Freezing and unfreezing method for long-term preservation of reagent red blood cells

Abstract

The invention belongs to the technical field of red blood cell preservation, and discloses a freezing and unfreezing method for long-term preservation of reagent red blood cells. According to the invention, a simple and convenient way for cryopreserving and unfreezing the red blood cells is obtained, so that the rare blood can be effectively cryopreserved for a long time, the yield after redissolution is high, and the stability of the surface antigen of the red blood cells is maintained. The method is very important for research on erythrocyte surface antigens, research on complex antibodies, establishment of special blood group sample libraries and quality control of blood group typing reagents of various brands, and also lays technical reserve and important resources for erythrocyte blood group serology and genotype consistency evaluation.

Description

Technical Field

[0001] The invention belongs to the technical field of red blood cell preservation, and in particular relates to a freezing and thawing method for long-term preservation of reagent red blood cells. Background Art

[0002] ABO blood type is crucial in transfusion medicine. ABO-incompatible transfusion may lead to serious consequences. Therefore, ABO blood type testing before clinical transfusion is a key link. However, with the development of transfusion medicine, many ABO subtypes have been discovered. These subtypes have abnormal glycosyltransferase function caused by gene mutations, resulting in weak antigen expression or inconsistency between positive and negative typing. The existing antibody detection reagents on the market have good effects on conventional ABO blood type detection, but the detection ability of ABO subtypes varies greatly, mainly because the antibody clones of different manufacturers have different recognition of antigen epitopes. In order to improve the detection ability of ABO subtypes and rare blood types, it is particularly important to establish a reagent red blood cell bank to quality control blood type detection reagents.

[0003] At present, there are two main methods for preserving red blood cells: liquid refrigeration and frozen storage. Liquid refrigeration is usually stored at 2-8°C, but the storage period is short (2-3 months), hemolysis is severe in the later stage, and false positive results are prone to occur. Cryopreservation reduces ice crystal formation by using high osmotic pressure cryopreservation protection fluid (such as glycerol), and stores red blood cells at -80°C or in liquid nitrogen. It can be stored for ten years or longer, but the thawing process is cumbersome and the cell yield is low.

[0004] There are many problems with existing cryopreservation technology. For example, high concentration of glycerol lowers the freezing point, which makes it impossible to completely freeze cells at -20°C, shortening the shelf life. During the thawing process, a large amount of saline is used to remove macromolecular substances, which can easily cause red blood cell membrane rupture and affect cell yield. In addition, although liquid nitrogen ultra-low temperature preservation is effective, it is dangerous and has high operating costs, making it unsuitable for widespread application. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a freezing and thawing method for long-term storage of reagent red blood cells.

[0006] The present invention is achieved in that a frozen red blood cell preservation solution is prepared from isotonic phosphate buffer, comprising the following components: 0.1 mol / L phosphate buffer, 1.6% sodium citrate, 0.03% potassium chloride and 30-35% glycerol.

[0007] The present invention also provides a method for freezing red blood cells by gradient cooling, which comprises: washing fresh hematocrit with a red blood cell preservation solution available on the market for 1-2 times, adding the frozen red blood cell preservation solution and the hematocrit red blood cells in a volume ratio of 1:1, mixing by inversion, placing at room temperature for about 10 minutes for equilibrium, placing at 4°C for about 1 hour, placing at -20°C overnight, and then placing in a refrigerator below -60°C for long-term storage. The commercial red blood cell preservation solution is characterized by: an isotonic solution containing carbohydrate components that can provide energy for cells; and containing antibacterial components that can inhibit the growth of microorganisms.

[0008] The present invention also provides a simple method for thawing red blood cells. The principle of the method is: using a special translucent membrane, the frozen red blood cells are placed in a bag and immersed in a buffer solution. The red blood cells are trapped in the bag, while glycerol and small molecules continuously diffuse and dialyze to the outside of the bag.

