Red Blood Cell Storage Lesion Research Articles

Upregulation of Mir-20a-5p as the Potential Microrna Marker in Red Blood Cell Storage Lesion

Upregulation of Mir-20a-5p as the Potential Microrna Marker in Red Blood Cell Storage Lesion

Storage Lesions in Red Blood Cell-Saline Adenine Glucose Mannitol

Background and Objectives: Indian studies on evaluation of storage lesions in red blood cells (RBCs) are either limited to 21 or 28 days or have evaluated limited parameters for 42 days. Moreover, issue of transfusion of “fresh” versus “old” RBC is far from settled. The study serially assesses, up to 42 days, in vitro and in vivo RBC storage lesion parameters, including di-(2-ethylhexyl) phthalate (DEHP) leaching and its comparison with published literature. Methods: The study serially assessed in vitro RBC storage lesion parameters including potassium, lactate, glucose, pH, supernatant hemoglobin, percentage-hemolysis, and DEHP leaching of RBC unit during storage till 42 days. The study also evaluated in vivo recovery of potassium after transfusion of “older” RBC. Results: Serial monitoring of in vitro biochemical parameters showed increase in potassium, lactate, supernatant Hb, and hemolysis% and reduction in glucose and pH. DEHP content of the RBC bag was within no-observed adverse effect limit on days 42. Measurement of serum potassium after transfusion of “older” RBC unit revealed that levels of potassium were within normal limit in all four patients. Sterility testing done on days 42 was negative for all 24 bags. Conclusion: Development of storage lesions is inevitable. Appropriate storage limits the RBC lesions to within normal limits. The increase in potassium, lactate, or hemolysis consequent to aging of blood has little clinical significance in routine transfusion practice.

Improving packed red blood cell storage with a high-viscosity buffered storage solution

BackgroundMassive transfusion with older packed red blood cells is associated with increased morbidity and mortality. As packed red blood cells age, they undergo biochemical and structural changes known as the storage lesion. We developed a novel solution to increase viscosity in stored packed red blood cells. We hypothesized that packed red blood cell storage in this solution would blunt storage lesion formation and mitigate the inflammatory response after resuscitation. MethodsBlood was obtained from 8- to 10-week-old C57BL/6 male donor mice or human volunteers and stored as packed red blood cell units for 14 days for mice or 42 days for humans in either standard AS-3 storage solution or EAS-1587, the novel packed red blood cell storage solution. Packed red blood cells were analyzed for microvesicles, cell-free hemoglobin, phosphatidylserine, band-3 protein, glucose utilization, and osmotic fragility. Additional mice underwent hemorrhage and resuscitation with packed red blood cells stored in either AS-3 or EAS-1587. Serum was analyzed for inflammatory markers. ResultsMurine packed red blood cells stored in EAS-1587 demonstrated reductions in microvesicle and cell-free hemoglobin accumulation as well as preserved band-3 expression, increase glucose utilization, reductions in phosphatidylserine expression, and susceptibility to osmotic stress. Serum from mice resuscitated with packed red blood cells stored in EAS-1587 demonstrated reduced proinflammatory cytokines. Human packed red blood cells demonstrated a reduction in microvesicle and cell-free hemoglobin as well as an increase in glucose utilization. ConclusionStorage of packed red blood cells in a novel storage solution mitigated many aspects of the red blood cell storage lesion as well as the inflammatory response to resuscitation after hemorrhage. This modified storage solution may lead to improvement of packed red blood cell storage and reduce harm after massive transfusion.

Storage of red blood cell concentrates: Clinical impact.

The storage of red blood cells for transfusion purposes induces modifications of biochemical and biological properties. Moreover, these modifications are modulated by the donors' characteristics and the cell processing. These ex vivo alterations were suspected to decrease the transfusion efficiency and even to induce adverse events. This short article will review the red blood cells storage lesions and the clinical data related to them. In particular, the questions regarding the donors and recipients sex will be discussed.

