特别感谢南非共和国的艾滋病毒相关的诊断服务流的国家组织的国家卫生实验室服务（NHLS）（HTTP：\ \ www.nhls.ac.za和http://www.plgcd4 NET）。在已进行的研究特色，威特沃特斯兰德大学血液学和分子医学部（南非约翰内斯堡，由系主任，教授温迪史蒂文斯支持）。作者有没有补助金的支持，这从商业公司或资助机构的出版物。所表达的观点是作者的，而不一定反映的金山或南非NHLS大学院校的意见。

简介现代科学在生物医学领域的努力带来的实际利益，包括引进传染病的有效诊断。高品质和快速诊断的需要，因为疾病的危险蔓延，由于全球旅行和目前在全球 1贫困地区的病毒和细菌感染的破坏性影响。 在资源贫乏的国家抗逆转录病毒疗法的HIV阳性患者的治疗（ART）已成功推出2,3。 ART的协议是目前上（一）对艾滋病毒+个人的血清学试验为基础，（二）遗传药物疗法的每日一次的组合，用来作为“第一，行”艺术和（三）的CD4和/或病毒的负载检测决定何时开始抗逆转录病毒。协议，但是，对艺术的范围的变化，现在可以进一步扩大较为宽松的指引早些时候开始在更高的 CD4艺术计数2。更重要的是，越来越多的HIV阳性患者接受ART更高的CD4 +细胞/微升血，甚至可能放缓，尽管目前缺乏艾滋病毒传播的艾滋病毒疫苗，4，5的数量。 因此，艾滋病毒感染的是做好准备，成为越来越多的幸存者的慢性疾病，但比以往任何时候都更需要组织医学检验医学在非洲可能状态不佳，仍然是一个有效的医疗保健的障碍6。有声音强调，提供短期补助金不提供可持续的解决方案7-9，并与艾滋病毒有关的西方国家提供的补助在目前的规模不能永远继续下去8,9。在2008年的156亿美元用于艾滋病方案在低收入和中等收入国家，而其中约50％来自外部，包括北美，来源。计算的趋势表明，这项支出是增加高2-3倍的水平，疫情仍然会与我们在2031，除非有一个变化，在目前的整体方法9。 因此，非洲国家必须作出贡献，更好的预防艾滋病毒和明智的成本节约9。在这里，我们建议的最近期的发明“榜样”的日常工作​​将建立在资源贫乏的国家（例如，在非洲或印度）介绍。这些都是在全球领域的科学家们将学会充分受益于当前的国际支持，然后利用他们的技能，进一步提高每一天的技术，通过一个全球性的发展作出贡献。 中西药大学术中心和医院的诊断活动往往“各部门各自为政”，即独立的“利益区”可能存在导致haemato的，viro，retroviro，细菌，血清碎片的不同学科，免疫及细胞学实验室，而不是进行一个统一的方法，在传染病实验室使用一个多学科的诊断方法的最小集合。此外，诊断测试，也可以过于复杂和昂贵。显然，这种服务将需要更好的协调，但这些统摄努力都不可能完全来自西部方向。因此，领先的实验室和研究机构，如国家卫生实验室服务（NHLS SA），在南非约翰内斯堡的威特沃特斯兰德大学的合作，正面临着一个额外的任务，进一步制定，修改，重新集中，并提高他们的基于现代技术的成本效益的诊断。 在当前的视频中，我们描述了流流式细胞仪的方法来监测抗逆转录病毒疗法（ART）的艾滋病毒感染者。这项技术是在六个模块结合分步进行有关haemato，免疫，和病毒学检测单管相结合，与基本的内部质量控制（QC），以确保测试10的可重复性。这里使用的单克隆抗体是所有科学完全建立。 11-13白细胞计数与CD4结合起来，以列举的“帮手”的T淋巴细胞是艾滋病毒的首要目标12-14，CD45的试剂。这是一个“建设性的把戏”，在同一管内CD8 + T细胞CD38活化标志物检测作为一个HIV病毒负荷（VL），相关的检测 15-17 。为了强调南非的贡献，我们指出，这些概念和他们的成本效益的实用组合都工作在基于技术领先 10,12,13,16,17西部的NHLS -斗智斗勇的合作。 这些分析，结合进行，要适当的监测患者正在经历长期的艺术需要。当成本，efficieNCY审议，这种协调的流式细胞仪检测技术已经被证明是经济 16,18,19 。这些单管检测，节约成本，比他们的西方变种，并且通过单独的文书和/或使用其他方法不同的代理测试进行类似的试验。这些储蓄逐项讨论。 对于接受这基本上是简单的技术，它采用四色流式细胞仪在单管，它是必不可少的显示技术在视频格式的步骤。专家顾问，实验室进行了有关引进现代医疗技术的决策责任的管理者和决策者在一个位置不经常看到的与工作负载的巨大负担大型区域实验室进行的最新的技术细节。