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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).

Background and Objectives: In many countries, whole blood (WB) donations with collection times between 12 and 15 min are not allowed to be used for platelet concentrates (PC). Since the development of guidelines, many process-related changes have been introduced. We aimed to determine the effect of WB with long collection times on PC quality. Materials and Methods: Five participating centres tested buffy coat (BC)-derived PC in platelet additive solution type E prepared from only WB collections lasting 12 min (study group, n = 8). One centre produced platelet-rich plasma (PRP)-derived PC from single donations (12 min). All PC were stored at 22 ± 2°C and sampled on Days 1, 6 and 8 post-collection for in vitro quality determination. Results: Average collection time was significantly longer in the study group compared to controls (8.9 ± 2.6 vs. 7.3 ± 1.3 min, p < 0.001). There were no differences in volume, platelet concentration, basal CD62P expression, soluble-CD62P and CCL5 levels, or nucleotide content between the groups. Stimulation with TRAP-6 resulted in comparable levels of cell surface CD62P. On Day 8, all PC fulfilled requirements for pH. The findings from single PRP-derived PC centre were similar. Conclusion: PC with one BC and single PRP derived from collections lasting >12 min had equivalent in vitro quality to controls during storage. This study provides evidence that 12–15 min donations should not be excluded for PC preparation and justifies to readdress the guidelines to

Objectives Our objectives were to determine the separation characteristics and blood product quality of a gravity-driven microfiltration blood separation system (HemoClear, The Netherlands). Background A range of centrifugal blood separation devices, including intraoperative cell salvage devices (cell savers) and apheresis machines, are available to assist in preparing both allogenic and autologous blood products. These devices are expensive to operate and require extensive training. Methods and materials Nine whole blood units were collected under standard conditions and analysed for haematological parameters, thromboelastographic properties, platelet morphology and activation, and red blood cell (RBC) deformability and morphology. Three whole blood units were separated by means of the HemoClear device, into a liquid and cellular component. The cellular component was diluted with SAGM and cold stored for 14 days. To simulate cell salvage six whole blood units were diluted with isotonic saline, followed by multiple HemoClear separation rounds. Results The recovery of both RBCs (100 ± 1.6%) and white blood cells (99 ± 4.5%) after undiluted filtration were very high, while platelet recovery was high (83 ± 3.0%). During the filtration, and cold storage after filtration storage both the non-deformable RBC fraction and the RBC maximum elongation remained stable. Parameters of thromboelastography indicated that platelets remain functional after filtration and after 7 days of cold storage. In the cell salvage simulation the total protein load in the cellular fraction was reduced by 65 ± 4.1% after one washing round and 84 ± 1.9% after two consecutive washing rounds. Conclusion The novel blood filter studied effectively separates whole blood into diluted plasma and platelet-rich RBCs. Moreover, the device effectively washed diluted whole blood, driving over 80% of proteins to the liquid component.

Item does not contain fulltext BACKGROUND AND OBJECTIVES: Di-ethyl-hexyl-phthalate (DEHP) is currently the main plasticizer used for whole blood collection systems. However, in Europe, after May 2025, DEHP may no longer be used above 0.1% (w/w) in medical devices. DEHP stabilizes red cell membranes, thereby suppressing haemolysis during storage. Here we compared in vitro quality parameters of red cell concentrates (RCCs) collected and stored in DEHP-, DINCH- or DINCH/BTHC-PVC hybrid blood bags with saline-adenine-glucose-mannitol (SAGM) or phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGSM) storage solution. Last, we performed haemovigilance surveillance for RCC collected in DINCH-PVC and stored in PAGGSM/BTHC-PVC. MATERIALS AND METHODS: In vitro quality parameters of RCC were determined during 42 days of storage. Haemovigilance surveillance was conducted to compare the frequency and type of transfusion reaction. RESULTS: Haemolysis levels were increased in SAGM/BTHC-PVC as compared to SAGM/DEHP-PVC (0.66% ± 0.18% vs. 0.36% ± 0.17%). PAGGSM storage solution was able to adequately suppress haemolysis to levels observed during storage in SAGM/DEHP-PVC, both in BTHC-PVC (0.38% ± 0.12%), and to a slightly lesser extent in DINCH-PVC (0.48% ± 0.17%). A total of 1650 PAGGSM/BTHC-PVC and 5662 SAGM/DEHP-PVC RCC were transfused yielding a transfusion reaction frequency of 0.24% (95% CI 0.0000-0.0048) and 0.44% (95% CI 0.0027-0.0061) respectively. CONCLUSION: The in vitro quality of RCC stored in PAGGSM/BTHC-PVC and SAGM/DEHP-PVC is comparable. There is no indication that transfusion of erythrocytes stored in PAGGSM/BTHC-PVC results in increased transfusion reaction frequency. These initial results provide a basis for further clinical evaluation to narrow down the confidence interval of transfusion reaction frequency.

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.

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.

Gamma irradiation of red cell concentrates (RCCs) is regularly used to prevent transfusion-associated graft-versus-host disease (TA-GvHD) in at-risk patients. While studies have indicated that irradiated RCCs exhibit increased hemolysis, there have been no efforts to differentiate between free- and microvesicle (MV)-bound hemoglobin (Hb). As an increase in the proportion of free-Hb in irradiated RCCs could alter vascular function, we sought to characterize differences in the state of extracellular Hb based on the timing of irradiation.Four separate pools of seven CPD/SAGM leukoreduced RCCs were produced and split into four sets of seven identical units. The units from each set were subject to irradiation (25 Gy) at six different points during storage, with one unit serving as a nonirradiated control. All testing was performed immediately following unit expiry on day 43.The earlier in storage that units were irradiated, the higher the hemolysis and the lower the proportion of MV-bound Hb. Units irradiated earlier in storage (1-8 days post collection) additionally had lower membrane rigidity (KOur findings indicate that irradiation timing can alter the state of extracellular Hb, with "early" irradiation promoting an increased proportion of cell-free Hb as well as mechanical damage to the RBC membrane.

