Name: standardization of transfer across labs between flow cytometers for detection of lymphocytes in japanese encephalitis vaccinated children
Uploaded: 2023-02-10T13:30:00
Description: Watch this Scientific Journal Video about Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children at JoVE.com

RW获得北京自然科学基金（第7222059号），国家自然科学基金（第82002130号），XZ获得中国医学科学院医学科学创新基金（编号：2019-I2M-5-026）的支持。

该研究已获得首都医科大学北京儿童医院伦理委员会批准（批准文号：2020-k-85）。本研究放弃了人类受试者的知情同意，因为仅使用临床试验后的残留样本。两个实验室参与了这项研究。 转移实验室 是使用一台流式细胞仪开发标准化方法的地方。本实验室的细胞仪以下简称 细胞仪A。测试方法实验室是使用另一个流式细胞仪接收方法的实验室，该 实验室 中?...

<ol>
	<li>Cabanski, M., 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=Flow+cytometric+method+transfer:+Recommendations+for+best+practice.">Flow cytometric method transfer: Recommendations for best practice.</a> Cytometry Part B. Clinical Cytometry. 100 (1), 52-62 (2021).</li><li>Le Lann, L., 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=Standardization+procedure+for+flow+cytometry+data+harmonization+in+prospective+multicenter+studies.">Standardization procedure for flow cytometry data harmonization in prospective multicenter studies.</a> Scientific Reports. 10 (1), 11567 (2020).</li><li>Linskens, E., 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=Improved+standardization+of+flow+cytometry+diagnostic+screening+of+primary+immunodeficiency+by+software-based+automated+gating.">Improved standardization of flow cytometry diagnostic screening of primary immunodeficiency by software-based automated gating.</a> Frontiers in Immunology. 11, 584646 (2020).</li><li>Glier, H., 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=Standardization+of+8-color+flow+cytometry+across+different+flow+cytometer+instruments:+A+feasibility+study+in+clinical+laboratories+in+Switzerland.">Standardization of 8-color flow cytometry across different flow cytometer instruments: A feasibility study in clinical laboratories in Switzerland.</a> Journal of Immunological Methods. 475, 112348 (2019).</li><li>Wang, 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+epidemiology+and+disease+burden+of+children+hospitalized+for+viral+infections+within+the+family+Flaviviridae+in+China:+A+national+cross-sectional+study.">The epidemiology and disease burden of children hospitalized for viral infections within the family Flaviviridae in China: A national cross-sectional study.</a> PLOS Neglected Tropical Diseases. 16 (7), e0010562 (2022).</li><li>Ding, Y., 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=Reference+values+for+peripheral+blood+lymphocyte+subsets+of+healthy+children+in+China.">Reference values for peripheral blood lymphocyte subsets of healthy children in China.</a> The Journal of Allergy and Clinical Immunology. 142 (3), 970-973 (2018).</li><li>Zhang, L., 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=Detection+of+polyfunctional+T+cells+in+children+vaccinated+with+Japanese+encephalitis+vaccine+via+the+flow+cytometry+technique.">Detection of polyfunctional T cells in children vaccinated with Japanese encephalitis vaccine via the flow cytometry technique.</a> Journal of Visualized Experiments. (187), e64671 (2022).</li><li>Yu, N., 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=CD4(+)+CD25+(+)+CD127+(low/-)+T+cells:+a+more+specific+Treg+population+in+human+peripheral+blood.">CD4(+) CD25 (+) CD127 (low/-) T cells: a more specific Treg population in human peripheral blood.</a> Inflammation. 35 (6), 1773-1180 (2012).</li><li>Kalina, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+of+flow+cytometry+through+standardization:+opportunities+and+challenges.">Reproducibility of flow cytometry through standardization: opportunities and challenges.</a> Cytometry Part A. 97 (2), 137-147 (2020).</li><li>Sommer, U., 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=Implementation+of+highly+sophisticated+flow+cytometry+assays+in+multicenter+clinical+studies:+considerations+and+guidance.">Implementation of highly sophisticated flow cytometry assays in multicenter clinical studies: considerations and guidance.</a> Bioanalysis. 7 (10), 1299-1311 (2015).</li><li>Glier, H., 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=Comments+on+EuroFlow+standard+operating+procedures+for+instrument+setup+and+compensation+for+BD+FACS+Canto+II,+Navios+and+BD+FACS+Lyric+instruments.">Comments on EuroFlow standard operating procedures for instrument setup and compensation for BD FACS Canto II, Navios and BD FACS Lyric instruments.</a> Journal of Immunological Methods. 475, 112680 (2019).</li></ol>

