Low-temperature extraction method of active plasma
By using cryogenically controlled blood collection equipment and containers, the coagulation mechanism is inhibited, enabling the extraction of active plasma without anticoagulants. This solves the problems of anticoagulant contamination and high-temperature coagulation, providing high-purity, biocompatible active plasma.
Patent Information
- Application Number
- CN202511714615.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-21
- Publication Date
- 2026-01-02
AI Technical Summary
Existing technologies require the use of anticoagulants when extracting blood components, which affects blood activity and component purity. Furthermore, extraction at high temperatures can trigger the coagulation function, making it impossible to extract high-purity active plasma (AP).
Using blood collection devices and containers with temperature control, the blood is pre-cooled to -4 to 20°C, which inhibits the coagulation mechanism during collection and centrifugation, and uses the difference in blood density for separation, avoiding anticoagulant contamination and maintaining blood activity.
It enables the extraction of active plasma without anticoagulants, preserving the natural state and bioactivity of blood components, making it suitable for sensitive clinical applications and reducing the risk of chemical residues and immune reactions.
Smart Images
Figure CN121243513A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the fields of biotechnology and pharmaceutical technology, and more specifically, it relates to a method for extracting active plasma (AP) containing leukocytes and platelets. Background Technology
[0002] Blood is a complex mixture of many components. Generally, blood can be described as comprising four main components: red blood cells, white blood cells, platelets, and plasma. The first three are cellular or cell-like components, while the fourth (plasma) is a liquid component containing a broad and variable mixture of salts, proteins, and many other elements essential for bodily functions. Centrifugation separates the components of blood from each other. Typically, centrifugation causes the large volume / mass of thick red blood cells to migrate to the bottom of the centrifuge tube. Plasma, white blood cells, and platelets can be completely separated at the top of the tube.
[0003] Red blood cells, also known as erythrocytes, are responsible for carrying oxygen from the lungs to cells for use in cellular metabolism and carrying waste carbon dioxide from cells to the lungs for expulsion. The percentage of blood volume composed of red blood cells is called hematocrit. For women, a normal hematocrit is 37%–47%, and for men, it is 40%–54%.
[0004] White blood cells, also known as leukocytes, are nucleated cells responsible for protecting the body from foreign substances. They typically function to fight pathogens such as bacteria, fungi, and viruses, or potentially harmful substances like protein toxins.
[0005] Platelets, also commonly known as clotting cells, are small, irregularly shaped blood components derived from megakaryocytes. They are formed in the bone marrow and are involved in the clotting process, thus helping to prevent excessive bleeding from the body not only due to external trauma or injury but also from normal physiological activities. In fact, platelets are crucial for normal hemostasis, providing the first line of defense against blood flowing out of damaged blood vessels. Platelets typically function by adhering to the inner walls of blood vessels and interacting with components of the clotting system present in the plasma or released by other cellular components of the blood. There are approximately 150,000 to 450,000 platelets per microliter (μl) of blood.
[0006] The liquid portion of blood, commonly known as plasma, is a complex solution containing various proteins and salts. Generally, plasma is the residue remaining after red blood cells, white blood cells, and platelets are removed from the blood. Because many proteins are present in high concentrations, plasma is a pale yellow liquid that is unstable at room temperature (i.e., plasma must be properly stored below room temperature to prevent the proteins from losing their activity). The main protein components of plasma are albumin, fibrinogen, antibodies, and various proteins essential for coagulation and hemostasis. This concise list of plasma proteins demonstrates that plasma performs a wide range of functions, from maintaining satisfactory blood pressure and providing volume to providing proteins crucial for blood clotting and immunity.
[0007] The main process of maintaining proper blood flow and closure is called hemostasis, which involves the formation and eventual degradation of blood clots and the repair of damaged tissue. Hemostasis consists of four main events: vasoconstriction; platelet aggregation at the site of injury, mediated by fibrinogen and activated by prothrombin; clot formation (also known as a thrombus or fibrin network) through the complex interactions of platelets and many coagulation factors; and finally, clot degradation and repair of damaged tissue.
[0008] This invention is a technique for separating and removing red blood cells from blood while retaining other components. At normal temperatures, blood will coagulate after blood vessels rupture, forming a blood clot, which will prevent the extraction process from being completed. Existing technologies use anticoagulants to prevent the blood from coagulating, which affects the activity and purity of the blood. This invention does not add any substances to the blood, including anticoagulants, but adopts a low-temperature technology throughout the process. During the extraction process, the blood does not coagulate, and the extraction requirements can be successfully met.
