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.

In Sickness and in Health: Erythrocyte Responses to Stress and Aging.

Mature red blood cells (RBC) are the most abundant host cell in our body [...].

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

BACKGROUND: 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. STUDY DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSION: 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).

Fatty acid desaturase activity in mature red blood cells and implications for blood storage quality.

BACKGROUND: Increases in the red blood cell (RBC) degree of fatty acid desaturation are reported in response to exercise, aging, or diseases associated with systemic oxidant stress. However, no studies have focused on the presence and activity of fatty acid desaturases (FADS) in the mature RBC. STUDY DESIGN AND METHODS: Steady state metabolomics and isotope-labeled tracing experiments, immunofluorescence approaches, and pharmacological interventions were used to determine the degree of fatty acid unsaturation, FADS activity as a function of storage, oxidant stress, and G6PD deficiency in human and mouse RBCs. RESULTS: In 250 blood units from the REDS III RBC Omics recalled donor population, we report a storage-dependent accumulation of free mono-, poly-(PUFAs), and highly unsaturated fatty acids (HUFAs), which occur at a faster rate than saturated fatty acid accumulation. Through a combination of immunofluorescence, pharmacological inhibition, tracing experiments with stable isotope-labeled fatty acids, and oxidant challenge with hydrogen peroxide, we demonstrate the presence and redox-sensitive activity of FADS2, FADS1, and FADS5 in the mature RBC. Increases in PUFAs and HUFAs in human and mouse RBCs correlate negatively with storage hemolysis and positively with posttransfusion recovery. Inhibition of these enzymes decreases accumulation of free PUFAs and HUFAs in stored RBCs, concomitant to increases in pyruvate/lactate ratios. Alterations of this ratio in G6PD deficient patients or units supplemented with pyruvate-rich rejuvenation solutions corresponded to decreased PUFA and HUFA accumulation. CONCLUSION: Fatty acid desaturases are present and active in mature RBCs. Their activity is sensitive to oxidant stress, storage duration, and alterations of the pyruvate/lactate ratio.

Leukoreduction makes a difference: A pair proteomics study of extracellular vesicles in red blood cell units.

Prestorage filtration of blood to remove contaminating donor leukocytes and platelets has substantially increased the safety level of transfusion therapy. We have previously shown that leukoreduction has a mitigating effect on the storage lesion profile by lowering the extent of hemolysis and of RBC aging and removal phenotypes, including surface signaling and microvesiculation. Even though protein composition may determine the fate of EVs in the recipient, the probable effect of leukoreduction on the EV proteome has been scarcely investigated. In the present paired study, we characterized the proteome of EVs released in prestorage leukoreduced (L) and nonleukoreduced (N) RBC units prepared from the same donors, by immunoblotting and qualitative proteomics analyses at two storage intervals. Apart from common proteofrms typically associated with the established EV biogenesis mechanisms, the comparative proteomics analyses revealed that both leukoreduction and storage duration affect the complexity of the EV proteome. Membrane and cytoskeleton-related proteins and regulators, metabolic enzymes and plasma proteins exhibited storage duration dependent variation in L- and N-EVs. Specific proteoforms prevailed in each EV group, such as transferrin in L-units or platelet glycoproteins, leukocyte surface molecules, MHC HLA, histones and tetraspanin CD9 in N-units. Of note, several unique proteins have been associated with immunomodulatory, vasoregulatory, coagulatory and anti-bacterial activities or cell adhesion events. The substantial differences between EV composition under the two RBC preparation methods shed light in the underlying EV biogenesis mechanisms and stimuli and may lead to different EV interactions and effects to target cells post transfusion.

Proteome of Stored RBC Membrane and Vesicles from Heterozygous Beta Thalassemia Donors.

Genetic characteristics of blood donors may impact the storability of blood products. Despite higher basal stress, red blood cells (RBCs) from eligible donors that are heterozygous for beta-thalassemia traits (ßThal+) possess a differential nitrogen-related metabolism, and cope better with storage stress compared to the control. Nevertheless, not much is known about how storage impacts the proteome of membrane and extracellular vesicles (EVs) in ßThal+. For this purpose, RBC units from twelve ßThal+ donors were studied through proteomics, immunoblotting, electron microscopy, and functional ELISA assays, versus units from sex- and aged-matched controls. ßThal+ RBCs exhibited less irreversible shape modifications. Their membrane proteome was characterized by different levels of structural, lipid raft, transport, chaperoning, redox, and enzyme components. The most prominent findings include the upregulation of myosin proteoforms, arginase-1, heat shock proteins, and protein kinases, but the downregulation of nitrogen-related transporters. The unique membrane proteome was also mirrored, in part, to that of ßThal+ EVs. Network analysis revealed interesting connections of membrane vesiculation with storage and stress hemolysis, along with proteome control modulators of the RBC membrane. Our findings, which are in line with the mild but consistent oxidative stress these cells experience in vivo, provide insight into the physiology and aging of stored ßThal+ RBCs.

