Name: bronchoalveolar lavage (bal) for research; obtaining adequate sample yield
Uploaded: 2014-03-24T19:45:00.000+00:00
Duration: 11 min 47 s
Description: Watch this Scientific Journal Video about Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield at JoVE.com

谢谢艾琳娜Mitsi（研究助理）和Sr阿玲汉考克（研究护士）和剧院工作人员对拍摄的贡献，并以邵大卫（研究护士），他与我们的研究支气管镜的支持。还要感谢我们的志愿者丽贝卡·邓菲允许此过程被拍摄下来。

1。本主题由呼吸研究护士在飞抵<ol><li>这个问题已经被禁食&gt; 4小时固体食物和2小时为透明液体1。 </li><li>该过程执行了一天的情况下用适当的预处理和后处理过程检查（按照标准的医院政策）。 </li><li>主题穿着医院的病号服。 </li><li>一个小规套管插入（理想为左手），并保持在原位 ，直至后程序恢复期间1的端部。 </li><li>操作前观察被当作（血压，心脏率，血氧饱和度[SATS]）。 </li><li>受试者被转移到支气管镜套装。 </li></ol> 2。本主题由呼吸研究临床医生在经历了支气管镜检查准备在套件的程序<ol><li>监控设备连接，其中包括：3导联心电图，脉搏血氧仪1,10和血压计。 Anesthet不需要IC支持，包括书面知情同意书。 </li><li>氧气可以通过鼻导管高达4升/分钟交付，实现SATS&gt; 90％1。 </li><li>操作员检查支气管镜，这包括通过抽吸无菌生理盐水确保有效的吸力。 </li><li>局部麻醉用利多卡因在鼻通道（使用Instillagel 1）和口腔粘膜实现（使用利多卡因） </li><li>如果镇静是适当的，由主体要求，咪达唑仑静脉注射给药1。适当的逆转剂（氟马西尼）是立即可用。 </li><li>复苏设备应随时可用1,10。 </li></ol> 3。支气管镜插入并定位<ol><li>插管通过鼻子通常是。如果这是不可能的，由于鼻息肉，鼻甲发炎，或任何不适那么受试者经口插管（口后卫是用来防止损坏的范围或受试者的牙齿）。 </li><li>局部麻醉在喉是使用4％利多卡因完成，通常总共4-6毫升被使用。这通常导致咳嗽的灌输。 </li><li>声带传递，并用2毫升分装2％利多卡因在隆突，在右下肺叶（RLL）及右肺中叶（RML）的划分，并在入口处RML进一步黏膜麻醉。 </li><li>支气管镜定位在RML内，最好在内侧段，在一个位置，它足以在一个安全的位置的远端，但不能太远侧使气道塌陷，当施加抽吸（&#39;表情试验“使用抽吸按钮）。 </li><li>良好的定位是，可以通过支气管镜和不完全关闭紧随温和的吸力气道被完全保持在一个位置支气管镜眨眼测试期间表示。 </li></ol> 4。 BAL中进行<ol><li>四60毫升注射器预填充有温热生理盐水 - 60毫升，50毫升，50毫升，40毫升连续注射器。当主体和支气管镜准备好生理盐水的第一注射器是由支气管镜辅助灌输，而支气管镜保持在RML的位置。 </li><li>然后，通过使用相同的端口和50 ml注射器辅助进行温柔的手吸。 </li><li>这个过程，然后再重复3次，用在我们的具体技术中使用200毫升的最大体积。 </li><li>检索BAL液轻轻挤入已经举行了融冰标签的容器。玻璃容器可以presiliconized以抑制细胞附着和细胞最大化回报。 </li><li> BAL液出现朦胧的反对与表面活性剂形成表面的肥皂泡的光。 </li><li>支气管镜慢慢地完全撤出。 </li><li> BAL液被输送无延迟冰上立即处理。 </li></ol> 5。待恢复</p&gt; <ol><li>这个问题是由一个病房的呼吸研究护士恢复2-4小时。 </li><li>在此期间，他们被允许休息，然后只吃喝&gt; 60分钟1手术后（当他们的燕子被评定为安全），为了减少误吸的危险。 </li><li>发表意见的程序进行监控和记录。临床检查时在出院前。 </li><li>这个题目是介绍出院前对常见的副作用（发热，轻度右submammary胸部不适，咽 ​​痛），并给出显著副作用发生1的情况下的联系人号码。 </li><li>这个题目是跟进在1-5天之后程序与临床联系。这可能是通过电话或亲自。 </li></ol> 6。细胞被隔离在实验室 - 执行对冰川消融，在可能的情况<ol><li> BAL液的体积是通过无菌纱布拭子双层记录和过滤去除黏液栓到预冷，无菌50ml离心管中。 </li><li>细胞通过离心分离，在500×g离心沉淀，在4℃下进行5-10分钟，并通过涡旋在50毫升冷生理盐水洗涤。离心分离重复一次。 </li><li>细胞再悬浮于培养基（RPMI-1640 +10％胎牛血清+ 2 2mM L-谷氨酰胺+青霉素[40国际单位/毫升]，链霉素[75国际单位/毫升]，和两性霉素B [0.5国际单位/毫升]）和使用台盼蓝等体积和血细胞计数器计数的差分进行。 </li><li>细胞悬浮液被归一化到所要求的密度（我们使用1×10 6个细胞/ ml），通过移液导入适当组织培养板中，并在37℃下进行3小时，使巨噬细胞的粘附。 1毫升每孔该细胞悬浮液适用于24孔培养板中。 </li><li>对于淋巴细胞工作，3小时温育后，将培养基用移液管轻轻地上下3倍，并收集。此培养基中含有淋巴细胞和非粘附的巨噬细胞，并且可以用来评估淋巴细胞的功能，它也可在纯化步骤中使用，例如与CD14磁珠除去巨噬细胞。 </li><li>对巨噬细胞的工作，3小时温育后，将培养基小心地用最少的移液除去，用不含抗生素的新鲜，温暖培养基（RPMI 1640 +10％胎牛血清+ 2 2mM L-谷氨酰胺）取代。巨噬细胞是粘附于组织培养板中（参见图1）。 </li></ol>

