Young Red Blood Cells Research Articles

Donor age contributes more to the rheological properties of stored red blood cells than donor sex and biological age distribution

Variation in the quality of stored red cell concentrates (RCCs) has been linked to the biological age distribution of red blood cell (RBCs) subpopulations within blood products. Teen male RCCs contain higher proportions of biologically old RBC subpopulations, with poorer quality. This study sought to assess the contribution of donor sex and age on the deformability characteristics of different ages of RBC subpopulations in stored RCCs. On days 5, 14, 21, and 42 of hypothermic storage, RCCs from healthy teen male, senior male, teen female and senior female donors were biologically age profiled. The deformability of the resulting young (Y-RBCs) and old (O-RBCs) RBC subpopulations in addition to unseparated RBCs was assessed using ektacytometry. Over storage, donor age influenced the rheological profile of RBC subpopulations, with teen male RCCs having the largest reduction in Ohyper (the osmolality in the hypertonic region corresponding to 50% of the EImax). The strongest correlations between Ohyper and MCHC (R2 > 0.5) were witnessed with O-RBCs from senior donors, and to a lesser extent with teen males. O-RBCs from teens, particularly males, had higher elongation indices both under isotonic conditions and in the presence of an increasing osmotic gradient. Consistent across RBCs regardless of their biological age, teen samples were discovered to be more rigid (higher KEI). The donor-to-donor variation in age distribution of RBC subpopulations and its downstream effect on deformability serves as further evidence that factors beyond chronological aging could potentially impact the quality of red cell concentrates and variations in transfusion outcomes.

Primaquine-5,6-Orthoquinone Is Directly Hemolytic to Older G6PD Deficient RBCs in a Humanized Mouse Model.

Primaquine and Tafenoquine are the only approved drugs that can achieve a radical cure for Plasmodium vivax malaria but are contraindicated in patients who are deficient in glucose 6-phosphate dehydrogenase (G6PDd) due to risk of severe hemolysis from reactive oxygen species generated by redox cycling of drug metabolites. 5-hydroxyprimaquine and its quinoneimine cause robust redox cycling in red blood cells (RBCs) but are so labile as to not be detected in blood or urine. Rather, the quinoneimine is rapidly converted into primaquine-5,6-orthoquinone (5,6-POQ) that is then excreted in the urine. The extent to which 5,6-POQ contributes to hemolysis remains unclear, although some have suggested that it is a minor toxin that should be used predominantly as a surrogate to infer levels of 5-hydroxyprimaquine. In this report, we describe a novel humanized mouse model of the G6PD Mediterranean variant (hG6PDMed-) that recapitulates the human biology of RBC age-dependent enzyme decay, as well as an isogenic matched control mouse with human nondeficient G6PD hG6PDND In vitro challenge of RBCs with 5,6-POQ causes increased generation of superoxide and methemoglobin. Infusion of treated RBCs shows that 5,6-POQ selectively causes in vivo clearance of older hG6PDMed- RBCs. These findings support the hypothesis that 5,6-POQ directly induces hemolysis and challenges the notion that 5,6-POQ is an inactive metabolic waste product. Indeed, given the extreme lability of 5-hydroxyprimaquine and the relative stability of 5,6-POQ, these data raise the possibility that 5,6-POQ is a major hemolytic primaquine metabolite in vivo. SIGNIFICANCE STATEMENT: These findings demonstrate that 5,6-POQ, which has been considered an inert waste product of primaquine metabolism, directly induces ROS that cause clearance of older G6PDd RBCs. As 5,6-POQ is relatively stable compared with other active primaquine metabolites, these data support the hypothesis that 5,6-POQ is a major toxin in primaquine induced hemolysis. The findings herein also establish a new model of G6PDd and provide the first direct evidence, to our knowledge, that young G6PDd RBCs are resistant to primaquine-induced hemolysis.

Transfusion of female blood in a rat model is associated with red blood cells entrapment in organs.

Transfusion of red blood cells (RBCs) has been associated with adverse outcomes. Mechanisms may be related to donor sex and biological age of RBC. This study hypothesized that receipt of female blood is associated with decreased post-transfusion recovery (PTR) and a concomitant increased organ entrapment in rats, related to young age of donor RBCs. Donor rats underwent bloodletting to stimulate production of new, young RBCs, followed by Percoll fractionation for further enrichment of young RBCs based on their low density. Control donors did not undergo these procedures. Male rats received either a (biotinylated) standard RBC product or a product enriched for young RBCs, derived from either male or female donors. Controls received saline. Organs and blood samples were harvested after 24 hours. This study found no difference in PTR between groups, although only the group receiving young RBCs from females failed to reach a PTR of 75%. Receipt of both standard RBCs and young RBCs from females was associated with increased entrapment of donor RBCs in the lung, liver, and spleen compared to receiving blood from male donors. Soluble ICAM-1 and markers of hemolysis were higher in recipients of female blood compared to control. In conclusion, transfusing RBCs from female donors, but not from male donors, is associated with trapping of donor RBCs in organs, accompanied by endothelial activation and hemolysis.

