A method of platelet preservation

Abstract

The present application discloses a method for preserving platelets. The method comprises suspending the platelets to be preserved in hypercilemic plasma and storing them in a gas-impermeable container at room temperature. The inventors have found that the simultaneous use of hypercilemic plasma suspension and gas-impermeable container storage inhibits the apoptosis, activation and impaired aggregation function of platelets during in vitro storage. In particular, the simultaneous use of hypercilemic plasma suspension and multi-layer gas-impermeable container storage also prolongs the in vivo survival of platelets after transfusion and protects their hemostatic function.

Description

Technical Field

[0001] This invention relates to platelet preservation technology, specifically to a method for preserving platelets using heavily chylous plasma and airtight containers. Background Technology

[0002] Clinically used platelets are primarily suspended in non-severely chylous plasma, stored in breathable bags, and kept at 20–24°C with continuous agitation. Their main function is to treat or prevent bleeding in patients. While there are several reasons for the platelet shortage, the excessively short shelf life (only 5 days) under standard storage conditions is a major contributing factor. The high rate of expired platelets, coupled with the high cost of specialized equipment and consumables required for platelet collection, makes it difficult for blood centers and blood banks to maintain large-scale platelet reserves, leading to the shortage. Furthermore, the high rate of expired platelets further exacerbates the shortage. Therefore, exploring new methods to extend the shelf life of platelets is urgently needed.

[0003] Plasma is extremely rare and in severe clinical shortage. Although major blood banks have attempted to improve the situation by reducing plasma waste rates, the results have been minimal. Summary of the Invention

[0004] In view of the defects or deficiencies of the existing technology, the purpose of this invention is to provide a new method for platelet preservation.

[0005] The present invention is implemented as follows: The platelet preservation method provided by the present invention includes: suspending the platelets to be preserved in heavily chylous plasma with the same ABO blood type and Rh(D) blood type as the platelets to obtain a platelet suspension, and then storing the platelet suspension in an airtight container at room temperature; wherein the heavily chylous plasma is plasma with a chyle index greater than or equal to 5; and the volume of the airtight container is greater than the volume of the suspension.

[0006] An alternative is that the volume of the airtight container is 3 to 8 times the volume of the platelet suspension.

[0007] Alternatively, the airtight container may be an airtight container made of polystyrene (PS).

[0008] The inventors discovered that suspending platelets to be preserved in heavily chylous plasma and storing them in an airtight container at room temperature can inhibit the apoptosis, activation, and impaired aggregation functions of platelets during in vitro preservation. It also prolongs their survival after transfusion and protects their hemostatic function, extending the shelf life by up to 5 days (e.g., 7 days). Therefore, this invention, by suspending platelets to be preserved in heavily chylous plasma and storing them in an airtight container at room temperature, effectively reduces platelet and plasma waste while achieving longer-term, higher-activity preservation of platelets, fully utilizing blood resources, and avoiding resulting economic losses. Attached Figure Description

[0009] Figure 1 This relates to Example 1 of the present invention, which describes the effect of heavily chylous plasma and an airtight container on the eversion of phosphatidylserine (PS) during platelet in vitro preservation. + Platelet percentage statistics; n=6, all data in the histogram were analyzed using mean±SEM; ns: no statistical difference; *p<0.05.

[0010] Figure 2 This invention's Example 2 illustrates the effect of heavily chylous plasma and airtight containers on CD62P expression during platelet in vitro preservation. + Platelet percentage statistics); n=6, all data in the histogram were analyzed using mean±SEM; ns: no statistical difference; *p<0.05.

[0011] Figure 3 The effect of heavily chylous plasma and airtight containers on platelet aggregation function during in vitro platelet preservation in Example 3 of this invention (MAR statistics); n=6, all data in the histogram are mean±SEM; ns: no statistical difference; *p<0.05; **p<0.01; ***p<0.001.

[0012] Figure 4 This relates to Example 4 of the present invention, which utilizes heavily chylous plasma and an airtight container to influence the in vivo survival time of platelets after transfusion (APC). + / APC + &FITC + Platelet analysis statistics); n=5, all data in the histogram were analyzed using mean±SEM; ns: no statistical difference; *p<0.05; **p<0.01.

