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

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

Bacterial contamination of blood products is currently one of the greatest microbiological risks of transfusion. Of all blood products, platelet concentrates (PCs) are most frequently implicated in transfusiontransmitted bacterial infections. Several methods have been developed to reduce this risk. The most frequently used method at the moment is a culturing system, the BacT/ Alert (bioMerieux, Boxtel, The Netherlands). Although the method is sensitive, it also has several drawbacks. Therefore, new methods are being developed to increase the safety of transfusion of platelet concentrates in the future.

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

BACKGROUND: 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 Pathogen inactivation technologies require continuous development for adjustment to different blood components and products. With Theraflex UV-Platelets, a system using shortwave ultraviolet C (UVC) light (254 nm), efficient mixing of platelet concentrates (PCs) during UVC treatment is essential to ensure homogeneous illumination of the blood components. In this study, we investigated the impact of increasing the agitation speed during UVC treatment on pathogen inactivation capacity and platelet quality. Material and Methods The pathogen inactivation efficacy of UVC treatment was evaluated at two agitation speeds (110 vs. 180 rpm) using four different transfusion-relevant bacteria strains and three model viruses. Using a pool-and-split design, the in vitro quality of buffy coat-derived PCs stored in SSP+ additive solution for up to 7 days was assessed in UVC-treated PCs agitated at either 110 rpm (standard speed) or 180 rpm (increased speed) and in untreated controls. Results The higher agitation speed improved bacterial inactivation but did not influence viral inactivation. Metabolic activity (glucose consumption and lactate accumulation) in UVC-treated platelets was slightly higher than in untreated controls. Increases in parameters such as CD62P expression and annexin A5 binding indicated moderate activation of UVC-treated platelets. Quality variables for UVC-treated platelets agitated at standard vs. increased agitation speed were comparable. Conclusion The mixing rate during illumination may be a process parameter for further development of UVC-based pathogen inactivation procedures for PLT concentrates.

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

If regular artificial tears are ineffective for treatment of ocular surface disorders (including extreme dry eye syndrome), serum eye drops (SEDs) may provide a way to relieve the symptoms. However, not all patients are eligible to donate blood to produce autologous SEDs. Therefore, the use of allogeneic SEDs (obtained from voluntary blood donors) should be explored as an alternative for autologous SEDs. The Dutch blood bank organization is currently looking into the possibilities to provide allogeneic SEDs, as (GMP) regulations become stricter, making it for hospitals more difficult to provide autologous SEDs. To demonstrate effectiveness of both autologous and allogeneic SEDs, a clinical trial is planned. The current status of SEDs in The Netherlands is described. This paper is based on summary of the presentation given at the DGTI meeting in Dresden.

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

Background 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

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

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

Bacterial contamination of platelet concentrates (PCs) poses the highest transfusion-associated infectious risk, with Staphylococcus epidermidis being a predominant contaminant. Herein, the growth dynamics of 20 S. epidermidis strains in PCs and regular media were characterized. Strains were categorized as fast (short lag phase) or slow (long lag phase) growers in PCs. All strains were evaluated for the presence of the biofilm-associated icaAD genes by PCR, their capability to produce extracellular polysaccharide (slime) on Congo red agar plates and their ability to form surface-attached aggregates (biofilms) in glucose-supplemented trypticase soy broth (TSBg) using a crystal violet staining assay. A subset of four strains (two slow growers and two fast growers) was further examined for the ability for biofilm formation in PCs. Two of these strains carried the icAD genes, formed slime and produced biofilms in TSBg and PCs, while the other two strains, which did not carry icaAD, did not produce slime or form biofilms in TSBg. Although the two ica-negative slime-negative strains did not form biofilms in media, they displayed a biofilm-positive phenotype in PCs. Although all four strains formed biofilms in PCs, the two slow growers formed significantly more biofilms than the fast growers. Furthermore, growth experiments of the two ica-positive strains in plasma-conditioned platelet bags containing TSBg revealed that a slow grower isolate was more likely to escape culture-based screening than a fast grower strain. Therefore, this study provides novel evidence that links S. epidermidis biofilm formation with slow growth in PCs and suggests that slow-growing biofilm-positive S. epidermidis would be more likely to be missed with automate culture.

