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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.

Background and Objectives: Transfusion-associated circulatory overload (TACO) is the primary cause of transfusion-related mortality. Speed and volume of transfusion are major risk factors. The aim of this study was to investigate the interaction of red blood cell (RBC) transfusion speed and volume on the development of TACO. Materials and Methods: A validated model for TACO in anaemic Lewis rats with an acute myocardial infarction was used. The effect on pulmonary hydrostatic pressure of one, two or four units of packed RBCs transfused in either 30 or 60 min was evaluated (3.3–26.6 ml·kg −1·hr −1). Pulmonary capillary pressure was measured as left ventricular end-diastolic pressure (LVEDP). Cardiac stress biomarkers atrial natriuretic-peptide (ANP) and N-terminal pro-brain natriuretic peptide (NT-proBNP) were measured 1-h post-transfusion. Results: Thirty animals were included (n = 5 per group). Transfusion of RBCs increased LVEDP in a volume-dependent manner (ΔLVEDP [mmHg]: −0.95, +0.50, +6.26, p < 0.001). Fast transfusion increased overall ΔLVEDP by +3.5 mmHg and up to +11.8 mmHg in the four units' group (p = 0.016). Doubling transfusion speed increased ΔLVEDP more than doubling volume in the larger volume groups. No difference in ANP or NT-proBNP were seen in high transfusion volume or groups. Conclusion: Transfusion volume dose-dependently increased LVEDP, with speed of transfusion rapidly elevating LVEDP at higher transfusion volumes. ANP and NT-proBNP were not impacted by transfusion volume or speed in this model. TACO is seen as purely volume overload, however, this study emphasizes that limiting transfusion speed, as a modifiable risk factor, might aid in preventing TACO.

At present, the role of platelet-rich plasma (PRP) in burn and wound management is undefined. We present some of the evidence for PRP in wound healing and other medical fields. Currently, the high variation in product composition, mode and timing of application prevent a clear definition of the position of PRP in wound healing. Perspectives on solving these issues are described.

Background This multi-national study evaluated changes in platelet (PLT) unit distributions at 12 national or regional blood collectors over a 10-year period. Methods Data on the total number of PLT distributions, the collection method, that is apheresis vs whole blood-derived (WBD), the PLT unit characteristics and post-collection modifications were obtained from 12 national or regional blood collectors from 2008 through 2017. Individual WBD PLT units were converted to apheresis equivalent units (i.e. a dose of PLTs) by dividing by 4, the typical pool size; WBD units that were pooled before distribution were counted as a single dose. Results Overall at these 12 blood collectors, the total number of PLTs distributed in 2008 was 1 373 200, which rose by 10·2% to 1 513 803 in 2017. The Japanese Red Cross, which distributes only apheresis PLTs, had a 13·4% increase in the number of distributions between the years 2008 and 2017, while the other 11 blood collectors combined demonstrated a 6·8% increase in distributions between these two years. Between the years 2008 and 2017, the changes in the proportion of apheresis, platelet-rich plasma and buffy coat PLT distributions were -29·9%, -70·7% and 80·0%, respectively. Conclusion The number of PLT distributions increased during the 10-year study period despite prophylactic PLT transfusion thresholds having remained fairly consistent over the last decade. Perhaps this increase is in part driven by increased administration of platelets to patients with massive haemorrhage or an increase in stem cell transplantation. The use of buffy coat PLTs is increasing at these collectors.

The current reference method in the United States for measuring in vivo population red blood cell (RBC) kinetics utilizes chromium-51 (51 Cr) RBC labeling for determining RBC volume, 24-hour posttransfusion RBC recovery, and long-term RBC survival. Here we provide evidence supporting adoption of a method for kinetics that uses the biotin-labeled RBCs (BioRBCs) as a superior, versatile method for both regulatory and investigational purposes. RBC kinetic analysis using BioRBCs has important methodologic, analytical, and safety advantages over 51 Cr-labeled RBCs. We critically review recent advances in labeling human RBCs at multiple and progressively lower biotin label densities for concurrent, accurate, and sensitive determination of both autologous and allogeneic RBC population kinetics. BioRBC methods valid for RBC kinetic studies, including successful variations used by the authors, are presented along with pharmacokinetic modeling approaches for the accurate determination of RBC pharmacokinetic variables in health and disease. The advantages and limitations of the BioRBC method-including its capability of determining multiple BioRBC densities simultaneously in the same individual throughout the entire RBC life span-are presented and compared with the 51 Cr method. Finally, potential applications and limitations of kinetic BioRBC determinations are discussed.