The Post-Storage Performance of RBCs from Beta-Thalassemia Trait Donors Is Related to Their Storability Profile.

Blood donors with beta-thalassemia traits (ßThal+) have proven to be good "storers", since their stored RBCs are resistant to lysis and resilient against oxidative/proteotoxic stress. To examine the performance of these RBCs post-storage, stored ßThal+ and control RBCs were reconstituted in plasma donated from transfusion-dependent beta-thalassemic patients and healthy controls, and incubated for 24 h at body temperature. Several physiological parameters, including hemolysis, were evaluated. Moreover, labeled fresh/stored RBCs from the two groups were transfused in mice to assess 24 h recovery. All hemolysis metrics were better in the group of heterozygotes and distinguished them against controls in the plasma environment. The reconstituted ßThal+ samples also presented higher proteasome activity and fewer procoagulant extracellular vesicles. Transfusion to mice demonstrated that ßThal+ RBCs present a marginal trend for higher recovery, regardless of the recipient's immune background and the RBC storage age. According to correlation analysis, several of these advantageous post-storage characteristics are related to storage phenotypes, like the cytoskeleton composition, low cellular fragility, and enhanced membrane proteostasis that characterize stored ßThal+ RBCs. Overall, it seems that the intrinsic physiology of ßThal+ RBCs benefits them in conditions mimicking a recipient environment, and in the circulation of animal models; findings that warrant validation in clinical trials.

Recipient's effects on stored red blood cell performance: the case of uremic plasma.

BACKGROUND: Despite universal administration of erythropoiesis-stimulating agents, patients with end-stage renal disease (ESRD) are at high risk for presenting persistent anemia. Due to ambiguities in optimal hemoglobin targets and evidence of recombinant human erythropoietin (EPO)-related toxicity, an increase in blood transfusions has been observed in chronic renal disease over the past years. The probable effects of uremic plasma on the performance of stored red blood cells (RBCs) after transfusion have not been investigated. STUDY DESIGN AND METHODS: Leukoreduced RBCs after short or long storage in CPD-SAGM (n = 5) were assessed for hemolysis, surface removal signaling, reactive oxygen species (ROS) accumulation, and shape distortions before and after reconstitution with healthy (n = 10) or uremic plasma from ESRD patients (n = 20) for 24 hours at physiologic temperature, by using a previously reported in vitro model of transfusion. RESULTS: Temperature and cell environment shifts from blood bag to plasma independently and in synergy affected the RBC physiology. Outcome measures at transfusion-simulating conditions might not be analogous to timing of storage lesion. The uremic plasma ameliorated the susceptibility of stored RBCs to hemolysis, phosphatidylserine externalization, and ROS generation after stimulation by oxidants, but negatively affected shape homeostasis versus healthy plasma. Creatinine, uric acid, and EPO levels had correlations with the performance of stored RBCs in ESRD plasma. CONCLUSION: Renal insufficiency and EPO supplementation likely affect the recovery of donor RBCs and the reactivity of RBCs after transfusion by exerting both toxic and cytoprotective influences on them. ESRD patients constitute a specific recipient group that deserves further examination.

Ex vivo generation of transfusable red blood cells from various stem cell sources: A concise revisit of where we are now.

Blood transfusion is an essential and irreplaceable part of modern medicine, as a therapeutic modality or additional support to other clinical therapies. Nevertheless, the entire procedure from blood collection to administration, absorbs a significant amount of resources and has a number of problems that need to be addressed. The paucity of donors, the transmission of pathogenic microorganisms and the overall costs of the process have switched the scientific interest to the quest of alternative transfusion methods. The industrial ex vivo production of transfusable red blood cells capable of replacing a unit of packed red blood cells is a very attractive prospect, let alone the idea of a massive production of such a biological material. Various scientific groups, by exploiting erythropoiesis, the stem cells' characteristics and the constantly renewed knowledge in the fields of collection, culture, preservation and expansion of stem cells, have made significant progress towards the realization of such an idea. All three major sources of stem cells, haematopoietic stem/progenitor cells, human embryonic stem cells and induced pluripotent stem cells are thought to be capable of generating adequate amounts of red blood cells. By further studying and refining the in vitro red cell production protocols, it is anticipated that the economic and biotechnological obstacles of the current methods will be overcome in the near future. This manuscript is a brief revisit of their current state of the art, potentials and obstacles that are associated with industrial and clinical application issues.

Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage.

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1-7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13C1-aspartate or 13C5-adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and - preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates.

Donor-specific individuality of red blood cell performance during storage is partly a function of serum uric acid levels.

BACKGROUND: Previous investigations in leukoreduced units of red blood cells (RBCs) in mannitol additive solution revealed the close association of uric acid (UA) levels in vivo with the susceptibility of RBCs to storage lesion markers. In this study, we examined whether UA has a similar correlation with the capability of RBCs to cope with the oxidative provocations of storage under different conditions, namely, in CPDA-1 and in the absence of leukoreduction. STUDY DESIGN AND METHODS: The UA-dependent antioxidant capacity of the supernatant was measured in nonleukoreduced units of RBCs in CPDA (n = 47). The possible effect of UA variability on the storage lesion profile was assessed by monitoring several physiologic properties of RBCs and supernatant, including cell shape, reactive oxygen species, and size distribution of extracellular vesicles, in units exhibiting the lowest or highest levels of UA activity (n = 16) among donors, throughout the storage period. RESULTS: In stored RBC units, the UA-dependent antioxidant activity of the supernatant declined as a function of storage duration but always in strong relation to the UA levels in fresh blood. Contrary to units of poor-UA activity, RBCs with the highest levels of UA activity exhibited better profile of calcium- and oxidative stress-driven modifications, including a significant decrease in the percentages of spherocytes and of 100- to 300-nm-sized vesicles, typically associated with the exovesiculation of stored RBCs. CONCLUSION: The antioxidant activity of UA is associated with donor-specific differences in the performance of RBCs under storage in nonleukoreduced CPDA units.

Red cell transfusion in paediatric patients with thalassaemia and sickle cell disease: Current status, challenges and perspectives.

Notwithstanding the high safety level of the currently available blood for transfusion and the decreasing frequency of transfusion-related complications, administration of labile blood products to paediatric patients still poses unique challenges and considerations. The incidence of thalassaemia and sickle cell disease in the paediatric population may be high enough under specific racial and geographical contexts. Red cell transfusion is the cornerstone of ß-thalassaemia treatment and one of the most effective ways to prevent or correct specific acute and chronic complications of sickle cell disease. However, this life-saving strategy comes with its own complications, such as additional iron overload, alloimmunization and haemolytic reactions, among others. In paediatrics, the dependency of the transfusion outcome upon disease and other recipient characteristics is more prominent compared with the adults, owing to differences in developmental maturity and physiology that render them more susceptible to common risks, exacerbate the host response to transfused cells, and modify the type or the clinical severity of the transfusion-related morbidity. The adverse branch of red cell transfusion is likely the overall effect of several factors acting synergistically to shape the clinical phenotype of this therapy, including inherent donor/blood unit variables, like antigenicity, red cell deformability and extracellular vesicles, as well as recipient variables, such as history of alloimmunization and inflammation level at time of transfusion. This review focuses on paediatric patients with ß-thalassaemia and sickle cell disease as a recipient group with distinct transfusion-related characteristics, and introduces new concepts for consideration, not adequately studied and elucidated so far.

Short-term effects of hemodiafiltration versus conventional hemodialysis on erythrocyte performance.

Hemodiafiltration (HDF) is a renal replacement therapy that is based on the principles of diffusion and convection for the elimination of uremic toxins. A significant and increasing number of end-stage renal disease (ESRD) patients are treated with HDF, even in the absence of definite and conclusive survival and anemia treatment data. However, its effects on red blood cell (RBC) physiological features have not been examined in depth. In this study, ESRD patients under regular HDF or conventional hemodialysis (cHD) treatment were examined for RBC-related parameters, including anemia, hemolysis, cell shape, redox status, removal signaling, membrane protein composition, and microvesiculation, in repeated paired measurements accomplished before and right after each dialysis session. The HDF group was characterized by better redox potential and suppressed exovesiculation of blood cells compared with the cHD group pre-dialysis. However, HDF was associated with a temporary but acute, oxidative-stress-driven increase in hemolysis, RBC removal signaling, and stomatocytosis, probably associated with the effective clearance of dialyzable natural antioxidant components, including uric acid, from the uremic plasma. The nature of these adverse short-term effects of HDF on post-dialysis plasma and RBCs strongly suggests the use of a parallel antioxidant therapy during the HDF session.

