Erythrocyte Morphology Research Articles

Anti-erythrocyte antibodies may contribute to anaemia in Plasmodium vivax malaria by decreasing red blood cell deformability and increasing erythrophagocytosis.

BackgroundPlasmodium vivax accounts for the majority of human malaria infections outside Africa and is being increasingly associated in fatal outcomes with anaemia as one of the major complications. One of the causes of malarial anaemia is the augmented removal of circulating non-infected red blood cells (nRBCs), an issue not yet fully understood. High levels of auto-antibodies against RBCs have been associated with severe anaemia and reduced survival of nRBCs in patients with falciparum malaria. Since there are no substantial data about the role of those antibodies in vivax malaria, this study was designed to determine whether or not auto-antibodies against erythrocytes are involved in nRBC clearance. Moreover, the possible immune mechanisms elicited by them that may be associated to induce anaemia in P. vivax infection was investigated.MethodsConcentrations of total IgG were determined by sandwich ELISA in sera from clinically well-defined groups of P. vivax-infected patients with or without anaemia and in healthy controls never exposed to malaria, whereas the levels of specific IgG to nRBCs were determined by cell-ELISA. Erythrophagocytosis assay was used to investigate the ability of IgGs purified from each studied pooled sera in enhancing nRBC in vitro clearance by THP-1 macrophages. Defocusing microscopy was employed to measure the biomechanical modifications of individual nRBCs opsonized by IgGs purified from each group.ResultsAnaemic patients had higher levels of total and specific anti-RBC antibodies in comparison to the non-anaemic ones. Opsonization with purified IgG from anaemic patients significantly enhanced RBCs in vitro phagocytosis by THP-1 macrophages. Auto-antibodies purified from anaemic patients decreased the nRBC dynamic membrane fluctuations suggesting a possible participation of such antibodies in the perturbation of erythrocyte flexibility and morphology integrity maintenance.ConclusionsThese findings revealed that vivax-infected patients with anaemia have increased levels of IgG auto-antibodies against nRBCs and that their deposition on the surface of non-infected erythrocytes decreases their deformability, which, in turn, may enhance nRBC clearance by phagocytes, contributing to the anaemic outcome. These data provide insights into the immune mechanisms associated with vivax malaria anaemia and may be important to the development of new therapy and vaccine strategies.

Gene disruption of dematin causes precipitous loss of erythrocyte membrane stability and severe hemolytic anemia

AbstractDematin is a relatively low abundance actin binding and bundling protein associated with the spectrin–actin junctions of mature erythrocytes. Primary structure of dematin includes a loosely folded core domain and a compact headpiece domain that was originally identified in villin. Dematin's actin binding properties are regulated by phosphorylation of its headpiece domain by cyclic adenosine monophosphate–dependent protein kinase. Here, we used a novel gene disruption strategy to generate the whole body dematin gene knockout mouse model (FLKO). FLKO mice, while born at a normal Mendelian ratio, developed severe anemia and exhibited profound aberrations of erythrocyte morphology and membrane stability. Having no apparent effect on primitive erythropoiesis, FLKO mice show significant enhancement of erythroblast enucleation during definitive erythropoiesis. Using membrane protein analysis, domain mapping, electron microscopy, and dynamic deformability measurements, we investigated the mechanism of membrane instability in FLKO erythrocytes. Although many membrane and cytoskeletal proteins remained at their normal levels, the major peripheral membrane proteins spectrin, adducin, and actin were greatly reduced in FLKO erythrocytes. Our results demonstrate that dematin plays a critical role in maintaining the fundamental properties of the membrane cytoskeleton complex.

