Osmotic Fragility Research Articles

Difficulty in Diagnosis of Hereditary Spherocytosis in the Neonate.

Difficulty in Diagnosis of Hereditary Spherocytosis in the Neonate.

The use of green apple and beet-root as natural radio-protectors against health effects of ionizing radiation

Objective: Evaluation of the antioxidant protective role of beetroot and apple to reduce the impact of ionizing radiation on the blood components. Subjects and Methods: Sixty adult white male rats were evenly divided into four experimental groups, Group 1 (n= 15) was used as a control group. Groups 2, 3, and 4 (n=15 each) were injected intravenously with radioactive material Tc-99m 130 ± 5 Mega Becquerel )MBq( (5 ± 0.5 Micro Curry )mCi)( as a source of ionizing radiation (gamma rays). Groups 2 and 4 were fed with apple fruit and beet seeds in addition to a small amount of commercial food. Rats in G3 were fed with commercial food pellets only and received the same volume of water. Rats in groups G1, G2, and G3 were sacrificed after7 days while those of G4 were sacrificed after 30 days. Osmotic fragility of RBCs and hematological parameters were investigated for each group. The structure characteristics of the blood cells for all groups were studied by recording the UV -Vis spectra. Results: The study revealed statistically significant changes regarding RBCs indices along with increased osmotic fragility among rats of group 3 compared to those in groups 1, 2, and 4. The U.V. spectrum revealed a partial irregularity in the RBCs. Conclusion: Our study pointed out the protective role of natural products namely, beetroots and green apple extracts, against the adverse impact of ionizing radiation on blood indices.

Assessment of Nanosilver Hemocompatibility in Prehypertensive Salt-Induced Animal Model

Aim: There are Conflicting reports on safety profile of nanoparticles on biological cells. This study evaluated the impact of nanosilver on hemocompatibility on salt-loaded rats. Materials and Methods: Sprague-Dawley rats [(inbred) (120-140 g)] randomly divided into of 4 groups, (n = 6) were studied. Group 1(control) received normal rat chow and tap water, Group 2 received rat chow containing 8% NaCl [(salt-loaded rats (SLRs)]. Group 3 received rat chow + Nanosilver Solution (NS) 0.18 mL 10 ppm/kg/day. Group 4 comprised SLRs + NS. After 6 weeks oral gavage treatments, measurements of Blood pressure (Bp) and Heart Rate (HR) were by pressure transducer via cannulation of left common carotid artery following anaesthesia with urethane. HR was computed by the number of arterial pulse per 60 seconds. 5 ml of blood for WBC, PLATELETS, RBC, PCV, HB, MCH, MCHC and MCV analyses using automated haematology analyser and Osmotic fragility reactivity with standard spectrophotometer at 540 nm wavelength. Results: Exposure of nanosilver to normotensive rats resulted in significantly lower RBC level compared with control, whereas RBC level in Salt-Loaded Co-Treated Nanosilver (SCNS) was comparable with the SLRs. The tenet was the same for HB, PCV, MCH and MCHC. Nanosilver induced leukopenia in normotensive compared with control and prevented WBC elevation in SCNS. Platelets significantly increased in Nanosilver-Treated Normotensive Rats (NTNRs) compared with control and decreased in SCNS. Osmotic burst resistance increased in NTNRs and decreased in cells from treated groups. Conclusion: Chronic exposure of nanosilver to salt loaded rats alters haematological parameters which may worsen circulatory function and activate risk factors of cardiovascular disorders.

Molecular detection of Theileria species, Anaplasma species, Candidatus Mycoplasma haemobos, Trypanosoma evansi and first evidence of Theileria sinensis-associated bovine anaemia in crossbred Kedah-Kelantan x Brahman cattle