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.

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.

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 For a clinical platelet (PLT) transfusion trial conducted in three countries, the production of PLT concentrates (PCs) that were pathogen inactivated with the Mirasol technology was set up and validated. While the Mirasol procedure is applied to an established PLT product, the PLT processing procedure still had to be modified to ensure a treated PC was of sufficient quality. Further, the effect of simulated transport conditions and the effect of ambient light on Mirasol-treated PCs was determined. Study Design and Methods Platelet concentrates in plasma were made from pooled buffy coats followed by Mirasol treatment. To mimic transport conditions, units were left unagitated for 6 h at room temperature. To mimic ambient light exposure, units were held unagitated for 4 h in direct fluorescent tube light. Results Measures had to be taken to allow 7-day storage of treated concentrates. In one site, PCs made from five buffy coats with >450 × 109 PLTs were removed from inventory. Another site went from five to four buffy coats per pool. Interruption of agitation for 6 h on day 3 did not induce meaningful changes in in vitro measures, even when stored up to 7 days. Exposure to ambient light for 4 h, either on day 3 or 6, had no effect on in vitro measures. Conclusion The Mirasol pathogen inactivation process can be implemented in routine, but changes to current PLT processing methods might be needed. Transport conditions and 4-h-long ambient light exposure have no negative effect on the in vitro quality of Mirasol-treated PCs.

BACKGROUND Transfusion of a single unit of stored red blood cells (RBCs) has been hypothesized to induce supra-physiological levels of non-transferrin bound iron (NTBI), which may enhance inflammation and act as a nutrient for bacteria. We investigated the relation between RBC storage time and iron levels in a clinically relevant “two-hit” human transfusion model. STUDY DESIGN AND METHODS Eighteen healthy male volunteers (ages 18-35 years) were infused with 2 ng lipopolysaccharide (LPS)/kg to induce systemic inflammatory response syndrome. Two hours later, each participant received either 1 unit of 2-day stored (2D) autologous RBCs, 35-day stored (35D) autologous RBCs, or an equal volume of saline. Every 2 hours up to 8 hours after LPS infusion, hemoglobin, hemolysis parameters, and iron parameters, including NTBI, were measured. RESULTS Transfusion of both 2D and 35D RBCs caused increases in hemoglobin, plasma iron, and transferrin saturation; whereas levels remained stable in the saline group. Transfusion of 35D RBCs did not result in hemolysis nor did it lead to increased levels of NTBI compared with 2D RBCs or saline. LPS induced increases in ferritin, haptoglobin, bilirubin, and lactate dehydrogenase that were similar in all three groups. CONCLUSION We conclude that 35D autologous RBCs do not cause hemolysis or increased levels of NTBI during human endotoxemia.

Background and Objectives In mice, loss of sialic acid resulting in shedding of glycoprotein (GP) Ibα and GPV has been linked to platelet survival. The aim of this study was to determine whether loss of sialic acid and the GPIb-IX-V complex contributes to development of the platelet storage lesion (PSL) in human platelet concentrates (PCs). Materials and methods PCs (stored in plasma (with or without Mirasol treatment); PAS-C or PAS-E) were stored at room temperature. Flow cytometry was used to monitor membrane expression of the GPIb-IX-V complex, CD62P, surface glycans and PS exposure. The functionality of stored platelets was determined employing aggregometry and ristocetin-induced VWF binding. Results Storage time of PCs in blood banks is limited to 7 days. During this time period, a minor but gradually increasing subpopulation of GPIbα-negative platelets was observed. Also, ristocetin-induced VWF binding was impaired in a small population of platelets. Mean surface expression of GPIbα and GPV remained stable until day 9, whereas CD62P expression increased; also a rapid decrease in ADP-induced aggregation was observed for PAS-C, PAS-E and Mirasol-treated PCs. Upon prolonged storage (>9 days), a slow decline in surface expression of GPIbα and GPV was observed; no major changes were observed in surface sialylation with the exception of Mirasol-treated platelets. Conclusion In a small population of stored platelets, changes in GPIbα occur from day 2 onwards. Loss of sialic acid and subsequent shedding of GPIbα and GPV is not an early event during the development of the PSL.

Background and Objectives: Previously it has been shown that platelet (PLT) storage performance is consistent by donor. Differences involved metabolic activity, which might be caused by mitochondrial (dys)function, associated with age and age-related diseases like Type 2 diabetes (T2D). We aimed to test PLTs from young donors in comparison with PLTs from older donors with or without diagnosis for T2D. Materials and methods: Fifteen whole blood donors 45 years with and without T2D. The sPC were stored for 8 days and analysed at regular intervals for in vitro quality. Results: Donors were 24 ± 3, 60 ± 7 (without T2D) and 59 ± 8 (with T2D) years old. All sPC groups had comparable volume and PLT content. On Day 8, sPC from young donors showed higher pH37°C than sPC from older donors (6.84 ± 0.15 vs. 6.40 ± 0.48, P

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

Whole blood is not usually collected close to the processing site, which results in a holding time between collection and processing. In some countries, the holding time is limited to 8 hours, after which the units are cooled, rendering them useless for platelet preparation. Other countries allow a 24-hour ("overnight") ambient hold to allow platelet preparation. The impact of this holding time on subsequent blood components will be reviewed in this article. In addition, there are various "in-process" holding times that further prolong the time before the final blood component is ready. Particularly, these in-process holding times are not well defined and poorly controlled, but can nevertheless affect the biochemical and functional characteristics of blood components. Furthermore, current, non–evidence-based, guidelines have restricted the length of some of these holding times. This article summarizes the evidence and fills gaps where evidence is lacking.

