Osmotic Fragility Of Red Blood Cells Research Articles

Hereditary spherocytosis due to a novel variant, p.Q1034X, in the beta subunit of the spectrin gene: A case report

BackgroundHeterozygous pathogenic variants of SPTB cause hereditary spherocytosis (HS) in a quarter of cases. Case reportA 14-day-old male presenting with persistent anemia and hyperbilirubinemia was diagnosed with HS by increased red blood cell osmotic fragility and decreased fluorescence on the eosin-5′-maleimide binding test. For his failure to thrive and hypotonia, genetic sequencing revealed a de novo variant of the SPTB gene (p.Q1034X) on exon 15. This variant is predicted to cause loss of normal protein function either through protein truncation or nonsense-mediated mRNA decay. A variant of uncertain significance (p.R438W) in the chondroitin sulfate synthase 1 (CHSY1) gene was incidentally found. Loss of CHSY1 is associated with autosomal recessive Temtamy preaxial brachydactyly syndrome (TPBS). However, this patient's heterozygosity and lack of typical TPBS phenotype make this variant less likely the cause of his symptoms. ConclusionFurther investigation can evaluate a potential link between the patient's presentation and these gene variants.

Effect of mangostin on the functional activity and integrity of the <i>Plasmodium falciparum</i> infected and Uninfected red blood cells membrane

Mangostin is a natural xanthanoid obtained from various parts of the mangosteen tree Garcinia mangostana with diverse medicinal effects, such as; antioxidant, antibacterial, anticancer and anti- inflammatory. This study aimed at exploring its impact on the integrity, and the physiological function of both Plasmodium falciparum-infected and uninfected red blood cells (RBCs). This was achieved by studying its impact on the RBCs morphology, integrity and osmotic fragility. On the one hand, its effect on the plasmodium-infected cells was achieved by screening its impact on the parasite-induced new permeation pathways (NPPs) and the plasmodial invasiveness to the RBCs and these were related to its growth inhibitory effect against the parasite. On the other hand, its molecular characteristics were determined using the In Silico Molispiration simulation software and its anti-oxidant activity was measured using conventional in vitro tests and the anti-lipid peroxidation assay. The results showed that mangostin had a good growth inhibitory effect against Plasmodium falciparum with a moderate selectivity as compared to its effect against RBCs. Furthermore, it showed a good inhibitory effect against merozoite invasion and the plasmodium-induced new permeation pathway at concentrations less than the cytotoxic dose against RBCs and the parasite. Besides it succeeded in hampering the oxidative stress- induced RBCs damage without affecting the RBC fragility when it was added at low doses. Overall, the results suggest that their anti-plasmodium effect is not related to their impact on the RBCs membrane. Further studies are recommended to explore their precise mechanism of action.

Novel mutation in alpha-spectrin gene in Saudi patients with hereditary spherocytosis

Hereditary spherocytosis (HS) is the most common hereditary hemolytic disorder induced by red blood cell (RBC) membrane defect. This study was undertaken to determine mutations in genes associated with RBC membrane defect in patients with HS such as α-spectrin gene (SPTA1), β-spectrin gene (SPTB), ankyrin gene (ANK1), band 3 anion transport gene (SLC4A1) and erythrocyte membrane protein band 4.1 gene (EPB41). Blood samples were collected from 23 unrelated patients with HS. Patients were diagnosed according to the guidelines from the British Society for Hematology. All hematological examinations for the determination of RBC abnormalities and osmotic fragility tests were conducted. Genomic DNA were extracted from peripheral blood cells and coding exons of known genes for hereditary spherocytosis were enriched using Roche/KAPA sequence capture technology and sequenced on an Illumina system via next-generation sequencing (NGS). The data showed that most of the HS patients confirmed splenomegaly and showed elevated reticulocytes and abnormal bilirubin values. NGS analysis identified the heterozygous variant c.5501G > A in the exon 39 of SPTA1 gene, resulted in a Trp1834*, which leads to a premature stop codon and subsequent mRNA degradation (nonsense- mediated decay) or truncation in α spectrin. Moreover, our data also revealed conventional mutations in genes SPTB, ANK, SLC4A1 and EBP41 in severe patients of HS. In short, this is the first report that determined a novel mutation c.5501G > A in SPTA1 gene in the Saudi population. To the best of our knowledge, this variant c.5501G > A has not been described in global literature so far. This novel mutation in SPTA1 gene is unique in the Saudi population.

