high risk of bleeding. Although the risk of pathogen contamination of platelet components has declined significantly over the last 40 years, it remains a concern for the patients, for blood banks and for physicians. Pathogen inactivation (PI) technologies have been developed to mitigate this risk. This review focuses on the residual risks of transfusion-transmitted bacterial infections by platelet transfusion after PI. We describe and assess the relationship between the bacterial load and the timing and capacity of reduction of the different PI technologies, as well as the risks that could represent spore-forming microorganisms and the possible introduction of microorganisms after PI.

Background: Transfusion-related acute lung injury (TRALI) is a severe complication of blood transfusion that is thought of as a two-hit event: first the underlying patient condition (e.g., sepsis), and then the transfusion. Transfusion factors include human leukocyte antigen antibodies or biologic response modifiers (BRMs) accumulating during storage. Preclinical studies show an increased TRALI risk with longer stored platelets, clinical studies are conflicting. We aim to discover whether longer platelet concentrate (PC) storage time increases TRALI risk in a controlled human experiment. Study Design and Methods: In a randomized controlled trial, 18 healthy male volunteers received a first hit of experimental endotoxemia (2 ng/kg lipopolysaccharide), and a second hit of fresh (2-day old) or aged (7-day old) autologous PC, or physiological saline. After 6 h, changes in TRALI pathways were determined using spirometry, chest X-ray, and bronchoalveolar lavage (BAL). Results: All subjects reacted adequately to lipopolysaccharide infusion and satisfied SIRS criteria (increased pulse [>90/min] and temperature [>38°C]). There were no differences between the saline, fresh, and aged PC groups in BAL-fluid protein (95 ± 33 μg/ml; 83 ± 21 μg/ml and 104 ± 29 μg/ml, respectively) and relative neutrophil count (1.5 ± 0.5%; 1.9 ± 0.8% and 1.3 ± 0.8%, respectively), nor in inflammatory BAL-fluid BRMs (Interleukin-6, CXCL8, TNFα (Formula presented.) and myeloperoxidase), clinical respiratory parameters, and spirometry results. All chest X-rays were normal. Conclusions: In a human endotoxemia model of autologous platelet transfusion, with an adequate first hit and platelet storage lesion, transfusion of 7-day-old PC does not increase pulmonary inflammation compared with 2-day-old PC.

Background: Platelets (PLTs) differ in glycolytic activity, resulting in rapid acidification of ‘poor’ storing PLT concentrates (PCs) in plasma, or depletion of glucose when stored in PLT additive solution (PAS). We aimed to understand why PLT glycolysis rates vary between donors and how this affects storage performance. Study Design and Methods: Buffy coats from donors 70 years were selected and single-donor PCs in plasma or PAS-E were prepared. PCs were stored for 8 days at 22 ± 2°C and sampled regularly for analysis. Mitochondrial activity was analyzed with an Oroboros oxygraph. Age groups, or subgroups divided into quartiles based on glucose consumption, were analyzed with ANOVA. Results: In each comparison, PCs of the different groups were not different in volume and cellular composition. PLTs with the highest glucose consumption had a higher initial mean platelet volume (MPV) and developed higher CD62P expression and Annexin A5 binding during storage. Higher glycolytic activity in these PLTs was not a compensation for lower mitochondrial ATP production, because mitochondrial ATP-linked respiration of fresh PLTs correlated positively with MPV (R2 = 0.71). Donors of high glucose-consuming PLTs had more health-related issues. Storage properties of PCs from donors over 70 were not significantly different compared to PCs from donors younger than 45 years. Conclusions: High glucose-consuming PCs developing higher activation levels, not only displayed enhanced mitochondrial activity but were also found to contain larger PLTs, as determined by MPV. Storage performance of PLTs was found to be associated with donor health, but not with donor age.

Sprouting angiogenesis is key to many pathophysiological conditions, and is strongly regulated by vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we report that the early endosomal GTPase Rab5C and its activator RIN2 prevent lysosomal routing and degradation of VEGF-bound, internalized VEGFR2 in human endothelial cells. Stabilization of endosomal VEGFR2 levels by RIN2/Rab5C is crucial for VEGF signaling through the ERK and PI3-K pathways, the expression of immediate VEGF target genes, as well as specification of angiogenic ‘tip’ and ‘stalk’ cell phenotypes and cell sprouting. Using overexpression of Rab mutants, knockdown and CRISPR/Cas9-mediated gene editing, and live-cell imaging in zebrafish, we further show that endosomal stabilization of VEGFR2 levels is required for developmental angiogenesis in vivo. In contrast, the premature degradation of internalized VEGFR2 disrupts VEGF signaling, gene expression, and tip cell formation and migration. Thus, an endosomal feedforward mechanism maintains receptor signaling by preventing lysosomal degradation, which is directly linked to the induction of target genes and cell fate in collectively migrating cells during morphogenesis. Supplementary Information The online version of this article contains supplementary material available (10.1007/s10456-021-09788-4).

