A Current Synopsis of the Emerging Role of Extracellular Vesicles and Micro-RNAs in Pancreatic Cancer: A Forward-Looking Plan for Diagnosis and Treatment.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies worldwide, while it persists as the fourth most prevalent cause of cancer-related death in the United States of America. Although there are several novel therapeutic strategies for the approach of this intensely aggressive tumor, it remains a clinical challenge, as it is hard to identify in early stages, due to its asymptomatic course. A diagnosis is usually established when the disease is already in its late stages, while its chemoresistance constitutes an obstacle to the optimal management of this malignancy. The discovery of novel diagnostic and therapeutic tools is considered a necessity for this tumor, due to its low survival rates and treatment failures. One of the most extensively investigated potential diagnostic and therapeutic modalities is extracellular vesicles (EVs). These vesicles constitute nanosized double-lipid membraned particles that are characterized by a high heterogeneity that emerges from their distinct biogenesis route, their multi-variable sizes, and the particular cargoes that are embedded into these particles. Their pivotal role in cell-to-cell communication via their cargo and their implication in the pathophysiology of several diseases, including pancreatic cancer, opens new horizons in the management of this malignancy. Meanwhile, the interplay between pancreatic carcinogenesis and short non-coding RNA molecules (micro-RNAs or miRs) is in the spotlight of current studies, as they can have either a role as tumor suppressors or promoters. The deregulation of both of the aforementioned molecules leads to several aberrations in the function of pancreatic cells, leading to carcinogenesis. In this review, we will explore the role of extracellular vesicles and miRNAs in pancreatic cancer, as well as their potent utilization as diagnostic and therapeutic tools.

The Arising Role of Extracellular Vesicles in Cholangiocarcinoma: A Rundown of the Current Knowledge Regarding Diagnostic and Therapeutic Approaches.

Cholangiocarcinomas (CCAs) constitute a heterogeneous group of highly malignant epithelial tumors arising from the biliary tree. This cluster of malignant tumors includes three distinct entities, the intrahepatic, perihilar, and distal CCAs, which are characterized by different epidemiological and molecular backgrounds, as well as prognosis and therapeutic approaches. The higher incidence of CCA over the last decades, the late diagnostic time that contributes to a high mortality and poor prognosis, as well as its chemoresistance, intensified the efforts of the scientific community for the development of novel diagnostic tools and therapeutic approaches. Extracellular vesicles (EVs) comprise highly heterogenic, multi-sized, membrane-enclosed nanostructures that are secreted by a large variety of cells via different routes of biogenesis. Their role in intercellular communication via their cargo that potentially contributes to disease development and progression, as well as their prospect as diagnostic biomarkers and therapeutic tools, has become the focus of interest of several current studies for several diseases, including CCA. The aim of this review is to give a rundown of the current knowledge regarding the emerging role of EVs in cholangiocarcinogenesis and their future perspectives as diagnostic and therapeutic tools.

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.

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).

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.

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.

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.

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.

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.

Uric acid variation among regular blood donors is indicative of red blood cell susceptibility to storage lesion markers: A new hypothesis tested.

BACKGROUND: Oxidative stress orchestrates a significant part of the red blood cell (RBC) storage lesion. Considering the tremendous interdonor variability observed in the "storability," namely, the capacity of RBCs to sustain the storage lesion, this study aimed at the elucidation of donor-specific factors that affect the redox homeostasis during the storage of RBCs in standard systems. STUDY DESIGN AND METHODS: The hematologic profile of regular blood donors (n = 78) was evaluated by biochemical analysis of 48 different variables, including in vivo hemolysis and plasma oxidant and antioxidant factors and statistical analysis of the results. The possible effect of the uric acid (UA) variable on RBC storability was investigated in leukoreduced CPD/SAGM RBC units (n = 8) collected from donors exhibiting high or low prestorage levels of UA, throughout the storage period. RESULTS: Among the hematologic variables examined in vivo, cluster analysis grouped the donors according to their serum UA levels. Plasma antioxidant capacity, iron indexes, and protein carbonylation represented covariants of UA factor. RBCs prepared by low- or high-UA donors exhibited significant differences between them in spheroechinocytosis, supernatant antioxidant activity, and other RBC storage lesion-associated variables. CONCLUSION: UA exhibits a storability biomarker potential. Intrinsic variability in plasma UA levels might be related to the interdonor variability observed in the storage capacity of RBCs. A model for the antioxidant effect of UA during the RBC storage is currently proposed.

