这项工作得到了列日大学医院的支持。我们承认 "瞬间生产" 的视频制作。我们还要感谢在电影中出现的那个病人, 塞德里克. 弗朗索瓦。

本节所描述的所有方法都已得到列日大学医院伦理委员会的批准, 所有健康的受试者都书面同意参与。
1.Sputum 感应
<ol>
	<li>预 post-bronchodilator 肺活量
	<ol>
		<li>根据美国胸腔学会 (ATS)/欧洲呼吸系统学会标准标准 12, 执行肺活量 (强制呼气音量在1秒 [FEV1] 和强制肺活量 [FVC] 机动)。</li>
		<li>管理400µg 吸入沙丁胺醇从计量吸入器 (MDI) 通过间隔装置。 注: 在成人中, 吸入沙丁胺醇的不良事件包括震颤和心动过速13。</li>
		<li>重复肺活量 (FEV1和 FVC 机动) 15 分钟后, 根据 ATS/再培训标准标准12。</li>
	</ol>
	</li>
	<li>超声波雾化器的研制 注: 喷雾器必须在每次使用前彻底清洗和消毒, 并按照制造商的指示进行。<ol>
		<li>用蒸馏水填满雾化器室, 直至推荐的水平。</li>
		<li>在雾化器室里放一个干净的杯子。</li>
		<li>用合适的盖子盖住杯子。</li>
		<li>用50毫升高渗盐水 (5%) 填充 post-bronchodilator FEV1和 #62 的患者; 65% 预测或50毫升生理盐水 (0.9%) 为 post-bronchodilator FEV1≤65% 预测的患者。</li>
		<li>加入1.75 毫升硫酸沙丁胺醇溶液 (5 毫克/毫升) 的杯子。</li>
		<li>按照制造商的说明连接管道和阀门。</li>
	</ol>
	</li>
	<li>雾化吸痰 注意: 在医疗监督下执行技术。<ol>
		<li>向病人解释程序。</li>
		<li>让病人用鼻夹。</li>
		<li>打开雾化器, 选择最低级别的气溶胶和风扇设置。</li>
		<li>要求病人吸入气雾剂通过口片与潮汐呼吸5分钟。 注: 气溶胶和风扇设置可能会增加取决于病人的耐受性。<ol>
			<li>在无法忍受的咳嗽或恶心的情况下, 停止手术并去1.3.5。</li>
			<li>如有胸闷或呼吸不舒服, 请到步1.3.8。</li>
		</ol>
		</li>
		<li>关掉雾化器</li>
		<li>请病人取出鼻夹, 用清水漱口, 然后在洗涤盆中漱口两次。 注: 唾液细胞可能污染痰样, 导致无法使用的样本。</li>
		<li>让病人把痰咳成塑料容器。</li>
		<li>执行肺活量 (强制呼气机动) 来测量 FEV1。</li>
		<li>评估 FEV1秋季的结果如下: (FEV1在步骤 3.8 [ml] 中测量)-(post-bronchodilator FEV1在步骤 1.3 [ml])/(post-bronchodilator FEV1在步骤 1.3 [ml]) * 100 中测量。<ol>
			<li>如果 FEV1从 post-bronchodilator 值中下降20% 或更多, 请停止该过程。再次测量 FEV1 10 分钟后。如果 FEV1下降20% 或以上, 请医生管理雾化托溴化物 (0.25 mg/2 毫升), 并让病人接受医学观察。转到步骤1.3.10。</li>
			<li>如果 FEV1不从 post-bronchodilator 值中下降20% 或以上, 请重复步骤3.2 到3.9 一至三次, 以达到总雾化时间10到20分钟。 注: 总雾化时间将取决于质量 (存在的插头或粘性部分) 和数量的痰样本。如果样品质量和/或数量是可怜的在10分钟的雾化后, 程序应扩大到达到总雾化时间15至20分钟。</li>
		</ol>
		</li>
		<li>将痰标本冷藏至加工。</li>
	</ol>
	</li>
</ol>
2. 痰液处理
注: 在取样3小时内处理痰液以获得最佳细胞活性。
注意: 穿上工作服、防护手套和护目镜。
<ol>
	<li>初步准备
	<ol>
		<li>按照制造商的说明, 将浓缩的糖溶液 (mucolytic 剂) 与无菌蒸馏水进行10倍稀释, 以获得10毫升的 6.5 mM 溶液。 注: 为了防止浓缩的糖溶液与周围空气混合, 用无菌注射器和针头从瓶子中取出溶液。一旦打开, 浓缩溶液瓶必须保持室温, 并在5天内使用。稀释的溶液每天准备。 注意: 由于眼部和皮肤刺激性危险, 穿戴防护设备 (衣物、手套和护目镜)。</li>
		<li>将台盼蓝溶液 (0.4%) 与 Dulbecco 的磷酸盐缓冲盐水 (DPBS) 进行5倍稀释, 以获得0.08% 的溶液。在室温下保持稀释溶液, 并在2周内使用。 警告: 由于致癌危害, 穿戴防护设备 (衣物、手套和护目镜)。</li>
	</ol>
	</li>
	<li>痰清液的收集
	<ol>
		<li>将整个痰液输送到50毫升的锥形底塑料管中, 并称量样品。</li>
		<li>添加一个3倍重的 DPBS 解决方案。</li>
		<li>三十年代的样品慢慢地涡旋。</li>
		<li>离心机在 800 x g 和4° c 为10分钟。</li>
		<li>通过2单层无菌纱布过滤样品, 并在50毫升锥形底塑料管中收集上清液。</li>
		<li>分2毫升塑料管中的上清液, 并将其贮存在-80 ° c。 注: 上清液的样品对分析液体相成分是有用的。</li>
	</ol>
	</li>
	<li>痰 Mucolysis
	<ol>
		<li>如有必要, 将 DPBS 添加到细胞颗粒中, 以达到5毫升的细胞悬浮液的总容积。</li>
		<li>稀释1卷6.5 毫米糖的细胞悬浮。使用一个板凳摇杆摇滚的细胞悬浮20分钟室温。<ol>
			<li>用 DPBS 至少重复3次。</li>
		</ol>
		</li>
		<li>离心稀释的细胞悬浮在 550 x g 和4° c 为 10 min。</li></ol></li></ol>丢弃上清液。
		<li>重1毫升的 DPBS 细胞颗粒。</li>
	
