Changes in Human Erythrocyte Membrane Exposed to Aqueous and Ethanolic Extracts from Uncaria tomentosa

Uncaria tomentosa (Willd.) DC is a woody climber species originating from South and Central America that has been used in the therapy of asthma, rheumatism, hypertension, and blood purification. Our previous study showed that U. tomentosa extracts altered human erythrocyte shape, which could be due to incorporation of the compounds contained in extracts into the erythrocyte membrane. The aim of the present study was to determine how the compounds contained in U. tomentosa extracts incorporate into the human erythrocyte membrane. The study has assessed the effect of aqueous and ethanolic extracts from leaves and bark of U. tomentosa on the osmotic resistance of the human erythrocyte, the viscosity of erythrocyte interior, and the fluidity of erythrocyte plasma membrane. Human erythrocytes were incubated with the studied extracts in the concentrations of 100, 250, and 500 µg/mL for 2, 5, and 24 h. All extracts tested caused a decrease in erythrocyte membrane fluidity and increased erythrocyte osmotic sensitivity. The ethanolic extracts from the bark and leaves increased viscosity of the erythrocytes. The largest changes in the studied parameters were observed in the cells incubated with bark ethanolic extract. We consider that the compounds from U. tomentosa extracts mainly build into the outer, hydrophilic monolayer of the erythrocyte membrane, thus protecting the erythrocytes against the adverse effects of oxidative stress.

Cytoskeletal changes in Erythrocytes during storage in banked blood

Introduction: Erythrocytes have flexible, non-nucleated bi-concave shape with lipid bilayer cytoskeleton. Any alterations of erythrocyte shape make it susceptible to hemolysis. Blood for transfusion purpose is routinely stored in Citrate Phosphate Dextrose Adenine (CPDA-1) containing blood bags. During storage, blood undergoes an array of different morphological changes termed as “storage lesions” which makes it more fragile. This study was aimed to determine the structural and functional modifications in erythrocytes in CPDA-1 blood stored in local blood bank of KPK. Material & Methods: Blood from twenty healthy volunteer donors was taken and kept in CPDA-1 containing blood bags at Institute of Basic Medical Sciences (IBMS), Khyber Medical University (KMU). Hb-levels and Erythrocyte, Reticulocyte counts, Mechanical Fragility Index (MFI) and immunofluorescence staining for ankiyrin1 protein were performed on fresh blood samples. Samples for reticulocyte count was taken for 5 consecutive days while for the remaining parameters, blood was taken at 5 days interval till day 20th. The light and fluorescence micrographs were obtained accordingly and osmotic fragility tests were performed. Results: A significant mean reduction in erythrocyte counts and Hb-level was observed from day 0 to 20 (p=0.001), while MFI increased from day 0 to day 20 (12.88%±7.58 p=0.001). Reticulocyte count also decreased from day 0 up to day 5 (p=0.001). A weak association was observed between changes in MFI and erythrocyte morphology from day 0 to 20 (r=0.349), while the intensity and pattern of ankyrin1 protein expression appeared to change from day 10. Conclusion: Blood stored for a week has same properties as fresh blood, however, important structural alterations start to appear after the first week of storage and worsen with time. Therefore, to gain better transfusion results, blood stored for up to one week can safely be transfused.

A method for the approximate determination of microparticle amount and mass in biological mixtures using a fractionation process in a centrifugal force field

The paper presents a simplified method of estimating the amount and specific mass of microparticles in complex biological mixtures using the fractionation process in the field of centrifugal forces. In the presented method, using the heaviest blood cells – erythrocytes – as an example, a geometric model of a binary fraction in a borderline equilibrium state formed in the process of blood fractionation was used. In this model, an approximated shape of the erythrocyte and a sample normal distribution of the size and number of microparticles in the examined model fraction filled with erythrocytes were used. Based on publicly available blood data, it has been shown that it is possible to estimate the amount and specific weight of microparticles. The accuracy of such estimation generally depends on the precision of representation of shapes, the degree of filling the fraction with microparticles and on the individual quantitative and dimensional distribution of these particles in the examined fraction. The above conclusions, which determine the accuracy of the method presented, were verified in the paper with the use of rheological blood data, commonly available in literature for various types of measuring containers, which affect the level of filling of a given fraction with microparticles. Keywords: fractionation of biological mixtures, fractionation of blood components, erythrocyte model, erythrocyte shape, blood components

Oxidative Stress in Autistic Children Alters Erythrocyte Shape in the Absence of Quantitative Protein Alterations and of Loss of Membrane Phospholipid Asymmetry

