Method for preserving platelets at normal temperature

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of platelet preservation, and particularly 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 the peripheral blood of normal people is (100 - 300)×10 9 / mL. Due to some diseases and traumas, the number of PLT in the peripheral circulation decreases. When the number is lower than 20×10 9 / mL, it will greatly increase the risk of internal bleeding and even endanger life. When clinical blood transfusion indications appear, the supply of concentrated PLT is required.

[0003] The commonly used method for platelet preservation in clinics is to preserve platelets by shaking at room temperature in plasma. Platelets collected by apheresis can be preserved for about 5 days. However, the plasma used as the platelet preservation solution will cause various transfusion-related adverse reactions after being transfused into the human body, including non-hemolytic fever, allergy, circulatory overload, etc., which bring great harm to the human body.

[0004] Therefore, in recent years, the development of platelet additives (PAS) to reduce plasma use has become one of the research hotspots. Since Rock et al. proposed PAS in 1985, PAS-A, PAS-B, PAS-C, PAS-D, PAS-E, PAS-F, PAS-G, etc., as well as other new types of platelet special additives such as M-sol (Mixture of solutions) and BRS-A (bicarbonated Ringer's solution - ACD-A) have been successively developed. However, these preservation methods all have certain problems, such as platelet preservation damage, increased platelet activation, and the induction of febrile transfusion adverse reactions.

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

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

[0007] In the first aspect of the present invention, a platelet preservation solution for room temperature preservation is provided, and the platelet preservation solution comprises the following components: (i)The first component, the first component comprising: 60 - 96 volume parts of sodium bicarbonate Ringer's injection (BRS solution), 2 - 20 volume parts of ACD-A solution, 2 - 20 volume parts of serum albumin solution; and (ii)The second component, the second component comprising the following additional compounds: NaHCO 3 and NaAc, and the additional concentration C1 of NaHCO 3 is 5 - 15 mM, and the additional concentration C2 of NaAc is 5 - 15 mM, and the additional concentrations are based on the volume of the platelet preservation solution.

[0008] In another preferred example, the ratio R0 of C1 to C2 is 1 / 2 - 2 / 1.

[0009] In another preferred example, the ratio R0 of C1 to C2 is 0.8:1 - 1.2:1, more preferably 1:1.

[0010] In another preferred example, the additional concentration C1 of NaHCO 3 is 7 - 13 mM, preferably 8 - 12 mM.

[0011] In another preferred example, the additional concentration C2 of NaAc is 7 - 13 mM, preferably 8 - 12 mM.

[0012] In another preferred example, the ratio V1 / V2 of the total volume parts V1 of the first component and the volume parts V2 of the total volume of the second component is ≥5, preferably ≥10, more preferably ≥20, and most preferably ≥25.

[0013] In another preferred example, the second component is added in solid form without changing the proportion of the first component in the platelet preservation solution.

[0014] In another preferred example, the normal temperature refers to 4 - 26 °C.

[0015] In another preferred example, the normal temperature is 10 - 26 °C, preferably 15 - 25 °C, and most preferably 20 - 24 °C.

[0016] In another preferred example, 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.

[0017] In another preferred example, the ACD-A solution comprises: 124.12 mM glucose, 37.42 mM citric acid, and 73.76 mM sodium citrate.

[0018] In another preferred example, the serum albumin solution is a serum albumin solution of mammalian origin.

[0019] In another preferred example, the serum albumin solution of animal origin comprises: human serum albumin solution, bovine serum albumin solution, ovine serum albumin solution, or a combination thereof.

[0020] In another preferred example, the serum albumin solution comprises serum.

[0021] In another preferred example, the serum albumin solution is a human serum albumin (HSA) solution.

[0022] In another preferred example, the first component comprises 2 - 20 parts by volume of HSA solution, preferably 3 - 18, more preferably 4 - 16, and most preferably 5 - 15.

[0023] In another preferred example, the first component comprises 10 parts by volume of HSA solution.

[0024] In another preferred example, 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.

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

[0026] In another preferred example, the preservative solution comprises the following components: (i) a first component, the first component comprising: 80 parts by volume of BRS solution, 10 parts by volume of ACD - A, 10 parts by volume of HSA solution; and (ii) a second component, the second component comprising the following additional compounds: NaHCO 3 and NaAc, and the additional concentration C1 of NaHCO 3 is 5 - 15 mM, and the additional concentration C2 of NaAc is 5 - 15 mM, and the additional concentrations are based on the volume of the platelet preservative solution.