Metabolic profile of irradiated whole blood by chemical isotope-labeling liquid chromatography-mass spectrometry

Irradiated blood is a new type of blood product used to prevent transfusion-associated graft-versus-host disease. However, the effects of irradiation on the metabolism of plasma, red blood cells (RBCs), and peripheral blood mononuclear cells (PBMCs) are largely unknown. We developed a workflow for testing metabolic changes in whole blood to determine the impact of irradiation by chemical isotope labeling liquid chromatography-mass spectrometry (CIL LC–MS). Blood parameters, PBMC proliferation and apoptosis were examined before and after irradiation. Next, the amine/phenol metabolites in the blood components were assayed by 12C- and13C-dansylation labeling LC–MS. We identified 1654, 1730, and 1666 peak pairs in plasma, RBCs, and PBMCs, respectively. We screened out 367, 177, and 219 significant metabolites in plasma, RBCs, and PBMCs, respectively, by principle component analyses, volcano plots, and Venn plots. Metabolic pathway analyses showed that irradiation modulated taurine and hypotaurine metabolism in plasma and purine metabolism in RBCs and PBMCs. Changes in potential biomarkers, including an increase in hypoxanthine level and a decrease in adenine level, may be related to the dysfunction of DNA synthesis in PBMCs. The decreased AMP level in RBCs may interfere with RBC storage lesions. Our research provides a more comprehensive perspective on blood metabolism associated with irradiation.

Effect of parachute delivery on red blood cell (RBC) and plasma quality measures of blood for transfusion.

BackgroundParachute airdrop offers a rapid transfusion supply option for humanitarian aid and military support. However, its impact on longer‐term RBC survival is undocumented. This study aimed to determine post‐drop quality of RBCs in concentrates (RCC), and both RBCs and plasma in whole blood (WB) during subsequent storage.Study design and methodsTwenty‐two units of leucodepleted RCC in saline, adenine, glucose, mannitol (SAGM) and 22 units of nonclinical issue WB were randomly allocated for air transportation, parachute drop, and subsequent storage (parachute), or simply storage under identical conventional conditions (4 ± 2°C) (control). All blood products were 6–8 days post‐donation. Parachute units were packed into Credo Cubes, (Series 4, 16 L) inside a PeliCase (Peli 0350) and rigged as parachute delivery packs. Packs underwent a 4‐h tactical flight (C130 aircraft), then parachuted from 250 to 400 ft before ground recovery. The units were sampled aseptically before and after airdrop at weekly intervals. A range of assays quantified the RBC storage lesion and coagulation parameters.ResultsBlood units were maintained at 2–6°C and recovered intact after recorded ground impacts of 341–1038 m s−2. All units showed a classical RBC storage lesion and increased RBC microparticles during 42 days of storage. Fibrinogen and clotting factors decreased in WB during storage. Nevertheless, no significant difference was observed between Control and Parachute groups. Air transportation and parachute delivery onto land did not adversely affect, or shorten, the shelf life of fresh RBCs or WB.DiscussionAppropriately packaged aerial delivery by parachute can be successfully used for blood supply.

No Association between Age of Transfused Red Cells and 30-Day Mortality in Massively Transfused Patients: Results from the Australian and New Zealand Massive Transfusion Registry