THE NANOSTRUCTURE OF ERYTHROCYTE MEMBRANES UNDER BLOOD INTOXICATION: AN ATOMIC FORCE MICROSCOPY STUDY

Актуальность. Действие токсинов на нано- структуру мембран клеток крови – одна из ключевых проблем биофизики и медицины. Главными показателями качества крови признаны морфология и структура мембран эритроцитов. В этой связи актуальной задачей представляется анализ дефектов мембран при действии токсинов. Цель – выявить характерные особенности наноструктуры мембран и установить закономерности ее изменения, возникающие при интоксикации гемином и при длительном хранении эритроцитарной взвеси. Материал и методы. Исследования проводили in vitro на цельной крови человека, в которую добавляли гемин, и на эритроцитарной взвеси с гемокон- сервантом CPD, которую хранили при 4 °С в течение 30 суток. Наноструктуру мембран эритроцитов изучали с помощью атомной силовой микроскопии. Результаты. Характерные размеры пространственных периодов между «зернами» составили 120–200 нм. Количество «зерен» в области топологического дефекта варьировало от 4–5 до нескольких десятков. Такие домены возникали практически на всех клетках эритроцитарной взвеси, а также при воздействии гемина на кровь. При повышении интоксикации гемином и при увеличении срока хранения возрастало количество эхиноцитов, которые впоследствии трансформировались в сфероэхиноциты. При воздействии гемином и при хранении эритроцитарной взвеси на 9–12-е сутки наблюдалось специфическое изменение наноструктуры мембран красных клеток крови. Образовывались структурные кластеры – домены, в которых проявлялась зернистая структура. Заключение. Экспериментально установлено, что гемин и окислительные процессы в крови оказывают специфическое воздействие на наноструктуру мембран эритроцитов, образуя домены на поверхности. Характерный размер зернистых структур в доменах составляет 100–200 нм, что совпадает с характерным размером спектринового матрикса. Данные результаты можно применять в фундаментальной и практической медицине, при проведении гемотрансфузии, при анализе действия токсина на организм человека. Биофизические механизмы образования доменов могут лечь в основу изучения взаимодействия токсинов с мембранами.

Measuring Erythrocyte Morphology by Electron Microscopy to Diagnose Hematuria

We have used scanning electron microscopy to study erythrocyte morphology in patients with hematuria. We have developed a procedure for studying cells and nanoparticles without sputter deposition of conductive coatings. In IgA nephropathy and nephrotic syndrome, nanosized particles have been observed on the surface of the erythrocytes, as well as dysmorphic erythrocytes and crystals.

Morphology and Phenotype of Peripheral Erythrocytes of Fish: A Rapid Screening of Images by Using Software

Many information of biological study as stained cells analysis under microscope cannot be obtained rich information like detail morphology, shape, size, proper intensity etc. but image analysis software can easily be detected all these parameters within short duration. The cells types can be yeast cells to mammalian cells. An attempt has been made to detect cellular abnormalities from an image of metronidazole (MTZ) treated compared to control images of peripheral erythrocytes of fish by using non-commercial, open-source, CellProfiler (CP) image analysis software (Ver. 2.1.0). The comparative results were obtained after analysis the software. In conclusion, this image based screening of Giemsa stained fish erythrocytes can be a suitable tool in biological research for primary toxicity prediction at DNA level alongwith cellular phenotypes. Moreover, still suggestions are needed in relation to accuracy of present analysis for Giemsa stained fish erythrocytes because previous works have been carried out images of cells with fluorescence dye.

Morphology and Phenotype of Peripheral Erythrocytes of Fish: A Rapid Screening of Images by Using Software

Many information of biological study as stained cells analysis under microscope cannot be obtained rich information like detail morphology, shape, size, proper intensity etc. but image analysis software can easily be detected all these parameters within short duration. The cells types can be yeast cells to mammalian cells. An attempt has been made to detect cellular abnormalities from an image of metronidazole (MTZ) treated compared to control images of peripheral erythrocytes of fish by using non-commercial, open-source, CellProfiler (CP) image analysis software (Ver. 2.1.0). The comparative results were obtained after analysis the software. In conclusion, this image based screening of Giemsa stained fish erythrocytes can be a suitable tool in biological research for primary toxicity prediction at DNA level alongwith cellular phenotypes. Moreover, still suggestions are needed in relation to accuracy of present analysis for Giemsa stained fish erythrocytes because previous works have been carried out images of cells with fluorescence dye.

Atomic force microscopy of the erythrocyte membrane in obstructive jaundice

The erythrocyte intracellular pressure was calculated using a biomechanical model of the erythrocyte and atomic-force-microscopy (AFM) data. The intracellular pressure was characterized as a function of the model erythrocyte morphology. Based on numerical modeling and data of erythrocyte imaging by AFM, a method was developed to estimate the erythrocyte intracellular pressure by comparing experimental data and the results of numerical calculations. Calculations were performed for erythrocytes of dwarf domestic pigs with and without obstructive jaundice that varied in severity. The erythrocyte membrane was affected with increasing disease severity and blood bilirubin concentration, i.e., the erythrocyte volume increased on average, and substantial changes were observed for erythrocyte intracellular pressure relative to its normal value. As an example, an increase in bilirubin concentration from 5 to 96 μmol/L was associated with an increase in intracellular pressure from 0 to 2.2 kPa. An examination of the erythrocyte membrane surface by high-resolution AMF showed that the membrane is disrupted with an increase in bilirubin concentration and displays lesions and an increasing rupture length.

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

Is postoperative encephalopathy with choreoathetosis an acquired form of neuroacanthocytosis?

