Red Blood Cell Storage Research Articles

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.

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

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.

Profiling and Bioinformatics Analysis Revealing Differential Circular RNA Expression about Storage Lesion Regulatory in Stored Red Blood Cells

Introduction: Circular RNA (circRNA) plays an important role in regulating metabolism of red blood cells (RBCs) and their storage lesions, but the study of how circRNA expression changes in stored RBCs has rarely been conducted. Methods: The expression change of circRNA was systemically evaluated via high-throughput sequencing on healthy RBCs on day 0, 20, and 40. And then we confirmed the reliability of the high-throughput sequencing analysis by RT-qPCR characterization on selected circRNAs. A higher parental gene enrichment was used to explore circRNA function in pathways. In addition, we deciphered a dysregulated circRNA-related ceRNAs network, and identified three circRNA-miRNA-mRNA regulatory axes related to storage lesion. Results: We identified 2,586 known and 6,216 putative novel circRNAs, more than 100 circRNAs expression levels were shifted, and the number of downregulated circRNAs was greater with longer storage time. Furthermore, a higher parental gene enrichment related to circRNA was found in pathways, including cAMP signaling pathway, ubiquitin-mediated proteolysis, apoptosis, adhesion, MAPK signaling pathway, cystine methionine metabolism, RNA degradation, RNA transport, TGF-β, and actin regulatory pathway. hsa_circ_0007127-miR-513a-5p-SMAD4, hsa_circ_0000033-miR-19a-3p-VAMP3, and hsa_circ_0005546-miR-4720-CCND3 regulatory axes related to storage lesion was found. Conclusions: Through investigation in circRNAs profile and circRNA-miRNA-mRNA interactions, this study provides insights on stored RBC circRNA expression changes, which closely relate to the storage lesion of RBCs and their physiological functions.

Blood bank storage of red blood cells increases RBC cytoplasmic membrane order and bending rigidity.

Blood banks around the world store blood components for several weeks ensuring its availability for transfusion medicine. Red blood cells (RBCs) are known to undergo compositional changes during storage, which may impact the cells’ function and eventually the recipients’ health. We extracted the RBC’s cytoplasmic membrane (RBCcm) to study the effect of storage on the membranes’ molecular structure and bending rigidity by a combination of X-ray diffraction (XRD), X-ray diffuse scattering (XDS) and coarse grained Molecular Dynamics (MD) simulations. Blood was stored in commercial blood bags for 2 and 5 weeks, respectively and compared to freshly drawn blood. Using mass spectrometry, we measured an increase of fatty acids together with a slight shift towards shorter tail lengths. We observe an increased fraction (6%) of liquid ordered (lo) domains in the RBCcms with storage time, and an increased lipid packing in these domains, leading to an increased membrane thickness and membrane order. The size of both, lo and liquid disordered (ld) lipid domains was found to decrease with increased storage time by up to 25%. XDS experiments reveal a storage dependent increase in the RBCcm’s bending modulus κ by a factor of 2.8, from 1.9 kBT to 5.3 kBT. MD simulations were conducted in the absence of proteins. The results show that the membrane composition has a small contribution to the increased bending rigidity and suggests additional protein-driven mechanisms.

Hypoxic Storage of Red Blood Cells: Assessment of Adhesion Properties Using a Standardized Endothelium-on-a-Chip Microfluidic Platform

