Preparation method and application of preoperative storage autoblood preparation composition

Abstract

The invention belongs to the technical field of medical treatment, and discloses a preparation method and application of a preoperative storage autoblood preparation composition, and the preparation method comprises the following steps: S1, collecting autologous whole blood; s2, carrying out first centrifugation on the whole blood; s3, transferring the platelet-rich plasma into a transfer bag; s4, introducing the erythrocyte preservative solution into the packed erythrocytes to obtain autologous suspension erythrocytes; s5, centrifuging the platelet-rich plasma for the second time; s6, the platelet-poor plasma is transferred into an empty bag to be frozen at a deep low temperature, and autologous frozen plasma is obtained; and S7, retaining the plasma in the blood bag with the concentrated platelets, standing the platelet-rich plasma, and uniformly shaking after natural depolymerization to obtain the autologous platelet-rich plasma. Through separation and preservation, the activity of red blood cells, blood coagulation factors and platelets is effectively reserved, intraoperative blood and PRP adjuvant therapy can be provided for patients, and tissue repair is promoted; the method is simple and convenient to operate and suitable for clinical popularization.

Description

Technical Field

[0001] This invention relates to the field of medical technology, specifically to a method for preparing a preoperative autologous blood preparation composition and its application. Background Technology

[0002] Blood consists of plasma and blood cells suspended in it. Currently, after collecting whole blood from donors, blood banks use centrifugation technology within a specified time and temperature range to separate the whole blood into individual blood components that are smaller in volume, higher in purity, have better clinical efficacy, and fewer adverse reactions. The principle is to select different centrifugal forces for separation based on the different specific gravities of the different blood components (plasma 1.025~1.030, platelets 1.030~1.060, lymphocytes 1.050~1.078, granulocytes 1.080~1.095, erythrocytes 1.090~1.111). In the process of separating whole blood to prepare different single blood components, the preparation environment is a closed system. The closed system is a disposable blood bag system (single bag capacity is 100~500 ml). Its contents are completely isolated from the external environment of the system during separation, dispensing and other processing. Several closed systems are aseptically fused together to form a new system. The new system is still a closed system, which can ensure that the blood component preparation process is aseptic.

[0003] However, the aforementioned mature and efficient blood component separation and preparation technologies are mostly applicable to blood banks' processing of allogeneic blood donations. For patients undergoing elective surgery, autologous blood storage is a recommended and safe strategy that effectively avoids the risks of allogeneic transfusions. However, current clinical practice often employs the simple method of storing the collected autologous whole blood as a whole. While red blood cells can survive for 35 days under storage conditions of 2-6°C, platelets and unstable clotting factors rapidly inactivate at this low temperature (usually losing all activity after about 5 days). This means that reinfusing autologous blood stored in existing methods can only replenish red blood cells and stable plasma proteins; it cannot provide effective clotting factor support for intraoperative and postoperative coagulation disorders, nor can it utilize autologous platelets for tissue repair.

[0004] On the other hand, platelet-rich plasma (PRP), a biological agent rich in various growth factors and produced by centrifuging and concentrating autologous platelets, has significant efficacy in promoting the repair of tissues such as bones, joints, tendons, ligaments, and wounds. However, the preparation of PRP currently mainly relies on commercial kits or expensive blood cell separators for single-donor apheresis, which means that patients face the dilemma of having to undergo both autologous blood preparation and PRP preparation before surgery, resulting in two blood collections, two separate procedures, and high costs. Summary of the Invention

[0005] This invention aims to provide a method for preparing a preoperative autologous blood preparation composition and its application, thereby addressing the problems in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for preparing a preoperative autologous blood preparation composition includes the following steps:

[0008] S1. Collect autologous whole blood into a disposable multi-unit blood bag containing its own red blood cell preservation solution, temporarily store it at 22±2℃, and separate and prepare it within 6 hours;

[0009] S2. Centrifuge the whole blood collected in step S1 for the first time to separate the upper layer of platelet-rich plasma and the lower layer of hematocrit red blood cells.

[0010] S3. Transfer the upper layer of platelet-rich plasma separated in step S2 into a transfer bag;

[0011] S4. The red blood cell preservation solution in the disposable multi-unit blood bag is introduced into the lower layer of packed red blood cells separated in step S2. After mixing, the mixture is heat-sealed and separated to obtain a bag of autologous suspended red blood cells, which are then preserved.

[0012] S5. Centrifuge the platelet-rich plasma packaged in step S3 a second time to separate the upper layer of anemic platelet plasma and the lower layer of concentrated platelets.

