CN120577541A - A whole blood quality control kit for automated testing of blood transfusion compatibility - Google Patents

Abstract

The invention relates to a whole blood quality control kit for blood transfusion compatibility automatic detection, which comprises 7 whole blood quality control products, namely a tube 1:A type RhD+C+e+c-E-, a tube 2:B type RhD+c+E+C-E-, a tube 3:O type RhD-C-E-c+e+, a tube 4:O type RhD+C+e+c-E-, a tube 5:O type RhD+c+E+C-E-, a tube 6:O type RhD+ and a tube 7:O type RhD+. The whole blood quality control kit has the advantages of reducing systematic and invisible risks of automatic detection equipment, improving blood transfusion compatibility detection quality, being compatible with different serological methodologies (such as a test tube method, a microplate method, a microcolumn gel method, a microcolumn glass bead method and the like), being compatible with different types of automatic detection equipment and being compatible with different brands of reagents.

Description

Whole blood quality control kit for automatic detection of blood transfusion compatibility

Technical Field

The invention belongs to the technical field of blood transfusion compatibility detection, and particularly relates to a whole blood quality control kit for blood transfusion compatibility automatic detection.

Background

Blood transfusion compatibility detection is the most important last defense line for guaranteeing clinical blood safety, and provides reliable basis for accurate diagnosis and effective treatment of clinical work. The blood transfusion compatibility detection range is 5 items of ABO blood type positive and negative setting, rhD blood type, antibody screening and cross matching detection, and the indoor quality control of blood transfusion compatibility detection is mainly carried out aiming at the key links of the 5 items of detection.

Along with the development of medical technology, an automated technology becomes an important means for detecting blood transfusion compatibility, and has obvious advantages in the aspects of reducing labor intensity of workers, reducing errors of detection results, improving detection efficiency, shortening result reporting time, reducing biohazard risks and the like. However, there are also some level of failure rate, detection sensitivity and reproducibility differences in the blood transfusion compatibility detection automation system.

Patent CN102183631a discloses a blood transfusion compatibility detection indoor quality control kit. The kit consists of 8 improved whole blood quality control products, namely, a tube 1:A type RhD positive, the blood plasma contains anti-B, a tube 2:B type RhD negative, the blood plasma contains anti-A and IgG anti-D, a tube 3:B type RhD negative, the blood plasma contains anti-A and IgG anti-D, a tube 4:B type RhD positive, the blood plasma contains anti-A, a tube 5:B type RhD negative, the blood plasma contains anti-A, a tube 6:B type RhD positive, the blood plasma contains anti-A, a tube 7:A type RhD positive, the blood plasma contains anti-B, a tube 8:O type RhD positive, and the blood plasma contains anti-A and anti-B. Patent CN107576807a discloses a blood group detection reagent quality control product, a preparation method thereof and application in blood group detection, wherein the blood group detection reagent quality control product comprises 6 bottles of ABO, rhD blood group detection quality control products, 5 bottles of cross matching blood quality control products and 4 bottles of irregular antibody screening quality control products. The patent CN109239372A discloses a method for evaluating the detection capability of ABO blood group antigen and a kit thereof, wherein the kit comprises quality control erythrocytes of weak A, B antigen and erythrocytes mixed with ABO weak antigen, and the quality control erythrocytes of weak A, B antigen are prepared by preparing a series of erythrocytes containing a relative amount of A, B antigen, wherein a certain amount of O-type erythrocytes possibly serve as negative control, and a capability verification product is prepared. The preparation method of the mixed ABO weak antigen comprises mixing O-type and A or B-type erythrocytes according to different ratios, adding erythrocyte preservation solution after mixing uniformly, and preparing an A or B mixed agglutination sample with a definite negative-positive ratio, wherein a certain amount of O-type erythrocytes can be contained as negative control. Patent CN117805401A discloses a low-value positive quality control composition for blood transfusion compatibility detection, a preparation method and application thereof, wherein the quality control composition comprises one selected from B/RhD (-) E (-) and A/RhD (+) E (+), A/RhD (-) E (-) and B/RhD (+) E (+), AB/RhD (-) E (-) and O/RhD (+) E (+), and O/RhD (-) E (-) and AB/RhD (+) E (+), and IgM type monoclonal anti-E antibodies and IgG type monoclonal anti-D antibodies.

In order to meet the expected use of the blood group quality control product proposed by each patent, different reagents need to be prepared during use, for example, ABO (human red reagent) anti-typing reagent is needed during development of anti-typing project, and red cell reagent needs to be screened by irregular antibody during development of irregular antibody screening project. When the quality control result is out of control, the detection system is out of control, the quality control product is out of control, and the quality control of an automatic instrument cannot be completed in one quality control product system. In addition, each of the above patents evaluates laboratory detection capability, either for the occurrence of reduced specificity and/or sensitivity of blood group reagents during transportation and storage, or for the quality control of pollution caused during preparation and use, or for quality control of reagent specificity and sensitivity differences of different laboratory manufacturers. But not for quality control of an automatic blood transfusion compatibility detection system, no whole blood quality control product with different detection methodologies or detection systems for different matched instruments at home and abroad at present.

Disclosure of Invention

The invention aims to provide a whole blood quality control kit for automatic detection of blood transfusion compatibility, which has the advantages of reducing systematic and invisible risks of automatic detection equipment and improving the detection quality of blood transfusion compatibility.

The invention solves the problems by adopting the technical scheme that the whole blood quality control kit for blood transfusion compatibility automatic detection comprises the following 7 whole blood quality control products:

the tube 1:A type RhD+C+e+c-E-, the blood plasma contains serum of A type healthy blood donors, anti-B monoclonal antibody IgG anti-E monoclonal antibody and erythrocyte preservation solution;

Tube 2:B type RhD+c+E+C-E-, the blood plasma contains B type healthy blood donor serum, anti-A monoclonal antibody and erythrocyte preservation solution;

the tube 3:O type RhD-C-E-c+e+ contains O type healthy blood donor serum, anti-A monoclonal antibody, anti-B monoclonal antibody, igG anti-D monoclonal antibody and erythrocyte preservation solution;

Tube 4, O-type RhD+C+e+c-E-, wherein the blood plasma contains O-type healthy blood donor serum, anti-A monoclonal antibody, anti-B monoclonal antibody and erythrocyte preservation solution;

the tube 5:O type RhD+c+E+C-E-, the blood plasma contains O type healthy blood donor serum, anti-A monoclonal antibody, anti-B monoclonal antibody and erythrocyte preservation solution;

tube 6:O type rhd+, igG sensitized cells, blood plasma containing serum of healthy blood donors of type O, red blood cell preservation solution;

Tube 7:O type rhd+, C ₃ d sensitized cells, and blood plasma contained O type healthy donor serum and red blood cell preservation solution.