There are few studies investigating the effect of irradiation on red blood cells (RBCs) during storage. This study analyzed changes in in vitro quality of RBCs irradiated at several points during storage with the aim of providing evidence to support current maximum pre- and postirradiation storage limits.Each of seven participating centers produced four pools of 7 standard RBC units (SAGM, AS-3, or PAGGSM), which were then split back into 7 units. All units in a pool were from sex-matched blood donors. Every week during 6 weeks of refrigerated storage, 1 unit was irradiated, while 1 unit was not irradiated (control). Units were tested weekly for biochemical variables, morphology, and mechanical fragility.The earlier during storage that units were irradiated, the higher the hemolysis and KThe method of blood component manufacturing determined the absolute levels of hemolysis and potassium in irradiated and nonirradiated units, but did not influence the effect that timing of irradiation had on the in vitro quality characteristics. This study provides support for the current Council of Europe guidelines on the time limitations for the irradiation of RBCs.

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).

Plastic blood bags improve the safety and effectiveness of blood component separation and storage. Progress towards optimal storage systems is driven by medical, scientific, business and environmental concerns and is limited by available materials, consumer acceptance and manufacturing and regulatory concerns. Blood bag manufacturers were invited to submit lists of the bags they manufacture. The lists were combined and sorted by planned use. The lists were analysed by experts to assess the degree to which the products attend to scientific problems. Specific issues addressed included the use of di-ethylhexyl phthalate (DEHP) as plasticizer for polyvinyl chloride (PVC) blood bags, the size, material and thickness of platelet bags, and the fracture resistance of plasma bags. Alternatives to DEHP for red blood cell (RBC) storage exist, but are mostly in a developmental stage. Plastic bags (DEHP-free, PVC-free) for platelet storage with better gas diffusion capabilities are widely available. Alternatives for plasma storage with better fracture resistance at low temperatures exist. Most RBC products are stored in DEHP-plasticized PVC as no fully satisfactory alternative exists that ensures adequate storage with low haemolysis. A variety of alternative platelet storage systems are available, but their significance - other than improved oxygen transport - is poorly understood. The necessity to remove DEHP from blood bags still needs to be determined.

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.

In Europe, red cell concentrates (RCC) are usually stored in SAGM (saline-adenine-glucose-mannitol). During storage, in vitro red cell quality declines, including lowered energy status and increased cell lysis. Recently, several additive solutions (ASs), designed to diminish the decline in in vitro quality during storage, have been developed. These new solutions have mainly been developed to better maintain red blood cell (RBC) 2,3-biphosphoglycerate (2,3 BPG) levels and energy status during storage. High levels of 2,3 BPG allow for better oxygen release while high energy status is necessary for function and survival of RBC in vivo. In a paired study design, RBC ASs were compared for their ability to provide improved in vitro quality during hypothermic storage.For each experiment, 5 whole blood units held overnight were pooled and split. The whole blood units were processed according to the buffy coat method. RBCs were resuspended in either SAGM, PAGGSM, PAG3M, E-Sol 5 or AS-7 and leucoreduced by filtration. RCCs were stored for eight weeks at 2-6 °C and sampled weekly for analysis of in vitro quality parameters.Red cell concentrates stored in PAG3M, E-Sol 5 and AS-7 showed significantly higher lactate production and higher levels of intracellular adenosine triphosphate (ATP) and total adenylate. 2,3 BPG levels rapidly declined during storage in SAGM and PAGGSM. The decline in 2,3 BPG was inhibited during storage in E-Sol 5 and AS-7, while in PAG3M, 2,3 BPG level increased above the initial level till day 35 and remained detectable till day 56. Haemolysis was comparable for all ASs until day 35, upon prolonged storage, haemolysis in SAGM was higher than with the other ASs. As compared to SAGM, storage in PAGGSM, PAG3M, E-Sol 5 and AS-7 better maintained morphological properties.Storage of RBCs in the new generation ASs yield RBCs with more stable metabolite levels and improved overall quality during storage as compared with RBCs stored in SAGM.

BACKGROUND: In retrospective studies, it has been shown that differences in storage variables of platelet (PLT) concentrates (PCs) are partially donor dependent. It was our aim to prospectively determine the donor effect on PLT quality. STUDY DESIGN AND METHODS: Based on quality control data of outdated apheresis PCs, male donors were selected with at least one PC with a pH value of more than 7.0 (“good,” n = 6) or one PC with a pH value of less than 6.7 (“poor,” n = 6) on Day 8. These donors donated a PC (Trima Accel, Terumo) and completed a short questionnaire about their health and lifestyle. PCs were stored for 12 days and analyzed at regular intervals for in vitro quality. RESULTS: Donor characteristics were comparable, except that zero of six good and four of six poor donors reported high blood pressure and/or high cholesterol/fat and/or use of medicines. Lactate production in good PCs was lower than that in poor PCs (0.09 ± 0.03 mmol/day/1011 PLTs vs. 0.13 ± 0.04 mmol/day/1011 PLTs, p < 0.05) resulting in a higher pH from Day 5 onward. At the end of storage, the good PCs showed lower CD62P expression, lower phosphatidylserine exposure, and higher mitochondrial membrane potential. PLT functional properties were only slightly different. Despite having lower pH, the poor PCs also fulfilled European Guidelines during 7-day storage. CONCLUSION: Platelet storage performance is consistent when donors are dichotomized as having good or poor storing PLTs. Metabolic differences are perhaps due to different functionality of the mitochondria. More research is needed to establish the underlying causes and the implications for donors and blood products.

