Red blood cell room-temperature preserving fluid as well as preparation method and application thereof

Abstract

The invention discloses a red blood cell room-temperature preservation solution as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The erythrocyte preservative comprises the following components in parts by weight: 1-200 parts of an anticoagulant, 1-200 parts of a blood cell stabilizer, 1-100 parts of a metabolic inhibitor, 1-100 parts of an electrolyte, 1-150 parts of an antioxidant and 350-1000 parts of deionized water, and the pH value of the erythrocyte preservative is 4.6-7.0. All the components in the preservative are mutually matched and have a synergistic effect, and the integrity of erythrocyte membranes can be kept through synergistic protection of the multiple components; the electrolyte equilibrium osmotic pressure is maintained, the antioxidant scavenges free radicals, and hemolysis is reduced; the preservation time is prolonged, and the cell morphology and function are good; the dependence on a cold chain is reduced, and the demand on deep hypothermia equipment is reduced.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically the field of blood sample preservation technology, and specifically relates to a red blood cell room temperature preservation solution, its preparation method, and its application. Background Technology

[0002] Current blood preservation faces multiple challenges: 1) Limited shelf life: Traditional preservation solutions can only preserve blood at low temperatures for a few days to a few weeks. For example, red blood cells can be preserved for 35 days in MAP preservation solution at 2-6°C, while room temperature storage is more likely to cause hemolysis; 2) Hemolysis and cell damage: Red blood cells are prone to rupture during preservation due to changes in osmotic pressure, oxidative stress, and other factors, affecting blood typing and transfusion safety; 3) Cold chain dependence and contamination risk: Deep cryopreservation requires a -80°C or liquid nitrogen environment, which is costly; freeze-drying technology has the problem of low red blood cell recovery rate and is susceptible to microbial contamination during preservation.

[0003] While existing technologies have attempted to extend preservation time by optimizing the formulation of preservatives or improving preservation processes, problems such as insufficient stability of the preservation solution and poor maintenance of cell biological characteristics still exist. There is an urgent need for a highly efficient preservation solution that can be applied to red blood cells. Summary of the Invention

[0004] In order to overcome the problems existing in the prior art, one of the objectives of the present invention is to provide a red blood cell room temperature preservation solution.

[0005] The second objective of this invention is to provide a method for preparing the above-mentioned red blood cell room temperature preservation solution.

[0006] The third objective of this invention is to provide the application of the above-mentioned red blood cell room temperature preservation solution.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A red blood cell preservation solution at room temperature comprises the following components in parts by weight: 1-200 parts anticoagulant, 1-200 parts blood cell stabilizer, 1-100 parts metabolic inhibitor, 1-100 parts electrolyte, 1-150 parts antioxidant, 350-1000 parts deionized water, pH 4.6-7.0.

[0008] Furthermore, the anticoagulant is one or more of sodium citrate, citric acid, sodium heparin, and dipotassium ethylenediaminetetraacetate.

[0009] Furthermore, the blood cell stabilizer is one or more of ethylenediaminetetraacetic acid, glucose, trehalose, glycine, mannitol, glutathione, dimethyl sulfoxide, and sorbitol.

[0010] Furthermore, the metabolic inhibitor is one or more of sodium fluoride, adenine, and tricarboxylic acid.

[0011] Furthermore, the electrolyte is calcium chloride and / or magnesium chloride, or calcium chloride and / or magnesium chloride plus one or more of sodium chloride, potassium chloride, magnesium phosphate, magnesium hydrogen phosphate, and phosphate buffer.

[0012] Furthermore, the antioxidant is one or more of vitamin C, vitamin E, and glutathione.

[0013] Furthermore, the anticoagulant is preferably 20 to 150 parts, more preferably 20 to 100 parts.

[0014] Furthermore, the blood cell stabilizer is preferably 50 to 200 parts, more preferably 50 to 150 parts.

[0015] Furthermore, the metabolic inhibitor is preferably 1 to 80 parts, more preferably 10 to 50 parts.

[0016] Furthermore, the electrolyte is preferably 20 to 100 parts, more preferably 20 to 60 parts.