Background: Blood transfusions are routine medical procedures in which stored blood or blood products (i.e., red blood cells, RBCs) are given to the patient to prevent adverse health outcomes due to acute or chronic anemia. The current regulations require RBCs to be stored at 4 ºC not more than 42 days before a transfusion. However, RBCs undergo extensive rheological changes during storage and may contribute to complications associated with transfusion. Hemanext has recently introduced an innovative storage system to ameliorate such storage lesions, in which RBCs are stored in a hypoxic environment, and thus they are exposed to much lower oxidative stress during storage. In this study, we report the changes in adhesion properties of stored RBCs to human endothelial cells following a 42-day storage in normoxia vs hypoxia, using a standardized endothelialized microfluidic platform: Endothelium-on-a-chip.Methods: Two units of 1-day old blood type O positive leukocyte reduced RBCs in additive solution were pooled and divided into equal aliquots (300mL each) A and B. Unit A was stored under conventional normoxic storage condition at 4°C for 42 days. Unit B was deoxygenated so that the percent oxygen saturation of the hemoglobin was less than 20%. The deoxygenated RBCs were stored in an oxygen impermeable storage bag for 42 days at 4°C. Adhesion levels were tested at the beginning (baseline, Week 1) and the end of the storage period (Week 6). To conduct the adhesion experiments, human umbilical vein endothelial cells (HUVECs) were first cultured within microfluidic channels under flow for at least 48 hours. Next, the HUVECs were treated with 40 µM heme for 4 hours at 37 ºC [1]. RBCs were centrifuged to remove the storage buffer and resuspended in basal cell culture medium (EGM, Lonza, Morristown, USA) supplemented with 10 mM HEPES at a hematocrit of 40%. A 15 µl of RBC solution was then injected over heme-activated HUVECs at a shear stress of 1 dyne/cm 2 followed by a 10-minute rinse with fresh basal culture medium to remove non-adherent RBCs. At the end of the experiment, adherent erythrocytes were manually quantified. Control experiments were conducted with non-activated HUVECs.Results: We found that RBCs that were stored under conventional normoxic condition displayed higher adhesion to heme-activated HUVECs than hypoxic condition as illustrated in Figure 1A and 1B. The baseline adhesion levels (Week 1) to non-activated HUVECs (control) were negligible (Fig. 1C, normoxia: 13±6; hypoxia: 18±10, p>0.05) while both cell populations had considerable adhesion levels to heme-activated HUVECs at baseline (Fig. 1C, normoxia: 372±59; hypoxia: 335±37). Following a 6-week storage, adhesion of RBCs stored in normoxia to heme-activated HUVECs was higher compared to those stored in hypoxic conditions while the p-value was not significant, which was likely due to the limited sample size (Fig. 1C, 1782±519 vs 594±55, p=0.08, N=3). These results suggest that storage-mediated RBC adhesion to heme-activated HUVECs may be ameliorated by the novel hypoxic-storage condition.Discussion: This study showed a decrease in the adhesion of hypoxic RBCs to heme-activated HUVECs when compared to conventionally stored RBCs for transfusion. This result suggests that hypoxic RBCs may reduce the risk of developing vaso-occlusion (VOC) after RBC transfusion in patients such as in sickle cell disease where adhesion to heme-activated HUVECs has been implicated in the pathogenesis of VOC [1].Acknowledgements: This work was funded by Hemanext. The authors would like to thank the Ohio Third Frontier Technology Validation and Start-up Fund (TVSF) and National Science Foundation Phase-I Small Business Technology Transfer (STTR) award, which supported this work in part. Stored RBCs were donated by Hemanext.Reference:1. Kucukal, E., et al., American Journal of Hematology, 2018. 93(8): p.1050-1060 [Display omitted] DisclosuresKucukal: BioChip Labs: Current Employment, Patents & Royalties. Kocevar: BioChip Labs: Current Employment. Nayak: BioChip Labs: Current Employment. Sowemimo-Coker: Hemanext: Current Employment. Zak: BioChip Labs: Current Employment, Current holder of stock options in a privately-held company; XaTek: Current Employment, Current holder of stock options in a privately-held company; TecTraum Inc: Current Employment, Current holder of stock options in a privately-held company. Omert: Hemanext: Current Employment. Gurkan: Biochip Labs: Patents & Royalties; Hemex Health, Inc.: Current Employment, Patents & Royalties; Dx Now Inc.: Patents & Royalties; Xatek Inc.: Patents & Royalties.

