Light microscopic morphology of blood cells of non-descript indigenous Zoar chicken of Mizoram, India.

Identifying and analysing distinct blood cells is crucial for the diagnosis and treatment of diseases in the field of biomedicine. The present study was undertaken to study the cytomorphological and cytochemical characteristics of the blood cells of Zoar, a non-descript indigenous breed of chicken extensively reared under backyard poultry farming in Mizoram, India. For this study, 2 mL of blood samples were aseptically collected from the wings veins of 12 chickens and were processed for light microscopic study under standard protocols. The matured erythrocytes were elliptical, while the immature erythrocytes appeared oval. The heterophils were positive for SBB (SBB), Periodic Acid Schiff (PAS), acid phosphatase, alkaline phosphatase and Arylsulphatase while the eosinophils were positive for SBB, PAS, alkaline phosphatase, cytochrome oxidase and peroxidase. The basophils of were positive for toluidine blue while the thrombocytes were positive for PAS. These cytochemical and cytoenzymatic staining properties plays a very important role in diagnosis, differentiation, and classification of leukaemias.

Cortex-wide transcranial localization microscopy with fluorescently labeled red blood cells.

Large-scale imaging of brain activity with high spatio-temporal resolution is crucial for advancing our understanding of brain function. The existing neuroimaging techniques are largely limited by restricted field of view, slow imaging speed, or otherwise do not have the adequate spatial resolution to capture brain activities on a capillary and cellular level. To address these limitations, we introduce fluorescence localization microscopy aided with sparsely-labeled red blood cells for cortex-wide morphological and functional cerebral angiography with 4.9 µm spatial resolution and 1 s temporal resolution. When combined with fluorescence calcium imaging, the proposed method enables extended recordings of stimulus-evoked neuro-vascular changes in the murine brain while providing simultaneous multiparametric readings of intracellular neuronal activity, blood flow velocity/direction/volume, and vessel diameter. Owing to its simplicity and versatility, the proposed approach will become an invaluable tool for deciphering the regulation of cortical microcirculation and neurovascular coupling in health and disease.

Optimization of single-needle red cell exchange in patients with sickle cell disease.

The hypercoagulable state associated with sickle cell disease (SCD) can be challenging for apheresis procedures. Among 62 single-needle red cell exchanges (SN-RCEs) performed over a 15-month period, 4 patients experienced 6 hemolytic events with a discolored plasma layer, elevated plasma/RBC interface in the centrifuge, and accompanying alarms of "Cells were detected in plasma line from centrifuge" or "AIM system detected RBC at top of connector." The hemolysis originated from the apheresis instrument because samples from the apheresis belt but not the patients' peripheral blood were positive for hemolysis. Further analysis showed the alarms occurred more often in SN-RCEs (20.4%) than double-needle RCEs (2.7%), and the hemolysis was probably secondary to clumping. To optimize SN-RCE, we increased the anticoagulant dosage by changing Inlet/AC ratio from 13 to 8 and lowered the inlet rate to the level comparable to double-needle RCE. The adjustments were well-tolerated with no more hemolysis.

Accurate and High-Resolution Particle Mass Measurement Using a Peak Filtering Algorithm.

Charge detection quadrupole ion trap mass spectrometry (CD-QIT MS) is an effective way of achieving the mass analysis of microparticles with ultrahigh mass. However, its mass accuracy and resolution are still poor. To enhance the performance of CD-QIT MS, the resolution Rpeak of each peak in the mass spectra resulting from an individual particle was assessed, and a peak filtering algorithm that can filter out particle adducts and clusters with a lower Rpeak was proposed. By using this strategy, more accurate mass information about the analyzed particles could be obtained, and the mass resolution of CD-QIT MS was improved by nearly 2-fold, which was demonstrated by using the polystyrene (PS) particle size standards and red blood cells (RBCs). Benefiting from these advantages of the peak filtering algorithm, the baseline separation and relative quantification of 3 and 4 µm PS particles were achieved. To prove the application value of this algorithm in a biological system, the mass of yeast cells harvested at different times was measured, and it was found that the mixed unbudded and budded yeast cells, which otherwise would not be differentiable, were distinguished and quantified with the algorithm.

[Progress in the analysis of hemolysis and coagulation models for interventional micro-axial flow blood pumps].

