## 3 Microfluidics Methods and Devices for Evaluation of RBC Deformability
(1) I.E.1 = L/W: The elongation index (IE = L/W, where L and W are the length and width (the major and minor axes) of the deformed cell, respectively, and the method described above was utilized). (2) I.E.2 = (L - W)/(L + W), n = 100: The length and width of 100 deformed erythrocytes were measured (Photoshop was used), obtaining the deformability index (I.E.2), which is a relation between the long and short axes of the stretched cell, where L and W are the major and minor axes of the deformed cell. A microfluidic device and method were developed [3; 17] to assess red blood cell (RBC) deformability, RBC aggregation, and white blood cell (WBC) aggregation. The system integrates a microscope with a digital camera, a pump with a manometer, and a  
Figure 1: Red blood cell (RBC) deformation: visualization and quantification using microfluidic chambers under shear flow conditions. (**1**): A non-deformed erythrocyte at rest, representing its natural biconcave shape in the absence of flow. (**2**): A deformed erythrocyte that is anchored at one point to the chamber wall and elongated due to shear stress from the flowing medium. (**3**): The method for calculating the elongation index (I.E. ), which quantifies RBC deformation. computer with custom software. Using highly diluted blood suspensions between two parallel slides (100 \(\upmu\)m apart), the motion of blood cells in the microchamber is observed and recorded. This setup enables the measurement of microrheological parameters--such as RBC deformability, RBC aggregation, and leukocyte adhesion--and allows for simultaneous analysis of the interactions between RBC aggregation and leukocyte adhesion in both healthy individuals and patients with type 2 diabetes mellitus (T2 DM). [PERSON] et al. [34] studied the relationship between blood rheology--assessed using the Micro Channel Array Flow Analyzer--and various hemorheological factors, in healthy individuals. They measured blood passage time as an indicator of rheology and found that factors such as increased age, body mass index, RBC and WBC counts, total and LDL cholesterol, and triglycerides, along with decreased HDL cholesterol, were associated with impaired blood flow. These findings suggest that aging, obesity, erythrocytes, leukocytosis, and dyslipidemia may contribute to hemorheological disorders, even in healthy subjects. [PERSON] et al. [35] simultaneously measured erythrocyte adhesion and deformability using a microfluidic device equipped with an inverted optical microscope, and advanced image analysis technologies. Red blood cells suspended in saline with varying concentrations of human serum albumin were studied under a low shear stress (0.04-0.25 Pa) that caused them to deform and detach from a glass surface. The maximum deformability index observed was 0.3, and the adhesion energy per unit area was 1.1 \(\times\) 10\({}^{-6}\) Pa-m, aligning with prior studies. Imaging showed that adhesion tends to localize at a single point--forming a \"spike-like tip\"--suggesting receptor migration on the cell membrane. Additionally, stronger adhesion in lower albumin concentrations implies that membrane receptor mobility influences both deformability and adhesion behavior. The measurements and visualizations of blood cell deformability in flows in microfluidic devices (point-of-care devices) can provide information that is vital to the diagnosis of early symptoms of blood diseases and consequently these devices could be used as a speedy clinical tool for the early detection of biomarkers [22]. For example, erythrocyte (RBCs) stiffness has been linked to myocardial infarction, diabetes mellitus, and hypertension, among other blood diseases. Analyzing blood rheology--strongly influenced by cell mechanics and interactions--has become a key focus in biomedical research. Additionally, creating blood analogs that replicate blood's rheological properties is essential for the effective design and optimization of the microfluidic systems used in personalized medicine.