CN106290921A - A blood type detection card and blood type detection system based on microporous membrane - Google Patents

Abstract

The invention provides a blood type detection card based on a microporous membrane, which consists of a plurality of microcolumn tubes and the microporous membrane fixed in the middle of the microcolumn tubes, wherein the microcolumn tubes are used for accommodating red blood cells to be detected and a detection reagent containing an antibody, the aperture of the microporous membrane is 0.1-10 microns, the pore density is 10⁵‑10⁸Per cm²According to the requirement of blood type detection, the pore diameter of the microporous membrane is selected so that the red blood cells which are not combined with the antibody are settled to the bottom of the microcolumn tube through the microporous membrane, the red blood cells which are combined with the antibody are blocked above the microporous membrane, and the blood type is judged according to the red position of the red blood cells. The blood type detection card has the characteristics of long quality guarantee period, low cost and capability of almost detecting all blood type related identification tests.

Description

A blood type detection card and blood type detection system based on microporous membrane

technical field

The invention relates to the field of blood type testing, in particular to a blood type testing card and a blood type testing system.

Background technique

Blood type is a method of classifying blood, usually referring to the typing of red blood cells, based on whether there are certain heritable antigenic substances on the surface of red blood cells. Antigenic substances can be proteins, carbohydrates, glycoproteins or glycolipids. Usually some antigens come from the alleles of the same gene or the coding products of several closely linked genes, and these antigens form a blood group system. There are 30 blood group systems that have been discovered in humans and recognized by the International Society of Blood Transfusion, such as ABO blood group system, Rh blood group system, MNS blood group system, P blood group system, etc., among which the ABO blood group system and Rh blood group system are the most important. At present, the most commonly used blood group identification methods in clinical laboratories are hemagglutination test and microcolumn gel test (MGT).

The ABO blood group system is the most important of all known blood group systems. The identified ABO blood group antigens are: A, B, AB, A1, and the red blood cell membrane with A antigen is called type A red blood cells. Type A can be further divided into subtypes A1 and A2. A1 subtype red blood cells contain both A and A1 antigens, while A2 red blood cells only contain A antigens; red blood cell membranes have B antigens called B red blood cells, and both A and B antigens are called AB red blood cells. , are called type O red blood cells. The Rh blood group is the most polymorphic blood group system. There are more than 40 antigens discovered so far, but there are mainly 5 antigens involved in clinical problems, namely D, C, c, E, e and their corresponding specific antibodies. The order of antigen strength is D>E>c>C>e. At the same time, this blood type is the most clinically significant blood group system besides the ABO blood type, and plays an important role in the diagnosis and treatment of hemolytic disease of the newborn caused by blood transfusion therapy and fetal-maternal immunization. In the Rh blood group system, the D antigen has the strongest antigenicity, and 50% to 75% of Rh-negative individuals are transfused with D-positive red blood cells to produce anti-D antibodies. The importance of the D antigen is second only to the A and B antigens. Under normal circumstances, the Rh blood type test only detects the D antigen in blood recipients, and further examination and determination of the weak D antigen in blood donors is required.

The blood received by patients during blood transfusion should not only be tested for compatibility between ABO and Rh blood or blood components, but also the inspection and screening of irregular antibodies, high-frequency and low-frequency antigens in the body is also very necessary. This is mainly because the blood of different people has different immunological antigens, and certain adverse reactions may occur during blood transfusion, especially delayed hemolytic transfusion reactions. In addition, for pregnant women, irregular antibodies can cause hemolytic disease of the newborn, affect the development of the newborn's organs, and harm its intellectual development, and in severe cases, it will endanger the life of the newborn. Therefore, antibody screening is necessary and necessary.

The so-called irregular antibodies refer to blood type antibodies that do not conform to the Landsteiner rule of the ABO blood type system, that is, blood type antibodies other than anti-A and anti-B. The subtypes in the ABO line, variant anti-A or certain anti-B antibodies, are also called irregular antibodies. Most of them are IgG antibodies. Red blood cells that cannot agglutinate but can only be sensitized to the corresponding antigen in saline medium must pass through a special medium (enzyme, antihuman globulin, polybrene, etc.) to cause agglutination of the sensitized red blood cells.

The purpose of antibody screening is to discover irregular antibodies with clinical significance, and to select suitable blood in a targeted manner, so as not to blindly match blood. At present, more and more hospitals conduct routine antibody screening tests on the serum of blood recipients.

