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首页> 外文期刊>Ukrainian Biochemical Journal >Novel approach for discrimination of eosinophilic granulocytes and evaluation of their surface receptors in a multicolor fluorescent histological assessment
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Novel approach for discrimination of eosinophilic granulocytes and evaluation of their surface receptors in a multicolor fluorescent histological assessment

机译：多色荧光组织学评估中嗜酸性粒细胞鉴别的新方法及其表面受体的评价

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Eosinophilic granulocytes mediate immune responses against multicellular parasites and are also the main contributor to such pathological conditions as allergy and asthma. Eosinophils are discriminated with eosin staining in conventional histology using light microscopy. However, molecular detection of antigens and the widely introduced automated analyzers usually require fluorescent markers to allow quantitative determination. Surprisingly, there is no selective CD marker to differentiate eosinophils and basophils. Recently reported analogs to replace hematoxylin and eosin staining for immune-histochemical applications such as DRAQ5 & eosin are also unsuitable due to wide fluorescent spectra. Different combinations of fluorescent dyes were tested using fluorescent microscopy aimed to develop a simple and specific method for detecting eosinophilic granules, DNA and surface receptors, the approach was used for evaluation of IgE levels (total and specific to casein) on cells of patients suffering from cow milk allergy. We were able to achieve selective visualization of eosinophil granules using aniline blue dye by modifying the method of Berretty & Cormane (1978) and detecting emission at 440nm; this allowed simultaneous staining of blood smears with anti-IgE-FITC (emission at 520 nm) and casein-FITC, detection of DNA with propidium iodide (em. 590 nm), and also provided specific metachromatic signal of eosinophils in the NIR region (em. ~700 nm) with subsequent quantification of fluorescent signal. Application of this approach to clinical cases revealed increased IgE levels and increased casein-binding targets on eosinophils in 3 patients with cow milk allergy compared to 2 healthy donors, demonstrating the general usefulness of the approach.
机译：嗜酸性粒细胞介导针对多细胞寄生虫的免疫应答，也是对患者和哮喘的病理条件的主要因素。使用光学显微镜与常规组织学中的eosin染色与eosin染色的嗜酸性粒细胞。然而，抗原的分子检测和广泛引入的自动分析仪通常需要荧光标记物以允许定量测定。令人惊讶的是，没有选择性CD标记来区分嗜酸性粒细胞和嗜碱粒细胞。最近报道的类似物以代替血毒素和曙红染色的免疫组织化学应用，例如DRAQ5和eosin的染色也是不合适的，因为宽荧光光谱也是不合适的。使用荧光显微镜测试荧光染料的不同组合，旨在开发用于检测嗜酸性颗粒，DNA和表面受体的简单且特异性的方法，该方法用于评估IgE水平（总和特异性酪蛋白）对患者的患者细胞进行评估牛奶过敏。我们能够通过改变苯丙胺和纤维蛋白（1978）的方法来实现使用苯胺蓝染料的嗜酸性粒细胞颗粒的选择性可视化，并在440nm处检测到发射;这允许使用抗IgE-FITC（520nm发射）和酪蛋白，用碘化丙酮（EM.590nm）检测DNA的血液涂片同时染色，并且还提供了NIR区域的嗜酸性粒细胞的特异性心动源性信号（ EM。〜700nm）随后定量荧光信号。这种方法在临床病例中的应用揭示了IgE水平增加，3例牛奶过敏与2例健康捐赠者相比，3例嗜酸性粒细胞增加了酪蛋白粒细胞的靶向靶，展示了这种方法的一般有用性。

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