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

Background The prevalence of bacterial contamination in whole-blood collections, either with immediate sampling or sampling after overnight storage as whole blood at 20 degrees C, is determined. Study design and methods Whole blood was collected under blood bank conditions in special five-bag systems, allowing sampling in a closed system for culture bottles. Samples were taken within 2 hours after collection (Group 1) or after overnight storage of the whole blood at 20 degrees C (Group 2). Culture bottles were incubated for 7 days, and positive samples were entered on agar plates for confirmation and determination. Results In Group 1, 9219 units were tested; 27 units were positive with positive subculture, that is, 0.29 percent with a 95% CI of 0.19 to 0.42 percent. In Group 2, 9038 units were tested; 36 units were positive with positive subculture, that is, 0.39 percent with a 95% CI of 0.28 to 0.55 percent. No significant difference could be found between the two test groups. The majority of bacteria were either Staphylococcus (all coagulase-negative) or Propionibacterium species. Conclusion For a total of 18,257 units, 0.34 percent (CI, 0.25-0.44) of whole-blood collections appeared to have bacterial contamination (mainly skin-derived). Overnight storage of whole blood at 20 degrees C did not have a significant effect on the prevalence of bacterial contamination.

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

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

Background and Objectives The study evaluated mixing performance for various commercial blood-mixing devices at different flow rates. Materials and Methods A glycerol solution with a density equal to blood was pumped into a blood bag (mounted on a mixer) that contained 70 ml of Toluidin Blue in citrate–phosphate–dextrose (CPD). After 500 ml of glycerol solution had been pumped into the blood bag, the bag was emptied in fractions and the absorbance at 640 nm of each fraction (expressed as percentage of mixture absorbance) was plotted against the fraction number. Results At a flow rate of 30 ml/min, the curves were very sigmoid, with high initial values that decreased during collection of the fractions. The absorbance values of the fractions collected later (after ≈ 250–300 ml) were 10–50% (i.e. percentage of absorbance for complete mixing) for the different mixers, indicating that the CPD solution was incompletely mixed with the incoming solution, resulting in a lower-than-expected ‘anticoagulant’ content for the fractions collected later. At a flow rate of 50 ml/min the mixing was improved, but only at 75 ml/min did all mixers show relatively good mixing. Manual mixing, by kneading the bag three times/min, gave at all rates an almost ideal mixing curve, 105% initially to 90% in the final fractions. Conclusion At relatively high bleeding rates, all mixers performed well, although complete mixing was only obtained with manual kneading. However, most blood-mixing devices still fail to mix efficiently at normal and low bleeding rates. Although the minimal degree of necessary mixing is unknown, further optimization of mixing devices seems warranted.

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

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

Background and Objectives Semi-automatic separation devices can be used for the separation of centrifuged whole blood into leucoreduced red cell concentrate (LR-RCC), plasma and buffy coat (BC) and to make platelet concentrates (PC) from pooled BCs. To improve and to obtain a more uniform and standardized process, the CompoMat G5 (Fresenius) was implemented, a new-generation semi-automated device. Materials and Methods Uniform programs for WB separation and preparation of PCs were validated, using collection and pooling systems with CompoFlow (CF) closures, which can be automatically opened by the G5. Cell counts were performed and compared with historic data of blood components obtained with the formerly used Compomat G4 and Optipress II. After implementation, different adjustments were made to improve product quality. Results Leucoreduced red cell concentrates (280 ± 15 ml, 53 ± 5 g haemoglobin) and plasma (317 ± 16 ml) met European guidelines. BCs (48 ± 2 ml, 0·42 ± 0·05 l/l, 93 ± 25 × 109 platelets) contained a similar platelet (PLT) content as BC prepared before with the Compomat G4. A relatively high percentage (4–6%) of PCs (330 ± 17 ml, 330 ± 50 × 109 PLT, 0·12 ± 0·21 × 106 leucocytes) contained

