Differential intracellular trafficking of extracellular vesicles in microglia and astrocytes.

Extracellular vesicles (EVs) have emerged as key players in cell-to-cell communication in both physiological and pathological processes in the Central Nervous System. Thus far, the intracellular pathways involved in uptake and trafficking of EVs within different cell types of the brain are poorly understood. In our study, the endocytic processes and subcellular sorting of EVs were investigated in primary glial cells, particularly linked with the EV-associated α-synuclein (α-syn) transmission. Mouse microglia and astrocytic primary cultures were incubated with DiI-stained mouse brain-derived EVs. The internalization and trafficking pathways were analyzed in cells treated with pharmacological reagents that block the major endocytic pathways. Brain-derived EVs were internalized by both glial cell types; however, uptake was more efficient in microglia than in astrocytes. Colocalization of EVs with early and late endocytic markers (Rab5, Lamp1) indicated that EVs are sorted to endo-lysosomes for subsequent processing. Blocking actin-dependent phagocytosis and/or macropinocytosis with Cytochalasin D or EIPA inhibited EV entry into glial cells, whereas treatment with inhibitors that strip cholesterol off the plasma membrane, induced uptake, however differentially altered endosomal sorting. EV-associated fibrillar α-Syn was efficiently internalized and detected in Rab5- and Lamp1-positive compartments within microglia. Our study strongly suggests that EVs enter glial cells through phagocytosis and/or macropinocytosis and are sorted to endo-lysosomes for subsequent processing. Further, brain-derived EVs serve as scavengers and mediate cell-to-glia transfer of pathological α-Syn which is also targeted to the endolysosomal pathway, suggesting a beneficial role in microglia-mediated clearance of toxic protein aggregates, present in numerous neurodegenerative diseases.

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.

Pathogen Reduction Technologies and Their Impact on Metabolic and Functional Properties of Treated Platelet Concentrates: A Systematic Review.

Pathogen reduction technologies (PRTs) such as Mirasol and Intercept were developed to eliminate transfusion-transmitted infections. The impact of PRTs on platelet function during the storage period, their effect on platelet storage lesions, and the optimal storage duration following PRTs have not been clearly defined. The aim of this study was to systematically review the existing literature and investigate the impact of PRTs on functional alterations of PRT-treated platelets during the storage period. The authors identified 68 studies suitable to be included in this review. Despite the high heterogeneity in the literature, the results of the published studies indicate that PRTs may increase platelet metabolic activity, accelerate cell apoptosis, and enhance platelet activation, which can subsequently lead to a late exhaustion of activation potential and reduced aggregation response. However, these effects have a minor impact on platelet function during the early storage period and become more prominent beyond the fifth day of the storage period. Large in vivo trials are required to evaluate the effectiveness of PRT-treated platelets during the storage period and investigate whether their storage can be safely extended to more than 5 days, and up to the traditional 7-day storage period.

The Utility of NATEM Assay in Predicting Bleeding Risk in Critically Ill Neonates.

We aimed to investigate the hemostatic status of diseased neonates using nonactivated rotational thromboelastometry (ROTEM) assay (NATEM) assay and, in addition, to evaluate the discriminative power of NATEM parameters in predicting the risk of bleeding in critically ill neonates and compare it to that of EXTEM (extrinsically activated ROTEM) parameters. This cohort study included 158 consecutive, critically ill neonates with presumed sepsis, perinatal hypoxia, or respiratory distress syndrome. The EXTEM and NATEM assays were performed on the first day of disease onset. The neonatal bleeding assessment tool was used to record and assess clinical bleeding events on the day of ROTEM analysis. Several EXTEM and NATEM ROTEM parameters differed between neonates with and without clinical bleeding events, indicating a hypo-coagulable state in neonates with clinical bleeding. NATEM parameters had comparable predictive performance for clinical bleeding events with EXTEM parameters for clotting time, clot formation time (CFT), A10 (clot amplitude at 10minutes), maximum clot firmness, lysis index at 60minutes, and maximum clot elasticity (p>0.05). However, NATEM A20, A30, and α angle demonstrated better predictive ability than EXTEM A20, A30, and α angle, respectively (p<0.05). A NATEM CFT value ≥147seconds presented 95.2% sensitivity (95% confidence interval [CI]: 76.1-99.8%) and 65.6% specificity (95% CI: 57.1-73.5%) to detect neonates with clinical bleeding, while a NATEM A10 value ≤42mm had 80.8% sensitivity (95% CI: 71.8-85.9%) and 76.0% specificity (95% CI: 52.8-91.7%) to detect neonates with clinical bleeding events. The NATEM assay has shown remarkable sensitivity in predicting bleeding in critically ill neonates, exceeding EXTEM performance in some selected parameters. The incorporation of NATEM test parameters in predictive models for neonatal hemorrhage seems promising.