BackgroundSerious disease outbreaks in cattle are usually associated with blood pathogens. This study aims to detect blood pathogens namely Theileria species, Anaplasma species, Candidatus Mycoplasma haemobos and Trypanosoma evansi, and determine their phylogenetic relationships and haemato-biochemical abnormalities in naturally infected cattle.MethodsMolecular analysis was achieved by PCR amplification and sequencing of PCR amplicons of 18SrRNA gene of Theileria species, 16SrRNA genes of Anaplasma and Mycoplasma species, MPSP genes of T. orientalis and T. sinensis, MSP4 gene of A. marginale, 16SrRNA gene of Candidatus Mycoplasma haemobos, and RoTat1.2 VSG gene of Trypanosoma evansi, in sixty-one (61) clinically ill Kedah-Kelantan x Brahman cattle in Pahang, Malaysia.ResultsA total of 44 (72.13%) cattle were infected with more than one blood pathogen. Theileria species was the blood pathogen with the highest molecular detection rate (72.13, 95% CI 59.83–81.81%). Nucleotide blast analyses of all sequences demonstrated high degree of molecular similarity (98–100%) in comparison with their respective reference sequences. Analysis of 18SrRNA gene sequences of Theileria species and 16SrRNA gene sequences of Anaplasma species revealed Theileria sinensis and Anaplasma platys respectively as additional species detected in these cattle. MPSP-PCR analysis was conducted for further confirmation of T. sinensis. The blood picture of eight infected cattle groups revealed poikilocytosis, anisocytosis, rouleaux formation and degenerative left shift. High mean erythrocyte fragility values were common in infected cattle groups. Anaemia of the macrocytic normochromic type and spherocytes were observed in the T. evansi and Anaplasma platys + Theileria sinensis double species co-infected cattle group. Normocytic normochromic anaemia was observed in the T. sinensis infected cattle group. Significant (p < 0.05) increases in serum liver and kidney parameters, total protein, globulin, total and unconjugated bilirubin and decreased albumin values were observed in the T. evansi infected cattle when compared to clinically healthy cattle.ConclusionWe present the first evidence of Theileria sinensis-associated bovine anaemia (TSABA) in Malaysian cattle. Because of the high occurrence of bovine theileriosis and detection of A. platys, there is an urgent need for appropriate preventive and control measures against these blood pathogens.

NESTROFT-A Cost-Effective Mass Screening Tool for the Detection of β-Thalassemia Carrier Status in Anemic Pregnant Women: A Step Toward Reducing the National Disease Burden.

Introduction β-Thalassemias are inherited hemoglobinopathies commonly encountered in practice. With chances of a promising cure being rare, the prevention of births with this disorder should assume priority, especially in low-resource countries. This can be achieved by the implementation of a mass screening program that is reliable and, at the same time, cost-effective. Objectives This study focuses on the utility of Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT) as a mass screening tool to detect thalassemia carriers. Hematological parameters that may predict carrier status were also evaluated. Materials and Methods Hemoglobin estimation was performed on all consented pregnant women. If the patient was found to have hemoglobin < 11 g/dL, the blood sample was subjected to other routine hematological tests along with peripheral smear examination. NESTROFT was performed using 0.36% saline solution. Confirmation was done using high-performance liquid chromatography (HPLC). Statistical Analysis Data obtained were tabulated using version 21 of the Statistical Package for Social Sciences. Means, standard deviations, and percentages were used to describe the sample. Chi-square test and Students’ t test were used to identify differences between the groups. Results Of 441 pregnant women enrolled, 206 were found to be anemic. Nineteen (9.2%) of the anemic pregnant women were detected to be carriers of hemoglobinopathies. Among the hematological parameters, mean red blood cell count and reticulocyte count were higher, while mean corpuscular hemoglobin concentration was lower in carriers. Also, carriers were more likely to present with microcytic hypochromic anemia. NESTROFT showed a sensitivity of 84.21%, specificity of 96.25%, a positive predictive value of 69.56%, and a negative predictive value of 98.36%. A false-positive result was seen in 3.74% of the tests, while a false negative result was seen in 15.78% of the tests. Conclusions NESTROFT (0.36%) can be used as a simple and cost-effective mass screening tool for the detection of carrier status. This should be followed by confirmation using HPLC or hemoglobin electrophoresis.

Minor Changes in Erythrocyte Osmotic Fragility in Trace Amine-Associated Receptor 5 (TAAR5) Knockout Mice.

Trace amine-associated receptors (TAARs) are a group of G protein-coupled receptors that are expressed in the olfactory epithelium, central nervous system, and periphery. TAAR family generally consists of nine types of receptors (TAAR1-9), which can detect biogenic amines. During the last 5 years, the TAAR5 receptor became one of the most intriguing receptors in this subfamily. Recent studies revealed that TAAR5 is involved not only in sensing socially relevant odors but also in the regulation of dopamine and serotonin transmission, emotional regulation, and adult neurogenesis by providing significant input from the olfactory system to the limbic brain areas. Such results indicate that future antagonistic TAAR5-based therapies may have high pharmacological potential in the field of neuropsychiatric disorders. TAAR5 is known to be expressed in leucocytes as well. To evaluate potential hematological side effects of such future treatments we analyzed several hematological parameters in mice lacking TAAR5. In these mutants, we observed minor but significant changes in the osmotic fragility test of erythrocytes and hematocrit levels. At the same time, analysis of other parameters including complete blood count and reticulocyte levels showed no significant alterations in TAAR5 knockout mice. Thus, TAAR5 gene knockout leads to minor negative changes in the erythropoiesis or eryptosis processes, and further research in that field is needed. The impact of TAAR5 deficiency on other hematological parameters seems minimal. Such negative, albeit minor, effects of TAAR5 deficiency should be taken into account during future TAAR5-based therapy development.

