Peroxiredoxin-2 as a candidate biomarker to test oxidative stress levels of stored red blood cells under blood bank conditions

BACKGROUND: Several researches on aging red blood cells (RBCs)—performed both in vivo and under blood bank conditions—revealed that RBC membrane proteins undergo a number of irreversible alterations, mainly due to oxidative stress. The individuation of proteins to be used as indicators of irreversible RBC injury and to be proposed as candidate biomarkers of oxidative damage or aging status during blood storage is therefore of great interest. STUDY DESIGN AND METHODS: Based on this purpose we performed proteomic analysis of the membranes of RBCs during various storage periods under blood bank conditions. Changes in protein composition of RBC membranes were monitored as a function of the storage period by means of polyacrylamide gel electrophoresis coupled with immunoblotting and mass spectrometry analyses. RESULTS: During storage, a progressive linkage of typical cytosolic proteins to the membrane was detected, including both antioxidant and metabolic enzymes (such as catalase, peroxiredoxin‐2 [Prx2], and 2,3‐bisphosphoglycerate‐mutase), as well as nonreducible cross‐linkings of probably oxidized or denatured hemoglobin. This phenomenon was unequivocally related to oxidative stress, since storage of RBCs under anaerobic conditions showed a suppression of these protein recruitments to the membrane. CONCLUSION: The detailed analysis of these protein associations to the membrane of aged RBCs allowed Prx2 to be suggested as a potential RBC oxidative stress marker for the sake of developing new approaches in quality assurance of blood components.     

Red blood cell aging markers during storage in citrate‐phosphate‐dextrose–saline‐adenine‐glucose‐mannitol

BACKGROUND: It has been suggested that red blood cell (RBC) senescence is accelerated under blood bank conditions, although neither protein profile of RBC aging nor the impact of additive solutions on it have been studied in detail. STUDY DESIGN AND METHODS: RBCs and vesicles derived from RBCs in both citrate‐phosphate‐dextrose (CPD)–saline‐adenine‐glucose‐mannitol (SAGM) and citrate‐phosphate‐dextrose‐adenine (CPDA) were evaluated for the expression of cell senescence markers (vesiculation, protein aggregation, degradation, activation, oxidation, and topology) through immunoblotting technique and immunofluorescence or immunoelectron microscopy study. RESULTS: A group of cellular stress proteins exhibited storage time– and storage medium–related changes in their membrane association and exocytosis. The extent, the rate, and the expression of protein oxidation, Fas oligomerization, caspase activation, and protein modifications in Band 3, hemoglobin, and immunoglobulin G were less conspicuous and/or exhibited significant time retardation under storage in CPD‐SAGM, compared to the CPDA storage. There was evidence for the localization of activated caspases near to the membrane of both cells and vesicles. CONCLUSIONS: We provide circumstantial evidence for a lower protein oxidative damage in CPD‐SAGM–stored RBCs compared to the CPDA‐stored cells. The different expression patterns of the senescence markers in the RBCs seem to be accordingly related to the oxidative stress management of the cells. We suggest that the storage of RBCs in CPD‐SAGM might be more alike the in vivo RBC aging process, compared to storage in CPDA, since it is characterized by a slower stimulation of the recognition signaling pathways that are already known to trigger the erythrophagocytosis of senescent RBCs.     

IMMUNOHEMATOLOGY: Storage of murine red blood cells enhances alloantibody responses to an erythroid‐specific model antigen

BACKGROUND: 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. STUDY DESIGN AND METHODS: 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 posttransfusion RBC survival were analyzed by flow cytometry. RBC alloimmunization was monitored by measuring circulating anti‐HEL immunoglobulin levels. RESULTS: 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. CONCLUSIONS: 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.     

