CN106727700B - Method for preparing platelet rich plasma PRP and application of platelet rich plasma - Google Patents

Method for preparing platelet rich plasma PRP and application of platelet rich plasma Download PDF

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CN106727700B
CN106727700B CN201611074599.9A CN201611074599A CN106727700B CN 106727700 B CN106727700 B CN 106727700B CN 201611074599 A CN201611074599 A CN 201611074599A CN 106727700 B CN106727700 B CN 106727700B
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plasma
platelet
blood
platelets
rich plasma
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CN106727700A (en
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李诣书
尚聪
梅俊
周丹
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Sinica Life Technology Co ltd
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Sinica Life Technology Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/19—Platelets; Megacaryocytes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D17/00—Separation of liquids, not provided for elsewhere, e.g. by thermal diffusion
    • B01D17/02—Separation of non-miscible liquids
    • B01D17/0217—Separation of non-miscible liquids by centrifugal force

Abstract

The present invention relates to a method for preparing platelet rich plasma PRP and uses of the platelet rich plasma. In particular, the present invention relates to a method for extracting platelet rich plasma from blood, comprising the steps of: (a) putting the collected whole blood into a container containing an anticoagulant, and fully and uniformly mixing the blood and the anticoagulant; (b) placing the blood mixed with the anticoagulant in a centrifuge tube, and carrying out first centrifugation to divide the blood into three layers basically; (c) the uppermost layer and most of the middle layers are pumped out and transferred to a new centrifuge tube, and the mixture is uniformly mixed and centrifuged for the second time; (d) discarding the plasma on the upper layer of the centrifugal tube, and resuspending the precipitated platelet by using the residual plasma to obtain the platelet-rich plasma. The invention also relates to the platelet-rich plasma obtained by the method according to the invention and to the use thereof for producing health products, for example for orthopedics, stomatology, maxillofacial surgery, sports medicine and cosmetic medicine.

Description

Method for preparing platelet rich plasma PRP and application of platelet rich plasma
Technical Field
The invention belongs to the technical field of clinical medicine, in particular relates to a method for preparing platelet-rich plasma, and more particularly relates to a method for preparing platelet-rich plasma with the advantages of high concentration of platelets, high platelet recovery rate and the like in one or more aspects. The invention also relates to the prepared platelet rich plasma PRP prepared by the method, and the application of the platelet rich plasma PRP prepared by the method in preparing health products. For example, the health products are applied in orthopedics, stomatology, maxillofacial surgery, sports medicine, and cosmetic medicine.
Background
Platelet rich plasma, also commonly referred to in the art as Platelet Rich Plasma (PRP), is plasma containing high volume fractions of platelets obtained by centrifugation of whole human or animal blood itself. Since David r.knighton et al discovered that platelet cells can promote vascular proliferation and collagen synthesis in the last 80 s, people have focused on the application of Platelet Rich Plasma (PRP) to the clinic and eagerly solved the problem of repairing damaged organs and tissues with low repair capacity. However, the preparation of PRP was difficult at that time, which limited the clinical application.
Several suitable methods for preparing platelet rich plasma have been explored after decades of research: manual methods (primary centrifugation, secondary centrifugation and tertiary centrifugation) and equipment preparation methods. Among them, the secondary centrifugation method has the highest extraction rate of PRP and is most widely used in clinical practice. The principle of preparing PRP by the secondary centrifugation method is as follows: (1) drawing venous blood and injecting the venous blood into a test tube containing anticoagulant; (2) the 1 st centrifugation divides the blood into 3 layers, the bottommost part being about 55% of the total volume of the blood in red blood cells; the top part is platelet-poor plasma (PPP) accounting for about 40% of the total volume fraction, and is mainly plasma components such as fibrinogen and the like; the middle layer is Platelet Concentrate (PC) which accounts for only 5% of the total volume, namely a commonly known yellow coating layer; (3) sucking PPP and PC and part of red blood cells adjacent to the PC by using a liquid transfer machine, and injecting the red blood cells into another test tube without anticoagulant; (2) centrifuging again at a speed and for a time to separate the plasma into 3 layers, the lowest of which is a small amount of remaining red blood cells and the top of which is PPP in an amount of about 80% of the total volume fraction, the two layers being enriched platelets; (5) most of the PPP is pipetted and enough serum is left to hold the enriched platelets suspended therein, resulting in PRP. In addition, the platelets in PRP are fragile and easily activated in vitro, too high a centrifugation speed may rupture the platelet membrane and reduce its biological activity, while lower a centrifugation speed may keep the platelet activity at a minimum level during the preparation process, so the centrifugation speed should not be too fast. Furthermore, the volume fraction of platelets and the amount and activity of various growth factors vary among PRP prepared at different centrifugation times, centrifugation forces and centrifugation times.
Although PRP with single component and up-to-standard concentration can be prepared at present, the preparation technology is not completely mature, and the secondary centrifugation method advocated by most people has the risks of complex operation, long process and product pollution. The PRP equipment sold in a certain company in China has the defect of low concentration of platelets in PRP, while the PRP equipment sold in the United states has the defect of high concentration of platelet cells but low purity because the concentration of hemoglobin in the PRP is not disclosed, and the PRP equipment sold in the market is quite expensive and is greatly limited in clinical application and popularization, particularly in application in developing countries. At present, the method for solving the preparation problem of PRP is worth further exploration, so that a convenient and rapid preparation method with high purity and concentration and low cost is obtained.
In addition to providing aggregation during hemostasis, platelets in humans also contain a number of growth factors involved in wound healing and bone regeneration. Such as Platelet Derived Growth Factor (PDGF), Transforming Growth Factor (TGF), Insulin-like Growth Factor (IGF), Epidermal Growth Factor (EGF), and Vascular Endothelial Growth Factor (VEGF), Platelet derived Growth factors promote cell differentiation and proliferation, and these Growth factors have a synergistic response to each other, interact and influence with other factors that promote cell activity, maintain the tissue environment in balance, and play an important role in wound healing, repair, and regeneration.
In addition, since PRP can be extracted from the blood of a patient and can be used for treating related diseases after extraction and enrichment treatment, the PRP has the advantages of rich sources, convenient material taking, simple preparation method and capability of being absorbed by the body. The use of self-PRP avoids the problems of viral transmission and immune rejection. PRP is therefore widely used in different medical fields. Such as orthopedics, stomatology, maxillofacial surgery, sports medicine, and cosmetic medicine.
There is no uniform preparation method for PRP so far, and the principle of PRP preparation is mainly to separate blood by centrifugation, taking advantage of the different sedimentation rates of various components in blood. Thereby obtaining plasma containing platelets and further concentrating the plasma by using the centrifugal principleObtaining high-concentration platelet plasma, wherein the concentration of platelets in human whole blood is generally 1-3 × 105And/ml. Studies suggest that the PRP platelet concentration after concentration should be 3-4 times the platelet concentration of whole blood. Research also finds that the increase of the platelet concentration in PRP can effectively improve the proliferation and differentiation capacity of stem cells and can obviously increase the proliferation of fibroblasts and the expression of type I collagen.
Thus, efficient isolation and increased platelet concentration in PRP becomes critical to the overall PRP treatment regimen.
Chinese Yushu Ru and the like (CN101402940A, China patent application No. 200810228726.5, the name of the invention is: a method for extracting mouse platelets) disclose a method for extracting mouse platelets, including steps of collecting blood from orbital veins of a mouse, placing the blood in an anticoagulation centrifuge tube, standing for 30MIN, centrifuging at 800RPM for 10MIN, collecting supernatant as platelet-rich plasma, sucking supernatant, placing the supernatant in a clean test tube, centrifuging at 3500RPM for 10MIN, discarding supernatant, collecting sediment at the bottom of the tube as platelets, adding 50-100M L mass% ammonium oxalate solution into the test tube, gently stirring with a glass rod, adding 2-3M L mass% ammonium oxalate solution, standing for 5MIN, dissolving erythrocytes, centrifuging at 3500RPM for 10MIN, discarding supernatant, adding platelet washing solution, repeatedly blowing to obtain platelet suspension, centrifuging at 3300RPM for 10MIN, collecting supernatant, adding a small amount of platelets, repeatedly blowing to obtain platelet suspension, using the platelet washing solution as an antigen, taking the platelets as a guinea pig, preparing serum, and obtaining platelet-rich plasma by a simple method, wherein the platelet-rich plasma concentration can be obtained by a simple method for obtaining high platelet-rich plasma10However, this method does not disclose the yield of platelets, and is complicated in operation and hardly applicable to the treatment of human blood.
