Blood group serological analysis card including blood group card and anti-human globulin card and preparation method thereof

Abstract

The application relates to the field of in-vitro diagnosis, and particularly discloses a blood type serological analysis card including a blood type card and an anti-human globulin card and a preparation method thereof. The blood type serological analysis card utilizes the mechanism of forming a molecular sieve by PMMA microspheres with a certain diameter to realize efficient analysis of blood type serology. Meanwhile, the blood type serological analysis card has high uniformity of particle size of a filler, good temperature resistance, small result interpretation interference, high human environment simulation degree, low external technology dependence and low price, and provides guarantee for blood type identification, irregular antibody screening and cross matching test.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, and more specifically to a blood typing and serological analysis card including a blood typing card and an anti-human globulin card, and a method for preparing the same. Background Technology

[0002] Blood transfusion is an essential clinical treatment method, primarily used to improve hypoxia and correct coagulation. Safe and effective transfusions can quickly alleviate symptoms and buy time for further treatment. However, improper handling can cause various adverse reactions, worsening the patient's condition and even threatening their life. Therefore, all patients must undergo serological analysis before transfusion, including blood typing, crossmatching, and screening for irregular antibodies, to ensure that the red blood cells transfused into the recipient and the recipient's own red blood cells are not damaged. Details are as follows:

[0003] Before a blood transfusion, it is essential to confirm the ABO and Rh(D) blood types of both the donor and recipient. This is because A and B antigens are the most antigenic in all blood group systems, followed by the D antigen. Almost every ABO incompatibility transfusion will produce specific alloantibodies. For D antigen incompatibility transfusions, two-thirds of recipients will develop anti-D antibodies. Therefore, it is crucial to confirm the ABO and Rh(D) blood types of both the donor and recipient before transfusion and select a compatible donor. Blood typing methods vary and are divided into forward typing and reverse typing. Forward typing involves reacting the recipient's red blood cells with blood group antibodies to detect the presence of the corresponding blood group antigen. Reverse typing involves reacting the recipient's serum with blood group antigens to detect the presence of the corresponding blood group antibodies. Article 15 of the Clinical Blood Transfusion Technical Specifications stipulates that: "The blood transfusion department (blood bank) shall check the blood transfusion application form, the blood sample of the recipient and the blood donor item by item, re-examine the ABO blood type (forward and reverse typing) of the recipient and the blood donor, and routinely check the patient's Rh(D) blood type (except for emergency blood transfusion of patients in emergency rescue). If the results are correct, crossmatching can be performed."

[0004] Crossmatching involves mixing donor red blood cells or serum with recipient red blood cells or serum separately, observing for agglutination or hemolysis. Transfusion is only permitted if no agglutination or hemolysis occurs on either side. The fundamental purpose of crossmatching is to predict blood compatibility in vivo before transfusion through in vitro testing, thus avoiding hemolytic transfusion reactions caused by blood type antigen-antibody reactions. Depending on the purpose, crossmatching is further divided into primary crossmatching and secondary crossmatching. Primary crossmatching involves incubating recipient serum with donor red blood cells to detect the presence of antibodies in the recipient serum that specifically react with donor red blood cells. Secondary crossmatching involves incubating donor serum with recipient red blood cells to detect the presence of antibodies in the donor serum that specifically react with recipient red blood cells. Article 16 of the Clinical Transfusion Technical Specifications stipulates that: "For patients receiving transfusions of whole blood, concentrated red blood cells, red blood cell suspension, washed red blood cells, frozen red blood cells, concentrated white blood cells, or manually separated concentrated platelets, crossmatching tests should be performed."

[0005] Human blood group antibodies can be divided into two main categories: irregular antibodies and regular antibodies. Irregular antibodies are mainly IgG with a small molecular weight. Although they can bind to antigens on red blood cells, they cannot cause red blood cell agglutination in saline medium. Regular antibodies can agglutinate the corresponding red blood cells in saline medium, so they are also called saline antibodies. They are mainly IgM with a larger molecular weight. Antibody screening involves routine testing of the recipient's serum to detect irregular antibodies that can cause adverse reactions to alloimmune transfusions. Article 17 of the "Clinical Transfusion Technical Specifications" stipulates that: "Antibody screening tests must be performed in accordance with the relevant provisions of the 'National Clinical Laboratory Operation Procedures' in the following situations: when crossmatching is incompatible; for patients with a history of transfusion, pregnancy, or who need to receive multiple transfusions in a short period of time."

[0006] While some manufacturers both domestically and internationally have invented blood typing cards for blood typing identification, as well as anti-human globulin cards for crossmatching and irregular antibody screening, these cards almost all use dextran gel particles, a dextran gel particle matrix, and a corresponding antibody mixture as fillers to separate agglutinated and non-agglutinated red blood cells. However, they have the following drawbacks:

[0007] (1) The mechanical strength is low and it is easy to deform during high-speed centrifugation. Therefore, to ensure the stability of the cell sieve pore size, the centrifugation effect can only be achieved by extending the centrifugation time at low speed, thereby reducing the detection speed.

[0008] (2) The density is low, the self-lubricating properties are poor and the coagulation effect is weak. Bumps or inversions during transportation can cause the gel particles to redistribute, which eventually leads to the cell sieve pore size becoming larger. It must be centrifuged again before it can be used, which increases the workload of users.

