Hydroformylated red blood cells capable of being stored for long time, application of hydroformylated red blood cells in blood type reverse typing and irregular antibody detection and detection system

Abstract

The invention discloses hydroformylated red blood cells capable of being stored for a long time, application of the hydroformylated red blood cells in blood type reverse typing and irregular antibody detection and a detection system.The hydroformylated red blood cells are prepared through the following steps that S1-1, red blood cells of a blood type A, a blood type B and a blood type O from healthy people are collected to serve as standard red blood cells, and the standard red blood cells are washed with normal saline and diluted; a standard erythrocyte suspension is obtained; s1-2, adding the standard red blood cell suspension into an hydroformylation reagent consisting of glutaraldehyde and formaldehyde, and treating at room temperature; s1-3, centrifuging to remove supernatant, and washing with normal saline to obtain hydroformylated red blood cells; and S1-4, diluting the hydroformylated red blood cells with a red blood cell preserving fluid to obtain a hydroformylated red blood cell suspension. Through hydroformylation process optimization, the preservation period of the red blood cells can reach one year (generally less than or equal to 3 months in the prior art), the transportation and storage problems of the fresh red blood cells can be solved, and the method is particularly suitable for long-term storage of blood banks and medical institutions in remote areas and long-term storage of the red blood cells.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, and in particular to an aldehyde-containing red blood cell that can be stored for a long time, its application in blood typing and irregular antibody detection, and a detection system thereof. Background Technology

[0002] In clinical blood typing, reverse typing (serological typing) is a crucial step in determining blood type by reacting the patient's serum or plasma with standard red blood cells. Irregular antibody screening and identification are important tests before transfusion, during pregnancy, and in the diagnosis of certain diseases. This involves reacting the patient's serum or plasma with screening red blood cells (usually containing 3 samples of type O red blood cells with known antigens) or spectrum cells (containing 10-16 samples of type O red blood cells with known antigens) and an anti-human globulin reagent. Analysis of the reaction pattern determines the presence of irregular antibodies and their specificity, aiming to identify clinically significant IgG blood group antibodies (such as anti-D, anti-Kell, etc.) to prevent hemolytic transfusion reactions and neonatal hemolytic disease.

[0003] Clinically used ABO reverse typing reagents, irregular antibody screening reagents, and spectrophotometers all use fresh red blood cells. Fresh red blood cells are prone to hemolysis, difficult to store, and have a very short shelf life for cellular antigens, making it difficult to guarantee reagent quality. The stability period of reverse typing red blood cells prepared in clinical laboratories is mostly between 3 and 60 days, while commercially available reagents only have a shelf life of 90 days. After the time spent on manufacturing, transportation, and supplier preparation, the shelf life is already quite short by the time they reach the user. Spectrophotometers, in particular, require specific blood group antigen combinations, have limited availability, are expensive, and are used infrequently, often exceeding their expiration date and hemolyzing after only a few uses, resulting in waste. Furthermore, the freshness of red blood cells within their shelf life directly affects the antigenic intensity. The structure and stability of the red blood cell membrane and membrane proteins change with storage time, often leading to hemolysis, weak agglutination, and false negative results in clinical applications, severely interfering with blood typing test results.

[0004] Microcolumn gel immunoassay (such as microcolumn gel cards) has become a mainstream detection method due to its high degree of automation. Its principle is based on the molecular sieving effect of gel particles, causing red blood cells to migrate to the bottom of the gel or a specific location under centrifugal force, and the blood type is determined by the reaction results.

[0005] In existing technologies, aldehyde treatment (such as glutaraldehyde cross-linking) is often used to stabilize the structure of red blood cells and extend their shelf life to several months in order to prolong their preservation. However, due to changes in physical properties (such as increased rigidity and changes in surface charge), aldehyde-treated red blood cells are prone to tailing during migration in micropillar gel cards, leading to false positive results. Therefore, aldehyde-treated red blood cells are only occasionally used in methods such as the test tube method and are not routinely used in clinical practice.

[0006] Defects and shortcomings of existing technology:

[0007] ①Shelf life limitation: Fresh red blood cells need to be prepared frequently, which is inconvenient to use and cannot meet the needs of long-term storage.

[0008] ② False positive problem: Aldehyde-oxidized red blood cells cannot completely pass through conventional microcolumn gel particles (such as Sephadex G-50 or polyacrylamide gel), and remain in the middle or upper layer of the gel, causing misjudgment of the test results.

