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1.
J Biomed Opt ; 26(9)2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34590447

RESUMO

SIGNIFICANCE: We introduce a model for better calibration of the trapping force using an equal but oppositely directed drag force acting on a trapped red blood cell (RBC). We demonstrate this approach by studying RBCs' elastic properties from deidentified sickle cell anemia (SCA) and sickle cell trait (SCT) blood samples. AIM: A laser trapping (LT) force was formulated and analytically calculated in a cylindrical model. Using this trapping force relative percent difference, the maximum (longitudinal) and minimum (transverse) radius rate and stiffness were used to study the elasticity. APPROACH: The elastic property of SCA and SCT RBCs was analyzed using LT technique with computer controlled piezo-driven stage, in order to trap and stretch the RBCs. RESULTS: For all parameters, the results show that the SCT RBC samples have higher elastic property than the SCA RBCs. The higher rigidity in the SCA cell may be due to the lipid composition of the membrane, which was affected by the cholesterol concentration. CONCLUSIONS: By developing a theoretical model for different trapping forces, we have also studied the elasticity of RBCs in SCT (with hemoglobin type HbAS) and in SCA (with hemoglobin type HbSS). The results for the quantities describing the elasticity of the cells consistently showed that the RBCs in the SCT display lower rigidity and higher deformability than the RBCs with SCA.


Assuntos
Anemia Falciforme , Traço Falciforme , Contagem de Eritrócitos , Eritrócitos , Eritrócitos Anormais , Humanos
2.
Sci Rep ; 11(1): 15898, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354145

RESUMO

The YPEL family genes are highly conserved across a diverse range of eukaryotic organisms and thus potentially involved in essential cellular processes. Ypel4, one of five YPEL family gene orthologs in mouse and human, is highly and specifically expressed in late terminal erythroid differentiation (TED). In this study, we investigated the role of Ypel4 in murine erythropoiesis, providing for the first time an in-depth description of a Ypel4-null phenotype in vivo. We demonstrated that the Ypel4-null mice displayed a secondary polycythemia with macro- and reticulocytosis. While lack of Ypel4 did not affect steady-state TED in the bone marrow or spleen, the anemia-recovering capacity of Ypel4-null cells was diminished. Furthermore, Ypel4-null red blood cells (RBC) were cleared from the circulation at an increased rate, demonstrating an intrinsic defect of RBCs. Scanning electron micrographs revealed an ovalocytic morphology of Ypel4-null RBCs and functional testing confirmed reduced deformability. Even though Band 3 protein levels were shown to be reduced in Ypel4-null RBC membranes, we could not find support for a physical interaction between YPEL4 and the Band 3 protein. In conclusion, our findings provide crucial insights into the role of Ypel4 in preserving normal red cell membrane integrity.


Assuntos
Proteínas de Transporte/genética , Membrana Eritrocítica/fisiologia , Eritropoese/genética , Anemia/metabolismo , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Proteínas de Transporte/metabolismo , Membrana Eritrocítica/genética , Eritrócitos/metabolismo , Eritrócitos Anormais/metabolismo , Eritropoese/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Policitemia/genética , Baço
4.
Biorheology ; 58(1-2): 51-60, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34219708

RESUMO

BACKGROUND: Measurement of abnormal Red Blood Cell (RBC) deformability is a main indicator of Sickle Cell Anemia (SCA) and requires standardized quantification methods. Ektacytometry is commonly used to estimate the fraction of Sickled Cells (SCs) by measuring the deformability of RBCs from laser diffraction patterns under varying shear stress. In addition to estimations from model comparisons, use of maximum Elongation Index differences (ΔEImax) at different laser intensity levels was recently proposed for the estimation of SC fractions. OBJECTIVE: Implement a convolutional neural network to accurately estimate rigid-cell fraction and RBC concentration from laser diffraction patterns without using a theoretical model and eliminating the ektacytometer dependency for deformability measurements. METHODS: RBCs were collected from control patients. Rigid-cell fraction experiments were performed using varying concentrations of glutaraldehyde. Serial dilutions were used for varying the concentration of RBC. A convolutional neural network was constructed using Python and TensorFlow. RESULTS AND CONCLUSIONS: Measurements and model predictions show that a linear relationship between ΔEImax and rigid-cell fraction exists only for rigid-cell fractions less than 0.2. The proposed neural network architecture can be used successfully for both RBC concentration and rigid-cell fraction estimations without a need for a theoretical model.