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安德烈M.柯林斯是由比尔和梅林达·盖茨基金会，也没有潜在的利益冲突提供资金。她收到的资金从默克（MSD）和葛兰素史克公司出席会议和讲课费。 50研究BAL中独立进行的。 杰米Rylance由威康信托基金资助，并具有无利益冲突。他没有收到任何其他资金。 70研究BAL中独立进行的。 丹尼尔G.伍顿是由NIHR博士研究生奖学金资助，并具有无利益冲突。 40研究BAL中独立进行的。 安吉拉·莱特有没有潜在的利益冲突。协助超过100个研究BAL中。 亚当KA Wright有没有潜在的利益冲突。他已经处理了超过50 BAL样本。 邓肯G.富勒顿 </strong&gt;有没有潜在的利益冲突。 100研究BAL中独立进行的。 斯蒂芬B.戈登有没有潜在的利益冲突。他曾获得的资金从默克（MSD）和诺华出席会议。 400研究BAL中独立进行的。

为了达到良好的效果，以下是有所帮助： <ol><li>好anesthetisation在舌和后口咽部的基极。这很重要，因为它提高了主体的程序宽容和整体舒适度。 </li><li>无论是儿科（外径为3.4-3.6毫米）或成年支气管镜（外径为4.8-5.9毫米）都可以使用。 </li><li>如果有一个在鼻腔气管插管任何不适，口腔插管是首选。一般来说鼻腔插管可以是一个简单的技术或应该放弃。 </li><li>利多卡因对口咽初始滴注后，受试者可能更喜欢支气管镜被撤回以待麻醉发作，尤其是如果支气管镜经口插入。 </li><li>确保至少有10×10毫升注射器预填充只有2-4毫升的利多卡因。空气应留在注射器使麻醉的改进提供直接到线和支气管黏膜SA。 </li><li>因为在操作过程中咳嗽显著降低BAL产率;用温热的盐水，并具有良好的粘膜麻醉是重要的，以减少此。 </li><li> 50ml的注射器必须预先填充用温盐水。这是常见的前50 ml生理盐水滴注，以产生由于死空间损耗和运行到终端呼吸道其余的10-20毫升。进一步等份返回流体的比例更大。 </li><li>操作人员应经常检查支气管镜前插管，包括确保有效吸吸用无菌生理盐水。吸力的损失可由于管或周围的支气管镜或抽吸装置的连接泄漏的扭结普遍发生。 </li><li>在手动吸，最好呼吸道应该不会崩溃。如果吸入循环地折叠该气道，BAL回报减少到一个阀机构，和局部炎症和出血的可能性更大：这反过来又使呼吸道米矿石烦躁和咳嗽的可能性较大。选择最合适的RML亚段分支，并使用低吸气压力，因此，这两个重要的。 </li><li>当空气被不断吸入重新检查注射器向气管镜的连接以及在所述远端气道的支气管镜的位置。如果这是一个经常出现的问题，后期的程序保证有在支气管镜本身没有漏洞。 </li><li> BAL从吸烟者往往较深由于细胞内颗粒的存在。吸烟者可能容忍支气管镜较差，并有咳嗽加重。 </li><li>优选的BAL量产量&gt; 100毫升。为&gt; 150 ml具有产量非常出色。我们没有经历过任何产量&gt; 170毫升还有就是细胞产量和BAL量产量之间没有线性关系。 </li><li>的&lt;100毫升收益率，根据我们的经验，更可能导致副作用，如咳嗽，胸痛和发烧。