因此，该视频显示在南非推出的新的发展，优化工作流程，每工作一天10,13,16,17检测临床标本&gt; 1000的细节。 协议流式细胞仪四色流式细胞仪进行了测试，但也可以用在两个平行管（见下文）2色的检测。贝克曼-库尔特XL - MCL的，稳定的流体 21流式细胞仪，使用，但相同的技术也可能适用于其他流式细胞仪。描述该技术处理大每天的工作量〜800-1100样品/天10,13。 模块1：文书成立每天都在内部维修清理执行每天两次：启动和完成最后的患者样本。这包括冲洗真空管线和收购Clenz（5％次氯酸钠;公元前，迈阿密，佛罗里达州）。此外，在每30管传送带Clenz和蒸馏水月底通过系统运行，以尽量减少蛋白质的建立。 在每天启动时真空和压力读数检查。据证实，背景计数仍然可以接受的（&lt;100事件，在100秒）。运行后的第一次血液样本，它是建立在鞘液等份的结转是&lt;1％。然后激光准直和荧光灵敏度监测每天使用的FlowCheck 21样本。据观察，平均通道数是一致的（500 ± 50）与低的变化（半峰的CV值&lt;2％）。的补偿，每周检查一次; FlowSet珠策划，看看是否在1预设的目标值 22％的平均通道值。直观地确认利维 - 詹宁斯地块，平均通道数和半峰值系数的变化（HPCV）每天都显示了四个荧光通道（FL1 - FL4）的值的精度。下一步的CD4细胞计数的准确性被证实在两个CD4 +水平。期间PLG - CD4是用来测试血液稳定的过程控制（IMMUNOTROL）低正常（450-700 CD4细胞/μL）和低的水平（100-200 CD4 +细胞/μL，上一批;文献23）。这些Immunotrol值须SD平均在0.5％的CV &lt;5％的国际BC - IQAP方案24，英国NEQAS 25和AFREQAS 26的要求。重要的是，这些简单的组织措施，在视频显示了强大的流式细胞仪可以保持为绝对细胞计数，如CD4 +测试和用于活化标志CD38的荧光强度测量的需要稳定。这种稳定，最近17详细记录在以往的研究，确认使用其他流式细胞仪15,27。 在每周例行维护列表模式数据（LMD）的文件被存档以供日后参考，这归档的材料，然后从流式细胞仪的硬盘驱动器中删除。 每月维护包括试剂容器，鞘，clenz和废物箱的清洁。传送带模式样本头清洗也将被删除，而所有的过滤器和洗涤。清洗也扩展到准备站TQPrep和PrepPlus2（公元前，佛罗里达州）。手册的编写所用的移液器内部验证，至少每3个月，但吸管吹打不准确，如果怀疑是立即采取停止服务的纠正措施。每半年保养计划由Beckman Coulter公司，SA。 模块2：准备样品对于CD45/CD4两个颜色PanLeucogated议定书“第5贝克曼FlowCARE套件28是用于在裂解洗程序（IMMUNOPREP，贝克曼，佛罗里达州）。此套件包含CD45的荧光素异硫氰酸酯（FITC，绿色）和CD4 -藻红蛋白（PE;橙色）。流计数珠29日在批量购买，并添加到发挥着特殊的运营商友好的作用称为numbe已知体积珠RS吸管当场进样30。这是通过使用提供的血量相同的吹打。因此，珠计数率在预期范围内（BCR）显示，在给定的样本的吹打也正是这样做的，在下面的模块3描述。更重要的是，这里简单的移液方法，简称为“向前吹打”，效果最好，提供珠和血液量是相同的吸管和小费和交付使用相同的处理30。我们发现，为运营商提出移液产生最佳的实验室，双方之间的整体统一性，实现内可接受的容忍限度和重复配药合作效率的变化（％CV）的10,31,32测量的可重复性吹打目标。这种方法，在32个地方人大提出的，是根据南非国家认可计划（SANAS，www.SANAS.co.za）的指导方针的批准。这种珠子的BCR使用从其他体积计数其中最常见的如BDS的Trucount制剂冻干珠产品使用含有珠已知的数字的方法是不同的。这些准备工作出色的文件流量仪表的流体流计数率（FCR）的方法20,31稳定。不过，使用现成的含珠管，使技术更复杂，因为在这里有必要提供这些管子最大的绝对精度的血容量。人们普遍认为，为此目的只有“反向移液”技术是可以接受的的，适用的培训。因此，我们的BCR方法，更便宜，更丰富的，更简单，更容易学习，如视频所示-替代10,31,32类似的方法相比，我们还发现，使用向前吹打的运营商是不太可能反向移液可能导致损害的内在机制，并需要重新校准，这是在偏远地区难以进入移液器吸样品。我们的BCR方法也省却了为小到中等大小的实验室（表1）自动移液系统的需要。 