The separation of leukocyte from erythrocytes was investigated as a function of oscillation pressure and funnel thickness, which varied from 14–20 kPa and 11–15 µm respectively. This operating range was established using previous experiments of acceptable operating conditions (see supplemental information). As shown in Fig. 3A–C, higher filtration pressures increased the frequency for cells to transit narrower openings, represented by a slight leftward shift of leukocytes’ outlet distributions for three different donors’ blood samples. Increasing the funnel thickness from 11 to 15 µm allows each cell to have greater vertical extension during deformation. However, as show in Fig. 3D–F, this change has minimal effect on the leukocyte distributions. RBCs were found exclusively in O1–3 in all scenarios except when the filtration pressure was decreased to 14 kPa, in which case, a small fraction of RBCs escaped into O4, leading to decreased leukocyte separation efficiency. Here, separation efficiency is defined as the pure fraction of leukocytes captured in outlets over the total number of leukocytes entering the sorting region (Fig. 4A). Since device thickness (11–15 µm) does not seem to have a strong influence on the efficiency of leukocytes separation from RBCs (Fig. 4B), we select a modest oscillation pressure of 17 kPa, as well as a device thickness of 11 µm to make the device robust to potential debris in the sample. Based on this configuration the microfluidic ratchet device is able to obtain a leukocyte separation efficiency of 98–99% (Fig. 4).Figure 3Leukocyte distribution at outlets as a function of filtration pressure applied at OSC2 and funnel thickness. (A–C) Leukocyte deformability profiles of three different donors at various filtration pressure with fixed funnel thickness of 11 µm. (D–F) Leukocyte deformability profiles of three donors at two different funnel thickness at a fixed filtration pressure of 17 kPa. Figure 4Performance of deformability-based separation of leukocytes from whole blood as a function of (A) filtration pressure and (B) funnel thickness. (P values determined using Student’s T-test).