In addition, Wangyue et al (CN 102078644A, China patent application No. 201110053979.5, the name of the invention: a simple and efficient extraction device and extraction method for autologous platelet-rich plasma) disclose a simple and efficient extraction device and extraction method for autologous platelet-rich plasma, wherein the extracted autologous platelet-rich plasma contains various composite growth factors, and can effectively promote tissue repair. The device consists of 1 injector, 1 venous transfusion device connecting pipe, 1 venous indwelling needle plastic card and 1 venous transfusion device needle head; the syringe is used for drawing venous blood and serves as a centrifugal tube; the intravenous infusion apparatus needle is connected with the outlet of the injector through an intravenous infusion apparatus connecting pipe and is used for puncturing skin blood vessels, extracting anticoagulant and activator and injecting the extracted autologous platelet-rich plasma to the required part of the human body; the venous transfusion device connecting pipe can be bent and fixed in the venous indwelling needle plastic card. It is believed that such a device is beneficial in improving the operability of platelet extraction.
Therefore, in order to obtain platelet-rich plasma having clinical therapeutic significance by enriching platelets from blood, it is highly desirable for those skilled in the art to search for a method having a simple operation process, a high yield, and a high platelet concentration in enriched plasma.
Disclosure of Invention
The object of the present invention is to find a simple and efficient method for separating and concentrating platelets from plasma; in particular, the invention seeks a method with simple operation process, high yield and high platelet concentration in the enriched plasma in order to obtain platelet-rich plasma with clinical therapeutic significance by enriching the platelets in the blood. The present inventors have found that plasma containing platelets at a high concentration can be obtained in a high yield under specific operating conditions by concentrating and separating platelets by gradient centrifugation.
To this end, the present invention provides in a first aspect a method for extracting platelet rich plasma from blood, comprising the steps of:
(a) putting the collected whole blood into a container containing an anticoagulant, and fully and uniformly mixing the blood and the anticoagulant;
(b) placing the blood mixed with the anticoagulant in a centrifuge tube, and carrying out first centrifugation to divide the blood into three layers basically;
(c) the uppermost layer and most of the middle layers are pumped out and transferred to a new centrifuge tube, and the mixture is uniformly mixed and centrifuged for the second time;
(d) the Plasma on the upper layer of the centrifuge tube is discarded, and the precipitated platelets are resuspended in the remaining Plasma, thus obtaining Platelet-Rich Plasma (PRP).
The method according to any embodiment of the first aspect of the present invention, wherein the whole blood in step (a) is fresh whole blood.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the whole blood in step (a) is whole blood of a subject, such as a patient or a healthy volunteer. Thus, the platelet rich plasma obtained according to the present invention can be conveniently re-used in the patient to treat the associated disease, or in the associated need of the healthy volunteer.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the anti-gelling agent in step (a) is selected from ethylenediaminetetraacetic Acid (EDTA) and salts thereof (e.g. disodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, calcium sodium ethylenediaminetetraacetate), sodium citrate, glucose citrate solution, or combinations thereof. The typical dosage of ethylenediaminetetraacetic acid or a salt thereof is 0.5-1.5 mg per 1ml of anticoagulation agent, for example, 0.75-1.25 mg per 1ml of anticoagulation agent, and the anticoagulation agent can be a dry powder directly coated on the inner wall of the anticoagulation tube, or a solution prepared with a suitable concentration (for example, a concentration of 1-2%) coated on the inner wall of the anticoagulation tube for use after drying. The sodium citrate is typically used in an amount of 3-5 mg per 1ml of blood anticoagulation, and may be applied directly to the inner wall of the anticoagulation tube as dry powder, or may be prepared into a solution with a suitable concentration (e.g., 3-5%) and applied to the inner wall of the anticoagulation tube after drying. A typical formulation for citric acid dextrose solution commonly used in the art comprises: 0.48g of citric acid, 1.32g of sodium citrate and 1.47g of glucose, and adding water to 100ml, wherein 0.15-0.2 ml of citric acid glucose solution is usually used for per 1ml of blood anticoagulation.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the container in step (a) may be a centrifuge tube or a blood collection bag. If the centrifuge tube is a centrifuge tube and the centrifuge tube has a volume suitable for direct centrifugation, the centrifuge tube does not need to be separately placed in the centrifuge tube in the step (b), and the centrifugation can be directly performed.
The method according to any one of the embodiments of the first aspect of the present invention, wherein step (a) further comprises taking an appropriate amount (e.g. less than 500ul, such as about 100ul) after mixing the blood and the anticoagulant for determining the number of platelets in the blood therein for subsequent process control and monitoring.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the first centrifugation in step (b) is performed to an extent that the blood is substantially separated into three layers. In one embodiment, wherein the uppermost layer is a platelet-containing plasma layer, the middle layer is a Buffy Coat layer (which contains platelets and leukocytes), and the lowermost layer is a red blood cell layer.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the centrifugation in step (b) is performed at a speed of 2000rpm to 3000rpm, such as 2200rpm to 2800rpm, such as 2300rpm to 2500rpm, such as about 2400 rpm.
The method according to any embodiment of the first aspect of the present invention, wherein the time of centrifugation in step (b) is 1-10min, such as 2-8min, such as 3-5min, such as about 4 min.
The process according to any one of the embodiments of the first aspect of the present invention, wherein the rotation speed of the centrifugation in the step (b) is 2300rpm to 2500rpm for 3 to 5 min.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the centrifugation in step (b) is performed at 2400rpm for a period of 4 min.
The method according to any embodiment of the first aspect of the present invention, wherein in step (c), said substantial part of the intermediate layer means that the intermediate layer is sucked out as completely as possible but without extracting red blood cells.
The method according to any embodiment of the first aspect of the present invention, wherein the centrifugation in step (c) is performed at a speed of 1000rpm to 2000rpm, such as 1200rpm to 1800rpm, such as 1400rpm to 1600rpm, such as about 1500 rpm.
The method according to any embodiment of the first aspect of the present invention, wherein the time of centrifugation in step (c) is between 10 and 30min, such as between 15 and 25min, such as between 18 and 22min, such as about 20 min.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the centrifugation in step (c) is performed at a rotation speed of 1400rpm to 1600rpm for a period of 18 to 22 min.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the rotation speed of the centrifugation in the step (c) is 1500rpm for 20 min.
The method according to any embodiment of the first aspect of the present invention, wherein the upper layer and the majority of the intermediate layer obtained in step (c) are mixed together, wherein the concentration of platelets is 1-5 times, such as 1.5-4 times, such as 2-3 times the concentration of platelets in whole blood before separation.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the upper layer and most of the intermediate layer obtained in step (c) are mixed uniformly, wherein the concentration of platelets is 2.5-3.5 times, such as 2.6-3.1 times, the concentration of platelets in whole blood before separation.
The method according to any embodiment of the first aspect of the present invention, wherein the step (c) further comprises taking an appropriate amount (e.g. less than 500ul, such as about 100ul) for determining the number of platelets therein after mixing the extracted uppermost and intermediate layers uniformly for subsequent process control and monitoring.