BackgroundClinical trials studying age of transfused red blood cells (RBCs) have found no evidence of benefit from transfusion of fresher RBCs. However, these studies have not included patients requiring massive transfusion for major bleeding, a patient population that may be more susceptible to adverse effects of the RBC storage lesion. Further, no clinical trials have specifically evaluated the safety of transfusing RBCs approaching expiry (30-42 days). We investigated the association between RBC storage time and 30-day mortality in a large cohort of massively transfused patients (5 RBCs within 4 hours, from any bleeding context).MethodsUsing 2007-2015 Australian/NZ Massive Transfusion Registry data from 25 hospitals linked with Australian and New Zealand National Death Index data, we modelled the association between storage time and 30-day mortality in massively transfused patients. Our logistic regression model adjusted for Charlson co-morbidity score, patient age, patient sex, number of RBC units transfused, and bleeding category (cardiothoracic, gastrointestinal, obstetric, surgical, trauma, vascular surgery, or other) with treating hospital as a random-effects grouping variable. Storage time was quantified as the mean storage time of all units each patient received for our primary analysis, and as a binary predictor indicating patients receiving only RBCs older than 30 days for our secondary analysis. As sensitivity analyses, we stratified patients by number of RBC units received and ran our model individually on each stratum containing more than 50 patients receiving equivalent numbers of RBCs in order to eliminate any confounding introduced by comparing patients receiving differing numbers of RBCs. In our stratified sensitivity analyses, in addition to mean storage time of RBC units received by each patient, we tested minimum and maximum storage times of transfused RBC units as predictors.Results4127 patients meeting the massive transfusion definition during the study period had RBC age available. Stratifying on number of RBC units transfused resulted in 19 strata containing more than 50 patients (those receiving 5,6,7,...23 RBC units), comprising 3641 patients (88% of study population). In the analysis of the entire cohort together, we found no significant association between mean storage duration and 30-day mortality (ORST=0.99 [95%CI: 0.98 to 1.01]; p=0.42) or patients transfused with only RBCs >30days old and 30-day mortality (ORST>30d=0.85 [95%CI: 0.52 to 1.37]; p=0.49). In our stratified sensitivity analyses, we found no association between mean storage time and 30-day mortality (OR for a 1-day increase in mean storage time [ORmeanST] range = 0.95 to 1.08 with p>0.05 for entire range). We likewise found no associations between maximum and minimum storage times and 30-day mortality (ORmaxST range = 0.93 to 1.05 with p>0.05 for all but one stratum; ORminST range = 0.94 to 1.04 with p>0.05 for all strata). For all three stratified sensitivity analyses, we observed no trend in ORST across the strata of patients receiving equivalent numbers of RBCs (figure 1 shows the stratified analysis with mean storage time as the predictor).ConclusionsWe found no association between storage time of transfused RBCs and 30-day mortality in a large cohort of massively transfused patients. Our findings were consistent both for the cohort as a whole and within strata of patients receiving equivalent numbers of RBC transfusions. Additionally, we observed no association when we modelled storage duration as maximum RBC age, minimum RBC age, or when we considered patients receiving only RBCs >30 days old. Our conclusions are consistent with recent large randomised trials which have not demonstrated mortality benefit from transfusing fresher RBCs. Differences in our findings and those of previous observational studies reporting correlations between storage time and mortality may be due to methodological differences: specifically, our stratified analysis eliminated the potential confounding effect of number of RBCs transfused, and we more accurately modelled the storage lesion as a continuous function by using mean storage age rather than comparing patients who received only RBCs above or below arbitrary storage time thresholds. In conclusion, we find no evidence that fresher blood leads to better clinical outcomes in massively transfused patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Osmotic hemolysis is a donor-specific feature of red blood cells under various storage conditions and genetic backgrounds.

Transfusion research has recently focused on the discovery of red blood cell (RBC) storage capacity biomarkers and the elucidation of donor variation effects. This shift of focus can further strengthen personalization of transfusion therapy, by revealing probable links between donor biology, RBC storage lesion profile, and posttransfusion performance. We performed a paired correlation analysis of osmotic fragility in freshly drawn RBCs and during cold storage in different preservative solutions at weekly intervals until unit's expiration date (n= 231), or following 24 h reconstitution in allogeneic plasma (n= 32) from healthy controls or transfusion-dependent beta-thalassemia patients. We observed exceptional correlation profiles (r > 0.700, p < 10-5 in most cases) of RBC osmotic fragility in the ensemble of samples, as well as in subgroups characterized by distinct genetic backgrounds (sex, beta-thalassemia traits, glucose-6-phosphate dehydrogenase deficiency) and storage strategies (additive solutions, whole blood, RBC concentrates). The mean corpuscular fragility (MCF) of fresh and stored RBCs at each storage time significantly correlated with the MCF of stored RBCs measured at all subsequent time points of the storage period (e.g., MCF values of storage day 21 correlated with those of storage days 28, 35 and 42). A similar correlation profile was also observed between the osmotic hemolysis of fresh/stored RBCs before and following in vitro reconstitution in plasma from healthy controls or beta-thalassemia patients. Our findings highlighted the potential of osmotic fragility to serve as a donor-signature on RBCs at every step of any individual transfusion chain (donor, blood product, and probably, recipient).

