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

Avaliação da hipercoagulação sanguínea do plasma rico em EV

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.

Autor em destaque: decifrando distúrbios de coagulação em pacientes com lesão cerebral traumática 

Determinação da atividade pró-coagulante de vesículas extracelulares (EV) utilizando o tempo de coagulação ativado por EV (EV-ACT)

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   

Isolamento de Plasma Rico em Vesículas Extracelulares (EV) de Sangue Humano e Detecção do Tempo de Coagulação Ativado 

Para começar, centrifugar o sangue humano a 120 G por 20 minutos à temperatura ambiente para remover as células sanguíneas. Em seguida, transfira a metade superior do sobrenadante para um novo tubo. Em seguida, centrifugar o plasma rico em plaquetas para remover as plaquetas e transferir a metade superior do sobrenadante para um novo tubo.Em seguida, remova os restos celulares centrifugando a plaqueta para o nosso plasma a 13.000 G por dois minutos. Transfira a metade superior do sobrenadante para um novo tubo. Ligue o analisador de coágulos para detectar a vesícula extracelular, ou o tempo de coagulação ativado pelo EV, e pré-aqueça o instrumento a 37 graus Celsius.Instale a sonda descartável e o copo de teste. Em seguida, inicie o procedimento de controle de qualidade da máquina. Após a execução do controle de qualidade, insira informações de amostra no sistema.Em seguida, adicione 200 microlitros de plasma rico em EV ao copo de teste, seguido por 20 milimolares, 170 microlitros de cloreto de cálcio. Clique no botão Iniciar, feche a tampa e permita que a sonda detecte a alteração na resistência da amostra. As amostras de EV não qualificadas exibem um cluster distinto com um tamanho de partícula ligeiramente maior perto da porta de EV.In contraste, amostras qualificadas ricas em EV mostraram a maioria dos sinais dentro da porta de EV como um único grupo.Um aumento na concentração de EV encurtou o tempo de coagulação ativado por EV, enquanto uma diminuição na concentração de EV prolongou o tempo de coagulação ativado por EV. Amostras de plasma rico em EV de pacientes com pré-eclâmpsia, fraturas de quadril e câncer de pulmão tiveram tempo de coagulação ativado por EV significativamente menor em comparação com voluntários saudáveis.

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Research

JoVE Journal

Medicine

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

73 visualizações.  Tianjin Medical University General Hospital. Para começar, centrifugar o sangue humano a 120 G por 20 minutos à temperatura ambiente para remover as células sanguíneas. Em seguida, transfira a metade superior do sobrenadante para um novo tubo. Em seguida, centrifugar o plasma rico em plaquetas para remover as plaquetas e transferir a metade superior do sobrenadante para um novo tubo.Em seguida, remova os restos celulares centrifugando a plaqueta para o nosso plasma a 13.000 G por dois minutos. Transfira a metade superior do s...

Vídeo: 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. ...

Medicina

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.