Red cell abnormalities characterized by ektacytometry in children with cholestasis.

Spur-cell anemia sometimes accompanies cholestasis. We postulated that even in the absence of spur-cells, cholestasis might alter red blood cell (RBC) osmotic fragility and deformability. Therefore, we assessed these RBC measures by ektacytometry in pediatric patients. We conducted a single center, prospective, cross-sectional investigation of RBC membrane characteristics by ektacytometry in pediatric patients with intra- and extrahepatic cholestasis followed at Cincinnati Children's Hospital Medical Center. We measured red cell membrane fragility and deformability in 17 patients with cholestasis and 17 age-matched controls without cholestasis. Patients with cholestasis had decreased RBC osmotic fragility compared to controls, with a significant left shift in Omin, indicating increased RBC surface-to-volume ratio. One showed spur cell morphology. However, the other 16 had no spurring, indicating that ektacytometry is a sensitive method to detect RBC membrane abnormalities. Left shift of Omin positively correlated with serum conjugated bilirubin levels and even more negatively with serum vitamin E concentration. This study suggests that subclinical red blood cell membrane abnormalities exist in most pediatric patients with cholestasis, increasing risk for hemolysis when subjected to oxidative stress. Hence minimizing pro-oxidants exposure and maximizing antioxidant exposure is advisable for this group. NCT05582447 https://clinicaltrials.gov/ct2/show/NCT05582447?cond=Cholestasis&cntry=US&state=US%3AOH&city=Cincinnati&draw=2&rank=2 . Spur cell anemia due to decreased red cell osmotic fragility and decreased deformability has been reported among patients with cholestasis. Ektacytometry is a reliable, reproducible method to measure red cell osmotic fragility and deformability. Few data describe red cell osmotic fragility or deformability in patients with cholestasis who may or may not have spur cell anemia. Ektacytometry shows that red cell osmotic fragility and deformability are decreased in many children with cholestasis even when spur cell anemia has not yet occurred. Factors associated with decreased osmotic fragility include elevated serum bilirubin, elevated serum bile acids, and decreased serum vitamin E.

Exploration of the mechanism of reinfusion of fresh autologous blood in type 2 diabetes mice to induce macrophage polarization and inhibit erythrocyte damage.

Type 2 diabetes triggers an inflammatory response that can damage red blood cells. M2 macrophages have inhibitory effects on inflammation, and play an important role in tissue damage repair and fibrosis. Autologous blood transfusion has the potential to inhibit red blood cell damage by mediating macrophage polarization. Swiss mice were used to establish a suitable type 2 diabetes model, and autologous blood transfusion was carried out. The mice were killed, the blood of the mice was collected and CD14+ monocytes were sorted. The expression levels of phenotypic molecules CD16, CD32 and CD206 in CD14+ monocytes were analyzed by flow cytometry. The proportion of M1 and M2 macrophages were analyzed by flow cytometry. The Q value, P50 , 2,3-diphosphoglycerate and Na+ -K+ -ATPase of red blood cells were detected. The red blood cell osmotic fragility test analyzed the red blood cell osmotic fragility. Western blot analysis was used to analyze the expression changes of erythrocyte surface membrane proteins or transporters erythrocyte membrane protein band 4.1, sphingosine-1-phosphate, glycolipid transfer protein and signal peptide peptidase-like 2A. Autologous blood transfusion induced a significant increase in the number of macrophages. The state and capacity of blood cells improved with autologous blood transfusion. Reinfusion of fresh autologous blood in type 2 diabetes mice made erythrocytes shrink. The expression of erythrocyte-related proteins proved that the erythrocyte injury in the reinfusion of fresh autologous blood + type 2 diabetes group was significantly reduced. The reinfusion of fresh autologous blood into the body of patients with type 2 diabetes can induce macrophage polarization to M2, thereby inhibiting red blood cell damage.

