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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 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. STUDY DESIGN AND METHODS 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). RESULTS 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. CONCLUSION 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.

Shortly after the introduction of component therapy in the 1960s, the first additives for red cell concentrates (RCC) were introduced. This development resulted in prolongation of RCC shelf life from 21 to 35 or 42 days. Over the years, some new solutions were developed, but still SAGM (Europe) and AS-1 (United States) are the standard, nearly exclusive in combination with CPD as anticoagulant. Most of these new solutions were minor variations on SAGM or AS-1, with limited improvement of in vitro quality of erythrocytes. Development of new concepts, resulting in improved quality of RCC, was in first instance focused on achieving longer storage time. However, as a result from the concern about possible negative side effects caused by transfusion of older units, the research on new additive solutions is more focused on having a better quality during the currently accepted maximum storage time of 35–42 days. Around 1990, Meryman developed the concept of the so-called chloride shift. Through replacement of chloride by impermeable solutes, it is possible to manipulate the intracellular pH of erythrocytes, resulting in high 2,3-DPG and/or ATP values throughout storage. During the past 15–20 years, several research groups, with the group of Greenwalt and Hess as pioneers, developed new additive solutions based on the Meryman concept. Recently, the first commercial product from these studies was launched, with an improved in vitro quality parameter profile and improved in vivo recovery data. Finally, some recent developments are discussed with respect to alkaline additive solutions and anaerobic storage of erythrocyte concentrates.

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: Apheresis is increasingly being applied to collect cells or plasma, even allowing the collection of multiple blood components during one procedure. Although the quality of the cellular and plasma products that are obtained by apheresis have been extensively studied and shown to be of high quality, the impact of apheresis on the red blood cells (RBCs) that are returned to the donor has not been investigated. STUDY DESIGN AND METHODS: The effect of the plasma- or plateletpheresis procedures by four different devices—MCS+ (Haemonetics), PCS2 (Haemonetics), Trima Accel (Terumo BCT), and Autopheresis-C (Auto-C, Fresenius Kabi)—on the RBCs that are returned to the donor was tested in a blinded, prospective trial in a cohort of 25 donors. RESULTS: A rheologic analysis of donor RBCs before and after plasma- or plateletpheresis showed no differences in outcome. However, a strong increase in hemolysis was found in samples from the Trima Accel devices after plateletpheresis, compared to all other machines tested. Furthermore, an increase in complement deposition on RBCs was seen after all plasmapheresis procedures (MCS+, PCS2, and Auto-C). Finally, a significant decrease in the expression of the complement-regulating protein CD59 was seen in all postapheresis samples as well as a significant decrease of the adhesion molecule CD147. CONCLUSION: The increase in complement deposition and the decrease in the expression of CD59 suggests that RBC clearance might be enhanced after return to the donor. Possible side effects due to an increase in hemolysis after Trima Accel plateletpheresis should be further investigated.

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 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. STUDY DESIGN AND METHODS 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. RESULTS 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. CONCLUSION 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 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. STUDY DESIGN AND METHODS 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). RESULTS 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. CONCLUSION 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.

Transfusion-transmitted infections (TTI) have been greatly reduced in numbers due to the strict donor selection and screening procedures, i.e. the availability of technologies to test donors for endemic infections, and routine vigilance of regulatory authorities in every step of the blood supply chain (collection, processing and storage). However, safety improvement is still a matter of concern because infection zero-risk in transfusion medicine is non-existent. Alternatives are required to assure the safety of the transfusion product and to provide a substitution to systematic blood screening tests, especially in less-developed countries or at the war-field. Furthermore, the increasing mobility of the population due to traveling poses a new challenge in the endemic screening tests routinely used, because non-endemic pathogens might emerge in a specific population. Pathogen reduction treatments sum a plethora of active approaches to eliminate or reduce potential threatening pathogen load from blood transfusion products. Despite the success of pathogen reduction treatments applied to plasma products, there is still a long way to develop and deploy pathogen reduction treatments to cellular transfusion products (such as platelets, RBCs or even to whole blood) and there is divergence on its acceptance worldwide. While the use of pathogen reduction treatments in platelets is performed routinely in a fair number of European blood banks, most of these treatments are not (or just) licensed in the USA or elsewhere in the world. The development of pathogen reduction treatments for RBC and whole blood is still in its infancy and under clinical trials. In this review, we discuss the available and emerging pathogen reduction treatments and their advantages and disadvantages. Furthermore, we highlight the importance of characterizing standard transfusion products with current and emerging approaches (OMICS) and clinical outcome, and integrating this information on a database, thinking on the benefits it might bring in the future toward personalized transfusion therapies.