我们建议受试者&lt;100毫升的产量是RECO维雷德在左侧卧位，这样可以帮助任何剩余肺液引流。 </li><li>临床接触应使与主体在1-5天，以确保没有副作用。 </li><li>咪达唑仑镇静用于&lt;50％的受试者在英国和&lt;5％，在马拉维。蓝精灵似乎并没有直接提高耐受性，一些经营者认为，镇静有时可能适得其反维持与主题一致的（和令人放心的）对话。 </li><li>根据我们的经验，有这种技术一个显著的学习曲线。例如医生和护士之间的协调需要时间和收益率往往较低在学习过程的开始。 </li><li>在HIV阳性者中，我们使用抗生素预防prebronchoscopy。 </li></ol>

背景纤维支气管镜在临床实践中形成的，它被广泛用于两个及诊断治疗1,2。支气管肺泡灌洗（BAL），去除未贴壁细胞，并从粘膜表面肺衬液，活检用于采样的粘膜和粘膜下组织的。在研究环境，BAL使肺部内采样的先天（肺巨噬细胞3-5），细胞（B-和T-细胞6）和体液（免疫球蛋白7）的反应。 BAL是国际公认的用于研究目的8，自1999年该技术已在&gt; 1000例受试者完成了在英国和马拉维由本集团。我们使用这种技术的肺炎球菌抗原与生俱来的，细胞和体液免疫反应，包括人体实验肺炎球菌马车9,17，生物质烟雾5，艾滋病毒和疫苗接种，并辅助治疗STU研究死成肺炎恢复。我们的技术使用滴注液轻柔手持式抽吸，这样的设计最大限度地返回BAL量和应用上的纤毛上皮细胞的最小剪切力，以BAL液中保存细胞的结构和功能。 在研究方面BAL利用从呼吸和重症监护医师（通常被称为支气管冲洗，洗涤，冲洗，或BAL）谁的目标是要获得诊断或治疗的好处实行不同的技术。研究方法被设计为收获细胞，并保持生存力，以促进细胞在体外培养中的生长。由于这些原因，研究技术使用较大体积的滴注液（通常在200毫升的数量级），并采用手动吸减少细胞损伤。患者给予局部麻醉剂，提供清醒镇静（咪达唑仑），并以最小的副作用耐受的过程良好。口头和书面的主观科技信息提高了宽容和书面知情同意书是强制1。 目标总的目标是，程序应该是安全和有效的。主题应该不会遇到任何生理干扰和运营商应该坚持收集超过从受试者百毫升BAL的。手术后，受试者应该经历最小的副作用。 安全受试者的安全是至关重要的。主题使用明确的纳入和排除标准精心挑选的。该协议包括的潜在风险，以及减轻他们所采取的步骤的描述。 禁忌症研究支气管镜可表示为绝对或相对的，都包括在我们的研究协议作为排斥和纳入标准。我们的研究对象都是筛选，以确保完全健康。 绝对禁忌症包括颈椎不稳，反应迟钝缺氧，不稳定型心绞痛，出血素质，恶性心律失常。 相对禁忌症，包括那些具有增加的并发症发生率有关的病症，包括： 一般：不好合作课题1，任何显著一般的医疗问题如癫痫，以前的耐受性很差支气管镜检查，已知的不良反应，利多卡因或咪达唑仑，怀孕，营养差。 呼吸系统：，高碳酸血症，哮喘不稳定1，显著损害呼吸功能1（FEV 1 &lt;1升），肺动脉高压缺氧[空气&lt;94％饱和度（SATS）]。 心血管：尿毒症，6周心肌梗塞1， 上腔静脉阻塞的范围内。 其他：免疫抑制（我们的小组会定期执行此多项式杜热在HIV阳性者）。 见表1 - BAL与研究相关 ​​的风险。