BACKGROUND AND OBJECTIVES: Di(2-ethylhexyl) phthalate (DEHP) must be removed from blood bag sets in Europe by 27 May 2025. DEHP is known to interact with the red blood cell (RBC) membrane, resulting in reduced haemolysis and thus prolonging shelf-life. Current non-DEHP alternatives result in increased haemolysis requiring reconsideration of the RBC shelf-life. Although the immediate impact of eliminating DEHP is to the European community, the non-DEHP movement could affect blood bag set availability globally. The purpose of this survey is to understand blood centre readiness regarding the transition to non-DEHP blood collection and storage systems. MATERIALS AND METHODS: A 24-question on-line survey was completed by members of the Biomedical Excellence for Safer Transfusion Collaborative research network. RESULTS: Responses were obtained from 16 blood collection or processing institutions. A majority of respondents (12/16) indicated that both shelf-life and haemolysis were equally important in selecting non-DEHP blood bag sets. Six respondents would accept a lower RBC product shelf-life compared to current practice. Respondents were not clear on the best non-DEHP vinyl material or RBC storage solution. Three European blood centres indicated they have developed non-DEHP transition plans. One challenge identified regarding the transition to non-DEHP is the extensive validation testing that will be required. CONCLUSION: Blood centres in Europe are concerned with meeting the sunset date for DEHP, considering that limited non-DEHP blood bag and RBC storage solutions are currently available. Banning DEHP in Europe, which may have global ramifications, represents a major challenge not yet fully understood by the transfusion medicine community.

Background and objectives Colloid osmotic pressure (COP) is a principal determinant of intravascular fluid homeostasis and a pillar of fluid therapy and transfusion. Transfusion-associated circulatory overload (TACO) is a leading complication of transfusion, and COP could be responsible for recruiting additional fluid. Study objective was to measure COP of blood products as well as investigate the effects of product concentration and storage lesion on COP. Materials and methods Three units of each product were sampled longitudinally. COP was measured directly as well as the determinants thereof albumin and total protein. Conventional blood products, that is red blood cell (RBC), fresh-frozen plasma (FFP) and platelet concentrates (PLTs), were compared with their concentrated counterparts: volume-reduced RBCs, hyperconcentrated PLTs, and fully and partially reconstituted lyophilized plasma (prLP). Fresh and maximally stored products were measured to determine changes in protein and COP. We calculated potential volume load (PVL) to estimate volume recruited using albumin's water binding per product. Results Colloid osmotic pressure varies widely between conventional products (RBCs, 1·9; PLTs, 7·5; and FFP, 20·1 mmHg); however, all are hypooncotic compared with human plasma COP (25·4 mmHg). Storage lesion did not increase COP. Concentrating RBCs and PLTs did not increase COP; only prLP showed a supraphysiological COP of 47·3 mm Hg. The PVL of concentrated products was lower than conventional products. Conclusion Colloid osmotic pressure of conventional products was low. Therefore, third-space fluid recruitment is an unlikely mechanism in TACO. Concentrated products had a lower calculated fluid load and may prevent TACO. Finally, storage did not significantly increase oncotic pressure of blood products.

Transfusion of whole blood rather than blood components is gaining popularity. It is easy to use, with one transfusion product to administer rather than 3, and is held at one storage temperature. It only contains anticoagulant-preservative solution, while components contain various storage solutions, which in theory may induce dilution coagulopathy. In this review, the quality of platelets in stored whole blood is summarized. In cold-stored whole blood, the platelet count declines by 1% to 2% per day. The responsiveness to various agonists declines during the storage time, but this appears to have a limited impact on clotting time or on clot strength as measured with thromboelastography. Animal studies have confirmed that platelets from stored whole blood participate equally well in clot formation. The recovery of platelets in stored whole blood is acceptable during at least 15 days of storage. The survival of platelets after transfusion is only 1 day, but this is likely to be sufficient for the intended patient group requiring massive transfusions, as the platelets are rapidly consumed in the wound area. In addition to the logistic benefits, there are drawbacks, most importantly having a sufficiently large inventory with an acceptable outdating rate, particularly since massive transfusions are rare, while requiring a lot of whole blood. The positive experience of the United States military with whole blood transfusion is often brought forward for introduction in the civilian blood bank, but patients with trauma are only a small fraction of the civilian population requiring massive transfusions. It needs to be determined whether in the resourceful environment of the hospital, these patients benefit from whole blood transfusions. Optimization of whole blood storage, with focus on platelet quality, needs to be performed to allow extension of the storage time beyond 15 days to a point where the number of units in inventory and outdating can be balanced.

Platelet concentrates (PCs) represent a blood transfusion product with a major concern for safety as their storage temperature (20-24°C) allows bacterial growth, and their maximum storage time period (less than a week) precludes complete microbiological testing. Pathogen inactivation technologies (PITs) provide an additional layer of safety to the blood transfusion products from known and unknown pathogens such as bacteria, viruses, and parasites. In this context, PITs, such as Mirasol Pathogen Reduction Technology (PRT), have been developed and are implemented in many countries. However, several studies have shown in vitro that Mirasol PRT induces a certain level of platelet shape change, hyperactivation, basal degranulation, and increased oxidative damage during storage. It has been suggested that Mirasol PRT might accelerate what has been described as the platelet storage lesion (PSL), but supportive molecular signatures have not been obtained. We aimed at dissecting the influence of both variables, that is, Mirasol PRT and storage time, at the proteome level. We present comprehensive proteomics data analysis of Control PCs and PCs treated with Mirasol PRT at storage days 1, 2, 6, and 8. Our workflow was set to perform proteomics analysis using a gel-free and label-free quantification (LFQ) approach. Semi-quantification was based on LFQ signal intensities of identified proteins using MaxQuant/Perseus software platform. Data are available via ProteomeXchange with identifier PXD008119. We identified marginal differences between Mirasol PRT and Control PCs during storage. However, those significant changes at the proteome level were specifically related to the functional aspects previously described to affect platelets upon Mirasol PRT. In addition, the effect of Mirasol PRT on the platelet proteome appeared not to be exclusively due to an accelerated or enhanced PSL. In summary, semi-quantitative proteomics allows to discern between proteome changes due to Mirasol PRT or PSL, and proves to be a methodology suitable to phenotype platelets in an unbiased manner, in various physiological contexts.