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

Bacterial contamination of platelet concentrates (PCs) poses the greatest infectious risk in modern transfusion medicine despite the implementation of measures such as improved skin disinfection and first aliquot diversion. The majority of PC contaminants are commensal skin flora introduced by venipuncture at the time of blood collection. The predominant organisms are Gram-positive coagulase-negative staphylococci such as Staphylococcus capitis. This bacterium has been implicated in numerous instances of infection and sepsis, likely for its ability to form surface-associated communities of micro-organisms encased in extracellular materials, known as biofilms. In the present study, five strains of S. capitis isolated from contaminated PCs were assessed for their ability to produce extracellular polysaccharide (slime), a canonical indicator of biofilm-formation ability, on Congo red agar plates. Biofilm formation was evaluated in both glucose-enriched trypticase soy broth (TSBg) and in PCs by using a crystal violet staining assay. The chemical nature of the biofilms was evaluated by disruption assays using sodium metaperiodate and proteinase K. In addition, biofilm architecture was observed by scanning electron microscopy. The presence of the biofilm-associated icaR and icaADBC genes was also examined by PCR. While only two out of the five S. capitis strains formed biofilms in TSBg, all strains formed biofilms in PCs. The ability of strains to produce extracellular polysaccharide and their possession of wild-type ica genes were not exclusive predictors of biofilm formation in TSBg or PCs; different profiles of biofilm markers were observed among isolates. This is likely due to the proteinaceous composition of the S. capitis biofilm matrix. Interestingly, an ica-negative, non-slime-producing isolate was capable of biofilm formation in PCs. Together, these data indicate that the platelet storage environment stimulates biofilm formation in S. capitis in the absence of extracellular polysaccharide production and that multiple bacterial factors and regulatory elements are likely involved in biofilm formation in this milieu.

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

SummaryPlatelet concentrates stored at room temperature deteriorate. The so-called storage lesion is characterised by morphological changes and a loss of functionality. To find an assay for early platelet activation in platelet concentrates the morphological score, β-TG release and P-selectin expression were determined, and compared with the amount of soluble P-selectin. An ELISA was used to quantify soluble P-selectin in the storage medium. We found a significant correlation between the amount of soluble P-selectin and the percentage of P-selectin positive platelets (flow-cytometric analysis) (r = 0.7449; p

Background Platelet concentrates (PCs) are stored at room temperature to preserve their biological activity. To minimize the risk of bacterial outgrowth, storage time is limited to 7 days. It is well-established that prolonged storage of PCs results in modifications that result in a decreased hemostatic efficacy. This loss of platelet functionality during storage is commonly referred to as the platelet storage lesion (PSL). Typical events linked to development of the PSL are shape changes, platelet activation and loss of receptors crucial for platelet functionality. Two-dimensional (2D) differential gel electrophoresis (DIGE), isotope tagging and isotope-coded affinity tagging (ICAT) have been previously used to monitor changes in protein composition during storage. These studies have provided valuable insights into the changes associated with the PSL, however, these studies generally focused on a limited set of proteins. Aim We aimed to generate an overview of changes in the platelet proteome during storage using label free quantitative mass spectrometry. Furthermore, we employed Gene ontology (GO) enrichment analysis to identify pathways and biological processes that were linked to development of the PSL. Methods Three independently pooled PCs were stored in plasma under standard blood bank conditions for 16 days. Tryptic peptides were separated by nanoscale C18 reverse phase chromatography coupled on line to an Orbitrap Fusion Tribrid mass spectrometer. The RAW mass spectrometry files were processed with the MaxQuant computational platform. The global changes in protein level during platelet storage were assessed employing the analysis-of-variance functions of PERSEUS. Gene ontology enrichment analysis of biological processes, molecular functions and cellular compartments of the significantly different proteins was performed using the Cytoscape plug-in BiNGO. Results A total number of 2501 proteins was detected in all 3 biological replicates in at least one of the time points analyzed. The analysis showed that 18 proteins were down-regulated over time, whereas the level of 3 proteins was found to increase. CytoScape BinGo analysis of these significantly downregulated proteins revealed that the majority of this set was linked to GO-terms platelet degranulation, secretion and regulated exocytosis. This set of proteins included von Willebrand factor (VWF), serglycin (SRGN), SPARC, amyloid beta A4 protein (APP), multimerin-1 (MMRN1) and platelet factor 4 (PF4). A significant decline in these protein levels was observed at day 5 of storage, suggesting that release of α-granules is a relatively early event during platelet storage. At day 5 also a marked decline in S100A9 was observed. S100A9 has been implicated in degranulation in neutrophils, and may therefore also be linked to platelet granule release. Levels of membrane surface platelet glycoproteins such as glycoprotein Ibα did not significantly change at day 5. Only one single protein, histone H2A, was found to be consistently decreased already at two days of storage, but the significance of this finding is not clear. Upon prolonged storage (13 and 16 days) an increase in the level of α-2-macroglobulin (A2M), immunoglobulin M (IGM) and glycogenin-1 (GYG1) was observed suggesting that platelets acquire an (increased) potential to bind and/or internalize proteins from their environment. Consistent with this notion we also detected significant levels of several serine protease inhibitors, although levels of these proteins did not change upon storage. Conclusions Overall, our findings highlight dynamic changes in protein composition of platelets during storage. Our data provide evidence for sustained release of α-granules over time which becomes significant at day 5. Our data also suggest that during storage, platelets can bind or ingest proteins from their environment which may have impact on the hemostatic properties of stored platelets. Disclosures Leebeek: CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxalta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Dutch Hemphilia Foundation: Research Funding.

