Method for solving difficult blood matching problem caused by irregular antibody

Abstract

The invention discloses a method for solving the difficult blood matching problem caused by irregular antibodies, and belongs to the technical field of medicine. The invention aims to provide a method for solving the difficult blood matching problem caused by irregular antibodies. According to the method, pore plate culture of mononuclear cells and sensitized red blood cells in a mononuclear cell monolayer culture experiment (MMA) is carried out in an incubation cavity on a gel column in a micro-column gel card prepared from a neutral gel reagent, after centrifugation, the situation that mononuclear-macrophages adhere to and swallow the sensitized red blood cells can be intuitively observed on the gel column, and the situation that the mononuclear-macrophages adhere to and swallow the sensitized red blood cells can be intuitively observed on the gel column. Therefore, the experimental process of the traditional MMA is simplified and optimized, the experimental time is shortened, the error of the result caused by counting under an artificial microscope is reduced, the visualization of the result interpretation is realized, and the accuracy and repeatability of the experimental result are improved. And a relatively simple, convenient, rapid and visual blood matching technology is provided for clinical difficult blood matching.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and particularly to a method for solving the problem of difficult blood matching caused by irregular antibodies. Specifically, the incubation of monocytes and sensitized red blood cells in a well plate of MMA is changed to be carried out in the incubation cavity on the gel column of a microcolumn gel card, which can not only simplify the cell culture process to complete the phagocytosis experiment of monocytes-macrophages on sensitized red blood cells, but also directly observe the adhesion and phagocytosis of monocytes-macrophages on sensitized red blood cells and the degree thereof on the gel column. Background Art

[0002] For acute severe anemia caused by various reasons, chronic anemia that is ineffective or has poor effects with drug treatment and the patient cannot tolerate, and invasive operations such as surgery, etc., it may be necessary to transfuse red blood cell components to correct anemia.

[0003] In order to avoid hemolytic reactions caused by different red blood cell blood group antigens between donors and patients, before blood transfusion, four blood transfusion compatibility tests, namely ABO blood group identification of donors and patients, RhD antigen detection, irregular antibody screening test (i.e., unexpected antibody screening test. If the experimental result is agglutination positive, it indicates that there may be alloantibodies or autoantibodies, and further antibody identification is required to clarify the presence or absence of antibody specificity), and cross-matching (including the main-side cross-matching agglutination reaction of the patient's serum and the donor's red blood cells, and the sub-side cross-matching agglutination reaction of the patient's red blood cells and the donor's serum. A negative main-side cross-matching agglutination indicates that the donor's red blood cells are compatible with the recipient, and the donor's red blood cells can be transfused to the recipient), are important prerequisites and guarantees for safe and effective blood transfusion. Among them, ABO blood group identification, RhD detection, and unexpected antibody screening of patients are basic tests to ensure the maximum compatibility of the blood of donors and recipients; while cross-matching is the final guarantee experiment to ensure the compatibility of the blood of donors / recipients.

[0004] In some cases, even when the blood types of the donor and the recipient are the same, the presence of red blood cell alloantibodies (antibodies against antigens lacking in one's own red blood cells, mostly IgG antibodies produced due to blood transfusion, pregnancy, transplantation, use of immunogenic substances, etc.) and autoantibodies (antibodies against one's own red blood cell blood group antigens. Some autoantibodies have specific reactivity, i.e., they show positive agglutination reactions with red blood cells positive for certain blood group antigens; some have no specificity and show agglutination reactions with both one's own red blood cells and other red blood cells) will cause the main side of the cross-match to be incompatible and thus blood transfusion cannot be carried out. If a patient has an alloantibody against a high-frequency antigen (a blood group antigen carried by more than 99% of the population), even if the blood type specificity can be identified, it is difficult to find antigen-negative donors because the vast majority of the population has the corresponding positive antigen; if the specificity of the alloantibody is difficult to clarify, it is also impossible to transfuse antigen-negative blood corresponding to it; if a patient has autoantibodies, the antibodies will agglutinate with both one's own and other donors' red blood cells, and it is also difficult to select suitable blood for transfusion; if a patient has a complex antibody (multiple alloantibodies coexist in the patient's serum, and it is generally difficult to identify the specificity of each antibody; or autoantibodies and alloantibodies coexist, and the autoantibodies often mask the specificity of the alloantibodies and make it difficult to identify), most of the main-side cross-matches between the donors and the patient show agglutination, and it is difficult to find a suitable donor for transfusion.

[0005] In summary, when there are alloantibodies against high-frequency antigens, alloantibodies with unclear antigen specificity, autoantibodies, complex alloantibodies against multiple antigens, or the coexistence of autoantibodies and alloantigen in the patient's blood, the cross-match between the recipient and ABO / RhD-compatible donor blood is generally incompatible, which is the so-called "difficult blood matching", and thus it is impossible to provide the patient with suitable blood for transfusion in a timely manner. "Difficult blood matching" has always been one of the blood transfusion problems that trouble the staff in the blood transfusion department and clinicians.

[0006] The current solutions for difficult blood cross-matching include the following: (1) For difficult blood cross-matching caused by alloantibodies against high-frequency antigens or composite alloantibodies against multiple antigens, the main approach is to screen for blood donors negative for the specific antigens corresponding to the alloantibodies and perform cross-matching; (2) For difficult blood cross-matching caused by autoantibodies, the main approach is to use autologous red blood cells or allogeneic red blood cells absorption method to absorb the autoantibodies and then perform major cross-matching, or use autologous serum dilution method to serially dilute the patient's serum until the autoantibodies disappear and then perform blood cross-matching; (3) When the specificity of alloantibodies cannot be identified or autoantibodies are present simultaneously, difficult blood cross-matching cannot be performed by the aforementioned methods. Cross-matching is performed by screening for blood donors with phenotypes consistent with the patient's multiple blood group antigen systems (ABO, RhD / C / c / E / e, Kell, Kidd, Duffy, etc.) (or with fewer blood group antigens than the patient's above antigen systems). Generally, the method of randomly cross-matching with blood donors of the same ABO / RhD blood type as the patient is used to screen for "the least incompatible cross-matching" blood donors; (4) For difficult blood cross-matching caused by the aforementioned multiple antibodies, individual foreign laboratories use the monocyte monolayer assay (MMA) to screen for blood donors with a percentage of monocyte-macrophage adhesion and phagocytosis of the patient's sensitized red blood cells <5%. The several methods mentioned above for solving difficult blood cross-matching through serological related techniques are complex, time-consuming, and there is a possibility of missing alloantibodies or autoantibodies with alloantibody-like specificity, thus causing post-transfusion hemolysis; while the MMA cross-matching method is also complex and time-consuming, and requires manual counting of experimental results under a microscope, thus there are problems with the timeliness, repeatability, and accuracy of the completion of the experiment.