Red blood cells (RBCs) from people affected by autism spectrum disorders (ASDs) are a target of oxidative stress. By scanning electron microscopy, we analyzed RBC morphology from 22 ASD children and show here that only 47.5 ± 3.33% of RBC displayed the typical biconcave shape, as opposed to 87.5 ± 1.3% (mean ± SD) of RBC from 21 sex- and age-matched healthy typically developing (TD) controls. Codocytes and star-shaped cells accounted for about 30% of all abnormally shaped ASD erythrocytes. RBC shape alterations were independent of the anticoagulant used (Na2-EDTA or heparin) and of different handling procedures preceding glutaraldehyde fixation, thus suggesting that they were not artefactual. Incubation for 24 h in the presence of antioxidants restored normal morphology in most erythrocytes from ASD patients. By Coomassie staining, as well as Western blotting analysis of relevant proteins playing a key role in the membrane-cytoskeleton organization, we were unable to find differences in RBC ghost composition between ASD and normal subjects. Phosphatidylserine (PS) exposure towards the extracellular membrane domain was examined in both basal and erythroptosis-inducing conditions. No differences were found between ASD and TD samples except when the aminophospholipid translocase was blocked by N-ethylmaleimide, upon which an increased amount of PS was found to face the outer membrane in RBC from ASD. These complex data are discussed in the light of the current understanding of the mode by which oxidative stress might affect erythrocyte shape in ASD and in other pathological conditions.

Morphometric variations between triploid and diploid Clarias gariepinus (Burchell, 1822)

Several scientific methods have been described in the identification of triploid fish. However, many of these methods are not applicable for routine management purposes due to their complexity and cost. In this study, the possibility of using morphological variation as a least cost and less complex method of distinguishing triploid and diploid African catfishClarias gariepinus(Burchell, 1822) was examined. Triploid catfish were produced by cold shock of fertilized eggs in 5°C for 20 mins (at approximately 3 mins after fertilization). The fish were incubated, hatched and raised for 3 months. Ploidy levels of the fish were then ascertained by observing the erythrocyte shape. Triploid erythrocyte was ellipsoidal in shape while diploid was round. Morphological characterization was then carried out on 100 samples each of triploid and diploid African catfish. Although significant differences were observed in many parameters, the principal morphometric difference between triploid and diploid African catfish could not be clearly distinguished. It was therefore concluded that morphological characteristics is not ideal for discriminating triploids and diploids of African catfish. The used of erythrocyte characteristics still remains the cheapest and relatively effective method for triploid and diploid determination in African catfish.

The Effect of Endogenous and Synthetic Estrogens on Whole Blood Clot Formation and Erythrocyte Structure

As erythrocyte and estrogens interact so closely and erythrocytes can indicate the healthiness of an individual, it is essential to investigate the effects of natural estrogens as well as synthetic estrogens on these cells. Whole blood samples were used for thromboelastography (TEG), light microscopy (LM), and scanning electron microscopy (SEM) investigation. Viscoelastic investigation with TEG revealed that estrogens affected the rate of clot formation without any significant effect on the strength or stability of the clot. Axial ratio analysis with LM showed a statistically significant increase in number of erythrocytes with decreased roundness. Morphological analysis with SEM confirmed the change in erythrocyte shape and revealed both ultrastructural membrane changes and erythrocyte interactions. As erythrocyte shape and membrane flexibility correlates to physiological functioning of these cells in circulation, these changes, indicative of possible eryptosis brought on by estrogens, when experienced by individuals with an underlying inflammatory or hematological illness, could impair erythrocyte functioning and even result in obstructions in circulation. In conclusion, we suggest that whole blood analysis with viscoelastic and morphological techniques could be used as assessment of the hematological healthiness of individuals using estrogens.

Effect of Lidocaine and Epinephrine on Human Erythrocyte Shape and Vesiculability of Blood Cells

The effect of local anesthetic composed of lidocaine and epinephrine on vesiculability of blood cells and erythrocyte shape was studied. Whole blood and plasma were incubated with lidocaine/epinephrine. Extracellular vesicles were isolated by centrifugation and washing and counted by flow cytometry. Lidocaine/epinephrine and each component alone were added to diluted blood. Shape changes were recorded by micrographs. An ensemble of captured frames was analyzed for populations of discocytes, echinocytes, and stomatocytes by using statistical methods. Incubation of whole blood and blood plasma with lidocaine/epinephrine considerably increased concentration of extracellular vesicles in isolates (for an average factor 3.4 in blood and 2.8 in plasma). Lidocaine/epinephrine caused change of erythrocyte shape from mainly discocytic to mainly stomatocytic (higher than 50%). Lidocaine alone had even stronger stomatocytic effect (the percent of stomatocytes was higher than 95%) while epinephrine had echinocytic effect (the percent of echinocytes was higher than 80%). The differences were highly statistically significantp<10-8with statistical powerP=1. Lidocaine/epinephrine induced regions of highly anisotropically curved regions indicating that lidocaine and epinephrine interact with erythrocyte membrane. It was concluded that lidocaine/epinephrine interacts with cell membranes and increases vesiculability of blood cellsin vitro.