[0017] Furthermore, the antioxidant is preferably 50 to 150 parts, more preferably 50 to 100 parts.

[0018] Furthermore, the deionized water is preferably 500-800 parts, more preferably 650-750 parts.

[0019] Furthermore, the red blood cell room temperature preservation solution comprises the following components in parts by weight: 50-70 parts sodium citrate, 10-30 parts citric acid, 40-60 parts glucose, 10-30 parts mannitol, 10-30 parts sorbitol, 10-30 parts adenine, 5-20 parts calcium chloride, 10-20 parts magnesium chloride, 10-30 parts sodium chloride, 5-20 parts potassium chloride, 40-60 parts phosphate buffer, 680-700 parts deionized water, pH 6.0-7.0.

[0020] The preparation method of the above-mentioned red blood cell room temperature preservation solution includes the following steps: (1) Weigh out the anticoagulant, blood cell stabilizer, metabolic inhibitor, electrolyte, antioxidant and deionized water by weight. (2) Take 40% to 60% of the total amount of deionized water, add electrolyte, and stir until completely dissolved; (3) Add anticoagulant, blood cell stabilizer, metabolic inhibitor and antioxidant in sequence, stirring until completely dissolved after each addition; (4) Add the remaining deionized water, stir well, and bring to a final volume; (5) Adjust the pH of the solution to 4.6–7.0 using a pH adjuster; (6) Sterilize and cool to obtain the red blood cell room temperature preservation solution.

[0021] Furthermore, the pH adjuster is preferably at least one of hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, sodium hydroxide, potassium hydroxide, ammonia, and tris(hydroxymethyl)aminomethane, more preferably at least one of hydrochloric acid, phosphoric acid, acetic acid, and ammonia.

[0022] Furthermore, the sterilization process involves sterilizing at 121°C for 20 minutes.

[0023] The above-mentioned red blood cell room temperature preservation solution is used for long-term preservation of red blood cells at room temperature.

[0024] A method for long-term preservation of red blood cells at room temperature involves mixing the red blood cell room temperature preservation solution with the cell sample to be preserved and storing it at room temperature for a long period of time.

[0025] Furthermore, the cell sample to be preserved is blood or red blood cells.

[0026] Furthermore, the room temperature is 10–35°C, preferably 20–30°C, and more preferably 25±2°C.

[0027] Furthermore, the storage time is 0 to 40 days, preferably 0 to 35 days.

[0028] The present invention has the following advantages and effects compared with the prior art: The red blood cell preservation solution provided by this invention has multi-component synergistic protection: the anticoagulant in the red blood cell preservation solution prevents coagulation, and the cell stabilizer maintains the integrity of the cell membrane; the red blood cell preservation solution balances osmotic pressure through electrolytes and removes free radicals through antioxidants, thereby reducing hemolysis.

[0029] The red blood cell preservation solution provided by this invention can extend the storage time of red blood cells at room temperature: it can stably preserve red blood cells at room temperature for more than 35 days, and can preserve red blood cells at 4°C for 65 days with a hemolysis rate of <1% and good cell morphology and function.

[0030] The red blood cell preservation solution provided by this invention can reduce reliance on the cold chain: it supports room temperature transportation, and red blood cells can be preserved for a long time at 4°C, reducing the need for cryogenic equipment. Attached Figure Description

[0031] Figure 1 The images shown are of fresh blood placed at room temperature for different times in Example 3. Figure 2 This is a graph showing the results of hemolysis rate determination of fresh blood after being placed at room temperature for different times in Example 3; Figure 3 Examples 3: Cell morphology diagrams of fresh blood after being placed at room temperature for different times; Figure 4 The image shows the results of 2,3-DPG measurement in erythrocytes. Figure 5 The graph shows the results of ATP measurement in erythrocytes. Figure 6 The graph shows the results of the measurement of oxygen uptake function of erythrocytes. Figure 7 This is a graph showing the results of the measurement of the deoxygenation function of erythrocytes. Detailed Implementation

[0032] To better understand the technical solution of the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0033] The performance testing method of this invention is as follows: Performance testing of red blood cell preservation solutions is a crucial step in ensuring the maintenance of red blood cell function, extending preservation period, and guaranteeing transfusion safety. The performance testing method is described in detail below from multiple dimensions, including morphology, cell function, preservation effect, and safety. (1) Structural testing of red blood cells Objective: To assess the ability of erythrocytes to pass through microvessels, as poor deformability can lead to microcirculatory obstruction.

