Red Blood Cell Morphology Research Articles

STUDY OF DHATU SARATA AND ITS ASSOCIATION WITH BLOOD INDICES

Ashta Sarata parikshana is explained especially to determine the strength of person. It is important to add modern parameter to giv e authenticity to the ancient subjective parameter . 50 healthy male students between the ages of 18 - 25 years were selected for study . Sarata parikshan was done with the help of standard sarata parikshan proforma speci ally prepared by AYUSOFT C - DAC Centre for Development of Advance Computing Pune , India to find out percentage of each dhatu separately. Blood indices hav e been formulated by modern science to detect type of anaemia by extracting information about structure, morphology of Red Blood Cells and about haemoglobin. Blood indices give complete information of Red Blood Cells. Blood sample were collected for analys is of blood indices and this was done by auto analyser . This was s urvey type co relational research . The s tatistical analysis shows that “ there is a relation between Rakta sarata and Blood indices” , it’s a positive relation. The person having higher percen tage of Ra kta sarata possesses good value of Blood indices .

Automated reticulocyte parameters for hereditary spherocytosis screening.

The laboratory diagnosis of hereditary spherocytosis (HS) is based on several screening and confirmatory tests; our algorithm includes clinical features, red blood cell morphology analysis and cryohaemolysis test, and, in case of positive screening, sodium dodecyl sulphate polyacrylamide gel electrophoresis as a diagnostic test. Using the UniCel DxH800 (Beckman Coulter) haematology analyser, we investigated automated reticulocyte parameters as HS screening tool, i.e. mean reticulocyte volume (MRV), immature reticulocyte fraction (IRF) and mean sphered cell volume (MSCV). A total of 410 samples were screened. Gel electrophoresis was applied to 159 samples that were positive for the screening tests. A total of 48 patients were diagnosed as HS, and seven were diagnosed as acquired autoimmune haemolytic anaemia (AIHA). Some other 31 anaemic conditions were also studied. From the receiver operating characteristic (ROC) curve analysis, both delta (mean cell volume (MCV)-MSCV) and MRV presented an area under the curve (AUC) of 0.98. At the diagnostic cut-off of 100 % sensitivity, MRV showed the best specificity of 88 % and a positive likelihood ratio of 8.7. The parameters IRF, MRV and MSCV discriminated HS not only from controls and other tested pathologies but also from AIHA contrary to the cryohaemolysis test. In conclusion, automated reticulocyte parameters might be helpful for haemolytic anaemia diagnostic orientation even for general laboratories. In combination with cryohaemolysis, they ensure an effective and time-saving screening for HS for more specialised laboratories.

Cellular imaging using phase holographic microscopy: for the study of pathophysiology of red blood cells and human umbilical vein endothelial cells

Digital holographic microscopy (DHM) for phase-contrast imaging is employed for live cell imaging to study pathophysiology of red blood cells (RBCs) and human umbilical vein endothelial cells (HUVECs). The morphology and membrane fluctuation of healthy RBCs are investigated by adapting post-image processing which automatically quantify the morphological properties of RBCs, such as perimeter, mean diameter, cell volume, cell thickness and membrane fluctuation. Moreover, morphological variation of malaria parasite-infected RBCs and hypertonic-shocked RBCs was observed. As another biological application, HUVECs cultured on collagen films and their abnormal deformations caused by collagenase are observed using DHM to understand the role of extracellular matrix (ECM) structure on atheroprotective effect and endothelial dysfunction.

Simultaneous Camouflage of Major and Minor Antigens on Red Blood Cell Surface With Activated mPEGs