[0007] At present, serological cross-matching methods include the polybrene medium test tube method, the indirect anti-human globulin medium test tube method, the indirect anti-human globulin medium microcolumn gel method, etc. These methods all have disadvantages such as false negatives and false positives. In addition, in vitro serological agglutination experiments cannot reflect the hemolytic and phagocytic functions of complement and monocytes on red blood cells in vivo. Therefore, even if the in vitro agglutination experiment shows very weak agglutination, the degree of hemolysis or red blood cell destruction in vivo may be different. Therefore, in difficult cross-matching, even if the "least incompatible cross-match" (i.e., the weakest cross-match agglutination) blood supply is selected through the above-mentioned several serological cross-matching methods, acute or delayed hemolysis may occur, aggravating the condition and resulting in ineffective or poor blood transfusion effects. As a compensatory measure, MMA reported in the existing literature (hereinafter referred to as traditional MMA) is used for cross-matching, which has good effects in reflecting the adhesion and phagocytosis of antibody-sensitized red blood cells by monocytes-macrophages in vivo. However, the disadvantage of this experiment is that the experiment process takes a long time, at least 12 - 24 hours, and the timeliness for emergency blood transfusion is poor; at the same time, it is more complex in operation than serological experiments, with many interfering factors; it is necessary to count the phagocytosis rate of monocytes-macrophages on sensitized red blood cells under the microscope, and the result observation has poor intuitiveness and repeatability, which limits its routine clinical application. Summary of the Invention

[0008] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a method for solving the difficult cross-matching problems caused by irregular antibodies. The well plate culture of monocytes and sensitized red blood cells in traditional MMA is changed to incubation in the incubation cavity on the gel column of a microcolumn gel card. After centrifugation, it can be very intuitively observed on the gel column the adhesion and phagocytosis of monocytes-macrophages to sensitized red blood cells, thereby simplifying and optimizing the experimental process of traditional MMA, shortening the experimental time, and reducing the error caused by manual counting of experimental results, so as to realize the visualization of result judgment, improve the accuracy and repeatability, and provide a relatively simple, fast and intuitive cross-matching technique for clinical difficult cross-matching.

[0009] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions.

[0010] The present invention discloses a method for solving the difficult cross-matching problems caused by irregular antibodies, which is characterized by including the following steps: Cultivate monocytes and sensitized red blood cells in the incubation cavity on the gel column of a microcolumn gel card; after centrifugation, the free sensitized red blood cells that are not adhered and phagocytosed by monocytes-macrophages pass through the micropores in the gel column and are deposited at the bottom of the gel column, while the adhered and phagocytosed sensitized red blood cells and monocytes-macrophages aggregate at the upper end of the gel column. Visually observe with the naked eye or through image analysis the adhesion and phagocytosis of monocytes-macrophages on the gel column to sensitized red blood cells; Select the suitable blood supply for transfusion according to the phagocytosis situation.

[0011] Furthermore, the gel column in the microcolumn gel card is prepared from a neutral gel reagent. The microcolumn cavity and the gel do not contain specific antibodies and anti-human globulin, and its structure is a microporous structure crosslinked by dextran gel, which can provide support for the growth and adhesion of monocytes-macrophages.

[0012] Furthermore, the isolation and culture of the monocytes include: Isolating monocytes from peripheral blood using density gradient centrifugation; Washing and resuspending the isolated monocytes in RPMI1640 medium; culturing in a 5% CO2 incubator at 37°C for 1 hour; Detecting the activity of monocytes using trypan blue staining to ensure that its activity is ≥95%.

[0013] Furthermore, the preparation of the sensitized red blood cells includes: Mixing the patient's plasma with donor red blood cells whose agglutination intensity screened by the polybrene crossmatching method is ≤2+ (2+, 1+, ±) and ≤ the agglutination intensity of the self-control, and incubating in a 37°C water bath for 1 hour to prepare sensitized red blood cells; at the same time, preparing positive control sensitized red blood cells and negative control sensitized red blood cells for in-batch experiment quality control; Detecting whether the red blood cells are sensitized by antibodies through the direct anti-human globulin test.

[0014] Furthermore, the incubation conditions of the monocytes and the sensitized red blood cells are: incubating in a 5% CO2 incubator at 37°C for 2 hours.

[0015] Furthermore, the centrifugation conditions of the microcolumn gel card are: using a gel card centrifuge, with a centrifugal force of 128.1 g and a centrifugation time of 9 minutes.

[0016] Furthermore, the result interpretation includes: Visually observing the number of red blood cells at the upper end of the gel column. The donor with the least number of red blood cells and closest to the negative control column is the most suitable for blood donation; when it is difficult to compare the number of red blood cells by visual observation, use Image J software to measure the gray value of the red blood cell aggregation part at the upper end of the gel column, and the one with the smallest gray value is the most suitable for blood donation.

[0017] Even further, the operation steps of the Image J software include: Importing the gel column image and converting it to 8-bit format; Using the "Subtract Background" function to subtract the background, and setting the rolling ball radius to 50 pixels; Selecting the red blood cell aggregation area at the upper end of the gel column and measuring its gray value; Exporting the gray value data and performing statistical analysis.