Antiglobulin methods include direct antiglobulin test (DAT) and indirect antiglobulin test (IAT). Antibody molecules and complement components are globulins, and immunization of animals with human globulins stimulates the production of antibodies against foreign proteins. Animal serum can react specifically with human globulin (AHG) after inactivating complement and absorbing and removing unwanted lectins. AHG reacts with human globulin bound to red blood cells or free in serum. AHG is added to a system containing coated erythrocytes and free globulin, which neutralizes AHG. After the AHG-coated red blood cells are washed, the Fab fragment of the AHG molecule binds to the Fc fragment of the human globulin molecule coated on the red blood cells. In this way, the red blood cells are agglutinated by bypassing AHG. The process of agglutination is directly proportional to the amount of globulin coated on red blood cells, and uncoated red blood cells will not agglutinate.

Before blood transfusion, be sure to check the blood types of the patient (recipient) and the donor (donor), and perform a cross-matching test. Blood transfusion is a safe and temporarily effective method to correct blood inversion. Cross-matching is the most important inspection item to determine whether blood transfusion is possible, and it is a necessary work before blood transfusion. Accurate blood typing and cross-matching are important guarantees for clinical safety and effective blood transfusion. In clinical medicine, in addition to blood transfusion and transplantation immunity, blood type knowledge and related technologies are also required for the examination of specific antibodies for hemolytic disease of the newborn and autoimmune hemolytic anemia. Blood type is not only important in blood transfusion, but also has important application value in ethnology, genetics, forensic science, transplant immunity, disease resistance (or susceptibility) and so on.

Common methods of blood typing are:

1. Hemagglutination test

Hemagglutination test refers to the visible agglutination reaction of blood group antigens and antibodies in red blood cells and serum in liquid medium. There are roughly three methods for ABO blood type identification with hemagglutination test: slide method, test tube method and automatic method. The experimental principles of the slide method and the test tube method are basically the same, and the common point of the two is that it is easy to operate. However, compared with the slide method only for positive typing, using the test tube method for ABO blood type identification. Positive and negative typing controls avoid blood type errors due to blood group antigens or antibodies, and because the test tube method is used for ABO blood type identification, the red blood cells to be tested are washed and diluted (2-5% concentration), reducing the risk of blood type False positives due to too high concentration of red blood cells. Although the traditional blood type identification slide method and test tube method are time-consuming, labor-intensive, poorly sensitive, difficult to automate, batch-scale, and difficult to store results, the test tube method for ABO blood type identification is easy to operate. , The result is accurate and reliable, and it is still widely used in ABO blood group identification work.

2. Microcolumn gel method

The micro-column gel technology was approved by the US FDA in 1994 and is an immunological detection technology based on traditional blood group serology. The micro-column gel method is an improved hemagglutination test. During the experiment, the reaction between the red blood cell antigen and the corresponding antibody is carried out in the micro-column gel medium. The micro-column gel has the function of a gel molecular sieve. , the formed clots cannot pass through the gel molecular sieve after centrifugation and remain on the upper layer of the gel medium; if the red blood cells are not combined with the antibody and no clots are formed after the reaction, the red blood cells can pass through the gel molecular sieve to precipitate the bottom of the gel layer after centrifugation , so as to achieve the purpose of identifying ABO blood type. The micro-column gel test omits operations such as washing red blood cells, and the working procedures and interpretation of results are easy to standardize and automate. The experimental method also has the advantages of less required specimens, accurate results, strong anti-interference ability, less influence of hemolysis and lipemia, and good repeatability. The ABO blood type identification method, more and more domestic units use it for ABO blood type identification.

Chinese patent application CN101718794A provides a preparation method of ABO/RhD blood typing detection reagent card, the reagent card has a microcolumn gel tube, and the gel tube contains polyacrylamide dextran gel, and the red blood cells without agglutination can pass through completely Agglutinated red blood cells cannot pass through the gap between gel particles. Chinese patent CN101701961A provides a method for preparing an ABO blood typing detection reagent card, and the gel tube also contains polyacrylamide dextran gel.

However, except for the antihuman globulin card, which can detect three kinds of blood group test card by microcolumn gel method, each other experiment can only be tested with a special test card. At present, the best micro-column gel blood type test card can only be stored at 2-25°C for 1 year.

