Screening of subclinical functional hemoglobin and red blood cell abnormalities among blood donors of Fayoum University Hospital in Egypt: Are RET-He, and IRF useful screening tools?

BACKGROUND: The effectiveness of red cell transfusion in a given blood unit that relied on both quantity and quality of donated cells undoubtedly affects prognostic outcomes. OBJECTIVE: We aimed to determine the frequency of subclinical functional hemoglobin and red cell abnormalities in donated blood of Fayoum University Hospital in Egypt. Additionally, to assess the usefulness of reticulocyte mean hemoglobin content (RET-He) and immature reticulocyte fraction (IRF) as screening measures for such abnormalities. MATERIAL AND METHODS: This cross-sectional study enrolled 200 volunteer blood donors who met the national standard criterion of blood donation. Complete blood count with reticulocyte parameters, serum ferritin, sickling test, G6PD assay, Mentzer index, and naked-eye single tube red cell osmotic fragility test were carried out. RESULTS: Functional red cell abnormalities represented 44 % of this cohort. Out of them, 4.5 % had iron deficiency, 11 % had a positive sickling test, 19 % had G6PD deficiency, and 9.5 % had suspicious thalassemia. The sensitivity and specificity test for RET-He in selective identification of functional hemoglobin abnormalities in donated blood were 83.3 % and 61.2 %, respectively at a cutoff value of 26.9. Though there was no statistically significant effect of RET-He on the selective detection of G6PD deficiency, IRF had a statistically significant high level with a p-value of 0.04. CONCLUSION: Subclinical functional red cell abnormalities seem to be prevalent among blood donors. Reticulocyte/ erythrocyte indices could be useful screening tools for red cell abnormalities. Further studies are required for assessing the impact of transfusing such abnormalities to neonates and other critical recipients.

Red cell membrane disorders: structure meets function.

The mature red blood cell (RBC) lacks a nucleus and organelles characteristic of most cells, but it is elegantly structured to perform the essential function of delivering oxygen and removing carbon dioxide from all other cells while enduring the shear stress imposed by navigating small vessels and sinusoids. Over the past several decades, the efforts of biochemists, cell and molecular biologists, and hematologists have provided an appreciation of the complexity of RBC membrane structure, while studies of the RBC membrane disorders have offered valuable insights into structure-function relationships. Within the last decade, advances in genetic testing and its increased availability have made it possible to substantially build upon this foundational knowledge. Although disorders of the RBC membrane due to altered structural organization or altered transport function are heterogeneous, they often present with common clinical findings of hemolytic anemia. However, they may require substantially different management depending on the underlying pathophysiology. Accurate diagnosis is essential to avoid emergence of complications or inappropriate interventions. We propose an algorithm for laboratory evaluation of patients presenting with symptoms and signs of hemolytic anemia with a focus on RBC membrane disorders. Here, we review the genotypic and phenotypic variability of the RBC membrane disorders in order to raise the index of suspicion and highlight the need for correct and timely diagnosis.

Electrical Impedance Characterization of Erythrocyte Response to Cyclic Hypoxia in Sickle Cell Disease.

Cell sickling is the process in which intracellular polymerization of deoxygenated sickle hemoglobin (HbS) leads to distorted, rigid cells, resulting in abnormal blood rheology and painful vaso-occlusion. Current methods for detection of this process mainly rely on optical microscopy of cellular morphology and measurements of cell deformability and blood rheology. As electrical impedance of cells is a sensitive indicator of changes in cellular structure and biophysical characteristics, it can be a promising marker for characterization of abnormal blood rheology and a means more convenient than optics to be integrated into point-of-care devices. In this work, a microfluidics-based electrical impedance sensor has been developed for characterizing the dynamic cell sickling-unsickling processes in sickle blood. The sensor is capable of measuring the continuous variation in the sickle cell suspension due to cyclic hypoxia-induced intracellular HbS polymerization and depolymerization. Simultaneous microscopic imaging of cell morphological change shows the reliability and repeatability of the electrical impedance-based measurements of cell sickling and unsickling processes. Strong correlation is found between the electrical impedance measurement and patients' hematological parameters such as levels of HbS and fetal hemoglobin. The combination of electrical impedance measurement and on-chip hypoxia control provides a promising method for rapid assessment of the dynamic processes of cell sickling and unsickling in patients with sickle cell disease.

The genotoxicity and cytotoxicity of tannery effluent in bullfrog (Lithobates catesbeianus).