Red Blood Cells Fragility among Sudanese Patients with Type 2 Diabetic Mellitus

Background: Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia and has become a major public health problem globally. Diabetes is the risk factor for osmotic fragility. Objective: The aim of the present study is to describe erythrocyte osmotic fragility changes in type II diabetes. The osmotic fragility test is a measure of the resistance of erythrocytes to hemolysis by osmotic stress. Material and method: In this study 50 diabetic type 2 patients were informed about the study and parameters selected for the study are : median corpuscular fragility (MCF), hemoglobin(HB) , hemoglobin A1c(HA1c) , Fasting blood glucose (FBG) . Result: The analyzed results showed: Patients with type 2 diabetes have a significantly higher erythrocyte mechanical fragility. And that fasting blood glucose and anemia are also strongest correlate of increased mechanical fragility of erythrocytes. Conclusion: The result of FBG, HBA1c and OFT were significantly increased in diabetic’s type 2 patient (p value &lt; 0.05), furthermore hemoglobin (HB) estimation was significantly decreased in diabetic’s type 2 patient (p value &lt;0.05). The correlation between OFT and duration of type 2 DM were positive significant correlation (p value &lt; 0.05).

Evaluation of low-grade In Vitro Oxidative Stress in Goat Erythrocytes ‎Exposed to Hydrogen Peroxide and Dexamethasone ‎

Under conditions of oxidative stress, erythrocytes of goats could be predisposed to haemolysis. This study was aimed at evaluating the effect of oxidant exposure to goat erythrocytes using an in vitro model. Blood samples from 10 goats were incubated with 0.06 – 0.18 mM hydrogen peroxide (H2O2) either singly or in combination with 0.02 µM dexamethasone for 60 min, and erythrocyte parameters such as packed cell volume (PCV), red blood cell count (RBC), mean corpuscular volume (MCV) and percentage haemolysis in hypotonic sucrose solution (250 mOsmol/L) were determined thereafter. No significant changes in the mean values of all parameters were observed. The in vitro model indicated that erythrocyte parameters remained stable under low-grade oxidant exposure in goats. Therefore, plasma H2O2 concentration of ≤ 0.18 mM, whether in the presence or absence of 0.02 µM dexamethasone, may not induce apparent oxidative damage in goat erythrocytes that could be estimated by PCV, RBC, MCV and sucrose-based osmotic fragility at low hypotonicity.