血液样本总是在II级生物危害柜处理。为了避免在拍摄过程中的广泛的噪声，显示视频是重新制定一个与这些橱柜以外的安全材料。上流式细胞仪PLG - CD4收购协议允许的时间停留在一个给定的时间限制，或继续收购5000淋巴细胞的活动，以改善低CD4计数的样品的准确性。 模块3：珠计数率（BCR） 监测珠计数率在我们的系统或每个控制样品（BCR）的计算公式珠事件/秒10,13,30,32,33。均值，SD和％CV值上使用电子表格来生成一个序列确定的BCR离群30图。这些确认的吹打，处理和分析的样品制备验证（SPV），被称为三大系统，适用于样品吸管手动或使用的PrepPlus2自动移液仪器 10的最佳操作。 在采集过程中的BCR结果是直接在监视器上实时观看。在公元前流式细胞仪的平均BCR的价值为80 ± 5个事件/秒。我们显示的异常BCR - S确定并重新运行前，病人结果公布。决策树算法如图1所示（见ref.10）。 在纵向精度验证（LPV）监测，以发现吹打趋势，模式和10的突然变化时，仪器故障表示BCR波动。射流精密验证（FPV）是一个可选的内部程序，单一的珠子稀释分装成10管串行采集，确认一个潜在的行为不端的仪器10的流体不稳定的迹象。 模块4：CD45 一个完整的血球计数分析仪LH - 750（Beckman Coulter公司），校准每月，对于在我们的研究例行绝对计数为白细胞计数的参考（WCC）的反对，流式细胞仪相比，每天使用。 CD45 -生成的大会上流式细胞仪也证实，每天稳定贝克曼5C全血细胞控制的三个层次的比较。 WCC协议之间的流动CD45的计数和全血计数分析仪可接受的限度&lt;5％〜10 。这表明，资源限制，更严重的比单一的仪器，如流式细胞仪，然后在我们的服务时，似乎要提供足够的临床适当的世界基督教协进会，包括CD4细胞计数。 白CE会差数上执行各种不同的血液分析仪，特别是在评估单核细胞计数。差CD45的表达模式是一个古典使用流技术差WCC 11,12，34。由于流流式细胞仪源性CD45的基督教协进会和差分计数被证明是准确，精确和可靠的，这些都可以进行常规纳入小组的CD4测试方法，也提供给临床医生在不增加成本。 CD45的异常模式指出PLG - CD4协议CD45/side散点图和随访上沾着血膜，以提示临床进一步调查。这种方法检测感染（细菌和病毒），白血病和淋巴瘤HIV阴性和HIV阳性患者34岁，35。在一次例行的实验室跟进在HIV阳性患者的全血计数经常没有要求从血液学实验室。因此异常CD45的模式是诊断时，坦率地在CD4细胞计数发送的样本观察。另外一个优点是即使在白血病或淋巴瘤血液中的35结果患者仍然可以作出准确的绝对的CD4计数。 非常适合流CD45的规范血液学。血液仪器显着性差异，取长补短。我们建议引入WBC的差异CD45的基于流式细胞仪的质量控制比较和规范10,36血液分析仪。 模块5：CD4 + PLG - CD4 +协议Beckman Coulter公司XL软件推出由8点图。这4个是用来确定的CD4细胞计数和质量监测12,13。其中，在图2所示的两个点图是重要的。使用CD45和侧向散射（地区积2点），而忽略碎片，红血细胞/鬼和血小板，白细胞确定。 CD45 +事件点积3分，然后转移到那里的CD4 +淋巴细胞是由明亮的CD4表达和低侧散射（C区）确定。绝对的CD4 +淋巴细胞计数的C区和世界基督教协进会的活动（在流动计数校准地区F）计算。从区域B点积2 CD4淋巴细胞％值获得。强烈的CD4 + T淋巴细胞和弱的CD4 +单核细胞CD4荧光强度和散射特性，而不需要使用一个额外的CD3 + T细胞特异性抗体试剂鸡尾酒（ref.13审查）歧视。 模块6：CD8/CD38 CD8 - CD38的染色是使用CD45-FITC/CD4-PE/CD8-ECD/CD38-PC5 4色染色的一部分。 CD4/CD45是与上面相同（流量护理PLG - CD4），并添加两个额外的试剂CD8-ECD/CD38-PC5 17。如果流式细胞仪运行只有两种颜色，添加额外的管执行CD8-FITC/CD38-PE两色染色，使用再次裂解，没有洗程序（IMMUNOPREP，贝克曼，佛罗里达州）。门控策略类似的CD4是用来识别CD8 + T细胞与CD8 - ECD与侧散射（图3）。平均荧光强度（MFI）与细胞计数16,17，对CD8 +淋巴细胞CD38的表达CD38 - PC5单参数直方图绘制。 