Interventional micro-axial flow blood pump is widely used as an effective treatment for patients with cardiogenic shock. Hemolysis and coagulation are vital concerns in the clinical application of interventional micro-axial flow pumps. This paper reviewed hemolysis and coagulation models for micro-axial flow blood pumps. Firstly, the structural characteristics of commercial interventional micro-axial flow blood pumps and issues related to clinical applications were introduced. Then the basic mechanisms of hemolysis and coagulation were used to study the factors affecting erythrocyte damage and platelet activation in interventional micro-axial flow blood pumps, focusing on the current models of hemolysis and coagulation on different scales (macroscopic, mesoscopic, and microscopic). Since models at different scales have different perspectives on the study of hemolysis and coagulation, a comprehensive analysis combined with multi-scale models is required to fully consider the influence of complex factors of interventional pumps on hemolysis and coagulation.

Red cell concentrates from teen male donors contain poor-quality biologically older cells.

BACKGROUND AND OBJECTIVES: Donor factors influence the quality characteristics of red cell concentrates (RCCs) and the lesions that develop in these heterogeneous blood products during hypothermic storage. Teen male donors' RCCs contain elevated levels of biologically old red blood cells (RBCs). The aim of this study was to interrogate the quality of units of different donor ages and sexes to unravel the complex interplay between donor characteristics, long-term cold storage and, for the first time, RBC biological age. MATERIALS AND METHODS: RCCs from teen males, teen females, senior males and senior females were density-separated into less-dense/young (Y-RBCs) and dense/old RBCs (O-RBCs) throughout hypothermic storage for testing. The unseparated and density-separated cells were tested for haematological parameters, stress (oxidative and osmotic) haemolysis and oxygen affinity (p50). RESULTS: The O-RBCs obtained from teen donor samples, particularly males, had smaller mean corpuscular volumes and higher mean corpuscular haemoglobin concentrations. While biological age did not significantly affect oxygen affinity, biologically aged O-RBCs from stored RCCs exhibited increased oxidative haemolysis and decreased osmotic fragility, with teenage male RCCs exhibiting the highest propensity to haemolyse. CONCLUSION: Previously, donor age and sex were shown to have an impact on the biological age distribution of RBCs within RCCs. Herein, we demonstrated that RBC biological age, particularly O-RBCs, which are found more prevalently in male teens, to be a driving factor of several aspects of poor blood product quality. This study emphasizes that donor factors should continue to be considered for their potential impacts on transfusion outcomes.

Structural basis of membrane skeleton organization in red blood cells.

The spectrin-based membrane skeleton is a ubiquitous membrane-associated two-dimensional cytoskeleton underneath the lipid membrane of metazoan cells. Mutations of skeleton proteins impair the mechanical strength and functions of the membrane, leading to several different types of human diseases. Here, we report the cryo-EM structures of the native spectrin-actin junctional complex (from porcine erythrocytes), which is a specialized short F-actin acting as the central organizational unit of the membrane skeleton. While an α-/ß-adducin hetero-tetramer binds to the barbed end of F-actin as a flexible cap, tropomodulin and SH3BGRL2 together create an absolute cap at the pointed end. The junctional complex is strengthened by ring-like structures of dematin in the middle actin layers and by patterned periodic interactions with tropomyosin over its entire length. This work serves as a structural framework for understanding the assembly and dynamics of membrane skeleton and offers insights into mechanisms of various ubiquitous F-actin-binding factors in other F-actin systems.

Membrane skeleton hyperstability due to a novel alternatively spliced 4.1R can account for ellipsoidal camelid red cells with decreased deformability.

