Senescent Red Blood Cells Research Articles

Associations between blood donor sex and age, and outcomes of transfused newborn infants.

It is controversial whether the sex or age of red blood cell (RBC) donors affects mortality or morbidities of transfused newborn infants. We assessed these issues using a multi-year, multi-hospital database linking specific outcomes of neonatal transfusion recipients with RBC donor sex and age. We performed retrospective analyses of all neonates receiving ≥ one RBC transfusion during a 12-year period in all Intermountain Healthcare hospitals, matching mortality and specific morbidities of each transfusion recipient with the sex and age of each donor. There were 6396 RBC transfusions administered to 2086 infants in 15 hospitals. A total of 825 infants were transfused exclusively with RBC from female donors, 935 infants were transfused exclusively with RBC from male donors, and 326 infants were transfused with RBC from both female and male donors. No differences in baseline characteristics were identified among the three groups. Infants who received blood from both male and female donors had more RBC transfusions (5.3 ± 2.9 transfusions if received both male and female donor blood vs. 2.6 ± 2.2 if received blood from only one sex, mean ± SD, p < .001). We identified no significant differences in mortality or morbidities associated with the sex or the age of blood donors. Similarly, an analysis of matched vs. mismatched donor/recipient sex revealed no associations with death or neonatal morbidities. These data support the practice of transfusing newborn infants with RBC obtained from donors of either sex and regardless of donor age.

Stereochemistry and functions of the new cysteinyl-resolvin, 4S,5R-RCTR1, in efferocytosis and erythrophagocytosis of human senescent erythrocytes.

Specialized pro-resolving lipid mediators play key functions in the resolution of the acute inflammatory response. Herein, we elucidate the stereochemical structure of the new 4S,5R-RCTR1, a cysteinyl-resolvin, recently uncovered in human leukocytes incubated with a 4S,5S-epoxy-resolvin intermediate, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and ultra-violet (UV) spectrophotometry. With this approach, the physical properties of the new mediator prepared by total organic synthesis were matched to enzymatically produced biogenic material. In addition, we confirmed the potent biological actions of 4S,5R-RCTR1 with human M2-like macrophage phagocytosis of live bacteria, efferocytosis of apoptotic neutrophils, and erythrophagocytosis of senescent human red blood cells in a concentration-dependent manner from 0.1 to 10 nM. Taken together, these results establish the complete stereochemistry of 4S,5R-RCTR1 as 5R-glutathionyl-4S,17S-dihydroxy-6E,8E,10Z,13Z,15E,19Z-docosahexaenoic acid and give evidence of its novel bioactivities in human phagocyte responses. Moreover, they confirm and extend the stereoselective functions of the 4S,5R-RCTR1 with isolated human phagocytes of interest in the resolution of inflammation.

Blocking the CD47-SIRPα interaction reverses the disease phenotype in a polycythemia vera mouse model

Polycythemia vera (PV) is a hematopoietic stem cell neoplasm driven by somatic mutations in JAK2, leading to increased red blood cell (RBC) production uncoupled from mechanisms that regulate physiological erythropoiesis. At steady-state, bone marrow macrophages promote erythroid maturation, whereas splenic macrophages phagocytose aged or damaged RBCs. The binding of the anti-phagocytic (“don’t eat me”) CD47 ligand expressed on RBCs to the SIRPα receptor on macrophages inhibits phagocytic activity protecting RBCs from phagocytosis. In this study, we explore the role of the CD47-SIRPα interaction on the PV RBC life cycle. Our results show that blocking CD47-SIRPα in a PV mouse model due to either anti-CD47 treatment or loss of the inhibitory SIRPα-signal corrects the polycythemia phenotype. Anti-CD47 treatment marginally impacted PV RBC production while not influencing erythroid maturation. However, upon anti-CD47 treatment, high-parametric single-cell cytometry identified an increase of MerTK+ splenic monocyte-derived effector cells, which differentiate from Ly6Chi monocytes during inflammatory conditions, acquire an inflammatory phagocytic state. Furthermore, in vitro, functional assays showed that splenic JAK2 mutant macrophages were more “pro-phagocytic,” suggesting that PV RBCs exploit the CD47-SIRPα interaction to escape innate immune attacks by clonal JAK2 mutant macrophages.

A novel model for hemolysis estimation in rotating impeller blood pumps considering red blood cell aging.

For blood pumps with a rotating vane-structure, hemolysis values are estimated using a stress-based power-law model. It has been reported that this method does not consider the red blood cell (RBC) membrane's shear resistance, leading to inaccurate estimation of the hemolysis value. The focus of this study was to propose a novel hemolysis model which can more accurately predict the hemolysis value when designing the axial flow blood pump. The movement behavior of a single RBC in the shear flow field was simulated at the mesoscale. The critical value of shear stress for physiological injury of RBCs was determined. According to the critical value, the equivalent treatment of RBC aging was studied. A novel hemolysis model was established considering the RBC's aging and the hemolysis' initial value. The model's validity was verified under the experimental conditions of shear stress loading and the conditions of the shear flow field of the blood pump. The results showed that compared with other hemolysis models for estimating the hemolysis value of blood pumps, the novel hemolysis model proposed in this paper could effectively reduce the estimation error of the hemolysis value and provide a reference for the optimal design of rotary vane blood pumps.