Preferably, the tube 1 whole blood quality control is prepared by the following method:

(1) Selecting raw materials:

A-type RhD positive red blood cells within 10 days of healthy blood donors are selected, the direct anti-human globulin experiment is negative, A-type serum within 10 days of healthy blood donors is negative in irregular antibody screening, the titer is more than or equal to 32 of monoclonal anti-E with IgG property, the titer is more than or equal to 256-512 of monoclonal anti-B with IgM property, and the agglutination strength is more than or equal to 3+ antibody highest dilution is more than or equal to 1:8;

(2) Preparing a quality control product:

① Centrifuging the erythrocytes, discarding the supernatant to obtain packed erythrocytes, adding 6 times of physiological saline to wash the packed erythrocytes to obtain supernatant, clarifying, and finally discarding the supernatant for standby;

② Preparing plasma:

a. Diluting an anti-B monoclonal antibody with IgM property by normal A type serum multiple ratio, performing titer determination, selecting a last tube with the agglutination intensity of more than or equal to 3+ by a test tube method and a last tube with the agglutination intensity of more than or equal to 2+ by a test tube method, determining the specific antibody content of an anti-B stock solution, the last tube with the agglutination intensity of more than or equal to 3+ and the last tube with the agglutination intensity of more than or equal to 2+ by a Human IGM ELISA KIT antibody concentration kit, adding and dividing the antibody concentration value of the last tube with the agglutination intensity of more than or equal to 3+ and the last tube with the agglutination intensity of more than or equal to 2+ by 2 to obtain an anti-B antibody content value with the agglutination intensity of more than or equal to 2+ by the anti-B stock solution, and dividing the antibody concentration value with the anti-B stock solution by the agglutination intensity of more than or equal to 2+ value with the anti-setting requirement to obtain dilution;

b. diluting an anti-E monoclonal antibody with IgG property by using a normal A type serum multiple ratio, performing titer titration, selecting a test tube method 2+ agglutination last tube, a 1+ agglutination first tube, measuring the specific antibody content of an anti-E stock solution, the 2+ agglutination last tube and the 1+ agglutination first tube through a Human IGM ELISA KIT antibody concentration kit, adding and dividing the antibody concentration value of the 2+ agglutination last tube and the 1+ agglutination first tube by 3 (including negative), obtaining an anti-E weak positive quality control antibody concentration value, dividing the anti-E stock solution antibody concentration value by the weak positive antibody concentration value, and diluting multiple;

c. Mixing blood donor A type serum and erythrocyte preservation solution in a volume ratio of 1:1, and adding an anti-B and an anti-E according to the proportion;

③ 1ml packed red blood cells were taken and 2ml plasma was added.

Preferably, the tube 2 whole blood quality control is prepared by the following method:

(1) Selecting raw materials, namely selecting B-type RhD positive erythrocytes within 10 days of healthy blood donors, wherein the direct anti-human globulin experiment is negative, B-type serum within 10 days of healthy blood donors is negative, and screening irregular antibodies, wherein the titer of monoclonal anti-A with IgM properties is 256-512, and the agglutination strength is more than or equal to 3+ and the highest antibody dilution is more than or equal to 1:8;

(2) Preparing a quality control product:

① Centrifuging the erythrocytes, discarding the supernatant to obtain packed erythrocytes, adding 6 times of physiological saline to wash the packed erythrocytes to obtain supernatant, clarifying, and finally discarding the supernatant for standby;

② Preparing plasma:

a. diluting an anti-A monoclonal antibody with IgM property by using a normal B type serum multiple ratio, performing titer measurement, selecting a last tube with the agglutination intensity of more than or equal to 3+ by a test tube method and a last tube with the agglutination intensity of more than or equal to 2+ by a test tube method, measuring the specific antibody content of an anti-A stock solution, the last tube with the agglutination intensity of more than or equal to 3+ and the last tube with the agglutination intensity of more than or equal to 2+ by using a Human IGM ELISA KIT antibody concentration kit, adding and dividing the antibody concentration value of the last tube with the agglutination intensity of more than or equal to 3+ and the last tube with the agglutination intensity of more than or equal to 2+ by 2 to obtain an anti-A antibody content value with the agglutination intensity of more than or equal to 2+ by an anti-A stock solution antibody concentration value divided by an anti-setting requirement agglutination intensity of more than or equal to 2+ value to obtain dilution times;

b. mixing blood donor type B serum and erythrocyte preservation solution in a volume ratio of 1:1, and adding an anti-A agent according to the proportion;

③ 1ml packed red blood cells were taken and 2ml plasma was added.

Preferably, the tube 3 whole blood quality control is prepared by the following method:

(1) The method comprises the steps of selecting O-type RhD negative erythrocytes within 10 days of healthy blood donors, directly resisting human globulin and testing negative, screening O-type serum within 10 days of healthy blood donors with irregular antibodies, resisting monoclonal antibodies-D with titer more than or equal to 1024 IgG, resisting monoclonal antibodies-A with titer between 256 and 512 IgM with agglutination strength more than or equal to 3+ antibody highest dilution more than or equal to 1:8, resisting monoclonal antibodies-B with titer between 256 and 512 IgM with agglutination strength more than or equal to 3+ antibody highest dilution more than or equal to 1:8;

(2) Preparing a quality control product:

① Centrifuging the erythrocytes, discarding the supernatant to obtain packed erythrocytes, adding 6 times of physiological saline to wash the packed erythrocytes to obtain supernatant, clarifying, and finally discarding the supernatant for standby;

② Preparing plasma:

a. Diluting an anti-A monoclonal antibody with IgM property and an anti-B monoclonal antibody with normal O-type serum multiple ratio, performing titer measurement, selecting a last tube with the agglutination strength of more than or equal to 3+ by a test tube method and a last tube with the agglutination strength of more than or equal to 2+ by a test tube method, measuring the specific antibody content of an anti-A stock solution, an anti-B stock solution, a last tube with the agglutination strength of more than or equal to 3+ and a last tube with the agglutination strength of more than or equal to 2+ by a Human IGM ELISA KIT antibody concentration kit, adding the antibody concentration values of the last tube with the agglutination strength of more than or equal to 3+ and the last tube with the agglutination strength of more than or equal to 2+ to 2, dividing the antibody concentration values of the anti-A and anti-B stock solution by the anti-A and anti-B required agglutination strength of more than or equal to 2+ to obtain dilution factors;

b. Quantitatively determining the anti-D monoclonal reference WHO standard substance IgG anti-D with the IgG property, wherein the final value is more than or equal to 1IU/ml, and dividing the quantitative value of the anti-D stock solution by 1 is the dilution;

c. mixing blood donor O-type serum and erythrocyte preservation solution in a volume ratio of 1:1, and adding an anti-A, an anti-B and an anti-D according to the proportion;

③ 1ml packed red blood cells were taken and 2ml plasma was added.