<table><tbody><tr><td>&lt;p&gt;Fiberoptic bronchoscope, light, and suction source&lt;/p&gt;</td><td>unspecified</td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Surgical gown, sterile gloves&lt;/p&gt; </td><td>unspecified</td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Sphygnomanometer&lt;/p&gt; </td><td>unspecified</td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;For continuous patient monitoring&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Pulse oximeter&lt;/p&gt; </td><td>unspecified</td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;For continuous patient monitoring&lt;/p&gt; </td></tr><tr><td>&lt;p&gt;3-lead ECG monitor&lt;/p&gt;</td><td>unspecified</td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;For continuous patient monitoring&lt;/p&gt; </td></tr><tr><td>&lt;p&gt;Nasal oxygen delivery 2-4 L/min&lt;/p&gt;</td><td>unspecified</td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Lidocaine 10% spray (Xylocaine)&lt;/p&gt; </td><td>AstraZeneca</td><td>&lt;p&gt;&lt;/p&gt;</td><td>&lt;p&gt;For topical anesthesia&lt;/p&gt;</td></tr><tr><td> &lt;p&gt;Lidocaine hydrochloride 2%&lt;/p&gt; </td><td>unspecified</td><td>&lt;p&gt;&lt;/p&gt;</td><td>&lt;p&gt;For topical anesthesia&lt;/p&gt;</td></tr><tr><td> &lt;p&gt;Lidocaine gel (Instillagel)&lt;/p&gt; </td><td>Farco-Pharma, Germany</td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;Alternative products available from other suppliers&lt;/p&gt; </td></tr><tr><td>&lt;p&gt;Normal saline, sterile, 200 ml&lt;/p&gt;</td><td>unspecified</td><td>&lt;p&gt;&lt;/p&gt;</td><td>&lt;p&gt;Warm to 30 &amp;#176;C&lt;/p&gt;</td></tr><tr><td>&lt;p&gt;10 ml taper-end syringe (x10)&lt;/p&gt;</td><td>unspecified</td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;For administration of lidocaine&lt;/p&gt; </td></tr><tr><td>&lt;p&gt;Intravenous cannula 18 G&lt;/p&gt;</td><td>unspecified</td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;For administration of midazolam&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Midazolam&lt;/p&gt; </td><td>unspecified</td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;For sedation&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Flumazenil&lt;/p&gt; </td><td>unspecified</td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;Reversal of benzodiazepine sedation (emergency use only)&lt;/p&gt; </td></tr><tr><td>&lt;p&gt;60 ml taper-end syringes (x4)&lt;/p&gt;</td><td>unspecified</td><td>&lt;p&gt;&lt;/p&gt;</td><td>&lt;p&gt;For normal saline injection and BAL fluid withdrawal.&lt;/p&gt; &lt;p&gt;May require connector to attach to the injection port of the bronchoscope.&lt;/p&gt;</td></tr><tr><td> &lt;p&gt;Sterile gauze swab&lt;/p&gt; </td><td>Vernaid, UK</td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;Alternative products available from other suppliers&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Sterile container for BAL fluid&lt;/p&gt; </td><td>See text and comments</td><td>&lt;p&gt;&lt;/p&gt;</td><td>&lt;p&gt;Siliconized glass bottles reduce macrophage adherence. Alternatively, 50 ml centrifuge tubes may be used (total capacity 200 ml)&lt;/p&gt;</td></tr><tr><td> &lt;p&gt;Sigmacote&lt;/p&gt; </td><td>Sigma, UK</td><td>&lt;p&gt;SL2&lt;/p&gt;</td><td> &lt;p&gt;Only if siliconised glass bottles used. NB: Harmful and flammable. If used, follow precautions detailed in manufacturer&amp;rsquo;s MSDS&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Centrifuge&lt;/p&gt; </td><td>unspecified</td><td></td><td>&lt;p&gt;At least 4 x 50 ml tube capacity. Refrigeration to 4 &amp;#176;C preferred.&lt;/p&gt;</td></tr><tr><td> &lt;p&gt;Shandon Cytospin centrifuge&lt;/p&gt; </td><td>Thermo Scientific, UK</td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;For differential count only&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;RPMI 1640 medium with L-glutamine&lt;/p&gt; </td><td>Sigma, UK</td><td>&lt;p&gt;R8758&lt;/p&gt;</td><td> &lt;p&gt;Alternative products available from other suppliers&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Fetal bovine serum&lt;/p&gt; </td><td>Sigma, UK</td><td>&lt;p&gt;F6178&lt;/p&gt;</td><td> &lt;p&gt;Alternative products available from other suppliers&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Penicillin-Streptomycin&lt;/p&gt; </td><td>Sigma, UK</td><td>&lt;p&gt;P4333&lt;/p&gt;</td><td> &lt;p&gt;Alternative products available from other suppliers&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Amphotericin B&lt;/p&gt; </td><td>Sigma, UK</td><td>&lt;p&gt;A2942&lt;/p&gt;</td><td> &lt;p&gt;Alternative products available from other suppliers&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Tissue culture plates&lt;/p&gt; </td><td>Greiner Bio-One, UK</td><td>&lt;p&gt;662160&lt;/p&gt;</td><td> &lt;p&gt;Alternative products available from other suppliers&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Glass microscope slides&lt;/p&gt; </td><td>unspecified</td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;For differential count only&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Shandon cytofunnel&lt;/p&gt; </td><td>Thermo Scientific, UK</td><td>&lt;p&gt;A78710003&lt;/p&gt; &lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;For differential count only&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Shandon cytoclip&lt;/p&gt; </td><td>Thermo Scientific, UK</td><td>&lt;p&gt;59910052&lt;/p&gt;</td><td> &lt;p&gt;For differential count only&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Hemacolor staining set (fixative, red and blue reagents)&lt;/p&gt; </td><td>Merck, Germany</td><td>&lt;p&gt;111661&lt;/p&gt;</td><td> &lt;p&gt;Use according to manufacturer&amp;rsquo;s instructions&lt;/p&gt; </td></tr></tbody></table>