BACKGROUND: Over a century of advancements in the field of additive solutions for red blood cell (RBC) storage has made transfusion therapy a safe and effective practice for millions of recipients worldwide. Still, storage in the blood bank results in the progressive accumulation of metabolic alterations, a phenomenon that is mitigated by storage in novel storage additives, such as alkaline additive solutions. While novel alkaline additive formulations have been proposed, no metabolomics characterization has been performed to date. STUDY DESIGN AND METHODS: We performed UHPLC-MS metabolomics analyses of red blood cells stored in SAGM (standard additive in Europe), (PAGGSM), or alkaline additives SOLX, E-SOL 5 and PAG3M for either 1, 21, 35 (end of shelf-life in the Netherlands), or 56 days. RESULTS: Alkaline additives (especially PAG3M) better preserved 2,3-diphosphoglycerate and adenosine triphosphate (ATP). Deaminated purines such as hypoxanthine were predictive of hemolysis and morphological alterations. Guanosine supplementation in PAGGSM and PAG3M fueled ATP generation by feeding into the nonoxidative pentose phosphate pathway via phosphoribolysis. Decreased urate to hypoxanthine ratios were observed in alkaline additives, suggestive of decreased generation of urate and hydrogen peroxide. Despite the many benefits observed in purine and redox metabolism, alkaline additives did not prevent accumulation of free fatty acids and oxidized byproducts, opening a window for future alkaline formulations including (lipophilic) antioxidants. CONCLUSION: Alkalinization via different strategies (replacement of chloride anions with either high bicarbonate, high citrate/phosphate, or membrane impermeant gluconate) results in different metabolic outcomes, which are superior to current canonical additives in all cases.

BACKGROUND: Platelet-rich plasma (PRP) is frequently used in the treatment of acute and chronic wounds. One of the major problems concerning the use of PRP is the absence of a well-characterized and standardized product, which leads to a high variety in study outcomes. Therefore, more studies on the composition and standardization of PRP in wound healing are needed. STUDY DESIGN AND METHODS: Platelet concentrates derived from healthy blood donors were made in plasma (PC-plasma) or platelet additive solution (PC-PAS). The effects of PC-plasma, PC-PAS, and plasma were then tested on proliferation, differentiation, and migration of fibroblasts, as well as sprouting of endothelial cells in fibrin gels and chemotaxis of white blood cells (WBCs). RESULTS: PC-plasma stimulates the migration and proliferation of human dermal fibroblasts more than plasma or platelets alone. Furthermore, platelet factors decrease the expression of α-smooth muscle actin in dermal fibroblast cultures. PC-plasma also stimulates sprouting of endothelial cells. Finally, PC-plasma also acts as a strong chemoattractant for WBCs. CONCLUSIONS: Allogeneic PC-plasma has beneficial effects on various aspects of wound healing in vitro and is superior to plasma or platelets alone. PC-plasma is an attractive candidate for further in vivo evaluation.

Burn injury has severe impact on the physiologic homeostasis. Platelet counts show a distinct course post-burn injury, with a nadir at day 3 followed by a thrombocytotic period with at peak at day 15, with a gradual return to normal. So far, it is unknown how the functionality and activational status of platelets develop post burn. In this study, we investigated if the function, activation and growth factor content of platelets of burn patients are affected and how this evolves in time. Six burn patients with over 15% total burned surface area were followed during 1 month. Standard hematological and coagulation analyses, thromboelastography (TEG), platelet-function analyzer-100 (PFA), several platelet activation parameters (CD62P-CD63, AnnexinV) and growth factors (TGFb1, VEGF, PDGF-AB/BB, EGF, TGFb2, FGF-2, PDGF-AA) analyses were performed. TEG analyses showed procoagulant changes. PFA-100 analyses were nearly all within normal range. CD62P and CD63 and Annexin-V indicated no clear activation of platelets. Growth factor content followed the same course as the platelet count, reflecting a constant growth factor per platelet ratio. Concluding, platelets post burn-injury appears to be functional and not overly activated. However, burn patients seem to remain in a procoagulant state for an extensive period, which may impact their pathology.

BACKGROUND: Labeling of platelets (PLTs) is required to measure the recovery and survival of transfused PLTs in vivo. Currently a radioactive method is used to label PLTs. However, application of those radiolabeling methods is limited by both safety issues and the inability to isolate transfused PLTs from the circulation. Biotin-labeled PLTs are an attractive nonradioactive option. However, no validated protocol to biotinylate PLTs is currently available for human studies. STUDY DESIGN AND METHODS: Six PLT concentrates (PCs) were subaliquoted and biotinylated on Days 1 and 7 of storage. To distinguish the effect of the processing steps from the effects of biotin incubation, two control groups were used: 1) “sham” samples were processed without the biotinylation reagent and 2) control samples were assessed without any processing other than the PC isolation. For the biotinylation procedure, 50 mL of PCs was washed twice and incubated with 5 mg/L biotin for 30 minutes in a closed system. As measures of PLT activation, phosphatidylserine exposure and CD62p expression were assessed. RESULTS: After biotinylation, 98.4% ± 0.9% of PLTs were labeled. PLT counts, pH, and “swirling” were within the range accepted by the Dutch blood bank for standard PLT products. Biotinylated PLTs were more activated compared than controles but not more than sham samples, but were more activated than the controls. CONCLUSION: We developed a standardized and reproducible protocol according to Good Practice Guidelines standards, for biotin labeling of PLTs for clinical purposes. This method can be applied as nonradioactive alternative assess survival and recovery of transfused PLTs in vivo.

