Normal Erythrocytes Research Articles

Structure-activity relationship of heme and its analogues in membrane damage and inhibition of fusion.

Under pathological conditions, such as sickle cell disease and malaria, heme concentration increases considerably, and it induces membrane damage. As sickled and normal erythrocytes contain high cholesterol: phospholipid ratio, we investigated the role of lipid composition, chain length, and unsaturation on the partitioning and leakage of hemin in phospholipid vesicles. To establish structure-activity relationship in membrane damage, experiments with two other analogues, protoporphyrin-IX and hematoporphyrin (HP) were also carried out. Hemin and its analogues localize differently in membranes and exhibit distinct roles in partitioning, leakage and fusion. Hemin and HP trigger more leakage in the presence of aminophospholipids, whereas cholesterol buffers the destabilizing effect remarkably. Inhibition of fusion by hemin further suggests its unexplored and important role in membrane trafficking, particularly under diseased conditions.

Differentiation of human pluripotent stem cells into red blood cells

At present, the experimental technique to produce human red blood cells in vitro is complicated, and in order to optimize the induction steps, human pluripotent stem cells were differentiated into red blood cells through two induction steps. First, human pluripotent stem cells (including Rh negative A type umbilical cord mesenchymal stem cells (hUCMSCRh-A) and human iPS cells (hiPS)) were differentiated into CD31+ and CD34+ cells in BVF medium. PCR and flow cytometry were used to exam the expression of CD31 and CD34. We found that hUCMSCRh-A derived CD31+ and CD34+ cells were 5.3% and 22.7%, respectively; hiPS derived CD31+ and CD34+ cells were 31.2% and 8.2%, respectively. For the second induction step, the obtained CD31+ and CD34+ cells were differentiated into mature erythrocytes for 36 days under the addition of various growth factors. Through Giemsa staining, we found that the obtained mature erythrocytes were similar in morphology and size to normal human erythrocytes, and some obtained erythrocytes were enucleated. Globin expression was detected by real time RT-PCR, and the expression of β-globin was more than 20%. The obtained erythrocytes are collected into the centrifuge tube, and then erythrocytes were naturally settled and showed the red color. Our findings provide a novel and effective method for the quantity generation of human red blood cells in vitro.

Geometría fractal aplicada para comparar los espacios ocupados por eritrocitos normales y esferocitos

Objetivo: desarrollar una nueva metodología que evalúe la forma del eritrocito normal y el esferocito a partir de los espacios ocupados por cada uno de ellos, medido con el método de Box Counting. Materiales y métodos: se analizaron 20 imágenes de extendido de sangre periférica, en las cuales se observaron un gran número de eritrocitos normales y esferocitos. Se superpusieron dos rejillas Kp de 5 x 5 pixeles y Kg de 10 x 10 pixeles, para calcular el espacio ocupado por dos regiones del eritrocito normal las cuales son, el disco y centro de este visto de manera frontal, así como el espacio ocupado por el esferocito mediante el método de Box Counting. Resultados: los espacios ocupados por el exterior e interior del eritrocito normal con la rejilla Kp variaron entre 47 y 55, y entre 8 y 16, respectivamente. En cambio, los espacios ocupados por los esferocitos variaron entre 25 y 36. Las proporciones entre la superficie y los valores de la rejilla Kp, de las regiones del eritrocito normal variaron entre 9,250 y 32.24, y para los esferocitos variaron entre 34,63 y 71.39. Conclusiones: las alteraciones en la forma del eritrocito normal pueden ser diferenciadas mediante los rangos hallados para el espacio ocupado por la superficie y las proporciones S/Kp de cada una de las regiones del eritrocito a partir de las nociones de la geometría fractal y euclidiana.