Background: Host immune system response against blood group antigens is a major problem in blood transfusions, especially for thalassemic patients. Thus, an approach was proposed coating the red blood cell (RBC) surface by polyethylene glycol. Objectives: This study aimed to obtain the optimal simultaneous camouflge of the major and minor antigens by activated methoxy polyethylene glycol (mPEG) with succinimidyl valerate (SVA) and succinimidyl carbonate (SC), separately. Materials and Methods: The degree of RBC agglutination by antibodies against the major and minor blood groups was used as a surrogate measurement for quantitative assessment of the effctiveness of the surface coating. Also, the RBC morphology was assessed using scanning electron microscope (SEM). In addition, to evaluate the host immune system response, the PEGylated RBCs were transferred between two diffrent mouse strains. Results: Statistical analysis of the results demonstrated that the optimal reaction conditions for simultaneous coating of the antigens by mPEG-SVA and mPEG-SC are as mPEG20 in the polymer mixture, 91.2 and 90.0%, and polymer concentration, 17.21 and 19.80 mg.mL-1, respectively. However, according to the SEM results, the maximum polymer concentration of 14.5 mg.mL-1 was suggested as the best condition for mPEG-SVA modifid human RBCs. Conclusions: It is concluded that the membrane PEGylation camouflges the blood group antigens. This effct is observed signifiantly for non-ABO/Rh(D) antigens. Also, it is found that the mPEG-SVA provide better coverage than mPEG-SC. The results of in vivo analysis showed that the immune reactions against PEGylated RBCs were considerably reduced, so that the levels of the relevant biochemical parameters in serum were similar to those of the normal hosts 24 hours after transfusion.

Causes of hemolysis in neonates with extreme hyperbilirubinemia

We instituted a quality improvement process to enhance our capacity to diagnose genetic hemolytic conditions in neonates with extreme hyperbilirubinemia. During a 1-year period, whenever the total serum bilirubin (TSB) was >25 mg dl(-1) a special evaluation was performed. If we deemed an erythrocyte membrane defect likely, based on red blood cell morphology, EMA-flow cytometry was performed. Otherwise 'next-generation' sequencing was performed using a panel of genes involved in neonatal hyperbilirubinemia. Ten neonates had a TSB ⩾ 25 mg dl(-1). Two others were evaluated as part of this process at the request of their attending neonatologists, because each had a TSB >14 mg dl(-1) in the first hours after birth and required phototherapy for ⩾ 1 week. Explanations for the jaundice were found in all 12 neonates. Five had hereditary spherocytosis, three of which also had ABO hemolytic disease. Two had pyruvate kinase deficiency. One had severe G6PD deficiency. The other four had ABO hemolytic disease. On the basis of the present small case series, we suggest that among neonates with extreme hyperbilirubinemia, it can be productive to pursue a genetic basis for hemolytic disease.

Reduced red blood cell membrane damage in streptozotocin-induced diabetic rats supplemented with roselle (UKMR-2) calyx extract.

The aim of this study was to evaluate the effects of aqueous extract of roselle on the membrane composition of red blood cells. A total of 32 male Sprague-Dawley rats (230-250 g) were divided randomly into four groups. Diabetic rats were induced streptozotocin (45 mg kg(-1), i.v). The normal and diabetes groups were administrated with distilled water. The other normal and diabetes group were administrated with roselle aqueous extracts (100 mg kg(-1)). After 28 days, the blood was drawn by sinus orbital for biochemical tests including membrane total protein, cholesterol and phosphatidylcholine and morphology of red blood cells was carried out through light microscope. In diabetic rats, the result showed the weight of rat, membrane total protein and Phosphatidylcholine (PCh) were significantly lower (p<0.05), while blood glucose and membrane cholesterol showed significantly higher (p<0.05) than control rats. In diabetic rats demonstrated with roselle, the result showed no significant difference (p>0.05) in weight and blood glucose compared to diabetic rats. The membrane total protein and PCh were significantly higher (p<0.05) than diabetic rats, whereas membrane cholesterol was significantly lower (p<0.05) compared to diabetic rats. The observation red blood cells morphology that showed echinocytes, schistocytes and Heinz body in diabetic rats was caused by oxidative stress damage. The morphology of red blood cells in diabetic rats supplemented with roselle is normal. Aqueous extract of roselle showed potential protective effects on membrane composition of damaged red blood cells.

Reporting and grading of abnormal red blood cell morphology

In spite of the continual standardization of test result formats, the improvements of laboratory technologies, publications of reference guidelines, and the advancements in hematology analyzers, the methods of reporting or grading abnormal red blood cell morphology still vary among laboratories everywhere. This article describes the methods or systems of reporting abnormal red cell morphology and the conditions associated with the abnormalities.