Postoperative encephalopathy with choreoathetosis (“postpump chorea”) is a rare complication of open-heart surgery and, in particular, the employment of a cardiopulmonary bypass pump. It almost exclusively occurs in young children. While risk factors and the underlying histopathology have been identified, the pathogenesis of postpump chorea, crucially, remains largely unknown. Transient cerebral hypoperfusion associated with cardiopulmonary bypass is considered a likely candidate mechanism, but the evidence is insufficient and inconclusive. It is hypothesized in this article, that postpump chorea may be caused by mechanical trauma to red blood cells and resulting acanthocytosis. These dysfunctional erythrocytes could then lead to damage to the globus pallidus and disease development akin to that presumed in neuroacanthocytosis. In patients with neuroacanthocytosis an association between acanthocytosis and basal ganglia pathology has been suggested. To test the mechanism hypothesized here, the effects of cardiopulmonary bypass on erythrocyte morphology and function could be systematically tested in children undergoing cardiac surgery. Ideally, the extent of erythrocyte damage could be correlated with the risk of developing postpump chorea. Finally, if the proposed hypothesis is supported by empirical findings, efforts to reduce blood cell damage during extracorporeal circulation in children might prevent this devastating complication.

Antioxidant activities of novel galloyl phytosterols evaluated by human erythrocytes with the aid of confocal microscopy imaging

Notable protective effect of the novel galloyl phytosterol antioxidant on human erythrocytes was disclosed in this study. 2,2′-Azo-bis (2-amidinopropane) hydrochloride (AAPH) induced haemolysis of erythrocytes, and depletion of cellular glutathione were effectively inhibited by the galloyl phytosterol antioxidant. It was revealed that the level of glutathione was a more sensitive indicator than haemolysis for cellular oxidative stress. The morphology of erythrocytes obtained through laser scanning confocal microscopy (LSCM) imaging provided structural details for haemolysis and glutathione investigation and was more sensitive than the level of glutathione. The liposome model exhibited that the novel antioxidant preferred to anchor in the membrane and had better performance for membrane protection than gallic acid. The strategy of integrated use of biochemical and visual methods developed in this study is valuable for investigation on mechanistic behaviours of antioxidants.

Evaluating the morphology of erythrocyte population: An approach based on atomic force microscopy and flow cytometry

Erythrocyte morphology is gaining importance as a powerful pathological index in identifying the severity of any blood related disease. However, the existing technique of quantitative microscopy is highly time consuming and prone to personalized bias. On the other hand, relatively unexplored, complementary technique based on flow cytometry has not been standardized till date, particularly due to the lack of a proper morphological scoring scale. In this article, we have presented a new approach to formulate a non-empirical scoring scale based on membrane roughness (Rrms) data obtained from atomic force microscopy. Subsequently, the respective morphological quantifier of the whole erythrocyte population, commonly known as morphological index, was expressed as a function of highest correlated statistical parameters of scattered signal profiles generated by flow cytometry. Feed forward artificial neural network model with multilayer perceptron architecture was used to develop the intended functional form. High correlation coefficient (R2=0.95), even for model-formulation exclusive samples, clearly indicates the universal validity of the proposed model. Moreover, a direct pathological application of the proposed model has been illustrated in relation to patients, diagnosed to be suffering from a wide variety of cancer.

Could “red cell distribution width” predict COPD severity?

Background and objectivesErythrocyte morphology changes not only by primary hematological diseases but also by systemic inflammation, ineffective erythropoiesis and nutritional deficiencies. Red blood cell distribution width (RDW) is a parameter reflecting erythrocyte morphology. We aimed to investigate the relationship of RDW with chronic obstructive pulmonary disease (COPD) stages, BODE index and survival in COPD patients. MethodsMedical records of 385 COPD patients between July 2004 and November 2005 were studied retrospectively. Demographic features, BODE index factors and oxygen saturation were recorded. Survival analysis of all patients by 2014 was performed. Measured RDW values at the time of the inclusion were evaluated. ResultsMean age of the patients was 65.6±9.6 years. Distribution of the COPD stages of the patients were stage 1: 16%, stage 2: 52%, stage 3: 26%, stage 4: 6%. RDW was found significantly different between stages. The highest RDW was observed in the very severe stage (p<0.001). Median of BODE index was 1 (0–3). As the BODE index increased RDW also increased (p<0.001). When the patients were grouped according to the laboratory upper limit of RDW, survival rate was 31% in the RDW >14.3% group and 75% in the RDW <14.3% group. ConclusionThe variability in the size of circulating erythrocytes increases as the COPD severity progresses. Therefore, a simple and noninvasive test, such as RDW, might be used as a biomarker in the evaluation of the severity of COPD. At the same time, there seems to be a correlation between the survival of COPD patients and RDW.