P‐CB‐24 | Young RBC Subpopulations Reduce the Hemoglobin‐Oxygen Affinity of Cord Blood from Preterm Neonates

P‐CB‐24 | Young RBC Subpopulations Reduce the Hemoglobin‐Oxygen Affinity of Cord Blood from Preterm Neonates

Experimental Study on Primary Bird Co-Infection with Two Plasmodium relictum Lineages-pSGS1 and pGRW11.

Simple SummaryCo-infections with several parasites in one host at the same time are common in the wild. It is conceivable that a host infected with several different parasites may be significantly more affected immunologically than a host infected with only one parasite. However, one plus one is not always two when talking about parasite infections. We followed the infection and some health parameters of twenty-four domestic canaries allocated to four groups: birds in two groups were infected with a single parasite (Plasmodium relictum genetic lineage pSGS1 or P. relictum pGRW11), in one group with both mentioned parasites, and birds in the fourth group were considered as the controls. After infection, the time of appearance of parasites in the blood and developed parasitemia did not differ between groups. Differences in hemoglobin levels and in the numbers of young red blood cells between the control and experimental groups were detected, but observed differences between the groups of infected birds were not statistically significant. This study shows that co-infection with two malarial parasites does not necessarily result in a greater number of parasites and more severe disease to the host.Background: Co-infections are common in the wild. Thus, studies focused on parasite interactions are essential. We aimed to (i) follow the development of two genetic lineages of Plasmodium relictum—pSGS1 and pGRW11—during single infections and co-infections and (ii) evaluate their impact on bird host health. Materials: Twenty-four domestic canaries were allocated to four groups: two groups were infected with parasites of a single genetic lineage, one group was infected with parasites of both genetic lineages, and one group was considered as the control group. Parasitemia, the number of polychromatophils, changes in body weight, and hemoglobin levels were all quantified up to 32 days post-infection. Results: Three birds infected with pSGS1 died within 20 days post-infection. The prepatent period and the peak of parasitemia did not differ significantly between groups. Differences in hemoglobin levels between the control and experimental groups were observed and there was an abnormal increase in the number of polychromatophils in infected birds. In all infected groups, correlations were detected between the number of polychromatophils and parasitemia (positive), and between the number of polychromatophils and hemoglobin levels (negative). Conclusion: This study shows that co-infection with two phylogenetically closely related P. relictum parasites does not alter overall parasitemia and does not cause higher virulence to the host.

Autologous Blood Doping Induced Changes in Red Blood Cell Rheologic Parameters, RBC Age Distribution, and Performance.

Simple SummaryAutologous blood doping (ABD) refers to the artificial increase in circulating red blood cell (RBC) mass by sampling, storage, and transfusion of one’s own blood. It is assumed that some athletes apply this prohibited technique to improve oxygen transport capacity and thus exercise performance. The primary aim of this study was to test whether RBC rheological and associated parameters significantly change due to ABD with the consideration of whether this type of measurement might be suitable for detecting ABD. Further, it was assessed whether those changes are translated into indices of endurance performance. Eight males underwent an ABD protocol combined with several blood parameter measurements and two exercise tests (pre and post transfusion). Results of this investigation suggest a change in the distribution of age-related RBC sub-populations and altered deformability of total RBC as well as of the respective sub-populations. Further, the identified changes in RBC also appear to improve sports performance. In conclusion, these data demonstrate significant changes in hematological and hemorheological parameters, which could be of interest in the context of new methods for ABD detection. However, additional research is needed with larger and more diverse study groups to widen the knowledge gained by this study.Autologous blood doping (ABD) refers to the transfusion of one’s own blood after it has been stored. Although its application is prohibited in sports, it is assumed that ABD is applied by a variety of athletes because of its benefits on exercise performance and the fact that it is not detectable so far. Therefore, this study aims at identifying changes in hematological and hemorheological parameters during the whole course of ABD procedure and to relate those changes to exercise performance. Eight healthy men conducted a 31-week ABD protocol including two blood donations and the transfusion of their own stored RBC volume corresponding to 7.7% of total blood volume. Longitudinal blood and rheological parameter measurements and analyses of RBC membrane proteins and electrolyte levels were performed. Thereby, responses of RBC sub-populations—young to old RBC—were detected. Finally, exercise tests were carried out before and after transfusion. Results indicate a higher percentage of young RBC, altered RBC deformability and electrolyte concentration due to ABD. In contrast, RBC membrane proteins remained unaffected. Running economy improved after blood transfusion. Thus, close analysis of RBC variables related to ABD detection seems feasible but should be verified in further more-detailed studies.