[0009] Further, the red blood cell thawing method:

[0010] 1) Path 1:

[0011] Prepare materials: 9% sodium chloride solution, 2.5% sodium chloride solution, 0.9% sodium chloride solution, red blood cell preservation solution;

[0012] Thaw at room temperature and use gradient elution method. When the frozen red blood cells are completely thawed at room temperature, measure the total volume after lysis, slowly add 9% sodium chloride solution at a ratio of 1:1, mix well, centrifuge and discard the supernatant;

[0013] Wash 2 to 3 times with 2.5% sodium chloride solution and packed red blood cells at a volume ratio of 6:1 until the supernatant is clear;

[0014] Wash 2 to 3 times with 0.9% sodium chloride solution and packed red blood cells at a volume ratio of 6:1 until the supernatant is clear;

[0015] Wash once with red blood cell preservation solution and packed red blood cells at a volume ratio of 6:1, and discard the supernatant after centrifugation;

[0016] Use red blood cell preservation solution to prepare 3% and 1% concentration of reagent red blood cells;

[0017] 2) Path 2:

[0018] Prepare materials: MD34 dialysis bag MW: 8000-14000, plastic clips, plastic boxes, 2.5% sodium chloride solution, 1.6% sodium chloride solution, 0.9% sodium chloride solution, red blood cell preservation solution;

[0019] Thaw at room temperature, use dialysis method, wait until the frozen red blood cells are completely thawed at room temperature, move them into dialysis bags, immerse them in 2.5% sodium chloride solution, and place them at room temperature for 1 hour, shaking them every 15 minutes, or place them at 2-8℃ for 2 hours;

[0020] Take out, measure the total volume collected after thawing, add 1.6% sodium chloride solution at a volume ratio of 1:1, mix, centrifuge and discard the supernatant;

[0021] Wash 1 to 2 times with 0.9% sodium chloride solution and packed red blood cells at a volume ratio of 6:1 until the supernatant is clear;

[0022] Wash once with red blood cell preservation solution and packed red blood cells at a volume ratio of 6:1, and discard the supernatant after centrifugation;

[0023] Use red blood cell preservation solution to prepare reagent red blood cells at 3% and 1% concentration.

[0024] The present invention also provides a freezing and thawing system for long-term storage of reagent red blood cells, comprising:

[0025] Red blood cell freezing system, used for freezing red blood cells;

[0026] The red blood cell thawing system is used to thaw frozen red blood cells.

[0027] Further, the red blood cell freezing system comprises:

[0028] Red blood cell processing equipment, used to centrifuge fresh red blood cell samples at 1000g for 5 minutes in a serological centrifuge, separate the upper plasma and buffy coat layers, mix with 0.9% sodium chloride solution at a volume ratio of 1:6, wash the red blood cells and centrifuge again;

[0029] Prepare red blood cell freezing solution: accurately weigh 300-350 g glycerol, 0.3 g potassium chloride, 16 g sodium citrate, 1.242 g disodium hydrogen phosphate, and 0.571 g sodium dihydrogen phosphate, dissolve in purified water and make up to 1 L, and store at 2-8°C;

[0030] The red blood cell freezing solution adding equipment is used to mix red blood cells and red blood cell preservation solution in a volume ratio of 1:6, and then mix them with the freezing solution in a ratio of 1:1 after centrifugation, and undergo a series of temperature treatments to finally store them at a low temperature below -60°C.

[0031] Further, the red blood cell thawing system comprises:

[0032] Path 1 thawing equipment, including room temperature thawing device, 9% sodium chloride solution, 2.5% sodium chloride solution, 0.9% sodium chloride solution, and storage and processing equipment for red blood cell preservation solution; it is capable of thawing red blood cells at room temperature, and gradually adding sodium chloride solutions of different concentrations for washing and centrifugation through gradient elution method, and finally preparing reagent red blood cells with concentrations of 3% and 1%.

[0033] Further, the red blood cell thawing system comprises:

[0034] Path 2 thawing equipment, including MD34 dialysis bags, plastic clamps, plastic boxes, 2.5% sodium chloride solution, 1.6% sodium chloride solution, 0.9% sodium chloride solution, and storage and processing equipment for red blood cell preservation solution; it is capable of thawing red blood cells at room temperature, placing the red blood cells in the dialysis bag, continuously diffusing glycerol to the outside of the dialysis bag, washing and centrifuging, and finally preparing reagent red blood cells with a concentration of 3% and 1%.

[0035] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0036] First, the present invention provides a simple and convenient way to freeze and thaw red blood cells, so that red blood cells can be effectively frozen for a long time, and the yield after re-dissolution is more than 80%, and the stability of red blood cell surface antigens is preserved. It is very important for the research of red blood cell surface antigens, the research of complex antibodies, the establishment of special blood type reagent red blood cell banks, and the quality control of blood typing reagents of various manufacturers. It also lays a technical reserve and important resources for the evaluation of red blood cell blood type serology and genotype consistency.