Rotational Thromboelastometry Predicts Transfusion Requirements in Total Joint Arthroplasties.

The frequency of red blood cell (RBC) transfusions is high in total joint arthroplasties, and the hemorrhagic risk is associated with both surgery- and patient-related factors. This study aims to assess the ability of rotational thromboelastometry (ROTEM) to identify patients at high risk for transfusion and excessive bleeding. A prospective observational study was conducted including 206 patients who underwent total knee or hip arthroplasties. Assessment of the coagulation status was performed preoperatively and immediately postoperatively using ROTEM analysis and conventional coagulation tests. The number of RBC transfusions and the postoperative hemoglobin drop were recorded. ROTEM findings were compared between transfused and nontransfused patients, and also between patients with and without excessive bleeding. Higher values of postoperative FIBTEM maximum clot firmness (MCF) were associated with lower risks of transfusion (odds ration [OR]: 0.66, 95% confidence interval [CI]: 0.57-0.78, p<0.001) and excessive bleeding (OR: 0.58, 95% CI: 0.36-0.94, p=0.028). A postoperative FIBTEM MCF value ≤10mm had 80.1% (95% CI: 73.1-85.9%) sensitivity with 75.5% (95% CI: 60.4-87.1%) specificity to predict transfusion requirements, and 70.5% (95% CI: 63.6-76.8%) sensitivity with 88.8% (95% CI: 51.7-99.7%) specificity to predict excessive bleeding. The estimated average probability of transfusion in patients with FIBTEM MCF values of 0 to 4mm is 86.3%. ROTEM assay demonstrated high predictive ability for transfusion and excessive bleeding. Identification of patients at risk for transfusion could allow blood banks to ensure adequate blood supply, while also more intense blood-salvaging strategies could be implemented in these patients.

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.

Assessment of agreement between EXTEM and NATEM thromboelastometry measurement assays in critically ill neonates.

OBJECTIVES: To investigate the agreement between the EXTEM and NATEM measurements. METHODS: In this retrospective observational study, EXTEM and NATEM analyses were performed on blood samples from 162 ill neonates, providing 324 paired measurements. The agreement between EXTEM and NATEM measurements was evaluated by the nonparametric spearman's rank correlation to assess the correlation between the paired measurements, by the Bland-Altman analysis for the graphical presentation of the agreement, and by the Deming regression model to assess the significance of the agreement. The agreement between the two methods for the detection of bleeding events was determined by kappa statistic. RESULTS: Strong correlations were found between EXTEM and NATEM measurements for A10, MCF. The Bland-Altman plots showed good agreement for A10, MCF, LI60, and alpha angle parameters, while CT showed a nearly linear slope indicating that bias increased with the mean. The highest agreement for bleeding events was found for the A10 parameter (κ = 0.70, p < .001), while the lowest for the CT parameter (κ = 0.36, p = .94). CONCLUSIONS: NATEM parameters that reflect clot firmness and fibrinolytic activity are strongly correlated with the corresponding EXTEM measurements with a good agreement between them, indicating that these two methods could be used interchangeably.

Economic crisis in Greece: The invisible enemy of blood donation or not?