Transfusion of stored red blood cells adhere in the rat microvasculature

BACKGROUND: Ex vivo storage of red blood cells (RBCS) for transfusions is associated with a “storage lesion,” which decreases RBC deformability and increases RBC adhesiveness to vascular endothelium. This may impair microcirculatory flow with deleterious effects on oxygen delivery after transfusion. Previous studies have shown that human RBCs adhere to endothelial monolayers in vitro with prolonged storage and is reduced by prestorage leukoreduction (LR). The objective of this study was to determine whether duration of RBC storage and LR influence RBC adhesion in vivo in capillaries. STUDY DESIGN AND METHODS: Rat RBCs were collected and stored in CPDA‐1 under standard blood bank conditions. Three RBC products were compared: 1) fresh RBCs, less than 24 hours of storage (n = 6); 2) nonleukoreduced (NLR) RBCs stored for 7 days (n = 6); and 3) prestorage LR RBCs stored for 7 days (n = 6). RBCs were labeled with fluorescein isothiocyanate (FITC) 24 hours before transfusion and reinjected in an isovolemic manner into healthy rats. The FITC‐labeled RBCs were visualized in the extensor digitorum longus muscle using intravital video microscopy (20× magnification). The number of RBCs adherent in capillaries was counted 1 hour after transfusion in 10 random fields and the median values were compared with one‐way analysis of variance. RESULTS: Stored RBCs showed increased levels of adherence in capillaries compared to their fresh counterparts (p < 0.05). Prestorage LR decreased RBC adherence to levels equivalent to those of fresh RBCs (p < 0.05 for stored LR vs. stored NLR). CONCLUSION: Rat RBCs stored under conditions that closely mimicked clinical transfusion adhere in capillaries. The decreased RBC adherence with LR suggest a direct effect of white blood cells or their byproducts on RBC deformability and/or adhesiveness to microvascular endothelium. Further study will examine the mechanism of adherence and the impact it has on microcirculatory flow and oxygen delivery in the critically ill host.     

Is red blood cell rheology preserved during routine blood bank storage?

BACKGROUND: Red blood cell (RBC) units stored for more than 2 weeks at 4°C are currently considered of impaired quality. This opinion has primarily been based on altered RBC rheologic properties (i.e., enhanced aggregability, reduced deformability, and elevated endothelial cell interaction), during prolonged storage of nonleukoreduced RBC units. In this study, the rheologic properties and cell variables of leukoreduced RBC units, during routine blood bank storage in saline‐adenine‐glucose‐mannitol, were investigated. STUDY DESIGN AND METHODS: Ten leukoreduced RBC units were stored at the blood bank for 7 weeks at 4°C. RBCs were tested weekly for aggregability, deformability, and other relevant variables. RESULTS: RBC aggregability was significantly reduced after the first week of storage but recovered during the following weeks. After 7 weeks aggregability was slightly, but significantly, reduced (46.9 ± 2.4‐44.3 ± 2.2 aggregation index). During storage the osmotic fragility was not significantly enhanced (0.47 ± 0.01% phosphate‐buffered saline) and the deformability at shear stress of 3.9 Pa was not significantly reduced (0.36 ± 0.01 elongation index [EI]). The deformability at 50 Pa was reduced (0.58 ± 0.01‐0.54 ± 0.01 EI) but remained within reference values (0.53 ± 0.04). During 5 weeks of storage, adenosine triphosphate was reduced by 54% whereas mean cell volume, pH, and mean cell hemoglobin concentration were minimally affected. CONCLUSIONS: RBC biochemical and physical alterations during storage minimally affected the RBC ability to aggregate and deform, even after prolonged storage. The rheologic properties of leukoreduced RBC units were well preserved during 7 weeks of routine blood bank storage.     

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

Introduction  

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.     

Clinical consequences of red cell storage in the critically ill

Red cell (RBC) transfusions are a potentially life-saving therapy employed during the care of many critically ill patients to replace losses in hemoglobin to maintain oxygen delivery to vital organs. During storage, RBCs undergo a series of biochemical and biomechanical changes that reduce their survival and function. Additionally, accumulation of other biologic by-products of RBC preservation may be detrimental to recipients of blood transfusions. Laboratory studies and an increasing number of observational studies have raised the possibility that prolonged RBC storage adversely affects clinical outcomes. In this article, the laboratory and animal experiments evaluating changes to RBCs during prolonged storage are reviewed. Subsequently, the clinical studies that have evaluated the clinical consequences of prolonged RBC storage are reviewed. These data suggest a possible detrimental clinical effect associated with the transfusion of stored RBCs; randomized clinical trials further evaluating the clinical consequences of transfusing older stored RBCs are required.     

