Density, heterogeneity and deformability of red cells as markers of clinical severity in hereditary spherocytosis.

Hereditary spherocytosis (HS) originates from defective anchoring of the cytoskeletal network to the transmembrane protein complexes of the red blood cell (RBC). Red cells in HS are characterized by membrane instability and reduced deformability and there is marked heterogeneity in disease severity among patients. To unravel this variability in disease severity, we analyzed blood samples from 21 HS patients with defects in ankyrin, band 3, α-spectrin or ß-spectrin using red cell indices, eosin-5-maleimide binding, microscopy, the osmotic fragility test, Percoll density gradients, vesiculation and ektacytometry to assess cell membrane stability, cellular density and deformability. Reticulocyte counts, CD71 abundance, band 4.1 a:b ratio, and glycated hemoglobin were used as markers of RBC turnover. We observed that patients with moderate/severe spherocytosis have short-living erythrocytes of low density and abnormally high intercellular heterogeneity. These cells show a prominent decrease in membrane stability and deformability and, as a consequence, are quickly removed from the circulation by the spleen. In contrast, in mild spherocytosis less pronounced reduction in deformability results in prolonged RBC lifespan and, hence, cells are subject to progressive loss of membrane. RBC from patients with mild spherocytosis thus become denser before they are taken up by the spleen. Based on our findings, we conclude that RBC membrane loss, cellular heterogeneity and density are strong markers of clinical severity in spherocytosis.

Cardiac N-methyl D-aspartate Receptors as a Pharmacological Target.

This study focuses on characterization of the cardiac N-methyl D-aspartate receptors (NMDARs) as a target for endogenous and synthetic agonists and antagonists. Using isolated perfused rat hearts, we have shown that intracoronary administration of the NMDAR agonists and antagonists has a pronounced effect on autonomous heart function. Perfusion of rat hearts with autologous blood supplemented with NMDAR agonists was associated with induction of tachycardia, sinus arrhythmia, and ischemia occurring within physiological plasma concentration range for glutamate and glycine. Intracoronary administration of the NMDAR antagonists exerted an antiarrhythmic effect and resulted in bradycardia and improvement of capillary perfusion. Action of antagonists eliprodil, Ro25-6981, memantine, ketamine, and MK-801 on autonomous heart function diverged strikingly from that of L-type Ca channel blockers. Cardiac NMDAR subunit composition differed from that of neuronal receptors and was age specific and chamber specific. Transcripts of the GluN3A and GluN2D were found in all heart chambers, whereas expression of GluN1 and GluN2A and 2C was restricted to the atria. Expression of the GluN2B protein in ventricles increased markedly with age of the animals. The obtained data reveal that NMDARs are expressed in rat heart contributing to the autonomic heart rate regulation and the function of the cardiac conduction system.

Washing stored red blood cells in an albumin solution improves their morphologic and hemorheologic properties.

BACKGROUND: Prolonged storage of red blood cells (RBCs) leads to storage lesions, which may impair clinical outcomes after transfusion. A hallmark of storage lesions is progressive echinocytic shape transformation, which can be partially reversed by washing in albumin solutions. Here we have investigated the impact of this shape recovery on biorheologic variables. STUDY DESIGN AND METHODS: RBCs stored hypothermically for 6 to 7 weeks were washed in a 1% human serum albumin (HSA) solution. RBC deformability was measured with osmotic gradient ektacytometry. The viscosity of RBC suspensions was measured with a Couette-type viscometer. The flow behavior of RBCs suspended at 40% hematocrit was tested with an artificial microvascular network (AMVN). RESULTS: Washing in 1% albumin reduced higher degrees of echinocytes and increased the frequency of discocytes, thereby shifting the morphologic index toward discocytosis. Washing also reduced RBC swelling. This shape recovery was not seen after washing in saline, buffer, or plasma. RBC shape normalization did not improve cell deformability measured by ektacytometry, but it tended to decrease suspension viscosities at low shear rates and improved the perfusion of an AMVN. CONCLUSIONS: Washing of stored RBCs in a 1% HSA solution specifically reduces echinocytosis, and this shape recovery has a beneficial effect on microvascular perfusion in vitro. Washing in 1% albumin may represent a new approach to improving the quality of stored RBCs and thus potentially reducing the likelihood of adverse clinical outcomes associated with transfusion of blood stored for longer periods of time.

