Morphologic appearance of peripheral blood cells from Zovawk pigs (Sus scrofa domesticus) visualized by transmission electron microscopy.

BACKGROUND: Ultrastructural information regarding the peripheral blood cells of local (Zovawk) pigs from Mizoram, India, is not available in the scientific literature. OBJECTIVES: The present study was designed to reveal the fine structural details of the blood cells from these local pigs using a transmission electron microscope (TEM). METHODS: Blood samples were collected from 12 healthy Zovawk pigs of either sex and processed according to a standard protocol. Processed blood samples were then sent to the All India Institute of Medical Sciences, New Delhi, for further processing and imaging under TEM. Different types of blood cells were viewed under TEM, and different characteristics of these cells were assessed. RESULTS: In the present study, erythrocytes are elongated, biconcave, and nucleated without cytoplasmic organelles. Neutrophils are round with 2-5 lobed nuclei surrounded by cytoplasm with an indistinct bilayered nuclear membrane. The cytoplasm is packed with membrane bound round, oval, and elongated cytoplasmic granules. Eosinophils are round to oval with 2-3 lobed nuclei with distinct nuclear membranes. Basophils are spherical and contained small, medium, and large electron-dense granules. Lymphocytes are small, medium, and large and contained all cellular components. Monocytes are irregularly spherical with slight nuclear indentations. The platelets are elongated, oval, or rounded, with a few pseudopods at the cell surface. CONCLUSIONS: From the present study, we can conclude that the ultrastructural morphology of blood cells from Zovawk pigs resembles those of other domestic animals. However, a few differences have been observed.

Intraerythrocytic Mycoplasma-like organism diagnosed ultrastructurally as an agent of anaemia in laboratory-reared cyprinid hybrids.

A study targeting the etiology of severe anaemia that sporadically occurred in laboratory-bred cyprinid hybrids resulted in a diagnosis of a Mycoplasma-like organism selectively invading the cytoplasm of erythrocytes. Despite the fact that there was a concurrent yeast infection in moribund anaemic hybrids, the primary role in the development of anaemia was assigned to the Mycoplasma-like organism due to its regular occurrence in erythrocytes of both the moribund hybrids and hybrids that were free of yeast infection yet showed early to advanced symptoms of the disease. Novel data on the Mycoplasma-like organism's cytoskeleton were obtained from ultrathin sections of affected erythrocytes. An ultrastructural study of the concurrent yeast infection in moribund hybrids manifesting the most advanced anaemia revealed the presence of Titan cells in ascitic fluid. The original findings presented in this study underline the diagnostic relevance of transmission electron microscopy in the research of similar infections.

A systematic analysis of ultrastructural lesions in the Plasmodium coatneyi splenectomized rhesus macaque model of severe malaria.

Plasmodium falciparum remains one of the world's deadliest diseases and with ongoing concerns of evolving drug resistance, there is a need for continued refinement of the Plasmodium coatneyi infection model in macaques to study severe malaria. As such, the systemic ultrastructural lesions associated with P. coatneyi infection in splenectomized rhesus macaques was evaluated in 6 animals. Autopsy samples from multiple areas of the central nervous system (CNS), kidneys, heart, liver, and lungs of all 6 animals were processed for electron microscopy. A systematic analysis of the ultrastructural changes associated with the plasmodium was undertaken by multiple pathologists to ensure consensus. All tissues exhibited marked sequestration of infected red blood cells comprised either of cytoadherence to endothelium or rosette formation, associated with variable degrees of host cell damage in a range of tissues that in severe cases resulted in necrosis. This is the first complete systemic evaluation of ultrastructural tissue lesions in P. coatneyi-infected rhesus macaques, and the findings have important implications evaluating of the use of this model for the study of severe malaria caused by P. falciparum in humans.

Omega-3 index is directly associated with a healthy red blood cell distribution width.

