Changes In Morphology Of Red Blood Cells Research Articles

Pursuing the elusive footsteps of malaria in peripheral blood smears utilizing artificial intelligence.

For over a century, the need to identify malaria in the peripheral blood has been the driving force behind the development of fundamental clinical microscopy techniques. In the study by Moysis etal., artificial intelligence-based model was utilized to identify and provide quantitative morphological characteristics of red blood cells typical to severe malaria anaemia, irrespective to the actual presence of visible parasites. Commentary on: Moysis etal. Leveraging deep learning for detecting red blood cell morphological changes in blood films from children with severe malaria anaemia. Br J Haematol 2024;205:699-710.

Leveraging deep learning for detecting red blood cell morphological changes in blood films from children with severe malaria anaemia.

In sub-Saharan Africa, acute-onset severe malaria anaemia (SMA) is a critical challenge, particularly affecting children under five. The acute drop in haematocrit in SMA is thought to be driven by an increased phagocytotic pathological process in the spleen, leading to the presence of distinct red blood cells (RBCs) with altered morphological characteristics. We hypothesized that these RBCs could be detected systematically and at scale in peripheral blood films (PBFs) by harnessing the capabilities of deep learning models. Assessment of PBFs by a microscopist does not scale for this task and is subject to variability. Here we introduce a deep learning model, leveraging a weakly supervised Multiple Instance Learning framework, to Identify SMA (MILISMA) through the presence of morphologically changed RBCs. MILISMA achieved a classification accuracy of 83% (receiver operating characteristic area under the curve [AUC] of 87%; precision-recall AUC of 76%). More importantly, MILISMA's capabilities extend to identifying statistically significant morphological distinctions (p < 0.01) in RBCs descriptors. Our findings are enriched by visual analyses, which underscore the unique morphological features of SMA-affected RBCs when compared to non-SMA cells. This model aided detection and characterization of RBC alterations could enhance the understanding of SMA's pathology and refine SMA diagnostic and prognostic evaluation processes at scale.

Analysis of non-physiological shear stress-induced red blood cell trauma across different clinical support conditions of the blood pump.

Systematic research into device-induced red blood cell (RBC) damage beyond hemolysis, including correlations between hemolysis and RBC-derived extracellular vesicles, remains limited. This study investigated non-physiological shear stress-induced RBC damage and changes in related biochemical indicators under two blood pump clinical support conditions. Pressure heads of 100 and 350mmHg, numerical simulation methods, and two in vitro loops were utilized to analyze the shear stress and changes in RBC morphology, hemolysis, biochemistry, metabolism, and oxidative stress. The blood pump created higher shear stress in the 350-mmHg condition than in the 100-mmHg condition. With prolonged blood pump operation, plasma-free hemoglobin and cholesterol increased, whereas plasma glucose and nitric oxide decreased in both loops. Notably, plasma iron and triglyceride concentrations increased only in the 350-mmHg condition. The RBC count and morphology, plasma lactic dehydrogenase, and oxidative stress across loops did not differ significantly. Plasma extracellular vesicles, including RBC-derived microparticles, increased significantly at 600min in both loops. Hemolysis correlated with plasma triglyceride, cholesterol, glucose, and nitric oxide levels. Shear stress, but not oxidative stress, was the main cause of RBC damage. Hemolysis alone inadequately reflects overall blood pump-induced RBC damage, suggesting the need for additional biomarkers for comprehensive assessments.

Red Blood Cell Deformability and Morphological Dynamic Sickling As a Metric to Monitor Pharmacodynamic Response to Voxelotor