[0009] ③ Equipment dependency issues: Existing solutions force the migration of aldehyde-treated red blood cells by increasing centrifugation force or extending centrifugation time, but this requires modification of the corresponding centrifuge parameters or customization of automated equipment, increasing the cost and complexity of clinical applications. In addition, some blood typing cards combine forward and reverse typing on one card. If the aldehyde-treated red blood cells used for reverse typing require changes in centrifugation force or time, this will not be suitable for tests that perform forward and reverse typing simultaneously.

[0010] ④ Insufficient functional equivalence: Red blood cells treated with high concentrations of aldehydes may have reduced ability to react with antibodies due to damage to antigen epitopes, and therefore cannot completely replace the detection efficacy of fresh red blood cells. Summary of the Invention

[0011] The technical problem this invention aims to solve is to address the shortcomings of the prior art by providing a long-term preservable aldehyde-modified erythrocyte, its application in blood typing and irregular antibody detection, and a detection system. This invention provides a system for blood typing or irregular antibody detection with a long shelf life, high detection accuracy, and without requiring changes to existing centrifugation equipment. It overcomes the problems of short shelf life of fresh erythrocytes and migration barriers and false positives associated with aldehyde-modified erythrocytes in microcolumn gel cards. The detection system of this invention comprises long-term preservable aldehyde-modified erythrocytes and a modified microcolumn gel card formulation; the synergistic effect of these two components achieves detection results equivalent to those of fresh erythrocytes.

[0012] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: In its first aspect, the present invention provides aldehyde-modified erythrocytes that can be stored for a long period of time, which are prepared through the following steps:

[0013] S1-1. Collect red blood cells of blood type A, blood type B, and blood type O from healthy individuals as standard red blood cells, wash with physiological saline, and then dilute with physiological saline to obtain a standard red blood cell suspension.

[0014] S1-2. Add the standard red blood cell suspension to an aldehyde reagent composed of glutaraldehyde and formaldehyde, and treat at room temperature for 5-30 minutes.

[0015] S1-3. Centrifuge to remove the supernatant and wash with physiological saline to obtain aldehyde-treated erythrocytes;

[0016] S1-4. Dilute the aldehyde-treated red blood cells with red blood cell preservation solution to obtain an aldehyde-treated red blood cell suspension;

[0017] Aldehyde-containing red blood cell suspensions prepared from blood types A, B, and O are respectively designated as blood type A aldehyde-containing red blood cell suspension, blood type B aldehyde-containing red blood cell suspension, and blood type O aldehyde-containing red blood cell suspension.

[0018] Preferably, the long-term preservable aldehyde-treated erythrocytes are prepared through the following steps:

[0019] S1-1. Collect red blood cells of blood type A, blood type B and blood type O from healthy people as standard red blood cells. Wash them with physiological saline 1-6 times to remove plasma components, and then dilute them with physiological saline to a volume concentration of 25-75% to obtain standard red blood cell suspension.

[0020] S1-2. Add the standard red blood cell suspension to an aldehyde reagent composed of glutaraldehyde and formaldehyde, so that the final volume concentration of glutaraldehyde in the resulting mixture is 0.0001-0.5% and the final volume concentration of formaldehyde is 0.001-1%, and treat at room temperature for 5-30 min.

[0021] S1-3. Centrifuge to remove the supernatant, and wash with physiological saline 1-8 times to remove excess aldehyde reagent to obtain aldehyde-treated red blood cells;

[0022] S1-4. Dilute the aldehyde-treated red blood cells to 0.8-5 wt% with red blood cell preservation solution to obtain an aldehyde-treated red blood cell suspension, and store at 2-8℃.

[0023] Aldehyde-containing red blood cell suspensions prepared from blood types A, B, and O are respectively designated as blood type A aldehyde-containing red blood cell suspension, blood type B aldehyde-containing red blood cell suspension, and blood type O aldehyde-containing red blood cell suspension.

[0024] Preferably, the long-term preservable aldehyde-treated erythrocytes are prepared through the following steps:

[0025] S1-1. Collect red blood cells of blood type A, blood type B and blood type O from healthy people as standard red blood cells. Wash them three times with physiological saline to remove plasma components, and then dilute them with physiological saline to a volume concentration of 50% to obtain a standard red blood cell suspension.

[0026] S1-2. Add the standard red blood cell suspension to an aldehyde reagent composed of glutaraldehyde and formaldehyde, so that the final volume concentration of glutaraldehyde in the resulting mixture is 0.01% and the final volume concentration of formaldehyde is 0.02%, and treat at room temperature for 15 min.

