Storage Lesion Research Articles

Targeted Proteomics Reveals That the Dominant Mechanism of Gpibα Loss during Platelet Storage Depends on Temperature of Storage

Background: Platelet transfusion is a potentially lifesaving procedure, used for both prophylactic and therapeutic indications. Platelets can be stored at room temperature (RT) for up to 7 days in air-permeable bags. Platelet function diminishes during storage, a phenomenon known as the storage lesion. We and others have shown that platelets can be stored for extended periods of time at 4°C and still show acceptable in vitro function while limiting bacterial growth. In the present study, we used proteomics to examine the changes in human platelets stored at RT and 4°C with a focus on the glycoprotein (GP) Ib-IX-V complex, the key receptor for platelet adhesion at sites of vessel injury. Study Design/Method: Platelet units from healthy donors were stored in 100% plasma with or without agitation (at 22°C or 4°C, respectively) at a concentration of 3x1011/L and sampled on days 0, 3, 7, and 14. Microparticles were detected by flow cytometry as described previously. For proteomic analysis, platelets were washed and digested with trypsin. Tryptic peptides were analyzed by nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nano LC-MS/MS). MS/MS spectra were searched against the human protein database using Proteome Discoverer 2.4 software. A student t-test test was used to determine significant differences in analytes amongst the different storage groups. Results/Finding: Under both storage conditions, GPIbα and GPV decreased significantly over storage time. However, comparison of the decline in these proteins to GPIbβ, GPIX, and other membrane proteins indicated that the mechanisms for this decline differ in the two conditions. At RT, the decrease in GPIbα and GPV appears to be largely proteolytic, given that only a minor concomitant decrease in surface level was seen in the protease-insensitive GPIX and a slight increase in GPIbβ. In addition, a comparable decrease in GPIbα level was not observed when a cytoplasmic GPIbα peptide was assayed, suggesting the extracellular portion had been proteolytically removed. In contrast, at 4°C the decline in GPIbα and GPV was accompanied by a modest decrease in GPIX, and only a small decrease in the ratio of extracellular to cytoplasmic to GPIbα peptide. These results suggested that, at 4°C, in addition to proteolysis, which was attenuated as compared to RT storage, another mechanism was responsible for removal of full-length GPIbα and other polypeptides. One such mechanism that could explain this would be loss of membrane from the platelets during 4°C storage. Indeed, we found that extracellular vesicles accumulated in the platelet supernatant during 4°C to a much higher level than at RT storage. Summary: One of the hallmarks of the platelet storage lesion at RT is shedding of surface membrane proteins including GPIbα. Previous studies in stored mouse and human platelets revealed a cleavage mechanism dependent on the metalloproteinase ADAM17. However, whether GPIbα is lost by the same mechanism in cold-stored human platelets was unknown. Our targeted proteomics analysis confirms that proteolysis is a major cause of GPIbα loss at RT, but is a less prominent mechanism at 4°C. However, another mechanism for membrane protein loss is more prominent at the lower temperature: microvesiculation. Thus, these studies provide new insights into the platelet storage lesion and suggest that measures to prevent them will have to be tailored to the dominant mechanism operating at a particular storage temperature. Disclosures No relevant conflicts of interest to declare.

Human Platelets Stored at Room Temperature Demonstrate Diminished Resistance to Oxidative Stress Compared to Those Stored at 4°C