[0037] Second, the expected benefits and commercial value of the technical solution of the present invention after transformation are as follows: After the technical transformation of this invention, not only can the small-scale laboratory subtype red blood cells and rare blood type red blood cell preparation be transformed into commercialized and marketable reagent red blood cells, but it can also become a conventional production method for other red blood cell reagents. This technology can also be applied to the preparation of reagent red blood cells for irregular antibody screening of red blood cells, spectrum cells, and quality control products. Red blood cells can be stored for a long time, so that many rare red blood cell resources found in clinical practice can be effectively preserved and fully utilized. It lays a technical foundation for the establishment of ABO subtype reagent libraries and rare blood type reagent libraries. Reagent red blood cells can also be prepared in a planned manner according to market demand, which greatly improves the utilization rate of blood and reduces waste.

[0038] The cryopreservation of red blood cells is simple and convenient. It does not require any special equipment and is highly operable and can handle large volumes. The frozen red blood cell preservation solution is simple to prepare, has no special chemical components, and is low cost. Red blood cells can be stored at -60°C for up to 10 years or longer, and only the operating cost of the low-temperature refrigerator is required during the storage period.

[0039] The original dialysis method for thawing red blood cells removes glycerol to the greatest extent through the principle of materials science. It has simple materials and is easy to operate. It replaces the currently conventional method of gradually reducing the osmotic pressure of red blood cell washing solution. It can maintain the integrity of the red blood cell membrane to the greatest extent, provide high-quality reagent red blood cells, reduce test time, and indirectly improve the level of immunohematological tests, providing favorable support for the effectiveness of blood transfusion and hematological research.

[0040] The technical solution of the present invention fills the technical gap in the industry at home and abroad: At present, blood immunology experimental technology is inseparable from antigen-antibody reaction, and the antibody reagents used in serology are easy to produce and purify, and have been commercialized and standardized in large quantities. However, the raw materials of reagent red blood cells must come from the human body and cannot be replaced by substances from other sources, so the problems faced are very complicated. The main problem is that the average life span of red blood cells in vivo is about 120 days, and the effective period after in vitro preparation is 35 days. Frozen red blood cells are a conventional way to preserve red blood cells for a long time. However, in the later thawing process, the operation process is complicated and there is a lot of glycerol residue. To remove glycerol as much as possible, hypertonic saline and multiple mechanized washing are required, which easily destroys red blood cells, resulting in low yields, and rapid hemolysis of red blood cells in the later stage of preservation. In conventional serological experiments, false positives and cell membrane spine adhesions are easily caused to interfere with experimental results, so thawing red blood cells is generally not used to prepare reagent red blood cells. The technology of the present invention uses the dialysis principle to trap cells in a translucent membrane, thereby avoiding cell damage to the greatest extent.

[0041] The technical solution of the present invention solves a technical problem that people have been eager to solve but have never succeeded in solving: it solves the damage to cells during the process of thawing and removing glycerol from cells. During the thawing process, people have been studying the addition of thawing protection fluid to maintain the balance of osmotic pressure inside and outside the cell membrane. The thawing protection fluid generally adopts a formula similar to that of the freezing preservation fluid to reduce the concentration of glycerol or macromolecules, but ignores that the thawing process is a process of removing additions, which requires more washing in the later stage, causing greater damage to cells. Hematology and immunology is an interdisciplinary subject with multiple disciplines, and the rational use of materials science can achieve twice the result with half the effort.

[0042] The technical solution of the present invention overcomes technical prejudice: Through a series of experimental studies in the later stage, the present invention overcomes the technical prejudice that frozen red blood cells cannot be used as raw materials for reagent red blood cells. Through later studies, the cell membrane is intact, and the red blood cell blood type carbohydrate antigens and transmembrane protein antigens are not significantly weakened. Frozen and thawed red blood cells can be used as a way to prepare reagent red blood cells.

[0043] Third, the technical problems of the prior art solved by the present invention are:

[0044] 1. Poor long-term storage stability of red blood cells: Traditional red blood cell preservation methods can easily lead to a decrease in the activity and function of red blood cells during long-term storage, affecting their application effect.