Assuntos
Anemia Falciforme , Aprendizado Profundo , Deformação Eritrocítica , Eritrócitos , Eritrócitos Anormais , Humanos
5.
Blood Adv ; 5(9): 2385-2390, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33944896

RESUMO

Sickle cell anemia (SCA) results from an abnormal sickle hemoglobin (HbS). HbS polymerizes upon deoxygenation, resulting in red blood cell (RBC) sickling and membrane damage that cause vaso-occlusions and hemolysis. Sickle RBCs contain less adenosine triphosphate and more 2,3-diphosphoglycerate than normal RBCs, which allosterically reduces hemoglobin (Hb) oxygen (O2) affinity (ie, increases the partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen [P50]), potentiating HbS polymerization. Herein, we tested the effect of investigational agent FT-4202, an RBC pyruvate kinase (PKR) activator, on RBC sickling and membrane damage by administering it to Berkeley SCA mice. Two-week oral FT-4202 administration was well tolerated, decreasing HbS P50 to levels similar to HbA and demonstrating beneficial biological effects. In FT-4202-treated animals, there was reduced sickling in vivo, demonstrated by fewer irreversibly sickled cells, and improved RBC deformability, assessed at varying shear stress. Controlled deoxygenation followed by reoxygenation of RBCs obtained from the blood of FT-4202-treated mice showed a shift in the point of sickling to a lower partial pressure of oxygen (pO2). This led to a nearly 30% increase in RBC survival and a 1.7g/dL increase in Hb level in the FT-4202-treated SCA mice. Overall, our results in SCA mice suggest that FT-4202 might be a potentially useful oral antisickling agent that warrants investigation in patients with SCA.


Assuntos
Anemia Falciforme , Hemoglobina Falciforme , Anemia Falciforme/tratamento farmacológico , Animais , Antidrepanocíticos , Eritrócitos Anormais , Humanos , Camundongos , Piruvato Quinase
6.
Clin Hemorheol Microcirc ; 79(2): 357-361, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34057137

RESUMO

Sickle cell anemia (SCA) is a genetic disorder characterized by chronic hemolysis and the presence of erythrocytes with low deformability, which may trigger vaso-occlusive crises. We tested the in-vitro effects of aqueous extract of chives (Allium schoenoprasum L.) on erythrocyte deformability of SCA patients. Blood samples from 6 apparently healthy volunteers and 5 SCA patients were collected into heparin coated tubes. Both apparently healthy and SCA patient blood samples were incubated with 80µg/mL chives plant aqueous extract at 37°C for 60 min and erythrocyte deformability was measured by ektacytometry (3 Pa and 30 Pa; 37°C). Results of incubation of apparently healthy blood samples with plant extract showed that incubation did not alter erythrocyte deformability significantly. However, for SCA blood samples, erythrocyte deformability decreased significantly with plant extract exposure at 3 Pa (p < 0.043) and 30 Pa (p < 0.043). In conclusion, although ex-vivo incubation with plant extract does not fully model gastrointestinal processing of onions, the decrease in SCA erythrocyte deformability following incubation with aqueous chives should stimulate further studies to test the in-vivo effects of this diet in sickle cell mice.


Assuntos
Anemia Falciforme , Cebolinha-Francesa , Anemia Falciforme/tratamento farmacológico , Animais , Deformação Eritrocítica , Eritrócitos , Eritrócitos Anormais , Humanos , Camundongos
7.
Sci Rep ; 11(1): 8513, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33875765

RESUMO

This work aimed to investigate the effect of different shim techniques, voxel sizes, and repetition time (TR) on using theT2 and T2* sequences to determine their optimum settings to investigate the quantification of iron in transfused dependent sickle cell patients. The effect of each of these parameters was investigated on phantoms of different Gadolinium (Gd) concentrations, on 10 volunteers and 25 patients using a1 5T MRI Philips scanner. No significant difference between the three shim techniques was noticed in either T2 or T2* sequence measurements. Pixel sizes of 1 × 1 and 2 × 2 mm provided optimum results for T2 measurements. At 1 × 1 mm pixel size the T2* measurements experienced less error in measurements than the size of 2.5 × 2.5 mm used in the literature. Even though the slice thickness variation did not provide any changes in T2 measurements, the 12 mm provided optimum T2* measurements. TR variation did not yield significant changes on either T2 or T2* measurements. These results indicate that both T2 and T2* sequences can be further improved by providing more reliable measurements and reducing acquisition time.