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

BACKGROUND Several circumstances require the accurate measurement of red blood cell (RBC) survival and clearance, such as determination of posttransfusion recovery of stored RBCs to investigate the effect of new additive solutions. To this end, biotin as a marker of RBCs to track donor RBCs in the blood of the recipient has been used in many studies. However, so far only experimental, nonvalidated, biotin-labeled red cell concentrates (RCCs) are transfused. The goal of this study was to produce a standardized biotin-labeled RCC product in a fast, simple, and sterile manner that can be used for clinical research and for the evaluation of new blood products according to Good Practice Guidelines (GPG) for blood establishments. STUDY DESIGN AND METHODS RCC fractions were labeled with two different concentrations of biotinylation reagent in a closed system, to prevent bacterial contamination of the end product. Using flow cytometry, the reproducibility and robustness of the biotin labeling was assessed, as well as the stability of the biotin label on the (un-)irradiated RCC fraction. Additionally, parameters such as phosphatidylserine (PS) exposure, sodium (Na), potassium (K), free hemoglobin, adenosine triphosphate (ATP), pH, and morphology were determined prior to and after biotin labeling to rule out detrimental effects of the labeling procedure on the RCC. RESULTS Our data show that RCCs can be labeled under sterile conditions in a closed system with two different biotinylation reagent concentrations, without affecting the biological activity. CONCLUSION An easy, rapid (

Background 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. Study design and methods 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. Results The earlier during storage that units were irradiated, the higher the hemolysis and K+ at end of storage. Irrespective of the timing of irradiation, there was a rapid increase in extracellular K+ , followed by a more gradual increase in hemolysis. ATP levels decreased faster in irradiated units and were reduced below accepted values if irradiated early. Irradiated female RBCs had an absolute lower hemolysis and K+ level compared to male RBCs at all time points. Conclusions The 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 Hyperfibrinolysis has been observed in patients heavily transfused with solvent/detergent-treated pooled plasma (S/D plasma). We compared coagulation and fibrinolytic variables in blood containing S/D plasma with blood containing fresh-frozen plasma (FFP), with and without α2-antiplasmin or tranexamic acid (TXA) supplementation. STUDY DESIGN AND METHODS Whole blood samples were reconstituted from red blood cells, platelet (PLT) concentrates, and varying mixtures of FFP and S/D plasma. Hematocrit and PLT count of reconstituted whole blood samples were varied. For a subset of runs, α2-antiplasmin or TXA was added to S/D plasma whole blood samples. Thromboelastography (TEG) analysis was performed to assess 50% clot lysis time (CLT50%), maximum amplitude (MA), and initial clotting time (R-time). RESULTS The change in CLT50% of whole blood as the plasma compartment transitions from FFP to S/D plasma was −52% (95% confidence interval [CI], −60% to −45%; p

Background 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). Study design and methods 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. Results 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. Conclusion 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.

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 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 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.

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.

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.

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.

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

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.

BACKGROUND: The sensitivity of a real-time polymerase chain reaction (PCR) assay detecting bacteria in platelet concentrates (PCs) was improved by detection of ribosomal RNA (rRNA) in addition to DNA. The real-time reverse transcription–PCR (RT-PCR) assay was compared with the BacT/ALERT culturing system (bioMerieux) to determine its value for routine screening of PCs for bacterial contamination. STUDY DESIGN AND METHODS: The sensitivity of the assay was determined by spiking PCs with serial dilutions of bacteria. RNA amplification was performed with real-time RT-PCR, using a universal primer and probe set based on the conserved 16S rRNA gene of bacteria. Routinely prepared PCs in plasma were spiked with low bacterial titers of four different bacteria to compare the real-time RT-PCR with the BacT/ALERT. For the BacT/ALERT, samples were taken directly after spiking. For the real-time RT-PCR, samples were taken daily during 7 days of storage. RESULTS: RNA detection improved the sensitivity of the PCR assay at least 10-fold. When PCs were spiked with low bacterial titers, all positive samples were detected by the real-time RT-PCR after 48 hours. The BacT/ALERT became positive for almost all samples within 24 hours. However, some positive PC samples remained negative in the BacT/ALERT. CONCLUSION: The sensitivity of the PCR assay was improved by detection of rRNA. A spiking study demonstrated the advantage of late sampling for PCR testing compared to early sampling for culturing with the BacT/ALERT system. A real-time RT-PCR assay that is performed on PCs during storage or shortly before transfusion can be a good alternative to culturing methods.

