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1.  Serial assessment of biochemical parameters of red cell preparations to evaluate safety for neonatal transfusions 
Background & Objectives:
Neonatologists often prefer fresh blood (<7 days) for neonatal transfusions. The main concerns for stored RBCs are ex vivo storage lesions that undermine red cell functions and may affect metabolic status of neonatal recipients. This study was designed to evaluate serial in vitro changes of biochemical parameters in different RBC preparations during storage to consider for neonatal transfusions even after storage beyond one week.
Twenty five units each of whole blood (CPDA-1 RBC, SAGM RBC) were selected for serial biochemical parameter assessment after each fulfilled the quality criteria (volume and haematocrit). These units were tested serially for supernatant potassium, pH, lactate, haemoglobin, glucose and red cell 2,3 diphosphoglycerate (2,3 DPG) up to 21 days of storage.
Within each group of RBC, rise in mean concentration of potassium, lactate and plasma haemoglobin from day 1 to 21 of storage was significant in CPDA-1 RBC having the highest levels at day 21. From day 3 to 21, SAGM RBC had higher mean pH value than CPDA-1 RBC though this difference was not statistically significant. SAGM RBC had highest mean glucose concentration during storage than other two types of red cell preparations (P<0.005). Within each group, fall in mean 2,3 DPG concentration from day 1 to 7 was significant (P<0.05). A positive correlation existed between mean plasma potassium and haemoglobin in all three types of red cells (r=0.726, 0.419, 0.605 for CPDA-1 RBC, SAGM RBC and whole blood respectively, P<0.005).
Interpretation & Conclusions:
All the three red cell preparations tested revealed biochemical changes within acceptable limits of safety till 21 days of storage. CPDA-1 RBCs had the highest degree of these changes.
PMCID: PMC3102460  PMID: 21245620
Biochemical changes; packed red cells; whole blood
2.  Quantification of surgical trauma: comparison of conventional and minimally invasive surgical techniques for pertrochanteric fracture surgery based on markers of inflammation (interleukins) 
Fixation of pertrochanteric fracture is undoubtedly an additional trauma after the fracture itself. In elderly patients, it might have an important impact on the whole organism. In the literature we find various techniques to perform this type of surgery. Up to now, there are no parameters validated for quantification of the invasiveness of a surgical procedure; it is therefore still not demonstrated that any method is less invasive than any other. In an effort to find a way to quantify the invasiveness of a surgical procedure, inflammatory markers were collected in patients undergoing fixation of trochanteric fracture with gliding hip screw [dynamic hip screw (DHS)] using either a conventional (DHS conv) or minimally invasive (DHS MIO) technique.
Two groups of patients were investigated prospectively; 36 of them were treated with conventional DHS technique and 32 with minimally invasive technique. Mean age was 84.7 ± 7.20 and 82.78 ± 7.71 years, respectively. Fracture type was classified according to the AO classification. Interleukin (IL)-6, IL-10, IL-8, and tumor necrosis factor (TNF)-α were measured 1 h before and 1 h after surgery. Student’s t test, chi-square test, and multivariate logistic regression were used for statistical analysis.
Preoperative levels of interleukins showed no significant differences between the two groups. In contrast, the postoperative blood level of IL-6 in patients operated with DHS conv technique (78.41 ± 67.04 pg/ml) was on average higher than in patients operated by DHS MIO technique (39.02 ± 37.36 pg/ml), the mean difference being 39.39 pg/ml [95 % confidence interval (CI) 12.65–66.13 pg/ml; p = 0.0045]. Multivariate logistic regression (backward method with limit of significance 0.05) confirmed that patients operated by conventional technique were significantly more likely to have increased IL-6 after surgery than those operated by MIO technique. IL-8 was measured in only 36 patients (20 for DHS conv, 16 for DHS MIO). No significant differences were found between the two groups; however, there was a drastic decrease postoperatively (p < 0.0001) regardless of the type of surgery performed. IL-10 and TNF-α were tested in all subjects, but did not show significant differences between the two groups. Average length of incision was significantly different (4.61 cm, 95 % CI 3.50–5.71 cm; p < 0.001) between the two groups, being 11.65 ± 2.64 cm for DHS conv and 7.05 ± 1.77 cm for DHS MIO. Similarly, average units of red blood cells (RBCs) transfused [performed for hemoglobin (Hb) <9 g/dl and/or hematocrit (HCT) <27 %] was higher (2.22 ± 0.99) in the DHS conv group compared with the DHS MIO group (1.09 ± 1.20), with average difference of 1.13 (95 % CI 0.59–1.66; p < 0.001).
This attempt to quantify the invasiveness of internal fixation for trochanteric fracture comparing two techniques (DHS conv versus DHS MIO) based on inflammatory markers (IL-6) has given encouraging results. Measurement of systemic inflammatory response to local tissue damage caused by osteosynthesis using IL-6 as marker seems to confirm the lower invasiveness of MIO techniques. These results for trauma cases seem in line with those published for hip prosthesis. Ongoing further studies analyzing the effect of nailing will confirm or invalidate these preliminary results.
PMCID: PMC3427700  PMID: 22688402
Quantification of surgical trauma; Measuring surgical trauma; Gliding hip screw; Minimally invasive technique; Interleukins
3.  Impaired ATP release from red blood cells promotes their adhesion to endothelial cells: A mechanism of hypoxemia after transfusion 
Critical care medicine  2011;39(11):2478-2486.
Transfusion of red blood cells (RBCs) has been linked to disappointing clinical outcomes in the critically ill, but specific mechanisms of organ dysfunction after transfusion remain poorly understood. We tested the hypothesis that RBC storage impairs the ability of RBCs to release ATP and that impaired ATP-release was injurious in vivo, in part through increased RBC adhesion.
Prospective, controlled, mechanistic study.
University research laboratory.
Human and mouse blood donors; nude mouse transfusion recipients.
Manipulation of ATP release, supplemental ATP, and antibodies to RBC and endothelial adhesion receptors were used in vitro and in vivo to probe the roles of released ATP and adhesion in responses to (transfused) RBCs.
