We would like to thank the patients and parents for their continuous support; the caregivers for their dedication; Pr Bierling, director of the French Blood Bank in the Paris area, for his support of the collaborative work between our hospital and his team to develop the continuous manual exchange transfusion method; and the University Paris Diderot and the hospital Robert-Debr&#233; for their support.

该协议遵循医院伦理委员会的指导方针。有在交流会3个步骤:病人准备;最初的等容放血（如适用）;和全血交换，由若干个周期，或连续全血放血，其与稀释PRBCs的输液相关联的。根据患者的血红蛋白水平的，中介等容放血的第四步骤可以中途通过交换阶段加入。在会议开始时，所有必要的材料（袋装RBC和5％血清白蛋白）的必须准备。另外，各步骤必须提前制备。除了精密刻度和双静脉通路，没有具体的设备是必需的。然而，就必须有超过整个过程不断的医疗监督 ，更应如此，如果市场交易量是很高的。 1.患者安装<ol><li>之前，ST进行的实验室测试arting换血会话。在会议之前在同一天执行它们，少于24小时。医生检查下面的测试结果之前，不要执行测试。 注:该测试包括一个完整的血球计数，网织红细胞计数，哈佛商学院率测定，间接抗人球蛋白试验（IAT），血钙和电解质测定，肝功能，凝血功能检查，由医院的血液及生化单位进行例行。 </li><li>有医师执行患者的全面和完整的身体检查，并特别注意的血液动力学参数，如心脏速率，血压，呼吸率，和氧饱和度。获取最近的体重。维持血氧饱和度&gt; 98％，在整个交流会。这样做，通过鼻插管给药患者用1L的氧，而在与脚仰卧位稍微升高。 </li></ol> 2.血液制品的制备</p&gt; <ol><li>计算并获得初始放血体积（ 见表1）。例如，如果患者在会话初始血红蛋白速率为10克/升，流血12毫升/公斤的血液中60分钟。对于一个30公斤的病人，出血360毫升血液（这些是适当的卷）。 注:由于初始放血量根据患者的血红蛋白水平（ 表1）计算的约8克血红蛋白率/前换血升必达。 如果血红蛋白水平低于8.5克/升事先没有放血是必需的。最初的放血不得在谁已经从近期遭受中风（最近3个月内）的患者的情况下超过5毫升/公斤体重。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>初始Hb水平（克/升） </td><td> 10 </td><td> 9.5 </td><td> 9 </td><td> 8.5 </td></tr><tr><td>体积要布莱德（毫升/公斤） </td> &lt;TD&gt; 12 </td><td> 10 </td><td> 8 </td><td>五</td></tr><tr><td>放血的最小时间（分钟） </td><td> 60 </td><td> 60 </td><td> 45 </td><td> 20 </td></tr></tbody></table> 表1.计算的初始放血的音量在手册换血。 初始放血体积是基于患者的初始Hb水平计算，与交换输血之前达到约8克/升的血红蛋白率的目标。初始出血的持续时间取决于放血的体积。如果血红蛋白水平低于8.5克/升事先没有放血是必需的。初始放血不能在谁已经从近期中风遭受患者的情况下超过5毫升/公斤体重。 <ol start="2"><li>计算交易量;总交换容量是35 - 45毫升/公斤体重（ 例如，如果患者是30公斤的子，该沃的换血的lume明将围绕1200毫升）。 注意:此体积是血液，将在交换步骤和将在交换步骤中输向病人稀释PRBCs的体积流血两者的音量。终体积取决于交换输血的体积，与患者体重的计算上。 </li><li>获得的（从药店）5％血清白蛋白溶液适当的音量，以便它准备在会议开始时使用。计算所需的体积:50 - 100 mL到之前发起放血注入，加上初始放血期间相同的体积，以补偿出血，再加上交流体积稀释PRBCs的1/3。 </li><li>获得表型匹配PRBCs的适当体积（ 即，从血库的计算交换体积的2/3）。 </li><li>稀释，用5％血清白蛋白的PRBCs从60％降低PRBCs的血细胞比容40％。 