[0034] Methods: Microscopic observation was used to examine the morphology, structure, and integrity of erythrocytes.

[0035] Reference standard: At the end of the storage period (e.g., 35 days), the deformability of red blood cells should be ≥ 70% of that of fresh blood.

[0036] (2) Erythrocyte hemolysis markers Objective: To detect free hemoglobin released from ruptured red blood cells in the preservation solution, reflecting the degree of cell damage caused by the preservation solution.

[0037] Methods: The concentration of free hemoglobin in the supernatant was determined by spectrophotometry (absorbance at 540 nm wavelength). The hemolysis rate was calculated as (free hemoglobin concentration / total hemoglobin concentration) × 100%.

[0038] Reference standard: The hemolysis rate at the end of the storage period should be <0.8% (as determined by national standard GB / T 16886.4-2022 / ISO10993-4:2017).

[0039] (3) Red blood cell function assay Objective: Erythrocyte oxygen uptake and release function assay is a key test to assess the ability of erythrocytes to carry oxygen to tissues and release oxygen, mainly reflecting the characteristics of hemoglobin (Hb) binding and releasing oxygen. This is crucial for diagnosing certain hematological diseases, respiratory diseases, and understanding tissue oxygenation status.

[0040] The blood used in this invention is all fresh blood collected from Panyu District, Guangzhou (from healthy volunteers, approved through ethical review).

[0041] Example 1 Red blood cell preservation solution formula (parts by weight): 50 parts sodium citrate, 20 parts glycine, 20 parts glucose, 30 parts mannitol, 20 parts adenine, 50 parts magnesium chloride, 80 parts glutathione, and 730 parts deionized water. Preparation of red blood cell preservation solution: (1) Weigh the above-mentioned components in parts by weight; (2) Take 350 parts of deionized water, add magnesium chloride, and stir until completely dissolved; (3) Add anticoagulant, blood cell stabilizer, metabolic inhibitor and antioxidant to the above solution in sequence, stirring until completely dissolved after each addition; (4) Add the remaining 380 parts of deionized water and stir well; (5) Adjust the pH value to 6.6 with hydrochloric acid; (6) Sterilize at 121℃ for 20 minutes, then cool and store for later use.

[0042] Red blood cell preservation: Take fresh blood (within 6 hours after collection) and mix it with the above red blood cell preservation solution at a volume ratio of 2:1. Gently invert the centrifuge tube 5 to 8 times to mix it evenly. Store the mixture in an environment of 25±2℃, observe the morphology of red blood cells, and take samples for testing at 7 days, 14 days, 21 days, 28 days and 35 days.

[0043] Example 2 Preparation of red blood cell preservation solution (parts by weight): Weigh out 40 parts sodium citrate, 10 parts citric acid, 20 parts glucose, 20 parts sorbitol, 5 parts adenine, 5 parts calcium chloride, 5 parts magnesium chloride, 20 parts sodium chloride, 10 parts potassium chloride, 60 parts phosphate buffer (0.01 mol / L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, pH 7.4), 5 parts vitamin E, and 705 parts deionized water. Adjust the pH to 6.6.

[0044] The preparation steps for the red blood cell preservation solution are the same as in Example 1 (the pH adjuster is hydrochloric acid).

[0045] Red blood cell preservation: Take fresh blood (within 6 hours after collection) and mix it with the above red blood cell preservation solution at a volume ratio of 2:1. Gently invert the centrifuge tube 5 to 8 times to mix it evenly. Store the mixture in an environment of 25±2℃, observe the morphology of red blood cells, and take samples for testing at 7 days, 14 days, 21 days, 28 days and 35 days.