The method according to any embodiment of the first aspect of the present invention, wherein the step (d) of discarding the plasma on the upper layer of the centrifuge tube means discarding at least 2/4 of the plasma on the upper layer of the centrifuge tube. In one embodiment, in step (d), the step of discarding the plasma on the upper layer of the centrifuge tube refers to discarding at least 3/4% of the plasma on the upper layer of the centrifuge tube. It should be noted here that the upper plasma contains only low concentrations of platelets.
The Platelet Rich Plasma (PRP) according to any embodiment of the first aspect of the present invention can be obtained by resuspending the precipitated platelets with the plasma remaining from the lower portion (about 2/4, or more preferably about 1/4) after discarding the plasma from the upper portion of the centrifuge tube in step (d).
The method according to any embodiment of the first aspect of the present invention, wherein the concentration of platelets in the Platelet Rich Plasma (PRP) obtained in step (d) is 5-10 times, such as 6-8 times, such as 6-7 times, such as 6.1-6.8 times the concentration of platelets in whole blood before separation.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the concentration of platelets in the Platelet Rich Plasma (PRP) obtained in step (d) is 8-10 times, such as 8.8-9.2 times, the concentration of platelets in whole blood before isolation.
The method according to any one of the embodiments of the first aspect of the present invention, wherein step (d) further comprises taking an appropriate amount (e.g. less than 500ul, such as about 100ul) for determining the number of platelets therein after obtaining Platelet Rich Plasma (PRP).
The method according to any one of the embodiments of the first aspect of the present invention, wherein said anticoagulant is further supplemented with two reagents, magnesium chloride and potassium sodium tartrate, in amounts of 50 μ g and 100 μ g, respectively, for each 1ml of blood anticoagulation.
The method according to any one of the embodiments of the first aspect of the invention, wherein the two reagents are added to the anticoagulant solution at the time of formulating the anticoagulant.
The Platelet Rich Plasma (PRP) obtained by the method has the characteristics of high concentration and high platelet recovery rate.
Further, the second aspect of the invention provides platelet rich plasma, which comprises 1-3 × 109Platelets in a concentration of 1.5 to 2.5 × 10/ml9Platelets at a concentration of/ml.
Platelet-rich plasma according to any embodiment of the second aspect of the invention substantially obtained by the method according to any embodiment of the first aspect of the invention.
The platelet-rich plasma according to any one of the embodiments of the second aspect of the present invention is prepared by a method comprising the steps of:
(a) putting the collected whole blood into a container containing an anticoagulant, and fully and uniformly mixing the blood and the anticoagulant;
(b) placing the blood mixed with the anticoagulant in a centrifuge tube, and carrying out first centrifugation to divide the blood into three layers basically;
(c) the uppermost layer and most of the middle layers are pumped out and transferred to a new centrifuge tube, and the mixture is uniformly mixed and centrifuged for the second time;
(d) the Plasma on the upper layer of the centrifuge tube is discarded, and the precipitated platelets are resuspended in the remaining Plasma, thus obtaining Platelet-Rich Plasma (PRP).
The platelet-rich plasma according to any one of the embodiments of the second aspect of the present invention, wherein the whole blood in the step (a) is fresh whole blood.
The platelet-rich plasma according to any embodiment of the second aspect of the present invention, wherein the whole blood in step (a) is whole blood of a subject such as a patient or a healthy volunteer. Thus, the platelet rich plasma obtained according to the present invention can be conveniently re-used in the patient to treat the associated disease, or in the associated need of the healthy volunteer.
The platelet rich plasma according to any embodiment of the second aspect of the present invention, wherein the anticoagulant in step (a) is selected from the group consisting of ethylenediaminetetraacetic Acid (EDTA) and salts thereof (e.g. disodium EDTA, dipotassium EDTA, calcium sodium EDTA), sodium citrate, glucose citrate solution, or combinations thereof. The typical dosage of ethylenediaminetetraacetic acid or a salt thereof is 0.5-1.5 mg per 1ml of anticoagulation agent, for example, 0.75-1.25 mg per 1ml of anticoagulation agent, and the anticoagulation agent can be a dry powder directly coated on the inner wall of the anticoagulation tube, or a solution prepared with a suitable concentration (for example, a concentration of 1-2%) coated on the inner wall of the anticoagulation tube for use after drying. The sodium citrate is typically used in an amount of 3-5 mg per 1ml of blood anticoagulation, and may be applied directly to the inner wall of the anticoagulation tube as dry powder, or may be prepared into a solution with a suitable concentration (e.g., 3-5%) and applied to the inner wall of the anticoagulation tube after drying. A typical formulation for citric acid dextrose solution commonly used in the art comprises: 0.48g of citric acid, 1.32g of sodium citrate and 1.47g of glucose, and adding water to 100ml, wherein 0.15-0.2 ml of citric acid glucose solution is usually used for per 1ml of blood anticoagulation.
The platelet-rich plasma according to any one of the embodiments of the second aspect of the present invention, wherein the container in the step (a) may be a centrifuge tube or a blood collection bag. If the centrifuge tube is a centrifuge tube and the centrifuge tube has a volume suitable for direct centrifugation, the centrifuge tube does not need to be separately placed in the centrifuge tube in the step (b), and the centrifugation can be directly performed.
The platelet rich plasma according to any embodiment of the second aspect of the present invention, wherein step (a) further comprises taking an appropriate amount (e.g., less than 500ul, such as about 100ul) after mixing the blood and anticoagulant for determining the number of platelets in the blood therein for subsequent process control and monitoring.
The platelet-rich plasma according to any one of the embodiments of the second aspect of the present invention, wherein the first centrifugation in step (b) is performed to the extent that the blood is substantially separated into three layers. In one embodiment, wherein the uppermost layer is a platelet-containing plasma layer, the middle layer is a Buffy Coat layer (which contains platelets and leukocytes), and the lowermost layer is a red blood cell layer.
The platelet rich plasma according to any embodiment of the second aspect of the present invention, wherein the rotation speed of centrifugation in step (b) is in the range of 2000rpm to 3000rpm, such as 2200rpm to 2800rpm, such as 2300rpm to 2500rpm, such as about 2400 rpm.
The platelet rich plasma according to any embodiment of the second aspect of the invention, wherein the time of centrifugation in step (b) is 1-10min, such as 2-8min, such as 3-5min, such as about 4 min.
The platelet-rich plasma according to any one of the embodiments of the second aspect of the present invention, wherein the rotation speed of the centrifugation in the step (b) is 2300rpm to 2500rpm for 3 to 5 min.
The platelet-rich plasma according to any of the embodiments of the second aspect of the present invention, wherein the centrifugation in step (b) is performed at 2400rpm for 4 min.
The platelet-rich plasma according to any embodiment of the second aspect of the present invention, wherein in step (c), the majority of the intermediate layer is the intermediate layer that is sucked out as completely as possible without sucking out red blood cells.
The platelet rich plasma according to any embodiment of the second aspect of the present invention, wherein the rotation speed of the centrifugation in step (c) is 1000rpm to 2000rpm, such as 1200rpm to 1800rpm, such as 1400rpm to 1600rpm, such as about 1500 rpm.
The platelet rich plasma according to any embodiment of the second aspect of the invention, wherein the time of centrifugation in step (c) is 10-30min, such as 15-25min, such as 18-22min, such as about 20 min.
The platelet-rich plasma according to any of the embodiments of the second aspect of the present invention, wherein the centrifugation in the step (c) is performed at 1400rpm to 1600rpm for 18 to 22 min.
The platelet-rich plasma according to any of the embodiments of the second aspect of the present invention, wherein the centrifugation in step (c) is performed at 1500rpm for 20 min.
The platelet-rich plasma according to any of the embodiments of the second aspect of the present invention, wherein the upper layer and most of the intermediate layer obtained in step (c) are mixed uniformly, wherein the concentration of platelets is 1-5 times, such as 1.5-4 times, such as 2-3 times, the concentration of platelets in whole blood before separation.
The platelet-rich plasma according to any of the embodiments of the second aspect of the present invention, wherein the upper layer and most of the intermediate layer obtained in step (c) are mixed uniformly, wherein the concentration of platelets is 2.5 to 3.5 times, for example 2.6 to 3.1 times, the concentration of platelets in the whole blood before separation.
