Calcium in Red Blood Cells—A Perilous Balance

Ca2+ is a universal signalling molecule involved in regulating cell cycle and fate, metabolism and structural integrity, motility and volume. Like other cells, red blood cells (RBCs) rely on Ca2+ dependent signalling during differentiation from precursor cells. Intracellular Ca2+ levels in the circulating human RBCs take part not only in controlling biophysical properties such as membrane composition, volume and rheological properties, but also physiological parameters such as metabolic activity, redox state and cell clearance. Extremely low basal permeability of the human RBC membrane to Ca2+ and a powerful Ca2+ pump maintains intracellular free Ca2+ levels between 30 and 60 nM, whereas blood plasma Ca2+ is approximately 1.8 mM. Thus, activation of Ca2+ uptake has an impressive impact on multiple processes in the cells rendering Ca2+ a master regulator in RBCs. Malfunction of Ca2+ transporters in human RBCs leads to excessive accumulation of Ca2+ within the cells. This is associated with a number of pathological states including sickle cell disease, thalassemia, phosphofructokinase deficiency and other forms of hereditary anaemia. Continuous progress in unravelling the molecular nature of Ca2+ transport pathways allows harnessing Ca2+ uptake, avoiding premature RBC clearance and thrombotic complications. This review summarizes our current knowledge of Ca2+ signalling in RBCs emphasizing the importance of this inorganic cation in RBC function and survival

RedTell: an AI tool for interpretable analysis of red blood cell morphology

Introduction: Hematologists analyze microscopic images of red blood cells to study their morphology and functionality, detect disorders and search for drugs. However, accurate analysis of a large number of red blood cells needs automated computational approaches that rely on annotated datasets, expensive computational resources, and computer science expertise. We introduce RedTell, an AI tool for the interpretable analysis of red blood cell morphology comprising four single-cell modules: segmentation, feature extraction, assistance in data annotation, and classification.Methods: Cell segmentation is performed by a trained Mask R-CNN working robustly on a wide range of datasets requiring no or minimum fine-tuning. Over 130 features that are regularly used in research are extracted for every detected red blood cell. If required, users can train task-specific, highly accurate decision tree-based classifiers to categorize cells, requiring a minimal number of annotations and providing interpretable feature importance.Results: We demonstrate RedTell’s applicability and power in three case studies. In the first case study we analyze the difference of the extracted features between the cells coming from patients suffering from different diseases, in the second study we use RedTell to analyze the control samples and use the extracted features to classify cells into echinocytes, discocytes and stomatocytes and finally in the last use case we distinguish sickle cells in sickle cell disease patients.Discussion: We believe that RedTell can accelerate and standardize red blood cell research and help gain new insights into mechanisms, diagnosis, and treatment of red blood cell associated disorders

Simple Assessment of Red Blood Cell Deformability Using Blood Pressure in Capillary Channels for Effective Detection of Subpopulations in Red Blood Cells

Assessment of red blood cell (RBC) deformability as a biomarker requires expensive equipment to induce and monitor deformation. In this study, we present a simple method for quantifying RBC deformability. We designed a microfluidic channel consisting of a micropillar channel and a coflowing channel connected in series. When blood (loading volume = 100 μL) was injected continuously into the device under constant pressure (1 bar), we monitored the boundary position of the blood and the reference flow in the coflowing channel. A decrease in the deformability of RBCs results in a growing pressure drop in the micropillar channel, which is mirrored by a decrease in blood pressure in the coflowing channel. Analysis of this temporal variation in blood pressure allowed us to define the clogging index (CI) as a new marker of RBC deformability. As a result of the analytical study and numerical simulation, we have demonstrated that the coflowing channel may serve as a pressure sensor that allows the measurement of blood pressure with accuracy. We have shown experimentally that a higher hematocrit level (i.e., more than 40%) does not have a substantial influence on CI. The CI tended to increase to a higher degree in glutaraldehyde-treated hardened RBCs. Furthermore, we were able to resolve the difference in deformability of RBCs between two different RBC density subfractions in human blood. In summary, our approach using CI provides reliable information on the deformability of RBCs, which is comparable to the readouts obtained by ektacytometry. We believe that our microfluidic device would be a useful tool for evaluating the deformability of RBCs, which does not require expensive instruments (e.g., high-speed camera) or time-consuming micro-PIV analysis