[0009] Related medical and biological research suggests that the temperature at which intracellular water begins to freeze and cause cell membrane rupture typically falls below -4 degrees Celsius before significant freezing necrosis occurs, resulting in severe damage to cell structure. Therefore, -4 to 37 degrees Celsius is a safe temperature range for blood cells and other blood components. Enzyme activity peaks at 37 degrees Celsius, as does prothrombin activity in the blood. For every 10 degrees Celsius decrease in blood temperature, prothrombin activity halves. Therefore, providing an environment that lowers the blood temperature to approximately -4 degrees Celsius and preventing it from dropping below -4 degrees Celsius to avoid cell damage is crucial. By lowering the temperature of the blood collection device, blood collection container, and syringe, with or without a temperature control device, the collected blood is rapidly cooled, inhibiting the triggering of the blood clotting mechanism. At the same time, the activity and function of various components in the blood are maintained normally, ensuring that the extract can normally initiate the clotting function at 37°C. When injected into the human body (except into blood vessels), the plasma at the injection site can undergo clotting, producing fibrin gel, which creates the same damage signal as a ruptured blood vessel, guiding tissue repair cells to repair the damage. At the same time, the repair factors contained in the blood without red blood cells are also slowly released and participate in tissue repair. Summary of the Invention
[0010] To extract an active plasma (AP) that is free from contamination and additives, contains all components except red blood cells, and has coagulation function.
[0011] This application provides a low-temperature extraction method for active plasma, employing the following technical solution:
[0012] A method for low-temperature extraction of active plasma, comprising the following steps:
[0013] S1. Pre-cool the blood collection device and / or blood collection container, or the blood collection device and / or blood collection container with a temperature control device, to between -4°C and 20°C.
[0014] S2. Collect arterial or venous blood from the body using the pre-cooled blood collection device or container described in S1, so that the temperature of the collected blood drops rapidly and the triggering of the coagulation mechanism in the blood is inhibited within a certain period of time.
[0015] S3. Place the blood collected in S2 along with the blood collection container into a centrifuge and centrifuge for 3-10 minutes with a centrifugal force of 70-1000g to separate the blood into upper active plasma (AP) and lower red blood cells.
[0016] S4. Extract active plasma (AP) using the pre-cooled blood collection device described in S1.
[0017] By adopting the above technical solution, the blood collection equipment and containers are pre-cooled to -4 to 20°C, resulting in a rapid drop in blood temperature during collection, which slows down the activation process of coagulation factors and maintains the fluid state of the blood before centrifugation. Separation is performed using a centrifugal force of 70-1000g, a parameter based on the density differences of blood components, avoiding damage to blood cells caused by excessive centrifugal force. Plasma is extracted using a cooled syringe to prevent the temperature from rising and activating the coagulation cascade reaction. Therefore, high-purity active plasma (AP) is obtained without relying on anticoagulants, improving biocompatibility.
[0018] Preferably, no anticoagulants or other substances are added during the extraction of active plasma (AP) to minimize the destruction of active substances in the active plasma (AP).
[0019] By adopting the above technical solution, the use of a low-temperature environment to replace chemical anticoagulants inhibits platelet aggregation and prothrombin conversion. According to the principles of blood coagulation, the low temperature slows down the rate of enzymatic reactions, thereby avoiding the introduction of contamination by external additives. The entire process relies on physical cooling to maintain the natural state of the blood. Therefore, active plasma (AP) without chemical residues is obtained, reducing the risk of immune reactions and making it suitable for sensitive clinical applications.
[0020] Preferably, blood is collected and active plasma (AP) is extracted using a pre-cooled blood collection device and / or blood collection container, so that the collected blood is rapidly cooled to between -4 and 20°C, and the collected blood is kept at -4 and 20°C before and after centrifugation and the extracted active plasma (AP) is kept at between -4 and 20°C for 30-120 minutes, so as not to damage blood cells and active substances and not to trigger the coagulation cascade reaction.
[0021] By adopting the above technical solution, blood collection devices pre-cooled to -4℃ to 20℃ absorb heat upon contact with blood, lowering the blood temperature to the target range. This low-temperature environment can inhibit the contact activation of coagulation factors and the overexpression of platelet surface receptors, thereby blocking the initiation of coagulation. At the same time, controlling subsequent processing steps within the low-temperature range of -4℃ to 20℃ for 30-120 minutes avoids damage to blood cell membrane structures caused by ice crystal formation and provides stable preservation conditions for active proteins in plasma. Therefore, active plasma (AP) can maintain its natural active state without the addition of exogenous anticoagulants, and coagulation function is under controllable inhibition.
[0022] Preferably, the collected blood is centrifuged to separate red blood cells at the bottom layer and active plasma (AP) at the top layer.
