Concentrated platelet product, production process, platelet preserving fluid and storage method

Abstract

The invention discloses a concentrated platelet product, a production process, a platelet preserving fluid and a storage method, and belongs to the technical field of biology. The production process comprises the following steps: taking fresh anticoagulant whole blood, shaking and depolymerizing; performing light centrifugation on the fresh anticoagulant whole blood subjected to shaking depolymerization to obtain upper-layer platelet-rich plasma I and lower-layer blood cells I; performing gradient centrifugation on the lower-layer blood cells I to obtain upper-layer and lower-layer red blood cells II containing an albuginea layer, plasma and a small amount of red blood cells; performing gradient centrifugation on the upper layer to obtain platelet-rich plasma II and lower-layer red blood cells III; combining the platelet-rich plasma I and the platelet-rich plasma II to obtain mixed platelet-rich plasma I; centrifuging the mixed platelet-rich plasma I to obtain upper-layer mixed platelet-rich plasma II and lower-layer red blood cells IV; centrifuging the upper-layer mixed platelet-rich plasma II to obtain upper-layer plasma and lower-layer platelets V; and adding a certain amount of plasma or platelet preserving fluid into the platelet V to obtain the concentrated platelet product.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to concentrated platelet products, production processes, platelet preservation solutions and storage methods. Background Art

[0002] Platelets (PLT) are one of the main components of blood. Their main functions are coagulation and hemostasis, and repairing damaged blood vessels. PLT is a blood cell released into the blood after the mature megakaryocytes degranulate. It has various irregular shapes, with diameters ranging from 1 to 4 μm to 7 to 8 μm, and there are great individual differences (5 to 12 cubic microns). It has no nucleus. PLT exists mixed with other cells in the blood, and is often distributed in groups between red blood cells. According to the quality requirements for concentrated platelets in the "Quality Requirements for Whole Blood and Component Blood" (GB 18469), the amount of red blood cells mixed must be ≤2.0×10 9 (derived from 400mL whole blood). In order to meet the requirements, the traditional preparation of concentrated platelets will reduce the platelet yield. Therefore, it is very necessary to study the high-purity and high-yield separation and preparation process of platelets.

[0003] At present, the concentrated platelet products used by most blood stations in China are stored in plasma, which contains allogeneic proteins such as antigens, antibodies, and thrombin. During the blood transfusion process, patients often experience fever, allergies, and other allergic reactions. Therefore, further research on platelet storage fluids that can replace plasma is extremely necessary. Summary of the invention

[0004] In view of the above-mentioned defects in the prior art, the present application first provides a production process for concentrated platelet products, and the prepared concentrated platelets have high purity, good yield, strong stability and long shelf life. On the other hand, the present application provides a storage solution for concentrated platelets and a storage method for platelets, which can maintain the stability of platelet storage for a long time, improve the quality of platelets during storage and reduce damage to platelets during storage.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present application provides a production process for concentrated platelet products, comprising:

[0007] S1: Take fresh anticoagulated whole blood and shake to disaggregate;

[0008] S2: lightly centrifuge the whole blood after shaking and disaggregation to obtain the upper layer of platelet-rich plasma I and the lower layer of blood cells I;

[0009] S3: performing gradient centrifugation on the lower layer of blood cells I to obtain an upper layer and a lower layer of red blood cells II containing a buffy coat layer, plasma and a small amount of red blood cells;

[0010] S4: performing gradient centrifugation on the upper layer containing the buffy coat layer, plasma and a small amount of red blood cells to obtain platelet-rich plasma II and lower layer red blood cells III;

[0011] S5: combining the platelet-rich plasma I and platelet-rich plasma II to obtain mixed platelet-rich plasma I;

[0012] S6: centrifuging the mixed platelet-rich plasma I to obtain an upper layer of mixed platelet-rich plasma II and a lower layer of red blood cells IV;

[0013] S7: centrifuging the upper mixed platelet-rich plasma II to obtain an upper plasma layer and a lower platelet layer V;

[0014] S8: After adding a certain amount of plasma or platelet preservation solution to the platelets V, a concentrated platelet product is obtained.