[0027] S1-3. Centrifuge to remove the supernatant, and wash with physiological saline four times to remove excess aldehyde reagent to obtain aldehyde-treated red blood cells;

[0028] S1-4. Dilute the aldehyde-treated red blood cells to 0.8 wt% with red blood cell preservation solution to obtain an aldehyde-treated red blood cell suspension, and store at 4°C.

[0029] Aldehyde-containing red blood cell suspensions prepared from blood types A, B, and O are respectively designated as blood type A aldehyde-containing red blood cell suspension, blood type B aldehyde-containing red blood cell suspension, and blood type O aldehyde-containing red blood cell suspension.

[0030] A second aspect of the present invention provides the use of aldehyde-modified erythrocytes as described above in blood typing or in the preparation of blood typing reagents.

[0031] A third aspect of the present invention provides a blood typing reverse typing system, comprising an aldehyde-modified erythrocyte suspension as described above and a modified reverse typing microcolumn gel card; the aldehyde-modified erythrocyte suspension includes blood type A aldehyde-modified erythrocyte suspension, blood type B aldehyde-modified erythrocyte suspension, and blood type O aldehyde-modified erythrocyte suspension.

[0032] The improved reverse-shaping micropillar gel card was prepared through the following steps:

[0033] S2-1. Prepare the gel buffer solution according to the following formula:

[0034] 15.4-61.6 mM sodium chloride, 0.75-3.0 mM disodium hydrogen phosphate, 0.75-3.0 mM sodium dihydrogen phosphate, 0.12-0.48 M glycine, and 0.5-2% bovine serum albumin to adjust the pH to 6.65-6.85;

[0035] S2-2. Dextran gel particles with pore sizes adapted to aldehyde erythrocytes are soaked and washed with gel buffer to obtain gel particles.

[0036] S2-3. After mixing the gel particles with the gel buffer, dispense them into the reaction wells of the microcolumn tube to obtain the modified reverse-sizing microcolumn gel card.

[0037] Preferably, the modified reverse-shaping micropillar gel card is prepared by the following steps:

[0038] S2-1. Prepare the gel buffer solution according to the following formula:

[0039] 30.8 mM sodium chloride, 1.5 mM disodium hydrogen phosphate, 1.5 mM sodium dihydrogen phosphate, 0.24 M glycine, and 1% bovine serum albumin were added to adjust the pH to 6.65-6.85.

[0040] S2-2. G25 dextran gel particles with a particle size of 50-150μm and a size exclusion molecular weight of 1-5KD are soaked in gel buffer and washed 3-5 times to obtain gel particles.

[0041] S2-3. Mix the gel particles and gel buffer at a volume ratio of 1:1 and dispense them into microcolumns to obtain the modified reverse-sizing microcolumn gel card. The number of reaction wells in the microcolumn is not less than 3.

[0042] A fourth aspect of the present invention provides a blood type reverse typing detection method, which is implemented using the blood type reverse typing system described above, and the method includes the following steps:

[0043] S3-1. Add the plasma or serum sample to be tested to the three reaction wells of the modified reverse-sizing microcolumn gel card, respectively.

[0044] S3-2. Add A-type aldehyde erythrocyte suspension, B-type aldehyde erythrocyte suspension, and O-type aldehyde erythrocyte suspension to each reaction well, respectively.

[0045] S3-3, Centrifugation;

[0046] S3-4. Observe the migration of red blood cells in the modified reverse-stylating microcolumn gel card and judge the test results.

[0047] Preferably, the method includes the following steps:

[0048] S3-1. Add 50 μL of the plasma / serum sample to be tested to each of the three reaction wells of the modified reverse-sizing microcolumn gel card;

[0049] S3-2. Add 50 μL of A-type aldehyde erythrocyte suspension, B-type aldehyde erythrocyte suspension, and O-type aldehyde erythrocyte suspension, each with a concentration of 0.8-5 wt%, to each reaction well.

[0050] S3-3, Centrifugation in a conventional centrifuge: Centrifuge at 900 rpm for 2 min, then centrifuge at 1500 rpm for 3 min;

[0051] S3-4. Observe the migration of red blood cells in the modified reverse-stylating microcolumn gel card and judge the test results.

[0052] A fifth aspect of the present invention provides the use of aldehyde-treated erythrocytes as described above in the detection of irregular antibodies or in the preparation of reagents for the detection of irregular antibodies.