The recent introduction of new therapies, such as hemoglobin (Hb) oxygen affinity modifiers, fetal Hb (HbF) inducers, and gene therapy (replacement of HbS with non-sickling Hb), that affect the Hb polymerization rate for sickle cell disease (SCD), highlight the need for new metrics to assess changes in sickling characteristics as therapeutic response. Primary outcomes in clinical trials often focus on indirect clinical improvement i.e. decrease in vaso-occlusive events (VOC), Hb increase, fatigue and pain score. There is currently a lack of direct biomarkers to assess the pharmacodynamic impact of these therapies. Two methods have been developed to assess downstream Hb polymerization, LoRRca (oxygenscan), a commercial oxygen gradient ektacytometry that measures deformability as a function of oxygen tension (pO 2), and dynamic sickling assay (DSA), an enzymatic oxygen scavenging system that directly assesses morphological sickling. In the present study, we investigate the relationship between red blood cell (RBC) deformability and kinetics of sickling morphology after a treatment with different doses of voxelotor (GBT440, Global Blood Therapeutics), the only FDA approved Hb oxygen modifier. Blood samples from eight SCD patients were collected following the IRB #041718MP2E &amp; IRB #Pro00066220 protocols. RBCs were incubated with voxelotor at five different doses based on sample hematocrit (Hct): 25% (0.32 ± 0.06 mM), 50% (0.65 ± 0.11 mM), 75% (0.97 ± 0.17 mM), 100% (1.29 ± 0.23 mM) and 125% (1.62 ± 0.28 mM), or with an equal volume of DMSO-containing buffer (vehicle). Oxygenscan is performed (LoRRca, RR Mechatronics, The Netherlands) as per manufacturer's instructions. The following parameters are calculated: i) elongation index (EImin), measured at minimum RBC deformability, ii) point of sickling (PoS), the pO 2 at which RBC deformability decreases below 5% of EImax during deoxygenation and iii) ΔEI, the difference between EImin and EImax. DSA uses an enzymatic reaction that allows for tight control of the rate and depth of hypoxia, resulting in RBC morphological changes observed through time-lapse photography. Sickling profiles were constructed to represent: i) morphological points of sickling at 50% of induced fraction (mPoS i@50%), ii) rate of sickling at 50% of induced fraction (rate i@50%) and iii) area under the curve at 15 minutes (AUC 15). Paired t-test analysis of the LoRRca elongation curves show a significant dose response in Elmin (Vehicle: 0.16 ± 0.16; 25%: 0.20 ± 0.16, p=0.0033; 50%: 0.23 ± 0.15, p=0.0001; 75%: 0.27 ± 0.15, p=0.0004; 100%: 0.30 ± 0.14, p=0.0009; 125%: 0.35 ± 0.13, p=0.0015), ΔEl (Vehicle: 0.26 ± 0.11; 25%: 0.22 ± 0.11, p=0.0016; 50%: 0.18 ± 0.11, p=0.0003; 75%: 0.15 ± 0.08, p=0.0002; 100%: 0.11 ± 0.06, p=0.0004; 125%: 0.09 ± 0.07, p=0.0011) and PoS (Figure 1; Vehicle: 35 ± 11 mmHg; 25%: 32 ± 6, p=0.0092; 50%: 28 ± 7, p=0.0038; 75%: 25 ± 6, p&amp;lt;0.0001; 100%: 17 ± 5, p=0.0007; 125%: 14 ± 4, p=0.0015). These findings indicate improvements in RBC deformability under hypoxic conditions when the drug is present. Furthermore, paired t-test of DSA data showed a significant dose response with AUC 15 (Vehicle: 958 ± 104; 25%: 909 ± 105, p=0.0061; 50%: 845 ± 146, p=0.0004; 75%: 768 ± 194, p=0.0014; 100%: 672 ± 179, p&amp;lt;0.0001; 125%: 623 ± 192, p=0.0002), rate i@50% (Vehicle: 0.87 ± 0.26; 25%: 0.77 ± 0.30, p=0.1236; 50%: 0.61 ± 0.29, p=0.0003; 75%: 0.33 ± 0.13, p&amp;lt;0.0001; 100%: 0.28 ± 0.10, p&amp;lt;0.0001; 125%: 0.25 ± 0.09, p=0.0004) and mPoS i@50% (Figure 2; Vehicle: 4.4 ± 0.7 minutes; 25%: 4.7 ± 0.9, p=0.0260; 50%: 5.0 ± 1.2, p=0.0407; 75%: 5.9 ± 1.7, p=0.0082; 100%: 7.0 ± 1.6, p=0.0002; 125%: 7.8 ± 1.8, p=0.0004). These results indicate a delay in morphological sickling due to drug treatment. Based on the results of our study there is a dose-dependent decrease in RBC deformability and in the kinetics of morphologic sickling based of DSA following voxelotor treatment. These two methods have demonstrated a comparable ability to assess downstream therapeutic effects on delaying/inhibiting HbS polymerization. The combination of these two unique properties, RBC deformability and morphology change, can possibly provide a comprehensive approach to describe the cellular level of phenotypes, which may predict SCD severity. With further development these complementary assays have the potential to become important tools for drug development and monitoring response to SCD therapies.