外周血淋巴细胞亚群的免疫分型有助于了解儿童接种疫苗后细胞介导的适应性免疫的变化。在临床应用中，会出现意外情况，如未能及时检测样本或更换流式细胞仪;因此，需要快速标准化的方法，以促进不同实验室中流式细胞仪之间的转移9，10，11。在这里，我们描述了一种快速标准化的方法，以确保不同仪器之间的结果一致...

流式细胞术的标准化有助于不同实验室和研究中心不同细胞仪结果的可比性，有利于结果的相互认可，提高工作效率。越来越多的方案需要标准化。在药物开发过程中，流式细胞术标准化非常重要，因为开发和验证的检测方法将支持从临床前到临床分析的整个药物开发过程。流式细胞术方法经常在制药行业和合作实验室之间转移1.此外，从多中心临床研究中获取可比数据至关重要。例如，在系统性自身免疫性疾病多中心临床研究项目中开发了一个标准化工作流程，以获得多中心流式细胞术2的可比数据。
流式细胞术方法的标准化具有挑战性。实验室面临的挑战归因于缺乏标准化材料、软件兼容性问题、仪器设置不一致以及不同流式细胞仪之间使用不同的配置以及中心之间的不同门控策略3，4。因此，在实验室之间进行差距分析很重要。必须审查样品访问、质量体系、人员资格和仪器配置，以确保满足要求。
目前，接种乙型脑炎（JE）疫苗的儿童乙型乙型肝炎5发病率显著降低。监测外周血免疫细胞有助于了解接种后细胞介导的适应性免疫的变化，以及外周血淋巴细胞亚群的变化与疫苗接种效果之间的相关性。由于全血样本的稳定性有限，疫苗功效的评估通常在多个中心进行。对于此分析，我们将幼稚CD8+或CD4 + T细胞定义为CD27 + CD45RA+，将中枢记忆T细胞（TCM）定义为CD27 + CD45RA-，效应记忆T细胞（TEM）定义为CD27-CD45RA-，终末分化的效应记忆T细胞（T EMRA）定义为CD27-CD45RA+。CD19+ B细胞可以分为表达CD27与IgD 6，7的群体，表达CD27n记忆的幼稚B细胞可以根据IgD 6的表达进行鉴定，调节性T细胞（Tregs）可以鉴定为CD4 + CD25++ CD127低8。为了建立标准化的流式细胞术实验，以实现多个中心实验结果的一致性和可比性，建立了一种快速可行的标准化方法，以促进不同流式细胞仪之间转移方案，以检测接种乙脑疫苗的儿童全血淋巴细胞。从首都医科大学附属北京儿童医院招募了6名健康儿童（2岁）。在不到6个月前接种了JE SA14-14-2减毒活疫苗后，从志愿者身上收集了外周血样本。按照标准化程序从不同仪器获得高度可比的数据，这有助于多中心评估。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD CompBeads Anti-Mouse Ig, &#954;/Negative Control Compensation Particles Set</td>
			<td>BD</td>
			<td>552843</td>
			<td>compensation</td>
		</tr>
		<tr>
			<td>BD FACSCanto</td>
			<td>BD</td>
			<td>FACSCanto</td>
			<td>flow Cytometry A in the transferring lab</td>
		</tr>
		<tr>
			<td>BD FACSDiva CS&#38;T Research Beads</td>
			<td>BD</td>
			<td>655051</td>
			<td>define flow cytometer baseline and track cytometer performance</td>
		</tr>
		<tr>
			<td>BD Horizon BV421 Mouse Anti-Human CD127</td>
			<td>BD</td>
			<td>562436</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BD Horizon BV605 Mouse Anti-Human CD27</td>
			<td>BD</td>
			<td>562656</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BD Horizon V500 Mouse Anti-Human CD45</td>
			<td>BD</td>
			<td>560777</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BD LSRFortessa</td>
			<td>BD</td>
			<td>LSRFortessa</td>
			<td>flow Cytometry B in the test method lab</td>
		</tr>
		<tr>
			<td>BD OptiBuild BB700 Mouse Anti-Human CD19</td>
			<td>BD</td>
			<td>745907</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BD OptiBuild R718 Mouse Anti-Human CD8</td>
			<td>BD</td>
			<td>751953</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BD Pharmingen APC Mouse Anti-Human CD45RA</td>
			<td>BD</td>
			<td>550855</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BD Pharmingen APC-H7 Mouse Anti-Human CD3</td>
			<td>BD</td>
			<td>560176</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BD Pharmingen FITC Mouse Anti-Human CD4</td>
			<td>BD</td>
			<td>566320</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BD Pharmingen PE Mouse Anti-Human CD25</td>
			<td>BD</td>
			<td>555432</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BD Pharmingen PE-Cy7 Mouse Anti-Human IgD</td>
			<td>BD</td>
			<td>561314</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>Brilliant Staining Buffer Plus</td>
			<td>BD</td>
			<td>566385</td>
			<td>Staining Buffer</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810</td>
			<td>Cell centrifugation</td>
		</tr>
		<tr>
			<td>Centrifuge Tube</td>
			<td>BD Falcon</td>
			<td>BD-35209715</td>
			<td>15 mL centrifuge tube</td>
		</tr>
		<tr>
			<td>CS&#38;T IVD Beads</td>
			<td>BD</td>
			<td>662414</td>
			<td>standard beads to setup cytometer settings in different flow cytometer</td>
		</tr>
		<tr>
			<td>Lysing Solution 10x Concentrate</td>
			<td>BD</td>
			<td>349202</td>
			<td>lysing red blood cells</td>
		</tr>
		<tr>
			<td>Phosphate-buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Round-bottom test tube</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td>5 mL test tube</td>
		</tr>
	</tbody>
</table>

