The effect of the transfusion of stored RBCs on intestinal microvascular oxygenation in the rat

BACKGROUND: Although it is known that the transfusion of stored RBCs does not always improve tissue O(2) consumption under conditions of limited tissue oxygenation, the efficiency of O(2) 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 PO(2) was determined with the O(2)-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 pO(2) were restored with the transfusion of both fresh and stored RBCs, except for CPD-stored RBCs. The intestinal microvascular pO(2) 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 O(2) unloading, but, except for CPD-stored RBCs, the storage-induced changes were not enough to impair intestinal VO(2) and mesenteric venous pO(2).     

The viability of autologous human red cells stored in additive solution 5 and exposed to 25°C for 24 hours

BACKGROUND: No data exist on the viability of red cells (RBCs) stored in modern additive solution systems and allowed to warm above 10 degrees C. STUDY DESIGN AND METHODS: In a randomized crossover study, 3 units of blood were collected at least 8 weeks apart from 11 volunteer donors and stored in additive solution 5 (AS-5). Of 3 units from each volunteer, 1 was stored for 6 weeks at 4 degrees C, 1 for 5 weeks at 4 degrees C except for 24 hours at 25 degrees C on Day 14, and 1 for 5 weeks at 4 degrees C except for 24 hours at 25 degrees C on Day 28. Units were sampled periodically during storage; at the end of storage, viability was measured by the 99mTc/51 Cr double-label method. RESULTS: RBC viability was not significantly different in the storage protocols. Less than 1 percent of stored cells hemolyzed. RBC ATP concentrations at the end of storage correlated with viability and were approximately equal in the warmed units after 30 days' storage and the conventionally stored units after 42 days. CONCLUSIONS: The data suggest that RBCs stored in AS-5 and allowed to warm to 25 degrees C for 24 hours lose about 12 days of their shelf life.     

Effect of rejuvenation and frozen storage on 42-day-old AS-1 RBCs

BACKGROUND: The FDA has approved a 42-day storage period for RBCs stored in ADSOL (AS-1). This study was undertaken to provide data for the FDA about the feasibility of salvaging AS-1 RBCs at the end of their storage period by rejuvenation and freezing. STUDY DESIGN AND METHOD: The investigation, consisting of a study (n = 10) and control (n = 6) arm, was carried out in two centers. In both centers, eight healthy volunteers donated a unit (450 mL) of whole blood. The RBC concentrates were stored at 4 degrees C in AS-1 for 42 days. The study units were rejuvenated, whereas the control units were not. All units were stored frozen at -80 degrees C, then deglycerolized and kept for an additional 24 hours at 4 degrees C. RESULTS: After the 42-day storage period, ATP had declined to 62 percent of the original value, 2,3 DPG was zero, and MCV was significantly larger than that of fresh RBCS: Following rejuvenation and deglycerolization, the mean ATP level was 141 percent, the mean 2,3 DPG level was 109 percent, and the MCV was normal. The freeze-thaw-wash recovery of the rejuvenated and nonrejuvenated RBCs was similar, 88.4 and 84.0 percent, respectively. There was no difference in hypoxanthine, inosine, and uric acid levels in the rejuvenated and nonrejuvenated units, which indicated that the chemicals in the rejuvenation solution and their by-products had been removed during processing. In both centers, the mean 24-hour survival of rejuvenated, deglycerolized RBCs exceeded 75 percent, whereas that of nonrejuvenated RBCs did not. The long-term survival rates of viable study and control RBCs were similar. CONCLUSION: Forty-two-day-old AS-1 RBCs that have been rejuvenated and then frozen have more than 75 percent viability and normal oxygen delivery function. Rejuvenation of RBCs does not introduce additional safety hazards to blood transfusion.     

Rejuvenation of stored human red blood cells reverses the renal microvascular oxygenation deficit in an isovolemic transfusion model in rats

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 (µPO₂) 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 µPO₂ 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.     