没有TE:稀释必须在血库在一个新的血袋进行。如果这是不可能的，则使用3路抽头同时输血的PRBCs和5％的血清白蛋白，尊重的2/3为PRBCs和1/3的流动比例为白蛋白溶液。 </li></ol> 3.病人准备<ol><li>准备在两个不同的肢体，一个用于放血和一个用于白蛋白溶液和PBRCs输液两个外围静脉线;对于放血静脉线就必须足够的血流量，以及一个用于输液要求的标准血流量。使用输液和静脉切开一个3路自来水单一的静脉通路，如果静脉通路受到严重限制。 </li><li>辖1个3g的操作系统钙前后交流会后，病人的;这可以防止低血钙症的发生由于钙螯合抗凝剂在输液袋的存在。 </li></ol> 4. MET的第一步:等容放血，如果适当<ol><li>开始的5％白蛋白的输注在一个静脉通路。注入大约20后 - 50毫升的白蛋白溶液，开始对第二静脉通路放血。 </li><li>以执行出血，安装在病人的臂连接到一个空的出血袋的周静脉通路。下方放置患者的床的水平的袋子。观察静脉血液逐渐填补出血袋。 <ol><li>如果血流量太低，抬起患者的床（或降低出血袋），以增加所述臂和出血袋之间的高度差，从而增加了血流量。任选地，在非常低的血流量的情况下，有护士手动绘制血液使用放置在静脉管路3路抽头的50mL注射器。 注:放血的流量必须是相同的输液的流量，以便严格保持等容的平衡。 </li></ol></li><li>称取出血袋上precisi上，以适应于实时输液流量，以补偿放血卷的放血期间规模。 注意:如果没有规模可用或如果只有一个单一的静脉通路，流血每次20毫升白蛋白输注20毫升血液。 </li><li>在放血步骤结束时，检查使用根据制造商的说明的血红蛋白点 - - 关心测试的Hb水平，并确保它是约8克/升。 </li><li>监测患者在初始等容放血步骤每隔5分钟。停止放血如果观察到相关的患者的年龄临床变化。 </li></ol> 5. MET的第二步:等容换血<ol><li>出于安全原因，首先启动稀释PRBCs的输血。输血前20毫升血液，然后开始放血。该步骤中的放血的计划总体积是相同的输血的体积（35 - 45毫升/公斤体重）。 注意:放血的速率必须相同的稀释PRBCs的输注速度，按照同样的方法，在上一步骤（ 即，称量在精密标度或备用20毫升周期）。 </li><li>通过交换步骤中途，检查血红蛋白水平，如步骤4.3。如果水平&gt;9.5克/升，执行另一轮放血的。如果没有，继续换血。 </li><li>监控患者在换血一步，每15分钟;护士必须保持对任何临床和/或血流动力学变化的发生密切关注。 </li></ol> 6.其他放血<ol><li>执行额外的出血放血（见步骤4.1），如果Hb水平中途交换步骤都是&gt;9.5克/升，因为有在会议结束时将达到血红蛋白的过高的水平的风险。 注意:附加放血步骤的体积取决于Hb水平（ 表2）。 补偿用5％白蛋白输注，如上所述，为了保持附加放血等容，就像在其他步骤。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>中途岛Hb水平（克/升） </td><td> 10.5 </td><td> 10 </td><td> 9.5 </td></tr><tr><td>体积要布莱德（毫升/公斤） </td><td> 8 </td><td> 6 </td><td> 3 </td></tr><tr><td>放血的最小时间（分钟） </td><td>三十</td><td> 20 </td><td> 15 </td></tr></tbody></table> 表2.计算中级放血的音量在手册换血。 如果通过交换步骤的Hb水平中间高超过95克/升，因为有在会议结束时将达到血红蛋白的一个过高的水平的风险进行额外的抽血。初次出血的持续时间仍取决于放血的体积。 <ol start="2"><li>就像初始放血，监视患者每隔5分钟。停止，如果立即发生临床变化放血。 </li><li>另外放血后，检查使用血红蛋白点护理测试Hb水平，然后用同样的方法继续交换过程。 </li></ol> 7.最后的测试实验室<ol><li>在换血结束后，进行实验室测试血红蛋白，哈佛商学院，和血钙。不允许患者对实验室测试的结果之前离开（或至少是Hb水平）已经由医生进行检查。 注:总的来说，而该过程的持续时间随体积流血和交换上，它持续约4小时，平均。 </li></ol>

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The authors have nothing to disclose.

