Initially, the device was evaluated of its ability to separate leukocytes from the highly abundant erythrocytes. To distinguish leukocytes from erythrocytes, blood samples were pre-stained with Hoechst 33342 DNA stain and infused into the sorting region (Fig. 2A). As shown in Fig. 2B–E, the sample formed the characteristic diagonal trajectory. Individual leukocytes (blue) that have reached their limiting funnel constrictions were found to transit horizontally as expected (Fig. 2E and supplemental video). As a result, RBCs were collected in fractions O1-O3 (Fig. 2F; 2, 2.5 and 3 µm pores) while leukocytes were preferentially segregated among outlets O4-O9 (Fig. 2G; 3.5, 4, 4.5, 5.5, 6.5 and 8 µm pores). These results indicated that 3 µm is a suitable cut-off pore size for the separation of erythrocytes and leukocytes, which is similar to 3.5 µm pores used by other microfilters with pillar, weir, and membrane configurations23. It is important to note that staining the leukocytes required washing the cells, which removed the plasma component of blood. In this case, the blood cells were re-suspended at ~45% hematocrit. Both stained blood cells and unstained whole blood were tested and observed to behave equivalently.Figure 2Microfluidic ratchets to separate leukocytes directly from whole blood. (A) Image of the sorting region of microfluidic ratchets infused with food color dyes illustrating the diagonal trajectory of the sample inlet flow. (B–E) Images of the leukocytes stained with Hoechst 33342 being separated from the whole blood and traversing diagonally through the funnel matrix. (F,G) Bright field and fluorescence images of the outlets showing the separate reservoirs into which RBCs and leukocytes are collected.