Red Blood Cell Morphology Research Articles

Two-Layer Red Blood Cell Membrane Model Using the Discrete Element Method

The red blood cell (RBC) membrane consists of a lipid bilayer and spectrin-based cytoskeleton, which enclose haemoglobin-rich fluid. Numerical models of RBCs typically integrate the two membrane components into a single layer, preventing investigation of bilayer-cytoskeleton interaction. To address this constraint, a new RBC model which considers the bilayer and cytoskeleton separately is developed using the discrete element method (DEM). This is completed in 2D as a proof-of-concept, with an extension to 3D planned in the future. Resting RBC morphology predicted by the two-layer model is compared to an equivalent and well-established composite (one-layer) model with excellent agreement for critical cell dimensions. A parametric study is performed where area reduction ratio and spring constants are varied. It is found that predicted resting geometry is relatively insensitive to changes in spring stiffness, but a shape variation is observed for reduction ratio changes as expected.

Chorea-acanthocytosis:Report of 1 case

Abstract: To report 1 case of chorea-acanthocytosis (ChAc). The case was studied with clinical symptoms,signs, biochemical,red blood cell morphology and gene sequencing observation.The patient’s main symptoms and signs were mouth-tongue dystonia, tongue bite, walking instability, seizures,abated gag reflex,decreased muscle tone,abated tendon reflex.Blood lactate dehydrogenase (LDH, 459 U/L) ,creatine kinase (CK, 2806 U/L), prolactin (PRL, 38.35 ng/ml)were increased.There were large number of acanthocyte in blood smear(50% of the red blood cell),and there were lot acanthocyte in blood smear of her parents(30% of father and 25% of mother).Electromyography showed peripheral nerve damage. Brain MRI scans showed caudate nucleus atrophy.Gene sequencing detected mutations in VPS13A. According to the clinical features of patients, blood LDH, CK, PRL,blood smear, brain MRI imaging characteristics, gene sequencing result, the patient diagnosed ChAc.The disease treatment is limited, need clinical tracking,and explore effective methods of diagnosis and treatment. Keywords: chorea;acanthocyteosis

Red Blood Cells Morphology Depending on Potential of Optically Transparent Electrode