Red blood cells of sickle cell disease patients exhibit abnormally high abundance of N-methyl D-aspartate receptors mediating excessive calcium uptake.

Recently we showed that N-methyl D-aspartate receptors (NMDARs) are expressed in erythroid precursors (EPCs) and present in the circulating red blood cells (RBCs) of healthy humans, regulating intracellular Ca(2+) in these cells. This study focuses on investigating the possible role of NMDARs in abnormally high Ca(2+) permeability in the RBCs of patients with sickle cell disease (SCD). Protein levels of the NMDAR subunits in the EPCs of SCD patients did not differ from those in EPCs of healthy humans. However, the number and activity of the NMDARs in circulating SCD-RBCs was substantially up-regulated, being particularly high during haemolytic crises. The number of active NMDARs correlated negatively with haematocrit and haemoglobin levels in the blood of SCD patients. Calcium uptake via these non-selective cation channels was induced by RBC treatment with glycine, glutamate and homocysteine and was facilitated by de-oxygenation of SCD-RBCs. Oxidative stress and RBC dehydration followed receptor stimulation and Ca(2+) uptake. Inhibition of the NMDARs with an antagonist memantine caused re-hydration and largely prevented hypoxia-induced sickling. The EPCs of SCD patients showed higher tolerance to memantine than those of healthy subjects. Consequently, NMDARs in the RBCs of SCD patients appear to be an attractive target for pharmacological intervention.

N-methyl-D-aspartate receptors in human erythroid precursor cells and in circulating red blood cells contribute to the intracellular calcium regulation.

The presence of N-methyl-d-aspartate receptor (NMDAR) was previously shown in rat red blood cells (RBCs) and in a UT-7/Epo human myeloid cell line differentiating into erythroid lineage. Here we have characterized the subunit composition of the NMDAR and monitored its function during human erythropoiesis and in circulating RBCs. Expression of the NMDARs subunits was assessed in erythroid progenitors during ex vivo erythropoiesis and in circulating human RBCs using quantitative PCR and flow cytometry. Receptor activity was monitored using a radiolabeled antagonist binding assay, live imaging of Ca(2+) uptake, patch clamp, and monitoring of cell volume changes. The receptor tetramers in erythroid precursor cells are composed of the NR1, NR2A, 2C, 2D, NR3A, and 3B subunits of which the glycine-binding NR3A and 3B and glutamate-binding NR2C and 2D subunits prevailed. Functional receptor is required for survival of erythroid precursors. Circulating RBCs retain a low number of the receptor copies that is higher in young cells compared with mature and senescent RBC populations. In circulating RBCs the receptor activity is controlled by plasma glutamate and glycine. Modulation of the NMDAR activity in RBCs by agonists or antagonists is associated with the alterations in whole cell ion currents. Activation of the receptor results in the transient Ca(2+) accumulation, cell shrinkage, and alteration in the intracellular pH, which is associated with the change in hemoglobin oxygen affinity. Thus functional NMDARs are present in erythroid precursor cells and in circulating RBCs. These receptors contribute to intracellular Ca(2+) homeostasis and modulate oxygen delivery to peripheral tissues.

S-glutathionylation of the Na,K-ATPase catalytic α subunit is a determinant of the enzyme redox sensitivity.