Assessing Real-Time Sickling Kinetics in Individual Cells from Erythrocyte Populations of Subjects with Different HbA, HbS and HbF Percentages

INTRODUCTION: Polymerization of deoxygenated sickle hemoglobin (HbS) leads to erythrocyte sickling. Treatment in sickle cell disease (SCD) aims to reduce the risks of red blood cell (RBC) sickling and associated complications including, but not limited to vaso-occlusive crises (VOC). There is substantial promise in developing therapies targeting HbS polymerization that consequently decrease RBC sickling, a fundamental pathophysiological driver of SCD. Nonetheless, there are limited options available for reliable diagnostic tools for the assessment of disease status and therapeutic outcomes. A robust and easy-to-use system to assess population-scale sickling kinetics and to monitor responses to Hb-modifying therapies in clinical trials for the clinical management of patients may be beneficial. We have developed a dynamic sickling assay (DSA) that uses an enzymatic oxygen scavenging system to provide tight control over the rate and depth of induced-hypoxia. DSA is able to differentiate samples based on endogenous Hb expression and can be used to monitor sickling kinetics pre- and post HbS-modifying therapies, including hematopoietic stem cell transplantation (HSCT). METHODS: SCD and sickle cell trait (SCT) subjects were recruited under the RBC Health Initiative IRB protocol number FF-RBC-003. Four hematopoietic stem cell transplant (HSCT) SCD subjects were enrolled in NIH haploidentical HSCT protocol 17-H-0069. Diluted blood samples were supplemented with protocatechuic acid (PCA), followed by protocatechuate 3,4-dioxygenase (PCD) enzyme inducing hypoxia at a predetermined rate. The sample was then injected into a microfluidic chamber, and resulting RBC morphological changes were observed and captured with time-lapse photography. Image analysis identified and quantified RBC using an AI-trained SICKLE image analysis software. HbAS (n=3), HbSS with HbA (post-transfusion, n=3) and HbSS samples containing 3.1 to 34.5% HbF (n=9) were processed with DSA to assess the effects of hemoglobin distribution on sickling kinetics.Pre- and post-HSCT samples were run to ascertain the effect on sickling kinetics at baseline, 3M, 6M and 12M post-HSCT. Sickling profiles were constructed enabling a comprehensive assessment of RBC sickling and treatment outcomes. The reported parameters include: mPOS@5% (time to reach 5% induced sickling, min), mPOS@50% (time to reach 50% maximum induced sickling,min), rate of sickling (the maximum sickling rate, %/min), maximum induced sickling (the highest induced sickling percentage, %), AUC at 10min (area under the curve@10min of DSA, %∙minute) RESULTS: DSA inter-run CVs are 6.9% for mPOS@5%, 3.8% for mPOS@50%, 2.7% for max sickling, 4.2% for AUC and 10.8% for the rate of sickling. Statistically significant correlations are observed between %HbS and rate of sickling, max sickling, mPOS@5%, and AUC at 10min. As percentages of Hb increased in transfused SCD patient samples, there were noticeable correlations between the delay in mPOS@5% and decreased rate of sickling, maximum sickling, and AUC10. Similarly, as HbA percentages increased in SCT samples, a decrease in AUC10 and max sickling were observed. Transfused and SCT samples did not show statistical significance due to sample size limitations. However, there was a strong correlation in delay of mPOS@5% and mPOS@50%, as well as a decrease in the rate of sickling and AUC10 with increasing percentages of HbF in samples (Table 1). Comparing pre-HSCT (BL) sample sickling kinetics to 3, 6 and 12 month post-HSCT samples showed decreases in the rate of sickling (from 44.4 to 0.6%), maximum sickling (from 45-23%), decreases in AUC and delays in mPOS (Table 2). CONCLUSIONS: This study found significant linear relationships between %HbS and sickling parameters.As HbA percentage increases in transfused SCD and SCT samples, improvements in sickling parameters are observed. There is a strong correlation between increased HbF percentage and improved DSA parameters. Comparative data analysis from patients pre- and post-HSCT also show improvement in sickling parameters. Overall, DSA provides a simple, robust method for assessing responses to Hb differences and monitoring SCD therapies such as gene-editing.