[0009] (3) Low temperature resistance. The swelling rate of dextran gel will change with the change of ambient temperature, causing the particle size of dextran gel particles to be easily changed, which directly leads to the cell sieve pore size fluctuating. In order to ensure quality stability, it must be transported or stored at 2-25℃, which brings inconvenience to storage and transportation.

[0010] (4) Poor particle size uniformity, high technical requirements for producing dextran gel, and many influencing factors lead to large differences in the size of dextran gel particles, which affects the stability of the experiment.

[0011] (5) The human body's pH level is poorly simulated, and the stability of dextran gel particles is easily affected by pH changes. Therefore, the pH of dextran gel particle systems currently used in blood typing and serological analysis is between 6.5 and 7.0, significantly lower than the normal human body's pH of 7.35 to 7.45. pH is one of the most important factors affecting antigen-antibody reactions. Therefore, antigen-antibody reactions in dextran gel particle systems cannot fully represent antigen-antibody reactions in the in vivo environment.

[0012] (6) The production process of dextran gel particles used for cell separation is complex. To date, this technology has only been mastered by a few countries in Europe and America. Domestic use is completely dependent on imports, which makes the foreign technology dependence and price of domestic blood type cards and anti-human globulin cards relatively high. Summary of the Invention

[0013] In view of this, the purpose of the present invention is to provide a blood typing and serological analysis card, including a blood typing card and an anti-human globulin card, and a method for preparing the same, so that the analysis card has fast detection speed, is easy to use, has simple storage and transportation conditions, has little interference in result interpretation, has high uniformity of filler particle size, stable quality, high simulation of human environment, low dependence on external technology, and low price.

[0014] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0015] A blood typing serological analysis card including a blood typing card and an anti-human globulin card, consisting of microcolumns for detecting blood type and / or microcolumns for screening irregular antibodies;

[0016] The microcolumn for blood typing includes microcolumn 1 filled with a mixture of a suspended matrix, monoclonal IgM anti-A antibody, and blocked PMMA (polymethylmethacrylate) microspheres; microcolumn 2 filled with a mixture of a suspended matrix, monoclonal IgM anti-B antibody, and blocked PMMA microspheres; microcolumn 3 filled with a mixture of a suspended matrix, monoclonal IgM anti-D antibody, and blocked PMMA microspheres; and microcolumn 4 filled with a mixture of a suspended matrix and blocked PMMA microspheres.

[0017] The microcolumn used for detecting anti-human globulin is a microcolumn 5 filled with a mixture of a suspended matrix, anti-human globulin antibody and blocked PMMA microspheres;

[0018] The PMMA microspheres in micropillars 1-4 have a diameter of 80-150 μm, and the PMMA microspheres in micropillar 5 have a diameter of 75-125 μm; the suspension matrix includes preservatives, antibody stabilizers, and buffer solutions, and its pH value is within the normal range for human use.

[0019] Unlike conventional methods that involve coating antibodies or antigens onto the surface of microspheres using surface groups, this invention utilizes the corresponding pore size formed by stacking microspheres of a certain diameter. Combined with centrifugation, this separates single red blood cells from clots formed by the aggregation of multiple red blood cells, thus functioning as a cell sieve. For example, when 100 μm diameter PMMA microspheres are stacked together, the pore size between the PMMA microspheres can reach approximately 15 μm. A single red blood cell has a diameter of 6-9 μm. Under the action of IgM blood group antibodies, single red blood cells can agglomerate into clots with a diameter of 50 μm or more, composed of aggregated red blood cells. Because the pore size of the PMMA microspheres is only about 15 μm, only single red blood cells with a diameter of less than 15 μm can pass through, while red blood cell clots with a diameter of more than 50 μm cannot pass through, thereby separating the two.

[0020] The principle behind the irregular antibody screening assay is similar. When 100μm diameter PMMA microspheres are stacked together, the pore size between them reaches approximately 15μm. Although the irregular antibody is of the IgG type, it can only bind to antigens on a single red blood cell and cannot bind to multiple red blood cells simultaneously. However, IgM type anti-human globulin antibodies can bind to irregular antibodies on multiple red blood cells simultaneously, thus linking multiple red blood cells bound to irregular antibodies together to form a clot with a diameter greater than 50μm. Because the pore size of the PMMA microspheres is only about 15μm, only single red blood cells with a diameter less than 15μm can pass through, while red blood cell clots with a diameter greater than 50μm cannot pass through, thus separating the two.

[0021] The PMMA microspheres in this invention require a blocking treatment before mixing with the suspension matrix and antibodies. The main reason is that unblocked PMMA microspheres will non-specifically bind to red blood cells, leading to false positive or false negative results. For example, theoretically, if an irregular antibody screening test is performed on a sample without irregular antibodies, the red blood cells will not aggregate due to the absence of these antibodies. Under centrifugal force, the unaggregated red blood cells will settle to the bottom of the microcolumn, resulting in a negative result. However, because the unblocked PMMA microspheres non-specifically bind to red blood cells, the red blood cells that should have settled to the bottom after centrifugation remain in the upper or middle layer of the microcolumn, resulting in a false positive result. Furthermore, even when performing an irregular antibody screening test on a sample containing irregular antibodies, the unblocked PMMA microspheres will non-specifically bind to red blood cells. Under centrifugal force, the red blood cells non-specifically bound to the PMMA microspheres will drift slightly downwards with the microspheres, resulting in a false sinking and a false negative result. To avoid the aforementioned false positive and false negative results, this invention uses a blocking solution to pretreat PMMA microspheres. This allows the proteins in the blocking solution to bind to the untreated sites on the PMMA microspheres, "occupying" non-specific binding sites and preventing untreated PMMA microspheres from binding to red blood cells, thus resolving the interference of false positive and / or false negative experimental results. Preferably, the blocking solution is skim milk powder, bovine serum albumin, fetal bovine serum, calf serum, or any combination thereof. This invention does not exclude other substances that can achieve the same blocking effect.