The red blood cells (RBCs) of vertebrates have evolved into two basic shapes, with nucleated nonmammalian RBCs having a biconvex ellipsoidal shape and anuclear mammalian RBCs having a biconcave disk shape. In contrast, camelid RBCs are flat ellipsoids with reduced membrane deformability, suggesting altered membrane skeletal organization. However, the mechanisms responsible for their elliptocytic shape and reduced deformability have not been determined. We here showed that in alpaca RBCs, protein 4.1R, a major component of the membrane skeleton, contains an alternatively spliced exon 14-derived cassette (e14) not observed in the highly conserved 80 kDa 4.1R of other highly deformable biconcave mammalian RBCs. The inclusion of this exon, along with the preceding unordered proline- and glutamic acid-rich peptide (PE), results in a larger and unique 90 kDa camelid 4.1R. Human 4.1R containing e14 and PE, but not PE alone, showed markedly increased ability to form a spectrin-actin-4.1R ternary complex in viscosity assays. A similar facilitated ternary complex was formed by human 4.1R possessing a duplication of the spectrin-actin-binding domain, one of the mutations known to cause human hereditary elliptocytosis. The e14- and PE-containing mutant also exhibited an increased binding affinity to ß-spectrin compared with WT 4.1R. Taken together, these findings indicate that 4.1R protein with the e14 cassette results in the formation and maintenance of a hyperstable membrane skeleton, resulting in rigid red ellipsoidal cells in camelid species, and suggest that membrane structure is evolutionarily regulated by alternative splicing of exons in the 4.1R gene.

Polo-like kinase inhibitor BI2536 induces eryptosis.

BI2536 is potent inhibitor of polo-like kinases PLK1, 2, and 3. The inhibition of PLKs in nucleated cells induces apoptosis by perturbing the cell cycle with consequent engagement of mitotic catastrophe. BI2536 is being tested as chemotherapy in various phase I/II/III clinical trials. Erythrocytes do not have a nucleus; however, they may undergo programmed suicide with characteristic hallmarks including cell shrinkage and phosphatidylserine translocation to the cell surface. This particular death is baptized eryptosis. Our study explored whether BI2536 induces eryptosis. We used flow cytometry to access death in red blood cells. We analyzed the cellular volume, the intracellular calcium concentration, the cell surface phosphatidylserine exposure, and the ceramide abundance. In addition, we analyzed the effect of BI2536 on hemolysis. Our investigation showed that after 48 h of incubation with PLK inhibitor BI2536, erythrocytes lost volume and were positive for annexin­V without any effect on hemolysis. Cells also showed an abundance of ceramide and an increase of intracellular calcium. All these finding suggest that BI2536 provokes eryptosis in red blood cells, ostensibly in part due to Ca2+ entry and ceramide accumulation.

Glassfrogs conceal blood in their liver to maintain transparency.

Transparency in animals is a complex form of camouflage involving mechanisms that reduce light scattering and absorption throughout the organism. In vertebrates, attaining transparency is difficult because their circulatory system is full of red blood cells (RBCs) that strongly attenuate light. Here, we document how glassfrogs overcome this challenge by concealing these cells from view. Using photoacoustic imaging to track RBCs in vivo, we show that resting glassfrogs increase transparency two- to threefold by removing ~89% of their RBCs from circulation and packing them within their liver. Vertebrate transparency thus requires both see-through tissues and active mechanisms that "clear" respiratory pigments from these tissues. Furthermore, glassfrogs' ability to regulate the location, density, and packing of RBCs without clotting offers insight in metabolic, hemodynamic, and blood-clot research.

Interactive Dynamics of Cell Volume and Cell Death in Human Erythrocytes Exposed to α-Hemolysin from Escherichia coli.

α-hemolysin (HlyA) of E. coli binds irreversibly to human erythrocytes and induces cell swelling, ultimately leading to hemolysis. We characterized the mechanism involved in water transport induced by HlyA and analyzed how swelling and hemolysis might be coupled. Osmotic water permeability (Pf) was assessed by stopped-flow light scattering. Preincubation with HlyA strongly reduced Pf in control- and aquaporin 1-null red blood cells, although the relative Pf decrease was similar in both cell types. The dynamics of cell volume and hemolysis on RBCs was assessed by electrical impedance, light dispersion and hemoglobin release. Results show that HlyA induced erythrocyte swelling, which is enhanced by purinergic signaling, and is coupled to osmotic hemolysis. We propose a mathematical model of HlyA activity where the kinetics of cell volume and hemolysis in human erythrocytes depend on the flux of osmolytes across the membrane, and on the maximum volume that these cells can tolerate. Our results provide new insights for understanding signaling and cytotoxicity mediated by HlyA in erythrocytes.

Storage conditions determine the characteristics of red blood cell derived extracellular vesicles.