Bisphenol S remodels red blood cell membrane lipids by altering plasma lipid levels, causing the risk of venous thrombosis in SD rats and zebrafish embryos

Bisphenol S (BPS) is a raw material that is used extensively in various manufacturing processes but possesses a high detection rate in human red blood cells (RBCs). Accordingly, BPS is a potential toxicant in disturbing the function of RBCs and causing RBC-related diseases. To date, the effects and mechanisms of BPS-induced RBC-related diseases have not been elucidated. Here, using different models, including rats, zebrafish embryos and RBCs, the underlying mechanism of RBC-related diseases induced by BPS was explored. The accumulation of BPS in tissue was colon > kidney > liver > plasma > testicle > heart > brain in SD rats orally administered BPS (10 and 50 mg/kg bw/day) for 32 days, which was similar in both 10 mg/kg bw/day and 50 mg/kg bw/day group. Rats given BPS orally developed hyperlipidemia and increased RBC membrane cholesterol, as well as changes in RBC morphology and function. Moreover, BPS at the concentrations measured in rats plasma caused oxidative stress and phosphatidylserine exposure in vitro RBCs. These combined factors led to RBC aggregation in blood and an increasing in the number of RBCs in the blood vessels of the liver in rats. The dynamic visual observation of RBCs in vein vessels of zebrafish embryos exposed to BPS at 0, 1, 10 and 100 μg/L further found that the flow of RBCs in the tail vein is slow or even immobile, posing the risk of venous thrombosis. The present study provides new insight into the links between environmental pollutants and venous thrombosis.

A novel missense variant in ATP11C is associated with reduced red blood cell phosphatidylserine flippase activity and mild hereditary hemolytic anemia.

Adenosine Triphosphatase (ATPase) Phospholipid Transporting 11C gene (ATP11C) encodes the major phosphatidylserine (PS) flippase in human red blood cells (RBCs). Flippases actively transport phospholipids (e.g., PS) from the outer to the inner leaflet to establish and maintain phospholipid asymmetry of the lipid bilayer of cell membranes. This asymmetry is crucial for survival since externalized PS triggers phagocytosis by splenic macrophages. Here we report on pathophysiological consequences of decreased flippase activity, prompted by a patient with hemolytic anemia and hemizygosity for a novel c.2365C > T p.(Leu789Phe) missense variant in ATP11C. ATP11C protein expression was strongly reduced by 58% in patient-derived RBC ghosts. Furthermore, functional characterization showed only 26% PS flippase activity. These results were confirmed by recombinant mutant ATP11C protein expression in HEK293T cells, which was decreased to 27% compared to wild type, whereas PS-stimulated ATPase activity was decreased by 57%. Patient RBCs showed a mild increase in PS surface exposure when compared to control RBCs, which further increased in the most dense RBCs after RBC storage stress. The increase in PS was not due to higher global membrane content of PS or other phospholipids. In contrast, membrane lipid lateral distribution showed increased abundance of cholesterol-enriched domains in RBC low curvature areas. Finally, more dense RBCs and subtle changes in RBC morphology under flow hint toward alterations in flow behavior of ATP11C-deficient RBCs. Altogether, ATP11C deficiency is the likely cause of hemolytic anemia in our patient, thereby underlining the physiological role and relevance of this flippase in human RBCs.

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.

Red Blood Cell Deformation and Progressive Anemia Following Therapeutic Intervention in Patients With Adult T-Cell Leukemia/Lymphoma.

