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.

Cardiometabolic factors and risk of non-arteritic anterior ischemic optic neuropathy: a systematic review and meta-analysis.

PURPOSE: The purpose of this systematic review and meta-analysis of the literature is to evaluate the association between cardiometabolic risk factors (hypertension, diabetes mellitus, hypercholesterolemia/dyslipidemia, HDL cholesterol, LDL cholesterol, lipoprotein(a), and triglycerides) and non-arteritic anterior ischemic optic neuropathy (NAION). METHODS: Pertinent publications were identified through a systematic search in PubMed and EMBASE databases, without language restrictions. The pooled odds ratios (OR) and standardized mean differences (SMD), with their 95% confidence intervals (95% CI) were estimated using random effects (DerSimonian Laird) models, as appropriate. A set of subgroup analyses and meta-regression analysis models were performed. RESULTS: Twenty-one studies (including 1560 patients with NAION and 2292 controls), examining the association between NAION and cardiometabolic risk factors, were eligible for the systematic review and meta-analysis. Hypertension (pooled OR = 1.50; 95% CI: 1.16-1.94), diabetes mellitus (pooled OR = 1.71; 95% CI: 1.33-2.21), and hypercholesterolemia/dyslipidemia (pooled OR = 2.00; 95% CI: 1.53-2.62) were associated with NAION. Among the components of dyslipidemia, higher serum triglycerides were associated with NAION, with a medium effect size (SMD = + 0.58, 95% CI: + 0.12 to + 1.04), whereas synthesis of four studies reporting on HDL and LDL cholesterol did not reveal any significant associations. A significant association between NAION and higher serum lipoprotein(a) levels (pooled OR = 2.88; 95%CI: 1.01-8.21) was also noted. CONCLUSIONS: This systematic review and meta-analysis found that NAION was associated with cardiometabolic factors, suggesting that vascular dysfunction may be implicated in the pathogenesis of the disease. Our findings may alert health care providers to try modifying these risk factors for NAION prevention.

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.

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).

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.

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.

Clinimetric properties of WOMAC Index in Greek knee osteoarthritis patients: comparisons with both self-reported and physical performance measures.

This observational study aimed to examine the clinimetric properties of the Greek for Greece translation of the Western Ontario and McMaster Osteoarthritis Index (WOMAC(®)). One hundred and twenty-three patients with knee osteoarthritis (mean age 69.5 years) participated in the study. An extensive reliability study was carried out to assess WOMAC's internal consistency and repeatability (8-day interval). In addition, we examined the construct (convergent, nomological and known-groups) and criterion-related (concurrent and predictive) validity of the index against both self-report [SF-36 and combined visual analog/faces pain scale-revised (VAS/FPS-R)] and physical performance measures [timed up and go test (TUG)]. The internal consistency of the WOMAC subscales ranged from high (0.804) to excellent (0.956). Intra-class correlation coefficients for test-retest reliability were excellent, ranging from 0.91 to 0.95. Partial correlation analysis, adjusted for age and use of an assistive device, showed that WOMAC scores were significantly associated with all validation criteria, presenting fair to strong (-0.33 to -0.86) correlation coefficients. WOMAC-function was strongly associated with SF36-function (-0.86) and TUG (0.71), WOMAC-pain to VAS/FPS-R (0.71) and SF36-pain (-0.67). Of all WOMAC outcomes, stiffness subscale had the lowest, though still significant, correlations with all validation criteria. Multiple linear regression analyses indicated that WOMAC-function was a significant factor for TUG, WOMAC-pain for VAS/FPS-R and both for SF36-function and SF36-pain. The WOMAC LK3.1 Greek for Greece Index is a reliable and valid assessment tool for the evaluation of individuals with knee osteoarthritis, showing excellent reliability and significant validity properties.

Exploring Aggressive Behaviors in Greek Secondary Schools: Prevalence, Sociodemographic Factors, and Comparative Analysis with Elementary School Students.

The main objectives of this study are to determine the prevalence of bullying in Greek secondary schools and detect the possible characteristics of bullies' profiles in Greek school settings. A structured questionnaire was given to one hundred ninety-two (n = 192) educators at Greek junior high schools in urban and rural areas. The educators were asked to report the frequencies and forms of aggressive behavior observed during the 2022-2023 school year, the bullies' sociodemographic characteristics, and ways of dealing with bullying episodes. The data are presented, after conducting statistical analyses, in comparison with data for elementary school students. The results revealed that higher rates of bullying were reported compared with elementary school children. Moreover, according to teachers' observations, aggressive behavior is independent of a pupil's diagnosis, but specific types are correlated significantly with a pupil's gender, nationality, low academic performance, and popularity. Factor analysis showed two main factors of aggression types, where common points and differences with elementary school students are mentioned. Implementations for the prevention of school bullying are discussed.