Pathophysiological aspects of red blood cells in end-stage renal disease patients resistant to recombinant human erythropoietin therapy.

OBJECTIVE: Modified, bioreactive red blood cells (RBCs) and RBC-derived microvesicles (MVs) likely contribute to the hematological and cardiovascular complications in end-stage renal disease (ESRD). This study assesses the physiological profile of RBCs in patients with ESRD receiving standard or high doses of recombinant human erythropoietin (rhEPO). METHOD: Blood samples from twenty-eight patients under sustained hemodialysis, responsive, or not to standard rhEPO administration were examined for RBC morphology, fragility, hemolysis, redox status, removal signaling, membrane protein composition, and microvesiculation before and after dialysis. Acute effects of uremic plasma on RBC features were examined in vitro through reconstitution experiments. RESULTS: Overall, the ESRD RBCs were characterized by pathological levels of shape distortions, surface removal signaling, and membrane exovesiculation, but reduced fragility compared to healthy RBCs. Irreversible transformation of RBCs was found to be a function of baseline Hb concentration. The more toxic uremic context in non-responsive patients compared to rhEPO responders was blunted in part by the antioxidant, antihemolytic, and anti-apoptotic effects of high rhEPO doses, and probably, of serum uric acid. A selective lower expression of RBC membrane in complement regulators (CD59, clusterin) and of CD47 "marker-of-self" was detected in non-responders and responders, respectively. Evidence for different short-term dialysis effects and probably for a different erythrocyte vesiculation mechanism in rhEPO responsive compared to non-responsive patients was also revealed. CONCLUSION: Deregulation of RBC homeostasis might involve diverse molecular pathways driving erythrocyte signaling and removal in rhEPO non-responders compared to responsive patients.

Red blood cell transfusion in surgical cancer patients: Targets, risks, mechanistic understanding and further therapeutic opportunities.

Anemia is present in more than half of cancer patients and appears to be an independent prognostic factor of short- and long-term adverse outcomes. It increases in the advanced period of cancer and perioperatively, in patients with solid tumors who undergo surgery. As a result, allogeneic red blood cell (RBC) transfusion is an indispensable treatment in cancer. However, its safety remains controversial, based on several laboratory and clinical data reporting a linkage with increased risk for cancer recurrence, infection and cancer-related mortality. Immunological, inflammatory and thrombotic reactions mediated by the residual leukocytes and platelets, the stored RBCs per se, the biological response modifiers and the plasticizer of the unit may underlie infection and tumor-promoting effects. Although the causality between transfusion and infection has been established, the effects of transfusion on cancer recurrence remain confusing; this is mainly due to the extreme biological heterogeneity that characterizes RBC donations and cancer context. In fact, the functional interplay between donation-associated factors and recipient characteristics, including tumor biology per se, inflammation, infection, coagulation and immune activation state and competence may synergistically and individually define the clinical impact of each transfusion in any given cancer patient. Our understanding of how the potential risk is mediated is important to make RBC transfusion safer and to pave the way for novel, promising and highly personalized strategies for the treatment of anemia in surgical cancer patients.

Erythrocyte-based drug delivery in Transfusion Medicine: Wandering questions seeking answers.

Red blood cells (RBCs) represent the most commonly used and best-studied natural carriers in the history of drug delivery. Their abundance and long circulation half-life, their great immune-biocompatibility and biodegradability profiles, along with the availability of well established protocols for their safe collection, ex vivo processing and quality control make them advantageous as drug delivery systems (DDS). As a result, several drug-loading techniques (including encapsulation and surface conjugation) have been developed in order to construct RBC-based or RBC-inspired drug delivery vehicles for the effective treatment of infections, cancer, chronic and autoimmune diseases in both pre-clinical protocols and clinical trials. Despite the fact that the collected laboratory (in vitro and in vivo) and clinical data exhibit variable potential for translation into transfusion-associated prototypes and feasible protocols with significant clinical impact, little is known and done in the direction of drug delivery through RBC transfusion. Accordingly, several wandering questions for the application and utility of RBC-based drug delivery in transfusion medicine seek answers. By focusing on the most prominent of them, namely, "why not the stored/transfused RBCs", this review quotes some thoughtful considerations based on the current applications of RBCs as DDS, and on the potential application of RBC-based DDS in transfusion therapy.