Antibacterial, antibiofilm, hemolytic activity and phytochemical study of various aerial parts of Moringa oleifera in Pakistan

Theamethanolicoextractsaofffiveeaerial parts ofaMoringa oleifera Lamwwereascreened for their antibacterial, antibiofilm and hemolytic activity.”Theaphytochemical screening of the methanolic extracts of aerial parts was also evaluated. The antibacterial, antibiofilmiactivityiof the extractssof various aerial parts of M. oleifera was evaluated by disc diffusion and micro titer plate assay method against two selected bacterial species. Hemolyticaactivity of the methanoliciextracts was screened against normal human erythrocytes. Comparatively the methanolic extracts of all aerial parts were more effective against Gram positive bacteria Bacillus subtilis and Gram negative bacteria Escherichia coli (E. coli) .The present study suggests that all aerial parts of this plant can be used as an antibacterial, anti-biofilm agent in treating ailments caused by the tested organisms (Bacillus subtilis and Escherichia coli). Methanolic extracts of aerial parts possessed minimum hemolytic activity. Significant antimicrobial, antibiofilm activity of the extract was found in this study.Asian J. Med. Biol. Res. March 2018, 4(1): 77-85

An Unusual Myeloma Case with Normal Erythrocyte Sedimentation Rate and Serum Immunoglobulins

Multiple Myeloma (MM) is a disease characterized by the increase of plasma cells in bone marrow or in other tissues and increased production of abnormal immunoglobulin (Ig) or Ig parts. In this report, we aimed to present a case of myeloma whom showed in our clinic with complaints of diarrhea, vomiting, fatigue and difficulty in walking. A 68- year-old male patient presented with diarrhea, fatigue, nausea, vomiting, and loss of appetite. He had splenomegaly in physical examination. Laboratory test revealed erythrocyte sedimentation rate: 4 mm/h and corrected calcium: 11.1 mg/dl. In conclusion, despite it is rare, multiple myeloma case may present with normal erythrocyte sedimentation rate and serum immunoglobulin levels. Anemia, bone lesions and hypercalcemia in an elderly may prompt diagnostic effort for multiple myeloma.

Functional Characterization of Plasmodium falciparum Surface-Related Antigen as a Potential Blood-Stage Vaccine Target.

Plasmodium falciparum erythrocyte invasion is a multistep process that involves a spectrum of interactions that are not well characterized. We have characterized a 113-kDa immunogenic protein, PF3D7_1431400 (PF14_0293), that possesses coiled-coil structures. The protein is localized on the surfaces of both merozoites and gametocytes, hence the name Plasmodium falciparum surface-related antigen (PfSRA). The processed 32-kDa fragment of PfSRA binds normal human erythrocytes with different sensitivities to enzyme treatments. Temporal imaging from initial attachment to internalization of viable merozoites revealed that a fragment of PfSRA, along with PfMSP119, is internalized after invasion. Moreover, parasite growth inhibition assays showed that PfSRA P1 antibodies potently inhibited erythrocyte invasion of both sialic acid-dependent and -independent parasite strains. Also, immunoepidemiological studies show that malaria-infected populations have naturally acquired antibodies against PfSRA. Overall, the results demonstrate that PfSRA has the structural and functional characteristics of a very promising target for vaccine development.

Neocytolysis: How to Get Rid of the Extra Erythrocytes Formed by Stress Erythropoiesis Upon Descent From High Altitude.

Neocytolysis is the selective destruction of those erythrocytes that had been formed during stress-erythropoiesis in hypoxia in order to increase the oxygen transport capacity of blood. Neocytolysis likely aims at decreasing this excess amount of erythrocytes and hemoglobin (Hb) when it is not required anymore and to decrease blood viscosity. Neocytolysis seems to occur upon descent from high altitude. Similar processes seem to occur in microgravity, and are also discussed to mediate the replacement of erythrocytes containing fetal hemoglobin (HbF) with those having adult hemoglobin (HbA) after birth. This review will focus on hypoxia at high altitude. Hemoglobin concentration and total hemoglobin in blood increase by 20–50% depending on the altitude (i.e., the degree of hypoxia) and the duration of the sojourn. Upon return to normoxia hemoglobin concentration, hematocrit, and reticulocyte counts decrease faster than expected from inhibition of stress-erythropoiesis and normal erythrocyte destruction rates. In parallel, an increase in haptoglobin, bilirubin, and ferritin is observed, which serve as indirect markers of hemolysis and hemoglobin-breakdown. At the same time markers of progressing erythrocyte senescence appear even on reticulocytes. Unexpectedly, reticulocytes from hypoxic mice show decreased levels of the hypoxia-inducible factor HIF-1α and decreased activity of the BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), which results in elevated mitochondrial activity in these cells. Furthermore, hypoxia increases the expression of miR-21, which inhibits the expression of catalase and thus decreases one of the most important mechanisms protecting against oxygen free radicals in erythrocytes. This unleashes a series of events which likely explain neocytolysis, because upon re-oxygenation systemic and mitochondrial oxygen radical formation increases and causes the selective destruction of those erythrocytes having impaired anti-oxidant capacity.