[0022] Furthermore, high-speed centrifugation does not affect the experimental results of the novel blood typing and serological analysis card of this invention. However, commercially available dextran gel analysis cards are affected by high-speed centrifugation. Therefore, dextran gel analysis cards generally have lower centrifugation speeds and longer centrifugation times. In comparison, the novel blood typing and serological analysis card of this invention, which can be centrifuged at high speeds, has a faster detection speed.

[0023] Meanwhile, commercially available dextran gel analysis cards must be centrifuged again before use after being shaken. However, the novel blood typing and serological analysis card of this invention can be reused after being shaken simply by being left to stand vertically for 2 minutes, without the need for centrifugation. Therefore, compared to traditional dextran gel analysis cards, the novel blood typing and serological analysis card is more convenient to use.

[0024] In temperature resistance tests at low temperatures, room temperature, and high temperatures, commercially available dextran gel analysis cards showed that storage temperatures as low as -20°C or as high as 40°C affected gel quality, resulting in false positive results. However, the blood typing and serological analysis card of this invention did not produce any false positive or false negative results. Furthermore, the analysis card of this invention provides a cleaner field of view and clearer color contrast. These results demonstrate that, compared to traditional dextran gel analysis cards, the novel blood typing and serological analysis card of this invention exhibits better temperature resistance and less interference during result interpretation.

[0025] Furthermore, the particle size uniformity test results of the microcolumn filler in the novel blood typing and serological analysis card of this invention showed that the particle size distribution of the traditional dextran gel analysis card filler was 20–120 μm, while the particle size distribution of the microcolumn filler in the analysis card of this invention was 70–120 μm. This result indicates that the particle size uniformity of the novel blood typing and serological analysis card filler is superior to that of the dextran gel analysis card filler.

[0026] Preferably, the suspension matrix comprises a preservative, an antibody stabilizer, and a buffer solution, with a pH value within the normal range for human use. In a specific embodiment of the present invention, the suspension matrix comprises 2-4 g / L NaN3, 2-5 g / L skim milk powder, 8-10 g / L Glycine, 1.8-2.0 g / L NaCl, 0.1-0.2 g / L KCl, and 80-120 ml Tris-HCl buffer solution, with a pH value of 7.35-7.45.

[0027] When determining the titer of antibodies for each blood type, antibodies diluted at different volume ratios are mixed with the corresponding red blood cells of each blood type, and the titer is determined by the presence of visible agglutination. In a specific embodiment of the present invention, monoclonal IgM anti-A antibody diluted at a volume ratio of 1:128 is mixed with type A red blood cells, centrifuged at 300g for 2 minutes, and shaken; visible agglutination is the standard for anti-A antibody. Monoclonal IgM anti-B antibody diluted at a volume ratio of 1:128 is mixed with type B red blood cells, centrifuged at 300g for 2 minutes, and shaken; visible agglutination is the standard for anti-B antibody. Monoclonal IgM anti-D antibody diluted at a volume ratio of 1:64 is mixed with Rh D positive red blood cells, centrifuged at 300g for 2 minutes, and shaken; visible agglutination is the standard for anti-D antibody. Therefore, the titer of IgM anti-A antibody is ≥128, the titer of IgM anti-B antibody is ≥128, and the titer of IgM anti-D antibody is ≥64.

[0028] In a specific embodiment of the present invention, each liter of mixture in microcolumn 1 contains 400-600g of blocked PMMA microspheres and 100-200ml of IgM anti-A antibody; each liter of mixture in microcolumn 2 contains 400-600g of blocked PMMA microspheres and 100-200ml of IgM anti-B antibody; each liter of mixture in microcolumn 3 contains 400-600g of blocked PMMA microspheres and 100-200ml of IgM anti-D antibody; and each liter of mixture in microcolumn 4 contains 400-600g of blocked PMMA microspheres.

[0029] In a specific embodiment of the present invention, the number of micropillars 1-3 is 1, and the number of micropillars 4 is 3.

[0030] When determining the titer of irregular IgG antibodies, IgM anti-human globulin antibodies of different volume ratios are mixed with IgG-sensitized red blood cells, and visible agglutination is used as the standard. In a specific embodiment of the present invention, IgM anti-human globulin antibodies diluted at a volume ratio of 1:64 are mixed with IgG-sensitized red blood cells diluted at a volume ratio of 1:8, centrifuged at 300g for 10 minutes and then shaken, and visible agglutination is used as the standard. Therefore, the titer of the anti-human globulin antibody is ≥64.

[0031] In a specific embodiment of the present invention, each liter of mixture in the microcolumn 5 contains 400-600g of blocked PMMA microspheres and 100-200ml of anti-human globulin antibody; the number of microcolumns 5 is 6.