During therapeutic intervention for adult T-cell leukemia-lymphoma (ATLL), transient red blood cell (RBC) deformations and rapid anemia progression are often observed. These RBC responses are characteristically observed during the treatment of ATLL, and we examined the details and significance of these RBC responses. Seventeen patients withATLL were enrolled. Peripheral blood smears and laboratory findings were collected during the first two weeks after treatment intervention. We examined the transition of erythrocyte morphology and the factors associated with the induction of anemia. RBC abnormalities (i.e., elliptocytes, anisocytosis, and schistocytes) rapidly progressed following therapeutic intervention in five of the six cases for whom evaluable consecutive blood smears were available, with significant improvement evident after two weeks. Changes in RBC morphology were significantly associated with the red cell distribution width (RDW). Laboratory findings from all 17 patients showed various levels of anemia progression. A transient increase in RDW values was observed in 11 cases after therapeutic intervention. The degree of progressive anemia during the two-week period was significantly correlated with increased lactate dehydrogenase and soluble interleukin-2 receptor levels and an increase in RDW (P <0.01). In cases of ATLL, transient progression of RBC morphological abnormalities and RDW value were observed early after therapeutic intervention. These RBC responses may be associated with tumor and tissue destruction. RBC morphology or RDW values may provide important information about the tumor dynamics and general condition of the patients.

Topological Relationships Cytoskeleton-Membrane Nanosurface-Morphology as a Basic Mechanism of Total Disorders of RBC Structures.

The state of red blood cells (RBCs) and their functional possibilities depend on the structural organization of the membranes. Cell morphology and membrane nanostructure are compositionally and functionally related to the cytoskeleton network. In this work, the influence of agents (hemin, endogenous oxidation during storage of packed RBCs, ultraviolet (UV) radiation, temperature, and potential of hydrogen (pH) changes) on the relationships between cytoskeleton destruction, membrane nanostructure, and RBC morphology was observed by atomic force microscope. It was shown that the influence of factors of a physical and biochemical nature causes structural rearrangements in RBCs at all levels of organization, forming a unified mechanism of disturbances in relationships “cytoskeleton-membrane nanosurface-cell morphology”. Filament ruptures and, consequently, large cytoskeleton pores appeared. The pores caused membrane topological defects in the form of separate grain domains. Increasing loading doses led to an increase in the number of large cytoskeleton pores and defects and their fusion at the membrane nanosurfaces. This caused the changes in RBC morphology. Our results can be used in molecular cell biology, membrane biophysics, and in fundamental and practical medicine.

Red Blood Cell Morphological Changes and Enlarged Platelets Found in Idiopathic Multicentric Castleman Disease