Labile iron pool as a parameter to monitor iron overload and oxidative stress status in β-thalassemic erythrocytes.

Labile iron pool (LIP) is intracellular nonprotein bound iron that can generate oxygen radicals via the Fenton reaction resulting in oxidative cell damage. Therefore, quantitative measurement of LIP will be helpful for detecting and monitoring the toxic iron status in iron overloaded patients. This study demonstrated LIP level and its correlation to oxidative stress status in β-thalassemic erythrocytes. LIP and reactive oxygen species (ROS) level, numbers of erythrocyte vesicles and apoptosis were assayed by flow cytometric methods in 30 blood samples from β-thalassemia/hemoglobin E patients and 17 blood samples from healthy volunteers with normal hemoglobin type. β-thalassemic erythrocytes showed higher LIP level, defined as the difference in calcein fluorescent intensity of the cells treated with or without deferiprone, than normal erythrocytes (mean ± 2SD as 62.39 ± 39.58 versus 44.65 ± 35.86, P = 0.003). The LIP level above 67, a cutoff value of LIP level obtained from receiver operating characteristic curve analysis, had a significant positive correlation with oxidative stress status for ROS level (r = 0.90, P < 0.001) and also the amount of erythrocyte vesicles (r = 0.79, P = 0.002). In contrast, the LIP level showed a significant negative correlation with the patients' hemoglobin level (r = -0.66, P = 0.028). The LIP assay is suggested as an alternative test to monitor the magnitude of iron overload and its consequent oxidative stress in β-thalassemia. LIP level may also be used as a marker for therapeutic response to iron chelation treatment. © 2018 International Clinical Cytometry Society.

Case 1: A Rare Case of Antenatal Hemorrhagic Stroke and Recurrent Purpura Fulminans in a Preterm Newborn

A female infant is born prematurely at 33 weeks of gestation to consanguineous parents (parents are first cousins). The mother has had 2 uneventful pregnancies and both children are well. There is no history of recurrent miscarriages, stillbirth or neonatal stroke, or family history of bleeding diathesis or clotting disorders. Antenatal scans had revealed the fetus to be growing well until 30 weeks of gestation, when the weight dropped from the 40th to the 3rd percentile. At that time, the amniotic fluid volume, Doppler scan, and placental blood flow were normal. The mother had been asymptomatic, with normal blood cell count and erythrocyte sedimentation rate. Now at 33 weeks of gestation, the mother presents to the emergency department with reduced fetal movements over the previous week. She is admitted for observation. Antenatal ultrasonography confirms the earlier findings of intrauterine growth restriction and documented normal amniotic fluid volume and Doppler scan. She remains clinically well. However, cardiotocographic monitoring the next day reveals the presence of repeated decelerations suggestive of nonreassuring fetal status, prompting an urgent cesarean delivery. The infant is well at birth, and has a good cry. Apgar scores are 6 and 9 at 1 and 5 minutes, respectively. Birthweight is 1,160 g (<3rd percentile), length 39 cm (3rd-10th percentile) and head circumference 27.5 cm (3rd percentile). Soon after birth, the infant develops respiratory distress secondary to partially compensated metabolic acidosis (pH, 7.32; partial pressure of carbon dioxide, 19.5 mm Hg [2.6 kPa]; partial pressure of oxygen, 111 mm Hg [15 kPa]; base excess, −12; and bicarbonate, 14 mEq/L [14 mmol/L]). Clinical examination findings are unremarkable and vital signs are normal. No hypoglycemia or electrolyte abnormality is noted. Laboratory investigations on day 1 reveal thrombocytopenia (platelet count 39×103/μL [39×109/L]) and coagulopathy (prothrombin time 60 seconds, activated …

