## 4 Methods and Devices for Assessing Erythrocyte (RBC) Aggregation
### Image Analysis Method in a Flow Chamber  
Microfluidic devices allow for precise control over flow rate, shear stress, and the chemical environment. They mimic capillary-sized vessels, making the technology ideal for studying RBC behavior in conditions similar to those in vivo. They enable real-time imaging and measurement of aggregation dynamics, including formation and disaggregation. Some of the devices and methods developed for microfluidic analyses of red blood cell aggregation are summarized in Table 2. [PERSON] et al. (2006) [33] developed a low-cost, disposable aggregometer for clinically measuring dynamic RBC aggregation using minimal blood volume. The device combines a vibration-based disaggregation mechanism, a microfluidic channel, and a laser-photodiode system to assess aggregation behavior. The aggregometer uniquely measures RBC aggregation and its influence on blood viscosity across a range of shear rates, a capacity which previous methods lacked. By monitoring backscattered light intensity \(I(t)\) and pressure \(p(t)\) over time, the device captures both aggregation dynamics and stress-shear rate data. The results showed that RBC aggregability is shear-dependent and correlates with shear-thinning blood viscosity. The method successfully links dynamic aggregation characteristics to viscosity changes, supporting its clinical relevance. \begin{table}
\begin{tabular}{p{113.8 pt} p{113.8 pt} p{113.8 pt} p{113.8 pt}} \hline \hline
**Technique** & **Main Features** & **Performance/Limitations** & **Disease Focus/Applications** \\ \hline Micropipette Aspiration & Aspirates individual RBCs through a small pipette to measure mechanical deformation. & Highly accurate for static/dynamic deformability, but low-throughput and labor-intensive. & Evaluates deformability changes in stored vs. fresh RBCs. \\ \hline Microfluidic High-Throughput Device & Uses flow around a micro-obstacle to assess the Young’s modulus of RBCs. & High-throughput; allows population heterogeneity analysis. & Scalable tool for personalized RBC stiffness screening. \\ \hline Ektacytometry & Measures elongation index vs. shear stress or osmolality (osmoscan). & Quantifies RBC deformability across various physiological conditions; sensitive to setup. & Useful in diagnosing disorders affecting cell morphology/deformability (e.g., hereditary diseases, oxidative stress). \\ \hline Rheoscope & Directly visualizes RBC deformation in flow using microscope-based system. & Enables real-time observation under physiological conditions; depends on setup precision. & Research on RBC behavior under controlled flow conditions. \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Comparison of RBC deformability measurement techniques. A comparison of traditional and microfluidic methods used to measure erythrocyte deformability is presented in Table 4.  
Table 5 presents various techniques and their corresponding methods for measuring erythrocyte aggregation. [PERSON] et al. [45] investigated the relationship between local red blood cell (RBC) aggregation, velocity, and hematocrit in a microfluidic channel--an area often overlooked in previous studies. Advanced image processing techniques were used to quantify local and bulk aggregation. The study found that aggregation intensity strongly correlates with local velocity variations, both in the center and near the walls of the microchannel. The results high