BACKGROUND Red blood cell (RBC) transfusion has been related to thromboembolic events. Microvesicles in the RBC product may support coagulation because they have procoagulant effects in vitro. We investigated whether transfusion of RBCs containing extracellular vesicles promotes coagulation in human recipients. As transfusion is mostly administered to ill patients, we used a model of endotoxemia. STUDY DESIGN AND METHODS Eighteen healthy volunteers were randomized to receive either saline or fresh (2 days stored) or stored autologous (35 days stored) RBC transfusion (Dutch Trial Register: NTR4455). Two hours after infusion of lipopolysaccharide (LPS, from Escherichia coli, 2 ng/kg body weight), subjects received either saline or fresh or stored RBCs. Blood was sampled every 2 hours up to 8 hours after LPS infusion. Vesicles were measured with a flow cytometer (A50-Micro, Apogee Flow Systems). RESULTS LPS resulted in increased thrombin generation compared to baseline. During storage, the total number of extracellular vesicles increased from 1.4 × 108 /mL (interquartile range [IQR], 8.3 × 107 -1.9 × 108 /mL) in the fresh product to 1.7 × 1010 /mL (IQR, 7.9 × 109 -2.3 × 1010 /mL; p

Background and Objectives There are concerns about the haemostatic function of platelets stored in platelet additive solution (PAS). Aim of this study was to compare the haemostatic function of PAS-C–platelets to plasma–platelets in reconstituted whole blood. Materials and Methods In our experiment, whole blood was reconstituted with red blood cells, solvent–detergent (SD) plasma and either PAS-C–platelets or plasma–platelets (n = 7) in a physiological ratio. On storage days 2, 5, 8 and 13, the agonist-induced aggregation (multiple electrode aggregometry), clot formation (thromboelastography) and agonist-induced CD62P responsiveness (flow cytometry) were measured. Results Samples with PAS-C–platelets showed significantly lower aggregation than plasma–platelets when induced with adenosine diphosphate, −6 U (95% confidence interval: −8; −4) or thrombin receptor-activating protein, −15 U (−19; −10). Also when activated with collagen and ristocetin, the PAS-C–platelets showed less aggregation, although not statistically significant. All samples with PAS-C–platelets showed significantly lower agonist-induced CD62P responsiveness than samples with plasma–platelets. However, there was no difference regarding all TEG parameters. Conclusion Our findings demonstrate that the function – aggregation and CD62P responsiveness – of PAS-C–platelets in reconstituted whole blood is inferior to that of plasma–platelets, which may have implications in the setting of massive transfusions.

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).

During storage, erythrocytes undergo changes that alter their clearance and function after transfusion and there is increasing evidence that these changes contribute to the complications observed in transfused patients. Stored erythrocytes were incubated overnight at 37°C to mimic the temperature after transfusion. After incubation, several markers for erythrocyte damage were analysed. After overnight incubation, stored erythrocytes showed increased potassium leakage, haemolysis, PS exposure and vesicle formation, and all these effects increased with increasing storage time. Furthermore, we demonstrated that long-term stored erythrocytes develop decreased flippase activity and increased scrambling activity after overnight incubation, leading to PS exposure and the release of vesicles. Reduced intracellular potassium was identified as the cause of the decreased flippase activity. Lastly, we provide evidence that erythrocytes can return to a PS-negative state by shedding parts of their membrane as PS-containing vesicles and that these vesicles can serve as a platform for the coagulation cascade. These findings reveal that potassium leakage, a well-known phenomenon of prolonged erythrocyte storage, primes erythrocytes for PS exposure. PS exposure will lead to vesicle formation and might have an important impact on the post-transfusion function and side effects of stored erythrocytes.

CD47 on erythrocytes inhibits phagocytosis through interaction with the inhibitory immunoreceptor SIRP alpha expressed by macrophages. Thus, the CD47-SIRP alpha interaction constitutes a negative signal for erythrocyte phagocytosis. However, we report here that CD47 does not only function as a "do not eat me" signal for uptake but can also act as an "eat me" signal. In particular, a subset of old erythrocytes present in whole blood was shown to bind and to be phagocytosed via CD47-SIRP alpha interactions. Furthermore, we provide evidence that experimental aging of erythrocytes induces a conformational change in CD47 that switches the molecule from an inhibitory signal into an activating one. Preincubation of experimentally aged erythrocytes with human serum before the binding assay was required for this activation. We also demonstrate that aged erythrocytes have the capacity to bind the CD47-binding partner thrombospondin-1 (TSP-1) and that treatment of aged erythrocytes with a TSP-1 derived peptide enabled their phagocytosis by human red pulp macrophages. Finally, CD47 on erythrocytes that had been stored for prolonged time was shown to undergo a conformational change and bind TSP-1. These findings reveal a more complex role for CD47-SIRP alpha interactions in erythrocyte phagocytosis, with CD47 acting as a molecular switch for controlling erythrocyte phagocytosis. (Blood. 2012;119(23):5512-5521)

