Name: rapid point-of-care assay of enoxaparin anticoagulant efficacy in whole blood
Uploaded: 2012-10-12T12:00:00
Duration: 11 min 17 s
Description: Watch this Scientific Journal Video about Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood at JoVE.com

仅与支持部门和机构资源进行调查。该的凝血装置（HEMOCHRON 801）被租借制造商不承担责任。在他们的零售价格购买该检测盒。在视频的介绍图修改Sanderink 等12，第二个数字的视频（乙酰肝素酶消化时间过程）与许可来自Inchiosa 等再现。1

1。肝素重建<ol><li>冻干肝素酶I的购得（西门子，纽瓦克特拉华，DadeHepzyme楼盘B4240-10）在小瓶中，含有1微克的纯化蛋白。的蛋白质溶解在0.25毫升的生理盐水（0.9％NaCl）中。 </li></ol> 2。采集血样<ol><li>两个血液样本成柠檬酸钠（0.109M;0.3毫升为3.2％），通过真空绘制缓冲Vacutainer管（流式细胞-Dickinson公司，新泽西州富兰克林湖）;管都是预尖刺与已知浓度的依诺肝素;血容量所绘制为2.7毫升。的总含量，然后3.0毫升。 </li></ol> 3。肝素酶消化<ol><li>的肝素酶，可以被吸入到一个注射器（约0.2​​毫升，80％的小瓶; 0.8μg蛋白）的小瓶，其全部内容被转移到1的Vacutainer管; 0.2毫升的生理盐水（0.9％NaCl）中被添加到第二管，其中的巴塞尔作为国家统计局前的状态，依诺肝素的效果消除。 </li><li>为5分钟的期间，两个Vacutainer管被放置在恒温井的凝血时间装置。 （5分钟的消化时间是否足够与肝素成立于我们此前发表的作品。1） </li></ol> 4。凝血分析<ol><li>的肝素酶孵育结束时，2.0毫升的血液转移到测定盒。在的MAA的管的情况下，它是预先充填了0.1毫升的250mM CaCl 2的启动凝血。商业APTT的测定盒（A104）被设计来启动已卷入Vacutainer管含有枸橼酸钠抗凝全血中的凝血。 </li><li> HEMOCHRON装置血液凝固的完成信号，并保留在几秒钟内的凝血时间。凝血时间的差异之间的肝素酶处理的样品和基线样品，除以凝血钛我为基准样品，得到消除的依诺肝素的百分比减少。正如下面所讨论的，强的线性关系已由chromgenic测定抗Xa％的减少，凝血时间和浓度的依诺肝素样品中添加的全血样本，以及依诺肝素与测量浓度的相关性之间的关系获得活动。抗Xa的依诺肝素浓度检测进行了商业化试剂盒为一个两阶段的显色分析（BIOPHEN; Aniara，梅森，OH）。 </li></ol>