[0018] The present invention also discloses a product for solving the difficult blood matching problems caused by irregular antibodies, which is characterized by including a microcolumn gel card prepared from a neutral gel reagent, and the microcolumn cavity and the gel do not contain specific antibodies and anti-human globulin. Each gel column has a microporous structure crosslinked by dextran gel that only allows free red blood cells to pass through, and is used for the experiment in the method for solving the difficult blood matching problems caused by irregular antibodies described in any one of the above.

[0019] Further, the product includes a kit.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0021] 1. Compared with the microcolumn gel method and the traditional MMA, the improved MMA has a higher diagnostic efficiency in providing safe blood supply for patients with difficult blood matching.

[0022] 2. The improved MMA simplifies the experimental operation and shortens the experimental time compared with the traditional MMA, and is more suitable for patients with difficult blood matching who are in urgent need of blood transfusion.

[0023] 3. The improved MMA realizes the intuitive visualization of the results, reduces the result error caused by manual cell counting in the traditional MMA, and improves the accuracy and repeatability of result judgment.

[0024] 4. The transfusion effectiveness of the most suitable blood donor screened based on the improved MMA is higher than that of the microcolumn gel method and the traditional MMA.

[0025] In summary, the present invention uses a microcolumn gel card prepared from a neutral gel reagent, which does not contain specific antibodies and anti-human globulin. Each gel column has a microporous structure crosslinked by dextran gel, which can provide support for the growth and adhesion of monocytes-macrophages. The present invention changes the incubation of monocytes and sensitized red blood cells in the well plate in the MMA method to the incubation cavity on the gel column of the microcolumn gel card, which can not only simplify the cell culture process to complete the adhesion and phagocytosis experiments of monocytes-macrophages on sensitized red blood cells, but also directly observe the situation of monocytes-macrophages adhering to and phagocytosing sensitized red blood cells on the gel column, and analyze the degree of phagocytosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the interpretation standard for the results of microcolumn gel blood matching.

[0027] Figure 2 It is the microscopic observation photo image of monocytes-macrophages adhering to and phagocytosing sensitized red blood cells in the traditional MMA.

[0028] Figure 3 It is the experimental result photo image of blood matching for a certain difficult blood matching case 1 using the traditional MMA.

[0029] Figure 4The photograph image of the experimental result of using the improved MMA of the present invention for blood matching in a certain difficult blood matching case 2.

[0030] Figure 5 The photograph image of the experimental result of using the improved MMA of the present invention for blood matching in a certain difficult blood matching case 3.

[0031] Figure 6 The exported graph and gray value measurement of the experimental result of using the improved MMA of the present invention for blood matching in a certain difficult blood matching case 3.

[0032] Figure 7 The ROC curve made according to the gray value measurement of the adhered and phagocytosed red blood cells in the suitable and unsuitable blood donors screened by the improved MMA of the present invention.

[0033] Figure 8 The ROC curve made according to the gray value measurement of the adhered and phagocytosed red blood cells in the suitable and unsuitable blood donors screened by microcolumn gel, traditional MMA and improved MMA. Detailed implementation manners

[0034] The following further describes the present invention in detail with specific examples. However, it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following examples, and all technologies implemented based on the content of the present invention belong to the scope of the present invention.

[0035] Unless otherwise specified, the reagents and materials used in the present invention are all commercially available.

[0036] Example 1, the specific implementation manner of the method described in the present invention.

[0037] I. Preparation before experiment.

[0038] 1. Collection of difficult blood matching specimens: When performing irregular antibody screening and identification experiments according to the reagent instructions, if there is agglutination with multiple panel cells or general agglutination, and / or agglutination occurs in the autologous control, referring to the panel cell pattern, the number and specificity of antibodies cannot be determined; randomly take multiple blood donors with the same ABO / RH blood type as the patient, use the polybrene method or the tube indirect antiglobulin test for blood matching, and agglutination occurs in the major cross-match of each blood donor's red blood cells, and this specimen is determined to be a difficult blood matching specimen.

[0039] 2. Collection of general information: Query and record the medical history of the subject, and record gender, age, weight, blood transfusion history, pregnancy history, etc.

[0040] 3. Record of relevant pre-transfusion tests: Record the patient's red blood cell count, hemoglobin value, total bilirubin and indirect bilirubin values.

[0041] II. Preliminary screening for suitable blood donors: Perform the major crossmatch by the polybrene method (operate according to the reagent instruction manual) with blood donors having the same ABO / RH blood types as the patient (or having fewer of the above blood type antigens than the patient), and select blood donors with an agglutination strength ≤ 2+ (2+, 1+, ±) and ≤ the agglutination strength of the autologous control.

[0042] III. Further screen for the most suitable blood donor using 3 different crossmatching methods.

[0043] 1. Microcolumn gel method.

[0044] Refer to the microcolumn gel card and the instruction manual of the supporting reagents, perform the major crossmatch with the patient's serum and the red blood cells of the blood donors preliminarily screened by the polybrene crossmatching method, and select the blood donor with the weakest agglutination strength and ≤ the agglutination strength of the autologous control as the blood for transfusion.

[0045] After the surface antigens of red blood cells bind to their corresponding IgG antibodies, they continuously sediment to the bottom of the tube under the action of centrifugal force, and during the sedimentation process, they bind to the anti-human globulin in the microcolumn to form red blood cell agglutination. The gel particles have the function of a molecular sieve, which can block the agglutinated red blood cells from passing downward through the gel particles and make them suspended at the upper end of the microcolumn, which is a positive reaction; the unagglutinated free red blood cells can pass through the gel particles to reach the bottom of the microcolumn, which is a negative reaction.

