A method for storing platelets at room temperature

Abstract

The present invention provides a serum-free platelet preservation solution and a method for preserving platelets at room temperature based on the preservation solution. The preservation solution comprises a first component and a second component, wherein the first component comprises 60-96 parts by volume of sodium bicarbonate Ringer's injection, 2-20 parts by volume of ACD-A solution, and 2-20 parts by volume of serum albumin solution; and the second component comprises the following supplementary compounds: NaHCO3 and NaAc. After seven days of preservation of platelets by the method, CD41 + CD42b + The ratio can still be maintained at more than 93%, and the activation ability is more than 5 times that of the control group preservation solution.

Description

Technical Field

[0001] The present invention belongs to the field of platelet preservation, and in particular relates to a method for preserving platelets at room temperature. Background Art

[0002] Platelets (PLT) are important blood cells that maintain the body's hemostatic function. The number of PLT in normal human peripheral blood is (100-300)×10 9 / mL. Due to some diseases and trauma, the number of PLT in peripheral circulation decreases. When the number is less than 20×10 9 / mL will greatly increase the risk of internal bleeding and even endanger life. When blood transfusion is clinically indicated, a supply of concentrated PLT is needed.

[0003] The commonly used clinical method for preserving platelets is to store them in plasma at room temperature with shaking. Machine-collected platelets can be stored for approximately five days. However, after transfusion into the human body, the platelet storage medium, plasma, can cause a variety of transfusion-related adverse reactions, including non-hemolytic fever, allergies, and circulatory overload, which pose a significant risk to the human body.

[0004] Therefore, the development of platelet additives (PAS) to reduce plasma usage has become a research hotspot in recent years. Since Rock et al. proposed PAS in 1985, several versions have been developed, including PAS-A, PAS-B, PAS-C, PAS-D, PAS-E, PAS-F, and PAS-G. Newer platelet additives, such as M-sol (Mixture of Solutions) and BRS-A (bicarbonated Ringer's solution-citrate anticoagulant), have also been developed. However, these storage methods all present certain challenges, such as platelet storage damage, increased platelet activation, and the development of febrile transfusion reactions.

[0005] Therefore, there is an urgent need to provide a platelet preservation solution with good preservation effect and fewer side effects, so as to achieve effective preservation of platelet activity and function. Summary of the Invention

[0006] The present invention provides a serum-free platelet preservation solution at room temperature, and provides a platelet preservation method at room temperature based on the platelet preservation solution.

[0007] In a first aspect of the present invention, a platelet storage solution for room temperature storage is provided, the platelet storage solution comprising the following components:

[0008] (i) a first component comprising: 60-96 parts by volume of sodium bicarbonate Ringer's injection (BRS solution), 2-20 parts by volume of ACD-A solution, and 2-20 parts by volume of serum albumin solution; and

[0009] (ii) a second component comprising the following supplementary compounds: NaHCO 3 and NaAc, wherein the supplementary concentration C1 of NaHCO 3 is 5-15 mM, and the supplementary concentration C2 of NaAc is 5-15 mM, the supplementary concentrations being calculated based on the volume of the platelet storage solution.

[0010] In another preferred embodiment, the ratio R0 of C1 to C2 is 1 / 2-2 / 1.

[0011] In another preferred embodiment, the ratio R0 of C1 to C2 is 0.8:1-1.2:1, more preferably 1:1.

[0012] In another preferred embodiment, the added concentration C1 of NaHCO3 is 7-13 mM, preferably 8-12 mM.

[0013] In another preferred embodiment, the supplemental concentration C2 of NaAc is 7-13 mM, preferably 8-12 mM.

[0014] In another preferred embodiment, the ratio V1 / V2 of the total volume fraction V1 of the first component to the volume fraction V2 of the total volume of the second component is ≥5, preferably ≥10, more preferably ≥20, and most preferably ≥25.

[0015] In another preferred embodiment, the second component is added in solid form without changing the proportion of the first component in the platelet storage solution.

[0016] In another preferred embodiment, the normal temperature refers to 4-26°C.