A comparison of biochemical and functional alterations of rat and human erythrocytes stored in CPDA-1 for 29 days: implications for animal models of transfusion

Animal models of transfusion are employed in many research areas yet little is known about the storage-related changes occurring in the blood used in these studies. This study assessed storage-related changes in red blood cell (RBC) biochemistry, function and membrane deformability in rat and human packed RBCs when stored in CPDA-1 at 4 degrees C over a 4-week period. Human blood from five volunteers and five bags of rat RBC concentrates (five donor rats per bag) were collected and stored at 4 degrees C. RBC function was assessed by post-transfusion viability and the ability to regenerate adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) when treated with a rejuvenation solution. Membrane deformability was determined by a micropipette aspiration technique. ATP in rat RBCs declined more rapidly than human RBCs; after 1 week rat ATP fell to the same level as human cells after 4 weeks of storage (rat, 2.2 +/- 0.2 micromol g(-1) Hb; human, 2.5 +/- 0.3 micromol g(-1) Hb). Baseline DPG concentrations were similar in rat and human RBCs (16.2 +/- 2.3 micromol g(-1) Hb and 13.7 +/- 2.4 micromol g(-1) Hb) and declined very rapidly in both species. Human RBCs fully regenerated ATP and DPG when treated with a rejuvenation solution after 4 weeks of storage. Rat RBCs regenerated ATP but not DPG. Post-transfusion viability in rat cells was 79%, 26% and 5% after 1, 2 and 4 weeks of storage, respectively. In rats, decreased membrane deformability became significant (- 54%) after 7 days. Human RBC deformability decreased significantly by 34% after 4 weeks of storage. The rejuvenation solution restored RBC deformability to control levels in both species. Our results indicate that rat RBCs stored for 1 week in CPDA-1 develop a storage lesion similar to that of human RBCs stored for 4 weeks and underscores significant species-specific differences in the structure and metabolism of these cells.     

Rejuvenation capacity of red blood cells in additive solutions over long-term storage

BACKGROUND: Red blood cells (RBCs) are Food and Drug Administration (FDA)‐approved for 42‐day storage with the use of additive solutions (ASs). However, adenosine triphosphate (ATP) and 2,3‐diphosphoglycerate (2,3‐DPG) levels in the RBCs decline over this time. These constituents may be restored by treatment with rejuvenation (REJ) solutions. This study was done to assess the response capability of RBCs from 30 to 120 days of storage in three FDA‐licensed RBC storage solutions after incubation with a rejuvenating solution of pyruvate, inosine, phosphate, and adenine. STUDY DESIGN AND METHODS: Three units each of RBCs in approved AS (AS‐1 [Adsol, Fenwal, Inc.], AS‐3 [Nutricel, Medsep Corp.], and AS‐5 [Optisol, Terumo Corp.]) were stored under standard conditions at 1 to 6°C for up to 120 days. Aliquots (4 mL) on Days 30, 42, 60, 80, 100, and 120 (±2 days) were REJ by incubating with Rejuvesol (Encyte Corp.). Control untreated and REJ aliquots were extracted using perchloric acid and stored at ?80°C until assayed for 2,3‐DPG and ATP. RESULTS: RBCs responded to REJ by increasing DPG and ATP contents. The response declined linearly at 0.070 ± 0.008 µmol DPG/g hemoglobin (Hb)/day and 0.035 ± 0.004 µmol ATP/g Hb/day with no differences between ASs. CONCLUSION: We conclude that Rejuvesol is able to restore ATP and 2,3‐DPG levels in RBCs stored up to 120 days in AS. The response diminishes as storage time increases. This rejuvenation (REJ) capability does not seem useful for routine assessment of RBC anabolic capacity in research programs, but may be useful to the investigator when studying unique and novel treatment methods.     

