Flow cytometry-based characterization of isolated human neutrophils: Evaluating viability, purity and defining a reference interval for basal activation in functional assays.

INTRODUCTION: Neutrophils are the most abundant circulating leukocytes and play a central role in innate immunity. As in vitro functional assays (NETosis, degranulation, phagocytosis, etc.) depend on cell viability, purity and basal activation, it is essential to validate neutrophil suspensions before experimental use. OBJECTIVES: To characterize isolated neutrophils using an in-house flow cytometry workflow for assessing viability, purity, and activation, and to establish a reference interval (RI) for neutrophil basal activation. METHODS: Twenty healthy volunteers were recruited, and neutrophils were isolated from whole blood by negative selection. Cell viability was determined by propidium iodide staining. Leukocyte and neutrophil percentage were evaluated using conjugated antibodies against CD45 and CD15 respectively. Basal activation was assessed by measuring mean fluorescence intensity (MFI) using a conjugated antibody against CD66b. To validate the RI of basal activation, neutrophils were also stimulated with TNF. RI were calculated as mean ± 2 standard deviations (SD). RESULTS: Neutrophil suspensions displayed high quality across all criteria. Viability averaged 99.4% (SD =0.4). Leukocyte purity reached 99.3% (SD =0.4), and neutrophil purity was elevated, with a mean of 98.6% (SD = 0.5). Isolated neutrophils presented a mean CD66b MFI of 37.4 (SD =10.8), and CD66b expression increased significantly following TNF stimulation. The calculated RI was 37.4 ± 21.6. CONCLUSION: This study demonstrated that neutrophils isolated by negative selection displayed high viability, purity, and minimal activation. It also allowed the definition of a RI for basal activation which could serve as objective criterion to validate neutrophil suspensions for functional assays.