Sepsis Induced Red Cell Dysfunction (SiRD): Physiology and Mechanisms

BACKGROUND: In sepsis, many red blood cell (RBC) defects are described: ↑ oxygen (O2) affinity, ↓ deformability, ↑ aggregation & adhesion, and dysregulated RBC-based vasoregulation. We suggest these comprise a unique class of organ (RBC) failure, which we term sepsis-induced RBC dysfunction (SiRD). We propose SiRD arises from RBC metabolic failure and depowered antioxidant systems - resulting in impaired RBC physiology that disables O2 transport. We employed a murine cecal ligation and puncture (CLP) sepsis model to characterize SiRD features and explore mechanisms of SiRD initiation.METHODS: Mice underwent sham (Gcont), mild (Gmild: D1 mortality 10%), or severe CLP (Gsev: D1 mortality 50%); N=4-8/group. D1 RBCs were collected. SiRD phenotype was characterized by analyzing: (1) O2 affinity (p50, Bohr effect), (2) deformability (LORRCA - elongation index, EI), (3) osmotic fragility (LORRCA - ΔOsm resilience), (4) aggregation (LORRCA - syllectogram) and (5) vasoregulation (organ chamber bioassay - RBC hypoxic vasodilaton). Mechanistic explanation for the SiRD phenotype was explored by evaluating: (6) Ca+ accumulation (flow cytometry), (7) caspase3 activation (flow cytometry), (8) cdB3 proteolysis (western blot), (9) RBC metabolomic analysis (13C1,2,3-glucose based MS, +/- exogenous oxidant stress), (10) nitric oxide content (photolysis:chemiluminescence - total RBC NO, FeNO, and SNO content), and (11) eryptosis:phosphatidylserine (PS) exposure (flow cytometry). SiRD physiologic impact was evaluated by (12) quantifying tissue O2 delivery impairment in vivo (HIF-1a bioluminescent mouse, IVIS).RESULTS: RBCs from septic mice demonstrated the following physiologic Δs: (1) ↑ O2 affinity (L-shifted HbO2 dissociation) at 3 pHs (7.2, 7.4, and 7.6, with ↓ Bohr effect), with ↑ p50 (Gsev vs Gcont) (pH7.2: 47.8±1.2* vs. 53.3±0.9 Torr; pH7.4: 37.2±1.1** vs. 42.9±0.7 Torr; and pH7.6: 29.0±0.6*** vs. 34.2±0.8 Torr); (2) ↓ RBC deformability - with similar EImax, shear stress (SS)1/2 was ↑ in Gsev vs Gcont (0.92 vs. 0.65 Pa); (3) ↑ osmotic fragility - min & max tolerated osmolality (Omin & Omax): Omin in Gcont, Gmild, and Gsev were 166.1±8.9, 158.8±4.5, and 159.1±2.8 mOsm/kg; and Omax were 336.0±21.5, 315.3±9.1, and 314.0±8.5 mOsm/kg, respectively; (4) ↑ aggregation - syllectogram backscatter intensity (Isc): in Gsev, Isc ↓ from 32.5±5.0 to 12.5±2.8 Au; but in Gcont, Isc only changed from 31.1±3.4 to 23.6±4.6 Au; (5) hypoxic vasodilation response ↓ in Gsev RBCs vs Gcont (8.9±4.2% vs 10.2±4.0%*, respectively). Exploration of potential mechanisms for SiRD revealed: (6) ↑ RBC Ca+ (Fluo3-based flow cytometry): ↑ mean florescence intensity (MFI) with sepsis severity (Gcont: 61.3±11.6; Gmild: 73.4±14.6, and Gsev: 105±36.0); (7) MIF of active caspase3 probe (CaspGlow-DEVD-FMK) did not change following CLP; however, after incubation with 0.1mM H2O2, MIF for Gsev ↑ significantly (Gcont: 549.3±28.8 vs. Gsev: 3123±782.2*); (8) ↑ cdB3 proteolysis was observed, Gsev > Gmild; (9) principal component and pathway analysis of metabolomic data identified sepsis severity-based constraints in the hexose monophosphate shunt pathway & linked antioxidant systems (NADPH and GSH); (10) RBC SNOHb & membrane SNO ↓ in Gsev vs Gcont (0.08 ± 0.04* & 0.04 ± 0.02* vs 0.22 ± 0.08 & 0.17 ± 0.07 SNOHb (SNO:Hb molar ratio) and membrane SNO mol/mg protein, respectively; (11) ↑ %age of eryptotic RBCs after CLP (annexin V-based flow cytometry): Gsev annexin V positive sub-population was 5.3±2.9%**, compared to Gcont 1.3±0.6% - following oxidative stress (0.1mM H2O2), annexin V positive sub- population in Gcont ↑ further to 11.5±4.1%; whilst Gsev, ↑ to 31.0±9.2%***. (12) O2 delivery impairment was quantified by imaging (IVIS) HIF-bioluminescent mice (FVB/Gt(ROSA)26Sor tm2(HIF1A/luc)kael): D1 normalized luciferase activity in Gsev was ↑ 3.4±0.8* fold vs baseline; a similar difference was observed after washed RBC exchange from Gsev → Gcont mice. (NB: * P<0.05; ** P<0.01; *** P<0.001)CONCLUSIONS: In sepsis, RBCs undergo major biochemical & physiologic alterations that impair O2 delivery, which we term SiRD. We propose SiRD arises from acquired RBC metabolic constraints, oxidative RBC injuries and impaired physiology, comprising a distinct form of organ (RBC) failure that may influence sepsis outcome. This well characterized model will allow further characterization, mechanistic study, and therapeutic exploration. DisclosuresDoctor:KaloCyte, Inc.: Equity Ownership, Research Funding; Washington University in St. Louis: Patents & Royalties; NIH: Research Funding; DoD: Research Funding; Biogen: Consultancy; Children's Discovery Institute: Research Funding.

Thalassemia and Hemoglobinopathies Screening by Osmotic Fragility (OF) and Dichlorophenol Indophenol Precipitation (DCIP) Tests Among Vocational Students in Ubon Ratchathani Province

Background: Thalassemia and hemoglobinopathies are the most common and clinically serious single gene disorders. Screening by osmotic fragility (OF) test and dichlorophenol indophenols precipitation (DCIP) test have been found to be effective and low-cost approaches to identify those who are carriers especially among young population.&#x0D; Objective: To study the prevalence rates of abnormal of OF test and/or abnormal DCIP test among vocational students in Ubon Ratchathani province.&#x0D; Methods: This cross-sectional research collected data from 311 students aged 15 to 19 years in a vocational school in Ubon Ratchathani, Thailand. OF and DCIP tests were done after participants signed consent to join the study. Knowledge and attitude towards thalassemia and thalassemia screening was obtained from self-administered questionnaires. Chi-square and Fisher exact tests were performed to examine the association between variables.&#x0D; Results: Of 311 students, 124 (39.9%) students had abnormal OF test or DCIP test or both, 72 (23.2%) students had both DCIP and OF tests positive. The final diagnosis was that 75.8% had hemoglobin E, 21.8% and 2.4% were α-thalassemia and β-thalassemia carriers respectively. Approximately, 91.4% of them considered thalassemia screening useful and necessary for premarital screening.&#x0D; Conclusions: High incidence of abnormal OF test and/or DCIP test was found among vocational students in Ubon Ratchathani province, showing a predominance of hemoglobin E. Most of them agreed that thalassemia screening is useful for adolescents to avoid marriage among carriers.&#x0D;