Effect of 24-hour storage at 25°C on the in vitro storage characteristics of CPDA- 1 packed red cells

BACKGROUND: Packed red cells (RBCs) warmed above 10 degrees C are generally discarded. Few data exist on the degree of accelerated metabolism and increased hemolysis of packed RBCs allowed to warm. STUDY DESIGN AND METHODS: Twenty-four CPDA-1 packed RBC units were combined in 3-unit pools and subdivided into 2 test units and a control unit. One test unit from each pool was warmed to 25 degrees C for 24 hours on Day 6 and the other test unit was warmed on Day 20; control units were maintained at 1 to 6 degrees C. RBC and supernatant chemistries and RBC morphology were measured weekly (Days 0, 7, 14, 21, and 28) and on the day before warming (Days 6 and 20). RESULTS: Warming CPDA-1 packed RBCs accelerated the catabolism of glucose 10-fold and produced concentrations of glucose, lactate, and ATP after 25 days of storage that were equivalent to those in unwarmed units at 35 days. Supernatant sodium and potassium concentrations were corrected partially with warming. RBC morphology transiently normalized with warming and without increased hemolysis; no bacteria growth was detected. CONCLUSION: One day of 25 degrees C storage of CPDA-1 packed RBCs accelerates essential metabolite break-down equivalent to 10 days of storage at 1 to 6 degrees C. It does not appear to matter whether the packed RBCs are warmed on Day 6 or Day 20. This information may be useful in determining the acceptability of blood allowed to warm above 10 ° C.     

RBC-derived vesicles during storage: ultrastructure, protein composition, oxidation, and signaling components

BACKGROUND: Red cells (RBCs) lose membrane in vivo, under certain conditions in vitro, and during the ex vivo storage of whole blood, by releasing vesicles. The vesiculation of the RBCs is a part of the storage lesion. The protein composition of the vesicles generated during storage of banked RBCs has not been studied in detail. STUDY DESIGN AND METHODS: Vesicles were isolated from the plasma of nonleukoreduced RBC units in citrate‐phosphate‐dextrose‐adenine, at eight time points of the storage period and shortly afterward. The degree of vesiculation, ultrastructure, oxidation status, and protein composition of the vesicles were evaluated by means of electron microscopy and immunoblotting. RBCs and ghost membranes were investigated as controls. RESULTS: The total protein content of the vesicle fraction and the size of the vesicles increased but their structural integrity decreased over time. The oxidation index of the vesicles released up to Day 21 of storage was greater than that of the membrane ghosts of the corresponding intact RBCs. The vesicles contain aggregated hemoglobin, band 3, and lipid raft proteins, including flotillins. They also contain Fas, FADD, procaspases 3 and 8, caspase 8 and caspase 3 cleavage products (after the 10th day), CD47 (after the 17th day), and immunoglobulin G. CONCLUSION: These data indicate that the vesicles released during storage of RBCs contain lipid raft proteins and oxidized or reactive signaling components commonly associated with the senescent RBCs. Vesiculation during storage of RBCs may enable the RBC to shed altered or harmful material.     

Improved maintenance of 2,3 DPG and ATP in RBCs stored in a modified additive solution

BACKGROUND : All currently used systems for the storage of RBCs result in loss of 2,3 DPG and an associated increase in affinity for oxygen. Previously, it was demonstrated that a hypotonic additive solution for RBC storage (Erythro‐Sol) ^{4 , 5} resulted in prolonged maintenance of 2,3 DPG when blood was collected in 0.5 CPD (half‐strength CPD), but not when full‐strength CPD was used. The present study aims at improving the quality of stored RBCs collected in ordinary CPD. STUDY DESIGN AND METHODS : A new formulation of Erythro‐Sol (Erythro‐Sol 2) (pH 8.8) in a larger volume (150 mL) was compared with Erythro‐Sol (Erythro‐Sol 1). In vitro measures during 49 days of storage in the two additives were compared using WBC‐depleted RBCs after whole‐blood collection in CPD and separation in an automated blood separation instrument (Optipress II, Baxter Healthcare). RESULTS : The maintenance of RBC ATP and 2,3 DPG was significantly better in Erythro‐Sol 2 than in Erythro‐Sol 1. The ATP concentration rose to approximately 30 percent above initial level in both systems; however, the maximum occurred on Day 21 in Erythro‐Sol 2 as compared with Day 14 in Erythro‐Sol 1. In RBCs stored in Erythro‐Sol 2, the mean RBC 2,3 DPG concentration increased to 14 percent above initial level on Day 7, then decreased to the initial level on Day 14, whereas in Erythro‐Sol 1, the 2,3 DPG had decreased to 85 and 50 percent on Days 7 and 14, respectively. Both intracellular pH and extracellular pH were slightly higher in Erythro‐Sol 2 than in Erythro‐Sol 1 units but decreased rapidly during the first storage week, which seems to have been the major reason for the limitation in the time of maintenance of 2,3 DPG. Hemolysis was very low in both systems, 0.14 to 0.17 percent on Day 49. The additional amount of inorganic phosphate submitted with Erythro‐Sol 2 did not raise concern because the phosphate content in the storage medium, being 1.3 ± 0.2 mmoL on Day 0, decreased to values below 1 mmoL during most of subsequent storage. CONCLUSION : Erythro‐Sol 2 is an improved additive solution for the storage of RBCs.     