Some of the most polluting activities occur in bovine skin processing. Tannery generates effluents containing high concentrations of heavy metals and organic compounds. The phases composing the leather production process generate a large volume of tannery effluents that are often discarded in aquatic environments without any previous treatment. However, the effect these xenobiotics have on adult representatives belonging to the class Amphibia remains unknown. Thus, the aim of the present study is to assess the geno- and cytotoxic effects of tannery effluent on adult male bullfrogs (Lithobates castesbeianus) exposed to it. Accordingly, the animals were divided into the following groups: negative control (tannery effluent-free water), positive control (cyclophosphamide), and effluent (water added with 5% tannery effluent). The animals were euthanized for blood collection, and erythrocyte analyses were conducted after 35 and 90 days of exposure. The micronuclei (MN) frequency and the frequency of other nuclear abnormalities in each of the animals in the experimental groups were assessed in 2000 erythrocytes. According to the present results, the exposure to tannery effluents increased MN frequency as well as other nuclear abnormalities (i.e., lobed nuclei, binucleated cell, kidney-shaped nuclei, notched nuclei, and apoptotic cell) in the erythrocytes of animals in the effluent group and in the positive control group after 35 and 90 exposure days. Thus, the current study corroborated the hypothesis that the tannery effluent has aneugenic and clastogenic potential in adult male bullfrogs (L. castesbeianus). The present study is the first to report such effect.

Single Molecule Studies of the Diffusion of Band 3 in Sickle Cell Erythrocytes.

Sickle cell disease (SCD) is caused by an inherited mutation in hemoglobin that leads to sickle hemoglobin (HbS) polymerization and premature HbS denaturation. Previous publications have shown that HbS denaturation is followed by binding of denatured HbS (a.k.a. hemichromes) to band 3, the consequent clustering of band 3 in the plane of the erythrocyte membrane that in turn promotes binding of autologous antibodies to the clustered band 3, and removal of the antibody-coated erythrocytes from circulation. Although each step of the above process has been individually demonstrated, the fraction of band 3 that is altered by association with denatured HbS has never been determined. For this purpose, we evaluated the lateral diffusion of band 3 in normal cells, reversibly sickled cells (RSC), irreversibly sickled cells (ISC), and hemoglobin SC erythrocytes (HbSC) in order to estimate the fraction of band 3 that was diffusing more slowly due to hemichrome-induced clustering. We labeled fewer than ten band 3 molecules per intact erythrocyte with a quantum dot to avoid perturbing membrane structure and we then monitored band 3 lateral diffusion by single particle tracking. We report here that the size of the slowly diffusing population of band 3 increases in the sequence: normal cells

Association of abnormal erythrocyte morphology with oxidative stress and inflammation in metabolic syndrome.

In carrying out their role of free radical scavenging, erythrocytes become damaged due to oxidation of membrane lipids and proteins. Such damage may change the morphology of the erythrocytes. The present study aims to demonstrate change in erythrocyte morphology in MetS and associate the changes with increased oxidative stress and inflammation that were shown in our recent study. One hundred participants were recruited from a rural town of Australia. Whole blood viscosity, erythrocyte aggregation, erythrocyte deformability, lipid profile and blood sugar level, oxidative stress markers (erythrocyte reduced glutathione, superoxide dismutase, urinary isoprostanes) and inflammatory markers (high sensitivity C-reactive protein) were measured. Erythrocyte morphological study was performed by scanning electron microscopy. Recruited participants were classified into MetS and non-MetS following the National Cholesterol Education Program Adult Treatment Panel III definition. Data were analyzed by IBM SPSS 20 software. The mean percentages of biconcave cells were decreased whereas acanthocytes, stomatocytes and echinocytes were increased in MetS group compared to healthy controls. Morphologically abnormal erythrocytes were significantly correlated with oxidative stress and chronic inflammation markers. Free radicals generated in increased concentration in MetS seem to damage erythrocyte changing its morphology which possibly could affect other hemorheological parameters.

2D DIGE based proteomics study of erythrocyte cytosol in sickle cell disease: altered proteostasis and oxidative stress.

Sickle cell disease (SCD) is a hemolytic disorder caused by a mutation in beta-globin gene and affects millions of people worldwide. Though clinical manifestations of the disease are quite heterogeneous, many of them occur due to erythrocyte sickling at reduced oxygen concentration and vascular occlusion mediated via blood cell adhesion to the vessel wall. We have followed proteomic approach to resolve the differentially regulated proteins of erythrocyte cytosol. The deregulated proteins mainly fall in the group of chaperone proteins such as heat shock protein 70, alpha hemoglobin stabilizing protein, and redox regulators such as aldehyde dehydrogenase and peroxiredoxin-2 proteoforms. Proteasomal subunits are found to be upregulated and phospho-catalase level also got altered. Severe oxidative stress inside erythrocyte is evident from the ROS analysis and Oxyblot(TM) experiments. Peroxiredoxin-2 shows significant dimerization in the SCD patients, a hallmark of oxidative stress inside erythrocytes. One interesting fact is that most of the differentially regulated proteins are also common for hemoglobinopathies such as Eß thalassemia. These could provide important clues in understanding the pathophysiology of SCD and lead us to better patient management in the future.