Recovery of donor hearts after circulatory death with normothermic extracorporeal machine perfusion

OBJECTIVES A severe donor organ shortage leads to the death of a substantial number of patients who are listed for transplantation. The use of hearts from donors after circulatory death could significantly expand the donor organ pool, but due to concerns about their viability, these are currently not used for transplantation. We propose short-term ex vivo normothermic machine perfusion (MP) to improve the viability of these ischaemic donor hearts. METHODS Hearts from male Lewis rats were subjected to 25 min of global in situ warm ischaemia (WI) (37°C), explanted, reconditioned for 60 min with normothermic (37°C) MP with diluted autologous blood and then stored for 4 h at 0-4°C in Custodiol cold preservation solution. Fresh and ischaemic hearts stored for 4 h in Custodiol were used as controls. Graft function was assessed in a blood-perfused Langendorff circuit. RESULTS During reconditioning, both the electrical activity and contractility of the ischaemic hearts recovered rapidly. Throughout the Langendorff reperfusion, the reconditioned ischaemic hearts had a higher average heart rate and better contractility compared with untreated ischaemic controls. Moreover, the reconditioned ischaemic hearts had higher tissue adenosine triphosphate levels and a trend towards improved tissue redox state. Perfusate levels of troponin T, creatine kinase and lactate dehydrogenase were not significantly lower than those of untreated ischaemic controls. The micro- and macroscopic appearance of the reconditioned ischaemic hearts were improved compared with ischaemic controls, but in both groups myocardial damage and oedema were evident. CONCLUSIONS Our results indicate that functional recovery from global WI is possible during short-term ex vivo reperfusion, allowing subsequent cold storage without compromising organ viability. We expect that once refined and validated, this approach may enable safe transplantation of hearts obtained from donation after circulatory deat

Continuous Percoll Gradient Centrifugation of Erythrocytes—Explanation of Cellular Bands and Compromised Age Separation

(1) Background: When red blood cells are centrifuged in a continuous Percoll-based density gradient, they form discrete bands. While this is a popular approach for red blood cell age separation, the mechanisms involved in banding were unknown. (2) Methods: Percoll centrifugations of red blood cells were performed under various experimental conditions and the resulting distributions analyzed. The age of the red blood cells was measured by determining the protein band 4.1a to 4.1b ratio based on western blots. Red blood cell aggregates, so-called rouleaux, were monitored microscopically. A mathematical model for the centrifugation process was developed. (3) Results: The red blood cell band pattern is reproducible but re-centrifugation of sub-bands reveals a new set of bands. This is caused by red blood cell aggregation. Based on the aggregation, our mathematical model predicts the band formation. Suppression of red blood cell aggregation reduces the band formation. (4) Conclusions: The red blood cell band formation in continuous Percoll density gradients could be explained physically by red blood cell aggregate formation. This aggregate formation distorts the density-based red blood cell age separation. Suppressing aggregation by osmotic swelling has a more severe effect on compromising the RBC age separation to a higher degree

Continuous Percoll Gradient Centrifugation of Erythrocytes—Explanation of Cellular Bands and Compromised Age Separation

(1) Background: When red blood cells are centrifuged in a continuous Percoll-based density gradient, they form discrete bands. While this is a popular approach for red blood cell age separation, the mechanisms involved in banding were unknown. (2) Methods: Percoll centrifugations of red blood cells were performed under various experimental conditions and the resulting distributions analyzed. The age of the red blood cells was measured by determining the protein band 4.1a to 4.1b ratio based on western blots. Red blood cell aggregates, so-called rouleaux, were monitored microscopically. A mathematical model for the centrifugation process was developed. (3) Results: The red blood cell band pattern is reproducible but re-centrifugation of sub-bands reveals a new set of bands. This is caused by red blood cell aggregation. Based on the aggregation, our mathematical model predicts the band formation. Suppression of red blood cell aggregation reduces the band formation. (4) Conclusions: The red blood cell band formation in continuous Percoll density gradients could be explained physically by red blood cell aggregate formation. This aggregate formation distorts the density-based red blood cell age separation. Suppressing aggregation by osmotic swelling has a more severe effect on compromising the RBC age separation to a higher degree

The protective effect of the spleen in sickle cell patients. A comparative study between patients with asplenia/hyposplenism and hypersplenism