Heme oxygenase 1 in erythropoiesis: an important regulator beyond catalyzing heme catabolism.

Heme oxygenase 1 (HO-1), encoded by the HMOX-1 gene, is the main heme oxygenase that catalyzes the degradation of heme into iron, carbon monoxide, and biliverdin. HMOX-1 gene expression is stimulated by oxidative stress and regulated at transcriptional and post-transcriptional levels. After translation, subcellular location and protein stability of HO-1 are also altered by different extracellular and intracellular stimuli. HO-1 plays a key role in regulating iron homeostasis and cell protection and has become a new target for disease treatment. Erythropoiesis is a tightly controlled, iron-dependent process that begins with hematopoietic stem cells and maturates to red blood cells. HO-1 is expressed in hematopoietic stem/progenitor cells, hematopoietic niche cells, erythroblasts, and especially erythroblastic island and phagocytic macrophages. HO-1 functions importantly in the entire erythroid development process by influencing hematopoietic stem cell proliferation, erythroid lineage engagement, terminal erythroid differentiation, and even senescent RBC erythrophagocytosis. HO-1 is also related to stress erythropoiesis and certain red blood cell diseases. Elucidation of HO-1 regulation and function in erythropoiesis will be of great significance for the treatment of related diseases.

Aging Injury Impairs Structural Properties and Cell Signaling in Human Red Blood Cells; Açaì Berry Is a Keystone.

Red blood cell (RBC) deformability is the ability of cells to modulate their shape to ensure transit through narrow capillaries of the microcirculation. A loss of deformability can occur in several pathological conditions, during natural RBC aging through an increase in membrane protein phosphorylation, and/or through the structural rearrangements of cytoskeletal proteins due to oxidative conditions, with a key role played by band 3. Due to the close relationship between aging and oxidative stress, flavonoid-rich foods are good candidates to counteract age-related alterations. This study aims to verify the beneficial role of Açaì extract in a d-Galactose (d-Gal)-induced model of aging in human RBCs. To this end, band 3 phosphorylation and structural rearrangements in membrane cytoskeleton-associated proteins, namely spectrin, ankyrin, and/or protein 4.1, are analyzed in RBCs treated with 100 mM d-Gal for 24 h, with or without pre-incubation with 10 μg/mL Açaì extract for 1 h. Furthermore, RBC deformability is also measured. Tyrosine phosphorylation of band 3, membrane cytoskeleton-associated proteins, and RBC deformability (elongation index) are analyzed using western blotting analysis, FACScan flow cytometry, and ektacytometry, respectively. The present data show that: (i) Açaì berry extract restores the increase in band 3 tyrosine phosphorylation and Syk kinase levels after exposure to 100 mM d-Gal treatment; and (ii) Açaì berry extract partially restores alterations in the distribution of spectrin, ankyrin, and protein 4.1. Interestingly, the significant decrease in membrane RBC deformability associated with d-Gal treatment is alleviated by pre-treatment with Açaì extract. These findings further contribute to clarify mechanisms of natural aging in human RBCs, and propose flavonoid substances as potential natural antioxidants for the treatment and/or prevention of oxidative-stress-related disease risk.

Iron in blood cells: Function, relation to disease, and potential for magnetic separation.

Iron in blood cells has several physiological functions like transporting oxygen to cells and maintaining iron homeostasis. Iron is primarily contained in red blood cells (RBCs), but monocytes also store iron as these cells are responsible for the recycling of senescent RBCs. Iron also serves an important role related to the function of different leukocytes. In inflammation, iron homeostasis is dependent on cytokines derived from T cells and macrophages. Fluctuations of iron content in the body lead to different diseases. Iron deficiency, which is also known as anemia, hampers different physiological processes in the human body. On the other hand, genetic or acquired hemochromatosis ultimately results in iron overload and leads to the failure of different vital organs. Different diagnoses and treatments are developed for these kinds of disorders, but the majority are costly and suffer from side effects. To address this issue, magnetophoresis could be an attractive technology for the diagnosis (and in some cases treatment) of these pathologies due to the paramagnetic character of the cells containing iron. In this review, we discuss the main functions of iron in blood cells and iron-related diseases in humans and highlight the potential of magnetophoresis for diagnosing and treating some of these disorders.

The impact of biological age of red blood cell on in vitro endothelial activation markers.