Low red blood cell (RBC) membrane content of EPA and DHA, i.e., the omega-3 index (O3I), and elevated RBC distribution width (RDW) are risk factors for all-cause mortality. O3I and RDW are related with membrane fluidity and deformability. Our objective was to determine if there is a relationship between O3I and RDW in healthy adults. Subjects without inflammation or anemia, and with values for O3I, RDW, high-sensitivity C-reactive protein (CRP), body mass index (BMI), age and sex were identified (n = 25,485) from a clinical laboratory dataset of  > 45,000 individuals. RDW was inversely associated with O3I in both sexes before and after (both p < 0.00001) adjusting models for sex, age, BMI and CRP. Stratification by sex revealed a sex-O3I interaction with the RDW-O3I slope (p < 0.00066) being especially steep in females with O3I ≤ 5.6%. In healthy adults of both sexes, the data suggested that an O3I of > 5.6% may help maintain normal RBC structural and functional integrity.

Mild COVID-19 and Impaired Blood Cell-Endothelial Crosstalk: Considering Long-Term Use of Antithrombotics?

BACKGROUND: Current coronavirus disease 2019 (COVID-19) pandemic reveals thrombotic, vascular, and endothelial dysfunctions at peak disease. However, the duration, degree of damage, and appropriate long-term use of antithrombotic strategies are unclear. Most COVID data are yielded from random clinical observations or autopsy of postmortem samples, while precise blood cellular data in survivors are insufficient. METHODS: We analyzed erythrocytes, circulating endothelial cells, and echinocytes by electron microscopy and flow cytometry in patients with confirmed COVID-19 (n = 31) and matched healthy controls (n = 32) on admission and at hospital discharge. RESULTS: All patients experienced mild disease, none required pulmonary support, and all survived. Admission number of circulating endothelial cells was significantly (40-100 times) higher in COVID-19 patients. Cells were massively damaged by multiple fenestrae in membranes with diameter comparable to the size of supercapsid in SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) virus. COVID-19 also provoked formation of stacked aggregated erythrocytes capable of clogging microvascular bed and of diminishing oxygen supply. In some patients, such abnormalities persisted at hospital discharge revealing remaining intracellular penetration of SARS-CoV-2 where it may be replicated and returned to circulation. CONCLUSION: These observational and descriptive data suggest that persistent viral cell injury may cause blood vessel damage; their increased permeability resulted in tissue edema, inflammation, platelet activation, and augmented thrombosis. There is a residual blood cell damage following the acute phase in some COVID-19 survivors. Controlled outcome-driven trials are urgently needed for exploring optimal use of long-term antithrombotics and vascular protection strategies even after mild COVID-19.

Energetic Lifestyle Drives Size and Shape of Avian Erythrocytes.

The size and shape of red blood cells (erythrocytes) is determined by key life history strategies in vertebrates. They have a fundamental role to deliver oxygen to tissues, and their ability to do so is shaped by the tissue's need and their shape. Despite considerable interest in how other components of blood are shaped by ecology and life history, few studies have considered erythrocytes themselves. We tested how erythrocyte size and shape varied in relation to energetically demanding activities using a dataset of 631 bird species. We found that in general, birds undergoing greater activities such as long distance migration had smaller and more elongated cells, while those with greater male-male competition had smaller and rounder cells. Smaller, more elongated erythrocytes allow more rapid oxygenation/deoxygenation and support greater aerobic activity. The rounder erythrocytes found in species with strong male-male competition may stem from younger erythrocytes deriving from androgen-induced erythropoiesis rates. Finally, diving species of bird had larger erythrocytes, indicating that erythrocytes are acting as a vital oxygen store. In summary, erythrocyte size and shape in birds are driven by the need to deliver oxygen during energetically costly activities.

Interactions between Babesia microti merozoites and rat kidney cells in a short-term in vitro culture and animal model.

Babesiosis is one of the most common infections in free-living animals and is rapidly becoming significant among human zoonoses. Cases of acute renal failure in humans caused by Babesia spp. have been described in the literature. The kidneys are characterised by intense blood flow through the blood vessels, which increases the likelihood of contact with the intra-erythrocyte parasite. The aim of this study was to observe the influence of B. microti (ATCC 30221) on renal epithelial cells in vitro cultured (NRK-52E line) and Wistar rats' kidney. Both NRK-52E cells and rats' kidney sections were analysed by light microscopy, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH). Necrotic changes in renal epithelial cells have been observed in vitro and in vivo. In many cross-sections through the rats' kidney, adhesion of blood cells to the vascular endothelium, accumulation of erythrocytes and emboli were demonstrated. In NRK-52E culture, elements with a distinctly doubled cell membrane resembling B. microti were found inside the cytoplasm and adjacent to the cell layer. The study indicates a chemotactic tendency for B. microti to adhere to the renal tubules' epithelium, a possibility of piroplasms entering the renal epithelial cells, their proliferation within the cytoplasm and emboli formation.

Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy.

Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4-10), osmolarity (50-1000 mOsm/L), temperature (15-60 °C), and surfactant Triton X-100 (10-500 µM)). Their stability was evaluated by LS by considering the hydrodynamic radius (Rh), intensity of scattered light (I), and the shape parameter (ρ). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS-citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50-1000 mOsm/L, pH 4-10) had no significant effect on the Rh (=100-130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in I of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material.

Erythrocyte morphological symmetry analysis to detect sublethal trauma in shear flow.

The viscoelastic properties of red blood cells (RBC) facilitate flexible shape change in response to extrinsic forces. Their viscoelasticity is intrinsically linked to physical properties of the cytosol, cytoskeleton, and membrane-all of which are highly sensitive to supraphysiological shear exposure. Given the need to minimise blood trauma within artificial organs, we observed RBC in supraphysiological shear through direct visualisation to gain understanding of processes leading to blood damage. Using a custom-built counter-rotating shear generator fit to a microscope, healthy red blood cells (RBC) were directly visualised during exposure to different levels of shear (10-60 Pa). To investigate RBC morphology in shear flow, we developed an image analysis method to quantify (a)symmetry of deforming ellipsoidal cells-following RBC identification and centroid detection, cell radius was determined for each angle around the circumference of the cell, and the resultant bimodal distribution (and thus RBC) was symmetrically compared. While traditional indices of RBC deformability (elongation index) remained unaltered in all shear conditions, following ~100 s of exposure to 60 Pa, the frequency of asymmetrical ellipses and RBC fragments/extracellular vesicles significantly increased. These findings indicate RBC structure is sensitive to shear history, where asymmetrical morphology may indicate sublethal blood damage in real-time shear flow.

Impact of olokizumab on platelets, leukocytes and erythrocytes during mild COVID-19.

No abstract present.

Adrenergically induced translocation of red blood cell ß-adrenergic sodium-proton exchangers has ecological relevance for hypoxic and hypercapnic white seabass.

White seabass (Atractoscion nobilis) increasingly experience periods of low oxygen (O2; hypoxia) and high carbon dioxide (CO2, hypercapnia) due to climate change and eutrophication of the coastal waters of California. Hemoglobin (Hb) is the principal O2 carrier in the blood and in many teleost fishes Hb-O2 binding is compromised at low pH; however, the red blood cells (RBC) of some species regulate intracellular pH with adrenergically stimulated sodium-proton-exchangers (ß-NHEs). We hypothesized that RBC ß-NHEs in white seabass are an important mechanism that can protect the blood O2-carrying capacity during hypoxia and hypercapnia. We determined the O2-binding characteristics of white seabass blood, the cellular and subcellular response of RBCs to adrenergic stimulation, and quantified the protective effect of ß-NHE activity on Hb-O2 saturation. White seabass had typical teleost Hb characteristics, with a moderate O2 affinity (Po2 at half-saturation; P50 2.9 kPa) that was highly pH-sensitive (Bohr coefficient -0.92; Root effect 52%). Novel findings from super-resolution microscopy revealed ß-NHE protein in vesicle-like structures and its translocation into the membrane after adrenergic stimulation. Microscopy data were corroborated by molecular and phylogenetic results and a functional characterization of ß-NHE activity. The activation of RBC ß-NHEs increased Hb-O2 saturation by ∼8% in normoxic hypercapnia and by up to ∼20% in hypoxic normocapnia. Our results provide novel insight into the cellular mechanism of adrenergic RBC stimulation within an ecologically relevant context. ß-NHE activity in white seabass has great potential to protect arterial O2 transport during hypoxia and hypercapnia but is less effective during combinations of these stressors.

Erythrocyte flow through the interendothelial slits of the splenic venous sinus.