与此过程相关的风险是放血和输血，这可能产生危险的后果之间的意外misbalance。一种快速枯竭会导致血容量减少和急性贫血，而没有出血过量输血会导致血液粘度增加危险。在这两种情况下，SCA患者可从血管闭塞的并发症，以及中风受损。为此，一名护士必须专用于每一个病人，并在整个过程中留在他的床边。此外，还必须密切医疗监督，医生需要在任何问题的情况下，迅速提供。任何意外的血流动力学改变的必要医务人员的干预和程序的中断，直到病人的病情稳定。为了避免发生任何事故，放血量的计算，白蛋白补偿量，而换血量必须进行非常CAutiously启动会话之前，应该仔细检查，如果有必要。一切都必须准备在病人床边的过程（PRBC和白蛋白）的开始，而静脉通路必须由护士进行定期检查。最后，前血红蛋白水平，中途，并在会议结束时，必须检查和由医生批准。 这种方法的局限性是获得两个静脉访问每个会话的必要性。尽管该技术可以只用一个静脉通路（由交替的短的20毫升出血/输血周期）下进行，优选的方案是有2个大口径血管，一个在每个臂。这种技术的另一局限在于，尽管ferritinemia的初始控制，仍然有在两年铁超负荷的以下使用这种方法的危险。事实上，通过在患者的铁蛋白水平密切关注，我们可以观察到手动EXCHANGES铅，在一定程度上，给铁超负荷。在先前的研究中，我们报道了是51 mo erythrapheresis的平均持续时间之后，铁蛋白水平保持稳定（从586微克/升[491; 709]至609微克/升[221; 1064]）没有铁螯合治疗。然而，39个月的MET平均持续时间之后，铁蛋白水平从327微克/ L [206增加; 535] 802微克/ L [146; 873] 6无铁螯合疗法，这仍然比反复输血低得多。然而，我们必须考虑到手动方法最好应通过erythrapheresis如果输血交流计划持续超过2年更换一次。在等待erythrapheresis的可用性，MET是非常有效的，并迈出了坚实的选择。时相比erythrapheresis，这种技术具有在HBS方面具有相似的效率每个会话降低，尽管略高PRBC消耗（约2.5毫升/千克SES的差锡永）。我们的程序是对所有患者是可行的，不论年龄或体重。为10公斤以下的儿童，主要的限制可能会是静脉接入，这可通过动脉进入，如果需要为紧急换血更换（在急性中风的情况下，例如）。 两种技术之间的最显着的区别在于设备，这是人类的监督和每个会话一次性用品是非常相似的erythrapheresis，成本高得多的成本之内。输血方法和持续时间的选择是直接影响铁超负荷7，其手动方法能够限制所发生的参数。在输血程序防止铁超负荷而不是将它在SCA患者的主要健康目标，经历了长期的输血程序。公布的数据表明，铁超负荷的长期作用可以潜在地严重。这些措施包括肝cirrhoSIS（约1/3病例在4年后的输血成人）8，心肌损害，糖尿病，性腺机能减退，和肺动脉高压9。对于儿童来说，主要的铁超负荷的结果是尚未确定。当螯合疗法是明，只有治疗的患者有一半是对它很敏感，如美国6等10日报道。这种治疗方法的疗效有限，建议治疗效果不佳达标10停止治疗，由于副作用归因于铁螯合疗法。适当的换血方法从而显得至关重要，因为螯合疗法疗效有限。 总之，尽管erythrapheresis似乎是在SCA长期输血治疗最安全和最有效的方法，它的技术和财务功能不是特别有利。连续MET方法是广泛适用，不需要特殊的设备，并且可以是安全和有效地限制铁过载，而慢性单纯性输血较少有效地控制该参数。对于孩子谁必须接受输血计划了好几年，这似乎是一个重要的挑战。连续MET方法为患者等待erythrapheresis一个很好的选择，并且应当优选简单输血。