[0053] In a sixth aspect, the present invention provides an irregular antibody detection system, comprising the O blood type aldehyde erythrocyte suspension and the modified anti-human globulin microcolumn gel card as described above.

[0054] The modified anti-human globulin microcolumn gel card was prepared by the following steps:

[0055] S4-1. Prepare the gel buffer solution according to the following formula:

[0056] 15.4-61.6mM sodium chloride, 0.75-3.0mM disodium hydrogen phosphate, 0.75-3.0mM sodium dihydrogen phosphate, 0.12-0.48M glycine, 0.5-2% bovine serum albumin, and anti-human globulin antibody; adjust pH to 6.65-6.85; final titer of anti-human globulin antibody is 64-128.

[0057] S4-2. The dextran gel particles with pore sizes adapted to aldehyde erythrocytes are soaked and washed with gel buffer to obtain gel particles.

[0058] S4-3. After mixing the gel particles with the gel buffer, dispense them into the reaction wells of the microcolumn to obtain the modified anti-human globulin microcolumn gel card.

[0059] Preferably, the modified anti-human globulin micropillar gel card is prepared by the following steps:

[0060] S4-1. Prepare the gel buffer solution according to the following formula:

[0061] 30.8mM sodium chloride, 1.5mM disodium hydrogen phosphate, 1.5mM sodium dihydrogen phosphate, 0.24M glycine, 1% bovine serum albumin, and anti-human globulin antibody were added to adjust the pH to 6.65-6.85. The final titer of the anti-human globulin antibody was 64-128.

[0062] S4-2. Soak and wash G25 dextran gel particles with a particle size of 50-150μm and a size exclusion molecular weight of 1-5KD in gel buffer 3-5 times to obtain gel particles.

[0063] S4-3. Mix the gel particles with the gel buffer and dispense them into the reaction wells of the microcolumn to obtain the modified anti-human globulin microcolumn gel card. The number of reaction wells in the microcolumn is not less than 3.

[0064] A seventh aspect of the present invention provides a method for detecting irregular antibodies, which is implemented using the irregular antibody detection system described above, the method comprising the following steps:

[0065] S5-1. Add the plasma or serum sample to be tested into the reaction well of the modified anti-human globulin microcolumn gel card;

[0066] S5-2. Add aldehyde-treated red blood cell suspension to the reaction well;

[0067] Incubate at 37℃ using S5-3;

[0068] S5-4, Centrifugation;

[0069] S5-5. Observe the migration of red blood cells in the modified anti-human globulin microcolumn gel card and determine the test results.

[0070] Preferably, the method includes the following steps:

[0071] S5-1. Add 50 μL of plasma or serum sample to be tested to the reaction well of the modified anti-human globulin microcolumn gel card.

[0072] S5-2. Add 50 μL of aldehyde-treated red blood cell suspension with a concentration of 0.8-5 wt% to the reaction well;

[0073] S5-3, incubate at 37℃ for 15 minutes;

[0074] S5-4. Centrifuge in a conventional centrifuge: centrifuge at 900 rpm for 2 min, then centrifuge at 1500 rpm for 3 min;

[0075] S5-5. Observe the migration of red blood cells in the modified anti-human globulin microcolumn gel card and determine the test results.

[0076] The beneficial effects of this invention are:

[0077] (1) The shelf life is significantly extended:

[0078] This invention optimizes the aldehyde process, enabling red blood cells to be stored for up to one year (compared to ≤3 months in existing technologies). This solves the problem of transporting and storing fresh red blood cells, and is especially suitable for long-term storage in blood banks and medical institutions in remote areas, as well as for the long-term preservation of a wide range of cells.

[0079] (2) Detection accuracy has been greatly improved:

[0080] Eliminating false positives: The G25 particles in the improved reverse-sizing microcolumn gel card and the improved anti-human globulin microcolumn gel card of the present invention have pore sizes that match the size of aldehyde erythrocytes. Combined with the lubrication system (BSA) in the suspension, aldehyde erythrocytes can easily pass through the gel gaps, which can solve the migration obstacles and false positive problems of aldehyde erythrocytes in the microcolumn gel card.

[0081] Antigen activity is stable: The intensity of ABO blood group antigens on red blood cells is basically stable after aldehyde treatment in this invention, and its ability to react with antibodies is equivalent to that of fresh red blood cells.

[0082] (3) Reduced clinical operation costs:

[0083] No equipment modification required: This invention uses a conventional centrifuge, avoiding investment in customized equipment and is compatible with existing automated testing lines.