We investigated whether improving the metabolic status of red blood cell concentrates before freezing could extend the postthaw shelf life beyond 14 days while still meeting the requirements for hemolysis (0.8%) and total adenylate (82% of original values).At Day 8 after collection, four leukoreduced red blood cell concentrates in saline-adenine-glucose-mannitol (SAGM) were pooled, mixed, and split (n = 4). Of these concentrates, two were rejuvenated in Rejuvesol. In addition, two leukoreduced red blood cell concentrates in phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM) were pooled, mixed, and split at Day 8 after collection (n = 4). All concentrates were glycerolized, frozen, and stored for at least 2 weeks at -80°C. After thawing and deglycerolization, from each pair, one red blood cell concentrate was resuspended in SAGM, and one was suspended in AS-3. During postthaw storage at 2 to 6°C for 35 days, all concentrates were sampled weekly and analyzed for hematologic, metabolic, and morphologic parameters.Both Rejuvesol and PAGGGM treatment produced increased adenosine triphosphate and total adenylate and 2,3-diphosphoglycerate levels compared with untreated red blood cell concentrates. Regardless of prefreeze Rejuvesol or PAGGGM treatment, postthaw hemolysis remained below 0.8% during 7 days in SAGM and during 35 days in AS-3. At Day 35 of postthaw storage in AS-3, total adenylate in nonrejuvenated red blood cell concentrates had decreased to 72% of the original values; whereas, in prefreeze Rejuvesol-treated and PAGGGM-treated concentrates, adenylate values were still were at 101% and 98%, respectively.Based on maximum allowable hemolysis of 0.8% and total adenylate content greater than 82% of the original value, thawed, prefreeze Rejuvesol-treated or PAGGGM-treated red blood cell concentrates can be stored for 35 days at 2 to 6ºC in AS-3.

Transfusion-related immunomodulation (TRIM) encompasses immunosuppressive and proinflammatory effects induced by red blood cell (RBC) transfusion. Changes that occur during storage in the RBC product have been hypothesized to underlie TRIM, mediated by tolerance of toll-like receptors (TLR). We investigated whether transfusion of 35-day-stored autologous RBCs alters cytokine production in response to stimulation with lipopolysaccharide (LPS) or lipotheic acid (LTA), in a clinically relevant model of endotoxemia.Eighteen volunteers received 2 ng/kg LPS intravenously, followed by normal saline or 2- or 35-day-stored autologous RBC transfusion. Before LPS, before transfusion, and 6 hours after transfusion blood was collected to measure cytokine gene expression. Whole blood was used for ex vivo stimulation with LPS and LTA, after which cytokine levels were measured with enzyme-linked immunosorbent assay.In vivo LPS induced a biphasic response in cytokine mRNA with peak values 2 hours after LPS infusion. Storage time of RBC transfusion did not influence cytokine mRNA levels. In vivo infusion of LPS resulted in tolerance for ex vivo stimulation with LPS and LTA. However, transfusion of either fresh or stored RBCs did not further affect the capacity to produce cytokines after ex vivo stimulation.In a clinically relevant model of human endotoxemia, autologous transfusion of 35-day-stored RBCs does not influence cytokine mRNA levels nor does it change the capacity of white blood cells in whole blood to produce cytokines after ex vivo stimulation with LPS or LTA.

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.

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.

Objective Transfusion-related acute lung injury is the leading cause of transfusion-related mortality. Preclinical studies have shown that aged RBCs can induce transfusion-related acute lung injury in the presence of a "first hit" (e.g., sepsis). Clinical studies, however, show conflicting results on this matter. We tested whether maximally stored RBCs are able to induce lung injury in the presence of a "first hit" in humans (Dutch Trial Register: NTR4455). Design Open-label, randomized controlled trial. Patients Healthy male volunteers. Interventions Eighteen healthy male volunteers donated one unit of autologous RBCs 2 or 35 days before the experiment. The experiment was started by infusion of 2 ng/kg lipopolysaccharide ("first hit"). After 2 hours, volunteers received normal saline (n = 6), 2-day stored transfusion (n = 6), or 35-day stored transfusion (n = 6) ("second hit"). Blood was sampled hourly. Six hours after transfusion, the diffusion capacity of the lungs for carbon monoxide was tested and volunteers underwent spirometry, chest x-ray study, and a bronchoalveolar lavage. Measurements and main results All volunteers fulfilled sepsis criteria after lipopolysaccharide injection. The stored blood transfusion did not result in significant changes in either hemodynamic or respiratory variables compared with the control groups. Furthermore, chest x-rays, lung function, and PaO2/FIO2 ratios did not differ between groups. Transfusion of stored autologous RBCs did not result in an increased level of protein in the lungs or neutrophil influx. Conclusions Transfusion of 35-day stored autologous RBCs in the presence of endotoxemia does not result in lung injury in humans.

It is thought that the clearance of transfused red blood cells (RBCs) is related both to the storage time of the transfusion product and to the inflammatory status of the recipient. We investigated these effects in a randomized, "two-hit," healthy volunteer transfusion model, comparing autologous RBCs that were stored for 35 days with those that were stored for 2 days.Healthy male volunteers donated 1 unit of autologous RBCs either 2 days (2D) or 35 days (35D) before the study date. The experiment was started by infusion of 2 ng/kg lipopolysaccharide ("first hit"). Two hours later, the stored RBCs ("second hit") were reinfused, followed by the labeling of RBCs with biotin. Clearance of biotin-labeled RBCs (BioRBCs) was measured during the 5-hour posttransfusion endotoxemia period along with measurements of phosphatidylserine (PS) exposure, lactadherin binding, and expression of CD47 (cluster of differentiation 47; a transmembrane protein encoded by the CD47 gene).In the 2D stored RBCs group, 1.5% ± 3.4% of infused BioRBCs were cleared from the circulation 5 hours posttransfusion versus 4.8% ± 4.0% in the 35D stored RBCs group (p = 0.1). There were no differences in PS exposure, lactadherin binding, or CD47 expression between fresh and stored RBCs or between pretransfusion and posttransfusion measurements.Our study shows a low clearance of RBCs even during endotoxemia. Furthermore, short-term clearance of BioRBCs during endotoxemia was not related to storage duration. Consistent with these observations, PS exposure, lactadherin binding, and CD47 expression did not differ between 2D and 35D stored cells before or after transfusion. We conclude that, in the presence of endotoxemia, clearance of 35D stored autologous RBCs is not increased compared with 2D stored fresh RBCs.