[0015] In some embodiments of the present invention, the fresh anticoagulated whole blood is obtained by collecting fresh blood from humans or animals and adding an anticoagulant.

[0016] In some embodiments of the present invention, the shaking depolymerization conditions are 20-24° C. and the time is 20-40 min.

[0017] In some embodiments of the present invention, the specific conditions of the light centrifugation in step S2 are: 150-250g, 15-20min; the conditions of the gradient centrifugation in step S3 are: 440-470g, 6-8min; 2400-2700g, 4-6min; the conditions of the gradient centrifugation in step S4 are: 260-320g, 5-7min; 450-500g, 5-7min.

[0018] In some embodiments of the present invention, the centrifugation conditions in step S6 are: 350-430g, 5-7min; the centrifugation conditions in step S7 are: 2250-2750g, 15-20min.

[0019] In some embodiments of the present invention, the composition and concentration of the platelet preservation solution are: sodium chloride 75-85mmol / L, potassium chloride 4-5mmol / L, magnesium chloride 1-3mmol / L, sodium acetate 20-24mmol / L, calcium chloride 1-2mmol / L, citric acid 3-5mmol / L, sodium bicarbonate 43-46mmol / L, glucose 12-16mmol / L, sodium citrate 9-11mmol / L, and trehalose 5-15mmol / L.

[0020] The second aspect of the present application provides a concentrated platelet product prepared by any of the above-mentioned production processes.

[0021] In some embodiments of the present invention, the platelet concentration of the concentrated platelet product is 800-1400E9 / L.

[0022] The third aspect of the present application provides a platelet preservation solution, the components and concentrations of the platelet preservation solution are: sodium chloride 75-85mmol / L, potassium chloride 4-5mmol / L, magnesium chloride 1-3mmol / L, sodium acetate 20-24mmol / L, calcium chloride 1-2mmol / L, citric acid 3-5mmol / L, sodium bicarbonate 43-46mmol / L, glucose 12-16mmol / L, sodium citrate 9-11mmol / L, trehalose 5-15mmol / L. The pH value of the platelet preservation solution is 7.2-7.4.

[0023] The fourth aspect of the present application provides a method for storing platelets, comprising: mixing a certain amount of platelets with a certain volume of the platelet preservation solution, and storing the mixture in a constant temperature oscillating box at 22±2°C.

[0024] In some embodiments of the present invention, the platelet storage method further comprises storing the platelets for 3-7 days.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides a method for separating and extracting platelets in the production process of concentrated platelet products, which can not only improve the yield but also improve the purity. The obtained platelets can be stored in the platelet storage solution prepared by the present invention to maintain the stability of platelet storage for a long time, improve the quality of platelets during storage and reduce damage during storage.

[0027] The present invention provides a novel platelet preservation solution that can effectively maintain platelet stability, improve platelet quality and reduce damage during storage, and can replace traditional plasma to preserve platelets. The platelet preservation solution contains trehalose, which is often used as a plasma substitute, is safe and harmless to the human body and can be absorbed and metabolized by the body. The preservation solution can reduce the probability of adverse reactions during blood transfusion and improve the activity and storage time of concentrated platelets;

[0028] The concentrated platelet product obtained by the process for separating and extracting platelets provided by the present invention has high purity, good yield, strong stability, long storage time, and good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The present invention is a process flow chart for producing concentrated platelet products. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described below in conjunction with specific embodiments.

[0031] Embodiment 1:

[0032] like Figure 1 As shown, the production process of concentrated platelet products provided in this application includes the following steps:

[0033] S1: Take fresh anticoagulated whole blood and shake to disaggregate;

[0034] The shaking depolymerization is carried out at 20-24°C for 20-40 minutes. Sterile fresh anticoagulated whole blood can be obtained by collecting venous blood from humans or animals and adding an anticoagulant, and the anticoagulant can be a commercially available citric acid-sodium citrate anticoagulant, or heparin.