bronchoalveolar lavage (bal) for research; obtaining adequate sample yield

我们描述了纤维支气管镜支气管肺泡灌洗（BAL）使用手动手持抽吸，以从粘膜表面除去非贴壁细胞和肺里流体的研究方法。在研究环境，BAL使先天（肺巨噬细胞）的采样，细胞（B-和T-细胞）和体液免疫（免疫球蛋白）肺中的反应。 BAL是国际公认的用于研究目的，自1999年该技术已在&gt; 1000例受试者完成了在英国和马拉维由本集团。 我们的技术采用滴注液的温和的手持抽吸，这是被设计来最大化BAL体积退回和应用上的纤毛上皮最小的剪切力，以维持细胞的结构和功能的BAL液中，并保持生存力，以促进生长细胞在体外培养的。因此，研究技术使用量较大插件tillate（通常在200毫升的数量级），并采用手动吸减少细胞损伤。 患者给予局部麻醉剂，提供清醒镇静（咪达唑仑），并以最小的副作用耐受的过程良好。口头和书面的主题信息提高了宽容和书面知情同意书是强制性的。受试者的安全是至关重要的。主题使用明确的纳入和排除标准精心挑选的。 该协议包括的潜在风险的描述，并减轻他们所采取的步骤，禁忌名单，前，后，过程检查，以及精确的支气管镜检查及实验室技术。