	
	<li>例计数法评估痰样 (细胞浓度、鳞状细胞污染和台盼蓝排斥方法的可行性) 的定性特性
	<ol>
		<li>评估并记录细胞悬浮液的确切容积。</li>
		<li>增加50µL 的细胞悬浮到50µL 0.08% 台盼蓝溶液和质。</li>
		<li>将样品置于例 (Thoma 室) 的片下, 置于光学显微镜下 (400X)。</li>
		<li>统计鳞状细胞、活非细胞 (无色细胞) 和死非细胞 (蓝色细胞)。 注意: 如果细胞密度过低或太高, 集中或稀释细胞悬浮物, 重复步骤 2.4. 1-2. 4.3。</li>
		<li>评估悬浮液的细胞浓度, 鳞状细胞的百分率, 以及非细胞的存活百分率。 注: 计算痰液总非细胞数/克: 悬浮液的细胞浓度 (步骤 5.4) * 细胞悬液容积 (步骤 4.8) * 非细胞的百分比/痰样的重量 (步骤 2.2.1)。</li>
	</ol>
	</li>
	<li>在需要的情况下, 用细胞悬浮和储存残留的细胞来制备染色的 cytospin 玻片 注: 如果细胞悬浮液中含有超过80% 鳞状细胞, 则该样品被认为质量较差, 不适合于 cytospin 滑动14。在这种情况下, 停止处理并认为此示例不成功。<ol>
		<li>用 DPBS 稀释细胞悬浮液的分, 以获得至少350µL 的细胞悬浮在50万细胞/毫升。</li>
		<li>按照制造商的说明组装电池浓缩器。</li>
		<li>填充细胞集中器的每3舱室与100µL 这个细胞悬浮。</li>
		<li>在 cytocentrifuge, 旋转2分钟, 在 150 x g 和室温。丢弃清。</li>
		<li>按照制造商的说明拆卸电池集中器。</li>
		<li>允许滑片风干。 注意: 在这个阶段, 如果需要进一步的实验, 剩余的细胞可以储存在足够的缓冲中。</li>
		<li>根据制造商的说明, 使用商业染色套件 (参见材料表) 对幻灯片着色。</li>
		<li>用合适的安装介质和盖板安装滑轨。</li>
		<li>将幻灯片置于光学显微镜 (600X) 下, 并浸入油中。</li>
		<li>计数至少500非细胞: 嗜中性、嗜酸性粒、巨噬、淋巴细胞和上皮细胞。 注: 差异细胞计数通常是由一个或两个专门和训练有素的观察员, 以限制者的差异。</li>
		<li>记录每个单元格类型的绝对值和百分比。</li>
	</ol>
	</li>