Background: Current blood banking practices dictate that platelets can only be stored at room temperature (RT) for up to seven days because of functional decline and risk of bacterial overgrowth. A lower storage temperature has the potential to slow platelet metabolism and reduce oxidative damage during storage. In the current study, we quantified by mass spectrometry small molecule thiols and disulfides as markers of oxidative stress in platelets stored in plasma at RT or 4°C. Because glutathione (GSH), γGlu-Cys-Gly, is the major thiol in platelets, we also analyzed metabolites (amino acids and dipeptides) involved in GSH synthesis and degradation cycles. Method: Platelets were acquired from five healthy donors and adjusted to 3 × 1011/L in plasma. The platelets from each donor were split into two bags and stored in plasma at RT or 4°C (with or without agitation, respectively) for up to 14 days. Aliquots from each group were taken at days 0, 3, 7, and 14, and N-ethylamaleimide (NEM) was added to block free thiols before storing the samples at -80°C until analysis. Total and reduced forms of GSH and cysteine including protein-bound disulfides were quantified by mass spectrometry as described previously [Fu X. et al. Scientific Reports. 2019; 9(115):1-9]. Paired t-tests were used to evaluate the differences between the groups. Results: GSH is the major intracellular antioxidant that maintains a reducing environment within cells. After 7 days of storage at RT, reduced GSH decreased drastically from 13.0±1.2 pre-storage to 1.7±0.6 µM (p <0.005), an 85% decrease. In contrast, GSH in platelets stored at 4°C decreased at a much slower rate, with over 60% and 50% GSH remaining after 7 and 14 days of storage, respectively, indicating that platelets stored at 4°C maintain higher reducing capacities than those stored at RT. The decrease in reduced GSH can be caused by oxidation and/or degradation. If it is primarily due to oxidation, total GSH ― the sum of reduced and oxidized (disulfides) forms ― should remain relatively constant. The total GSH in the stored platelets decreased in a manner similar to the reduced GSH at both RT and 4°C, suggesting that degradation, not oxidation, was responsible for the decrease in reduced GSH. We then determined the quantities of metabolites related to GSH synthesis and degradation and found significant increases in the total amounts of the amino acids Glu, Cys, and Gly, suggesting that more GSH was degraded than synthesized in platelets during storage. We also examined the changes of Cys during storage at RT and 4°C because Cys is the main thiol in plasma and the platelets were stored in 100% plasma. Protein-bound Cys was significantly elevated in the samples stored at RT compared to those stored at 4°C, the differences being observed as early as day 3 and continuing to day 14 day (p<0.01). We previously identified protein-bound Cys as a marker for oxidative stress in plasma [Fu X. et al. Scientific Reports. 2019; 9(115):1-9]; its elevation is therefore indicative of increased oxidation of the platelet sample during RT storage compared to 4°C storage. Summary: In this study, we found that the ability of platelets to withstand oxidative stress during storage is severely compromised in platelets stored at RT as opposed to 4°C. This storage lesion affected both intracellular (GSH) and extracellular (Cys) antioxidant molecules and involved 2 mechanisms: GSH degradation and Cys oxidation. We are currently attempting to determine whether the differences observed result from increased metabolic rate in the RT-stored platelets or from greater exposure to ambient air caused by the need for agitation. These studies will identify targets for improving the quality and shelf-life of platelets stored for transfusion. Disclosures No relevant conflicts of interest to declare.

A Single Injection of an Optimized Adeno-Associated Viral Vector into Cerebrospinal Fluid Corrects Neurological Disease in a Murine Model of GM1 Gangliosidosis.

GM1 gangliosidosis is a rare neurodegenerative lysosomal storage disease caused by loss-of-function mutations in the gene encoding beta-galactosidase (β-gal). There are no approved treatments for GM1 gangliosidosis. Previous studies in animal models have demonstrated that adeno-associated viral (AAV) vector-mediated gene transfer to the brain can restore β-gal expression and prevent the onset of neurological signs. We developed an optimized AAV vector expressing human β-gal and evaluated the efficacy of a single intracerebroventricular injection of this vector into the cerebrospinal fluid (CSF) of a murine disease model. The AAV vector administration into the CSF increased β-gal activity in the brain, reduced neuronal lysosomal storage lesions, prevented the onset of neurological signs and gait abnormalities, and increased survival. These findings demonstrate the potential therapeutic activity of this vector and support its subsequent development for the treatment of GM1 gangliosidosis.

Ovine red cell concentrates for transfusion research – is the storage lesion comparable to human red cell concentrates?