Prognostic factors in cancer of unknown primary site

Patients with cancer of an unknown primary site (CUP) usually have a poor outcome. The identification of prognostic factors that affect survival can help clinicians find a better approach to such cases in terms of diagnostic and therapeutic management. We conducted a retrospective study including the cases of CUP recorded at the University Hospital 12 de Octubre Tumor Registry between 1999 and 2003. CUP was diagnosed in 265 patients during the analyzed period. One hundred and seventy-one were men (64.5%) and the mean age of the patients was 66.9 years (range 32-98 years). The median survival was 2.5 months, and the survival rate was 35.1% 6 months from diagnosis (95% CI: 28.9-41.3) and 24.5% 1 year from diagnosis (95% CI: 18.7-30.3). Univariate analysis revealed as significant predictive variables of a better outcome age under 70 years; involvement of a single organ; normal serum levels of alkaline phosphatase and albumin; normal erythrocyte sedimentation rate; normal levels of the serum tumor markers CEA, CA 19.9 and CA 15.3; squamous carcinoma histology; clinical presentation as lymph node enlargement; and the administration of treatment. Multivariate analysis showed that albumin and alkaline phosphatase levels, squamous carcinoma histology, age and treatment were the most important prognostic factors. Other variables analyzed (liver, bone or lung involvement, lactate dehydrogenase levels, gender) did not affect survival. CUP has a poor prognosis. Some prognostic factors that affect survival in these patients, however, may be identified.

Phytosynthesis of Silver Nanoparticles: Characterization, Biocompatibility Studies, and Anticancer Activity.

Silver nanoparticles (SNPs), owing to their wide range of biomedical applications, have recently attracted remarkable interest for use in cancer nanomedicine. The present research work investigated the anticancer activity of phytosynthesized SNPs against human cancer cell lines. Phytosynthesis of SNPs was achieved by using an aqueous extract of Salacia chinensis (SC) bark as a green source to reduce silver nitrate to silver nanoparticles. Characterization of synthesized nanoparticles demonstrated a UV-visible peak at 443 nm, ζ-potential (zetasizer) of -25.6 ± 0.34 and particle size (transmission electron microscopy analysis) in the range of 40-80 nm, which validates formation of stable silver nanoparticles. The absence of cytotoxicity against normal human fibroblasts and blood erythrocytes confirms the biocompatible nature of green synthesized SNPs. In vitro anticancer assay demonstrated IC50 values of 6.31, 4.002, 5.228, 8.452, 14.37, 7.46, and 6.55 μg/mL against liver (Hep G2), lungs (L-132), pancreas (MIA-Pa-Ca-2), breast (MDA-MB-231), oral (KB cells), prostate (PC-3), and cervical (HeLa) cancer cell lines respectively, which confirms its potent anticancer action. The results of the present study give an experimental proof that the SC mediated green synthesized SNPs could serve as a promising anticancer agent to overcome limitations of existing conventional cancer chemotherapeutics.

Effect of inherited red cell defects on growth of Plasmodium falciparum: An in vitro study.

