Aim: We determined the long-term role of increased RDW (red blood cell distribution width) detected during cardiac decompensation.Methods: We followed 3697 patients [mean age 71.4 years (±SD 10.1), 59.1% males] hospitalized for acute heart failure (HF) and assessed the five-year all-cause mortality risk associated with tertiles of RDW.Results: Patients with RDW in the top tertile showed roughly twofold higher 5-year mortality risk than those in the bottom tertile. The association remained significant not only after adjustments for potential covariates but even if we excluded patients who deceased during the first year of follow-up [HRR 1.76 (95% CIs:1.42-2.18), p < 0.0001].Conclusion: The high degree of anisocytosis represents an independent predictor of poor prognosis in HF patients, even long-term after an acute manifestation.
[Box: see text].

Microscopic inspection of thin-film blood smears is widely used to identify red blood cell (RBC) pathologies, including malaria parasitism and hemoglobinopathies, such as sickle cell disease and thalassemia. Emerging research indicates that non-pathologic changes in RBCs can also be detected in images, such as deformability and morphological changes resulting from the storage lesion. In transfusion medicine, cell deformability is a potential biomarker for the quality of donated RBCs. However, a major impediment to the clinical translation of this biomarker is the difficulty associated with performing this measurement. To address this challenge, we developed an approach for biophysical profiling of RBCs based on cell images in thin-film blood smears. We hypothesize that subtle cellular changes are evident in blood smear images, but this information is inaccessible to human expert labellers. To test this hypothesis, we developed a deep learning strategy to analyze Giemsa-stained blood smears to assess the subtle morphologies indicative of RBC deformability and storage-based degradation. Specifically, we prepared thin-film blood smears from 27 RBC samples (9 donors evaluated at 3 storage time points) and imaged them using high-resolution microscopy. Using this dataset, we trained a convolutional neural network to evaluate image-based morphological features related to cell deformability. The prediction of donor deformability is strongly correlated to the microfluidic scores and can be used to categorize images into specific deformability groups with high accuracy. We also used this model to evaluate differences in RBC morphology resulting from cold storage. Together, our results demonstrate that deep learning models can detect subtle cellular morphology differences resulting from deformability and cold storage. This result suggests the potential to assess donor blood quality from thin-film blood smears, which can be acquired ubiquitously in clinical workflows.

The growing world population and increasing life expectancy are driving the need to improve the quality of blood transfusion, organ transplantation, and preservation. Here, to improve the ability of red blood cells (RBCs) for normothermic machine perfusion, a biocompatible blood silicification approach termed \"shielding-augmenting RBC-in-nanoscale amorphous silica (SARNAS)\" has been developed. The key to RBC surface engineering and structure augmentation is the precise control of the hydrolysis form of silicic acid to realize stabilization of RBC within conformal nanoscale silica-based exoskeletons. The formed silicified RBCs (Si-RBCs) maintain membrane/structural integrity, normal cellular functions (e.g., metabolism, oxygen-carrying capability), and enhance resistance to external stressors as well as tunable mechanical properties, resulting in nearly 100% RBC cryoprotection. In vivo experiments confirm their excellent biocompatibility. By shielding RBC surface antigens, the Si-RBCs provide universal blood compatibility, the ability for allogeneic mechanical perfusion, and more importantly, the possibility for cross-species transfusion. Being simple, reliable, and easily scalable, the SARNAS strategy holds great promise to revolutionize the use of engineered blood for future clinical applications.

We aimed to study the influence of preventing methemoglobin (metHb) formation, in the roles of peroxiredoxin 2 (Prx2), glutathione peroxidase (GPx) and catalase (CAT) on the erythrocyte antioxidant defense system. We performed in vitro assays using healthy erythrocytes, with and without inhibition of autoxidation of Hb (saturation with carbon monoxide), followed by H2O2-induced oxidative stress. We assessed the enzyme activities and amounts of CAT, GPx and Prx2 in the red blood cell (RBC) cytosol and membrane and several biomarkers of oxidative stress, such as the reduced and oxidized glutathione levels, thiobarbituric acid reactive substances (TBARS) levels, membrane bound hemoglobin and total antioxidant status. When autoxidation of Hb was inhibited, no significant changes were found for GPx and CAT; Prx2 was observed only in the monomeric form in the cytosol and none bound to the membrane. Blocking the function of Hb as a pseudo-peroxidase does not seem to have an impact on the function of the RBC peroxidases.