[0046] Example 3 Preparation of Red Blood Cell Preservation Solution (Reagent 3) (parts by weight): Weigh out 60 parts sodium citrate, 20 parts citric acid, 50 parts glucose, 20 parts mannitol, 20 parts sorbitol, 10 parts adenine, 10 parts calcium chloride, 15 parts magnesium chloride, 20 parts sodium chloride, 10 parts potassium chloride, 50 parts phosphate buffer (0.01 mol / L, pH 7.4 sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, pH 7.4), 15 parts glutathione, and 695 parts deionized water. Adjust the pH to 6.6 with hydrochloric acid.

[0047] The preparation steps for the red blood cell preservation solution are the same as in Example 1 (the pH adjuster is hydrochloric acid).

[0048] Red blood cell preservation: Take fresh blood (within 6 hours after collection) and mix it with the above red blood cell preservation solution at a volume ratio of 2:1. Gently invert the centrifuge tube 5 to 8 times to mix it evenly. Store the mixture in an environment of 25±2℃, observe the morphology of red blood cells, and take samples for testing at 7 days, 14 days, 21 days, 28 days and 35 days.

[0049] Comparative Example 1 10 mL of blood was collected directly using commercial purple-capped EDTA vacuum tubes. After gently inverting and mixing, the tubes were placed in an environment of 25±2℃, and the hemolysis of red blood cells in the tubes was detected at 7, 14, 21, 28 and 35 days.

[0050] Comparative Example 2 10 mL of blood was collected directly using commercial MAP preservation solution (formula: trisodium citrate 1.5 g, citric acid 0.2 g, glucose 7.93 g, sodium dihydrogen phosphate 0.94 g, adenine 0.14 g, sodium chloride 4.97 g, mannitol 14.57 g). After gently inverting to mix, the solution was placed in an environment of 25±2℃, and the hemolysis of red blood cells in the tube was detected at 7, 14, 21, 28 and 35 days.

[0051] Comparative Example 3 10 mL of blood was collected directly using a standard commercial vacuum tube (without preservation solution). After gently inverting and mixing, the tube was placed in an environment of 25±2℃, and the hemolysis of red blood cells in the tube was detected at 7, 14, 21, 28 and 35 days.

[0052] Comparative Example 4 (without added antioxidants) Preparation of red blood cell preservation solution (parts by weight): 60 parts sodium citrate, 20 parts citric acid, 50 parts glucose, 20 parts mannitol, 20 parts sorbitol, 10 parts adenine, 10 parts calcium chloride, 15 parts magnesium chloride, 20 parts sodium chloride, 10 parts potassium chloride, 50 parts phosphate buffer (0.01 mol / L, pH 7.4 sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, pH 7.4), adjust the pH to 6.6 with hydrochloric acid, and the remainder is deionized water.

[0053] The preparation steps, red blood cell preservation, and performance testing are the same as in Example 1.

[0054] Comparative Example 5 (without cell stabilizer) Preparation of red blood cell preservation solution (parts by weight): 60 parts sodium citrate, 20 parts citric acid, 20 parts mannitol, 20 parts sorbitol, 10 parts adenine, 10 parts calcium chloride, 15 parts magnesium chloride, 20 parts sodium chloride, 10 parts potassium chloride, 50 parts phosphate buffer (0.01 mol / L, pH 7.4 sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, pH 7.4), 15 parts glutathione. Adjust the pH to 6.6 with hydrochloric acid, and the remainder is deionized water.

[0055] The preparation steps, red blood cell preservation, and performance testing are the same as in Example 1.

[0056] Comparative Example 6 (The electrolyte does not contain calcium chloride and magnesium chloride) Preparation of red blood cell preservation solution (parts by weight): 60 parts sodium citrate, 20 parts citric acid, 50 parts glucose, 20 parts mannitol, 20 parts sorbitol, 10 parts adenine, 20 parts sodium chloride, 10 parts potassium chloride, 50 parts phosphate buffer (0.01 mol / L, pH 7.4 sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, pH 7.4), 15 parts glutathione. Adjust the pH to 6.6 with hydrochloric acid, and the remainder is deionized water.