The platelet-rich plasma according to any embodiment of the second aspect of the present invention, wherein the step (c) further comprises taking an appropriate amount (e.g. less than 500ul, e.g. about 100ul) for determining the number of platelets therein after mixing the extracted uppermost layer and the middle layer uniformly for subsequent process control and monitoring.
The platelet-rich plasma according to any embodiment of the second aspect of the present invention, wherein the step (d) of discarding the plasma on the upper layer of the centrifuge tube means discarding at least 2/4 of the plasma on the upper layer of the centrifuge tube. In one embodiment, in step (d), the step of discarding the plasma on the upper layer of the centrifuge tube refers to discarding at least 3/4% of the plasma on the upper layer of the centrifuge tube. It should be noted here that the upper plasma contains only low concentrations of platelets.
The platelet-rich plasma (PRP) according to any embodiment of the second aspect of the present invention can be obtained by discarding the plasma in the upper layer of the centrifuge tube in step (d), and resuspending the precipitated platelets with the plasma remaining in the lower portion (about 2/4, or more preferably about 1/4).
The platelet rich plasma according to any embodiment of the second aspect of the present invention, wherein the concentration of platelets in the Platelet Rich Plasma (PRP) obtained in step (d) is 5-10 times, such as 6-8 times, such as 6-7 times, such as 6.1-6.8 times the concentration of platelets in whole blood before separation.
The platelet rich plasma according to any embodiment of the second aspect of the present invention, wherein the concentration of platelets in the Platelet Rich Plasma (PRP) obtained in step (d) is 8-10 times, such as 8.8-9.2 times, the concentration of platelets in whole blood before separation.
The platelet rich plasma according to any embodiment of the second aspect of the present invention, wherein step (d) further comprises taking an appropriate amount (e.g. less than 500ul, e.g. about 100ul) for determining the number of platelets therein after obtaining the Platelet Rich Plasma (PRP).
The platelet-rich plasma according to any one of the embodiments of the second aspect of the present invention, wherein the anticoagulant is further supplemented with two reagents of magnesium chloride and potassium sodium tartrate in amounts of 50 μ g and 100 μ g, respectively, for their use in anticoagulation of blood per 1 ml.
The platelet rich plasma according to any one of the embodiments of the second aspect of the present invention, wherein the two reagents are added to the anticoagulant solution at the time of formulating the anticoagulant.
It is well known that platelet rich plasma PRP can be used to prepare health products. For example, the health products are applied in orthopedics, stomatology, maxillofacial surgery, sports medicine, and cosmetic medicine.
Thus, a third aspect of the invention provides the use of platelet rich plasma PRP for the preparation of a health product. For example, the health products are applied in orthopedics, stomatology, maxillofacial surgery, sports medicine, and cosmetic medicine.
The use according to any embodiment of the third aspect of the invention, wherein the platelet rich plasma comprises 1-3 × 109Platelets in a concentration of 1.5 to 2.5 × 10/ml, for example, in the platelet-rich plasma9Platelets at a concentration of/ml.
The use according to any embodiment of the third aspect of the invention, wherein the platelet rich plasma is substantially obtained by the method according to any embodiment of the first aspect of the invention.
The use according to any of the embodiments of the third aspect of the present invention, wherein the platelet rich plasma is prepared by a method comprising the steps of:
(a) putting the collected whole blood into a container containing an anticoagulant, and fully and uniformly mixing the blood and the anticoagulant;
(b) placing the blood mixed with the anticoagulant in a centrifuge tube, and carrying out first centrifugation to divide the blood into three layers basically;
(c) the uppermost layer and most of the middle layers are pumped out and transferred to a new centrifuge tube, and the mixture is uniformly mixed and centrifuged for the second time;
(d) the Plasma on the upper layer of the centrifuge tube is discarded, and the precipitated platelets are resuspended in the remaining Plasma, thus obtaining Platelet-Rich Plasma (PRP).
The use according to any embodiment of the third aspect of the invention, wherein the whole blood in step (a) is fresh whole blood.
The use according to any embodiment of the third aspect of the invention, wherein the whole blood in step (a) is whole blood of a subject, e.g. a patient or a healthy volunteer. Thus, the platelet rich plasma obtained according to the present invention can be conveniently re-used in the patient to treat the associated disease, or in the associated need of the healthy volunteer.
The use according to any one of the embodiments of the third aspect of the present invention, wherein the anticoagulant in step (a) is selected from ethylenediaminetetraacetic Acid (EDTA) and salts thereof (e.g. disodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, calcium sodium ethylenediaminetetraacetate), sodium citrate, glucose citrate solution, or a combination thereof. The typical dosage of ethylenediaminetetraacetic acid or a salt thereof is 0.5-1.5 mg per 1ml of anticoagulation agent, for example, 0.75-1.25 mg per 1ml of anticoagulation agent, and the anticoagulation agent can be a dry powder directly coated on the inner wall of the anticoagulation tube, or a solution prepared with a suitable concentration (for example, a concentration of 1-2%) coated on the inner wall of the anticoagulation tube for use after drying. The sodium citrate is typically used in an amount of 3-5 mg per 1ml of blood anticoagulation, and may be applied directly to the inner wall of the anticoagulation tube as dry powder, or may be prepared into a solution with a suitable concentration (e.g., 3-5%) and applied to the inner wall of the anticoagulation tube after drying. A typical formulation for citric acid dextrose solution commonly used in the art comprises: 0.48g of citric acid, 1.32g of sodium citrate and 1.47g of glucose, and adding water to 100ml, wherein 0.15-0.2 ml of citric acid glucose solution is usually used for per 1ml of blood anticoagulation.
The use according to any of the embodiments of the third aspect of the present invention, wherein the container in step (a) may be a centrifuge tube or a blood collection bag. If the centrifuge tube is a centrifuge tube and the centrifuge tube has a volume suitable for direct centrifugation, the centrifuge tube does not need to be separately placed in the centrifuge tube in the step (b), and the centrifugation can be directly performed.
The use according to any of the embodiments of the third aspect of the present invention, wherein step (a) further comprises taking an appropriate amount (e.g. less than 500ul, such as about 100ul) after mixing the blood and the anticoagulant for determining the number of platelets in the blood therein for subsequent process control and monitoring.
The use according to any of the embodiments of the third aspect of the present invention, wherein the first centrifugation in step (b) is performed to an extent that the blood is substantially separated into three layers. In one embodiment, wherein the uppermost layer is a platelet-containing plasma layer, the middle layer is a Buffy Coat layer (which contains platelets and leukocytes), and the lowermost layer is a red blood cell layer.
Use according to any embodiment of the third aspect of the invention, wherein the rotation rate of the centrifugation in step (b) is in the range of 2000rpm to 3000rpm, such as 2200rpm to 2800rpm, such as 2300rpm to 2500rpm, such as about 2400 rpm.
Use according to any embodiment of the third aspect of the invention, wherein the time of centrifugation in step (b) is from 1 to 10min, such as from 2 to 8min, such as from 3 to 5min, such as about 4 min.
Use according to any of the embodiments of the third aspect of the present invention, wherein the rotation speed of the centrifugation in step (b) is 2300rpm to 2500rpm for 3 to 5 min.
Use according to any of the embodiments of the third aspect of the present invention, wherein the rotation speed of the centrifugation in step (b) is 2400rpm for 4 min.
Use according to any embodiment of the third aspect of the present invention, wherein in step (c), the majority of the intermediate layer is drawn as much as possible without drawing out red blood cells.
Use according to any embodiment of the third aspect of the invention, wherein the rotation rate of the centrifugation in step (c) is between 1000rpm and 2000rpm, such as between 1200rpm and 1800rpm, such as between 1400rpm and 1600rpm, such as around 1500 rpm.