WITHDRAWN: Exploration of the mechanism of reinfusion of fresh autologous blood in type 2 diabetic body to induce macrophage polarization and inhibit erythrocyte damage

BackgroundType 2 diabetes (T2D) triggers an inflammatory response that can damage red blood cells. M2 macrophages have inhibitory effects on inflammation and play an important role in tissue damage repair and fibrosis. Autologous blood transfusion has the potential to inhibit red blood cell damage by mediating macrophage polarization. MethodsSwiss mice were used to establish a suitable type 2 diabetes model and autologous blood transfusion was performed. The mice were executed, the blood of the mice was collected, and CD14+ monocytes were sorted. The expression levels of phenotypic molecules CD16, CD32 and CD206 in CD14+ monocytes were analyzed by flow cytometry. The proportion of M1 and M2 macrophages were analyzed by flow cytometry. The Q value, P50, 2,3-DPG and Na+-K+-ATPase of red blood cells were detected. The red blood cell osmotic fragility test analyzed the RBC osmotic fragility. Western Blot analysis was used to analyze the expression changes of erythrocyte surface membrane proteins or transporters EPB41, S1P, GLTP and SPPL2A. ResultsAutologous blood transfusion induced a significant increase in the number of macrophages. The state and capacity of blood cells improved with autologous blood transfusion. Reinfusion of fresh autologous blood in T2D body made erythrocytes shrank. The expression of erythrocyte-related proteins proved that the erythrocyte injury in the Fresh+T2D group was significantly reduced. ConclusionThe reinfusion of fresh autologous blood into the body of patients with type 2 diabetes can induce macrophage polarization to M2, thereby inhibiting red blood cell damage.

Red blood cells from patients with sitosterolemia exhibit impaired membrane lipid composition and distribution and decreased deformability

Sitosterolemia is a metabolic disorder leading to excessive accumulation of phytosterols. Hemolytic stomatocytosis and macrothrombocytopenia are part of the clinical picture. However, the impact of phytosterols on red blood cell (RBC) deformability, membrane lipid composition and distribution and the efficiency of the reference treatment, Ezetimibe, are largely unknown. This study addresses these issues using RBCs from three patients with sitosterolemia and healthy RBCs exposed to β-sitosterol. Patients presented an increased proportion of stomatocytes, decreased RBC deformability and increased RBC hydration and osmotic fragility compared to healthy donors. At the membrane level, patient RBCs showed (i) very high content in β-sitosterols, (ii) increased proportions of saturated fatty acids and polyunsaturated fatty acid species with long and unsaturated carbon chains, and (iii) decreased content in phosphatidylethanolamine species. These lipid changes were accompanied by an almost complete abrogation of cholesterol-enriched domains, which could result from: (i) the reduced phosphatidylethanolamine content which positively correlated with domain abundance; and (ii) the fatty acid modifications and increased phytosterol content, both compatible with higher membrane stiffness. The role of β-sitosterol was supported by comparable changes in RBC morphology and cholesterol-enriched domains upon β-sitosterol integration at the healthy RBC membrane. Finally, Ezetimibe treatment combined with a sterol restricted diet lowered phytosterols and improved anemia and RBC deformability and hydration. However, this treatment had no or limited effect on RBC morphology and cholesterol-enriched domain abundance. This study reveals for the first time that phytosterols affect RBC membrane lipid composition and distribution but also RBC morphology, hydration, deformability and fragility.

Effects of Dietary Glycine Supplementation on Growth Performance, Immunological, and Erythrocyte Antioxidant Parameters in Common Carp, Cyprinus carpio.