[0027] In another preferred example, the ratio R0 of C1 to C2 is 1 / 2 - 2 / 1 In another preferred example, the preservative 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.

[0028] In another preferred example, 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.

[0029] In another preferred example, 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.

[0030] In a second aspect of the present invention, there is provided the use of the platelet preservation solution described in the first aspect of the present invention for preserving platelets.

[0031] In another preferred example, it is used for washing platelets.

[0032] In another preferred example, the platelets include: platelets derived from umbilical cord blood, platelets derived from peripheral blood, in vitro regenerated platelets, or a combination thereof.

[0033] In a third aspect of the present invention, there is provided a method for preparing the platelet preservation solution described in the first aspect of the present invention, comprising the following steps: (S1) Weigh, dissolve and mix the components of the preservation solution to obtain a mixed solution; (S2) Sterilize the mixed solution to obtain the preservation solution described in the first aspect of the present invention.

[0034] In a fourth aspect of the present invention, there is provided a method for preserving platelets at room temperature, comprising the following steps: after adding the platelet preservation solution described in the first aspect of the present invention to the platelets, storing the resulting platelet mixture at room temperature.

[0035] In another preferred example, the platelets include: platelets derived from umbilical cord blood, platelets derived from peripheral blood, in vitro regenerated platelets, or a combination thereof.

[0036] In another preferred example, the room temperature is 10 - 26 °C, preferably 15 - 25 °C, and most preferably 20 - 24 °C.

[0037] In another preferred example, the preservation solution is added to the platelets until the final concentration is 1E8 / mL - 2E9 / mL.

[0038] In another preferred example, the preservation solution is added to the platelets until the final concentration is 1E8 / mL - 1E9 / mL.

[0039] In another preferred embodiment, the storage time is ≥3 days, preferably ≥4 days, more preferably ≥5 days, and most preferably ≥7 days.

[0040] In another preferred embodiment, the method further comprises the following step: characterizing the characteristics of platelets after storage at room temperature.

[0041] In another preferred embodiment, the storage at room temperature is carried out at 22°C for 7 days, and the characteristics include: (Z1) The ratio Y0 of CD41 + CD42b + of platelets before storage and the ratio Y1 of CD41 + CD42b + of platelets after storage, and the difference Y0 - Y1 ≤ 8%, preferably ≤ 6%, more preferably ≤ 4%; and / or (Z2) The ratio N1 / N0 of the activation ability N1 of platelets after storage to the activation ability N0 without an activator is ≥5, preferably ≥10; more preferably ≥15, and most preferably ≥20.

[0042] In another preferred embodiment, the activation ability is the expression level of platelet PAC-1.

[0043] In the fifth aspect of the present invention, there is provided a platelet mixture, which comprises: the platelet preservation solution described in the first aspect of the present invention and platelets.

[0044] 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 specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Brief Description of the Drawings

[0045] Figure 1 Shows the change in the CD42b expression level of platelets after 7 days of storage in different preservation solutions. The BRS-A group decreased the fastest. While the other three groups could maintain a high expression of CD42b well. Detailed Description of the Embodiments

[0046] The present inventors have conducted extensive and in-depth research and unexpectedly discovered for the first time a serum-free platelet preservation solution, which contains BRS, ACD-A, HSA, NaHCO 3 、and NaAc. The room temperature storage of platelets based on this platelet preservation solution can reduce glucose consumption and lactic acid production during storage. Moreover, after storage at room temperature for seven days, CD41 + CD42b +The proportion can still maintain about 96%, and the activation ability can still remain above 88%. Based on this, the present invention is completed.

[0047] Term To more easily understand the present disclosure, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0048] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the related listed items.

[0049] As used herein, the terms "comprising," "including," and "containing" may be used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of" and "consisting essentially of."

[0050] When a numerical range is provided, unless the context clearly indicates otherwise, it should be understood that each intermediate integer of that value, each tenth of each intermediate integer of that value, and any other intermediate value between the upper and lower limits of the range and within the specified range are included in the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also covered by the present invention, subject to any express exclusionary limitations within the specified range. For example, "1 to 50" includes "2 to 25," "5 to 20," "25 to 50," "1 to 10," etc.