Sheep are increasingly being used as a large in vivo animal model of blood transfusion because they provide several advantages over small animals. Understanding the effects of storage duration on ovine (ov) red cell concentrates (RCCs) and how these changes compare with stored human (hu) RCCs is necessary to facilitate clinical translation of research findings. OvRCCs (n=5) collected and processed in standard human blood collection packs, and equivalent huRCCs provided by Australian Red Cross Lifeblood (n=5), were stored at 2-6°C for 42days, with samples collected weekly. Haemolysis index was determined by measuring supernatant haemoglobin concentration. Biochemical parameters were evaluated using a blood gas analyser. Energy metabolites and biologically active lipids were measured using commercial assays. Osmotic fragility was determined by lysis in various saline concentrations. Extracellular vesicles were characterized by nanoparticle tracking analysis. Ovine red blood cells (RBCs) are double in number, smaller in size and more fragile than human RBCs. Haematological values were unchanged throughout storage. In contrast, biochemical and metabolic values, and haemolysis index in three of the five ovRCCs exceeded huRCCs licensing criteria by day 42. Accumulation of extracellular vesicles and biologically active lipids was comparable between huRCCs and ovRCCs. This study documents similarities and differences in the storage lesion of ovRCCs and huRCCs. This new information will guide the design of ovine transfusion models to enhance translation of findings to human transfusion settings.

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.

Hypoxia and hypocapnia storage of γ-irradiated red cell concentrates.

γ-irradiation is used to treat red blood cell (RBC) concentrates (RCCs) transfused to immunosuppressed patients. This treatment damages RBCs and increases storage lesions. Several studies have shown the beneficial effect of reducing O2 content during RBC storage. The present research work investigated the effect of γ-irradiation on RCCs stored under normal and hypoxia/hypocapnia conditions. O2 concentration (measured as oxyhaemoglobin fraction, sO2) and ABO-matched RCCs from whole blood donations, leukoreduced and prepared in phosphate, adenine, glucose, guanosine, saline and mannitol (PAGGSM) were pooled and split in two identical RCCs within 24 h post donation. One bag (Hx) was submitted to O2 and CO2 adsorption for 3 h on an orbital shaker at 22±2 °C and then transferred to a storage bag impermeable to gas. The other bag (Ctrl) was left as it was. The two bags were then stored at 4 °C. γ-irradiation (25 Gy) was applied at day 2 or 14, and the RCCs were stored until day 43. Different parameters (metabolites, haemolysis, morphology) were measured. Starting sO2 values were 63.7±18.4% (n=12) in Ctrl and 20.8±9.8% (n=12) in Hx bags, and reached 90.8±9.1% and 6.6±5.9% at day 43, respectively. As expected, an increase in glycolysis rate was observed after deoxygenation. Extracellular potassium concentrations were identical and reached around 70 mM at expiry with an irradiation-dependent kinetic release. No difference in haemolysis was observed after irradiation on day 2 in either group (<0.40%, p>0.9999). When irradiated at day 14, haemolysis was lower (p=0.033) in RCCs under hypoxia at the end of storage (day 28, 0.67±0.16%) compared to control (1.06±0.33%). Percentages of spherocytes were lower under hypoxia. The storage under hypoxia provided equivalent storage when RCCs were irradiated at day 2 and was advantageous when irradiated at day 14. In summary, O2-depletion of RCCs enable a better storage of RBCs, particularly when late irradiation is applied.

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.

Donor-dependent aging of young and old red blood cell subpopulations: Metabolic and functional heterogeneity.