SEM observation of the live morphology of human red blood cells under high vacuum conditions using a novel RTIL

In this study, we succeed in visualizing a ‘living’ morphology of red blood cells (RBCs) by a rapid and simple scanning electron microscope (SEM) pretreatment using a hydrophilic room temperature ionic liquid (RTIL) with asymmetrical structure. The conventional pretreatment induces size shrinkage of the cells, and the diameter observed in an SEM (less than 6 µm) was smaller than that in an optical microscope (8 µm). Though RBCs are well‐known to be easily deformed with the environmental conditions, the cells did not show the deformation when they were pretreated with our RTILs. Since the chemical structure of our RTIL resembles a ‘choline’, which is a typical bioactive material, and has biocompatibilities, they may make it avoid from the deformation of RBCs. In fact, only immersing RBCs, our novel RTIL can provide a clear SEM image without size shrinkage and deformation. Interestingly, the obtained SEM image shows 8 µm in the diameter, almost same size as that in optical microscope. Therefore, this method is expected for novel SEM pretreatment for various biological samples observation as ‘living’ matter. Copyright © 2014 John Wiley &amp; Sons, Ltd.

High Control, Fast Growth OEG-Based Dendron Synthesis via a Sequential Two-Step Process of Copper-Free Diazo Transfer and Click Chemistry

Dendrons up to generation 3 were quickly synthesized using two sequential steps of diazo transfer and click chemistry reactions. The building blocks for dendron synthesis were made by incorporating monodisperse oligoethylene glycol (OEG) chains of exact length onto diethyelenetriaminepentaacetic acid (DTPA)-derived moieties to obtain first-generation dendrons. Afterward, the amine groups of the first-generation core dendron were converted to azides by means of the diazo transfer reaction. The second- and third-generation dendrons were constructed using the copper-catalyzed azide–alkyne cycloaddition (CuAAC). Hemocompatibility studies including hemolysis, red blood cell morphology, cell count and size distribution, hemostasis, and complement system activation were performed to evaluate their safety in blood.

English

BACKGROUND: This study was planned with an aim to identify the nature and distribution of cranial lesions on magnetic resonance imaging (MRI) and its correlation with clinical and laboratory data in eclampsia and severe preeclampsia. MATERIAL AND METHODS: 40 patients admitted for indication of severe preeclampsia or eclampsia with or without neurological signs were first stabilized and then underwent cranial MRI. Following MRI they were divided into two groups; Group MP (n=24) including patients with positive finding on the cranial MRI and Group MN (n=16) which included patients with normal Cranial MR imaging. Nature and distribution of the lesions were documented and statistical comparison was made on the basis of clinical findings, arterial blood pressure and laboratory data in both the groups. Patient with cerebral changes in the MRI were also called back for repeat MRI postnatal after two 2 months. RESULTS: Out of the 40 patients who underwent cranial MRI 24 patients had cerebral changes (Group MP) whereas 16 patients had normal scan (Group MN). In 21 out of 24 (87.5%) MRI finding positive patients the finding was consistent with diagnosis of posterior reversible encephalopathy syndrome (PRES). The most commonly involved areas in patients with PRES were parietal (85.7%,), frontal (71.42%) and occipital lobe (71.42), followed by temporal lobe (38.09) and basal ganglia (33.33) and cerebellum. All the patients who were diagnosed with PRES had a normal MR scan on the follow up at two months after the initial presentation. There was a significantly greater incidence of seizures and neurological disturbances in patients with positive MRI findings as compared to patients with no MRI findings (p<0.001). There was no statistical difference between the blood pressure measurements of the two groups. Markers of endothelial dysfunction like Serum LDH (p=0.002) Serum creatinine (p=0.006) and abnormal red blood cell morphology (0.002) was significantly higher in patients with positive MRI findings compared to MRI Finding Negative group. CONCLUSION: Our study suggests that PRES is the core component of the pathogenesis of cerebral findings of eclampsia and development of PRES is associated with endothelial dysfunction and not elevated blood pressure alone.