Storage Differentially Impacts Immunization to Red Cell Antigens

Introduction: The impact of red blood cell (RBC) storage on alloimmunization rates after transfusion remains controversial, with clinical experience demonstrating conflicting results. Indeed, some reports suggest increased rates of alloimmunization associated with longer storage of RBC units, while others suggest no association between alloimmunization rates and length of storage. We hypothesized that this discrepancy may reflect the differential impact of storage on alloimmunization related to each unique antigen and the immune response elicited. Here we define the effect of red cell storage on two distinct antigens, including the Hel-Ova-Duffy (HOD) and KEL antigens, using a preclinical murine model of RBC storage and transfusion.Methods: RBCs singly expressing HOD (HOD RBC) or KEL (KEL RBC) antigens, in addition to those dually expressing both HOD and KEL (HOD x KEL RBC) antigens, were collected from respective transgenic donor mice into CPDA. Units of HOD, KEL or HOD x KEL RBC were generated and used immediately (fresh RBC), or stored for 8 (D8) or 14 (D14) days at 4C before transfusion into wildtype recipients. Recipient cytokine profiles were assessed 2 hours post-transfusion using a multiplex inflammation panel. Donor RBC survival at 24 hours post-transfusion was assessed by flow cytometry. Serum was collected from recipients on days 5 and 14 post-transfusion and analyzed for IgM or IgG development by flow-cytometric crossmatch using HOD or KEL RBC targets.Results: Post-transfusion RBC survival was decreased with increased storage, but was not significantly different between donor cell types (HOD, KEL or HOD x KEL). Recipient cytokine profiles demonstrated a distinct inflammatory profile associated with storage. Development of IgM antibodies against HOD in mice transfused fresh versus D8 or D14 HOD or HOD x KEL RBCs were not significantly different; however, significantly increased (p<0.05) IgG antibodies were detected in recipients transfused with D8 and D14 HOD or HOD x KEL RBCs as compared to those transfused fresh HOD or HOD x KEL RBCs. In contrast, and while the development of anti-KEL IgM antibodies was not significantly different in mice transfused fresh versus D8 or D14 KEL or HOD x KEL RBCs, the IgG response to KEL was abrogated with longer storage, with recipients generating significantly less (p<0.05) anti-KEL IgG following transfusion with stored KEL or HOD x KEL RBCs as compared to fresh HOD or HOD x KEL RBCs.Conclusions: Although the impact of RBC storage on various measures of transfusion efficacy have been determined, the impact of storage on alloimmunization remains incompletely understood. Using a murine model of transfusion, we demonstrate that storage differentially impacts immunization to red cell antigens, increasing the response to certain antigens, like HOD, while decreasing the response to others, like KEL. Although it is unclear whether these findings apply to other RBC antigens and what relevance this holds for human patients, our study provides insight into the impact of storage on red cell alloimmunization and may enlighten future storage protocols. DisclosuresStowell: Argenx: Speakers Bureau; Grifols: Speakers Bureau; Alexion: Consultancy.

Cleavage of High Molecular Weight Kininogen and Bradykinin Release By Red Blood Cell Microvesicles As a Putative Mechanism for Hypotensive Transfusion Reactions