Beta thalassemia minor is a beneficial determinant of red blood cell storage lesion.

Blood donor genetics and lifestyle affect the quality of red blood cell (RBC) storage. Heterozygotes for beta thalassemia (bThal+) constitute a non-negligible proportion of blood donors in the Mediterranean and other geographical areas. The unique hematological profile of bThal+ could affect the capacity of enduring storage stress, however, the storability of bThal+ RBC is largely unknown. In this study, RBC from 18 bThal+ donors were stored in the cold and profiled for primary (hemolysis) and secondary (phosphatidylserine exposure, potassium leakage, oxidative stress) quality measures, and metabolomics, versus sex- and age-matched controls. The bThal+ units exhibited better levels of storage hemolysis and susceptibility to lysis following osmotic, oxidative and mechanical insults. Moreover, bThal+ RBC had a lower percentage of surface removal signaling, reactive oxygen species and oxidative defects to membrane components at late stages of storage. Lower potassium accumulation and higher uratedependent antioxidant capacity were noted in the bThal+ supernatant. Full metabolomics analyses revealed alterations in purine and arginine pathways at baseline, along with activation of the pentose phosphate pathway and glycolysis upstream to pyruvate kinase in bThal+ RBC. Upon storage, substantial changes were observed in arginine, purine and vitamin B6 metabolism, as well as in the hexosamine pathway. A high degree of glutamate generation in bThal+ RBC was accompanied by low levels of purine oxidation products (IMP, hypoxanthine, allantoin). The bThal mutations impact the metabolism and the susceptibility to hemolysis of stored RBC, suggesting good post-transfusion recovery. However, hemoglobin increment and other clinical outcomes of bThal+ RBC transfusion deserve elucidation by future studies.

The effect of N-acetyl-cysteine (NAC) on RBC oxidative damage and RBC metabolism during storage of red blood cell product in blood bank condition

Background: Oxidative damage is one of the main causes of Red blood cell (RBC) storage lesion which can reduce RBC survival during RBC storage in blood bank condition. In this study, we evaluate the effect of N-acetyl cysteine (NAC) as an anti-oxidant compound on RBC oxidative damage and RBC metabolism during storage of this product. Methods: In this experimental study, 10 bag of packed RBC were provided to the Iranian Blood Transfusion Organization's Innovation Center were randomly selected and effect of NAC was investigated on metabolism, oxidative status and hematologic variables of RBC during RBC storage. The results of this study were compared between two groups of NAC treated RBC and untreated RBC (without NAC). All of the data were analyzed with SPSS statistical program (version 22). Results: In this study, the concentration of lactate and LDH enzyme activity in the NAC treated RBC were lower than the control group (without NAC). The concentration of malondialdehyde (MDA) as a lipid peroxidation marker in the NAC treated RBC was lower increase than the untreated RBC. Also antioxidant capacity was so higher in the NAC - RBC than in the control group, especially in the 28th day of RBC storage (Pday28 &lt;0.05). Conclusion: The results of this study indicated that the use of NAC as an additive solution could decrease oxidative damage via maintaining of the RBC oxidant capacity during RBC storage. In the future NAC may be used as an additive for maintaining of the RBC survival and RBC quality during storage of RBC in blood bank condition.