Advances in Urine Microscopy.

Urine microscopy is an important tool for the diagnosis and management of several conditions affecting the kidneys and urinary tract. In this review, we describe the automated instruments, based either on flow cytometry or digitized microscopy, that are currently in use in large clinical laboratories. These tools allow the examination of large numbers of samples in short periods. We also discuss manual urinary microscopy commonly performed by nephrologists, which we encourage. After discussing the advantages of phase contrast microscopy over bright field microscopy, we describe the advancements of urine microscopy in various clinical conditions. These include persistent isolated microscopic hematuria (which can be classified as glomerular or nonglomerular on the basis of urinary erythrocyte morphology), drug- and toxin-related cystalluria (which can be a clue for the diagnosis of acute kidney injury associated with intrarenal crystal precipitation), and some inherited conditions (eg, adenine phosphoribosyltransferase deficiency, which is associated with 2,8-dihydroxyadenine crystalluria, and Fabry disease, which is characterized by unique urinary lamellated fatty particles). Finally, we describe the utility of identifying "decoy cells" and atypical malignant cells, which can be easily done with phase contrast microscopy in unfixed samples.

Serum Ferritin and Severity Scores in Sickle Cell Disease Patients in Nnewi (South East Nigeria)

Background: Sickle cell disease (SCD) patients have mechanisms that are thought to protect them more than apparently normal individuals from iron deficiency. However, evidence exists that in SCD, hypoferritinaemia may be more prevalent than hyperferitinaemia, especially in developing countries. Methods: Serum ferritin (SF) levels were measured - using an ELISA based kit (Biocheck, USA), and disease severity calculated in fifty- two asymptomatic steady state (ASS) SCD patients; who were iron chelation naive and both parameters correlated. Erythrocyte morphology and malaria parasitaemia were assessed, patients with parasitaemia were excluded. 64 apparently normal individuals in the same environment and socioeconomic group were also assessed as above and served as controls. Statistical analysis was done using SPSS version 20. Results were expressed

Role of Membrane Lipids in the Regulation of Erythrocytic Oxygen-Transport Function in Cardiovascular Diseases.

The composition and condition of membrane lipids, the morphology of erythrocytes, and hemoglobin distribution were explored with the help of laser interference microscopy (LIM) and Raman spectroscopy. It is shown that patients with cardiovascular diseases (CVD) have significant changes in the composition of their phospholipids and the fatty acids of membrane lipids. Furthermore, the microviscosity of the membranes and morphology of the erythrocytes are altered causing disordered oxygen transport by hemoglobin. Basic therapy carried out with the use of antiaggregants, statins, antianginals, beta-blockers, and calcium antagonists does not help to recover the morphofunctional properties of erythrocytes. Based on the results the authors assume that, for the relief of the ischemic crisis and further therapeutic treatment, it is necessary to include, in addition to cardiovascular disease medicines, medication that increases the ability of erythrocytes' hemoglobin to transport oxygen to the tissues. We assume that the use of LIM and Raman spectroscopy is advisable for early diagnosis of changes in the structure and functional state of erythrocytes when cardiovascular diseases develop.

White Rot Fungus (Pleurotus pulmonarius) Cultivated on Lead Contaminated Rice Straw Induced Haematotoxicity and Lead Accumulation in Liver and Kidney of Wistar Rats

White rot fungi (Pleurotus species) are edible mushrooms known for their ability to bioaccumulate contaminants including metals from the environment. There is scarcity of information on the toxic effects of the bioaccumulated metals on organisms at higher trophic levels. In this study, we investigated alterations in haematological indices and erythrocyte morphology and lead bioaccumulation potentials in liver and kidney of Wistar rats fed aqueous extract of Pleurotus pulmonarius cultivated on lead (0, 10, 100, 200, 500 and 1000 mg/L) contaminated rice straw substrate. After 8 weeks of cultivation, mushroom did not germinate in the 1000 mg/L Pb contaminated rice straw. 1 g of mushroom harvested from each of the remaining concentrations showed significant (p<0.001) increase in Pb concentration. Rats exposed to 0.5 mL of the contaminated mushroom extracts for 30 days, showed decreased leucocyte, erythrocyte, haematocrit, platelet and haemoglobin concentrations. Abnormal erythrocyte morphologies like acanthocytes, schizocytes and tear drop were significantly higher in the Pb treated mushroom fed rats than the control. The kidney and liver of treated rats showed significant (p<0.05) increase in Pb concentration with higher Pb bioaccumulation factor in the kidney than the liver. Also insignificant decrease in body and organ weight was observed in treated rats than the control. Pb accumulation in P. pulmonarius increased liver and kidney Pb bioaccumulation and induce alterations in haematological indices and erythrocyte morphology in rats. This poses public health threat to humans and other tertiary consumers foraging white rot fungi from metal contaminated sites.