Measurements and main results
The ability of stored RBCs to release ATP declined markedly within 14 days after collection, despite relatively stable levels of ATP within the RBCs. Inhibiting ATP release promoted the adhesion of stored RBCs to endothelial cells in vitro and RBC sequestration in the lungs of transfused mice in vivo. Unlike transfusion of fresh human RBCs, stored-RBC transfusion in mice decreased blood oxygenation and increased extravasation of RBCs into the lung’s alveolar airspaces. Similar findings were seen with transfusion of fresh RBCs treated with the ATP-release inhibitors glibenclamide and carbenoxolone. These findings were prevented by either co-infusion of an ATP analog or pre-transfusion incubation of the RBCs with an antibody against the erythrocyte adhesion receptor LW (Landsteiner-Wiener; ICAM-4).
The normal flow of RBCs in pulmonary microvessels depends in part on the release of anti-adhesive ATP from RBCs, and storage-induced deficiency in ATP release from transfused RBCs may promote or exacerbate microvascular pathophysiology in the lung, in part through increased RBC adhesion.
PMCID: PMC3196852  PMID: 21765360
erythrocyte; oxygenation; acute lung injury; intercellular adhesion molecule-4 (ICAM-4); Landsteiner-Weiner (LW); endothelium; blood storage
4.  Red blood cell transfusion and increased length of storage are not associated with deep vein thrombosis in medical and surgical critically ill patients: a prospective observational cohort study 
Critical Care  2011;15(6):R263.
With prolonged storage times, cell membranes of red blood cells (RBCs) undergo morphologic and biochemical changes, termed 'RBC storage lesions'. Storage lesions may promote inflammation and thrombophilia when transfused. In trauma patients, RBC transfusion was an independent risk factor for deep vein thrombosis (DVT), specifically when RBC units were stored > 21 days or when 5 or more units were transfused. The objective of this study was to determine if RBC transfusions or RBC storage age predicts incident DVT in medical or surgical intensive care unit (ICU) patients.
Using a database which prospectively enrolled 261 patients over the course of 1 year with an ICU stay of at least 3 days, we analyzed DVT and RBC transfusions using Cox proportional hazards regression. Transfusions were analyzed with 4 thresholds, and storage age using 3 thresholds. DVTs were identified by twice-weekly proximal leg ultrasounds. Multivariable analyses were adjusted for 4 significant DVT predictors in this population (venous thrombosis history, chronic dialysis, platelet transfusion and inotropes).
Of 261 patients, 126 (48.3%) had at least 1 RBC transfusion; 46.8% of those transfused had ≥ 5 units in ICU. Patients receiving RBCs were older (68.8 vs 64.1 years), more likely to be female (47.0 vs 30.7), sicker (APACHEII 26.8 vs 24.4), and more likely to be surgical (21.4 vs 8.9) (P < 0.05). The total number of RBCs per patient was 1-64, mean was 6.3 (SD 7.5), median was 4 (IQR 2,8). In univariate analyses, there was no association between DVT and RBC exposure (1 day earlier, 3 days earlier, 7 days earlier, or ever) or RBC storage (≤ 7 or > 7 days, ≤ 14 or > 14 days, ≤ 21 or > 21 days). Among patients transfused, no multivariable analyses showed that RBC transfusion or storage age predicted DVT. Trends were counter to the hypothesis (e.g., RBC storage for ≤ 7 days suggested a higher DVT risk compared to > 7 days (HR 5.3; 95% CI 1.3-22.1).
We were unable to detect any association between RBC transfusions or prolonged red cell storage and increased risk of DVT in medical or surgical ICU patients. Alternate explanations include a lack of sufficient events or patients' interaction, between patient groups, a mixing of red cell storage times creating differential effects on DVT risk, and unmeasured confounders.
PMCID: PMC3388665  PMID: 22044745
5.  Red Cell Apheresis with Automated In-Line Filtration 
The aim of this study was to provide data on concurrent red blood cell (RBC) and platelet (PLT) apheresis with RBC in-line leukoreduction and automated addition of saline-adenine-glucose-mannitol (SAGM) using the new version (V6.0) of Trima Accel®.
In this two-center paired study, each subject completed a test and a control procedure with an interval of 9 weeks between procedures. In the test arm, single RBC and PLT units were collected on the Trima Accel V6.0 (in-line leukofiltration and automated addition of SAGM). In the control arm, they were collected on Trima Accel V5.1/V5.2 (post-collection leukoreduction, manual SAGM addition). RBC percent hemolysis, potassium concentration and adenosine triphosphate over storage, hemoglobin (Hb) yield, and residual white blood cells (WBC) were determined.
34 subjects successfully completed both test and control procedures. Post-storage hemolysis was similar in both groups, and all values were less than 0.8% for both arms. Residual WBC counts in all RBC units were less than 1 × 106/unit. In-line processed RBC units (V6.0) have a significantly higher volume and more Hb/unit due to filtration recovery improvements. All procedures were well tolerated by the subjects.
In-line filtration and automated addition of storage solution on Trima Accel V6.0 allows collection of ready-to-use RBC units that meet EU requirements.
PMCID: PMC4025052  PMID: 24847185
Red blood cell; Apheresis; In-line filtration; Leukoreduction; Multicomponent collection; Blood storage
6.  Storage of murine red blood cells enhances alloantibody responses to an erythroid-specific model antigen 
Transfusion  2009;50(3):642-648.
Red blood cell (RBC) alloimmunization can be a serious complication of blood transfusion, but factors influencing the development of alloantibodies are only partially understood. Within FDA-approved time limits, RBCs are generally transfused without regard to length of storage. However, recent studies have raised concerns that RBCs stored for more than 14 days have altered biologic properties that may affect medical outcomes. To test the hypothesis that storage time alters RBC immunogenicity, we utilized a murine model of RBC storage and alloimmunization.
Blood from transgenic HOD donor mice, which express a model antigen (hen egg lysozyme [HEL]) specifically on RBCs, was filter leukoreduced and stored for 14 days under conditions similar to those used for human RBCs. Fresh or 14-day-stored RBCs were transfused into wild-type recipients. The stability of the HOD antigen and post-transfusion RBC survival were analyzed by flow cytometry. RBC alloimmunization was monitored by measuring circulating anti-HEL immunoglobulin levels.
Transfusion of 14-day-stored, leukoreduced HOD RBCs resulted in 10- to 100-fold higher levels of anti-HEL alloantibodies as detected by enzyme-linked immunosorbent assay than transfusion of freshly collected, leukoreduced RBCs. RBC expression of the HOD antigen was stable during storage.