[0032] Meanwhile, the present invention also provides a method for preparing the analytical card, wherein the filling composition of microcolumns 1-5 is prepared by mixing a suspension matrix, antibodies of each blood type, blocked PMMA microspheres and anti-human globulin antibodies, and filling each microcolumn.

[0033] Preferably, the preparation method is divided into microcolumn preparation for blood typing and microcolumn preparation for screening irregular antibodies;

[0034] The preparation of microcolumns for blood typing includes the following steps:

[0035] (1) Preparation of PMMA microsphere suspension matrix: Dissolve NaN3, skim milk powder, Glycine, NaCl, KCl and Tris-HCl buffer in distilled water in proportion to prepare PMMA microsphere suspension matrix;

[0036] (2) Screening of target PMMA microspheres: Target PMMA microspheres were screened using a sieve;

[0037] (3) Blocking of PMMA microspheres: First, PMMA microspheres were soaked in blocking solution (2-10% skim milk powder solution, 2-10% bovine serum albumin solution, 2-10 ml / L fetal bovine serum or 2-10 ml / L calf serum, etc.) at 25°C for 1 hour. Then, they were centrifuged at 1000g for 5 minutes and the supernatant was discarded. Next, they were washed three times with PMMA microsphere suspension matrix under the same conditions and then used for later use.

[0038] (4) Selection of blood type antibodies: Target monoclonal IgM anti-A antibody, monoclonal IgM anti-B antibody and monoclonal IgM anti-D antibody were selected by using type A red blood cells, type B red blood cells and Rh D positive red blood cells respectively;

[0039] (5) Preparation of mixtures of antibodies and PMMA microspheres for each blood type: Mix the prepared PMMA microsphere suspension matrix, blocked PMMA microspheres and corresponding blood type antibodies in a certain proportion to prepare mixtures of antibodies and PMMA microspheres for different blood types.

[0040] (6) Preparation of PMMA microsphere suspension: The prepared PMMA microsphere suspension matrix and the sealed PMMA microspheres are mixed in a certain proportion to prepare PMMA microsphere suspension;

[0041] (7) Canning and sealing.

[0042] The preparation of microcolumns for screening irregular antibodies includes the following steps:

[0043] (1) Preparation of suspension matrix: Same as step (1) of microcolumn preparation for blood typing;

[0044] (2) Screening of target PMMA microspheres: Target PMMA microspheres were screened using a sieve;

[0045] (3) Blocking of PMMA microspheres: Same as step (3) of microcolumn preparation for blood typing;

[0046] (4) Selection of anti-human globulin antibodies: IgG-sensitized red blood cells are used to select target anti-human globulin antibodies;

[0047] (5) Preparation of PMMA microspheres and anti-human globulin antibody mixture: The prepared PMMA microsphere suspension matrix, the blocked PMMA microspheres and the screened anti-human globulin antibody are mixed in a certain proportion to prepare PMMA microspheres and anti-human globulin antibody mixture;

[0048] (6) Canning and sealing.

[0049] As can be seen from the above technical solutions, this invention utilizes the mechanism of forming molecular sieves with PMMA microspheres of a certain diameter to achieve efficient analysis of blood type serology. At the same time, the blood type serology analysis card of this invention has high particle size uniformity, good temperature resistance, small interference in result interpretation, high simulation of human environment, low dependence on external technology and low price, providing a guarantee for blood type identification, irregular antibody screening and crossmatching test. Attached Figure Description

[0050] Figure 1 The following are the experimental results of high-speed centrifugation verification of the novel anti-human globulin card of this invention: A. Experimental results of centrifugation of dextran gel against human globulin card under normal conditions; B. Experimental results of high-speed centrifugation of dextran gel against human globulin card; C. Experimental results of high-speed centrifugation of the novel anti-human globulin card.

[0051] Figure 2 The following are experimental results verifying the blocking effect of PMMA microspheres of the present invention: A. Experimental results of anti-human globulin card using dextran gel; B. Experimental results of novel anti-human globulin card prepared using PMMA microspheres without blocking pretreatment; C. Experimental results of novel anti-human globulin card prepared using PMMA microspheres with blocking pretreatment.

[0052] Figure 3 The following are the experimental results of the anti-bumping verification of the novel anti-human globulin card of the present invention: A. Normal distribution and shape of the filling material inside the dextran gel anti-human globulin card; B. Distribution and shape of the filling material inside the dextran gel anti-human globulin card after rotation and vertical standing for 2 minutes; C. Distribution and shape of the filling material inside the dextran gel anti-human globulin card after rotation and vertical standing for 24 hours; D. Distribution and shape of the filling material inside the dextran gel anti-human globulin card after rotation and re-centrifugation; E. Normal distribution and shape of the filling material inside the novel anti-human globulin card; F. Distribution and shape of the filling material inside the novel anti-human globulin card after rotation and vertical standing for 2 minutes; G. Distribution and shape of the filling material inside the novel anti-human globulin card after rotation and vertical standing for 24 hours.

[0053] Figure 4 The following are the experimental results for temperature resistance verification of the novel anti-human globulin card of this invention: A. Experimental results of placing the anti-human globulin card in dextran gel at 10℃; B. Experimental results of placing the anti-human globulin card in dextran gel at 40℃; C. Experimental results of placing the anti-human globulin card in dextran gel at -20℃; D. Experimental results of placing the novel anti-human globulin card at 10℃; E. Experimental results of placing the novel anti-human globulin card at 40℃; F. Experimental results of placing the novel anti-human globulin card at -20℃.