Sepsis is a life threatening organ dysfunction caused by a dysregulated host response to infection. It is a global health care problem and the leading cause of death in ICU patients. A rather unknown but effective mechanism to protect ourselves from such pathogen invasion is immune-adherence clearance (IAC). During IAC pathogens (including bacteria, fungi and viruses) can bind to red blood cells (RBCs) through complement opsonization and subsequent binding to complement receptor 1 (CR1, CD35) expressed on their cell surface. After binding of the pathogen to CR1, the RBCs deliver the pathogens to macrophages of the spleen and liver, where they are phagocytosed and degraded. To study the underlying mechanisms of IAC in more detail, we have developed an assay to monitor the transfer of opsonized pathogens bound to RBCs to human monocytes and/or macrophages under flow conditions, using confocal microscopy. The transfer of a variety of different pathogens, including S. aureus, E. coli and C. albicans, was studied in this assay. When looking at the transfer process in detail, it was noticed that RBC attach to the phagocyte prior to the transfer and remain attached shortly after the transfer, suggesting that RBC might be interacting with phagocytes through adhesion molecules such as integrins to establish firm binding between these two cell types. To test this hypothesis several adhesion molecules, on the phagocytes as well as on the RBC, were blocked by monoclonal antibodies and IAC was quantified. First, complement receptor 3 (CR3, amb2 CD11b/CD18 integrin) an important receptor for adhesion, migration and phagocytosis was blocked by monoclonal antibodies on the phagocytes, after which bacterial transfer was impaired. This indicated that CR3 is crucial for efficient IAC. To confirm the involvement of CR3 in IAC, we analyzed the monocytes of a known LAD-1 patient. This rare immunodeficiency is characterized by an inherited molecular defect of the β2integrin subunit (CD18) which results in impaired adhesion and migration of the patient's leukocytes and clinically manifests in recurrent infections. Our results showed a highly reduced interaction between the phagocytes and the RBCs, resulting in a decrease in bacterial transfer of >70%. Next, we tested the effects of blocking antibodies directed against several RBC adhesion molecules on the transfer process. When blocking CD147 (Basigin, Ok blood group) and ICAM-4 (CD242, Landsteiner-Wiener), but not Glycophorin A (GPA, CD235a) on the RBC the transfer process was largely inhibited. These findings demonstrate the involvement of direct cell-cell interactions between RBC and macrophages in IAC and provide evidence that RBC adopt a "sticky" phenotype after binding a pathogen through CR1, which enables phagocytes to bind them under flow. We anticipated that blood bank filters (currently used for leukocyte depletion) might be used to bind and filter RBC-pathogen complexes from blood due to their "sticky" phenotype. This was found to be possible in a series of experiments, not only using in vitrogenerated RBC-pathogen complexes, but also using blood from septic patients. Filtration using a standard leukocyte reduction filter showed reduction of RBC-pathogen complexes close to 100%, independent of the type of pathogen. We foresee that this knowledge can be used to develop a generic approach to deplete RBCs carrying pathogens specifically from the blood stream, and thereby filter RBC-pathogen complexes from the blood of patients suffering from sepsis. Disclosures No relevant conflicts of interest to declare.

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

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.