Introduction: Blood donor characteristics influence red blood cell transfusion outcomes. As donor sex affects the distribution of young to old RBCs in the circulation, we hypothesized that the amount of circulating young RBCs in the blood product are associated with immune suppression. Materials and Methods: Blood samples were collected from healthy volunteers and density fractionated into young and old subpopulations. In an activated endothelial cell model, RBC adhesion to endothelium and secretion of endothelial activation markers were assessed. The impact of RBC biological age was also assessed in a T cell proliferation assay and in a whole blood stimulation assay. Results: After Percoll fractionation, young RBCs contained more reticulocytes compared to old RBCs. Young RBCs associated with lower levels of E-selectin, ICAM-1, and vWF from activated endothelial cells compared to old RBCs. RBC subpopulations did not affect T cell proliferation or cytokine responses following whole blood stimulation. Conclusion: Young RBCs contain more reticulocytes which are associated with lower levels of endothelial activation markers compared to old RBCs.

Prediction of red blood cell transfusion after orthopedic surgery using an interpretable machine learning framework

Postoperative red blood cell (RBC) transfusion is widely used during the perioperative period but is often associated with a high risk of infection and complications. However, prediction models for RBC transfusion in patients with orthopedic surgery have not yet been developed. We aimed to identify predictors and constructed prediction models for RBC transfusion after orthopedic surgery using interpretable machine learning algorithms. This retrospective cohort study reviewed a total of 59,605 patients undergoing orthopedic surgery from June 2013 to January 2019 across 7 tertiary hospitals in China. Patients were randomly split into training (80%) and test subsets (20%). The feature selection method of recursive feature elimination (RFE) was used to identify an optimal feature subset from thirty preoperative variables, and six machine learning algorithms were applied to develop prediction models. The Shapley Additive exPlanations (SHAP) value was employed to evaluate the contribution of each predictor towards the prediction of postoperative RBC transfusion. For simplicity of the clinical utility, a risk score system was further established using the top risk factors identified by machine learning models. Of the 59,605 patients with orthopedic surgery, 19,921 (33.40%) underwent postoperative RBC transfusion. The CatBoost model exhibited an AUC of 0.831 (95% CI: 0.824-0.836) on the test subset, which significantly outperformed five other prediction models. The risk of RBC transfusion was associated with old age (>60 years) and low RBC count (<4.0 × 1012/L) with clear threshold effects. Extremes of BMI, low albumin, prolonged activated partial thromboplastin time, repair and plastic operations on joint structures were additional top predictors for RBC transfusion. The risk score system derived from six risk factors performed well with an AUC of 0.801 (95% CI: 0.794-0.807) on the test subset. By applying an interpretable machine learning framework in a large-scale multicenter retrospective cohort, we identified novel modifiable risk factors and developed prediction models with good performance for postoperative RBC transfusion in patients undergoing orthopedic surgery. Our findings may allow more precise identification of high-risk patients for optimal control of risk factors and achieve personalized RBC transfusion for orthopedic patients.

Impaired iron recycling from erythrocytes is an early hallmark of aging.

Aging affects iron homeostasis, as evidenced by tissue iron loading and anemia in the elderly. Iron needs in mammals are met primarily by iron recycling from senescent red blood cells (RBCs), a task chiefly accomplished by splenic red pulp macrophages (RPMs) via erythrophagocytosis. Given that RPMs continuously process iron, their cellular functions might be susceptible to age-dependent decline, a possibility that has been unexplored to date. Here, we found that 10- to 11-month-old female mice exhibit iron loading in RPMs, largely attributable to a drop in iron exporter ferroportin, which diminishes their erythrophagocytosis capacity and lysosomal activity. Furthermore, we identified a loss of RPMs during aging, underlain by the combination of proteotoxic stress and iron-dependent cell death resembling ferroptosis. These impairments lead to the retention of senescent hemolytic RBCs in the spleen, and the formation of undegradable iron- and heme-rich extracellular protein aggregates, likely derived from ferroptotic RPMs. We further found that feeding mice an iron-reduced diet alleviates iron accumulation in RPMs, enhances their ability to clear erythrocytes, and reduces damage. Consequently, this diet ameliorates hemolysis of splenic RBCs and reduces the burden of protein aggregates, mildly increasing serum iron availability in aging mice. Taken together, we identified RPM collapse as an early hallmark of aging and demonstrated that dietary iron reduction improves iron turnover efficacy.

Effects of a Maximal Exercise Followed by a Submaximal Exercise Performed in Normobaric Hypoxia (2500 m), on Blood Rheology, Red Blood Cell Senescence, and Coagulation in Well-Trained Cyclists.

