Impact of sequential bifurcations on the cell-free layer of healthy and rigid red blood cells.

In the microcirculation, red blood cells (RBCs) tend to move away from vessel walls, creating a central flow of cells and a peripheral cell-free layer (CFL). The CFL significantly affects blood flow and is important for lab-on-a-chip applications, such as cell-plasma separation. This study investigates how the length of the feeding branch before bifurcations affects RBC distribution and CFL formation, especially in sequential T-bifurcations. We conducted experiments to study RBC flow in microfluidic bifurcating channels of different lengths (2.5-7.5 mm) at a fixed hematocrit of 5% using both healthy and artificially rigidified RBCs. Our findings show that a minimum branch length is required before a bifurcation to achieve a steady state in the CFL. If the channel length before a second bifurcation is shorter than this minimum, reaching an equilibrium CFL in sequential bifurcations is impossible. We observe that short channels after the first bifurcation lead to increased CFL asymmetry in the daughter branches after the second bifurcation, while longer channels better maintain symmetry. Additionally, we explored the impact of RBC rigidity on CFL development. Rigid and healthy RBCs showed similar behavior at the first bifurcation, but their CFL development patterns differed significantly by the second bifurcation, affecting RBC partitioning. These results emphasize the importance of considering branch length in the study and design of bifurcations for lab-on-a-chip devices and provide insights into how impaired RBC deformability can affect blood flow.