微流体流钱伯斯再造使用血止血建模和血小板输注<em>体外</em
Summary
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.
Abstract
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.
Introduction
止血需要的细胞，蛋白质，离子和组织在有限的时空背景下1组合和受规管活动。不受控制的活性可能导致出血或血栓形成和发病率或死亡率有关凝血障碍的光谱。一种微流体流室的实验是一个具有挑战性的技术， 在体外模拟止血。这种方法允许的参与止血与血小板的主导作用过程中的复杂的相互作用的研究。
血管损伤后，血小板粘附到暴露的内皮下基质（糖）的蛋白质，以防止失血。以下粘附，血小板激活和聚集响应于自动和旁分泌信号并最终导致形成血小板网络的，由纤维蛋白稳定化，并导致一公司，伤口密封血栓2。与大多数其他的血小板功能检测，experi与流动室ments顾及血流的物理参数，因此，流变学对参与细胞和生物分子的3,4的影响。
流动腔实验通过改变影响止血（分）关键参数过程，包括粘合剂基质，流变和流动剖面，蜂窝组合物，毒素或药物，离子强度等等的存在产生在止血和血栓形成里程碑式的见解。在过去的二十年中，低通量需要大样本量（10-100毫升）流动腔实验已经发展到微流控室往往由小平行板室，并包括现代技术控制在墙上剪切条件下5灌注全血。 Microscaling已显著增加检测的吞吐量主要是因为硬件设置简化并需要更少的（血）量，使实验更加方便和versati勒。例如，来自小实验动物的血液，现在可以无需牺牲动物使用。因此，转基因小鼠的血样已经在帮助促进或抑制止血的关键分子的鉴定，并在新的基本见解6。
专门研究实验室还经常使用，例如定制流室从聚二甲基硅氧烷（PDMS）7上，可以通过软件blueprinted石印模具聚合。所得室是便宜，一次性和可容易地拆卸为事后分析。此外，基本上船只，包括分叉或急转弯的任何设计可以建立在命令。这种优势也是其缺点，因为标准化已经与流室实验的首要问题，而PDMS定制室都没有帮助这一点。在这个特殊问题的顶部，涂层（条件），荧光探针，抗凝，临时erature和时间采样和分析之间都很差标准化8。这些变量标准化是具有挑战性的，但仍然需要允许实验室间的结果进行比较。本主题是血栓与止血国际社会在对生物流变学9,10科学和标准化分委员会的主要议题。
血小板浓缩物（PC）进行输血在从引起血小板减少和/或出血的各种疾病的患者。但血小板在个人计算机是众所周知的脱敏，特别是在放置时间11功能，劣化进程链接到老化和通常被称为血小板储存损伤。据说有时，这种血小板输血一次发行12恢复，但对于这方面的证据是稀缺的。此外，血小板构成的PC的功能不是常规测试，因为这样的测定之间的关系的治疗或预防效力是13不清楚。微流体流动腔室提供给调查在个人电脑的血小板功能，以优化收集和发行之间操作的链的装置。它是PC，因为我们先前公布的14,15和这里描述的直接（配对）比较强大的研究工具。
Protocol
Representative Results
Discussion
微流体流室的实验是研究在流动的血液中血小板的功能一个很好的工具，用于在不同的实验环境，以评估在体外止血。尽管可怜的实验室间标准化9，我们证明了我们的实验室内的实验误差是可以接受的。这允许一个给定的研究中比较可靠（配对）的样品。这是使用血小板存储病变，这是血小板保存在血库条件11的不利后果的有案可稽现象验证。此外，我们最近公布的三个可…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors have no acknowledgements.
Materials
| BD vacutainer tube with EDTA | Becton, Dickinson and Company | 368856 | |
| BD vacutainer tube with Heparin | Becton, Dickinson and Company | 368480 | |
| BD vacutainer tube with Sodium Citrate | Becton, Dickinson and Company | 366575 | |
| Hirudin Blood tube | Roche | 6675751 001 | |
| BD vacutainer Eclipse | Becton, Dickinson and Company | 368650 | Blood collection needle with preattached holder |
| Pipette tips 100-1000 | Greiner bio-one | 740290 | |
| Pipette tips 2-200 | Greiner bio-one | 739280 | |
| Pipette tips 1-10 | Eppendorf | A08928 | |
| Tube 5mL | Simport | 11691380 | |
| Conical tube 15mL | Greiner bio-one | 1888271 | |
| Conical tube 50mL | Greiner bio-one | 227261 | |
| 10 mL Syringe | BD | 309604 | |
| Precision wipes | Kimtech | 5511 | |
| Vena8 Fluoro+ Biochips | Cellix | 188V8CF-400-100-02P10 | Named in figure S1 A as 'Biochip' |
| Vena8 Tubing | Cellix | TUBING-TYGON-B1IC-B1OC-ROLL 100F | Named in figure S1 B as 'Disposable tubing' |
| Vena8 Needles | Cellix | SS-P-B1IC-B1OC-PACK200 | Named in figure S1 B as 'Pin' |
| Connectors for single inlet cables of biochips | Cellix | CONNECTORS-B1IC-PACK100 | |
| Multiflow8 connect | Cellix | MF8-CONNECT-BIC3-N-THROMBOSIS | Named in figure S1 B as 'Reusable tubing' and 'Splitter' |
| Humidified box | Cellix | HUMID-BOX | |
| Software microfluidic pump | Cellix | N/A | Venaflux Assay |
| Horm Collagen | Takeda/Nycomed | 1130630 | Native equine tendon collagen (type I) Isotonic glucose solution to dilute collagen is supplemented |
| HEPES buffered saline (HBS) | in house preparation | in house preparation | 10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (0.9% (w/v) NaCl, pH 7.4 |
| Blocking buffer | in house preparation | in house preparation | 1.0% (w/v) bovine serum albumin and 0.1% (w/v) glucose in HBS |
| Calcein AM | Molecular probes | C1430 | |
| Bleach 10% | in house preparation | in house preparation | |
| 0.1M NaOH | in house preparation | in house preparation | |
| Denaturated alcohol | Fiers | T0011.5 | |
| Mirus Evo Nanopump | Cellix | 188-MIRUS-PUMP-EVO | with Multiflow8. Named in figure S1 A as 'Pump' and 'Manifold' |
| Microscope | Zeiss | Axio Observer Z1 | equipped with a colibri-LED and high resolution CCD camera |
| Software microscope | Zeiss | N/A | ZEN 2012 |
| Hematology analyzer | Sysmex | N/A | |
| Table Top Centrifuge | Eppendorf | 521-0095 | |
| Platelet incubater | Helmer | PF-48i | |
| Incubation water bath | GFL | 1013 | |
| Pipette | Brand | A03429 | |
| Tube Roller | Ratek | BTR5-12V | |
| Sterile docking device | Terumo BCT | TSCD | |
| Tubing Sealer | Terumo BCT | AC-155 | |
| Vortex | VWR | 58816-121 |
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