[0017] In another preferred embodiment, the normal temperature is 10-26°C, preferably 15-25°C, and most preferably 20-24°C.

[0018] In another preferred embodiment, the BRS solution comprises: 97.58 mM sodium chloride, 3.99 mM potassium chloride, 27.07 mM sodium bicarbonate, 1.5 mM calcium chloride, 1 mM magnesium chloride, and 1.35 mM sodium citrate.

[0019] In another preferred embodiment, the ACD-A solution comprises: 124.12 mM glucose, 37.42 mM citric acid, and 73.76 mM sodium citrate.

[0020] In another preferred embodiment, the serum albumin solution is a serum albumin solution of mammalian origin.

[0021] In another preferred embodiment, the animal-derived serum albumin solution comprises: human serum albumin solution, bovine serum albumin solution, goat serum albumin solution, or a combination thereof.

[0022] In another preferred embodiment, the serum albumin solution contains serum.

[0023] In another preferred embodiment, the serum albumin solution is a human serum albumin (HSA) solution.

[0024] In another preferred embodiment, the first component comprises 2-20 parts by volume of HSA solution, preferably 3-18, more preferably 4-16, and most preferably 5-15.

[0025] In another preferred example, the first component comprises 10 parts by volume of HSA solution.

[0026] In another preferred embodiment, the first component comprises 2-20 parts by volume of ACD-A solution, preferably 3-18, more preferably 4-16, and most preferably 5-15.

[0027] In another preferred example, the first component comprises 10 parts by volume of ACD-A solution.

[0028] In another preferred embodiment, the preservation solution comprises the following components:

[0029] (i) a first component comprising: 80 parts by volume of a BRS solution, 10 parts by volume of an ACD-A solution, and 10 parts by volume of an HSA solution; and

[0030] (ii) a second component comprising the following supplementary compounds: NaHCO 3 and NaAc, wherein the supplementary concentration C1 of NaHCO 3 is 5-15 mM, and the supplementary concentration C2 of NaAc is 5-15 mM, the supplementary concentrations being calculated based on the volume of the platelet storage solution.

[0031] In another preferred embodiment, the ratio R0 of C1 to C2 is 1 / 2-2 / 1

[0032] In another preferred embodiment, the preservation solution comprises the following components: 10-15 mM glucose, 75-85 mM sodium chloride, 1-5 mM potassium chloride, 25-35 mM sodium bicarbonate, 0.5-1.5 mM calcium chloride, 0.6-1 mM magnesium chloride, 1-8 mM citric acid, 4-12 mM sodium citrate, 5-15 mM sodium acetate, and 5%-15% HSA.

[0033] In another preferred embodiment, 12 mM glucose, 78 mM sodium chloride, 3 mM potassium chloride, 32 mM sodium bicarbonate, 1 mM calcium chloride, 0.8 mM magnesium chloride, 4 mM citric acid, 8 mM sodium citrate, 10 mM sodium acetate, and 10% HSA.

[0034] In another preferred embodiment, the preservation solution comprises the following components: 12.41 mM glucose, 78.06 mM sodium chloride, 3.19 mM potassium chloride, 31.66 mM sodium bicarbonate, 1.2 mM calcium chloride, 0.8 mM magnesium chloride, 3.74 mM citric acid, 8.46 mM sodium citrate, 10 mM sodium acetate, and 10% HSA.

[0035] In a second aspect of the present invention, there is provided a use of the platelet preservation solution according to the first aspect of the present invention for preserving platelets.

[0036] In another preferred embodiment, it is used for washing platelets.

[0037] In another preferred embodiment, the platelets include: platelets derived from umbilical cord blood, platelets derived from peripheral blood, in vitro regenerated platelets, or a combination thereof.

[0038] In a third aspect of the present invention, there is provided a method for preparing the platelet preservation solution according to the first aspect of the present invention, comprising the following steps:

[0039] (S1) weighing, dissolving, and mixing components of the preservation solution to obtain a mixed solution;

[0040] (S2) Sterilizing the mixed solution to obtain the preservation solution according to the first aspect of the present invention.