Background The speed of blood donation is affected by the resistance induced by the needle and tubing during collection. To increase blood donation speed, the connection between the needle and the tubing was modified. The aim was to evaluate the effect of this modification on various donation parameters. Methods Apart from the modified needle inset, the study systems were identical to reference bag systems routinely used in our centre, and only identifiable by their unique lot number. Blood collections were done at fixed sites under similar conditions using automated mixers. In total 8541 evaluable donations were performed in the reference group and 7430 in the study group. Electronically stored donation data were obtained from the mixers and used for analysis. Results The average donation speed increased from 63 ± 12 in the reference group to 75 ± 15 ml/min in the study group (P

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

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.

BACKGROUND: The ability to deform is important for circulating RBCs in vivo, and earlier studies showed that this property can objectively be measured in vitro by the LORCA. In this study it was investigated whether photodynamic treatment of human RBCs (meant to inactivate contaminating pathogens) affects deformability. STUDY DESIGN AND METHODS: WBC-reduced RBC suspensions (30% Hct) were treated with 1,9dimethylmethylene blue (DMMB) and red light. Changes in deformability were analyzed by LORCA measurements, in which elongation of the cells is measured at increasing shear stress. The effect of DMMB concentration and light dose was determined as well as the interfering effect of two scavengers of reactive oxygen species, that is, dipyridamole and Trolox. RESULTS: Photodynamic treatment with DMMB resulted in clear changes in RBC deformability. Deformability changes occurred before onset of hemolysis. Under relatively mild treatment conditions, especially deformability at low shear stress was decreased, whereas deformability changes at high shear stress only occurred under harsher treatment conditions. Inclusion of dipyridamole and/or Trolox primarily prevented deformability changes at high shear stress. CONCLUSION: LORCA measurements can effectively be used to detect changes in deformability that are induced by photodynamic treatment of human RBCs. A change in deformability represents an early marker of RBC damage under these conditions. ecently, the interest to develop appropriate sterilization methods for RBC concentrates has increased. Photodynamic treatment is one of the very few methods that may be applicable on RBCs. 1 When photosensitizer solutes are illuminated with light of the appropriate wavelength, reactive oxygen species (ROS) are formed that can inactivate nucleic acids, but which may have also a damaging effect on proteins or lipids. Because RBCs, in contrast to most pathogens, possess defense mechanisms against attack by ROS, they may be protected to some extent against this damaging action. For RBCs, it is desirable to use a sensitizer that can be

Background and Objectives In a previous study we established a reliable setpoint for the prevalence of bacteria in whole blood. In the present study we investigated the possible preventive effect, of diversion of the first 10 ml of a blood donation, on the bacterial contamination rate. Materials and Methods To divert the first 10 ml of a whole-blood donation, we used a special five-bag system equipped with a Composampler® device. After venepuncture, the first 10 ml of a donation was sampled into a vacutainer tube. This was followed by the collection of the whole-blood unit. The extra bag allowed direct sampling of the final donation in a closed system for BacT/Alert. Whole-blood samples were taken after storage (2–14 h at 20 °C) and subsequent mixing. BacT/Alert culture bottles were incubated until positive, or for 7 days if negative. Confirmation and identification of positive cultures was performed according to internationally recognized standard reference methods. Results The prevalence of bacteria in whole blood, as determined by using standard collection techniques, was 0·35% (95% confidence interval 0·27–0·44%, n = 18 257). After diversion of the first 10 ml this value was significantly lower: 0·21% (P