Contents of the invention

In order to solve the above-mentioned problems, the object of the present invention is to overcome the above-mentioned shortcomings, and provide a blood type detection card and a blood type detection system with a long shelf life, low cost, and almost all blood type-related identification tests.

The purpose of the present invention is to provide a blood type detection card based on a microporous membrane, the microporous membrane in the detection card allows a single free red blood cell to pass through, but the red blood cells that have agglutinated due to binding with antibodies cannot pass through, and will be trapped on the surface of the membrane A red layer of erythrocytes forms. Red blood cells and antibodies are added to the microcolumn tube in the detection card, and gradient centrifugation is performed after the red blood cells react with the antibodies. If the antibody reacts with erythrocytes and agglutinates, it will be trapped on the surface of the microporous membrane to form a red erythrocyte layer, which is judged as a positive result; if the antibody does not react with erythrocytes, the free erythrocytes will pass through the filter membrane and gather in the microcolumn tube Bottom, judged as a negative result.

Another object of the present invention is to provide a blood type detection system based on a microporous membrane.

According to one aspect of the present invention, a blood type test card is composed of a plurality of microcolumn tubes and a microporous membrane fixed in the middle of the microcolumn tubes, and the microcolumn tubes are used to accommodate red blood cells to be tested and contain antibodies for detection Reagents, the pore size of the microporous membrane is 0.1 micron to 10 micron, and the pore density is 10 ⁵ -10 ⁸ cells/cm ² , according to the needs of blood type detection, the pore size of the microporous membrane is selected so that the red blood cells not bound to the antibody The microporous membrane settles to the bottom of the microcolumn tube, the red blood cells combined with the antibody are blocked above the microporous membrane, and the blood type is determined according to the red position of the red blood cells.

Wherein, the microporous membrane is selected from polyethersulfone, nylon, polypropylene, polyethylene, polyethylene terephthalate, polycarbonate, polytetrafluoroethylene, polyvinylidene fluoride, polytetrafluoroethylene Dispersion resin, polyurethane, polyvinyl chloride, polyimide, glass fiber membrane, mixed cellulose and silicone membrane materials.

The microporous membrane is preferably selected from polyethylene terephthalate, polycarbonate, polypropylene and polyimide.

The microporous membrane is prepared by the following method:

1) Base film imaging: the film of polyethylene terephthalate, polycarbonate, polypropylene, and polyimide materials with a thickness of 6 μm to 50 μm is used as the base film;

2) Irradiation: choose a heavy ion beam with an energy of 1MeV-500MeV and a current intensity of 0.1 nanoampere-10 microamperes to irradiate the basement membrane from one side, and the irradiation time is 0.1 seconds to 9 hours;

3) Sensitization: use an ultraviolet lamp with a wavelength of 200-365nm to irradiate the irradiated basement film, the power of the ultraviolet lamp is 50 watts to 2000 watts, the distance between the lamp and the basement film is 0.1 cm to 50 cm, and the sensitization time is 10 seconds - 180 minutes;

4) Etching: Put the sensitized base film into a strong alkali or strong acid solution with a concentration of 0.1 equivalents to 30 equivalents for etching, and the etching time is 1 to 180 minutes;

5) Drying: drying in an oven at a temperature of 10°C to 150°C to obtain the microporous membrane.

The multiple microcolumn tubes include detection tubes and quality control tubes.

The micro column tube is made of polypropylene, polyethylene or polyvinyl chloride.

The detection reagents are ABO positive type, reverse type, Rh blood type, direct antiglobulin test, indirect antiglobulin test, cross-match, Rh blood type typing, direct antiglobulin classification test, A2 subtype identification Reagents for testing, neonatal blood typing.

According to one aspect of the present invention, a blood type detection system is provided, including the above-mentioned blood type detection card and supporting reagents, the supporting reagents include antibodies for detecting blood types and corresponding antibody diluents, and the antibody diluents contain phosphate buffered liquid, bovine serum albumin (BSA), polyethylene glycol (PEG) as a lubricant, NaN ₃ as a preservative, and sucrose, BSA as a stabilizer.

Wherein, the antibody diluent is composed of: Na ₂ HPO ₄ ·12H ₂ O 0.715g, KH ₂ PO ₄ 0.408g, NaCl 1.75g, EDTA-2Na 2.0g, BSA 10g, PEG6000 0.5g, sucrose 40g and NaN ₃ 0.1g, fully dissolved in purified water to 1000ml, and stored at 2-25°C.