[0045] 2. Unstable preservation solution formula: Existing preservation solution formulas may not provide adequate protection, making red blood cells susceptible to damage during freezing and thawing.

[0046] 3. The thawing process is complicated and inefficient: Many existing thawing methods are cumbersome and involve many steps, which can easily lead to contamination and misoperation, affecting the quality and safety of red blood cells.

[0047] 4. High cell loss rate: During the freezing and thawing process, the red blood cell loss rate is high, resulting in an insufficient number of red blood cells obtained, affecting experimental or clinical applications.

[0048] Significant technological advancements in technical solutions:

[0049] 1. Improving the storage stability of red blood cells: The present invention significantly improves the stability of red blood cells during long-term low-temperature storage by optimizing the formula of red blood cell freezing solution, thereby reducing cell damage and activity decline.

[0050] 2. Efficient red blood cell freezing method: The present invention proposes a systematic red blood cell freezing method, which adopts multi-step washing and precise solution ratio configuration to ensure that the red blood cells are fully protected during the freezing process, thereby greatly reducing the cell loss rate.

[0051] 3. Simplified thawing steps: The present invention provides two red blood cell thawing paths (gradient elution method and dialysis method), which greatly simplify the thawing operation steps, improve the thawing efficiency, and effectively reduce the risk of contamination during the operation.

[0052] 4. Maintaining the activity of red blood cells: Through improved preservation solution and scientific thawing process, the present invention can maintain the high activity and functional integrity of red blood cells after thawing, ensuring its effectiveness in subsequent applications.

[0053] 5. Controllable thawing process: The method of the present invention allows flexible control of the red blood cell recovery speed and effect during the thawing process by adjusting the concentration and ratio of the solution, thereby improving the controllability and repeatability of the thawing process.

[0054] The present invention proposes a series of improvement measures to address the technical difficulties in long-term storage and thawing of red blood cells, significantly improving the effect and efficiency of red blood cell storage and thawing. These technological advances not only improve the stability and activity of red blood cells, but also simplify the operation steps and reduce the cell loss rate, providing a more reliable and efficient solution for the long-term storage and practical application of red blood cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 It is a flow chart of a freezing and thawing method for long-term storage of reagent red blood cells provided by an embodiment of the present invention.

[0056] Figure 2 This is a finished product inspection diagram provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0058] The present invention innovatively proposes a freezing and thawing method for long-term storage of red blood cells. By optimizing the freezing solution formula, a systematic multi-step washing procedure and simplified thawing steps, the storage stability and activity of red blood cells are significantly improved, the cell loss rate is reduced, and two efficient and controllable thawing paths (gradient elution and dialysis) are provided, thereby ensuring the integrity and functionality of red blood cells during the freezing and thawing process, providing a reliable guarantee for its effectiveness in practical applications.

[0059] like Figure 1 As shown, a freezing and thawing method for long-term storage of reagent red blood cells provided by an embodiment of the present invention comprises the following steps:

[0060] S101, red blood cell cryopreservation;

[0061] S102, red blood cells thawing.

[0062] The red blood cell freezing method provided by the embodiment of the present invention:

[0063] (1) Red blood cell freezing solution:

[0064] Accurately weigh 300-350g glycerol, 0.3g potassium chloride, 16g sodium citrate, 1.242g disodium hydrogen phosphate, and 0.571g sodium dihydrogen phosphate, dissolve in purified water and dilute to 1L; store at 2-8℃;

[0065] (2) Cryopreservation of red blood cells:

[0066] Place the fresh sample in a serological centrifuge at 1000g for 5 minutes, separate the upper plasma and store it separately, separate the buffy coat layer for nucleic acid extraction, mix the red blood cells and red blood cell preservation solution at a volume ratio of 1:6, pipette and mix, place in a serological centrifuge at 1000g for 1 minute, remove the supernatant, wash the red blood cells 1 to 2 times, add the freezing solution to the cryopreservation tube, and slowly add the red blood cells to the freezing solution at a ratio of 1:1 to the packed red blood cells, and mix evenly by inverting;

[0067] Place at room temperature for about 10 minutes, then at 4°C for about 1 hour, then place at -20°C overnight, and finally place in a refrigerator below -60°C for long-term storage.