Impact of lipid peroxidation and antioxidants on erythrocytes during blood storage

Background: Blood transfusion plays important role in the management of certain clinical conditions like acute blood loss, injury and anemia. The red blood cells (RBCs) for transfusion can be stored for 35 to 42 days at 2–6°C. It has been reported that some biochemical changes occur during the course of storage. During storage, progressive morphological and biochemical changes occur which are often related to the reduction of ATP, 2,3-diphosphoglycerate, and NADH in RBCs. These changes are referred to as the “storage lesions”. Oxidative damage is the most important factor causing RBC storage lesion. Free radicals can damage RBC products by lipid and protein oxidation affecting cell quality. The present study is aimed to study the impact of lipid peroxidation and potential role of enzymatic antioxidants in stored blood. Material and methods: The present study was observational study carried out in healthy blood donors at KEM hospital, Mumbai. Thirty healthy donors, who were fulfilling the inclusion and exclusion criteria, were enrolled in the study. Estimation of hemoglobin, levels of lipid peroxidation and some enzymatic antioxidants like glutathione peroxidase, catalase and superoxide dismutase activity was carried out in a properly stored blood samples at 40C. Enzyme levels estimation was carried out at every 7 days interval. Blood grouping of all the samples was also done to check if there is any change in the levels of lipid peroxidation and antioxidant levels across the groups. Results: Malondialdehyde (MDA) is an indirect marker of lipid peroxidation that can modify proteins. Increased MDA levels in the study indicate that lipid peroxidation in red cells has occurred during the preservation period. Throughout storage period, the levels of glutathione peroxidase and catalase declined. Statistically significant negative correlation existed between lipid peroxidation and glutathione peroxidase. Whereas study established positive correlation between lipid peroxidation and superoxide dismutase (SOD). On day 8, day 15 and day 22, lipid peroxidation was found to be positively correlated with SOD. But on Day 30, there was negative correlation between lipid peroxidation and SOD. Blood grouping of all samples indicate no significant susceptibility to lipid peroxidation when the different blood groups were compared. Methemoglobin levels in stored blood were increased over a period of 30 days. Conclusion: Red cell storage lesions due to oxidative injury during storage are now the reported fact, confirmed by the findings of the present study. This also indicates that antioxidant enzymatic machinery of the system comes into play adequately to circumvent the damage done. To investigate further therapeutic role of antioxidants in preventing oxidative damage to red cells during storage, large sample studies will be required.

Oxygen in Red Blood Cell Concentrates: Influence of Donors' Characteristics and Blood Processing.

Objective: Unexpectedly wide distribution (<10 to >90%) of hemoglobin oxygen saturation (sO2) within red cell concentrates (RCCs) has recently been observed. Causes of such variability are not yet completely explained whereas the roles of oxygen and oxidative lesions during the storage of RCCs are known. The objectives of the present study are to characterize sO2 distribution in RCCs produced in a Swiss blood center and to investigate the influence of processing and donors’ characteristics.Methods: The level of sO2 was measured in 1701 leukocyte-depleted RCCs derived from whole blood donations in both top–bottom (TB; component filtered, SAGM) and top–top (TT; whole blood filtration, PAGGSM) RCCs. The sO2 value was measured non-invasively through the PVC bag prior to storage by resonance Raman spectroscopy. Gender, age, blood type, hemoglobin level, and living altitude of donors, as well as process method and time-to-process were recorded.Results: Overall, the sO2 exhibited a wide non-Gaussian distribution with a mean of 51.2 ± 18.5%. Use of top-top kits resulted in a 16% higher sO2 (P < 0.0001) than with top-bottom ones. Waiting time before processing only had a modest impact, but the blood processing itself reduced the sO2 by almost 12% (P < 0.0001). sO2 was also significantly affected by some donors’ characteristics. RCCs from men exhibited 25% higher sO2 (P < 0.0001) than those donated by women. Multivariate analysis revealed that the apparent correlation observed with hemoglobin level and age was actually due to multicollinearity with the sex variable. Finally, we noticed no significant differences across blood type but found that altitude of residence was associated with the sO2 (i.e., higher in higher living place).Conclusion: These data confirm wide sO2 distribution in RCCs reported recently. The sO2 was impacted by the processing and also by donors’ characteristics such as the gender and the living altitude, but not by the hemoglobin level, blood group and donor age. This study provides new hints on the factors influencing red blood cells storage lesions, since they are known to be related to O2 content within the bags, giving clues to better process and to better store RCCs and therefore potentially improve the efficacy of transfusion.