Background & objectives:High prevalence of certain polymorphic alleles of erythrocytes in malaria endemic area has been linked to the resistance provided by these alleles against parasitic infestations. Numerous studies undertaken to demonstrate this correlation have generated conflicting results. This study was undertaken to investigate the abilities of various polymorphic erythrocytes to support in vitro growth of Plasmodium falciparum parasites.Methods:In this study under in vitro condition the ability of P. falciparum parasites to grow was assessed in the erythrocytes obtained from a total of 40 patients with various haemoglobinopathies, such as β-thalassaemia (β-Thal), sickle cell anaemia, erythroenzymopathy-like glucose-6-phosphate dehydrogenase deficiency and membranopathy-like hereditary spherocytosis.Results:Significantly reduced in vitro invasion and growth of parasites was seen in the cultures containing abnormal erythrocytes than in control cultures containing normal erythrocytes (P< 0.05). The mean per cent parasitaemia comparison was also carried out among the three polymorphic erythrocyte groups, i.e. β-Thal, sickle cell anaemia and enzyme-membranopathies.Interpretation & conclusions:Erythroenzymopathies and membranopathies were found to provide a more hostile environment for parasites, as the least parasitaemia was observed in these erythrocytes. The present in vitro study showed that P. falciparum did not grow well and did not invade well in erythrocytes obtained from common inherited red cell disorders.

Features of 921 Patients With Spinal Tuberculosis: A 16-Year Investigation of a General Hospital in Southwest China.

Southwest China has a high burden of spinal tuberculosis (TB). Few large case studies of spinal TB in southwest China have been conducted. This study investigated the features of 921 patients who were treated for spinal TB at a general hospital in southwest China between 2001 and 2016. Demographic data, clinical data, laboratory data, imaging findings, treatment methods, and outcomes of patients who were admitted to the hospital for spinal TB were reviewed retrospectively. The annual incidence of spinal TB increased throughout the study period. The greatest number of patients were 41 to 50 years old (22.04%). Local pain was the most common symptom (97.8%). A significant difference in the duration of symptoms was observed between rural populations (28.40 months) and urban populations (10.17 months) (P=.041). Of the patients, 32.68% had a normal erythrocyte sedimentation rate and 25.84% had a normal C-reactive protein level. The lumbar spine was the most commonly involved spinal site (44.77%), followed by the thoracic spine (43.60%). All of the patients underwent chemotherapy, and 77.10% of patients underwent surgery. Most of the patients were cured, with the exception of 2.19% of patients who discontinued chemotherapy after early improvement of clinical status. Spinal TB is still a major public health problem in southwest China. Chemotherapy and surgery can yield satisfactory outcomes with timely diagnosis and long-term treatment. It is urgent to increase the attention paid to spinal TB and improve knowledge of this disease among the general public, especially in southwest China. [Orthopedics. 2017; 40(6):e1017-e1023.].

Cell Axis Ratio a Standardized Approach for Normal Erythrocyte Shape Determination in Fishes

Cell Axis Ratio a Standardized Approach for Normal Erythrocyte Shape Determination in Fishes

Lipidomic analysis of biological samples: Comparison of liquid chromatography, supercritical fluid chromatography and direct infusion mass spectrometry methods

Lipidomic analysis of biological samples in a clinical research represents challenging task for analytical methods given by the large number of samples and their extreme complexity. In this work, we compare direct infusion (DI) and chromatography – mass spectrometry (MS) lipidomic approaches represented by three analytical methods in terms of comprehensiveness, sample throughput, and validation results for the lipidomic analysis of biological samples represented by tumor tissue, surrounding normal tissue, plasma, and erythrocytes of kidney cancer patients. Methods are compared in one laboratory using the identical analytical protocol to ensure comparable conditions. Ultrahigh-performance liquid chromatography/MS (UHPLC/MS) method in hydrophilic interaction liquid chromatography mode and DI-MS method are used for this comparison as the most widely used methods for the lipidomic analysis together with ultrahigh-performance supercritical fluid chromatography/MS (UHPSFC/MS) method showing promising results in metabolomics analyses. The nontargeted analysis of pooled samples is performed using all tested methods and 610 lipid species within 23 lipid classes are identified. DI method provides the most comprehensive results due to identification of some polar lipid classes, which are not identified by UHPLC and UHPSFC methods. On the other hand, UHPSFC method provides an excellent sensitivity for less polar lipid classes and the highest sample throughput within 10min method time. The sample consumption of DI method is 125 times higher than for other methods, while only 40μL of organic solvent is used for one sample analysis compared to 3.5mL and 4.9mL in case of UHPLC and UHPSFC methods, respectively. Methods are validated for the quantitative lipidomic analysis of plasma samples with one internal standard for each lipid class. Results show applicability of all tested methods for the lipidomic analysis of biological samples depending on the analysis requirements.