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 Interventions to prevent and detect bacterial contamination of platelet concentrates (PCs) have reduced, but not eliminated the sepsis risk. Standardized bacterial strains are needed to validate detection and pathogen reduction technologies in PCs. Following the establishment of the First International Reference Repository of Platelet Transfusion-Relevant Bacterial Reference Strains (the ‘repository’), the World Health Organization (WHO) Expert Committee on Biological Standardisation (ECBS) endorsed further repository expansion. Materials and Methods Sixteen bacterial strains, including the four repository strains, were distributed from the Paul-Ehrlich-Institut (PEI) to 14 laboratories in 10 countries for enumeration, identification and growth measurement on days 2, 4 and 7 after low spiking levels [10–25 colony-forming units (CFU)/PC bag]. Spore-forming (Bacillus cereusPEI-B-P-07-S, Bacillus thuringiensisPEI-B-P-57-S), Gram-negative (Enterobacter cloacaePEI-B-P-43, Morganella morganiiPEI-B-P-74, PEI-B-P-91, Proteus mirabilisPEI-B-P-55, Pseudomonas fluorescensPEI-B-P-77, Salmonella choleraesuisPEI-B-P-78, Serratia marcescensPEI-B-P-56) and Gram-positive (Staphylococcus aureusPEI-B-P-63, Streptococcus dysgalactiaePEI-B-P-71, Streptococcus bovisPEI-B-P-61) strains were evaluated. Results Bacterial viability was conserved after transport to the participating laboratories with one exception (M. morganiiPEI-B-P-74). All other strains showed moderate-to-excellent growth. Bacillus cereus, B. thuringiensis, E. coli, K. pneumoniae, P. fluorescens, S. marcescens, S. aureus and S. dysgalactiae grew to >106 CFU/ml by day 2. Enterobacter cloacae, P. mirabilis, S. epidermidis, S. bovis and S. pyogenes achieved >106 CFU/ml at day 4. Growth of S. choleraesuis was lower and highly variable. Conclusion The WHO ECBS approved all bacterial strains (except M. morganiiPEI-B-P-74 and S. choleraesuisPEI-B-P-78) for repository enlargement. The strains were stable, suitable for spiking with low CFU numbers, and proliferation was independent of the PC donor.

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).

Background 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. Study Design and Methods 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. Results 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. Conclusions 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.

Background 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. Study Design and Methods Hypoxia-induced NO bioavailability and methemoglobin formation were measured in vitro after nitrite administration to fresh (

Background 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. Study Design and Methods 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. Results 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. Conclusion 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.

Anaerobic Propionibacterium acnes and Staphylococcus saccharolyticus are frequently isolated during platelet screening with anaerobic culture methods. Although neither P. acnes nor S. saccharolyticus proliferates during platelet storage, both species survive well in this environment. This study was aimed at determining whether strains of P. acnes and/or S. saccharolyticus form surface-attached bacterial cell aggregates, known as biofilms, under platelet storage conditions. We report that these organisms are able to adhere to the inner surface of platelet containers in tight interaction with activated platelets.

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.

Donor graft washout can be impaired by colloids in organ preservation solutions that increase the viscosity and agglutinative propensity of red blood cells (RBCs) and potentially decrease organ function. The colloid-induced agglutinative effects on RBCs and RBC retention after liver washout with Ringer's lactate (RL), histidine tryptophan ketoglutarate solution, University of Wisconsin solution, and Polysol were determined as a function of the washout pressure (15 or 100 mm Hg) and temperature (4 or 37°C) in a rat liver washout model with (99m) Tc-pertechnetate-labeled RBCs. Colloids (polyethylene glycol in Polysol and hydroxyethyl starch in University of Wisconsin) induced RBC agglutination, regardless of the solution's composition. RL was associated with the lowest degree of (99m) Tc-pertechnetate-labeled RBC retention after simultaneous arterial and portal washout at 37°C and 100 mm Hg. RL washout was also associated with the shortest washout time. A single portal washout with any of the solutions did not result in differences in the degree of RBC retention, regardless of the temperature or pressure. In conclusion, no differences were found in portal washout efficacy between colloidal solutions, histidine tryptophan ketoglutarate, and RL. Simultaneous arterial and portal washout with RL at 37°C and 100 mm Hg resulted in the least RBC retention and the shortest washout time.

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

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 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. Study Design and Methods 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. Results 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. Conclusion 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.