A new membrane formulation for modelling the flow of stomatocyte, discocyte, and echinocyte red blood cells

In this work, a numerical model that enables simulation of the deformation and flow behaviour of differently aged Red Blood Cells (RBCs) is developed. Such cells change shape and decrease in deformability as they age, thus impacting their ability to pass through the narrow capillaries in the body. While the body filters unviable cells from the blood naturally, cell aging poses key challenges for blood stored for transfusions. Therefore, understanding the influence RBC morphology and deformability have on their flow is vital. While several existing models represent young Discocyte RBC shapes well, a limited number of numerical models are developed to model aged RBC morphologies like Stomatocytes and Echinocytes. The existing models are also limited to shear and stretching simulations. Flow characteristics of these morphologies are yet to be investigated. This paper aims to develop a new membrane formulation for the numerical modelling of Stomatocyte, Discocytes and Echinocyte RBC morphologies to investigate their deformation and flow behaviour. The model used represents blood plasma using the Lattice Boltzmann Method (LBM) and the RBC membrane using the discrete element method (DEM). The membrane and the plasma are coupled by the Immersed Boundary Method (IBM). Previous LBM-IBM-DEM formulations represent RBC membrane response based on forces generated from changes in the local area, local length, local bending, and cell volume. In this new model, two new force terms are added: the local area difference force and the local curvature force, which are specially incorporated to model the flow and deformation behaviour of Stomatocytes and Echinocytes. To verify the developed model, the deformation behaviour of the three types of RBC morphologies are compared to well-characterised stretching and shear experiments. The flow modelling capabilities of the method are then demonstrated by modelling the flow of each cell through a narrow capillary. The developed model is found to be as accurate as benchmark Smoothed Particle Hydrodynamics (SPH) approaches while being significantly more computationally efficient.

Hemoglobin Oxidation Reactions in Stored Blood.

Hemoglobin (Hb) inside and outside the red blood cells (RBCs) undergoes constant transformation to an oxidized form in a process known as autoxidation. The ferrous heme iron (Fe2+) of the prosthetic group is spontaneously transformed into an oxidized ferric (Fe3+) form, but under oxidative stress conditions a higher oxidation ferryl heme (Fe4+) is also formed. Although Fe3+ is a non-functional form of Hb, the Fe4+ is also extremely reactive towards other biological molecules due to its high redox potential. The RBC contains an effective reductive machinery that maintains Hb in the functional form with little oxidation during its life span. The redox transformation of Hb occurs to a lesser extent in young RBCs; it may, however, have detrimental effects on the integrity of these cells during ex vivo storage or when RBCs are subjected to pathogen reduction processes. In this review, Hb oxidation reactions (“oxidative lesion”) will be described, including details of how these reactions might impact the clinical use of stored or processed blood for therapeutic purposes.

How the spleen reshapes and retains young and old red blood cells: A computational investigation.

The spleen, the largest secondary lymphoid organ in humans, not only fulfils a broad range of immune functions, but also plays an important role in red blood cell’s (RBC) life cycle. Although much progress has been made to elucidate the critical biological processes involved in the maturation of young RBCs (reticulocytes) as well as removal of senescent RBCs in the spleen, the underlying mechanisms driving these processes are still obscure. Herein, we perform a computational study to simulate the passage of RBCs through interendothelial slits (IES) in the spleen at different stages of their lifespan and investigate the role of the spleen in facilitating the maturation of reticulocytes and in clearing the senescent RBCs. Our simulations reveal that at the beginning of the RBC life cycle, intracellular non-deformable particles in reticulocytes can be biomechanically expelled from the cell upon passage through IES, an insightful explanation of why this peculiar “pitting” process is spleen-specific. Our results also show that immature RBCs shed surface area by releasing vesicles after crossing IES and progressively acquire the biconcave shape of mature RBCs. These findings likely explain why RBCs from splenectomized patients are significantly larger than those from nonsplenectomized subjects. Finally, we show that at the end of their life span, senescent RBCs are not only retained by IES due to reduced deformability but also become susceptible to mechanical lysis under shear stress. This finding supports the recent hypothesis that transformation into a hemolyzed ghost is a prerequisite for phagocytosis of senescent RBCs. Altogether, our computational investigation illustrates critical biological processes in the spleen that cannot be observed in vivo or in vitro and offer insights into the role of the spleen in the RBC physiology.