[0013] S6. Transfer the upper layer of anemic platelet plasma separated in step S5 to a clean and sterile empty bag, heat seal and separate it to obtain a bag of autologous frozen plasma, and preserve it; retain 20-60ml of plasma in a concentrated platelet blood bag, heat seal and separate it to obtain a bag of autologous platelet-rich plasma.

[0014] S7. The platelet-rich plasma obtained in step S6 is allowed to stand at 22±2℃ for 1-2 hours. After natural deaggregation, it is shaken well to obtain autologous platelet-rich plasma. The autologous platelet-rich plasma is then aseptically connected and aliquoted into multiple bags of several milliliters to tens of milliliters.

[0015] Furthermore, in S1, the amount of autologous whole blood collected is 200ml~400ml.

[0016] Furthermore, in S1, the disposable multi-unit blood bag includes a blood collection bag, a red blood cell preservation solution bag, and at least two transfer bags.

[0017] Furthermore, in S2, the ambient temperature for the first centrifugation is 18℃~25℃, the centrifugal force is 150g~250g, the centrifugation time is 10~20 minutes, and the centrifuge temperature is 22±2℃; where g is the acceleration due to gravity.

[0018] Furthermore, the centrifugal force for the first centrifugation was 250g, and the centrifugation time was 10 minutes.

[0019] Furthermore, in S4, the storage temperature of autologous suspended red blood cells is 2~6℃, and the shelf life is 35 days.

[0020] Furthermore, in S5, the ambient temperature for the second centrifugation is 18℃~25℃, the centrifuge temperature is 22±2℃, the centrifugal force is 1500g~3000g, and the centrifugation time is 10~20 minutes; where g is the acceleration due to gravity.

[0021] Furthermore, the centrifugal force for the second centrifugation was 1500g, and the centrifugation time was 10 minutes.

[0022] Furthermore, in S6, the method for preserving autologous frozen plasma is as follows: it is rapidly frozen at a deep low temperature and then stored in an environment below -20°C; in addition, if the amount of autologous whole blood collected in step S1 is 200ml, then 20~30ml of plasma is retained in the concentrated platelet bag; if the amount of autologous whole blood collected is 400ml, then 40~60ml of plasma is retained.

[0023] Furthermore, in S7, autologous platelet-rich plasma is stored at 22±2℃ and has a shelf life of 5 days. If autologous platelet-rich plasma needs to be aliquoted and stored for a long time, the storage temperature should be below -80℃, and it can be thawed before use.

[0024] The above-mentioned method for preparing a preoperative autologous blood preparation composition results in a blood preparation composition consisting of one bag of autologous suspended red blood cells, one bag of autologous frozen plasma, and one bag of autologous platelet-rich plasma.

[0025] The above-mentioned method for preparing autologous blood preparations for preoperative storage is used in the preparation of drugs for promoting tissue repair in surgical patients.

[0026] Furthermore, the tissue repair includes bone injury repair, joint ligament injury repair, wound healing, or postoperative scar repair.

[0027] The autologous suspended red blood cells and autologous frozen plasma prepared by the above-mentioned method for preoperative storage of autologous blood preparations are used for intraoperative reinfusion to meet the blood requirements of the surgery.

[0028] The beneficial effects of the technical solution are:

[0029] 1. The present invention provides a method for preparing a preoperative autologous blood preparation composition. Through an optimized two-stage centrifugation process, three blood components can be prepared simultaneously from a single collection of autologous whole blood: autologous suspended red blood cells, autologous frozen plasma, and autologous platelet-rich plasma (PRP). This combines the originally separate processes of "preoperative blood preparation" and "PRP preparation" into one, improving operational efficiency and blood resource utilization.

[0030] 2. The present invention provides a method for preparing a preoperative autologous blood preparation composition, which utilizes common disposable multi-unit blood bags and centrifugation equipment, avoiding the need for expensive dedicated PRP apheresis kits or blood cell separators, and reducing the overall cost for patients to prepare autologous blood and undergo PRP treatment.

[0031] 3. The present invention provides a method for preparing a preoperative autologous blood preparation composition, which uses different conditions to preserve each component (suspended red blood cells at 2~6℃, plasma frozen at -20℃, PRP shaken at 22±2 degrees or deep cryogenic storage at -80°), effectively preserving the oxygen-carrying function of red blood cells, the activity of coagulation factors in plasma, and the biological activity and growth factor concentration of platelets, overcoming the technical defects of existing simple whole-blood autologous storage where platelets and unstable coagulation factors are rapidly inactivated.