BACKGROUND: Avocado intake improves dietary fat quality, but the subsequent impact on red blood cell (RBC) saturated (SFA), monounsaturated (MUFA), polyunsaturated (PUFA), and trans-fatty acid (TFA) composition and association with cardiometabolic health, has not been elucidated.
OBJECTIVE: To compare the effect of consuming 1 avocado/d relative to habitual diet (HAB) on RBC-FA profiles, and their association with visceral adiposity and cardiometabolic risk factors (CMRFs) in individuals with abdominal obesity.
METHODS: RBC-FA profiling at baseline, 3- and 6 mo was conducted in participants (n = 994) from the Habitual Diet and Avocado Trial (HAT). HAT was a multisite, free-living, parallel-arm intervention study in which participants were randomly assigned to either the avocado-supplemented group (AVO, usual diet with 1 avocado/d) or the HAB group (usual diet with limited avocado intake) for 6 mo. Changes in RBC-FA profiles, a secondary outcome measure, were determined within and between groups using linear regression and mixed effect models, adjusting for age, sex, BMI, clinical site, smoking status, and percentage of energy intake from fat at baseline. The association between changes in RBC-FAs with visceral adiposity measures and CMRFs was assessed after covariate and False Discovery Rate (FDR <0.05) adjustment.
RESULTS: No major differences in RBC-FA profiles were observed between groups, with the exception of MUFA cis-vaccenic [18:1n-7c], which was significantly higher in AVO (β: 0.11 [0.05, 0.17]) compared with the HAB (β: 0.03 [-0.03, 0.08]) participants. In the HAB but not AVO group, increases in MUFA cis (18:1n-7c, oleic [18;1n-9c], erucic [22:1n-9c]) and MUFA trans (palmitelaidic [16:1n-7t], vaccenic [18:1n-7t], elaidic [18:1n-9t], and petroselaidic [18;1n-10-12t), as well as PUFA γ-linolenic [18:3n-6], dihomo-γ-linolenic [20:3n-6], arachidonic [20:4n-6], and α-linolenic [18:3n-3] were associated with unfavorable changes in visceral adiposity measures, lipid profiles, glucose, insulin and high sensitivity C-reactive protein concentrations.
CONCLUSIONS: Daily avocado intake over 6-mo modified RBC-MUFA composition, notably 18:1n-7c, and potentially mitigated some of the unfavorable individual RBC-FA-CMRF associations observed over time in the HAB group. This trial was registered at https://clinicaltrials.gov/study as NCT03528031.

Enucleated erythrocytes are characteristic of adult mammals. In contrast, fish, amphibians, reptiles, birds, and fetal mammals possess nucleated erythrocytes in their circulation. Erythroid maturation is regulated by erythropoietin (EPO) and its receptor (EPOR), which are conserved among vertebrates. In mammals, EPOR on the erythroid progenitor membrane disappears after terminal differentiation. However, in western clawed frog, Xenopus tropicalis, mature erythrocytes maintain EPOR expression, suggesting that they have non-canonical functions of the EPO-EPOR axis rather than proliferation and differentiation. In this study, we investigated the non-canonical functions of EPOR in Xenopus mature erythrocytes. EPO stimulation of peripheral erythrocytes did not induce proliferation but induced phosphorylation of intracellular proteins, including signal transducer and activator of transcription 5 (STAT5). RNA-Seq analysis of EPO-stimulated peripheral erythrocytes identified 45 differentially expressed genes (DEGs), including cytokine inducible SH2 containing protein gene (cish) and suppressor of cytokine signaling 3 gene (socs3), negative regulators of the EPOR-Janus kinase (JAK)-STAT pathway. These phosphorylation studies and pathway analysis demonstrated the activation of the JAK-STAT pathway through EPO-EPOR signaling in erythrocytes. Through comparison with EPO-responsive genes in mouse erythroid progenitors obtained from a public database, we identified 31 novel EPO-responsive genes indicating non-canonical functions. Among these, we focused on ornithine decarboxylase 1 gene (odc1), which is the rate-limiting enzyme in polyamine synthesis and affects hematopoietic progenitor differentiation and the endothelial cell response to hypoxic stress. An EPO-supplemented culture of erythrocytes showed increased odc1 expression followed by a decrease in polyamine-rich erythrocytes, suggesting EPO-responsive polyamine excretion. These findings will advance our knowledge of the unknown regulatory systems under the EPO-EPOR axis and functional differences between vertebrates\' nucleated and enucleated erythrocytes.