Background Transfusion of erythrocytes is associated with increased morbidity in certain patient groups. Storage time of erythrocytes may contribute to respiratory complications. Using a syngeneic in vivo transfusion model, we investigated whether transfusion of stored rat erythrocytes causes lung injury in healthy and in lipopolysaccharide-primed rats in a "two-hit" model of lung injury. Methods Rats were infused with aged rat erythrocytes (14 days of storage) and washed aged erythrocytes or supernatant of aged erythrocytes. Controls received fresh rat erythrocytes (0 days of storage) or saline. In the "two-hit" model of lung injury, lipopolysaccharide was used as a "first hit" before transfusion. Rat and control human erythrocyte products were analyzed for lysophosphatidylcholine accumulation. Results In healthy rats, transfusion of aged erythrocytes caused mild pulmonary inflammation but no coagulopathy. In lipopolysaccharide-pretreated rats, transfusion of aged erythrocytes augmented lung injury by inducing coagulopathy, both in the pulmonary and systemic compartment, when compared with transfusion with fresh erythrocytes. When transfused separately, supernatant of aged erythrocytes, but not washed aged erythrocytes, mediated coagulopathy in the "two-hit" model. Analysis of the supernatant of aged erythrocytes (rat and human) showed no lysophosphatidylcholine accumulation. Conclusions Transfusion of aged erythrocytes induces lung injury in healthy rats. In a "two-hit" model, injury induced by aged erythrocytes was characterized by coagulopathy and was abrogated by washing. Washing of aged erythrocytes may decrease pulmonary complications in patients with an inflammatory condition who are exposed to a blood transfusion.

BACKGROUND: Storage of red blood cells (RBCs) results in various biochemical changes, including a decrease in cellular adenosine triphosphate and 2,3-diphosphoglycerate acid. Previously it was shown that stored human RBCs show a deficit in the oxygenation of the microcirculation in the gut of anesthetized rats. In this study, the effect of RBCs on rat kidney oxygenation and the effect of rejuvenation of stored RBCs on their ability to deliver oxygen were investigated. STUDY DESIGN AND METHODS: Washed RBCs, derived from leukoreduced RBCs stored in saline-adenine-glucose-mannitol, were tested in an isovolemic transfusion model in rats after hemodilution until 30 percent hematocrit (Hct). The cells were derived from RBCs stored for up 3 days or from RBCs stored for 5 to 6 weeks with or without incubation in Rejuvesol to rejuvenate the cells. Renal microvascular oxygen concentrations (microPO(2)) were determined by Pd-porphyrin phosphorescence lifetime measurements. RESULTS: Isovolemic transfusion exchange of 5- to 6-week-stored RBCs resulted in a significantly larger decrease in renal microPO(2) than RBCs stored for up to 3 days: 16.1 +/- 2.3 mmHg versus 7.1 +/- 1.5 mmHg, respectively (n = 5). Rejuvenation of stored RBCs completely prevented this deficit in kidney oxygenation. The differences in oxygen delivery were not due to different recoveries of the human RBCs in the rat circulation. CONCLUSION: This study shows that the storage-induced deficit of human RBCs to oxygenate the rat kidney microcirculation at reduced Hct is completely reversible. Prevention of metabolic changes during storage is therefore a valid approach to prevent this deficit.

UV-C irradiation has been shown to be effective for pathogen reduction in platelet concentrates, but preliminary work indicated that UV-C irradiation of platelets can induce platelet aggregation. In this study, the mechanism underlying this phenomenon was investigated. Irradiation of platelets with UV-C light (1500 J/m(2)) caused platelet aggregation, which was dependent on integrin alphaIIbbeta3 activation (GPIIb/IIIa). This activation occurred despite treatment with several signal transduction inhibitors known to block platelet activation. UV-C also induced activation of recombinant alphaIIbbeta3 in Chinese hamster ovary (CHO) cells, an environment in which physiologic agonists fail to activate. Activation of alphaIIbbeta3 requires talin binding to the beta3 tail, yet alphaIIbbeta3-Delta724 (lacking the talin binding site) was activated by UV-C irradiation, excluding a requirement for talin binding. The UV-C effect appears to be general in that beta(1) and beta(2) integrins are also activated by UV-C. To explain these findings, we investigated the possibility of UV-C-induced photolysis of disulfide bonds, in analogy with the activating effect of reducing agents on integrins. Indeed, UV-C induced a marked increase in free thiol groups in platelet surface proteins including alphaIIbbeta3. Thus, UV-C appears to activate alphaIIbbeta3 not by affecting intracellular signal transduction, but by reduction of disulfide bonds regulating integrin conformation.

Background Animal models are valuable in transfusion research. Use of human red blood cells (RBCs) in animal models facilitates extrapolation of the impact of storage conditions to the human condition but may be hampered by the use of cross species. Methods Investigation of clearance and posttransfusion recovery in a rat model using fresh and stored human RBCs. Results Directly following transfusion, human RBCs could be detected in the circulation of all recipients, with higher recovery rates for stored RBCs than for fresh RBCs. After 24 h following transfusion, no donor RBCs could be detected in the circulation, but donor RBCs could be detected in all organs of all recipients. Conclusion The use of human donor RBCs in a rat transfusion model resulted in clearance from cells from the circulation. Donor cells were found in different organs of the recipients. Rat transfusion models are thus not appropriate to study the efficacy of human RBC transfusion.