Direct electron transfer from native human hemoglobin using a glassy carbon electrode modified with chitosan and a poly(N,N-diethylacrylamide) hydrogel containing red blood cells

We report on a new whole cell biosensor for hydrogen peroxide. A chitosan-coated glassy carbon electrode (GCE) was modified with poly(N,N-diethylacrylamide) (PDEA) hydrogel containing human red blood cells (RBCs). The morphology of RBCs in the hydrogel was investigated using scanning electron microscopy (SEM). Fourier transform infrared spectroscopy and SEM were applied to study the association of the PDEA chains and RBCs. Uncompromised bioactivity of native human hemoglobin in the RBCs on the modified GCE was confirmed by cyclic voltammetry. The modified electrode showed a faster electron transfer rate and better electrocatalytic activity in the reduction of H2O2 than previously reported sensors. A linear relationship is found between the response to H2O2 and its concentration in the range from 0.11 μM to 12.7 mM. The detection limit is 55 nM at an SNR of 3. It is assumed that the improvement of the biosensor results from the porosity and conductivity of the PDEA hydrogel.

Evaluation of red cell membrane cytoskeletal disorders using a flow cytometric method in South iran.

Objective: The diagnosis of hereditary red blood cell (RBC) membrane disorders, and in particular hereditary spherocytosis (HS) and Southeast Asian ovalocytosis (SAO), is based on clinical history, RBC morphology, and other conventional tests such as osmotic fragility. However, there are some milder cases of these disorders that are difficult to diagnose. The application of eosin-5’-maleimide (EMA) was evaluated for screening of RBC membrane defects along with some other anemias. We used EMA dye, which binds mostly to band 3 protein and to a lesser extent some other membrane proteins, for screening of some membrane defects such as HS. Materials and Methods: Fresh RBCs from hematologically normal controls and patients with HS, SAO, hereditary elliptocytosis, hereditary spherocytosis with pincered cells, severe iron deficiency, thalassemia minor, and autoimmune hemolytic anemia were stained with EMA dye and analyzed for mean fluorescent intensity (MFI) using a flow cytometer. Results: RBCs from patients with HS and iron deficiency showed a significant reduction in MFI compared to those from normal controls (p<0.0001 and p<0.001, respectively), while macrocytic RBCs showed a significant increase in MFI (p<0.01). A significant correlation was shown between mean corpuscular volume and MFI, with the exceptions of HS and thalassemia minor.Conclusion: Our results showed that the flow cytometric method could be a reliable diagnostic method for screening and confirmation, with higher sensitivity and specificity (95% and 93%, respectively) than conventional routine tests for HS patients prior to further specific membrane protein molecular tests.

A morphology-based method for the diagnosis of red blood cells parasitized by Plasmodium malariae and Plasmodium ovale

The morphology of red blood cells (RBCs) is altered significantly during the maturation stages of malaria parasites, which include ring, trophozoite, and schizont. There is dissimilarity in terms of the morphological characteristics of parasitized RBCs infected by the 4 species of Plasmodium, including falciparum, vivax, malariae, and ovale. This makes the process of diagnosis very difficult, which may lead to a wrong treatment method and substantial damage to the health of the patient. An innovative technique in introduced that accurately defines the shape of parasitized RBCs at each stage of infection as a potential method of diagnosis. Giemsa-stained thin blood films were prepared using blood samples collected from healthy donors as well as patients infected with P. malariae and P. ovale. The diameter and thickness of healthy and infected RBCs at each stage of infection were measured from their optical images using Olysia and Scanning Probe Image Processor (SPIP) software, respectively. A shape equation was fitted based on the morphological characteristics of RBCs, and their relative 2-dimensional shapes were plotted using Wolfram Mathematica. At the ring stage, the thicknesses of RBCs parasitized by P. malariae (Pm-RBCs) and P. ovale (Po-RBCs) increased by 42% and 51%, respectively. Both Pm-RBCs and Po-RBCs remained nearly biconcave throughout parasite development even though their volumes increased. It is proposed that the morphology-based characterization technique introduced here could be used to intensify the accuracy of the Giemsa staining diagnosis method for the detection of the Plasmodium genus and infection stage. Based on the significant morphological alterations induced by different Plasmodium species, the results may also find practical use for faster prediction and treatment of human malaria.