[0013] Figure 5The effect of heavily chylous plasma and an airtight container on in vivo hemostasis after platelet transfusion in Example 5 of this invention (statistical analysis of carotid artery thrombosis time in mice); n=6, ns: no statistical difference; ***p<0.001. Detailed Implementation

[0014] Unless otherwise specified, the scientific and technical terms used in this article are intended for understanding by those skilled in the art.

[0015] The severely chylous plasma applicable to this invention refers to plasma components prepared in accordance with the relevant content of the "Blood Station Technical Operation Procedures (2019 Edition)" regarding component preparation and blood testing, and whose test results are qualified. Specifically, it includes: fresh frozen plasma, virus-inactivated fresh frozen plasma, frozen plasma, virus-inactivated frozen plasma, and single-donor fresh frozen plasma. At the same time, it refers to the blood chyle degree detection method in Appendix A of WS / T550-2017 "Guidelines for Quality Monitoring of Whole Blood and Blood Components", and selects plasma components with a chyle index greater than 5.

[0016] Chyloproteinases can be obtained from references GB 18467-2011 Requirements for Health Examination of Blood Donors Healthy voluntary blood donors.

[0017] Platelets can suffer from platelet storage lesions (PSLs) during preservation, which manifest as apoptosis, activation, and functional impairment with prolonged preservation time, as well as shortened in vivo survival and impaired hemostatic function after platelet transfusion. Therefore, this invention first analyzes the effects of heavily chylous plasma and airtight containers on the eversion of human platelet PS, demonstrating that the preservation method of this invention can alleviate apoptosis during in vitro platelet preservation.

[0018] Then, the effects of heavily chylous plasma and airtight containers on the expression of CD62P in human platelets were analyzed, demonstrating that the preservation method of the present invention can alleviate the activation of platelets during in vitro preservation.

[0019] Next, the effects of heavily chylous plasma and airtight containers on human platelet aggregation function were analyzed, demonstrating that the preservation method of the present invention can alleviate the damage to platelet aggregation function during in vitro preservation.

[0020] Based on the above in vitro experiments, the inventors further analyzed the ratio of heavily chylous plasma and human platelets stored in airtight containers in NOD scid mice using a NOD scid mouse platelet transfusion model, as well as the carotid artery thrombosis time in NOD scid mice that received both heavily chylous plasma and human platelets stored in airtight containers. This verified that the preservation method of the present invention can improve the in vivo survival time of platelets after transfusion while protecting their hemostatic function.

[0021] The present invention will be further illustrated by the following examples. The healthy plasma, platelets, and severely chylous plasma used in the following examples were all collected from healthy voluntary blood donors. Platelets were collected directly using a blood cell separator (Amicus 4R4580, Fenwal). Severely chylous plasma was obtained by centrifuging the collected whole blood (CL6R, Xiangyi), then transferring the qualified severely chylous plasma to another blood storage bag, and then freezing it at ≤-20℃ using a plasma freezer (XSD-24FL, Haier). Before use, the frozen severely chylous plasma was thawed in a plasma thawing device.

[0022] The airtight containers (5-layer cell culture flasks) used in the following examples were purchased from Nice, made of polystyrene, model 731002; Annexin V-FITC Apoptosis Staining / Detection was purchased from BD; APC anti-human CD62PAntibody was purchased from BD; Platelet aggregation function (adenosine diphosphate) detection kit (turbidimetric method) was purchased from Sysmex; The 20-25g male NOD scid mice used were purchased from Cyagen Biosciences; APC anti-human CD41 Antibody and FITC anti-mouse CD41 Antibody were both purchased from Biolegend; Anti-mice CD42b Antibody was purchased from Emfret; and Rhodamine was purchased from Sigma.

[0023] Example 1: The inventors discovered that severely chylous plasma and airtight containers can alleviate apoptosis during the in vitro preservation of human platelets.