Serological Screening and Risk Factors Associated with Leishmania infantum Positivity in Newly Diagnosed HIV Patients in Greece.

A serological screening was conducted to detect IgG antibodies against Leishmania infantum (L. infantum) in newly diagnosed human immunodeficiency virus (HIV) patients in Greece. The study also examined potential risk factors and the agreement of commercially available serological methods. IgG antibodies against L. infantum were detected using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence antibody test (IFAT), and Western blot (WB). Out of 155 samples, 14 (9.0%) tested positive for IgG antibodies against L. infantum using at least two methods. Statistical analysis showed substantial agreement between WB and IFAT methods (Cohen's kappa = 0.75) but moderate overall agreement among the three methods (Fleiss' kappa = 0.42). Additionally, HIV+ intravenous drug users faced 3.55 times (p = 0.025) higher risk of testing positive for L. infantum IgG, positing that anthroponotic transmission between these patients is a plausible hypothesis based on existing literature. Non-invasive and cost-effective techniques are preferred to detect asymptomatic infections, and leishmaniasis screening should be conducted immediately after HIV diagnosis in endemic regions to enable prophylactic treatment for leishmaniasis in addition to antiretroviral therapy. To maximize sensitivity, performing at least two different serological methods for each patient is recommended.

Effect of silica-based mesoporous nanomaterials on human blood cells.

Different mesoporous nanomaterials (MSNs) are constantly being developed for a range of therapeutic purposes, but they invariably interact with blood components and may cause hazardous side effects. Therefore, when designing and developing nanoparticles for biomedical applications, hemocompatibility should be one of the primary goals to assess their toxicity at the cellular level of all blood components. The aim of this study was to evaluate the compatibility of human blood cells (erythrocytes, platelets, and leukocytes) after exposure to silica-based mesoporous nanomaterials that had been manufactured using the sol-gel method, with Ca and Ce as doping elements. The viability of lymphocytes and monocytes was unaffected by the presence of MSNs at any concentration. However, it was found that all nanomaterials, at all concentrations, reduced the viability of granulocytes. P-selectin expression of all MSNs at all concentrations was statistically significantly higher in platelet incubation on the first day of storage (day 1) compared to the control. When incubated with MSNs, preserved platelets displayed higher levels of iROS at all MSNs types and concentrations examined. Ce-containing MSNs presented a slightly better hemocompatibility, although it was also dose dependent. Further research is required to determine how the unique characteristics of MSNs may affect various blood components in order to design safe and effective MSNs for various biomedical applications.

Precision Oncology: Circulating Microvesicles as New Biomarkers in a Very Early Stage of Colorectal Cancer.

BACKGROUND: The release of microvesicles (MVs) is an essential phenomenon for inter-cellular signaling in health and disease. The role of MVs in cancer is multidimensional and includes cancer cell survival, proliferation, and invasion. In this prospective study, we analyzed MV levels in colorectal cancer patients and assessed the importance of MV release in early-stage colorectal cancer and survival. METHODS: This study included 98 patients and 15 controls. The characterization of MVs from human plasma was performed by flow cytometry using monoclonal antibodies. RESULTS: The levels of total MVs and MUC-1-positive, tissue factor (TF)-positive, and endothelial cell-derived MVs (EMVs) were statistically significantly higher in the colon cancer patients than in the controls (p < 0.001). Furthermore, the subgroup of patients with very early-stage colorectal cancer also had statistically significant differences in the levels of the abovementioned MVs compared to the controls (p < 0.01). Highly differentiated tumors had lower levels of MUC-1-positive MVs (p < 0.02), EMVs (p < 0.002), and EMV/TF combinations (p < 0.001) versus those with tumors with low/intermediate differentiation. CONCLUSIONS: Our data demonstrate that the analysis of circulating MV levels in plasma could possibly become a tool for the early diagnosis of colon cancer at a very early stage of the disease.

Prevalence of Aggressive Behavior in Greek Elementary School Settings from Teachers' Perspectives.