These findings demonstrate that HOD murine RBCs become more immunogenic with storage and generate the rationale for clinical trials to test if the same phenomenon is observed in humans. Length of storage of RBCs may represent a previously unappreciated variable in whether or not a transfusion recipient becomes alloimmunized.
PMCID: PMC3568949  PMID: 19906034
7.  The quality of stored umbilical cord and adult-donated whole blood in Mombasa, Kenya 
Transfusion  2009;50(3):611-616.
In sub-Saharan Africa umbilical cord blood may be a useful source of blood for transfusion. Before clinical trials, evidence is needed that cord blood donations, which vary greatly in volume, can be collected and stored into a fixed volume of anticoagulant-preservative solution obviating the need for prestorage processing.
Twenty-four umbilical cord whole blood (UC-WB) donations were collected into 21 mL of CPDA-1 and refrigerated for 35 days. The Kenya Blood Transfusion Service provided 12 adult-donated whole blood (AD-WB) controls. Supernatant hemoglobin (Hb) and potassium were assayed at 7-day intervals.
UC-WB red blood cell hemolysis and potassium loss increased throughout storage but did not differ significantly with cord blood volume. Hemolysis rates did not differ significantly between UC-WB and AD-WB but UC-WB potassium loss was slightly but significantly greater than AD-WB on Days 2, 7, and 14 (p < 0.05). In the AD-WB controls, eight were low volume (<405 mL), two had total Hb of less than 45 g, and two showed hemolysis greater than 0.8% by Day 28.
Variable volumes of UC-WB can be stored for 35 days without prestorage processing and further work into its suitability for transfusion to children is justified. The quality of conventional AD-WB is a concern and needs further evaluation.
PMCID: PMC2948540  PMID: 19912583
8.  The quality of stored umbilical cord and adult-donated whole blood in Mombasa, Kenya 
Transfusion  2010;50(3):611-616.
In sub-Saharan Africa umbilical cord blood may be a useful source of blood for transfusion. Before clinical trials, evidence is needed that cord blood donations, which vary greatly in volume, can be collected and stored into a fixed volume of anticoagulant-preservative solution obviating the need for prestorage processing.
Twenty-four umbilical cord whole blood (UC-WB) donations were collected into 21 mL of CPDA-1 and refrigerated for 35 days. The Kenya Blood Transfusion Service provided 12 adult-donated whole blood (AD-WB) controls. Supernatant hemoglobin (Hb) and potassium were assayed at 7-day intervals.
UC-WB red blood cell hemolysis and potassium loss increased throughout storage but did not differ significantly with cord blood volume. Hemolysis rates did not differ significantly between UC-WB and AD-WB but UC-WB potassium loss was slightly but significantly greater than AD-WB on Days 2, 7, and 14 (p < 0.05). In the AD-WB controls, eight were low volume (<405 mL), two had total Hb of less than 45 g, and two showed hemolysis greater than 0.8% by Day 28.
Variable volumes of UC-WB can be stored for 35 days without prestorage processing and further work into its suitability for transfusion to children is justified. The quality of conventional AD-WB is a concern and needs further evaluation.
PMCID: PMC2948540  PMID: 19912583
9.  Red blood cells stored for increasing periods produce progressive impairments in nitric oxide–mediated vasodilation 
Transfusion  2013;53(11):2619-2628.
Clinical outcomes in transfused patients may be affected by the duration of blood storage, possibly due to red blood cell (RBC)-mediated disruption of nitric oxide (NO) signaling, a key regulator of vascular tone and blood flow.
AS-1 RBC units stored up to 42 days were sampled at selected storage times. Samples were added to aortic rings ex vivo, a system where NO-mediated vasodilation could be experimentally controlled.
RBC units showed storage-dependent changes in plasma hemoglobin (Hb), RBC 2,3-diphosphoglycerate acid, and RBC adenosine triphosphate conforming to expected profiles. When freshly collected (Day 0) blood was added to rat aortic rings, methacholine (MCh) stimulated substantial NO-mediated vasodilation. In contrast, MCh produced no vasodilation in the presence of blood stored for 42 days. Surprisingly, the vasoinhibitory effects of stored RBCs were almost totally mediated by RBCs themselves: removal of the supernatant did not attenuate the inhibitory effects, while addition of supernatant alone to the aortic rings only minimally inhibited MCh-stimulated relaxation. Stored RBCs did not inhibit vasodilation by a direct NO donor, demonstrating that the RBC-mediated vasoinhibitory mechanism did not work by NO scavenging.
These studies have revealed a previously unrecognized vasoinhibitory activity of stored RBCs, which is more potent than the described effects of free Hb and works through a different mechanism that does not involve NO scavenging but may function by reducing endothelial NO production. Through this novel mechanism, transfusion of small volumes of stored blood may be able to disrupt physiologic vasodilatory responses and thereby possibly cause adverse clinical outcomes.
PMCID: PMC4140194  PMID: 23480490
10.  Storage of whole blood overnight in different blood bags preceding preparation of blood components: in vitro effects on red blood cells 
Blood Transfusion  2009;7(3):210-215.
Routines for the storage of whole blood (WB) overnight for the preparation of blood components on the following day are of increasing interest primarily for logistic reasons. The present study focuses on in vitro effects during storage for 6 weeks on red blood cells (RBC) prepared in different blood containers after being held overnight.
Study design and methods
Five different blood collection systems were used with either inline leucocyte reduction red cell filters for the preparation of RBC, buffy coat (BC) and plasma or WB filters for the preparation of RBC and plasma. A new container with an integrated WB filter removing leucocytes but not platelets was also included for the preparation of leucocyte-reduced RBC, BC and plasma units. Standard CPD solution (63 or 70 mL) and SAG-M solution (100 or 110 mL) were used for the collection of either 450 or 500 mL blood. All WB units were stored at room temperature, either overnight for 18–24 hours (test groups, n=104) or for up to 8 hours (reference groups, n=20). In addition, five test units were stored overnight under refrigeration.
In test groups (overnight storage at room temperature) we found significantly lower levels of extracellular potassium, 2,3-DPG and pH (up to day 28). During storage, higher levels of ATP (Terumo, CaridianBCT until day 35, Fresenius until day 14, Fenwal throughout storage) were seen in test groups than in reference groups. When WB was stored overnight at 2–6°C before WB filtration, the levels of ATP and haemolysis were higher than in the corresponding reference.