Characteristics of red blood cells (RBCs) are influenced by donor variability. This study assessed quality and metabolomic variables of RBC subpopulations of varied biologic age in red blood cell concentrates (RCCs) from male and female donors to evaluate their contribution to the storage lesion. Red blood cell concentrates from healthy male (n = 6) and female (n = 4) donors were Percoll separated into less dense ("young", Y-RCCs) and dense ("old", O-RCCs) subpopulations, which were assessed weekly for 28 days for changes in hemolysis, mean cell volume (MCV), hemoglobin concentration (MCHC), hemoglobin autofluorescence (HGB), morphology index (MI), oxygen affinity (p50), rigidity, intracellular reactive oxygen species (ROS), calcium ([Ca2+ ]), and mass spectrometry-based metabolomics. Young RCCs having disc-to-discoid morphology showed higher MCV and MI, but lower MCHC, HGB, and rigidity than O-RCCs, having discoid-to-spheroid shape. By Day 14, Y-RCCs retained lower hemolysis and rigidity and higher p50 compared to O-RCCs. Donor sex analyses indicated that females had higher MCV, HGB, ROS, and [Ca2+ ] and lower hemolysis than male RBCs, in addition to having a decreased rate of change in hemolysis by Day 28. Metabolic profiling indicated a significant sex-related signature across all groups with increased markers of high membrane lipid remodeling and antioxidant capacity in Y-RCCs, whereas O-RCCs had increased markers of oxidative stress and decreased coping capability. The structural, functional, and metabolic dissimilarities of Y-RCCs and O-RCCs from female and male donors demonstrate RCC heterogeneity, where RBCs from females contribute less to the storage lesion and age slower than males.

Treatment of platelet concentrates with the L-carnitine modulates platelets oxidative stress and platelet apoptosis due to mitochondrial reactive oxygen species reduction and reducing cytochrome C release during storage.

Platelet concentrate (PC) transfusion is administrated to reduce the hemostatic complications in patients with thrombocytopenia. Strength platelet against oxidative stress conditions lead to decrease in platelet storage lesion (PSL). This study was aimed to evaluate L-carnitine (LC) effects on platelet oxidative stress and platelet apoptosis during storage time. PC bags were randomly selected and each bag was divided into two equal parts. L-carnitine was added to test groups. Normal saline was added to control groups. Platelets count, mean platelet volume (MPV), pH, Platelet aggregation, nitric oxide metabolism (nitric/nitrate), total antioxidant capacity (TAC), malondealdehyde concentration (MDA), lactate dehydrogenase (LDH) enzyme activity, mitochondrial reactive oxygen species (ROS) and cytochrome C releasing were assayed by standard methods in 1, 3, 5 and 7days of platelet storage. LDH enzyme activity was increased during storage but it had lower level in L-carnitine-treated platelets. LC treatment led to reduction in MDA concentration (3.35 ± 0.98 vs 5.3 ± 1.32, p = 0.003 and 6.52 ± 1.88 vs 5.67 ± 1.25, p = 0.005 for day 5 and day 7 respectively). Increased level of TAC was detected in LC-treated platelets in comparison to control (0.29 ± 0.06 vs 0.21 ± 0.05, p = 0.008 and 0.22 ± 0.03 vs 0.16 ± 0.03, p = 0.003 for day 5 and day 7 respectively). Interestingly, mitochondrial ROS and cytochrome C releasing was significantly lower in LC-treated versus control group during platelet storage. L-carnitine not only decreases mitochondrial ROS but also reduces cytochrome C releasing in PCs during storage. It might be considered as safe additive to decrease PSL in the future.

Computational Identification and Characterization of New microRNAs in Human Platelets Stored in a Blood Bank.

Platelet concentrate (PC) transfusions are widely used to save the lives of patients who experience acute blood loss. MicroRNAs (miRNAs) comprise a class of molecules with a biological role which is relevant to the understanding of storage lesions in blood banks. We used a new approach to identify miRNAs in normal human platelet sRNA-Seq data from the GSE61856 repository. We identified a comprehensive miRNA expression profile, where we detected 20 of these transcripts potentially expressed in PCs stored for seven days, which had their expression levels analyzed with simulations of computational biology. Our results identified a new collection of miRNAs (miR-486-5p, miR-92a-3p, miR-103a-3p, miR-151a-3p, miR-181a-5p, and miR-221-3p) that showed a sensitivity expression pattern due to biological platelet changes during storage, confirmed by additional quantitative real-time polymerase chain reaction (qPCR) validation on 100 PC units from 500 healthy donors. We also identified that these miRNAs could transfer regulatory information on platelets, such as members of the let-7 family, by regulating the YOD1 gene, which is a deubiquitinating enzyme highly expressed in platelet hyperactivity. Our results also showed that the target genes of these miRNAs play important roles in signaling pathways, cell cycle, stress response, platelet activation and cancer. In summary, the miRNAs described in this study, have a promising application in transfusion medicine as potential biomarkers to also measure the quality and viability of the PC during storage in blood banks.