BACKGROUND: Plasma can be removed from platelet (PLT) concentrates (PCs) when volume reduction for PLT transfusion is indicated. Volume-reduced PCs are currently produced from pooled buffy coat (BC) PCs or apheresis PCs by pretransfusion volume reduction, followed by transfer to a syringe for immediate transfusion. We evaluated the maximal storage time of the volume-reduced PCs in gas-permeable containers. STUDY DESIGN AND METHODS: Volume-reduced PCs were produced from BC-derived and apheresis PCs by hard-spin centrifugation. Supernatant was removed and the PLTs were resuspended in 20 mL of retained original PC and had PLT concentrations ranging from 10.8 × 109 to 13.8 × 109 PLTs/mL. Volume-reduced PCs were stored either in syringes or in containers made from diethylhexyl phthalate (DEHP)-polyvinylchloride (PVC) or butyryl trihexyl citrate (BTHC)-PVC plastic. Units were sampled at t = 0, 1, 3, and 6 hours for in vitro measurements. RESULTS: When prepared from 2-day-old PCs (n = 4), volume-reduced PCs from BCs in a syringe had a pH37°C of 5.76 ± 0.04 at t = 6 hours after volume reduction. In the DEHP-PVC container, pH was 5.85 ± 0.15 (not significant), and in the BTHC-PVC, 6.34 ± 0.16 (p

For platelets to maintain their in vitro quality and in vivo effectiveness, they need to be stored at room temperature with gentle agitation in gas-permeable containers. The mode of agitation affects the quality of the platelets, and a gentle method of agitation, either a circular or a flat bed movement, provides the best results. Tumblers or elliptical agitators induce platelet activation and subsequent damage. As long as the platelets remain in suspension, the agitation speed is not important. Agitation of the platelet concentrates ensures that the platelets are continuously oxygenated, that sufficient oxygen can enter the storage container and that excess carbon dioxide can be expelled. During transportation of platelet concentrates, nowadays over long distances where they are held without controlled agitation, platelets may tolerate a certain period without agitation. However, evidence is accumulating that during the time without agitation, local hypoxia surrounding the platelets may induce irreversible harm to the platelets. Over the decades, more gas-permeable plastics have been used to manufacture platelet containers. The use of different plastics and their influence on the platelet quality both in vitro and in vivo is discussed. The improved gas-permeability has allowed the extension of platelet storage from 3 days in the early 1980s, to currently at least 7 days. In the light of new developments, particularly the introduction of pathogen reduction techniques, the use of platelet additive solutions and the availability of improved automated separators, further (renewed) research in this area is warranted.

BACKGROUND: The introduction of platelet (PLT) additive solutions (PASs) and pathogen reduction (PR) technologies possibly allow extension of PLT shelf life. It was our aim to compare in vitro quality of leucocyte-reduced PLT concentrates (PCs) stored in various PASs, including PR, with those in plasma during 8 days of storage. The study was performed in four blood centres where each tested four conditions. STUDY DESIGN AND METHODS: In paired experiments (n = 12), buffy coat pools were made to which various storage media were added. Plasma served as reference; two centres used InterSol followed by PR (InterSol+PR) and InterSol without PR; T-sol, SSP+ and Composol were also studied. RESULTS: All PCs fulfilled release criteria (pH(37 degrees C)>6.6; swirl present) until Day 8. Marked differences were seen for other parameters, including CD62P expression: 28 +/- 5; 31 +/- 7; and 39 +/- 9% for T-sol, Intersol+PR and without PR, respectively, which were higher as found for Composol (12 +/- 3%), SSP+ (15 +/- 5%) and plasma (15 +/- 6%). Three parameters (CD62P, Annexin A5, and lactate concentration) were collapsed into one rating value (6 = good quality, 0 = poor quality); PLTs in plasma had a rating of 2.8 +/- 1.0, which was higher as for T-Sol (1.5 +/- 0.5), InterSol+PR (1.3 +/- 0.6) and without PR (1.7 +/- 0.5). PLTs in potassium- and magnesium-containing PASs showed higher ratings as plasma, 4.3 +/- 0.5 for Composol and 3.8 +/- 0.8 for SSP+. CONCLUSION: PLT concentrates in plasma, SSP+ and Composol scored better using an arbitrary rating system as PLTs stored in T-Sol or InterSol; PR further impaired rating parameters. The applicability of these differences in rating for clinical effects needs a clinical study.

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.

BACKGROUND: The use of a functionally closed system (ACP215, Haemonetics) for the glycerolization and deglycerolization of red blood cell (RBC) units allows for prolonged postthaw storage. In this study, the postthaw quality of previously frozen, deglycerolized RBCs resuspended in saline-adenine-glucose-mannitol (SAGM) or additive solution AS-3 was investigated. STUDY DESIGN AND METHODS: Leukoreduced RBC units were frozen with 40 percent glycerol and stored at -80 degrees C for at least 14 days. The thawed units were deglycerolized with the ACP215, resuspended in SAGM or AS-3, and stored at 2 to 6 degrees C for up to 21 days. RESULTS: The mean +/- standard deviation in vitro freeze-thaw-wash recovery was 81 +/- 5 percent. During storage, hemolysis of deglycerolized cells remained below 0.8 percent for 2 days in SAGM and for 14 days in AS-3. This difference was explained by the protective effect of citrate, which is present in AS-3. Cells stored in AS-3 showed a lower glycolytic activity and a faster decline in adenosine 5'-triphosphate (ATP) than cells in SAGM. Increasing the internal pH of cells before storage in AS-3 by use of phosphate-buffered saline (PBS) in the deglycerolization procedure resulted in elevated lactate production and better maintenance of intracellular ATP content. After 3 weeks of storage, the ATP content of PBS-washed cells amounted to 2.5 +/- 0.5 micromol per g of hemoglobin (Hb), whereas for saline/glucose-washed cells this value was decreased to 1.0 +/- 0.3 micromol per g of Hb. CONCLUSIONS: Leukoreduced, deglycerolized RBCs can be stored for 48 hours in SAGM. Improved ATP levels during refrigerated storage can be observed with thawed cells, resuspended in AS-3, when PBS is used as a washing solution