Significant differences in in vitro parameters were observed between RBC prepared within 8 hours and 18–24 hours after blood collection. The results were consistent irrespective of the blood container used. New alkaline solutions may decrease the differences.
PMCID: PMC2719273  PMID: 19657485
red blood cells; overnight; storage; in vitro; ATP; 2,3-DPG
11.  Impact of Transfusion of Autologous 7 versus 42 day old AS-3 Red Blood Cells on Tissue Oxygenation and the Microcirculation in Healthy Volunteers 
Transfusion  2012;52(11):2459-2464.
Stored RBCs accumulate biochemical and biophysical changes. Maximum storage duration is based on acceptable in vitro characteristics and 24-hour survival, but not RBC function. Relatively little is known about the impact of RBC-storage duration on oxygenation and the microcirculation.
Study Design and Methods
Eight healthy subjects donated a double RBC apheresis, which were prestorage leukocyte-reduced, and processed in AS-3. Subjects were transfused one unit of RBCs at 7- and 42-days following blood collection. Measurements of percent (%) tissue oxygenation in the thenar eminence muscle (StO2) and brain (SctO2) were recorded with FDA-cleared non-invasive devices. Sublingual microvascular blood flow (MFI) was quantified prior to and after RBC transfusion using a video microscope. Raw electronic data for all measurements were analyzed by a blinded observer at a core laboratory.
The only pre-versus-posttransfusion change observed in measurements of SctO2, StO2, or MFI was a very small increase in SctO2, from 70.4 to 71.8 (means, p=0.032) at 7 days. There was no significant difference in the amount of pre-post change at 7 days versus 42 days for any of the measures.
Transfusion of one unit of 42-day stored RBCs to healthy subjects has no overt detrimental effect on tissue oxygenation or the microcirculation assessed by clinically available monitors.
PMCID: PMC3387324  PMID: 22452273
transfusion; packed red blood cells (RBCs); tissue oxygenation; microvascular blood flow; near infrared spectroscopy (NIRS); microcirculatory flow index (MFI); age of blood
12.  Quality of Red Blood Cells Isolated from Umbilical Cord Blood Stored at Room Temperature 
Journal of blood transfusion  2011;2012:102809.
Red blood cells (RBCs) from cord blood contain fetal hemoglobin that is predominant in newborns and, therefore, may be more appropriate for neonatal transfusions than currently transfused adult RBCs. Post-collection, cord blood can be stored at room temperature for several days before it is processed for stem cells isolation, with little known about how these conditions affect currently discarded RBCs. The present study examined the effect of the duration cord blood spent at room temperature and other cord blood characteristics on cord RBC quality. RBCs were tested immediately after their isolation from cord blood using a broad panel of quality assays. No significant decrease in cord RBC quality was observed during the first 65 hours of storage at room temperature. The ratio of cord blood to anticoagulant was associated with RBC quality and needs to be optimized in future. This knowledge will assist in future development of cord RBC transfusion product.
PMCID: PMC3777252  PMID: 24089645
13.  Fresh and Stored Red Blood Cell Transfusion Equivalently Induce Subclinical Pulmonary Gas Exchange Deficit in Normal Humans 
Anesthesia and analgesia  2012;114(3):511-519.
Transfusion can cause severe acute lung injury, although most transfusions do not appear to induce complications. We tested the hypothesis that transfusion can cause mild pulmonary dysfunction that has not been noticed clinically and is not sufficiently severe to fit the definition of transfusion-related acute lung injury.
We studied 35 healthy normal volunteers who donated one unit of blood 4 weeks and another 3 weeks before two study days separated by one week. On study days two units of blood were withdrawn while maintaining isovolemia, followed by transfusion with either the volunteer’s autologous fresh red cells (RBCs) removed 2 hours earlier or their autologous stored RBCs (random order). The following week each volunteer was studied again, transfused with the RBCs of the other storage duration. The primary outcome variable was the change in alveolar to arterial difference in oxygen partial pressure (AaDO2) from before to 60 min after transfusion with fresh or older RBCs.
Fresh RBCs and RBCs stored for 24.5 days equally (P=0.85) caused an increase of AaDO2 (fresh: 2.8 mmHg [95% CI: 0.8 - 4.8; (P=0.007)]; stored 3.0 mmHg [1.4 - 4.7; (P=0.0006)]). Concentrations of all measured cytokines, except for interleukin-10 (P=0.15), were less in stored leukoreduced (LR) than stored non-LR packed RBCs; however, vascular endothelial growth factor was the only measured in vivo cytokine that increased more after transfusion with LR than non-LR stored packed RBCs. Vascular endothelial growth factor was the only cytokine tested with in vivo concentrations that correlated with AaDO2.
RBC transfusion causes subtle pulmonary dysfunction, as evidenced by impaired gas exchange for oxygen, supporting our hypothesis that lung impairment after transfusion includes a wide spectrum of physiologic derangements and may not require an existing state of altered physiology. These data do not support the hypothesis that transfusion of RBCs stored for >21 days is more injurious than that of fresh RBCs.
PMCID: PMC3927839  PMID: 22262647
14.  Anaerobic Storage of Red Blood Cells in a Novel Additive Solution Improves In vivo Recovery 
Transfusion  2009;49(3):458-464.
In preliminary studies, anaerobic RBC storage reduced oxidative damage and phosphatidylserine exposure while maintaining ATP levels. The purpose of this study was to compare the 24-hour recovery and lifespan of autologous RBCs stored 6 and 9 weeks using OFAS3 additive solution in an anaerobic environment, compared to control RBCs aerobically stored in AS-3 for 6 weeks.
Eight subjects were entered into a randomized, cross-over study. Whole blood was collected from each subject twice separated by ≥12 wk into CP2D and leukocyte reduced. Controls were stored in AS-3. Test units in OFAS3 were oxygen depleted with Argon then stored 9 wk in an anaerobic chamber at 1–6°C. At the end of each storage period, RBC were labeled with 51Cr and 99mTc and reinfused to the subject following standard methods to determine double label recovery and lifespan. Hypotheses tests were conducted using paired, repeated-measures ANOVA.
Recovery for the anaerobically stored test RBC was significantly better than control at 6 weeks (p=0.023). Test units at 9 wk were not different than the 6 wk control units (p=0.73). Other in vitro measures of RBC characteristics followed the same trend. Two test units at 9 wk had hemolysis > 1%.