Assuntos
Anemia Falciforme/metabolismo , Ferro/metabolismo , Imageamento por Ressonância Magnética/métodos , Eritrócitos Anormais/metabolismo , Gadolínio/metabolismo , Humanos , Imagens de Fantasmas
8.
Biophys J ; 120(11): 2138-2147, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33861996

RESUMO

We have characterized the imbibed horizontal flow of sickle blood into 100-µm-diameter glass capillaries. We find that blood containing sickled cells typically traverses the capillaries between three and four times as slowly as oxygenated cells from the same patient for all genotypes tested, including SS, AS, SC and Sß+ thalassemia blood. Blood from SS patients treated with hydroxyurea has a viscosity intermediate between the SS and AA values. Blood containing cells that are not rigidified, such as normal red cells or oxygenated sickle cells, follows a simple Lucas-Washburn flow throughout the length of the 3-cm capillary. By fitting the flexible-cell data to the Lucas-Washburn model, a viscosity can be derived that is in good agreement with previous measurements over a range of volume fractions and is obtained using an apparatus that is far more complex. Deoxygenation sickles and thus rigidifies the cells, and their flow begins as Lucas-Washburn, albeit with higher viscosity than flexible cells. However, the flow further slows as a dense mass of cells forms behind the meniscus and increases in length as flow progresses. By assuming that the dense mass of cells exerts a frictional force proportional to its length, we derive an equation that is formally equivalent to vertical imbibition, even though the flow is horizontal, and this equation reproduces the observed behavior well. We present a simple theory using activity coefficients that accounts for this viscosity and its variation without adjustable parameters. In the course of control experiments, we have found that deoxygenation increases the flexibility of normal human red cells, an observation only recently published for mouse cells and previously unreported for human erythrocytes. Together, these studies form the foundation for an inexpensive and rapid point-of-care device to diagnose sickle cell disease or to determine blood viscosity in resource-challenged settings.


Assuntos
Anemia Falciforme , Capilares , Anemia Falciforme/tratamento farmacológico , Animais , Viscosidade Sanguínea , Eritrócitos , Eritrócitos Anormais , Humanos , Camundongos , Oxigênio
9.
J Ethnopharmacol ; 273: 113992, 2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-33677007

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Evaluation of plants such as Combretum racemosum with claimed traditional use in the management of sickle cell anaemia in Nigeria and other parts of West Africa could serve as a useful research strategy in the search for potential anti-sickling drugs and templates. AIM OF THE STUDY: This study aimed at evaluating the antisickling potential of C. racemosum by activity-guided purification and isolation of its active constituents. MATERIALS AND METHODS: Crude methanol extract of the root of C. racemosum and the fractions obtained by partitioning with chloroform, ethyl acetate, and aqueous were investigated for anti-sickling activity against sodium metabisulphite induced sickling of sickle cell haemoglobin (HbSS). Repeated chromatographic separations were conducted on the most active chloroform fraction to purify and isolate bioactive compounds for further tests for anti-sickling activity. The characterization of the isolated compounds was done by mass spectrometry (FD+MS) and nuclear magnetic resonance (1HNMR) spectroscopy. RESULTS: The chloroform fraction (FA) (% sickled erythrocyte ranged from 3.0 to 34.1) exhibited better anti-sickling activity than aqueous (% sickled erythrocyte ranged from 38.9 to 51.5) as well as the crude methanol (% sickled erythrocyte ranged from 19.1 to 30.4). Hence, the phytochemical investigation was focused on the chloroform fraction, which led to the identification of two ellagic acid derivatives (3,3',4'-tri-O-methyl ellagic acid (A) and 3,3'-di-O- methyl ellagic acid (B). The two isolated compounds possessed good, comparable anti-sickling activities with compound A exhibiting a slightly better in vitro activity. CONCLUSION: This paper reports for the first time anti-sickling principles from C. racemosum and therefore, provided some justification for the ethnomedicinal use of the plant in the management of sickle cell disease.


Assuntos
Anemia Falciforme/sangue , Combretum/química , Eritrócitos Anormais/efeitos dos fármacos , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Medicina Tradicional Africana , Compostos Fitoquímicos , Extratos Vegetais/química , Adulto Jovem
10.
Environ Toxicol Pharmacol ; 85: 103637, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33753236

RESUMO

Pesticide contamination is an important factor in the global decline of amphibians. The herbicides glyphosate and 2,4-D are the most applied worldwide. These herbicides are often found in surface waters close to agricultural areas. This study aims at evaluating the chronic effects caused by glyphosate + 2,4-D mixture in Boana faber and Leptodactylus latrans tadpoles. The combined solution of the glyphosate and 2,4-D, in 5 different concentrations, was applied for 168 h. Herbicide mixtures did not affect the survival of the exposed tadpoles but growth and swimming activity were altered; besides causing several damages in the mouth and intestine. The erythrocytes showed micronuclei and other nuclear abnormalities. There is an ecological risk in the exposure of tadpoles of B. faber and L. latrans from the mixture of glyphosate + 2,4-D. Therefore, the approach used in this study provides important information on how commonly used pesticides can affect non-target organisms.


