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Coagulation Assay
血细胞凝集试验   

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参见作者原研究论文

本实验方案简略版
PLOS Pathogens
Dec 2013

Abstract

Clotting times can be measured by using citrate plasma. The intrinsic pathway of coagulation is measured by the activated partial thromboplastin time (aPTT), the extrinsic pathway of coagulation, monitored by measuring the prothrombin time (PT), and thrombin-induced fibrin-network formation (thrombin clotting time; TCT).

Materials and Reagents

  1. Citrated plasma (fresh or frozen)
  2. Eppendorf tubes
  3. Coagulation reagents
    1. Thrombin reagent (Technoclone, catalog number: 5100005 )
    2. TriniCLOT PT Excel reagent (Trinity Biotech, catalog number: T1105 / T1106 )
    3. DAPTTIN TC (Technoclone, catalog number: 5035060 )
  4. 30 mM CaCl2 (freshly made)
  5. Test agent (e.g. antimicrobial peptide LL-37)

Equipment

  1. BD vacutainer® plus blood collection tubes (BD, catalog number: 364305 )
  2. Coagulometer (Mc10 Plus merlin medical)
  3. Cuvettes and balls macro (merlin medical, catalog number: Z05100 )

Procedure

  1. Warm up the coagulation machine (at least 10 min before you begin the experiment switch on the machine).
  2. Prepare all reagents (should have room temperature before use).
  3. First place the special cuvette with a steel ball on the measuring positions in instrument related racks.
    Principle behind this technique: Once the cuvette is kept in the rack it starts rotating and due to gravity the metal ball inside the cuvette always remains. When the plasma/blood is in solution the ball remains in the position and if the plasma/blood starts clotting the clot pulls the ball out of the basic position and the sensor detects the disturbance and measures the clotting time.
  4. In order to measure the clotting times, add the reagents to the cuvette according to the schedule:
    1. For whole blood assay
      1. Add 100 µl citrate blood to the coagulometer and press incubation.
      2. After 60 sec incubation (helps to bings the sample to 37 °C).
      3. Add 100 µl of 30 mM CaCl2 → then immediately press manual start button to measure coagulation.
    2. Prothrombin time (PT)
      1. Add 100 µl citrate plasma to the coagulometer and press incubation.
      2. After 60 sec incubation.
      3. Add 100 µl TriniCLOT PT Excel → then immediately press manual start button to measure coagulation.
    3. Thrombin time (TT)
      1. Add 100 µl citrate plasma to the coagulometer and press incubation.
      2. After 60 sec incubation, add 100 µl Thrombin reagent → then immediately press manual start button to measure coagulation.
    4. Activated partial thromboplastin time (APTT)
      1. Add 100 µl citrate plasma to the coagulometer and press incubation.
      2. After 60 sec incubation.
      3. Add 100 µl DAPTTIN TC.
      4. Alter 200 sec incubation, add 100 µl 30 mM CaCl2→ then immediately press manual start button to measure coagulation.

Notes

  1. This assay is very sensitive, so precise sample pipetting and timing of each step is crucial!
  2. Prior testing coagulation time, test agent should be mixed with plasma or blood in an Eppendorf tube and incubate for a desired time.
  3. 50 µl citrate plasma is the minimal volume to use or the optimum in 100 µl.
  4. Use 1:1 volume ratio of the reagents or samples e.g. 100 µl plasma and 100 µl reagent/CaCl2.
  5. For coagulometer operation manual visit this link: http://www.merlinmedical.net/fileadmin/media/pdf/anleitungen/MC_10_OPERATION_MANUAL_ENGLISH.pdf.

Acknowledgments

This protocol is adapted from Papareddy et al. (2013).

References

  1. Papareddy, P., Kalle, M., Sorensen, O. E., Malmsten, M., Morgelin, M. and Schmidtchen, A. (2013). The TFPI-2 derived peptide EDC34 improves outcome of gram-negative sepsis. PLoS Pathog 9(12): e1003803.