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: 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. STUDY DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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

BACKGROUND: Fibrin sealant is a human blood product consisting of two components: cryoprecipitate and thrombin. Commercial fibrin sealants are produced from multidonors, increasing the viral risk, and contain fibrinolytic inhibitors such as tranexamic acid or bovine aprotinin. Autologous fibrin sealants reduce the viral risk and are mostly produced during a surgical procedure or well in advance. Alternatively, the allogeneic single-donor fibrin sealant cryoseal can be used. In this study cryoseal was characterized and the manufacturing consistency of the production process was investigated. STUDY DESIGN AND METHODS: Cryoseal was produced from plasma collected on apheresis machines using a commercial device. In a research setting the protein composition and recovery were determined. Also, the manufacturing consistency of the production process was tested in a research setting as well as in a routine setting. RESULTS: In the research setting all produced cryoseal met the quality control requirements of a clotting time of less than 10 seconds and the presence of Factor (F)XIII (qualitative). In the routine setting, one procedure per year did not meet these requirements. The protein composition showed the following mean ± standard deviation (%recovery) results: thrombin 25.7 ± 11.1 IU/mL, fibrinogen 19.9 ± 4.6 (15%) mg/mL, FVIII 15.6 ± 5.4 (44%) IU/mL, FXIII 2.7 ± 0.7 (6%) IU/mL, and plasminogen 1.8 ± 0.2 (4%) U/mL. In both research and routine settings the production process resulted in a consistent product. CONCLUSION: The cryoseal manufacturing process resulted in a consistent product, which meets the predetermined specifications. The single-donor origin and the absence of fibrinolytic inhibitors make cryoseal a good alternative for multidonor and autologous fibrin sealants.

BACKGROUND: The platelets (PLTs) in PLT concentrates are counted with hematology analyzers, but varying results among different hematology analyzers are observed, making comparisons very difficult. Due to the absence of red blood cells in PLT concentrates, the International Council for Standardization in Hematology (ICSH) reference method was modified to be used for PLT concentrates and validated in an international comparative study. STUDY DESIGN AND METHODS: Five PLT samples were shipped to eight participating centers of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative and counted on the same day. PLTs were stained with fluorescein isothiocyanate–labeled anti-CD41a in tubes (TruCount, BD Biosciences), measured on a flow cytometer, and analyzed with a uniform template. These samples were also counted on 15 hematology analyzers. RESULTS: The ICSH method and newly developed BEST method yielded PLT counting results with less than 1% difference (not significant). The intercenter coefficient of variation (CV) of the BEST method was on average 6.3% versus 7.6% on average for hematology analyzers. The CV of individual hematology analyzers was on average 0.9%, which was considerably lower than for the flow cytometers with a mean of 3.7%. CONCLUSION: The BEST flow cytometric method has a smaller intercenter CV and a smaller center-to-center deviation from the group mean compared to hematology analyzers. Conversely, individual hematology analyzers are more precise than the flow cytometric method. Thus, the flow cytometric method provides a calibration tool to allow comparisons between centers, but there is no need to replace routine counting with hematology analyzers.

Background Bacterial contamination of platelet concentrates (PCs) still remains a significant problem in transfusion with potential important clinical consequences, including death. The International Society of Blood Transfusion Working Party on Transfusion-Transmitted Infectious Diseases, Subgroup on Bacteria, organised an international study on Transfusion-Relevant Bacteria References to be used as a tool for development, validation and comparison of both bacterial screening and pathogen reduction methods. Material and Methods Four Bacteria References (Staphylococcus epidermidis PEI-B-06, Streptococcus pyogenes PEI-B-20, Klebsiella pneumoniae PEI-B-08 and Escherichia coli PEI-B-19) were selected regarding their ability to proliferate to high counts in PCs and distributed anonymised to 14 laboratories in 10 countries for identification, enumeration and bacterial proliferation in PCs after low spiking (0·3 and 0·03 CFU/ml), to simulate contamination occurring during blood donation. Results Bacteria References were correctly identified in 98% of all 52 identifications. S. pyogenes and E. coli grew in PCs in 11 out of 12 laboratories, and K. pneumoniae and S. epidermidis replicated in all participating laboratories. The results of bacterial counts were very consistent between laboratories: the 95% confidence intervals were for S. epidermidis: 1·19–1·32 × 107 CFU/ml, S. pyogenes: 0·58–0·69 × 107 CFU/ml, K. pneumoniae: 18·71–20·26 × 107 CFU/ml and E. coli: 1·78–2·10 × 107 CFU/ml. Conclusion The study was undertaken as a proof of principle with the aim to demonstrate (i) the quality, stability and suitability of the bacterial strains for low-titre spiking of blood components, (ii) the property of donor-independent proliferation in PCs, and (iii) their suitability for worldwide shipping of deep frozen, blinded pathogenic bacteria. These aims were successfully fulfilled. The WHO Expert Committee Biological Standardisation has approved the adoption of these four bacteria strains as the first Repository for Transfusion-Relevant Bacteria Reference Strains and, additionally, endorsed as a project the addition of six further bacteria strain preparations suitable for control of platelet contamination as the next step of enlargement of the repository.

