Chronic lymphocytic leukemia (CLL), the most common type of leukemia, remains incurable with conventional therapy. Despite advances in therapies targeting Bruton’s tyrosine kinase and anti-apoptotic protein BCL-2, little is known about their effect on red blood cell (RBC) aggregation in blood flow. In this study, we applied a microfluidic device and a newly developed Software Image Flow Analysis to assess the extent of RBC aggregation in CLL patients and to elucidate the hemorheological effects of the commonly applied therapeutics Obinutuzumab/Venetoclax and Ibrutinib. The results revealed that, in RBC samples from untreated CLL patients, complex 3D clusters of large RBC aggregates are formed, and their number is significantly increased compared to healthy control samples. The application of the Obinutuzumab/Venetoclax combination did not affect this aspect of RBCs’ rheological behavior. In contrast, targeted therapy with Ibrutinib preserves the aggregation state of CLL RBCs to levels seen in healthy controls, demonstrating that Ibrutinib mitigates the alterations in the rheological properties of RBCs associated with CLL. Our findings highlight the alterations in RBC aggregation in CLL and the impact of different targeted therapies on RBCs’ rheological properties, which is critical for predicting the potential complications and side effects of CLL treatments, particularly concerning blood flow dynamics.

Red blood cell (RBC) aggregation and disaggregation are key factors in microcirculatory flow, and their disturbance can lead to alterations in the rheological properties of blood in disorders such as chronic lymphocytic leukemia (CLL). This study aimed to determine the critical shear rate required to fully disaggregate RBC aggregates using samples from healthy individuals, providing a reference point for evaluating pathological changes. Using a microfluidic system and software-image-based flow analysis, RBC disaggregation was assessed by the Aggregation-Area Indicator at a high shear rate (AAI_H) changes and the number of undestroyed aggregates. The defined critical shear rate at 446 s⁻¹ was applied to assess RBC disaggregation in CLL patients, both untreated and treated with Obinutuzumab/Venetoclax or Ibrutinib. CLL samples exhibited significantly elevated AAI_H values compared to controls, indicating a greater resistance to shear-induced dispersion. Although both treatments reduced the number of stable aggregates, neither therapy fully normalized RBC disaggregation to the levels observed in healthy controls. Moreover, there was a notable heterogeneity among Ibrutinib-treated patients, revealing different therapeutic effects on RBC rheology. These findings suggest alterations in the RBC rheology in CLL despite therapy and support the use of a shear-dependent disaggregation analysis as a complementary tool for understanding and monitoring CLL-related hematologic abnormalities.

Preeclampsia is a pregnancy-related disease with poor placentation and presents itself through hypertension and proteinuria. The disease is also associated with the oxidative modification of proteins in maternal blood plasma. In this work, we combine differential scanning calorimetry (DSC), capillary electrophoresis, and atomic force microscopy (AFM) to evaluate the changes in the plasma denaturation profiles of patients with preeclampsia (PE) as compared with those of pregnant controls. Our results demonstrate that the last trimester of pregnancy substantially affects the main calorimetric characteristics of blood plasma from pregnant controls relative to nonpregnant women. These variations correlate well with the changes in protein levels determined by electrophoresis. DSC analysis revealed significant deviations in the plasma heat capacity profiles of preeclamptic patients from those of pregnant controls. These alterations are expressed mainly in a substantial reduction in albumin-assigned transitions and an upward shift in its denaturation temperature, lower calorimetric enthalpy changes, and a reduced ratio of heat capacity in the albumin/globulin-assigned thermal transitions, which are more pronounced in severe PE cases. The in vitro oxidation model shows that the alteration of PE thermograms is partly related to protein oxidation. AFM data detected numerous aggregate formations in the plasma of PE samples and fewer small ones in the pregnant controls, which are not found in healthy nonpregnant samples. These findings could serve as a basis for further investigations to reveal the possible relationship between albumin thermal stabilization, the increased inflammatory state and oxidative stress, and protein misfolding in preeclampsia.

Chronic lymphocytic leukemia (CLL) has systemic effects that extend beyond malignant lymphocytes, potentially altering the structure and function of circulating red blood cells (RBCs). In this study, atomic force microscopy (AFM) was combined with complementary calorimetric analysis to investigate the membrane ultrastructure, nanomechanical characteristics, and thermodynamic behavior of RBCs from untreated CLL patients and those receiving targeted therapies (Obinutuzumab/Venetoclax or Ibrutinib). RBCs from untreated patients exhibited pronounced reduction in membrane roughness, increased stiffness and adhesion forces, and altered thermal unfolding of cytoskeletal and membrane proteins, indicative of impaired structural flexibility and stability. Treatment with Obinutuzumab/Venetoclax partially restored surface topography, but stiffness and adhesion forces remained elevated, suggesting persistent cytoskeletal rigidity. The obscured spectrin and Band 2–4 thermal transitions and the elevated total enthalpy change revealed by differential scanning calorimetry indicated a modified conformation or binding state of membrane proteins. In contrast, Ibrutinib therapy produced near-normal nanomechanical and thermal characteristics, reflecting a more comprehensive restoration of RBC integrity. These findings demonstrate that CLL and its therapies distinctly influence erythrocyte morphology and mechanics, underscoring the systemic impact of the disease. The strong correspondence between AFM and calorimetric data highlights the potential of integrated biophysical approaches to detect subtle RBC alterations and to serve as complementary indicators for therapeutic monitoring.

Red blood cell (RBC) deformability is a key determinant of microcirculatory flow and can be altered in hematological disorders such as chronic lymphocytic leukemia (CLL). This study aimed to evaluate RBC deformability under controlled microfluidic flow conditions and to assess the influence of software platform choice on deformability quantification. RBC suspensions from healthy individuals and untreated CLL patients were analyzed using a microfluidic imaging system across a range of shear rates. A dedicated image-processing algorithm was developed and implemented in two software environments (LabVIEW and Python) to automatically detect deformed cells, measure major and minor cell axes, and calculate the deformability index (DI). Both analytical approaches demonstrated a shear-dependent increase in DI in healthy controls, whereas RBCs from CLL patients exhibited reduced deformability and a blunted response to increasing shear rates, particularly at intermediate shear rates. Although LabVIEW produced consistently higher absolute DI values than Python, both platforms showed strong correlation and preserved the same relative trends and group discrimination. These findings demonstrate that microfluidic image flow analysis provides a robust approach for assessing RBC biomechanics and highlight the importance of standardized image-processing workflows for reliable deformability quantification across software platforms.

The physicochemical behavior of gold nanoparticles (AuNPs) functionalized with two-antennary oligoglycines is studied by analyzing the colloidal stability and size distribution via dynamic light scattering, the surface-charge reconfiguration through zeta-potential, the nanoscale structure using atomic force microscopy, and the thermodynamic interactions with blood serum using differential scanning calori­metry. These analyses provide an initial assessment of the suitability of the two-antennary oligo­glycines as a supramolecular surface-engineering ligand for AuNP-based biomedical applications. The two-antennary oligoglycines form stable complexes with AuNPs, creating a coating layer and reversing surface charge. The interaction between bare nanoparticles and blood serum is weak, primarily affecting albumin. On the other hand, the oligoglycines interact strongly with the blood serum proteins altering their stability. Together, these results demonstrate that two-antennary oligoglycines serve as both a stabilizing and functionalizing layer, thereby enhancing nanoparticle stability and modulating biological interactions. This highlights their potential for the design of well-defined nanomaterials and for applications in drug delivery and other biomedical fields.