Background: Hypotensive transfusion reactions are adverse events typified by a sudden decrease in blood pressure that usually occurs within the first minutes after the initiation of a transfusion and resolves shortly after the transfusion is stopped. Due to current passive reporting practices, the incidence is likely underreported, but recent studies estimate an incidence of 1.3 cases per 10000 RBC units. The pathophysiology of these reactions are not fully understood. One hypothesis proposed is that increased bradykinin (BK), a nonapeptide released from cleavage of high molecular weight kininogen (HK), as seen with the use of negatively charged leukoreduction filters and the use of angiotensin-converting enzyme inhibitors, is a major contributor to the pathophysiology. We have recently demonstrated that red blood cell derived microvesicles (RBCMVs) from aging red blood cell (RBC) units are able to trigger thrombin generation via kallikrein activation - a predominant enzyme to cleave high molecular weight kininogen (Noubouossie, Blood, 2020). Thus, we hypothesize that the same RBCMVs would lead to bradykinin generation and might explain these hypotensive events.Objectives: To determine if RBC storage lesion-derived microvesicles are able to facilitate HK cleavage and BK release.Methods: RBCMVs were prepared from 4 recently expired RBC units (42 or 43 day old, AS-3 preserved, prestorage leukoreduced, all A+) via a series of centrifugations and washes. RBCMVs were quantified and characterized using nanoparticle tracking analysis. Obtained RBCMVs were first assessed for the capacity to initiate thrombin generation in microvesicle free human plasma via a substrate cleavage assay. Next, RBCMVs were added to a buffer reaction containing prekallikrein and HK, and kininogen cleavage was assessed via western blot. RBCMVs were also mixed with microvesicle-free human plasma and analyzed for evidence of kallikrein activation, cleavage of high molecular weight kininogen, and bradykinin production by ELISA. Cohn fractionation of plasma was used to enrich for BK.Results: RBCMVs were enumerated and concentrated to 7.5 ± 1.4 x 10 12 per mL (mean±SD size 160 ± 29µm). RBCMVs were able to initiate thrombin generation principally via contact pathway activation, independently of tissue factor. In a buffer system RBCMVs demonstrated activity to generate kallikrein with a sequential high molecular weight kininogen cleavage in a dose-dependent manner. Exclusion of kallikrein from the buffer system or addition of the small molecule inhibitor of kallikrein - ecallantide - halted cleavage of kininogen. A dose-dependent cleavage of high molecular weight kininogen indicated that RBCMVs could cause BK release in plasma; this was confirmed via an independent assay of Cohn -fractionated samples.Conclusions: Results of this current study demonstrate that RBCMVs are leading to high HK cleavage via kallikrein activation in vitro. We suspect that the same mechanisms could lead to BK generation in patients receiving older RBC units, possibly increasing the risk for hypotensive events from transfusion. DisclosuresKarafin: Westat, Inc.: Consultancy. Key: Sanofi: Consultancy; BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Takeda: Research Funding; Grifols: Research Funding; Uniqure: Consultancy, Other: Participation as a clinical trial investigator.

Red blood cell transfusion-induced non-transferrin-bound iron promotes Pseudomonas aeruginosa biofilms in human sera and mortality in catheterized mice.

Transfusion of storage-damaged red blood cells (RBCs) increases non-transferrin-bound iron (NTBI) levels in humans. This can potentially enhance virulence of microorganisms. In this study, Pseudomonas aeruginosa replication and biofilm production in vitro correlated with NTBI levels of transfused subjects (R2 = 0·80; P < 0·0001). Transfusion of stored RBCs into catheterized mice enhanced P. aeruginosa virulence and mortality in vivo, while pre-administration of apotransferrin reduced NTBI levels improving survival (69% vs 27% mortality; P < 0·05). These results suggest that longer RBC storage, by modulating the bioavailability of iron, may increase the risk of P. aeruginosa biofilm-related infections in transfused patients.

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.

Hemolytic anemia blunts the cytokine response to transfusion of older red blood cells in mice and dogs.

Transfusion of red blood cells (RBCs) stored for longer durations induces hemolysis and inflammatory cytokine production in murine and canine models. Despite immune system activation by stored RBCs, human randomized trials suggest that fresher RBC transfusions do not improve clinical outcomes. We hypothesized that underlying recipient hemolysis may affect cytokine responses to older RBC transfusions. C57BL/6 mouse cohorts were infused with anti-TER119 antibody to induce hemolysis, rabbit anti-platelet antiserum to induce immune thrombocytopenia (ITP), or appropriate control antibodies. Two days later, mice were transfused with fresh or stored RBCs. Furthermore, in a prospective, randomized, blinded trial, 38 client-owned dogs with primary autoimmune hemolytic anemia (AIHA) and two dogs with ITP, requiring RBC transfusion, were enrolled and randomized to receive fresh (≤7 days) or old (≥21 days) stored RBC transfusions. Monocyte chemoattractant protein (MCP)-1 levels were assessed at defined times after transfusion. Prior immune-mediated hemolysis blunted the MCP-1 response to stored RBC transfusion in mice (361 ± 111 pg/ml vs. 6836 ± 1528 pg/ml in mice with immune hemolysis vs. ITP, respectively; mean ± SD; p< .0001). Although hemolysis markers increased after transfusion of older RBCs, the cytokine response was also muted in dogs with AIHA. No differences in morbidity or mortality were evident comparing dogs randomized to fresh or old RBCs. These data suggest that underlying hemolysis blunts inflammatory responses to old RBC transfusions. The canine data support randomized trial results suggesting a lack of clinical benefit with fresh RBC transfusions in subjects with underlying, baseline hemolysis.