Extracellular vesicles (EVs) are released during the storage of red blood cell (RBC) concentrates and might play adverse or beneficial roles throughout the utilization of blood products (transfusion). Knowledge of EV release associated factors and mechanism amends blood product management. In the present work the impact of storage time and medium (blood preserving additive vs isotonic phosphate buffer) on the composition, size, and concentration of EVs was studied using attenuated total reflection infrared (ATR-IR) spectroscopy, microfluidic resistive pulse sensing (MRPS) and freeze-fraction combined transmission electron micrography (FF-TEM). The spectroscopic protein-to-lipid ratio based on amide and the C-H stretching band intensity ratio indicated the formation of various vesicle subpopulations depending on storage conditions. After short storage, nanoparticles with high relative protein content were detected. Spectral analysis also suggested differences in lipid and protein composition, too. The fingerprint region (from 1300 to 1000 cm-1) of the IR spectra furnishes additional information about the biomolecular composition of RBC-derived EVs (REVs) such as adenosine triphosphate (ATP), lactose, glucose, and oxidized hemoglobin. The difference between the vesicle subpopulations reveals the complexity of the REV formation mechanism. IR spectroscopy, as a quick, cost-effective, and label-free technique provides valuable novel biochemical insight and might be used complementary to traditional omics approaches on EVs.

Hemolytic Response of Iron Oxide Magnetic Nanoparticles at the Interface and in Bulk: Extraction of Blood Cells by Magnetic Nanoparticles.

Surface-active and water-soluble magnetic nanoparticles (NPs) were synthesized in the presence of a series of amphiphilic molecules of different functional groups to determine the hemolytic response and their ability to extract blood cells across the interface and aqueous bulk while maintaining minimum hemolysis. Amphiphilic molecules such as Gemini surfactants of strong hydrophobicity and low hydrophilic-lipophilic balance produced surface-active magnetic NPs, which were highly cytotoxic even when placed at the blood suspension (aqueous)-air interface. A similar behavior was shown by water-soluble magnetic NPs produced using monomeric ionic and nonionic surfactants and different amino acids. The NPs produced using mild biological surfactants and mono- and oligosaccharides of the same functional group proved to be excellent blood cell extractors with minimum hemolysis. α/ß-cyclodextrin and dextrose-stabilized magnetic NPs induced negligible hemolysis and extracted more than 50% of blood cells. The results showed that nontoxic magnetic NPs are excellent blood cell extractors from the blood suspension when tagged with amphiphilic molecules possessing good biocompatibility with cell membranes without inducing hemolysis. The work highlights the biological applicability of nontoxic magnetic NPs at biointerfaces and in blood suspensions.

DRAMP 3.0: an enhanced comprehensive data repository of antimicrobial peptides.

Stapled antimicrobial peptides are an emerging class of artificial cyclic peptide molecules which have antimicrobial activity and potent structure stability. We previously published the Data Repository of Antimicrobial Peptides (DRAMP) as a manually annotated and open-access database of antimicrobial peptides (AMPs). In the update of version 3.0, special emphasis was placed on the new development of stapled AMPs, and a subclass of specific AMPs was added to store information on these special chemically modified AMPs. To help design low toxicity AMPs, we also added the cytotoxicity property of AMPs, as well as the expansion of newly discovered AMP data. At present, DRAMP has been expanded and contains 22259 entries (2360 newly added), consisting of 5891 general entries, 16110 patent entries, 77 clinical entries and 181 stapled AMPs. A total of 263 entries have predicted structures, and more than 300 general entries have links to experimentally determined structures in the Protein Data Bank. The update also covers new annotations, statistics, categories, functions and download links. DRAMP is available online at http://dramp.cpu-bioinfor.org/.

Red blood cell distribution width as a prognostic biomarker for viral infections: prospects and challenges.

Viral diseases remain a significant global health threat, and therefore prioritization of limited healthcare resources is required to effectively manage dangerous viral disease outbreaks. In a pandemic of a newly emerged virus that is yet to be well understood, a noninvasive host-derived prognostic biomarker is invaluable for risk prediction. Red blood cell distribution width (RDW), an index of red blood cell size disorder (anisocytosis), is a potential predictive biomarker for severity of many diseases. In view of the need to prioritize resources during response to outbreaks, this review highlights the prospects and challenges of RDW as a prognostic biomarker for viral infections, with a focus on hepatitis and COVID-19, and provides an outlook to improve the prognostic performance of RDW for risk prediction in viral diseases.