[0024] 1. Preparation of fresh frozen plasma for severely chylous blood

[0025] After voluntary blood donors donate whole blood, in accordance with the relevant contents of the "Blood Station Technical Operation Procedures (2019 Edition)" on component preparation and blood testing, and with reference to Appendix A of WS / T 550-2017 "Guidelines for Quality Monitoring of Whole Blood and Component Blood" on blood chyle degree testing method, fresh frozen plasma with qualified blood test results (chyle index greater than 5) is prepared for use.

[0026] 2. Platelet collection, preparation, preservation, and grouping

[0027] Healthy adults who had not taken any medication within the past 10 days were recruited as volunteers. Two therapeutic doses of platelets were collected using a blood cell separator (Amicus 4R4580, Fenwal) and divided equally into four 0.5 therapeutic dose aliquots, randomly labeled as 1, 2, 3, and 4.

[0028] No. 1: 0.5 therapeutic dose of platelets was suspended in healthy plasma from the corresponding blood donor, with a final suspension volume of 125 mL, and was placed in a standard platelet storage bag (volume of 125 mL) as the control group;

[0029] 2: 0.5 therapeutic dose of platelets were suspended in pre-prepared heavily chylous plasma with the same ABO and Rh(D) blood type as the platelets. The final volume of the suspension was 125 mL, which was placed in a standard platelet storage bag (volume of 125 mL) and served as experimental group 1 (test1).

[0030] No. 3: 0.5 therapeutic dose of platelets were suspended in the healthy plasma of the corresponding blood donor. The final volume of the suspension was 125 mL, which was placed in a 5-layer cell culture flask (volume of 725 mL) as experimental group 2 (test2).

[0031] 4: 0.5 therapeutic dose of platelets were suspended in pre-prepared severely chylous plasma with the same ABO and Rh(D) blood types. The final volume of the suspension was 125 mL, which was placed in a 5-layer cell culture flask (volume 725 mL) as experimental group 3 (test3).

[0032] All platelets were placed in a platelet shaker and stored at 20°C–24°C with continuous shaking.

[0033] 3. PS-positive platelet detection

[0034] On days 3, 5, and 7 of storage, appropriate amounts of the suspension were taken, washed three times with PBS (centrifuged at 214g for 5 minutes), and resuspended. PS were labeled according to the Annexin V-FITC Apoptosis Staining / Detection instructions, and the percentage of Annexin V-positive platelets was detected by flow cytometry (BD FACS Canto).

[0035] Statistical analysis of Annexin V positive platelet percentage, as follows: Figure 1 As shown, the percentages of Annexin V-positive platelets on days 3, 5, and 7 in the control group were 6.6%, 9.7%, and 13.0%, respectively; in experimental group 1, they were 10.4%, 12.9%, and 16.6%; in experimental group 2, they were 4.3%, 6.3%, and 9.3%; and in experimental group 3, they were 4.3%, 6.3%, and 9.3%. Compared with the control group, the percentages in experimental group 1 were higher at the same storage period; in experimental group 2, they were higher at the same storage period; and in experimental group 3, they were lower at the same storage period, with statistically significant differences. Furthermore, there was no statistically significant difference between the 9.3% in experimental group 3 on day 7 and the 9.7% in the control group on day 5.

[0036] The results indicate that using only heavily chylous plasma suspension or only airtight containers increases the percentage of Annexin V-positive platelets. However, using both heavily chylous plasma suspension and multi-layered airtight containers simultaneously decreases the percentage of Annexin V-positive platelets. Annexin V is a fluorescent probe for PS, and PS eversion is a classic indicator of apoptosis.

[0037] Therefore, the results of this embodiment confirm that the preservation method of the present invention can alleviate apoptosis of human platelets during in vitro preservation, and can extend the preservation period of platelets from the conventional 5 days to 7 days.

[0038] Example 2: The inventors discovered that heavily chylous plasma and airtight containers can delay the activation of human platelets during in vitro preservation.

[0039] Platelet collection, preparation, preservation, and grouping were the same as in Example 1. On days 3, 5, and 7 of preservation, appropriate amounts of suspension were taken, washed three times with PBS (centrifuged at 214g for 5 minutes), and resuspended. Then, after FITC anti-human CD62P labeling, the percentage of CD62P positive platelets was detected by flow cytometry (BD FACS Canto).