[0041] In a fourth aspect of the present invention, a method for storing platelets at room temperature is provided, comprising the following steps: adding the platelet storage solution described in the first aspect of the present invention to the platelets, and storing the resulting platelet mixture at room temperature.

[0042] In another preferred embodiment, the platelets include: platelets derived from umbilical cord blood, platelets derived from peripheral blood, in vitro regenerated platelets, or a combination thereof.

[0043] In another preferred embodiment, the normal temperature is 10-26°C, preferably 15-25°C, and most preferably 20-24°C.

[0044] In another preferred embodiment, the preservation solution is added to a final platelet concentration of 1E8 / mL-2E9 / mL.

[0045] In another preferred embodiment, the preservation solution is added to a final platelet concentration of 1E8 / mL-1E9 / mL.

[0046] In another preferred embodiment, the storage time is ≥3 days, preferably ≥4 days, more preferably ≥5 days, and most preferably ≥7 days.

[0047] In another preferred embodiment, the method further comprises the following step: characterizing the characteristics of the platelets after storage at room temperature.

[0048] In another preferred embodiment, the room temperature storage is storage at 22°C for 7 days, and the characteristics include:

[0049] (Z1) Preservation of proplatelet CD41 + CD42b + The ratio of Y0 and CD41 in platelets after storage + CD42b + The difference Y0-Y1 of the ratio Y1 is ≤8%, preferably ≤6%, more preferably ≤4%; and / or

[0050] (Z2) The ratio N1 / N0 of the activation capacity N1 of the platelets after storage with the addition of an activator to the activation capacity N0 without the addition of an activator is ≥5, preferably ≥10; more preferably ≥15, and most preferably ≥20.

[0051] In another preferred embodiment, the activation ability is the platelet PAC-1 expression level.

[0052] In a fifth aspect of the present invention, a platelet mixture is provided, comprising: the platelet preservation solution according to the first aspect of the present invention and platelets.

[0053] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This figure shows the changes in CD42b expression levels in platelets stored in different storage solutions for 7 days. The BRS-A group showed the fastest decline, while the other three groups all maintained high CD42b expression. DETAILED DESCRIPTION

[0055] After extensive and in-depth research, the inventors unexpectedly discovered a serum-free platelet preservation solution for the first time. The platelet preservation solution contains BRS, ACD-A, HSA, NaHCO3, and NaAc. Platelets stored at room temperature based on this platelet preservation solution can reduce glucose consumption and lactic acid production during storage. Moreover, after seven days of storage at room temperature, CD41 + CD42b +The ratio can still maintain about 96%, and the activation ability still maintains more than 88%. On this basis, the present invention has been completed.

[0056] the term

[0057] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0058] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0059] As used herein, the terms "comprises," "includes," and "comprising" are used interchangeably to encompass not only closed definitions but also semi-closed and open definitions. In other words, the terms encompass "consisting of," "consisting essentially of."

[0060] Where a numerical range is provided, it is understood that every intermediate integer of that value, every tenth of each intermediate integer of that value, between the upper and lower limits of that range, and any other intermediate values in the specified range are encompassed within the present invention, unless the context clearly indicates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the present invention, subject to any express exclusions in the specified range. For example, "1 to 50" includes "2 to 25," "5 to 20," "25 to 50," "1 to 10," etc.

[0061] Platelet storage

[0062] Platelet storage is crucial. It directly impacts the efficacy of platelet transfusions. Effective platelet storage maintains platelet activity and function, ensuring timely hemostasis and life-saving for patients with thrombocytopenia and bleeding. It also prevents bacterial contamination and loss of function caused by improper storage, reducing transfusion risks. Proper storage optimizes resource utilization and ensures safe and efficient clinical blood use.