The antibody used for detecting blood type is selected from anti-A monoclonal antibody, anti-B monoclonal antibody, anti-D monoclonal antibody, anti-human IgG antibody, anti-C3d antibody titer, anti-human IgG+C3d antibody, anti-A1 antibody, Anti-C antibody, anti-c antibody, anti-E antibody and anti-e antibody.

Compared with the micro-column gel method, the beneficial effect of the blood type detection card of the present invention is:

1. The microcolumn tube of the blood type detection card of the present invention can be made of materials such as polypropylene, polyethylene, polyvinyl chloride, and the microporous membrane can be made of polyethersulfone, nylon, polypropylene, polyethylene, polycarbonate, polytetrafluoroethylene, etc. Vinyl fluoride, polyvinylidene fluoride, polyethylene terephthalate, polytetrafluoroethylene dispersion resin, polyurethane, polyvinyl chloride, polyimide, glass fiber film, mixed cellulose, silicone film and other materials These materials have very high stability, there is no special requirement for storage temperature, and the validity period is more than 10 years. At present, the best micro-column gel blood type test card can only be stored at 2-25°C for 1 year.

2. No gel/glass beads are added to the blood type test card of the present invention, and the cost is much lower than that of the microcolumn gel method blood type test card.

3. The supporting reagents for the blood type test card of the present invention can detect almost all blood type-related identification tests, including ABO positive typing, reverse typing, Rh blood type, direct antiglobulin test, indirect antiglobulin test, cross-matching blood , Rh blood typing, direct antiglobulin classification test, A2 subtype identification test, neonatal blood type detection test, etc. In addition to the three types of antiglobulin cards that can be detected by the micro-column gel blood type test card, each other experiment can only be tested with a special test card. The supporting reagents for the blood type testing card of the present invention greatly facilitate medical operators and improve the working efficiency of medical testing institutions.

4. The microporous membrane of the present invention is made by track etching method, the pore diameter of the microporous membrane can be from 0.1 micron to 10 micron, the pore diameter is uniform and controllable, the pore density can be controlled from 10 ⁵ -10 ⁸ /cm ² , the micropore The film stability and uniformity are very good.

5. The supporting reagents for the blood type detection card of the present invention can be used in an automatic blood type analyzer to realize full automation of detection.

The beneficial effect of supporting reagent of the present invention is:

1. The antibody diluent of the present invention adopts phosphate buffer solution, which has good buffer capacity and can protect the antibody well; the NaCl concentration added in the buffer solution is lower, compared with the NaCl concentration of normal saline, it can reduce Antibodies gain resistance when reacting with red blood cells, enhancing reaction sensitivity.

2. The antibody diluent of the present invention has added bovine serum albumin (BSA) and polyethylene glycol (PEG) as a lubricant, which can protect red blood cells well, so that red blood cells are not easily broken, and free red blood cells pass through the microporous membrane more easily. Improve the specificity of the reaction.

3. The antibody diluent of the present invention has added NaN ₃ as a preservative, which can avoid the growth of bacteria in the diluent and improve the stability of each reagent.

4. Adding sucrose and BSA to the antibody diluent of the present invention as stabilizers can well protect the stability of various antibodies in the solution. Experimental results show that the validity period of various antibody reagents prepared with the antibody diluent of the present invention is not less than 12 months under the condition of 2-8°C.

5. All the supporting reagents for various blood type cards described in the present invention are packaged independently, which is easy to store. It is easy to use and can also be used in automatic blood type analyzers to realize full automation of detection.

Description of drawings

Figure 1 is a schematic diagram of a blood type card;

Figure 2 is a schematic diagram of a single hole of the blood type card, 1: microcolumn tube; 2: aluminum foil composite membrane sealing; 3: microporous membrane, the microporous membrane allows red blood cells to pass through, but red blood cells that have agglutinated due to binding to antibodies cannot pass through;

Figure 3 is a positive result;

Figure 4 is a negative result.

detailed description

The present invention will be further described in detail below in combination with specific embodiments.

Source of materials: Unless otherwise specified, the following materials are commercially available.