The flow patterns of red blood cells through the spleen are intimately linked to clearance of senescent RBCs, with clearance principally occurring within the open flow through the red pulp and slits of the venous sinus system that exists in humans, rats, and dogs. Passage through interendothelial slits (IESs) of the sinus has been shown by MacDonald et al. (Microvasc Res 33:118-134, 1987) to be mediated by the caliber, i.e., slit opening width, of these slits. IES caliber within a given slit of a given sinus section has been shown to operate in an asynchronous manner. Here, we describe a model and simulation results that demonstrate how the supporting forces exerted on the sinus by the reticular meshwork of the red pulp, combined with asymmetrical contractility of stress fibers within the endothelial cells comprising the sinus, describe this vital and intriguing behavior. These results shed light on the function of the sinus slits in species such as humans, rats, and dogs that possess sinusoidal sinuses. Instead of assuming a passive mechanical filtering mechanism of the IESs, our proposed model provides a mechanically consistent explanation for the dynamically modulated IES opening/filtering mechanism observed in vivo. The overall perspective provided is also consistent with the view that IES passage serves as a self-protective mechanism in RBC vesiculation and inclusion removal.

Tadalafil impacts the mechanical properties of Plasmodium falciparum gametocyte-infected erythrocytes.

Plasmodium falciparum gametocytes modify the mechanical properties of their erythrocyte host to persist for several weeks in the blood circulation and to be available for mosquitoes. These changes are tightly regulated by the plasmodial phosphodiesterase delta that decreases both the stiffness and the permeability of the infected host cell. Here, we address the effect of the phosphodiesterase inhibitor tadalafil on deformability and permeability of gametocyte-infected erythrocytes. We show that this inhibitor drastically increases isosmotic lysis of gametocyte-infected erythrocytes and impairs their ability to circulate in an in vitro model for splenic retention. These findings indicate that tadalafil represents a novel drug lead potentially capable of blocking malaria parasite transmission by impacting gametocyte circulation.

Type II Grass Carp Reovirus Infects Leukocytes but Not Erythrocytes and Thrombocytes in Grass Carp (Ctenopharyngodon idella).

Grass carp reovirus (GCRV) causes serious losses to the grass carp industry. At present, infectious tissues of GCRV have been studied, but target cells remain unclear. In this study, peripheral blood cells were isolated, cultured, and infected with GCRV. Using quantitative real-time polymerase chain reaction (qRT-PCR), Western Blot, indirect immunofluorescence, flow cytometry, and transmission electron microscopy observation, a model of GCRV infected blood cells in vitro was established. The experimental results showed GCRV could be detectable in leukocytes only, while erythrocytes and thrombocytes could not. The virus particles in leukocytes are wrapped by empty membrane vesicles that resemble phagocytic vesicles. The empty membrane vesicles of leukocytes are different from virus inclusion bodies in C. idella kidney (CIK) cells. Meanwhile, the expression levels of IFN1, IL-1ß, Mx2, TNFα were significantly up-regulated in leukocytes, indicating that GCRV could cause the production of the related immune responses. Therefore, GCRV can infect leukocytes in vitro, but not infect erythrocytes and thrombocytes. Leukocytes are target cells in blood cells of GCRV infections. This study lays a theoretical foundation for the study of the GCRV infection mechanism and anti-GCRV immunity.

Erythroid enucleation: a gateway into a "bloody" world.

Erythropoiesis is an intricate process starting in hematopoietic stem cells and leading to the daily production of 200 billion red blood cells (RBCs). Enucleation is a greatly complex and rate-limiting step during terminal maturation of mammalian RBC production involving expulsion of the nucleus from the orthochromatic erythroblasts, resulting in the formation of reticulocytes. The dynamic enucleation process involves many factors ranging from cytoskeletal proteins to transcription factors to microRNAs. Lack of optimum terminal erythroid maturation and enucleation has been an impediment to optimum RBC production ex vivo. Major efforts in the past two decades have exposed some of the mechanisms that govern the enucleation process. This review focuses in detail on mechanisms implicated in enucleation and discusses the future perspectives of this fascinating process.

MALDI TOF Mass Spectrometry Imaging of Blood Smear: Method Development and Evaluation.