[0063] Platelet concentrates are prepared by collecting whole blood with whole blood preservatives such as ACD (citric acid-sodium citrate-dextrose), CPD (citric acid-sodium citrate-sodium dihydrogen phosphate-dextrose), or CPDA-1 or CPDA-2. The plasma from these concentrates is an excellent preservative for platelet concentrates. They should be stored at (22±2)°C with shaking. However, transfusion may cause adverse reactions such as allergic reactions to plasma and febrile transfusion reactions in some patients.

[0064] The commonly used clinical method for preserving platelets is to store them in plasma at room temperature with shaking. Machine-collected platelets can be stored for approximately five days. However, after transfusion into the human body, the platelet storage medium, plasma, can cause a variety of transfusion-related adverse reactions, including non-hemolytic fever, allergies, and circulatory overload, which pose a significant risk to the human body.

[0065] Therefore, the development of platelet additives (PAS) to reduce plasma usage has become a research hotspot in recent years. Since Rock et al. proposed PAS in 1985, various versions have been developed, including PAS-A, PAS-B, PAS-C, PAS-D, PAS-E, PAS-F, and PAS-G. Newer platelet-specific additives, such as M-sol (Mixture of Solutions) and BRS-A (bicarbonated Ringer's solution-ACD-A), have also been developed. However, these preservation methods all present certain challenges.

[0066] M-sol is typically prepared by manually mixing commercially available infusion solutions. However, the preparation process is tedious and involves complex steps, and its final composition contains plasma, which may affect the accuracy of its composition and, consequently, the quality of cryopreserved platelets. BRS-A, a simple preparation, is a mixture of the commonly used clinically available BRS and a citrate anticoagulant. However, its final composition inevitably contains plasma, and high plasma concentrations (25%-35%) can cause side effects of plasma transfusion, such as hemolysis. Importantly, when these solutions are used to store platelets, the glucose content in the solution decreases sharply, while the lactate content increases sharply in the late storage period (3-7 days). This leads to platelet activation during storage and increased CD62P expression, which can affect the normal physiological function of platelets after transfusion.

[0067] Platelet additives such as AS-Ⅱ, PAS-Ⅲ, PAS-ⅢM, and Composol are widely used clinically in Europe and the United States. They can reduce platelet activation and apoptosis during storage, reduce platelet storage damage caused by pathogen inactivation technology, and promote the detection of pathogenic microorganisms. The storage conditions are also (22±2)℃ oscillation storage. However, different platelet additives have different effects on pH and average platelet volume. Some additives may increase platelet activation. Over time, glucose decreases, lactate and lactate dehydrogenase increase, etc.

[0068] In summary, the short storage time also limits the platelet collection and preparation, and large-scale storage easily leads to expiration and scrapping. Therefore, the present invention provides a safer platelet storage method that does not contain plasma and can maintain more than 93% of CD41 after seven days of storage.+ CD42b + The platelet preservation solution has a high specific activation capacity and a high ratio of platelets.

[0069] Platelet storage solution of the present invention

[0070] The platelet storage solution of the present invention comprises a first component and a second component. The first component comprises 80% sodium bicarbonate Ringer's solution (BRS solution), 10% ACD-A solution, and 10% serum albumin solution. The second component comprises NaHCO3, NaAc, or a combination thereof. The percentages are by volume.

[0071] In a specific embodiment, the BRS solution comprises: 97.58 mM sodium chloride, 3.99 mM potassium chloride, 27.07 mM sodium bicarbonate, 1.5 mM calcium chloride, 1 mM magnesium chloride, and 1.35 mM sodium citrate.

[0072] In a specific embodiment, the ACD-A solution comprises: 124.12 mM glucose, 37.42 mM citric acid, and 73.76 mM sodium citrate.

[0073] In specific embodiments, the serum albumin solution is a human serum albumin (HSA) solution.

[0074] In a specific embodiment, the first component comprises a 10% HSA solution.

[0075] In a specific embodiment, the first component comprises a 10% ACD-A solution.

[0076] In a specific embodiment, the concentration of NaHCO3 in the second component is 10 mM.

[0077] In a specific embodiment, the concentration of NaAc in the second component is 10 mM.