Novel single-cell functional analysis of red blood cells using laser tweezers Raman spectroscopy: application for sickle cell disease.

Laser tweezers Raman spectroscopy was used to characterize the oxygenation response of single normal adult, sickle, and cord blood red blood cells (RBCs) to an applied mechanical force. Individual cells were subjected to different forces by varying the laser power of a single-beam optical trap, and the intensities of several oxygenation-specific Raman spectral peaks were monitored to determine the oxygenation state of the cells. For all three cell types, an increase in laser power (or mechanical force) induced a greater deoxygenation of the cell. However, sickle RBCs deoxygenated more readily than normal RBCs when subjected to the same optical forces. Conversely, cord blood RBCs were able to maintain their oxygenation better than normal RBCs. These results suggest that differences in the chemical or mechanical properties of fetal, normal, and sickle cells affect the degree to which applied mechanical forces can deoxygenate the cell. Populations of normal, sickle, and cord RBCs were identified and discriminated based on this mechanochemical phenomenon. This study demonstrates the potential application of laser tweezers Raman spectroscopy as a single-cell, label-free analytical tool to characterize the functional (e.g., mechanical deformability, oxygen binding) properties of normal and diseased RBCs.

Additional erythrocytic and reticulocytic parameters helpful for diagnosis of hereditary spherocytosis: results of a multicentre study.

Hereditary spherocytosis (HS) is characterised by weakened vertical linkages between the membrane skeleton and the red blood cell's lipid bilayer, leading to the release of microparticles. All the reference tests suffer from specific limitations. The aim of this study was to develop easy to use diagnostic tool for screening of hereditary spherocytosis based on routinely acquired haematological parameters like percentage of microcytes, percentage of hypochromic cells, reticulocyte counts, and percentage of immature reticulocytes. The levels of haemoglobin, mean cell volume, mean corpuscular haemoglobin concentration, reticulocytes (Ret), immature reticulocytes fraction (IRF), hypochromic erythrocytes (Hypo-He) and microcytic erythrocytes (MicroR) were determined on EDTA samples on Sysmex instruments from a cohort of 45 confirmed SH. The HS group was then compared with haemolytical disorders, microcytic anaemia, healthy individuals and routine samples (n = 1,488). HS is characterised by a high Ret count without an equally elevated IRF. All 45 HS have Ret >80,000/µl and Ret(10(9)/L)/IRF (%) greater than 7.7 (rule 1). Trait and mild HS had a Ret/IRF ratio greater than 19. Moderate and severe HS had increased MicroR and MicroR/Hypo-He (rule 2). Combination of both rules gave predictive positive value and negative predictive value of respectively 75% and 100% (n=1,488), which is much greater than single parameters or existing rules. This simple and fast diagnostic method could be used as an excellent screening tool for HS. It is also valid for mild HS, neonates and ABO incompatibilities and overcomes the lack of sensitivity of electrophoresis in ankyrin deficiencies.

Discriminant value of % microcytic/% hypochromic ratio in the differential diagnosis of microcytic anemia.

BACKGROUND: % Microcytic and % hypochromic red cells show an inverse trend in beta thalassemia trait and in iron deficiency anemia (IDA). The aim of this study was to assess the discriminant value of % microcytic/% hypochromic ratio, as can be obtained from the Advia 2120 (Siemens) analyzer, in the differential diagnosis of microcytic anemia, compared to the conventional England and Fraser, Green and King, Mentzer, Ricerca, Shine and Lal and Srivastava indices. METHODS: Receiver operating characteristic (ROC) curves, sensitivity, specificity and Youden index of indices were calculated for a set of 148 IDA patients and 170 beta thalassemia carriers. RESULTS: The Green and King index showed the best area under the curve (AUC, 0.962), with a Youden index of 80.9%; the % microcytic/% hypochromic ratio ranked second (AUC 0.943), with a Youden index of 76.3% and a sensitivity of 99.2%. CONCLUSIONS: This index, with high sensitivity for beta thalassemia screening, can be a useful tool in the differential diagnosis of microcytic anemia, so samples can be chosen for HbA(2) analysis, to confirm the presumptive diagnosis of the disease. As specificity is 77.1%, we assume it will be 22.9% for false positives, but these samples will have HbA(2) values within the reference range, for correct diagnosis.