Acute normoxic exercise impacts the rheological properties of red blood cells (RBC) and their senescence state; however, there is a lack of data on the effects of exercise performed in hypoxia on RBC properties. This crossover study compared the effects of acute hypoxia vs. normoxia on blood rheology, RBC senescence, and coagulation during exercise. Nine trained male cyclists completed both a session in normoxia (FiO2 = 21%) and hypoxia (FiO2 = 15.3% ≈ 2500 m). The two sessions were randomly performed, separated by one week, and consisted of an incremental and maximal exercise followed by a 20 min exercise at the first ventilatory threshold (VT1) on a home-trainer. Blood samples were taken before and after exercise to analyze hematological parameters, blood rheology (hematocrit, blood viscosity, RBC deformability and aggregation), RBC senescence markers (phosphatidylserine (PS) and CD47 exposure, intraerythrocyte reactive oxygen species (ROS), and calcium content), and blood clot viscoelastic properties. Hemoglobin oxygen saturation (SpO2) and blood lactate were also measured. In both conditions, exercise induced an increase in blood viscosity, hematocrit, intraerythrocyte calcium and ROS content, and blood lactate concentration. We also observed an increase in blood clot amplitude, and a significant drop in SpO2 during exercise in the two conditions. RBC aggregation and CD47 exposure were not modified. Exercise in hypoxia induced a slight decrease in RBC deformability which could be related to the slight increase in mean corpuscular hemoglobin concentration (MCHC). However, the values of RBC deformability and MCHC after the exercise performed in hypoxia remained in the normal range of values. In conclusion, acute hypoxia does not amplify the RBC and coagulation changes induced by an exercise bout.

Age-related changes in antigen-specific natural antibodies are influenced by sex.

Natural antibody (NAb) derived from CD5+ B-1 cells maintains tissue homeostasis, controls inflammation, aids in establishing long-term protective responses against pathogens, and provides immediate protection from infection. CD5+ B-1 cell NAbs recognize evolutionarily fixed epitopes, such as phosphatidylcholine (PtC), found on bacteria and senescent red blood cells. Anti-PtC antibodies are essential in protection against bacterial sepsis. CD5+ B-1 cell-derived NAbs have a unique germline-like structure that lacks N-additions, a feature critical for providing protection against infection. Previously, we demonstrated the repertoire and germline status of PtC+CD5+ B-1 cell IgM obtained from male mice changes with age depending on the anatomical location of the B-1 cells. More recently, we demonstrated serum antibody from aged female mice maintains protection against pneumococcal infection, whereas serum antibody from male mice does not provide protection. Here, we show that aged female mice have significantly more splenic PtC+CD5+ B-1 cells and more PtC specific serum IgM than aged male mice. Furthermore, we find both age and biological sex related repertoire differences when comparing B cell receptor (BCR) sequencing results of PtC+CD5+ B-1 cells. While BCR germline status of PtC+CD5+ B-1 cells from aged male and female mice is similar in the peritoneal cavity, it differs significantly in the spleen, where aged females retain germline configuration and aged males do not. Nucleic acid sensing toll-like receptors are critical in the maintenance of PtC+ B-1 cells; therefore, to begin to understand the mechanism of differences observed between the male and female PtC+CD5+ B-1 cell repertoire, we analyzed levels of cell-free nucleic acids and found increases in aged females. Our results suggest the antigenic milieu differs between aged males and females, leading to differential selection of antigen-specific B-1 cells over time. Further elucidation of how biological sex differences influence the maintenance of B-1 cells within the aging environment will be essential to understand sex and age-related disparities in the susceptibility to bacterial infection and will aid in the development of more effective vaccination and/or therapeutic strategies specific for males and females.

Transfusion trigger in the critically ill with sepsis or septic shock: A prospective study.

Current practice around transfusion trigger in critically ill sepsis patients is not clear. Moreover, any association of haemoglobin trigger and other transfusion parameters such as age of red blood cells (RBCs) at transfusion and number of units of RBCs transfused with mortality and other adverse outcomes need further assessment. In this prospective study, patients aged 18-70 yr and admitted to intensive care with a diagnosis of sepsis were included (n=108). Baseline demographic, clinical and laboratory parameters were noted and various transfusion data, i.e., haemoglobin trigger, number of units of RBCs and the age of RBCs were recorded. Following outcome data were collected: 28 and 90 day mortality, duration of mechanical ventilation, vasopressor therapy, intensive care unit (ICU) and hospital stay and requirement of renal replacement therapy. Of the total 108 participants, 78 (72.2%) survived till 28 days and 66 (61.1%) survived till 90 days. Transfusion trigger was 6.9 (6.7-7.1) g/dl [median (interquartile range)]. On multivariable logistic regression analysis, acute physiology and chronic health evaluation (APACHE) II [adjusted odds ratio (aOR) (95% confidence interval {CI}): 0.86 (0.78, 0.96); P=0.005], cumulative fluid balance (CFB) [aOR (95% CI): 0.99 (0.99, 0.99); P=0.005] and admission platelet count [aOR (95% CI): 1.69 (1.01, 2.84); P=0.043] were the predictors of 28 day mortality [model area under the receiver operating characteristics (AUROC) 0.81]. APACHE II [aOR (95% CI): 0.88 (0.81, 0.97); P=0.013], CFB [a OR (95% CI): 0.99977 (0.99962, 0.99993); P=0.044] and transfusion trigger [aOR (95% CI): 3 (1.07, 8.34); P=0.035] were the predictors of 90 day mortality (model AUROC: 0.82). In sepsis, patients admitted to the ICU, current practice suggests transfusion trigger is below 7 g/dl and it does not affect any adverse outcome including 28 day mortality.