Assuntos
Ácido 2,4-Diclorofenoxiacético/toxicidade , Glicina/análogos & derivados , Herbicidas/toxicidade , Mutagênicos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Anuros/crescimento & desenvolvimento , Anuros/fisiologia , Dano ao DNA , Eritrócitos Anormais , Glicina/toxicidade , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/fisiologia , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Medição de Risco , Natação
11.
Klin Lab Diagn ; 66(1): 22-25, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33567169

RESUMO

The morphofunctional state of erythrocytes significantly changes by the onset and progression of various diseases, as well as in critical conditions, injuries, accompanied by severe tissue damage. This leads to a violation of microcirculation and has an effect on hemorheology in general. In connection with this, there is a need for the development of adequate methods for assessing the functional activity of erythrocytes. The aim of the work was to study the possibilities of laser interference microscopy in the analysis of the functional state of erythrocytes. The results allowed us to quantify the state of the phase height and phase diameter of erythrocytes. Analysis of the surface nanostructure gave a qualitative assessment of the functional activity of cells. The following patterns were revealed: the action of adrenaline causes the appearance of echinocytes with an increase in phase height and decrease phase diameter and the appearance of spicules on the surface of cells. The action of cortisol leads to a decrease in the phase height and the phase diameter with the appearance of numerous loosening of the structure of the cell surface. Moreover, the traditional method of analyzing the distribution of erythrocytes in a population by morphology (discocytes, stomatocytes, and degeneratively altered cell forms) did not reveal statistically significant differences. Thus, the results indicate that the interference microscopy method allows a qualitative assessment of the morphofunctional activity of native cells. This increases the information content of the analysis and leads to the objectification of data on the functional capabilities of erythrocytes.


Assuntos
Eritrócitos Anormais , Eritrócitos , Contagem de Eritrócitos , Humanos , Lasers , Microscopia de Interferência
14.
Clin Lab ; 67(1)2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33491415

RESUMO

BACKGROUND: This study investigated the feasibility and accuracy of an automated hematology analyzer in the detection of schistocytes. METHODS: In total, 1,026 peripheral blood samples were collected. Schistocytes were morphologically diagnosed by manual examination of digital microscopic red blood cell images captured by a Sysmex DI-60. Automated diagnoses were performed using a Sysmex XN-3000. RESULT: The accuracy of automated diagnosis using the XN-3000 with the default algorithm "fragments?" was determined through comparison with the findings of morphological examination. The comparison showed a sensitivity of 100% and a specificity of 41.6% for automated diagnosis. To improve the low specificity, a two-step analysis was performed. Use of the algorithm "fragments?" in XN-3000 followed by an off-line analysis using the cell parameter %FRC (percent fragmented red blood cells) yielded a sensitivity of 86.5% and a specificity of 70.3%. CONCLUSIONS: Our study indicated that combined use of the %FRC parameter with the default algorithm of the Sysmex XN-3000 automated hematology analyzer can improve the low specificity of the default algorithm in rapid screening for schistocytes.


Assuntos
Hematologia , Algoritmos , Contagem de Eritrócitos , Eritrócitos , Eritrócitos Anormais
16.
Ann Lab Med ; 41(1): 44-50, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32829578