[0054] Figure 5The images show the particle size uniformity test results of the novel anti-human globulin card filler of this invention; A. Microscopic observation of the filler in the dextran gel anti-human globulin card; B. Microscopic observation of the filler in the novel anti-human globulin card. Detailed Implementation

[0055] This invention discloses a blood typing and serological analysis card, including a blood typing card and an anti-human globulin card, and its preparation method. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The analysis card and its preparation method described in this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the analysis card and its preparation method described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0056] In the specific embodiments of this invention, the raw materials and reagents used in each treatment group of the comparative experiment are kept consistent with other experimental conditions, except for necessary differences. Unless otherwise specified, all raw materials and reagents involved in this invention can be obtained commercially available.

[0057] The present invention will be further illustrated below with reference to the embodiments.

[0058] Example 1: Preparation of the blood type serological analysis card of the present invention

[0059] 1. Preparation of blood typing cards (for ABO and RhD blood typing)

[0060] The blood typing card consists of six microcolumns. The first three microcolumns are sequentially filled with a mixture of IgM anti-A antibody and polymethyl methacrylate (PMMA) microspheres, a mixture of IgM anti-B antibody and PMMA microspheres, and a mixture of IgM anti-D antibody and PMMA microspheres. The last three microcolumns are each filled with a PMMA microsphere suspension. The specific implementation includes the following steps:

[0061] (1) Preparation of PMMA microsphere suspension matrix

[0062] Dissolve the following solutes in distilled water according to their respective proportions:

[0063]

[0064] Adjust the pH to 7.35-7.45 and mix well;

[0065] (2) Screening of PMMA microspheres with diameters of 80-150 μm

[0066] PMMA microspheres with diameters of 80-150 μm were screened using 180-mesh and 100-mesh sieves.

[0067] (3) Encapsulation treatment of PMMA microspheres

[0068] A blocking solution was prepared using a PMMA microsphere suspension matrix.

[0069] PMMA microspheres were immersed in a blocking solution at 25°C for 1 hour;

[0070] Centrifuge at 1000g for 5 minutes and discard the supernatant;

[0071] Wash the PMMA microsphere suspension matrix three times (centrifuge at 1000g for 5 minutes) before use;

[0072] (4) Selection of blood type antibodies

[0073] The standard for anti-A antibodies is to mix monoclonal IgM anti-A antibodies diluted 1:128 (v / v) with type A erythrocytes, centrifuge at 1000g for 2 min and then shake. Visible agglutination is the result of anti-A antibody treatment.

[0074] The standard for anti-B antibodies is to mix monoclonal IgM anti-B antibodies diluted 1:128 (v / v) with type B erythrocytes, centrifuge at 1000g for 2 min and then shake. Visible agglutination is the result of the anti-B antibody.

[0075] The standard for anti-D antibody is to mix monoclonal IgM anti-D antibody diluted 1:64 (v / v) with RhD positive red blood cells, centrifuge at 1000g for 2 min and shake. Visible agglutination is the standard for anti-D antibody.

[0076] (5) Preparation of mixtures of antibodies for each blood type and PMMA microspheres

[0077] The following substances were mixed in their respective proportions using a PMMA microsphere suspension matrix:

[0078]

[0079] (6) Preparation of PMMA microsphere suspension

[0080] The PMMA microsphere suspension matrix was mixed with blocked PMMA microspheres (400-600 g / L) with a diameter of 80-150 μm.

[0081] (7) Packaging of blood type antibodies and PMMA microsphere mixtures, and PMMA microsphere suspensions

[0082] Using a syringe, draw 80 μL of the mixture of monoclonal IgM anti-A antibody and PMMA microspheres, monoclonal IgM anti-B antibody and PMMA microspheres, and monoclonal IgM anti-D antibody and PMMA microspheres prepared in step (4) and inject them sequentially into the first three microcolumns from the bottom of the tube. Using a syringe, draw 80 μL of the PMMA microsphere suspension prepared in step (6) and inject it into the last three microcolumns from the bottom of the tube.

[0083] (8) Seal

[0084] The top of the microcolumn is sealed using aluminum foil through a pressing process.

[0085] 2. Preparation of anti-human globulin cards (for screening irregular antibodies)

[0086] The anti-human globulin card consists of six microcolumns, each containing a mixture of anti-human globulin antibody and PMMA microspheres. The specific implementation includes the following steps:

[0087] (1) Preparation of PMMA microsphere suspension matrix

[0088] The preparation of the PMMA microsphere suspension matrix is ​​the same as described above;

[0089] (2) Screening of PMMA microspheres with diameters of 75-125 μm

[0090] PMMA microspheres with diameters of 75-125 μm were screened using 200-mesh and 120-mesh sieves;

[0091] (3) Enclosure of PMMA microspheres

[0092] The same as the sealing of the PMMA microspheres mentioned above;

[0093] (4) Selection of anti-human globulin antibodies

[0094] IgM anti-human globulin antibody diluted 1:64 (v / v) was mixed with IgG sensitized red blood cells diluted 1:8 (v / v), centrifuged at 1000g for 10 min and shaken. Visible agglutination was taken as the standard.