[0084] Simplified operation process: The detection steps of this invention are the same as those of the traditional microcolumn gel method, requiring no additional parameter settings and reducing the training cost for medical staff.

[0085] (4) Clear industrialization value:

[0086] This invention enables the formation of a standardized detection combination of "aldehyde-derived erythrocytes + modified microcolumn gel card", which can be mass-produced, has a long shelf life, and has broad market application prospects. Attached Figure Description

[0087] Figure 1 The test results are for example 1.

[0088] Figure 2 The results of reverse blood typing test for example 2;

[0089] Figure 3 The detection results are for test example 3;

[0090] Figure 4 The results are from the accelerated stability test at 37°C for Test Example 4. Detailed Implementation

[0091] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0092] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0093] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0094] Example 1

[0095] A type of aldehyde-modified erythrocyte that can be stored for a long time is prepared by the following steps:

[0096] S1-1. Collect red blood cells of blood type A, blood type B and blood type O from healthy people as standard red blood cells. Wash them three times with physiological saline to remove plasma components, and then dilute them with physiological saline to a volume concentration of 50% to obtain a standard red blood cell suspension.

[0097] S1-2. Add the standard red blood cell suspension to an aldehyde reagent composed of glutaraldehyde and formaldehyde, so that the final volume concentration of glutaraldehyde in the resulting mixture is 0.01% and the final volume concentration of formaldehyde is 0.02%, and treat at room temperature for 15 min.

[0098] S1-3. Centrifuge to remove the supernatant, and wash with physiological saline four times to remove excess aldehyde reagent to obtain aldehyde-treated red blood cells;

[0099] S1-4. Dilute the aldehyde-treated red blood cells to 0.8 wt% with conventional red blood cell preservation solution (Changchun Boxun Biotechnology Co., Ltd., cell preservation solution, catalog number BX3002-100) to obtain an aldehyde-treated red blood cell suspension, and store at 4°C.

[0100] Aldehyde-containing red blood cell suspensions prepared from blood types A, B, and O are respectively designated as blood type A aldehyde-containing red blood cell suspension, blood type B aldehyde-containing red blood cell suspension, and blood type O aldehyde-containing red blood cell suspension.

[0101] Example 2

[0102] A blood typing reverse typing system, comprising an aldehyde-modified erythrocyte suspension and a modified reverse typing microcolumn gel card as described in Example 1; wherein the aldehyde-modified erythrocyte suspension includes blood type A aldehyde-modified erythrocyte suspension, blood type B aldehyde-modified erythrocyte suspension, and blood type O aldehyde-modified erythrocyte suspension;

[0103] The improved reverse-shaping micropillar gel card was prepared through the following steps:

[0104] S2-1. Prepare the gel buffer solution according to the following formula:

[0105] 30.8 mM sodium chloride, 1.5 mM disodium hydrogen phosphate, 1.5 mM sodium dihydrogen phosphate, 0.24 M glycine, and 1% bovine serum albumin (BSA) were added to adjust the pH to 6.65-6.85.

[0106] S2-2. G25 dextran gel particles with a particle size of 50-150μm and a size exclusion molecular weight of 1-5KD are soaked in gel buffer and washed 5 times to remove broken gel fragments and obtain gel particles.

[0107] S2-3. Mix the gel particles and suspension at a volume ratio of 1:1 and then dispense them into microcolumns to obtain the improved reverse-shaping microcolumn gel card. The number of reaction pores in the microcolumn is not less than 3.

[0108] Example 3

[0109] A blood typing reverse typing method, implemented using the blood typing reverse typing system of Example 2, includes the following steps:

[0110] S3-1. Add 50 μL of the plasma / serum sample to be tested to each of the three reaction wells of the modified reverse-sizing microcolumn gel card;

[0111] S3-2. Add 50 μL of 0.8 wt% A blood type aldehyde erythrocyte suspension, B blood type aldehyde erythrocyte suspension, and O blood type aldehyde erythrocyte suspension to each reaction well.

[0112] S3-3, Centrifugation in a conventional centrifuge: Centrifuge at 900 rpm for 2 min, then centrifuge at 1500 rpm for 3 min;

[0113] S3-4. Observe the migration of red blood cells in the modified reverse-stylating microcolumn gel card and judge the test results.

[0114] Example 4

[0115] An irregular antibody detection system includes the O-type aldehyde erythrocyte suspension and the modified anti-human globulin microcolumn gel card from Example 1;

[0116] The modified anti-human globulin microcolumn gel card was prepared by the following steps:

[0117] 30.8mM sodium chloride, 1.5mM disodium hydrogen phosphate, 1.5mM sodium dihydrogen phosphate, 0.24M glycine, 1% bovine serum albumin, and anti-human globulin antibody were added to adjust the pH to 6.65-6.85. The final titer of the anti-human globulin antibody was 64-128.

