CN114324888A - Micro-column agglutination method indirect anti-human globulin test method - Google Patents

Abstract

The invention provides a method for testing indirect anti-human globulin by a microcolumn agglutination method, which comprises the following steps: adding red blood cells and a first antibody into a test tube to perform red blood cell sensitization reaction, wherein the red blood cells are donor/receiver red blood cells or reagent red blood cells in a matching test, and the first antibody is receiver/donor serum in the matching test, or an antibody to be detected or an autoantibody; after the erythrocyte sensitization reaction is finished, the product after the reaction is added into a micro-column gel tube of the anti-human globulin card for agglutination reaction. According to the method provided by the invention, the red blood cell sensitization reaction is finished in the test tube, the first antibody is not contacted with the second antibody and is not neutralized, the uniform mixing is easy, and the number of times of uniform mixing can be increased in an experiment to increase the contact and reaction of the antigen and the antibody, so that the red blood cell sensitization reaction is more sufficient, and the reaction sensitivity is improved; after the erythrocyte sensitization reaction is finished, a microcolumn reagent is added for agglutination reaction, so that the first antibody is not needed to be neutralized, and the operation difficulty is greatly reduced.

Description

A kind of indirect anti-human globulin test method by microcolumn agglutination method

technical field

The invention relates to the technical field of medical detection, in particular to an indirect anti-human globulin test method by a microcolumn agglutination method.

Background technique

Indirect anti-human globulin test is widely used in blood transfusion compatibility testing (alloantibody screening, cross-matching blood test) and serum or plasma free autoantibody detection. Diagnosis is of great significance. At present, the indirect anti-human globulin test is mainly carried out by microcolumn agglutination method.

The microcolumn agglutination anti-human globulin test uses an integrated reagent cartridge (anti-human globulin) for the detection of clinical specimens. The reagent card consists of an upper reaction chamber and a micro-column connected to it. The reaction chamber is used for red blood cell sensitization reaction, and the micro-column is filled with anti-human globulin reagent (containing a second antibody). The experiment is divided into two parts. First, the reagent red blood cells (or the donor and recipient red blood cells of the blood matching test) and the serum to be tested (the primary antibody, or the recipient and donor serum of the blood matching test) react in the reaction chamber (red blood cell sensitization reaction). ), followed by agglutination, centrifugation after the sensitization reaction of red blood cells is completed, the red blood cells slowly pass through the gel micro-column under the action of low centrifugal force, if the red blood cells are sensitized by the primary antibody, they will agglutinate with the secondary antibody and be retained in the micro-column At the upper end or in the middle of the microcolumn, unsensitized red blood cells sink to the bottom of the microcolumn.

When the sensitization reaction is carried out in the reaction chamber, the second antibody in the liquid-phase reagent will neutralize the free first antibody to be detected and prevent it from binding to the red blood cells, thereby reducing the sensitivity of the reaction. In order to reduce the neutralization reaction, when adding reagent red blood cells, the testing personnel will try to form a separation "membrane" between the liquid-phase reagent and the red blood cell reagent, which is difficult to operate; in order to protect this "membrane", after adding the serum to be tested, The mixing method is relatively gentle and the mixing is not sufficient, and the small reaction chamber also increases the difficulty of mixing. Therefore, the operation is difficult, the primary antibody is neutralized, the sensitization reaction is insufficient, and the sensitivity is reduced.

SUMMARY OF THE INVENTION

In view of the above situation, the present invention provides an indirect anti-human globulin test method by microcolumn agglutination to solve the problems of difficult operation, neutralization of the primary antibody, insufficient sensitization reaction and reduced sensitivity in the prior art.

The technical scheme of the present invention is:

A microcolumn agglutination indirect anti-human globulin test method, comprising:

Add red blood cells and primary antibodies into the test tube for red blood cell sensitization reaction, the red blood cells are donor/recipient red blood cells or reagent red blood cells in the blood matching test, and the first antibody is the recipient/donor serum of the blood matching test, or Test alloantibodies, or autoantibodies;

After the red blood cell sensitization reaction is completed, the reacted product is added to the anti-human globulin microcolumn gel tube for agglutination reaction.

According to the indirect anti-human globulin test method provided by the microcolumn agglutination method, the red blood cell sensitization reaction is completed in the test tube, without contact with the second antibody, without being neutralized, the mixing is easy, and the mixing times can be increased in the experiment In order to increase the contact and reaction of the antigen and antibody, the red blood cell sensitization reaction is more sufficient, and the reaction sensitivity is improved; after the red blood cell sensitization reaction is completed, the microcolumn reagent is added to perform the agglutination reaction, and there is no need to worry about the neutralization of the primary antibody, and the operation difficulty is greatly reduced.

Description of drawings

Fig. 1 is a photo of the experimental result of the clinical sample 1 to be tested;

Figure 2 is a photo of the experimental results of the clinical sample 2 to be tested.