[0068] The red blood cell thawing method provided by the embodiment of the present invention:

[0069] 1) Path 1:

[0070] Prepare materials: 9% sodium chloride solution, 2.5% sodium chloride solution, 0.9% sodium chloride solution, red blood cell preservation solution;

[0071] Thaw at room temperature and use gradient elution method. When the frozen red blood cells are completely thawed at room temperature, measure the total volume after lysis, slowly add 9% sodium chloride solution at a ratio of 1:1, mix well, centrifuge at 1000g / min for 1min, and discard the supernatant;

[0072] Wash 2 to 3 times with 2.5% sodium chloride solution and packed red blood cells at a volume ratio of 6:1 until the supernatant is clear;

[0073] Wash 2 to 3 times with 0.9% sodium chloride solution and packed red blood cells at a volume ratio of 6:1 until the supernatant is clear;

[0074] Wash once with red blood cell preservation solution and packed red blood cells at a volume ratio of 6:1, and discard the supernatant after centrifugation;

[0075] Use red blood cell preservation solution to prepare 3% and 1% concentration of reagent red blood cells;

[0076] 2) Path 2:

[0077] Prepare materials: MD34 dialysis bag MW: 8000-14000, plastic clips, plastic boxes, 2.5% sodium chloride solution, 1.6% sodium chloride solution, 0.9% sodium chloride solution, red blood cell preservation solution;

[0078] Thaw at room temperature, use dialysis method, wait until the frozen red blood cells are completely thawed at room temperature, move them into dialysis bags, immerse them in 2.5% sodium chloride solution, and place them at room temperature for 1 hour, shaking them every 15 minutes, or place them at 2-8℃ for 2 hours;

[0079] Take out, measure the total volume of cells collected after dialysis, add 1.6% sodium chloride solution at a ratio of 1:1, mix, centrifuge and discard the supernatant;

[0080] Wash 1 to 2 times with 0.9% sodium chloride solution and packed red blood cells at a volume ratio of 6:1 until the supernatant is clear;

[0081] Wash once with red blood cell preservation solution and packed red blood cells at a volume ratio of 6:1, and discard the supernatant after centrifugation;

[0082] Use red blood cell preservation solution to prepare reagent red blood cells at 3% and 1% concentration.

[0083] The present invention is specifically implemented:

[0084] 1. The present invention provides a simple and convenient way to freeze and thaw red blood cells, so that red blood cells can be effectively frozen for a long time, and the yield after re-dissolution is more than 80%, and the stability of red blood cell surface antigens is maintained. It is very important for the research of red blood cell surface antigens, the research of complex antibodies, the establishment of special blood type reagent red blood cell banks, and the quality control of blood typing reagents of various manufacturers. It also lays a technical reserve and important resources for the evaluation of red blood cell blood type serology and genotype consistency.

[0085] 2. Cryopreservation of red blood cells

[0086] 2.1 Red blood cell freezing solution:

[0087] Accurately weigh 300-350g glycerol, 0.3g potassium chloride, 16g sodium citrate, 1.242g disodium hydrogen phosphate, and 0.571g sodium dihydrogen phosphate, dissolve in purified water and make up to 1L. Store at 2-8℃.

[0088] 2.2 Cryopreservation of red blood cells:

[0089] 2.2.1 Centrifuge the fresh sample at 1000g for 5 minutes in a serological centrifuge, separate the upper plasma and store it separately, separate the buffy coat layer for nucleic acid extraction, mix the red blood cells and red blood cell preservation solution at a volume ratio of 1:6, pipette and mix, centrifuge at 1000g for 1 minute in a serological centrifuge, remove the supernatant, wash the red blood cells 1 to 2 times and add them to the freezing solution.

[0090] 2.2.2 Place at room temperature for about 10 minutes, then at 4℃ for about 1 hour, then place at -20℃ overnight, and finally place in a refrigerator below -60℃ for long-term storage.

[0091] 2.3 Notes:

[0092] 2.3.1 During the red blood cell washing process, it is not advisable to wash multiple times. Washing with 0.9% sodium chloride twice is sufficient. The force of blowing and mixing should be controlled. Excessive mechanical force will destroy the red blood cell surface antigens.

[0093] 2.3.2 Before freezing, red blood cells need to be washed once with red blood cell preservation solution to provide the red blood cells with the corresponding nutrients and isotonic buffer environment.