The effect of iron overload on red blood cell morphology

A 76-year-old man with an extremely high serum ferritin concentration (33 790 μg/L) was referred to the National Reference Laboratory for further laboratory testing to determine the cause. Genetic analysis showed that he was a carrier of a haemo chromatosis-associated allele (H63D/hetero zygous). Pathology con fi rmed renal insuffi ciency and liver damage, with an exceptionally high bilirubin concen tration of 730 μmol/L and an unconjugated bilirubin concentration of 272 μmol/L. This light micro scopy smear showed that most of the red blood cells have a changed morphology (fi gure). Scanning electron micro scopy, by which high magnifi cations of the red blood cells can be viewed, showed that the cells form pointed extensions with large membrane pores (fi gure). This abnormality is not seen in red blood cells from healthy individuals (fi gure). We suggest that the presence of the H63D haemochromatosisassociated allele is partly the cause of the exceptionally high serum ferritin concentrations, which brings about the abnormal red blood cell morphology.

As a Rare Disease Bernard–Soulier Syndrome in Differential Diagnosis of Immune Thrombocytopenic Purpura: A Case Report

Introduction: Bernard–Soulier syndrome is a rare autosomal recessive disease that causes a deficiency of glycoprotein Ib, the receptor for von Willebrand factor, which is important for clot formation. It is estimated to occur in fewer than 1 per 1 million persons. The differential diagnosis includes von Willebrand disease, immune thrombocytopenic purpura, May–Hegglin anomaly, thrombocytopenia-absent radius syndrome, grey platelet syndrome, and other inherited giant platelet disorders. Case Presentation: A 30-year-old Turkish woman was admitted to the Department of Heamatology for evaluation of thrombocytopenia. Because she had repeated epistaxis during admission, she was assessed to evaluate for haemorrhagic diathesis. She had been diagnosed with immune thrombocytopenic purpura and given steroid therapy at different times. Peripheral blood smear was characterized by neutrophils 75%, lymphocytes 20%, monocytes 5%, giant platelets and platelets forming three to four clusters, a normal red blood cell morphology. A decreased ristocetin-induced platelet aggregation was detected in low and high concentration. On the basis of these findings, Bernard–Soulier syndrome was screened with flow cytometry and genetic mutation. CD41a 86.2%, CD42a 92.9%, CD42b 92.5%, and CD61 87.8% were detected in flow cytometry. Normal platelet GPIb/IX levels by flow cytometry turned down the suspected diagnosis. genetic analysis, we have applied to our patient and her parents. We detected same mutation in patient and her mother. Laboratuar parameters and flow cytometry of mother did not support diagnosis of Bernard–Soulier syndrome. Conclusion: Bernard–Soulier syndrome should be considered before a young patient is diagnosed with immune thrombocytopenia purpura and peripheral blood smear should be examined for giant platelets.

Alterations in red blood cell deformability during storage: a microfluidic approach.

Red blood cells (RBCs) undergo extensive deformation when travelling through the microcapillaries. Deformability, the combined result of properties of the membrane-cytoskeleton complex, the surface area-to-volume ratio, and the hemoglobin content, is a critical determinant of capillary blood flow. During blood bank storage and in many pathophysiological conditions, RBC morphology changes, which has been suggested to be associated with decreased deformability and removal of RBC. While various techniques provide information on the rheological properties of stored RBCs, their clinical significance is controversial. We developed a microfluidic approach for evaluating RBC deformability in a physiologically meaningful and clinically significant manner. Unlike other techniques, our method enables a high-throughput determination of changes in deformation capacity to provide statistically significant data, while providing morphological information at the single-cell level. Our data show that, under conditions that closely mimic capillary dimensions and flow, the capacity to deform and the capacity to relax are not affected during storage in the blood bank. Our data also show that altered cell morphology by itself does not necessarily affect deformability.

Evaluation of complement regulatory components in patients with atypical hemolytic uremic syndrome.