Potential Consequences of the Red Blood Cell Storage Lesion on Cardiac Electrophysiology.

BackgroundThe red blood cell (RBC) storage lesion is a series of morphological, functional, and metabolic changes that RBCs undergo following collection, processing, and refrigerated storage for clinical use. Since the biochemical attributes of the RBC unit shifts with time, transfusion of older blood products may contribute to cardiac complications, including hyperkalemia and cardiac arrest. We measured the direct effect of storage age on cardiac electrophysiology and compared it with hyperkalemia, a prominent biomarker of storage lesion severity.Methods and ResultsDonor RBCs were processed using standard blood‐banking techniques. The supernatant was collected from RBC units, 7 to 50 days after donor collection, for evaluation using Langendorff‐heart preparations (rat) or human induced pluripotent stem cell–derived cardiomyocytes. Cardiac parameters remained stable following exposure to “fresh” supernatant from red blood cell units (day 7: 5.8±0.2 mM K+), but older blood products (day 40: 9.3±0.3 mM K+) caused bradycardia (baseline: 279±5 versus day 40: 216±18 beats per minute), delayed sinus node recovery (baseline: 243±8 versus day 40: 354±23 ms), and increased the effective refractory period of the atrioventricular node (baseline: 77±2 versus day 40: 93±7 ms) and ventricle (baseline: 50±3 versus day 40: 98±10 ms) in perfused hearts. Beating rate was also slowed in human induced pluripotent stem cell–derived cardiomyocytes after exposure to older supernatant from red blood cell units (−75±9%, day 40 versus control). Similar effects on automaticity and electrical conduction were observed with hyperkalemia (10–12 mM K+).ConclusionsThis is the first study to demonstrate that “older” blood products directly impact cardiac electrophysiology, using experimental models. These effects are likely caused by biochemical alterations in the supernatant from red blood cell units that occur over time, including, but not limited to hyperkalemia. Patients receiving large volume and/or rapid transfusions may be sensitive to these effects.

Sex-related aspects of the red blood cell storage lesion.

Several factors contribute to the manifestation of red blood cell (RBC) storage lesions, with one of the most interesting being the "donor variation effect". Since many haematological characteristics of blood donors are sex-dependent, sex hormones and their age-dependent variation may affect the storage profile of RBCs. Fresh blood from 200 healthy male and female donors underwent haematological, biochemical and physiological analysis. Three selected groups of donors (men, n=8; pre-menopausal women, n=8; and post-menopausal women, n=4) exhibiting as similar as possible baseline values were recruited for blood donation in leukoreduced CPD/SAGM units. RBC indices, haemolysis and propensity for haemolysis, reactive oxygen species (ROS) and plasma antioxidant capacity were measured bi-weekly. Female blood was characterised by lower plasma antioxidant capacity and free haemoglobin (Hb) levels in vivo, in spite of the higher RBC osmotic fragility, compared to male blood. Comparatively low Hb concentration was also measured in stored RBCs from female donors, as in vivo. Mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), and plasma antioxidant capacity were also lower in female donors throughout storage, even though baseline levels were equal to those of the male group. There was no difference in propensity of stored RBCs for haemolysis between male and female units but intracellular ROS levels were significantly lower in female RBCs. Increased end-of-storage extracellular potassium and recruitment of protein stress markers (clusterin, Hb) to the RBC membrane were observed in the units of post- vs pre-menopausal female donors at mid-storage onwards. Donor's sex has an impact on Hb concentration and redox parameters of stored RBCs. In addition, menopause seems to promote RBC membrane remodelling, at least during prolonged storage. Our pilot study provides new insights on the different effects on RBC storage lesion according to sex.

