isolation of extracellular vesicle (ev)-rich plasma from human blood and detection of activated clotting time 

EV-Rich Plasma의 혈액 과응고 평가

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. Several studies have identified EVs as a promising predictor of thrombosis risk6,7. The procoagulant activity of EVs depends on the expression of coagulation factors, primarily tissue factor (TF) and phosphatidylserine (PS). EVs with robust procoagulant activity significantly enhance the catalytic efficiency of tenase and prothrombin complex, thereby promoting thrombin-mediated fibrinogen and local thrombosis8. Elevated levels of EVs and their causal relationship with hypercoagulability have been observed in numerous diseases9. Consequently, standardizing the detection of EVs and reporting their procoagulant activity is an important area of investigation10.
To date, only a few commercial kits are available for detecting the procoagulant activity of EVs. The MP-Activity assay and the MP-TF assay, produced by a commercial company, are functional assays used to measure EV&#39;s procoagulant activity in plasma11. These assays employ a principle similar to that of enzyme-linked immunosorbent assays to detect PS and TF on EVs. However, these kits are expensive and limited to a few high-level research institutions. The process is complex and time-consuming, making it challenging to implement them in clinical settings. Additionally, a commercially developed procoagulant phospholipid (PPL) assay mixes PS-free plasma with test plasma, measuring clotting time to quantitatively detect levels of PS-positive EVs12. However, these assays primarily focus on PS and TF on EVs, overlooking other clotting pathways that circulating EVs may be involved in12.
The plasma coagulation system is intricate and comprises both &#34;invisible&#34; and &#34;visible&#34; components, including coagulants, anticoagulants, fibrinolytic systems, and EVs suspended in the plasma. Physiologically, these components maintain a dynamic balance. In pathological conditions, significantly increased EVs in circulation contribute to hypercoagulability, particularly in patients with brain trauma, preeclampsia, fractures, and various types of cancer13. Currently, the evaluation of coagulation status in clinical laboratories primarily involves assessing the coagulation system, anticoagulation system, and fibrinolysis14,15,16,17. Prothrombin time, activated partial thromboplastin time, thrombin time, and international normalized ratio are commonly used to evaluate coagulation factor levels in the coagulation system18. However, recent studies have revealed that these tests do not fully reflect the hypercoagulability of certain diseases19. Other assay methods, such as thromboelastometry (TEG), rotational TEG, and Sonoclot analysis, measure whole-blood viscoelastic changes20,21. Since whole blood samples contain numerous blood cells and platelets, these tests are more likely to indicate the clotting status of the sample as a whole. Some researchers have reported on the role of blood cells and platelets in procoagulant activity22,23. A recent study also discovered that previous coagulation function tests face difficulties in detecting changes in microparticles&#39; procoagulant activity24. Therefore, a hypothesis has been proposed that the procoagulant function of EVs can be evaluated by viscoelastic measurements of the activated clotting time (ACT) in EV-rich plasma.

저자 스포트라이트: 외상성 뇌 손상 환자의 응고 장애 해독 

EV-ACT(Activated Clotting Time)를 사용한 세포외 소포(EV)의 응고 활성 측정

Our primary focus is on coagulation disorders in patients who have suffered brain trauma. In this study, we try to monitor changes in the clotting function with extracellular vesical activity clotting time. Our teams put attention on the research of brain injury.And in the brain injury, they are a severe condition we call the hyper coagulated state. It's very difficult to measure. And we find in the blood of patient there are some micro vesicle we called extracellular vesicles, including the apoptosis body, micro vesicle, and exosome.And they are different in the pro coagulated ability. So we must find a method to measure the ability. And this method, we can acute quickly to find the difference of the extracellular vesicles.That's very important. Depend on this measure, we can do many things. We have make sure it in the TPI mice, and the clinical patient, especially in the peripheral circulation, it will cost some embolization.That is very, very serious in the clinical. Our protocol offered a significant advantage compared to other techniques due to its simplicity, cost effectiveness, and speed. It allows for a blood clotting test to be conducted at the bedside, eliminating the need for extensive laboratory processing.Our research have established a new method that can measure the hyper coagulated states of the peripheral blood. It can be used not only in the TPI, we can also used in other disease such as sepsis, pre-eclampsia, tumor, and hematology disease. In the future, we have planned to investigated the of in scanning for clinical hyper a condition that increases the risk of blood clot.