[0095] (5) Preparation of a mixture of anti-human globulin antibody and PMMA microspheres

[0096] The following substances were mixed in their respective proportions using a PMMA microsphere suspension matrix:

[0097] 400-600 g / L of sealed PMMA microspheres with a diameter of 75-125 μm

[0098] Anti-human globulin antibody with a titer ≥64, 100-200 ml / L

[0099] (6) Filling of the mixture of anti-human globulin antibody and PMMA microspheres

[0100] The mixture of anti-human globulin antibody and PMMA microspheres prepared in step (5) was drawn up with a syringe and injected into 6 microcolumns from the bottom of the tube at a rate of 80 μL per well.

[0101] (7) Seal

[0102] The top of the microcolumn is sealed with aluminum foil using a pressing method for later use.

[0103] Example 2: Application verification of the novel anti-human globulin card in high-speed centrifugation

[0104] The novel anti-human globulin card was prepared by the method in Example 1. The traditional dextran gel anti-human globulin card was purchased from the market, and all the dextran gel anti-human globulin cards used were of the same type.

[0105] (1) Irregular antibody screening positive and negative test

[0106] Normal centrifugation: Take one dextran gel anti-human globulin card as the control group, add positive plasma and corresponding irregular antibody screening cells confirmed by Coombs test to the first two wells as positive controls, and add negative plasma and corresponding irregular antibody screening cells confirmed by Coombs test to the last two wells as negative controls. After incubation at 37°C for 15 minutes, centrifuge according to the manufacturer's recommended conditions (100g, centrifuge for 9 minutes).

[0107] High-speed centrifugation: Take another dextran gel anti-human globulin card as experimental group 1. Add the same irregular antibody screening positive plasma and irregular antibody screening cells as in step (1) to the first two wells as positive controls. Add the same irregular antibody screening negative plasma and irregular antibody screening cells as in step (1) to the last two wells as negative controls. After incubating at 37°C for 15 minutes, perform high-speed centrifugation (300g, centrifugation for 2 minutes).

[0108] Take a novel anti-human globulin card as experimental group 2. The first two wells are used as positive controls. Add the same irregular antibody screening positive plasma and irregular antibody screening cells as in step (1). Add the same irregular antibody screening negative plasma and irregular antibody screening cells as in step (1) as in step (1). Incubate at 37°C for 15 minutes and then centrifuge at high speed (300g, centrifuge for 2 minutes).

[0109] (2) Experimental Results and Analysis

[0110] The results of the high-speed centrifugation verification experiment of the novel anti-human globulin card are as follows: Figure 1 As shown. Compared with the control group ( Figure 1 Compared to A), although the red blood cells in the positive wells of experimental group 1 were located in the upper layer of the gel, the red blood cells in the negative wells were distributed in the middle and bottom of the gel. Figure 1 B), this result suggests that under high-speed centrifugation conditions, using dextran gel anti-human globulin cards for irregular antibody screening may result in false positives. The results for experimental group 2 were the same as the control group; the red blood cells in the positive wells remained on the upper layer of the PMMA microspheres, and the red blood cells in the negative wells remained at the bottom of the PMMA microspheres. Figure 1 C) This result demonstrates that high-speed centrifugation does not affect the irregular antibody screening assay using the novel anti-human globulin card. A comprehensive analysis of these experimental results indicates that the novel anti-human globulin card can withstand high-speed centrifugation, while the dextran gel anti-human globulin card cannot. Because increasing the centrifugation speed reduces centrifugation time to achieve the same centrifugation effect, the novel anti-human globulin card offers a faster detection speed for irregular antibody screening compared to the traditional dextran gel anti-human globulin card.

[0111] Example 3: Verification of the sealing effect of PMMA microspheres

[0112] The novel anti-human globulin card was prepared by the method in Example 1. The traditional dextran gel anti-human globulin card was purchased from the market, and all the dextran gel anti-human globulin cards used were of the same type.

[0113] (1) Irregular antibody screening positive and negative test

[0114] One dextran gel anti-human globulin card was used as the control group. The first well was used as the positive control and was filled with plasma positive for screening irregular antibodies and the corresponding irregular antibody screening cells confirmed by the Coombs test. The second well was used as the negative control and was filled with plasma negative for screening irregular antibodies and the corresponding irregular antibody screening cells confirmed by the Coombs test. After incubation at 37°C for 15 minutes, the cells were centrifuged according to the manufacturer's recommended conditions (100g, centrifuged for 9 minutes).

[0115] Take a novel anti-human globulin card prepared by PMMA microspheres that has been sealed as experimental group 1. The first well is used as a positive control and irregular antibody screening positive plasma and irregular antibody screening cells are added in the same way as in step (1). The second well is used as a negative control and irregular antibody screening negative plasma and irregular antibody screening cells are added in the same way as in step (1). After incubation at 37°C for 15 minutes, high-speed centrifugation is performed (300g, centrifugation for 2 minutes).

[0116] Take another novel anti-human globulin card prepared from PMMA microspheres that has not been sealed but has the same process as other experimental groups 2. The first well is used as a positive control and irregular antibody screening positive plasma and irregular antibody screening cells are added in the same way as in step (1). The second well is used as a negative control and irregular antibody screening negative plasma and irregular antibody screening cells are added in the same way as in step (1). After incubation at 37°C for 15 minutes, high-speed centrifugation is performed (300g, centrifugation for 2 minutes).