BACKGROUND: Although it is known that the transfusion of stored RBCs does not always improve tissue O2 consumption under conditions of limited tissue oxygenation, the efficiency of O2 delivery to the microcirculation by stored RBCs has never been determined. STUDY DESIGN AND METHODS: In a rat hemorrhagic shock model, the effects of resuscitation with fresh or 28-day-old RBCs stored in CPD plasma, saline-adenine-glucose-mannitol, and CPDA-1 plasma were investigated. Systemic hemodynamic and intestinal oxygenation measures were monitored. Intestinal microvascular PO2 was determined with the O2-dependent quenching of palladium-porphyrin phosphorescence, and the RBC deformability was measured with a Laser-assisted optic rotational cell analyzer. RESULTS: Hemodynamic and oxygenation measures were significantly decreased during hemorrhagic shock. Intestinal oxygen consumption and mesenteric venous pO2 were restored with the transfusion of both fresh and stored RBCs, except for CPD-stored RBCs. The intestinal microvascular pO2 improved only with the transfusion of fresh RBCs. Deformability of the stored RBCs was significantly decreased. CONCLUSION: In contrast to that of fresh RBCs, the transfusion of stored RBCs did not restore the microcirculatory oxygenation, possibly because of impaired O2 unloading, but, except for CPD-stored RBCs, the storage-induced changes were not enough to impair intestinal VO2 and mesenteric venous pO2.

Non-woven poly[ethylene terephthalate] (NW-PET) filter fabric, usually used for leucocyte removal of red cells, was modified by water vapour glow discharge (WVGD) treatment to improve platelet compatibility. Modified filter material was evaluated with different kinds of platelet concentrates (PCs). In addition, modified filter materials were gamma-sterilized and tested after different time intervals at different storage conditions. Modification of the filter material resulted in an improved platelet recovery after filtration of PC from 57 to about 80%. No significant difference in platelet recovery was observed when filtering either freshly prepared (79 +/- 3.5%, mean +/- SD), overnight-stored single BC-PC (78 +/- 3.3%), overnight-stored single PRP-PC (75 +/- 8.8%) or overnight-stored pooled BC-PC (79 +/- 8.9%). However, freshly prepared pooled BC-PC gave a significantly higher platelet recovery (84 +/- 3.5%). Leukocyte depletion did not differ significantly between the different types of PC. gamma-Sterilization and subsequent storage of the modified filter material for 5, 14 and 26 weeks at 20 degrees C or 37 degrees C had no significant influence on the filtration results of overnight-stored pooled BC-PC. The results of the present study show that WVGD-treated NW-PET is platelet compatible and can be used for leucocyte removal from preferably BC-PC. It can be gamma-sterilized and stored for at least 6 months prior to filtration without affecting the platelet recovery and leucocyte removal.

The role of cytidine triphosphate (CTP) synthetase (EC 6.3.4.2.) m the pyrimidine ribonucleotide metabolism of MOLT-3 human T-ALL cell line cells and normal human T lymphocytes during the cell cycle traverse was studied. Highly pure G1-phase samples and samples enriched in S-phase cells were obtained by counterflow centrifugation. The activity of CTP synthetase in situ , measured in pulse-chase experiments, was similar in the G1-phase and S-phase MOLT-3 cells. In contrast, in S-phase T lymphocytes, an increased activity of CTP synthetase was observed compared with G1-phase T lymphocytes. Nevertheless, the MOLT-3 samples showed an increased activity of CTP synthetase in comparison with either G1-phase or S-phase enriched samples of normal T lymphocytes. Therefore, the increased activity of CTP synthetase of MOLT-3 cells is a cell cycleindependent feature, whereas among normal T lymphocytes, the increase in activity of CTP synthetase that arises after a growth stimulus is more prominent in the S-phase.

Anemia is a frequently encountered problem in the critically ill patient. The inability to compensate for anemia includes several mechanisms, collectively referred to as anemia of inflammation: reduced production of erythropoietin, impaired bone marrow response to erythropoietin, reduced iron availability, and increased red blood cell (RBC) clearance. This review focuses on mechanisms of RBC clearance during inflammation. We state that phosphatidylserine (PS) expression in inflammation is mainly enhanced due to an increase in ceramide, caused by an increase in sphingomyelinase activity due to either platelet activating factor, tumor necrosis factor-α, or direct production by bacteria. Phagocytosis of RBCs during inflammation is mediated via RBC membrane protein band 3. Reduced deformability of RBCs seems an important feature in inflammation, also mediated by band 3 as well as by nitric oxide, reactive oxygen species, and sialic acid residues. Also, adherence of RBCs to the endothelium is increased during inflammation, most likely due to increased expression of endothelial adhesion molecules as well as PS on the RBC membrane, in combination with decreased capillary blood flow. Thereby, clearance of RBCs during inflammation shows similarities to clearance of senescent RBCs, but also has distinct entities, including increased adhesion to the endothelium.Anämie ist ein häufig auftretendes Problem bei schwerkranken Patienten. Die Unfähigkeit, die Anämie zu kompensieren, umfasst mehrere Mechanismen, die unter dem Begriff Entzündungsanämie zusammengefasst werden: reduzierte Erythropoetin-Produktion, vermindertes Ansprechen des Knochenmarks auf Erythropoetin, reduzierte Eisenverfügbarkeit sowie erhöhte Erythrozyten-Clearance. Diese Übersichtsarbeit beschäftigt sich mit den Mechanismen der Erythrozyten-Clearance im Zustand der Entzündung. Wir behaupten, dass die gesteigerte Phosphatidylserin(PS)-Expression im Zuge von Entzündungsprozessen hauptsächlich durch einen Anstieg des Ceramid-Spiegels bedingt wird, der wiederum die Folge einer erhöhten Sphingomyelinase-Aktivität ist, die durch den plättchenaktivierenden Faktor, den Tumor-nekrosefaktor-α oder die direkte Produktion durch Bakterien generiert wird. Phagozytose von Erythrozyten bei Entzündungsvorgängen wird durch das Erythrozyten-membranprotein, Bande 3 vermittelt. Verminderte Deformierbarkeit der Erythrozyten scheint ein wichtiges Entzündungsmerkmal zu sein, das ebenfalls von Bande 3 sowie Stickoxid, reaktiven Sauerstoffradikalen und Sialinsäure-Resten vermittelt wird. Des Weiteren ist die Haftung der Erythrozyten am Endothel bei Entzündungsvorgängen gesteigert, was höchstwahrscheinlich die Folge einer erhöhten Expression von endothelialen Adhesionsmolekülen sowie PS an der Erythrozytenmembran in Kombination mit verminderter kapillärer Durchblutung ist. Damit zeigt die Erythrozyten-Clearance im Zustand der Entündung Ähnlichkeiten mit der Clearance alternder Erythrozyten, aber auch individuelle Eigenschaften wie die gesteigerte Endotheladhäsion.

