To investigate whether deformability based sorting could separate granulocyte and lymphocyte subpopulations directly from whole blood, we pre-stained donor blood for CD3 and CD19 in order to identify the majority of lymphocytes (T cells and B cells) as well as for CD66b to identify granulocytes. Following microfluidic ratchet enrichment, leukocytes were distributed among O4–O9. Lymphocytes were preferentially distributed around O6 (retained between 4.5 to 5.5 µm pore size), where such cells can be obtained at 62–68% purity (Fig. 5A–F). Granulocytes were distributed around outlets O8 or O9 (retained between 6.5 to 8 µm funnel opening), where such cells can be obtained at 88–95% purity in these outlets (Fig. 5A–F). The images in Fig. 5G and H demonstrates that lymphocytes and granulocytes comprise the majority of the cells in O6 and O8 respectively. Based on their distribution into outlets, it was observed that leukocytes experience significantly less deformation than would be expected within smaller capillaries, such as pulmonary capillaries, which has circular constrictions that are ~5 µm in diameter39, 40. Specifically, lymphocytes (average diameter 7 µm) are typically blocked by the 4.5 × 11 µm constriction while neutrophils (average diameter 7.5 µm) are blocked by the 6.5 × 11 µm constriction.Figure 5Separation of lymphocytes and granulocytes from whole blood. (A–C) Deformability profiles of lymphocytes and granulocytes enriched from blood of three different donors. (D–F) Relative abundance of lymphocytes and granulocytes in each outlet. (G,H) Images of the immunostained lymphocytes (green) in O6 and granulocytes (red) in O8. (I) Comparison of distributions of leukocytes in O4–9 of three different samples: (1) whole blood without stain, (2) with Hoechst 33342 and (3) with anti-CD markers conjugated with fluorescence tags.