<ol>
	<li>Inchiosa, M. A. J. r., Pothula, S., Kubal, K., Sanchala, V. T., Navarro, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+development+of+a+point-of-care+assay+of+enoxaparin+anticoagulant+activity+in+whole+blood.">Toward development of a point-of-care assay of enoxaparin anticoagulant activity in whole blood.</a> J. Thromb. Thrombolysis. 32, 47-53 (2011).</li><li>Despotis, G. J., Filos, K. S., Levine, V., Alsoufiev, A., Spitznagel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aprotinin+prolongs+activated+and+nonactivated+whole+blood+clotting+time+and+potentiates+the+effect+of+heparin+in+vitro.">Aprotinin prolongs activated and nonactivated whole blood clotting time and potentiates the effect of heparin in vitro.</a> Anesth. Analg. 82, 1126-1131 (1996).</li><li>Pothula, S., Inchiosa, M. A., Sanchala, V. T., Sarabu, M., Inchiosa, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+effect+of+protamine+excess+on+blood+coagulation+assays.">In vitro effect of protamine excess on blood coagulation assays.</a> Anesth Analg. 96, SCA39 (2010).</li><li>Inchiosa, M., Pothula, S., Sanchala, V., Kubal, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+low+plasma+levels+of+low+molecular+weight+heparin.">Detection of low plasma levels of low molecular weight heparin.</a> Anesth Analg. 102, SCA10 (2010).</li><li>Gogarten, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+new+antithrombotic+drugs+on+regional+anesthesia.">The influence of new antithrombotic drugs on regional anesthesia.</a> Curr. Opin. Anaesthesiol. 19, 545-550 (2006).</li><li>Gouin-Thibault, I., Pautas, E., Siguret, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+profile+of+different+low-molecular+weight+heparins+used+at+therapeutic+dose.">Safety profile of different low-molecular weight heparins used at therapeutic dose.</a> Drug Saf. 28, 333-349 (2005).</li><li>Bazinet, A., Almanric, K., Brunet, C., Turcotte, I., Martineau, J., Caron, S., Blais, N., Lalonde, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dosage+of+enoxaparin+among+obese+and+renal+impairment+patients.">Dosage of enoxaparin among obese and renal impairment patients.</a> Thromb. Res. 116, 41-50 (2005).</li><li>El Rouby, S., Cohen, M., Gonzales, A., Hoppensteadt, D., Lee, T., Zucker, M. L., Khalid, K., LaDuca, F. M., Fareed, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+a+HEMOCHRON+JR.+HEMONOXTM+point+of+care+test+in+monitoring+the+anticoagulant+effect+of+enoxaparin+during+interventional+coronary+procedures.">The use of a HEMOCHRON JR. HEMONOXTM point of care test in monitoring the anticoagulant effect of enoxaparin during interventional coronary procedures.</a> J. Thromb. Thrombolysis. 21, 137-145 (2006).</li><li>Yang, V. C., Bernstein, H., Cooney, C. L., Kadam, J. C., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+the+anticoagulant+activities+of+the+low+molecular+weight+heparin+fractions+and+fragments+with+flavobacterial+heparinase.">Removal of the anticoagulant activities of the low molecular weight heparin fractions and fragments with flavobacterial heparinase.</a> Thromb Res. 44, 599-610 (1986).</li><li>Maddineni, J., Walenga, J. M., Jeske, W. P., Hoppensteadt, D. A., Fareed, J., Wahi, R., Bick, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Product+individuality+of+commercially+available+low-molecular-weight+heparins+and+their+generic+versions:+therapeutic+implications.">Product individuality of commercially available low-molecular-weight heparins and their generic versions: therapeutic implications.</a> Clinical and Applied Thrombosis/Hemostasis. 12, 267-276 (2006).</li><li>Wijnhoven, T. J., van de Westerlo, E. M., Smits, N. C., Lensen, J. F., Rops, A. L., vander Vlag, J., Berden, J. H., vanden Heuvel, L. P., van Kuppevelt, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+anticoagulant+heparinoids+by+immunoprofiling.">Characterization of anticoagulant heparinoids by immunoprofiling.</a> Glycoconj. J. 25, 177-185 (2008).</li><li>Sanderink, G. -. J. C. M., Guimart, C. G., Ozoux, M. -. L., Jariwala, N. U., Shukla, U. A., Boutouyrie, B. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+and+pharmacodynamics+of+the+prophylactic+dose+of+enoxaparin+once+daily+over+4+days+in+patients+with+renal+impairment.">Pharmacokinetics and pharmacodynamics of the prophylactic dose of enoxaparin once daily over 4 days in patients with renal impairment.</a> Thromb. Res. 105, 225-231 (2002).</li></ol>

作者有没有透露。

此法的依诺肝素的疗效，可作为一个重要的选择，以确定相对安全的进行侵入性手术，有一个随之而来的风险，出血或血肿的发展。正在考虑硬膜外注射时，这是特别重要的。大多数的指引，建议等待一段时间注射依诺肝素和一种侵入性手术开始12小时之间。5肾功能受损，肥胖和妊娠可能影响计量的要求，依诺肝素。6,7由于内就可以完成检测10 -分钟的时间内，它也可以被用来在诊所或医生的办公室，在那里将提供的信息是否有足够的抗凝治疗的患者，是在发展的静脉血栓栓塞的风险。即时调整剂量，根据需要，可以制定。 可以测量由一个生色一个，依诺肝素血浆中的浓度SSAY的凝血因子Xa的抑制程度。然而，这仅仅得到它的浓度，特别是其抗凝血剂的功效，个别患者有很大的不同。6-8 我们的研究只进行了使用依诺肝素。然而，可以预计，该方法的，可以适用于任何的低分子肝素（LMWH），是由肝素酶I。降解这已经是已知的情况下，达肝素，低分子量肝素和亭扎肝素9-11我们设计的检测有关的功能凝血时间凝血时间的仪器。该仪器是价格相对低廉，一次性使用血液收集管，肝素安瓿，试验盒，也完成分析一个病人约15亿美元，。因此，可以预计的是适合在不同的临床设置监测抗凝状态。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Hepzyme </td><td>西门子</td><td> B4240 </td><td>肝素我</td></tr><tr><td> “MAA”墨盒</td><td> Technidyne </td><td> FTK-ACT </td><td>高岭土去除修改</td></tr><tr><td> APTT墨盒</td><td> Technidyne </td><td> A104 </td><td></td></tr></tbody></table>

