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.

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.

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.