Differential profiling of human red blood cells during storage for 52 selected microRNAs

BACKGROUND: MicroRNAs (miRNAs), the negative regulators of cellular mRNAs, are present in mature red blood cells (RBCs) in abundance relative to other blood cells. So far, there are no studies aimed at identifying large‐scale miRNA profiles during storage of RBCs. STUDY DESIGN AND METHODS: RNA samples from each RBC bag stored at 4°C were collected on Days 0, 20, and 40 and subjected to miRNA profiling by using a membrane‐based array. Fifty‐two selected miRNAs of cellular apoptotic pathway represent the array. Through bioinformatics analyses, we identified potential target genes for selected miRNAs. RESULTS: Differential profiling of RBCs for 52 miRNAs revealed two distinguishable patterns during storage: Forty‐eight miRNAs demonstrated no trend at all, while four miRNAs, miR‐96, miR‐150, miR‐196a, and miR‐197, demonstrated an increase up to Day 20 and subsequently decreased during storage. We selected miR‐96 and subjected it to standard bioinformatics analyses for target gene predictions, which identified several mRNAs including the RBC proapoptotic calpain small subunit‐1 (CAPNS1) as potential targets of miR‐96. To validate these predictions, we selected CAPNS1 mRNA as an example and confirmed its presence in the RBCs. Future experimental verification would help define miR‐96–CAPNS1 interaction, if any, in the stored RBCs. CONCLUSIONS: This study for the first time provided a differential profile of stored RBCs for selected miRNAs related to cellular apoptotic pathway and opened new avenues toward identification of novel in vitro RBC biomarkers of storage lesions. Future studies focusing on target gene‐miRNA interactions in stored RBCs would also unravel underlying mechanisms of storage lesions.     

The release of prion protein from platelets during storage of apheresis platelets

BACKGROUND: Recent studies using a time-resolved fluoroimmunoassay method (dissociation-enhanced lanthanide fluoroimmunoassay) showed that platelets and plasma are the main reservoir of the normal isoform of cell-associated prion protein (PrPc) in human blood. The aims of the present study were to monitor PrPc levels in various fractions of apheresis platelets during storage by using the DELFIA method and to assess the association of this release with alpha-granule protein ss-thrombo-globulin and cytoplasmic LDH. STUDY DESIGN AND METHODS: Units of apheresis platelets (n = 6) were obtained from volunteer donors by the use of a cell separator and stored up to 10 days. Samples (7-9 mL) were aseptically collected from each unit on storage Days 1, 2, 3, 4, 5, 8, and 10. Platelet-poor plasma and apheresis platelets were prepared and the former split into two fractions, one centrifuged at 40,000 x g for 2 hours at 4 degrees C to remove microparticles. The spun microparticles, apheresis platelets and platelet samples, platelet-poor plasma, and high-spun plasma fractions were stored in a frozen state until they were tested. RESULTS: The results showed that the mean overall levels of PrPc throughout storage remained within 15 percent of Day 1 levels. In contrast, the mean cellular levels in platelets significantly decreased to 46 percent of Day 1 levels by Day 10 of storage (p<0.01), while the corresponding levels in plasma significantly rose as much as 329 percent (p<0.01). Moreover, although microparticle-bound PrPc was released during storage, it was increasingly superseded by soluble protein. PrPc and ss-thrombo-globulin release exhibited very similar patterns (p<0.01). In contrast, LDH showed a significant increase in high-spun plasma only toward the end of the storage period (p<0.01). CONCLUSION: These results indicate that PrPc is released from platelets during the storage of apheresis platelets and that this release is probably due mainly to platelet activation and alpha-granule release in the first few days of storage. Moreover, the released PrPc is increasingly composed of soluble proteins, as the storage period exceeds 5 days.     