Background: Posttransfusion complications can be prevented by pretransfusion removal of donor white cells from platelet concentrate. The filtration used for this removal seems to have little effect on platelet function and activation, but more information is needed on its effect on function during subsequent long-term storage of concentrate. Study Design and Methods: The effect of prestorage filtration of buffy coat-prepared platelet concentrates (PCs) on platelet function, metabolism, and activation was investigated. A pool of three PCs, each made of four buffy coats, was split into three equal volumes; two were filtered over two different filters and the third served as a control. Variables monitored immediately after filtration and during the subsequent 8-day storage period at 22°C included aggregation upon stimulation with collagen and/or ADP, platelet adhesion capacity to collagen and fibrinogen in flowing blood, nucleotide content of and nucleobase release by the platelets, expression of activation-dependent antigens, and β-thromboglobulin release by the platelets. Results: No differences were observed between the PCs filtered over two different filters and the nonfiltered control PCs immediately after filtration and during storage, except for a selective removal (20%) of β-thromboglobulin by one filter. Conclusion: PCs prepared from a pool of four buffy coats can be filtered and subsequently stored for 8 days (starting +/− 24 hours after whole blood collection) without detriment to platelet function, metabolism, or activation.

Transfusion-related acute lung injury is suggested to be a “2-hit” event resulting from priming and activation of pulmonary neutrophils. Activation may result from infusion of lysophosphatidylcholines (LysoPCs), which accumulate during storage of blood products. In the present study, we developed a syngeneic in vivo transfusion model to test whether storage of platelet concentrates (PLTs) results in lung injury in healthy rats as well as in a “2-hit” model using lipopolysaccharide-pretreated rats. In addition, the effect of washing of platelets was studied. In healthy rats, transfusion of aged PLTs caused mild lung inflammation. In LPS-pretreated rats, transfusion of aged PLTs, but not fresh PLTs, augmented pulmonary systemic coagulopathy. When PLTs components were transfused separately, supernatant of aged PLTs, but not washed aged platelets, induced pulmonary injury in the “2-hit” model. Supernatants of aged PLTs contained increased concentrations of LysoPCs compared with fresh PLTs, which enhanced neutrophil priming activity in vitro. We conclude that transfusion of aged PLTs induces lung inflammation in healthy rats. In a “2-hit” model, aged PLTs contribute to pulmonary and systemic coagulopathy, which may be mediated by LysoPCs, which accumulate in the supernatant of PLTs during storage.

BACKGROUND: The BacT/ALERT system for bacterial monitoring of platelet concentrates (PCs) was introduced in the Netherlands in 2001. Samples are cultured for 7 days, and as a result of the short shelf-life of PCs, they are usually released as 'negative to date'. Therefore, some of the PCs have already been transfused at the moment of a positive signal in continued cultures in the BacT/Alert. It is unclear, however, whether these PCs are associated with more transfusion reactions.METHODS: During a 2-year period clinical data were collected from all patients who received PCs released as 'negative to date' but with a positive bacterial culture after being transfused.RESULTS: Data of 158 patients who received PCs with confirmed positive bacterial culture tests were analysed. Two patients developed a transfusion reaction. In both PCs, Propionibacterium was cultured. The imputability as related to the transfusion was classified as unlikely in both patients.CONCLUSION: Two of 158 transfusions of PCs released as 'negative to date', but with a confirmed positive BacT/ALERT result, were initially associated with transfusion reactions. However, the imputability of both reactions was low.

BACKGROUND AND OBJECTIVES: Photodynamic treatment (PDT) with the cationic porphyrin, mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin chloride [Tri-P(4)], has previously been shown to be effective at inactivating vesicle stomatitis virus (VSV) in red cell concentrates (RCC) with limited damage to red blood cells (RBC). The aim of this study was to determine the pathogen-inactivating capacity of PDT with Tri-P(4) for a broader range of pathogens and to establish the associated effect on in vitro RBC quality. MATERIALS AND METHODS: A series of viruses and bacteria was spiked into 60% RCC. Pathogen inactivation was determined after PDT with 25 microm Tri-P(4) and red light up to 360 kJ/m2. Human immunodeficiency virus (HIV)-infected cells were evaluated for cell death induction, and RCC were analysed for the induction of haemolysis and ATP content. RESULTS: For the lipid-enveloped viruses bovine viral diarrhoea virus, HIV and pseudorabies virus, and for the Gram positive bacterium, Staphylococcus aureus, and the Gram-negative bacteria, Pseudomonas aeruginosa and Yersinia enterolitica, inactivation of > or = 5 log10 was measured after 60 min of PDT with Tri-P(4). The required treatment time to achieve this level of inactivation was four times longer than required for VSV. For cell-associated HIV, only 1.7 log10 of inactivation was found, despite clear induction of cell death of HIV-infected cells. The non-enveloped virus, canine parvovirus, was completely resistant to the treatment. PDT of RCC with Tri-P(4) for 60 min, and subsequent storage in AS-3, resulted in 4% haemolysis after 35 days of storage. The ATP content of untreated and treated RBC declined with similar kinetics during storage. CONCLUSION: PDT of RCC with Tri-P(4) for 60 min inactivates a wide range of pathogens, but not cell-associated HIV and a non-enveloped virus, and compromises RBC quality. This reduces the suitability of PDT with Tri-P(4) for red cell sterilization. Therefore, further improvements in the treatment procedures to potentiate pathogen inactivation and to preserve RBC integrity will be required to generate an effective treatment for sterilizing RCC