Survey of the RhD selection and issuing practices for uncrossmatched blood products at pediatric trauma hospitals in the United States: The BEST collaborative study.

As evidence demonstrating the importance of early transfusions in trauma resuscitation accumulates, when RhD-negative products might not be available, it is important to understand the nature of the RhD-type of products provided to bleeding pediatric patients of potentially unknown RhD-type. A survey link was electronically sent to the transfusion service medical director and/or laboratory manager at American pediatric Level I and Level II hospitals inquiring about their practices for selecting RhD-type of uncrossmatched red blood cells (RBC) or low titer group O whole blood (LTOWB) for boys and girls. There were 55/117 (47.0%) analyzable responses; 43/55 (78.2%) from Level I and 12/55 (21.8%) from Level II hospitals. For in hospital transfusions, 51/55 (92.7%) of centers use only RhD-negative blood products to resuscitate girls ≤18 years old while 30/55 (54.5%) of centers do the same for boys ≤18 years old. Most centers 41/55(74.5%) store RBCs and/or LTOWB in in-hospital remote refrigerators; 27 store only RhD-negative RBCs and 2 store only RhD-negative LTOWB units in these refrigerators. A total of 24/55 (43.6%) centers have RBCs and/or LTOWB available on road ambulances or helicopters for prehospital transfusion; 12 transport only RhD-negative RBCs and two transport only RhD-negative LTOWB. Most centers, 35/55 (63.6%), address the prophylaxis of an RhD-negative female recipient of RhD-positive transfusion on a case-by-case basis. While there is some variability, most of the responding pediatric trauma centers routinely utilized RhD-negative RBCs for emergency transfusion for patients ≤18 years old of unknown RhD-type.

Association between the length of storage of transfused leukoreduced red blood cell units and hospital-acquired infections in critically ill children: A secondary analysis of the TRIPICU study.

Evaluate the association between leukoreduced red blood cell (RBC) storage length and hospital-acquired infection (HAI) incidence rate in critically ill children. RBC transfusions are common in critically ill children. Despite their benefits, observational studies suggest an association between them and HAIs. One possible mechanism for increased HAI is transfusion-related immunomodulation due to bioactive substances' release as transfused blood ages. In this secondary analysis of the 'Transfusion Requirement in Paediatric Intensive Care Units' (TRIPICU) study, we analysed a subset of 257 participants that received only one pre-storage leukoreduced RBC transfusion. RBC storage length was classified as 1) transfusion of 'fresh' RBCs (≤10 days), 2) transfusion of 'stored' RBCs (21-34 days), and 3) transfusion of 'long-stored' RBCs (≥35 days). All were compared to a 'golden' period (11-20 days), representing the time between 'fresh' and 'stored'. We used quasi-Poisson multivariable regression models to estimate the HAI incidence rate ratio (IRR) and corresponding 95% confidence interval (CI). We found that the association between the length of storage time of leukoreduced RBCs and HAIs was not significant in the 'fresh' group (IRR 1.23; 95% CI 0.55, 2.78) and the 'stored' group (IRR 1.61; 95% CI 0.63, 4.13) when compared to the 'golden' period. However, we observed a statistically significant association between the 'long-stored' group and an increase in the HAI incidence rate (IRR 3.66; 95% CI 1.22, 10.98). Transfusion of leukoreduced RBC units stored for ≥35 days is associated with increased HAI incidence rate in haemodynamically stable, critically ill children.