[0078] In a specific embodiment, the platelet storage solution of the present invention comprises the following components: 12 mM glucose, 78 mM sodium chloride, 3 mM potassium chloride, 32 mM sodium bicarbonate, 1 mM calcium chloride, 0.8 mM magnesium chloride, 4 mM citric acid, 8 mM sodium citrate, 10 mM sodium acetate, and 10% HSA.

[0079] Method of the present invention

[0080] The method of the present invention is a method for storing platelets at room temperature based on the storage solution of the present invention. The method comprises adding the platelet storage solution of the present invention to platelets and storing the resulting platelet mixture at room temperature.

[0081] The method of the present invention eliminates the reliance on plasma, significantly improves the ability of the preservation solution to buffer pH drops, and significantly reduces lactic acid production. Therefore, it can better maintain platelet activity and achieve stable storage of platelets at room temperature.

[0082] The main advantages of the present invention include:

[0083] (a) The present invention provides a serum-free platelet preservation solution, which contains BRS, ACD-A, HSA, NaHCO3, and NaAc. Platelets stored at room temperature based on this platelet preservation solution can reduce glucose consumption and lactate production during storage.

[0084] (b) After storage at room temperature for seven days, CD41 + CD42b + The ratio can still be maintained at about 96%, the specific activation ability can still be maintained at more than 88%, and it still has good platelet aggregation ability.

[0085] (c) The platelet preservation solution of the present invention does not contain plasma, has clear components, and can be used for direct infusion or injection. Therefore, the established PLT preservation method is safe and easy to operate. It can be applied to the room temperature storage of umbilical cord blood, peripheral blood-derived, and in vitro regenerated PLTs, and can better preserve their activity and function.

[0086] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are percentages by volume and parts by volume.

[0087] Example 1: Configuration of the platelet storage solution of the present invention.

[0088] The platelet preservation solution of this invention is based on sodium bicarbonate Ringer's solution (BRS), supplemented with citrate anticoagulant (ACD-A), clinical-grade human serum albumin (HSA), sodium bicarbonate (NaHCO3), and sodium acetate (NaAc). The addition of HSA eliminates the final platelet preservation solution's reliance on plasma. NaHCO3 is an effective pH buffer, while sodium acetate can, to a certain extent, replace glucose as a metabolic substrate. Acetyl-CoA synthetase generates acetyl-CoA, which enters the tricarboxylic acid cycle directly, bypassing the glycolysis pathway and reducing lactate production. The specific formula is shown in Table 1.

[0089] Table 1 Platelet storage solution formula

[0090]

[0091] Specifically, the base solution shown in Table 1 contains 12.41 mM glucose, 78.06 mM sodium chloride, 3.19 mM potassium chloride, 21.66 mM sodium bicarbonate, 1.20 mM calcium chloride, 0.8 mM magnesium chloride, 3.74 mM citric acid, 8.46 mM sodium citrate, and 10% HSA.

[0092] For different room temperature platelet storage formulations, various parameters and properties of the platelets before and after storage were compared in Examples 2 to 6.

[0093] Platelets from healthy human peripheral blood were resuspended in platelet storage medium at a density of 5E8 / mL and stored at 22±2°C with shaking for 7 days. Samples were collected at various time points during storage (i.e., test samples).

[0094] Example 2 Glucose and lactate changes.

[0095] First, measure changes in glucose and lactate in platelet storage fluid. Add 500 μL of the sample to be tested to a 1.5 mL centrifuge tube. Centrifuge at 1000 g for 10 minutes, discard the pellet, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0096] Place the open end of the centrifuge tube containing the supernatant into a test slot on the Silman Biochemical Analyzer and name the corresponding slot on the touchscreen. Once the slot is named, tap the "Start Test" tab on the touchscreen. The instrument will automatically begin calibration and, after calibration, test the sample.

[0097] The results of glucose changes are shown in Table 2.

[0098] Table 2 Changes in glucose concentration (g / L) of platelets in different storage solutions at different time points.

