Red blood cells respond to fluctuations in blood plasma pH by changing the rate of biochemical and physical processes that affect the specific functions of individual cells. This study aimed to analyze the effect of pH changes on red blood cell morphology and structure. The findings revealed that an increase or decrease in pH above or below the physiological level of pH 7.4 results in the transformation of discocytes into echinocytes and causes significant alterations in the membrane, including its roughness, cytoskeleton structure, and the cell’s elastic modulus. Furthermore, the study shown a strong connection between critical acidosis and alkalosis with increased intracellular reactive oxygen species production.

The transcription factor IRF5 maintains macrophages in the pro-inflammatory M1 state. We assessed the effects of siRNA-mediated knockdown of Irf5 on murine bone marrow-derived macrophages (BMDM) in M0, M1 and M2 states. Knockdown of Irf5 in M1 macrophages made them phenotypically similar to M2 macrophages, which was reflected in the decreased expression of the M1 marker iNOS, increased expression of the M2 marker CD206, increased mitochondrial content and respective morphological changes. Interestingly, the M2 phenotype was also affected by the reduction in Irf5. Using atomic force microscopy (AFM), we showed that Irf5 knockdown increases plasma membrane roughness, particularly in M2 macrophages. AFM-based stiffness measurements indicated that Irf5 knockdown altered macrophage elasticity, potentially influencing their functional behavior. Our data suggest a complex role of IRF5 in macrophage polarization, supporting its dual role as a transcriptional activator and repressor both in M1 and M2 states, and highlight the importance of IRF5 in the maintenance of metabolic and functional properties of macrophages.

Background/Objectives: Leukoreduced low-titer group O whole blood (LTOWB) is a promising option for early transfusion in massive hemorrhages, by providing red blood cells (RBCs), plasma, and platelets in a single unit. This study aimed to assess the effects of pathogen reduction (PR) on the LTOWB quality and its suitability for emergency transfusions, including its post-thaw RBC usability. Methods: Whole blood from 24 donors was processed and analyzed over seven days, including post-thaw assessments. Hematological and coagulation parameters, along with atomic force microscopy, were used to assess the RBCs’ morphology, cytoskeletal integrity, and nanomechanical properties. Results: The PR reduced the leukocyte content while preserving platelet counts at the level of the control group. Although certain clotting factors, such as fibrinogen (p < 0.001) and factor VIII (p < 0.001), were diminished after the PR, thromboelastometry results remained within reference ranges, indicating a maintained hemostatic potential. Morphological changes in RBCs were observed, but the membrane elasticity remained stable throughout storage and after thawing, indicating preserved macromechanical properties essential for hemostasis. Conclusions: These findings demonstrate that LTOWB treated with partial a leukoreduction and PR maintains functional and structural integrity, supporting its potential utility as a safe and effective transfusion product for managing acute blood loss.

This study aimed to identify neutrophil membrane characteristics that could serve as clinical biomarkers for the development of infectious complications in newborns.Materials and Methods. Neutrophils isolated from healthy donors were used as a model system. The cells were incubated with plasma samples (S) isolated from blood of newborns categorized into three groups: apparently healthy (normal) (NS) (N=6), with localized infection (LIS) (N=7), and with generalized infection (GIS) (N=8). We assessed cellular morphology and membrane roughness before and after stimulation with phorbol 12-myristate 13-acetate (PMA) using fluorescence and atomic force microscopy. We quantified nuclear and membrane surface areas, the intensity of neutrophil extracellular trap (NET) formation, and membrane arithmetic average roughness (Ra).Results. A standardized protocol for neutrophil preparation and evaluation was developed. Optimal incubation conditions were established; 1% bovine serum albumin (BSA) yielded minimal background activation. Dose-dependent activation of neutrophils by PMA was observed in the presence of 1% plasma. PMA stimulation significantly increased nuclear area (P0.001), membrane area (P0.001), and Ra (P0.001), regardless of plasma sample group. The most significant changes occurred in neutrophils incubated with plasma from the GIS group. Generalized infection was associated with enhanced NET activation, which may contribute to the pathogenesis of thrombotic complications in neonatal sepsis.Conclusion. Microscopy-based neutrophil characteristics are promising biomarkers for evaluating infection including sepsis in newborns.

INTRODUCTION: Currently, whole (preserved) blood is a promising tool for transfusion therapy in cases of massive blood loss. However, the lack of universal technological solutions that minimize immunological and infectious risks of preserved blood limits its widespread use. OBJECTIVE: To study the impact of modified collection methods and pathogen reduction technology on the properties of preserved blood that determine its suitability for the correction of massive blood loss. MATERIALS AND METHODS: Whole blood of group O was collected from 44 donors (34 men and 10 women). Four groups were studied: the control group without leukoreduction (Control), leukoreduced (LR), collected using a modified technology with partial leukoreduction (PLR), and with subsequent pathogen reduction (PLR + PR). Blood samples were stored at +4–6 °C for 7 days. On days 1 and 7 hematological parameters, coagulation factors, and viscoelastic tests were evaluated. Atomic force microscopy (AFM) was used to assess erythrocyte morphology, cytoskeletal structure, and cell elasticity modulus. RESULTS: Partial leukoreduction reduced leukocyte counts to 0.36 × 109/L, with similar levels observed in PLR + PR and no significant changes during storage. Platelet levels remained stable in both groups: 139 (102; 164) × 109/L and 86 (43; 107) × 109/L in PLR, and 114 (94; 142) × 109/L and 70 (55; 96) × 109/L in PLR + PR on days 1 and 7, respectively. Coagulation properties (NATEM test) remained within reference ranges; fibrinogen levels were lower in the pathogen-reduced group, while Factor XIII remained stable throughout storage. AFM analysis showed that erythrocyte morphological changes after pathogen reduction did not exceed those observed in other groups; no negative effects on cytoskeletal structure or cell elasticity were detected. The Young’s modulus remained at 5.5 ± 2 kPa across all groups. CONCLUSIONS: Modified blood collection technologies preserve the hemostatic potential of preserved blood and do not significantly affect the structure of erythrocytes. Partial leukoreduction and pathogen reduction could become universal technological solutions that enhance the safety of using preserved blood for correcting massive blood loss.

Scanning probe microscopy (SPM) is a versatile tool for studying a wide range of materials. It is well suited for investigating living matter, for example, in single-cell neutrophil studies. SPM has been extensively utilized to analyze cell physical properties, providing detailed insights into their structural and functional characteristics at the nanoscale. Its long-standing application in this field highlights its essential role in cell biology and immunology research, significantly contributing to understanding cellular mechanics and interactions. In this review, we discuss the application of SPM techniques, specifically atomic force microscopy (AFM) and scanning ion-conductance microscopy (SICM), to study the fundamental functions of neutrophils. In addition, recent advances in the application of SPM in single-cell immunology are discussed. The application of these techniques allows for obtaining data on the morphology, topography, and mechanical and electrochemical properties of neutrophils with high accuracy.