The performance of the microfluidic ratchet mechanism can be compared to other filtration methods using several metrics, including separation efficiency (the fraction of input leukocytes captured), leukocyte purity (the proportion of leukocytes in the output cell suspension), RBC depletion rate and overall sample throughput. In comparison with microfiltration, the key advantages of microfluidic ratchet sorting are the separation efficiency and leukocyte purity. While previous microfiltration methods typically report poor separation efficiency or fail to report this metric (Table 1), microfluidic ratchet consistently generates pure leukocyte isolates from whole blood with only 1–2% loss. The microfluidic ratchet also compares favorably with other biophysical separation (Table 2), including magnetophoresis, dielectrophoresis, and leukocyte margination. Magnetophoresis uses paramagnetic properties of RBCs to deplete these cells from the leukocyte suspension19–21. While RBC depletion using this approach exceeds 95%, the separated leukocytes remain at low relative purity because of the large number of starting RBCs (106/µl) in whole blood. Conversely, size-based sorting strategies, such as dielectrophoresis17, DLD10–12 and leukocyte margination14–16 can achieve superior leukocyte separation efficiency. However, a key advantage of the microfluidic ratchet mechanism is that it combines both size and deformability based selection to not only generate pure leukocyte suspensions but also to segregate leukocyte subpopulations, despite significant overlap in the sizes of different leukocyte subpopulations22.Table 1Performances of recent research in leukocytes separation from whole blood using microfiltration.Filtration Strategies and Filter Types*Performance MetricsLeukocyte Separation EfficiencyLeukocyte PurityRBC Depletion RateThroughputRequirement for Blood dilutionMicrofluidic Ratchet Current Study 98–99%100%100%5 µL hour−1 NoDead end; Pillar46 18–25%—(<1%)84–89%15–50 µL min−1 Yes (1:2 in PBS)Dead end; weir26 71%~10% (210-fold enrichment)n.d.4000 cells s−1 Yes (1:10 in PBS)Dead end; weir47 60%n.d.n.d.3–15 µL hour−1 NoDead end; pore27 >90%n.d.n.d.Only process 1.5 µL whole bloodNoDead end; pore23 72–85%n.d.n.d.Only process 200 µL whole bloodNoDead end; weir23 ~70%n.d.n.d.Only process <50 µL whole bloodNoDead end; pillar23 70–95%n.d.n.d.Only process 300 µL whole bloodNoCross flow; weir29 98%72%n.d.3.6 µL hour−1 YesCross flow; pore31 27.4 ± 4.9%93.5 ± 0.5%n.d.~17 µL min−1 NoCross flow; pillar 30 90%<1%90%10 µL min−1 NoCross flow; weir30 20%<1%40%10 µL min−1 Yes (1:100 in PBS)Cross flow; pillar23 70–95%n.d.n.d.20 µL min−1 NoCross flow; pillar28 97%<1%50%5 µL min−1 n.d.n.d. Data not available.*References in uppercase. Table 2Performances specifications of recent microfluidic research in leukocytes separation from whole blood using biophysical methods.Method*Performance MetricsLeukocyte Separation EfficiencyLeukocyte PurityRBC Depletion RateThroughputRequirement for Blood DilutionMicrofluidic Ratchet Current Study 98–99%100%100%5 µL hour−1 NoMagnetophoresis19 n.d.n.d93.5%5 µL hour−1 Yes (1:10 in PBS)Magnetophoresis20 n.d.n.d95%0.5–0.7 mL hour−1 Yes (1:20 in PBS)Magnetophoresis21 n.d.n.d93.7%0.12–0.92 µL min−1 Yes (1:40 in PBS)Dielectrophoresis (DEP)17 76.9–92.1%n.d.n.d.50 µL hour−1 Yes (1:5 in PBS)DLD10 99%n.dn.d.1 µL min−1 NoLeukocyte margination14 80%90%n.d.600 µL hour−1 Yes (0.5–2% hematocrit)Leukocyte margination15 97%100%n.d.1 µL min−1 Yes (1:1000 in PBS)Leukocyte margination16 67%1%n.d.1 µL hour−1 Non.d. Data not available.*References in uppercase.