[0099]

[0100] Table 2 shows that after 7 days of storage, the BRS-A (20A) group decreased the slowest, with 1.41 g / L remaining, and the BRS-A (20B) group decreased faster, with only 0.35 g / L remaining after 7 days of storage. However, in general, glucose was not completely consumed in each group.

[0101] The results of lactic acid changes are shown in Table 3.

[0102] Table 3 Changes in lactate concentration (g / L) of platelets in different storage solutions at different time points.

[0103]

[0104] The results showed that after 7 days of storage, the lactate concentration in each group increased slightly. The BRS-A (20A) group increased the slowest, with a lactate concentration of only 1.12 g / L. The BRS-A (20B) group increased faster, but still at a low level (1.82 g / L).

[0105] Example 3 Osmotic pressure and pH changes.

[0106] To detect changes in osmotic pressure, take 50 μL of the sample to be tested and place it at the bottom of a test tube specifically for osmotic pressure detection. Be careful not to have bubbles. Install the test tube onto the test probe and lower the test probe into the cooling tank of the cooling system. The instrument will automatically start detection.

[0107] The results are shown in Table 4.

[0108] Table 4 Changes in platelet osmotic pressure (mOsm / kg) in different storage solutions and at different time points.

[0109]

[0110] The results showed that all groups were within the normal physiological osmotic pressure range and did not exceed the normal physiological osmotic pressure even at the highest point.

[0111] Detect pH changes. Before use, calibrate the instrument using the three-point method. Wipe the pH electrode clean and place it in the sample to be tested. Click the "Read" icon, and the instrument will automatically measure and print the results.

[0112] The results are shown in Table 5.

[0113] Table 5 Changes in pH value of platelets in different storage solutions at different time points.

[0114]

[0115] The results showed that, with the exception of the BRS-A group, the pH values of all other groups were above 6.5. The results from the 20B and 10B+10A groups demonstrated that the addition of sodium bicarbonate to the platelet storage solution significantly enhanced the buffering capacity of the platelet storage solution to cope with decreased pH.

[0116] Example 4CD41 + CD42b + changes in expression.

[0117] Platelets were subjected to flow cytometry before and after storage. Specifically, the sample to be tested was centrifuged at 1000 g for 5 minutes, the supernatant was discarded, 500 μL of flow buffer was added, and the platelets were washed once; the sample to be tested was then resuspended with 50 μL of flow buffer; anti-human CD41 and anti-human CD42b antibodies were added, and the platelets were incubated at room temperature in the dark for 30 minutes; after the antibody incubation, the platelets were washed twice with 500 μL of flow buffer; after washing, the cells were resuspended with flow buffer, and data were acquired using Beckman's CytoFlex flow cytometer and analyzed using FlowJo software.

[0118] CD41 expression of platelets in different storage solutions and at different time points + CD42b + The results of the proportion change are shown in Table 6.

[0119] Table 6 CD41 of platelets in different storage solutions and at different time points + CD42b + proportion changes.

[0120]

[0121] The results showed that after 7 days of storage, the CD41 + CD42b + The proportion of cells decreased from 88.5% to 81.26% (a decrease of 7.24%). + CD42b + The decrease in the proportion of cells slowed down (decreased by 5.34%), while the addition of NaAc increased the decrease (decreased by 10.13%).

[0122] Unexpectedly, the addition of specific concentrations of NaHCO3 and NaAc to BRS-A (10B+10A) resulted in the + CD42b + The decrease in the proportion of cells was significantly reduced (only 3.62%), and the relative decrease percentage was about 50%.

[0123] The changes in the mean fluorescence intensity of CD42b expression at different time points are shown in Figure 2. Figure 1 and as shown in Table 7.

[0124] Table 7 Changes in mean fluorescence intensity (MFI) of CD42b expression in platelets stored in different storage solutions for 7 days.

[0125]

[0126] The results showed that the platelets in each group came from the same donor, and the mean fluorescence intensity of CD42b expression in each group before storage was the same, at 69616. After 7 days of storage, the MFI value in the BRS-A group decreased by 53507.