简介

凝血时间可以通过使用柠檬酸盐血浆来测量。 通过测量凝血酶原时间(PT)和凝血酶诱导的纤维蛋白网络形成(凝血酶凝血时间; TCT)监测的凝血的外在途径,通过活化的部分凝血活酶时间(aPTT)测量凝固的内在途径。

材料和试剂

  1. 柠檬酸等离子体(新鲜或冷冻)
  2. Eppendorf管
  3. 凝血试剂
    1. 凝血酶试剂(Technoclone,目录号:5100005)
    2. TriniCLOT PT Excel试剂(Trinity Biotech,目录号:T1105/T1106)
    3. DAPTTIN TC(Technoclone,目录号:5035060)
  4. 30mM CaCl 2(新鲜制备)
  5. 测试剂(例如抗微生物肽LL-37)

设备

  1. BD vacutainer ®加采血管(BD,目录号:364305)
  2. 凝血计(Mc10 Plus merlin医疗)
  3. 比色杯和球宏(merlin medical,目录号:Z05100)

程序

  1. 预热凝固机(至少在开始实验开关前10分钟)。
  2. 准备所有试剂(使用前应具有室温)。
  3. 首先将具有钢球的特殊比色杯放在仪器相关机架的测量位置。
    这种技术的原理:一旦比色杯放在架子上 它开始旋转,并且由于重力,比色皿内的金属球 总是保持。 当血浆/血液在溶液中时,球保持在其中   位置,如果血浆/血液开始凝结,凝块拉动   球离开基本位置,传感器检测到扰动 并测量凝血时间
  4. 为了测量凝血时间,根据计划将试剂添加到比色杯中:
    1. 用于全血测定
      1. 加入100微升柠檬血液到凝血计,并按培养
      2. 60秒孵育后(帮助样品37℃)。
      3. 加入100μl的30mM CaCl 2→然后立即按手动开始按钮以测量凝固。
    2. 凝血酶原时间(PT)
      1. 向凝血计中加入100μl柠檬酸盐血浆,并按培养
      2. 孵育60秒后。
      3. 加入100μlTriniCLOT PT Excel→然后立即按手动启动按钮测量凝血。
    3. 凝血酶时间(TT)
      1. 向凝血计中加入100μl柠檬酸盐血浆,并按培养
      2. 孵育60秒后,加入100μl凝血酶试剂→然后 立即按手动启动按钮测量凝血。
    4. 活化部分凝血活酶时间(APTT)
      1. 向凝血计中加入100μl柠檬酸盐血浆,并按培养
      2. 孵育60秒后。
      3. 加入100μlDAPTTIN TC。
      4. 改变200秒孵育,加入100μl30mM CaCl 2→然后立即按手动开始按钮以测量凝固。

笔记

  1. 该测定非常灵敏,因此精确的样品吸取和每步的时间是至关重要的!
  2. 先前   测试凝固时间,待测试剂应与等离子体混合 血液在Eppendorf管中并孵育所需的时间。
  3. 50微升柠檬酸盐血浆是使用的最小体积或最佳100微升
  4. 使用1:1体积比的试剂或样品,例如100μl血浆和100μl试剂/CaCl 2。
  5. 对于凝血计操作手册,请访问此链接: http://www.merlinmedical.net /fileadmin/media/pdf/anleitungen/MC_10_OPERATION_MANUAL_ENGLISH.pdf

致谢

该协议改编自Papareddy等人(2013)。

参考文献

  1. Papareddy,P.,Kalle,M.,Sorensen,O.E.,Malmsten,M.,Morgelin,M。和Schmidtchen,A。(2013)。 TFPI-2衍生肽EDC34可改善革兰氏阴性脓毒症的结果。 PLoS Pathog 9(12):e1003803。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Papareddy, P., Kalle, M. and Schmidtchen, A. (2014). Coagulation Assay. Bio-protocol 4(19): e1247. DOI: 10.21769/BioProtoc.1247.
  2. Papareddy, P., Kalle, M., Sorensen, O. E., Malmsten, M., Morgelin, M. and Schmidtchen, A. (2013). The TFPI-2 derived peptide EDC34 improves outcome of gram-negative sepsis. PLoS Pathog 9(12): e1003803.
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