[0117] (2) Experimental Results and Analysis

[0118] The experimental results verifying the sealing effect of PMMA microspheres are as follows: Figure 1 As shown. Compared with the control group ( Figure 2 Compared to A), in experimental group 1, the red blood cells in the positive wells were located in the upper and middle layers of the PMMA microspheres, while the red blood cells in the negative wells were distributed in the middle and bottom layers of the PMMA microspheres. Figure 2 B) This result suggests that using the novel anti-human globulin card prepared with unblocked PMMA microspheres for irregular antibody screening experiments may result in false positives and false negatives. The results of experimental group 2 were the same as the control group; the red blood cells in the positive wells remained on the upper layer of the PMMA microspheres, and the red blood cells in the negative wells remained at the bottom of the PMMA microspheres. Figure 2 C) This result demonstrates that the blocking pretreatment can resolve the issues of false negative and false positive experimental results mentioned above.

[0119] Example 4: Anti-bump test of the novel anti-human globulin card of the present invention

[0120] The novel anti-human globulin card was prepared by the method in Example 1. The traditional dextran gel anti-human globulin card was purchased from the market, and all the dextran gel anti-human globulin cards used were of the same type.

[0121] (1) Bump simulation

[0122] One dextran gel anti-human globulin card was centrifuged under normal conditions and used as control group 1. One novel anti-human globulin card was placed vertically for 5 minutes and used as control group 2. Another dextran gel anti-human globulin card and the novel anti-human globulin card were used as experimental group 1 and experimental group 2, respectively. The anti-human globulin cards of experimental group 1 and experimental group 2 were fixed together on a multi-purpose rotary shaker and rotated 60 times per minute for 30 seconds.

[0123] (2) Observation of the shape and distribution of the filler

[0124] The distribution of the filler material in the cards of control group 1 and control group 2 was observed by taking photographs. The anti-human globulin cards of experimental group 1 and experimental group 2 were removed from the multi-purpose rotary shaker and left to stand vertically for 2 minutes and 24 hours, respectively, for observation. The anti-human globulin card of experimental group 1 was then centrifuged normally and photographed for observation.

[0125] (3) Experimental Results and Analysis

[0126] The results of the novel anti-human globulin card anti-bump verification experiment are as follows: Figure 2 As shown. Compared with control group 1 ( Figure 3 Compared to A), after being rotated and left to stand vertically for 2 minutes, the dextran gel antiglobulin card filler changed from a regular columnar shape perpendicular to the bottom of the tube to an irregular sheet-like shape randomly adhered to the tube wall. Figure 3 B), and even when the vertical standing time was extended from 2 minutes to 24 hours, the position of the irregularly shaped gel pieces randomly attached to the tube wall remained unchanged. Figure 3 C) Only after the analysis card is re-centrifuged can it be restored to its previous regular column shape. Figure 3 D). This result indicates that vibration causes the filler material in the dextran gel anti-human globulin card to separate, and centrifugation is required for it to recombine into a regular column shape.

[0127] Unlike the above experimental results, compared with control group 2 ( Figure 3 Compared to E), not only after being rotated and left to stand vertically for 24 hours, the filler in the novel anti-human globulin card can recover to its previous regular cylindrical shape. Figure 3 F), and after standing vertically for 2 minutes, the filling material inside the card had returned to its previous regular cylindrical shape. Figure 3 G). This result indicates that centrifugation is not required; simply standing vertically for 2 minutes is sufficient to restore the card's filler to its previous regular cylindrical shape.

[0128] These experimental results suggest that dextran gel anti-human globulin cards must be centrifuged again before use after being shaken. In contrast, the novel anti-human globulin cards can be reused after being shaken and simply left to stand vertically for 2 minutes without centrifugation. Therefore, the novel anti-human globulin cards are more convenient to use than traditional dextran gel anti-human globulin cards.

[0129] Example 5: Temperature resistance verification of the novel anti-human globulin card of the present invention

[0130] The novel anti-human globulin card was prepared by the method in Example 1. The traditional dextran gel anti-human globulin card was purchased from the market, and all the dextran gel anti-human globulin cards used were of the same type.

[0131] (1) Preliminary preparation of dextran gel anti-human globulin card and novel anti-human globulin card

[0132] Three dextran gel anti-human globulin cards were used as control group 1 (placed at 10℃ for 24 hours), experimental group 1 (placed at -20℃ for 24 hours), and experimental group 2 (placed at 40℃ for 24 hours), respectively. Three novel anti-human globulin cards were also used as control group 2 (placed at 10℃ for 24 hours), experimental group 3 (placed at -20℃ for 24 hours), and experimental group 4 (placed at 40℃ for 24 hours), respectively.

[0133] (2) Irregular antibody screening positive and negative test

[0134] Six anti-human globulin cards (control group 1, control group 2, experimental group 1, experimental group 2, experimental group 3, and experimental group 4) were placed at 25°C for equilibration for 30 minutes. The first two wells of each card served as positive controls, with Coombs-confirmed irregular antibody screening positive plasma and corresponding irregular antibody screening cells added respectively. The last two wells served as negative controls, with Coombs-confirmed irregular antibody screening negative plasma and corresponding irregular antibody screening cells added respectively. After incubation at 37°C for 15 minutes, the dextran gel anti-human globulin cards were centrifuged under the manufacturer's recommended conditions (100g, 9 minutes), while the novel anti-human globulin cards were centrifuged at high speed (300g, 2 minutes).

