This protocol investigates the use of extracellular vesicle (EV)-rich plasma as an indicator of the coagulative ability of EV. EV-rich plasma is obtained through a process of differential centrifugation and subsequent recalcification.
方法文章
This protocol investigates the use of extracellular vesicle (EV)-rich plasma as an indicator of the coagulative ability of EV. EV-rich plasma is obtained through a process of differential centrifugation and subsequent recalcification.
The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. However, there is an urgent need for a bedside test to assess the procoagulant activity of EV in clinical settings. This study proposes the use of thrombin activation time of EV-rich plasma as a measure of EV's procoagulant activity. Standardized procedures were employed to obtain sodium-citrated whole blood, followed by differential centrifugation to obtain EV-rich plasma. The EV-rich plasma and calcium chloride were added to the test cup, and the changes in viscoelasticity were monitored in real-time using an analyzer. The natural coagulation time of EV-rich plasma, referred to as EV-ACT, was determined. The results revealed a significant increase in EV-ACT when EV was removed from plasma obtained from healthy volunteers, while it significantly decreased when EV was enriched. Furthermore, EV-ACT was considerably shortened in human samples from preeclampsia, hip fracture, and lung cancer, indicating elevated levels of plasma EV and promotion of blood hypercoagulation. With its simple and rapid procedure, EV-ACT shows promise as a bedside test for evaluating coagulation function in patients with high plasma EV levels.
Thrombosis, which is caused by hypercoagulability, plays a significant role in various diseases, including brain trauma1, pre-eclampsia2, tumors3, and fracture patients4. The mechanism underlying hypercoagulability is complex, and recent emphasis has been placed on the role of extracellular vesicles (EV) in coagulation disorders. EVs are vesicle-like bodies with a bilayer structure that detach from the cell membrane, ranging in diameter from 10 nm to 1000 nm. They are associated with a variety of disease processes, particularly coagulation disorders5....
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The collection of human samples was approved by the Medical Ethics Committee of Tianjin Medical University General Hospital. The collection of human venous blood strictly followed the guideline issued by the National Health Commission of China, namely WS/T 661-2020 Guideline for Collection of Venous Blood Specimens. Briefly, blood was collected from healthy individuals with informed consent from the anterior brachial area vein, and the samples were mixed using 3.2% sodium citrate anticoagulant in a ratio of 1:9. When only sodium citrate anticoagulant specimens were collected, the first collection vessel was discarded. The processing flow was initiated within 0.5 h of ....
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The thrombin activation time of EV-rich plasma was measured using a viscoelastic method analyzer for plasma coagulation time measurement. The machine consists of four main components: an electronic signal converter, a probe, a detection tank, and a heating element (Figure 1A,B). The probe utilizes high-frequency and low-amplitude oscillations to detect changes in plasma viscosity. Daily quality control primarily involves air quality control to assess the stability of the tes.......
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In this study, the preparation of EV-rich plasma was described, and the method's rationality was verified using flow cytometry. Subsequently, the recalcified plasma samples were analyzed for ACT time using a clot analyzer based on viscoelasticity principles24. As shown in Figure 3A, the concentration of EVs obtained through ultracentrifugation was found to shorten the EV-ACT time, while the supernatant after ultracentrifugation, which had reduced EV levels, exhibi.......
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All authors declared that there are no potential conflicts of interest.
This work was supported by grants from the National Natural Science Foundation of China, grant no. 81930031, 81901525. In addition, we thank Tianjin Century Yikang Medical Technology Development Co., Ltd. for providing us with machines and technical guidance.
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| 姓名 | 公司 | 目录编号 | 评论 |
|---|---|---|---|
| AccuCount Ultra Rainbow 荧光微粒 | 3.8 μm;Spherotech， Lake Forest， IL， USA | 用于MP | |
| Werfen （china） | 0020006800 | 20 mM | |
| Century Clot分析仪 | 天津世纪益康医疗科技发展有限公司 | 原理是用粘弹性法测量血浆粘度 | |
| 一次性探针和试杯 | 天津世纪益康医疗科技发展有限公司 | ||
| LSR Fortessa 流式细胞仪 | BD，美国 | 用于检测 MP | |
| Megamix 聚苯乙烯微珠 | Biocytex，马赛，法国 | 7801 | Megamix 由选定直径的微珠混合物组成：0.5 和微量;m、0.9 和微量;m 和 3 µm。 |
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