Preferably, the tube 4 whole blood quality control is prepared by the following method:

(1) Selecting raw materials, namely selecting O-type RhD positive erythrocytes within 10 days of healthy blood donors, wherein the direct anti-human globulin experiment is negative, O-type serum within 10 days of healthy blood donors is negative, and screening irregular antibodies is negative, wherein the titer of monoclonal anti-A with IgM properties is more than or equal to 3+ antibodies with the highest dilution of more than or equal to 1:8, and the titer of monoclonal anti-B with IgM properties is more than or equal to 256-512, and the agglutination strength of monoclonal anti-B with the highest dilution of more than or equal to 3+ antibodies with the titer of more than or equal to 1:8;

(2) Preparing a quality control product:

① Centrifuging the erythrocytes, discarding the supernatant to obtain packed erythrocytes, adding 6 times of physiological saline to wash the packed erythrocytes to obtain supernatant, clarifying, and finally discarding the supernatant for standby;

② Preparing plasma:

a. Diluting an anti-A monoclonal antibody with IgM property and an anti-B monoclonal antibody with normal O-type serum multiple ratio, performing titer measurement, selecting a last tube with the agglutination strength of more than or equal to 3+ by a test tube method and a last tube with the agglutination strength of more than or equal to 2+ by a test tube method, measuring the specific antibody content of an anti-A stock solution, an anti-B stock solution, a last tube with the agglutination strength of more than or equal to 3+ and a last tube with the agglutination strength of more than or equal to 2+ by a Human IGM ELISA KIT antibody concentration kit, adding the antibody concentration values of the last tube with the agglutination strength of more than or equal to 3+ and the last tube with the agglutination strength of more than or equal to 2+ to 2, dividing the antibody concentration values of the anti-A and anti-B stock solution by the anti-A and anti-B required agglutination strength of more than or equal to 2+ to obtain dilution factors;

b. Mixing blood donor O-type serum and erythrocyte preservation solution according to the volume ratio of 1:1, and adding an anti-A and an anti-B according to the proportion;

③ 1ml packed red blood cells were taken and 2ml plasma was added.

Preferably, the tube 5 whole blood quality control is prepared by the following method:

(1) Selecting raw materials, namely selecting O-type RhD positive erythrocytes within 10 days of healthy blood donors, wherein the direct anti-human globulin experiment is negative, O-type serum within 10 days of healthy blood donors is negative, and screening irregular antibodies is negative, wherein the titer of monoclonal anti-A with IgM properties is more than or equal to 3+ antibodies with the highest dilution of more than or equal to 1:8, and the titer of monoclonal anti-B with IgM properties is more than or equal to 256-512, and the agglutination strength of monoclonal anti-B with the highest dilution of more than or equal to 3+ antibodies with the titer of more than or equal to 1:8;

(2) Preparing a quality control product:

① Centrifuging the erythrocytes, discarding the supernatant to obtain packed erythrocytes, adding 6 times of physiological saline to wash the packed erythrocytes to obtain supernatant, clarifying, and finally discarding the supernatant for standby;

② Preparing plasma:

a. Diluting an anti-A monoclonal antibody with IgM property and an anti-B monoclonal antibody with normal O-type serum multiple ratio, performing titer measurement, selecting a last tube with the agglutination strength of more than or equal to 3+ by a test tube method and a last tube with the agglutination strength of more than or equal to 2+ by a test tube method, measuring the specific antibody content of an anti-A stock solution, an anti-B stock solution, a last tube with the agglutination strength of more than or equal to 3+ and a last tube with the agglutination strength of more than or equal to 2+ by a Human IGM ELISA KIT antibody concentration kit, adding the antibody concentration values of the last tube with the agglutination strength of more than or equal to 3+ and the last tube with the agglutination strength of more than or equal to 2+ to 2, dividing the antibody concentration values of the anti-A and anti-B stock solution by the anti-A and anti-B required agglutination strength of more than or equal to 2+ to obtain dilution factors;

b. Mixing blood donor O-type serum and erythrocyte preservation solution according to the volume ratio of 1:1, and adding an anti-A and an anti-B according to the proportion;

③ 1ml packed red blood cells were taken and 2ml plasma was added.

Preferably, the tube 6 whole blood quality control is prepared by the following method:

(1) Selecting raw materials, namely selecting O-type RhD positive red blood cells within 10 days of healthy blood donors, namely directly resisting human globulin and testing negative, O-type serum within 10 days of healthy blood donors, screening negative by irregular antibodies, and resisting monoclonal antibody D with the IgG property of titer more than or equal to 1024;

(2) Preparing a quality control product:

① Centrifuging the erythrocytes, discarding the supernatant to obtain packed erythrocytes, adding 6 times of physiological saline to wash the packed erythrocytes to obtain supernatant, clarifying, and finally discarding the supernatant for standby; diluting the anti-D with AB type human serum multiple ratio, taking the last tube with agglutination strength of 1+ as dilution multiple of the anti-D by test tube method, adding O type RhD positive red blood cells into diluted anti-D according to volume ratio of 1:10, incubating at 37deg.C for 30min, taking out, washing with normal saline for 3-4 times, and removing supernatant to obtain cell volume for use;

② Preparing blood plasma, namely mixing blood donor O-type serum and red blood cell preservation solution according to a volume ratio of 1:1;

③ 1ml packed red blood cells were taken and 2ml plasma was added.