[0046] Interpretation of experimental results: If the autologous control is negative for agglutination, the major crossmatch is negative for agglutination and there is no hemolysis, it indicates that the donor / recipient crossmatch is compatible and the blood donor can be transfused; if the major crossmatch is positive for agglutination or there is hemolysis, it indicates that there are irregular antibodies in the patient's serum against the red blood cell blood type antigens of the blood donor, and the donor / recipient crossmatch is incompatible, and this blood donor cannot be transfused. A positive agglutination or hemolysis in the minor crossmatch indicates that there are antibodies in the donor's serum against the red blood cell blood type antigens of the patient; or the patient's red blood cells have been sensitized by alloantibodies or autoantibodies in the patient's own serum. The specific interpretation criteria are shown in Table 1 and Figure 1 。

[0047] Table 1 Interpretation criteria for the crossmatching experiment by the microcolumn gel method 。

[0048] 2. Use the traditional MMA to screen for the most suitable blood donor.

[0049] 2.1. Isolation of monocytes: (1) Draw 10 mL of the patient's venous blood using an EDTA anticoagulant tube. If it is difficult to draw the patient's blood, or if the patient is severely anemic and it is difficult to isolate a sufficient number of monocytes, blood can also be drawn from a voluntary healthy donor with the same ABO blood type as the patient (approved by the institutional ethics committee. The blood specimen should be stored at room temperature (18 - 22°C) and the storage time should not exceed 24 hours; (2)Dilute whole blood at a ratio of 1:1 v / v with RPMI 1640 medium (supplemented with 5% fetal bovine serum) at room temperature (18 - 22°C); (3)Slowly add 10 mL of the diluted blood along one side of a 15 mL centrifuge tube containing 3 mL of density gradient separation solution, so that the blood is placed on top of the density gradient solution; (4)Centrifuge the mixture at 700 g for 30 minutes. The centrifuged mixture should be divided into 5 layers from top to bottom: plasma, buffy coat containing PBMCs, density gradient material, granulocytes, and red blood cells. Aspirate most of the plasma with a pipette, and carefully remove the buffy coat containing PBMCs to a new 15 mL sterile centrifuge tube with a sterile pipette; (5)Wash the separated PBMCs three times with phosphate - buffered saline (PBS) at pH 7.3 (centrifuge at 350 g for 10 minutes), and resuspend the PBMCs with 3 - 7 mL of RPMI 1640 medium to make the cell concentration approximately 1,750,000 cells / mL; (6)Count viable PBMCs: Mix the PBMCs suspension with trypan blue staining solution at a ratio of 1:1, stain for 3 minutes, observe under a microscope using a hemocytometer. Dead cells are blue, and live cells are colorless. Viable cell rate (%) = number of viable cells / (number of viable cells + number of dead cells) × 100%. Monocyte viability ≥ 95% can be used for subsequent experiments.

[0050] 2.2 Sensitization of red blood cells.

[0051] 2.2.1 Preparation of donor red blood cell sensitization: Thoroughly mix 600 µL of patient plasma with 200 µL of the blood donor red blood cells preliminarily screened by the aforementioned polybrene method and washed three times with normal saline (each washing centrifugal force is 350 g, 5 minutes), incubate in a 37°C water bath for 1 hour, and mix once every 15 minutes. After the water bath, centrifuge (350 g, 5 minutes), completely remove the supernatant, label it, and reserve it for the direct antiglobulin test to detect the sensitization situation; if agglutination appears in the result of this test, it indicates that the red blood cells have been sensitized by antibodies.

[0052] 2.2.2 Preparation of positive control sensitized red blood cells: 2.2.2.1 Preliminary experiment on the optimal concentration and volume of anti - D: Perform an indirect antiglobulin test on RhD - positive red blood cells with different dilution ratios and volumes of human polyclonal anti - D (if there is no human polyclonal anti - D, monoclonal anti - D can also be used), and require the immune agglutination intensity of red blood cells and anti - D to be 3+ - 4+; using the traditional MMA described below, require the phagocytosis rate of sensitized red blood cells by mononuclear macrophages to reach more than 10%. The optimal dilution ratio of human polyclonal anti - D used in this laboratory for sensitizing positive control red blood cells is 1:1, and the volume is 200 µL; 2.2.2.2 Mix 200 μL of the aforementioned human polyclonal IgG anti-D with 60 μL of packed red blood cells from an ABO-compatible, RhD-positive blood donor whose cells have been washed three times with normal saline (centrifugation at 350 g for 5 min each time). Mix well, and incubate in a 37°C water bath for 1 h, mixing every 15 min. After the water bath, centrifuge (350 g, 5 min), completely remove the supernatant, and label. The direct antiglobulin test (performed according to the reagent instructions) of the sensitized red blood cells should show a positive agglutination result.

[0053] 2.2.3 Preparation of negative control sensitized red blood cells: Mix 200 μL of AB plasma with a negative irregular antibody screening result with 200 μL of packed red blood cells from a blood donor who is ABO / RH-compatible with the patient and whose cells have been washed three times with normal saline (centrifugation at 350 g for 5 min each time). Mix well, and incubate in a 37°C water bath for 1 h, mixing every 15 min. After the water bath, centrifuge (350 g, 5 min), completely remove the supernatant, and label. The direct antiglobulin test (performed according to the reagent instructions) of the red blood cells after incubation should show a negative (-) agglutination result.

[0054] 2.2.4 Monocyte culture: Place glass slides cut to the appropriate size in each well of an 8-well culture plate. Seed 400 μL of PBMC (approximately 700,000 cells) in each well. Incubate in a 5% CO2 incubator for 1 h.