Anaerobically stored RBC in OFAS-3 has superior recovery at 6 weeks compared to the Controls and equivalent recovery at 9 weeks with no change in lifespan. Anaerobic storage of RBC may provide an improved RBC for transfusion at 6 wk of storage and may enable extending storage beyond the current 42 day limit.
PMCID: PMC2710818  PMID: 19171002
15.  Evaluation of haemoglobin, haematocrit, haemolysis, residual protein content and leucocytes in 345 red blood cell concentrates used for the treatment of patients with β-thalassaemia 
Blood Transfusion  2012;10(1):39-44.
The aim of this study was to evaluate the quality of red blood cell concentrates obtained from donated whole blood, selected for transfusion therapy of thalassaemic patients, by measuring the following parameters: haemoglobin, haematocrit, percentage haemolysis, residual leucocyte count and residual protein content.
Materials and methods
Overall 345 red cell concentrates were evaluated, of which 205 had been filtered in-line pre-storage and washed and 140 were buffy coat-depleted and used within 2 days of collection. Of the buffy coat-depleted concentrates, 62 were leucodepleted and 78 washed and leucodepleted post-storage all within 2 days of collection. The off-line filters used for the leucodepletion were gamma-irradiated polyester with a pore size of 200 μm. The washing procedure was automated (Haemonetics ACP 215, Braintree, MA, USA). The haematological parameters were evaluated by a blood cell counter (Coulter, Ramsey, IL, USA) and the white blood cell count by cytofluorimetry (FACScan).
Ninety-five percent (194/205) of the red cell concentrates that had been filtered pre-storage and washed, 92% (57/62) of the red cell concentrates that had been leucodepleted post-storage and 94% (73/78) of the those subjected to both treatments had normal values of haemoglobin (>40 g/unit), haematocrit (between 50–70%), percentage haemolysis (<0.8/unit), white cell count (<1×106) and residual protein content (<0.5 g/L). Five percent (11/205) of the red cell concentrates that had been filtered pre-storage and washed, 8% (5/62) of those leucodepleted post-storage after 2 days and 6% (5/78) of those that underwent both procedures had a haemoglobin content <40 g/unit and a haematocrit <50%.
The preparation procedures had been carried out satisfactorily; nevertheless, transfusion therapy with some “low dose” normal units could be less effective and might, therefore, result in greater transfusion requirements in patients receiving such units.
PMCID: PMC3258988  PMID: 22153690
post-storage leucodepletion; washed red blood cells; haemoglobin
16.  Process Improvement by Eliminating Mixing of Whole Blood Units after an Overnight Hold Prior to Component Production Using the Buffy Coat Method 
Journal of blood transfusion  2013;2013:154838.
The elimination of a thorough manual mixing of whole blood (WB) which takes place following the overnight hold, but before the first centrifugation step, during buffy coat component production at Canadian Blood Services (CBS) was investigated. WB was pooled after donation and split. Pairs of platelet, red blood cell (RBC), and plasma components were produced, with half using the standard method and half using a method in which the mixing step was eliminated. Quality assessments included yield, pH, CD62P expression and morphology for platelets, hemoglobin, hematocrit, hemolysis, and supernatant K+ for RBCs, and volume and factor VIII activity levels for plasma. All components, produced using either method, met CBS quality control criteria. There were no significant differences in platelet yield between components produced with and without mixing. A significant difference was seen for RBC hemolysis at expiry (P = 0.03), but for both groups, levels met quality control requirements. Noninferiority of components produced without mixing was confirmed for all parameters. Manual mixing is laborious and has a risk of repetitive strain for production staff and its significance is unclear. Elimination of this step will improve process efficiencies without compromising quality.
PMCID: PMC3771126  PMID: 24066260
17.  Red Blood Cell Storage Duration Is Associated with Various Clinical Outcomes in Pediatric Cardiac Surgery 
Recommendations on the use of fresh red blood cells (RBCs) in pediatric patients undergoing cardiac surgery are based on limited information. Furthermore, the RBC storage time cut-off of fresh units remains unknown.
Data from 139 pediatric patients who underwent cardiac surgery and received RBCs from a single unit within 14 days of storage were analyzed. To identify the optimal cut-off storage time of RBCs for transfusion, multiple multivariate analyses aimed at different outcome parameters were performed.
26 patients received RBC units stored for ≤3 days, while 126 patients received RBCs that were stored for 4-14 days. The latter group required more RBC transfusions and fresh frozen plasma (FFP) than the former group (19 vs. 25 ml/kg, p = 0.003 and 73% vs. 35%, p = 0.0006, respectively). In addition, the odds for the administration of FFP increased with the transfusion of RBCs stored for more than 4 days. The optimal cut-off for post-operative morbidity was observed with a storage time of ≤6 days for length of ventilation (p = 0.02) and peak of C-reactive protein (CRP; p = 0.008).
The obtained results indicate that the hazard of blood transfusion increased with increasing storage time of RBCs. The results of this study suggest that transfusion of fresh RBCs with a storage time of ≤2 or 4 days (concerning transfusion requirements) or ≤6 days (concerning postoperative morbidity) may be beneficial in pediatric patients undergoing cardiac surgery. However, further prospective randomized studies are required in order to draw any final conclusions.
PMCID: PMC4025050  PMID: 24847191
Red blood cell storage; Erythrocytes; Blood; Transfusion; Pediatric cardiac surgery; Clinical outcome; Inflammation
18.  Association between red blood cell storage duration and clinical outcome in patients undergoing off-pump coronary artery bypass surgery: a retrospective study 
BMC Anesthesiology  2014;14(1):95.
Prolonged storage of red blood cells (RBCs) leads to fundamental changes in both the RBCs and the storage media. We retrospectively evaluated the relationship between the RBC age and in-hospital and long-term postoperative outcomes in patients undergoing off-pump coronary artery bypass.
The electronic medical records of 1,072 OPCAB patients were reviewed and information on the transfused RBCs and clinical data were collected. The effects of RBCs age (mean age, oldest age of transfused RBCs, any RBCs older than 14 days) on various in-hospital postoperative complications and long-term major adverse cardiovascular and cerebral events over a mean follow-up of 31 months were investigated. Correlations between RBCs age and duration of intubation, intensive care unit, or hospital stay, and base excess at the first postoperative morning were also analyzed.