[0040] Statistical analysis of the percentage of CD62P-positive platelets, such as Figure 2 As shown in the figure, the percentages of CD62P-positive platelets on days 3, 5, and 7 in the control group were 10.3%, 14.9%, and 21.9%, respectively; in experimental group 1, they were 15.4%, 18.8%, and 27.0%; in experimental group 2, they were 13.8%, 17.3%, and 25.5%; and in experimental group 3, they were 7.1%, 9.9%, and 15.1%. Compared with the control group, the percentages in experimental group 1 and experimental group 2 were all higher at the same storage period; and in experimental group 3, they were all lower at the same storage period, with statistically significant differences. Furthermore, there was no statistically significant difference between the 15.1% percentage in experimental group 3 on day 7 and the 15.4% percentage in the control group on day 5.

[0041] The results indicate that using only heavily chylous plasma suspension or only airtight containers increases the percentage of CD62P-positive platelets. However, using both heavily chylous plasma suspension and airtight containers simultaneously decreases the percentage of CD62P-positive platelets, as CD62P production is a classic indicator of platelet activation. Therefore, these results confirm that the preservation method of this invention can delay the activation of human platelets during in vitro preservation and extends the platelet storage period from the conventional 5 days to 7 days.

[0042] Example 3: The inventors found that severely chylous plasma and airtight containers can delay the functional impairment of platelet preservation.

[0043] Platelet collection, preparation, preservation, and grouping were the same as in Example 1. On days 3, 5, and 7 of preservation, appropriate amounts of suspension were taken and platelet aggregation function was analyzed using a fully automated coagulation analyzer (Sysmex CA1500) according to the instructions of the platelet aggregation function (adenosine diphosphate) test kit (turbidimetric method).

[0044] Statistical analysis of the maximum platelet aggregation rate using adenosine diphosphate as an inducer, as follows: Figure 3 As shown in the figure, the maximum aggregation rates on days 3, 5, and 7 in the control group were 31.3%, 19.8%, and 5.0%, respectively; in experimental group 1, they were 26.3%, 16.3%, and 3.7%; in experimental group 2, they were 24.0%, 15.3%, and 1.8%; and in experimental group 3, they were 61.2%, 43.8%, and 27.1%. Compared with the control group, there were no statistically significant differences among experimental groups with the same storage days. Similarly, there were no statistically significant differences among experimental groups with the same storage days. However, compared with the control group, the maximum aggregation rates in experimental group 3 were all significantly higher for the same storage days, and these differences were statistically significant. Furthermore, the 27.1% aggregation rate in experimental group 3 on day 7 was higher than the 19.8% aggregation rate in the control group on day 5, and this difference was statistically significant.

[0045] The results indicate that using only heavily chylous plasma suspension or only airtight containers does not increase the maximum platelet aggregation rate. However, using both heavily chylous plasma suspension and airtight containers simultaneously increases the maximum platelet aggregation rate. Maximum aggregation rate is a classic indicator of platelet function. Therefore, these results confirm that the preservation method of the present invention can delay functional impairment of human platelets during in vitro preservation and further suggest that the preservation period of platelets may be extended from the conventional 5 days to 7 days.

[0046] Example 4: The inventors discovered that heavily chylous plasma and airtight containers can prolong the in vivo survival period of platelets after transfusion.

[0047] Platelet collection, preparation, and storage were the same as in Example 1. The groups were divided into control and experimental groups 3, with only the control group and experimental group 3 from Example 1 retained. Furthermore, on day 5 of storage, 4 × 10⁴ platelets from the control group were collected. 8 Each, and on the 5th and 7th day of storage, 4 × 10⁴ platelets were collected from the experimental group. 8One sample was injected into NOD scid mice via the tail vein. 50 μL of peripheral blood was collected from the tail tip at 0.5, 2, and 5 hours. Red blood cells were lysed and labeled with APC anti-human CD41 Antibody and FITC anti-mouse CD41 Antibody. APC levels were detected by flow cytometry (BD FACS Canto). + / APC + &FITC + percentage.