将β珠蛋白基因的单点突变是负责生产异常血红蛋白（血红蛋白S，HBS）。这会导致镰状细胞性贫血（SCA），全球1的最常见的疾病之一。 SCA患者的急性症状和一些慢性并发症可以通过堆积的红血细胞（PRBCs）的输血治疗。事实上，正常红细胞的输血纠正贫血，同时稀释镰刀红细胞。其结果是，它可以增加氧的运输能力，同时减少溶血和血管闭塞事件。为了避免慢性并发症或用于治疗急性并发症，输血与镰状红细胞的耗竭相结合，或者通过放血或erythrapheresis，是限制在血红蛋白和血粘度危险增加的有效方法，同时减少循环的SS红细胞的数量2。 一个精神的主要原因障碍和神经认知缺陷与SCA 3个孩子是脑血管病变，这种疾病严重的并发症。在SCA儿童与经颅多普勒异常高的速度，长期输血能有效地防止第一冲程4的发生。为了减少复发的那些已经从缺血性卒中患者遭受的风险，输血治疗是最适当的方法5。在慢性治疗的情况下，红细胞的交换输血是比简单的RBC输注更好，因为它消除镰刀细胞，并增加了正常细胞的同时降低血液粘度和限制性铁超负荷。然而，简单的红细胞输注仍然被广泛用作脑宏观血管病治疗。虽然它迅速导致铁超负荷4，这种选择经常发生，因为它在技术上是简单，最大限度地提高输血CA的患者人数回覆。事实上，即使erythrapheresis已报道为SCA患者的慢性输血的最有效的方法，它不能被到处执行;它不适合所有患者，尤其是年幼的孩子;和它势必需要特定和昂贵的设备。 在超过20年了，我们一直在治疗儿童SCA展示脑血管病和谁是与连续手动换血（MET）方法erythrapheresis暂时不合格。在2016年，我们的团队公布了患者随访已经经历连续手动输了好几年，表明我们的方法与令人满意的哈佛商学院下降，有效预防中风和铁过载相媲美的erythrapheresis 6的限制相关。 MET的会话可在没有特定装置任何医院环境，并通过使用所需要的erythrapheresis相同体积下进行。一个这种技术的显着优点是，它可以帮助防止或至少减少，副作用（特别是铁过载）连接到重复输血的患者谁不能够经受erythrapheresis。这篇文章的目的是一步，如何才能让没有任何单采机，或有病人谁没有资格erythrapheresis，使用该医疗中心进行连续MET的会话来形容，一步方法为他们的SCD患者，特别是儿童。

<table><tbody><tr><td>Precision scale</td><td></td><td></td><td></td></tr><tr><td>Cannula (x2)</td><td>Macopharma</td><td></td><td></td></tr><tr><td>Transfusion tubing (x2)</td><td>Macopharma</td><td></td><td></td></tr><tr><td>Bleeding bag (x4)</td><td>Macopharma</td><td></td><td></td></tr><tr><td>3 Way tap</td><td></td><td></td><td></td></tr><tr><td>Syringe (x4)</td><td></td><td></td><td></td></tr><tr><td>Hemoglobin test</td><td>HemoCue</td><td>Hb 201&lt;sup&gt;+&lt;/sup&gt;&amp;nbsp;System</td><td></td></tr></tbody></table>

continuous manual exchange transfusion for patients with sickle cell disease: an efficient method to avoid iron overload

镰状细胞性贫血（SCA）的儿童可能在脑血管病和中风，这可以通过慢性输注方案可以防止的危险。堆积的红血细胞（PRBCs）反复输血是目前用于治疗慢性输血方案的最简单和最常用的技术。然而，铁超负荷是这种治疗的主要副作用之一。较发达的方式存在，RBC（erythrapheresis），这是目前最安全和最有效的方法，尤其是单采。然而，这是昂贵的，复杂的，并且不能到处执行，它也不是适合于所有的患者。手册换血结合一个或多个手动静脉切开一个PRBC输血。 在镰刀的参考中心细胞病，我们设置了手动换血的连续方法，它是所有医院的设置是可行的，要求没有具体的设备，并广泛适用。在HBS减少方面，中风上一页ention和铁过载防止，这种方法显示出可比较的效率erythrapheresis。在erythrapheresis不可用的情况下，这个方法可以是为患者和护理中心一个很好的选择。