standardization of transfer across labs between flow cytometers for detection of lymphocytes in japanese encephalitis vaccinated children

越来越多的实验室需要从多个流式细胞仪收集数据，特别是对于跨多个中心进行的研究项目。在不同实验室使用两台流式细胞仪的挑战包括缺乏标准化材料、软件兼容性问题、仪器设置不一致以及不同流式细胞仪使用不同的配置。为了建立标准化的流式细胞术实验，实现实验结果在多个中心的一致性和可比性，建立了一种快速可行的标准化方法，在不同流式细胞仪之间传递参数。 
本研究中开发的方法允许在不同实验室的两个流式细胞仪之间转移实验设置和分析模板，以检测接种乙型脑炎（JE）的儿童的淋巴细胞。使用荧光标准微球在两个细胞仪之间获得一致的荧光强度，以建立细胞仪设置。在两个使用不同类型仪器的实验室中获得了可比的结果。利用该方法，我们可以对不同实验室使用不同仪器评估JE接种儿童免疫功能的分析进行标准化，减少多个中心流式细胞仪之间数据和结果的差异，为实验室结果的相互认证提供可行的方法。流式细胞仪实验的标准化方法将确保跨多个中心的研究项目的有效性能。

图1显示了CST亮珠的目标值模板的全局工作表。使用 FSC/SSC 图绘制多边形门以选择 CST 亮珠。获得CST亮珠的10个荧光通道的直方图：FITC，PE，BB700，PE-Cy7，APC，R718，APC-H7，BV421，V500和BV605。通过显示 表2中直方图门内的中位数来显示每个参数的目标值。细胞仪A和细胞仪B软件中的模板和参数设置截图见 补充图S1。
图 2 显示了仪器标?...