Nitric oxide donors offer protection to RBC from storage lesion.

Red blood cell (RBC), which is the most commonly transfused blood component, due to its ability to save a life in absence of any other blood components, can be stored up to maximum 6 weeks by following standard preservation procedure. During storage, RBC undergoes various biophysical and biochemical changes (commonly known as storage lesion) for which blood transfusion with "old RBC" shows a lot of clinical problems especially relevant to critically ill patients. Recent research on S-nitrosylation of haemoglobin to improve oxygen delivery of banked blood revealed the important role of nitric oxide (NO) in protecting storage lesion. In the present study, we used various "NO donating" chemicals with different NO release dynamics and chemistries in RBC storage cocktails to test the effects of NO on storage lesion. Changes in different storage markers were evaluated after 7 days storage of pre-treated RBC. All the NO donors have shown protection against hemolysis. However, S-nitroso glutathione (GSNO) ranks first in shielding RBCs from storage lesion and additionally, it helps in elevating the value of 2, 3-di phosphoglycerate (2, 3-DPG), improving the RBC membrane fluidity and decreasing the adhesion towards endothelial monolayer. Present study reveals that NO released from NO donors confers protection against storage lesions of the RBC. Further, the study confirms that pre-treatment with GSNO, a NO donor and a nitrosylating agent, ensures the best protection to RBC during low temperature storage, when compared to other NO donor treatments.

Influence of AS-7 on the storage lesion in young and old circulating erythrocytes

Blood and its components are stored to meet the demands of blood transfusion. Erythrocytes undergo progressive modifications during storage known as storage lesions. Storage solutions were developed to improve shelf life and extend red cell viability. Therefore, the objective of this study is to analyze the effects of AS-7 on young and old erythrocytes during storage. Blood was collected from the blood bank at Kempegowda Institute of Medical Sciences (KIMS) hospital, Bengaluru. Erythrocytes were isolated from whole blood and separated based on its age using Percoll density gradient. The young and old erythrocytes were stored in AS-7 for 35 days and every 5th day, oxidative stress markers – Hemoglobin (Hb), Oxidative Hemolysis, Mechanical Fragility, Sialic Acid, Superoxides, Glucose, Lactate Dehydrogenase (LDH), Glutathione, antioxidant capacity (TACCUPRAC), Plasma Membrane Redox System (PMRS), antioxidant enzymes, lipid peroxidation, and protein oxidation products were assessed. Hb, glucose, TACCUPRAC, and superoxide dismutase reduced, while oxidative hemolysis, mechanical fragility, protein oxidation, and lipid peroxidation products increased in young and old cells over storage. LDH, PMRS, catalase, advanced oxidation protein products, and conjugate dienes were significant in old cells from day 5 itself, whereas in young cells towards the end of storage (from day 25). Oxidative insult was higher in old cells compared to young cells. AS-7 was beneficial to young erythrocytes during storage and thus laying the foundation for the possibilities of utilizing young cells as models for storage studies.

Damage to red blood cells during whole blood storage.

Transfusion with stored whole blood (WB) is increasingly routine practice to resuscitate bleeding trauma patients. Storage of packed red blood cells (pRBC) results in multiple biochemical, structural, and metabolic changes, referred to as to the storage lesion that may mediate adverse effects associated with transfusion of older RBC units. These include increased hemolysis, oxidative stress, and accelerated scavenging of nitric oxide (NO). Whether similar changes occur to stored WB is unclear and are characterized in this study. Ten WB units, in citrate-phosphate-dextrose, were purchased from the American Red Cross and changes in hemolysis (increased free hemoglobin, heme, and microparticles), oxidative stress indexed by redox cycling of peroxiredoxin-2 (Prx-2) and NO-scavenging kinetics were determined at different storage times until expiration. Microparticle number and free hemoglobin, but not heme, increased in a storage time-dependent manner. When normalized to the initial number of RBCs in stored WB units, hemolysis rates were similar to those reported for pRBCs. Prx-2 recycling kinetics were slower at expiration compared with earlier storage times. Rates of NO dioxygenation did not change with storage, but were decreased compared with freshly isolated RBCs. Storage of WB results in changes associated with the pRBC storage lesion but not for all parameters tested. The relative rate of hemolysis (indexed by free hemoglobin and microparticles) and oxidative stress was similar to that of pRBCs. However, the absolute level of hemolysis products were lower due to lower hematocrit of stored WB units. The clinical significance of these findings requires further investigation.