[0035] S2: lightly centrifuge the fresh anticoagulated whole blood after shaking and disaggregation to obtain the upper layer of platelet-rich plasma I and the lower layer of blood cells I;

[0036] The specific conditions of the light centrifugation may be: 150-250 g, 15-20 min. Under the light centrifugation conditions, as many platelets as possible can be effectively retained in the plasma, while other blood cells such as red blood cells and lymphocytes can be separated.

[0037] S3: The lower layer of blood cells I is subjected to gradient centrifugation to obtain the upper layer and the lower layer of red blood cells II containing the buffy coat, plasma and a small amount of red blood cells.

[0038] The conditions for gradient centrifugation may be: 440-470 g, 6-8 min; 2400-2700 g, 4-6 min.

[0039] Specifically, the specific conditions for the first stage centrifugation of blood cells are: 440-470g, 8-6min. The first stage centrifugation is to initially separate platelets from red blood cells; the second stage centrifugation of blood cells is 2400-2700g, and the centrifugation time is 6-8min, which can make the initially separated red blood cells settle quickly and reduce platelet loss.

[0040] The applicant has found that if non-gradient centrifugation is used in this step, platelets are easily co-sedimented with red blood cells. When the centrifugal force is low, red blood cells cannot be effectively removed, and when the centrifugal force is high, more platelets are lost.

[0041] S4: performing gradient centrifugation on the upper layer containing the buffy coat layer, plasma and a small amount of red blood cells to obtain platelet-rich plasma II and lower layer red blood cells III.

[0042] The conditions for gradient centrifugation may be: 260-320 g, 5-7 min; 450-500 g, 5-7 min.

[0043] The centrifugation conditions of the first stage are 260-320g, 5-7min, to gently separate the red blood cells from the white film layer. The centrifugation conditions of the second stage are 450-500g, 5-7min, to effectively remove the red blood cells. Under the gradient centrifugation conditions, the components of the white film layer can be better maintained in the upper layer.

[0044] The applicant has found that if non-gradient centrifugation is used in step S4, the buffy coat layer will sink together with the red blood cells, resulting in a large loss of platelets.

[0045] S5: PRP I and PRP II are combined to obtain mixed PRP I, which is used for the next centrifugation step.

[0046] S6: Centrifuge the mixed platelet-rich plasma I to obtain mixed platelet-rich plasma II and red blood cells.

[0047] The centrifugation conditions may be: 350-430 g, 5-7 min. This step is to remove red blood cells from the mixed platelet-rich plasma I.

[0048] S7: Centrifuge the upper mixed platelet-rich plasma II to obtain an upper plasma layer and a lower platelet layer V.

[0049] The centrifugal conditions may be: 2250-2750 g, 15-20 min.

[0050] S8: After adding a certain amount of plasma or platelet preservation solution to the platelets V, a concentrated platelet product is obtained. The appropriate concentration of the concentrated platelet product is: 800-1400E9 / L.

[0051] Optionally, a platelet preservation solution can be prepared in advance before step S8. The composition and concentration of the platelet preservation solution provided in this application are: sodium chloride 75-85mmol / L, potassium chloride 4-5mmol / L, magnesium chloride 1-3mmol / L, sodium acetate 20-24mmol / L, calcium chloride 1-2mmol / L, citric acid 3-5mmol / L, sodium bicarbonate 43-46mmol / L, glucose 12-16mmol / L, sodium citrate 9-11mmol / L, trehalose 5-15mmol / L. The pH value of the platelet preservation solution is 7.2-7.4. The platelet preservation solution provided in this application can effectively maintain platelet stability, improve platelet quality and reduce damage during storage, and can replace traditional plasma to preserve platelets. The method for preparing a platelet preservation solution is: weigh each component according to the composition and concentration of the platelet preservation solution, mix them evenly, dissolve them with a certain amount of water for injection, then divide them into packages and sterilize them with high-pressure steam, and cool them for use.