Platelet-white blood cell (WBC) interaction, WBC apoptosis, and procoagulant activity in stored red blood cells

BACKGROUND: Nonleukoreduced units of red blood cells (RBCs) contain activated platelets (PLTs) that interact with white blood cells (WBCs) and may promote inflammation and thrombosis in the recipient. The aim of this study was to characterize PLT‐WBC interactions (PLT‐WBC aggregates [PLAs]), WBC apoptosis, WBC death, and the development of procoagulant activity in RBCs during storage. STUDY DESIGN AND METHODS: RBCs were prepared from volunteer donor blood and stored. Samples were analyzed with flow cytometry between Days 1 and 15 to measure PLT‐monocyte aggregate (PMA) and PLT‐neutrophil aggregate (PNA) formation, WBC apoptosis (annexin V binding), and cell death (binding of 7‐aminoactinomycin D). Procoagulant activity in the supernatant of four RBC preparations was assessed between Days 1 and 39 using a clotting assay with and without the addition of an inhibitory anti‐tissue factor (TF) antibody, αTF‐5. RESULTS: PLA formation was extensive and maximal on Day 3 of storage (PNA, 23 ± 13%; PMA, 93 ± 4%; n = 6). Apoptosis was progressive throughout storage, with 95 ± 4% of neutrophils and 73 ± 19% of monocytes binding annexin V on Day 15. Cell death became measurable after apoptosis. Procoagulant activity was observed in all RBCs but with varying temporal patterns. It was partially TF dependent and removed with high‐speed centrifugation, suggestive of an association with microparticles. CONCLUSION: The activation of PLTs during the storage of RBCs induces PLA formation that precedes WBC apoptosis and death. Procoagulant activity, likely associated with microparticles derived from apoptotic WBCs, may contribute to adverse effects of stored, nonleukoreduced RBCs.     

Rejuvenation of irradiated AS-1 red cells

BACKGROUND: Gamma irradiation of blood components is used to prevent transfusion-associated graft-versus-host disease. The demand for irradiated blood components is increasing because of the increase in directed donation by family members. Irradiated units currently have a recommended maximum storage life of 28 days. Since in vivo recovery is related to red cell ATP levels, rejuvenation of stored irradiated units using a pyruvate-inosine phosphate-adenine additive was explored. STUDY DESIGN AND METHODS: Units of AS-1 red cells from 16 volunteer donors were divided into two equal volumes and one split unit from each was irradiated with 25 Gy. Ten units were irradiated on Day 5, 6, or 7 of 4 degrees C storage and 6 units were irradiated on Day 1 of 4 degrees C storage. All units were rejuvenated for 1 hour at 37 degrees C using a pyruvate-inosine-phosphate-adenine additive on Day 42 of 4 degrees C storage. Units were assayed for ATP, 2, 3 DPG and supernatant sodium, potassium, and glucose. RESULTS: ATP and 2, 3 DPG levels were restored equally well in irradiated and non-irradiated units. The previously reported irradiation-induced red cell potassium-sodium shift was demonstrated. Supernatant potassium and sodium levels did not reverse 1 hour after rejuvenation was completed. There was no significant difference in results between units irradiated on Day 1 or Day 5, 6, or 7. CONCLUSION: Red cell ATP and 2, 3 DPG levels were restored in irradiated AS-1 units stored at 4 degrees C for 42 days using a pyruvate-inosine-phosphate-adenine rejuvenation additive.     