CD38的MFI值绘制顺序为同一病人每次访问。第一次访问（治疗前）的“基线”的MFI值，每同一个人的CD38的小额信贷机构的其他访问相比，16 。 在模块1中所述的控制和仪器的稳定性是基于流量每天收购检查监测激光准直和电压稳定。流量设定集所有四个荧光通道（见上文）的色彩补偿。 Immunotrol是“负”的过程控制样品。 “积极的”控制在正常的血液单核细胞，每个细胞中表达的9 × 10 3 CD38分子，和已知的HIV阳性样品未经处理的17。 南非国家卫生实验室服务（SA - NHLS）网络实验室已通过标准化的70个实验室，13，33，一个集中的数据库，包含标准作业程序和培训材料的质量控制方法。在网络实验室的讨论，进一步的培训和定期的试镜，包括审议的移液技术32,33帮助。每月监测质量评估方案。这样，质量管理权力下放区域，但保持高标准的SA - NHLS质量保证部13，26的贡献。 表1：可能在HIV疾病的协调流式细胞仪所节省的成本。 <ol><li>一个熟悉和服务合同的单台仪器的使用，而不是AP行走多个血液学，免疫学和病毒学文书</li><li>表现在单管检测，无需额外的成本，并没有送样，并从不同的地点，包括两个世界基督教协进会，白细胞分类计数CD4检测和CD38MFI监测，预测响应抗逆转录病毒治疗</li><li>与CD38的MFI报告艺术的反应立即报告，显着改善的周转时间，相对传统的艾滋病毒VL的报告（实时） </li><li>结果考核相结合的清晰度和临床上使用相同的报告形式</li><li>自动移液工具的需要可能是否定的吹打“前瞻性”的使用，是由于BCR样品监测的简单方法， </li><li>在操作过程中，标识不准确的样品处理和纠正问题的测试结果，结果是授权前</li><li>仪器故障，导致严重的崩溃之前提前预约服务的积极的预防性管理</li><li>白血病/​​淋巴瘤的诊断过程中的日常服务活动不受怀疑的情况下</li><li>类似流技术扩展为白血病分型和监测</li><li>协调与CD38的干血点，所有患者的艾滋病毒VL测试，并节省成本派遣评估星展（DBS）的存储测试，如果有临床或CD38的迹象表明，这样做</li><li>类似的流技术，以取代干扰素γELISPOT方法在结核病检测</li><li>为执行“保健点”的CD4测试的外设实验室的质量控制</li><li> “不必要的”，如抗体试剂的保存成本CD3和子集标记（但购买CD38的，而不是） </li><li> BCR测试（而不是购买昂贵的预吸管计数管）大量购买廉价的珠子</li><li>在一个网络，共同为有利的散装价格谈判与商业公司</li></ol><img src="/files/ftp_upload/2312/2312fig1.jpg" alt="图1" /> 图1。一个树/算法测试样品时使用的决定表明珠计数率（BCR）外围的结果。其主要任务是排除吹打与间歇体积失败的错误。图中显示错误的可能原因。它也表示，协议直方图和统计，其中受影响的 10 。 <img src="/files/ftp_upload/2312/2312fig2.jpg" alt="图2" /> 图2该小组的CD4 +协议，结合CD45 - FITC（绿色; X轴）的一个例子。与侧散射（复杂性; Y -轴）和CD4 - PE;（橙色; X轴）和侧散射（的复杂性; y轴）。门“A”是总的白细胞与淋巴细胞“B”选择门的人口。门“A”被转移到右侧的第二个图，以确定的CD4 +淋巴细胞门“C”。 <img src="/files/ftp_upload/2312/2312fig3.jpg" alt="图3" /> 图3 CD45/CD4 panleucogating检测相结合（图&#39;2&#39;和&#39;3&#39;;也参见图2）与CD8/CD38法（图表&#39;4&#39;和&#39;6） 。当总的白细胞（门“A”）被转移到图&#39;4&#39;，这里的CD8 +淋巴细胞门的CD8 - ECD的基础上（“x&#39; -轴）和侧散射（”y&#39;轴，如图所示门“D”）。 CD8 +细胞，然后转移到图&#39;6&#39;在一个单一的参数图CD38 - PC5染色强度可视化。这是显示为“G”参数图&#39;6&#39;。 CD38 - PC5平均荧光强度（MFI）被记录下来。对艺术的价值逐渐降低16,17。

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+North+American+multilaboratory+study+of+CD4+counts+using+flow+cytometric+panLeukogating+(PLG):+a+NIAID-DAIDS+Immunology+Quality+Assessment+Program+Study.">A North American multilaboratory study of CD4 counts using flow cytometric panLeukogating (PLG): a NIAID-DAIDS Immunology Quality Assessment Program Study.</a> Cytometry B Clin Cytom. 74, S52-S64 (2008).</li><li>Janossy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+changing+pattern+of+&#34;smart&#34;+flow+cytometry+(S-FC)+to+assist+the+cost-effective+diagnosis+of+HIV,+tuberculosis,+and+leukemias+in+resource-restricted+conditions.">The changing pattern of &#34;smart&#34; flow cytometry (S-FC) to assist the cost-effective diagnosis of HIV, tuberculosis, and leukemias in resource-restricted conditions.</a> Biotechnol J. 3, 32-42 (2008).</li></ol>

这里描述的是由南非NHLS和许可证的BCI，美国佛罗里达州迈阿密，目前正在举行的国际专利的权利的CD4方法。

近年来，慈善驱动和赠款分配方案已经帮助建立一些优秀的临床实践在全球资源有限地区艾滋病高负担。文艺节目已经成功地降低了死亡率，现正发起更高的CD4计数2,3。已经有迹象表明，这些新措施将减少艾滋病毒感染4,5的蔓延，尽管缺乏一个专门针对艾滋病毒感染者的疫苗。我们的视频，我们的文件，监测患者接受抗逆转录病毒治疗时，这些技术可以更好地集中，成为更有效的成本比在西部实验室（表1）执行的常规做法。 这样一个非洲主导的方式很可能是感染艾滋病毒，抗逆转录病毒治疗，正在成为一种慢性疾病时，必须同时支持实验室服务的升级换代。这种最先进的监视，需要精确的CD4计数和病毒负荷相关的技术，更艰巨的任务比简单地执行一个放置到任意组的患者，以协助决定第16条开始的CD4计数。随着这些诊断的帮助下，即使用CD8/CD38测试，它是可能的，以减少&gt; 60％的患者抗逆转录病毒16,19 VL相关的监督成本。同样重要的是要强调这是CD8/CD38测试16,17，而不是CD4细胞计数37，这是可能拿起VL的篮板和/或缺乏患者的依从性，在服药的早期迹象。 一个协调的成本效益的中央服务的一个基本要素，是一个大型的国家或地区，由熟悉操作技术和医疗方面的专家和学术顾问支持网络的可用性。这表明南非NHLS网络活动和CD4 - PLG.net培训计划，33，36，提供为鼓励规模较小的中心，教育和质量评估，这些可能还包括“点保健”服务。 更重要的是，中央和医疗点实验室的运作是相辅相成的。而前者则提供具有成本效益的大型服务操作以及基本/高级培训的外设实验室33,38的双重作用，后者有助于检查HIV阳性患者的人有资格招收艺术传播艺术从一个更大的基地，包括乡村诊所。这里有些谨慎是必要的的，因为点护理诊断，可昂贵得多，不会自动保证精度38。因此，合理的保留两级制，并促进两层之间的协调，而不让无论是手臂萎缩。 总之，诊断监测传染病的临床管理领域的一个必要的成本节约可以通过使用更便宜和更好地协调的现代技术的一个显着改善的质量13,39 APT服务实现。精心组织国家（地区）的诊断服务条款应更好地支持没有预先小心避免的情景时，几乎所有的赠款支持，因为这些来自富裕国家的进口浪费昂贵和/或未经试验的方法，尽快6-8先发制人建设性的审议。同样，一个更灵活的设计，与当地的医生和医疗人员的充分参与的临床治疗试验应减少不必要的频繁和/或超过雄心勃勃的实验室测试3。这项政策将给予更多的机会更充分地利用了准确诊断结果，以一种协调的抗逆转录病毒治疗患者的临床/实验室评估。这些智能诊断也可以率先为资源贫乏的国家在结核病和白血病的地区，使用现有熟悉的流式细胞仪平台40 8。