Optimizing interpretation of survival studies of fresh and aged transfused biotin-labeled RBCs.

Ex vivo labeling with 51 chromium represents the standard method to determine red blood cell (RBC) survival after transfusion. Limitations and safety concerns spurred the development of alternative methods, including biotinylated red blood cells (BioRBC). Autologous units of whole blood were divided equally into two bags and stored under standard blood bank conditions at 2 to 6°C (N=4 healthy adult volunteers). One bag was biotinylated (15 μg/ml) on storage days 5 to 7 (fresh) and the other was biotinylated (3μg/ml) on days 35 to 42 (aged). The proportion of circulating BioRBC was measured serially, and cell-surface biotin was quantified with reference to molecules of equivalent soluble fluorochrome. Clearance kinetics were modeled by RBC age distribution at infusion (Gaussian vs. uniform) and decay over time (constant vs. exponential). Data were consistent with biphasic exponential clearance of cells of uniform age. Our best estimate of BioRBC clearance (half-life [T1/2 ]) was 49.7 ± 1.2 days initially, followed by more rapid clearance 82 days after transfusion (T1/2 =15.6 ± 0.6 days). As BioRBC aged in vivo, molecules of equivalent soluble fluorochrome declined with a T1/2 of 122 ± 9 days, suggesting gradual biotin cleavage. There were no significant differences between the clearance of fresh and aged BioRBC. Similar clearance kinetics of fresh and aged BioRBC may be due to the extensive washing required during biotinylation. Survival kinetics consistent with cells with uniform rather than Gaussian or other non-uniform age distributions suggest that washing, and potentially RBC culling, may extend the storage life of RBC products.

Platelets Bind Senescent Red Blood Cells and Mediate Their Clearance from the Circulation

The bloodstream of mice and humans contain consistent proportions of red blood cells (RBC) which are bound to platelets (1-2% and 0.1-0.3%, respectively). This study identifies senescent RBC as the main constituent of platelet-RBC complexes (Plt-RBC) and their importance in RBC clearance. RBC have a defined circulation lifespan and upon reaching senescence are eliminated by phagocytes, thereby ensuring homeostasis and preventing accumulation of dead cells and toxic hemolytic byproducts. Senescent RBC are characterized by cellular and molecular changes, including reduced deformability, externalization of membrane phosphatidylserine (PS), and altered expression of membrane proteins such Fas ligand (CD95; upregulated) and integrin associated protein (CD47; downregulated), which act as pro-phagocytic signals. Similar changes to deformability and membrane composition occur in RBC compromised by sickle cell disease or Plasmodium infection. These pathologic cells also display a 10x higher platelet-binding capacity which is related to disease pathogenesis. Thus, we investigated how platelets may interact with senescent RBC and the purpose of Plt-RBC. Platelet binding to senescent RBC was studied using mice pulse-labelled with biotin-NHS, which enabled tracking of RBC age and clearance over time. The biotin+ RBC remaining for longer times represented older cells; the oldest detected were aged ≥58 days consistent with the reported maximal RBC lifespan in mice. During the first 45 days of observation Plt-RBC were similar in biotin+ versus newly replenished biotin- RBC (1.6-2.2%), but were significantly higher in biotin+ cells detected at 52 and 58 days (average ± SD: 6.7 ± 5.1% and 15 ± 8.1%, respectively; p < 0.001). These observations were replicated when biotin-NHS was replaced with CFSE. The aged RBC and Plt-RBC also contained significantly increased PS and CD95 compared to the replenished and non-complexed cells, although CD47 levels were not different. Flow cytometry imaging revealed Plt-RBC were composed of single RBC bound by 1-2 platelets. Collectively this indicated that platelets preferentially bind senescent RBC. To investigate if platelet binding affected senescent RBC clearance, platelet-depleting antibodies (anti-GPIbα or anti-GPIIb/IIIa) were given to temporarily reduce Plt-RBC when biotin+ Plt-RBC levels first began to diverge (Day 46-49 in multiple experiments). The treatment caused a maximal 10-fold reduction in Plt-RBC levels sustained for 2-3 days, and a marked retention of biotin+ RBC which coincided with Plt-RBC reductions. Consistent with these results, the clearance of transfused RBC in platelet-depleted mice was also significantly inhibited compared to control mice. Erythrophagocytes in the spleen, but not the liver, from the platelet-depleted mice also contained fewer transfused cells, and erythrophagocytes containing platelets were observed using flow cytometry and microscopy. Thus, platelets are important for the clearance and/or phagocytosis of both senescent and transfused RBC. We also studied the Plt-RBC clearance mechanism by transfusing ex vivo generated and labelled Plt-RBC and RBC. The Plt-RBC were cleared at least 5x faster than RBC (p=0.0014) suggesting that once formed, their lifespan in the circulation is transient. Similar transfusions in mice depleted of phagocytes (using clodronate liposomes) impeded Plt-RBC clearance, indicating a phagocyte-dependent pathway for their removal. An additional mechanistic insight, Plt-RBC displayed an increased retention in a microsphiltration assay system, indicating platelet binding may hinder the passage of cells through the microcirculation. Our studies have thus revealed an unexpected contribution by platelets in senescent RBC clearance. In our proposed model (Figure 1) senescent RBC have an enhanced ability to bind platelets, possibly due to upregulated PS and CD95, which accelerates their removal via splenic retention and phagocytosis. Platelets may also present additional pro-phagocytic signals to stimulate erythrophagocytosis. A normal platelet count provides surplus platelets to maintain RBC clearance, but in conditions of acute platelet loss, reduction of plt-RBC may cause accumulation of senescent RBC to pathological levels. Conversely, conditions where more RBC bind platelets such as in malarial infection will increase plt-RBC and result in thrombocytopenia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effects of Pyruvate Kinase Activators on Red Blood Cell Rheology, Sickling and Senescence in Sickle Cell Disease