[0032] 4. The formulation composition provided by this invention (autologous suspended red blood cells, autologous frozen plasma, and autologous PRP) can be used separately or in combination according to the actual needs of patients during and after surgery. It can not only meet the basic needs of replenishing blood volume, improving oxygen carrying capacity and coagulation function, but also actively promote tissue repair and regeneration through the local application of PRP. This upgrades the current common preoperative blood preparation method for elective surgery patients, "stored autologous blood" and intraoperative single autologous whole blood reinfusion, to the rational and effective utilization of various blood components and reduces the medical costs for patients. Attached Figure Description

[0033] Figure 1 This is a flowchart of a method for preparing a preoperative autologous blood preparation composition according to the present invention;

[0034] Figure 2 This is a schematic diagram illustrating the preparation of one bag of autologous suspended red blood cells, one bag of autologous frozen plasma, and multiple bags of autologous platelet-rich plasma using autologous whole blood according to the present invention. Detailed Implementation

[0035] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments:

[0036] Example 1: As Figure 1 As shown, a method for preparing a preoperative autologous blood preparation composition includes the following steps:

[0037] S1. Collect autologous whole blood from a full cubital vein into a disposable multi-unit blood bag containing red blood cell preservation solution, temporarily store at 22±2℃, and separate and prepare the blood within 6 hours; the volume of autologous whole blood collected is 200ml or 400ml; the disposable multi-unit blood bag contains a blood collection bag (containing citrate anticoagulant), a red blood cell preservation solution bag, and at least two transfer bags;

[0038] S2. The whole blood collected in step S1 is centrifuged for the first time to separate the upper layer of platelet-rich plasma and the lower layer of hematocrit red blood cells. The centrifugation environment temperature is 18℃~25℃, the centrifugal force is 150g~250g, the centrifugation time is 10~20 minutes, and the centrifuge temperature is 22±2℃. Here, g is the acceleration due to gravity. In this embodiment, a centrifugal force of 250g and a centrifugation time of 10 minutes are further preferred.

[0039] S3. Transfer the upper layer of platelet-rich plasma separated in step S2 into a transfer bag;

[0040] S4. The red blood cell preservation solution in the disposable multi-unit blood bag is introduced into the lower layer of packed red blood cells separated in step S2. After mixing, the mixture is heat-sealed and separated to obtain a bag of autologous suspended red blood cells. The shelf life is 35 days under the condition of 2~6℃.

[0041] S5. The platelet-rich plasma packaged in step S3 is centrifuged a second time to separate the upper layer of anemic platelet plasma and the lower layer of concentrated platelets; wherein the centrifugal force of the second centrifugation is 1500g~3000g, and the centrifugation time is 10~20 minutes; where g is the acceleration due to gravity. In this embodiment, it is further preferred that the centrifugal force of the second centrifugation is 1500g and the centrifugation time is 10 minutes;

[0042] S6. Transfer the upper layer of anemic platelet plasma separated in step S5 to another empty bag, heat-seal and separate it to obtain a bag of autologous plasma. Quick-freeze it and store it in an environment below -20°C to obtain autologous frozen plasma. Keep 20-60ml of plasma in the concentrated platelet blood bag, heat-seal and separate it to obtain a bag of platelet-rich plasma. If the amount of autologous whole blood collected in step S1 is 200ml, then keep 20-30ml of plasma in the concentrated platelet bag; if the amount of autologous whole blood collected is 400ml, then keep 40-60ml of plasma.

[0043] S7. Let the platelet-rich plasma obtained in step S6 stand at 22±2℃ for 1~2 hours, and shake well after natural depolymerization to prepare autologous platelet-rich plasma; place one bag of autologous PRP in a storage environment at 22±2℃ for 5 days.

[0044] The autologous blood preparation obtained in this embodiment will be used for the following purposes:

[0045] Target audience: Patients with spinal cord injuries.

[0046] Methods / Applications: Autologous red blood cells and autologous frozen plasma are used for intraoperative reinfusion to ensure the patient's blood needs during surgery; autologous platelet-rich plasma is used for local one-time injection at the surgical site and for vertebral fusion filling to promote repair.

[0047] Results: No adverse reactions occurred during the patient's autologous suspended red blood cell and autologous frozen plasma reinfusion process and during the local treatment with autologous platelet-rich plasma. Vertebral fusion was good.