Human erythrocytes are affected in vitro by flavonoids of Aristotelia chilensis (Maqui) leaves.

Aristotelia chilensis (Mol.) Stuntz (A. chilensis), also known as maqui, is a plant of the Elaeocarpaceae family that grows in central and southern Chile as well as southwestern Argentina. Infusions of its leaves have long been used in the traditional native herbal medicine to treat different ailments. Phytochemical studies of the plant's chemical composition of the plant indicate the presence of indolic alkaloids, flavonoids, cianidine glucosides, delfidine, malvidine, petunidine, cumarines and triterpenes. These compounds, particularly the flavonoids, have antioxidant properties. In order to evaluate the mechanisms of its toxicity and their antioxidant properties, the leaves' aqueous extracts were induced to interact with human red cells, their isolated unsealed membranes (IUM), and molecular models of the human erythrocyte membrane. These consisted of multibilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipids classes located in the outer and inner monolayers of the human erythrocyte membrane, and large unilamellar vesicles (LUV) of DMPC. The capacity of A. chilensis aqueous extracts to perturb the bilayer structure of DMPC and DMPE was evaluated by X-ray diffraction, DMPC LUV and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). Results of the present study indicate that aqueous extracts of A. chilensis induced an alteration of human erythrocyte morphology from the normal discoid shape to an echinocytic form, changes that are explained in terms of the extract interaction with the membrane's outer phospholipid monolayer.

Association between myeloid malignancies and acquired deficit in protein 4.1R: a retrospective analysis of six patients.

Constitutional deficit in the erythroid protein 4.1 (4.1R), a structural component of the erythrocyte membrane, is implicated in hereditary elliptocytosis. Acquired deficit in protein 4.1R have been rarely described in myelodysplastic syndromes. Here, we report a series of six patients presenting a myelodysplastic or a myeloproliferative disease in association with an elliptocytosis curve on osmotic gradient ektacytometry and a significant decrease in protein 4.1R level. We confirm that deficit in protein 4.1R is recurrent in myeloid malignancies and should be particularly investigated when deletion del (20 q) is present, since we found this chromosomal abnormality in four out of six patients.

Case report of HbC/beta(0)-thalassemia from India.

This 22-year-old women presented to the ante-natal clinic of this hospital for prenatal screening for beta-thalassemia. Cation exchange high performance liquid chromatography (HPLC) using 'Beta Thalassemia Short Program' on Bio-Rad 'Variant' system revealed HbC value of 81.6%. The CBC showed microcytic hypochromic anemia. The HPLC and CBC suggested the possibility of compound heterozygote state for HbC/beta-thalassemia. The alkali and acid electrophoresis findings were consistent with the above diagnosis. The DNA analysis confirmed compound heterozygote state for HbC/beta(0)-thalassemia (Fr 8/9 mutation). The studies on the parents showed that mother was a compound heterozygote for HbD(Punjab) and HbC while father had beta-thalassemia trait. To the best of our knowledge, this is the first confirmed report of HbC from India. The paper discusses the hematological findings in this subject and her mother (a compound heterozygote for HbD(Punjab) and HbC).

Protein profiling of sickle cell versus control RBC core membrane skeletons by ICAT technology and tandem mass spectrometry.

A proteomic approach using a cleavable ICAT reagent and nano-LC ESI tandem mass spectrometry was used to perform protein profiling of core RBC membrane skeleton proteins between sickle cell patients (SS) and controls (AA), and determine the efficacy of this technology. The data was validated through Peptide/Protein Prophet and protein ratios were calculated through ASAPratio. Through an ANOVA test, it was determined that there is no significant difference in the mean ratios from control populations (AA1/AA2) and sickle cell versus control populations (AA/SS). The mean ratios were not significantly different from 1.0 in either comparison for the core skeleton proteins (alpha spectrin, beta spectrin, band 4.1 and actin). On the natural-log scale, the variation (standard deviation) of the method was determined to be 14.1% and the variation contributed by the samples was 13.8% which together give a total variation of 19.7% in the ratios.

Does higher red blood cell (RBC) lactate transporter activity explain impaired RBC deformability in sickle cell trait?