[0051] Platelet preservation Platelet preservation is crucial. It directly affects the efficacy of platelet transfusion. Effective preservation can maintain platelet activity and function, providing guarantee for timely hemostasis and saving the lives of patients with thrombocytopenia and bleeding; it can also avoid problems such as bacterial contamination and loss of function caused by improper preservation, reducing the risk of blood transfusion. At the same time, reasonable preservation can optimize resource utilization and ensure the safety and efficiency of clinical blood use.

[0052] Concentrated platelets prepared by separating whole blood collected with whole blood preservatives such as ACD (citric acid - sodium citrate - glucose), CPD (citric acid - sodium citrate - sodium dihydrogen phosphate - glucose), or CPDA - 1, CPDA - 2, etc., have plasma as a better preservative for storing concentrated platelets. The storage condition is oscillatory storage at (22 ± 2) °C. The disadvantage is that it may cause side effects such as allergy to plasma and febrile transfusion adverse reactions in some patients during transfusion.

[0053] The commonly used method for platelet preservation in clinics is to preserve platelets by shaking them at room temperature in plasma. Apheresis platelets can be preserved for about 5 days. However, the plasma in the platelet preservation solution can cause various transfusion-related adverse reactions after being transfused into the human body, including non-hemolytic fever, allergy, circulatory overload, etc., which pose great harm to the human body.

[0054] Therefore, in recent years, the development of platelet additives (PAS) to reduce plasma use has become one of the research hotspots. Since Rock et al. proposed PAS in 1985, PAS-A, PAS-B, PAS-C, PAS-D, PAS-E, PAS-F, PAS-G, etc., as well as other new platelet special additives such as M-sol (Mixture of solutions) and BRS-A (bicarbonated Ringer's solution-ACD-A) have been successively developed. However, there are certain problems with these preservation methods.

[0055] Typically, M-sol is prepared by manually mixing commercially available infusion solutions. However, the preparation process of M-sol is cumbersome and involves complex operation steps, and its final composition contains plasma, which may affect the accuracy of its components, thereby affecting the quality of cryopreserved platelets. BRS-A is composed of BRS commonly used in clinics and citrate anticoagulant, and its preparation is simple. However, the presence of plasma is also inevitable in its final composition, and high-concentration plasma (25%-35%) can cause side effects caused by plasma transfusion, such as hemolysis. More importantly, when these preservation solutions are used to preserve platelets, in the later stage of platelet preservation (3-7 days), the glucose component in the preservation solution drops sharply, and the lactic acid content rises sharply, resulting in the activation of platelets during preservation and an increase in CD62P expression, thereby affecting the normal physiological functions of platelets after transfusion.

[0056] Platelet additives such as AS-II, PAS-III, PAS-IIIM, Composol, etc. are widely used clinically in European and American countries. They can reduce platelet activation and apoptosis during preservation, reduce platelet preservation damage caused by pathogen inactivation technology, and promote the detection of pathogenic microorganisms. The preservation condition is also shaking preservation at (22±2)°C. However, different platelet additives have different effects on factors such as pH and mean platelet volume. Some additives may increase platelet activation. As time goes by, glucose decreases, and lactic acid and lactate dehydrogenase increase, etc. In summary, the short preservation time limit also restricts platelet collection and preparation. Large-scale storage is likely to lead to expiration and scrapping. Therefore, the present invention provides a platelet preservation solution with higher safety, no plasma, and still able to maintain more than 93% of CD41 + CD42b+ and a platelet preservation solution with a high specific activation ability.

[0057] The platelet preservation solution of the present invention The platelet preservation solution of the present invention comprises a first component and a second component. The first component comprises 80% sodium bicarbonate Ringer's injection (BRS solution), 10% ACD-A solution, and 10% serum albumin solution; the second component comprises: NaHCO 3 , NaAc, or a combination thereof. Wherein, each percentage is a volume percentage.

[0058] 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.

[0059] In a specific embodiment, the ACD-A solution comprises: 124.12 mM glucose, 37.42 mM citric acid, and 73.76 mM sodium citrate.

[0060] In a specific embodiment, the serum albumin solution is a human serum albumin (HSA) solution.

[0061] In a specific embodiment, the first component comprises 10% HSA solution.

[0062] In a specific embodiment, the first component comprises 10% ACD-A solution.