BackgroundAtypical hemolytic uremic syndrome (aHUS), a rare disorder characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure, is associated with mutations and polymorphisms in various components and regulators of the complement alternative pathway (AP), including factor H, factor I, membrane cofactor protein (MCP or CD46) and factor B. This impaired regulation of the alternative pathway leads to a procoagulant state with microthrombi formation in the renal vasculature, which influences disease onset and progression.Aim of the studyTo evaluate the role of complement regulatory factors in occurrence of aHUS; we also included evaluation of ADAMTS13 activity and autoantibody against ADAMTS13 in order to exclude thrombotic thrombocytopenic purpura (TTP) cases, which might have overlapping clinical and laboratory findings.Material and methodsThis study was conducted on 273 individuals with aHUS. Diagnosis was based on clinical manifestations, kidney function tests, red blood cell count, morphology and reticulocyte count. Then, the ADAMTS 13 autoantibody and activity and also complement factor B, complement factor H (CFH) and complement factor-I (CIF) were analyzed. Finally, the statistical analysis was performed by SPSS software.ResultsThe mean age of our patients was 27.3 years, 55% were female and 45% were male. The mean levels of urea and creatinine concentration were 92.9 mg/dl and 5.1 mg/dl, respectively. The mean levels of RBC count, Hb and HCT in these patients were lower than normal but the mean percentage of reticulocyte count was higher than normal (2.5%). The assessment of complement regulatory factors revealed that the B and H factors levels were normal except in two cases but the level of factor I was higher than normal.ConclusionsAccording to the results of this study, it seems that up regulation of factor I had a significant role in occurrence of aHUS in our study group.

The Observation Report of Red Blood Cell Morphology in Thailand Teenager by Using Data Mining Technique

It is undeniable that laboratory information is important in healthcare in many ways such as management, planning, and quality improvement. Laboratory diagnosis and laboratory results from each patient are organized from every treatment. These data are useful for retrospective study exploring a relationship between laboratory results and diseases. By doing so, it increases efficiency in diagnosis and quality in laboratory report. Our study will utilize J48 algorithm, a data mining technique to predict abnormality in peripheral blood smear from 1,362 students by using 13 data set of hematological parameters gathered from automated blood cell counter. We found that the decision tree which is created from the algorithm can be used as a practical guideline for RBC morphology prediction by using 4 hematological parameters (MCV, MCH, Hct, and RBC). The average prediction of RBC morphology has true positive, false positive, precision, recall, and accuracy of 0.940, 0.050, 0.945, 0.940, and 0.943, respectively. A newly found paradigm in managing medical laboratory information will be helpful in organizing, researching, and assisting correlation in multiple disciplinary other than medical science which will eventually lead to an improvement in quality of test results and more accurate diagnosis.

Automated quantitative analysis of 3D morphology and mean corpuscular hemoglobin in human red blood cells stored in different periods

Quantitative phase (QP) images of red blood cells (RBCs), which are obtained by off-axis digital holographic microscopy, can provide quantitative information about three-dimensional (3D) morphology of human RBCs and the characteristic properties such as mean corpuscular hemoglobin (MCH) and MCH surface density (MCHSD). In this paper, we investigate modifications of the 3D morphology and MCH in RBCs induced by the period of storage time for the purpose of classification of RBCs with different periods of storage by using off-axis digital holographic microscopy. The classification of RBCs based on the duration of storage is highly relevant because a long storage of blood before transfusion may alter the functionality of RBCs and, therefore, cause complications in patients. To analyze any changes in the 3D morphology and MCH of RBCs due to storage, we use data sets from RBC samples stored for 8, 13, 16, 23, 27, 30, 34, 37, 40, 47, and 57 days, respectively. The data sets consist of more than 3,300 blood cells in eleven classes, with more than 300 blood cells per class. The classes indicate the storage period of RBCs and are listed in chronological order. Using the RBCs donated by healthy persons, the off-axis digital holographic microscopy reconstructs several quantitative phase images of RBC samples stored for eleven different periods. We employ marker-controlled watershed transform to remove the background in the RBC quantitative phase images obtained by the off-axis digital holographic microscopy. More than 300 single RBCs are extracted from the segmented quantitative phase images for each class. Such a large number of RBC samples enable us to obtain statistical distributions of the characteristic properties of RBCs after a specific period of storage. Experimental results show that the 3D morphology of the RBCs, in contrast to MCH, is essentially related to the aging of the RBCs.

Cover Picture: J. Biophotonics 11–12/2013

AbstractHyperspectral imagery with enhanced Darkfield microscopy was used for probing and characterizing changes in blood cell components. Unique spectral signatures of ordinary and most common deformed morphologies of red blood cells were identified, showing a preliminary but promising application of the hyperspectral analysis to blood cell screening for clinical use.(Picture: G. Sacco Verebes et al., pp. 960–967 in this issue)