[0055] 2.2.5 Determination of macrophage phagocytosis rate: 2.2.5.1 Aspirate the culture medium supernatant from the culture dish to remove non-adherent lymphocytes. Add 1.25% v / v, 400 μL of donor sensitized red blood cells (3 wells are needed for subsequent calculation of the average phagocytosis rate), positive control sensitized red blood cells, and negative control sensitized red blood cells. If complement-dependent antibodies are suspected, 50 μL of AB-type healthy human serum can be added to each well as a fresh complement source. Incubate the culture plate at 37°C for 2 h. Remove the glass slides, add PBS with a pH of 7.3 to a 100 mL beaker, immerse the glass slides in the PBS, and slowly move the glass slides back and forth about 30 - 40 times to wash away most of the non-phagocytosed red blood cells; 2.2.5.2 Remove the glass slides from the PBS. Wipe off the excess PBS with a tissue paper, and then air-dry the slides. Stain with Wright-Giemsa. 2.2.5.3 Under a microscope (40x objective), count 200 mononuclear-macrophages and the number of cells that have adhered to and phagocytosed red blood cells among these cells. Calculate the PI for the negative control wells, positive control wells, and experimental wells (take the average PI of the 3 experimental wells for the same experimental specimen) respectively.

[0056] Percentage of monocytes-macrophages that adhere to and phagocytize red blood cells (Percentage index, PI) = number of monocytes-macrophages that adhere to and phagocytize / total number of monocytes-macrophages × 100%.

[0057] 2.2.5.4 Result interpretation: (1) Microscopic appearance of monocytes-macrophages adhering to and phagocytizing sensitized red blood cells: Specific identification rules can be found in relevant literature. Figure 2 This is the microscopic appearance of monocytes-macrophages adhering to and phagocytizing sensitized red blood cells. The stained cells are monocytes-macrophages, and the unstained, round and translucent cells pointed by the arrow are the red blood cells adhered to and phagocytized by monocytes-macrophages; (2) Positive control PI > 10%, negative control PI 0 - 3%, and the quality control of this batch of experiments is qualified (positive and negative controls can be seen under the microscope); Figure 3 (3) Experimental specimen PI < 5% (under the microscope, it can be seen that Figure 3 in the case of blood donor 1), when the patient is transfused with antigen-positive blood donors corresponding to the specific antibody, it is predicted that there is no obvious hemolytic reaction, and it is regarded as a suitable blood donor; conversely, if the experimental specimen PI > 5%, there will be an obvious hemolytic reaction, and it is regarded as an unsuitable blood donor (see Figure 3 blood donor 2, blood donor 3). The higher the PI ratio, the more obvious the hemolysis and the worse the blood transfusion effect.

[0058] 2.2.5.5 Screening criteria for the most suitable blood donor: PI < 5% and the smallest PI value (under the microscope, the result can be seen in Figure 3 blood donor 1).

[0059] 3. The present invention uses the improved MMA method to screen the most suitable blood donor.

[0060] 3.1. The specific operation methods of monocyte isolation and activity determination, initial screening of blood donor red blood cell sensitization, positive control red blood cell sensitization, and negative control red blood cell sensitization are the same as those described in the traditional MMA blood matching.

[0061] 3.2. Monocyte culture, monocytes-macrophages adhesion to and phagocytosis of sensitized red blood cells: (1) Mark the experimental well, negative control well, and positive control well on the gel column of the microcolumn gel card respectively; (2) Add 100 µL of the prepared monocytes to the incubation chambers of each well respectively; (3) Add 50 µL of the prepared 1% sensitized donor red blood cell suspension, 1% positive sensitized red blood cells, and 1% prepared negative control red blood cell suspension (the above cell suspensions are all diluted and prepared with RPMI 1640 medium) to the incubation chambers of the experimental well, positive control well, and negative control well respectively; after adding the samples, gently mix them with a pipette; ​(4) Seal the sample addition end of the microcolumn gel card with tissue culture sealing film and incubate in a 5% CO2 incubator at 37 °C for 2 hours; (5) Place the microcolumn gel card on a dedicated centrifuge for gel cards, with a centrifugal force of 128.1 g and centrifuge for 9 minutes; (6) Observe the number of red blood cells at the upper end and bottom of each negative control well column, positive control well column, and experimental well column with the naked eye, and determine whether the batch experiment quality control is qualified, and conduct a comparative analysis of the adhesion and phagocytosis of each blood donor's sensitized red blood cells.

[0062] 3.3 Quality control evaluation of this batch of experiments: The top of the positive control well column shows an obvious dense and thick red band or large mass, or a large number of red particles appear from top to bottom (especially in the upper 1 / 3 section) of the gel column, but there are no red blood cells or fewer red blood cells at the bottom of the gel column; there are no red blood cells at the upper end of the negative control well column, or the top of the column shows a thin, light-colored strip (or small mass, or granular), but a large number of red blood cells are deposited at the bottom of the gel column. The difference in the number of red blood cells at the upper end and bottom of the positive control well column and the negative control well column is significant. The quality control of this experiment is qualified, as shown in Figure 4 the positive control and negative control in. If the appearance of the upper end and bottom of the negative control well column is close to that of the positive control well column, or the appearance of the upper end and bottom of the positive control well column is close to that of the negative control well column, or the difference in the number of red blood cells at the upper end and bottom of both well columns is not significant, the quality control of this batch of experiments is unqualified.

[0063] 3.4 Screening for the most suitable blood donors: (1) Observe with the naked eye that the well column with the least number of red blood cells at the top or upper end and the most red blood cells deposited at the bottom, and the one closest to the negative control well column is the most suitable for blood donation, as shown in Figure 4 blood donation No. 1155 in; conversely, the well column with a large number of red blood cells at the top or upper end of the column, a small number or no red blood cells deposited at the bottom, and close to the positive control well column is not suitable for blood donation, as shown in Figure 5 blood donations 2 and 3 in; (2) When the number of red blood cells at the top or upper end of each experimental well column is close and it is difficult to compare the difference in quantity with the naked eye, semi-quantitative analysis of the adhesion and phagocytosis degree is required, that is, output the experimental result image of the microcolumn gel card, and use Image J software to measure the gray value of the red blood cell part at the top and upper end of each well column, and estimate the relative number of red blood cells with the gray value, as shown in Figure 5 、 Figure 6 . Among them, the blood donation with the smallest gray value, blood donation 1, is the most suitable for blood donation, as shown in Figure 6 blood donation 1.