[0057] The performance test data of the red blood cell preservation solutions of Examples 1-3 and Comparative Examples 1-6 are shown in Table 1.

[0058] Table 1 Performance parameters of red blood cell preservation solutions in Examples 1-3 and Comparative Examples 1-6

[0059] As can be seen from the data in Table 1, the red blood cell preservation solution prepared in Example 3 showed the best results, with a hemolysis rate of less than 0.27% after 28 days of storage at room temperature, and the red blood cells maintained their original morphology (e.g., Figure 1 , Figure 2 , Figure 3 (As shown).

[0060] Further comparison of the preservation effects of the red blood cell preservation solution prepared in this example at room temperature and 4°C revealed that the hemolysis rate of red blood cells preserved at 4°C was significantly lower than that at room temperature. After 35 days, the hemolysis rate at 4°C was less than 0.3%, while that at room temperature was 0.4%. Furthermore, the red blood cells preserved at 4°C had more intact morphology and better maintained deformability.

[0061] Red blood cells preserved in the red blood cell preservation solution prepared in Example 3 for 35 days at room temperature were used to determine 2,3-DPG, ATP, oxygen uptake capacity, and deoxygenation capacity. The procedure is as follows: 2,3-DPG Assay: A 2,3-diphosphoglycerate assay kit (purchased from Yuanju Biotechnology Co., Ltd.) was used. The procedure is as follows: Step 1: Add 100 μL of standard or test sample to the wells, cover with a membrane, and incubate at 37°C for 90 minutes. Wash the plate twice. Do not soak. Step 2: Add 100 μL of biotin-antibody working solution, cover with a membrane, and incubate at 37°C for 60 minutes. Wash the plate three times. Soak for 1 minute each time. Step 3: Add 100 μL of HRP-streptavidin (SABC) working solution, cover with a membrane, and incubate at 37°C for 30 minutes. Wash the plate five times. Soak for 1 minute each time. Step 4: Add 90 μL of TMB chromogenic substrate. Cover with a membrane and incubate at 37°C for 10-20 minutes. Step 5: Add 50 μL of reaction stop solution. Immediately read the OD at 450 nm. 450 Value and calculate (e.g.) Figure 4 (As shown).

[0062] ATP Assay: The ATP assay kit (purchased from Yuanju Biotechnology Co., Ltd.) was used. The procedure is as follows: Step 1: Add 100 μL of standard or test sample to the wells, cover with a membrane, and incubate at 37°C for 90 minutes. Wash the plate twice. Do not soak. Step 2: Add 100 μL of biotin-antibody working solution, cover with a membrane, and incubate at 37°C for 60 minutes. Wash the plate three times. Soak for 1 minute each time. Step 3: Add 100 μL of HRP-streptavidin (SABC) working solution, cover with a membrane, and incubate at 37°C for 30 minutes. Wash the plate five times. Soak for 1 minute each time. Step 4: Add 90 μL of TMB chromogenic substrate. Cover with a membrane and incubate at 37°C for 10-20 minutes. Step 5: Add 50 μL of reaction stop solution. Immediately read the OD at 450 nm. 450 Value and calculate (e.g.) Figure 5 (As shown).

[0063] Oxygen uptake capacity assay: Take 0.5 mL of erythrocyte suspension (hematocrit 30%), add 2 mL of oxygen-saturated buffer (preheated to 37℃), and collect the absorbance at 300-700 nm using a microplate reader (e.g., ...). Figure 6 (As shown).

[0064] Deoxygenation function assay: Take 0.5 mL of oxygen-enriched red blood cell suspension (equilibrated with pure oxygen at 37℃ for 30 minutes), add 2 mL of hypoxia buffer (purged with nitrogen until PO2 < 10 mmHg), and collect the absorbance at 300-700 nm using a microplate reader (e.g., ...). Figure 7 (As shown).

[0065] The results showed that: the 2,3-DPG content of red blood cells preserved for 35 days in Example 3 was more than 75% of that of fresh red blood cells, and the ATP content was more than 80% of that of fresh red blood cells; the oxygen uptake function (PO2 decrease rate) was 85% of that of fresh red blood cells, and the oxygen removal function (PO2 increase rate) was 82% of that of fresh red blood cells. This indicates that the preservation solution can effectively maintain the metabolic activity and oxygen-carrying and oxygen-releasing functions of red blood cells during room temperature storage.