Pathophysiologic Implications of Type-2 Phosphatidylserine-Positive Erythrocytes in Hemoglobin SS Disease (sickle cell anemia) and β-Thalassemia

Background: Phosphatidylserine (PS), a cell membrane phospholipid present in the inner layer of the plasma membrane, is expressed on the surface of red blood cells (RBCs) from individuals with congenital and acquired hemoglobinopathies including sickle cell disease (SCD) and thalassemia. These cells are involved in the genesis of several pathobiologic processes including perturbed hemostasis. Based on the amount of cell surface PS, Yasin et al (Blood, 2003) have grouped PS+ RBCs into low level PS type-1 (PS1) and high level PS type-2 (PS2) cells. In SCD, the majority of transferrin receptor-positive (CD71+) stress reticulocytes (retics) are PS1+, whereas the majority of PS+ dense cells are PS2+and are specific to SCD. PS exposure on retics in subjects with Hemoglobinopathies appears to be physiologically relevant and may play a role in retic maturation. In contrast, dense cells may play a role in the pathogenesis of several SCD-related complications including skin ulcers, pulmonary hypertension and renal disease.Aim: To assess whether PS2+ red cells are present in other hemoglobinopathies besides SCD, and to evaluate which PS fraction associates with hemolysis and hemostatic activation.Methods: Sixty-one children with SCD (38 HbSS and 23 HbSC, age range 1.5 to 22 years), 23 age- and race-matched controls, and 11 with β-thalassemia were evaluated for the presence of PS+ RBCs by flow cytometry using RBCs labeled with annexin V-FITC (PS marker) and anti-glycophorin A-PE (red cell marker). The data was analyzed for PS1+ and PS2+ RBCs, and also for PS per RBC assessed as mean cell fluorescence (MCF). PS levels among groups and between groups were compared for statistical differences. Representative markers of hemostatic perturbation (D-dimer) and hemolysis (LDH) were measured, and relationships with PS1+ and PS2+ RBCs evaluated. In in vitro assays, osmotic fragility of PS2+ RBCs was tested. In a subset of SCD and controls, PS+ RBCs were further characterized using red cells labeled with annexin V-FITC and anti-CD71-PE (a stress retic marker) to delineate PS1+ and PS2+ stress retics and non-retics. Data was analyzed in HbSS based on stress retic levels (low vs high).Results: In contrast to Yasin, we found that PS2 cells were not specific for HbSS, since they were present at similar levels in subjects with β-thalassemia (Figure 1). Both PS1+ and PS2+ red cells (percent and absolute number) as also PS/cell were significantly increased in HbSS compared to HbSC and controls (Table 1). In HbSS, PS2+ RBCs correlated positively with D-dimer (p<0.02). No D-dimer correlation was found with PS1+ RBCs. Neither PS1+ nor PS2+ RBCs in HbSC correlated with D-dimer. Additional analysis demonstrated that PS2+ RBCs in HbSS correlated with the hemolytic marker LDH (p<0.001). In a complementary in vitro assay, we found that PS2+ RBCs were osmotically fragile and readily hemolyze. To confirm the previous demonstration by Yasin of minimal to no PS2+ stress retics in HbSS, we further undertook an analysis of this biomarker in HbSS subjects with low vs high stress retics. We demonstrate that while PS2+ stress retics were present minimally in subjects with low stress retics, they were significantly elevated in individuals with high stress retics (median PS2+ stress retics of 827 and 3269 /μl blood, p<0.03). A positive correlation was also noted between PS2+ stress retics and CD71+ RBCs (r=0.50, p<0.01, n=26).Conclusion: We demonstrate that PS2+ RBCs, which are elevated in HbSS compared to HbSC and controls, are not specific to SCD as they were also present at similarly elevated levels in β-thalassemia. We also demonstrate the presence of PS2+ stress retics in HbSS subjects with high retic levels. Previous studies in HbSS implicated PS+ RBCs in modulating hemostasis. Our results delineate that PS2+ red cells are the PS sub-group involved in this activation. We further demonstrate that PS2+ red cells are associated with elevated LDH levels. These cells are osmotically fragile releasing multiple inflammatory mediators including heme, which is a modulator of thrombin generation. These findings taken together suggest a pathophysiologic role for PS2+ red cells linking hemolysis with hemostasis in hemoglobinopathies including HbSS and β-thalassemia.Acknowledgments: supported by NIH grants P60HL62148, U54HL70585 and P20GM109021Data was collected at St Christopher's Hospital for Children and Thomas Jefferson University, Philadelphia PA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