[0064] The specific operations are as follows: First, open Image J and import the image to be analyzed. Then, select 8-bit in Image J; set the rolling ball radius = 50 pixels in Process - Subtract Background, and then check the light background; select Analyze - Set Measurements and check the parameter Set Measurements; set the Unit of length in Analyze - Set Scale to pixel; then click Edit - Invert, select the appropriate shape tool to select the target analysis area - the red band at the top of the microcolumn gel; click Analyze - Measure to obtain the IntDen value; finally, export the result to GraphPad for statistical analysis.

[0065] 3.5 Criteria for screening the most suitable blood donor by improved MMA: Visually observe that the least amount of red blood cells is at the top or upper end of the experimental well column, the largest number of red blood cells is at the bottom of the gel column, and it is the closest to the negative control in terms of visual appearance. See Figure 4 Donate blood from the one numbered 1155 in Figure 6 or the one with the smallest gray value of red blood cells at the upper end of the well column analyzed by Image J software. See

[0066] Example 2: Clinical application experiments have proven feasible.

[0067] I. Clinical characteristics of 60 patients with difficult blood matching.

[0068] 1. Case inclusion criteria: ① Age ≥ 18 years old; ② Difficult blood matching patients with internal and surgical chronic anemia or postoperative anemia who meet the following blood transfusion application indications: Internal medicine blood transfusion indications: Hb < 60 g / L or Hct < 0.20 can be transfused; Hb 70 - 100 g / L accompanied by: Poor cardiopulmonary compensatory function (coronary heart disease, ventilator, > 70 years old), increased metabolic rate (high fever, severe infection); Severe hypoxia, active gastrointestinal bleeding > 400 ml, etc. can be transfused. Surgical blood transfusion indications: Hb < 70 g / L or Hct < 0.22 can be transfused; Hb 70 - 100 g / L accompanied by: Persistent bleeding at the wound surface, DIC, poor cardiopulmonary compensatory function (coronary heart disease, ventilator, > 65 years old), increased metabolic rate (high fever, severe infection), etc. can be transfused.

[0069] 2. Case exclusion criteria: ① Patients who are undergoing autologous blood transfusion while receiving allogeneic blood transfusion; ② Active bleeding patients; ③ Infants and adolescents under 18 years old; ④ Patients applying for blood transfusion who are currently using CD38 monoclonal antibody and CD47 monoclonal antibody; ⑤ Blood transfusion before the start of surgery and blood transfusion during surgery.

[0070] Among 60 patients with difficult blood matching, from the perspective of gender, there were 34 male patients, accounting for 56.67%, and 26 female patients, accounting for 43.33%. The difference in gender among the 60 patients was not statistically significant ( P >0.05); from the perspective of age, there were 41 patients aged ≥60 years, accounting for 68.33%, and 19 patients aged <60 years, accounting for 31.67%. The difference in age among the 60 patients was not statistically significant ( P >0.05); from the perspective of disease distribution, there were 10 patients with hematological system tumors (including multiple myeloma, myelodysplastic syndrome, leukemia, etc.), accounting for 16.67%, 19 patients with autoimmune system diseases (including autoimmune diseases and systemic lupus erythematosus, etc.), accounting for 31.67%, 6 patients with digestive system diseases (including upper gastrointestinal bleeding, etc.), accounting for 10.00%, 3 patients with cardiovascular system diseases (including coronary heart disease and acute myocardial infarction, etc.), 9 patients with unexplained anemia, accounting for 15.00%, 5 patients with gynecological system diseases (including uterine fibroids and ovarian tumors, etc.), accounting for 8.33%, and 8 patients with other conditions (including renal insufficiency, dizziness, etc.), accounting for 13.33%. The difference in disease types among the 60 patients was not statistically significant ( P >0.05); there were 33 patients with a history of blood transfusion, accounting for 55.00%. The difference in blood transfusion history among the 60 patients was not statistically significant ( P >0.05), as shown in Table 2.

[0071] Table 2 Clinical characteristics of 60 patients with difficult blood matching .

[0072] Note: ^, P =0.12, #, P =0.07, *, P =0.11, $, P =0.12.

[0073] II. Modified MMA screening for suitable blood donors.

[0074] According to the gray value results of the blood matching experiments for 60 patients using the modified MMA, the ROC curve was plotted for the gray value results of the negative control and positive control using SPSS 23.0 software (see Figure 7). The optimal cut-off value was determined based on sensitivity and specificity, that is, the point with the maximum sum of sensitivity and specificity was found. The maximum sum of sensitivity and specificity was calculated to be 2, the maximum Youden index was 1, and the optimal cut-off value was 100025.29. The diagnostic threshold obtained from the ROC curve was 100025.29, that is, those with the gray value of blood-supplying red blood cell aggregation < 100025.29 were suitable for blood donation, and vice versa. The sensitivity, specificity, negative predictive value, and positive predictive value of the improved MMA test for screening suitable blood donors were all 100%, and the Youden index was 1.0. In the diagnosis of suitable blood donation, the area under the ROC curve was 0.732, and the 95% confidence interval (CI) was 0.701 - 0.723, as shown in Tables 3 and 4 specifically.

[0075] Table 3 Diagnostic results of the improved MMA for screening suitable blood donors 。

[0076] Table 4 Area under the ROC curve of the improved MMA for screening suitable blood donors 。

[0077] III. Analysis of the diagnostic efficacy of three blood matching methods: microcolumn gel method, traditional MMA, and improved MMA.

[0078] Taking the effectiveness after patient blood transfusion as the gold standard, the number of cases diagnosed as suitable for transfusion by the microcolumn gel method, traditional MMA, and improved MMA according to their respective diagnostic criteria were 42 cases, 45 cases, and 53 cases respectively. The sensitivity, specificity, and other performance of the microcolumn gel method, traditional MMA, and improved MMA were compared, as shown in Table 5.