It is known that normal erythrocytes (discocytes) may undergo transformation in degenerative forms (e.g. echinocytes or stomatocytes) due to changes in temperature, electrolyte concentration, pH, and the addition of certain agents [1]. It is also important that the morphology of red blood cells depends on transmembrane potential [2].On the other hand, direct contact with the charged solid surfaces, such as the wall of the vessel or foreign materials (such as metal stents or carbon hemosorbents during the detoxification of the body) can lead to the morphological changes of red blood cells.For the first time the influence of the surface potential of the solid phase to the behavior of blood cells have been proposed in the investigation of thrombus formation. This hypothesis was confirmed by the data of [3], it was found a direct correlation between the decrease of blood cells and the potential magnitude of activated carbon for process of hemosorption.The goal of this work was to study the effect of potential of an optically transparent ITO electrode on the morphology of red blood cells in real time.The three-electrode cell was used, the bottom of it was optically transparent ITO working electrode (Sigma-Aldrich, USA), area of the electrode was 28.3 mm2, auxiliary electrode was a carbon black electrode, a reference electrode was silver chloride electrode. Potentiostat IPC Pro L (ZAO «Kronas», Russia) was used in the potentiodynamic mode, the scanning rate was 10 mV/s. The suspension of erythrocytes was prepared by diluting volunteer red blood cells in isotonic saline (0.15 M NaCl), concentration of erythrocyte was 8∙109 cells/L. The morphology of erythrocytes was examined by an inverted light microscope Eclipse TS100 (Nikon, Japan), the lens CFI Achromat LWD 40x/0.55 (Nikon, Japan) in real time depending on the electrode potential. It was found that the morphological forms of red blood cells have been changed depending on ITO electrode potential. It turned out that, initial normal erythrocytes (discocytes) transformed into various degenerative forms (echinocytes, spherocytes, stomatocytes) depending on value of ITO electrode potential (Fig. 1). It is important that each of morphological form formed and existed in certain ranges of potentials. For example at potentials around - 250 mV discocytes turned into echinocytes, than at more negative potential values echinocytes turned into spherocytes. The mentioned cathodic morphological changes of erythrocytes occurred in the cathode potential region from -250 mV to -400 mV.The anodic transformation of red blood cells was not obtained to the potential of about 600 mV. A very important effect was found in a potential range 600 mV to 1200 mV. At the region transformation echinocytes in discocytes occurred. In the positive potential more 1200 mV the transformation discocytes into stomatocytes was observed.Finally, it was found that the potential ranges more negative than -400 mV and more positive than 1300 correspond to cell destruction. It is very important that the transformation discocytes into degenerative forms are usually reversible. The most likely reason of obtained transformations of morphological forms of red blood cells in the cell/electrode system at different electrode potentials may be the processes of electroreduction or electrooxidation of functional groups on the surface of erythrocyte membranes. Undoubtedly electron transfer cell from electrode to membrane or opposite direction is due to sign and ratio of charge density of cell membrane and electrode. It was proposed the breakdown of the membrane at potentials more negative than -400 mV and a positive 1300 mV lead to destruction of cellThus, the observed in reversible and irreversible changes in morphological forms of erythrocytes and cell destruction for different values of electrode potential was obtained. These effects can be used to develop diagnostic methods of erythrocytes state and perhaps to improve their quality during prolonged storage. 1. Tachev K.D., Danov K.D., Kralchevsky P.A. Colloids Surf B Biointerfaces 2004 34:123–140.2. Glaser R. Stud Biophys 1978 74: 57-58.3. Goldin Mark M., Volkov A.G., Goldfarb Yu.S., Goldin Mikhail M. J Electrochem Soc 2006 153(8): J91-J99. Fig. 1. Erythrocyte morphology depending on electrode potential. The arrows identified three initial echinocytes and their morphological change depending on electrode potential. Figure 1

Rheological Property of Tapioca Maltodextrin‐based Plasma Expander and Effect on Red Blood Cells Morphology

Rheological Property of Tapioca Maltodextrin‐based Plasma Expander and Effect on Red Blood Cells Morphology

Multispectral quantitative phase imaging of human red blood cells using inexpensive narrowband multicolor LEDs.

We report multispectral phase-shifting interference microscopy for quantitative phase imaging of human red blood cells (RBCs). A wide range of wavelengths are covered by means of using multiple color light emitting diodes (LEDs) with narrow spectral bandwidth ranging from violet to deep red color. The multicolor LED light source was designed and operated sequentially, which works as a multispectral scanning light source. Corresponding to each color LED source, five phase-shifted interferograms were recorded sequentially for the measurement of phase maps, as well as the refractive index of RBCs within the entire visible region. The proposed technique provides information about the effect of wavelengths on the morphology and refractive index of human RBCs. The system does not require expensive multiple color filters or any wavelength scanning mechanism along with broadband light source.

In Situ Loading of Drugs into Mesoporous Silica SBA-15.

In a new strategy for loading drugs into mesoporous silica, a hydrophilic (heparin) or hydrophobic drug (ibuprofen) is encapsulated directly in a one-pot synthesis by evaporation-induced self-assembly. In situ drug loading significantly cuts down the preparation time and dramatically increases the loaded amount and released fraction of the drug, and appropriate drug additives favor a mesoporous structure of the vessels. Drug loading was verified by FTIR spectroscopy and release tests, which revealed much longer release with a larger amount of heparin or ibuprofen compared to postloaded SBA-15. Besides, the in vitro anticoagulation properties of the released heparin and the biocompatibility of the vessels were carefully assessed, including activated partial thromboplastin time, thrombin time, hemolysis, platelet adhesion experiments, and the morphologies of red blood cells. A concept of new drug-release agents with soft core and hard shell is proposed and offers guidance for the design of novel drug-delivery systems.

