流循环模型中血液接触植入物的血液相容性测试模拟人类血流
Summary
该协议描述了使用激光切割神经血管植入物对血液接触装置的全面血液相容性评估。采用新鲜肝化人血的流循环模型模拟血流。灌注后，对各种造血标记进行分析，并将与采血后直接获得的值进行比较，对被测装置进行血液相容性评价。
Abstract
医疗设备（例如血管移植物、支架和心脏导管）越来越多地用于暂时或永久目的，这些装置仍留在人体的循环系统中，需要一种可靠和多参数化的方法，评估这些设备（即血液成分的激活和销毁）可能造成的造血并发症。血液接触植入物的体外血相容性综合测试是成功实施体内的第一步。因此，在临床应用之前，必须根据国际标准化组织 10993-4 （ISO 10993-4） 进行广泛的分析。所述的流回路描述了一个敏感模型，用于分析支架的止血性能（在本例中为神经血管），并揭示不利影响。使用新鲜的人类全血和温和的血液取样对于避免血液的预活化至关重要。血液通过含测试标本的肝化管中渗透，在37°C下以150 mL/min的速度使用蠕动泵60分钟。灌注前后， 血液学标记（即血细胞计数、血红蛋白、血细胞和血浆标记），指示白细胞（多态核[PMN]-弹性酶）、血小板（β-血栓球蛋白[-TG]）、凝血系统（通宾-抗血栓III[TAT]）和补数级联（SC5b-9）的。最后，在临床应用之前，我们提出了支架和其他血液接触装置广泛血液相容测试的基本可靠模型。
Introduction
植入物和生物材料与人的血液相互作用，在体内应用，需要严格的临床前测试，重点是调查止血系统的各种标记。国际标准化组织 10993-4 （ISO 10993-4） 规定了血液接触装置（即支架和血管移植物）评估的核心原则，并考虑了设备设计、临床效用和所需材料1。
人血是一种含有各种血浆蛋白和细胞的液体，包括白细胞（白细胞[WBCs]）、红细胞（红血球[RBC]）和血小板，在人体中执行复杂的功能2。异物与血液的直接接触可引起不良影响，如免疫或凝固系统的激活，可导致炎症或血栓并发症和植入后严重问题3、4、5。因此，体外血合一验证提供了在植入前发现和排除血液接触时可能引起的任何血液并发症的机会。
提出的流回路模型是为评估神经血管支架和类似装置的血管相容性而建立的，在管材中应用150 mL/min的流速（直径为3.2毫米），以模拟大脑流动条件和动脉直径2、7。除了需要一个最佳的体外模型外，血液来源是分析生物材料8的血液相容性时获得可靠和不变结果的重要因素。采集的血液应在取样后立即使用，以防止长时间储存引起的变化。一般来说，应使用21 G针进行无停滞的温和采集血液，以尽量减少血小板的预活化和血脉叠。此外，捐赠者排除标准包括吸烟、怀孕、健康状况不佳或在过去14天内服用口服避孕药或止痛药的人。
本研究描述了在流动条件下对支架植入物进行广泛溶容性测试的体外模型。当比较未涂覆的纤维布林-肝素涂层支架时，全面的溶性测试结果反映了纤维蛋白-肝素涂层支架9的溶性增强。相反，未涂层支架诱导凝固级联的激活，如血小板附着到支架表面引起的汤宾-抗血栓三 （TAT） 浓度增加和血小板数损失。总体而言，建议将此血效相容性模型集成到临床前测试中，以检测设备对止血系统造成的任何不利影响。
Protocol
血液取样程序经图宾根大学医学院道德委员会批准（项目识别代码：270/2010BO1）。所有科目均提供书面、知情同意，供在参与前加入。 1. 准备肝素装载的单体 将未稀释的肝素 （5，000 IU/mL） 与氯化钠（NaCl，0.9%） 混合溶液，并准备一个溶液，结果浓度为15 IU/mL的肝素。 在每个中性单体（9 mL）中加入900μL的稀释肝素溶液，在血液取样后获得最终肝素浓度为1.5 IU/m…
Representative Results
简要总结，人类整个血液被收集在肝素加载的单体，然后汇集，并用于评估细胞计数的基线水平以及血浆血相容性标记。 随后，装有神经血管植入样本的管子被填充，血液在150mL/min和37°C使用蠕动泵被灌注60分钟。再次，对所有组细胞数进行分析，并为ELISA分析制备血浆样本（图1）。血细胞和血液参数的定量，如血红蛋白和血细胞，直接在采血后，以及?…
Discussion
该协议描述了一种全面可靠的方法，用于在模拟人类血流的剪切流模型中，根据ISO 10993-4对血液接触植入物进行血液相容性测试。这项研究基于激光切割神经血管植入物的测试，但可以通过各种样本进行。结果表明，该方法能够广泛分析各种参数，如血细胞计数、几种血合性标记的患病率以及血液接触后设备表面的微观可视化。使用此协议，可以检测到不同设备与系统相容性的潜在差异。
<p cla…Disclosures
The authors have nothing to disclose.