Preferably, the tube 7 whole blood quality control is prepared by the following method:

(a) Selecting raw materials, namely selecting O-type RhD positive red blood cells of healthy blood donors within 10 days, and directly resisting human globulin negative experiments;

(b) Preparing a quality control product:

① Centrifuging erythrocytes, discarding supernatant to obtain packed erythrocytes, adding 6 times of physiological saline to clean the supernatant, finally discarding the supernatant for later use, weighing 23.8ml of sucrose sensitization solution, placing the supernatant in a container with a proper temperature, pre-cooling the container in an ice bath for 0-1 ℃, adding 1.2ml of O-type RhD positive erythrocytes into the sensitization solution, immediately adding 0.1ml of MgCl ₂ solution, uniformly mixing, ice-bath for 1 hour, washing for 4 times with the physiological saline to obtain the packed volume of C3b sensitization erythrocytes, weighing 0.1mol/L of phosphate buffer solution and 1% wt/vol trypsin solution, preparing cell transformation solution according to the proportion of 9:1, weighing the packed volume of C3b sensitization cells and the cell transformation solution, uniformly mixing the packed volume of the cells according to the proportion of 1:2, placing the container in a 37 ℃ water bath for 30min, taking out and washing for 3-4 times with the physiological saline, discarding the packed volume of the supernatant for later use;

② Preparing blood plasma, namely mixing blood donor O-type serum and red blood cell preservation solution according to a volume ratio of 1:1;

③ 1ml packed red blood cells were taken and 2ml plasma was added.

Compared with the prior art, the invention has the advantages that:

(1) The whole blood quality control kit for blood transfusion compatibility automatic detection can be used for setting ABO positive and negative typing, rh typing, irregular antibody screening and cross matching conventional projects, and can also be used for developing direct antibody typing, MNS, kell and other blood type system detection, antibody titer and antibody identification projects.

(2) The whole blood quality control product can completely replace clinical samples, is compatible with different serological methodologies (such as a test tube method, a microplate method, a microcolumn gel method, a microcolumn glass bead method and the like), is also compatible with different types of automatic detection equipment, and is compatible with different brands of reagents.

Detailed Description

The present invention is described in further detail below with reference to examples.

Example 1

A whole blood quality control kit for blood transfusion compatibility automated detection, the whole blood quality control kit comprising the following 7 whole blood quality controls:

the tube 1:A type RhD+C+e+c-E-, and the blood plasma contains serum of A type healthy blood donors, anti-B monoclonal antibody IgG anti-E monoclonal antibody and erythrocyte preservation solution.

Tube 2:B, rhD+c+E+C-E-, contained B-type healthy donor serum, anti-A monoclonal antibody, and red blood cell stock solution in the plasma.

The tube 3:O contains RhD-C-E-c+e+ and blood plasma of O-type healthy blood donors, anti-A monoclonal antibody, anti-B monoclonal antibody, igG anti-D monoclonal antibody and erythrocyte preservation solution.

Tube 4-O-type RhD+C+e+c-E-plasma contains serum from an O-type healthy donor, anti-A monoclonal antibody, anti-B monoclonal antibody, and red blood cell stock solution.

The tube 5:O-RhD+c+E+C-E-contains serum from an O-type healthy donor, an anti-A monoclonal antibody, an anti-B monoclonal antibody, and a red blood cell preservation solution.

Tube 6:O type rhd+, igG sensitized cells, and blood plasma contained serum from healthy blood donors of type O, red blood cell stock.

Tube 7:O type rhd+, C ₃ d sensitized cells, and blood plasma contained O type healthy donor serum and red blood cell preservation solution.

Wherein, the tube 1 whole blood quality control product is prepared by the following method:

(1) Selecting raw materials:

A type RhD positive red blood cells of healthy blood donors within 10 days are selected, the direct anti-human globulin experiment is negative, A type serum of healthy blood donors within 10 days is negative, irregular antibody screening is negative, the titer is more than or equal to 32 IgG monoclonal anti-E, the titer is more than or equal to 256-512 IgM monoclonal anti-B, and the agglutination strength is more than or equal to 3+ antibody highest dilution is more than or equal to 1:8.

(2) Preparing a quality control product:

① Centrifuging the erythrocytes, discarding the supernatant to obtain packed erythrocytes, adding 6 times of physiological saline to wash the supernatant to be clear, and finally discarding the supernatant for standby.

② Preparing plasma:

a. Diluting an anti-B monoclonal antibody with IgM property by using a normal A type serum multiple ratio, performing titer measurement, selecting a last tube with the agglutination intensity of more than or equal to 3+ by a test tube method and a last tube with the agglutination intensity of more than or equal to 2+ by a test tube method, measuring the specific antibody content of an anti-B stock solution, the last tube with the agglutination intensity of more than or equal to 3+ and the last tube with the agglutination intensity of more than or equal to 2+ by using a Human IGM ELISA KIT antibody concentration kit, adding and dividing the antibody concentration value of the last tube with the agglutination intensity of more than or equal to 3+ and the last tube with the agglutination intensity of more than or equal to 2+ by 2 to obtain an anti-B antibody content value with the agglutination intensity of more than or equal to 2+ by a reverse determination requirement, and dividing the anti-B stock solution antibody concentration value by the reverse determination requirement agglutination intensity of more than or equal to 2+ value to obtain dilution.

B. Diluting an anti-E monoclonal antibody with IgG property by using a normal A type serum multiple ratio, performing titer titration, selecting a test tube method 2+ agglutination last tube, a 1+ agglutination first tube, measuring the specific antibody content of an anti-E stock solution, the 2+ agglutination last tube and the 1+ agglutination first tube by using a Human IGM ELISA KIT antibody concentration kit, adding and dividing the antibody concentration value of the 2+ agglutination last tube and the 1+ agglutination first tube by 3 (including negative), obtaining an anti-E weak positive quality control antibody concentration value, dividing the anti-E stock solution antibody concentration value by the weak positive antibody concentration value, and diluting multiple.

C. the blood donor A serum and the erythrocyte preservation solution are mixed according to the volume ratio of 1:1, and then the anti-B and the anti-E are added according to the proportion.

③ 1Ml packed red blood cells were taken and 2ml plasma was added.

Wherein, the tube 2 whole blood quality control product is prepared by the following method:

(1) The method comprises the steps of selecting B-type RhD positive erythrocytes within 10 days of healthy blood donors, directly resisting human globulin and testing negative, screening B-type serum within 10 days of healthy blood donors with irregular antibodies, and carrying out monoclonal anti-A with the titer of 256-512 IgM, wherein the agglutination strength is more than or equal to 3+ and the highest antibody dilution is more than or equal to 1:8.

(2) Preparing a quality control product:

① Centrifuging the erythrocytes, discarding the supernatant to obtain packed erythrocytes, adding 6 times of physiological saline to wash the supernatant to be clear, and finally discarding the supernatant for standby.