After adjusting for confounders, there was no relationship between the RBCs age and in-hospital and long-term clinical outcomes except for postoperative wound complications. A significant linear trend was observed between the oldest age quartiles of transfused RBCs and the postoperative wound complications (quartile 1 vs. 2, 3 and 4: OR, 8.92, 12.01 and 13.79, respectively; P for trend = 0.009). The oldest transfused RBCs showed significant relationships with a first postoperative day negative base excess (P = 0.021), postoperative wound complications (P = 0.001), and length of hospital stay (P = 0.008).
In patients undergoing off-pump coronary artery bypass, the oldest age of transfused RBCs were associated with a postoperative negative base excess, increased wound complications, and a longer hospital stay, but not with the other in-hospital or long-term outcomes.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2253-14-95) contains supplementary material, which is available to authorized users.
PMCID: PMC4210472  PMID: 25352766
RBC storage age; Old stored RBC; Postoperative outcome; Wound complication; Cardiac surgery
19.  Tenofovir, Emtricitabine, and Tenofovir Diphosphate in Dried Blood Spots for Determining Recent and Cumulative Drug Exposure 
Tenofovir (TFV) disoproxil fumarate (TDF)±emtricitabine (FTC) are widely used for HIV treatment and chemoprophylaxis, but variable adherence may lead to suboptimal responses. Methods that quantify adherence would allow for interventions to improve treatment and prevention outcomes. Our objective was to characterize the pharmacokinetics of TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP) in red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs); to extend the RBC analysis to dried blood spots (DBSs); and to model how RBC/DBS monitoring could inform recent and cumulative drug exposure/adherence. Blood samples were collected from 17 HIV-negative adults at 5 visits over a 30-day pharmacokinetics study of daily oral TDF/FTC. Dosing was discontinued on day 30 and blood was collected on days 35, 45, and 60 during the washout period. Plasma/RBCs/PBMCs/DBSs were all quantified by liquid chromatography/tandem mass spectrometry. DBSs were paired with RBCs and plasma for comparisons. The median (interquartile range) RBC TFV-DP half-life was 17.1 (15.7–20.2) versus 4.2 (3.7–5.2) days in PBMCs. At steady state, TFV-DP was 130 fmol/106 RBCs versus 98 fmol/106 PBMCs. FTC-TP was not quantifiable in most RBC samples. TFV-DP in RBCs versus DBSs yielded an r2=0.83. TFV-DP in DBSs was stable at −20°C. Simulations of TFV-DP in RBCs/DBSs, when dosed from one to seven times per week, demonstrated that each dose per week resulted in an average change of approximately 19 fmol/106 RBCs and 230 fmol/punch. TFV and FTC in plasma versus DBSs was defined by y=1.4x; r2=0.96 and y=0.8x; r2=0.99, respectively. We conclude that DBSs offer a convenient measure of recent (TFV/FTC) and cumulative (TFV-DP in RBCs) drug exposure with potential application to adherence monitoring.
PMCID: PMC3552442  PMID: 22935078
20.  Duration of Red Cell Storage Influences Mortality After Trauma 
The Journal of trauma  2010;69(6):1427-1432.
Although previous studies have identified an association between the transfusion of relatively older red blood cells (RBCs) (storage ≥14 days) and adverse outcomes, they are difficult to interpret because the majority of patients received a combination of old and fresh RBC units. To overcome this limitation, we compared in-hospital mortality among patients who received exclusively old versus fresh RBC units during the first 24 hours of hospitalization.
Patients admitted to a Level I trauma center between January 2000 and May 2009 who received ≥1 unit of exclusively old (≥14 days) vs. fresh (<14 days) RBCs during the first 24 hours of hospitalization were identified. Risk ratios (RRs) and 95% confidence intervals (CIs) were calculated for the association between mortality and RBC age, adjusted for patient age, Injury Severity Score, gender, receipt of fresh frozen plasma or platelets, RBC volume, brain injury, and injury mechanism (blunt or penetrating).
One thousand six hundred forty-seven patients met the study inclusion criteria. Among patients who were transfused 1 or 2 RBC units, no difference in mortality with respect to RBC age was identified (adjusted RR, 0.97; 95% CI, 0.72–1.32). Among patients who were transfused 3 or more RBC units, receipt of old versus fresh RBCs was associated with a significantly increased risk of mortality, with an adjusted RR of 1.57 (95% CI, 1.14–2.15). No difference was observed concerning the mean number of old versus fresh units transfused to patients who received 3 or more units (6.05 vs. 5.47, respectively; p = 0.11).
In trauma patients undergoing transfusion of 3 or more RBC units within 24 hour of hospital arrival, receipt of relatively older blood was associated with a significantly increased mortality risk. Reservation of relatively fresh RBC units for the acutely injured may be advisable.
PMCID: PMC3136808  PMID: 21150522
Transfusion; Blood storage; Trauma
21.  A Novel Mouse Model of Red Blood Cell Storage and Post-Transfusion in Vivo Survival 
Transfusion  2009;49(8):1546-1553.
Storage of RBCs is necessary for an adequate blood supply. However, reports have identified potential negative sequelae of transfusing stored RBCs. An animal model would be useful to investigate the pathophysiology of transfusing stored RBCs. However, it has been reported that storage of rat RBCs in CPDA-1 resulted in an unexpected sudden decline in post-transfusion survival. We developed a mouse model of RBC storage and transfusion to assess survival kinetics of mouse RBCs.
Study Design and Methods
RBCs expressing green fluorescent protein were collected in CPDA-1, filter leukoreduced, adjusted to a 75% hematocrit, and stored at 4°C. At weekly intervals, stored RBCs were transfused into C57BL/6 recipients. RBC survival was measured by flow cytometry and 51Chromium labeling. Phosphatidylserine externalization and CD47 expression was also evaluated.
Mean 24-hour survival of transfused RBCs was 99%, 91%, 64%, 54%, 30%, and 18% following 0, 7, 14, 21, 28, and 35 days of storage, respectively. Stored RBCs showed an initial rapid clearance with subsequent extended survival. Increased surface phosphatidylserine and decreased CD47 expression was also observed.
Mouse RBCs showed a progressive decline in survival, as a function of storage time, unlike the precipitous loss of viability previously reported for rat RBCs. Moreover, changes in the measured surface markers were analogous to trends reported for human RBCs. Together, these findings provide an initial characterization of a novel mouse model of RBC storage with the potential to serve as an experimental platform for studying the pathophysiological consequences of transfusing stored RBCs.