[0023] By adopting the above technical solution, the centrifugation process utilizes the density gradient of blood components, causing red blood cells to settle and plasma to supernatant under a centrifugal force of 70-1000g, thus avoiding hemolysis caused by excessive centrifugation; the separated layers are clear, making them easy to identify and extract; therefore, a high yield of active plasma (AP) is obtained, reducing cell contamination and improving product purity.
[0024] Preferably, the extracted active plasma (AP) can be injected into the body tissues through a 34G injection needle within 30-120 minutes.
[0025] By adopting the above technical solution, the extracted plasma maintains low viscosity and bioactivity within a specific time window, thus ensuring that no gel forms before injection; while 34G needles require plasma with high fluidity, and low-temperature preservation delays fibrin polymerization; therefore, plasma that can be directly used for minimally invasive injection is obtained, expanding its clinical applicability.
[0026] Preferably, the prothrombin and coagulation factors in the extracted active plasma (AP) can be activated and can form a blood clot when injected into body tissues within 30-120 minutes or in a specific in vitro environment.
[0027] By adopting the above technical solution, the extraction process inhibits the coagulation mechanism through low temperature, but prothrombin and coagulation factors are naturally activated when exposed to body temperature or an in vitro simulated environment after injection. This method controls the activation timing to avoid premature coagulation. Therefore, the plasma can remain in a fluid state during injection and coagulate at the target site, thereby promoting regeneration and healing.
[0028] Preferably, the extracted active plasma (AP), after being activated in the body tissues before inactivation to form a blood clot, can release bioactive substances and trigger inflammatory responses and tissue repair processes.
[0029] By adopting the above technical solution, since low-temperature extraction is used to preserve the natural conformation and functional integrity of active components in plasma, when the active plasma (AP) is injected into the tissue wound, prothrombin is activated into thrombin, which then catalyzes the conversion of fibrinogen into fibrin and intertwines into a three-dimensional network structure to form a temporary blood clot matrix. This matrix not only plays a role in mechanical hemostasis, but also acts as a biological scaffold to adsorb and release a variety of active substances, thereby orderly initiating and promoting the natural repair process of inflammation resolution, granulation tissue formation and epithelial regeneration.
[0030] Preferably, the extracted active plasma (AP) can be made into lyophilized plasma and retain its activity under low-temperature storage.
[0031] By adopting the above technical solution, the centrifuged active plasma (AP) is made into freeze-dried plasma. Since freeze-dried plasma can be stored for a long time while ensuring the efficacy of active plasma (AP), it is convenient for medical staff to use it later.
[0032] In summary, this application has the following beneficial effects:
[0033] Because this application uses a blood collection device with a temperature control mechanism, it achieves low-temperature control throughout the entire process from blood collection to plasma extraction. This device system can ensure that the blood is rapidly cooled at the moment of collection and maintained at a low temperature of -4 to 20°C throughout the process, thereby inhibiting platelet activation and the initiation of the coagulation mechanism. It maintains the natural state and biological activity of the blood without relying on any anticoagulants. This not only allows for the use of smaller diameter needles to reduce patient pain, but also avoids the risk of contamination and immunogenicity problems introduced by chemical additives. It is also beneficial to obtain high-purity, biocompatible active plasma (AP), providing a guarantee for subsequent clinical applications. Attached Figure Description
[0034] Figure 1 This is a flowchart of a low-temperature extraction method for active plasma proposed in this application. Detailed Implementation
[0035] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.
[0036] Technical concept:
[0037] Related medical and biological research suggests that the temperature at which intracellular water begins to freeze and causes cell membrane rupture is generally below -4°C, but typically below -5°C to -7°C for significant freeze necrosis and severe damage to cell structure to occur. The critical temperature for cell death is -40°C, at which point homogeneous ice crystals form inside the cell, leading to cell membrane rupture. Within the range of -5°C to -21°C, ice crystals begin to form on the cell periphery, and dehydration damage begins, but the cell membrane does not necessarily rupture immediately. When the temperature drops below -40°C, a large number of intracellular ice crystals form and expand, mechanically damaging the cell membrane, causing it to rupture, leaking cell contents, and resulting in cell death.
[0038] In this application, the blood collection device and / or blood collection container are pre-cooled to no less than -4°C, and the initial temperature of the ex vivo blood is between 36.5-37.7°C. When the ex vivo blood enters the pre-cooled and continuously cooled blood collection device and / or blood collection container, the blood temperature will decrease and remain at a low temperature for a short time. The activity of prothrombin and coagulation factors will decrease accordingly, and the coagulation system will not be triggered for a certain period of time. At the same time, it ensures that blood cells and blood components are not damaged by low temperature and the integrity of coagulation function is maintained. This invention overcomes the shortcomings of current APC technology, including the reduced therapeutic effect due to the use of anticoagulants, the inability to inject platelet-rich plasma due to in vitro triggering of the coagulation system, the shortened injection time, and the pain caused to patients by the need to use larger syringe needles. It provides a more advanced solution for regenerative medicine.