[0135] (3) Experimental Results and Analysis

[0136] The results of the thermostability verification experiment of dextran gel against human globulin card are as follows: Figure 4 As shown. Compared with control group 1 ( Figure 4 Compared to A), although the red blood cells in the positive wells of experimental group 1 were located in the upper layer of the gel, the red blood cells in the negative wells were distributed in the middle and bottom of the gel. Figure 4 B), and similar to experimental group 1, in experimental group 2, the red blood cells in the positive wells were located in the upper layer of the gel, while the red blood cells in the negative wells were distributed in the middle and bottom of the gel. Figure 4 C), this result suggests that storage temperatures as low as -20℃ or as high as 40℃ can affect gel quality and lead to false positive results. However, compared to control group 1, not only were the red blood cells in the positive wells of control group 2 located on the upper layer of PMMA microspheres, but the red blood cells in the negative wells were located at the bottom of the PMMA microspheres. Figure 4 D), and experimental group 3 ( Figure 4 E) and experimental group 4 ( Figure 4 F) The red blood cells in the positive wells were all located on the upper layer of the PMMA microspheres, and the red blood cells in the negative wells were all located at the bottom of the PMMA microspheres. No false positive or false negative results were observed. Furthermore, compared to control group 1, control group 2, experimental group 3, and experimental group 4 showed clearer fields of view and more pronounced color contrast. These experimental results indicate that, compared to the traditional dextran gel anti-human globulin card, the novel anti-human globulin card exhibits better temperature resistance and less interference during result interpretation.

[0137] Example 6: Detection of particle size uniformity of the novel anti-human globulin card filler of the present invention

[0138] The novel anti-human globulin card was prepared by the method in Example 1. The traditional dextran gel anti-human globulin card was purchased from the market, and all the dextran gel anti-human globulin cards used were of the same type.

[0139] (1) Observation of filling material

[0140] The fillers in the dextran gel anti-human globulin card (control group) and the novel anti-human globulin card (experimental group) were removed, smeared, and photographed under a microscope for observation.

[0141] (2) Experimental Results and Analysis

[0142] The experimental results of particle size uniformity detection of the novel anti-human globulin card filler are as follows: Figure 4 As shown. The particle size distribution of the control group filler was 20–120 μm. Figure 5 A), while the particle size distribution of the filler in the experimental group was 70–120 μm. Figure 5 B). This result indicates that the particle size uniformity of the novel anti-human globulin card filler is superior to that of the dextran gel anti-human globulin card filler.

[0143] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A blood typing and serological analysis card including a blood typing card and an anti-human globulin card, characterized in that, It consists of microcolumns for detecting blood type and microcolumns for detecting anti-human globulin; The microcolumn for blood typing includes microcolumn 1 filled with a mixture of a suspended matrix, monoclonal IgM anti-A antibody, and blocked PMMA microspheres. Microcolumn 2, filled with a mixture of suspended matrix, monoclonal IgM anti-B antibody, and blocked PMMA microspheres. Microcolumn 3, filled with a mixture of suspended matrix, monoclonal IgM anti-D antibody, and blocked PMMA microspheres. And microcolumns 4 filled with a mixture of suspended matrix and sealed PMMA microspheres; The microcolumn used for detecting anti-human globulin is a microcolumn 5 filled with a mixture of a suspended matrix, anti-human globulin antibody and blocked PMMA microspheres; The diameter of the PMMA microspheres in micropillars 1-4 is 80-150 μm, and the diameter of the PMMA microspheres in micropillar 5 is 75-125 μm. The PMMA microspheres in micropillars 1-5 have all undergone sealing treatment; The suspension matrix comprises 2-4 g / L NaN3, 2-5 g / L skim milk powder, 8-10 g / L Glycine, 1.8-2.0 g / L NaCl, 0.1-0.2 g / L KCl, and 80-120 ml Tris-HCl buffer solution with a pH of 7.35-7.

45.

2. The analysis card according to claim 1, characterized in that, The titer of the IgM anti-A antibody is ≥128, the titer of the IgM anti-B antibody is ≥128, and the titer of the IgM anti-D antibody is ≥64.

3. The analysis card according to claim 1 or 2, characterized in that, Each liter of mixture in microcolumn 1 contains 400-600 g of blocked PMMA microspheres and 100-200 ml of IgM anti-A antibody; each liter of mixture in microcolumn 2 contains 400-600 g of blocked PMMA microspheres and 100-200 ml of IgM anti-B antibody; each liter of mixture in microcolumn 3 contains 400-600 g of blocked PMMA microspheres and 100-200 ml of IgM anti-D antibody; and each liter of mixture in microcolumn 4 contains 400-600 g of blocked PMMA microspheres.

4. The analysis card according to claim 1, characterized in that, The titer of the anti-human globulin antibody is ≥64.

5. The analysis card according to claim 1 or 4, characterized in that, Each liter of the mixture in microcolumn 5 contains 400-600 g of blocked PMMA microspheres and 100-200 ml of anti-human globulin antibody.

6. The method for preparing the analytical card according to claim 1, characterized in that, According to the filling composition of microcolumns 1-5, a mixture of suspension matrix, antibodies of various blood types, blocked PMMA microspheres and anti-human globulin antibodies is prepared and filled into each microcolumn.

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