The effects of dietary glycine supplementation, 0 (control), 5 (5 GL), and 10 (10 GL) g/kg, have been investigated on growth performance, hematological parameters, erythrocyte antioxidant capacity, humoral and mucosal immunity in common carp, Cyprinus carpio. After eight weeks feeding, the 5 GL treatment exhibited significant improvement in growth performance and feed efficacy, compared to the control treatment. Red blood cell (RBC) and white blood cell (WBC) counts, hemoglobin, hematocrit, neutrophil and monocyte counts/percentages, RBC reduced glutathione (GSH) content, and skin mucosal alkaline phosphatase, peroxidase, protease, and lysozyme activities were similar in the glycine-treated fish and significantly higher than the control treatment. Blood lymphocyte percentage decreased in the glycine-treated fish, but lymphocyte count increased, compared to the control fish. RBC glutathione reductase activities in the glycine-treated fish were similar and significantly lower than the control treatment. The highest plasma lysozyme and alternative complement activities were observed in GL treatment. The glycine-treated fish, particularly 5 GL, exhibited significant improvement in RBC osmotic fragility resistance. Dietary glycine had no significant effects on RBC glutathione peroxidase activity, plasma immunoglobulin, eosinophil percentage/count, and hematological indices. In conclusion, most of the benefits of dietary glycine supplementation may be mediated by increased glutathione synthesis and antioxidant power.

Corpuscular Fragility and Metabolic Aspects of Freshly Drawn Beta-Thalassemia Minor RBCs Impact Their Physiology and Performance Post Transfusion: A Triangular Correlation Analysis In Vitro and In Vivo.

The clarification of donor variation effects upon red blood cell (RBC) storage lesion and transfusion efficacy may open new ways for donor–recipient matching optimization. We hereby propose a “triangular” strategy for studying the links comprising the transfusion chain—donor, blood product, recipient—as exemplified in two cohorts of control and beta-thalassemia minor (βThal+) donors (n = 18 each). It was unraveled that RBC osmotic fragility and caspase-like proteasomal activity can link both donor cohorts to post-storage states. In the case of heterozygotes, the geometry, size and intrinsic low RBC fragility might be lying behind their higher post-storage resistance to lysis and recovery in mice. Moreover, energy-related molecules (e.g., phosphocreatine) and purine metabolism factors (IMP, hypoxanthine) were specifically linked to lower post-storage hemolysis and phosphatidylserine exposure. The latter was also ameliorated by antioxidants, such as urate. Finally, higher proteasomal conservation across the transfusion chain was observed in heterozygotes compared to control donors. The proposed “triangularity model” can be (a) expanded to additional donor/recipient backgrounds, (b) enriched by big data, especially in the post-transfusion state and (c) fuel targeted experiments in order to discover new quality biomarkers and design more personalized transfusion medicine schemes.