BACKGROUNDIdiopathic multicentric Castleman disease (iMCD) is a rare polyclonal lymphoproliferative disorder characterized by systemic inflammatory symptoms, cytopenias, generalized lymphadenopathy, and multiple organ system dysfunction. iMCD is subclassified into iMCD-TAFRO, with thrombocytopenia, anasarca, fever/elevated C-reactive protein, reticulin myelofibrosis/renal failure, and organomegaly, and iMCD-NOS (not otherwise specified), with high platelet counts and hypergammaglobulinemia. Despite recent diagnostic guidelines, iMCD diagnosis remains challenging because many of the histopathologic features and clinical/laboratory abnormalities are non-specific. Characterization of easily measurable findings from minimally invasive procedures, such as peripheral blood smear, could help to improve the accuracy of diagnosis as well as potentially providing insights into disease pathogenesis. Peripheral blood smears are often performed during routine clinical evaluation and enable morphologic evaluation of red blood cells (RBCs) and white blood cells (WBCs). In this study, we systematically characterized peripheral blood smear morphologic findings in iMCD for the first time.METHODSPeripheral blood smear morphologic findings in medical records from 68 iMCD patients, who were enrolled in an international registry for CD patients and had been confirmed to meet iMCD criteria by an expert panel, were reviewed and analyzed. Patients were also categorized into iMCD-TAFRO (N=39) or iMCD-NOS (N=29) based on clinical and laboratory data. Two-sample proportion tests were used to compare the frequency of features between iMCD-TAFRO and iMCD-NOS, when the abnormality was present in ≥ 5 subjects in both subtypes. P-value correction was not performed as findings are preliminary and hypothesis generating.RESULTSAmong the 68 iMCD patients, average age was 35.8 years (14-61); 46% were female, 54% were male; racial distribution was 62% white; 10% Black; 15% Asian; 13% other. The most common peripheral blood smear findings across all iMCD patients included abnormalities in RBC size and color, with anisocytosis (58.8%), polychromasia (57.4%), hypochromia (55.9%), and microcytosis (50.0%) being the most prevalent findings in the overall cohort (Table 1). Other RBC abnormalities were observed but less frequently, including abnormalities in shape such as poikilocytosis (44.1%). Giant or large platelets were found in 47% of iMCD cases. Abnormalities in WBCs, including toxic granulation, atypical lymphocytes, and toxic vacuolization, were detected less frequently. When comparing the two clinical subtypes, patients with iMCD-TAFRO had more frequent anisocytosis (p=0.043), hypochromia (p=0.002), polychromasia (p=0.022), and giant/large platelets (p<0.001) versus iMCD-NOS.DISCUSSIONHere, we have characterized peripheral blood morphologic abnormalities in iMCD for the first time. The most common findings in this cohort included abnormalities in RBC size and color. These abnormalities can be found in a number of conditions, including myelofibrosis, autoimmune diseases, and other inflammatory conditions. In iMCD, they are likely related to cytokine-driven anemia of chronic inflammation. The presence of large or giant platelets in nearly half of iMCD patients, with significantly more in iMCD-TAFRO than iMCD-NOS, is particularly notable since this has not been described in iMCD. These enlarged platelets, which can be found in immune thrombocytopenic purpura, myeloproliferative disorders, pseudothrombocytopenia, and inherited platelet disorders, may reflect hyperactivity of megakaryocytes and/or early release from the bone marrow in response to peripheral platelet sequestration. Though less common in this cohort, there are additional changes in RBC shape and WBC features that may be more specific to iMCD and potentially informative in increasing the index of suspicion for iMCD. These findings suggest that the peripheral blood smear may be able to support the diagnosis of iMCD and result in faster treatment administration. Future work is needed to determine whether the constellation of findings in the peripheral blood identified herein is unique to iMCD or seen in various other infectious, malignant, and autoimmune diseases. [Display omitted] DisclosuresFajgenbaum: Pfizer: Other: Study drug for clinical trial of sirolimus; EUSA Pharma: Research Funding; N/A: Other: Holds pending provisional patents for ‘Methods of treating idiopathic multicentric Castleman disease with JAK1/2 inhibition’ and ‘Discovery and validation of a novel subgroup and therapeutic target in idiopathic multicentric Castleman disease’.

What determines blood viscosity at the highest city in the world?

What determines blood viscosity at the highest city in the world?

Abnormal red blood cell morphological changes in thalassaemia associated with iron overload and oxidative stress

AimsIron overload is a major factor contributing to the overall pathology of thalassaemia, which is primarily mediated by ineffective erythropoiesis and shorter mature red blood cell (RBC) survival. Iron accumulation...

Quantitative phase microscopy of red blood cells during planar trapping and propulsion.

Red blood cells (RBCs) have the ability to undergo morphological deformations during microcirculation, such as changes in surface area, volume and sphericity. Optical waveguide trapping is suitable for trapping, propelling and deforming large cell populations along the length of the waveguide. Bright field microscopy employed with waveguide trapping does not provide quantitative information about structural changes. Here, we have combined quantitative phase microscopy and waveguide trapping techniques to study changes in RBC morphology during planar trapping and transportation. By using interference microscopy, time-lapsed interferometric images of trapped RBCs were recorded in real-time and subsequently utilized to reconstruct optical phase maps. Quantification of the phase differences before and after trapping enabled study of the mechanical effects during planar trapping. During planar trapping, a decrease in the maximum phase values, an increase in the surface area and a decrease in the volume and sphericity of RBCs were observed. QPM was used to analyze the phase values for two specific regions within RBCs: the annular rim and the central donut. The phase value of the annular rim decreases whereas it increases for the central donut during planar trapping. These changes correspond to a redistribution of cytosol inside the RBC during planar trapping and transportation.