Na,K-ATPase is highly sensitive to changes in the redox state, and yet the mechanisms of its redox sensitivity remain unclear. We have explored the possible involvement of S-glutathionylation of the catalytic α subunit in redox-induced responses. For the first time, the presence of S-glutathionylated cysteine residues was shown in the α subunit in duck salt glands, rabbit kidneys, and rat myocardium. Exposure of the Na,K-ATPase to oxidized glutathione (GSSG) resulted in an increase in the number of S-glutathionylated cysteine residues. Increase in S-glutathionylation was associated with dose- and time-dependent suppression of the enzyme function up to its complete inhibition. The enzyme inhibition concurred with S-glutathionylation of the Cys-454, -458, -459, and -244. Upon binding of glutathione to these cysteines, the enzyme was unable to interact with adenine nucleotides. Inhibition of the Na,K-ATPase by GSSG did not occur in the presence of ATP at concentrations above 0.5 mm. Deglutathionylation of the α subunit catalyzed by glutaredoxin or dithiothreitol resulted in restoration of the Na,K-ATPase activity. Oxidation of regulatory cysteines made them inaccessible for glutathionylation but had no profound effect on the enzyme activity. Regulatory S-glutathionylation of the α subunit was induced in rat myocardium in response to hypoxia and was associated with oxidative stress and ATP depletion. S-Glutathionylation was followed by suppression of the Na,K-ATPase activity. The rat α2 isoform was more sensitive to GSSG than the α1 isoform. Our findings imply that regulatory S-glutathionylation of the catalytic subunit plays a key role in the redox-induced regulation of Na,K-ATPase activity.

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.

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.

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.

Absence of neocytolysis in humans returning from a 3-week high-altitude sojourn.

AIMS: Total haemoglobin mass (tot-Hb) increases during high-altitude acclimatization. Normalization of tot-Hb upon descent is thought to occur via neocytolysis, the selective destruction of newly formed erythrocytes. Because convincing experimental proof of neocytolysis is lacking, we performed a prospective study on erythrocyte survival after a stay at the Jungfraujoch Research Station (JFJRS; 3450 m). METHODS: Newly formed erythrocytes of 12 male subjects (mean age 23.3 years) were age cohort labelled in normoxia (110 m) and during a 19-day high-altitude sojourn by ingestion of 13 C2- and 15 N-labelled glycine respectively. Elimination dynamics for erythrocytes produced in normoxia and at high altitude were measured by isotope ratio mass spectrometry of haem, by determining tot-Hb, reticulocyte counts, erythrocyte membrane protein 4.1a/4.1b ratio and by mathematical modelling. RESULTS: Tot-Hb increased by 4.7% ± 2.7% at high altitude and returned to pre-altitude values within 11 days after descent. Elimination of 13 C- (normoxia) and 15 N- (high altitude) labelled erythrocytes was not different. Erythropoietin levels and counts of CD71-positive reticulocytes decreased rapidly after descent. The band 4.1a/4.1b ratio decreased at altitude and remained low for 3-4 days after descent and normalized slowly. There was no indication of haemolysis. CONCLUSION: We confirm a rapid normalization of tot-Hb upon descent. Based on the lack of accelerated removal of age cohorts of erythrocytes labelled at high altitude, on patterns of changes in reticulocyte counts and of the band 4.1a/4.1b ratio and on modelling, this decrease did not occur via neocytolysis, but by a reduced rate of erythropoiesis along with normal clearance of senescent erythrocytes.

Functional NMDA receptors in rat erythrocytes.

N-methyl-d-aspartate (NMDA) receptors are ligand-gated nonselective cation channels mediating fast neuronal transmission and long-term potentiation in the central nervous system. These channels have a 10-fold higher permeability for Ca(2+) compared with Na(+) or K(+) and binding of the agonists (glutamate, homocysteine, homocysteic acid, NMDA) triggers Ca(2+) uptake. The present study demonstrates the presence of NMDA receptors in rat erythrocytes. The receptors are most abundant in both erythroid precursor cells and immature red blood cells, reticulocytes. Treatment of erythrocytes with NMDA receptor agonists leads to a rapid increase in intracellular Ca(2+) resulting in a transient shrinkage via Gardos channel activation. Additionally, the exposure of erythrocytes to NMDA receptor agonists causes activation of the nitric oxide (NO) synthase facilitating either NO production in l-arginine-containing medium or superoxide anion (O(2)(.-)) generation in the absence of l-arginine. Conversely, treatment with an NMDA receptor antagonist MK-80, or the removal of Ca(2+) from the incubation medium causes suppression of Ca(2+) accumulation and prevents attendant changes in cell volume and NO/O(2)(.-) production. These results suggest that the NMDA receptor activity in circulating erythrocytes is regulated by the plasma concentrations of homocysteine and homocysteic acid. Moreover, receptor hyperactivation may contribute to an increased incidence of thrombosis during hyperhomocysteinemia.