Use according to any embodiment of the third aspect of the invention, wherein the time of centrifugation in step (c) is from 10 to 30min, such as from 15 to 25min, such as from 18 to 22min, such as about 20 min.
Use according to any of the embodiments of the third aspect of the present invention, wherein the centrifugation in step (c) is performed at a speed of 1400rpm to 1600rpm for a period of 18 to 22 min.
Use according to any of the embodiments of the third aspect of the present invention, wherein the rotation speed of the centrifugation in step (c) is 1500rpm for 20 min.
The use according to any of the embodiments of the third aspect of the present invention, wherein the upper layer and most of the intermediate layer obtained in step (c) are mixed uniformly, wherein the concentration of platelets is 1-5 times, such as 1.5-4 times, such as 2-3 times, the concentration of platelets in whole blood before separation.
Use according to any of the embodiments of the third aspect of the invention, wherein the upper layer and the majority of the intermediate layer obtained in step (c) are mixed uniformly, wherein the concentration of platelets is 2.5-3.5 times, such as 2.6-3.1 times, the concentration of platelets in whole blood before separation.
The use according to any of the embodiments of the third aspect of the present invention, wherein the step (c) further comprises taking an appropriate amount (e.g. less than 500ul, such as about 100ul) for determining the number of platelets therein after mixing the extracted uppermost layer and the extracted intermediate layer uniformly for subsequent process control and monitoring.
The use according to any embodiment of the third aspect of the present invention, wherein the step (d) of discarding the plasma on the upper layer of the centrifuge tube means discarding at least 2/4 of the plasma on the upper layer of the centrifuge tube. In one embodiment, in step (d), the step of discarding the plasma on the upper layer of the centrifuge tube refers to discarding at least 3/4% of the plasma on the upper layer of the centrifuge tube. It should be noted here that the upper plasma contains only low concentrations of platelets.
The Platelet Rich Plasma (PRP) according to the third aspect of the present invention can be obtained by resuspending the precipitated platelets with the plasma remaining in the lower portion (about 2/4, or more preferably about 1/4) after discarding the plasma in the upper layer of the centrifuge tube in step (d).
The use according to any of the embodiments of the third aspect of the present invention, wherein the concentration of platelets in the Platelet Rich Plasma (PRP) obtained in step (d) is 5-10 times, such as 6-8 times, such as 6-7 times, such as 6.1-6.8 times the concentration of platelets in whole blood before separation.
The use according to any of the embodiments of the third aspect of the present invention, wherein the concentration of platelets in the Platelet Rich Plasma (PRP) obtained in step (d) is 8-10 times, such as 8.8-9.2 times, the concentration of platelets in whole blood before isolation.
The use according to any of the embodiments of the third aspect of the present invention, wherein step (d) further comprises taking an appropriate amount (e.g. less than 500ul, such as about 100ul) for determining the number of platelets therein after obtaining Platelet Rich Plasma (PRP).
The use according to any of the embodiments of the third aspect of the present invention, wherein said anticoagulant is further supplemented with two reagents, magnesium chloride and potassium sodium tartrate, in amounts of 50 μ g and 100 μ g, respectively, for anticoagulation of blood per 1 ml.
The use according to any of the embodiments of the third aspect of the invention, wherein the two reagents are added to the anticoagulant solution at the time of formulating the anticoagulant.
Any technical feature possessed by any one aspect of the invention or any embodiment of that aspect is equally applicable to any other embodiment or any embodiment of any other aspect, so long as they are not mutually inconsistent, although appropriate modifications to the respective features may be made as necessary when applicable to each other. Various aspects and features of the disclosure are described further below.
All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.
In one embodiment of the invention, the whole blood is human whole blood.
In the present invention, a method for platelet enrichment using specific treatment conditions is provided. However, it will be understood by those skilled in the art that the conditions for disposal according to the present invention may be suitably modified, for example, the centrifuge device used therein. For example, a device comprising a syringe, an intravenous set connection tube, a plastic card for an intravenous needle and a needle of an intravenous set, which is used in Wangyue et al (CN 102078644A, Chinese patent application No. 201110053979.5, title of the invention: a simple and efficient autologous platelet-rich plasma extraction device and extraction method), can be used, and the device described in the document can be used in the method of the present invention, and makes the method of the present invention advantageous in keeping the cleanliness of the operation process.
Surprisingly, the invention has found that in the process of researching the concentration and separation of the platelets by the gradient centrifugation method, a method with simple operation process, high yield and high concentration of the platelets in the enriched plasma is obtained.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
In the following specific examples, when referring to the anticoagulant used, ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate and dipotassium ethylenediaminetetraacetate are used after being dried by preparing a solution having a concentration of 1 to 2% on the inner wall of the anticoagulant tube, and anticoagulated with 1ml of blood per 0.8 mg; the sodium citrate is prepared into a solution with the concentration of 4 percent and is coated on the inner wall of the anticoagulation tube for use after being dried, and every 4mg of the sodium citrate is used for anticoagulation of 1ml of blood; the formula of the citric acid glucose solution comprises: citric acid 0.48g, sodium citrate 1.32g, glucose 1.47g, adding water to 100ml, per 1ml blood anticoagulation usually using the citric acid glucose solution 0.18 ml.
Example 1: extraction of Platelet Rich Plasma (PRP)
(1) Whole blood of a subject is collected into a container containing an anticoagulant, and the container is a blood collection anticoagulation tube capable of being centrifuged. Ethylenediaminetetraacetic acid was used as an anticoagulant.
(2) The blood and the anticoagulant are fully and uniformly mixed, so that the coagulation is avoided. 100 microliters were extracted to calculate the platelet count.
(3) The blood was dispensed into centrifuge tubes (15 ml).
(4) The tube was placed in a centrifuge and centrifuged at 2400rpm for 4 minutes.
(5) The centrifuged blood should be divided into three layers: the bottom layer is red blood cells, the top layer is plasma containing platelets, and the middle layer is buffer Coat containing platelets and white blood cells.
(6) The uppermost plasma and most of the buffy coat were removed by pipette and transferred to a new centrifuge tube to avoid extracting red blood cells as much as possible. 100 microliters were extracted to calculate the platelet count.
(7) The tube containing the mixture of plasma and buffy coat was placed in a centrifuge and centrifuged at 1500rpm for 20 minutes.
(8) Low Platelet Plasma (PPP) at 3/4 concentration was removed, and the precipitated platelets were resuspended in the remaining 1/4 Plasma to obtain Platelet Rich Plasma (PRP).
(9) 100 microliters of PRP was extracted for platelet counting.
And (3) calculating test data:
(1) platelet concentration in Whole blood before separation 250 × 106The total platelet count is 2.5 × 10, the whole blood volume is 10ml9。
(2) Platelet concentration in plasma after first centrifugation 570 × 106Perml (2.28 fold concentration), plasma volume 4ml total platelet count 2.26 × 109. The recovery was 90.4%.
(3) Platelet concentration in plasma (PRP) after second centrifugation 1680 × 106(6.72-fold concentration, which means that the platelet concentration in the obtained platelet-rich plasma is a multiple of the platelet concentration in whole blood before separation, which may be referred to as final fold enrichment in the present invention), and the plasma volume is 1.2ml to obtain platelet-rich plasma the total platelet count is 2.016 × 109. The final recovery rate was 80.6% (this final recovery rate means that the number of platelets in the resulting platelet-rich plasma was a percentage of the number of platelets in the whole blood before separation).
Example 2: extraction of Platelet Rich Plasma (PRP)
(1) Whole blood of a subject is collected into a container containing an anticoagulant, and the container is a blood collection anticoagulation tube capable of being centrifuged. Disodium edetate is used as anticoagulant.
(2) The blood and the anticoagulant are fully and uniformly mixed, so that the coagulation is avoided. 100 microliters were extracted to calculate the platelet count.
(3) The blood was dispensed into centrifuge tubes (15 ml).
(4) The tube was placed in a centrifuge and centrifuged at 2400rpm for 4 minutes.
