Name: Isolation of Extracellular Vesicle (EV)-Rich Plasma from Human Blood and Detection of Activated Clotting Time
Uploaded: 2023-08-04T13:40:00
Description: 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 

הערכת קרישת יתר בדם של פלזמה עשירה ברכב חשמלי

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.

מחבר זרקור: פענוח הפרעות קרישה בנפגעי מוח טראומטיים 

קביעת פעילות הקרישה של שלפוחית חוץ-תאית (EV) באמצעות זמן קרישה המופעל על ידי 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   

בידוד פלסמה חוץ-תאית עשירה בשלפוחית (EV) מדם אנושי וזיהוי זמן קרישה פעיל 

כדי להתחיל, צנטריפוגה את הדם האנושי ב 120 G במשך 20 דקות בטמפרטורת החדר כדי להסיר את תאי הדם. לאחר מכן מעבירים את החצי העליון של הסופרנאטנט לצינור חדש. לאחר מכן, צנטריפוגו את הפלזמה העשירה בטסיות כדי להסיר את הטסיות, והעבירו את החצי העליון של הסופרנאטנט לצינור חדש.לאחר מכן להסיר את פסולת התא על ידי צנטריפוגה טסיות לפלזמה שלנו ב 13, 000 G במשך שתי דקות. מעבירים את החצי העליון של הסופרנאטנט לצינור חדש. הפעל את מנתח הקריש כדי לזהות שלפוחית חוץ-תאית, או זמן קרישה המופעל על ידי EV, וחמם מראש את המכשיר ל -37 מעלות צלזיוס.התקינו את הבדיקה החד פעמית ואת הבדיקה. לאחר מכן התחל את הליך בקרת האיכות של המכונה. לאחר הפעלת בקרת האיכות, הזן מידע לדוגמה במערכת.לאחר מכן הוסיפו 200 מיקרוליטר של פלזמה עשירה ברכב חשמלי לכוס הבדיקה, ואחריה 20 מילימולר, 170 מיקרוליטר סידן כלורי. לחץ על לחצן התחל, סגור את הכיסוי ואפשר לגשושית לזהות את השינוי בהתנגדות הדגימה. דגימות ה-EV הבלתי מוסמכות מציגות צביר מובחן עם גודל חלקיקים מעט גדול יותר ליד השער של ניגודיות EV.In, דגימות מוסמכות עשירות ב-EV הראו את רוב האותות בתוך שער הרכב החשמלי כקבוצה אחת.עלייה בריכוז הרכב החשמלי קיצרה את זמן הקרישה המופעל על ידי EV, בעוד שירידה בריכוז EV האריכה את זמן הקרישה המופעל על ידי EV. דגימות פלזמה עשירות ב-EV מחולים עם רעלת הריון, שברים בירך וסרטן ריאות היו בעלות זמן קרישה קצר משמעותית של EV בהשוואה למתנדבים בריאים.

Research

JoVE Journal

Medicine

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. ...