[0039] Example 1
[0040] This embodiment provides a low-temperature extraction method for active plasma, comprising the following steps:
[0041] S1. Pre-cool the blood collection device and blood collection container to -4°C;
[0042] S2. Collect venous blood from the body using the pre-cooled blood collection device described in S1, so that the temperature of the collected blood drops rapidly and the triggering of the coagulation mechanism in the blood is inhibited within a certain period of time.
[0043] S3. Place the blood collected in S2 along with the blood collection container into a centrifuge and centrifuge for 3 minutes with a centrifugal force of 70g to separate the blood into upper active plasma (AP) and lower red blood cells.
[0044] S4. Extract active plasma (AP) using the pre-cooled blood collection device described in S1.
[0045] Example 2
[0046] This embodiment provides a low-temperature extraction method for active plasma, comprising the following steps:
[0047] S1. Pre-cool the blood collection device and blood collection container to 20°C;
[0048] S2. Collect venous blood from the body using the pre-cooled blood collection device described in S1, so that the temperature of the collected blood drops rapidly and the triggering of the coagulation mechanism in the blood is inhibited within a certain period of time.
[0049] S3. Place the blood collected in S2 along with the blood collection container into a centrifuge and centrifuge for 10 minutes with a centrifugal force of 1000g to separate the blood into upper active plasma (AP) and lower red blood cells.
[0050] S4. Extract active plasma (AP) using the pre-cooled blood collection device described in S1.
[0051] Example 3
[0052] This embodiment provides a low-temperature extraction method for active plasma, comprising the following steps:
[0053] S1. Pre-cool the blood collection device and blood collection container to 8°C;
[0054] S2. Collect venous blood from the body using the pre-cooled blood collection device described in S1, so that the temperature of the collected blood drops rapidly and the triggering of the coagulation mechanism in the blood is inhibited within a certain period of time.
[0055] S3. Place the blood collected in S2 along with the blood collection container into a centrifuge and centrifuge for 6 minutes at a centrifugal force of 500g to separate the blood into upper active plasma (AP) and lower red blood cells.
[0056] S4. Extract active plasma (AP) using the pre-cooled blood collection device described in S1.
[0057] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.
Claims
1. A method for cryo-extraction of active blood plasma, characterized in that, Includes the following steps: S1. Pre-cool the blood collection device and / or blood collection container, or the blood collection device and / or blood collection container with a temperature control device, to between -4°C and 20°C. S2. Collect arterial or venous blood from the body using the pre-cooled blood collection device or container described in S1, so that the temperature of the collected blood drops rapidly and the triggering of the coagulation mechanism in the blood is inhibited within a certain period of time. S3. Place the blood collected in S2 along with the blood collection container into a centrifuge and centrifuge for 3-10 minutes with a centrifugal force of 70-1000g to separate the blood into upper active plasma and lower red blood cells. S4. Extract active plasma using the pre-cooled blood collection device described in S1.
2. The method of claim 1, wherein, No anticoagulants or other substances are added during the extraction of active plasma, so as to minimize the destruction of active substances in the active plasma.
3. The method of claim 1, wherein, Blood is collected and active plasma is extracted using a pre-cooled blood collection device and / or blood collection container. The collected blood is rapidly cooled to between -4 and 20°C, and the collected blood is kept at -4 and 20°C before and after centrifugation, as well as the extracted active plasma, for 30-120 minutes. This process does not damage blood cells or active substances and does not trigger the coagulation cascade reaction.
4. The method of claim 1, wherein, The collected blood is centrifuged and separated into red blood cells at the bottom layer and active plasma at the top layer.
5. The method according to claim 1, characterized in that, The extracted active plasma can be injected into the body tissues through a 34G injection needle within 30-120 minutes.
6. The method according to claim 1, characterized in that, The prothrombin and coagulation factors in the extracted active plasma can be activated and can form blood clots when injected into body tissues within 30-120 minutes or in a specific in vitro environment.
7. The method according to claim 1, characterized in that, The extracted active plasma, after being activated in the body tissues before being deactivated, forms a blood clot and releases bioactive substances, triggering an inflammatory response and tissue repair process.
8. The method according to claim 1, characterized in that, The extracted active plasma can be made into freeze-dried plasma and retain its activity under low-temperature storage.