[0052] The present application also provides a method for storing platelets prepared by the above process, wherein a certain amount of platelets is mixed with a certain volume of platelet preservation solution, and stored in a constant temperature oscillating box at 22±2°C. The platelets can be stored for 3-7 days while still maintaining good activity.

[0053] Embodiment 2:

[0054] 1. Preparation of platelet storage solution

[0055] The pH value of the platelet preservation solution of the present invention is 7.2-7.4, and the specific components are shown in Table 1 below.

[0056] Weigh each component in three batches according to the following table, mix them evenly and dissolve them in water for injection to make up to 1000 mL, then package them and sterilize them with high-pressure steam, cool them and set them aside.

[0057] Table 1 Platelet storage solution ratio composition table

[0058]

[0059] 2. Comparison of centrifugal platelet extraction processes

[0060] See also Figure 1, take 400mL of fresh sterile anticoagulated whole blood from pigs and shake it at 22℃ for 30min to disaggregate platelets. After shaking, sample and weigh the whole blood. 200g, 20min light centrifugation to obtain the upper platelet-rich plasma I and the lower blood cell I; the lower blood cell I is subjected to gradient centrifugation: 460g, 6min; 2650g, 4min, to obtain the upper layer containing buffy coat, plasma and a small amount of red blood cells and the lower red blood cell II; retain the upper layer containing buffy coat, plasma and a small amount of red blood cells obtained after centrifugation, and the lower red blood cell II can be used as raw materials for the red blood cell preparation process. The upper layer containing buffy coat, plasma and a small amount of red blood cells is gradient centrifuged again: 290g, 6min; 460g, 5min, to obtain the upper platelet-rich plasma II and the lower red blood cell III, remove the lower red blood cell III obtained by centrifugation, and retain the upper platelet-rich plasma II. The platelet-rich plasma I and platelet-rich plasma II were combined into mixed platelet-rich plasma I by a sterile pipette machine and centrifuged for the second time: 390g, 5min, to obtain the upper mixed platelet-rich plasma II and the lower red blood cells IV, retaining the obtained upper mixed platelet-rich plasma II and removing the lower red blood cells IV. After the mixed platelet-rich plasma II was centrifuged at 2500g, 15min, the upper plasma and the lower platelet V were obtained.

[0061] Repeat the above process 3 times to extract platelets, and add an equal volume of platelet preservation solution to the lower platelet V for preservation, as three example groups. Repeat the traditional centrifugation process 3 times: 150-200g, 20-30min centrifugation to separate platelets from 400mL of fresh anticoagulated whole blood of pigs, and then add an equal volume of platelet preservation solution to the lower platelet V for preservation, as three comparative groups. The volume of platelet preservation solution used in the example group and the comparative group is the same, and the results of sampling and testing are shown in Table 2 below:

[0062] Table 2 Comparison of platelet concentrate preparation processes

[0063]

[0064] Note: Platelet yield / % = volume after preparation * platelet count after preparation / (whole blood volume * whole blood platelet count)

[0065] The results show that the yield of concentrated platelets prepared by the embodiment group of the present invention is significantly higher than that of the comparative group, and the residual red blood cells, residual albumin and hemoglobin are much lower than those of the comparative group, indicating that the yield of concentrated platelets prepared by this process is high and the purity is high.