Metabolomics of ADSOL (AS-1) Red Blood Cell Storage

Population-based investigations suggest that red blood cells (RBCs) are therapeutically effective when collected, processed, and stored for up to 42 days under validated conditions before transfusion. However, some retrospective clinical studies have shown worse patient outcomes when transfused RBCs have been stored for the longest times. Furthermore, studies of RBC persistence in the circulation after transfusion have suggested that considerable donor-to-donor variability exists and may affect transfusion efficacy. To understand the limitations of current blood storage technologies and to develop approaches to improve RBC storage and transfusion efficacy, we investigated the global metabolic alterations that occur when RBCs are stored in AS-1 (AS1-RBC). Leukoreduced AS1-RBC units prepared from 9 volunteer research donors (12 total donated units) were serially sampled for metabolomics analysis over 42 days of refrigerated storage. Samples were tested by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry, and specific biochemical compounds were identified by comparison to a library of purified standards. Over 3 experiments, 185 to 264 defined metabolites were quantified in stored RBC samples. Kinetic changes in these biochemicals confirmed known alterations in glycolysis and other pathways previously identified in RBCs stored in saline, adenine, glucose and mannitol solution (SAGM-RBC). Furthermore, we identified additional alterations not previously seen in SAGM-RBCs (eg, stable pentose phosphate pathway flux, progressive decreases in oxidized glutathione), and we delineated changes occurring in other metabolic pathways not previously studied (eg, S-adenosyl methionine cycle). These data are presented in the context of a detailed comparison with previous studies of SAGM-RBCs from human donors and murine AS1-RBCs. Global metabolic profiling of AS1-RBCs revealed a number of biochemical alterations in stored blood that may affect RBC viability during storage as well as therapeutic effectiveness of stored RBCs in transfusion recipients. These results provide future opportunities to more clearly pinpoint the metabolic defects during RBC storage, to identify biomarkers for donor screening and prerelease RBC testing, and to develop improved RBC storage solutions and methodologies.     

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.     

Advanced glycation end products on stored red blood cells increase endothelial reactive oxygen species generation through interaction with receptor for advanced glycation end products

BACKGROUND: Recent evidence suggests that storage‐induced alterations of the red blood cell (RBC) are associated with adverse consequences in susceptible hosts. As RBCs have been shown to form advanced glycation end products (AGEs) after increased oxidative stress and under pathologic conditions, we examined whether stored RBCs undergo modification with the specific AGE N‐(carboxymethyl)lysine (N^ε‐CML) during standard blood banking conditions. STUDY DESIGN AND METHODS: Purified, fresh RBCs from volunteers were compared to stored RBCs (35‐42 days old) obtained from the blood bank. N^ε‐CML formation was quantified using a competitive enzyme‐linked immunosorbent assay. The receptor for advanced glycation end products (RAGE) was detected in human pulmonary microvascular endothelial cells (HMVEC‐L) by real‐time polymerase chain reaction, Western blotting, and flow cytometry. Intracellular reactive oxygen species (ROS) generation was measured by the use of 5‐(and 6‐)chloromethyl‐2′,7′‐dichlorodihydrofluorescein diacetate, acetyl ester–based assays. RESULTS: Stored RBCs showed increased surface N^ε‐CML formation when compared with fresh RBCs. HMVEC‐L showed detectable surface RAGE expression constitutively. When compared to fresh RBCs, stored RBCs triggered increased intracellular ROS generation in both human umbilical vein endothelial cells and HMVEC‐L. RBC‐induced endothelial ROS generation was attenuated in the presence of soluble RAGE or RAGE blocking antibody. CONCLUSIONS: The formation of the AGE N^ε‐CML on the surface of stored RBCs is one functional consequence of the storage lesion. AGE‐RAGE interactions may be one mechanism by which transfused RBCs cause endothelial cell damage.     

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.     

Chronological storage age and metabolic age of stored red blood cells: are they the same?

• Red blood cell (RBC) storage in the blood bank is characterized by the progressive loss of metabolic regulation, a phenomenon that targets energy and antioxidant metabolism;
• While the progression of the storage lesion is inevitable, the rate at which this phenomenon occurs varies from donor to donor;
• Red blood cells from some donors at the end of storage are metabolically superior to RBCs from other donors at the beginning of storage, suggesting that the age of blood alone may not be a sufficiently accurate predictor of stored blood quality; and
• Animal studies and large‐scale omics screening in blood donors have helped identify mechanistic contributors to the metabolic heterogeneity of stored blood units.

