The authors have no acknowledgements.

1.菌悬液的制备： <ol><li>从划线培养板，在无菌条件下接种单个菌落到含有无菌接种环的方式将100毫升合适的生长培养基（表1）的250毫升无菌瓶中。 </li><li>将瓶子放入轨道摇床在适当的条件下（表1）为1-2天，定期检查的增长。将摇床转速为140 RPM。 </li><li>无菌地在23℃下转移细菌悬浮液分成两无菌50ml锥形管中并离心管在3000×g离心20分钟。 </li><li>吸出上清，悬浮每个细菌沉淀用15毫升无菌蛋白胨水。必要时旋涡。结合悬浮细菌培养成一个管/容器。 </li><li>滴度的股票文化，每节4一式三份，以确定该股文化滴度。 </li><li>冷藏长达2周，在477; 2°C，直到需要。重新滴度文化在使用时，请参阅步骤4.8。 </li></ol> 2.血小板产品的制备： <ol><li>收集标准的单采人血小板产品在经认可的血库。血小板产品必须满足以下集合规格：90-360毫升的体积和0.8 -3.8×10 6的血小板/μL的浓度。 <ol><li>确保血小板产品已经休息之前处理至少2小时的病原体减少。本产品存放在22±2℃的恒温箱血小板不搅拌这些2小时。两小时后确保所述血小板产物搅拌在22±2℃的血小板培养箱。使用产品长达22小时后集合。 </li></ol></li><li>用3毫升注射器取出2ml样品以测量血小板产品的pH 22C。使用血气分析仪，保证了产品的pH&gt; 6.6。使用相同的样品来测量使用血液分析仪的血小板制品的血小板浓度。确保产品符合0.80-3.8×10 6的血小板/μL收集的浓度要求。 </li><li>目视评价的血小板制品的漩涡。如果产品有一个“0”的漩涡，产品将被丢弃。 </li><li>握住血小板包下了一个直接的白光光源和挤压血小板袋简单。目视观察血小板样品的涡流图案。得分使用以下等级的产品。 注：2 - 正旋：袅袅的不均匀性在整个包包可见有强烈的对比。 1 - 中级漩涡：有些不均匀性可见的，但只有在少数地方和对比度差。 0 - 负漩涡：前后挤压血小板袋后剩余浑浊同质化</li></ol> 3，核黄素和紫外线过程： <ol><li>陈德良sfer使用无菌管道对接设备的血小板产品送至核黄素和UV光照射和储存袋。转让完成后取出使用管封口机空血小板单位收集袋，丢弃空袋的生物危害性的废物。 </li><li>删除的35ml袋500微米核黄素从它的光保护外部包装原液。无菌码头核黄素包到治疗和储物袋的入口管路。允许内容排入血小板单元。 </li><li>挂核黄素小袋/处理和储存袋从核黄素袋设置并轻轻表达残余空气出处理和储存袋并进入核黄素袋。确保少量的血小板产品也体现到核黄素袋冲洗残留的核黄素到血小板产品。允许核黄素和血小板溶液回流到处理和储存袋，然后夹紧入口管线。取出空核黄素包我们荷兰国际集团一管封口机和丢弃的空包作为生物危害性的废物。 </li><li>无菌码头与女性注射器连接到照明和储物袋的进气管路6“的管线。附上10ml的注射器管（删除柱塞）上的阴连接器的注射器。 </li><li>无菌添加5 ml储备细菌进入注射器筒经由吸移管。打开在入口线夹紧，并允许该细菌排放到血小板产品。通过允许一些血小板产物流回10ml注射器冲洗注射器筒。取出10ml注射器。 </li><li>无菌附上30ml的注射器和冲洗入口端口最少两次以确保库存菌混入的血小板制品。之后，产品将充分混合，用一个干净的5〜10毫升的注射器，并删除2毫升的前处理的样品。使用新的注射器，确保从接种步骤中引入任何空气或采样步骤被删除。 <ol><li>滴定每部4的前处理的样品。 </li></ol></li><li>拆下处理和储存袋的进线和权衡的产物。 </li><li>将产品投入照明，并按照屏幕上的命令来启动一个标准血小板治疗。 <ol><li>输入操作人员编号。 </li><li>安全和安装包的照明器压板。 </li><li>输入样品编号为使用任何条形码阅读器或手动输入键盘的照明。 </li><li>扫描产品类型。 </li><li>关闭石英夹。 </li><li>该照明装置将自动提供能量的6.24焦耳/毫升。典型的处理时间需要5-10分钟。 </li></ol></li><li>治疗后不育码头与女性注射器连接器6“的管线上处理和储存袋的样品球线。确保产品的充分混合，然后再连接5毫升注射器，取出2ml的后处理样品。 <ol><li>滴度后处理SAM每节4 PLE。 </li></ol></li></ol> 4.细菌滴度<ol><li>估计前处理和后处理的样品的效价，并准备来执行各样品的终点系列稀释需要5 ml的稀释管中的适当数量。无菌填充所需的管与1.8毫升蛋白胨水。 </li><li>滴度用10倍系列稀释方案各样品。无菌转移0.2 ml的从未经稀释的样品到第一稀释管（10 -1）。涡这种稀释管和转让0.2毫升下一个稀释管。通过稀释液的所需数量继续连续稀释。 <ol><li>无菌转移100μl的每种稀释液的被镀到合适的琼脂平板（ 表1）。 </li></ol></li><li>用无菌细胞吊具，均匀地将样品分配到每个琼脂平板上。 </li><li>反转和孵化板在适当的条件下（ <stroNG&gt;表1），1-2天定期检查增长。 </li><li>计数板的30-300殖民地。含有大于300菌落板被认为是不计其数（TNTC）。含有少于30菌落板被认为是低于定量限（LOQ）的限制。 </li><li>计算各试验样品的滴度并记录结果以CFU /毫升。 <ol><li>使用下面的公式来计算CFU /毫升： <img alt="式（1）" fo:content-width="2.5in" src="/files/ftp_upload/52820/52820eq1.jpg" width="250" /></li><li>如果有上最低的稀释板没有殖民地，用以下公式来计算检测限（LOD）的限制： <img alt="公式（2）" fo:content-width="1in" src="/files/ftp_upload/52820/52820eq2.jpg" width="100" /> 和 <img alt="公式3" fo:content-width="1in" src="/files/ftp_upload/52820/52820eq3.jpg" width="100" /> 其中： N =表示最低的粒子数目（CFU）以P可以有把握地检测产品为准。 P =概率微生物会漏检;为了检测微生物95％的置信，P = 0.05。 在毫升的V =总产品数量 板的V =体积铺板（毫升）×稀释水平</li></ol></li><li>为阴性对照，板0.5 ml的是，用于为连续稀释到每两个琼脂平板的蛋白胨水的。使用相同的板型为样本进行测试。反转和孵化板在适当的条件下1-2天定期检查增长。 <ol><li>如果增长要么琼脂平板上观察到的，瓶用蛋白胨水的必须丢弃;该研究的结果也无效。 </li></ol></li><li>对于阳性对照，再滴度细菌所使用的储用培养物。反转和孵化板在适当的条件下1-2天定期检查增长。效价应为±1.0日志记录的CFU /毫升ED股票滴度（见第1.5节）为研究对象，以被视为有效。 </li></ol>

<ol>
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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=Photochemical+inactivation+of+chikungunya+virus+in+plasma+and+platelets+using+the+Mirasol+pathogen+reduction+technology+system.">Photochemical inactivation of chikungunya virus in plasma and platelets using the Mirasol pathogen reduction technology system.</a> Transfusion. 53 (2), 284-290 (2013).</li><li>Walther-Wenke, G., 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=Monitoring+bacterial+contamination+of+blood+components+in+Germany:+effect+of+contamination+reduction+measures.">Monitoring bacterial contamination of blood components in Germany: effect of contamination reduction measures.</a> Vox Sang. 100 (4), 359-366 (2011).</li></ol>

本文的所有作者泰尔茂BCT，开发商的Mirasol PRT系统和制造商正在采用。此视频文章出版费用已经支付泰尔茂BCT。

细菌筛查已成为血液银行机构普遍的做法，并为许多地方它作为主要的方法，以防止细菌污染血小板输血。虽然细菌筛查通常interdicts广大喜欢S.快速增长的革兰氏阴性菌沙雷氏菌，大肠杆菌大肠杆菌和E.阴沟肠杆菌 ，它的斗争，以检测像S.生长缓慢的革兰氏阳性菌黄色葡萄球菌，链球菌属和 S. 表皮8。不幸的是，这三个革兰氏阳性菌有牵连的所有受污染的血小板单位和含有这三种生物血小板输注产品的50％以上已经导致病人死亡1。 细菌筛选依赖于细菌滴度血小板产物是足够高到足以使至少一种生物体中包含的典型4-8毫升筛选样本内。如果细菌滴度不够高的细菌在4-8毫升的样品体积被捕获，该单元被错误地标记为负，它被释放用于输血。另一种方法，以细菌的筛选可以采用可以在所有血小板单位来执行，并可能使污染微生物非功能主动过程。 在这项研究中，细菌的高滴度故意加入血小板单位来测试核黄素和紫外线病原体还原过程的总减少量。虽然细菌载量在本研究中测试的远远超过通常在捐赠的时候发现在血液产品，这种研究有助于证明核黄素和UV光处理能够通过以显著减少污染的血小板单位活菌滴度2.6到5.4的日志（99.7-99.9996％减少污染细菌的）（ 图2）。为了获得ACCURA德数减少值，至关重要的是，在执行细菌滴度步骤当操作者具有沿着与精确移液技能良好无菌操作。执行串行稀释时，不精确的移液器可能会导致错误的积累，因此是不准确的效价测定。 收集在此研究的数据支持以前的工作与核黄素和UV光，其挑战系统免受低的水平，临床相关菌血症（&lt;20 CFU /单位）。以前的研究是设计用来模拟一个实际的临床污染事件，结果表明核黄素和紫外线是有效地防止细菌生长超过7天的血小板保存期。基于来自先前研究的数据模型表明，血小板产品细菌污染的当前风险估计可以由多达98％，从目前水平8减小。核黄素和紫外线的过程相比，毫不逊色于细菌屏幕ING，它仅表现出66％的有效性时，用临床相关的细菌负荷8的挑战，以检测细菌污染物在血小板单位。 与所有的技术，PRT治疗血小板制品，以防止细菌污染的血小板单位有其局限性相关的并发症。 PRT系统不是有效对抗细菌芽孢和不灭活内毒素，因此即使PRT系统可以灭活的革兰氏阴性细菌高滴度的其余内毒素尚可引起脓毒性休克中的收件人。储存期间PRT最好及早进行之前，细菌可以传播到高滴度。 总之，就证明了这两项研究的组合，核黄素和UV光处理是在减少两个革兰氏阴性和革兰氏阳性生物体的细菌载量有效，并且可以提供一个可行的替代细菌筛选。按惯例treatin克血小板制品与核黄素和紫外线也具有降低的像病毒和寄生虫等血源性剂的潜在传输的额外的好处。

血小板输血，血浆，袋装RBC和全血制品起到在现代医学中的主要作用，可用于治疗各种医学病症，更换重要的流体，并最终挽救生命。血小板是，它们或者从全血中分离并汇集成transfusable剂量或通过单采血小板的过程中收集的蜂窝产物。在体内血小板的主要作用是阻止在伤口部位止血，并帮助维持止血。患者从低血小板计数（血小板减少症）的痛苦是容易自发性出血事件，并输注了血小板，使他们的血小板细胞计数返回到正常范围。收集血小板保存最长为5-7天，并储存在22±2℃，同时持续搅拌。 尽管救生血小板输注的性质仍然存在轻微的风险受血者因合作ntamination这些产品由寄生虫，细菌和病毒11。病毒核酸检测的执行器（NAT）已显著降低病毒传播主要血源性剂，如丙型肝炎病毒（HCV），乙型肝炎病毒（HBV）和人免疫缺陷病毒（HIV）的风险5。最近公布的加拿大血液服务估计剩余风险，这些药物是1元800万捐款为HIV，1％的670万捐款为HCV和1％的170万捐款为HBV 15。 虽然细菌通常争取较少注意在一般公众，细菌污染的血小板产品的频率已被估计为高达1：1000 7和因为百万血小板制品每年输血许多收件人暴露于潜在的危及生命的并发症像败血症6。研究表明，该细菌负荷的时候的污染低，&lt;100菌落形成单位（CFU）/产物2,16，然而营养丰富的环境中和室温保存允许污染细菌增殖到危险的高滴度输血前。目前，可用来防止细菌污染的产品到达血小板收件人的唯一认可的方法是通过使用基础的文化系统和快速床旁检测的。简要地说，基于培养系统血小板产品上存储的搅拌器在22℃下12-24小时，从而使细菌在产品的激增，在其上4-8毫升样品从血小板产物中除去，并接种到瓶含有营养培养基。将该瓶放入仪器，它监视瓶中细菌生长。如果仪器检测在瓶中细菌生长它被标记和相应的血小板单元被丢弃。虽然这个过程是相当成功的，在检测速度快GRO翼生物体，许多生长缓慢的种类不生长到足够高的效价被检测，从而为假阴性单元的电势被释放用于输血7,12,14,16,22。与基于文化的检测系统，通常在血小板保存期之后进行的快速护理点测试时，细菌负荷已显著增加。需要较高的效价，因为点的护理测试比的基础培养系统较不敏感的，并且只可靠地检测细菌一旦达到滴度≥1×10 3 CFU / ml的17。然而，这种测试可以提供100小时采样具有内结果。变异在这些测试中的性能已导致假阴性产物的释放，引起在接受者9致命脓毒症反应。 打击血小板制品细菌污染的问题的另一种方法是通过常规使用的病原体减少进程内的ESS可以灭活细菌污染，而不是试图探测到它们。使用核黄素作为光敏剂，在用UV光组合，已显示减少一系列广泛致病血源性污染物的感染性，包括细菌3,4,8,10,19-21。使用核黄素和紫外线的光为减少病原体是无毒和非致突变，和核黄素和UV光处理过的组件已被证明是安全的受血者以及为那些处理血液制品18。简言之，核黄素分子可与细菌，寄生虫，病毒和任何有核细胞（如白细胞）的核酸（DNA和RNA）相关联。暴露于UV光激活核黄素，引起化学改变到的核酸（主要是鸟嘌呤碱基）的官能团，从而防止复制的核酸和/或转录和离开灭活13的生物体。无核细胞样板允许和红血细胞不由于缺乏核酸的受核黄素化学。 以前的工作与核黄素和紫外线技术的使用评价实验设计意在模仿沾染临床相关的细菌负荷（&lt;20 CFU /产品）8血小板产品的一组选定的细菌。本研究的目的是评估抗高滴度细菌污染的核黄素和UV光处理（&gt; 1.0×10 5 CFU / ml）的，以便测量系统的总细菌减少容量处理血浆的血小板制品。基于从hemovigilance研究1中收集的数据，通常发生的革兰氏阴性和革兰氏阳性生物体的小组被选定为评价本研究中，并包括下列物种： 表皮葡萄球菌，金黄色葡萄球菌，缓症链球菌，酿脓链球菌，粘质沙雷氏菌，小肠结肠炎耶尔森菌，布鲁氏菌neotomae，蜡样芽胞杆菌（营养型），大肠埃希菌，绿脓杆菌和肺炎克雷伯菌。所有的生物，除了B.蜡状 ，得自ATCC，并优先放在获得已被确定为从血液成分经分离的细菌菌株。该B.蜡状在此研究中测试是一种临床菌株内部分离从污染的血小板制品。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mirasol Illuminator</td>
			<td>Terumo BCT</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mirasol Treatment/Storage Bag</td>
			<td>Terumo BCT</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematology Analyzer</td>
			<td>Beckman-Coulter</td>
			<td>Ac&#8226;T diff&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Gas Analyzer</td>
			<td>Siemens</td>
			<td>RapidLab 1265&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Docking Device</td>
			<td>Terumo BCT</td>
			<td>TSCD</td>
			<td></td>
		</tr>
		<tr>
			<td>Platelet Incubator</td>
			<td>Helmer</td>
			<td>PC2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Incubator Shaker</td>
			<td>Lab-Line</td>
			<td align="right">4628</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Incubator</td>
			<td>Fisher</td>
			<td>Iso-temp</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II A/B3 Biosafety Cabinet</td>
			<td>Thermo-forma</td>
			<td align="right">1286</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing Sealer</td>
			<td>Sebra</td>
			<td>Smart Sealer II</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Centrifuge</td>
			<td>IEC</td>
			<td>Centra GP8R</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Syringe</td>
			<td>BD</td>
			<td align="right">309604</td>
			<td></td>
		</tr>
		<tr>
			<td>30 mL Syringe</td>
			<td>BD</td>
			<td align="right">309650</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Dilution Tube</td>
			<td>VWR</td>
			<td>211-0058</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical Tube</td>
			<td>Corning</td>
			<td align="right">430290</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>Gilson</td>
			<td>P-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>Rainin</td>
			<td>R-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Repeat Pipette</td>
			<td>Eppendorf</td>
			<td align="right">4780</td>
			<td></td>
		</tr>
		<tr>
			<td>1-200 &#181;L Filter Tip</td>
			<td>Costar</td>
			<td align="right">4810</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Disposable Serrological Pipette</td>
			<td>Costar</td>
			<td align="right">4489</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Disposable Serrological Pipette</td>
			<td>Costar</td>
			<td align="right">4487</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mL 500 &#181;M Riboflavin</td>
			<td>Terumo BCT</td>
			<td>35 mL 500 &#181;M&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Brucella Agar with 5% Horse Blood</td>
			<td>BBL</td>
			<td align="right">221547</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion</td>
			<td>Teknova</td>
			<td>B9993</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone Water</td>
			<td>BD</td>
			<td align="right">257087</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypticase Soy Broth</td>
			<td>BD</td>
			<td align="right">299113</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypticase Soy Agar</td>
			<td>Remel</td>
			<td>R01917</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated Horse Serum</td>
			<td>Gibco</td>
			<td align="right">26050</td>
			<td></td>
		</tr>
	</tbody>
</table>

treatment of platelet products with riboflavin and uv light: effectiveness against high titer bacterial contamination

血小板单位被细菌污染一直被公认为一个显著输血的风险，由于其后期的捐赠储存条件。产品通常存储在22℃上的搅拌罐，能够促进细菌生长的条件。虽然据信被导入血小板产品的细菌总数极低，这些细菌可繁殖到一个非常高的效价在输血前，可能造成严重的不良事件。本研究的目的是评估针对该已被确定为对血小板制品常见的污染物的细菌的一个面板基于核黄素减少病原体的过程。该小组成员包括以下生物：S。表皮，金黄色葡萄球菌，缓症链球菌，化脓性链球菌，粘质沙雷氏菌，小肠结肠炎耶尔森菌，B。neotomae，蜡状芽孢杆菌，大肠杆菌，绿脓杆菌和 K. 肺炎 。每个血小板单位接种具有高细菌负荷和除去样品B治疗前后OTH。甲集落形成测定法中，使用一个终点稀释方案，用于测定治疗前和治疗后的细菌效价。数减少了计算和减去预处理滴度后处理滴度。以下数减少观察：S.表皮 4.7日志（99.998％），金黄色葡萄球菌 4.8日志（99.998％），缓症链球菌 3.7日志（99.98％），化脓性链球菌 2.6日志（99.7％），粘质沙雷氏菌 4.0日志（99.99％），Y。菌 3.3日志（99.95％），B。neotomae 5.4日志（99.9996％），蜡状芽孢杆菌 2.6日志（99.7％），大肠杆菌≥5.4日志（99.9996％），铜绿假单胞菌 4.7日志（99.998％）和K 。肺炎 2.8日志（99.8％）。从该研究的结果表明该过程可以帮助降低与细菌污染相关的严重不良事件输血的风险。