Antibody raw materials were purchased from Immucor, and the specific information is shown in the table below:

The standard red blood cells used in the test were purchased from Shanghai Blood Biomedicine Co., Ltd., and the specific information is shown in the following table:

Item No. name regulations use 5012053 Red blood cell kit for human ABO blood group reverse typing 3x10mL Standard red blood cells for ABO reverse typing 5012070 Antibody Screening Red Blood Cell Kit 3x5mL Irregular Antibody Screening

[Example 1] Preparation of test card

Step 1. A processing method for a microporous membrane, comprising the following steps:

(1) Basement film imaging: polyethersulfone, nylon, polypropylene, polyethylene, polyethylene terephthalate, polycarbonate, polytetrafluoroethylene, polyvinylidene fluoride with a thickness of 6-50 μm can be used , polytetrafluoroethylene dispersion resin, polyurethane, polyvinyl chloride, polyimide, glass fiber membrane, mixed cellulose, organic silicon membrane is a base membrane;

(2) Irradiation: choose a heavy ion beam with an energy of 1-500 MeV and a current intensity of 0.1 nanoampere-10 microamperes to irradiate the basement membrane from one side, and the irradiation time is 0.1 seconds to nine hours;

(3) Sensitization: Utilize an ultraviolet lamp with a wavelength of 200-365nm to irradiate the irradiated basement film, the power of the ultraviolet lamp is 50-2000 watts, the distance between the lamp and the basement film is 0.1-50 cm, and the sensitization time is 10 seconds - 180 minutes;

(4) Etching: put the sensitized base film into a strong alkali or strong acid solution with a concentration of 0.1-30 equivalents for etching, and the etching time is 1-180 minutes;

(5) Drying: After drying in an oven at a temperature of 10 degrees to 150 degrees, a microporous membrane that meets the requirements is produced.

Step 2: Fix the microporous membrane in the center of each microcolumn tube of the detection card.

Step 3: Seal with aluminum foil composite film to ensure that the seal is tight and complete.

[Example 2] Preparation of detection card supporting reagents: anti-A reagent, anti-B reagent, anti-D reagent, anti-human IgG reagent, anti-C3d reagent, anti-human IgG+C3d reagent, anti-A1 reagent, anti-C reagent, anti-c Reagent, Anti-E Reagent, Anti-E Reagent

(1) Preparation of Antibody Diluent

Weigh 0.715g of Na ₂ HPO ₄ ·12H ₂ O, 0.408g of KH ₂ PO ₄ , 1.75g of NaCl, 2.0g of EDTA-2Na, 10g of BSA, 0.5g of PEG6000, 40g of sucrose and 0.1g of NaN ₃ , and fully dissolve them in purified water to 1000ml, store at 2-25°C.

(2) Selection of antibodies

Anti-A monoclonal antibody titer ≥ 128.

Anti-B monoclonal antibody titer ≥128.

Anti-D monoclonal antibody titer ≥64.

Anti-human IgG antibody titer ≥ 50.

Anti-C3d antibody titer ≥ 50.

Anti-human IgG+C3d antibody titer ≥50.

Anti-A1 antibody titer ≥ 50.

Anti-C antibody titer ≥50.

Anti-c antibody titer ≥ 50.

Anti-E antibody titer ≥50.

Anti-e antibody titer ≥ 50.

(3) Preparation of antibody reagents

Add the anti-A monoclonal antibody to the antibody diluent according to the determined titer to make the anti-A reagent.

Add the anti-B monoclonal antibody to the antibody diluent according to the determined titer to make the anti-B reagent.

Add the anti-D monoclonal antibody to the antibody diluent according to the determined titer to make the anti-D reagent.

Add the anti-human IgG antibody to the antibody diluent according to the determined titer to make anti-human IgG reagent.

The anti-C3d antibody is added to the antibody diluent according to the determined titer to prepare the anti-C3d reagent.

Add the anti-human IgG+C3d antibody to the antibody diluent according to the determined titer to prepare the anti-human IgG+C3d reagent.

Add the anti-A1 antibody to the antibody diluent according to the determined titer to prepare the anti-A1 reagent.

Add the anti-C antibody to the antibody diluent according to the determined titer to make the anti-C reagent.

The anti-c antibody is added to the antibody diluent according to the determined titer to make the anti-c reagent.

Add the anti-E antibody to the antibody diluent according to the determined titer to make the anti-E reagent.