rapid point-of-care assay of enoxaparin anticoagulant efficacy in whole blood

是有需要的临床试验，以确定病人的血液在何种程度上有效的低分子量肝素（LMWH），依诺肝素抗凝。临床上也迫切的情况下，这将是重要的，如果能迅速确定。本试验的目的是实现这一目标。我们只检测人体血液样本，与已知浓度的依诺肝素飙升。本测定法的基本特征是通过降解它来完成与肝素的不活动的病人的血液试样中的依诺肝素的疗效的量化。两个血液样本被卷入Vacutainer管（流式细胞迪肯森新泽西州富兰克林湖），依诺肝素飙升;一个样品与肝素酶消化在37℃下5分钟，其他的样品代表患者的基线抗凝固状态。 ％缩短凝血时间在肝素酶处理的样品相比，到baseline状态，取得了依诺肝素的抗凝血剂的贡献。我们使用的便携式，电池供电的HEMOCHRON 801装置的凝血时间的测量（邮费Technidyne公司，新泽西州Edison）。该仪器具有恒温控制（37°C）测定油井管。在两种类型的检测仪器的制造商提供的墨盒，我们进行了分析。一个墨盒被修改，以增加其灵敏度。我们删除了高岭土FTK-ACT卡匣广泛用蒸馏水冲洗，离开仅在玻璃表面的管子，和可能的检测磁铁，作为活化剂。我们叫这是我们的最低限度的激活试验（MAA）。我们和其他人已研究使用最小激活的测定2-4进行了研究的第二盒是活化部分凝血活酶时间（APTT）测定法（A104）。这被用于从制造商提供。恒温井仪器WERE采用肝素酶消化和凝固分析。该法可在10分钟内完成。 ，MAA检测结果显示强劲的依诺肝素酶消化后，在一个典型的临床浓度范围内变化凝血时间。 0.2抗Xa IU每毫升的血液样本，依诺肝素，肝素酶消化引起的凝血时间平均下降了9.8％（20.4秒）;在1.0 IU每毫升的依诺肝素下降41.4％（148.8秒）。本报告只介绍了实验的分析，还必须建立它的价值，在临床上的应用。

图1给出的结果之间的关系，减少百分之凝血时间，消除了一系列的添加浓度的依诺肝素（0.2，0.4和1.0抗Xa国际单位每毫升）与肝素依诺肝素。结果的MAA和aPTT检测墨盒。依诺肝素从抗-Xa活性的发色底物法测得的浓度，还包括在每个图的横坐标的。 图2给出图相同的数据作为减少百分之生色衡量抗-Xa活性的凝血时间vs。 强的线性相关性，具有较高的统计学意义，发现为所有四个关系。 <img src="/files/ftp_upload/3852/3852fig1.jpg" alt="图1" /> 点的护理决定的依诺肝素对照图1。ibution的培养与肝素后凝血时间的百分比变化（缩短）的基础上加标全血凝血时间。的加标依诺肝素浓度添加到全血中，并测得的抗Xa因子的血浆浓度（平均值±SE，在括号中）放置在各曲线图中的横坐标。凝血时间的所有变化百分比中提出作为装置和SE，连同与平均缩短凝血时间，以秒为单位。来自相同的病人的数据是由虚线连接。用星号在剧情患者的数据一个（MAA结果）的患者在图B，活化部分凝血活酶时间（APTT检测）是相同的。所有的结果代表单一的测定。的Pearson相关系数为R = 0.869（p值= 1.9×10 -5）和r = 0.916（p值= 2.8×10 -5）的与MAA和aPTTassay的分别。转载的许可来自Inchiosa 等。1 图2的查询结果在护理点示于图1A和B的测定凝血时间的变化百分比对血浆的抗因子Xa的浓度作图。，MAA结果，B，APTT的测定。所有的结果都是从单一的凝血时间测定和单一测定抗 - 因子Xa浓度。的Pearson相关系数分别为r = 0.910（p值= 2.5×10 -6）和r = 0.922（p值= 2.0×10 -5），转载许可距离Inchiosa 等分别与MAA和APTT测定的关系1

Watch this Scientific Journal Video about Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood at JoVE.com

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

示范低分子重量肝素，依诺肝素抑制血液凝固的快速定量测定。消除它的影响力，通过消化与肝素依诺肝素的贡献进行评估。在我们发表的论文中详细介绍较详细地说明检测。<sup&gt; 1</sup&gt;该法还需要临床确认。

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