(5) The centrifuged blood should be divided into three layers: the bottom layer is red blood cells, the top layer is plasma containing platelets, and the middle layer is buffer Coat containing platelets and white blood cells.
(6) The uppermost plasma and most of the buffy coat were removed by pipette and transferred to a new centrifuge tube to avoid extracting red blood cells as much as possible. 100 microliters were extracted to calculate the platelet count.
(7) The tube containing the mixture of plasma and buffy coat was placed in a centrifuge and centrifuged at 1500rpm for 20 minutes.
(8) Low Platelet Plasma (PPP) at 3/4 concentration was removed, and the precipitated platelets were resuspended in the remaining 1/4 Plasma to obtain Platelet Rich Plasma (PRP).
(9) 100 microliters of PRP was extracted for platelet counting.
And (3) calculating test data:
(1) platelet concentration in Whole blood before separation 170 × 106The total platelet count is 1.7 × 10, the whole blood volume is 10ml9。
(2) Platelet concentration in plasma 448 × 10 after first centrifugation6Perml (2.64 fold concentration), plasma volume 3.4ml total platelet count 1.52 × 109. The recovery was 89.4%.
(3) Platelet concentration in plasma (PRP) after second centrifugation 1160 × 106(6.82 times concentrated) to a plasma volume of 1.2ml to give platelet rich plasma total platelet count of 1.39 × 109. The final recovery was 81.8%.
Example 3: extraction of Platelet Rich Plasma (PRP)
(1) Whole blood of a subject is collected into a container containing an anticoagulant, and the container is a blood collection anticoagulation tube capable of being centrifuged. The anticoagulant is citric acid glucose solution.
(2) The blood and the anticoagulant are fully and uniformly mixed, so that the coagulation is avoided. 100 microliters were extracted to calculate the platelet count.
(3) The blood was dispensed into centrifuge tubes (15 ml).
(4) The tube was placed in a centrifuge and centrifuged at 2400rpm for 4 minutes.
(5) The centrifuged blood should be divided into three layers: the bottom layer is red blood cells, the top layer is plasma containing platelets, and the middle layer is buffer Coat containing platelets and white blood cells.
(6) The uppermost plasma and most of the buffy coat were removed by pipette and transferred to a new centrifuge tube to avoid extracting red blood cells as much as possible. 100 microliters were extracted to calculate the platelet count.
(7) The tube containing the mixture of plasma and buffy coat was placed in a centrifuge and centrifuged at 1500rpm for 20 minutes.
(8) Low Platelet Plasma (PPP) at 3/4 concentration was removed, and the precipitated platelets were resuspended in the remaining 1/4 Plasma to obtain Platelet Rich Plasma (PRP).
(9) 100 microliters of PRP was extracted for platelet counting.
And (3) calculating test data:
(1) platelet concentration in Whole blood before separation 210 × 106The total platelet count is 2.1 × 10, the whole blood volume is 10ml9。
(2) Platelet concentration in plasma after first centrifugation 540 × 106Perml (2.57 fold concentration), plasma volume 3.5ml total platelet count 1.89 × 109. The recovery rate was 90%.
(3) Platelet concentration in plasma (PRP) after second centrifugation 1360 × 106(6.48 times concentrated) to a plasma volume of 1.2ml to give platelet rich plasma total platelet count of 1.63 × 109. The final recovery was 77.6%.
Example 4: extraction of Platelet Rich Plasma (PRP)
(1) Whole blood of a subject is collected into a container containing an anticoagulant, and the container is a blood collection anticoagulation tube capable of being centrifuged. Sodium citrate is used as anticoagulant.
(2) The blood and the anticoagulant are fully and uniformly mixed, so that the coagulation is avoided. 100 microliters were extracted to calculate the platelet count.
(3) The blood was dispensed into centrifuge tubes (15 ml).
(4) The tube was placed in a centrifuge and centrifuged at 2300rpm for 5 minutes.
(5) The centrifuged blood should be divided into three layers: the bottom layer is red blood cells, the top layer is plasma containing platelets, and the middle layer is buffer Coat containing platelets and white blood cells.
(6) The uppermost plasma and most of the buffy coat were removed by pipette and transferred to a new centrifuge tube to avoid extracting red blood cells as much as possible. 100 microliters were extracted to calculate the platelet count.
(7) The tube containing the mixture of plasma and buffy coat was placed in a centrifuge and centrifuged at 1600rpm for 18 minutes.
(8) Low Platelet Plasma (PPP) at 3/4 concentration was removed, and the precipitated platelets were resuspended in the remaining 1/4 Plasma to obtain Platelet Rich Plasma (PRP).
(9) 100 microliters of PRP was extracted for platelet counting.
And (3) calculating test data:
(1) platelet concentration in Whole blood before separation 200 × 106The total platelet count is 2 × 109。
(2) Platelet concentration in plasma after first centrifugation 510 × 106Perml (2.55 fold concentration), plasma volume 3.5ml total platelet count 1.78 × 109. The recovery was 89.2%.
(3) Platelet concentration in plasma (PRP) 1220 × 10 after second centrifugation6(6.1-fold concentration) and a plasma volume of 1.2ml, to obtain platelet-rich plasma the total platelet count was 1.46 × 109. The final recovery was 73.2%.
Example 5: extraction of Platelet Rich Plasma (PRP)
(1) Whole blood of a subject is collected into a container containing an anticoagulant, and the container is a blood collection anticoagulation tube capable of being centrifuged. The anticoagulant is ethylene diamine tetraacetic acid dipotassium.
(2) The blood and the anticoagulant are fully and uniformly mixed, so that the coagulation is avoided. 100 microliters were extracted to calculate the platelet count.
(3) The blood was dispensed into centrifuge tubes (15 ml).
(4) The tube was placed in a centrifuge and centrifuged at 2500rpm for 3 minutes.
(5) The centrifuged blood should be divided into three layers: the bottom layer is red blood cells, the top layer is plasma containing platelets, and the middle layer is buffer Coat containing platelets and white blood cells.
(6) The uppermost plasma and most of the buffy coat were removed by pipette and transferred to a new centrifuge tube to avoid extracting red blood cells as much as possible. 100 microliters were extracted to calculate the platelet count.
(7) The tube containing the mixture of plasma and buffy coat was placed in a centrifuge and centrifuged at 1400rpm for 20 minutes.
(8) Low Platelet Plasma (PPP) at 3/4 concentration was removed, and the precipitated platelets were resuspended in the remaining 1/4 Plasma to obtain Platelet Rich Plasma (PRP).
(9) 100 microliters of PRP was extracted for platelet counting.
And (3) calculating test data:
(1) platelet concentration in Whole blood before separation 270 × 106The total platelet count is 2.7 × 10, the whole blood volume is 10ml9。
(2) Platelet concentration in plasma after first centrifugation 610 × 106Perml (2.26 fold concentration), plasma volume 4ml total platelet count 2.44 × 109. The recovery was 90.4%.
(3) Platelet concentration in plasma (PRP) after second centrifugation 1830 × 106(6.78 times concentrated) to a plasma volume of 1.2ml to give platelet rich plasma total platelet count of 2.2 × 109. The final recovery was 81.3%.