Effects of GlyT1 inhibition on erythropoiesis and iron homeostasis in rats.

Glycine is a key rate-limiting component of heme biosynthesis in erythropoietic cells, where the high intracellular glycine demand is primarily supplied by the glycine transporter 1 (GlyT1). The impact of intracellular glycine restriction after GlyT1 inhibition on hematopoiesis and iron regulation is not well established. We investigated the effects of a potent and selective inhibitor of GlyT1, bitopertin, on erythropoiesis and iron homeostasis in rats. GlyT1 inhibition significantly affected erythroid heme biosynthesis, manifesting as microcytic hypochromic regenerative anemia with a 20% steady-state reduction in hemoglobin. Reduced erythropoietic iron utilization was characterized by down-regulation of the transferrin receptor 1 (TfR1) on reticulocytes and modest increased iron storage in the spleen. Hepatic hepcidin expression was not affected. However, under the condition of reduced heme biosynthesis with reduced iron reutilization and increased storage iron, hepcidin at the lower and higher range of normal showed a striking role in tissue distribution of iron. Rapid formation of iron-positive inclusion bodies (IBs) was observed in circulating reticulocytes, with an ultrastructure of iron-containing polymorphic mitochondrial remnants. IB or mitochondrial iron accumulation was absent in bone marrow erythroblasts. In conclusion, GlyT1 inhibition in rats induced a steady-state microcytic hypochromic regenerative anemia and a species-specific accumulation of uncommitted mitochondrial iron in reticulocytes. Importantly, this glycine-restricted anemia provides no feedback signal for increased systemic iron acquisition and the effects reported are pathogenetically distinct from systemic iron-overload anemias and erythropoietic disorders such as acquired sideroblastic anemia.

Circulatory markers of oxidative stress and dyslipidemia in male patients of chronic plaque psoriasis

Context: Psoriasis is one of the common chronic and recurrent inflammatory skin disorders. The inflammatory exudates in psoriasis are responsible for various lipid abnormalities as well as trigger a pro-oxidant and antioxidant imbalance resulting in copious generation of oxygen metabolites and proteases which may induce oxidative and proteolytic damage to plasma constituents and circulating red blood cells (RBCs) Aims: The aim was to evaluate dyslipidemia and erythrocyte oxidative stress as markers in plaque psoriasis. Materials and Methods: The study was performed on 120 male subjects, out of which 60 were patients of a moderate form of plaque psoriasis and 60 healthy age-matched controls. We evaluated lipid profile, RBC morphological indices such as total RBC count, hematocrit, hemoglobin concentration, hematimetric indices, osmotic fragility, and reticulocyte count. We also evaluated antioxidant defenses catalase, superoxide dismutase, glutathione peroxidase (GPX), and oxidant malondialdehyde (MDA) levels. Statistical Analysis Used: Independent sample t-test was used to compare the means between two groups. Results: Psoriasis patients showed a significant rise (P Conclusions: Evaluation of the changes in RBC morphology, lipid profile, and antioxidant enzymes may be considered as biomarkers in predicting the severity of plaque psoriasis.

Temporal sequence of major biochemical events during blood bank storage of packed red blood cells.

