In summary, the microfluidic ratchet mechanism enables highly selective sorting of leukocytes directly from whole blood. We showed this mechanism can achieve clear separation of lymphocytes and granulocytes, and we believe with further refinement, the separation of other leukocyte phenotypes can be achieved. Another important application of this technology is the interrogation of the phenotypic plasticity of leukocytes to identify biomechanical changes associated with leukocyte activation, such as neutrophil activation during cancer progression43 and viral infection44. This approach represents a potential alternative to flow cytometry to enrich for leukocytes that adopt specific activation states. After sorting, the fractionated cells are immediately available for downstream molecular characterization, such as immunofluorescence, transcriptome analysis, or cytokine secretion assays. Together, the microfluidic ratchet mechanism represents a simple and compelling approach for biophysical separation and characterization of leukocytes as part of biological assays.