Glucose-6-phosphate dehydrogenase activity in individuals with and without malaria: Analysis of clinical trial, cross-sectional and case-control data from Bangladesh.

Glucose-6-phosphate dehydrogenase (G6PD) activity is dependent upon G6PD genotype and age of the red blood cell (RBC) population, with younger RBCs having higher activity. Peripheral parasitemia with Plasmodium spp. induces hemolysis, replacing older RBCs with younger cells with higher G6PD activity. This study aimed to assess whether G6PD activity varies between individuals with and without malaria or a history of malaria. Individuals living in the Chittagong Hill Tracts of Bangladesh were enrolled into 3 complementary studies: (i) a prospective, single-arm clinical efficacy trial of patients (n = 175) with uncomplicated malaria done between 2014 and 2015, (ii) a cross-sectional survey done between 2015 and 2016 (n = 999), and (iii) a matched case-control study of aparasitemic individuals with and without a history of malaria done in 2020 (n = 506). G6PD activity was compared between individuals with and without malaria diagnosed by microscopy, rapid diagnostic test (RDT), or polymerase chain reaction (PCR), and in aparasitemic participants with and without a history of malaria. In the cross-sectional survey and clinical trial, 15.5% (182/1,174) of participants had peripheral parasitemia detected by microscopy or RDT, 3.1% (36/1,174) were positive by PCR only, and 81.4% (956/1,174) were aparasitemic. Aparasitemic individuals had significantly lower G6PD activity (median 6.9 U/g Hb, IQR 5.2-8.6) than those with peripheral parasitemia detected by microscopy or RDT (7.9 U/g Hb, IQR 6.6-9.8, p < 0.001), but G6PD activity similar to those with parasitemia detected by PCR alone (submicroscopic parasitemia) (6.1 U/g Hb, IQR 4.8-8.6, p = 0.312). In total, 7.7% (14/182) of patients with malaria had G6PD activity < 70% compared to 25.0% (248/992) of participants with submicroscopic or no parasitemia (odds ratio [OR] 0.25, 95% CI 0.14-0.44, p < 0.001). In the case-control study, the median G6PD activity was 10.3 U/g Hb (IQR 8.8-12.2) in 253 patients with a history of malaria and 10.2 U/g Hb (IQR 8.7-11.8) in 253 individuals without a history of malaria (p = 0.323). The proportion of individuals with G6PD activity < 70% was 11.5% (29/253) in the cases and 15.4% (39/253) in the controls (OR 0.7, 95% CI 0.41-1.23, p = 0.192). Limitations of the study included the non-contemporaneous nature of the clinical trial and cross-sectional survey. Patients with acute malaria had significantly higher G6PD activity than individuals without malaria, and this could not be accounted for by a protective effect of G6PD deficiency. G6PD-deficient patients with malaria may have higher than expected G6PD enzyme activity and an attenuated risk of primaquine-induced hemolysis compared to the risk when not infected.

CD47 blockade reduces the pathologic features of experimental cerebral malaria and promotes survival of hosts with Plasmodium infection

CD47 is an antiphagocytic "don't eat me" signal that inhibits programmed cell removal of self. As red blood cells (RBCs) age they lose CD47 expression and become susceptible to programmed cell removal by macrophages. CD47-/- mice infected with Plasmodium yoelii, which exhibits an age-based preference for young RBCs, were previously demonstrated to be highly resistant to malaria infection. Our study sought to test the therapeutic benefit of CD47 blockade on ameliorating the clinical syndromes of experimental cerebral malaria (ECM), using the Plasmodium berghei ANKA (Pb-A) murine model. In vitro we tested the effect of anti-CD47 mAb on Plasmodium-infected RBC phagocytosis and found that anti-CD47 treatment significantly increased clearance of Plasmodium-infected RBCs. Infection of C57BL/6 mice with Pb-A is lethal and mice succumb to the clinical syndromes of CM between days 6 and 10 postinfection. Strikingly, treatment with anti-CD47 resulted in increased survival during the cerebral phase of Pb-A infection. Anti-CD47-treated mice had increased lymphocyte counts in the peripheral blood and increased circulating levels of IFN-γ, TNF-α, and IL-22. Despite increased circulating levels of inflammatory cytokines, anti-CD47-treated mice had reduced pathological features in the brain. Survival of ECM in anti-CD47-treated mice was correlated with reduced cellular accumulation in the cerebral vasculature, improved blood-brain barrier integrity, and reduced cytotoxic activity of infiltrating CD8+ T cells. These results demonstrate the therapeutic benefit of anti-CD47 to reduce morbidity in a lethal model of ECM, which may have implications for preventing mortality in young African children who are the highest casualties of CM.

