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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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smoking+and+airway+inflammation+in+patients+with+mild+asthma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comprehensive+Evaluation+of+Nasal+and+Bronchial+Cytokines+and+Chemokines+Following+Experimental+Rhinovirus+Infection+in+Allergic+Asthma:+Increased+Interferons+(IFN-&#947;+and+IFN-&#955;)+and+Type+2+Inflammation+(IL-5+and+IL-13).">A Comprehensive Evaluation of Nasal and Bronchial Cytokines and Chemokines Following Experimental Rhinovirus Infection in Allergic Asthma: Increased Interferons (IFN-&#947; and IFN-&#955;) 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+technical+factors+influencing+the+induction+of+sputum+for+cell+analysis).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+sputum+composition+between+two+inductions+performed+on+consecutive+days.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+processing+on+inflammatory+markers+in+induced+sputum.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+Biochemical+Analysis+of+Induced+Sputum+from+Asthmatic+and+from+Healthy+Subjects.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+inflammatory+indices+in+induced+sputum:+effects+of+selection+of+sputum+to+minimize+salivary+contamination.">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。

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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

27.2K 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

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

表演和加工为淀粉样蛋白前脂肪垫活检

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

科研

医学

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

如何从MR图像测量皮质折叠:一个循序渐进的教程，以计算本地Gyrification指数

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

加工的主要脑肿瘤组织的干细胞检测和流量排序

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

处理人减少乳房整形和乳房切除术的组织细胞培养

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

浸润性膀胱移行细胞癌中的完整的人MUC1转基因小鼠免疫诱导免疫治疗发展模型

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

胆管结扎小鼠:感应炎性肝损伤和纤维化的阻塞性胆汁淤积

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

小鼠加速动脉硬化的诱导:"电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

鼻腔和支气管液的吸收: 人体呼吸道黏膜的精确取样和样品的实验室处理

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

猪生物医学模型生物库组织样品的取样策略与处理

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

视网膜器官诱导系统，用于从人类多能干细胞中提取3D视网膜组织

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...