Red Blood Cell Proteasome in Beta-Thalassemia Trait: Topology of Activity and Networking in Blood Bank Conditions.

Proteasomes are multi-catalytic complexes with important roles in protein control. Their activity in stored red blood cells (RBCs) is affected by both storage time and the donor’s characteristics. However, apart from their abundancy in the membrane proteome, not much is known about their topology, activity, and networking during the storage of RBCs from beta-thalassemia trait donors (βThal+). For this purpose, RBC units from fourteen βThal+ donors were fractionated and studied for proteasome activity distribution and interactome through fluorometric and correlation analyses against units of sex- and aged-matched controls. In all the samples examined, we observed a time-dependent translocation and/or activation of the proteasome in the membrane and a tight connection of activity with the oxidative burden of cells. Proteasomes were more active in the βThal+ membranes and supernatants, while the early storage networking of 20S core particles and activities showed a higher degree of connectivity with chaperones, calpains, and peroxiredoxins, which were nonetheless present in all interactomes. Moreover, the βThal+ interactomes were specially enriched in kinases, metabolic enzymes, and proteins differentially expressed in βThal+ membrane, including arginase-1, piezo-1, and phospholipid scramblase. Overall, it seems that βThal+ erythrocytes maintain a considerable “proteo-vigilance” during storage, which is closely connected to their distinct antioxidant dynamics and membrane protein profile.

Toxic masculinity in red blood cell units? Testosterone therapy in blood donors revisited.

FDA guidelines limit the use of blood from donors taking testosterone replacement therapy (TRT) to red blood cell (RBC) concentrates, whereas plasma and platelets are discarded. The purpose of this study is to bring awareness to above-average free testosterone concentrations in RBC units from TRT donors. We quantified the concentrations of free (bioavailable; pg/ml) and total (protein bound and free; ng/dl) testosterone in plasma (frozen within 24 h) and supernatants from 42-day stored leukocyte-reduced RBC units from 17 TRT male donors and 17 matched controls (no TRT). Total testosterone concentrations were determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Free testosterone concentrations were quantified in the same samples using equilibrium dialysis/LC-MS/MS. Plasma free and total testosterone concentrations in TRT donors were 2.9 and 1.8 times higher than that of controls. Total testosterone concentrations in RBC supernatants were about 30% of that of plasma. In contrast, free testosterone concentrations in RBC supernatants were 80%-100% of that of plasma and were significantly (p=.005) higher in TRT compared with controls (252.3 ± 245.3 vs. 103.4 ± 88.2pg/ml). Supraphysiological free testosterone concentrations (>244 pg/ml) in RBC supernatants were observed in five TRT donors and two control donors. RBC units from TRT donors may contain supraphysiological concentrations of free testosterone. This may be resolved by avoiding blood collections soon after testosterone dosing and by enhanced screening of TRT donors. These data establish a rationale for new studies and reexamination of the current guidelines concerning the utilization of blood components from TRT donors.

DEHT is a suitable plasticizer option for phthalate-free storage of irradiated red blood cells.

Due to increasing concerns about possible endocrine-disrupting properties, the use of the plasticizer di(2-ethylhexyl) phthalate (DEHP) will be banned in future blood storage. Di(2-ethylhexyl) terephthalate (DEHT) provides sufficient red blood cell (RBC) quality during conventional blood bank storage. It is important that a new plasticizer also maintains acceptable quality during exposure to high cell stress, such as irradiation, which is commonly used to prevent graft-versus-host disease. A total of 59 RBC units were collected and processed in polyvinyl chloride (PVC)-DEHT or PVC-DEHP blood bags combined with either saline-adenine-glucose-mannitol (SAGM) or phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGSM) additive solution. All units were X-ray irradiated on day 2 post-collection. Sampling for assessment of parameters of storage lesion was performed on day 2 pre-irradiation and day 14 and 28 post-irradiation. Though irradiation increased cell stress, DEHT/PAGGSM and current common European preference DEHP/SAGM were equally affected up to 14 days post-irradiation for all measured parameters. At day 28, haemolysis and microvesicle count were slightly increased in DEHT, whereas extracellular potassium ions, glucose, lactate, pH, mean corpuscular volume and microvesicle phosphatidylserine remained unaffected by plasticizer choice throughout storage. No individual unit exceeded 0.8% haemolysis, not even in DEHT/SAGM, the combination overall most affected by irradiation. Of the four combinations, membrane stability was least impacted in DEHP/PAGGSM. We demonstrate that DEHT is a suitable plasticizer for storage of RBCs after X-ray irradiation cell stress. This strengthens the option of DEHT as a viable non-phthalate substitute for DEHP.