Sickle cell disease (SCD) is caused by a point mutation in the beta-globin gene. SCD is characterized by chronic hemolytic anemia, vaso-occlusive events leading to tissue ischemia, and progressive organ failure. Chronic inflammatory state is part of the pathophysiology of SCD. Patients with SCD have extremely variable phenotypes, from mild disease to severe complications including early age death. The spleen is commonly injured in SCD. Early splenic dysfunction and progressive spleen atrophy are common. Splenomegaly and hypersplenism can also occur with the loss of the crucial splenic function. Acute, life-threatening spleen-related complications in SCD are well studied. The association of laboratory parameters with the spleen status including hyposplenism, asplenia, and splenomegaly/hypersplenism, and their implication in vaso-occlusive crisis and long-term complications in SCD remain to be determined. We evaluated the association between the spleen status with clinical and laboratory parameters in 31 SCD patients: Group a) Patients with asplenia/hyposplenism (N = 22) (including auto-splenectomy and splenectomized patients) vs. Group b) patients with splenomegaly and or hypersplenism (N = 9). Laboratory studies included: Complete Blood Count, reticulocyte count, iron metabolism parameters, C Reactive Protein (CRP), Hb variant distribution, and D-dimer. Metabolic and morphological red blood cell (RBC) studies included: density gradient (by Percoll), glucose consumption, lactate release, and K$^{+}$ leakage, fetal RBC (F-Cells) and F-Reticulocytes, annexinV+, CD71$^{+}$, oxidative stress measured by GSH presence in RBC and finally Howell Jolly Bodies count were all analyzed by Flow Cytometry. Scanning electron microscopy analysis of RBC was also performed. Patients with asplenia/hyposplenism showed significantly higher WBC, platelet, Hematocrit, hemoglobin S, CRP, D-dimer, Gamma Glutamyl Transferase (GGT), cholesterol, transferrin, annexin V+ RBCs, CD71$^{+}$ RBCs, together with a markedly lower F Reticulocyte levels in comparison with splenomegaly/hypersplenism patients. In summary, important differences were also found between the groups in the studied RBCs parameters. Further studies are required to elucidate the effect of the spleen including hyper and hypo-splenia on laboratory parameters and in clinical manifestations, vascular pathology, and long-term complications of SCD. The benefits and risks of splenectomy compared to chronic transfusion need to be evaluated in clinical trials and the standard approach managing hypersplenism in SCD patients should be re-evaluated

Red Blood Cell Membrane Conductance in Hereditary Haemolytic Anaemias

Electrophysiology; Haemolytic anemia; Hereditary spherocytosisElectrofisiologia; Anemia hemolítica; Esferocitosi hereditariaElectrofisiologia; Anèmia hemolítica; Esferocitosi hereditàriaCongenital haemolytic anaemias are inherited disorders caused by red blood cell membrane and cytoskeletal protein defects, deviant hemoglobin synthesis and metabolic enzyme deficiencies. In many cases, although the causing mutation might be known, the pathophysiology and the connection between the particular mutation and the symptoms of the disease are not completely understood. Thus effective treatment is lagging behind. As in many cases abnormal red blood cell cation content and cation leaks go along with the disease, by direct electrophysiological measurements of the general conductance of red blood cells, we aimed to assess if changes in the membrane conductance could be a possible cause. We recorded whole-cell currents from 29 patients with different types of congenital haemolytic anaemias: 14 with hereditary spherocytosis due to mutations in α-spectrin, β-spectrin, ankyrin and band 3 protein; 6 patients with hereditary xerocytosis due to mutations in Piezo1; 6 patients with enzymatic disorders (3 patients with glucose-6-phosphate dehydrogenase deficiency, 1 patient with pyruvate kinase deficiency, 1 patient with glutamate-cysteine ligase deficiency and 1 patient with glutathione reductase deficiency), 1 patient with β-thalassemia and 2 patients, carriers of several mutations and a complex genotype. While the patients with β-thalassemia and metabolic enzyme deficiencies showed no changes in their membrane conductance, the patients with hereditary spherocytosis and hereditary xerocytosis showed largely variable results depending on the underlying mutation