② Preparing plasma:

a. Diluting an anti-A monoclonal antibody with IgM property by using a normal B type serum multiple ratio, performing titer measurement, selecting a last tube with the agglutination intensity of more than or equal to 3+ by a test tube method and a last tube with the agglutination intensity of more than or equal to 2+ by a test tube method, measuring the specific antibody content of an anti-A stock solution, the last tube with the agglutination intensity of more than or equal to 3+ and the last tube with the agglutination intensity of more than or equal to 2+ by using a Human IGM ELISA KIT antibody concentration kit, adding and dividing the antibody concentration value of the last tube with the agglutination intensity of more than or equal to 3+ and the last tube with the agglutination intensity of more than or equal to 2+ by 2 to obtain an anti-A antibody content value with the agglutination intensity of more than or equal to 2+ by a reverse determination requirement, and dividing the anti-A stock solution antibody concentration value by the reverse determination requirement agglutination intensity of more than or equal to 2+ value to obtain dilution.

B. The blood donor B type serum and the erythrocyte preservation solution are mixed according to the volume ratio of 1:1, and then the anti-A is added according to the proportion.

③ 1Ml packed red blood cells were taken and 2ml plasma was added.

Wherein, the tube 3 whole blood quality control product is prepared by the following method:

(1) The method comprises the steps of selecting O-type RhD negative erythrocytes within 10 days of healthy blood donors, directly resisting human globulin and testing negative, screening O-type serum within 10 days of healthy blood donors with irregular antibodies, resisting monoclonal antibodies-D with titer more than or equal to 1024 IgG, resisting monoclonal antibodies-A with titer between 256 and 512 IgM with agglutination intensity more than or equal to 3+ antibody highest dilution more than or equal to 1:8, resisting monoclonal antibodies-B with titer between 256 and 512 IgM with agglutination intensity more than or equal to 3+ antibody highest dilution more than or equal to 1:8.

(2) Preparing a quality control product:

① Centrifuging the erythrocytes, discarding the supernatant to obtain packed erythrocytes, adding 6 times of physiological saline to wash the supernatant to be clear, and finally discarding the supernatant for standby.

② Preparing plasma:

a. Diluting an anti-A monoclonal antibody with IgM property and an anti-B monoclonal antibody with normal O-type serum multiple ratio, performing titer measurement, selecting a last tube with the agglutination strength of more than or equal to 3+ by a test tube method and a last tube with the agglutination strength of more than or equal to 2+ by a test tube method, measuring the specific antibody content of an anti-A stock solution, an anti-B stock solution, a last tube with the agglutination strength of more than or equal to 3+ and a last tube with the agglutination strength of more than or equal to 2+ by a Human IGM ELISA KIT antibody concentration kit, adding the antibody concentration values of the last tube with the agglutination strength of more than or equal to 3+ and the last tube with the agglutination strength of more than or equal to 2+ to 2, dividing the antibody concentration values of the anti-A and anti-B stock solution by the anti-A and anti-B required agglutination strength of more than or equal to 2+ to obtain dilution factors;

b. Quantitatively determining the anti-D monoclonal reference WHO standard substance IgG anti-D with the IgG property, wherein the final value is more than or equal to 1IU/ml, and dividing the quantitative value of the anti-D stock solution by 1 is the dilution;

c. mixing blood donor O-type serum and erythrocyte preservation solution in a volume ratio of 1:1, and adding an anti-A, an anti-B and an anti-D according to the proportion;

③ 1ml packed red blood cells were taken and 2ml plasma was added.

Wherein, the tube 4 whole blood quality control product is prepared by the following method:

(1) The method comprises the steps of selecting O-type RhD positive erythrocytes within 10 days of healthy blood donors, directly performing anti-human globulin experiment to obtain negative results, performing screening to obtain negative results of O-type serum and irregular antibodies within 10 days of healthy blood donors, wherein the titer of monoclonal anti-A with IgM properties is more than or equal to 3+ antibodies with the highest dilution of more than or equal to 1:8, and the titer of monoclonal anti-B with IgM properties is more than or equal to 256-512 with the highest dilution of more than or equal to 3+ antibodies with the titer of more than or equal to 1:8.

(2) Preparing a quality control product:

① Centrifuging the erythrocytes, discarding the supernatant to obtain packed erythrocytes, adding 6 times of physiological saline to wash the supernatant to be clear, and finally discarding the supernatant for standby.

② Preparing plasma:

a. Diluting an anti-A monoclonal antibody with IgM property and an anti-B monoclonal antibody with normal O-type serum multiple ratio, performing titer measurement, selecting the last tube with the agglutination intensity of more than or equal to 3+ by a test tube method and the last tube with the agglutination intensity of more than or equal to 2+ by a test tube method, measuring the specific antibody content of an anti-A stock solution, an anti-B stock solution, the last tube with the agglutination intensity of more than or equal to 3+ and the last tube with the agglutination intensity of more than or equal to 2+ by a Human IGM ELISA KIT antibody concentration kit, adding the antibody concentration values of the last tube with the agglutination intensity of more than or equal to 3+ and the last tube with the agglutination intensity of more than or equal to 2+ to 2, dividing the antibody concentration values of the anti-A and anti-B stock solution by the anti-A and anti-B stock solution required agglutination intensity of more than or equal to 2+ to obtain dilution factors.

B. Mixing blood donor O-type serum and erythrocyte preservation solution according to the volume ratio of 1:1, and adding an anti-A and an anti-B according to the proportion;

③ 1ml packed red blood cells were taken and 2ml plasma was added.

Wherein, the tube 5 whole blood quality control product is prepared by the following method:

(1) The method comprises the steps of selecting O-type RhD positive erythrocytes within 10 days of healthy blood donors, directly performing anti-human globulin experiment to obtain negative results, performing screening to obtain negative results of O-type serum and irregular antibodies within 10 days of healthy blood donors, wherein the titer of monoclonal anti-A with IgM properties is more than or equal to 3+ antibodies with the highest dilution of more than or equal to 1:8, and the titer of monoclonal anti-B with IgM properties is more than or equal to 256-512 with the highest dilution of more than or equal to 3+ antibodies with the titer of more than or equal to 1:8.

(2) Preparing a quality control product:

① Centrifuging the erythrocytes, discarding the supernatant to obtain packed erythrocytes, adding 6 times of physiological saline to wash the supernatant to be clear, and finally discarding the supernatant for standby.