[0066] 3. Comparison of the platelet preservation effects of plasma and platelet preservation fluid

[0067] See also Figure 1, take 400mL of fresh sterile anticoagulated whole blood from pigs and place it at 22℃ to shake and disaggregate platelets for 30min. After shaking, sample and weigh the whole blood. 200g, 20min light centrifugation to obtain the upper platelet-rich plasma I and the lower blood cell I; the lower blood cell I is subjected to gradient centrifugation: 460g, 6min; 2650g, 4min, to obtain the upper layer containing buffy coat, plasma and a small amount of red blood cells and the lower red blood cell II; retain the upper layer containing buffy coat, plasma and a small amount of red blood cells obtained after centrifugation, and the lower red blood cell II can be used as raw materials for the red blood cell preparation process. The upper layer containing buffy coat, plasma and a small amount of red blood cells is gradient centrifuged again: 290g, 6min; 460g, 5min, to obtain the upper platelet-rich plasma II and the lower red blood cell III, remove the lower red blood cell III obtained by centrifugation, and retain the upper platelet-rich plasma II. The platelet-rich plasma I and platelet-rich plasma II are combined into mixed platelet-rich plasma I by a sterile pipette machine and centrifuged for a second time: 390g, 5min, to obtain an upper mixed platelet-rich plasma II and a lower red blood cell IV, retain the obtained upper mixed platelet-rich plasma II, and remove the lower red blood cell IV. After the mixed platelet-rich plasma II is centrifuged at 2500g, 15min, an upper plasma and a lower platelet V are obtained, the plasma is removed, the lower platelet V is retained, and a certain amount of platelet preservation solution or plasma is added by a sterile pipette machine, and the concentration of the concentrated platelet product finally obtained is in the appropriate range of 800-1400E9 / L.

[0068] The concentrated platelet products were prepared with the three batches of platelet storage solutions shown in Table 1, as three parallel samples (n=3) of the example group, and the concentrated platelet products prepared with plasma were used as the comparative example group. The concentrated platelet products of the example group and the comparative example group had the same volume and concentration, and were all stored in a constant temperature oscillating box at 22±2°C. The recovery rate, pH value, and platelet aggregation function of each group were tested on days 0, 1, 5, and 7, and the test results of the three parallel samples of the example group were averaged.

[0069] Table 3 Comparison of platelet recovery rate at different storage times % (n=3)

[0070] 0d 1d 5d 7d Comparative group 100 97.2±1.5 75.45±3.1 60.34±5.32 Example Group 100 98.3±0.9 83.23±2.2 72.23±3.44

[0071] Platelets are disc-shaped and often present multiple forms because they can move and deform. They often aggregate during storage, resulting in a decrease in the number of platelets. The platelet recovery rate can be used to obtain the number and aggregation of platelets in each group during storage. The above statistical results show that the use of the platelet preservation solution of the present application has a better protective effect on the platelet morphology, and the recovery rate is higher than that of the comparative group preserved with plasma.

[0072] Table 4 pH values ​​of storage solutions at different storage times (n=3)

[0073] 0d 1d 5d 7d Comparative group 7.3±0.3 7.3±0.22 6.8±0.31 6.5±0.09 Example Group 7.28±0.2 7.26±0.18 7.00±0.08 6.84±0.12

[0074] During storage, platelets will undergo glycolysis, which will lead to an increase in lactate and a decrease in pH. When the pH drops to 6.0, platelets will lose activity. The platelet preservation solution of the present application contains glucose, citrate, acetate and phosphate to provide and maintain sufficient energy metabolism, buffering capacity and anticoagulant effect and inhibit the production of lactic acid. The pH value can be stabilized during platelet storage, which is beneficial to platelet storage and can effectively prevent storage damage of platelets.

[0075] Table 5 Platelet aggregation rate at different storage times (n=3)

[0076] 0d 1d 5d 7d Comparative group 97.27±10.23 88.34±9.44 64.32±10.63 44.45±12.22 Example Group 98.22±9.45 90.45±7.46 78.74±13.54 63.89±8.43

[0077] The platelet aggregation test can reflect the platelet aggregation function. Table 5 shows that the platelet aggregation of the comparative group was significantly reduced to 65% on the 5th day, while that of the example group was maintained at 78%. There was a significant difference between the two groups on the 7th day, indicating that the platelet preservation solution has a good effect in protecting the platelet function.