[0063] In a specific embodiment, in the second component, the concentration of the NaHCO 3 is 10 mM.

[0064] In a specific embodiment, in the second component, the concentration of the NaAc is 10 mM.

[0065] In a specific embodiment, the platelet preservation 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.

[0066] The method of the present invention The method of the present invention is a method for preserving platelets at room temperature based on the preservation solution of the present invention. The method includes adding the platelet preservation solution of the present invention to platelets and then preserving the obtained platelet mixture at room temperature.

[0067] The method of the present invention gets rid of the dependence on plasma, greatly improves the ability of the preservation solution to buffer the decline of pH value, and greatly reduces the production of lactic acid. Therefore, it can better maintain platelet activity and achieve stable room-temperature preservation of platelets.

[0068] The main advantages of the present invention include: (a) The present invention provides a serum-free platelet preservation solution, which contains BRS, ACD-A, HSA, NaHCO 3 , and NaAc. The room-temperature preservation of platelets based on this platelet preservation solution can reduce glucose consumption and lactic acid generation during storage.

[0069] (b) After storing at room temperature for seven days by the method of the present invention, the proportion of CD41 + CD42b + can still be maintained at about 96%, the specific activation ability still remains above 88%, and it still has good platelet aggregation ability.

[0070] (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, and can be applied to the room-temperature preservation of umbilical cord blood, peripheral blood-derived, and in vitro regenerated PLT, and can better preserve its activity and function.

[0071] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions indicated in the following embodiments are usually carried out 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 stated, percentages and parts are volume percentages and volume parts.

[0072] Example 1: Preparation of the platelet preservation solution of the present invention.

[0073] The platelet preservation solution of the present invention is based on sodium bicarbonate Ringer's injection (BRS), and additionally contains citrate anticoagulant (ACD-A), clinically injectable human serum albumin (HSA), sodium bicarbonate (NaHCO 3 ), and sodium acetate (NaAc). The addition of HSA avoids the dependence of the final platelet preservation solution on plasma, and NaHCO 3is an effective pH buffer, and sodium acetate can replace glucose as a metabolic substrate to a certain extent. Under the action of acetyl-CoA synthetase, it generates acetyl-CoA, which directly enters the tricarboxylic acid cycle without passing through the glycolysis pathway, reducing lactic acid production. The specific formula is shown in Table 1.

[0074] Table 1 Formulation of platelet preservation solution

[0075] Specifically, in the basal solution shown in Table 1, it 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.

[0076] For different room-temperature platelet preservation formulations prepared, the parameters and performance before and after platelet preservation were compared in Examples 2 to 6.

[0077] Platelets derived from healthy human peripheral blood were resuspended with platelet preservation solution at a density of 5E8 / mL, and then stored with oscillation at a temperature of 22 ± 2 °C for 7 days. At different time points during storage, samples (i.e., samples to be tested) were collected for testing.

[0078] Example 2 Glucose and lactate changes.

[0079] First, the changes in glucose and lactate in the platelet preservation solution were detected. Take 500 μL of the sample to be tested and add it to a 1.5 mL centrifuge tube. After centrifuging at 1000 g for 10 min, discard the precipitate, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0080] Place the centrifuge tube containing the supernatant with the opening in the detection slot of the Sillman biochemical analyzer, and name the corresponding slot on the touch screen. After naming, click the "Start Detection" tab on the touch screen. After clicking, the instrument will automatically start calibration and detect the sample after calibration is completed.

[0081] The results of glucose changes are shown in Table 2.

[0082] Table 2 Changes in glucose concentration (g / L) of platelets in different preservation solutions at different time points.

[0083]

[0084] Table 2 shows that after 7 days of storage, the BRS-A (20A) group decreased the slowest, with 1.41 g / L remaining. The BRS-A (20B) group decreased faster, and only 0.35 g / L remained after 7 days of storage. Generally speaking, the glucose in each group was still not completely depleted.

[0085] The results of lactic acid change are shown in Table 3.

[0086] Table 3 Changes in lactic acid concentration (g / L) of platelets in different storage solutions at different time points.

[0087]

[0088] The results show that after 7 days of storage, the lactic acid concentration in each group increased slightly. The BRS-A (20A) group increased the slowest, with a lactic acid concentration of only 1.12 g / L. The BRS-A (20B) group increased relatively faster, but still remained at a low level (1.82 g / L).