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.

Do We Store Packed Red Blood Cells under "Quasi-Diabetic" Conditions?

Red blood cell (RBC) transfusion is one of the most common therapeutic procedures in modern medicine. Although frequently lifesaving, it often has deleterious side effects. RBC quality is one of the critical factors for transfusion efficacy and safety. The role of various factors in the cells’ ability to maintain their functionality during storage is widely discussed in professional literature. Thus, the extra- and intracellular factors inducing an accelerated RBC aging need to be identified and therapeutically modified. Despite the extensively studied in vivo effect of chronic hyperglycemia on RBC hemodynamic and metabolic properties, as well as on their lifespan, only limited attention has been directed at the high sugar concentration in RBCs storage media, a possible cause of damage to red blood cells. This mini-review aims to compare the biophysical and biochemical changes observed in the red blood cells during cold storage and in patients with non-insulin-dependent diabetes mellitus (NIDDM). Given the well-described corresponding RBC alterations in NIDDM and during cold storage, we may regard the stored (especially long-stored) RBCs as “quasi-diabetic”. Keeping in mind that these RBC modifications may be crucial for the initial steps of microvascular pathogenesis, suitable preventive care for the transfused patients should be considered. We hope that our hypothesis will stimulate targeted experimental research to establish a relationship between a high sugar concentration in a storage medium and a deterioration in cells’ functional properties during storage.

GSTP1 rs1695 and rs1871042, and SOD2 rs4880 as molecular markers of lipid peroxidation in blood storage.

Red blood cells (RBC) are subject to oxidative stress by reactive oxygen species (ROS) during storage. Molecular characterisation of oxidative stress in stored RBC, which may also occur in other blood components during long periods of storage, is rare. Our study included 45 healthy RBC donors recruited in Brazil. Blood was collected into standard Grifols® Triple Bags containing CPD SAG-M. Haematological values, biochemical data, and oxidative stress markers were assessed weekly during storage until 42 days after collection. GSTM1 and GSTT1 were determined by multiplex-polymerase chain reaction (PCR), while GSTP1 rs1695 and rs1871042, CAT rs1001179, and SOD2 rs4880 were evaluated by real-time PCR. A direct proportional relationship was found between storage time and levels of ROS and thiobarbituric acid reactive substances (TBARS, indicators of lipid peroxidation) (p<0.001). These parameters were indirectly proportional to ABTS values (p<0.001). The plasma concentration of TBARS was associated with GSTP1 303AG/GG, GSTP1 -16CT/TT, and SOD2 47CT/TT genotypes. Single-nucleotide polymorphisms at the CAT C-262T gene were not associated with TBARS, nor were oxidative markers of ROS. Prolonged storage may result in the onset of erythrocyte deterioration. Our results clearly indicate that erythrocytes are capable of attenuating ROS for 2 weeks of storage. We observed an association between elevated TBARS levels and the presence of GSTP1 and SOD2 variants in stored RBC. Although notable for heterozygous variants, this association was even stronger for the homozygous variants GSTP1 rs1695 (303GG), GSTP1 rs1871042 (-16TT), and SOD2 rs4880 (47TT). These findings accentuate the importance of genetic factors in storage lesions and will expand our understanding and consideration of endogenous and exogenous causes in improving clinical treatment with blood transfusions.

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.