Objective assessment of stored blood quality by deep learning

Stored red blood cells (RBCs) are needed for life-saving blood transfusions, but they undergo continuous degradation. RBC storage lesions are often assessed by microscopic examination or biochemical and biophysical assays, which are complex, time-consuming, and destructive to fragile cells. Here we demonstrate the use of label-free imaging flow cytometry and deep learning to characterize RBC lesions. Using brightfield images, a trained neural network achieved 76.7% agreement with experts in classifying seven clinically relevant RBC morphologies associated with storage lesions, comparable to 82.5% agreement between different experts. Given that human observation and classification may not optimally discern RBC quality, we went further and eliminated subjective human annotation in the training step by training a weakly supervised neural network using only storage duration times. The feature space extracted by this network revealed a chronological progression of morphological changes that better predicted blood quality, as measured by physiological hemolytic assay readouts, than the conventional expert-assessed morphology classification system. With further training and clinical testing across multiple sites, protocols, and instruments, deep learning and label-free imaging flow cytometry might be used to routinely and objectively assess RBC storage lesions. This would automate a complex protocol, minimize laboratory sample handling and preparation, and reduce the impact of procedural errors and discrepancies between facilities and blood donors. The chronology-based machine-learning approach may also improve upon humans' assessment of morphological changes in other biomedically important progressions, such as differentiation and metastasis.

Washing packed red blood cells decreases red blood cell storage lesion formation

BackgroundTransfusion of blood products is the ideal resuscitative strategy after hemorrhage. Unfortunately, older packed red blood cells have been associated with increased morbidity and mortality after massive transfusion. These packed red blood cells accumulate biochemical and structural changes known as the red blood cell storage lesions. The effect of washing on the formation of red blood cell storage lesions is unknown. We hypothesized that washing packed red blood cells during storage would decrease the development of the red blood cell storage lesions. MethodsBlood from 8- to 10-week-old male mice donors was stored as packed red blood cells for 14 days. A subset of packed red blood cells were washed with phosphate-buffered saline on storage day 7 and resuspended in AS-1 solution for an additional 7 days as washed packed red blood cells. Subsequently, the packed red blood cells were analyzed for microvesicle release, band-3 erythrocyte membrane integrity protein (Band-3), expression of phosphatidylserine, cell viability (calcein), accumulation of cell-free hemoglobin, and osmotic fragility. ResultsIn the washed packed red blood cells group, there was less microvesicle accumulation, greater Band-3 expression, less phosphatidylserine expression, a decrease in cell-free hemoglobin accumulation, and a decrease in osmotic fragility, but no differences in red blood cells viability. ConclusionWashing packed red blood cells during storage decreases the accumulation of red blood cell storage lesions. This strategy may lessen the sequelae associated with transfusion of older packed red blood cells.

Expired But Not Yet Dead: Examining the Red Blood Cell Storage Lesion in Extended-Storage Whole Blood.