② Preparing plasma:

a. Diluting an anti-A monoclonal antibody with IgM property and an anti-B monoclonal antibody with normal O-type serum multiple ratio, performing titer measurement, selecting the last tube with the agglutination intensity of more than or equal to 3+ by a test tube method and the last tube with the agglutination intensity of more than or equal to 2+ by a test tube method, measuring the specific antibody content of an anti-A stock solution, an anti-B stock solution, the last tube with the agglutination intensity of more than or equal to 3+ and the last tube with the agglutination intensity of more than or equal to 2+ by a Human IGM ELISA KIT antibody concentration kit, adding the antibody concentration values of the last tube with the agglutination intensity of more than or equal to 3+ and the last tube with the agglutination intensity of more than or equal to 2+ to 2, dividing the antibody concentration values of the anti-A and anti-B stock solution by the anti-A and anti-B stock solution required agglutination intensity of more than or equal to 2+ to obtain dilution factors.

B. Mixing blood donor O-type serum and erythrocyte preservation solution according to the volume ratio of 1:1, and adding an anti-A and an anti-B according to the proportion;

③ 1ml packed red blood cells were taken and 2ml plasma was added.

Wherein, the tube 6 whole blood quality control product is prepared by the following method:

(1) The raw materials are selected from O-type RhD positive red blood cells within 10 days of healthy blood donors, direct anti-human globulin experiment is negative, O-type serum within 10 days of healthy blood donors is negative, irregular antibody screening is negative, and the titer of monoclonal anti-D with IgG property is more than or equal to 1024.

(2) Preparing a quality control product:

① Centrifuging erythrocytes, discarding supernatant to obtain packed erythrocytes, adding 6 times of physiological saline to wash the supernatant to clarify, discarding the supernatant for standby, diluting the anti-D with AB type human serum multiple ratio, generating a final tube with the agglutination strength of 1+ by a test tube method as the dilution multiple of the anti-D, adding O type RhD positive erythrocytes into the diluted anti-D according to the volume ratio of 1:10, incubating for 30min at 37 ℃, taking out, washing with physiological saline for 3-4 times, and finally removing the supernatant to obtain the packed erythrocytes for standby.

② Plasma preparation, namely mixing blood donor O-type serum and red blood cell preservation solution in a volume ratio of 1:1.

③ 1Ml packed red blood cells were taken and 2ml plasma was added.

Wherein, the tube 7 whole blood quality control product is prepared by the following method:

(a) The raw materials are selected from O-type RhD positive red blood cells within 10 days of healthy blood donors, direct anti-human globulin test negative, O-type serum within 10 days of healthy blood donors and irregular antibody screening negative.

(B) Preparing a quality control product:

① The method comprises the steps of centrifuging erythrocytes, discarding supernatant to obtain packed erythrocytes, adding 6 times of physiological saline to clean the packed erythrocytes, finally discarding the supernatant for later use, weighing 23.8ml of sucrose sensitization solution, placing the packed erythrocytes into a container with a proper temperature, pre-cooling the packed erythrocytes in an ice bath for 0-1 ℃, adding 1.2ml of O-type RhD positive erythrocytes into the sensitization solution, immediately adding 0.1ml of MgCl ₂ solution, uniformly mixing the packed erythrocytes, washing the packed erythrocytes with the physiological saline for 4 times for 1 hour in the ice bath, weighing 0.1mol/L of phosphate buffer solution and 1% wt/vol trypsin solution, preparing cell transformation solution according to the proportion of 9:1, weighing the C3b sensitization cell compression solution and the cell transformation solution, uniformly mixing the packed erythrocytes with the physiological saline for 30min at the temperature of 37 ℃, taking out the packed erythrocytes for 3-4 times, and discarding the packed erythrocytes for later use.

② Plasma preparation, namely mixing blood donor O-type serum and red blood cell preservation solution in a volume ratio of 1:1.

③ 1Ml packed red blood cells were taken and 2ml plasma was added.

Example 2

Application of Whole blood quality control kit in example 1

Quality control for blood transfusion compatibility testing is a standardized substance used to ensure the accuracy and reliability of the test system. The application model of the quality control product for blood transfusion compatibility detection generally comprises the following steps:

Blood group quality control, a sample containing known A, B, O and AB blood groups and Rh positive and Rh negative, was used to verify the accuracy in blood group testing.

Irregular antibody screening quality control, a specimen comprising known antibodies, is used to detect whether antibodies that may be present in a recipient can be accurately identified to prevent transfusion reactions.

The cross matching quality control product comprises known antibodies and red blood cell samples, and is used for verifying the accuracy in the cross matching test and ensuring that the incompatibility among different blood types is correctly identified.

ABO, rh blood typing and Rh typing (microcolumn gel method) experiments were as follows:

irregular antibody screening and rare blood typing experiments were as follows:

the experimental combination and the test result of the cross-matching quality control (microcolumn gel method) are as follows:

the direct resistance typing experiment is as follows:

In addition to the above embodiments, the present invention also includes other embodiments, and all technical solutions that are formed by equivalent transformation or equivalent substitution should fall within the protection scope of the claims of the present invention.

Claims (8)