Mature red blood cells: from optical model to inverse light-scattering problem.

We propose a method for characterization of mature red blood cells (RBCs) morphology, based on measurement of light-scattering patterns (LSPs) of individual RBCs with the scanning flow cytometer and on solution of the inverse light-scattering (ILS) problem for each LSP. We considered a RBC shape model, corresponding to the minimal bending energy of the membrane with isotropic elasticity, and constructed an analytical approximation, which allows rapid simulation of the shape, given the diameter and minimal and maximal thicknesses. The ILS problem was solved by the nearest-neighbor interpolation using a preliminary calculated database of 250,000 theoretical LSPs. For each RBC in blood sample we determined three abovementioned shape characteristics and refractive index, which also allows us to calculate volume, surface area, sphericity index, spontaneous curvature, hemoglobin concentration and content.

Blood compatibility evaluations of poly(ethylene glycol)-poly(lactic acid) copolymers.

Poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) copolymers have been widely used for various biomedical applications. However, their hemocompatibility has not been clarified, which would lag their developments and clinical applications. In this work, we studied the effect of PEG-PLA copolymers on key human blood components in terms of their structure and bio-functions, including morphology and lysis of red blood cells, fibrinogen structure and conformation, and plasma and blood coagulation. To elucidate a structure-activity relationship, we used diblock PEG-PLA copolymers with different molecular weight, PEG(5 kDa)-PLA(25 kDa) and PEG(2 kDa)-PLA(2 kDa), abbreviated as PEG5k-PLA25k and PEG2k-PLA2k, respectively. The results show that the PEG-PLA copolymers at the concentration range studied in this work neither caused morphological alteration and lysis of red blood cells nor affected the oxygen delivery function and fibrinogen conformation. PEG5k-PLA25k from 10 to 100 mg/mL and PEG2k-PLA2k from 1.5 to 5 mg/mL disturbed the local microenvironments of fibrinogen molecules. PEG5k-PLA25k at up to 0.1 mg/mL did not interfere in the coagulation process of plasma or whole blood, while PEG2k-PLA2k from 0.1 mg/mL significantly interfered in the intrinsic plasma coagulation pathway and impaired whole blood coagulation. The results provide important information for the molecular design and clinical applications of PEG-PLA copolymers.

Specific features of red blood cell morphology in hemolytic disease neonates undergoing intrauterine intravascular blood transfusion

The paper presents data on the characteristics of red blood cell morphology in infants who have undergone intrauterine intravascular blood transfusion for hemolytic disease of the fetus. The infants are shown to have a reduction in the mean volume of red blood cells and in their mean level of hemoglobin, a decrease in the fraction of fetal hemoglobin and an increase in oxygen tension at half saturation. The above morphological characteristics of red blood cells remain decreased during the neonatal period after exchange transfusion or others, as clinically indicated, which seems to suggest that the compensatory-adaptive mechanisms to regulate hematopoiesis are exhausted and a donor’s red blood cells continue to be predominant.

Activity of essential oils of Piper aduncum anf and Cinnamomum zeylanicum by evaluating osmotic and morphologic fragility of erythrocytes

IntroductionFree radicals generated in vivo and peroxidation of the unsaturated bonds of membrane lipids increases fragility and cellular lysis of red blood cells. The essential oils of Piper aduncum and Cinnamomum zeylanicum present antioxidant activity and are popular in some countries to treat gastrointestinal diseases and anxiolytic symptoms respectively. Osmotic fragility assays and morphometric analyses have been used to assess cytotoxic effects and to verify the interaction of compounds present in the essential oils using the membranes of red blood cells (RBC). The aim of this work was to evaluate the effects of the essential oils of P. aduncum and C. zeylanicum on osmotic fragility using osmotic stress and morphology of RBC. MethodologyThe essential oils were analysed by GC–MS. Blood samples were treated with essential oils in different concentrations; osmotic fragility was determined using saline stress assays and morphological analyses were carried out using light microscopy. ResultsIn the presence of the oils, the data obtained indicated (i) a significant increase in hemolysis and (ii) modifications to the morphology of RBC with consequent structural modifications, increasing the permeability and diffusion of essential oils and provoking toxic effects over the red blood cells membranes. ConclusionIn conclusion, the presence of essential oils from the leaves from P. aduncum and the bark of C. zeylanicum, significantly increased the osmotic fragility curve of red blood cells, and inducing toxic effects on red blood cell membranes.

