Detection of activated neutrophils by reactive oxygen species production using a hematology analyzer
Detection of activated neutrophils by reactive oxygen species production using a hematology analyzer
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DOI：
10.1016/j.jim.2018.10.004
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发表时间：
2018-12-01
影响因子：
2.2
通讯作者：
Kawano, Seiji
中科院分区：
文献类型：
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作者：
Kono, Mari;Saigo, Katsuyasu;Kawano, Seiji
中性粒细胞被募集到感染部位，并通过吞噬作用和活性氧（ROS）的产生杀死细菌。据报道，空泡存在于产生ROS的中性粒细胞中，并且大量存在于细菌感染患者的血涂片中。Sysmex全自动血液分析仪的白细胞分化功能通过流式细胞术对白细胞进行分类。本研究探讨了用血液分析仪检测产生ROS的中性粒细胞中细胞膜空泡或内陷的可能性，用佛波醇肉豆蔻酸酯（PMA）或甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）活化全血和多形核白细胞（PMN）细胞组分，用Sysmex XE-2100全自动血液分析仪进行分析。用共聚焦激光扫描显微镜（CLSM）、电子显微镜（EM）和通用流式细胞仪对PMN组分进行形态学分析，在该分析中获得的白色血细胞分化散点图中，在侧向散射（SSC）强度较高的区域中的嗜酸性粒细胞区域中出现了与原始中性粒细胞簇分离的新簇。PMN组分的流式细胞术分析显示，在这个新的集群中的细胞是CD 16 b和APF阳性的，表明这些细胞是产生ROS的活化的中性粒细胞。CLSM和EM结果显示，活性氧产生于胞浆，活化的中性粒细胞胞浆内有网状空泡样结构或胞膜内陷，产生活性氧的中性粒细胞胞浆内有胞浆内的S样结构。这些中性粒细胞在嗜酸性粒细胞区域中被检测为具有比白色血细胞分化散点图上传统簇中的中性粒细胞所示的更高SSC强度的独立簇，这可能是因为空泡样结构增加了SSC强度。
Neutrophils are recruited to infection sites and kill bacteria by phagocytosis and reactive oxygen species (ROS) production. It has been reported that vacuoles are present in neutrophils that produce ROS and are present in large numbers in blood smears of patients with bacterial infections. The leukocyte differentiation function on the Sysmex automated hematology analyzer classifies leukocytes by flow cytometry. Particularly, side-scattered light is known to reflect the quantity of organelles.This study investigated the possibility of detecting vacuoles or invagination of cell membrane in neutrophils producing ROS using a hematology analyzer.Whole blood and polymorphonuclear (PMN) cell fractions were activated with phorbol myristate acetate (PMA) or formylmethionylleucylphenylalanine (fMLP) and analyzed using the Sysmex XE-2100 automated hematology analyzer. PMN fractions were morphologically analyzed with a confocal laser scanning microscope (CLSM), electron microscope (EM), and general purpose conventional flow cytometer.In the white blood cell differentiation scattergram obtained in this analysis, a new cluster separate from the original neutrophil cluster appeared in the eosinophil area in an area of higher side-scattering (SSC) intensity. Flow cytometry analysis of the PMN fractions revealed that the cells in this new cluster were CD16b- and APF-positive, indicating that the cells were activated neutrophils that produced ROS. CLSM and EM findings revealed that ROS production occurred in the cytoplasm and that the activated neutrophils contained seine vacuole-like structures of vacuole or invagination of cell membrane.Vacuole-like Sstructures were found within the cytoplasm of neutrophils producing ROS. These neutrophils were detected as an independent cluster in the eosinophil area with higher SSC intensity than that shown by neutrophils in the traditional cluster on the white blood cell differentiation scattergram, likely because the vacuole-like structures increased the SSC intensity.