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: Bioactive lipids (lysophosphatidylcholines [lysoPCs]) accumulating during storage of cell-containing blood products are thought to be causative in onset of transfusion-related acute lung injury through activation of neutrophils. LysoPCs are thought to be derived from cell membrane degradation products such as phosphatidylcholines (PC) by partial hydrolysis of PC, a process that is catalyzed by phospholipase A2 (PLA2). STUDY DESIGN AND METHODS: We investigated the underlying mechanisms of lysoPC generation and its contribution to in vitro neutrophil-priming capacity during storage of red blood cells (RBCs), platelet (PLTs) concentrates, and cell-free plasma. Blood from healthy volunteers was drawn, processed, and stored according to Sanquin Blood Bank protocols. RESULTS: Storage of RBCs in saline-adenine-glucose-mannitol (SAGM) did not result in accumulation of lysoPCs or neutrophil-priming capacity. Replacement of SAGM by plasma as RBC storage medium caused elevated lysoPC levels on Day 0, which did not further increase during storage. Cell-free plasma stored at 22°C showed accumulation of lysoPCs during storage, which was not present at 4°C. Addition of a soluble PLA2 or cytosolic PLA2 inhibitor did not prevent accumulation of lysoPCs in plasma. In PLTs, lysoPC accumulation during storage was plasma dependent, but lysoPCs did not explain the observed neutrophil-priming effect as preventing accumulation of lysoPCs by removing the plasma fraction did not prevent the neutrophil-priming capacity. CONCLUSION: Accumulation of lysoPCs during storage is not cell but plasma derived and storage temperature dependent and does not explain the neutrophil-priming effect of aged products.

BACKGROUND: In this study it was investigated whether Propionibacterium acnes present in platelet concentrates (PCs) and related red blood cells (RBCs), originate from the skin of the donor. STUDY DESIGN AND METHODS: P. acnes that were cultured throughout 2007 and 2008 from PCs and their accompanying RBCs and in 2010 from the phlebotomy site of a selection of the respective donors (n = 22) were typed by amplified fragment length polymorphism. A part of the strains was also determined to species level by sequencing of the 16S rRNA and recA genes. RESULTS: Three different phylogenetic groups of P. acnes were found. The distribution of the P. acnes in three groups was confirmed by sequencing of the recA gene. All strains that were found in PCs and their accompanying RBCs were identical, which indicates that the strain is already present in the whole blood donation. P. acnes could be found on the skin of almost all screened donors. In eight of 22 cases (36.4%), one of the strains from the donor skin was identical to the strains found in PCs and their accompanying RBCs. In two other cases the strains belonged to the same phylogenetic group. CONCLUSION: This study supports the theory that the source of P. acnes contamination is in many cases the skin of the donor. However, further study is necessary to rule out other sources of contamination. Because it is difficult to prevent bacterial contamination by P. acnes completely, it is necessary to further investigate the clinical significance of blood products contaminated with P. acnes. C ontamination of platelets (PLTs) with bacteria is the major microbiologic risk of blood transfusion. This applies especially for PLT concentrates (PCs) because their storage conditions, at room temperature and under constant agitation, support bacterial growth. From all whole blood–derived PCs in the Netherlands, approximately 0.37% are tested positive for bacterial growth with the BacT/ALERT (bioMerieux, Marcy l’Etoile, France) culture system. 1 From all the bacteria that are found with the BacT/ALERT more than half are Propionibacterium acnes, 2,3 Gram-positive, anaerobic bacteria that are part of the normal resident skin flora. 4

Three EBA specified blood bag configurations (‘Eurobloodpack’) are described which are capable of meeting >80% of its member’s requirements. These include a ‘top-and-top’ and two ‘bottom-and-top’ packs enabling aseptic, pre-donation collection of up to 40 ml of samples, 427·5–522·5 ml of whole blood and the preparation of an extensive range of blood components. Features currently beyond the scope of ISO standardisation have been controlled including: anticoagulant and additive volumes; collection needle and sampling system; transfer tubing; cross-match line; base label; leucodepletion filter performance; compatibility of access ports and transfusion sets. Eurobloodpack has significant advantages for blood services and blood bag manufacturers.