[0048] Example 2: Figure 1 and Figure 2 As shown, a method for preparing a preoperative autologous blood preparation composition includes the following steps:

[0049] S1. Collect autologous whole blood from a full cubital vein into a disposable multi-unit blood bag containing red blood cell preservation solution, temporarily store at 22±2℃, and separate and prepare the blood within 6 hours; the volume of autologous whole blood collected is 200ml or 400ml; the disposable multi-unit blood bag contains a blood collection bag (containing citrate anticoagulant), a red blood cell preservation solution bag, and at least two transfer bags;

[0050] S2. The whole blood collected in step S1 is centrifuged for the first time to separate the upper layer of platelet-rich plasma and the lower layer of hematocrit red blood cells. The centrifugation environment temperature is 18℃~25℃, the centrifugal force is 150g~250g, the centrifugation time is 10~20 minutes, and the centrifuge temperature is 22±2℃. Here, g is the acceleration due to gravity. In this embodiment, a centrifugal force of 250g and a centrifugation time of 10 minutes are further preferred.

[0051] S3. Transfer the upper layer of platelet-rich plasma separated in step S2 into a transfer bag;

[0052] S4. The red blood cell preservation solution in the disposable multi-unit blood bag is introduced into the lower layer of packed red blood cells separated in step S2. After mixing, the mixture is heat-sealed and separated to obtain a bag of autologous suspended red blood cells. The shelf life is 35 days under the condition of 2~6℃.

[0053] S5. The platelet-rich plasma packaged in step S3 is centrifuged a second time to separate the upper layer of anemic platelet plasma and the lower layer of concentrated platelets; wherein the centrifugal force of the second centrifugation is 1500g~3000g, and the centrifugation time is 10~20 minutes; where g is the acceleration due to gravity. In this embodiment, it is further preferred that the centrifugal force of the second centrifugation is 1500g and the centrifugation time is 10 minutes;

[0054] S6. Transfer the upper layer of anemic platelet plasma separated in step S5 to another empty bag, heat seal and separate it to obtain a bag of autologous plasma. Quick-freeze it and store it in an environment below -20°C to obtain autologous frozen plasma. Keep 20-60ml of plasma in the concentrated platelet blood bag, heat seal and separate it to obtain a bag of platelet-rich plasma. If the autologous whole blood collection volume in step S1 is 200ml, then keep 20-30ml of plasma; if the autologous whole blood collection volume is 400ml, then keep 40-60ml of plasma.

[0055] S7. The platelet-rich plasma obtained in step S6 is allowed to stand at 22±2℃ for 1~2 hours. After natural deagglomeration, it is shaken well to prepare autologous platelet-rich plasma. After aseptic connection, the autologous platelet-rich plasma can be aliquoted into multiple bags ranging from a few milliliters to tens of milliliters. The aliquoted autologous platelet-rich plasma is stored at -80℃ or below and should be used within six months. It can be thawed before use.

[0056] The autologous blood preparation obtained in this embodiment will be used for the following purposes:

[0057] Target audience: Patients with leg fractures and facial abrasions resulting from car accidents.

[0058] Methods / Applications: Autologous red blood cells and autologous frozen plasma are used for intraoperative reinfusion to ensure the patient's blood needs during surgery; autologous platelet-rich plasma is used for local injection at the surgical site, once every 1-2 weeks after surgery, for 3-5 consecutive times; autologous platelet-rich plasma is injected into the dermis of facial abrasions after the scab falls off, once every 1 month, for 2-3 consecutive times.

[0059] Results: No adverse reactions were observed during the reinfusion of autologous suspended red blood cells and autologous frozen plasma; no adverse reactions were observed during the injection of autologous platelet-rich plasma; the fracture site and the facial abrasion site recovered well after 3 months, with no scarring or pigmentation.

[0060] Example 3: Two patients who underwent preoperative autologous blood preparation were selected. The results of routine blood tests before collection, the amount of blood components after collection and preparation, and the results of routine blood tests are shown in Table 1.

[0061] Table 1 Comparison of whole blood volume and complete blood count before and after collection.

[0062] As shown in Table 1, the hematocrit (HCT) of autologous suspended red blood cells from Patient 1 was 0.59, and that of Patient 2 was 0.63. In this field, the HCT of suspended red blood cells should be between 0.50 and 0.65, and both met this standard. Regarding autologous platelet-rich plasma, Patient 1 showed a 6.2-fold enrichment of platelets compared to whole blood, while Patient 2 showed a 7.98-fold enrichment. The platelet count in the prepared autologous platelet-rich plasma from both patients was above 1000 × 10⁻⁶. 9 All of the above concentrations meet the current general requirements for PRP treatment.