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<li>Brooks, C. R., Gibson, P. G., Douwes, J., Dalen, C. J. V., Simpson, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relationship+between+airway+neutrophilia+and+ageing+in+asthmatics+and+non-asthmatics%3A+Airway+neutrophilia+and+ageing%5BTitle%5D)+AND+%22Respirology%22%5BJournal%5D)">Relationship between airway neutrophilia and ageing in asthmatics and non-asthmatics: Airway neutrophilia and ageing.</a> Respirology. 18 (5), 857-865 (2013).</li>
<li>Thomas, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+age+on+induced+sputum+differential+cell+counts+in+normal+subjects%5BTitle%5D)+AND+%22Chest%22%5BJournal%5D)">The influence of age on induced sputum differential cell counts in normal subjects.</a> Chest. 126 (6), 1811-1814 (2004).</li>
<li>Chalmers, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Smoking+and+airway+inflammation+in+patients+with+mild+asthma%5BTitle%5D)+AND+%22Chest%22%5BJournal%5D)">Smoking and airway inflammation in patients with mild asthma.</a> Chest. 120 (6), 1917-1922 (2001).</li>
<li>Hansel, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Comprehensive+Evaluation+of+Nasal+and+Bronchial+Cytokines+and+Chemokines+Following+Experimental+Rhinovirus+Infection+in+Allergic+Asthma%3A+Increased+Interferons+%28IFN-%CE%B3+and+IFN-%CE%BB%29+and+Type+2+Inflammation+%28IL-5+and+IL-13%29%5BTitle%5D)+AND+%22EBioMedicine%22%5BJournal%5D)">A Comprehensive Evaluation of Nasal and Bronchial Cytokines and Chemokines Following Experimental Rhinovirus Infection in Allergic Asthma: Increased Interferons (IFN-γ and IFN-λ) and Type 2 Inflammation (IL-5 and IL-13).</a> EBioMedicine. 19, 128-138 (2017).</li>
<li>Popov, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Some+technical+factors+influencing+the+induction+of+sputum+for+cell+analysis%29%5BTitle%5D)+AND+%22European+Respiratory+Journal%22%5BJournal%5D)">Some technical factors influencing the induction of sputum for cell analysis).</a> European Respiratory Journal. 8 (4), 559-565 (1995).</li>
<li>Holz, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+sputum+composition+between+two+inductions+performed+on+consecutive+days%5BTitle%5D)+AND+%22Thorax%22%5BJournal%5D)">Changes in sputum composition between two inductions performed on consecutive days.</a> Thorax. 53 (2), 83-86 (1998).</li>
<li>Louis, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+processing+on+inflammatory+markers+in+induced+sputum%5BTitle%5D)+AND+%22European+Respiratory+Journal%22%5BJournal%5D)">The effect of processing on inflammatory markers in induced sputum.</a> European Respiratory Journal. 13 (3), 660-667 (1999).</li>
<li>Fahy, J. V., Liu, J., Wong, H., Boushey, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+and+Biochemical+Analysis+of+Induced+Sputum+from+Asthmatic+and+from+Healthy+Subjects%5BTitle%5D)+AND+%22American+Review+of+Respiratory+Disease%22%5BJournal%5D)">Cellular and Biochemical Analysis of Induced Sputum from Asthmatic and from Healthy Subjects.</a> American Review of Respiratory Disease. 147 (5), 1126-1131 (1993).</li>
<li>Pizzichini, E., Pizzichini, M. M., Efthimiadis, A., Hargreave, F. E., Dolovich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+inflammatory+indices+in+induced+sputum%3A+effects+of+selection+of+sputum+to+minimize+salivary+contamination%5BTitle%5D)+AND+%22The+European+Respiratory+Journal%22%5BJournal%5D)">Measurement of inflammatory indices in induced sputum: effects of selection of sputum to minimize salivary contamination.</a> The European Respiratory Journal. 9 (6), 1174-1180 (1996).</li>
</ol>