During storage, the activity of the red blood cell (RBC) antioxidant system decreases. Glucose-6-phosphate dehydrogenase (G6PD) is essential for protection against oxidative stress by producing NADPH. G6PD function of RBC transfusion products is reported to remain stable during storage, but activity was measured in hemolysates and not in individual RBCs. We hypothesized that analysis of G6PD activity in individual RBC identifies storage-dependent changes in G6PD function.Seven units of stored leukoreduced RBCs, stored in saline-adenine-glucose-mannitol, were sampled every week up to 6 weeks of storage. G6PD activity was determined with the cytofluorometric method and expressed as mean fluorescent intensity (MFI) per RBC.During storage, G6PD activity decreased significantly. Mean MFI after 3 days of storage was 27.8 ± 8.8 and gradually decreased significantly to 18.0 ± 8.3 after 42 days.G6PD activity decreases during storage of leukoreduced RBCs. Our results may form a new target to improve storage conditions of RBCs and subsequently improve the quality of transfusion products.

Background and Objectives A pathogen-reduction technology (PRT) system using riboflavin and light has been developed for the treatment of platelet concentrates (PC) obtained by either buffy coat preparation (BCPC) or apheresis procedures (APPC). The aim of this study was to evaluate the effects of the treatment process on in vitro cell quality and on riboflavin conversion in PC. Materials and Methods BCPC were prepared with the Compomat G4™ from whole blood which had been stored overnight after collection. APPC were obtained using the TRIMA™ apheresis procedure. Both PC products had been stored for 18–24 h prior to PRT treatment. BCPC and APPC were treated with PRT on day 2 and day 1 of shelf-life, respectively. The treated PCs were then maintained for an additional 5 days after the PRT treatment. A panel of cell quality assays and high-performance liquid chromatography (HPLC) analysis were performed. Results Cell counts and plasma lactate dehydrogenase (LDH) levels during storage indicated that PRT did not induce significant cell lysis. Acceleration of a decrease in glucose and an increase in lactate was observed for treated PCs, but no significant differences were observed between treated BCPC and APPC. The pH of treated samples remained above 7·0, although was lower than that of the control. Platelet morphology of BCPC and APPC was well preserved. P-selectin expression indicated significant platelet activation when compared with control PC (BCPC on day 6: 39% vs. 12%; APPC on day 5: 35% vs. 18%). Both P-selectin expression and microparticle formation were not significantly different between treated BCPC and APPC during storage. The JC-1 assay also displayed no loss of mitochondria integrity during the storage of treated products. Approximately 20% of riboflavin converted into photoproducts, including lumichrome. Conclusions PRT treatment had an effect on the development of the normal platelet storage lesion at a level which seems tolerable for clinical usage.

Pathogen reduction technologies (PRTs) increase the safety of the blood supply, but are also associated with cell damage. Our aim was to investigate the effect of Mirasol PRT on platelet (PLT) concentrates stored in plasma and whether the use of a PLT additive solution (PAS) is able to improve in vitro quality.Twenty-two buffy coats (BCs) were pooled and split into two equal parts. To one half, 2 units of plasma were added, and to the other, 2 units of SSP+ PAS were added. Each part was equally split in half again (to resemble pooling five BCs) and PLT concentrates were prepared. One plasma PLT concentrate was Mirasol treated, and the other served as control; similarly, one SSP+ PLT concentrate was Mirasol treated, and the other not. PLT concentrates were stored for 8 days (n = 12).Mirasol PRT led to elevated lactate production in PLT concentrates in plasma, giving lower pH values throughout storage. The use of SSP+ mostly abrogated this effect, and Mirasol-treated PLT concentrates in SSP+ had only slightly higher lactate production rates and annexin A5 binding as control PLT concentrates in plasma. However, irrespective whether plasma or SSP+ was used, Mirasol PRT led to higher CD62P expression and lower hypotonic shock response (HSR) scores.Mirasol treatment leads to higher PLT activation and lower HSR scores both when stored in plasma or SSP+. However, if Mirasol-treated PLTs are stored in SSP+, lactate metabolism and annexin A5 binding are lower, showing that PAS can partly mitigate the effect of PRT. The clinical relevance of this finding needs to be demonstrated.

The plasticizer di(2-ethylhexyl)phthalate (DEHP) is a common component in blood bags. DEHP is noncovalently bound to polyvinylchloride (PVC) polymer and can leach into the blood product. There are public concerns that exposure to DEHP might induce developmental and reproductive toxicity in humans. The aim of this study was to evaluate an alternative plasticizer, di(isononyl) cyclohexane-1,2-dicarboxylate (Hexamoll DINCH, BASF SE), for its use in blood bags.Whole blood (WB) was collected into DEHP-containing and DEHP-free collection systems. After overnight hold, WB was centrifuged and separated in plasma, buffy coat, and red blood cells (RBCs). Buffy coats and plasma were used to make platelet (PLT) concentrates in DEHP-free systems. After addition of additive solution (AS), SAG-M, PAGGS-M, AS-3, or PAGGG-M, RBCs were leukoreduced and analyzed for in vitro characteristics and plasticizer levels during storage.The use of DINCH-based systems had no effect on WB composition, blood processing, and plasma quality. PLT in vitro quality variables were maintained during storage in DEHP-free systems. During storage in SAG-M, hemolysis was significantly higher in DINCH-PVC while potassium leakage and adenosine triphosphate content were comparable. During storage in alternative ASs, hemolysis was reduced compared to storage in SAG-M.The complete absence of DEHP in the collection system had no effect on WB composition, processing, or plasma and PLT quality. During storage in SAG-M, the absence of DEHP resulted in increased hemolysis. With alternative ASs like PAGGS-M, AS-3, or PAGGG-M, the absence of DEHP had no effect on hemolysis. Leakage of DINCH into the blood product was less pronounced than that of DEHP.