We used sensitive spectroscopic techniques to measure changes in Band 3 oligomeric state during storage of packed red blood cells (RBC); these changes were compared to metabolic changes, RBC morphology, cholesterol and membrane protein loss, phospholipid reorganisation of the RBC membrane, and peroxidation of membrane lipid. The aim of the study was to temporally sequence major biochemical events occurring during cold storage, in order to determine which changes may underlie the structural defects in stored RBC. Fifteen RBC units were collected from normal volunteers and stored under standard blood bank conditions; both metabolic changes and lipid parameters were measured by multiple novel assays including a new mass spectrometric measurement of isoprostane (lipid peroxidation) and flow cytometric assessment of CD47 expression. Band 3 oligomeric state was assessed by time-resolved phosphorescence anisotropy, and RBC morphology by microscopy of glutaraldehyde-fixed RBC. Extracellular pH decreased and extracellular potassium increased rapidly during cold storage. Band 3 on the RBC membrane aggregated into large oligomers early in the storage period and coincident with changes in RBC morphology. Membrane lipid changes, including loss of unesterified cholesterol, lipid peroxidation and expression of CD47, also changed early during the storage period. In contrast loss of acetylcholinesterase activity and haemolysis of RBC occurred late during storage. Our results demonstrate that changes in the macromolecular organisation of membrane proteins on the RBC occur early in storage and suggest that lipid peroxidation and/or oxidative damage to the membrane are responsible for irreversible morphological changes and loss of function during red cell storage.

Low micromolar intravascular cell-free hemoglobin concentration affects vascular NO bioavailability in sickle cell disease: a computational analysis

In sickle cell disease, the changes in RBC morphology destabilize the red blood cell (RBC) membrane and lead to hemolysis. Several experimental and clinical studies have associated intravascular hemolysis with pulmonary hypertension in sickle cell disease. Cell-free hemoglobin (Hb) from intravascular hemolysis has high affinity for nitrixc oxide (NO) and can affect the NO bioavailability in the sickle cell disease, which may eventually lead to pulmonary hypertension. To study the effects of intravascular hemolysis related cell-free Hb concentrations on NO bioavailability, we developed a two-dimensional mathematical model of NO biotransport in 50-μm arteriole under steady-state sickle cell disease conditions. We analyzed the effects of flow-dependent NO production and axial and radial transport of NO, a recently reported much lower NO-RBC reaction rate constant, and cell-free layer thickness on NO biotransport. Our results show that the presence of cell-free Hb concentrations as low as 0.5 μM results in an approximately three- to sevenfold reduction in the predicted smooth muscle cell NO concentrations compared with those under physiological conditions. In addition, increasing the diffusional resistance for NO in vascular lumen from cell-free layer or reducing NO-RBC reaction rate did not improve the NO bioavailability at the smooth muscle cell layer significantly for cell-free Hb concentrations ≥1 μM. These results suggest that lower NO bioavailability due to low micromolar cell-free Hb can disturb NO homeostasis and cause insufficient bioavailability at the smooth muscle cell layer. Our results supports the hypothesis that hemolysis-associated reduction in NO bioavailability may play a role in the development of pathophysiological complications like pulmonary hypertension in sickle cell disease that are observed in several clinical and experimental studies.

Anemia in sepsis: the importance of red blood cell membrane changes

SUMMARYAnemia is a common problem in acutely ill patients, especially in those who develop sepsis. There are many factors contributing to the development of anemia in these patients, including blood sampling and other losses, decreased red blood cell (RBC) synthesis, and possibly increased destruction. Increased RBC uptake may be due to changes in RBC morphology and the RBC membrane during inflammatory processes. In particular, a rapid increase in RBC sphericity correlated with a decreased surface carbohydrate membrane content and alterations in the lipid bilayer with increased peroxidation and phosphatidylserine exposure in the outer leaflet of the membrane. Better understanding of these alterations could facilitate new strategies to prevent the development of anemia in sepsis.

Chemical Preservation of Urine Sediment for Phase-Contrast Microscopic Examination

A major difficulty in diagnostic urinary microscopy is the need to observe a freshly passed urine specimen. This study illustrates the use of glutaraldehyde (GA) and formaldehyde (FA) to preserve urinary sediment for diagnostic microscopy. GA and FA in whole urine cause a precipitate to form if there is a trace or more of proteinuria. This can be eliminated by centrifugation and fixation of urine sediment. Red blood cells (RBC) and casts are adequately preserved for diagnostic microscopy for at least 3 months. However, subtle changes in RBC morphology consisting of hexagonal distortion and wrinkling of cell margins are seen in some cells. White blood cells (WBC) are less reliably preserved. Diagnostic phase-contrast microscopy can be performed on urine sediment stored at room temperature for up to 3 months after fixation with GA or FA.