BACKGROUND: In this study the applicability of a 16S rRNA real-time reverse transcriptase polymerase chain reaction (RT-PCR) and a Staphylococcus genus–specific PCR for screening of bacterial contamination in platelet concentrates (PCs) was determined. STUDY DESIGN AND METHODS: A total of 336 sample bags, from PCs that were routinely tested in the BacT/ALERT (bioMerieux), were collected and frozen until testing by the PCR assays. Based on the BacT/ALERT results, 107 PCs were positive and 229 were negative for bacterial growth. RESULTS: The analytical sensitivity of the 16S rRNA real-time RT-PCR ranged from 5 to 40 colony-forming units (CFUs)/mL. The PCR detected five positive samples, four of which were also positive in the BacT/ALERT. The sensitivity of the test was 3.8%, and the specificity was 99.5%. The analytical sensitivity of the Staphylococcus genus–specific PCR ranged from 5 to 15 CFUs/mL. Thirty-nine units that were BacT/ALERT positive for staphylococci were tested with this PCR. Six samples were positive with the PCR, five of which were also BacT/ALERT positive. The sensitivity of the Staphylococcus genus–specific PCR was 12.8%, and the specificity was 98.8%. CONCLUSION: Despite the rapid availability of results compared to the BacT/ALERT, the analytical sensitivity of a generic or specific PCR assay is not high enough to be an alternative for the BacT/ALERT when PCs are screened on the day of production.

BACKGROUND: It is unknown whether the use of volumetric infusion pumps for the transfusion of red blood cells (RBCs) or platelet (PLT) concentrates (PCs) affects the quality of the blood components. We therefore investigated the in vitro quality of these components after use of infusion pumps. STUDY DESIGN AND METHODS: Ten different volumetric infusion pumps were used to simulate transfusion with RBCs and PCs. To prevent donor-dependent differences multiple units were pooled and divided into equal portions. The storage time of RBCs was 30 to 35 days (n = 10 experiments), and for PCs, either 2 (n = 5) or 7 days (n = 5). For RBCs an infusion rate of 100 or 300 mL/hr was used, and for PCs, 600 mL/hr. Transfusions without an infusion pump served as a reference. RESULTS: None of the infusion pumps induced an increase of free hemoglobin, annexin A5 binding, or formation of echinocytes in RBCs compared to reference units. In 2- and 7-day-old PCs no effect was shown on PLT concentration, annexin A5 binding, mean PLT volume, and morphology score compared to the reference. The CD62P expression of 2-day-old PCs was significantly lower after transfusion compared to the reference, that is, 11.7 ± 2.1% versus 8.1 ± 1.3% (p

SUMMARY: BACKGROUND: Contamination of platelets with bacteria is the major microbiological risk of blood transfusion. Screening for bacterial contamination can reduce the frequency of bacterial transmission considerably. In the present paper, the results of 10-year screening in the Netherlands are described. METHODS: All platelet concentrates were cultured with the BacT/Alert culturing system with large volume (7.5 ml) cultures in either an aerobic or an anaerobic bottle. Products were released on a 'negative-to-date' basis. RESULTS: After introduction of the diversion of the first milliliters of collected blood, the number of positive screening cultures decreased significantly from 0.85% to 0.37%. The frequency of transfusion-transmitted bacterial infections by platelet concentrates is currently less than 1 per 2 years in the Netherlands. CONCLUSION: Over a period of 10 years the bacterial screening system for platelet concentrates proved to result in a safe system with respect to microbiological infection as a result of platelet transfusions.

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

BACKGROUND: Many countries use cooling plates to actively cool collected whole blood (WB) to room temperature. Until now, no paired comparison had been performed, and it was our aim to compare the effect of active versus no active cooling on the in vitro quality of WB and subsequently prepared blood components. STUDY DESIGN AND METHODS: Two units of WB were pooled and divided shortly after donation. One unit was placed under a butane-1,4-diol plate to obtain active cooling; the other was placed in an insulated box with other warm units to mimic worst-case holding conditions. WB was held overnight and processed into a white blood cell (WBC)-reduced red blood cells (RBCs), buffy coat (BC), and plasma. The BCs were further processed into platelet (PLT) concentrates. RBCs were stored for 42 days, and PLT concentrates for 8 days (n = 12 paired experiments). RESULTS: After overnight storage, ATP content of the RBCs was 4.9 ± 0.3 µmol/g Hb for actively cooled WB versus 4.5 ± 0.4 µmol/g Hb for not actively cooled WB (p

BACKGROUND: Bacterial contamination or platelet (PLT) metabolism can change the pH of stored PLT concentrates (PCs). Measurement of pH for quality control is currently done on a limited basis. An easy noninvasive method was developed to obtain sequential pH measurements over time, without risking contamination and/or consuming PCs.

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.