Watch this Scientific Journal Video about Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children at JoVE.com

Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children

在这项研究中，开发了一种方法来促进两个实验室中两个流式细胞仪之间实验设置和分析模板的转移，以检测接种日本脑炎疫苗的儿童的淋巴细胞。流式细胞仪实验的标准化方法将使研究项目能够在多个中心有效进行。

流式细胞仪之间实验室转移的标准化，用于检测接种日本脑炎疫苗的儿童淋巴细胞

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple ...

流式细胞仪的标准化方法为实验室结果的相互认证提供了可行的方法，并确保了结果与多个中心的研究项目的一致性。该方法将时间消耗最小化，易于执行，配备自动化，分析模板，并且可以显着降低数据分析的要求。通过单击细胞仪按钮并选择视图配置，从转移实验室开始在细胞仪 A 的软件中创建新配置。右键单击配置列表中的基本配置文件夹，选择新建文件夹并重命名。接下来，通过右键单击基本配置并复制它来创建新配置。右键单击新文件夹并粘贴基本配置，然后再重命名新配置，标准化方法转移“将参数名称（例如FETC）从参数列表中拖到相应的检测器530/30上，同时编辑新配置的附加参数。为了在转移实验室中标准化实验，请使用此细胞仪A设置运行性能检查并跟踪磁珠以验证细胞仪A是否表现良好。右键单击软件浏览器窗口中的细胞仪 A 设置，然后通过选择应用程序设置来创建工作表。然后使用未染色的样品调整前向散射区域（FSC）和侧散射区域（SSC）的光电倍增管或PMT电压以及所有荧光参数。通过右键单击实验细胞仪设置并选择应用程序设置来保存应用程序设置。若要自动添加补偿控件，请单击实验按钮并选择补偿设置菜单，然后选择“创建补偿控件”。记录所有补偿控制磁珠的数据。完成后，单击实验并选择补偿设置，自动计算补偿，然后记录单细胞染色对照的数据。使用CD45 RAA APC将R7-18的补偿值修改为15%APC，使用CD19 BB700将APC H7的补偿值修改为14%BB700，使用CD8 R7-18将APC H7的补偿值修改为60%R7-18。记录CST磁珠的数据后，为CST亮珠创建目标值模板的全局工作表，完成后，在流式细胞仪阵列上运行样品并收集25， 000个淋巴细胞。为样品创建分析模板的全局工作表，并将实验模板保存在细胞仪上 A.使用 CD-ROM 导出实验模板。要将实验模板转移到测试实验室中的细胞仪B，请使用CST微球进行性能检查，以验证细胞仪B是否表现良好。从细胞仪 A 导入实验模板，并使用该模板为细胞仪 B 创建实验。使用相同批次的CST微球调整每个荧光通道的荧光参数电压，以匹配以前的MFI仪器。如果需要，使用未染色的样品调节FSC电压。右键单击实验细胞仪设置，然后选择应用程序设置以保存应用程序设置。接下来，使用 CD45 RA APC 将 R7-18 的补偿值修改为 15%APC。使用CD19 BB700将APC H7的补偿值修改为24%BB700。并使用CD8 R7-18将APC H7的补偿值修改为60%R7-18。补偿值修改后，在流式细胞仪B上运行样品并收集25， 000个淋巴细胞。在用于细胞仪设置和跟踪的目标值模板或CST亮珠的全局工作表中，获得了10个荧光通道的直方图，通过显示直方图门内的中位数来显示每个参数的目标值。使用细胞仪A和B获得的样品的点阵图经过仪器标准化，证明了使用自动分析模板的不同仪器之间的数据一致性。在两个不同的仪器模型中比较六个样品的结果时，未观察到显着差异。使用标准化方法从不同仪器获得的15个淋巴亚群的结果获得了高度可比的数据。相同的配置名称、创建模板以及在不同仪器中制作安全珠的生态技术值是该协议中的关键步骤。

Research

JoVE Journal

Immunology and Infection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

锁核酸流式细胞仪，荧光原位杂交（LNA流FISH）：细菌小RNA检测方法

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

小鼠小肠淋巴细胞的分离和流式细胞表征

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

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

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 ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...