作者没有什么可透露的。

诱导痰法是研究气道间隙的有效工具。这项技术有几种可能的应用。首先, 它可以改善有关各种呼吸道疾病的免疫细胞和机制的知识。例如, 这项技术允许对大量患者的气道炎症进行调查, 并且已经表明大约半数哮喘患者的特征是异常气道嗜酸性炎症15,16,17, 而 COPD 患者通常表现为痰中性粒细胞计数升高18。这项技术还有助于更好地表征特发性肺纤维化患者气道炎症和可能导致这种疾病的证据调解者19。第二, 诱导痰技术可用于预测治疗反应。例如, 痰嗜酸性粒细胞异常百分率的存在已被证明是皮质类固醇反应性的预测标记11,18。在哮喘和 COPD, 裁缝剂量的糖皮质激素, 以规范的百分比痰嗜酸性粒细胞被证明是更有效地减少恶化的数量比调整治疗根据目前的临床指南11 ,20,21。第三, 痰液分析有助于制定有针对性的治疗方法。例如, COPD 和一些哮喘患者出现的痰中性粒细胞异常数目导致了 antineutrophilic 治疗的发展11。第四, 该技术可能在诊断中起到作用。例如, 存在痰嗜是必要的诊断 non-asthmatic 嗜酸性支气管炎11。
除了获得一个差异细胞计数, 诱导痰技术也可以通过研究痰清液或痰细胞, 使许多额外的分析的表现。痰液上清的例子包括对介质的分析8、19、22以及对嗜酸性粒细胞的样品的趋活性的评估22。从痰细胞, RNA 可以提取和用于 rna 或基因表达分析19,23 。痰细胞也可以通过流式细胞术分析9,23 , 其中允许免疫和排序单元格。此外, 痰细胞可以被培养10, 并且他们的调解人生产可以被测量在体外24。值得注意的是, 在这种情况下, 调解人的内容是不同的, 可以发现在痰液上清。事实上, 痰液中的介质可能会受到呼吸道居民细胞分泌物和血浆渗出的影响, 与痰细胞培养模型24相反。最后, 免疫细胞化学和原位杂交也可以使用痰液室7进行。
诱导痰技术有一定的局限性。诱导必须在医疗监督下进行。此外, 操作人员必须向病人提供详尽的指示。其他限制包括病人的合作和医疗条件, 因为需要呼吸的努力。关于这项技术在儿童中的可行性, 有几项研究报告说, 它在6岁以上的儿童中是成功和安全的25。关于和 #60 的儿童的数据; 6 岁的人缺乏25, 但很可能在这些儿童中很难进行痰诱导.14。该技术目前仅限于研究服务和专业中心, 因为它在技术上要求很高, 耗时, 而且需要训练有素的工作人员1。另一个限制是, 痰诱导和分析技术并不总是成功的, 这意味着一个可读的 cytospin 并不总是获得26。然而, 成功率通常是大约80% 在不同的患者队列中4,26,27,28,29。最后, 在比较不同组患者的年龄匹配时必须谨慎, 因为它被证明会影响痰细胞计数30,31。同样, 其他的参数, 如性别和烟草的习惯, 必须匹配, 因为他们也可能会干扰痰细胞计数29,32。当病人的配对是不可能的, 另一种方式, 采取年龄, 性别或吸烟的状态考虑到的是调整统计分析的这些特点。
与其它允许从呼吸道采集细胞的技术, 如活检和支气管肺泡 lavages, 诱导痰法具有简单、耐受、安全、重现性、cost-effective 和无创的优点。这些优点使诱导痰技术能够在很大程度上反复进行, 并使其成为气道取样的替代选择。Bronchosorption 是另一种技术, 允许收集黏膜衬里流体, 以评估气道调解人33。虽然这种技术 (需要支气管镜) 比诱导痰更有侵入性, 但它的优点是避免任何唾液污染和获得更高浓度的调解比在支气管肺泡灌洗33。
关键步骤需要在协议中注意。首先, 必须注意盐水浓度和诱导时间, 因为这两个参数会影响结果, 因此必须标准化34,35。第二, mucolysis 与德勤是一个重要步骤的处理。与 PBS 相比, 该片显示出更好的分散痰细胞7, 这提高了 cytopsin 的滑动质量, 并且必须有一个可重现的细胞计数2。然而, mucolytic 剂可能会干扰生化成分的测量。在测量新的生物化学化合物36时, 应进行扣球试验。最后, 该过程的另一个关键步骤是细胞计数步骤, 必须由训练有素的技术人员执行, 以确保可靠和解释的结果。
另一种方法使用痰塞 (黏或高密度的部分), 而不是整个痰, 也描述了文献7。这两种技术都有优缺点。整个痰技术允许更快速的处理7, 但经常被唾液污染, 稀释了样品, 并可以降低 cytospins 的质量2,7。随着插头选择技术, 唾液污染减少, 这意味着样品往往是更好的质量的液体相介质的剂量和 cytospin 幻灯片7。然而处理是更长的7 , 并且选择的插座可能不是整体样品的代表37。同样值得注意的是, 并非所有的样品都含有堵塞, 这是可以限制的。这两种方法都是经过验证和重现的, 目前没有证据表明从这两种方法中获得的差异单元计数是不同的14,38。
在未来, 诱导痰可以作为一种临床工具, 为各种炎症性呼吸道疾病的调查、诊断和治疗提供生物标志物。