1. A whole blood quality control kit for automated testing of blood transfusion compatibility, characterized in that the whole blood quality control kit includes the following seven whole blood quality control products: Tube 1: Type A RhD+C+e+c-E-, plasma contains serum from a healthy blood donor of type A, anti-B monoclonal antibody IgG-type anti-E monoclonal antibody, and red blood cell preservation fluid; Tube 2: Type B RhD+c+E+C-e-, plasma contains serum from a healthy blood donor of type B, anti-A monoclonal antibody, and red blood cell preservation solution; Tube 3: O type RhD-C-E-c+e+, plasma contains O type healthy blood donor serum, anti-A monoclonal antibody, anti-B monoclonal antibody, IgG anti-D monoclonal antibody, and red blood cell preservation fluid; Tube 4: O type RhD+C+e+c-E-, plasma contains O type healthy blood donor serum, anti-A monoclonal antibody, anti-B monoclonal antibody, and red blood cell preservation fluid; Tube 5: O type RhD+c+E+C-e-, plasma contains O type healthy blood donor serum, anti-A monoclonal antibody, anti-B monoclonal antibody, and red blood cell preservation solution; Tube 6: Type O RhD+, IgG-sensitized cells, plasma contains serum from type O healthy blood donors and red blood cell preservation solution; Tube 7: O type RhD+, C ₃ d sensitized cells, plasma contains serum from O type healthy blood donors and red blood cell preservation fluid. 2. The whole blood quality control kit for automated blood transfusion compatibility testing according to claim 1, wherein the whole blood quality control product in tube 1 is prepared by the following method: (1) Selection of raw materials: Select healthy blood donors with RhD-positive red blood cells within 10 days and negative direct antiglobulin test results; healthy blood donors with type A serum within 10 days and negative irregular antibody screening results; IgG monoclonal anti-E with a titer ≥32; IgM monoclonal anti-B with a titer between 256 and 512 and an agglutination strength ≥3, and a maximum antibody dilution ≥1:8. (2) Preparation of quality control products: ① Cells: Centrifuge the red blood cells and discard the supernatant to obtain packed red blood cells. Add 6 times the volume of normal saline to wash until the supernatant is clear. Finally, discard the supernatant for later use. ② Preparation of plasma: a. The anti-B monoclonal antibody of IgM nature was diluted with normal type A serum in multiples for titer determination. The last tube with in vitro agglutination strength ≥3+ and the last tube with in vitro agglutination strength ≥2+ were selected. The specific antibody content of the anti-B stock solution, the last tube with agglutination strength ≥3+ and the last tube with agglutination strength ≥2+ were determined by the Human IgM ELISA Kit antibody concentration kit. The antibody concentration values of the last tube with agglutination strength ≥3+ and the last tube with agglutination strength ≥2+ were added and divided by 2 to obtain the anti-B antibody concentration value with the required agglutination strength ≥2+. The anti-B stock solution antibody concentration value was then divided by the required agglutination strength ≥2+ value to obtain the dilution multiple. b. Dilute the IgG anti-E monoclonal antibody serially with normal type A serum for titration. Select the last tube with 2+ agglutination and the first tube with 1+ agglutination by the test tube method. Determine the specific antibody content of the anti-E stock solution, the last tube with 2+ agglutination, and the first tube with 1+ agglutination using the Human IgM ELISA Kit. Add the antibody concentration values of the last tube with 2+ agglutination and the first tube with 1+ agglutination and divide by 3 (including negative values) to determine the anti-E weak-positive quality control antibody concentration. Then, divide the anti-E stock solution antibody concentration by the weak-positive antibody concentration value to determine the dilution factor. c. Mix the donor's type A serum and red blood cell preservation solution in a volume ratio of 1:1, and then add anti-B and anti-E according to the above ratios; ③Take 1ml of packed red blood cells and add 2ml of plasma. 3. The whole blood quality control kit for automated blood transfusion compatibility testing according to claim 1, wherein the whole blood quality control product in tube 2 is prepared by the following method: (1) Selection of raw materials: Select B-type RhD-positive red blood cells from healthy blood donors within 10 days, with negative direct anti-human globulin test; B-type serum from healthy blood donors within 10 days, with negative irregular antibody screening; monoclonal anti-A with IgM titer between 256 and 512, and agglutination strength ≥3+ antibody maximum dilution ≥1:8; (2) Preparation of quality control products: ① Cells: Centrifuge the red blood cells and discard the supernatant to obtain packed red blood cells. Add 6 times the volume of normal saline to wash until the supernatant is clear. Finally, discard the supernatant for later use. ② Preparation of plasma: a. The anti-A monoclonal antibody of IgM nature was diluted with normal type B serum in multiples for titer determination. The last tube with in vitro agglutination strength ≥3+ and the last tube with in vitro agglutination strength ≥2+ were selected. The specific antibody content of the anti-A stock solution, the last tube with agglutination strength ≥3+ and the last tube with agglutination strength ≥2+ were determined by the Human IgM ELISA Kit antibody concentration kit. The antibody concentration values of the last tube with agglutination strength ≥3+ and the last tube with agglutination strength ≥2+ were added and divided by 2 to obtain the anti-A antibody concentration value with the required agglutination strength ≥2+. The anti-A stock solution antibody concentration value was then divided by the required agglutination strength ≥2+ value to obtain the dilution multiple. b. Mix the donor's type B serum and red blood cell preservation solution in a volume ratio of 1:1, and then add anti-A according to the above ratio; ③Take 1ml of packed red blood cells and add 2ml of plasma. 4. The whole blood quality control kit for automated blood transfusion compatibility testing according to claim 1, wherein the whole blood quality control product in tube 3 is prepared by the following method: (1) Selection of raw materials: Select O-type RhD-negative red blood cells from healthy blood donors within 10 days, with negative direct anti-human globulin test; O-type serum from healthy blood donors within 10 days, with negative irregular antibody screening; IgG monoclonal anti-D with a titer ≥1024; IgM monoclonal anti-A with a titer between 256 and 512, and agglutination strength ≥3+, and the highest antibody dilution ≥1:8; IgM monoclonal anti-B with a titer between 256 and 512, and agglutination strength ≥3+, and the highest antibody dilution ≥1:8; (2) Preparation of quality control products: ① Cells: Centrifuge the red blood cells and discard the supernatant to obtain packed red blood cells. Add 6 times the volume of normal saline to wash until the supernatant is clear. Finally, discard the supernatant for later use. ② Preparation of plasma: a. The anti-A monoclonal antibody of IgM nature and the anti-B monoclonal antibody of IgM nature were diluted with normal O-type serum for titer determination. The last tube with in vitro agglutination strength ≥3+ and the last tube with in vitro agglutination strength ≥2+ were selected. The specific antibody content of the anti-A stock solution, anti-B stock solution, the last tube with agglutination strength ≥3+ and the last tube with agglutination strength ≥2+ was determined by the Human IgM ELISA Kit antibody concentration kit. The antibody concentration values of the last tube with agglutination strength ≥3+ and the last tube with agglutination strength ≥2+ were added and divided by 2 to obtain the anti-A and anti-B antibody concentration values with agglutination strength ≥2+, and then the anti-A and anti-B stock solution antibody concentration values were divided by the anti-agglutination strength ≥2+ value to obtain the dilution multiple. b. Quantitative determination of IgG anti-D monoclonal antibody was performed according to the WHO standard IgG anti-D assay. The final value should be ≥ 1 IU/ml. The dilution factor is the quantitative value of the anti-D stock solution divided by 1. c. Mix the donor's type O serum and red blood cell preservation solution in a volume ratio of 1:1, and then add anti-A, anti-B, and anti-D according to the above proportions; ③Take 1ml of packed red blood cells and add 2ml of plasma. 