The results of examples 1 to 5 above show that the final enrichment factor of platelets is in the range of 6.1 to 6.8, and the final recovery rate of platelets is in the range of 73 to 82%. In the following examples 11 to 15, the anticoagulant in each example was supplemented with two reagents, namely, magnesium chloride and potassium sodium tartrate, and the amounts of the two reagents added to each anticoagulant were 50 μ g and 100 μ g, respectively, for each 1ml of blood anticoagulation score, and were added to the anticoagulant solution when preparing the anticoagulant. The results of examples 11 to 15 show that the final enrichment factor of platelets is within the range of 8.92 to 9.14, the final recovery rate of platelets is within the range of 91.9 to 93.6%, and both important extraction effect indexes are obviously higher than those of examples 1 to 5 without adding two reagents. However, in the supplementary experiments, it was found that if only one of the magnesium chloride and the potassium sodium tartrate was added to the anticoagulants of examples 11 to 15, the final enrichment factor was within a range of 5.8 to 6.7, and the final platelet recovery rate was within a range of 68 to 84%, which was significantly lower than the effect obtained when both of the reagents were added. In addition, in the supplementary tests according to examples 11 to 15, when the amount of ethylenediaminetetraacetic acid or a salt thereof is in the range of 0.5 to 1.5mg per 1ml of blood anticoagulation, or when the amount of sodium citrate is in the range of 3 to 5mg per 1ml of blood anticoagulation, or when the amount of citric acid glucose solution is in the range of 0.15 to 0.2ml per 1ml of blood anticoagulation, the final platelet enrichment factor is in the range of 8.9 to 9.2, and the final platelet recovery rate is in the range of 91 to 934%. Indicating that anticoagulant dosages within these ranges are suitable.
Example 11: extraction of Platelet Rich Plasma (PRP)
(1) Whole blood of a subject is collected into a container containing an anticoagulant, and the container is a blood collection anticoagulation tube capable of being centrifuged. Ethylenediaminetetraacetic acid was used as an anticoagulant.
(2) The blood and the anticoagulant are fully and uniformly mixed, so that the coagulation is avoided. 100 microliters were extracted to calculate the platelet count.
(3) The blood was dispensed into centrifuge tubes (15 ml).
(4) The tube was placed in a centrifuge and centrifuged at 2400rpm for 4 minutes.
(5) The centrifuged blood should be divided into three layers: the bottom layer is red blood cells, the top layer is plasma containing platelets, and the middle layer is buffer Coat containing platelets and white blood cells.
(6) The uppermost plasma and most of the buffy coat were removed by pipette and transferred to a new centrifuge tube to avoid extracting red blood cells as much as possible. 100 microliters were extracted to calculate the platelet count.
(7) The tube containing the mixture of plasma and buffy coat was placed in a centrifuge and centrifuged at 1500rpm for 20 minutes.
(8) Low Platelet Plasma (PPP) at 3/4 concentration was removed, and the precipitated platelets were resuspended in the remaining 1/4 Plasma to obtain Platelet Rich Plasma (PRP).
(9) 100 microliters of PRP was extracted for platelet counting.
And (3) calculating test data:
(1) platelet concentration in Whole blood before separation 250 × 106The total platelet count is 2.5 × 10, the whole blood volume is 10ml9。
(2) Platelet concentration in plasma after first centrifugation 640 × 106Perml (2.56 fold concentration), plasma volume 3.8ml total platelet count 2.43 × 109. The recovery rate was 97.3%.
(3) Platelet concentration in plasma after second centrifugation (PRP) 2230 × 106(8.92-fold concentration) and a plasma volume of 1.05ml to give platelet rich plasma the total platelet count was 2.34 × 109. The final recovery was 93.6%.
Example 12: extraction of Platelet Rich Plasma (PRP)
(1) Whole blood of a subject is collected into a container containing an anticoagulant, and the container is a blood collection anticoagulation tube capable of being centrifuged. Disodium edetate is used as anticoagulant.
(2) The blood and the anticoagulant are fully and uniformly mixed, so that the coagulation is avoided. 100 microliters were extracted to calculate the platelet count.
(3) The blood was dispensed into centrifuge tubes (15 ml).
(4) The tube was placed in a centrifuge and centrifuged at 2400rpm for 4 minutes.
(5) The centrifuged blood should be divided into three layers: the bottom layer is red blood cells, the top layer is plasma containing platelets, and the middle layer is buffer Coat containing platelets and white blood cells.
(6) The uppermost plasma and most of the buffy coat were removed by pipette and transferred to a new centrifuge tube to avoid extracting red blood cells as much as possible. 100 microliters were extracted to calculate the platelet count.
(7) The tube containing the mixture of plasma and buffy coat was placed in a centrifuge and centrifuged at 1500rpm for 20 minutes.
(8) Low Platelet Plasma (PPP) at 3/4 concentration was removed, and the precipitated platelets were resuspended in the remaining 1/4 Plasma to obtain Platelet Rich Plasma (PRP).
(9) 100 microliters of PRP was extracted for platelet counting.
And (3) calculating test data:
(1) platelet concentration in Whole blood before separation 170 × 106The total platelet count is 1.7 × 10, the whole blood volume is 10ml9。
(2) Platelet concentration in plasma after first centrifugation 515 × 106Perml (3.03-fold concentration), plasma volume 3.2ml total platelet count 1.65 × 109. The recovery rate was 97.0%.
(3) Platelet concentration in plasma (PRP) after second centrifugation 1495 × 106(8.79 times concentrated) and a plasma volume of 1.05ml to give platelet rich plasma total platelet count of 1.57 × 109. The final recovery was 92.4%.
Example 13: extraction of Platelet Rich Plasma (PRP)
(1) Whole blood of a subject is collected into a container containing an anticoagulant, and the container is a blood collection anticoagulation tube capable of being centrifuged. The anticoagulant is citric acid glucose solution.
(2) The blood and the anticoagulant are fully and uniformly mixed, so that the coagulation is avoided. 100 microliters were extracted to calculate the platelet count.
(3) The blood was dispensed into centrifuge tubes (15 ml).
(4) The tube was placed in a centrifuge and centrifuged at 2400rpm for 4 minutes.
(5) The centrifuged blood should be divided into three layers: the bottom layer is red blood cells, the top layer is plasma containing platelets, and the middle layer is buffer Coat containing platelets and white blood cells.
(6) The uppermost plasma and most of the buffy coat were removed by pipette and transferred to a new centrifuge tube to avoid extracting red blood cells as much as possible. 100 microliters were extracted to calculate the platelet count.
(7) The tube containing the mixture of plasma and buffy coat was placed in a centrifuge and centrifuged at 1500rpm for 20 minutes.
(8) Low Platelet Plasma (PPP) at 3/4 concentration was removed, and the precipitated platelets were resuspended in the remaining 1/4 Plasma to obtain Platelet Rich Plasma (PRP).
(9) 100 microliters of PRP was extracted for platelet counting.
And (3) calculating test data:
(1) platelet concentration in Whole blood before separation 210 × 106The total platelet count is 2.1 × 10, the whole blood volume is 10ml9。
(2) Platelet concentration in plasma after first centrifugation 650 × 106Perml (3.1 fold concentration), plasma volume 3.15ml total platelet count 2.05 × 109. The recovery rate was 97.5%.
(3) Platelet concentration in plasma (PRP) after second centrifugation 1920 × 106(9.14-fold concentration) and a plasma volume of 1.02ml, to obtain platelet-rich plasma, the total platelet count was 1.96 × 109. The final recovery was 93.3%.
Example 14: extraction of Platelet Rich Plasma (PRP)
(1) Whole blood of a subject is collected into a container containing an anticoagulant, and the container is a blood collection anticoagulation tube capable of being centrifuged. Sodium citrate is used as anticoagulant.
(2) The blood and the anticoagulant are fully and uniformly mixed, so that the coagulation is avoided. 100 microliters were extracted to calculate the platelet count.
(3) The blood was dispensed into centrifuge tubes (15 ml).
(4) The tube was placed in a centrifuge and centrifuged at 2300rpm for 5 minutes.
(5) The centrifuged blood should be divided into three layers: the bottom layer is red blood cells, the top layer is plasma containing platelets, and the middle layer is buffer Coat containing platelets and white blood cells.
(6) The uppermost plasma and most of the buffy coat were removed by pipette and transferred to a new centrifuge tube to avoid extracting red blood cells as much as possible. 100 microliters were extracted to calculate the platelet count.
(7) The tube containing the mixture of plasma and buffy coat was placed in a centrifuge and centrifuged at 1600rpm for 18 minutes.