The aim of this study was to develop and evaluate matrix assisted LASER desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry imaging (MSI) of blood smear. Integrated light microscope and MALDI IT-TOF mass spectrometer, together with a matrix sublimation device, were used for analysis of blood smears coming from healthy male donors. Different blood plasma removal, matrix deposition, and instrumental settings were evaluated using the negative and positive ionization modes while agreement between the light microscopy images and the lateral distributions of cellular marker compounds served as the MSI quality indicator. Red and white blood cells chemical composition was analyzed using the differential m/z expression. Five seconds of exposure to ethanol followed by the 5 min of 9-aminoacridine or α-cyano-4-hydroxycinnamic acid deposition, together with two sets of instrumental settings, were selected for the MALDI TOF MSI experiments. Application of the thin and transparent matrix layers assured good correspondence between the LASER footprints and the preselected regions of interest. Cellular marker m/z signals coincided well with the appropriate cells. A metabolite databases search using the differentially expressed m/z produced hits which were consistent with the respective cell types. This study sets the foundations for application of blood smear MALDI TOF MSI in clinical diagnostics and research.

Application of atomic force microscopy to assess erythrocytes morphology in early stages of diabetes. A pilot study.

The study aim was to assess the application of atomic force microscopy (AFM) to evaluate erythrocyte morphology in early stages of type 2 diabetes mellitus, and the association with biochemical, anthropometric, diet, and physical activity indicators. This was a pilot cross-sectional study with four groups: healthy individuals, people with prediabetes (PDG), metabolic syndrome (MSG), and diabetes mellitus group (DMG). Blood samples were obtained to assess the erythrocyte morphology and biochemical parameters. Anthropometrical measurements were taken. Besides, a diet and a physical activity questionnaire were applied. The evaluation of the erythrocyte morphology through the AFM showed quantitative and qualitative alterations in the cell's form and size. Compared to the healthy group, the PDG had a reduction in height (-0.80 µm, p < 0.05), and an increase in axial ratio (-0.09 µm, p < 0.05); the MSG had lower concave depth (-0.19 µm, p < 0.05); and the DMG had a decreased height (-0.46 µm, p < 0.05) and concave depth (-0.29 µm, p < 0.05), and higher axial ratio (+0.08 µm) and thickness (+0.32 µm, p < 0.05). The PDG vs. DMG had a statistically significant difference in concave depth (+0.23 µm, p < 0.05) and thickness (-0.26 µm, p < 0.05). The MSG was different than the DMG in variables like axial ratio (-0.05 µm) and thickness (-0.25 µm). Besides, higher values of age, HbA1c, triglycerides, body mass index, waist-to-hip ratio, and physical inactivity were associated with altered erythrocyte morphology. AFM is a promising instrument to assess early but subtle changes in erythrocyte morphology (height, axial ratio, concave depth, thickness) before significant pathological conditions, such as type 2 diabetes mellitus. HbA1c might have a major effect in altered morphology, vs. metabolic parameters like high triglycerides, body mass index, waist, and physical inactivity.

Fibrin and Platelet-Rich Composition in Retrieved Thrombi Hallmarks Stroke With Active Cancer.

BACKGROUND AND PURPOSE: We aim to investigate whether histopathologic examination of thrombi retrieved from acute ischemic stroke patients undergoing endovascular treatment could distinguish cancer-related stroke from other etiologies. METHODS: Thrombi from patients undergoing endovascular treatment were analyzed. The etiology of stroke was divided into cardioembolism, large artery atherosclerosis, and active cancer groups. All selected thrombi were subjected to hematoxylin and eosin staining. The percentages of fibrin/platelets, red blood cells, and white blood cells within a thrombus were quantified. RESULTS: One-hundred fifty-two patients (active cancer, 19; cardioembolism, 107; large artery atherosclerosis, 26) were included. Thrombi from the active cancer group exhibited a higher fibrin/platelet composition than did those from the cardioembolism and large artery atherosclerosis groups (median, 85.7% versus 43.9% and 42.5%; P<0.001). Fibrin/platelet composition was the only independent factor (odds ratio, 1.05 [95% CI, 1.02-1.08]) in differentiating cancer-related stroke from stroke caused by cardioembolism and large artery atherosclerosis. A fibrin/platelet proportion of ≥65% accurately predicted cancer-related stroke (area under the curve, 0.84; P<0.001). CONCLUSIONS: In thrombi retrieved from patients undergoing endovascular treatment, a high fibrin/platelet composition was a probable indicator of cancer-related stroke.