Anisotropic diffusion in stretched hydrogels containing erythrocytes: evidence of cell-shape distortion recorded by PGSE NMR spectroscopy.

The remarkable flexibility of human red blood cells (RBCs) allows them to assume a range of shapes in normal and disease states. Biochemical mechanisms and energetic requirements associated with changes in RBC geometry are not well understood because of a lack of experimental procedures to fix and study cells in different morphological forms. By incorporating RBCs into stretchable gelatin hydrogels, we created conditions for adjustable elongation of their normal discocytic shape in all orientations. As the RBC-containing gels were stretched or compressed, the changes in the cell morphology were studied by using 1 H-PGSE-NMR spectroscopy. Measurements of the apparent diffusion coefficient of water along the three orthogonal directions revealed tuneable anisotropy in the environment of the hydrogel samples. Light microscopy was also used for recording the extent to which RBCs were distorted in a stretched gel that had been set around them. Having demonstrated the applicability of NMR diffusometry to detect morphological changes of immobilised cells, we have laid the groundwork for future investigations of controllably distorted RBCs. Specifically, we expect studies of metabolic and biophysical properties of the physically deformed cells, thus inferring the connection between intracellular physico-chemical processes and RBC morphology. Copyright © 2016 John Wiley & Sons, Ltd.

In vitro alteration of hematological parameters and blood viscosity by the perfluorocarbon: Oxycyte

While perfluorocarbons (PFCs) may be useful in some clinical situations, previous studies have shown that interferences with chemistry analytes can occur with blood samples containing PFCs. This in vitro study focused on how the PFC Oxycyte may affect hematology measurements in blood samples. Swine blood diluted with Oxycyte or saline (Controls) were analyzed for Hemoglobin (Hb), Mean Corpuscular Volume (MCV),Hematocrit (Hct) and Fluorocrit (Fct) using a HemaVet, ABL-735 (ABL), or microhematocrit. Ancillary tests (blood viscosity, electrolytes, cell counts, and red blood cell morphology) were performed secondarily. Increasing Oxycyte resulted in increases in MCV, Hct, and visible cell shape change and morphology vs. Effects correlated with lower sodium in Oxycyte samples vs. With increasing Oxycyte, Hb became higher than Controls or became unpredictable depending on the instrument (HemaVet or ABL, respectively). Fct was smaller than predicted and likely represented the heaviest components of Oxycyte. At ≥50 % Oxycyte, RBC hemolysis rendered further measurements impractical. Viscosity first increased then decreased with increasing Oxycyte, peaking at ~40 % Oxycyte. Hct, MCV, Hb, and RBC morphology may be affected by Oxycyte. These observations correlated with lower sodium and increasing Oxycyte, causing hemolysis at high Oxycyte concentrations. These changes were due to alterations in the blood samples in vitro and this should be considered when interpreting hematology parameters from in vivo studies.