The present study aims to estimate the prevalence of bullying in Greek elementary schools and to study the risk factors that lead to bullying episodes. A structured questionnaire was given to 221 teachers of elementary schools and 71 kindergarten teachers from urban and rural Greek schools. They were asked to note the forms and the frequency of aggressive behaviors that they had witnessed during the school years 2020-2021 and 2021-2022, as well as the sociodemographic characteristics of the aggressive children involved. Statistical analyses of the data were conducted, and the obtained results show that specific forms of aggression are significantly correlated with gender and low academic performance. In addition, there is no form of aggressive behavior that is associated with the perpetrator's age, nationality or family status. Further, the results of the factor analysis revealed four dominant factors in the aggressive behavior observed by teachers. The forms of bullying and the prevailing factors of aggressive behavior that dominate in Greek school settings are reported in the present study. Furthermore, a novel evaluation tool for teachers could potentially be developed based on the results of the present study.

Incorporating Medical Museum Specimens Into the Training of Environmental Health Students.

Xenobiotics, radiation, and other environmental health risk factors leave their mark on human organs. This can be demonstrated through the use of pathology museum specimens. Upon completing two semesters of postgraduate studies in environmental health, a tour of the Museum of Pathology is offered to postgraduate students at Athens Medical School who are being trained in environmental health. A structured questionnaire is employed to assess the specimens' impact on several aspects: improving students' observational skills, reinforcing the taught material, acquiring new relevant knowledge, and cultivate the social-cognitive ability of empathy. Additionally, students are asked to evaluate the necessity of preserving metadata associated mainly with the social context of the specimens. This research-educational initiative, a component of an ongoing larger project, underscores the significant educational and research value of museum specimens pertaining to environmental health. Furthermore, effectively utilizing such exhibits can enrich the museum experience for visitors and increase public awareness of environmental health issues.

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.

Prevalence and clinical implications of respiratory viruses in asthma during stable disease state and acute attacks: Protocol for a meta-analysis.

INTRODUCTION: Viruses are detected in over 50% of acute asthma attacks and in a notable proportion of patients with asthma during stable disease state They are associated with worse outcomes. We will conduct a series of systematic reviews and meta-analyses to quantify the prevalence and clinical burden of various respiratory viruses in stable asthma and acute asthma attacks. In addition, we will assess the viral loads of respiratory viruses during stable and acute asthma, to explore whether viral load could differentiate attacks triggered by viruses versus those where viruses are present as "innocent bystanders". MATERIALS AND METHODS: Based on a prospectively registered protocol (PROSPERO, ID: CRD42023375108) and following standard methodology recommended by Cochrane, we will systematically search Medline/PubMed, EMBASE, the Cochrane Library and relevant conference proceedings for studies assessing the prevalence or clinical burden of respiratory viruses in asthma. Methodological rigour of the included studies will be appraised using a tool specific for prevalence studies and the Newcastle-Ottawa Scale respectively. In anticipation of significant clinical and methodological heterogeneity, we will conduct random effect meta-analyses. For evaluating the prevalence of viruses, we will perform meta-analyses of proportions using the inverse variance method, and the Freeman-Tukey transformation. We will conduct meta-regression analyses for exploring heterogeneity. CONCLUSION: We envisage that these systematic reviews and meta-analyses will quantify the prevalence and burden of respiratory viruses in stable and acute asthma and will drive future research and clinical practice.

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.

Climate Changes Exacerbate the Spread of Ixodes ricinus and the Occurrence of Lyme Borreliosis and Tick-Borne Encephalitis in Europe-How Climate Models Are Used as a Risk Assessment Approach for Tick-Borne Diseases.

Climate change has influenced the transmission of a wide range of vector-borne diseases in Europe, which is a pressing public health challenge for the coming decades. Numerous theories have been developed in order to explain how tick-borne diseases are associated with climate change. These theories include higher proliferation rates, extended transmission season, changes in ecological balances, and climate-related migration of vectors, reservoir hosts, or human populations. Changes of the epidemiological pattern have potentially catastrophic consequences, resulting in increasing prevalence of tick-borne diseases. Thus, investigation of the relationship between climate change and tick-borne diseases is critical. In this regard, climate models that predict the ticks' geographical distribution changes can be used as a predicting tool. The aim of this review is to provide the current evidence regarding the contribution of the climatic changes to Lyme borreliosis (LB) disease and tick-borne encephalitis (TBE) and to present how computational models will advance our understanding of the relationship between climate change and tick-borne diseases in Europe.

Blood Cell-Derived Microvesicles in Hematological Diseases and beyond.