[0118] S4-2. G25 dextran gel particles with a particle size of 50-150μm and a size exclusion molecular weight of 1-5KD were soaked in gel buffer and washed 5 times to obtain gel particles.

[0119] S4-3. After mixing the gel particles with the suspension, dispense them into the reaction wells of the microcolumn to obtain the modified anti-human globulin microcolumn gel card. The number of reaction wells in the microcolumn is not less than 3.

[0120] Example 5

[0121] An irregular antibody detection method, implemented using the irregular antibody detection system of Example 4, includes the following steps:

[0122] S5-1. Add 50 μL of plasma or serum sample to be tested to the reaction well of the modified anti-human globulin microcolumn gel card.

[0123] S5-2. Add 50 μL of 0.8 wt% aldehyde-modified erythrocyte suspension to the reaction well;

[0124] S5-3, incubate at 37℃ for 15 minutes;

[0125] S5-4. Centrifuge in a conventional centrifuge: centrifuge at 900 rpm for 2 min, then centrifuge at 1500 rpm for 3 min;

[0126] S5-5. Observe the migration of red blood cells in the modified anti-human globulin microcolumn gel card, and determine the results based on the reaction pattern of the screened red blood cells.

[0127] Test Example 1

[0128] 1. Serially dilute the anti-A antibody reagent with PBS from 1:2 to 1:1024;

[0129] 2. In the modified reverse-sizing microcolumn gel card of Example 2, add 50 μL of anti-A reagent diluted 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, and 1:1024 respectively from left to right, along with the original anti-B reagent and physiological saline. Then add 50 μL of A blood type aldehyde erythrocyte suspension with a concentration of 0.8 wt% prepared in Example 1.

[0130] 3. Place in a conventional centrifuge and centrifuge using the standard centrifugation program (centrifuge at 900 rpm for 2 min, centrifuge at 1500 rpm for 3 min), and observe the results.

[0131] The results are as follows Figure 1 As shown, the anti-A results showed a gradient of positive reactions, while the anti-B and saline results were both negative.

[0132] Test Example 2

[0133] 1. Add 50 μL of the plasma / serum sample to be tested to each of the three reaction wells of the modified reverse-sizing microcolumn gel card in Example 2.

[0134] 2. Add 50 μL of each of the following 0.8 wt% blood type A aldehyde erythrocyte suspensions (prepared in Example 1): blood type A aldehyde erythrocyte suspension, blood type B aldehyde erythrocyte suspension, and blood type O aldehyde erythrocyte suspension.

[0135] 3. Place in a conventional centrifuge and centrifuge using the standard centrifugation program (centrifuge at 900 rpm for 2 min, then centrifuge at 1500 rpm for 3 min).

[0136] 4. Observe the migration of red blood cells in the microcolumn gel to determine the blood type result.

[0137] The results are as follows Figure 2 As shown, the results of reverse blood typing are consistent with the results of forward typing.

[0138] Test Example 3

[0139] 1. Serially dilute the intravenous immunoglobulin (pH4) with PBS from 1:2 to 1:64.

[0140] 2. In the modified anti-human globulin microcolumn gel card of Example 4, add 50 μL of intravenous human immunoglobulin diluted 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64 from left to right, and then add 50 μL of A blood type aldehyde erythrocyte suspension prepared in Example 1 with a concentration of 0.8 wt%.

[0141] 3. Incubate at 37°C for 15 minutes.

[0142] 4. Place in a conventional centrifuge and centrifuge using the standard centrifugation program (centrifuge at 900 rpm for 2 min, centrifuge at 1500 rpm for 3 min), and observe the results.

[0143] The results are as follows Figure 3 As shown, the results indicate that the intravenous immunoglobulin contains anti-A at a titer of 1:16 and anti-B at a titer of 1:4, demonstrating that aldehyde-treated red blood cells and the modified anti-human globulin test card can be used for the detection of IgG blood group antibodies. In this test example, intravenous immunoglobulin was used as a model to replace irregular antibody screening cells or spectrophotometers for detection based on a similar principle. While IgM antibodies are removed from intravenous immunoglobulin during production, it may still contain IgG anti-A or anti-B. If its titer is higher than 64, it may cause serious adverse transfusion reactions. Therefore, the pharmacopoeia stipulates that it must undergo quality control for anti-A and anti-B titers. This test example aims to demonstrate the feasibility of using aldehyde-treated red blood cells and the modified anti-human globulin test card for the detection of IgG blood group antibodies.