Watch this Scientific Journal Video about Isolation of Extracellular Vesicle EV-Rich Plasma from Human Blood and Detection of Activated Clotting Time at JoVE.com

Isolation of Extracellular Vesicle (EV)-Rich Plasma from Human Blood and Detection of Activated Clotting Time   

인간 혈액에서 세포외 소포(EV)가 풍부한 혈장 분리 및 활성화된 응고 시간 검출 

시작하려면 실온에서 120분 동안 20G의 인간 혈액을 원심분리하여 혈액 세포를 제거합니다. 그런 다음 상층액의 상반부를 새 튜브로 옮깁니다. 다음으로, 혈소판이 풍부한 혈장을 원심분리하여 혈소판을 제거하고 상층액의 상반부를 새 튜브로 옮깁니다.그 때 13에 우리의 플라스마에 혈소판을 분리해서 세포 파편을 제거하십시오, 2 분 동안 000 G. 상층액의 상반부를 새 튜브로 옮깁니다. 혈전 분석기를 켜서 세포외 소포 또는 EV 활성화 응고 시간을 감지하고 기기를 섭씨 37도로 예열합니다.일회용 프로브와 테스트 컵을 설치합니다. 그런 다음 기계의 품질 관리 절차를 시작하십시오. 품질 관리 실행 후 샘플 정보를 시스템에 입력합니다.그런 다음 EV가 풍부한 혈장 200마이크로리터를 테스트 컵에 넣은 다음 20밀리몰, 염화칼슘 170마이크로리터를 추가합니다. 시작 버튼을 클릭하고 덮개를 닫은 다음 프로브가 샘플 저항의 변화를 감지하도록 합니다. 자격이 없는 EV 샘플은 EV.In 대비 게이트 근처에서 약간 더 큰 입자 크기를 가진 뚜렷한 클러스터를 나타내며, 자격을 갖춘 EV가 풍부한 샘플은 EV 게이트 내에서 대부분의 신호를 단일 그룹으로 보여주었습니다.EV 농도가 증가하면 EV 활성화 응고 시간이 단축되고, EV 농도가 감소하면 EV 활성화 응고 시간이 연장됩니다. 자간전증, 고관절 골절 및 폐암 환자의 EV가 풍부한 혈장 샘플은 건강한 지원자에 비해 EV 활성화 응고 시간이 현저히 짧았습니다.

isolation-extracellular-vesicle-ev-rich-plasma-from-human-blood

Research

JoVE Journal

Medicine

Isolation of Extracellular Vesicle (EV)-Rich Plasma from Human Blood and Detection of Activated Clotting Time 

73 조회수.  Tianjin Medical University General Hospital. 시작하려면 실온에서 120분 동안 20G의 인간 혈액을 원심분리하여 혈액 세포를 제거합니다. 그런 다음 상층액의 상반부를 새 튜브로 옮깁니다. 다음으로, 혈소판이 풍부한 혈장을 원심분리하여 혈소판을 제거하고 상층액의 상반부를 새 튜브로 옮깁니다.그 때 13에 우리의 플라스마에 혈소판을 분리해서 세포 파편을 제거하십시오, 2 분 동안 000 G. 상층액의 상반부를 새 튜?...

비디오: Isolation of Extracellular Vesicle (EV)-Rich Plasma from Human Blood and Detection of Activated Clotting Time   

Determination of the Procoagulant Activity of Extracellular Vesicle (EV) Using EV-Activated Clotting Time (EV-ACT)

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.

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. ...

연구

의학

To begin, centrifuge the human blood at 120 G for 20 minutes at room temperature to remove the blood cells. Then transfer the upper half of the supernatant to a new tube. Next, centrifuge the platelet rich plasma to remove the platelets, and transfer the upper half of the supernatant to a new tube.Then remove the cell debris by centrifuging the platelet to our plasma at 13, 000 G for two minutes. Transfer the upper half of the supernatant to a new tube. Turn on the clot analyzer to detect extracellular vesicle, or EV activated clotting time, and preheat the instrument to 37 degrees Celsius.Install the disposable probe and test cup. Then start the quality control procedure of the machine. After the quality control run, enter sample information into the system.Then add 200 microliters of EV-rich plasma to the test cup, followed by 20 millimolar, 170 microliters of calcium chloride. Click the start button, close the cover, and allow the probe to detect the change in sample resistance. The unqualified EV samples exhibit a distinct cluster with a slightly larger particle size near the gate of EV.In contrast, qualified EV-rich samples showed most signals within the gate of EV as a single group.An increase in EV concentration shortened EV activated clotting time, while a decrease in EV concentration prolonged EV activated clotting time. EV-rich plasma samples from patients with preeclampsia, hip fractures, and lung cancer had significantly shorter EV activated clotting time compared to healthy volunteers.