[0127] The addition of 20 mM NaHCO3 resulted in a decrease in CD42b in platelet storage solution after 7 days of storage. + The decrease in CD42b expression levels of the cells was slowed down (down 39550), and the addition of 20mM NaAc also led to a decrease in CD42b in the platelet storage solution after 7 days of storage. + The reduction in CD42b expression levels in the cells was also slowed down (decreased by 46461).

[0128] Unexpectedly, the 10mM NaHCO3 and 10mM NaAc in BRS-A (10B+10A) resulted in a decrease in CD42b in platelet storage solution after 7 days of storage. + The decrease in CD42b expression levels of cells was significantly slower (decreased by only 35845), with a relative decrease percentage of approximately 67%.

[0129] Example 5 Comparison of platelet aggregation properties.

[0130] In this example, changes in platelet aggregation were detected using the following method: 5E+07 cells were taken from each sample to be tested, transferred to a 1.5 mL centrifuge tube, and centrifuged at 1000 g for 5 minutes. After centrifugation, each tube was resuspended in 250 μL of human plasma and set aside. 225 μL of the sample was transferred to a glass tube. The glass tube was placed in a Helena Platelet Aggregation Assay System and assayed according to the manufacturer's recommended protocol.

[0131] The experimental results are shown in Table 8. The platelet aggregation ability of the BRS-A group decreased most significantly, while the aggregation ability of the 20B group, 20A group, and 10B-10A group changed relatively little after 7 days of storage, among which the 10B+10A group had the smallest change.

[0132] Table 8 Changes in platelet aggregation ability in different storage solutions and at different time points.

[0133]

[0134] In the BRS-A group, platelet aggregation capacity decreased from 71.4 to 5.9 (a decrease of 65.5). The addition of NaHCO3 slowed the decline in aggregation capacity (a decrease of 46.4), and the addition of NaAc slowed the decline (a decrease of 61).

[0135] Unexpectedly, the addition of specific concentrations of NaHCO3 and NaAc to BRS-A (10B+10A) resulted in a significantly reduced decrease in aggregation ability (only a 41.8% decrease), with a percentage decrease of approximately 64% relative to the BRS-A group.

[0136] Example 6 Comparison of platelet activation

[0137] In this example, platelet activation was investigated before and after storage. The method was as follows: 1 mL of the sample to be tested was centrifuged at 1000 g for 10 minutes, and the supernatant was discarded. 50 μL of a staining mixture was added to each sample and thoroughly mixed. The staining mixture included the activators thrombin receptor-activating peptide 6 (TRAP-6) and adenosine diphosphate (ADP), human CD42b antibodies, and human PAC-1 antibodies. The samples were incubated at room temperature in the dark for 30 minutes. Each sample was terminated by adding 400 μL of benchtop buffer. Data were acquired using a Beckman CytoFlex flow cytometer and analyzed using FlowJo software.

[0138] The results are shown in Table 9.

[0139] Table 9 Platelet activation capacity (PAC-1 expression percentage) in different storage solutions and at different time points.

[0140]

[0141] Note: R = activation capacity with activator added / activation capacity without activator added, the activation capacity is calculated as the percentage of PAC-1+ expression.

[0142] Results showed that platelets in the BRS-A group showed some activation even without the addition of an activator. However, after the addition of an activator, activation capacity decreased most significantly with increasing storage time. By the seventh day of storage, the specific activation capacity (activation capacity after the addition of an activator) had decreased from 95.77 before storage to 15.58 (a decrease of 80.19). PAC-1 expression after the addition of an activator (15.58) was only 1.35 times higher than that without the addition of an activator (11.52).

[0143] The addition of NaHCO₃ minimized the decline in activation capacity after the addition of the activator. By the seventh day of storage, the specific activation capacity (activation capacity after the addition of the activator) had only decreased from 95.77 before storage to 81.93 (a decrease of 13.84). PAC-1 expression after the addition of the activator was 14.42 times higher than without the activator.