[0144] Test Example 4

[0145] 1. A batch of aldehyde-retyped red blood cell suspension prepared using the method in Example 1 was stored at 37°C, and one sample was taken out daily for testing in steps 2-5 below.

[0146] 2. Serially dilute the intravenous immunoglobulin (pH4) with PBS from 1:2 to 1:64.

[0147] 3. In the modified anti-human globulin microcolumn gel card of Example 4, add 50 μL of intravenous human immunoglobulin diluted 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64 from left to right, and then add 50 μL of type A aldehyde-containing red blood cell suspension with a concentration of 0.8 wt%.

[0148] 4. Incubate at 37°C for 15 minutes.

[0149] 5. Place in a conventional centrifuge and centrifuge using the standard centrifugation program (centrifuge at 900 rpm for 2 min, centrifuge at 1500 rpm for 3 min), and observe the results.

[0150] The results are as follows Figure 4 As shown, the results are from the accelerated stability test at 37℃. According to the Arrhenius equation, the aldehyde-modified red blood cells can be preserved for at least one year, and the strength of the A and B blood type antigens on the surface of the red blood cells is basically stable.

[0151] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A type of aldehyde-modified erythrocyte that can be stored for a long period of time, characterized in that, It is prepared through the following steps: S1-1. Collect red blood cells of blood type A, blood type B, and blood type O from healthy individuals as standard red blood cells, wash with physiological saline, and then dilute with physiological saline to obtain a standard red blood cell suspension. S1-2. Add the standard red blood cell suspension to an aldehyde reagent composed of glutaraldehyde and formaldehyde, and treat at room temperature for 5-30 minutes. S1-3. Centrifuge to remove the supernatant and wash with physiological saline to obtain aldehyde-treated erythrocytes; S1-4. Dilute the aldehyde-treated red blood cells with red blood cell preservation solution to obtain an aldehyde-treated red blood cell suspension; Aldehyde-containing red blood cell suspensions prepared from blood types A, B, and O are respectively designated as blood type A aldehyde-containing red blood cell suspension, blood type B aldehyde-containing red blood cell suspension, and blood type O aldehyde-containing red blood cell suspension.

2. The aldehyde-containing erythrocytes that can be stored for a long time according to claim 1, characterized in that, It is prepared through the following steps: S1-1. Collect red blood cells of blood type A, blood type B and blood type O from healthy people as standard red blood cells. Wash them with physiological saline 1-6 times to remove plasma components, and then dilute them with physiological saline to a volume concentration of 25-75% to obtain standard red blood cell suspension. S1-2. Add the standard red blood cell suspension to an aldehyde reagent composed of glutaraldehyde and formaldehyde, so that the final volume concentration of glutaraldehyde in the resulting mixture is 0.0001-0.5% and the final volume concentration of formaldehyde is 0.001-1%, and treat at room temperature for 5-30 min. S1-3. Centrifuge to remove the supernatant, and wash with physiological saline 1-8 times to remove excess aldehyde reagent to obtain aldehyde-treated red blood cells; S1-4. Dilute the aldehyde-treated red blood cells to 0.8-5 wt% with red blood cell preservation solution to obtain an aldehyde-treated red blood cell suspension, and store at 2-8℃. Aldehyde-containing red blood cell suspensions prepared from blood types A, B, and O are respectively designated as blood type A aldehyde-containing red blood cell suspension, blood type B aldehyde-containing red blood cell suspension, and blood type O aldehyde-containing red blood cell suspension.

3. The use of aldehyde-modified erythrocytes as described in any one of claims 1-2 in blood typing or in the preparation of blood typing reagents.

4. A blood type reverse typing system, characterized in that, Includes the aldehyde-modified erythrocyte suspension and the modified reverse-type microcolumn gel card as described in any one of claims 1-2; the aldehyde-modified erythrocyte suspension includes blood type A aldehyde-modified erythrocyte suspension, blood type B aldehyde-modified erythrocyte suspension, and blood type O aldehyde-modified erythrocyte suspension; The improved reverse-shaping micropillar gel card was prepared through the following steps: S2-1. Prepare the gel buffer solution according to the following formula: 15.4-61.6 mM sodium chloride, 0.75-3.0 mM disodium hydrogen phosphate, 0.75-3.0 mM sodium dihydrogen phosphate, 0.12-0.48 M glycine, and 0.5-2% bovine serum albumin to adjust the pH to 6.65-6.85; S2-2. Dextran gel particles with pore sizes adapted to aldehyde erythrocytes are soaked and washed with gel buffer to obtain gel particles. S2-3. After mixing the gel particles with the gel buffer, dispense them into the reaction wells of the microcolumn tube to obtain the modified reverse-sizing microcolumn gel card.