The proteome of red cell membranes and vesicles during storage in blood bank conditions

BACKGROUND: During storage of red cells (RBCs) for transfusion, RBCs undergo a number of biochemical and morphologic changes. To be able to identify the mechanisms underlying these storage lesions, a proteomic analysis of the membranes of RBCs and their vesicles was performed during various periods of storage in blood bank conditions. STUDY DESIGN AND METHODS: RBCs and vesicles were isolated from RBCs after various storage periods. The proteins of RBC membranes and vesicles were separated by gel electrophoresis and identified by a semiquantitative proteomic analysis. RESULTS: Our findings confirm previous data, such as a storage-associated increase in hemoglobin binding to the membrane and aggregation and degradation of the integral membrane protein band 3, suggesting a remodeling of the RBC membrane during storage. Our data also show storage-dependent changes in the membrane association of proteasome and chaperone proteins, metabolic enzymes, small G proteins, and signal transduction proteins. Vesicles display similar changes in their protein composition during storage. CONCLUSION: The results of this analysis indicate that the storage-related changes in the RBC membrane are the results of disturbance and/or acceleration of physiologic processes such as cellular aging, including vesicle formation. The latter may serve to remove damaged membrane patches that would otherwise lead to accelerated RBC removal. These data provide a framework for future studies toward the development of better storage conditions and a reduction of the side effects of RBC transfusion.     

Influence of prestorage leukoreduction and subsequent irradiation on in vitro red blood cell (RBC) storage variables of RBCs in additive solution saline-adenine-glucose-mannitol

BACKGROUND: There exists only very few data on in vitro and in vivo effects of gamma irradiation of red blood cells (RBCs) that have been leukoreduced by filtration before a subsequent irradiation. Reported studies reflect neither the current Food and Drug Administration (FDA) nor the European recommendations on timing of irradiation and subsequent storage.
STUDY DESIGN AND METHODS: We studied 40 RBC units that were prepared from inline filtered whole blood and 40 RBC units that were filtered after component separation. All RBCs were stored in the additive solution saline-adenine-glucose-mannitol and leukoreduced on the collection day. In both groups, 20 components were irradiated on Day +14 with 30 Gy, and 20 served as nonirradiated controls. In vitro evaluation of both irradiated and nonirradiated RBC units was performed before and after irradiation on Days +1, +7, +14, +21, +28, +35, and +42 from the collection day.
RESULTS: Gamma irradiation induced enhanced leakage of potassium ions and lactate dehydrogenase and an enhanced in vitro hemolysis rate in the irradiated components. However, in vitro hemolysis rate of both nonirradiated and irradiated components was remarkably lower than 0.8 percent, and the preservation of adenosine triphosphate over 42 days was satisfying.
CONCLUSIONS: This study reflects the current FDA and European recommendations on timing of irradiation and subsequent storage. Our findings together with recent results of other investigations on the effect of gamma irradiation on leukoreduced RBCs allow the proposal that a storage time up to 28 days after irradiation is allowable.     

Addition of L-carnitine to additive solution-suspended red cells stored at 4 degrees C reduces in vitro hemolysis and improves in vivo viability

BACKGROUND: The role of L-carnitine (LC) as the requisite carrier of long-chain fatty acids into mitochondria is well established. Human red cells (RBCs), which lack mitochondria, possess a substantial amount of LC and its esters. In addition, carnitine palmitoyl transferase, an enzyme that catalyzes the reversible transfer of the acyl moiety from acyl-coenzyme A to LC is found in RBCs. It has recently been shown that LC and carnitine palmitoyl transferase play a major role in modulating the pathway for the turnover of membrane phospholipid fatty acids in intact human RBCs, and that LC improved the membrane stability of RBCs subjected to high shear stress. RBC membrane lesions occur during storage at 4 degrees C; this study investigated whether the addition of LC (5 mM) to a standard RBC preservative solution (AS-3) affected cellular integrity with 42 days' storage. STUDY DESIGN AND METHODS: A paired (n = 10) crossover design was used for RBCs stored in AS-3 with and without LC. Both in vitro RBC properties reflective of metabolic and membrane integrity and in vivo measures of cell viability (24-hour percentage of recovery and circulating lifespan) were measured at the end of the storage. In addition, the turnover of membrane phospholipid and long-chain acylcarnitine fatty acids and the carnitine content of control and LC-stored RBCs were measured. RESULTS: It was shown that LC was irreversibly taken up by RBCs during storage, with a fourfold increase at 42 days. Furthermore, as found by the use of radiolabeled palmitate, the stored RBCs were capable of generating long-chain acylcarnitine. The uptake of LC during storage was associated with less hemolysis and higher RBC ATP levels and by a significantly greater in vivo viability for LC-stored RBCs than for control-stored RBCs: a mean 24-hour percentage of recovery of 83.9 +/? 5.0 vs. 80.1 +/? 6.0 percent and a mean lifespan of 96 +/? 11 vs. 86 +/? 14 days, respectively (p < 0.05). CONCLUSION: A beneficial effect of the addition of LC to RBCs stored at 4 degrees C was evident. This effect may be related to both biophysical and metabolic actions on the cell membrane.     