Dehydrated Hereditary Stomatocytosis: A Rare Inherited Hemolytic Anemia

Background: Dehydrated hereditary stomatocytosis (DHSt), also known as hereditary xerocytosis, is a rare congenital hemolytic anemia with an autosomal dominant inheritance. It is often misdiagnosed for other hemolytic conditions, such as hereditary spherocytosis. Herein, we present the case of a young female presenting with hemolytic anemia, who was found to have a mutation in PIEZO1 gene and was subsequently diagnosed with DHSt.Case Presentation: A 39-year-old woman of Asian origin presented to the hematology clinic for evaluation of anemia diagnosed on blood work performed by primary care physician for symptoms of fatigue. She was adopted and had no information about her family history. A complete blood count revealed: hemoglobin 8.2 g/dL, mean corpuscular volume (MCV) 121.6 fL, absolute retic counts 0.08 M/mcL, lactate dehydrogenase 851 units/L and a negative coombs test. Iron profile revealed iron saturation 85% and ferritin 1961.4 ng/mL (see table 1 for laboratory work up). The peripheral blood smear showed anisopoikilocytosis, macrocytes, spherocytes and several stomatocytes along with polychromatophils. An ultrasound of the abdomen was subsequently performed, and which revealed hepatomegaly and biliary stones. Enzyme assay for glucose-6-phosphate dehydrogenase and flow cytometry for paroxysmal nocturnal hemoglobinuria were also sent and were negative. Red blood cells osmotic fragility was decreased. The bone marrow biopsy showed full spectrum trilineage hematopoiesis with no mutations on molecular testing. Based on the blood smear and clinical presentation, a diagnosis of DHSt was suspected. Genetic testing was performed and which revealed Sc.2842C>T; p.Arg948Cys mutation in the PIEZO1 gene by massively parallel sequencing and confirmation by Sanger sequencing. This confirmed the diagnosis of DHSt. Patient was started on high dose folic acid with improvement in her hemoglobin in one month. She did not require any blood transfusions. MRI liver T2* scan measured quantitative liver iron of 31 mM/g, which was at the high normal range.Discussion: DHSt is caused by gain of function mutation in PIEZO1 gene or KCCN4 gene which encode the transmembrane cation ion channel and Gardo's channel respectively on red blood cell membrane. This results in delayed inactivation of the channel. The disease presents as a spectrum from asymptomatic anemia to massive hemolysis, and many patients present later in life. Patients may manifest clinical signs of jaundice, pallor, fatigue, splenomegaly, gallstones and iron overload. Labs are typically significant for elevation in mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW) and MCV, with classic slit cells red blood cells seen on peripheral blood smear (see image 1). PIEZO1 is expressed early in erythroid progenitor cells and may delay erythroid differentiation and reticulocyte maturation, which may be the cause of low reticulocyte count such as in our patient. While treatment is supportive with blood transfusions, only a minority of DHSt patients ever require regular transfusions. Interestingly, hyperferritinemia, high transferrin saturation or clinical iron overload are quite frequent in DHSt and iron chelation is recommended. Splenectomy is contraindicated due to increased risk of thrombosis.Conclusions: DHSt as a rare inherited hemolytic anemia and its diagnosis warrants maintaining a high index of clinical suspicion based on supportive laboratory findings. Diagnosis involves thorough testing earlier in the disease as patients may be asymptomatic until adulthood. Delaying the diagnosis may lead to severe iron overload and consequent organ damage. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Jaundice in a Preterm Infant Twelve Hours After Birth.

Jaundice in a Preterm Infant Twelve Hours After Birth.

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.

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).

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.

Impaired Cytoskeletal and Membrane Biophysical Properties of Acanthocytes in Hypobetalipoproteinemia - A Case Study.

Familial hypobetalipoproteinemia is a metabolic disorder mainly caused by mutations in the apolipoprotein B gene. In its homozygous form it can lead without treatment to severe ophthalmological and neurological manifestations. In contrast, the heterozygous form is generally asymptomatic but associated with a low risk of cardiovascular disease. Acanthocytes or thorny red blood cells (RBCs) are described for both forms of the disease. However, those morphological changes are poorly characterized and their potential consequences for RBC functionality are not understood. Thus, in the present study, we asked whether, to what extent and how acanthocytes from a patient with heterozygous familial hypobetalipoproteinemia could exhibit altered RBC functionality. Acanthocytes represented 50% of the total RBC population and contained mitoTracker-positive surface patches, indicating the presence of mitochondrial fragments. While RBC osmotic fragility, calcium content and ATP homeostasis were preserved, a slight decrease of RBC deformability combined with an increase of intracellular free reactive oxygen species were observed. The spectrin cytoskeleton was altered, showing a lower density and an enrichment in patches. At the membrane level, no obvious modification of the RBC membrane fatty acids nor of the cholesterol content were detected but the ceramide species were all increased. Membrane stiffness and curvature were also increased whereas transversal asymmetry was preserved. In contrast, lateral asymmetry was highly impaired showing: (i) increased abundance and decreased functionality of sphingomyelin-enriched domains; (ii) cholesterol enrichment in spicules; and (iii) ceramide enrichment in patches. We propose that oxidative stress induces cytoskeletal alterations, leading to increased membrane stiffness and curvature and impaired lipid lateral distribution in domains and spicules. In addition, ceramide- and spectrin-enriched patches could result from a RBC maturation defect. Altogether, the data indicate that acanthocytes are associated with cytoskeletal and membrane lipid lateral asymmetry alterations, while deformability is only mildly impaired. In addition, familial hypobetalipoproteinemia might also affect RBC precursors leading to disturbed RBC maturation. This study paves the way for the potential use of membrane biophysics and lipid vital imaging as new methods for diagnosis of RBC disorders.