In Vitro Protective Effect of Phikud Navakot Extraction on Erythrocyte.

Phikud Navakot (PN), Thai herbal remedy in National List of Essential Medicines, has been claimed to reduce many cardiovascular symptoms especially dizziness and fainting. Apart from blood supply, erythrocyte morphology, in both shape and size, is one of the main consideration factors in cardiovascular diseases and may be affected by vascular oxidative stress. However, little is known about antioxidative property of PN on erythrocyte to preserve red blood cell integrity. In this study, 1,000 μM hydrogen peroxide-induced oxidative stress was conducted on sheep erythrocyte. Three doses of PN (1, 0.5, and 0.25 mg/mL) and 10 μM of ascorbic acid were compared. The released hemoglobin absorbance was measured to demonstrate hemolysis. Electron microscopic and immunohistochemical studies were also performed to characterize dysmorphic erythrocyte and osmotic ability in relation to aquaporin- (AQP-) 1 expression, respectively. The results revealed that all doses of PN and ascorbic acid decreased the severity of dysmorphic erythrocyte, particularly echinocyte, acanthocyte, knizocyte, codocyte, clumping, and other malformations. However, the most effective was 0.5 mg/mL PN dosage. In addition, hydrostatic pressure may be increased in dysmorphic erythrocyte in association with AQP-1 upregulation. Our results demonstrated that PN composes antioxidative effect to maintain the integrity and osmotic ability on sheep erythrocyte.

Influence of Novosibirsk Terahertz free Electron Laser Radiation on the Morphology of Chicken Hepatocytes

The present report examines the impact of terahertz radiation of Novosibirsk free electron laser on the state of membrane and morphology of hepatocytes and erythrocytes in vitro. The aim of this work is a detailed study of the effects of terahertz laser radiation on cells, identification of the specific membrane effects, and comparison with similar effects to the erythrocytes.

Phase measurements of erythrocytes affected by metal ions with quantitative interferometric microscopy

Erythrocyte morphology is an important factor in disease diagnosis, however, traditional setups as microscopes and cytometers cannot provide enough quantitative information of cellular morphology for in-depth statistics and analysis. In order to capture variations of erythrocytes affected by metal ions, quantitative interferometric microscopy (QIM) is applied to monitor their morphology changes. Combined with phase retrieval and cell recognition, erythrocyte phase images, as well as phase area and volume, can be accurately and automatically obtained. The research proves that QIM is an effective tool in cellular observation and measurement.

A comprehensive analysis of membrane and morphology of erythrocytes from patients with glucose-6-phosphate dehydrogenase deficiency

Acute hemolytic anemia could be triggered by oxidative stress in the patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. However, the underlying hemolytic mechanism is unknown. To make clear the hemolytic mechanisms, a systematic study on membrane ultrastructure had been undertaken. A comprehensive method was used including atomic force microscopy, scanning electron microscopy, flow cytometer and fluorescence microscopy to analyze the membrane ultrastructure, externalized phosphatidylserine (PS), intracellular Ca(2+) concentration, morphology and the distributions of band 3 protein in G6PD deficient red blood cells (RBCs) after tert-butyl-hydroperoxide (t-BHP) oxidation. The results showed that erythrocyte shrinkage, annexin-V binding to externalized PS on the membrane of early-stage apoptotic cells, the increased membrane roughness and intracellular Ca(2+) concentration, as well as the change of distributions of band 3 protein in RBCs. Compared with the control RBCs, as the concentration of t-BHP up to 0.1mM, the membrane roughness of G6PD deficient RBCs showed significant difference (p<0.05) and as the concentration of t-BHP up to 0.3mM, externalized PS showed significant difference (p<0.05). Furthermore, the population types of RBCs showed dramatic difference between control groups and G6PD deficient groups. Oxidative stress induced more serious erythrocyte apoptosis and resulted in increased roughness of erythrocyte membrane and abnormal distributed band 3 protein in G6PD deficient RBCs. Echinocytes are the predominant abnormal erythrocyte shape occurring in the peripheral blood from patients with G6PD deficiency, which may shorten the RBCs lifespan. The results in the present study will give an increased understanding for the hemolytic mechanism of G6PD deficiency.