## 3 Microfluidics Methods and Devices for Evaluation of RBC Deformability
Integrating hemorheology with microfluidic technology enables single-cell analysis, supporting the development of new diagnostic tools and personalized treatment strategies. [PERSON] et al. [5] demonstrated the use of microfluidic devices to assess the vascular deformability of erythrocytes and agranular leukocytes. Their study showed that, compared to older methods such as micropipette aspiration, ektacytometry, and micropore cell-transit assays, microfluidic techniques provide a low-cost, high-throughput alternative. This highlights the efficiency and practicality of modern microfluidics in the study of blood cell mechanics. [PERSON] et al. [38] developed a microfluidic physiometer that measures blood viscosity, hematocrit, and RBC deformability on a chip. It combines a hydrodynamic unit (for viscosity, using reference fluid and microchannel arrays) and an electronic unit (for electrical properties like plasma resistance and membrane capacitance). This device is an advanced microfluidic-electronic system used to analyze whole-blood rheology and electrical properties under flow conditions. [PERSON] et al. [37] reviewed measurable physical quantities in microfluidics, distinguishing direct and indirect viscosity measurements, and outlined future challenges and research directions for clinical applications. [PERSON] et al. [36] created a microfluidic system that uses electrical signals to assess RBC deformability by analyzing changes in current as cells pass through microchannels. The system can differentiate RBCs with varying stiffness at a rate of \(\sim\)10 cells per second. Analyzing the data, we can make the following summary of the possibilities for standardizing the measurement of the EI index. There are several challenges in standardizing elongation index (EI) measurements across different platforms. One of them arises due to differing measurement techniques, as various platforms use distinct methods to assess cell deformability, such as optical tweezers, laser diffractometry, microfluidic stretching, or electrical impedance. These different physical principles can lead to variations in EI values, even for the same sample. Other reasons could relate to the variability in shear stress or strain application. The magnitude and type of the mechanical forces applied to cells vary across devices (e.g., controlled shear flow vs. stretching by optical traps). Since the EI is determined by the manner in which cells deform under specific forces, inconsistencies in applied stress make direct comparisons difficult. Differences in sample preparation and conditions and factors such as temperature, medium viscosity, cell concentration, and incubation time can influence cell deformability measurements. A lack of standardized sample handling protocols leads to variability in EI results. Data analysis and calculation methods are another factor. EI is often derived from image analysis or light-scattering patterns, but algorithms and parameters used for shape quantification differ between platforms. This can cause discrepancies in the ways in which elongation is quantified. Cell populations can be heterogeneous in size, shape, and mechanical properties, causing platform-dependent sensitivity differences. Some methods may be biased towards specific subpopulations, skewing EI outcomes. As a whole, there is a lack of universally accepted calibration standards and reference materials for EI measurements. Without standard references, alignment of data across platforms remains challenging.