In the past decade, studies suggesting a reduced oxygen delivery by stored red blood cell concentrates (RBCCs) have initiated a discussion about the use of fresh versus old blood. We determined whether old RBCCs represent a significant part of the total of RBCCs issued. The age of RBCCs at the time of transfusion was determined in 74 084 units during a 5-year period in the Academic Medical Center, a main Dutch University Hospital. The mean (+/-SD) storage time of the total number of transfused RBCC was 19.4 +/- 7.0 days, and 37% were older than 3 weeks. As more than one-third of the transfused RBCC units are stored for longer than 3 weeks, the research examining differences in oxygen delivery between fresh and stored RBCC is relevant for packed RBC transfusion practice.

Background and Objectives Photodynamic treatment (PDT) of red blood cell (RBC) suspensions has been reported to result in virus inactivation, but also in deterioration of cell quality. Recently, we have demonstrated the potential usefulness of the reactive oxygen species scavenger dipyridamole in selectively protecting RBCs against the harmful side-effects of PDT. Unfortunately, dipyridamole-conferred protection against long-term photohaemolysis was incomplete. In the present study, dipyridamole was applied in combination with Trolox (a hydrophilic vitamin E analogue) in order to augment RBC protection. Materials and Methods Leucodepleted RBC suspensions (30% haematocrit) were treated with 1,9-dimethylmethylene blue (DMMB) and red light, and the effect of inclusion of dipyridamole and Trolox was assessed on potassium leakage as well as on short-term and long-term photohaemolysis. Possible interference of the scavenger cocktail with virus inactivation was examined using extracellular pseudorabies virus (PRV). Results Treatment of RBC with DMMB and red light resulted in enhanced potassium leakage and both short- and long-term haemolysis. Dipyridamole and Trolox showed additive protective effects against induction of potassium leakage and photohaemolysis, suggesting different protection mechanisms for the two scavengers. Combined inclusion of dipyridamole and Trolox did not interfere with efficacy of PRV inactivation. Conclusions Combined inclusion of dipyridamole and Trolox results in substantially improved selectivity of photodynamic treatment of RBC suspensions.

BACKGROUND AND OBJECTIVES: Leucoreduced platelet concentrates (LR-PCs) can be stored at 20-24 degrees C for 5-7 days. When LR-PCs are cryopreserved they can be stored for several years. For cryopreservation to become applicable in blood-bank practice, an off-the-shelf cryoprotectant is needed that can be added to the LR-PC in a sterile manner. For this, we varied the composition of the cryopreservation medium and studied various parameters of cryopreserved LR-PCs for up to 24 h after thawing at room temperature. MATERIALS AND METHODS: LR-PCs in plasma or Composol were concentrated and divided into 2 units. To each unit, an equal part of 10% dimethylsulfoxide (DMSO) in plasma, Composol with or without 5% albumin, or GPO (pasteurized plasma-protein solution) was added. Freezing occurred at 1 degrees C/min and LR-PCs were placed in the vapour phase of nitrogen. LR-PCs were thawed at 37 degrees C and stored at room temperature. LR-PCs were tested for morphology, platelet recovery, swirling effect, and activation antigens at various time-points thereafter. RESULTS: LR-PCs in 100%, 65% and 50% plasma supplemented with Composol showed good morphology scores (>250), limited CD62P expression ( 80% under all conditions and CD42b expression varied between 70 and 85%. Results of LR-PCs in 15% plasma and Composol, with or without plasma substitutes, were not acceptable at 24 h after thawing, i.e. the morphology score was 40%. CONCLUSIONS: A minimum of 50% plasma in the cryopreserved LR-PC is necessary to maintain an acceptable in vitro quality of platelets up to 24 h after thawing. Composol is a good candidate for using to prepare an off-the-shelf cryoprotectant

The effect of filtration on the quality of platelet concentrates (PC) during storage was investigated. Two leukocyte depletion filters (Pall PL50HF and Sepacell PL-10A) were applied to filter PC made from a pool of 4 buffy coats. For each experiment 3 PC were pooled and divided into 3 identical PC to eliminate differences between the PC. Two PC were filtered, and the third PC served as an unfiltered control. A total of 12 experiments was performed. Before filtration, volumes of the PC were 263 +/- 11.7 ml (mean +/- SD). Platelet and leukocyte counts per PC were 241 +/- 25.9 x 10(9) and 7.2 +/- 1.8 x 10(6), respectively. After filtration leukocyte counts did not exceed 5 x 10(4) in any of the PC. In the PC filtered with the Pall PL50HF the mean platelet loss was approximately 14% and with the Sepacell PL-10A, 17%. During a 9-day storage period the pH, PO2, PCO2, bicarbonate, lactate and glucose concentration and LDH release as well as the morphology, examined by the swirling effect and microscopically, were not significantly different in filtered and unfiltered units. Filtration through the 2 investigated leukocyte depletion filters for PC did not adversely affect in vitro viability of the platelets during storage.