[0089] Example 3 Osmotic pressure and pH changes.

[0090] To detect the change in osmotic pressure, specifically, take 50 μL of the sample to be tested and place it at the bottom of the test tube dedicated for osmotic pressure detection, taking care not to have air bubbles. Install the test tube on the test probe, lower the test probe into the cooling tank of the cooling system, and the instrument will automatically start the detection.

[0091] The results are shown in Table 4.

[0092] Table 4 Changes in osmotic pressure (mOsm / kg) of platelets in different storage solutions at different time points.

[0093]

[0094] The results show that all groups are within the normal physiological osmotic pressure range, and even at the highest point, they do not exceed the normal physiological osmotic pressure.

[0095] To detect the change in pH value. Specifically, before using the instrument, calibrate it using the three-point method. Then wipe the pH electrode clean and place it in the sample to be tested, and click the "Read" icon, then the instrument will start automatic measurement and print the results.

[0096] The results are shown in Table 5.

[0097] Table 5 Changes in pH value of platelets in different storage solutions at different time points.

[0098]

[0099] The results show that except for the BRS-A group, the pH values of other groups are above 6.5. Among them, the results of the 20B and 10B + 10A groups show that adding sodium bicarbonate to the platelet storage solution can significantly enhance the buffering capacity of the platelet storage solution to cope with the decrease in pH.

[0100] Example 4 CD41 + CD42b + Expression changes.

[0101] Flow cytometry was used to detect platelets before and after storage. Specifically, the test sample was centrifuged at 1000 g for 5 minutes, then the supernatant was discarded, 500 μL of flow cytometry buffer was added, and it was washed once; then the sample to be detected was resuspended with 50 μL of flow cytometry buffer; anti-human CD41 and anti-human CD42b antibodies were added and incubated at room temperature in the dark for 30 minutes; after the antibody incubation, it was washed twice with 500 μL of flow cytometry buffer; after washing, the cells were resuspended with flow cytometry buffer, data was collected using a Beckman CytoFlex flow cytometer, and data analysis was performed using FlowJo software.

[0102] The proportion changes of CD41 + CD42b + in platelets in different storage solutions at different time points are shown in Table 6.

[0103] Table 6 Proportion changes of CD41 + CD42b + in platelets in different storage solutions at different time points.

[0104]

[0105] The results showed that on the 7th day of storage, the proportion of CD41 + CD42b + cells in the BRS-A group decreased from 88.5% to 81.26% (a decrease of 7.24%). The addition of NaHCO 3 slowed down the decrease in the proportion of CD41 + CD42b + cells (a decrease of 5.34%), while the addition of NaAc led to an increased decrease (a decrease of 10.13%).

[0106] Surprisingly, in BRS-A (10B + 10A), the addition of specific concentrations of NaHCO 3 and NaAc significantly reduced the decrease in the proportion of CD41 + CD42b + cells (only a decrease of 3.62%), and the relative percentage decrease was approximately 50%.

[0107] The changes in the mean fluorescence intensity of CD42b expression at different time points are shown in Figure 1 and Table 7.

[0108] Table 7 Changes in the mean fluorescence intensity (MFI) of CD42b expression in platelets in different storage solutions on the 7th day of storage.

[0109]

[0110] The results showed that the platelets in each group were from the same donor, and the mean fluorescence intensity of CD42b expression in each group before preservation was the same, all being 69,616. After 7 days of preservation, the MFI value in the BRS-A group decreased by 53,507.

[0111] The addition of 20 mM NaHCO 3 slowed down the reduction in the CD42b expression level of CD42b cells in the platelet preservation solution after 7 days of preservation (decreased by 39,550). The addition of 20 mM NaAc also led to a slowdown in the reduction in the CD42b expression level of CD42b cells in the platelet preservation solution after 7 days of preservation (decreased by 46,461). + +

[0112] Unexpectedly, in BRS-A (10B + 10A), the addition of 10 mM NaHCO 3 and 10 mM NaAc significantly slowed down the reduction in the CD42b expression level of CD42b cells in the platelet preservation solution after 7 days of preservation (only decreased by 35,845), and the relative percentage decrease was approximately 67%. +

[0113] Example 5 Comparison of platelet aggregation performance.