Red Blood Cell Membrane Conductance in Hereditary Haemolytic Anaemias

Congenital haemolytic anaemias are inherited disorders caused by red blood cell membrane and cytoskeletal protein defects, deviant hemoglobin synthesis and metabolic enzyme deficiencies. In many cases, although the causing mutation might be known, the pathophysiology and the connection between the particular mutation and the symptoms of the disease are not completely understood. Thus effective treatment is lagging behind. As in many cases abnormal red blood cell cation content and cation leaks go along with the disease, by direct electrophysiological measurements of the general conductance of red blood cells, we aimed to assess if changes in the membrane conductance could be a possible cause. We recorded whole-cell currents from 29 patients with different types of congenital haemolytic anaemias: 14 with hereditary spherocytosis due to mutations in α-spectrin, β-spectrin, ankyrin and band 3 protein; 6 patients with hereditary xerocytosis due to mutations in Piezo1; 6 patients with enzymatic disorders (3 patients with glucose-6-phosphate dehydrogenase deficiency, 1 patient with pyruvate kinase deficiency, 1 patient with glutamate-cysteine ligase deficiency and 1 patient with glutathione reductase deficiency), 1 patient with β-thalassemia and 2 patients, carriers of several mutations and a complex genotype. While the patients with β-thalassemia and metabolic enzyme deficiencies showed no changes in their membrane conductance, the patients with hereditary spherocytosis and hereditary xerocytosis showed largely variable results depending on the underlying mutation

Functional NMDA receptors in red blood cells and heart

N-methyl D-aspartate (NMDA) receptor is a nonselective cation channel formed four out of seven known subunits capable of binding glutamate or glycine. These amino acids are the agonists of the NMDA receptors in the brain where this receptor is involved in neurotransmission and intracellular signaling. Recently NMDA receptor expression and function were shown in several non-neuronal tissues. NMDA receptors were found in human bone marrow in osteoclasts, osteoblasts and hematopoietic precursor lineages. Expression of several of the NMDA receptor subunits was reported in mammalian myocardial tissue, however, the NMDA receptor function in the heart was never investigated. The aim of the present study was to characterize NMDA receptor expression and its physiological and pathophysiological roles in the heart, and in red blood cells (RBC) as well as in erythroid precursor cells (EPCs). We were the first to report the presence of NMDA receptors in RBCs of rats and humans and in human and rat EPCs. Subunit composition of the erythroid receptors varied depending on the differentiation stage in human EPCs and differed substantially from that in the brain with NR2D and NR3B were the dominating over the neuronal NR1 and NR2B common in neurons. Erythroid receptors could be activated by the NMDA and glutamate. Stimulation of the NMDA receptors in humans EPCs as well as in human and rat RBCs resulted in massive transient Ca2+ uptake. Plasma-born glutamate and glycine were sufficient to maintain basal receptor activity which was controlled by the plasma agonists levels and, most likely, other humoral factors. We have demonstrated that abnormally high numbers of receptor copies in RBCs of sickle cell disease patients translate into excessive permeability of RBC membranes for Ca2+. As calcium is a master regulator of various processes in RBCs, its uptake upon stimulation of the erythroid NMDA receptors resulted in the alterations of cytoskeletal structure and cell volume, modulation of oxidative state, intracellular pH, and RBC half-life in the circulation. In the EPCs NMDA receptors are required for survival of precursor cells, particular at the earlier differentiation stages. Our motivation to investigate the expression and function of the NMDA receptors in the heart was based on the fact that elevation of the plasma levels of the agonist of these receptors, homocysteine, was reported to be associated with several heart diseases including heart failure. We have characterized expression of the NMDA receptor subunits in different parts of rat myocardium and elaborated on the relation between the subunit composition of cardiac NMDA receptors and their pharmacology using “brainless rat heart model” (isolated blood-perfused heart) and sarcolemmal membrane preparations. Similar to that in RBCs, subunit composition of cardiac NMDA receptors differed substantially from that in the brain and showed remarkable chamber-dependent heterogeneity with NR3A and NR2D dominating. Expression of the NR2B subunit was age-dependent and most likely associated with hypertrophic remodeling. Expression of the subunits NR2A and 2C was restricted to atria. We found that NMDA receptor agonists and antagonists regulated autonomous heart rate and rhythmicity. Myocardial repolarization and depolarization rate were prolonged by the NMDA receptor antagonists. Our data indicate that cardiac NMDA receptors are an attractive target for pharmacological interventions to treat arrhythmias and other cardiovascular disorders