Add the anti-e antibody to the antibody diluent according to the determined titer to make the anti-e reagent.

[Example 3] Positive typing experiment

(1) Label the test card, and every 4 microcolumn tubes are 1 serving. Set 1-4 microcolumn tubes as tube A, tube B, tube D, and tube Ctl. respectively. Each test card can detect 2 people.

(2) Add anti-A reagent to tube A, anti-B reagent to tube B, anti-D reagent to tube D, and antibody diluent to tube Ctl, 50 μl per tube.

(3) Add 4% erythrocyte suspension diluted with normal saline to 1-4 microcolumn tubes successively, 10 μl per tube.

(4) Put the card into a medical centrifuge and centrifuge at 800rpm for 2 minutes, 1500rpm for 3 minutes, and take out the visual judgment result.

(5) Result judgment

Red blood cells agglutinate on the surface of the microporous membrane, which is a positive result;

Red blood cells condense at the bottom of the microcolumn tube, which is a negative result;

The results of blood type determination are shown in the table below:

Note: "+" is positive, "-" is negative.

[Example 4] positive and negative stereotypes experiment

(1) Label the test card, and each test card can test 1 person. Set 1-8 microcolumn tubes as tube A, tube B, tube D, tube Ctl., tube Ac, tube Bc, tube Oc, and tube itself. 1-4 microcolumn tubes are used for positive stereotype detection, and 5-8 microcolumn tubes are used for reverse stereotype detection.

(2) Add anti-A reagent to tube A, anti-B reagent to tube B, anti-D reagent to tube D, and antibody diluent to Ctl. tube, 50 μl for each tube; The serum/plasma to be tested was added to the tube and its own tube in sequence, 50 μl per tube.

(3) Add 4% erythrocyte suspension diluted with normal saline to tubes A, B, D, Ctl. For standard red blood cells, add 2-3% standard red blood cells of type B to tube Bc, add 2-3% standard red blood cells of type O to tube Oc, 15-20 μl per tube.

(4) Put the card into a medical centrifuge and centrifuge at 800rpm for 2 minutes, 1500rpm for 3 minutes, and take out the visual judgment result.

(5) Result judgment

Red blood cells agglutinate on the surface of the microporous membrane, which is a positive result;

Red blood cells condense at the bottom of the microcolumn tube, which is a negative result;

The results of blood type determination are shown in the table below:

Note: "+" is positive, "-" is negative.

[Example 5] Direct anti-human globulin experiment

(1) Label the test card, and each microcolumn tube can test 1 person.

(2) Add anti-human IgG+C3d reagent to 1-8 microcolumn tubes, 50 μl per tube.

(3) Add 4% erythrocyte suspension diluted with normal saline to 1-8 microcolumn tubes successively, 10 μl per tube.

(4) Put the card into a medical centrifuge and centrifuge at 800rpm for 2 minutes, 1500rpm for 3 minutes, and take out the visual judgment result.

(5) Result judgment

Red blood cells agglutinate on the surface of the microporous membrane, which is a positive result;

Red blood cells condense at the bottom of the microcolumn tube, which is a negative result;

[Example 6] Cross-match experiment

(1) Label the test card, and each 2 microcolumn tubes can test 1 serving. Label 1-2 microcolumn tubes "Primary Side" and "Secondary Side".

(2) Add 40 μl of serum or plasma from the recipient to the main side well, and then add 10 μl of 4% red blood cell suspension from the donor.

(3) 40 μl of blood donor’s serum or plasma was first added to the secondary side well, and then 10 μl of 4% erythrocyte suspension of blood recipient was added.

(4) Stand the test card upright on the table, tap the lower left and right corners of the card, mix the serum or plasma and red blood cells in the reaction chamber, and place it in an immunocolumn incubator at 37°C for 15 minutes after mixing.

(5) Put the card into a medical centrifuge and centrifuge at 800rpm for 2 minutes, 1500rpm for 3 minutes, and take out the visual judgment result.

(6) Result judgment

Red blood cells agglutinate on the surface of the microporous membrane, which is a positive result;

Red blood cells condense at the bottom of the microcolumn tube, which is a negative result;

[Example 7] Irregular antibody screening experiment

(1) Label the test card, and each 3 microcolumn tubes can test 1 person. Label 1-3 microcolumn tubes as I, II, and III, respectively.