[0048] APC + / APC + &FITC + Percentage statistical analysis such as Figure 4 As shown, the results indicated that after platelets stored in the control group for 5 days were transfused into mice, APC levels at 0.5, 2, and 5 hours were lower. + / APC + &FITC + The percentages were 23.4%, 18.8%, and 14.8%, respectively. In experimental group 3, platelets stored for 5 days were transfused into mice, and the APCs at 0.5, 2, and 5 hours were... + / APC + &FITC + The percentages were 41.6%, 31.0%, and 26.6% respectively, representing the APC at the same time point after infusion. + / APC + &FITC + All levels increased, and statistical differences were observed between the same time periods. Furthermore, after platelets from experimental group 3 were preserved for 7 days and transfused into mice, APC levels at 0.5, 2, and 5 hours were significantly higher. + / APC + &FITC + The percentages were 26.2%, 19.5%, and 17.8%, respectively. Compared with the control group stored for 5 days, the APCs at the same time point were... + / APC + &FITC + No statistically significant difference. APC + / APC + &FITC + The percentage represents the proportion of human platelets injected into NOD scid mice, which is directly proportional to the in vivo survival time of platelets after transfusion. Therefore, the preservation method of the present invention can prolong the in vivo survival time of platelets after transfusion, thus extending the preservation period of platelets.

[0049] Example 5: The inventors discovered that heavily chylous plasma and airtight containers can protect the hemostatic function of platelets.

[0050] 1. Knockout of platelets in NOD scid mice

[0051] Prepare NOD scid mice and inject Anti-mice CD42b Antibody monoclonal antibody via the tail vein according to the instructions to pre-deplete autologous platelets in the mice.

[0052] 2. Platelet transfusion

[0053] Platelet collection, preparation, and storage were the same as in Example 1. Only the control group and experimental group 3 from Example 1 were retained. On day 5 of storage, an appropriate amount of platelets from the control group was collected. On days 5 and 7 of storage, appropriate amounts of platelets from experimental group 3 were collected, 1×10⁻⁶ per animal. 9 Platelets were infused into the corresponding mice via the tail vein.

[0054] 3. Establishment of a mouse model of Fecl3 carotid artery thrombosis

[0055] ① Prepare a 0.5 mg / mL Rhodamine 6G solution and store it in the dark; ② Prepare a 100 mg / mL FeCl3 solution for later use; ③ After anesthetizing the mice treated in step 2 with 10 g / L sodium pentobarbital solution, inject 200 μL of Rhodamine 6G solution into the inner canthus vein of the mice; ④ Place the mice in a supine position, cut the skin along the midline of the neck, separate the sublingual gland under a stereomicroscope, turn it upwards, and fully expose the carotid artery; ⑤ Soak a 1 mm long and wide filter paper with 100 mg / mL FeCl3 solution and apply it to the carotid artery of the mice for 1 min; ⑥ Remove the filter paper, rinse with physiological saline, observe the thrombus formation process under a stereomicroscope, and record the thrombus formation time.

[0056] Statistical analysis of carotid artery thrombosis time in mice, as follows: Figure 5 As shown, the average thrombus formation time in experimental group 3 on day 5 was 424 seconds, shorter than the 754 seconds in the control group on day 5, with a statistically significant difference. Furthermore, the average thrombus formation time in experimental group 3 on day 7 was 733 seconds, similar to the 754 seconds in the control group on day 5, with no statistically significant difference. Thrombus formation time is inversely proportional to platelet hemostatic function; therefore, this result indicates that the method of this invention can protect the hemostatic function of platelets, further confirming that the new preservation method can extend the platelet storage period from the conventional 5 days to 7 days.

Claims

1. A method for preserving platelets, characterized in that, Platelets to be preserved are suspended in heavily chylous plasma with the same ABO and Rh(D) blood types as the platelets to obtain a platelet suspension. The platelet suspension is then stored in an airtight container at room temperature. The heavily chylous plasma is plasma with a chyle index greater than or equal to 5. The volume of the airtight container is larger than the volume of the suspension. The volume of the airtight container is 3 to 8 times the volume of the platelet suspension.

2. The platelet preservation method according to claim 1, characterized in that, The airtight container is made of polystyrene.

3. The platelet preservation method according to claim 1, characterized in that, The storage time is greater than 5 days.

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