Woltmann G, Ward R J, Symon F A, Rew D A, Pavord I D, Wardlaw A J
Division of Respiratory Medicine, University of Leicester, Glenfield Hospital, UK.
Thorax. 1999 Feb;54(2):124-30. doi: 10.1136/thx.54.2.124.
Sputum induction is an important non-invasive technique for measuring airway inflammation in asthma. Cell numbers are often too low for flow cytometric analysis. Laser scanning cytometry (LSC) is a novel technique that allows objective multicolour fluorescence analysis of cells on a microscope slide.
LSC was used to determine sputum eosinophil and bronchial epithelial cell counts. We first confirmed that we could measure eosinophil counts accurately in peripheral blood using alpha-major basic protein (MBP) immunofluorescent staining. Sputum induction was performed according to standard protocols. Sputum samples from eight normal controls and 12 asthmatic patients were analysed by LSC and manual counting by two independent observers. Octospot cytospins were fixed and stained with mouse-alpha-human-MBP monoclonal antibody or mouse-alpha-human-cytokeratin antibody and goat-alpha-mouse Oregon Green conjugated second antibody.
Sputum induction provided a mean (SE) of 0.99 (0.2) x 10(6) cells per donor. More than 3000 cells on three cytospins per slide were analysed per cell type. The intraclass correlation coefficient (R) and standard deviation (SD) of differences in eosinophils determined by manual counting and LSC were 0.9 and 2.1, respectively, and for bronchial epithelial cell counts they were 0.7 and 2.0. Selective detection of labelled cells was confirmed visually after relocation.
Eosinophils and bronchial epithelial cells can be accurately and reproducibly counted in an objective manner. LSC is therefore a potentially powerful new method for immunophenotyping leucocytes and epithelial cells objectively in induced sputum in patients with asthma.
痰液诱导是测量哮喘气道炎症的一项重要非侵入性技术。细胞数量通常过低，无法进行流式细胞术分析。激光扫描细胞术（LSC）是一种能够对显微镜载玻片上的细胞进行客观多色荧光分析的新技术。
采用LSC测定痰液嗜酸性粒细胞和支气管上皮细胞计数。我们首先证实，使用α-主要碱性蛋白（MBP）免疫荧光染色能够准确测量外周血中的嗜酸性粒细胞计数。按照标准方案进行痰液诱导。对来自8名正常对照者和12名哮喘患者的痰液样本进行LSC分析，并由两名独立观察者进行手工计数。将八孔涂片离心机制备的涂片固定，并用小鼠抗人MBP单克隆抗体或小鼠抗人细胞角蛋白抗体以及山羊抗小鼠俄勒冈绿偶联二抗进行染色。
每位供体的痰液诱导平均提供了0.99（0.2）×10⁶个细胞。每张载玻片上三个涂片上每种细胞类型分析的细胞数超过3000个。手工计数和LSC测定的嗜酸性粒细胞差异的组内相关系数（R）和标准差（SD）分别为0.9和2.1，支气管上皮细胞计数的分别为0.7和2.0。重新定位后，通过视觉确认了标记细胞的选择性检测。
嗜酸性粒细胞和支气管上皮细胞能够以客观的方式准确且可重复地计数。因此，LSC是一种潜在的强大新方法，可用于客观地对哮喘患者诱导痰液中的白细胞和上皮细胞进行免疫表型分析。