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

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

Recently, an important role for CD47, a well-known 'don't eat me' signal, in the clearance of aged erythrocytes was revealed. Experimental data support the conversion of CD47 from a 'don't eat me' to an 'eat me' signal through a conformational change in CD47. Intriguingly, erythrocyte phagocytosis after this switch seems to be mediated by the same receptor that normally signals inhibition of phagocytosis, SIRPα. In this review, the possible molecular mechanisms leading to this conformational change in CD47 as well as the possible signal transduction events leading to phagocytosis after this switch are discussed. Lastly, the consequences of this newly identified mode of erythrocyte phagocytosis for the clearance of aged erythrocytes during normal turnover and after erythrocyte transfusion are addressed.Vor kurzem ist eine wichtige Rolle für CD47, einem bekannten ‘Don't-Eat-Me’-Signal, bei der Beseitigung gealterter Erythrozyten aufgedeckt worden. Experimentelle Daten belegen die Umwandlung von CD47 von einem ‘Don't-Eat-Me’- zu einem ‘Eat-Me’-Signal durch konformative Veränderungen. Interessanterweise scheint die Erythrozytenphagozytose nach der Umwandlung von dem gleichen Rezeptor (SIRPα) vermittelt zu werden, der normalerweise die Hemmung der Phagozytose signalisiert. In dieser Übersichtsarbeit werden sowohl die potentiellen molekularen Mechanismen, die zu dieser Umwandlung von CD47 führen, als auch die möglichen Signaltransduktionsvorgänge, die nach der Umwandlung die Phagozytose einleiten, diskutiert. Des Weiteren werden die Konsequenzen dieses neu identifizierten Modus der Erythrozytenphagozytose für die Beseitigung gealterter Erythrozyten während des normalen Turnovers sowie nach Erythrozytentransfusion beleuchtet.

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

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

SummaryThe influence of storage of platelet concentrates (PC) on the adhesion capacity of platelets was studied. Twenty-four PC, 12 prepared by the buffy coat (BC) method and 12 by the platelet-rich plasma (PRP) method, were stored for 7 days at room temperature. On days 1,3 and 7 of storage, the platelet adhesion capacity to subendothelial matrix (SEM) and collagen was studied in a rectangular perfusion system under flow conditions in conjunction with the platelet aggregation capacity after stimulation and the adenine nucleotide content. The platelet adhesion capacity to collagen was constant until day 3 of storage and decreased to about 80% of the starting value on day 7 of storage. The adhesion capacity to SEM, however, had already decreased on day 3 to about 75% of the value of day 1 and was even more decreased on day 7 to about 45% of the starting value. On day 1, platelets prepared by the BC method displayed a higher adhesion capacity to collagen and a higher aggregation capacity after stimulation by collagen alone or in combination with ADP, compared to platelets prepared by the PRP method. No other significant differences in adhesion or aggregation capacity were observed between the PC prepared by the two different methods. Both platelet adhesion and aggregation response decreased during storage, as did the total adenine nucleotide content. This study shows that platelet function, as measured by the aggregation and adhesion capacity, of platelets prepared by the PRP method is more severely impaired during the first 3 days of storage as compared to the function of platelets prepared by the BC method.

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

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

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.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.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.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.

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.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 × 10(9) to 13.8 × 10(9) 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.When prepared from 2-day-old PCs (n = 4), volume-reduced PCs from BCs in a syringe had a pH(37°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 (p0.001), at t = 6 hours. When made from 7-day-old PCs, pH was lower for all storage conditions: 5.68 ± 0.06 in the syringe, 5.70 ± 0.09 in the DEHP-PVC container (not significant), and 6.07 ± 0.24 in the BTHC-PVC container (p0.01) at t = 6 hours. Volume-reduced 2-day-old apheresis PCs had a pH of 6.47 ± 0.20 at t = 6 hours.Adult-dose PCs derived from BC or apheresis can be volume-reduced to approximately 20 mL in a closed gas-permeable system. Volume-reduced PCs in BTHC-PVC containers retain a mean pH of more than 6.0 up to 6 hours after production. Syringes allow only 3 hours of storage.

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.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.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.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.

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.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.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%.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.

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 A(2) (PLA(2) ).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.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 PLA(2) or cytosolic PLA(2) 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.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.

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.A total of 336 sample bags, from PCs that were routinely tested in the BacT/ALERT (bioMérieux), 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.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%.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.

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.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 300mL/hr was used, and for PCs, 600mL/hr. Transfusions without an infusion pump served as a reference.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% (p0.01).There was no adverse effect on the in vitro quality of RBCs or PCs after simulated transfusion using volumetric infusion pumps. A decrease in PLT activation was observed, which can probably be explained by capturing of activated or damaged PLTs in the 200-µm filter present in the infusion system.