Synthesis, characterisation and preliminary investigation of the haemocompatibility of polyethyleneimine-grafted carboxymethyl chitosan for gene delivery

The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. In the present study, carboxymethyl chitosan (CMCS) was prepared by chitosan (CS) alkalisation and carboxymethylation reactions. Then polyethyleneimine (PEI) was grafted to the backbone of CMCS by an amidation reaction. The CMCS–PEI copolymer showed strong complexation capability with DNA to form nanoparticles, and achieved lower cytotoxicity and higher transfection efficiency compared with PEI (25kDa) towards 293T and 3T3 cells. Moreover, the haemocompatibility of the CMCS–PEI copolymer was investigated through the aggregation, morphology and lysis of human red blood cells (RBCs), along with the impact on the clotting function with activated partial thromboplastin time (APTT), prothrombin time (PT) and thromboelastographic (TEG) assays. The results demonstrated that the CMCS–PEI copolymer with a concentration lower than 0.05mg/mL had little impact on the aggregation, morphology or lysis of RBCs, or on blood coagulation. Therefore, the copolymer may be a strong alternative candidate as an effective and safe non-viral vector.

Hemocompatibility evaluation in vitro of methoxy polyethyleneglycol-polycaprolactone copolymer solutions.

Amphiphilic block copolymer methoxy polyethyleneglycol-polycaprolactone (mPEG-PCL) has attracted interest in the biomedical field, due to its water solubility and biodegradability. Nevertheless, the blood safety of mPEG-PCL copolymers has not been investigated in detail. Because mPEG-PCL copolymers introduced in vivo would inevitably interact with blood tissue, an investigation of possible interactions of mPEG-PCL with key blood components is crucial. We studied the effects of two mPEG-PCL copolymer solutions on blood coagulation, the morphology and lysis of human red blood cells (RBCs), the structure of plasma fibrinogen, complement activation, and platelet aggregation. We found that higher concentrations of the mPEG-PCL copolymers impaired blood clotting, and the copolymers had little impact on the morphology or lysis of RBCs. From the spectroscopy results, the copolymers affected the local microstructure of fibrinogen. The copolymers significantly activated the complement system in a concentration-dependent way. At higher concentrations, the copolymers impaired platelet aggregation, which may have been mediated by an inhibition of the arachidonic acid pathway. These findings provide important information that may be useful for the molecular design and biomedical applications of mPEG-PCL copolymers. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 802-812, 2016.

Calculation of Intracellular Pressure of Red Blood Cells at Jaundice According to Atomic Force Microscopy Data

The present work is devoted to the analysis of three-dimensional data of atomic force microscopy for research of the morphology of red blood cells. In this paper we built a biomechanical model of the erythrocyte, which allowed calculating the intracellular pressure of erythrocyte based on data of atomic force microscopy. As a result, we obtained the dependence intracellular pressure on the morphology of red blood cell. We have proposed a method of estimating of intracellular pressure of erythrocytes based on numerical modeling and data of atomic force microscopy of erythrocytes scan, which involves a comparison of the experimental data with the results of numerical calculation. The method is applied to the data of atomic force microscopy of erythrocytes of experimental animals - dwarf domestic pigs with different degrees of obstructive jaundice and normal. It is shown that with increasing severity of the disease and the concentration of bilirubin in the blood there is an infringement erythrocyte membranes, by an average increasing their volume and intracellular pressure.

Understanding the roles of intrinsic disorder in subunits of hemoglobin and the disease process of sickle cell anemia.

One of the common genetic disorders is sickle cell anemia, in which 2 recessive alleles must meet to allow for destruction and alteration in the morphology of red blood cells. This usually leads to loss of proper binding of oxygen to hemoglobin and curved, sickle-shaped erythrocytes. The mutation causing this disease occurs in the 6th codon of the HBB gene encoding the hemoglobin subunit β (β-globin), a protein, serving as an integral part of the adult hemoglobin A (HbA), which is a heterotetramer of 2 α chains and 2 β chains that is responsible for binding to the oxygen in the blood. This mutation changes a charged glutamic acid to a hydrophobic valine residue and disrupts the tertiary structure and stability of the hemoglobin molecule. Since in the field of protein intrinsic disorder, charged and polar residues are typically considered as disorder promoting, in opposite to the order-promoting non-polar hydrophobic residues, in this study we attempted to answer a question if intrinsic disorder might have a role in the pathogenesis of sickle cell anemia. To this end, several disorder predictors were utilized to evaluate the presence of intrinsically disordered regions in all subunits of human hemoglobin: α, β, δ, ε, ζ, γ1, and γ2. Then, structural analysis was completed by using the SWISS-MODEL Repository to visualize the outputs of the disorder predictors. Finally, Uniprot STRING and D2P2 were used to determine biochemical interactome and protein partners for each hemoglobin subunit along with analyzing their posttranslational modifications. All these properties were used to determine any differences between the 6 different types of subunits of hemoglobin and to correlate the mutation leading to sickle cell anemia with intrinsic disorder propensity.