[0079] As can be seen from Table 5: The diagnostic sensitivity of the improved MMA (95.33%) was significantly higher than that of the microcolumn gel method (86.92%) and traditional MMA (89.72%); the diagnostic specificity of the improved MMA (97.26) was significantly higher than that of the microcolumn gel method (95.89%) and traditional MMA (83.56%); the diagnostic coincidence rate of the improved MMA (96.11%) was significantly higher than that of the microcolumn gel method (85.56%) and traditional MMA (92.22%); the positive predictive value of the improved MMA (98.08%) was significantly higher than that of the microcolumn gel method (88.57%) and traditional MMA (92.22%); the negative predictive value of the improved MMA (93.42%) was significantly higher than that of the microcolumn gel method (81.33%) and traditional MMA (86.42%); the Youden index of the improved MMA (0.93) was significantly higher than that of the microcolumn gel method (0.70) and traditional MMA (0.86).

[0080] Table 5 Diagnostic efficacy of microcolumn gel method, traditional MMA, and modified MMA in screening suitable blood donors 。

[0081] IV. ROC curves of three blood matching techniques: microcolumn gel method, traditional MMA, and modified MMA

[0082] Taking the effectiveness after patient transfusion as the gold standard, ROC curves of the three blood matching techniques were drawn (see Figure 8 ). In terms of diagnosing the effectiveness after patients received suitable blood transfusions, using the respective screening criteria of the microcolumn gel method, traditional MMA, and modified MMA, the number of cases diagnosed as having suitable blood transfusions for 60 patients were 42 cases, 45 cases, and 53 cases respectively. The areas under the ROC curves were 0.818, 0.836, and 0.940 respectively, and the 95% confidence intervals (CI) were 0.751 - 0.885, 0.772 - 0.899, and 0.899 - 0.981 respectively. See Table 6 for details.

[0083] Table 6 Areas under the ROC curves of three blood matching techniques: microcolumn gel method, traditional MMA, and modified MMA 。

[0084] V. Time costs of random blood matching using the microcolumn gel method, traditional MMA, and modified MMA for 60 patients with difficult blood matching

[0085] When using the microcolumn gel cross - matching method to screen for suitable blood donors, according to the results of unexpected antibody screening and antibody identification, experiments such as autologous red blood cell absorption, allogeneic red blood cell absorption, or autologous serum dilution should be carried out as appropriate to exclude the interference of autoantibodies, and then blood matching should be performed with blood donors that are ABO - compatible (or type O) with the patient, or RhD (or C / c / E / e) - compatible (or with fewer antigen specificities than the patient), or antigen - matched with the clinically significant blood group system antigens of the patient (or with fewer antigen specificities than the patient). If autoantibodies cannot be excluded, only random blood matching can be carried out with blood donors that are ABO - compatible (or type O) with the patient and RhD - compatible (or C / c / E / e - compatible, or with fewer antigen specificities than the patient) to screen for the blood donor with the weakest main - side agglutination and not stronger than the agglutination intensity of the autologous control (the minimum incompatibility in blood matching) as the suitable blood donor.

[0086] In this study, the time costs of using three blood matching experiments to screen for suitable blood donors for 60 patients with difficult blood matching were recorded. The time required for traditional MMA (13.10 ± 0.82) was greater than that for the microcolumn gel method (11.71 ± 1.16), and the difference was statistically significant ( P(<0.05), the time required for the improved MMA (6.0 ± 0.45) was less than that required for the microcolumn gel method (11.71 ± 1.16), and the difference was statistically significant ( P <0.05), the time required for the improved MMA (6.0 ± 0.45) was less than that required for the traditional MMA (13.10 ± 0.82), and the difference was statistically significant ( P <0.05), as shown in Table 7 specifically.

[0087] Table 7 Time costs for screening suitable blood donors for 60 patients with difficult blood matching using three blood matching techniques: microcolumn gel method, traditional MMA, and improved MMA 。

[0088] VI. Safety analysis of screening suitable blood donors for patients with difficult blood matching using the microcolumn gel method, traditional MMA, and improved MMA.

[0089] Using the respective screening criteria for suitable blood donors of the microcolumn gel method, traditional MMA, and improved MMA, the number of cases where suitable blood donors could be screened out for 60 patients were 42 cases, 45 cases, and 53 cases respectively. According to the hemolysis index actually detected after blood transfusion in 60 patients, the safety differences of the three blood matching experimental techniques for screening suitable blood donors were analyzed. There was no statistically significant difference in the TBIL and IBIL levels of 60 patients before blood transfusion ( P >0.05). After the suitable blood donors screened by the microcolumn gel method for patients with difficult blood matching were transfused, the TBIL and IBIL levels increased compared with those before blood transfusion (both P <0.05), as shown in Table 8; after the suitable blood donors screened by the traditional method for patients with difficult blood matching were transfused, the TBIL and IBIL levels did not change significantly (both P >0.05), as shown in Table 9; after the suitable blood donors screened by the improved MMA for patients with difficult blood matching were transfused, the TBIL and IBIL levels decreased compared with those before blood transfusion (both P <0.05), as shown in Table 10.

[0090] Table 8 Changes in hemolysis index before and after transfusion of leukocyte-reduced red blood cell suspension for patients with difficult blood matching using the microcolumn gel method ( n =42) 。

[0091] Table 9 Changes in hemolysis index before and after transfusion of leukocyte-reduced red blood cell suspension for patients with difficult blood matching using the traditional MMA method ( n =45) 。

[0092] Table 10 Changes in hemolysis index before and after transfusion of leukocyte-reduced red blood cell suspension for patients with difficult blood matching using the improved MMA method ( n =53) 。

[0093] VII. Analysis of the effectiveness of the microcolumn gel method, traditional MMA, and modified MMA in screening suitable blood donors for difficult patients.