Race as Determinant of Red Blood Cell Osmotic Stress Haemolysis in South Indian and African Populations

The study was to compare the red blood cell osmotic fragility between South Indian and African ethnicities. A cross sectional study was performed in the Department of Physiology, Kasturba Medical College, Manipal. The study involved apparently healthy young adults between 18 to 40 years old. The whole study's total sample size was 50 healthy individuals, 25 South Indians (13 female and 12 male) and 25 Africans (13 female and 12 male). The results showed an increase in the percentage haemolysis that was statistically significant (p=0.0001) in South Indians (32.16 ±5.048%) compared to Africans (20.01 ± 3.151%), respectively. The present study has identified and quantified race's impact as one of the determinant factors of red blood cell osmotic stress haemolysis. Therefore, we conclude that the percentage of haemolysis is higher for the South Indian population than the African population.

Red Blood Cell Morphodynamics: A New Potential Marker in High-Risk Patients.

In the last years, a substantial contribution of red blood cells (RBCs) in cardiovascular homeostasis has been evidenced, as these cells are able to regulate cardiovascular function by the export of adenosine triphosphate and nitric oxide as well as to maintain redox balance through a well-developed antioxidant system. Recently a link between high-risk plaque (HRP) features and myocardial ischemia, in the absence of severe lumen stenosis, has been evidenced. Nonobstructive coronary artery disease (nonob CAD) has been associated in fact with a greater 1-year risk of myocardial infarction and all-cause mortality compared with no apparent CAD. This new evidence increases interest in searching new triggers to identify these high-risk patients, in the absence/or on top of traditional hazard markers. In this study, we investigated the existence of any association between RBC morphodynamics and HRP features in individuals with different grades of coronary stenosis detected by coronary computed tomography angiography (CCTA). Ninety-one consecutive individuals who underwent CCTA [33 no CAD; 26 nonobstructive (nonob), and 32 obstructive (ob) CAD] were enrolled. RBC morphodynamic features, i.e., RBC aggregability and deformability, were analyzed by means of Laser Assisted Optical Rotation Cell Analyzer (LoRRca MaxSis). The putative global RBC morphodynamic (RMD) score and the related risk chart, associating the extent of HRP (e.g., the non-calcified plaque volume) with both the RMD score and the max % stenosis were computed. In nonob CAD group only positive correlations between RBC rigidity, osmotic fragility or aggregability and HRP features (plaque necrotic core, fibro-fatty and fibro-fatty plus necrotic core plaque volumes) were highlighted. Interestingly, in this patient cohort three of these RBC morphodynamic features result to be independent predictors of the presence of non-calcified plaque volume in this patients group. The risk chart created shows that only in nonob CAD plaque vulnerability increases according to the score quartile. Findings of this work, by evidencing the association between erythrocyte morphodynamic characteristics assessed by LoRRca and plaque instability in a high-risk cohort of nonob CAD, suggest the use of these blood cell features in the identification of high-risk patients, in the absence of severe coronary stenosis.