     

Reduced deformability of stored red blood cells is associated with generation of extracellular vesicles

Throughout storage, red blood cells (RBCs) undergo detrimental changes in viability and their ability to effectively transport oxygen. RBC storage lesions are mediated, in part, by a progressive loss of cell deformability, and associated with the release of extracellular vesicles (EVs). Accumulation of EVs during the storage of RBCs correlates with a decrease in RBC surface area to volume ratio. Similarly, the loss of RBC-deformability is associated with loss of RBC surface area to volume ratio. In this study we thus tested whether loss of RBC-deformability is associated with increased RBC-EV production during blood storage.EVs obtained by differential centrifugation of stored RBCs (non-leukoreduced non-irradiated or leukoreduced γ-irradiated RBCs stored 35 or 28 days respectively) were enumerated by high-sensitivity flow cytometry. RBC deformability was quantified, using a cell-flow-properties-analyzer, by measuring the median cell elongation ratio (MER) and percentage of low and high deformable cells in the population (%, LDFC, and HDFC, respectively).The number of EVs was inversely correlated with the MER and positively correlated with the %LDFC with both measures showing highly significant logarithmic dependence with EV levels in stored RBCs. Considering how highly deformable cells did not correlate with EV formation as compared with low deformable RBCs we propose that the formation of EVs is a key factor leading to increased RBC-rigidity.     

RBC storage for 11 weeks

BACKGROUND: Increasing the length of RBC storage can increase both RBC availability and quality. This work addresses 11-week RBC storage in experimental ASs (EASs). STUDY DESIGN AND METHODS: Three studies were performed. In the first, 24-hour in vivo recovery of (51)Cr-labeled autologous RBCs was measured in nine volunteers after storage of their RBCs for 11 weeks in EAS 67. In the second study, 4 units of blood were divided and stored in aliquots with an EAS containing 0, 15, 30, or 45 mmol per L of mannitol; then hemolysis, RBC morphology, and microvesicle protein were measured. In the third study, 6 full units were stored for 12 weeks in the EAS containing 30 mmol per L of mannitol, with weekly sampling for morphologic and biochemical measures of RBC quality. RESULTS: RBCs stored for 11 weeks in EAS-67 had a mean 24-hour in vivo recovery of 79 +/- 5 percent, but the hemolysis was 1.35 +/- 0.68 percent. Increasing mannitol content of the EAS reduced hemolysis but increased microvesiculation. EAS-76, with 30 mmol per L of mannitol allowed 11-week storage with 0.48 +/- 0.10 percent hemolysis at 11 weeks and 0.62 +/- 0.14 percent hemolysis at 12 weeks. CONCLUSION: It is possible to store RBCs for 11 weeks in EAS with greater than 75 percent recovery and less than 1 percent hemolysis.     

Artificial microvascular network: a new tool for measuring rheologic properties of stored red blood cells

BACKGROUND: The progressive deterioration of red blood cell (RBC) rheologic properties during refrigerated storage may reduce the clinical efficacy of transfusion of older units. STUDY DESIGN AND METHODS: This article describes the development of a microfluidic device designed to test the rheologic properties of stored RBCs by measuring their ability to perfuse an artificial microvascular network (AMVN) comprised of capillary‐size microchannels arranged in a pattern inspired by the real microvasculature. In the AMVN device, the properties of RBCs are evaluated by passing a 40% hematocrit suspension of RBCs through the network and measuring the overall perfusion rate. RESULTS: The sensitivity of the AMVN device to the storage‐induced change in rheologic properties of RBCs was tested using five prestorage leukoreduced RBC units stored in AS‐1 for 41 days. The AMVN perfusion rate for stored RBCs was 26 ± 4% (19%‐30%) lower than for fresh RBCs. Washing these stored RBCs in saline improved their performance by 41 ± 6% (the AMVN perfusion rate for washed stored RBCs was still 15 ± 2% lower than for fresh RBCs). CONCLUSIONS: The measurements performed using the AMVN device confirm a significant decline in the rheologic properties of RBCs in units nearing expiration and demonstrate the sensitivity of the device to these storage‐induced changes. The AMVN device may be useful for testing the effect of new storage conditions, additive solutions, and rejuvenation strategies on the rheologic properties of stored RBCs in vitro.     