Visualizing red blood cell sickling and the effects of inhibition of sphingosine kinase 1 using soft X-ray tomography.

Sickle cell disease is a destructive genetic disorder characterized by the formation of fibrils of deoxygenated hemoglobin, leading to the red blood cell (RBC) morphology changes that underlie the clinical manifestations of this disease. Using cryogenic soft X-ray tomography (SXT), we characterized the morphology of sickled RBCs in terms of volume and the number of protrusions per cell. We were able to identify statistically a relationship between the number of protrusions and the volume of the cell, which is known to correlate to the severity of sickling. This structural polymorphism allows for the classification of the stages of the sickling process. Recent studies have shown that elevated sphingosine kinase 1 (Sphk1)-mediated sphingosine 1-phosphate production contributes to sickling. Here, we further demonstrate that compound 5C, an inhibitor of Sphk1, has anti-sickling properties. Additionally, the variation in cellular morphology upon treatment suggests that this drug acts to delay the sickling process. SXT is an effective tool that can be used to identify the morphology of the sickling process and assess the effectiveness of potential therapeutics.

Novel triphosphorylation polyurethane nanoparticles for blood-contacting biomaterials' coating.

Improving hemocompatibility of biomaterials and devices contacting the human blood has been the subject of intensive research. In this study, we synthesized a novel excellent blood compatible polyurethane/sodium triphosphate nanoparticle (PU/STPP). Characterization of polyurethane/sodium triphosphate (PU/STPP) nanoparticles was carried out by transmission electron microscopy (TEM), scanning electron microscopy (SEM), dynamic light scattering (DLS), nuclear magnetic resonance (NMR), and energy dispersive spectroscopy (EDS). Blood compatibility assessment of PU/STPP nanoparticles was performed by in vitro coagulation time, plasma clotting time, hemolysis rate, and red blood cell morphology tests. Cell compatibility evaluations of PU/STPP nanoparticles were obtained by MTT cell viability tests. The PU/STPP nanoparticles also were used to modify vascular prostheses with cosedimentation. Platelet adhesion tests showed that blood compatibility of vascular prostheses coated with PU/STPP nanoparticles is better than that of pure vascular prostheses.