Parathyroid Hormone as a Marker for the Red Cell Fragility in Different Stages of Chronic Kidney Disease

Objective: The aim of the work is to study the relationship between Red blood cell osmotic fragility and level of parathyroid hormone in patients with different stages of Chronic Kidney Disease including End Stage Renal Disease. Background: Anaemia is one of the common complications associated with Chronic Kidney Disease (CKD) responsible for the increase in the morbidity and mortality in such patients. Several factors have been attributed to causing renal anaemia, amongst which hyperparathyroidism is one of the less recognised reasons. The level of PTH in early stages of chronic kidney disease has not been much studied. The excess amount of Parathyroid Hormone (PTH) secondary to CKD has been suggested to be a causative factor for anaemia. Method: A number of chronic kidney disease patients were studied for the relationship between Red cell osmotic fragility and level of parathyroid hormone. Results: This study was conducted on a number of 111 patients with chronic kidney disease classified into three groups. The study revealed a significant fall in Hb%, along with a rise in Median Osmotic Fragility (MOF) and PTH in the CKD patients. iPTH and MOF were significantly lower in group 3 as compared with cases in group 1. Also, iPTH and MOF were significantly lower in cases in group 2 as compared with cases in group 1. Conclusions: Based on our findings, secondary hyperparathyroidism has considerable effects on erythrocyte survival, contributing to increased fragility and anemia.

Sex-related aspects of the red blood cell storage lesion.

Several factors contribute to the manifestation of red blood cell (RBC) storage lesions, with one of the most interesting being the "donor variation effect". Since many haematological characteristics of blood donors are sex-dependent, sex hormones and their age-dependent variation may affect the storage profile of RBCs. Fresh blood from 200 healthy male and female donors underwent haematological, biochemical and physiological analysis. Three selected groups of donors (men, n=8; pre-menopausal women, n=8; and post-menopausal women, n=4) exhibiting as similar as possible baseline values were recruited for blood donation in leukoreduced CPD/SAGM units. RBC indices, haemolysis and propensity for haemolysis, reactive oxygen species (ROS) and plasma antioxidant capacity were measured bi-weekly. Female blood was characterised by lower plasma antioxidant capacity and free haemoglobin (Hb) levels in vivo, in spite of the higher RBC osmotic fragility, compared to male blood. Comparatively low Hb concentration was also measured in stored RBCs from female donors, as in vivo. Mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), and plasma antioxidant capacity were also lower in female donors throughout storage, even though baseline levels were equal to those of the male group. There was no difference in propensity of stored RBCs for haemolysis between male and female units but intracellular ROS levels were significantly lower in female RBCs. Increased end-of-storage extracellular potassium and recruitment of protein stress markers (clusterin, Hb) to the RBC membrane were observed in the units of post- vs pre-menopausal female donors at mid-storage onwards. Donor's sex has an impact on Hb concentration and redox parameters of stored RBCs. In addition, menopause seems to promote RBC membrane remodelling, at least during prolonged storage. Our pilot study provides new insights on the different effects on RBC storage lesion according to sex.

Red blood cell osmotic fragility in healthy loggerhead and green sea turtles.

Loggerhead (Caretta caretta; Cc) and green sea (Chelonia mydas; Cm) turtles admitted to rehabilitation facilities may require blood transfusions for supportive treatment of disorders resulting in life-threatening anemia, but, considering the unique erythrocyte chemistry of sea turtles, standardized donor red blood cell (RBC) storage protocols have not been established. Prolonged cold storage and the effects of various anticoagulant-preservative solutions have been associated with increased RBC osmotic fragility across a broad range of species. Increased RBC fragility in stored RBC products has been associated with acute transfusion reactions. The osmotic fragility test is used to measure erythrocyte resistance to hemolysis while being exposed to a series of dilutions of a saline solution. We obtained baseline measurements for osmotic fragility in healthy Cc and Cm. Osmotic fragility testing was performed on samples from 10 Cc to 10 Cm. Fifty percent (50%) RBC hemolysis was identified at a mean NaCl concentration of 0.38% in both species. Results of our study will help guide future studies evaluating optimal storage solutions for sea turtle blood products.