Introduction: Pyruvate Kinase Activators (PKA) are a new class of pharmacological agents that enhance pyruvate kinase (PKR) activity and glycolytic flux in red blood cells (RBC), resulting in increased intracellular adenosine triphosphate (ATP) and decreased 2,3-diphosphoglycerate (2,3-DPG) contents. In patients with sickle cell disease (SCD), decreased PKR activity and thermostability result in decreased ATP/2,3-DPG ratio, which promotes HbS polymerization and sickling. Previous ex-vivo studies have shown that AG-348 (mitapivat) could represent a therapeutic option for those patients by restoring PK activity and thermostability and by increasing RBC deformability and the ATP/2,3-DPG ratio, thus reducing the propensity of RBC to sickle under deoxygenation (1). Recently, a new PKA molecule has been developed (AG-946) but its effects on RBC deformability and sickling have not been fully studied (2). Moreover, the effects of the two molecules in a context of enhanced oxidative stress - a situation frequently encountered in SCD - have not been investigated. Materials and Methods: Blood samples from non-transfused adults and children with SCD (3 to 53 years-old) were collected in EDTA tubes. HbF levels ranged from 1.3 to 47.1%. Washed RBC were resuspended in phosphate buffer saline after buffy-coat removal and incubated for 4 hours at 37°C, with or without 0.4 mM of cumene hydroperoxide (CH), a molecule that causes a considerable oxidative stress to the cells. Three different incubation conditions (AG-348 at 50 µM, AG-946 at 5 µM and a vehicle control) were systematically compared for the following ex-vivo experiments on RBC: (i) determination of the partial pressure of oxygen at which the hemoglobin is 50% saturated with oxygen (p50) with the Hemox® device; (ii) RBC deformability at normoxia (EImax and SS1/2 parameters), in hypoxia (EImin) and the partial pressure of oxygen at which sickling starts during deoxygenation (PoS; Point of Sickling) by ektacytometry; (iii) analyses of RBC senescence markers (i.e. Phosphatidylserine externalization, reactive oxygen species, intra-cellular calcium (Ca2+) and membrane CD47) by flow cytometry. One-way ANOVA with the Newman-Keuls method was performed for multiple comparisons and the significant level was set at 0.05. Results: A significant decrease of the p50 and PoS values was observed for both AG-molecules, with and without CH, compared to the control condition (Fig. 1-A). Without CH, both AG-molecules decreased SS1/2 (i.e., the shear stress for half-maximal deformation of RBCs) and AG-946 increased EImin and EImax. With CH, the two molecules still decreased the PoS compared to the control condition and a trend through an increase of EImin was observed with the AG-946 molecule. A slight increase of intra-cellular Ca2+ with the two AG-molecules (trend for AG-946) was observed in absence of oxidative stress (Fig. 2). The other RBC senescence markers were not modified, regardless of the incubation condition. Conclusion: Our results show that both PKAs increase the oxygen affinity and reduce the sickling propensity of RBCs from patients with SCD in vitro, in the presence or absence of enhanced oxidative stress. The observed small increase of intracellular Ca2+ upon drug treatment may be a result of increased overall cellular metabolic activity, as a consequence of PKR activation. Interestingly, the increased intracellular Ca2+ is not accompanied by enhanced RBC senescence. Overall, these data support testing PKA in prospective clinical trials for the reduction of vaso-occlusive crisis in patients with SCD. Bibliography: (1): Decreased activity and stability of pyruvate kinase in sickle cell disease: a novel target for mitapivat therapy. Rab MAE, Bos J, van Oirschot BA, van Straaten S, Kosinski PA, Chubukov V, Kim H, Mangus H, Schutgens REG, Pasterkamp G, Dang L, Kung C, van Beers EJ, van Wijk R.Blood. 2021 PMID: 33690814. (2): Pharmacodynamic effects of AG-946, a highly potent next-generation activator of pyruvate kinase, in ex vivo treatment of red blood cells from sickle cell disease patients. Rab, MAE, Van Dijk MJ, Van Oirschot BA, Gerrits J Kosinski PA, Kung C, Jans JJM, Van Beers EJ, Dang L and Van Wijk R. 2021 American Society of Hematology Congress, Abstract 113. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Fetal Hemoglobin Induced Improvement in Relative Age of Sickle Red Blood Cells Determined through Glycated Hemoglobin Assessment By Flow Cytometry