[0078] The above experiments verify that the present invention can better extract high-purity platelets and improve platelet yield. In addition, the new platelet preservation solution provided can replace traditional plasma preservation, provide energy for the metabolism of concentrated platelets during storage, maintain pH stability, and stabilize platelet status and activity.

Claims

1. A production process for concentrated platelet products, It is characterized in that The steps include: S1: Take fresh anticoagulated whole blood and shake to disaggregate; S2: lightly centrifuge the fresh anticoagulated whole blood after shaking and disaggregation to obtain the upper layer of platelet-rich plasma I and the lower layer of blood cells I; S3: performing gradient centrifugation on the lower layer of blood cells I to obtain an upper layer and a lower layer of red blood cells II containing a buffy coat layer, plasma and a small amount of red blood cells; S4: performing gradient centrifugation on the upper layer containing the buffy coat layer, plasma and a small amount of red blood cells to obtain platelet-rich plasma II and lower layer red blood cells III; S5: combining the platelet-rich plasma I and platelet-rich plasma II to obtain mixed platelet-rich plasma I; S6: centrifuging the mixed platelet-rich plasma I to obtain an upper layer of mixed platelet-rich plasma II and a lower layer of red blood cells IV; S7: centrifuging the upper mixed platelet-rich plasma II to obtain an upper plasma layer and a lower platelet layer V; S8: After adding a certain amount of plasma or platelet preservation solution to the platelets V, a concentrated platelet product is obtained.

2. The production process of concentrated platelet products according to claim 1, It is characterized in that The fresh anticoagulated whole blood is obtained by collecting fresh blood from humans or animals and adding an anticoagulant.

3. The production process of concentrated platelet products according to claim 1, It is characterized in that The shaking depolymerization is carried out at a temperature of 20-24°C and for a time of 20-40 minutes.

4. The production process of concentrated platelet products according to claim 1, It is characterized in that The specific conditions of the light centrifugation in step S2 are: 150-250g, 15-20min; the conditions of the gradient centrifugation in step S3 are: 440-470g, 6-8min; 2400-2700g, 4-6min; the conditions of the gradient centrifugation in step S4 are: 260-320g, 5-7min; 450-500g, 5-7min.

5. The production process of concentrated platelet products according to claim 1, It is characterized in that The centrifugation conditions in step S6 are: 350-430 g, 5-7 min; the centrifugation conditions in step S7 are: 2250-2750 g, 15-20 min.

6. The production process of concentrated platelet products according to claim 1, It is characterized in that The components and concentrations of the platelet preservation solution are: sodium chloride 75-85mmol / L, potassium chloride 4-5mmol / L, magnesium chloride 1-3mmol / L, sodium acetate 20-24mmol / L, calcium chloride 1-2mmol / L, citric acid 3-5mmol / L, sodium bicarbonate 43-46mmol / L, glucose 12-16mmol / L, sodium citrate 9-11mmol / L, and trehalose 5-15mmol / L.

7. A concentrated platelet product prepared by the production process according to any one of claims 1 to 6, wherein the platelet concentration of the concentrated platelet product is 800-1400E9 / L.

8. A platelet preservation solution, It is characterized in that The components and concentrations of the platelet preservation solution are: sodium chloride 75-85mmol / L, potassium chloride 4-5mmol / L, magnesium chloride 1-3mmol / L, sodium acetate 20-24mmol / L, calcium chloride 1-2mmol / L, citric acid 3-5mmol / L, sodium bicarbonate 43-46mmol / L, glucose 12-16mmol / L, sodium citrate 9-11mmol / L, trehalose 5-15mmol / L, and the pH value of the platelet preservation solution is 7.2-7.

4.

9. A method for storing platelets, It is characterized in that include: A certain amount of platelets is mixed with a certain volume of platelet preservation solution, and stored in a constant temperature oscillating box at 22±2°C. The platelet preservation solution is the platelet preservation solution according to claim 8.

10. The platelet storage method according to claim 9, It is characterized in that It also includes storing the platelets for 3-7 days.

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