几种方法用于研究气道炎症: 直接测量 (如支气管活检或支气管 lavages) 和间接方法 (如症状评估, 血液样本分析和肺功能测试)1。直接技术具有可靠评估气道炎症的优点, 但由于患者的不适和发生的危险, 它们是侵入性的, 不可行的, 而且有1。至于间接方法, 它们与气道炎症的直接评估1有很差的相关性。
痰收集是另一种方法, 样本细胞从呼吸道和允许直接评估气道炎症。然而, 自发产生的痰可能导致质量低劣的样本, 并且不可能为所有患者2。这一问题已经克服了使用超声雾化高渗盐水诱导痰生产的2。该方法最初用于感染人体免疫缺陷病毒 (HIV) 的患者, 以诊断为卡氏肺囊虫病肺炎3 , 并在 1992年4中为健康的受试者和哮喘病人进行了调整。在更严重的患者中使用等渗盐水5也可以进行痰诱导。虽然一般耐受, 吸入盐水可能导致支气管患者的 hyperresponsive 气管5。因此, 建议在过程1之前管理短效 beta 激动剂。此外, 我们以前已经表明, 加入沙丁胺醇的盐水溶液在超声雾化器进一步降低了这种风险6。痰诱导的优点是, 它是非侵入性的2和安全时, 适当的预防措施是采取5。
对于痰液标本的处理, 目前文献中采用了两种方法: 全痰法和插头选择法7。在我们的实验室, 整个痰技术的执行。痰液处理的主要目的是获得一个微分细胞计数来研究气道腔内炎症的类型。然而, 还有许多额外的分析也可以进一步研究炎症过程和免疫机制, 通过研究在痰液上清中的调解者8或通过对痰细胞进行详细调查 (例如, 流式细胞仪9, 细胞培养10, 基因组学10, 蛋白质组学10, 免疫组织化学7,原位杂交7,等.
痰诱导和分析技术目前仅限于临床实践中的研究服务和专业中心, 因为它在技术上要求很高, 耗时, 需要经过培训的工作人员1。气道炎症可以用这种方法对各种呼吸道疾病如哮喘、慢性阻塞性肺病、慢性咳嗽或特发肺纤维化的11进行调查。