Supernates from stored red blood cells inhibit platelet aggregation

BACKGROUND: Previously, we reported that red blood cells (RBCs) stored in AS‐5 accumulated proinflammatory substances during storage. We observed in those studies that supernates from nonleukoreduced (NLR) RBCs reduced mean anti‐CD41a‐fluorescein isothiocyanate (FITC) fluorescence on platelets (PLTs), indicative of decreased expression of glycoprotein (GP)IIb/IIIa on the PLT membrane. The objective of this study was to determine if supernates from stored RBCs impaired PLT aggregation as a consequence of reduction in GPIIb/IIIa expression. STUDY DESIGN AND METHODS: Leukoreduced (LR) and NLR RBC units were prepared in AS‐5 and stored at 1 to 6°C for 6 weeks. Supernates from RBC samples collected every 2 weeks were mixed with freshly collected type‐matched blood and incubated for 30 minutes at 37°C. PLTs in each incubated blood sample were evaluated for GPIIb/IIIa expression by flow cytometry and for aggregation response to collagen by whole blood aggregometry. RESULTS: Supernates from stored NLR RBCs reduced CD41a‐FITC fluorescence on PLTs by 15% to 31%. A reduction in fluorescence was induced by supernates of RBCs stored for 14 days and increased as storage time increased. Supernates from Day 42 NLR RBCs reduced the mean amplitude of PLT aggregation by 31% compared to Day 0 supernates and lengthened the time before onset of aggregation by 21%. In addition, amplitude correlated directly and lag time correlated inversely with CD41a‐FITC fluorescence in all samples. Supernates from prestorage LR RBCs did not affect PLT CD41a‐FITC fluorescence or aggregation response. CONCLUSIONS: Substances that decrease expression of GPIIb/IIIa and inhibit PLT aggregation accumulate in NLR RBCs. Accumulation of this material is prevented by leukoreduction.     

Posttransfusion survival (24-hour) and hemolysis of previously frozen, deglycerolized RBCs after storage at 4 degrees C for up to 14 days in sodium chloride alone or sodium chloride supplemented with additive solutions

BACKGROUND: Previously frozen human RBCs currently are glycerolized and deglycerolized by the use of open systems that limit storage of the deglycerolized RBCs at 4 degrees C to only 24 hours. STUDY DESIGN AND METHODS: Healthy male volunteers who met AABB requirements for blood donors (n = 38) were studied. A volume of 450 mL of blood was collected into CPDA-1. The RBC concentrates were stored at 4 degrees C for 3 to 6 days before being frozen with 40-percent (wt/vol) glycerol and stored at -80 degrees C. The RBCs were deglycerolized, resuspended in 0.9-percent sodium chloride and 0.2-percent glucose (SG) solution or SG solution supplemented with AS-1, AS-3, or AS-5, and stored in the resuspension medium at 4 degrees C for 14 days. RESULTS: The mean +/- SD freeze-thaw-wash process recovery was 90.0 +/- 4.0 percent for all 38 units. The mean 24-hour posttransfusion survival value was 79 percent for deglycerolized RBC stored at 4 degrees C for 7 days in SG alone, SG plus AS-3, or SG plus AS-5. Deglycerolized RBC that were stored at 4 C for 14 days in SG supplemented with AS-1, AS-3, or AS-5 had a mean 24-hour posttransfusion survival of 74 percent. After 7 days of storage of deglycerolized RBCs in SG alone, the mean hemolysis was 3. 7 percent. After 14 days of storage of deglycerolized RBCs in SG supplemented with AS-1, AS-3, or AS-5, the mean hemolysis was 2.5 percent. CONCLUSIONS: The levels of hemolysis did not correlate with the 24-hour posttransfusion survival values.     