在这里，我们将比较安全，成本和MET的方法与erythrapheresis 6，这是降低的HbS的SCD的患者百分比的最有效的方法的效率。要做到这一点，我们记录在SCD的基准中心1,353输血交流会，包括AET 333会议并会见了1,020会议，都在SCD儿童脑血管病和/或中风的痛苦。对于患者，我们选择了下25千克MET患儿体重和/或那些缺乏高血流静脉通路，这是需要erythrapheresis（外周静脉的足够的大小，动静脉瘘，或留置的中心静脉导管） 。相反地​​，AET了系统提供给儿童提供一个体重25公斤，只要适当的静脉通路是可利用的。 关于交换的效率，我们有观察到的18.8％[15.2哈佛商学院中位数下降; 23.0]一个MET会话与 21.5％[17.8后; 25.1]一个erythrapheresis会议结束后，有一个稍高的消费PRBC用手工方法（31.7毫升/公斤[28.0; 35.2]每MET会话与 29.2毫升/公斤[26.7; 32.7]每erythrapheresis会话）。因为哈佛商学院降低是SCA患者输血会议的主要目标，这些结果显示良好的效率，并允许5周的输血会话之间的平均间隔，既为MET和erythrapheresis。铁过载，慢性输血的主要并发症，是直接关系到输液方法和持续时间。 Lee 等人。在STOP研究已经描述了SCA患者的队列反复接受有输血治疗脑血管病变。在这项研究中，铁超负荷是实质性的，在12个月的慢性输血后1,804微克/升的平均铁蛋白水平24个月后4达到2,509微克/升</sup&gt;。 Erythrapheresis被认为是一种有效的方法来限制铁超负荷的经历换血7 SCA的患者。正如预期的那样，在队列，谁收到孩子只有erythrapheresis其实是有很稳定的ferritinemia（ 图1）。如果我们考虑谁只收到会见了连续法的病人，我们观察到ferritinemia的相当类似的稳定性，见证铁超负荷几乎等效控制与erythrapheresis（ 图1）。 关于这两种方法的副作用，在这项研究中，我们只报告（erythrapheresis期间3 MET期间和3）描述为迷走神经昏厥出1,353会话六个事件。任何会议期间没有报告严重的血流动力学的事件，从而使我们考虑这两种方法是安全的患者。 最后，为了<html>与MET会话比较的erythrapheresis会议的费用，我们会考虑一个30公斤重的孩子谁，在会话的开始，为9克/升的Hb水平。考虑到一次性物品（包括PRBCs和解决方案的成本），该患者的MET会议的费用约为601欧元，在护士的存在4小时的顶部。在另一方面，erythrapheresis只需要半个护士（ 即一名护士为2例），但它势必需要更密切的医疗监督为3小时的时间，将花费大约628欧元。总而言之，这两种方法是在一次性产品人员开支方面相当。最后，设备的成本是每年8886欧元为erythrapheresis 对每120年前欧元MET（ 即，为erythrapheresis更高的74倍，是根据假定的单采血液成分机等设备（精度尺度和输注注射器的寿命）是10年）。 "FO:保together.within页="1"&gt; <img alt="图1" src="/files/ftp_upload/55172/55172fig1.jpg" /> 图1.铁超负荷的比较期间会见和Erythrapheresis程序。从经历长期输血的患者SCA结果是从亚当斯等人拍摄。从经历MET和erythrapheresis SCA患者的结果与先前发表的作品拍摄（Koehl 等 ，2016）。 Ferritinemias表示为平均值±标准差。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55172/55172fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload at JoVE.com

Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload

我们已经概述了在患者的治疗镰状细胞病的连续手动换血的方法。这个安全协议的目的是要有效地限制在患者需要长期输血的铁过载，并且可以没有任何特殊的设备得到广泛使用。

与镰状细胞疾病患者连续手动换血:的有效方法避免铁超负荷

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. ...

continuous-manual-exchange-transfusion-for-patients-with-sickle-cell

Universidad San Sebastian

Research

JoVE Journal

Medicine

19.2K 次观看.  Robert Debré Hospital, AP-HP. 我们已经概述了在患者的治疗镰状细胞病的连续手动换血的方法。这个安全协议的目的是要有效地限制在患者需要长期输血的铁过载，并且可以没有任何特殊的设备得到广泛使用。 

视频: Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

Hemoglobinopathies 患者造血干细胞移植后残余供红祖细胞的检测

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

科研

发育生物学

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

免疫与感染

<meta charset="utf8"></head><body>用于检测新型铁螯合剂的荧光检测

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

<meta charset="utf8"></head><body>作者聚焦:评估新型铁螯合剂对癌细胞代谢的影响

We describe a fluorescent assay that can quickly and inexpensively confirm the ability of novel compounds to chelate iron. The assay measures the ability of compounds to outcompete the iron binding activity of the weak iron chelating fluorescent probe Calcein, resulting in a quantifiable increase in fluorescence when chelation occurs.