Folate deficiency in patients seeking treatment of alcohol use disorder

IntroductionNutritional deficiency is frequent in patients with an alcohol use disorder (AUD). We aimed to analyze serum and erythrocyte folate deficiency in a case series of patients that initiated treatment of AUD. Patients and methodsA cross-sectional study in patients admitted for detoxification between 2007 and 2015 was performed. Sociodemographic characteristics, history of alcohol consumption, type of alcohol, and medical co-morbidity were assessed at admission. Blood samples for biochemistry and hematological parameters were collected at admission. Logistic regression models were used to establish predictors of folate deficiency. Results211 patients (79.1% men) were eligible; age at admission was 46 years [IQR:40-51], and the amount of alcohol consumption was of 160g/day [IQR:120-200]. Thirty four percent of patients had macrocytosis (MCV>100fL), 12.8% had anemia, 23% of cases presented with serum folate deficiency and 7% presented with erythrocyte folate deficiency. Most (69%) of the patients with serum folate deficiency had normal erythrocyte folate levels. In univariate analysis, macrocytosis (OR=3.4, 95%CI:1.7-6.6), alcohol-related liver disease (ARLD) (OR=2.5, 95%CI:1.0-6.1) and drinking alcoholic beverages other than beer (OR=3.3, 95%CI:1.5-7.3) were associated with folate deficiency. However, only macrocytosis was significantly associated with serum folate deficiency in multivariate analysis (OR=3.1, 95%CI:1.1-8.9). Macrocytosis (P<0.001), ARLD (P=0.01) and the type of alcohol consumption (P<0.001) were factors associated with erythrocyte folate deficiency in univariate analysis. In multivariate analysis only macrocytosis remained significantly associated to erythrocyte folate deficiency (P=0.037). ConclusionFolate deficiency is a relatively frequent finding in contemporary, middle-aged patients with AUD, and macrocytosis is significantly associated with the deficiency.

Apigenin-induced ABCC1-mediated efflux of glutathione from mature erythrocytes inhibits the proliferation of Plasmodium falciparum

Plasmodium falciparum infection of mature erythrocytes leads to heightened oxidative stress that is tolerated in normal erythrocytes but not in erythrocytes from sickle cell, β-thalassaemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency hosts. In this report, it was of interest to perturb the redox homeostasis of normal erythrocytes through drug-induced active efflux of glutathione via erythrocyte ABCC1, a member of the C-subfamily of the human ATP-binding cassette (ABC) transporters. To achieve this objective, we made use of apigenin (API), shown previously to activate ABCC1 glutathione efflux in mature erythrocytes. Our results show that API increased ABCC1-mediated efflux of calcein-AM from uninfected erythrocytes, which was reversed with MK571, an inhibitor of ABCC1 drug efflux. Similarly, addition of API to uninfected normal erythrocytes led to a dramatic increase in reactive oxygen species coupled with a significant decrease in intracellular glutathione. Moreover, using P. falciparum-infected normal erythrocytes, we demonstrate that increasing concentrations of API inhibited the proliferation of chloroquine-susceptible (3D7) and -resistant (Dd2) parasites in culture with similar 50% inhibitory concentration (IC50) values (36.02 ± 2.4 µM and 34.45 ± 2.4 µM, respectively). Interestingly, the presence of API (25 µM) led to a three-fold decrease in the IC50 of artemisinin compared with artemisinin alone (2.8 ± 0.7 nM vs. 7.09 ± 1.5 nM, respectively). Taken together, the findings of this study demonstrate the feasibility of our proposed approach for the development of novel antimalarials by modulating host protein functions that leads to heightened oxidative stress in P. falciparum-infected erythrocytes and inhibits parasite proliferation.