5. The blood type reverse typing system according to claim 4, characterized in that, The improved reverse-shaping micropillar gel card was prepared through the following steps: S2-1. Prepare the gel buffer solution according to the following formula: 30.8 mM sodium chloride, 1.5 mM disodium hydrogen phosphate, 1.5 mM sodium dihydrogen phosphate, 0.24 M glycine, and 1% bovine serum albumin were added to adjust the pH to 6.65-6.

85. S2-2. G25 dextran gel particles with a particle size of 50-150μm and a size exclusion molecular weight of 1-5KD are soaked in gel buffer and washed 3-5 times to obtain gel particles. S2-3. Mix the gel particles and gel buffer at a volume ratio of 1:1 and dispense them into microcolumns to obtain the modified reverse-sizing microcolumn gel card. The number of reaction wells in the microcolumn is not less than 3.

6. A blood type reverse typing method, characterized in that, It is implemented using the blood type reverse typing system as described in claim 4 or 5, and the method includes the following steps: S3-1. Add the plasma or serum sample to be tested to the three reaction wells of the modified reverse-sizing microcolumn gel card, respectively. S3-2. Add A-type aldehyde erythrocyte suspension, B-type aldehyde erythrocyte suspension, and O-type aldehyde erythrocyte suspension to each reaction well, respectively. S3-3, Centrifugation; S3-4. Observe the migration of red blood cells in the modified reverse-stylating microcolumn gel card and judge the test results.

7. The use of aldehyde-modified erythrocytes as described in any one of claims 1-2 in the detection of irregular antibodies or in the preparation of reagents for the detection of irregular antibodies.

8. An irregular antibody detection system, characterized in that, Includes the O-type aldehyde erythrocyte suspension and the modified anti-human globulin microcolumn gel card as described in any one of claims 1-2; The modified anti-human globulin microcolumn gel card was prepared by the following steps: S4-1. Prepare the gel buffer solution according to the following formula: 15.4-61.6mM sodium chloride, 0.75-3.0mM disodium hydrogen phosphate, 0.75-3.0mM sodium dihydrogen phosphate, 0.12-0.48M glycine, 0.5-2% bovine serum albumin, and anti-human globulin antibody; adjust pH to 6.65-6.85; final titer of anti-human globulin antibody is 64-128. S4-2. The dextran gel particles with pore sizes adapted to aldehyde erythrocytes are soaked and washed with gel buffer to obtain gel particles. S4-3. After mixing the gel particles with the gel buffer, dispense them into the reaction wells of the microcolumn to obtain the modified anti-human globulin microcolumn gel card.

9. The irregular antibody detection system according to claim 8, characterized in that, The modified anti-human globulin microcolumn gel card was prepared by the following steps: S4-1. Prepare the gel buffer solution according to the following formula: 30.8mM sodium chloride, 1.5mM disodium hydrogen phosphate, 1.5mM sodium dihydrogen phosphate, 0.24M glycine, 1% bovine serum albumin, and anti-human globulin antibody were added to adjust the pH to 6.65-6.

85. The final titer of the anti-human globulin antibody was 64-128. S4-2. Soak and wash G25 dextran gel particles with a particle size of 50-150μm and a size exclusion molecular weight of 1-5KD in gel buffer 3-5 times to obtain gel particles. S4-3. After mixing the gel particles with the suspension, dispense them into the reaction wells of the microcolumn to obtain the modified anti-human globulin microcolumn gel card. The number of reaction wells in the microcolumn is not less than 3.

10. A method for detecting irregular antibodies, characterized in that, It is implemented using the irregular antibody detection system as described in claim 8 or 9, and the method includes the following steps: S5-1. Add the plasma or serum sample to be tested into the reaction well of the modified anti-human globulin microcolumn gel card; S5-2. Add aldehyde-treated red blood cell suspension to the reaction well; Incubate at 37℃ using S5-3; S5-4, Centrifugation; S5-5. Observe the migration of red blood cells in the modified anti-human globulin microcolumn gel card and determine the test results.

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