[0066] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A red blood cell preservation solution at room temperature, characterized in that: It comprises the following components by weight: 1-200 parts anticoagulant, 1-200 parts blood cell stabilizer, 1-100 parts metabolic inhibitor, 1-100 parts electrolyte, 1-150 parts antioxidant, 350-1000 parts deionized water, pH 4.6-7.0; The electrolyte is calcium chloride and / or magnesium chloride, or calcium chloride and / or magnesium chloride plus one or more of sodium chloride, potassium chloride, magnesium phosphate, magnesium hydrogen phosphate, and phosphate buffer. The antioxidant mentioned is one or more of vitamin C, vitamin E, and glutathione.

2. The red blood cell room temperature preservation solution according to claim 1, characterized in that: The anticoagulant is one or more of sodium citrate, citrate, sodium heparin, and dipotassium ethylenediaminetetraacetate. The blood cell stabilizer is one or more of the following: ethylenediaminetetraacetic acid, glucose, trehalose, glycine, mannitol, glutathione, dimethyl sulfoxide, and sorbitol; The metabolic inhibitor is one or more of sodium fluoride, adenine, and tricarboxylic acid.

3. The red blood cell room temperature preservation solution according to claim 1 or 2, characterized in that: The anticoagulant is in quantities of 20 to 100 parts; The blood cell stabilizer is present in quantities of 50–150 parts; The metabolic inhibitor is 10-50 parts; The electrolyte is in the form of 20-60 parts; The antioxidant is 50-100 parts; The amount of deionized water is 650-750 parts.

4. The red blood cell room temperature preservation solution according to claim 1 or 2, characterized in that: The red blood cell room temperature preservation solution comprises the following components by weight: 50-70 parts sodium citrate, 10-30 parts citric acid, 40-60 parts glucose, 10-30 parts mannitol, 10-30 parts sorbitol, 10-30 parts adenine, 5-20 parts calcium chloride, 10-20 parts magnesium chloride, 10-30 parts sodium chloride, 5-20 parts potassium chloride, 40-60 parts phosphate buffer, and 680-700 parts deionized water, with a pH of 6.0-7.

0.

5. The method for preparing the red blood cell room temperature preservation solution according to any one of claims 1-4, characterized in that: Includes the following steps: (1) Weigh out the anticoagulant, blood cell stabilizer, metabolic inhibitor, electrolyte, antioxidant and deionized water by weight. (2) Take 40% to 60% of the total amount of deionized water, add electrolyte, and stir until completely dissolved; (3) Add anticoagulant, blood cell stabilizer, metabolic inhibitor and antioxidant in sequence, stirring until completely dissolved after each addition; (4) Add the remaining deionized water, stir well, and bring to a final volume; (5) Adjust the pH of the solution to 4.6–7.0 using a pH adjuster; (6) Sterilize and cool to obtain the red blood cell room temperature preservation solution.

6. The method for preparing the red blood cell room temperature preservation solution according to claim 5, characterized in that: The pH adjuster is at least one of hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, sodium hydroxide, potassium hydroxide, ammonia, and tris(hydroxymethyl)aminomethane. The sterilization process involves sterilizing at 121°C for 20 minutes.

7. The use of the red blood cell room temperature preservation solution according to any one of claims 1-4 in the long-term preservation of red blood cells at room temperature.

8. A method for long-term preservation of red blood cells at room temperature, characterized in that: Mix the red blood cell room temperature preservation solution described in any one of claims 1-4 with the cell sample to be preserved and store it at room temperature for a long period of time.

9. The method for long-term preservation of red blood cells at room temperature according to claim 8, characterized in that: The cell sample to be preserved is blood or red blood cells.

10. The method for long-term preservation of red blood cells at room temperature according to claim 8 or 9, characterized in that: The room temperature is 10–35°C; The storage time is 0 to 40 days.

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