RESUMO

BACKGROUND: The Advanced RBC Application of the CellaVision DM9600 system (CellaVision AB, Lund, Sweden) automatically characterizes and classifies red blood cells (RBCs) into 21 morphological categories based on their size, color, shape, and inclusions. We evaluated the diagnostic performance of the CellaVision Advanced RBC Application with respect to the classification and grading of RBC morphological abnormalities in accordance with the 2015 International Council for Standardization in Haematology (ICSH) guidelines. METHODS: A total of 223 samples, including 123 with RBC morphological abnormalities and 100 from healthy controls, were included. Seven RBC morphological abnormalities and their grading obtained with CellaVision DM9600 pre- and post-classification were compared with the results obtained using manual microscopic examination. The grading cut-off percentages were determined in accordance with the 2015 ICSH guidelines. The sensitivity and specificity of the CellaVision DM9600 system were evaluated using the manual microscopic examination results as a true positive. RESULTS: In pre-classification, >90% sensitivity was observed for target cells, tear drop cells, and schistocytes, while >90% specificity was observed for acanthocytes, spherocytes, target cells, and tear drop cells. In post-classification, the detection sensitivity and specificity of most RBC morphological abnormalities increased, except for schistocytes (sensitivity) and acanthocytes (specificity). The grade agreement rates ranged from 35.9% (echinocytes) to 89.7% (spherocytes) in pre-classification and from 46.2% (echinocytes) to 90.1% (spherocytes) in post-classification. The agreement rate of samples with within-one grade difference exceeded 90% in most categories, except for schistocytes and echinocytes. CONCLUSIONS: The Advanced RBC Application of CellaVision DM9600 is a valuable screening tool for detecting RBC morphological abnormalities.


Assuntos
Eritrócitos Anormais/citologia , Microscopia/métodos , Acantócitos/classificação , Acantócitos/citologia , Área Sob a Curva , Estudos de Casos e Controles , Eritrócitos Anormais/classificação , Humanos , Microscopia/instrumentação , Curva ROC , Estudos Retrospectivos , Esferócitos/classificação , Esferócitos/citologia
17.
Int J Hematol ; 113(2): 163-174, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33074480

RESUMO

Hereditary red blood cell (RBC) membranopathies are characterized by mutations in genes encoding skeletal proteins that alter the membrane complex structure. Hereditary spherocytosis (HS) is the most common inherited RBC membranopathy leading to hereditary hemolytic anemia with a worldwide distribution and an estimated prevalence, in Europe, of about 1:2000 individuals. The recent availability of targeted next generation sequencing (t-NGS) and its combination with RBC deformability measured with a laser-assisted optical rotational ektacytometer (LoRRca) has demonstrated to be the most powerful contribution to lower the percentage of hereditary hemolytic anemia undiagnosed cases. In order to know the kind and frequency of RBC membrane mutations in our geographical area (Catalonia) and to better understand their pathophysiology, 42 unrelated, non-transfusion-dependent (NTD) patients with hereditary hemolytic anemia have been studied by combining t-NGS and LoRRca. The osmoscan module of LoRRca provides three rheological profiles that reflect the maximal deformability (EImax), osmotic fragility (Omin), and hydration state (Ohyper) of RBCs and contribute to a better understanding of the contribution RBC rheology to the severity of anemia. From the 42 patients studied, 37 were suspected to be a RBC membrane defect due to phenotypic characteristics and abnormal RBC morphology and, from these, in 31 patients (83.8% of cases) the mutation was identified by t-NGS. No definite diagnosis was achieved in 11 patients (26.2% of cases), including 6 out of 37 cases, with suspected membranopathy, and 5 with unclassifiable HHA. In all these undiagnosed patients, the existence of hemoglobinopathy and/or enzymopathy was ruled out by conventional methods.


Assuntos
Anemia Hemolítica Congênita/diagnóstico , Anemia Hemolítica Congênita/etiologia , Deformação Eritrocítica/genética , Membrana Eritrocítica/genética , Membrana Eritrocítica/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Fragilidade Osmótica/genética , Adolescente , Adulto , Idoso , Alelos , Anemia Hemolítica Congênita/sangue , Biomarcadores , Criança , Membrana Eritrocítica/patologia , Eritrócitos Anormais/patologia , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Pressão Osmótica , Esferocitose Hereditária/sangue , Esferocitose Hereditária/diagnóstico , Esferocitose Hereditária/genética , Esferocitose Hereditária/metabolismo , Adulto Jovem
19.
Nat Chem Biol ; 17(2): 196-204, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33106661

RESUMO

The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α1 and CC1α2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.


Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Proteínas de Neoplasias/genética , Molécula 1 de Interação Estromal/genética , Transtornos Plaquetários/genética , Clonagem Molecular , Dislexia/genética , Eritrócitos Anormais , Células HEK293 , Humanos , Ictiose/genética , Espectroscopia de Ressonância Magnética , Transtornos de Enxaqueca/genética , Miose/genética , Modelos Moleculares , Fadiga Muscular/genética , Mutação/genética , Conformação de Ácido Nucleico , Proteína ORAI1/genética , Técnicas de Patch-Clamp , Baço/anormalidades
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