Introduction: Sickle cell disease (SCD) is characterized by acute vaso-occlusive events and chronic hemolytic anemia, originating from the polymerization of the abnormal hemoglobin S (HbS) under low oxygen tension. Hemolysis is defined as an imbalance between the rate of red blood cell (RBC) production and destruction. It results in an overall reduced lifespan of RBCs, as they are prematurely removed from the circulation. While the normal RBC lifespan is approximatively 120 days, those of SCD is only a sixth of it (Hebbel, 2011). Fetal Hb (HbF) is the main clinical modulator of SCD severity by inhibiting HbS polymerization. Its protective effect relies on its quantity and distribution among the RBC population. However, HbF is often heterogeneously distributed in RBC from SCD patients. Among the numerous improvements triggered by HbF, a significant increase in RBC lifespan has been measured in high-HbF containing cells, as compared to low-HbF containing cells (Franco et al., 2006). As they age, normal RBCs exhibit physiological changes, such as dehydration, a decrease of the intracellular enzymes activities (pyruvate kinase and hexokinase) and an increase in Hb concentration. Glycated Hb (the most abundant being HbA1c form) also increases, as RBC age. The glycation of Hb is a non-enzymatical reaction that is affected by the plasmatic concentration of glucose and the time RBCs last in the circulation. In non-diabetic subjects, the Hb glycation only depends on the RBC lifespan. To date, current methods to assess RBC lifespan are invasive and time consuming, requiring infusion of autologous labeled RBCs and long follow-up. Objectives: This present work aims at 1/ evaluating the RBC relative age of SCD patients using glycated Hb measured by flow cytometry and 2/ estimating the HbF-induced increase in RBC lifespan of SCD patients. Methods: Glycated Hb was measured and compared between five healthy donors (AA), seven non-diabetic and non-transfused homozygous SCD patients (SS) and five non-diabetic, transfused SS patients. Total glycated Hb was measured by reference biochemical methods, cation-exchange high-pressure liquid chromatography or rapid glycohaemoglobin immunoassay (DCA test) used for AA-RBC and SS-RBC, respectively. The single cell glycation was assessed by flow cytometry using mouse anti-human HbA1c antibody. HbA, HbS and HbF were assessed by flow cytometry using anti βA-globin, anti βS-globin and anti g-globin antibodies, respectively. Results: The glycated Hb was significantly lower in SS patients than in healthy donors (glycated HbS =3.7%, glycated HbA =5.3%, p=0.0025 by DCA and HPLC respectively, and by flow cytometry p=0.0061). In transfused SS patients, results showed that glycated Hb was lower in autologous SS-RBCs than in AA-RBCs by a factor of four (P= 0.008), thus confirming the characterized reduced lifespan of SS RBCs. Glycated Hb levels were measured in RBC sub-populations based on their HbF level (high HbF or low HbF subfractions) in non-transfused SS patients. Glycated Hb level was lower in low HbF RBCs, as compared to high HbF RBCs (P=0.0023). This difference was not detected in healthy donors, thereby indicating that HbF has an effect on relative age only in SCD patients. Conclusion: Single cell glycated Hb assessed by flow cytometry allows to simultaneously estimate the relative age of different RBC sub-populations. This new tool is noninvasive and requires only one single time point measurement. It should also be considered to evaluate the efficacy of treatments that aim at improving hemolysis. Figure. Glycated Hb levels assessed by HPLC and DCA biochemical methods, and flow cytometry in different participant groups and RBC subpopulations. Results were expressed as median, percentage or mean fluorescence intensity (MFI) when appropriate. Glycated Hb level was compared between two groups using a nonparametric Mann-Whitney t-test. (A) Total glycated Hb measured by HPLC in AA healthy donors (n=5) and by DCA test in SS patients (n=7). (B) Single cell glycated Hb measured by flow cytometry in SS RBCs (n=7), compared to AA healthy donors (n=4). (C) Single cell glycated Hb measured in low HbF RBCs (n=7) and high HbF RBCs (purple, n=7) subpopulations in SS patients and AA healthy donors (n=4). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Contraction of the rigor actomyosin complex drives bulk hemoglobin expulsion from hemolyzing erythrocytes