R. N. I. Pietersz, H. W. Reesink, S. Panzer, S. Oknaian, S. Kuperman, C. Gabriel, A. Rapaille, M. Lambermont, V. Deneys,D. Sondag, S. Ramirez-Arcos, M. Goldman, G. Delage, F. Bernier, M. Germain, T. Vuk, J. Georgsen, P. Morel, C. Naegelen,L. Bardiaux, J.-P. Cazenave, J. Dreier, T. Vollmer, C. Knabbe, E. Seifried, K. Hourfar, C. K. Lin, M. Spreafico, L. Raffaele,A. Berzuini, D. Prati, M. Satake, D. de Korte, P. F. van der Meer, J. L. Kerkhoffs, L. Blanco, J. Kjeldsen-Kragh,A.-M. Svard-Nilsson, C. P. McDonald, I. Symonds, R. Moule, S. Brailsford, R. Yomtovian & M. R. JacobsSeptic reactions after transfusion, particularly of plateletconcentrates, still occur and belong to the most serioustransfusion reactions. From a previous InternationalForum [1] on the subject, it could be concluded that inpart of the countries that participated in the forum, plate-let concentrates (PCs) were tested for bacterial contamina-tion and that culture-based methods, particularly theBacT/Alert system, were used.In recent years, several rapid bacterial detection meth-ods, such as surrogate measurements of the pH or glu-cose, the detection of bacteria with a scan system orPCR tests that detect bacterial RNA, have been devel-oped. These tests can either be performed immediatelyprior to transfusion of the PC or at a variety of testmoments at which culture and release tests are com-bined.Pathogen inactivation (PI) methods also affect bacterialcontamination of PCs. In 2007 [1], in some countries, theIntercept method of PI of PCs was implemented insteadof bacterial screening.It seemed of interest to evaluate the present state ofthe art of this subject. In order to obtain the desiredinformation, the following questions were sent to expertsin the field.Question 1: How long do you store PC and is there adifference between whole-blood-derived PC and apheresisPC? Which method of preparation do you use for whole-blood-derived PC? Are PCs leuco-reduced?Question 2: Do you use a culture method to detect bac-terial contamination of PC? If so

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.

In this study we investigated whether storage of red blood cells (RBCs) leads to alterations in nitrite reductase activity, hence in altered hypoxia-induced nitric oxide (NO) bioavailability and methemoglobin formation.Hypoxia-induced NO bioavailability and methemoglobin formation were measured in vitro after nitrite administration to fresh (1 week of storage) and aged (5-6 weeks of storage) human RBC units and in blood samples of hemodiluted rats subjected to hypoxic ventilation after transfusion with fresh or aged human RBCs.In vitro, NO and methemoglobin levels 10 minutes after nitrite administration were lower in the fresh RBC samples compared to the aged RBC samples (p = 0.026 and p = 0.022, respectively). In vivo, NO bioavailability was also significantly lower in the rats receiving fresh RBCs compared to the group receiving aged RBCs (p = 0.003). In line with NO bioavailability, methemoglobin levels were higher, albeit not significantly, in the group receiving aged RBCs compared to in the group receiving fresh RBCs (p = 0.154). The difference in methemoglobin formation after nitrite administration between fresh and aged RBCs was only present under deoxygenated conditions and not under oxygenated conditions. There were no differences in methemoglobin reductase activity between fresh and aged RBCs.Storage of RBCs leads to an increased rate of hypoxia-induced nitrite reduction to NO and this is associated with increased methemoglobin formation. The increased methemoglobin formation and consequent decrease in oxygen delivery capacity might contribute to the storage-related impairment of aged RBCs to oxygenate the microcirculation.

Recent studies showed that Mirasol pathogen reduction treatment (PRT) leads to increased P-selectin expression and increased oxygen and glucose consumption in resting platelets (PLTs). This study investigates the effect of PRT on PLT activation.Untreated or Mirasol-treated PLTs were analyzed at different time points during storage. Microaggregation upon stimulation with phorbol myristate acetate (PMA), convulxin, and ristocetin was measured. Alpha granule contents and release upon thrombin stimulation were assessed by flow cytometry and Western blotting. PLT spreading was determined on collagen-coated glass slides.Mirasol PRT led to spontaneous aggregation (hyperreactivity), as measured by flow cytometry in the absence of agonist throughout storage time. PMA-induced aggregation was significantly higher in Mirasol PRT PLTs compared to controls. Aggregation in response to convulxin and ristocetin was significantly lower and directly influenced by storage time after Mirasol PRT, compared to untreated stored PLT concentrates. Despite the reported hyperreactivity of resting PLTs, PLT activation with thrombin on Day 8 after Mirasol PRT resulted in less P-selectin-positive PLTs. Furthermore, platelet factor 4 (PF4) secretion was reduced upon thrombin stimulation on Day 8 after PRT compared to controls. Significantly decreased spreading of Mirasol PRT PLTs over collagen-coated slides was observed directly after PRT and persisted throughout storage.Mirasol PRT leads to hyperreactive PLTs, probably caused by continuous basal degranulation through storage time. This results in a reduction in the degranulation capacity upon acute stimulation, which influences PLT spreading, but not overtly microaggregation. The clinical relevance needs to be investigated.

Thromboelastography (TEG) is widely used in hospitals but less commonly in blood banks for evaluation of platelet (PLT) concentrates (PCs). A TEG-PC assay for testing fresh or stored PLTs must reflect the quality of the PLTs. The added value could be measurement of donor-dependent PLT quality.Whole blood (WB) normal values were generated from 100 donors, using standard tests. Nineteen single-donor PCs were evaluated with a TEG-PC assay, using Octaplas as microparticle-free diluent and kaolin or collagen as activator, stored up to 12 days, and also sampled for additional in vitro tests.WB values showed larger reaction rates (R- and K-times, angle) compared to the reference values and almost similar maximum amplitude (MA). PCs showed usual storage lesion and TEG-PC results showed significant decreasing R- and K-times and increasing angle. Mean MA values remained constant but individual measurements were affected by clot retraction. TEG tracings of two PCs with good quality on Day 12 showed weak to strong clot retraction, while two PCs with poor quality showed moderate clot retraction on Day 1, no clot retraction on Days 5 to 12, and a decreased MA on Day 12. Clot strength (MA) and especially clot retraction represent possibly donor-specific effects.A TEG-PC assay has been developed that is sensitive to storage effects. The assay has the potential to be helpful in selection of PLT donors but needs improvement to be more sensitive, reproducible, and distinctive to determine whose PLTs store poorly and whose store well.