Viability and in vitro properties of AS-1 red cells after gamma irradiation

BACKGROUND: Irradiation has been shown to adversely affect both in vivo 24-hour recovery (recovery [%]) and in vitro properties of stored red cells (RBCs). There is uncertainty as to how these changes are related to the day of irradiation and the length of storage after irradiation. STUDY DESIGN AND METHODS: Four protocols used day of irradiation and storage time after irradiation as the independent variables. At the conclusion of the storage period, viability was measured with radiolabeled RBCs as the recovery and the long-term survival time for RBCs that were circulating beyond 24 hours. In addition, in vitro values including RBC ATP, hemolysis level, and supernatant potassium were measured. Each subject donated 2 units of whole blood (CPD) and received autologous irradiated and untreated control RBCs (AS-1) on two separate occasions. RESULTS: Reduced recovery in irradiated units was noted when compared to that in control units, and the reduction was most apparent with long periods of storage after irradiation, irrespective of the day of irradiation. With irradiation on Day 1 of storage and a total storage period of 28 days, mean +/- SD recovery (single label) was 84.2 +/- 5.1 percent for control RBCs and 78.6 +/- 5.9 percent for irradiated RBCs (n = 16; p<0.01). With irradiation on Day 14 and storage through Day 42, the recoveries were 76.3 +/- 7.0 percent for control RBCs and 69.5 +/- 8.6 percent for irradiated RBCs (n = 16; p<0.01). Less reduction in recovery was observed with shortening of the postirradiation storage time. When the total storage period was reduced to 28 days after Day 14 irradiation, the recoveries were not significantly different. With an additional 2-day storage period after irradiation on Day 26, the recoveries were also comparable. Long-term survival times for control and irradiated RBCs were not significantly different in any of the four protocols. RBC ATP levels and hemolysis were minimally, but significantly influenced by irradiation. Supernatant potassium levels, however, were substantially increased after irradiation in each of the four protocols. CONCLUSION: Irradiation has only a small effect on the properties of RBCs treated and stored according to the utilized protocols. Longer storage times after irradiation resulted in progressively reduced recovery while long-term survival remained unaffected.     

A single assay for multiple storage‐sensitive red blood cell characteristics by means of infrared spectroscopy

BACKGROUND: To maintain a high quality of red blood cells (RBCs), RBC characteristics must be followed during storage under blood bank conditions. By means of infrared (IR) spectroscopy, several characteristics can be measured simultaneously. STUDY DESIGN AND METHODS: IR spectra were acquired for samples from RBCs that were collected and stored according to Dutch blood bank procedures for a period of up to 50 days. Spectra of the soluble cell components were acquired separately after hypotonic lysis of the cells, followed by centrifugation. Characteristic vibrational bands were analyzed with respect to storage time–dependent changes in peak position and in intensity. RESULTS: A decrease in corresponding peak intensities indicates that RBCs lose protein and lipid during storage. Changes in protein secondary structure during storage are largely confined to integral membrane proteins and membrane‐associated proteins. A concurrent decrease in lipid packing density probably reflects the gradual change in cellular shape from discoidal to globular. By integration over a narrow range, storage‐dependent changes in intracellular adenosine triphosphate (ATP) and glucose levels could be estimated. ATP levels decrease during storage, but stay above the required 75% of the initial level after 35 days of storage. Glucose concentrations stay well above 5 mmol/L over the entire storage period. CONCLUSION: IR spectroscopy is a promising technique to follow structural and metabolic changes in RBCs during storage under blood bank conditions. Several variables can be determined rapidly in a single measurement.