Name: enumeration of major peripheral blood leukocyte populations for multicenter clinical trials using a whole blood phenotyping assay
Uploaded: 2012-09-16T14:00:00
Description: Watch this Scientific Journal Video about Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay at JoVE.com

我们感谢他们协助发展这种方法，手稿和视频杰西卡·琼斯，埃里卡·克拉克，康斯坦茨Ducar，唐娜·史密斯，罗伊·刘易斯，百合Apedaile，乔安妮·威斯纳，德文·亚当斯，科里·麦克贝恩和斯蒂芬Voght。 这项工作是由比尔和梅林达·盖茨基金会CAVD授予38645（MJM）和美国国立卫生补助UM1 AI068618和U01 AI069481（MJM）的支持。 EA-N。由美国国立卫生研究院资助T32 AI007140支持。我们感谢詹姆斯·B·彭德尔顿慈善信托基金的慷慨设备捐赠。

注：为了保护从光的荧光标记的抗体在生物安全柜的灯不亮，执行所有步骤。 1。抗体染色面板准备<ol><li>于表1中可以发现的抗体染色面板。抗体浓度应与全血通过滴定定义，并使用相同的流式细胞仪，将用于获得染色表型样本的设备和程序。 </li><li>一旦确定适当的染色滴度，所有的抗体结合成一个单一的混合物，的锁帽筒。添加流洗涤缓冲液（2％热?...

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在这份报告中，我们提出了一个珠列举流式细胞仪新鲜全血中的白细胞人口为基础的方法，并覆盖所需的参数的多中心临床试验中使用的集中样品分析。这种方法的基础上，优化的BD Trucount协议，并允许其可靠的多中心临床试验中使用的设置。染色法很简单，大约需要45分钟来执行，使血液处理实验室技术人员来执行它，并冻结和运送样本到中央分析实验室进行分析是可行的。该试验仅需要100微升...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> Trucount绝对计数管</td><td> BD Biosciences公司</td><td> 340334 </td></tr><tr><td> 10X FACS裂解解决方案</td><td> BD Biosciences公司</td><td> 349202 </td></tr><tr><td> B类及豁免的运输系统，绝缘</td><td> SAF-T-PAK </td><td> STP-320 </td></tr><tr><td>单克隆抗体CD45 AmCyan </td><td> BD Biosciences公司</td><td> 339192 </td></tr><tr><td> CD3 FITC单克隆抗体</td><td> BD Biosciences公司</td><td> 349201 </td></tr><tr><td> CD8 PerCp-CY 5.5单克隆抗体</td><td> BD Biosciences公司</td><td> 341051 </td></tr><tr><td> CD4的Alexa Fluor 700单克隆antibo的DY </td><td> BD Biosciences公司</td><td> 557922 </td></tr><tr><td> HLA-DR ECD单克隆抗体</td><td> Beckman Coulter公司</td><td> IM3636 </td></tr><tr><td>单克隆抗体CD14 V450 </td><td> BD Biosciences公司</td><td> 560349 </td></tr><tr><td>单克隆抗体CD19 PE </td><td> BD Biosciences公司</td><td> 555413 </td></tr><tr><td> CD16 APC-H7单克隆抗体</td><td> BD Biosciences公司</td><td> 560195 </td></tr><tr><td> CD56 PE-CY7单克隆抗体</td><td> BD Biosciences公司</td><td> 335791 </td></tr><tr><td>表面CD11c APC单克隆抗体</td><td> BD Biosciences公司</td><td> 559877 </td></tr><tr><td> CD123 PE-Cy5标记的单克隆抗体</td><td> BD Biosciences公司</td><td> 551065 </td></tr></tbody></table>

enumeration of major peripheral blood leukocyte populations for multicenter clinical trials using a whole blood phenotyping assay

外周血白细胞的超低温保存的被广泛用来保存在临床试验中的细胞的免疫应答的评估，为便于和标准化的免疫学评估提供了许多优点，但在此过程中的有害​​影响已被观察到一些细胞亚群，如粒细胞，B细胞和树突状细胞1-3。含量测定新鲜的白 ​​细胞在体内的细胞状态，给出了更准确的图片，但往往是难以执行的背景下，大规模的临床试验。新鲜的细胞检测是依赖于志愿者的承诺和时限，如果费时，他们的应用程序可以是不切实际的，由于实验室工作人员的工作时间要求。此外，在多个中心进行试验时，与要执行的检测的资源和培训实验室可能不会被位于足够接近临床站点。为了解决这些问题，我们已经开发开发了一个11-色抗体染色面板，可以使用Trucount管（Becton Dickinson公司，圣何塞，加利福尼亚州）的表型和外周血内列举主要的白细胞种群，得到更强大的细胞类型特异性的信息比检测诸如全血细胞计数（CBC）或分析与市售面板设计Trucount管，染色只有少数细胞类型。染色过程很简单，只需要100微升新鲜全血，需时约45分钟，使其成为可行的标准​​的血液处理实验室执行。它改编自BD Trucount管技术数据表（ <a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">8/2010</a> ）。在中央检测实验室，可以在散装提前准​​备染色的抗体鸡尾酒，运到现场处理实验室。可以是固定的彩绘管冻结装运中央多色流式细胞仪分析检测实验室。从这种染色面板生成的数据可以被用来追踪干预有关的白细胞浓度随着时间的推移的变化，并可以很容易地得到进一步的发展，以评估特定细胞类型的兴趣的激活状态。在这份报告中，我们展示了使用血液处理实验室技术人员进行染色新鲜的全血和步骤来分析这些染色样品在中央检测实验室支持的多中心临床试验的程序。视频详细信息的过程，因为它是在艾滋病疫苗试验网络（HVTN）的临床试验抽血的背景下进行。

Watch this Scientific Journal Video about Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay at JoVE.com

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

在这份报告中，我们展示了染色和分析步骤，新鲜全血进行列举主要的先天免疫和适应性白细胞种群的表型分析。执行这些程序在多中心临床试验的背景下，我们强调的注意事项。

枚举主要外周血白细胞人口多中心临床试验，使用全血表型分析

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Research

JoVE Journal

Immunology and Infection

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The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

科研

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Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

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在细菌粘附的研究剪应力

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Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

在富含白细胞的血液样本卤化过氧化物酶活性的快速，具体的评估

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