良好的临床效果都超过100毫升BAL体积和谁经历顶多一个主题，轻微不适（ 见表1）。这需要一个宽松，合作的主题。临床医生必须有信心，做好充分的准备（ 材料 ），并了解对志愿者。 在细胞产量（包括差分计数）之间和内部学科的变化进行了描述。从健康受试者细胞分类计数通常是&gt; 90％的巨噬细胞与少量的中性粒细胞（&lt;2％）和淋巴细胞（5-10％）。 BAL细胞离心到显微镜载片可以用一个差分核染色剂进行染色，以获得该信息（ 图1）。在图1中，BAL细胞已被固定，并根据制造商的说明使用Hemacolor染色染色。至少300个细胞应该算做获得可靠的结果巨噬细胞，中性粒细胞和淋巴细胞; 500个细胞的罕见的细胞类型。 <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> 风险 </td><td> 风险 </td><td> ：通过减少在这个协议 </td></tr><tr><td colspan="3"> 支气管镜和BAL 1，14 的影响 </td></tr><tr><td>轻度不适</td><td> &lt;25％14，＃ </td><td>适当的镇痛和镇静作用。 自信和关爱的方式的问题。 </td></tr><tr><td>鼻出血</td><td> &lt;1％＃ </td><td>轻度压力只有经鼻插管。 如果主体不适或缩小光圈 - 口腔插管代替。 </td></tr><tr><td>支气管出血（咯血） </td><td> &lt;0.1％* 15 </td><td>无取活检。 支气管镜检查过程中严格控制 - 避免呼吸道黏膜。 </td></tr><tr><td> Naus谈EA，呕吐及吸入性肺炎</td><td> &lt;0.02％14，＃ </td><td>禁食固体食物&gt; 4小时操作前1。 充足的局部麻醉。 过程中，半卧位。 仔细后期过程的观察。 </td></tr><tr><td>发烧</td><td> 0.01 14 - 1％＃16 </td><td>涉及炎症，并且可以通过BAL的最大集合最小化。 只有单瓣BAL。＃ </td></tr><tr><td>痛鼻/喉及声音嘶哑</td><td> &lt;25％14，＃ </td><td>鼻子，喉咙和喉部（减少咳嗽）有足够的局部麻醉。 </td></tr><tr><td>感染</td><td> &lt;在HIV阴性的0.1％， 在HIV阳性14，＃1％ </td><td>支气管标准洗涤程序1。 只有单瓣BAL。＃ 早期发现感染：在1小时内的临床检查支气管镜检查后1-5天付款交单的临床接触。 </td></tr><tr><td>胸痛/咳嗽</td><td> &lt;0.2％14，＃ </td><td>单瓣只BAL。最大BAL集合。＃ </td></tr><tr><td colspan="3"> 影响药物：利多卡因，咪达唑仑 </td></tr><tr><td>心律失常</td><td>非常罕见的* </td><td rowspan="3">最多5毫克/千克用14利多卡因。 温生理盐水注入。＃ 脉搏血氧饱和度（SATS&gt; 90％氧气补充），在整个过程心脏监测。 苯二氮卓受体拮抗剂（氟马西尼）立即可用。 </td></tr><tr><td>定向力障碍/搅拌</td><td> &lt;0.1％†＃ </td></tr><tr><td>有氧呼吸抑制</td><td>非常罕见的* </td></tr></tbody></table> *没有经历过在我们的小组中超过1,500研究程序。 †Idiosyncratic而不是剂量依赖性反应。 ＃在超过1500名研究程序本集团的经验。 表1中。 BAL与研究相关 ​​的风险 。这包括危险或风险，这是如何减少使用所描述的技术。纤支镜/ BAL，也是用任何药物药物的作用效果均包括在内。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/4345/4345fig1highres.jpg" width="500" /> 图1。显微镜幻灯片显示BAL巨噬细胞和中性粒细胞偶见 。 BAL细胞已被固定在甲醇并用Hemacolor红（曙红Y）和蓝色（天青B）。

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Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

我们描述了使用低压吸入纤维支气管镜检查和支气管肺泡研究技术。该技术被用于从肺支气管肺泡表面收获的免疫细胞。局部麻醉剂和轻度镇静（咪达唑仑）被使用。主题容忍的程序以及经验和最小的副作用。