GWAS of Osmotic Hemolysis in 12,352 Healthy Blood Donors Identifies Red Cell Genetic Variants Associated with Steady State Hemolysis in Patients with Sickle Cell Disease

Background: The evolutionary pressure of endemic malaria has produced highly variable genes encoding erythrocyte proteins and enzymes, many of which modulate cellular structure and function. We hypothesized that a high throughput screen of red blood cell susceptibility to osmotic hemolysis, coupled with a GWAS study, would identify rare and common genetic variants associated with cellular structure and function during red blood cell storage and in hemolytic diseases like sickle cell and malaria.Methods: The REDS III RED Blood Cell Omics (RBC-Omics) study enrolled 13,403 successful blood donors at 4 major hubs. Two labs were used for red blood cell phenotyping of leukoreduced RBC unit-derived samples stored for ~42 days under blood bank conditions. Red blood cell osmotic fragility was assayed in 12,352 donors, including 1,483 African American, 1,477 Asian and 960 Hispanic donors. DNA was isolated and genotyped using a custom Affymetrix Axiom TM-Array, which contained approximately 875,000 SNPs enriched for blood and transfusion related polymorphisms.Results: Genome wide association analysis in the complete RBC-omics data set revealed 18 genes/loci to be associated with osmotic hemolysis at a genome wide significance threshold of P<5x10-8. The genes included candidates with clear internal validity such as HBB (specifically rs344 the HbS variant) and promotor regions of HBA2 (thalassemic variants), as well as SNPs in logical candidates, such as Ankyrin, Spectrin, and Aquaporin 1, mutations in which are responsible for spherocytosis and red cell membrane water transport defects, among others. To test the potential relevance of these finding to human disease, we evaluated the top 100 regions in the RBC-Omics studies in the 852 pediatric and adult patients with sickle cell disease enrolled in the PUSH and Walk-PHASST screening studies with genome SNP data, and analyzed the association of the SNPs with the intensity of steady state hemolysis, as measured by a principle component analysis of reticulocyte count, bilirubin, lactate dehydrogenase and aspartate amino transferase. After multiple testing correction, numerous genes were associated with the intensity of hemolysis in both studies, including SNPs in red cell metabolism enzymes genes (HK1, TKTL1 MTOR signaling), ion channels (PIEZO1), as well as regulatory gene (ZNF573) and less obvious genes (FLN, SBF2, GAB3, and TMEM116).Conclusion: An analysis of the genetic variability underlying normal human red cell susceptibility to osmotic stress hemolysis identified new candidate genes that are also associated with the severity of red blood cell hemolysis during the disease stress of human SCD. We hypothesize that many of these candidate SNPs may modulate the severity of red cell hemolysis in other diseases like malaria and potential outcomes after routine red blood cell storage and transfusion. These studies highlight the discovery of a number of new metabolic and ion/water transport variants that are associated with the severity of steady state hemolysis and under disease stress. DisclosuresMast:Novo Nordisk: Research Funding. Gordeuk:Emmaus Life Sciences: Consultancy.

Antisickling effect of chrysin is associated with modulation of oxygenated and deoxygenated haemoglobin via alteration of functional chemistry and metabolic pathways of human sickle erythrocytes.

Sickle cell disease (SCD) treatment and management remain a challenging puzzle especially among developing Nations. Chrysin's sickling-suppressive properties in human sickle (SS) erythrocytes in addition to its effect on AA-genotype erythrocytes were evaluated. Sickling was induced (76%) with 2% sodium metabisulphite at 3 h. Chrysin prevented (81.19%) the sickling and reversed same (84.63%) with strong IC50s (0.0257 µM and 0.00275 µM, respectively). The levels of oxygenated haemoglobin in the two groups (before and after induction approaches) were similar but significantly (P < 0.05) higher than that of SS erythrocytes (the 'induced' control), with chrysin-treated AA-genotype showing no effects relative to the untreated. The level of deoxygenated haemoglobin in the 'induced' control group was significantly (P < 0.05) higher than those of the chrysin-treated SS erythrocytes. Normal and chrysin-untreated erythrocytes (AA-untreated) were significantly more resistant to osmotic fragility than the SS-untreated. However, treatment with chrysin significantly reduced the osmotic fragility of the cells relative to the untreated cells. Furthermore, chrysin treatment significantly lowers the high level of 2,3-diphosphoglycerate (2,3-DPG) observed in the sickle erythrocytes, with no effects on AA-genotype erythrocytes. Based on functional chemistry, chrysin treatment alters the functional groups in favour of its antisickling effects judging from the observed bends and shifts. From metabolomics analysis, it was observed that chrysin treatment favors fatty acid alkyl monoesters (FAMEs) production with concomitant shutting down-effects on selenocompound metabolism. Thus, sickling-suppressive effects of chrysin could potentially be associated with modulation of oxygenated and deoxygenated haemoglobin via alteration of human sickle erythrocyte's functional chemistry and metabolic pathways implicated in SCD crisis.