Erythrocyte ghost formation via hemolysis is a key event in the physiological clearance of senescent red blood cells (RBCs) in the spleen. The turnover rate of millions of RBCs per second necessitates a rapid efflux of hemoglobin (Hb) from RBCs by a not yet identified mechanism. Using high-speed video-microscopy of isolated RBCs, we show that electroporation-induced efflux of cytosolic ATP and other small solutes leads to transient cell shrinkage and echinocytosis, followed by osmotic swelling to the critical hemolytic volume. The onset of hemolysis coincided with a sudden self-propelled cell motion, accompanied by cell contraction and Hb-jet ejection. Our biomechanical model, which relates the Hb-jet-driven cell motion to the cytosolic pressure generation via elastic contraction of the RBC membrane, showed that the contributions of the bilayer and the bilayer-anchored spectrin cytoskeleton to the hemolytic cell motion are negligible. Consistent with the biomechanical analysis, our biochemical experiments, involving extracellular ATP and the myosin inhibitor blebbistatin, identify the low abundant non-muscle myosin 2A (NM2A) as the key contributor to the Hb-jet emission and fast hemolytic cell motion. Thus, our data reveal a rapid myosin-based mechanism of hemolysis, as opposed to a much slower diffusive Hb efflux.

Influence of 24 h Simulated Altitude on Red Blood Cell Deformability and Hematological Parameters in Patients with Fontan Circulation.

Patients with Fontan circulation are particularly dependent on low pulmonary vascular resistance because their lungs are passively perfused. Hypoxia drives pulmonary vasoconstriction; thus, red blood cell (RBC) deformability and stability of hematological parameters might be of particular importance, because alterations during hypoxia might further influence circulation. This study aimed to measure respective parameters in patients with Fontan circulation exposed to normobaric hypoxia. A total of 18 patients with Fontan circulation (16 to 38 years) were exposed to normobaric hypoxia (15.2% ambient oxygen). Blood samples were taken in normoxia, after 24 h in hypoxia, and 60 min after return to normoxia. Blood count, RBC age distribution, EPO, RBC deformability, marker of RBC nitric oxide, oxidative state, and RBC ATP were measured. Hypoxia increased oxidative stress in RBC, but without affecting RBC deformability. RBC age distribution remained unaffected, although EPO concentrations increased, followed by a rise in reticulocyte count at an already high hematocrit. NO metabolism was not affected by hypoxia. Modest normobaric hypoxia for 24 h did not impair RBC deformability in patients with Fontan circulation; however, the oxidative system seemed to be stressed. Given the high baseline Hct in these patients, hypoxia-induced erythropoiesis could adversely affect rheology with more prolonged hypoxia exposure.

Prediction and prognosis of reintubation after surgery for Stanford type A aortic dissection.

BackgroundReintubation is a serious adverse respiratory event after Stanford type A aortic dissection surgery (AADS), however, published studies focused on reintubation after AADS are very limited worldwide. The objectives of the current study were to establish an early risk prediction model for reintubation after AADS and to clarify its relationship with short-term and long-term prognosis.MethodsPatients undergoing AADS between 2016–2019 in a single institution were identified and divided into two groups based on whether reintubation was performed. Independent predictors were identified by univariable and multivariable analysis and a clinical prediction model was then established. Internal validation was performed using bootstrap method with 1,000 replications. The relationship between reintubation and clinical outcomes was determined by univariable and propensity score matching analysis.ResultsReintubation were performed in 72 of the 492 included patients (14.6%). Three preoperative and one intraoperative predictors for reintubation were identified by multivariable analysis, including older age, smoking history, renal insufficiency and transfusion of intraoperative red blood cells. The model established using the above four predictors showed moderate discrimination (AUC = 0.753, 95% CI, [0.695–0.811]), good calibration (Hosmer-Lemeshow χ2 value = 3.282, P = 0.915) and clinical utility. Risk stratification was performed and three risk intervals were identified. Reintubation was closely associated with poorer in-hospital outcomes, however, no statistically significant association between reintubation and long-term outcomes has been observed in patients who were discharged successfully after surgery.ConclusionsThe requirement of reintubation after AADS is prevalent, closely related to adverse in-hospital outcomes, but there is no statistically significant association between reintubation and long-term outcomes. Predictors were identified and a risk model predicting reintubation was established, which may have clinical utility in early individualized risk assessment and targeted intervention.