comprehensive &amp; cost effective laboratory monitoring of hiv/aids: an african role model

我们目前约有APT实验室服务协助抗逆转录病毒疗法（ART）的视频 - 代表南非的经济大规模的诊断测试的示范作用。在低收入国家的廉价艺术转化为艾滋病毒血清阳性患者的生存前景，但也有怀疑是否有最先进的实验室监测，并需要在哪些费用 - 的情况时，病理学服务的整体素质仍然可以非常低。适当的答案是要建立完善的服务与更好的协调和严格的内部质量评估比西方国家的经济。这在国家卫生实验室服务，约翰内斯堡金山大学，南非（NHLS SA）的位置拍摄的视频，提供了这样一个协调的计划，扩大原来的2色的CD4 - CD45的PanLeucoGating战略（PLG）。因此六个模块的视频演示，揭示了一个四色流式细胞仪检测的简单结合在一个单一的管血液学，免疫学和病毒学相关测试。这些视频模块是：（一）设置的文书;（二）样品的准备工作;（三）测试绝对计数，每个样品的质量监测珠计数率;（四）heamatological CD45的白细胞测试计数和差距;（V）的CD4细胞计数，（六）激活的CD8 +测量显示CD38的T细胞，病毒载量相关参数。潜在的成本节省是显着的的。这样的安排是一个最好的例子，执行严格的质量控制比西部实验室应用&gt; 800-1000每天测试的可行性，并与一个NHLS - SA网络内的其他实验室的技术转让。专家顾问，实验室进行了有关引进现代医疗技术的决策责任的管理者和决策者在一个位置不经常看到在巨大的工作量负担的大型区域性实验室进行的最新的技术细节。因此，这个视频显示了这些新的事态发展的详细资料。

Watch this Scientific Journal Video about Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model at JoVE.com

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

抗逆转录病毒疗法治疗艾滋病毒/艾滋病在南非大规模监测。流式细胞仪是结合血液学（CD45），免疫学（CD4 +）和病毒负荷挂钩CD38的检测。 NHLS - SA实验室，约翰内斯堡的记录，这些现代化的方法是提高本地内部质量控制与成本效益的服务，角色模型，为资源有限的诊断。

艾滋病毒/艾滋病的综合成本有效的实验室监测：非洲的角色模型

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

comprehensive-cost-effective-laboratory-monitoring-hivaids-an-african

Research

JoVE Journal

Immunology and Infection

16.6K 次观看.  National Health Laboratory Services (NHLS-SA). 抗逆转录病毒疗法治疗艾滋病毒/艾滋病在南非大规模监测。流式细胞仪是结合血液学（CD45），免疫学（CD4 +）和病毒负荷挂钩CD38的检测。 NHLS - SA实验室，约翰内斯堡的记录，这些现代化的方法是提高本地内部质量控制与成本效益的服务，角色模型，为资源有限的诊断。

视频: Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

科研

免疫与感染

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

口腔生物膜的生物化学，结构和转录综合评价分析工具盒介导变形链球菌

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

微生物源使用特定的人类和动物病毒的追踪成本效益的方法

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

利用荧光蛋白监视非洲锥虫Glycosome动态

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

安全注意事项和操作步骤在（A）BSL-4实验室：1.生物安全4级实验室西服套房出入境手续

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

安全注意事项和操作步骤在（A）BSL-4实验室：3，大气生物学

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

生物发光成像检测到非洲锥虫病的晚期感染

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

头孢哌酮治疗小鼠模型的临床相关的<em&gt;艰难梭菌</em&gt;应变R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...