Acknowledgements
对于扫描电子显微镜的性能，我们感谢来自图宾根大学医院医学材料科学和技术部分的恩斯特·施魏泽。这项研究得到了国家可持续发展方案二（BIOCEV-FAR LQ1604项目）和捷克科学基金会第18-01163S项目（BIOCEV-FAR LQ1604）中CR的青年和体育部的支持。
Materials
| aqua ad iniectabilia | Fresenius-Kabi, Bad-Homburg, Germany | 1088813 | |
| beta-TG ELISA | Diagnostica Stago, Duesseldorf, Germany | 00950 | |
| Centrifuge Rotana 460 R | Andreas Hettich, Tuttlingen, Germany | – | |
| Citrat monovettes (1.4 mL) | Sarstedt, Nümbrecht, Germany | 6,16,68,001 | |
| CTAD monovettes (2.7 mL) | BD Biosciences, Heidelberg, Germany | 367562 | |
| EDTA monovettes (1.2 mL) | Sarstedt, Nümbrecht, Germany | 6,16,62,001 | |
| Ethanol p.A. (1000 mL) | AppliChem, Darmstadt, Germany | 1,31,08,61,611 | |
| Glutaraldehyde (25 % in water) | SERVA Electrophoresis, Heidelberg, Germany | 23114.01 | |
| Heparin coating for tubes | Ension, Pittsburgh, USA | – | |
| Heparin-Natrium (25.000 IE/ 5 mL) | LEO Pharma, Neu-Isenburg, Germany | PZN 15261203 | |
| Multiplate Reader Mithras LB 940 | Berthold, Bad Wildbad, Germany | – | |
| NaCl 0,9% | Fresenius-Kabi, Bad-Homburg, Germany | 1312813 | |
| Neutral monovettes (9 mL) | Sarstedt, Nümbrecht, Germany | 2,10,63,001 | |
| PBS buffer (w/o Ca2+/Mg2+) | Thermo Fisher Scientific, Darmstadt, Germany | 70011044 | |
| Peristaltic pump ISM444B | Cole Parmer, Wertheim, Germany | 3475 | |
| Pipette (100 µL) | Eppendorf, Wesseling-Berzdorf, Germany | 3124000075 | |
| Pipette (1000 µL) | Eppendorf, Wesseling-Berzdorf, Germany | 3123000063 | |
| Plastic container (100 mL) | Sarstedt, Nümbrecht, Germany | 7,55,62,300 | |
| PMN-Elastase ELISA | Demeditec Diagnostics, Kiel Germany | DEH3311 | |
| Polyvinyl chloride tube | Saint-Gobain Performance Plastics Inc., Courbevoie France | – | |
| Reaction Tubes (1.5 mL) | Eppendorf, Wesseling-Berzdorf, Germany | 30123328 | |
| neurovascular laser-cut implants | Acandis GmbH, Pforzheim | 01-0011x | |
| SC5b-9 ELISA | TECOmedical, Buende, Germany | A029 | |
| Scanning electron microscope | Cambridge Instruments, Cambridge, UK | – | |
| Sealing tape (96 well plate) | Thermo Fisher Scientific, Darmstadt, Germany | 15036 | |
| Syringe 10/12 mL Norm-Ject | Henke-Sass-Wolf, Tuttlingen, Germany | 10080010 | |
| TAT micro kit | Siemens Healthcare, Marburg, Germany | OWMG15 | |
| Waterbath Type 1083 | Gesellschaft für Labortechnik, Burgwedel, Germany | – |
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Cite This Article
Link, A., Cattaneo, G., Brynda, E., Riedel, T., Kucerova, J., Schlensak, C., Wendel, H. P., Krajewski, S., Michel, T. Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow. J. Vis. Exp. (157), e60610, doi:10.3791/60610 (2020).