[0094] 2.3.3 Gradient cooling and freezing allows cells to be gradually dehydrated and will not be harmed by the production of large ice crystals.

[0095] 3. Red blood cell thawing (resuscitation):

[0096] 3.1 Path 1:

[0097] Prepare materials: 9% sodium chloride solution, 2.5% sodium chloride solution, 0.9% sodium chloride solution, and red blood cell preservation solution.

[0098] 3.1.1 Thaw at room temperature and use gradient elution method. When the frozen red blood cells are completely thawed at room temperature, measure the total volume after lysis, slowly add 9% sodium chloride solution in a 1:1 ratio, mix well, centrifuge and discard the supernatant.

[0099] 3.1.2 Wash 2 to 3 times with 2.5% sodium chloride solution and packed red blood cells in a volume ratio of 6:1 until the supernatant is clear.

[0100] 3.1.3 Wash 2 to 3 times with 0.9% sodium chloride solution and packed red blood cells in a volume ratio of 6:1 until the supernatant is clear.

[0101] 3.1.4 Wash once with red blood cell preservation solution and packed red blood cells at a volume ratio of 6:1, and discard the supernatant after centrifugation.

[0102] 3.1.5 Use red blood cell preservation solution to prepare reagent red blood cells at 3% and 1% concentration.

[0103] 3.2 Path 2:

[0104] Prepare materials: MD34 dialysis bag (MW:8000-14000), plastic clips, plastic boxes, 2.5% sodium chloride solution, 1.6% sodium chloride solution, 0.9% sodium chloride solution, and red blood cell preservation solution.

[0105] 3.1.1 Thaw at room temperature. Use dialysis method. When the frozen red blood cells are completely thawed at room temperature, transfer them into dialysis bags, immerse them in 2.5% sodium chloride solution, and place them at room temperature for 1 hour. During this period, shake them every 15 minutes or place them at 2-8℃ for 2 hours.

[0106] 3.1.2 Take out, measure the total volume of cells collected after dialysis, add 1.6% sodium chloride solution in a ratio of 1:1, mix, centrifuge and discard the supernatant.

[0107] 3.1.3 Wash 1 to 2 times with 0.9% sodium chloride solution and packed red blood cells in a volume ratio of 6:1 until the supernatant is clear.

[0108] 3.1.4 Wash once with red blood cell preservation solution and packed red blood cells at a volume ratio of 6:1, and discard the supernatant after centrifugation.

[0109] 3.1.5 Use red blood cell preservation solution to prepare reagent red blood cells at 3% and 1% concentration.

[0110] 4. Verification experiment:

[0111] 4.1 Verification of red blood cells: ZJ001-A 1 Type, ZJ002-B, ZJ003-O, ZJ004-A 1 Type B, ZJ005-A 2Type, ZJ006-Bel, ZJ007-AB 3 Type, ZJ008-Bw type,. The red blood cells were frozen and stored as described above and thawed after 18 months.

[0112] 4.2 Verification items:

[0113] The red blood cells were studied in accordance with the "Guidelines for Registration Review of Human Red Blood Cell Reverse Typing Reagents" (2023 revised version).

[0114] Red blood cell antigen detection: anti-A (IgM), anti-A 1 , anti-B (IgM), anti-AB (IgM), anti-H, anti-D (IgM), anti-E (IgM), anti-C (IgM). Combined with the serological test before red blood cell antigen cryopreservation and the "Guidelines for Registration and Review of Human Red Blood Cell Reverse Typing Reagents", the test results are required to meet the following table:

[0115] Anti-A <![CDATA[Anti-A 1 > Anti-B Anti-AB Anti-H Anti-D Anti-E Anti-C <![CDATA[ZJ001-A 1 Type]]> 4+ 4+ 0 4+ 0 Strong Strong Strong ZJ002-B 0 0 4+ 4+ 0 Strong 0 Strong ZJ003-O type 0 0 0 0 Strong Strong + Strong <![CDATA[ZJ004-A 1 Type B]]> 4+ 4+ 4+ 4+ 0 Strong 0 Strong <![CDATA[ZJ005-A 2 Type]]> 4+ 0 0 4+ + Strong Strong Strong ZJ006-Bel 0 0 0 0 + Strong 0 Strong <![CDATA[ZJ007-AB 3 Type]]> 4+ 4+ + / mf 4+ + Strong Strong Strong ZJ008-Bw 0 0 + / mf 0 + Strong 0 Strong

[0116] Determination of red blood cell antigen strength: Combined with the serological test of red blood cell antigens before freezing and the "Guidelines for Registration and Review of Human Red Blood Cell Reverse Typing Reagents", the test results are required to meet the following table:

[0117]

[0118] Direct antiglobulin test: required to be negative.

