Optimized protocol for isolation of neutrophils from small-volume human blood samples via Percoll density gradient centrifugation

Neutrophils are critical first-line innate immune defenders. Conventional Percoll-based isolation protocols perform reliably only with large blood volumes (e.g., 10 mL) and yield inconsistent results from small-volume samples (≤2 mL), such as those from pediatric individuals. Here, we present an optimized protocol tailored for minimal-volume specimens, with systematic adjustments to centrifugation speed, duration, and gradient setup to ensure neutrophil isolation with high purity and viability. Among healthy donor samples (n = 19), the protocol yielded 1.74 ± 0.57 × 10⁶ granulocytes per mL of blood. Across all tested samples (n = 22), granulocyte purity was 91.58% ± 3.70%, and neutrophils comprised 96.48% ± 1.75% of recovered granulocytes in six evaluated isolates. In comparison, conventional protocols yielded granulocytes with low purity of 33.25% ± 13.99%. This robust, reproducible method enables reliable neutrophil isolation, expanding the accessibility of neutrophil-related research to scenarios with limited blood volume.