<table><tbody><tr><td>Spirometer - Spirobank</td><td>MIR France</td><td></td><td></td></tr><tr><td>Salbutamol MDI - Ventolin</td><td>GLAXOSMITHKLINE</td><td>CNK: 0135-913</td><td>See drug available in the country</td></tr><tr><td>Nebulizer UltraNeb</td><td>DeVilbiss Healthcare USA</td><td>UltraNeb U3000</td><td></td></tr><tr><td>Devilbiss UltraNeb Disposable Cups &amp;amp; Lids</td><td>DeVilbiss Healthcare USA</td><td>100HD-647</td><td></td></tr><tr><td>DeVilbiss Bacterial Filter For UltraNeb Nebulisers</td><td>DeVilbiss Healthcare USA</td><td>1001005879</td><td></td></tr><tr><td>One-way Valves</td><td>Hudson RCI USA</td><td>41664</td><td></td></tr><tr><td>One-way Valves</td><td>Hudson RCI USA</td><td>41665</td><td></td></tr><tr><td>Aerosol T-connector&amp;nbsp;</td><td>Hudson RCI USA</td><td>41077</td><td></td></tr><tr><td>Ventilation circuit monobranch corrugated</td><td>Int&#039;Air Medical France</td><td>TA30A</td><td></td></tr><tr><td>NaCl 5 %&amp;nbsp;</td><td>/</td><td>/</td><td>Produced by our hospital pharmacy</td></tr><tr><td>Mini-Plasco NaCl 0.9%</td><td>B.Braun Medical&amp;nbsp; Diegem Belgium</td><td></td><td></td></tr><tr><td>Salbutamol sulfate 5 mg/mL - Ventolin</td><td>GLAXOSMITHKLINE</td><td>CNK: 0094-987</td><td>See drug available in the country</td></tr><tr><td>Ipratropium bromide 0.25 mg/2 mL - Atrovent</td><td>Boehringer Ingelheim Germany</td><td>CNK: 1543-305</td><td>See drug available in the country</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline</td><td>LONZA Belgium</td><td>BE17512F</td><td></td></tr><tr><td>Sputolysin reagent</td><td>Calbiochem</td><td>56000</td><td></td></tr><tr><td>Trypan blue 0.4% solution 100 mL</td><td>LONZA Bio Whittaker Belgium</td><td>17-942E</td><td></td></tr><tr><td>Hemocytometer - Thoma chamber</td><td>Labor Optik Lancing UK</td><td>1500000</td><td></td></tr><tr><td>Hemacolor Stainig set</td><td>Merck Belgium</td><td>1116610001</td><td>Alternative product&amp;nbsp; : Diff Quik Staining Set&lt;br/&gt; &amp;nbsp;Medion Diagnostic&amp;nbsp;</td></tr><tr><td>Mounting medium - Entellan</td><td>Merck Belgium</td><td>107960</td><td></td></tr><tr><td>Statspin Cytofuge 12&amp;nbsp;</td><td>Beckman Coulter Belgium</td><td>X00-003066-001</td><td></td></tr></tbody></table>

methodology for sputum induction and laboratory processing

痰液的诱导和处理技术是一种公认的非侵入性的方法, 允许收集和分析呼吸道的细胞, 这是有趣的各种呼吸系统疾病, 如哮喘, 慢性阻塞性肺疾病 (COPD),慢性咳嗽, 或特发性肺纤维化。这项技术是良好的耐受性, 安全和无创的, 但目前仅限于研究服务和专业中心的临床实践, 因为它是技术要求, 耗时, 并要求训练有素的工作人员。痰液诱导和分析成功率约为80%。
在这里, 我们描述了痰标本的诱导和实验室处理。吸入高渗或生理盐水与沙丁胺醇致痰。对于加工, 我们采用全痰技术。糖 (德勤) 是用来允许 mucolysis 的痰样本。痰液处理的主要目的是获得一个微分细胞计数来研究气道腔中存在的细胞类型。还可以对痰液和痰细胞进行进一步的分析, 这可以进一步研究炎症过程和免疫机制。例子包括研究痰液中的介质, 并对流式细胞仪、基因组学或蛋白质组学等痰细胞进行大量的分析。
最后, 介绍了痰液分析在健康控制、支气管哮喘和 COPD 患者中的代表性结果。