Donor variation effect on red blood cell storage lesion: a multivariable, yet consistent, story.

BACKGROUND: Previous studies have shown that baseline hematologic characteristics concerning or influencing red blood cell (RBC) properties might affect storage lesion development in individual donors. This study was conducted to evaluate whether variation in hemolysis, microparticle accumulation, phosphatidylserine (PS) exposure, and other storage lesion-associated variables might be a function of the prestorage hematologic and biologic profiles of the donor. STUDY DESIGN AND METHODS: Ten eligible, regular blood donors were paired and studied before donation (fresh blood) and during storage of RBCs in standard blood banking conditions. Plasma and cellular characteristics and modifications were evaluated by standard laboratory and biochemical or biologic analyses as well as by statistical and network analysis tools. RESULTS: Nitrate/nitrite and other bioactive factors exhibited high interdonor variability, which further increased during storage in a donor-specific manner. Storage lesion evaluators, including RBC fragility and PS exposure, fluctuated throughout the storage period in proportion to their values in fresh blood. Donors' levels of phosphatidylserine exposure and hemoglobin F correlated with stored cells' mean cell (RBC) Hb concentration, oxidative stress markers, and cellular fragility. DISCUSSION: Storage lesion indicators change in an orderly fashion, namely, by following donor-related prestorage attributes. These correlations are illustrated for the first time in "prestorage versus storage" biologic networks, which might help determine the best candidates for in vivo biomarkers of storage quality and provide deeper insight into the apparently complex donor variation effect on the RBC storage lesion.

Update on extracellular vesicles inside red blood cell storage units: Adjust the sails closer to the new wind.

Release of vesicles from cells is a universal biological system, an adaptive cellular response to endogenous or external physiological or stressful stimuli and a genius means for intercellular, inter-organ and even inter-organism communication. These secreted vesicles that are collectively designated extracellular vesicles (EVs) have increasingly attracted the interest of cell biologists due to their imaginable interactions with every piece of the known biological systems in both health and disease states. Although EVs isolation and characterization are challenges, owing to their particular physicochemical features and complex biology, recent technological innovation has offered better understanding and inevitably, driven the revision of previously established theories on them. However, a crucial question remains unsolved: the physiological relevance of EVs in vivo. Since membrane vesiculation is an integral part of red blood cell (RBC) aging and homeostatic machinery and a prominent feature of RBC storage lesion, the characterization of storage EVs and their probable clinical relevance with the therapeutic or adverse effects of transfusions are extremely important targets in the research fields of transfusion biology and medicine. The scientists involved should transfer nascent knowledge and state-of-the-art technological tools in the packed RBC unit in order to: (i) update the inventory of biochemical and biophysical features of storage EVs; (ii) gain insight into the molecular pathways/signals underlying their generation; and (iii) clarify their dependence on blood donor, storage strategies and analytical variations, in order to step forward on understanding their interactions with stored or recipient target cells.

Insights into red blood cell storage lesion: Toward a new appreciation.

Red blood cell storage lesion (RSL) is a multifaceted biological phenomenon. It refers to deterioration in RBC quality that is characterized by lethal and sub-lethal, reversible and irreversible defects. RSL is influenced by prestorage variables and it might be associated with variable clinical outcomes. Optimal biopreservation conditions are expected to offer maximum levels of RBC survival and acceptable functionality and bioreactivity in-bag and in vivo; consequently, full appraisal of RSL requires understanding of how RSL changes interact with each other and with the recipient. Recent technological innovation in MS-based omics, imaging, cytometry, small particle and systems biology has offered better understanding of RSL contributing factors and effects. A number of elegant in vivo and in vitro studies have paved the way for the identification of quality control biomarkers useful to predict RSL profile and posttransfusion performance. Moreover, screening tools for the early detection of good or poor "storers" and donors have been developed. In the light of new perspectives, storage time is not the touchstone to rule on the quality of a packed RBC unit. At least by a biochemical standpoint, the metabolic aging pattern during storage may not correspond to the currently fresh/old distinction of stored RBCs. Finally, although each unit of RBCs is probably unique, a metabolic signature of RSL across storage variables might exist. Moving forward from traditional hematologic measures to integrated information on structure, composition, biochemistry and interactions collected in bag and in vivo will allow identification of points for intervention in a transfusion meaningful context.