(2) Add 50 μl of corresponding 0.8-1% antibody screening erythrocyte suspension into the reaction chamber of each microcolumn tube according to the mark.

(3) Add 50 μl of serum/plasma of the subject to be tested into the reaction chamber of each marked microcolumn tube.

(4) Stand the test card upright on the table, tap the lower left and right corners of the card, mix the serum or plasma and red blood cells in the reaction chamber, and place it in an immunocolumn incubator at 37°C for 15 minutes after mixing.

(5) Put the card into a medical centrifuge and centrifuge at 800rpm for 2 minutes, 1500rpm for 3 minutes, and take out the visual judgment result.

(6) Result judgment

Red blood cells agglutinate on the surface of the microporous membrane, which is a positive result;

Red blood cells condense at the bottom of the microcolumn tube, which is a negative result;

[Example 8] Classification experiment of direct antihuman globulin

(1) Label the test card, and each 2 microcolumn tubes can test 1 serving. Label 1-2 microcolumn tubes as IgG tube and C3d tube respectively.

(2) Add anti-human IgG reagent to the IgG tube, and add anti-C3d reagent to the C3d tube, 50 μl per tube.

(3) Add 4% erythrocyte suspension diluted with normal saline to each microcolumn tube sequentially, 10 μl per tube.

(4) Put the card into a medical centrifuge and centrifuge at 800rpm for 2 minutes, 1500rpm for 3 minutes, and take out the visual judgment result.

(5) Result judgment

Red blood cells agglutinate on the surface of the microporous membrane, which is a positive result;

Red blood cells condense at the bottom of the microcolumn tube, which is a negative result;

[Example 9] A2 subtype identification test

(1) Label the test card, and each 2 microcolumn tubes can test 1 serving. Mark 1-2 microcolumn tubes as tube A and tube A1 respectively.

(2) Add anti-A reagent to tube A, and add anti-A1 reagent to tube A1, 50 μl per tube.

(3) Add 4% erythrocyte suspension diluted with normal saline to each microcolumn tube sequentially, 10 μl per tube.

(4) Put the card into a medical centrifuge and centrifuge at 800rpm for 2 minutes, 1500rpm for 3 minutes, and take out the visual judgment result.

(5) Result judgment

Red blood cells agglutinate on the surface of the microporous membrane, which is a positive result;

Red blood cells condense at the bottom of the microcolumn tube, which is a negative result;

The results of blood type determination are shown in the table below:

blood type Tube A A1 tube A1 + + A2 + -

Note: "+" is positive, "-" is negative.

[Example 10] Rh blood group typing experiment

(1) Label the test card, and every 5 microcolumn tubes can test 1 person. Label tubes 1-5 as tube D, tube C, tube c, tube E, and tube e.

(2) Add anti-D reagent to tube D, add anti-C reagent to tube C, add anti-c reagent to tube c, add anti-E reagent to tube E, and add anti-e reagent to tube E, each tube 50 μl.

(3) Add 4% erythrocyte suspension diluted with normal saline to each microcolumn tube sequentially, 10 μl per tube.

(4) Put the card into a medical centrifuge and centrifuge at 800rpm for 2 minutes, 1500rpm for 3 minutes, and take out the visual judgment result.

(5) Result judgment

Red blood cells agglutinate on the surface of the microporous membrane, which is a positive result;

Red blood cells condense at the bottom of the microcolumn tube, which is a negative result;

The results of blood type determination are shown in the table below:

Note: "+" is positive, "-" is negative.

[Example 11] Newborn blood type detection experiment

(1) Label the test card, and every 5 microcolumn tubes can test 1 person. Label 1-5 microcolumn tubes as tube A, tube B, tube D, tube Ctl., and tube IgG+C3d.

(2) Add anti-A reagent to tube A, add anti-B reagent to tube B, add anti-D reagent to tube D, add antibody diluent to tube Ctl, add anti-human IgG+C3d reagent to tube IgG+C3d , 50 μl per tube.

(3) Add 4% erythrocyte suspension diluted with normal saline to each microcolumn tube sequentially, 10 μl per tube.

(4) Put the card into a medical centrifuge and centrifuge at 800rpm for 2 minutes, 1500rpm for 3 minutes, and take out the visual judgment result.