The Spectrum of Alpha-Spectrin Associated Hereditary Spherocytosis

▪Hereditary spherocytosis (HS) is a genetically and phenotypically heterogeneous hemolytic anemia caused by deficiency in red blood cell (RBC) cytoskeleton proteins leading to disruptions in the vertical association of the cytoskeleton with the RBC lipid bilayer. Monoallelic mutations in the genes encoding ankyrin (ANK1), beta-spectrin (SPTB) and band 3 (SLC4A1) or biallelic mutations in the genes encoding alpha-spectrin (SPTA1), ankyrin, and protein 4.2 (EPB42) result in HS. Autosomal recessive HS due to compound heterozygous defects in SPTA1 is typically severe and diagnosis based on phenotypic assays like RBC morphology, osmotic fragility or ektacytometry is complicated by transfusion dependence resulting in most of the circulating RBCs to be of donor origin. We have developed a rapid comprehensive next-generation sequencing-based assay that evaluates 27 genes with published disease-causing mutations for RBC cytoskeletal disorders, enzymopathies, and CDAs. We describe here patients with hemolytic anemia due to SPTA1 mutations, identified utilizing this assay, and their phenotype-genotype correlation. Each of these cases, when possible, has been also evaluated with ektacytometry and immunoblotting of RBC ghosts for alpha-spectrin quantitation.Wichterle et al in 1996 had estimated that alphaLEPRA(Low Expression PRAgue) mutation (c.4339-99C>T) occurs in SPTA1 gene in about 5% of Caucasians. This mutation leads to activation of an alternate acceptor splice site at position -70 of intron 30, causing frame shift and premature termination, thereby leading to decrease in alpha-spectrin production in this allele to about 16% of normal. We have found a cohort of three transfusion-dependent hereditary hemolytic anemia cases where a nonsense mutation in SPTA1 gene has occurred in trans to alphaLEPRA mutation, resulting in premature termination (see Table 1). Transfusion dependence was alleviated in two of these patients after splenectomy; the third one did not have splenectomy yet. RBC phenotype explored after splenectomy revealed an ektacytometry curve indicating spherocytosis (Figure 1A) and severely decreased alpha-spectrin on immunoblotting along with significant decrease of the associated beta-spectrin (Figure 1B).A patient with moderately severe form of HS, maintaining a hemoglobin value greater than 7 g/dL and requiring only occasional transfusions during periods of illness or stress, was found to have alphaLEPRA occurring in trans to an intronic splicing mutation c.1351-1G>TG where there is substitution at nucleotide-1 of intron positioned between nucleotides 1350 and 1351 of the SPTA1 mRNA. This splicing mutation may allow for some expression of functional alpha-spectrin protein from this allele in contrast to no protein expression in the previous cases of premature termination. Alternatively, other gene mutations, not identified by the next-generation sequencing panel we used, may contribute to this patient's milder phenotype.A couple with history of two fetal losses associated with hydrops fetalis seeked genetic counseling and gave consent to have diagnostic evaluation of genes associated with non-immune hemolytic anemia using targeted next-generation sequencing. Results of the panel revealed a heterozygous frameshift SPTA1 mutation in each of the parents (c.4206delG in the father and c.4180delT in the mother). These mutations in compound heterozygous state in the offspring likely caused total absence of alpha spectrin and fatal hemolytic anemia by the time of birth.Hereditary Spherocytosis is characterized by wide phenotypic variability that will be better understood with studies of genotype-phenotype association. While complete absence of alpha-spectrin expression due to null mutations of both SPTA1 alleles is incompatible with life, a nonsense or splicing SPTA1 mutation in trans to an alphaLEPRA low expression allele causes severe or moderately severe recessive HS, respectively. Targeted next-generation sequencing can be an effective diagnostic tool particularly for patients requiring frequent transfusions that preclude meaningful phenotypical testing of their red blood cells. [Display omitted] [Display omitted] DisclosuresBegtrup:GeneDx: Employment.

Involvement of acetylcholinesterase and protein kinase C in the protective effect of caffeine against β-amyloid-induced alterations in red blood cells.

It is well known the role of oxidative stress in the pathophysiology of Alzheimer's disease (AD) and of other neurodegenerative pathologies. We have previously documented that Amyloid beta peptide (1-42) (Abeta) dependent-oxidative modifications affect red blood cell (RBC) morphology and function. Experimental studies show that caffeine (CF) consumption is inversely correlated with AD. In this study, we investigated the role played by RBC in the protective mechanism elicited by CF against Abeta mediated toxicity. PS exposure levels by FACS analysis, as well as protein band 3 functionality analysis, indicated that CF at 100μM protected against Abeta-mediated membrane alterations, which are known to occur in AD. Moreover, CF counteracts inhibition of ATP release from RBC by Abeta, restoring its ability to modulate vasodilation. Concurrently, analysis of protein kinase C (PKC) and caspase 3 activities, responsible for cytoskeleton alterations, revealed that unlike to caspase 3, PKCα activation induced by Abeta was fully abolished by CF through a mechanism involving Acetylcholinesterase (AChE), located on external face of RBC plasma membrane. These results provide support for the hypothesis concerning the protective role of CF in AD patients could include also a peripheral mechanism involving RBC.