支气管肺泡灌洗（BAL）的研究，获得适当的采样率

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove ...

bronchoalveolar-lavage-bal-for-research-obtaining-adequate-sample

Research

JoVE Journal

Medicine

Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield

73.4K 次观看.  National Institute for Health Research. 我们描述了使用低压吸入纤维支气管镜检查和支气管肺泡研究技术。该技术被用于从肺支气管肺泡表面收获的免疫细胞。局部麻醉剂和轻度镇静（咪达唑仑）被使用。主题容忍的程序以及经验和最小的副作用。 

视频: Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

科研

生物工程

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

免疫与感染

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

采用雾化脂多糖一种产生肺中性粒细胞增多

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals.
The aim was to establish in vivo sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions.
Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host.

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

This article describes bronchoscopic techniques in the bovine lung under experimental conditions, i.e. bronchoscopically guided inoculation, bronchoalveolar lavage, bronchial brushing, and transbronchial lung biopsy.

牛龙在生物医学研究：视觉引导支气管镜，支气管内接种和<em&gt;在体内</em&gt;抽样技术

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as ...

医学

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

支气管肺泡灌洗小鼠肺分析炎症细胞浸润

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

利用超滤离心技术分离小鼠支气管肺泡灌洗液中的细胞外囊

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing bronchoalveolar lavage (BAL) fluid. The BAL fluid is rich in cells and can thus provide a 'snapshot' of the pulmonary immune milieu. CD4 T cells are the best characterized HIV reservoirs, while there is strong evidence to suggest that tissue macrophages, including alveolar macrophages (AMs), also serve as viral reservoirs. However, much is still unknown about the role of AMs in the context of HIV reservoir establishment and maintenance. Therefore, developing a protocol for processing BAL fluid to obtain cells that may be used in virological and immunological assays to characterize and evaluate the cell populations and subsets within the lung is relevant for understanding the role of the lungs as HIV reservoirs. Herein, we describe such a protocol, employing standard techniques such as simple centrifugation and flow cytometry. The CD4 T cells and AMs may then be used for subsequent applications, including immunophenotyping and HIV DNA and RNA quantification.

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

We describe a method for processing bronchoalveolar lavage fluid and matched peripheral blood from chronically HIV-infected individuals on antiretroviral therapy to assess pulmonary HIV reservoirs. These methods result in the acquisition of highly pure CD4 T cells and alveolar macrophages that may subsequently be used for immunophenotyping and HIV DNA/RNA quantifications by ultrasensitive polymerase chain reaction.

用于阿尔韦拉尔巨噬细胞和CD4和T细胞免疫造血和HIV储液评估的支气管性洗浴液和匹配血液的处理

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing ...

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

在臭氧暴露后阿尔韦拉巨噬细胞的Vivo评估中

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

Experimental Model to Evaluate Resolution of Pneumonia

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to high morbidity and mortality. Currently, there are no pre-clinical models that recapitulate the complexity of human ARDS. However, infectious models of pneumonia (PNA) can replicate the main pathophysiological features of ARDS. Here, we describe a model of PNA induced by the intratracheal instillation of live Streptococcus pneumoniae and Klebsiella pneumoniae in C57BL6 mice. In order to evaluate and characterize the model, after inducing injury, we carried out serial measurements of body weight and bronchoalveolar lavage (BAL) for measuring markers of lung injury. Additionally, we harvested lungs for cell count and differentials, BAL protein quantification, cytospin, bacterial colony-forming unit counts, and histology. Lastly, high dimensional flow cytometry was performed. We propose this model as a tool to understand the immune landscape during the early and late resolution phases of lung injury.

This manuscript describes the establishment of an infectious model of pneumonia in mice and the respective characterization of injury resolution along with methods for growing bacteria and intratracheal instillation. A novel approach using high-dimensional flow cytometry to evaluate the immune landscape is also described.

评估肺炎消退的实验模型

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to ...

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines.
Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. 
The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

从切除的肺组织中分离支气管上皮细胞用于生物样本库并建立分化的气液界面培养物

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled ...