Name: treatment of platelet products with riboflavin and uv light: effectiveness against high titer bacterial contamination
Uploaded: 2015-08-24T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination at JoVE.com

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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><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&amp;bull;T diff&amp;nbsp;</td><td></td></tr><tr><td>Blood Gas Analyzer</td><td>Siemens</td><td>RapidLab 1265&amp;nbsp;</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>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>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>309604</td><td></td></tr><tr><td>30 ml Syringe</td><td>BD</td><td>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>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>4780</td><td></td></tr><tr><td>1-200 &amp;micro;l Filter Tip</td><td>Costar</td><td>4810</td><td></td></tr><tr><td>25 ml Disposable Serrological Pipette</td><td>Costar</td><td>4489</td><td></td></tr><tr><td>5 ml Disposable Serrological Pipette</td><td>Costar</td><td>4487</td><td></td></tr><tr><td>35 ml 500 &amp;micro;M Riboflavin</td><td>Terumo BCT</td><td>35 mL 500 &amp;micro;M&amp;nbsp;</td><td></td></tr><tr><td>Brucella Agar with 5% Horse Blood</td><td>BBL</td><td>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>257087</td><td></td></tr><tr><td>Trypticase Soy Broth</td><td>BD</td><td>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>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

Preparation and Pathogen Inactivation of Double Dose Buffy Coat Platelet Products using the INTERCEPT Blood System

Blood centers are faced with many challenges including maximizing production yield from the blood product donations they receive as well as ensuring the highest possible level of safety for transfusion patients, including protection from transfusion transmitted diseases. This must be accomplished in a fiscally responsible manner which minimizes operating expenses including consumables, equipment, waste, and personnel costs, among others.
Several methods are available to produce platelet concentrates for transfusion. One of the most common is the buffy coat method in which a single therapeutic platelet unit (&ge; 2.0 x1011 platelets per unit or per local regulations) is prepared by pooling the buffy coat layer from up to six whole blood donations. A procedure for producing "double dose" whole blood derived platelets has only recently been developed.
Presented here is a novel method for preparing double dose whole blood derived platelet concentrates from pools of 7 buffy coats and subsequently treating the double dose units with the INTERCEPT Blood System for pathogen inactivation. INTERCEPT was developed to inactivate viruses, bacteria, parasites, and contaminating donor white cells which may be present in donated blood. Pairing INTERCEPT with the double dose buffy coat method by utilizing the INTERCEPT Processing Set with Dual Storage Containers (the "DS set"), allows blood centers to treat each of their double dose units in a single pathogen inactivation processing set, thereby maximizing patient safety while minimizing costs. The double dose buffy coat method requires fewer buffy coats and reduces the use of consumables by up to 50% (e.g. pooling sets, filter sets, platelet additive solution, and sterile connection wafers) compared to preparation and treatment of single dose buffy coat platelet units. Other cost savings include less waste, less equipment maintenance, lower power requirements, reduced personnel time, and lower collection cost compared to the apheresis technique.

This article describes the process used by &#214;rebro University Hospital to produce double dose buffy coat platelet concentrates prepared from whole blood donations and treated with the INTERCEPT Blood System for pathogen inactivation. The in vitro quality of the final platelet units are evaluated over 7 days of storage.

巴菲大衣血小板制品双倍剂量使用INTERCEPT血液系统的制备和病原体灭活

Blood centers are faced with many challenges including maximizing production yield from the blood product donations they receive as well as ensuring the ...

科研

医学

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

PRP作为一种新的方法，以防止感染的制备及<em在体外</em&gt;抗菌性能的PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

免疫与感染

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

生物工程

LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic inactivation of bacteria (aPDI). This process involves the administration of a photosensitizer (PS) activated by a suitable visible light source, producing exacerbated levels of reactive oxygen species (ROS) that damage crucial cellular biomolecules, ultimately causing bacterial cell death. It is crucial to create standardized, easy-to-use, and reproducible initial tests to assess and compare the effectiveness of light-induced phototoxicity of potential photosensitizers. This study introduces a simple and efficient in vitro technique for assessing photodynamic activity against planktonic bacterial cells. By employing a 96-well microplate format along with a large LED panel, the system facilitates the systematic evaluation of various compounds. Such a configuration allows for high-throughput screening of potential photosensitizers in a controlled and consistent environment, simplifying the process of identifying promising candidates for further development. This flexible platform serves as an important step in advancing the development of innovative photodynamic therapies for managing antibiotic-resistant infections.

This protocol describes a straightforward and economical method for evaluating the effectiveness of potential photosensitizers in antibacterial photodynamic inactivation (aPDI), using a 96-well plate format combined with an LED panel light source. This approach enables the simultaneous testing of multiple experimental conditions, including different concentrations, compounds, and bacterial strains.

基于 LED 的体外 筛选用于评估细菌光动力灭活中的光激活分子

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic ...

生物化学

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

红光光疗法调节白色念珠菌生物膜生长

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

血小板输血中微粒的常规筛选方法

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" />) generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" /> species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Here, we present a protocol to inactivate pathogenic bacteria with reactive oxygen species produced during photolysis of flavin mononucleotide (FMN) under blue and violet light irradiation of low intensity. FMN photolysis is demonstrated to be a simple and safe method for sanitary processes.

通过可见光光解 核 黄素-5′-磷酸灭活病原体

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, mouth, vagina, and skin. Despite the availability of antifungal medication, the mortality rate of invasive candidiasis remains ~50%. Unfortunately, the incidence of drug-resistant C. albicans is increasing globally. Antimicrobial photodynamic therapy (aPDT) may offer an alternative or adjuvant treatment to inhibit C. albicans biofilm formation and overcome drug resistance. Rose bengal (RB)-mediated aPDT has shown effective cell killing of bacteria and C. albicans. In this study, the efficacy of RB-aPDT on multidrug-resistant C. albicans is described. A homemade green light-emitting diode (LED) light source is designed to align with the center of a well of a 96-well plate. The yeasts were incubated in the wells with different concentrations of RB and illuminated with varying fluences of green light. The killing effects were analyzed by the plate dilution method. With an optimal combination of light and RB, 3-log growth inhibition was achieved. It was concluded that RB-aPDT might potentially inhibit drug-resistant C. albicans.

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

The growing incidence of drug-resistant Candida albicans is a serious health issue worldwide. Antimicrobial photodynamic therapy (aPDT) may offer a strategy to fight against drug-resistant fungal infections. The present protocol describes Rose bengal-mediated aPDT efficacy on a multidrug-resistant C. albicans strain in vitro.

孟加拉玫瑰介导的光动力疗法抑制 白色念珠菌

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, ...

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.

用体外模型研究 5-氨基乙酰丙酸介导光动力疗法对金黄色葡萄球菌生物膜的影响

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

生物学

<meta charset="utf8"></head><body>分析血小板磷脂酰丝氨酸和微泡释放

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and releasing PS-expressing microvesicles that retain the original membrane architecture and cytoplasmic components of their originating cells. The accessibility of phosphatidylserine facilitates the binding of major coagulation factors, significantly amplifying the catalytic efficiency of coagulation enzymes, while microvesicle release acts as a pivotal mediator of intercellular signaling. Procoagulant platelets play a crucial role in clot stabilization during hemostasis, and their increased proportion in the bloodstream correlates with an increased risk of thrombosis. It has also been shown that platelet microvesicles are rich in growth factors that promote wound healing and inflammatory modulation. Analyzing phosphatidylserine exposure and microvesicle release using flow cytometry poses significant challenges due to their small size and the limited number of positive events for markers of interest. Despite considerable advances in the last decade, methods for assessing phosphatidylserine exposure and microvesicle release remain a work in progress. Unfortunately, no single universally applicable protocol exists, and several factors must be evaluated to determine the most appropriate methodology for each specific application. Here, we describe a detailed protocol for isolating washed platelets from human blood, followed by collagen and/or thrombin activation, to measure the exposure of phosphatidylserine and microvesicle release that characterize procoagulant platelets. This protocol is designed to facilitate the initial preparation of platelet-rich plasma and the isolation of washed platelets. Finally, phosphatidylserine exposure and microvesicle release are quantified by flow cytometry, enabling the identification of procoagulant platelets.

<meta charset="utf8"></head><body>通过测量人纯化血小板中的磷脂酰丝氨酸暴露和微泡释放来表征促凝血小板

Procoagulant platelet formation has been correlated with an increased risk of thrombosis. Presented here is a precise protocol for isolating washed platelets from human blood, intended to quantify the exposure of phosphatidylserine and microvesicle release, which are distinctive features of procoagulant platelets.

通过测量人纯化血小板中的磷脂酰丝氨酸暴露和微泡释放来表征促凝血小板

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and ...

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

摘要

探索更多视频

此视频中的章节

巴菲大衣血小板制品双倍剂量使用INTERCEPT血液系统的制备和病原体灭活

PRP作为一种新的方法，以防止感染的制备及

微流体流钱伯斯再造使用血止血建模和血小板输注

基于 LED 的体外筛选用于评估细菌光动力灭活中的光激活分子

红光光疗法调节白色念珠菌生物膜生长

血小板输血中微粒的常规筛选方法

通过可见光光解核黄素-5′-磷酸灭活病原体

孟加拉玫瑰介导的光动力疗法抑制白色念珠菌

用体外模型研究 5-氨基乙酰丙酸介导光动力疗法对金黄色葡萄球菌生物膜的影响

通过测量人纯化血小板中的磷脂酰丝氨酸暴露和微泡释放来表征促凝血小板

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

摘要

探索更多视频

此视频中的章节

巴菲大衣血小板制品双倍剂量使用INTERCEPT血液系统的制备和病原体灭活

PRP作为一种新的方法，以防止感染的制备及

微流体流钱伯斯再造使用血止血建模和血小板输注

基于 LED 的体外 筛选用于评估细菌光动力灭活中的光激活分子

红光光疗法调节白色念珠菌生物膜生长

血小板输血中微粒的常规筛选方法

通过可见光光解 核 黄素-5′-磷酸灭活病原体

孟加拉玫瑰介导的光动力疗法抑制 白色念珠菌

用体外模型研究 5-氨基乙酰丙酸介导光动力疗法对金黄色葡萄球菌生物膜的影响

通过测量人纯化血小板中的磷脂酰丝氨酸暴露和微泡释放来表征促凝血小板

基于 LED 的体外筛选用于评估细菌光动力灭活中的光激活分子

通过可见光光解核黄素-5′-磷酸灭活病原体

孟加拉玫瑰介导的光动力疗法抑制白色念珠菌