例 一个典型的图像显示在图 1中, 使用例的显微镜可视化。鳞状细胞很容易识别, 因为它们比非细胞大得多。这些鳞状细胞是来自口腔的上皮细胞。两种类型的细胞在死时都被台盼蓝染色。必须注意避免计数酵母, 细菌和废料。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56612/56612fig1.jpg"> 
图 1: 例图片显示 (a) 一个活的非细胞, (B) 一个死的非细胞, 和 (C) 一个鳞状细胞。缩放栏 = 50 µm.<a href="//ecsource.jove.com/files/ftp_upload/56612/56612fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
Cytospin 幻灯片:图 2显示了痰液处理后获得的 Cytospin 幻灯片的代表性图像。不同的细胞类型 (嗜中性粒细胞、嗜酸性血、巨噬细胞、淋巴细胞和上皮) 可以通过它们的形态学和着色来区分。在某些情况下, 鳞状细胞的污染可能很重要, 如果鳞状细胞的百分比大于 80%, 则认为样本不成功 (图 3)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56612/56612fig2.jpg"> 
图 2:Cytospin 幻灯片图片显示 (a) 一个嗜中性粒细胞, (B) 巨噬细胞, (C) 一个嗜酸性细胞, (D) 淋巴细胞, 和 (E) 一个上皮组织。缩放栏 = 20 µm.<a href="//ecsource.jove.com/files/ftp_upload/56612/56612fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56612/56612fig3.jpg"> 
图 3:质量较差的 cytospin 幻灯片 #62 的示例; 80% 的鳞状细胞。缩放栏 = 20 µm.<a href="//ecsource.jove.com/files/ftp_upload/56612/56612fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
成功率 在我们的部门, 该程序的成功率 (结合成功的诱导和可读的 cytospin), 根据样本的1129患者 (健康科目, 哮喘病人, 或 COPD 患者), 是 82% (924/1129)。sub-analysis 根据患者的类型, 成功率为 75% (57/76) 的健康科目, 82% (827/1004) 的哮喘患者, 和 82% (40/49) 的 COPD 病人。
结果在健康的主题 在回顾性分析我们系的289健康科目中, 中位数 (分范围) 的痰重为3.72 克 (分范围为2.46 克-5.54 g), 痰液中位总非细胞数/克为 0.59 x 106 (分范围为 0.37 x 106 -1.29 x 106)。
在那些健康的科目, 鳞状细胞的比例是低的在 19% (10%-34%) 和生存能力是高的 66% (54-78%)。对于不同单元格类型的百分比, 将在图 4A中总结结果。我们可以观察到, 巨噬细胞的百分比 (49% [31-68%]) 高于中性粒细胞 (34% [14%-60%]) 的百分比, 而淋巴细胞的百分比 (2% [1-3%]), 嗜酸性粒细胞 (0% [0%-0%]) 和上皮 (4% [2-11%]) 是低的。当数据以绝对值 (图 4B) 表示时, 这些结果是相似的。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56612/56612fig4.jpg"> 
图 4:有代表性的结果的差异细胞计数观察到健康的主题表示为 (A) 百分比或 (B) 绝对值。结果显示为中位数 (分范围)。<a href="//ecsource.jove.com/files/ftp_upload/56612/56612fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
考虑到病人的年龄也是很重要的。事实上, 患者的年龄与痰样中中性粒细胞比例之间存在强烈的相关性 (图 5)。同样, 根据10岁年龄组 (图 6) 对患者进行分类时, 我们观察到, 随着年龄组的增加, 中性粒细胞百分比显著增加。因此, 在比较不同队列的结果时, 必须考虑此变量, 并且必须谨慎地对主题进行匹配。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56612/56612fig5.jpg"> 
图 5:年龄与痰中性粒细胞百分比的相关性。用矛测试法计算了相关度。<a href="//ecsource.jove.com/files/ftp_upload/56612/56612fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56612/56612fig6.jpg"> 
图 6:根据年龄类别的中性粒细胞百分率的演变.方差分析的 p 值是和 #60; 0.0001 用于比较年龄阶层的中性粒细胞百分比。对邓恩的多重比较试验进行了多次比较。p 值的表示如下: * p 和 #60; 0.05, *** 和 #60; 0.01, 和 *** 和 #60; 0.001。结果显示为中位数 (分范围)。<a href="//ecsource.jove.com/files/ftp_upload/56612/56612fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
呼吸道疾病患者的结果 诱导痰技术是评价哮喘患者炎症细胞分布的常用方法。这种技术也可以应用于患有 COPD, 另一种炎症性呼吸道疾病的患者。当比较健康的对象, 哮喘患者和 COPD (3 组被匹配的年龄, 性别, 和烟草习惯), 我们观察到炎症细胞剖面是相当不同的这些人群 (图 7)。事实上, 哮喘患者的特征通常是痰中嗜酸性粒细胞增多, 而 COPD 患者的痰嗜中性的比例通常更高, 这与疾病的严重程度有关。
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56612/56612fig7.jpg"> 
图 7:健康受试者的痰炎细胞剖面 (n = 45), 哮喘患者 (n = 108) 和 COPD 患者 (n = 54).这三组与性别、年龄和烟草习惯相匹配。方差分析的 p-值分别为 #60; 0.05、#60; 0.0001、#60; 0.0001 为比较中性粒细胞、嗜酸细胞和巨噬群之间的差异。对邓恩的多重比较试验进行了多次比较。p 值表示如下: * p 和 #60; 0.05 和 *** 和 #60; 0.001。结果显示为中位数 (分范围)。<a href="//ecsource.jove.com/files/ftp_upload/56612/56612fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Methodology for Sputum Induction and Laboratory Processing at JoVE.com