Heterogeneity of Red Blood Cells: Causes and Consequences.

Mean values of hematological parameters are currently used in the clinical laboratory settings to characterize red blood cell properties. Those include red blood cell indices, osmotic fragility test, eosin 5-maleimide (EMA) test, and deformability assessment using ektacytometry to name a few. Diagnosis of hereditary red blood cell disorders is complemented by identification of mutations in distinct genes that are recognized "molecular causes of disease." The power of these measurements is clinically well-established. However, the evidence is growing that the available information is not enough to understand the determinants of severity of diseases and heterogeneity in manifestation of pathologies such as hereditary hemolytic anemias. This review focuses on an alternative approach to assess red blood cell properties based on heterogeneity of red blood cells and characterization of fractions of cells with similar properties such as density, hydration, membrane loss, redox state, Ca2+ levels, and morphology. Methodological approaches to detect variance of red blood cell properties will be presented. Causes of red blood cell heterogeneity include cell age, environmental stress as well as shear and metabolic stress, and multiple other factors. Heterogeneity of red blood cell properties is also promoted by pathological conditions that are not limited to the red blood cells disorders, but inflammatory state, metabolic diseases and cancer. Therapeutic interventions such as splenectomy and transfusion as well as drug administration also impact the variance in red blood cell properties. Based on the overview of the studies in this area, the possible applications of heterogeneity in red blood cell properties as prognostic and diagnostic marker commenting on the power and selectivity of such markers are discussed.

N-Methyl-D-Aspartate Receptors in Hematopoietic Cells: What Have We Learned?

The N-methyl-D-aspartate receptor (NMDAR) provides a pathway for glutamate-mediated inter-cellular communication, best known for its role in the brain but with multiple examples of functionality in non-neuronal cells. Data previously published by others and us provided ex vivo evidence that NMDARs regulate platelet and red blood cell (RBC) production. Here, we summarize what is known about these hematopoietic roles of the NMDAR. Types of NMDAR subunits expressed in megakaryocytes (platelet precursors) and erythroid cells are more commonly found in the developing rather than adult brain, suggesting trophic functions. Nevertheless, similar to their neuronal counterparts, hematopoietic NMDARs function as ion channels, and are permeable to calcium ions (Ca2+). Inhibitors that block open NMDAR (memantine and MK-801) interfere with megakaryocytic maturation and proplatelet formation in primary culture. The effect on proplatelet formation appears to involve Ca2+ influx-dependent regulation of the cytoskeletal remodeling. In contrast to normal megakaryocytes, NMDAR effects in leukemic Meg-01 cells are diverted away from differentiation to increase proliferation. NMDAR hypofunction triggers differentiation of Meg-01 cells with the bias toward erythropoiesis. The underlying mechanism involves changes in the intracellular Ca2+ homeostasis, cell stress pathways, and hematopoietic transcription factors that upon NMDAR inhibition shift from the predominance of megakaryocytic toward erythroid regulators. This ability of NMDAR to balance both megakaryocytic and erythroid cell fates suggests receptor involvement at the level of a bipotential megakaryocyte-erythroid progenitor. In human erythroid precursors and circulating RBCs, NMDAR regulates intracellular Ca2+ homeostasis. NMDAR activity supports survival of early proerythroblasts, and in mature RBCs NMDARs impact cellular hydration state, hemoglobin oxygen affinity, and nitric oxide synthase activity. Overexcitation of NMDAR in mature RBCs leads to Ca2+ overload, K+ loss, RBC dehydration, and oxidative stress, which may contribute to the pathogenesis of sickle cell disease. In summary, there is growing evidence that glutamate-NMDAR signaling regulates megakaryocytic and erythroid cells at different stages of maturation, with some intriguing differences emerging in NMDAR expression and function between normal and diseased cells. NMDAR signaling may provide new therapeutic opportunities in hematological disease, but in vivo applicability needs to be confirmed.

Aging Markers in Equine Red Blood Cells.