Transfusion of fresh murine red blood cells reverses adverse effects of older stored red blood cells

BACKGROUND: Although a subset of recent studies have suggested that red blood cell (RBC) storage length is associated with adverse patient outcomes, others have shown no such relationship. Adults may be transfused with RBC units of different storage lengths, and existing studies do not take into consideration that fresh RBCs may alter responses to concurrently transfused stored RBCs. To test this possibility, we utilized a murine model and investigated transfusion outcomes of fresh, stored, or fresh‐plus‐stored RBCs. STUDY DESIGN AND METHODS: Fresh, 14‐day‐stored or fresh plus 14‐day‐stored leukoreduced RBCs from HOD‐transgenic donors (with RBC‐specific expression of hen egg lysozyme, ovalbumin, and human Duffy^b) were transfused into naïve C57BL/6 recipients. Serum cytokines and anti‐HOD alloimmunization were evaluated after transfusion. RESULTS: In six of six experiments (n = 90 mice total), a proinflammatory serum cytokine storm of interleukin‐6, keratinocyte‐derived chemokine/CXCL1, and monocyte chemoattractant protein‐1 was observed in transfusion recipients of stored but not fresh RBCs, along with high degrees of anti‐HOD alloimmunization. However, concurrent transfusion of fresh HOD RBCs along with stored HOD RBCs significantly decreased these adverse outcomes (p < 0.05). CONCLUSIONS: These results are consistent with fresh murine HOD RBCs losing protective properties during storage, and introduce a previously unrecognized variable in RBC storage studies. If translatable to humans, uniform “old blood” groups may be needed in future clinical studies to more accurately investigate the biologic effects of older RBC units.     

Insufficient nitric oxide bioavailability: a hypothesis to explain adverse effects of red blood cell transfusion

While transfusion of red blood cells (RBCs) is effective at preventing morbidity and mortality in anemic patients, studies have indicated that some RBC components have functional defects ("RBC storage lesions") that may actually cause adverse events when transfused. For example, in some studies patients transfused with RBCs stored more than 14 days have had statistically worse outcomes than those receiving "fresher" RBC units. Recipient-specific factors may also contribute to the occurrence of these adverse events. Unfortunately, these events have been difficult to investigate because up to now they have existed primarily as "statistical occurrences" of increased morbidity and mortality in large data sets. There are currently no clinical or laboratory methods to detect or study them in individual transfusion recipients. We propose a unifying hypothesis, centered on insufficient nitric oxide bioavailability (INOBA), to explain the increased morbidity and mortality observed in some patients after RBC transfusion. In this model, variables associated with RBC units (storage time; 2,3-diphosphoglycerate acid concentration) and transfusion recipients (endothelial dysfunction) collectively lead to changes in nitric oxide (NO) levels in vascular beds. Under certain circumstances, these variables are "aligned" such that NO concentrations are markedly reduced, leading to vasoconstriction, decreased local blood flow, and insufficient O(2) delivery to end organs. Under these circumstances, the likelihood of morbidity and mortality escalates. If the key tenets of the INOBA hypothesis are confirmed, it may lead to improved transfusion methods including altered RBC storage and/or processing conditions, novel transfusion recipient screening methods, and improved RBC-recipient matching.     

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.     

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.     

Twelve-week RBC storage

BACKGROUND: Better storage can improve RBC availability and safety. Optimizing RBC ATP production and minimizing hemolysis has allowed progressively longer storage. STUDY DESIGN AND METHODS: In the first study, 24 units of packed CPD RBCs were pooled in groups of four, realiquoted, and added to 300 mL of one of four variants of experimental additive solution 76 (EAS-76) containing 45, 40, 35, or 30 mEq per L NaCl. Units were sampled weekly for 12 weeks for morphologic and biochemical measures. In the second study, 10 volunteers donated 2 units of RBCs for a crossover comparison of Tc/Cr 24-hour in vivo recovery of 6-week storage in AS-1 versus 12-week storage in EAS-76 variant 6 (EAS-76v6) having 30 mEq per L NaCl. RESULTS: RBCs stored in the lower salt variants of EAS-76 had higher concentrations of RBC ATP with less hemolysis and microvesiculation. RBC 2,3 DPG was preserved for two weeks. RBCs stored for 12 weeks in EAS-76v6 exhibited 78 +/- 4 percent 24-hour in vivo recovery. CONCLUSIONS: It is possible to store RBCs for 12 weeks with acceptable recovery and 0.6 percent hemolysis and with normal 2,3 DPG concentrations for 2 weeks.