(8) Low Platelet Plasma (PPP) at 3/4 concentration was removed, and the precipitated platelets were resuspended in the remaining 1/4 Plasma to obtain Platelet Rich Plasma (PRP).
(9) 100 microliters of PRP was extracted for platelet counting.
And (3) calculating test data:
(1) platelet concentration in Whole blood before separation 200 × 106The total platelet count is 2 × 109。
(2) Platelet concentration in plasma after first centrifugation 606 × 106Perml (3.03-fold concentration), plasma volume 3.22ml total platelet count 1.95 × 109. The recovery rate was 97.6%.
(3) Platelet concentration in plasma (PRP) after second centrifugation 1778 × 106(8.89 times concentrated) and a plasma volume of 1.04ml to give platelet rich plasma total platelet count of 1.85 × 109. The final recovery was 92.5%.
Example 15: extraction of Platelet Rich Plasma (PRP)
(1) Whole blood of a subject is collected into a container containing an anticoagulant, and the container is a blood collection anticoagulation tube capable of being centrifuged. The anticoagulant is ethylene diamine tetraacetic acid dipotassium.
(2) The blood and the anticoagulant are fully and uniformly mixed, so that the coagulation is avoided. 100 microliters were extracted to calculate the platelet count.
(3) The blood was dispensed into centrifuge tubes (15 ml).
(4) The tube was placed in a centrifuge and centrifuged at 2500rpm for 3 minutes.
(5) The centrifuged blood should be divided into three layers: the bottom layer is red blood cells, the top layer is plasma containing platelets, and the middle layer is buffer Coat containing platelets and white blood cells.
(6) The uppermost plasma and most of the buffy coat were removed by pipette and transferred to a new centrifuge tube to avoid extracting red blood cells as much as possible. 100 microliters were extracted to calculate the platelet count.
(7) The tube containing the mixture of plasma and buffy coat was placed in a centrifuge and centrifuged at 1400rpm for 20 minutes.
(8) Low Platelet Plasma (PPP) at 3/4 concentration was removed, and the precipitated platelets were resuspended in the remaining 1/4 Plasma to obtain Platelet Rich Plasma (PRP).
(9) 100 microliters of PRP was extracted for platelet counting.
And (3) calculating test data:
(1) platelet concentration in Whole blood before separation 270 × 106The total platelet count is 2.7 × 10, the whole blood volume is 10ml9。
(2) Platelet concentration in plasma after first centrifugation 796 × 106Perml (2.95 fold concentration), plasma volume 3.3ml total platelet count 2.63 × 109. The recovery was 97.4%.
(3) Platelet concentration in plasma (PRP) after second centrifugation 2435 × 106(9.02 times concentrated) and the plasma volume was 1.02ml, giving platelet rich plasma total platelet count of 2.48 × 109. The final recovery was 91.9%.
Platelet-rich plasma was prepared from blood samples of three clients (client A, client B, client C) by the method of example 15, and the amounts (pg/ml) of three cytokines (i.e., FGF (fibroblast growth factor), TGF- β 1 (transforming growth factor- β 1), PDGF (platelet-derived growth factor)) were measured, and the results are shown in Table 1.
Table 1: measurement results of three cytokines in platelet-rich plasma
Figure BDA0001165973150000191
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims (24)

1. A method for extracting platelet rich plasma from blood comprising the steps of:
(a) putting the collected whole blood into a container containing an anticoagulant, and fully and uniformly mixing the blood and the anticoagulant;
(b) putting the blood mixed with the anticoagulant in a centrifuge tube, and carrying out primary centrifugation to divide the blood into three layers;
(c) the uppermost layer and most of the middle layers are pumped out and transferred to a new centrifuge tube, and the mixture is uniformly mixed and centrifuged for the second time;
(d) discarding the plasma on the upper layer of the centrifuge tube, and resuspending the precipitated platelets by using the residual plasma to obtain platelet-rich plasma;
the method is characterized in that:
in the step (a), the anticoagulant is selected from ethylenediamine tetraacetic acid and salts thereof, sodium citrate, citric acid glucose solution or combination thereof, and two reagents of magnesium chloride and potassium sodium tartrate are additionally added into the anticoagulant, and the dosage of the two reagents is respectively 50 mu g and 100 mu g when the two reagents are used for per 1ml of blood anticoagulation.
2. The method according to claim 1, wherein the whole blood in step (a) is fresh whole blood.
3. The method according to claim 1, wherein the whole blood in step (a) is whole blood of a subject selected from the group consisting of a patient and a healthy volunteer.
4. The process according to claim 1, wherein the salt of ethylenediaminetetraacetic acid is selected from the group consisting of: disodium ethylene diamine tetraacetate, dipotassium ethylene diamine tetraacetate and calcium sodium ethylene diamine tetraacetate.
5. The method according to claim 1, wherein the amount of ethylenediaminetetraacetic acid or a salt thereof is 0.5 to 1.5mg per 1ml of blood anticoagulation solution.
6. The method according to claim 1, wherein the amount of sodium citrate is about 3-5 mg per 1ml of blood anticoagulation solution.
7. The method of claim 1, the citric acid dextrose solution comprising: 0.48g of citric acid, 1.32g of sodium citrate and 1.47g of glucose, and adding water to 100ml, wherein 0.15-0.2 ml of citric acid glucose solution is used for per 1ml of blood anticoagulation.
8. The method according to claim 1, wherein the container in step (a) is a centrifuge tube or a blood collection bag.
9. The method of claim 1, further comprising the step of (a) taking an appropriate amount of the blood after mixing the blood and the anticoagulant for determining the number of platelets in the blood for subsequent process control and monitoring.
10. The method according to claim 1, wherein the separation in step (b) is into three layers, the uppermost layer being a platelet-containing plasma layer, the middle layer being a buffy coat layer, and the lowermost layer being a red blood cell layer.
11. The method according to claim 1, wherein the rotation speed of the centrifugation in the step (b) is 2000rpm to 3000 rpm.
12. The method according to claim 1, wherein the centrifugation in step (b) is performed at 2300rpm to 2500rpm for 3 to 5 min.
13. The method according to claim 1, wherein the centrifugation in step (b) is carried out at 2400rpm for a period of 4 min.
14. The method according to claim 1, wherein the centrifugation in step (c) is performed at 1400rpm to 1600rpm for 18 to 22 min.
15. The method according to claim 1, wherein the upper layer and the majority of the intermediate layer obtained in step (c) are mixed to a concentration of platelets that is 1-5 times the concentration of platelets in whole blood prior to separation.
16. The method according to claim 1, wherein the upper layer and the majority of the intermediate layer obtained in step (c) are mixed to a concentration of platelets that is 2.5 to 3.5 times the concentration of platelets in whole blood prior to separation.
17. The method of claim 1, wherein step (c) further comprises taking an appropriate amount of the platelets for subsequent process control and monitoring after the extracted uppermost and intermediate layers are mixed to homogeneity.
18. The method of claim 1, wherein the step (d) of discarding the plasma on the top of the centrifuge tube is to discard at least 2/4 of the plasma on the top of the centrifuge tube.
19. The method of claim 1, wherein the plasma in the upper layer of the centrifuge tube is discarded and the precipitated platelets are resuspended in the plasma remaining in the lower portion of the centrifuge tube in step (d).
20. The method according to claim 1, wherein the concentration of platelets in the platelet rich plasma obtained in step (d) is 5-10 times the concentration of platelets in whole blood prior to isolation.
21. The method according to claim 1, wherein step (d) further comprises taking an appropriate amount for determining the number of platelets in the platelet rich plasma after it has been obtained.
22. The method according to claim 1, wherein the two reagents are added to the anticoagulant solution at the time of formulating the anticoagulant.
23. Platelet rich plasma obtained by the method of any one of claims 1 to 22.
24. Use of platelet rich plasma prepared by the method of any one of claims 1 to 22 for the preparation of a health product for use in orthopedics, stomatology, maxillofacial surgery, sports medicine and cosmetic medicine.
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