Microvesicles or ectosomes represent a major type of extracellular vesicles that are formed by outward budding of the plasma membrane. Typically, they are bigger than exosomes but smaller than apoptotic vesicles, although they may overlap with both in size and content. Their release by cells is a means to dispose redundant, damaged, or dangerous material; to repair membrane lesions; and, primarily, to mediate intercellular communication. By participating in these vital activities, microvesicles may impact a wide array of cell processes and, consequently, changes in their concentration or components have been associated with several pathologies. Of note, microvesicles released by leukocytes, red blood cells, and platelets, which constitute the vast majority of plasma microvesicles, change under a plethora of diseases affecting not only the hematological, but also the nervous, cardiovascular, and urinary systems, among others. In fact, there is evidence that microvesicles released by blood cells are significant contributors towards pathophysiological states, having inflammatory and/or coagulation and/or immunomodulatory arms, by either promoting or inhibiting the relative disease phenotypes. Consequently, even though microvesicles are typically considered to have adverse links with disease prognosis, progression, or outcomes, not infrequently, they exert protective roles in the affected cells. Based on these functional relations, microvesicles might represent promising disease biomarkers with diagnostic, monitoring, and therapeutic applications, equally to the more thoroughly studied exosomes. In the current review, we provide a summary of the features of microvesicles released by blood cells and their potential implication in hematological and non-hematological diseases.

Deciphering the Relationship Between Free and Vesicular Hemoglobin in Stored Red Blood Cell Units.

Red blood cells (RBCs) release hemoglobin (Hb)-containing extracellular vesicles (EVs) throughout their lifespan in the circulation, and especially during senescence, by spleen-facilitated vesiculation of their membrane. During ex vivo aging under blood bank conditions, the RBCs lose Hb, both in soluble form and inside EVs that accumulate as a part of storage lesion in the supernatant of the unit. Spontaneous hemolysis and vesiculation are increasingly promoted by the storage duration, but little is known about any physiological linkage between them. In the present study, we measured the levels of total extracellular and EV-enclosed Hb (EV-Hb) in units of whole blood (n = 36) or packed RBCs stored in either CPDA-1 (n = 99) or in CPD-SAGM additive solution (n = 46), in early, middle, and late storage. The spectrophotometry data were subjected to statistical analysis to detect possible correlation(s) between storage hemolysis and EV-Hb, as well as the threshold (if any) that determines the area of this dynamic association. It seems that the percentage of EV-Hb is negatively associated with hemolysis levels from middle storage onward by showing low to moderate correlation profiles in all strategies under investigation. Moreover, 0.17% storage hemolysis was determined as the potential cut-off, above which this inverse correlation is evident in non-leukoreduced CPDA units. Notably, RBC units with hemolysis levels > 0.17% are characterized by higher percentage of nanovesicles (<100 nm) over typical microvesicles (100-400 nm) compared with the lower hemolysis counterparts. Our results suggest an ordered loss of Hb during RBC accelerated aging that might fuel targeted research to elucidate its mechanistic basis.

Early and Late-Phase 24 h Responses of Stored Red Blood Cells to Recipient-Mimicking Conditions.

The 24-hour (24 h) post-transfusion survival of donor red blood cells (RBCs) is an important marker of transfusion efficacy. Nonetheless, within that period, donated RBCs may encounter challenges able to evoke rapid stress-responses. The aim of the present study was to assess the effect of exposure to plasma and body temperature upon stored RBCs under recipient-mimicking conditions in vitro from the first hours "post-transfusion" up to 24 h. For this purpose, packed RBCs from seven leukoreduced CPD/SAGM units were reconstituted with plasma of twenty-seven healthy individuals and incubated for 24 h at 37oC. Three units were additionally used to examine stress-responses in 3-hour intervals post mixing with plasma (n = 5) until 24 h. All experiments were performed in shortly-, medium-, and long-stored RBCs. Hemolysis, redox, morphology, membrane protein binding and vesiculation parameters were assessed. Even though spontaneous hemolysis was minimal post-reconstitution, it presented a time-dependent increase. A similar time-course profile was evident for the concentration of procoagulant extracellular vesicles and the osmotic fragility (shortly-stored RBCs). On the contrary, mechanical fragility and reactive oxygen species accumulation were characterized by increases in medium-stored RBCs, evident even from the first hours in the recipient-mimicking environment. Finally, exposure to plasma resulted in rapid improvement of morphology, especially in medium-stored RBCs. Overall, some RBC properties vary significantly during the first 24 h post-mixing, at levels different from both the storage ones and the standard end-of-24 h. Such findings may be useful for understanding the performance of RBCs and their possible clinical effects -especially on susceptible recipients- during the first hours post-transfusion.