Donor Age and Red Cell Age Contribute to the Variance in Lorrca Indices in Healthy Donors for Next Generation Ektacytometry: A Pilot Study

The ability of red blood cells (RBCs) to transport gases, their lifespan as well as their rheological properties invariably depend on the deformability, hydration, and membrane stability of these cells, which can be measured by Laser optical rotational red cell analyser (Lorrca® Maxsis, RR Mechatronics). The osmoscan mode of Lorrca is currently used in diagnosis of rare anemias in clinical laboratories. However, a broad range of normal values for healthy subjects reduces the sensitivity of this method for diagnosis of mild disease phenotype. In this pilot study, we explored the impact of age and gender of 45 healthy donors, as well as RBC age on the Lorrca indices. Whereas gender did not affect the Lorrca indices in our study, the age donors had a profound effect on the O_hyper parameter. To study the impact of RBC age on the osmoscan parameters, we have isolated low (L)-, medium (M)-, or high (H)- density fractions enriched with young, mature, and senescent RBCs, respectively, and evaluated the influence of RBC age-related properties, such as density, morphology, and redox state, on the osmoscan indices. As before, O_hyper was the most sensitive parameter, dropping markedly with an increase in RBC density and age. Senescence was associated with a decrease in deformability (EI_max) and tolerability to low and high osmolatites (Area). L-fraction was enriched with reticulocytes and cells with high projected area and EMA staining, but also contained a small number of cells small in projected area and most likely, terminally senescent. L-fraction was on average slightly less deformable than mature cells. The cells from the L-fraction produced more oxidants and NO than all other fractions. However, RBCs from the L-fraction contained maximal levels of reduced thiols compared to other fractions. Our study suggests that reference values for O_hyper should be age-stratified, and, most probably, corrected for the average RBC age. Further multi-center study is required to validate these suggestions before implementing them into clinical practice.

Sub-Fractions of Red Blood Cells Respond Differently to Shear Exposure Following Superoxide Treatment.

Red blood cell (RBC) deformability is an essential component of microcirculatory function that appears to be enhanced by physiological shear stress, while being negatively affected by supraphysiological shears and/or free radical exposure. Given that blood contains RBCs with non-uniform physical properties, whether all cells equivalently tolerate mechanical and oxidative stresses remains poorly understood. We thus partitioned blood into old and young RBCs which were exposed to phenazine methosulfate (PMS) that generates intracellular superoxide and/or specific mechanical stress. Measured RBC deformability was lower in old compared to young RBCs. PMS increased total free radicals in both sub-populations, and RBC deformability decreased accordingly. Shear exposure did not affect reactive species in the sub-populations but reduced RBC nitric oxide synthase (NOS) activation and intriguingly increased RBC deformability in old RBCs. The co-application of PMS and shear exposure also improved cellular deformability in older cells previously exposed to reactive oxygen species (ROS), but not in younger cells. Outputs of NO generation appeared dependent on cell age; in general, stressors applied to younger RBCs tended to induce S-nitrosylation of RBC cytoskeletal proteins, while older RBCs tended to reflect markers of nitrosative stress. We thus present novel findings pertaining to the interplay of mechanical stress and redox metabolism in circulating RBC sub-populations.

Enterococcus faecalis causes osteitis deformans in a Golden Lancehead snake (Bothrops insularis): a case report

Osteitis deformans (Paget’s disease) is a chronic bone disorder characterized by excessive osteoclast-mediated bone resorption followed by new bone formation. The present paper reports this condition in an 18-year-old captive golden lancehead (Bothrops insularis) from Brazil. This patient initially exhibited anorexia and swelling in the middle third of the spine associated with locomotor disability. For diagnosis, radiography, ultrasound, computed tomography, cytology, and microbiological culture were performed. Diagnostic imaging showed bone changes, vertebral fusion, and bone proliferation. Cytology revealed blood cells how toxic heterophiles, reactive monocytes, young red blood cells, and polychromasia compatible with an infectious process. A bacterial culture identified an ampicillin-susceptible strain of Enterococcus faecalis. Antibiotic treatment was promptly started, but the snake died 25 days later. Histopathologically, the bone tissue showed a generalized thickening of the vertebral trabeculae. For the first time, the presence of E. faecalis associated with the development of osteitis deformans in snakes was presented.