[0063] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific technical solutions or characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a preoperative autologous blood preparation composition, characterized in that, Includes the following steps: S1. Collect autologous whole blood into a disposable multi-unit blood collection bag containing red blood cell preservation solution, temporarily store it at 22±2℃, and separate and prepare it within 6 hours; S2. Centrifuge the whole blood collected in step S1 for the first time to separate the upper layer of platelet-rich plasma and the lower layer of hematocrit red blood cells. S3. Transfer the upper layer of platelet-rich plasma separated in step S2 into a transfer bag; S4. The red blood cell preservation solution in the disposable multi-unit blood bag is introduced into the lower layer of packed red blood cells separated in step S2. After mixing, the mixture is heat-sealed and separated to obtain a bag of autologous suspended red blood cells, which are then preserved. S5. Centrifuge the platelet-rich plasma packaged in step S3 a second time to separate the upper layer of anemic platelet plasma and the lower layer of concentrated platelets. S6. Transfer the upper layer of anemic platelet plasma separated in step S5 to a clean and sterile empty bag, heat seal and separate it to obtain a bag of autologous frozen plasma, and preserve it; retain 20-60ml of plasma in the concentrated platelet blood bag according to the total amount of blood collected, heat seal and separate it to obtain a bag of platelet-rich plasma. S7. The platelet-rich plasma obtained in step S6 is left to stand at 22±2℃ for 1~2 hours. After natural deagglomeration, it is shaken well to obtain autologous platelet-rich plasma. The autologous platelet-rich plasma is then aseptically connected and packaged into multiple bags of autologous platelet-rich plasma ranging from a few milliliters to tens of milliliters.

2. The method for preparing a preoperative autologous blood preparation composition according to claim 1, characterized in that, In S1, the amount of autologous whole blood collected is 200ml~400ml.

3. The method for preparing a preoperative autologous blood preparation composition according to claim 1, characterized in that, In S1, the disposable multi-unit blood bag includes a blood collection bag, a red blood cell preservation solution bag, and at least two transfer bags.

4. The method for preparing a preoperative autologous blood preparation composition according to claim 1, characterized in that, In S2, the ambient temperature for the first centrifugation is 18℃~25℃, the centrifugal force is 150g~250g, the centrifugation time is 10~20 minutes, and the centrifuge temperature is 22±2℃; where g is the acceleration due to gravity.

5. The method for preparing a preoperative autologous blood preparation composition according to claim 4, characterized in that, The centrifugal force for the first centrifugation was 250g, and the centrifugation time was 10 minutes.

6. The method for preparing a preoperative autologous blood preparation composition according to claim 1, characterized in that, In S4, the storage temperature of autologous suspended red blood cells is 2~6℃, and the shelf life is 35 days.

7. The method for preparing a preoperative autologous blood preparation composition according to claim 1, characterized in that, In S5, the ambient temperature for the second centrifugation is 18℃~25℃, the centrifugal force is 1500g~3000g, the centrifugation time is 10~20 minutes, and the centrifuge temperature is 22±2℃; where g is the acceleration due to gravity.

8. The method for preparing a preoperative autologous blood preparation composition according to claim 7, characterized in that, The second centrifugation was performed with a centrifugal force of 1500g and a centrifugation time of 10 minutes.

9. The method for preparing a preoperative autologous blood preparation composition according to claim 2, characterized in that, In S6, the method for preserving autologous frozen plasma is as follows: it is flash-frozen and then stored in an environment below -20°C; in addition, if the amount of autologous whole blood collected in step S1 is 200ml, then 20~30ml of plasma is retained in the concentrated platelet bag; if the amount of autologous whole blood collected is 400ml, then 40~60ml of plasma is retained.

10. A method for preparing a preoperative autologous blood preparation composition according to claim 1, characterized in that, In S7, autologous platelet-rich plasma is stored at 22±2℃ and has a shelf life of 5 days. If autologous platelet-rich plasma needs to be aliquoted and stored for a long time, the storage temperature should be below -80℃, and it can be thawed before use.

11. The formulation composition prepared by the method for preparing a preoperative autologous blood preparation composition according to any one of claims 1 to 10, characterized in that, The formulation composition consists of one bag of autologous suspended red blood cells, one bag of autologous frozen plasma, and one bag of autologous platelet-rich plasma.

12. The use of autologous platelet-rich plasma prepared by the method for preparing a preoperative reserve autologous blood preparation composition according to any one of claims 1 to 10 in the preparation of a drug for promoting tissue repair in surgical patients.

13. The use of autologous platelet-rich plasma prepared by the method for preparing a preoperative reserve autologous blood preparation composition according to claim 12 in the preparation of a medicament for promoting tissue repair in surgical patients, characterized in that, The tissue repair includes bone injury repair, joint ligament injury repair, wound healing, or postoperative scar repair.

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