5. The whole blood quality control kit for automated blood transfusion compatibility testing according to claim 1, wherein the whole blood quality control product in tube 4 is prepared by the following method: (1) Selection of raw materials: Select O-type RhD-positive red blood cells from healthy blood donors within 10 days, with negative direct anti-human globulin test; O-type serum from healthy blood donors within 10 days, with negative irregular antibody screening; monoclonal anti-A of IgM nature with a titer between 256 and 512, and agglutination strength ≥3+, and the highest antibody dilution ≥1:8; monoclonal anti-B of IgM nature with a titer between 256 and 512, and agglutination strength ≥3+, and the highest antibody dilution ≥1:8; (2) Preparation of quality control products: ① Cells: Centrifuge the red blood cells and discard the supernatant to obtain packed red blood cells. Add 6 times the volume of normal saline to wash until the supernatant is clear. Finally, discard the supernatant for later use. ② Preparation of plasma: a. The anti-A monoclonal antibody of IgM nature and the anti-B monoclonal antibody of IgM nature were diluted with normal O-type serum for titer determination. The last tube with in vitro agglutination strength ≥3+ and the last tube with in vitro agglutination strength ≥2+ were selected. The specific antibody content of the anti-A stock solution, anti-B stock solution, the last tube with agglutination strength ≥3+ and the last tube with agglutination strength ≥2+ was determined by the Human IgM ELISA Kit antibody concentration kit. The antibody concentration values of the last tube with agglutination strength ≥3+ and the last tube with agglutination strength ≥2+ were added and divided by 2 to obtain the anti-A and anti-B antibody concentration values with agglutination strength ≥2+, and then the anti-A and anti-B stock solution antibody concentration values were divided by the anti-agglutination strength ≥2+ value to obtain the dilution multiple. b. Mix the blood donor's type O serum and red blood cell preservation solution in a volume ratio of 1:1, and then add anti-A and anti-B according to the above ratio; ③Take 1ml of packed red blood cells and add 2ml of plasma. 6. The whole blood quality control kit for automated blood transfusion compatibility testing according to claim 1, wherein the whole blood quality control product in tube 5 is prepared by the following method: (1) Selection of raw materials: Select O-type RhD-positive red blood cells from healthy blood donors within 10 days, with negative direct anti-human globulin test; O-type serum from healthy blood donors within 10 days, with negative irregular antibody screening; monoclonal anti-A of IgM nature with a titer between 256 and 512, and agglutination strength ≥3+, and the highest antibody dilution ≥1:8; monoclonal anti-B of IgM nature with a titer between 256 and 512, and agglutination strength ≥3+, and the highest antibody dilution ≥1:8; (2) Preparation of quality control products: ① Cells: Centrifuge the red blood cells and discard the supernatant to obtain packed red blood cells. Add 6 times the volume of normal saline to wash until the supernatant is clear. Finally, discard the supernatant for later use. ② Preparation of plasma: a. The anti-A monoclonal antibody of IgM nature and the anti-B monoclonal antibody of IgM nature were diluted with normal O-type serum for titer determination. The last tube with in vitro agglutination strength ≥3+ and the last tube with in vitro agglutination strength ≥2+ were selected. The specific antibody content of the anti-A stock solution, anti-B stock solution, the last tube with agglutination strength ≥3+ and the last tube with agglutination strength ≥2+ was determined by the Human IgM ELISA Kit antibody concentration kit. The antibody concentration values of the last tube with agglutination strength ≥3+ and the last tube with agglutination strength ≥2+ were added and divided by 2 to obtain the anti-A and anti-B antibody concentration values with agglutination strength ≥2+, and then the anti-A and anti-B stock solution antibody concentration values were divided by the anti-agglutination strength ≥2+ value to obtain the dilution multiple. b. Mix the blood donor's type O serum and red blood cell preservation solution in a volume ratio of 1:1, and then add anti-A and anti-B according to the above ratio; ③Take 1ml of packed red blood cells and add 2ml of plasma. 7. The whole blood quality control kit for automated blood transfusion compatibility testing according to claim 1, wherein the whole blood quality control product in tube 6 is prepared by the following method: (1) Selection of raw materials: Select O-type RhD-positive red blood cells from healthy blood donors within 10 days, with negative direct anti-human globulin test; O-type serum from healthy blood donors within 10 days, with negative irregular antibody screening; monoclonal anti-D with IgG properties and titer ≥1024; (2) Preparation of quality control products: ① Cells: Centrifuge red blood cells and discard the supernatant to obtain a packed red blood cell. Add 6 volumes of normal saline to wash until the supernatant is clear. Finally, discard the supernatant and set aside. Dilute anti-D with AB type human serum in multiples. The last tube that produces an agglutination strength of 1+ by the test tube method is the dilution multiple of anti-D. Take type O RhD positive red blood cells and add them to the diluted anti-D at a volume ratio of 1:10. Incubate at 37°C for 30 minutes, remove, and wash with normal saline 3-4 times. Finally, remove the supernatant to obtain a packed cell and set aside. ② Preparation of plasma: Mix the blood donor's O-type serum and red blood cell preservation solution in a volume ratio of 1:1; ③Take 1ml of packed red blood cells and add 2ml of plasma. 8. The whole blood quality control kit for automated blood transfusion compatibility testing according to claim 1, wherein the whole blood quality control product in tube 7 is prepared by the following method: (a) Raw material selection: O-type RhD-positive red blood cells from healthy blood donors within 10 days, with negative direct anti-human globulin test results; O-type serum from healthy blood donors within 10 days, with negative irregular antibody screening results; (b) Preparation of quality control products: ① Cells: Centrifuge red blood cells and discard the supernatant to obtain packed red blood cells. Add 6 volumes of normal saline to wash until the supernatant is clear, and finally discard the supernatant for use. Measure 23.8 ml of sucrose sensitization solution and place it in a suitable container. Pre-cool it in an ice bath at 0-1°C. Add 1.2 ml of type O RhD-positive red blood cells to the sensitization solution and immediately add 0.1 ml of _MgCl2 solution. Mix well, incubate on ice for 1 hour, and wash four times with normal saline to obtain C3b-sensitized packed red blood cells. Then, measure 0.1 mol/L phosphate buffer and 1% wt/vol trypsin solution in a ratio of 9:1 to prepare cell transformation solution. Then, measure the C3b-sensitized packed cell volume and cell transformation solution, mix them evenly in a ratio of 1:2, incubate in a 37°C water bath for 30 minutes, remove the solution, wash it 3-4 times with normal saline, discard the supernatant packed cell volume, and set aside. ② Preparation of plasma: Mix the blood donor's O-type serum and red blood cell preservation solution in a volume ratio of 1:1; ③Take 1ml of packed red blood cells and add 2ml of plasma.