RNA Seq Profiles and Bioinformatics Validation in a Large Sample of Sickle Cell Disease Patients

Background Sickle Cell Disease (SCD) is a genetic disorder caused by a single amino acid substitution in the ß-hemoglobin chain. Clinical manifestations of SCD are multisystemic and heterogeneous, with a wide range of organ damage between patients despite identical genetic mutations. The sub-phenotypes of SCD reflect damage to different organs arising from the pathophysiology of the disease. Biomarkers, such as N-terminal-pro-Brain natriuretic peptide (NT-proBNP), tricuspid regurgitant velocity (TRV), and hemolytic parameters provide readouts of the degree of different organ damage and are known risk markers. Dissecting its pathophysiology along known biological pathways has led to development of therapeutic targets, but we still do not fully understand the pathogenesis for much of the organ damage. Given the complexity of the pathophysiology of SCD that is likely to involve multiple overlapping and interacting biological pathways, an agnostic, network-based approach using gene expression data with annotated risk markers provides an attractive alternative. We assessed the plausibility of using such an approach with bilirubin levels as a phenotype. Bilirubin is a breakdown product of red blood cells (RBCs) that is measured in routine labs and a validated hemolytic and disease severity marker of SCD. Methods Using RNA-Seq data from 224 patients with SCD (80% HbSS and HbSBeta0 thalassemia combined, 15% HbSC, 5% HbSBeta+ thalassemia, and 0.4% HbSD), we performed a differential gene expression (DGE) analysis and weighted gene co-expression network analysis (WGCNA) of 12,450 genes. For the DGE analysis we used total bilirubin levels as a continuous variable for the main phenotype of interest, adjusting for age, sex, and hemoglobin genotype. We then conducted WGCNA to identify modules of genes with similar coexpression patterns, which we functionally annotated (via the ClusterProfiler R package) for interesting biological themes. Results Of the 12,450 genes, 10,604 were protein coding, and 1,560 were lncRNAs, and 286 were other non-coding transcripts. 3,509 (2,975 coding, 447 lncRNA, and 87 other non-coding) were considered differentially expressed (unadjusted p-value &amp;lt; 0.05) according to bilirubin level. Through the WCGNA, the genes clustered into 11 co-expression modules, representing a range of biological pathways. Four of these modules had representative eigengenes that were significantly correlated with bilirubin (p &amp;lt; 0.05), and we focused on 2 of these that had biologically interesting functions. The first module of interest (turquoise) consisted of 3,636 genes (3,371 protein-coding and 210 lncRNA) (25% differentially expressed, and 62% upregulated). Genes in this module have been associated with cellular functioning of younger RBCs, validating the presence of the significant Gene Ontology (GO) biological pathways listed in the bar graph (mitochondria gene expression, ribonucleotide processes, etc.), and aligning with expected processes as the young RBCs replace the degraded RBCs that had caused the high bilirubin. Notably, this module contained the biliverdin reductase A (BLVRA) gene (differentially expressed at p &amp;lt; 0.008), which reduces biliverdin to bilirubin. The other highlighted module (red) consisted of 914 genes (846 protein-coding and 57 lncRNA) (42% differentially expressed, and 97% upregulated). Multiple pathways in the red module were related to catabolic processes. As previously stated, bilirubin is the byproduct of hemoprotein catabolism, suggesting an interesting correlation. Additionally, this module contained 2 other pathways associated with porphyrin, which is a component of RBCs that contains the heme group and is involved in heme biosynthesis. Conclusion Taken together, these results provide interesting insights into the biological pathways driving one facet of sickle cell disease's many clinical manifestations. Figure 1 Disclosures No relevant conflicts of interest to declare.

Aging-associated changes in CD47 arrangement and interaction with thrombospondin-1 on red blood cells visualized by super-resolution imaging.

CD47 serves as a ligand for signaling regulatory protein α (SIRPα) and as a receptor for thrombospondin‐1 (TSP‐1). Although CD47, TSP‐1, and SIRPα are thought to be involved in the clearance of aged red blood cells (RBCs), aging‐associated changes in the expression and interaction of these molecules on RBCs have been elusive. Using direct stochastic optical reconstruction microscopy (dSTORM)‐based imaging and quantitative analysis, we can report that CD47 molecules on young RBCs reside as nanoclusters with little binding to TSP‐1, suggesting a minimal role for TSP‐1/CD47 signaling in normal RBCs. On aged RBCs, CD47 molecules decreased in number but formed bigger and denser clusters, with increased ability to bind TSP‐1. Exposure of aged RBCs to TSP‐1 resulted in a further increase in the size of CD47 clusters via a lipid raft‐dependent mechanism. Furthermore, CD47 cluster formation was dramatically inhibited on thbs1 −/− mouse RBCs and associated with a significantly prolonged RBC lifespan. These results indicate that the strength of CD47 binding to its ligand TSP‐1 is predominantly determined by the distribution pattern and not the amount of CD47 molecules on RBCs, and offer direct evidence for the role of TSP‐1 in phagocytosis of aged RBCs. This study provides clear nanoscale pictures of aging‐associated changes in CD47 distribution and TSP‐1/CD47 interaction on the cell surface, and insights into the molecular basis for how these molecules coordinate to remove aged RBCs.