使用基于核黄素和UV光PRT过程的十一种不同的细菌物种的还原进行了评价。这些试剂的一些代表的血小板产品较常见的污染物和包括五个革兰氏阳性和六革兰氏阴性生物体。至少六个血小板制品每生物体进行评价，每个产品中接种足菌，得到&gt; 1.0×10 5 CFU / ml的最终细菌滴度在血小板制品。在治疗前的样品除去，以确定治疗前的滴度，然后将血小板产物与核黄素和UV处理及时治疗。治疗后的血小板制品以下数减少观察：S.表皮 4.7日志（99.998％），金黄色葡萄球菌 4.8日志（99.998％），缓症链球菌 3.7日志（99.98％），化脓性链球菌 2.6日志（99.7％），粘质沙雷氏菌 4.0日志（99.99％），Y。菌 3.3日志（99.95％），B。neotomae 5.4日志（99.9996％），蜡状芽孢杆菌 2.6日志（99.7％），大肠杆菌≥5.4日志（99.9996％），P。绿脓杆菌4.7日志（99.998％）和肺炎克雷伯菌2.8日志（99.8％）（图2）。各单位符合系统规格以上的核黄素和UV过程说明。 与 S进行额外减少测试用白膜层的血小板，化脓性链球菌 。无显著差异，观察在S的减少在血沉棕黄层的血小板与采血小板酿脓 （数据未显示）。 <img alt="表1" src="/files/ftp_upload/52820/52820table1.jpg" /> 表1：生长条件使用核黄素和UV光评估细菌物种 。已显示污染血小板制品既革兰氏阴性和革兰氏阳性菌的面板被选定为这项研究。用于繁殖的生长条件（温度和介质类型）和滴度r处的生物体被示出。 <img alt="图1" src="/files/ftp_upload/52820/52820fig1.jpg" /> 图1：核黄素和UV光减少病原体的过程。核黄素溶解于0.9％盐水，在500微米的标称浓度。将溶液无菌连接并排入每个血小板产品。菌中加入无菌每个血小板单元，然后用紫外线能量（约6-10分钟）的6.2焦耳/毫升处理。在这种体外研究后处理血小板单位进行采样，以确定最终的细菌滴度。 <img alt="图2" src="/files/ftp_upload/52820/52820fig2.jpg" /> 图2：还原时与核黄素和UV光处理过的核黄素和UV光被用于减少血小板产品的常见细菌污染物的细菌载量不同的细菌物种。日志10降低区别平均预处理效价，平均治疗后滴度之间。 <a href="https://www.jove.com/files/ftp_upload/52820/52820fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination at JoVE.com

Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

The storage conditions of platelet products create an ideal environment for bacterial contaminants to multiply to dangerous levels. The goal of this study was to use a colony forming assay to evaluate a riboflavin based pathogen reduction process against high titer bacterial contamination in human platelet products.

治疗核黄素血小板制品和紫外线光：对有效性高滴度细菌污染

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. ...

treatment-platelet-products-with-riboflavin-uv-light-effectiveness

Research

JoVE Journal

Immunology and Infection

13.5K 次观看.  Scientific Affairs. The storage conditions of platelet products create an ideal environment for bacterial contaminants to multiply to dangerous levels. The goal of this study was to use a colony forming assay to evaluate a riboflavin based pathogen reduction process against high titer bacterial contamination in human platelet products.

视频: Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

斑马鱼的胚胎细胞内细菌病原体感染

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

科研

免疫与感染

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Antileishmanial筛选对细胞内的寄生虫救援和转化实验<em&gt;杜氏利什曼原虫</em无鞭毛体在THP1人急性单核细胞白血病细胞株

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

四环素调节细胞株产生高滴度的慢病毒载体，专门针对树突状细胞

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

针对天然产物的单克隆抗体的产生

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

评估细菌病原体免疫反应的声吞噬杀灭试验

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

功能金报告系统测量特定分枝杆菌误调速率

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

从高致病性H5N1病毒和禽流感病毒中生产含有包络糖蛋白的高蒂特感染性流感伪型颗粒

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue.

建立猪外维沃角膜模型研究药物治疗细菌性角膜炎

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...