[0144] The addition of NaAc reduced the decline in activation ability after the addition of the activator. By the seventh day of storage, the specific activation ability had only decreased from 95.77 before storage to 47.81 (a decrease of 47.96). PAC-1 expression after the addition of the activator was 7.39 times higher than without the activator.

[0145] Unexpectedly, on the seventh day of storage, the addition of specific concentrations of NaHCO3 and NaAc to BRS-A (10B+10A) resulted in a significant reduction in the decline in specific activation ability (from 95.77 to 84.36, a decrease of only 11.41). After adding the activator, the expression of PAC-1 was 23.5 times that of the case without the activator, making it the most favorable preservation solution formula for retaining activation ability among the three groups.

[0146] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A platelet storage solution for room temperature storage, characterized in that: The platelet preservation solution comprises the following components: (i) a first component comprising: 60-96 parts by volume of sodium bicarbonate Ringer's injection, 5-15 parts by volume of ACD-A solution, and 5-15 parts by volume of serum albumin solution; and (ii) a second component comprising the following supplementary compounds: NaHCO 3 and NaAc, wherein the supplementary concentration C1 of NaHCO 3 is 5-15 mM, and the supplementary concentration C2 of NaAc is 5-15 mM, the supplementary concentrations being calculated based on the volume of the platelet storage solution; The sodium bicarbonate Ringer's injection contains: 97.58 mM sodium chloride, 3.99 mM potassium chloride, 27.07 mM sodium bicarbonate, 1.5 mM calcium chloride, 1 mM magnesium chloride, and 1.35 mM sodium citrate; The ACD-A solution contains: 124.12 mM glucose, 37.42 mM citric acid, and 73.76 mM sodium citrate; The serum albumin solution is a human serum albumin (HSA) solution.

2. The platelet storage solution according to claim 1, wherein The normal temperature refers to 4-26°C.

3. The platelet storage solution according to claim 1, wherein The preservation solution contains the following components: 12.41 mM glucose, 78.06 mM sodium chloride, 3.19 mM potassium chloride, 31.66 mM sodium bicarbonate, 1.2 mM calcium chloride, 0.8 mM magnesium chloride, 3.74 mM citric acid, 8.46 mM sodium citrate, 10 mM sodium acetate, and 10% v / v HSA solution.

4. The platelet storage solution according to claim 1, wherein The preservation solution comprises the following components: (i) a first component comprising: 80 parts by volume of sodium bicarbonate Ringer's injection, 10 parts by volume of ACD-A, and 10 parts by volume of HSA solution; and (ii) a second component comprising the following supplementary compounds: NaHCO 3 and NaAc, wherein the supplementary concentration C1 of NaHCO 3 is 5-15 mM, and the supplementary concentration C2 of NaAc is 5-15 mM, the supplementary concentrations being calculated based on the volume of the platelet storage solution.

5. The platelet storage solution according to claim 1, wherein The preservation solution contains the following components: 10-15 mM glucose, 75-85 mM sodium chloride, 1-5 mM potassium chloride, 25-35 mM sodium bicarbonate, 0.5-1.5 mM calcium chloride, 0.6-1 mM magnesium chloride, 1-8 mM citric acid, 4-12 mM sodium citrate, 5-15 mM sodium acetate, and 5%-15% v / v HSA solution.

6. The use of the platelet storage solution according to any one of claims 1 to 5, characterized in that: Used to preserve platelets.

7. The method for preparing the platelet storage solution according to any one of claims 1 to 5, wherein: The following steps are involved: (S1) weighing, dissolving, and mixing components of the preservation solution to obtain a mixed solution; (S2) Sterilizing the mixed solution to obtain the preservation solution according to any one of claims 1 to 5.

8. A method for storing platelets at room temperature, characterized in that: The following steps are involved: After adding the platelet storage solution according to any one of claims 1 to 5 to the platelets, the obtained platelet mixture is stored at room temperature.

9. The method according to claim 8, wherein The preservation solution is added to a final platelet concentration of 1E8 / mL-2E9 / mL.

10. A platelet mixture, characterized in that The platelet mixture comprises: the platelet storage solution according to claim 1 and platelets.

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