[0119] Hemolysis index: Hemoglobin concentration of the sample to be tested (mg / L) ≤ 0.29g / L.

[0120] 4.3 Detection classification:

[0121] 1% concentration of reagent red blood cells was used for microcolumn gel card detection, 3% concentration of reagent red blood cells was used for test tube detection. The saline gradient elution method and dialysis method of thawed red blood cells were tested separately.

[0122] 5. Related icons

[0123] Thaw cells and prepare 3% and 1% red blood cell reagents. Figure 2 As shown. Finished product testing:

[0124] The results of the red blood cell antigen detection are shown in Table 1:

[0125]

[0126]

[0127] Table 2 Red blood cell antigen strength detection:

[0128]

[0129] Direct antiglobulin test: all results were negative.

[0130] Table 3 Hemolysis index:

[0131] ZJ001 ZJ002 ZJ003 ZJ004 ZJ005 ZJ006 ZJ007 ZJ008 Salt 1% 0.017 0.019 0.013 0.016 0.017 0.014 0.014 0.013 Salt 3% 0.023 0.027 0.033 0.027 0.015 0.017 0.024 0.029 1% 0.019 0.011 0.013 0.013 0.015 0.023 0.021 0.018 3% 0.024 0.027 0.018 0.014 0.035 0.027 0.038 0.017

[0132] The reagent red blood cells prepared by the above four thawing methods were subjected to long-term stability experiments and open-bottle stability experiments to verify the effectiveness of their red blood cell surface antigens.

[0133] According to experimental data, the long-term stability of the 3% red blood cell reagent prepared by dialysis is valid for 63 days, and the stability after opening the bottle is valid for 42 days. The long-term stability of the 1% red blood cell reagent is valid for 91 days, and the stability after opening the bottle is valid for 63 days.

[0134] The long-term stability of the 3% red blood cell reagent prepared by the saline gradient elution method is valid for 42 days, and the stability after opening the bottle is valid for 28 days). The long-term stability of the 1% red blood cell reagent prepared is valid for 56 days; the stability after opening the bottle is valid for 35 days.

[0135] The experimental results are as follows:

[0136] Table 4-1. Long-term stability of 3% red blood cell reagent prepared by dialysis

[0137]

[0138] Table 4-2. Long-term stability of 1% red blood cell reagent prepared by dialysis.

[0139]

[0140]

[0141] Table 4-3. Open-bottle stability of 3% red blood cell reagent prepared by dialysis.

[0142]

[0143]

[0144] Table 4-4. Long-term stability of 3% red blood cell reagent prepared by saline gradient elution method.

[0145]

[0146] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A cryopreservation solution for long-term storage of red blood cells, characterized in that: It is composed of isotonic phosphate buffer, which contains the following components: 0.1 mol / L phosphate buffer, 1.6% sodium citrate, 0.03% potassium chloride and 30-35% glycerol.

2. The method for using the cryopreservation solution in long-term storage of red blood cells according to claim 1, characterized in that: The following steps are involved: (1) Wash fresh hematocrit 1-2 times with commercially available red blood cell preservation solution; (2) Mix the above freezing solution and packed red blood cells in a volume ratio of 1:1, invert to mix, and leave at room temperature for about 10 minutes to equilibrate; (3) Place the equilibrated red blood cells at 4°C for about 1 hour; (4) Place the red blood cells at -20°C overnight; (5) Transfer the red blood cells to a refrigerator below -60°C for long-term storage.

3. A method for thawing reagent red blood cells, characterized in that: Using a special translucent membrane, frozen red blood cells are placed in a bag and immersed in a buffer solution, so that the red blood cells are trapped in the bag, while glycerol and small molecules are dialyzed to the outside of the bag by diffusion.