INTRODUCTION: Volunteering presupposes having free time and refers to the provision of services without the motivation of material reward, for the benefit of society. In this study, we aimed to provide insight into the impact of economic crisis on blood donors and their motivation to donate blood during that period. STUDY DESIGN AND METHODS: We asked blood donors about their blood donation activity and motivation to donate using a standardized, anonymous questionnaire (n = 3000). Descriptive analysis was performed for the consideration of donor turnout during this economic period. The results were analyzed using the χ2 test and Spearman's correlation coefficient. RESULTS: Regarding gender, 68.2% were males, while 31.8% were females. Most blood donors donated voluntarily (75.8%) and only 24.2% were replacement or family blood donors. The economic crisis has affected the inhabitants of Athens more than the inhabitants of the province (χ2 = 9.910,p = 0.007). The influence of economic crisis on the regular blood donors' quality of life was greater than the non-regular donors (χ2 = 16.227,p < 0.001). According to our results, the economic crisis reduced the quality of life, but it did not affect the frequency of blood donations in a percentage of 87,3%. Not any significant difference was found between employment status, economic crisis and blood donation. CONCLUSION: Although the economic crisis has affected the lives of blood donors, it does not seem to affect the frequency of blood donation. We suggest that blood collection services should consider specialist campaigns that focus on the altruistic motivation of donors during an economic crisis.

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 [...].

The Emerging Role of Extracellular Vesicles and Autophagy Machinery in NASH-Future Horizons in NASH Management.

Non-alcoholic fatty liver disease (NAFLD) is considered the most frequent chronic hepatic disease in the general population, while it is the first cause of liver transplantation in the US. NAFLD patients will subsequently develop non-alcoholic steatohepatitis (NASH), which is characterized by aberrant hepatocellular inflammation with or without the presence of fibrosis. The lack of specific biomarkers and therapeutic strategies makes non-alcoholic steatohepatitis (NASH) management a difficult task for clinicians. Extracellular vesicles (EVs) constitute a heterogenic population of vesicles produced by inward or outward plasma-membrane budding. There is an emerging connection between autophagy EVs production, via an unconventional non-degradative procedure. Alterations in the amount of the secreted EVs and the cargo they carry are also involved in the disease progression and development of NASH. Autophagy constitutes a multistep lysosomal degradative pathway that reassures cell homeostasis and survival under stressful conditions, such as oxygen and energy deprivation. It prevents cellular damage by eliminating defected proteins or nοn-functional intracellular organelles. At the same time, it reassures the optimal conditions for the cells via a different mechanism that includes the removal of cargo via the secretion of EVs. Similarly, autophagy machinery is also associated with the pathogenetic mechanism of NAFLD, while it has a significant implication for the progression of the disease and the development of NASH. In this review, we will shed light on the interplay between autophagy and EVs in NASH, the emerging connection of EVs production with the autophagy pathway, and their possible manipulation for developing future therapeutic strategies for NASH.

Rotational Thromboelastometry in Neonates Admitted to a Neonatal Intensive Care Unit: A Large Cross-sectional Study.

The aim of the present study was to assess the coagulation profile in neonatal critical illness using rotational thromboelastometry (ROTEM), and to investigate its association with disease severity and its potential prognostic role in this clinical setting. Over a period of 67 months (July 2014-February 2020) 423 critically ill neonates with confirmed or suspected sepsis, perinatal hypoxia, or respiratory distress syndrome, hospitalized in our neonatal intensive care unit were included in the study. Demographic, clinical, and laboratory data were recorded on admission day and arterial blood was analyzed on ROTEM analyzer using the standard extrinsically activated rotational thromboelastometry assay (EXTEM). Neonatal illness severity scores (Modified NEOMOD [Neonatal Multiple Organ Dysfunction] and SNAPPE [Score for Neonatal Acute Physiology with Perinatal Extension]) were calculated at the same time as ROTEM analysis. Mortality during in-hospital stay was the main outcome measure. Multivariable analyses showed that a 10 mm decrease in EXTEM clot amplitude recorded at 10 minutes (A10) is significantly associated with a higher mortality (odds ratio [OR] = 1.69, 95% confidence interval [CI]: 1.33-2.08). Higher modified NEOMOD (OR = 1.36, 95% CI: 1.26-1.47) and higher SNAPPE scores (OR = 1.06, 95% CI: 1.04-1.08) were also associated with increased mortality. The CT and A10 variables demonstrated the best prognostic performance among the EXTEM parameters for mortality (area under the curve [AUC] = 0.78; 95% CI: 0.69-0.86 and AUC = 0.76; 95% CI: 0.66-0.85, respectively), showing an optimal cut-off CT ≥63 seconds and A10 ≤37 mm. Using optimal cut-off values of the EXTEM parameters for prediction of mortality, neonates with CT ≥63 seconds were 7.4 times more likely to die (OR = 7.40, 95% CI: 3.50-15.65), while neonates with A10 ≤37 mm were 5.8 times more likely to die (OR = 5.88, 95% CI: 2.94-12.50). An EXTEM hypocoagulable profile on disease onset was shown to be an independent risk factor for in-hospital mortality in neonatal critical illness.