(5) Result judgment

Red blood cells agglutinate on the surface of the microporous membrane, which is a positive result;

Red blood cells condense at the bottom of the microcolumn tube, which is a negative result;

The results of blood type determination are shown in the table below:

Note: "+" is positive, "-" is negative.

Claims (10)

1. a blood type test card, by multiple microtrabeculae pipes be fixed on the microporous membrane in the middle part of described microtrabeculae pipe and form, described microtrabeculae Pipe is for accommodating erythrocyte to be measured and the detectable comprising antibody, and the aperture of described microporous membrane is 0.1 micron to 10 microns, Hole density is 10⁵-10⁸Individual/cm², according to the needs of Blood grouping, select the aperture of described microporous membrane make not with antibodies Erythrocyte be settled down to, bottom described microtrabeculae pipe, be blocked on described microporous membrane with the erythrocyte after antibodies by microporous membrane Top, judges blood group according to described erythrocytic red locations.

2. blood type test card as claimed in claim 1, it is characterised in that described microporous membrane by selected from polyether sulfone, nylon, poly-third Alkene, polyethylene, polyethylene terephthalate, Merlon, politef, Kynoar, politef disperse The material of resin, polyurethane, polrvinyl chloride, polyimides, glass fibre membrane, cellulose mixture and organosilicon membrane is made.

3. blood type test card as claimed in claim 2, it is characterised in that described microporous membrane is by selected from poly terephthalic acid second two The material of alcohol ester, Merlon, polypropylene and polyimides is made.

4. blood type test card as claimed in claim 3, it is characterised in that described microporous membrane is prepared by following method:

1) basement membrane imaging: be that 6 μm～the polyethylene terephthalate of 50 μm, Merlon, polypropylene, polyamides are sub-with thickness The film of amine material is basement membrane；

2) irradiation: selection energy is 1MeV-500MeV, stream is by force that the heavy ion beam current of 0.1 na-10 microamperes is from an irradiation Basement membrane, exposure time is 0.1 second to 9 hours；

3) sensitization: utilizing the Burdick lamp of wavelength 200-365nm to be irradiated the basement membrane after irradiation, Burdick lamp power is 50 Watts-2000 watts, lamp is 0.1 centimetre-50 centimetres with the distance of basement membrane, and sensitization time is 10 seconds-180 minutes；

4) etching: put into the basement membrane after sensitization to enter in the highly basic or strong acid solution that concentration is-30 equivalents of 0.1 equivalent Row etching, etching period is 1-180 minute；

5) dry: through the oven for drying of excess temperature 10 degree to 150 degree, prepare described microporous membrane.

5. blood type test card as claimed in claim 1, it is characterised in that the plurality of microtrabeculae pipe includes detection pipe and Quality Control pipe.

6. blood type test card as claimed in claim 1, it is characterised in that described microtrabeculae pipe is by polypropylene, polyethylene or polychlorostyrene second Alkene is made.

7. blood type test card as claimed in claim 1, it is characterised in that described detectable is ABO positive definite form, reverse type, Rh Blood group, Direct antiglobulin test, indirect antihuman globulin test, cross matching, Rh blood grouping, direct antihuman globulin Classification experiments, A2 blood subgroup qualification test, the reagent of neonate Blood grouping.

8. a Blood grouping system, including the blood type test card described in claim 1 and matched reagent, described matched reagent bag Including the antibody for detecting blood group and corresponding antibody diluent, described antibody diluent contains phosphate buffer, as lubrication The bovine serum albumin of agent, Polyethylene Glycol, as the NaN of preservative₃, and as the sucrose of stabilizer, bovine serum albumin.

9. Blood grouping system as claimed in claim 8, it is characterised in that described antibody diluent consists of: Na₂HPO₄· 12H₂O 0.715g、KH₂PO₄0.408g, NaCl 1.75g, EDTA-2Na 2.0g, BSA 10g, PEG6000 0.5g, sucrose 40g and NaN₃0.1g, is fully dissolved to 1000ml, 2～25 DEG C of preservations by purified water.

10. Blood grouping system as claimed in claim 8, it is characterised in that the described antibody for detecting blood group is selected from anti-A Monoclonal antibody, anti-B monoclonal antibody, anti-D monoclonal antibody, anti-human IgG antibodies, anti-C3d antibody titer, anti-human igg+C3d Antibody, anti-A1 antibody, anti-C antibody, anti-c antibody, anti-E antibody and anti-e antibody.