Nueva metodología geométrica para evaluar la morfología del eritrocito normal

Objetivo. Desarrollar una nueva metodología para caracterizar la estructura del eritrocito normal mediante el espacio ocupado por el anillo del eritrocito normal caracterizado con el método de Box Counting. Método. se analizaron las imágenes de 20 extendidos de sangre periférica, cuyos eritrocitos fueron evaluados por un experto como normales. Se superpusieron dos rejillas Kp de 5 x 5 pixeles y Kg de 10 x 10 pixeles, para calcular el espacio ocupado por dos regiones del eritrocito estos son, el disco y centro de este, visto de manera frontal mediante el método de Box Counting. Resultados. Los espacios ocupados por la región del disco con la rejilla Kp variaron entre 47 y 56, la región del centro del eritrocito, varió entre 9 y 14. La dimensión fractal de estas dos regiones varió entre 0,941 y 1,115 para el disco, entre 0,652 y 1,222 para el centro. Conclusiones. La estructura del eritrocito normal puede ser caracterizada mediante el espacio ocupado por cada una de las regiones del eritrocito a partir de la geometría fractal.

Stasis Promotes Erythrocyte Adhesion to von Willebrand Factor.

Venous thromboembolism is a major contributor to global disease burden. Leukocytes and platelets initiate thrombogenesis on blood stasis and initiate the formation of a fibrin, VWF (von Willebrand factor), and neutrophil extracellular trap scaffold for erythrocytes. However, there is little knowledge on how erythrocytes become stably incorporated into this scaffold. Recently, we described the adhesion of calcium-loaded erythrocytes to endothelial-derived VWF strings. Because VWF is part of the scaffold of venous thrombi, we questioned whether reduced flow or stasis promotes the adhesion of normal erythrocytes to VWF and whether venous thrombi show evidence of erythrocyte-VWF interactions. In the present work, we perfused, under controlled shear conditions, washed, normal erythrocytes over surface-immobilized plasma and extracellular matrix proteins and showed that normal erythrocytes specifically bind to VWF. The interaction between erythrocytes and VWF significantly increased when the wall shear stress was reduced. Next, we investigated whether erythrocyte-VWF interactions support the structure of venous thrombi. High-resolution immunofluorescence imaging of human venous thrombi showed a striking pattern between erythrocytes, VWF, and fibrin, which suggests that VWF plays a supporting role, linking erythrocytes to fibrin in the thrombus. Our data suggest that erythrocyte retention in venous thrombi is mediated by erythrocyte-VWF or erythrocyte-VWF-fibrin interactions. Targeting erythrocyte retention could be a new strategy in the treatment or prevention of venous thrombosis.

P29 The in vitro use of natural antioxidant oils (Cyperus esculentus; Nigella sativa) to reduce cell sickling in sickle cell anaemia

Sickle cell anaemia is a genetically inherited red blood cell disorder, currently treated with time-consuming hospital treatments such as blood transfusions, which carry side effects such as iron overload. Sickle patients suffer from painful vaso-occlusive crises, whereby sickle erythrocytes adhere to blood vessel endothelium and block the natural flow of blood. A factor that exacerbates sickling is the low level of antioxidants in sickle cells, along with an increase in reactive oxygen species (ROS) generation. Tiger nut oil ( Cyperus esculentus ) and black seed oil ( Nigella sativa ) are natural products that have been anecdotally said to reduce crises in sickle cell sufferers, through their antioxidant properties. This research focuses on the potential reversal of sickle cells into normal spherical erythrocytes following treatment with these antioxidant oils. Morphological observation, as well as monitoring the potential reduction of ROS/increase in antioxidant presence, will be used to assess the treatment efficacy. The benefits of these natural oils as supplementary treatments for sickle cell patients include their accessibility to vast populations globally, as well as minimal risk of side effects. Children in particular would benefit as a decrease in cell sickling would require fewer hospital visits and ultimately would lead to improved school attendance. It was found that the oil treatments resulted in an increase in the antioxidant presence of sickle cell samples when tested in vitro , as well as a morphological decrease of sickle cells and increase of spherical erythrocytes. In vivo testing will be the next point of focus within this research.