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.

Rotational Thromboelastometry Findings Are Associated with Symptomatic Venous Thromboembolic Complications after Hip Fracture Surgery.

BACKGROUND: Venous thromboembolism is a common complication after hip fractures. However, there are no reliable laboratory assays to identify patients at risk for venous thromboembolic (VTE) events after major orthopaedic surgery. QUESTION/PURPOSES: (1) Are rotational thromboelastometry (ROTEM) findings associated with the presence or development of symptomatic VTE after hip fracture surgery? (2) Were any other patient factors associated with the presence or development of symptomatic VTE after hip fracture surgery? (3) Which ROTEM parameters were the most accurate in terms of detecting the association of hypercoagulability with symptomatic VTE? METHODS: This retrospective study was conducted over a 13-month period. In all, 354 patients with femoral neck and peritrochanteric fractures who underwent hip hemiarthoplasty or cephallomedullary nailing were assessed for eligibility. Of those, 99% (349 of 354) were considered eligible for the study, 1% (3 of 354) of patients were excluded due to coagulation disorders, and another 1% (2 of 354) were excluded because they died before the postoperative ROTEM analysis. An additional 4% (13 of 354) of patients were lost before the minimum study follow-up of 3 months, leaving 95% (336 of 354) for analysis. A ROTEM analysis was performed in all patients at the time of their hospital admission, within hours of the injury, and on the second postoperative day. The patients were monitored for the development of symptoms indicative of VTE, and the gold standard tests for diagnosing VTE, such as CT pulmonary angiography or vascular ultrasound, were selectively performed only in symptomatic patients and not routinely in all patients. Therefore, this study evaluates the association of ROTEM with only clinically evident VTE events and not with all VTE events. ROTEM results did not affect the clinical surveillance of the study group and the decision for further work up. To determine whether ROTEM findings were associated with the presence or development of symptomatic VTE, ROTEM parameters were compared between patients with and without symptomatic VTE. To establish whether any other patient factors were associated with the presence or development of symptomatic VTE after hip fracture surgery, clinical parameters and conventional laboratory values were also compared between patients with and without symptomatic VTE. Finally, to determine which ROTEM parameters were the most accurate in terms of detecting the association of hypercoagulability with symptomatic VTE, the area under the curve (AUC) for certain cut off values of ROTEM parameters was calculated. RESULTS: We found several abnormal ROTEM values to be associated with the presence or development of symptomatic VTE. The preoperative maximum clot firmness was higher in patients with clinically evident VTE than in patients without these complications (median [interquartile range] 70 mm [68 to 71] versus 65 mm [61 to 68]; p < 0.001). The preoperative clot formation time was lower in patients with clinically evident VTE than those without clinically evident VTE (median 61 seconds [58 to 65] versus 70 seconds [67 to 74]; p < 0.001), and also the postoperative clot formation time was lower in patients with clinically evident VTE than those without these complications (median 52 seconds [49 to 59] versus 62 seconds [57 to 68]; p < 0.001). Increased BMI was also associated with clinically evident VTE (odds ratio 1.26 [95% confidence interval 1.07 to 1.53]; p < 0.001). We found no differences between patients with and without clinically evident VTE in terms of age, sex, smoking status, comorbidities, and preoperative use of anticoagulants. Lastly, preoperative clot formation time demonstrated the best performance for detecting the association of hypercoagulability with symptomatic VTE (AUC 0.89 [95% CI 0.81 to 0.97]), with 81% (95% CI 48% to 97%) sensitivity and 86% (95% CI 81% to 89%) specificity for clot formation time ≤ 65 seconds. CONCLUSION: ROTEM's performance in this preliminary study was promising in terms of its association with symptomatic VTE. This study extended our earlier work by demonstrating that ROTEM has a high accuracy in detecting the level of hypercoagulability that is associated with symptomatic VTE. However, until its performance is validated in a study that applies a diagnostic gold standard (such as venography, duplex/Doppler, or chest CT) in all patients having ROTEM to confirm its performance, ROTEM should not be used as a regular part of clinical practice. LEVEL OF EVIDENCE: Level IV, diagnostic study.

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.