Reference intervals for neutrophil reactive oxygen species production capacity in Japanese adults

Abstract Innate immunity provides the first line of defense against pathogens; however, quantitative benchmarks for assessing its function remain limited. We developed optical sensing systems that enable real-time measurement of neutrophil reactive oxygen species (ROS) production by simply diluting 3 µL of whole blood into a reaction mixture. Here, we estimated reference intervals for innate immune responses of circulating neutrophils in healthy Japanese adults, primarily those in their 40s–60s, using these optical platforms. Neutrophil superoxide production and hypochlorous acid production (myeloperoxidase activity) were measured simultaneously following chemical stimulation. Data were obtained from 329 rigorously screened individuals selected from 7,263 participants (predominantly Japanese) who underwent comprehensive health examinations between February 2020 and March 2025. Reference intervals were calculated for six neutrophil activity indices, including total ROS production, ROS production per unit neutrophils, balance between the two ROS species, and neutrophil responsiveness to stimulation. Two corresponding hypochlorous acid-related indices obtained using simplified optical systems were also evaluated. Although the existence of sex-based differences in neutrophil activity indices remains unclear, sex-specific reference intervals were calculated for indices showing statistically significant sex differences and the observed differences are discussed from the perspective of future research. We further examined relationships among the indices, and their associations with age and conventional inflammatory markers. These findings provide an assay-specific quantitative framework for assessing innate immune function and may support future studies on subclinical immune variation. The gap between the current laboratory-based assay and broader clinical application, as well as future challenges, are also discussed.