The biodistribution of many therapeutics is controlled by the immune system. In addition, some molecules are cytotoxic when not encapsulated inside of larger cellular structures, such as hemoglobin (Hb) encapsulation inside of red blood cells (RBCs). To counter immune system recognition and cytotoxicity, drug delivery systems based on red blood cell membrane fragments (RBCMFs) have been proposed as a strategy for creating immunoprivileged therapeutics. However, the use of RBCMFs for drug delivery applications requires purification of RBCMFs at large scale from lysed RBCs free of their intracellular components. In this study, we were able to successfully use tangential flow filtration (TFF) to remove >99% of cell-free Hb from lysed RBCs at high concentrations (30%-40% v/v), producing RBCMFs that were 2.68 ± 0.17 μm in diameter. We were also able to characterize the RBCMFs more thoroughly than prior work, including measurement of particle zeta potential, along with individual TFF diacycle data on the cell-free Hb concentration in solution and time per diacycle, as well as concentration and size of the RBCMFs. In addition to purifying RBCMFs from lysed RBCs, we utilized a hypertonic solution to reseal purified RBCMFs encapsulating a model protein (Hb) to yield resealed Hb-encapsulated RBC ghosts (Hb-RBCGs). TFF was then compared against centrifugation as an alternative method for removing unencapsulated Hb from Hb-RBCGs, and the effects that each washing method on the resulting Hb-RBCG biophysical properties was assessed.

OBJECTIVE: Alloimmunization against red blood cell (RBC) antigen is an important concern in myelodysplastic syndromes (MDSs) patients with chronic transfusion, causing potential risk for hemolytic reaction and limited supply of compatible blood. However, there is little data addressing RBC alloimmunization in this patient cohort among the Chinese population. This study aims to evaluate the incidence, specificity of antibodies, and RBC units transfused before antibody formation and its significance in a population of patients consistently receiving RhD-matched RBC units.
METHODS: We retrospectively reviewed the transfusion and clinical information of all transfused patients with MDS enrolled in our hospital from 2012 to 2022. The cumulative incidence of alloimmunization was analyzed by a Kaplan-Meier plot. Alloimmunization incidence was compared based on different transfused RBC units using the log-rank test.
RESULTS: A total of 103 patients with MDS were included in this study; alloantibody formed in 8 (7.8%) patients. Before reaching 32 RBC units, 87.5% of the alloimmunized patients had developed their alloantibodies. All but 1 of the alloantibodies developed were antibodies to Rh antigens. The RBC transfusion intensity and frequency were significantly higher following alloimmunization in the alloimmunized patients (P = .008, P = .008, respectively).
CONCLUSIONS: The antibodies detected mostly involve the Rh system among MDS patients in China. The alloimmunization tended to occur early prior to reaching 32 RBC units in patients with MDS. Rh antigen matching should be considered early in the patient\'s transfusion history and completed before receiving 32 RBC units.

There is growing evidence that inflammation impairs erythrocyte structure and function. We assessed the impact of mild systemic inflammation on erythrocyte fragility in three different settings. In order to investigate causation, erythrocyte osmotic fragility was measured in mice challenged with a live attenuated bacterial strain to induce low-grade systemic inflammation; a significant increase in erythrocyte osmotic fragility was observed. To gather evidence that systemic inflammation is associated with erythrocyte fragility in humans, two observational studies were conducted. First, using a retrospective study design, the relationship between reticulocyte-based surrogate markers of haemolysis and high-sensitivity C-reactive protein was investigated in 9292 healthy participants of the UK Biobank project. Secondly, we prospectively assessed the relationship between systemic inflammation (measured by the urinary neopterin/creatinine ratio) and erythrocyte osmotic fragility in a mixed population (n = 54) of healthy volunteers and individuals with long-term medical conditions. Both human studies were in keeping with a relationship between inflammation and erythrocyte fragility. Taken together, we conclude that mild systemic inflammation increases erythrocyte fragility and may contribute to haemolysis. Further research is needed to assess the molecular underpinnings of this pathway and the clinical implications in inflammatory conditions.

Red blood cells (RBCs) express the nucleic acid-binding toll-like receptor 9 (TLR9) and bind CpG-containing DNA. However, whether human RBCs express other nucleic acid-binding TLRs is unknown. Here we show that human RBCs express the RNA sensor TLR7. TLR7 is present on the red cell membrane and is associated with the RBC membrane protein Band 3. In patients with SARS-CoV2-associated sepsis, TLR7-Band 3 interactions in the RBC membrane are increased when compared with healthy controls. In vitro, RBCs bind synthetic ssRNA and RNA from ssRNA viruses. Thus, RBCs may serve as a previously unrecognized sink for exogenous RNA, expanding the repertoire of non-gas exchanging functions performed by RBCs.