PMCID: PMC2888981  PMID: 19573176
22.  Red Blood Cell Transfusion and Mortality in Trauma Patients: Risk-Stratified Analysis of an Observational Study 
PLoS Medicine  2014;11(6):e1001664.
Using a large multicentre cohort, Pablo Perel and colleagues evaluate the association of red blood cell transfusion with mortality according to the predicted risk of death for trauma patients.
Please see later in the article for the Editors' Summary
Haemorrhage is a common cause of death in trauma patients. Although transfusions are extensively used in the care of bleeding trauma patients, there is uncertainty about the balance of risks and benefits and how this balance depends on the baseline risk of death. Our objective was to evaluate the association of red blood cell (RBC) transfusion with mortality according to the predicted risk of death.
Methods and Findings
A secondary analysis of the CRASH-2 trial (which originally evaluated the effect of tranexamic acid on mortality in trauma patients) was conducted. The trial included 20,127 trauma patients with significant bleeding from 274 hospitals in 40 countries. We evaluated the association of RBC transfusion with mortality in four strata of predicted risk of death: <6%, 6%–20%, 21%–50%, and >50%. For this analysis the exposure considered was RBC transfusion, and the main outcome was death from all causes at 28 days. A total of 10,227 patients (50.8%) received at least one transfusion. We found strong evidence that the association of transfusion with all-cause mortality varied according to the predicted risk of death (p-value for interaction <0.0001). Transfusion was associated with an increase in all-cause mortality among patients with <6% and 6%–20% predicted risk of death (odds ratio [OR] 5.40, 95% CI 4.08–7.13, p<0.0001, and OR 2.31, 95% CI 1.96–2.73, p<0.0001, respectively), but with a decrease in all-cause mortality in patients with >50% predicted risk of death (OR 0.59, 95% CI 0.47–0.74, p<0.0001). Transfusion was associated with an increase in fatal and non-fatal vascular events (OR 2.58, 95% CI 2.05–3.24, p<0.0001). The risk associated with RBC transfusion was significantly increased for all the predicted risk of death categories, but the relative increase was higher for those with the lowest (<6%) predicted risk of death (p-value for interaction <0.0001). As this was an observational study, the results could have been affected by different types of confounding. In addition, we could not consider haemoglobin in our analysis. In sensitivity analyses, excluding patients who died early; conducting propensity score analysis adjusting by use of platelets, fresh frozen plasma, and cryoprecipitate; and adjusting for country produced results that were similar.
The association of transfusion with all-cause mortality appears to vary according to the predicted risk of death. Transfusion may reduce mortality in patients at high risk of death but increase mortality in those at low risk. The effect of transfusion in low-risk patients should be further tested in a randomised trial.
Trial registration NCT01746953
Please see later in the article for the Editors' Summary
Editors' Summary
Trauma—a serious injury to the body caused by violence or an accident—is a major global health problem. Every year, injuries caused by traffic collisions, falls, blows, and other traumatic events kill more than 5 million people (9% of annual global deaths). Indeed, for people between the ages of 5 and 44 years, injuries are among the top three causes of death in many countries. Trauma sometimes kills people through physical damage to the brain and other internal organs, but hemorrhage (serious uncontrolled bleeding) is responsible for 30%–40% of trauma-related deaths. Consequently, early trauma care focuses on minimizing hemorrhage (for example, by using compression to stop bleeding) and on restoring blood circulation after blood loss (health-care professionals refer to this as resuscitation). Red blood cell (RBC) transfusion is often used for the management of patients with trauma who are bleeding; other resuscitation products include isotonic saline and solutions of human blood proteins.
Why Was This Study Done?
Although RBC transfusion can save the lives of patients with trauma who are bleeding, there is considerable uncertainty regarding the balance of risks and benefits associated with this procedure. RBC transfusion, which is an expensive intervention, is associated with several potential adverse effects, including allergic reactions and infections. Moreover, blood supplies are limited, and the risks from transfusion are high in low- and middle-income countries, where most trauma-related deaths occur. In this study, which is a secondary analysis of data from a trial (CRASH-2) that evaluated the effect of tranexamic acid (which stops excessive bleeding) in patients with trauma, the researchers test the hypothesis that RBC transfusion may have a beneficial effect among patients at high risk of death following trauma but a harmful effect among those at low risk of death.
What Did the Researchers Do and Find?
The CRASH-2 trail included 20,127 patients with trauma and major bleeding treated in 274 hospitals in 40 countries. In their risk-stratified analysis, the researchers investigated the effect of RBC transfusion on CRASH-2 participants with a predicted risk of death (estimated using a validated model that included clinical variables such as heart rate and blood pressure) on admission to hospital of less than 6%, 6%–20%, 21%–50%, or more than 50%. That is, the researchers compared death rates among patients in each stratum of predicted risk of death who received a RBC transfusion with death rates among patients who did not receive a transfusion. Half the patients received at least one transfusion. Transfusion was associated with an increase in all-cause mortality at 28 days after trauma among patients with a predicted risk of death of less than 6% or of 6%–20%, but with a decrease in all-cause mortality among patients with a predicted risk of death of more than 50%. In absolute figures, compared to no transfusion, RBC transfusion was associated with 5.1 more deaths per 100 patients in the patient group with the lowest predicted risk of death but with 11.9 fewer deaths per 100 patients in the group with the highest predicted risk of death.
What Do These Findings Mean?
These findings show that RBC transfusion is associated with an increase in all-cause deaths among patients with trauma and major bleeding with a low predicted risk of death, but with a reduction in all-cause deaths among patients with a high predicted risk of death. In other words, these findings suggest that the effect of RBC transfusion on all-cause mortality may vary according to whether a patient with trauma has a high or low predicted risk of death. However, because the participants in the CRASH-2 trial were not randomly assigned to receive a RBC transfusion, it is not possible to conclude that receiving a RBC transfusion actually increased the death rate among patients with a low predicted risk of death. It might be that the patients with this level of predicted risk of death who received a transfusion shared other unknown characteristics (confounders) that were actually responsible for their increased death rate. Thus, to provide better guidance for clinicians caring for patients with trauma and hemorrhage, the hypothesis that RBC transfusion could be harmful among patients with trauma with a low predicted risk of death should be prospectively evaluated in a randomised controlled trial.