Osmotic hemolysis is a donor-specific feature of red blood cells under various storage conditions and genetic backgrounds.

Transfusion research has recently focused on the discovery of red blood cell (RBC) storage capacity biomarkers and the elucidation of donor variation effects. This shift of focus can further strengthen personalization of transfusion therapy, by revealing probable links between donor biology, RBC storage lesion profile, and posttransfusion performance. We performed a paired correlation analysis of osmotic fragility in freshly drawn RBCs and during cold storage in different preservative solutions at weekly intervals until unit's expiration date (n= 231), or following 24 h reconstitution in allogeneic plasma (n= 32) from healthy controls or transfusion-dependent beta-thalassemia patients. We observed exceptional correlation profiles (r > 0.700, p < 10-5 in most cases) of RBC osmotic fragility in the ensemble of samples, as well as in subgroups characterized by distinct genetic backgrounds (sex, beta-thalassemia traits, glucose-6-phosphate dehydrogenase deficiency) and storage strategies (additive solutions, whole blood, RBC concentrates). The mean corpuscular fragility (MCF) of fresh and stored RBCs at each storage time significantly correlated with the MCF of stored RBCs measured at all subsequent time points of the storage period (e.g., MCF values of storage day 21 correlated with those of storage days 28, 35 and 42). A similar correlation profile was also observed between the osmotic hemolysis of fresh/stored RBCs before and following in vitro reconstitution in plasma from healthy controls or beta-thalassemia patients. Our findings highlighted the potential of osmotic fragility to serve as a donor-signature on RBCs at every step of any individual transfusion chain (donor, blood product, and probably, recipient).

Effect of Ethyl Acetate Fraction of P. amarus Leaf on Hematological and Biochemical Parameters in Albino Rat with Arsenic Induced Toxicity

Therapeutic effect of ethyl acetate fraction of P. amarus leaf was probed on hematological and biochemical indices in albino rats induced with arsenic poison. Acute toxicity of 100% ethyl acetate fraction was evaluated at dose range of 100 to 5000mg/kgbw for 24hrs and no apparent signs of toxicity were observed. However, exposure for 10days, death was observed for dose of 1000mg/kg and above. Thus LD50 of sub-acute toxicity is 707.11mg/kg. Significant decreased in hematological indices observed, indicate alterations that affect the physiology and hematopoiesis of blood by arsenate administered and with normal levels of MCV and MCH, this suggest normocytic anemia. Furthermore, decrease level of HGB could implies that arsenic might have affected the incorporation of hemoglobin in the red blood cells or the morphology and osmotic fragility in red blood cells and this can affect oxygen carrying capacity of blood, although with MCV and RDW both normal, the RBCs are likely the same normal size. Falls in Platelets count with low MPV and irregular morphology as indicated by PDW infer that the bone marrow isn't producing enough new platelets which may result in low clotting capacity of blood. This is not caused by infection from bacteria as procalcitonin (PCT) in all groups showed no significant difference but the effect was that of arsenic poison. WBCs drop significantly, suggesting susceptibility to opportunistic infections. Result also showed increased bilirubin, an indication of cholestasis. However, these effects were significantly (P&lt;0.05) ameliorated in the treatment groups, as levels of these parameters were reversed. Conclusively, this might be as a result of phenolics in P. amarus. Phenolics excite their mechanism of therapeutics either by ways of antioxidant activities as free radical scavengers or chelators of metal ions.&#x0D;

Haematological profiles and erythrocyte fragility of Arabian stallions subjected to a 2000-m maximal race following administration of ergothioneine

Haematological profiles and erythrocyte fragility of Arabian stallions subjected to a 2000-m maximal race following administration of ergothioneine

Vitamin C and E Supplementation Can Mitigate NaF Mediated Hematological and Hepatic Disorders in Adult Wistar Rats