4. The method for thawing red blood cells according to claim 3, characterized in that: The following steps are involved: (1) Thawing at room temperature, using gradient elution method, until the frozen red blood cells are completely thawed at room temperature; (2) Measure the total volume after dissolution, slowly add 9% sodium chloride solution in a ratio of 1:1, mix well, centrifuge, and discard the supernatant; (3) Wash 2 to 3 times with 2.5% sodium chloride solution and packed red blood cells at a volume ratio of 6:1 until the supernatant is clear; (4) Wash 2 to 3 times with 0.9% sodium chloride solution and packed red blood cells at a volume ratio of 6:1 until the supernatant is clear; (5) Wash once with red blood cell preservation solution and packed red blood cells at a volume ratio of 6:1, and discard the supernatant after centrifugation; (6) Prepare reagent red blood cells at concentrations of 3% and 1% using red blood cell preservation solution.

5. The method for thawing red blood cells according to claim 3, characterized in that: The following steps are involved: (1) Thawing at room temperature, using dialysis method, until the frozen red blood cells are completely thawed at room temperature; (2) Transfer the thawed red blood cells into a dialysis bag, immerse them in a 2.5% sodium chloride solution, and place them at room temperature for 1 hour, shaking them every 15 minutes or placing them at 2-8°C for 2 hours; (3) Take out the dialyzed red blood cells, measure the total volume collected after dialysis, add 1.6% sodium chloride solution at a volume ratio of 1:1, mix, centrifuge, and discard the supernatant; (4) Wash 1 to 2 times with 0.9% sodium chloride solution and packed red blood cells at a volume ratio of 6:1 until the supernatant is clear; (5) Wash once with red blood cell preservation solution and packed red blood cells at a volume ratio of 6:1, and discard the supernatant after centrifugation; (6) Prepare reagent red blood cells at concentrations of 3% and 1% using red blood cell preservation solution.

6. The method for long-term storage of red blood cells according to claim 2, characterized in that: The commercial red blood cell preservation solution is an isotonic solution, contains carbohydrate components to provide energy, and contains antibacterial components to inhibit the growth of microorganisms.

7. A freezing and thawing system for long-term storage of reagent red blood cells, characterized in that: include: Red blood cell freezing system, used for freezing red blood cells; The red blood cell thawing system is used to thaw frozen red blood cells.

8. The freezing and thawing system for long-term storage of reagent red blood cells according to claim 7, characterized in that: The red blood cell freezing system comprises: The equipment for preparing red blood cell freezing solution can accurately weigh 300-350g glycerol, 0.3g potassium chloride, 16g sodium citrate, 1.242g disodium hydrogen phosphate, 0.571g sodium dihydrogen phosphate, dissolve them with purified water and make up to 1L, and store at 2-8°C; Red blood cell processing equipment, used for placing fresh red blood cell samples in a serological centrifuge and centrifuging at 1000g for 5 minutes to separate red blood cells, plasma and buffy coat, and mixing with 0.9% sodium chloride solution at a volume ratio of 1:6, and centrifuging and washing red blood cells again; The red blood cell freezing solution adding equipment is used to mix red blood cells and red blood cell preservation solution in a volume ratio of 1:6, centrifuge to retain the red blood cells, mix them with the freezing solution in a ratio of 1:1, and perform a series of temperature treatments to finally store them below -60°C.

9. The freezing and thawing system for long-term storage of reagent red blood cells according to claim 7, characterized in that: The red blood cell thawing system comprises: Path 1 thawing equipment, including room temperature thawing device, 9% sodium chloride solution, 2.5% sodium chloride solution, 0.9% sodium chloride solution, and storage and processing equipment for red blood cell preservation solution; it is capable of thawing red blood cells at room temperature, and gradually adding sodium chloride solutions of different concentrations for centrifugation and washing through gradient elution method, and finally preparing reagent red blood cells with concentrations of 3% and 1%.

10. The freezing and thawing system for long-term storage of reagent red blood cells according to claim 7, characterized in that: The red blood cell thawing system comprises: Path 2 thawing equipment, including MD34 dialysis bags, plastic clamps, plastic boxes, 2.5% sodium chloride solution, 1.6% sodium chloride solution, 0.9% sodium chloride solution, and storage and processing equipment for red blood cell preservation solution; it is capable of thawing red blood cells at room temperature, placing the red blood cells in the dialysis bag through dialysis, and gradually shaking, centrifuging and washing with sodium chloride solutions of different concentrations, and finally preparing reagent red blood cells with concentrations of 3% and 1%.

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