Erythropoietic properties of human induced pluripotent stem cells-derived red blood cells in immunodeficient mice.

Transfusion of red blood cells (RBCs) is a life-saving intervention for anemic patients. Human induced pluripotent stem cells (iPSC) have the capability to expand and differentiate into RBCs (iPSC-RBCs). Here we developed a murine model to investigate the in vivo properties of human iPSC-RBCs. iPSC lines were produced from human peripheral blood mononuclear cells by transient expression of plasmids containing OCT4, SOX2, MYC, KLF4, and BCL-XL genes. Human iPSC-RBCs were generated in culture supplemented with human platelet lysate, and were CD34- CD235a+ CD233+ CD49dlow CD71low ; about 13% of iPSC-RBCs were enucleated before transfusion. Systemic administration of clodronate liposomes (CL) and cobra venom factor (CVF) to NOD scid gamma (NSG) mice markedly promoted the circulatory survival of human iPSC-RBCs following transfusion. While iPSC-RBCs progressively decreased with time, 90% of circulating iPSC-RBCs were enucleated 1 day after transfusion (CD235a+ CD233+ CD49d- CD71- ). Surprisingly, human iPSC-RBCs reappeared in the peripheral circulation at 3 weeks after transfusion at levels more than 8-fold higher than at 1 h after transfusion. Moreover, a substantial portion of the transfused nucleated iPSC-RBCs preferentially homed to the bone marrow, and were detectable at 24 days after transfusion. These results suggest that nucleated human iPSC-derived cells that homed to the bone marrow of NSG mice retained the capability to complete differentiation into enucleated erythrocytes and egress the bone marrow into peripheral blood. The results offer a new model using human peripheral blood-derived iPSC and CL/CVF-treated NSG mice to investigate the development and circulation of human erythroid cells in vivo.

Imaging flow cytometry data analysis using convolutional neural network for quantitative investigation of phagocytosis.

Macrophages play an important role in the adaptive immune system. Their ability to neutralize cellular targets through Fc receptor-mediated phagocytosis has relied upon immunotherapy that has become of particular interest for the treatment of cancer and autoimmune diseases. A detailed investigation of phagocytosis is the key to the improvement of the therapeutic efficiency of existing medications and the creation of new ones. A promising method for studying the process is imaging flow cytometry (IFC) that acquires thousands of cell images per second in up to 12 optical channels and allows multiparametric fluorescent and morphological analysis of samples in the flow. However, conventional IFC data analysis approaches are based on a highly subjective manual choice of masks and other processing parameters that can lead to the loss of valuable information embedded in the original image. Here, we show the application of a Faster region-based convolutional neural network (CNN) for accurate quantitative analysis of phagocytosis using imaging flow cytometry data. Phagocytosis of erythrocytes by peritoneal macrophages was chosen as a model system. CNN performed automatic high-throughput processing of datasets and demonstrated impressive results in the identification and classification of macrophages and erythrocytes, despite the variety of shapes, sizes, intensities, and textures of cells in images. The developed procedure allows determining the number of phagocytosed cells, disregarding cases with a low probability of correct classification. We believe that CNN-based approaches will enable powerful in-depth investigation of a wide range of biological processes and will reveal the intricate nature of heterogeneous objects in images, leading to completely new capabilities in diagnostics and therapy.

The regulation roles of Ca2+ in erythropoiesis: What have we learned?

Calcium (Ca2+) is an important second messenger molecule in the body, regulating cell cycle and fate. There is growing evidence that intracellular Ca2+ levels play functional roles in the total physiological process of erythroid differentiation, including the proliferation and differentiation of erythroid progenitor cells, terminal enucleation, and mature red blood cell aging and clearance. Moreover, recent research on the pathology of erythroid disorders has made great progress in the past decades, indicating that calcium ion hemostasis is closely related to ineffective erythropoiesis and increased sensitivity to stress factors. In this review, we summarized what is known about the functional roles of intracellular Ca2+ in erythropoiesis and erythrocyte-related diseases, with an emphasis on the regulation of the intracellular Ca2+ homeostasis during erythroid differentiation. An understanding of the regulation roles of Ca2+ homeostasis in erythroid differentiation will facilitate further studies and eventually molecular identification of the pathways involved in the pathological process of erythroid disorders, providing new therapeutic opportunities in erythrocyte-related disease.