The study aimed to investigate the effect of combined vitamin E and C, on hematological and hepatic systems of adult male Wistar rats with fluoride toxicity. Eighteen rats were randomly divided into three groups such as control, treated (given NaF, 20 mg/kg bw/rat/day), and supplemented (NaF and vitamin E-200 mg/kg bw + C- 400 mg/kg bw). Altered hematological parameters like erythrocyte count, leukocyte count and hemoglobin concentration were seen in NaF groups. Estimation of osmotic fragility of erythrocyte showed a marked increase fragile erythrocytes in fluoride-treated groups compared to control and supplemented groups. Total thiol concentration was also decreased due fluoride fluoride-induced oxidative stress and it was protected in supplementary groups. The oxidative imbalance induced by NaF in liver was evaluated by estimating the activity of antioxidant enzyme superoxide dismutase and catalase and with estimation of lipid peroxidation and protein oxidation product malondialdehyde and protein carbonyl. All enzymatic and non-enzymatic markers of oxidative imbalance were significantly altered (increased/decreased) due to fluoride toxicity, which was again observed to be protected in the supplemented group. The histopathological analysis of the liver also proved the mitigatory effect of the combined vitamin E+C on fluorideinduced hepatic insult.

Inhibition of acrylamide toxicity in vivo by arginine-glucose maillard reaction products

Acrylamide has a variety of toxicities, including carcinogenicity, and can be present in food via the Maillard reaction in processing of certain foods. Previous studies have demonstrated that co-existing Maillard reaction products (MRPs) ameliorated acrylamide-induced abnormal physiological status in mice. This study is focused on the effects on hematological parameters, erythrocyte osmotic fragility, oxidative stress in plasma and liver, and contents of 8-hydroxy-2-deoxyguanosine (8-OHdG) in mice exposed to acrylamide and to acrylamide and MRPs derived from arginine and glucose. Acrylamide alone caused significant increases in liver indexes, erythrocyte osmotic fragility, malonaldehyde level in liver and 8-OHdG level in testis, and significant decreases in weight gain, hematological parameters, levels of glutathione, glutathione peroxidase and total superoxide dismutase in plasma. Whether MRPs and acrylamide were physically mixed or when the solution is prepared from heating the mixture of arginine, glucose and acrylamide, the presence of MRPs effectively reduced the adverse changes caused by acrylamide. These results suggest that the toxicity of acrylamide to mice can be ameliorated by MRPs, the common compositions simultaneously generated with acrylamide in food matrix.

Clinical Features and Outcome of Children With Hereditary Spherocytosis.

The aim of this study was to evaluate the demographics, clinical, and laboratory findings and treatment responses of patients with hereditary spherocytosis (HS). Data of children with HS were examined. Diagnosis was based on clinical history, physical examination, family history, presence of spherocytes on peripheral blood smear, and osmotic fragility test. A total of 101 patients were included. The median (range) age at diagnosis was 38.0 (1 to 188) months. Mild, moderate, and severe forms of HS were present in 29 (28.7%), 15 (14.9%), and 57 (56.4%) patients, respectively. Family history was available in 73 patients and 56 of these (76.7%) had a positive family history for HS. Forty-five (44.5%) patients needed regular transfusions and all of these had severe disease. Although most patients did not require transfusion postsplenectomy, 2 of 45 (4.4%) patients continued to require transfusion. Transfusion dependence was significantly (P<0.001) higher in patients with severe spherocytosis. In HS, splenomegaly, pallor, and jaundice are the most common clinical features. Splenectomy dramatically reduces hemolysis in most cases and virtually abolishes further requirement for transfusion.

Protein kinase A activity and NO are involved in the regulation of crucian carp (Carassius carassius) red blood cell osmotic fragility.

Activation of the cAMP pathway by β-adrenergic stimulation and cGMP pathway by activation of guanylate cyclase substantially affects red blood cell (RBC) membrane properties in mammals. However, whether similar mechanisms are involved in RBC regulation of lower vertebrates, especially teleosts, is not elucidated yet. In this study, we evaluated the effects of adenylate cyclase activation by epinephrine and forskolin, guanylate cyclase activation by sodium nitroprusside, and the role of Na+/H+-exchanger in the changes of osmotic fragility and regulatory volume decrease (RVD) response in crucian carp RBCs. Western blot analysis of protein kinase A and protein kinase G substrate phosphorylation revealed that changes in osmotic fragility were regulated via the protein kinase A, but not protein kinase G signaling pathway. At the same time, the RVD response in crucian carp RBCs was not affected either by activation of adenylate or guanylate cyclase. Adenylate cyclase/protein kinase A activation significantly decreased RBC osmotic fragility, i.e., increased cell rigidity. Inhibition of Na+/H+-exchanger by amiloride had no effect on the epinephrine-mediated decrease of RBC osmotic fragility. NO donor SNP did not activate guanylate cyclase, however affected RBCs osmotic fragility by protein kinase G-independent mechanisms. Taken together, our data demonstrated that the cAMP/PKA signaling pathway and NO are involved in the regulation of crucian carp RBC osmotic fragility, but not in RVD response. The authors confirm that the study has no clinical trial.