Additional Information
Please access these websites via the online version of this summary at
This study is further discussed in a PLOS Medicine Perspective by Druin Burch
The World Health Organization provides information on injuries and on violence and injury prevention (in several languages)
The US Centers for Disease Control and Prevention has information on injury and violence prevention and control
The National Trauma Institute, a US-based non-profit organization, provides information about hemorrhage after trauma and personal stories about surviving trauma
The UK National Health Service Choices website provides information about blood transfusion, including a personal story about transfusion after a serious road accident
The US National Heart, Lung, and Blood Institute also provides detailed information about blood transfusions
MedlinePlus provides links to further resources on injuries, bleeding, and blood transfusion (in English and Spanish)
More information in available about CRASH-2 (in several languages)
PMCID: PMC4060995  PMID: 24937305
23.  Plasma levels of sphingosine 1-phosphate are strongly correlated with haematocrit, but variably restored by red blood cell transfusions 
Clinical Science (London, England : 1979)  2011;121(Pt 12):565-572.
Anaemia and RBC (red blood cell) transfusion may be associated with worse clinical outcomes, especially with longer blood storage duration prior to transfusion. The mechanisms underlying these harmful effects are unknown. RBCs have been proposed to buffer plasma S1P (sphingosine 1-phosphate), a lysophospholipid essential for the maintenance of endothelial integrity and important in the regulation of haematopoietic cell trafficking. The present study examined the effect of anaemia, RBC transfusion and RBC storage duration on plasma S1P levels. Plasma S1P from 30 individuals demonstrated a linear correlation with Hct (haematocrit; R2=0.51, P<0.001) with no evidence for a plateau at Hct values as low as 19%. RBC transfusion in 23 anaemic patients with baseline mean Hct of 22.2±0.34% (value is the mean±S.D.) increased Hct to 28.3±0.6% at 72 h. Despite an Hct increase, RBC transfusion failed to elevate plasma S1P consistently. A trend towards an inverse correlation was observed between RBC storage duration and the post-transfusion increase in plasma S1P. After 30 days of storage, RBC S1P decreased to 19% of that observed in fresh (3–7-day-old) RBC segments. RBC membranes contain low levels of both S1P phosphatase and S1P lyase activities that may account for the decline in S1P levels with storage. Our results support a role for RBCs in buffering plasma S1P and identify a disturbance in the capacity after transfusion. Changes in S1P content may contribute to an RBC storage lesion. Further studies should investigate the clinical significance of alterations in circulating S1P levels and the potential value of enriching stored RBCs with S1P.
PMCID: PMC3174054  PMID: 21749329
anaemia; blood storage; lysophospholipid; sphingosine 1-phosphate; transfusion; Apo, apolipoprotein; ESI, electrospray ionization; Hct, haematocrit; HDL, high-density lipoprotein; LPC, lysophosphatidylcholine; MRM, multiple reaction monitoring; MS/MS, tandem MS; RBC, red blood cell; S1P, sphingosine 1-phosphate; DH-S1P, dihydro-S1P
24.  Storage Duration of Red Blood Cell Transfusion and Clostridium difficile Infection: A Within Person Comparison 
PLoS ONE  2014;9(2):e89332.
Randomized controlled trials demonstrated that red blood cell (RBC) transfusion elevates the risk of infection, and trials are underway to evaluate whether RBC storage affects outcomes. We previously reported that transfusion predicts Clostridium difficile infection (CDI) and, therefore, planned an investigation to examine this further using a more robust design.
Within-person case-crossover study. Hospitalizations in which CDI developed (n = 406) were compared to hospitalizations for the same individuals in which CDI did not occur (n = 949). Transfusion volume and storage duration were assessed prior to the onset of CDI.
University of Michigan Health System.
Participants were individuals with a diagnosis of CDI from July 2009 through June 2012.
Measurements and Main Results
During the hospitalizations when CDI occurred, 34.7% of the patients received allogeneic RBC transfusions (mean volume, 688 ml) compared to 19.0% of patients in hospitalizations without CDI (mean volume, 180 ml). The odds of healthcare-associated CDI increased by 76% (95% CI 1.39–2.23) for every liter of RBCs transfused and was elevated in both nonsurgical (OR = 1.90) and surgical (OR = 1.86) hospitalizations. In patients who received RBC transfusions, the odds of developing CDI increased by 6% for every additional day of RBC stored and by 53% for every week of additional storage (P = 0.002).
Hospitalizations in which a patient received a greater volume of RBC transfusions were more likely to be associated with the development of CDI. RBC units stored for a longer duration were associated with the development of healthcare-associated CDI after adjustment for RBC volume.
PMCID: PMC3931729  PMID: 24586694
25.  Erythrocyte storage increases rates of NO- and Nitrite scavenging: Implications for transfusion related toxicity 
The Biochemical journal  2012;446(3):499-508.
Storage of erythrocytes in blood banks is associated with biochemical and morphological changes to the RBC. It has been suggested that these changes have a potential negative clinical effects characterized by inflammation and microcirculatory dysfunction which add to other transfusion related toxicities. However, mechanisms linking RBC storage and toxicity remain unclear. In this study we tested the hypothesis that storage of leukodepleted RBC result in cells that inhibit nitric oxide (NO)-signaling more so than younger cells. Using competition kinetic analyses and protocols that minimized contributions from hemolysis or microparticles, our data indicate that NO-consumption rates increased ~40-fold and NO-dependent vasodilation was inhibited 2-4 fold with 42d old vs. 0d RBC. These results are likely due to the formation of smaller RBC with increased surface area: volume as a consequence of membrane loss during storage. The potential for older RBC to affect NO-formation via deoxygenated RBC mediated nitrite reduction was also tested. RBC storage did not affect deoxygenated RBC-dependent stimulation of nitrite-induced vasodilation. However, stored RBC did increase the rates of nitrite oxidation to nitrate in vitro. Significant loss of whole blood nitrite was also observed in stable trauma patients after transfusion with 1 RBC unit, with the decrease in nitrite occurring after transfusion with RBC stored for >25d, but not with younger RBC. Collectively, these data suggest that increased rates of reactions between intact RBC and NO and nitrite may contribute to mechanisms that lead to storage lesion-related transfusion risk
PMCID: PMC3572541  PMID: 22720637
hemoglobin; vasoconstriction; kinetics

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