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.

Global CNS correction in a large brain model of human alpha-mannosidosis by intravascular gene therapy

Intravascular injection of certain adeno-associated virus vector serotypes can cross the blood-brain barrier to deliver a gene into the CNS. However, gene distribution has been much more limited within the brains of large animals compared to rodents, rendering this approach suboptimal for treatment of the global brain lesions present in most human neurogenetic diseases. The most commonly used serotype in animal and human studies is 9, which also has the property of being transported via axonal pathways to distal neurons. A small number of other serotypes share this property, three of which were tested intravenously in mice compared to 9. Serotype hu.11 transduced fewer cells in the brain than 9, rh8 was similar to 9, but hu.32 mediated substantially greater transduction than the others throughout the mouse brain. To evaluate the potential for therapeutic application of the hu.32 serotype in a gyrencephalic brain of larger mammals, a hu.32 vector expressing the green fluorescent protein reporter gene was evaluated in the cat. Transduction was widely distributed in the cat brain, including in the cerebral cortex, an important target since mental retardation is an important component of many of the human neurogenetic diseases. The therapeutic potential of a hu.32 serotype vector was evaluated in the cat homologue of the human lysosomal storage disease alpha-mannosidosis, which has globally distributed lysosomal storage lesions in the brain. Treated alpha-mannosidosis cats had reduced severity of neurological signs and extended life spans compared to untreated cats. The extent of therapy was dose dependent and intra-arterial injection was more effective than intravenous delivery. Pre-mortem, non-invasive magnetic resonance spectroscopy and diffusion tensor imaging detected differences between the low and high doses, and showed normalization of grey and white matter imaging parameters at the higher dose. The imaging analysis was corroborated by post-mortem histological analysis, which showed reversal of histopathology throughout the brain with the high dose, intra-arterial treatment. The hu.32 serotype would appear to provide a significant advantage for effective treatment of the gyrencephalic brain by systemic adeno-associated virus delivery in human neurological diseases with widespread brain lesions.

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.

Protective effect of L-carnitine on platelet apoptosis during storage of platelet concentrate.

Platelet apoptosis is considered as one of the important factors involved in platelet storage lesion (PSL) and affect the quality of platelets during storage. The beneficial effect of L-carnitine (LC) on platelet apoptosis during platelet concentrates (PCs) storage has not been fully investigated. The aim of this study was to evaluate the effects of LC on platelets of PC regarding their apoptosis markers during storage. Ten PCs from healthy donors were investigated in this study. PCs were prepared by platelet rich plasma (PRP) method and stored at 22±2°C with gentle agitation during storage. The effects of LC (15mM) on the platelet apoptosis were assessed by analyzing different indicative presence or absence of LC. Sampling was performed to evaluate apoptosis markers during platelet storage. The results indicated significantly higher mitochondrial membrane potential for LC-treated platelets than the untreated on the days 2 and 5 of storage (Pday2=0.001, Pday5=0.001). Phosphatidylserine (PS) exposure significantly increased on the untreated compared with LC-treated platelets on the second and third days of storage (Pday2=0.014, Pday3=0.012). Also, active caspase 3 was lower in the LC- treated platelets than the control group on the day 5 of storage (Pday5=0.004). Cytosolic cytochrome C was so significantly lower in LC-treated compared to the untreated platelets during storage time (Pday2=0.002, Pday3=0.001, Pday5=0.001). The results of this study indicate that the use of LC as an additive solution in platelets may be useful to reduce PSL by decreasing platelet apoptosis via mitochondrial pathway and increase platelet quality during storage.