可量化且廉价的无细胞荧光方法，用于确认新型化合物螯合铁的能力 

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target ...

癌症研究

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

用于小型细胞处理的自动逆流离心系统

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...

生物工程

A Precision Medicine Tool for Measurement and Monitoring of Hemoglobin S in Sickle Cell Disease Patients Receiving Transfusion Therapy

Sickle cell disease (SCD) causes many severe health complications, including anemia, stroke, and acute chest syndrome. Red blood cell transfusion is the most commonly used therapy to treat or prevent these devastating complications. Other therapies include hydroxyurea therapy as well as bone marrow transplantation. Chronic intermittent transfusion is especially indicated to prevent recurrent strokes. However, transfusion therapy is associated with significant adverse effects (e.g., alloimmunization and iron overload). The point-of-care (POC) lateral flow immunoassay used here quantifies the %HbS in 15 min using a small patient blood sample. Utilizing this information, the healthcare provider is able to adjust blood transfusion volume for SCD patients to achieve the desired target HbS (most often &#60;30%), while reducing the risk of transfusion-related complications. When compared to laboratory hemoglobin electrophoresis data for 38 whole blood samples, the POC test performed with very high correlation and linear fit (slope = 0.9949, R2 = 0.9751). The strong agreement between the two hemoglobin S percentage (%HbS) quantifying methods shows that 89.5% of samples fall within &#177;5.2% HbS bias. The calibration for quantifying %HbS is built into the device to allow for an automated quantification of %HbS. This time- and cost-effective POC test thus allows the healthcare provider to make timely informed decisions when treating SCD patients, using accurate and updated data.

We have developed a point-of-care immunoassay to rapidly quantify hemoglobin S (HbS) levels during transfusion therapy for sickle cell disease (SCD) patients. By applying a small amount of treated blood to the device, the healthcare provider can determine the %HbS in a SCD patient to immediately inform clinical decisions.

Sickle cell disease (SCD) causes many severe health complications, including anemia, stroke, and acute chest syndrome. Red blood cell transfusion is the ...

医学

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient&#8217;s pO2 at which sickling starts and the minimal deformability of a patient&#8217;s red blood cells, respectively. PoS is associated with an individual patient&#8217;s hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

Here, we present oxygen gradient ektacytometry, a rapid and reproducible method to measure red blood cell deformability in samples from patients with sickle cell disease under controlled deoxygenation and reoxygenation. This technique provides a way to study red blood cell sickling and to monitor sickle cell disease treatment efficacy.

受控自动脱氧过程中与氧梯度压质测定的病带特征

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When ...

Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The use of experimental animal models of iron overload or deficiency has been instrumental to advance knowledge of the mechanisms involved in the systemic and cellular regulation of iron homeostasis. The measurement of total iron levels in animal tissues is commonly performed with atomic absorption spectroscopy or with a colorimetric assay based on the reaction of non-heme iron with a bathophenanthroline reagent. For many years, the colorimetric assay has been used for the measurement of the non-heme iron content in a wide range of animal tissues. Unlike atomic absorption spectroscopy, it excludes the contribution of heme iron derived from hemoglobin contained in red blood cells. Moreover, it does not require sophisticated analytical skills or highly expensive equipment, and can thus be easily implemented in most laboratories. Finally, the colorimetric assay can be either cuvette-based or adapted to a microplate format, allowing higher sample throughput. The present work provides a well-established protocol that is suited for the detection of alterations in tissue iron levels in a variety of experimental animal models of iron overload or iron deficiency.

Here, a protocol for the measurement of the non-heme iron content in animal tissues is provided, using a simple, well-established colorimetric assay that can be easily implemented in most laboratories.

使用基于菲咯啉的比色法测量组织非血红素铁含量

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.

与镰状细胞疾病患者连续手动换血:的有效方法避免铁超负荷

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A Precision Medicine Tool for Measurement and Monitoring of Hemoglobin S in Sickle Cell Disease Patients Receiving Transfusion Therapy

受控自动脱氧过程中与氧梯度压质测定的病带特征

使用基于菲咯啉的比色法测量组织非血红素铁含量

造血干细胞和祖细胞的CRISPR/Cas9基因编辑用于基因治疗应用