[0094] Using the screening criteria of the microcolumn gel method, traditional MMA, and modified MMA respectively, the number of cases where the blood donors were suitable for transfusion diagnosed in 60 patients were 42 cases, 45 cases, and 53 cases respectively. According to the comparison and analysis of the actual blood routine indexes detected after the transfusion of 60 patients, the differences in the effectiveness of the three blood matching experimental techniques for screening suitable blood donors were analyzed.

[0095] Before the transfusion of 60 patients, there were no statistically significant differences in the levels of Hb, RBC, and Hct (all P > 0.05). The changes in the red blood cell indexes after the transfusion of the suitable blood donors screened by the microcolumn gel method for difficult blood matching were significant, and the levels of Hb, RBC, and Hct were significantly increased compared with those before the transfusion (all P < 0.05), as shown in Table 11; the changes in the red blood cell indexes after the transfusion of the suitable blood donors screened by traditional MMA for difficult blood matching were significant, and the levels of Hb, RBC, and Hct were significantly increased compared with those before the transfusion (all P < 0.05), as shown in Table 12; the changes in the red blood cell indexes after the transfusion of the suitable blood donors screened by modified MMA for difficult blood matching were significant, and the levels of Hb, RBC, and Hct were significantly increased compared with those before the transfusion (all P < 0.05), and the increase in the red blood cell indexes after the transfusion in the modified MMA group was more obvious, as shown in Table 13.

[0096] Table 11 Changes in red blood cell indexes before and after the transfusion of the suitable blood donors screened by the microcolumn gel method for difficult blood matching ( n = 42) 。

[0097] Table 12 Changes in red blood cell indexes before and after the transfusion of the suitable blood donors screened by traditional MMA for difficult blood matching (n = 45) 。

[0098] Table 13 Changes in red blood cell indexes before and after the transfusion of the suitable blood donors screened by modified MMA method for difficult blood matching (n = 53) 。

[0099] In summary, the method disclosed by the present invention changes the well plate culture of monocytes-macrophages and sensitized red blood cells in the MMA method to incubation in the incubation cavity on the gel column of a microcolumn gel card. After centrifugation, the adhesion and phagocytosis of monocytes-macrophages to sensitized red blood cells can be visually observed on the gel column, thus simplifying and optimizing the experimental process of traditional MMA, shortening the experimental time, reducing the error caused by manual counting in the experimental results, realizing the visualization of result interpretation, improving the accuracy and repeatability of experimental results, and providing a relatively simple, fast and intuitive blood matching technique for clinical difficult blood matching.

[0100] The above are only the preferred embodiments of the present invention and are not used to limit the patent scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for solving the difficult blood matching problem caused by irregular antibodies, characterized in that: The following steps are involved: The monocytes and sensitized red blood cells are cultured in the incubation chamber on the microcolumn of the gel card; after centrifugation, the sensitized red blood cells that are not adhered and phagocytosed are deposited at the bottom of the gel column through the micropores in the gel column, while the erythrocytes and monocytes-macrophages that are adhered and phagocytosed are gathered at the top or upper end of the gel column; the adhesion and phagocytosis of the monocytes-macrophages on the gel column are judged by naked eye observation or image analysis; Donated blood is selected for transfusion based on adhesion and phagocytosis.

2. The method according to claim 1, characterized in that The gel column in the microcolumn gel card is prepared by neutral gel reagent, that is, the microcolumn cavity and gel do not contain specific antibodies and anti-human globulin; the gel column structure is a microporous structure of dextran gel cross-linking, which can provide support for the growth and adhesion of monocytes-macrophages.

3. The method according to claim 1, characterized in that The separation and cultivation of monocytes comprises: Mononuclear cells were isolated from peripheral blood using density gradient centrifugation; The isolated monocytes were washed and resuspended in RPMI1640 medium; cultured in a 5% CO2 incubator at 37°C for 1 hour; The activity of monocytes was detected by trypan blue staining to ensure that the activity was ≥95%.

4. The method according to claim 1, characterized in that The preparation of the sensitized red blood cells comprises: Patient plasma was mixed with donor red blood cells with agglutination strength ≤2+ (2+, 1+, ±) and ≤ self-control agglutination strength in the initial screening by coagulation amine blood matching method, and the mixture was placed in a water bath at 37°C for 1 hour to prepare sensitized red blood cells; positive control sensitized red blood cells and negative control sensitized red blood cells were also prepared for internal quality control of the batch experiment; The direct antiglobulin test is used to detect whether red blood cells are sensitized by antibodies.

5. The method according to claim 1, characterized in that The incubation conditions of the monocytes and sensitized red blood cells are: incubation at 37° C. for 2 hours in a 5% CO 2 incubator.

6. The method according to claim 1, characterized in that The centrifugal conditions of the microcolumn gel card are: using a gel card centrifuge, the centrifugal force is 128.1g, and the centrifugal time is 9 minutes.

7. The method according to claim 1, characterized in that The result interpretation includes: Observe the number of red blood cells at the top of the gel column with the naked eye. The one with the least number of red blood cells and close to the negative control column is the most suitable for blood donation; When it is difficult to compare the number of red blood cells by naked eye observation, use Image J software to measure the gray value of the red blood cells at the top of the gel column, and the one with the smallest gray value is the most suitable for blood supply.

8. The method according to claim 7, characterized in that The operation steps of the Image J software include: Import gel column images and convert to 8-bit format; Use the "Subtract Background" function to subtract the background and set the rolling ball radius to 50 pixels; Select the red blood cell aggregation area at the upper end of the gel column and measure its gray value; Export gray value data and perform statistical analysis.

9. A product for solving the difficult blood matching problem caused by irregular antibodies, characterized in that: The invention comprises a commercially available microcolumn gel card, which is prepared from a neutral gel reagent, wherein the microcolumn cavity and the gel do not contain specific antibodies and anti-human globulin; each gel column structure is a microporous structure of cross-linked dextran gel that only allows free red blood cells to pass through, and is used for conducting experiments in the method for solving difficult blood matching problems caused by irregular antibodies as described in any one of claims 1 to 8.

10. The product according to claim 9, characterized in that The products include kits.

Images