Methodology for Sputum Induction and Laboratory Processing

痰液诱导和实验室处理方法

在这里, 我们描述的痰诱导技术。该协议还解释了痰液的处理, 以执行一个差异细胞计数和收集痰清液和细胞进一步分析。

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

methodology-for-sputum-induction-and-laboratory-processing

Research

JoVE Journal

Medicine

27.5K 次观看.  University of Liege. 在这里, 我们描述的痰诱导技术。该协议还解释了痰液的处理, 以执行一个差异细胞计数和收集痰清液和细胞进一步分析。

视频: Methodology for Sputum Induction and Laboratory Processing

<meta charset="utf8"></head><body>通过诱导痰液分析定量肺巨噬细胞中的碳

Measuring Carbon Content in Airway Macrophages Exposed to Carbon-Containing Particulate Matters

Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with sputum, rendering macrophages and particles visible and quantifiable under light microscopy. Notably, elemental carbon within the mammalian body originates exclusively from external contaminants. Consequently, the carbon content in airway macrophages (CCAM) serves as a valid exposure biomarker, accurately estimating individual exposure to carbon-containing particulate matter (PM). This article delineates a protocol involving sputum collection, preservation, processing, slide preparation, and staining, as well as macrophage photo acquisition and analysis. After removing the macrophage nuclei, the proportion of cytoplasm area occupied by carbon particles (PCOC) was calculated to quantify carbon content in each macrophage. The results indicate an elevation in CCAM levels after exposure to carbon-containing PM. In summary, this non-invasive, precise, reliable, and standardized method enables the direct measurement of carbon particles within target cells and is utilized for large-scale quantification of individual CCAM through induced sputum.

<meta charset="utf8"></head><body>测量暴露于含碳颗粒物的气道巨噬细胞中的碳含量

This article describes a detailed experimental protocol for measuring the carbon content of airway macrophages with the aim of assessing the internal exposure dose at the level of individual particulate matter exposure.

测量暴露于含碳颗粒物的气道巨噬细胞中的碳含量

Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with ...

科研

环境科学

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the host&#39;s microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples.
Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

通过真空辅助吸附剂提取从人类相关样品中捕获活性产生的微生物挥发性有机化合物

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the ...

生物化学

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

免疫与感染

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

囊性纤维化患者痰液中 铜绿假单胞菌 的可视化

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

使用等温扩增进行人类呼吸道细菌感染诊断

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

作者聚焦:使用微流控芯片推进呼吸道病原体的快速检测 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

痰液诱导和实验室处理方法

摘要

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测量暴露于含碳颗粒物的气道巨噬细胞中的碳含量

测量暴露于含碳颗粒物的气道巨噬细胞中的碳含量

测量暴露于含碳颗粒物的气道巨噬细胞中的碳含量

通过真空辅助吸附剂提取从人类相关样品中捕获活性产生的微生物挥发性有机化合物

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

可视化痰生物膜发展影响使用腔室盖玻片型号

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

囊性纤维化患者痰液中铜绿假单胞菌的可视化

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

痰液诱导和实验室处理方法

摘要

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测量暴露于含碳颗粒物的气道巨噬细胞中的碳含量

测量暴露于含碳颗粒物的气道巨噬细胞中的碳含量

测量暴露于含碳颗粒物的气道巨噬细胞中的碳含量

通过真空辅助吸附剂提取从人类相关样品中捕获活性产生的微生物挥发性有机化合物

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

可视化痰生物膜发展影响使用腔室盖玻片型号

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

囊性纤维化患者痰液中 铜绿假单胞菌 的可视化

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

囊性纤维化患者痰液中铜绿假单胞菌的可视化