Donor-dependent aging of young and old red blood cell subpopulations: Metabolic and functional heterogeneity.

Characteristics of red blood cells (RBCs) are influenced by donor variability. This study assessed quality and metabolomic variables of RBC subpopulations of varied biologic age in red blood cell concentrates (RCCs) from male and female donors to evaluate their contribution to the storage lesion. Red blood cell concentrates from healthy male (n = 6) and female (n = 4) donors were Percoll separated into less dense ("young", Y-RCCs) and dense ("old", O-RCCs) subpopulations, which were assessed weekly for 28 days for changes in hemolysis, mean cell volume (MCV), hemoglobin concentration (MCHC), hemoglobin autofluorescence (HGB), morphology index (MI), oxygen affinity (p50), rigidity, intracellular reactive oxygen species (ROS), calcium ([Ca2+ ]), and mass spectrometry-based metabolomics. Young RCCs having disc-to-discoid morphology showed higher MCV and MI, but lower MCHC, HGB, and rigidity than O-RCCs, having discoid-to-spheroid shape. By Day 14, Y-RCCs retained lower hemolysis and rigidity and higher p50 compared to O-RCCs. Donor sex analyses indicated that females had higher MCV, HGB, ROS, and [Ca2+ ] and lower hemolysis than male RBCs, in addition to having a decreased rate of change in hemolysis by Day 28. Metabolic profiling indicated a significant sex-related signature across all groups with increased markers of high membrane lipid remodeling and antioxidant capacity in Y-RCCs, whereas O-RCCs had increased markers of oxidative stress and decreased coping capability. The structural, functional, and metabolic dissimilarities of Y-RCCs and O-RCCs from female and male donors demonstrate RCC heterogeneity, where RBCs from females contribute less to the storage lesion and age slower than males.

Роль тканевого эритропоэтина и механизмов обратной связи в локальной регуляции эритропоэза

Erythroblastic islets (EI) of rat bone marrow were cultured for 24, 48, or 72 h together with various erythrocytes, simulating the state of erythron hyper- or hyporegeneration in the in vitro system. The concentration of erythropoietin was determined in the control and experimental cultures of EI by two-site enzyme-linked immunosorbent assay. With the addition of reticulocytes/young red blood cells (erythron hyperregeneration model) at a dose of 30 or 60 cells / 1 EI, after 24 h of cultivation, the concentration of erythropoietin in the culture medium increased by 37% compared to control cultures. In cultures with 120/1 EI reticulocytes / young red blood cells, the concentration of erythropoietin decreased by 25% by 48 h. The addition of mature/old red blood cells (an erythron hyporegeneration model) caused inhibition of erythropoietin synthesis in EI culture. By 48 h, the content of this hormone in the cultivation medium significantly decreased in all series of the experiment: in cultures with 30 cells / 1 EI - by 18%, and in cultures with a large erythrocyte load - by 28%. In 72-h cultures containing 30, 60, or 120 red blood cells / 1 EI, the concentration of erythropoietin decreased by 26%, 40%, and 53%, respectively, compared with the control. We found that local regulation of erythropoiesis is directly related to the synthesis of erythropoietin in EI and depends on the number and maturity of cells produced by red bone marrow. The data obtained suggest that it is tissue erythropoietin that is the most important link in the implementation of feedback mechanisms that provide an adequate response of the erythron to an oxygen demand in physiological conditions and support erythropoiesis in pathological conditions accompanied by small production of erythropoietin in the kidneys.