[0114] In this example, the changes in platelet aggregation were detected. The method was as follows: 5E+07 cells were taken from each test sample and transferred to a 1.5 mL centrifuge tube, and centrifuged at 1000 g for 5 minutes; after centrifugation, each tube was resuspended with 250 μL of human plasma and set aside; 225 μL of the sample was taken and transferred to a glass test tube; the glass test tube was placed in the platelet aggregation analysis system instrument of Helena, and the detection was carried out according to the method recommended by the manufacturer.

[0115] 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 preservation, and among them, the 10B + 10A group changed the least.

[0116] Table 8 Changes in the aggregation ability of platelets in different preservation solutions at different time points.

[0117]

[0118] The aggregation ability of platelets in the BRS-A group decreased from 71.4 to 5.9 (decrease of 65.5). The addition of NaHCO 3 slowed down the decrease in aggregation ability (decrease of 46.4), and the addition of NaAc led to a slowdown in the decrease (decrease of 61).​​​

[0119] Unexpectedly, in BRS-A (10B + 10A), adding a specific concentration of NaHCO 3 and NaAc resulted in a significantly reduced decrease in aggregation ability (only a 41.8% decrease), and the percentage decrease relative to the BRS-A group was approximately 64%.

[0120] Comparison of platelet activation in Example 6 In this example, the activation of platelets before and after storage was investigated. The method was as follows: Take 1 mL of the sample to be tested, centrifuge at 1000 g for 10 minutes, and discard the supernatant; add 50 μL of the staining mixture to each sample and mix the sample thoroughly. Among them, the staining mixture included: the activator thrombin receptor activating peptide 6 (TRAP-6) and adenosine diphosphate (ADP), human CD42b antibody and human PAC-1 antibody; incubate at room temperature in the dark for 30 minutes; add 400 μL of tabletop solution to each sample to terminate the incubation; collect data using a Beckman CytoFlex flow cytometer and analyze the data using FlowJo software.

[0121] The results are shown in Table 9.

[0122] Table 9 Activation ability of platelets in different storage solutions at different time points (expression percentage of PAC-1) and.

[0123]

[0124] Note: R = activation ability with activator added / activation ability without activator added, and the activation ability is calculated based on the expression percentage of PAC-1+.

[0125] The results showed that platelets in the BRS-A group showed a certain degree of activation without adding an activator, and after adding an activator, as the storage time increased, the activation ability decreased most significantly; on the seventh day of storage, the specific activation ability (activation ability after adding an activator) decreased from 95.77 before storage to 15.58 (a decrease of 80.19). The expression of PAC-1 after adding an activator (15.58) was only 1.35 times that without adding an activator (11.52).

[0126] The addition of NaHCO 3 made the decrease in activation ability after adding an activator smaller. On the seventh day of storage, the specific activation ability (activation ability after adding an activator) decreased from 95.77 before storage to only 81.93 (a decrease of 13.84). The expression of PAC-1 after adding an activator was 14.42 times that without adding an activator.

[0127] The addition of NaAc reduced the degree of decline in the activation ability after the addition of the activator. On the seventh day of storage, the specific activation ability only decreased from 95.77 before storage to 47.81 (a decrease of 47.96). The expression of PAC-1 after the addition of the activator was 7.39 times that without the activator.

[0128] Surprisingly, on the seventh day of storage, in BRS-A (10B + 10A), the addition of specific concentrations of NaHCO 3 and NaAc led to a significant reduction in the degree of decline in specific activation ability (from 95.77 to 84.36, only a decrease of 11.41). The expression of PAC-1 after the addition of the activator was 23.5 times that without the activator, making it the preservation solution formulation that was most conducive to retaining the activation ability among the three groups.

[0129] All the documents mentioned in the present invention are cited herein by reference as if each individual document was specifically cited. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A platelet storage solution for storage at room temperature, characterized in that: The platelet preservation solution comprises the following components: (i) a first component, the 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 (ii) a second component, the 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.

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

3. The platelet storage solution according to claim 1, characterized in that The serum albumin solution is a human serum albumin (HSA) solution.

4. The platelet storage solution according to claim 1, characterized in that The preservation solution comprises the following components: (i) a first component, the 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 (ii) a second component, the 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, characterized in that 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% HSA.

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, characterized in that: The following steps are involved: (S1) weighing, dissolving and mixing components of a 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 platelets, the obtained platelet mixture is stored at room temperature.

9. The method according to claim 8, characterized in that The storage 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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