Background Developing new quality control methods for platelet concentrates (PCs) can contribute to increasing transfusion safety and efficiency. The aim of this study was to investigate in a large field test the quality of expired PCs and whether 100% noninvasive pH monitoring can be used to predict PC quality. Study Design and Methods The pH of 13,693 PCs produced for transfusion was monitored daily using Blood Storage, Inc.'s pH sterile, automated fluoroscopic evaluation technology. Upon indication of compromised quality or expiration, PCs were returned and in vitro tests were performed. Results A total of 998 PCs were returned, of which 962 outdated, 26 had a positive BacT/ALERT reaction, seven had aggregates, one was without swirl, one had low pH, and one had high pH. BacT/ALERT was faster in identifying bacterial contamination than pH measurements. The pH at the end of the storage period was significantly lower than at the beginning. In vitro tests indicated that while the PC quality was acceptable upon expiration, it rapidly declined after expiration. Conclusion In this setting where the vast majority of PCs were of good quality and within acceptable pH limits, daily, noninvasive routine pH measurement has limited added value in identifying quality-compromised PCs.

Developing new quality control methods for platelet concentrates (PCs) can contribute to increasing transfusion safety and efficiency. The aim of this study was to investigate in a large field test the quality of expired PCs and whether 100% noninvasive pH monitoring can be used to predict PC quality.The pH of 13,693 PCs produced for transfusion was monitored daily using Blood Storage, Inc.'s pH sterile, automated fluoroscopic evaluation technology. Upon indication of compromised quality or expiration, PCs were returned and in vitro tests were performed.A total of 998 PCs were returned, of which 962 outdated, 26 had a positive BacT/ALERT reaction, seven had aggregates, one was without swirl, one had low pH, and one had high pH. BacT/ALERT was faster in identifying bacterial contamination than pH measurements. The pH at the end of the storage period was significantly lower than at the beginning. In vitro tests indicated that while the PC quality was acceptable upon expiration, it rapidly declined after expiration.In this setting where the vast majority of PCs were of good quality and within acceptable pH limits, daily, noninvasive routine pH measurement has limited added value in identifying quality-compromised PCs.

A donation of whole blood is most commonly collected in acidic citrate-phosphate-dextrose (CPD) variants with pH 5.2 to 6.2 as anticoagulants. Previously, we have shown that the initial pH after red blood cell (RBC) preparation can have an effect on RBCs during storage. First, we investigated the effect of the pH of the anticoagulant on RBCs. Second, we investigated the possibility of decreasing the pH of our new additive solution (AS) phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM) from pH 8.2 to 7.4 in combination with an anticoagulant with a physiologic pH.Whole blood was collected in CPD (pH 5.6) or trisodiumcitrate (TNC; pH 7.4), and leukoreduced units were prepared using saline-adenine-glucose-mannitol as AS. Second, whole blood was collected in TNC (pH 7.4), and leukoreduced units were prepared using PAGGGM (pH 7.4) or PAGGGM (pH 8.2) as AS. During cold storage, several in vitro characteristics were analyzed.In agreement with our previous findings, the initial pH of whole blood has an effect during storage of RBCs. In the second part we show that there are no differences between PAGGGM (pH 7.4) and PAGGGM (pH 8.2) units when an anticoagulant with a physiologic pH was used.These results indicate that the pH of the anticoagulant used during whole blood collection has an effect during storage of RBCs. When an anticoagulant with a physiologic pH is used during whole blood collection, the pH of PAGGGM can be decreased to physiologic levels, while maintaining adenosine triphosphate and 2,3-diphosphoglycerate levels.

Platelet concentrates (PC) were stored for 6 days in either polyolefin (PO) or polyvinylchloride/di-(2-ethylhexyl)phtalate (PVC/DEHP) bags in 100% plasma or in a synthetic medium with 35 or 10% plasma. For all conditions studied the usual in vitro parameters were well maintained, with a pH above 6.8. In both bag types platelets can be satisfactorily stored for 6 days in a synthetic medium with minimal amounts of residual plasma. For this medium, the PO bag offers a slight advantage with respect to the preservation of platelet ATP content (> 80 versus > 70% in the PVC bags) and aggregation and adhesion capacity. The adhesion capacity increased in the PO bags, while it decreased in the PVC bags.

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.

The Reveos system (Terumo BCT) is a fully automated device able to process four whole blood (WB) units simultaneously into a plasma unit, a red blood cell (RBC) unit, and an interim platelet (PLT) unit (IPU). Multiple IPUs can be pooled to form a transfusable PLT product. The aim of our study was to evaluate the quality of components made with the Reveos system from either fresh (2-8 hr) or overnight-held WB.A prototype of the Reveos system was used to process WB. RBCs were resuspended in SAGM, leukoreduced, and assayed for in vitro quality variables during a 42-day storage period at 2 to 6 °C. Twenty-four-hour in vivo recovery was determined on Day 42. Plasma was assayed for cellular contamination and activation variables. IPUs were pooled with SSP+ additive solution for in vitro quality assessments during a 7-day storage period at room temperature.Reveos-produced RBCs and plasma units met the predefined requirements. RBC recovery was superior to control units. On Day 42, hemolysis was below 0.8% and in vivo recovery was above 75% for all RBCs. Cellular contamination was lower for Reveos-produced plasma. PLT yield was higher with overnight-stored WB. PLT quality was well maintained during storage with no significant differences between the two groups.Blood components prepared with the Reveos from fresh or overnight-held WB meet quality criteria without any relevant difference between the two groups. The Reveos system has the potential to increase efficacy and standardization of blood component preparation.