Toxic effect of copper-zinc pyrite ore on erythropoiesis in chronic experiment

Natural ores contain a large number of harmful components for human health. Workers of mining and processing enterprises, who have long-term contact with these natural elements, are often diagnosed with anemia, that pathogenesis is not sufficiently studied. Aim of the study was to detail the mechanisms of rats’ erythron disorders in the long-termintoxication by natural complex of heavy metal compounds of copper-zinc pyrite ore. Material and methods. The work was performed on 60 white non-linear male rats aged 3-4 months weighing 220,52 ± 15,51 g. The experimental group of animals (n = 40) were injected orally with water suspension of copper-zinc powder in a bread crumb an hour before standard feeding during 75-120 days. The blood and bone marrow of experimental groups of rats were carried out on the 75-th, 90-th, 105-th and 120-th days of the experiment. The blood and bone marrow of rats’ control groups were studied on the 75-th and 105-th day. The central part of erythron was assessed by the number and composition of the erythroblastic islets (EI) of bone marrow, the number of free macrophages, the coefficients of involvement of colonyforming units of red blood cells (CFU-E) and macrophages into erythropoiesis. Results . In the peripheral blood of experimental rats the number of red blood cells and the content of hemoglobin were reliably less than the control values on the 90-th and 120-th day, the number of reticulocytes was exceeded the control group on the 75-th, 105-th and 120-th day. In the bone marrow of experimental rats there were only a single EI1 and EI2 classes of maturity. Throughout the experiment, the content of free macrophages and EI with reticulocytes «crown» in the bone marrow of rats was elevated, and the concentration of iron and erythropoietin in the blood, on the contrary, was reduced. The intensity of CFU-E involvement in the erythropoiesis was less than the control level by 2 times on 105th and 120th days of the experiment. Conclusion . With long-term introduction of copper-zinc pyrite ore in the bone marrow of rats the process of formation of EI de novo is suppressed by destabilizing the contact of free bone macrophages with young red blood cells. There is reduces in the content of serum erythropoietin and bound iron, that combines with the change of erythropoiesis nature, leads to decreasing of the number of mature red blood cells and content of hemoglobin in the peripheral blood.

Frequency of Lentz Bodies Inclusion in Whole Blood Erythrocytes and Expanded Buffy Coat Smears

Background: Canine distemper has been classified as highly contagious for most of domestic and wild carnivores, and the infection can be fatal. Canine distemper inclusion bodies, also denominated Lenz inclusion bodies, are large aggregates of viral nucleocapsid particles that can be form in red blood cells (RBCs), white blood cells (WBCs) and epithelial cells in many tissues during the acute phase of infection. Their presence in blood is transient and rarely encountered in light microscopy but are pathognomonic when identified in blood smears. The objective of this study was to investigate the frequency of distemper inclusions in erythrocytes according to the fraction of the sample used for blood smears. Materials, Methods &amp; Results: The study was conducted with routine blood sample provided by the Veterinary Laboratory of Clinical Analysis from the Veterinary Teaching Hospital of Universidade Federal do Rio Grande do Sul. The EDTA-K2 blood sample of a 40 days old male dog, mixed breed, no immunization records, presenting diarrhea, hyporexia, myoclonus and pustules in the abdomen, was selected. In a routine peripheral blood smear examination, several distemper inclusions were observed in the erythrocytes. From this sample, ten smears were performed using a whole blood (WB) and top erythrocyte fraction combined with buffy coat, denominated of expanded buffy coat (EBC). The EBC fraction was obtained after centrifugation of EDTA whole blood in microhematocrit tubes at 9600 x g for 5 min to obtained the packed cell volume (PCV) and buffy coat. After centrifugation, the blood cells are separated into three layers based on density: platelets (adjacent to supernatant), WBCs, and RBCs in the bottom. The PCV was measured and the microhematocrit tube was ruptured 2% below the interface between leukocytes and plasma, deposited into a plastic microtubes, homogenized and used for blood smear preparation. All smears were stained with Diff-Quick Stain. The frequency of observation of RBCs with distemper inclusions bodies was performed under optical microscopy, in the immersion objective (100x), accounting for complete fields up to a minimum of 1000 RBCs, and compared between WB and EBC. In comparison between blood smears obtained from WB and EBC, a highly significant difference (P = 0.0004) was observed in the frequency distribution of distemper inclusion. The median of frequency of RBCs with distemper inclusions in a WB smears was 12.68/1000 RBCs (10.1 - 16.1/1000 RBCs), with a coefficient of variation (CV) of 12%. Median of frequency of distemper inclusions from EBC smears was 54.23/1000 RBCs (45-77.9/1000 RBCs), CV of 18% were observed. The median frequency of inclusions found in EBC smears was 4.27 times higher than the WB smears. Discussion: Buffy coat smear providing a concentrated preparation of nucleated cells and this procedure is useful to looking for low-incidence infectious organisms or other hematologic alterations. The upper fraction of the RBC column, below the buffy coat, is composed of young RBCs. Selection of these portion, and their possible formed in the bone marrow viral replication phase, could justified the increase in the frequency of RBCs containing viral inclusions in EBC, which would also increase the sensitivity of the technique. EBC was homogenized previously to make the smears, certifying the adequate cell distribution in the slide surface without interfere with the frequency of distemper inclusion in RBCs observation. These results were confirmed with the coefficients of variation. In conclusion, distemper inclusions bodies in RBCs from EBC is a recommended diagnosis method in patients suspected of canine Distemper infection. The observation being more frequent in the EBC in comparison with WB, commonly used in veterinary hematology.