Detection of hematopoietic activity in horses is a challenge due to the lack of cells carrying reticulocyte markers such as RNA remnants or CD71 in the circulation. In this study, we fractionated equine red cells according to their density and analyzed the cells forming low (L), medium (M), and high (H) density fractions for markers of aging such as membrane loss, oxidation, and alterations in the intracellular free Ca2+ levels. Cells forming L and M fraction were highly heterogeneous in projected areas and shapes, and had higher propensity to swell in response to hypo-osmotic challenge than the cells from the H fraction. The densest cells were deprived of band 3 protein compared to the cells within L or M fraction. Furthermore, the equine red cells from the H fraction were hyper-oxidized compared to the cells within M and L fractions as follows from an increase in autofluorescence characteristic for oxidized damaged hemoglobin and from thiol oxidation as detected using monobromobimane. The lightest cells showed lower free thiol content compared to the red blood cells from the M fraction, but did not contain oxidized hemoglobin. Finally, the majority of red blood cells forming L, M, and H fraction prominently differed from each other in intracellular free Ca2+ levels and its distribution within the cells. Based on the obtained findings, we suggest that intraerythrocytic Ca2+ levels and its subcellular distribution, eosin-5-maleimide binding test for band 3 abundance, and autofluorescence of cells along with the changes in red blood cell indices, distribution width and creatine levels may become potential markers of regenerative erythropoiesis in horses. Validation of the power of these potential markers of red cell aging is pending.

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, ß-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 ß-thalassemia and 2 patients, carriers of several mutations and a complex genotype. While the patients with ß-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.

A Previously Unrecognized Ca2+-inhibited Nonselective Cation Channel in Red Blood Cells.

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NMDA Receptor Activity in Circulating Red Blood Cells: Methods of Detection.

Abundance and activity of N-methyl-D-aspartate (NMDA) in circulating red blood cells contributes to the maintenance of intracellular Ca2+ in these cells and, by doing that, controls red cell volume, membrane stability, and O2 carrying capacity. Detection of the NMDA receptor activity in red blood cells is challenging as the number of its copies is low and shows substantial cell-to-cell heterogeneity. Receptor abundance is reliably assessed using the radiolabeled antagonist ([3H]MK-801) binding technique. Uptake of Ca2+ following the NMDA receptor activation is detected in cells loaded with Ca2+-sensitive fluorescent dye Fluo-4 AM. Both microfluorescence live-cell imaging and flow cytometry may be used for fluorescence intensity detection. Automated patch clamp is currently used for recording of electric currents triggered by the stimulation of the NMDA receptor. These currents are mediated by the Ca2+-sensitive K+ (Gardos) channels that open upon Ca2+ uptake via the active NMDA receptor. Furthermore, K+ flux through the Gardos channels induced by the NMDA receptor stimulation in red blood cells may be detected using unidirectional K+(86Rb+) influx.

Is Increased Intracellular Calcium in Red Blood Cells a Common Component in the Molecular Mechanism Causing Anemia?

For many hereditary disorders, although the underlying genetic mutation may be known, the molecular mechanism leading to hemolytic anemia is still unclear and needs further investigation. Previous studies revealed an increased intracellular Ca2+ in red blood cells (RBCs) from patients with sickle cell disease, thalassemia, or Gardos channelopathy. Therefore we analyzed RBCs' Ca2+ content from 35 patients with different types of anemia (16 patients with hereditary spherocytosis, 11 patients with hereditary xerocytosis, 5 patients with enzymopathies, and 3 patients with hemolytic anemia of unknown cause). Intracellular Ca2+ in RBCs was measured by fluorescence microscopy using the fluorescent Ca2+ indicator Fluo-4 and subsequent single cell analysis. We found that in RBCs from patients with hereditary spherocytosis and hereditary xerocytosis the intracellular Ca2+ levels were significantly increased compared to healthy control samples. For enzymopathies and hemolytic anemia of unknown cause the intracellular Ca2+ levels in RBCs were not significantly different. These results lead us to the hypothesis that increased Ca2+ levels in RBCs are a shared component in the mechanism causing an accelerated clearance of RBCs from the blood stream in channelopathies such as hereditary xerocytosis and in diseases involving defects of cytoskeletal components like hereditary spherocytosis. Future drug developments should benefit from targeting Ca2+ entry mediating molecular players leading to better therapies for patients.