Whole blood is a powerful resuscitation strategy for trauma patients but has a shorter shelf life than other blood products. The red blood cell storage lesion in whole blood has not previously been investigated beyond the standard storage period. In the present study, we hypothesized that erythrocytes in stored whole blood exhibit similar aspects of the red blood cell storage lesion and that transfusion of extended storage whole blood would not result in a more severe inflammatory response after hemorrhage in a murine model. To test this hypothesis, we stored low-titer, O-positive, whole blood units, and packed red blood cells (pRBCs) for up to 42 days, then determined aspects of the red blood cell storage lesion. Compared with standard storage pRBCs, whole blood demonstrated decreased microvesicle and free hemoglobin at 21 days of storage and no differences in osmotic fragility. At 42 days of storage, rotational thromboelastometry demonstrated that clotting time was decreased, alpha angle was increased, and clot formation time and maximum clot firmness similar in whole blood as compared with pRBCs with the addition of fresh frozen plasma. In a murine model, extended storage whole blood demonstrated decreased microvesicle formation, phosphatidylserine, and cell-free hemoglobin. After hemorrhage and resuscitation, TNF-a, IL-6, and IL-10 were decreased in mice resuscitated with whole blood. Red blood cell survival was similar at 24 h after transfusion. Taken together, these data suggest that red blood cells within whole blood stored for an extended period of time demonstrate similar or reduced accumulation of the red blood cell storage lesion as compared with pRBCs. Further examination of extended-storage whole blood is warranted.

The dielectric spectroscopy of human red blood cells during 37-day storage: β-dispersion parameterization

This study exploits dielectric spectroscopy to monitor the kinetics of red blood cells (RBC) storage lesions, focusing on those processes linked to cellular membrane interface known as β-dispersion. The dielectric response of RBC suspensions, exposed to blood-bank cold storage for 37 days, was studied using time-domain dielectric spectroscopy in the frequency range 500 kHz to 200 MHz. The measured dielectric processes are characterized by their dielectric strength (Δε) and their relaxation times (τ). Changes in the dielectric properties of the RBC suspensions, due to storage-related biophysical changes, were evaluated. For a quantitative characterization of RBC vitality, we characterized the shape of fresh and stored RBC and measured their deformability as expressed by their average elongation ratio, which was achieved under a shear stress of 3.0 Pa. During the second week of storage, an increment in the evolution of the relaxation times and in the dielectric permittivity strength of about 25% was observed. We propose that the characteristic increment of ATP, during the second and third weeks of storage, is responsible for the raise of the specific capacitance of cell membrane, which in turn explains the changes observed in the dielectric response when combined with the influence of the shape changes.

Save it-don't waste it! Maximizing utilization of erythrocytes from previously stored whole blood.

Recent military and civilian experience suggests that fresh whole blood may be the preferred for treatment of hemorrhagic shock, but its use is limited by its 21-day shelf life. The red blood cell storage lesion and coagulation status of packed red blood cells (pRBCs) salvaged from expired whole blood are unknown. We hypothesized that pRBCs can be salvaged from previously stored whole blood. Cold stored, low-titer, O-positive, nonleukoreduced, whole blood units were obtained at 21 days of storage. Erythrocytes were separated by centrifugation, resuspended in AS-3, and stored for 21 additional days as salvaged pRBCs. The red blood cell storage lesion parameters of microvesicles, Band-3, free hemoglobin, annexin V, and erythrocyte osmotic fragility were measured and compared with pRBCs prepared at the time of donation and stored in AS-3 for 42 days (standard pRBCs). In additional experiments, murine pRBCs were prepared from expired whole blood units and compared with those stored under standard conditions. Mice underwent hemorrhage and resuscitation with standard and salvaged pRBC units, and serum cytokines and free hemoglobin were determined. There were no significant differences in microvesicle formation or cell-free hemoglobin concentration between salvaged and standard pRBCs. There was decreased Band-3 and increased phosphatidylserine in the salvaged units as well as greater osmotic fragility. Salvaged pRBCs maintained consistent clot firmness. After hemorrhage and resuscitation in a murine model, salvaged pRBCs did not demonstrate increased serum cytokine levels. Salvaged pRBCs from previously stored whole blood accumulate the red blood cell storage lesion in a similar fashion to standard pRBCs and maintain consistent coagulability when reconstituted with plasma. Salvaged pRBCs are not associated with an increased inflammatory response when used for resuscitation in a murine model. Salvaged pRBCs may be a viable product for utilization in the treatment of traumatic hemorrhagic shock.

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.