Lactate and H(+) are suggested to promote the sickling process in red blood cells (RBCs) containing hemoglobin S. We demonstrated greater activity of the RBC monocarboxylate cotransporter MCT-1, lower RBC deformability and impaired hematological indices in sickle cell trait (SCT) carriers compared to control subjects, suggesting an involvement of MCT-1 in hemorheological disturbances in SCT carriers.

Polymerization and sickle cell disease: a molecular view.

The present molecular-level understanding of polymerization and sickling is reviewed for 2 central questions in sickle hemoglobin pathophysiology, viz., what determines when cells sickle, and what determines when cells get stuck. The description of sickling includes the central aspects of the double nucleation mechanism, as well as recent results on the effects of crowding, with an emphasis on the physiological applicability of this fundamental knowledge. In considering when cells get stuck, new measurements of individual fiber stiffness and the processes of depolymerization are also considered. Finally, a fundamental connection is shown between thermodynamics and rheology.

Update on the clinical spectrum and genetics of red blood cell membrane disorders.

Disorders of the red blood cell membrane, such as hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis, are characterized by heterogeneity in their clinical and laboratory manifestations. Advances in molecular biology have allowed determination of the precise genetic defect in many cases of membrane-associated anemia and have revealed significant genetic heterogeneity. Six genetic loci have been identified and many defects, including gene deletions and insertions, missense and nonsense mutations, and splicing mutations, have been found. Analysis of these defects has provided a better understanding of the pathogenesis of these disorders and allowed a better understanding of the structure/function relationships of the proteins of the erythrocyte membrane.

Effects of N-acetylcysteine on dense cell formation in sickle cell disease.

The extent to which dense and irreversible sickle cells (ISCs) contribute to vaso-occlusive episodes in sickle cell disease remains unclear. N-Acetylcysteine (NAC) inhibits dense cell and ISC formation in sickle erythrocytes in vitro and restores glutathione levels toward normal. A phase II double-blind randomized clinical trial was completed to determine the efficacy of NAC in decreasing dense cell and ISC formation, and vaso-occlusive episodes in sickle cell disease. Twenty-one subjects with a history of at least two vaso-occlusive episodes per year and 6% dense cells were enrolled. Four treatment groups were analyzed; NAC at a dose of 2,400 mg per day decreased the percent dense cells from 20.1 +/- 2.9 to 12.6 +/- 2.1 (P < 0.05) and increased red cell glutathione levels from 292.8 +/- 74.5 to 576.7 +/- 155.1 (P < 0.05). In addition, we observed a decrease in vaso-occlusive episodes from 0.03 to 0.006 episodes per person-days and a decreased in relative risk to R = 0.39. Although NAC did not significantly decrease the number of ISCs, there was a downward trend at all doses tested. In summary, NAC inhibited dense cell formation, restored glutathione levels toward normal, and decreased vaso-occlusive episodes at a well-tolerated dose of 2,400 mg per day. To determine the long-term efficacy and safety of NAC, a multicenter phase III clinical trial is required.

Inhibitory effect of local ischaemic preconditioning in total body irradiated rats.

The aim of this study was to explore the relationship between local ischaemic preconditioning and the effectiveness of fractionated radiotherapy. The rat serum, bone marrow, and small intestine were examined for oxidative changes induced by total body irradiation with gamma rays with applied local ischaemic preconditioning immediately before irradiation. Serum concentrations of TBA-RS examined 12 hours after the last irradiation did not reveal any differences among the groups of animals analyzed. Twenty-four hours after the last dose of irradiation, the serum concentrations of TBA-RS varied in particular groups (P<0.0001). The concentration of triglycerides in the serum of local preconditioned ischaemia and irradiated animals showed a reversed shape similar to the TBA-RS fluctuation (P<0.003). The level of uric acid in the serum of animals treated only with radiation is slightly higher than the level of this acid in the serum of the local preconditioned ischaemia radiation group (P<0.58). The number of bone marrow polychromatic erythrocytes did not appear to differ substantially in both irradiated groups. At the first 12 hours after irradiation, the frequency of micronucleated polychromatic erythrocytes is significantly different in the bone marrow of both groups either in combination with ischaemic preconditioned radiation or with radiation alone (P<0.0002). In irradiated animals without ischaemic preconditioning, on the 3rd day after irradiation the number of crypts increased and in the next days decreased achieving the level of the control group on the 7th day. Irradiated rats with local ischaemic preconditioning did not reveal an increase in the number of crypts. The difference was statistically significant (P<0.05). These data indicate that the local ischaemic preconditioning modifies the radiation peroxidising effects through inhibition of free radical-dependent lipid peroxidation and, probably, other unrecognized mechanisms.