The time-course linkage between hemolysis, redox, and metabolic parameters during red blood cell storage with or without uric acid and ascorbic acid supplementation.

Oxidative phenomena are considered to lie at the root of the accelerated senescence observed in red blood cells (RBCs) stored under standard blood bank conditions. It was recently shown that the addition of uric (UA) and/or ascorbic acid (AA) to the preservative medium beneficially impacts the storability features of RBCs related to the handling of pro-oxidant triggers. This study constitutes the next step, aiming to examine the links between hemolysis, redox, and metabolic parameters in control and supplemented RBC units of different storage times. For this purpose, a paired correlation analysis of physiological and metabolism parameters was performed between early, middle, and late storage in each subgroup. Strong and repeated correlations were observed throughout storage in most hemolysis parameters, as well as in reactive oxygen species (ROS) and lipid peroxidation, suggesting that these features constitute donor-signatures, unaffected by the diverse storage solutions. Moreover, during storage, a general "dialogue" was observed between parameters of the same category (e.g., cell fragilities and hemolysis or lipid peroxidation and ROS), highlighting their interdependence. In all groups, extracellular antioxidant capacity, proteasomal activity, and glutathione precursors of preceding time points anticorrelated with oxidative stress lesions of upcoming ones. In the case of supplemented units, factors responsible for glutathione synthesis varied proportionally to the levels of glutathione itself. The current findings support that UA and AA addition reroutes the metabolism to induce glutathione production, and additionally provide mechanistic insight and footing to examine novel storage optimization strategies.

Red Blood Cell Metabolism In Vivo and In Vitro.

Red blood cells (RBC) are the most abundant cell in the human body, with a central role in oxygen transport and its delivery to tissues. However, omics technologies recently revealed the unanticipated complexity of the RBC proteome and metabolome, paving the way for a reinterpretation of the mechanisms by which RBC metabolism regulates systems biology beyond oxygen transport. The new data and analytical tools also informed the dissection of the changes that RBCs undergo during refrigerated storage under blood bank conditions, a logistic necessity that makes >100 million units available for life-saving transfusions every year worldwide. In this narrative review, we summarize the last decade of advances in the field of RBC metabolism in vivo and in the blood bank in vitro, a narrative largely influenced by the authors' own journeys in this field. We hope that this review will stimulate further research in this interesting and medically important area or, at least, serve as a testament to our fascination with this simple, yet complex, cell.

Stratification of ßSß+ Compound Heterozygotes Based on L-Glutamine Administration and RDW: Focusing on Disease Severity.

Sickle cell disease (SCD) is heterogeneous in terms of manifestation severity, even more so when in compound heterozygosity with beta-thalassemia. The aim of the present study was to stratify ßSß+ patient blood samples in a severity-dependent manner. Blood from thirty-two patients with HbS/ß-thalassemia compound heterozygosity was examined for several parameters (e.g., hemostasis, inflammation, redox equilibrium) against healthy controls. Additionally, SCD patients were a posteriori (a) categorized based on the L-glutamine dose and (b) clustered into high-/low-RDW subgroups. The patient cohort was characterized by anemia, inflammation, and elevated coagulation. Higher-dose administration of L-glutamine was associated with decreased markers of inflammation and oxidation (e.g., intracellular reactive oxygen species) and an altered coagulation profile. The higher-RDW group was characterized by increased hemolysis, elevated markers of inflammation and stress erythropoiesis, and oxidative phenomena (e.g., membrane-bound hemoglobin). Moreover, the levels of hemostasis parameters (e.g., D-Dimers) were greater compared to the lower-RDW subgroup. The administration of higher doses of L-glutamine along with hydroxyurea seems to attenuate several features in SCD patients, probably by enhancing antioxidant power. Moreover, anisocytosis may alter erythrocytes' coagulation processes and hemolytic propensity. This results in the disruption of the redox and pro-/anti-inflammatory equilibria, creating a positive feedback loop by inducing stress erythropoiesis and, thus, the occurrence of a mixed erythrocyte population.