Detailed ways

To facilitate understanding of the present invention, the present invention will be more fully described below with reference to various embodiments, however, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The present invention provides a microcolumn agglutination indirect anti-human globulin test method, comprising:

Add red blood cells and primary antibodies into the test tube for red blood cell sensitization reaction, the red blood cells are donor/recipient red blood cells or reagent red blood cells in the blood matching test, and the first antibody is the recipient/donor serum of the blood matching test, or Test alloantibodies, or autoantibodies;

After the red blood cell sensitization reaction is completed, the reacted product is added to the anti-human globulin microcolumn gel tube for agglutination reaction.

The above method will be described below with specific examples.

1. Materials and methods

1.1 Instruments BS-C12 low-speed centrifuge and BS-I24 reagent card incubator, Zhuhai Besso Biotechnology Co., Ltd. CU-600 Electric Heating Constant Temperature Water Tank, Shanghai Yiheng Scientific Instrument Co., Ltd. The instrument is set up by the engineer according to the instructions.

1.2 Source of clinical samples Two positive clinical samples for irregular antibody screening (donated by a hospital), clinical sample 1: a 76-year-old male who screened positive for type A irregular antibodies, and clinical sample 2: a 72-year-old female who screened positive for type O irregular antibodies. One fresh A-type and O-type RHD positive irregular antibody screening negative serum (hereinafter referred to as "A-type serum" and "O-type serum") (the remaining serum of the sample sent by our hospital).

1.3 Reagents Anti-D (IgG) (monoclonal antibody) reagent (batch number: 20210401), Shanghai Blood Bio-Pharmaceutical Co., Ltd.; Human ABO Blood Type Reversal Red Blood Cell Kit (batch number: 20215319), Shanghai Blood Bio-Pharmaceutical Co., Ltd.; Antibody screening red blood cell kit (batch number: 20217042), Shanghai Blood Biopharmaceutical Co., Ltd.; anti-human globulin card (batch number A210201), Zhuhai Besso Biotechnology Co., Ltd.

1.4 Experimental method

1.4.1 Use anti-D (IgG) reagent as the standard antibody to be tested. Dilute the anti-D (IgG) reagent with physiological saline into gradient standard test samples of 1:32, 1:64, 1:128, 1:256, 1:512 (hereinafter referred to as "gradient standard test samples"); Physiological saline was used to dilute the O erythrocytes in the erythrocyte kit to a concentration of 1% for human ABO blood typing as antigen cells (hereinafter referred to as "antigen cells"); the antibody screening erythrocyte reagents I and II were diluted to 1% with physiological saline. The concentration is used as the screening cell (hereinafter referred to as "screening cell I", "screening cell II").

1.4.2 Microcolumn agglutination method Anti-human globulin test adopts two red blood cell sensitization reaction scenarios (respectively: method 1 and method 2) to detect the above-mentioned gradient standard test samples by micro-column agglutination method.

Method 1: Strictly follow the instructions (i.e. the traditional method), add 50ul of antigenic cells and 25ul of the sample to be tested to each well of the anti-human globulin card, mix well, incubate at 37°C for 15 minutes, and read the results by centrifugation.

Method 2: The red blood cell sensitization reaction is completed in a test tube (that is, the method of the present invention), that is, ① Take 5 test tubes, add samples to each tube at a ratio of 2/1 of the antigen cells/sample to be tested as a reaction solution, mix well and set aside Incubate in a water bath at 37°C for 15 minutes, and mix once every 5 minutes; ② Pipet 75ul of the reaction solution into each tube and add the anti-human globulin card, and centrifuge immediately to read the results.

Each gradient standard sample to be tested was tested in parallel for 5 batches by the above two methods.

1.4.3 Clinical sample detection The above-mentioned clinical sample 1 is double-diluted with A-type serum, and clinical sample 2 is double-diluted with O-type serum to form the original, 1:2, 1:4 graded clinical samples to be tested.

The above-mentioned screening cells I and screening cells II are used as screening cells, and the above-mentioned clinical samples to be tested are detected by method 1 and method 2.

1.4.4 Erythrocyte sedimentation test Take an anti-human globulin card, add 50ul of antigenic cells and 25ul of type O serum to each well, mix well, and incubate in a water bath at 37°C for 15 minutes, respectively, at 0, 5, 10, and 15 minutes. The sedimentation of erythrocytes in the microcolumn reaction chamber was recorded and compared.

1.5 Interpretation of results

Interpret the results according to the reagent instructions, and record the reaction intensity of each well.

2. Results

2.1 The agglutination strength of each well of the standard sample to be tested is shown in Table 1. At two dilutions of 1:32 and 1:64, the agglutination strength of both methods is 5, and the agglutination strength of two dilutions of 1:128 and 1:256 is the agglutination of method one. The intensity is weaker than that of method 2, the dilution of 1:512 is negative for method 1, and the agglutination strength of method 2 is 2.

Table 1 The agglutination strength of each well of the standard sample to be tested

Note: No. 1-5 is method 1, No. 6-10 is method 2; 4+ is recorded as 5, 3+ is recorded as 4, 2+ is recorded as 3, 1+ is recorded as 2, ± is recorded as 1, - is recorded as 0.

2.2 The agglutination strength of each well of the clinical sample 1 is shown in Table 2, and the photos of the experimental results are shown in Figure 1. The agglutination intensity of the positive wells of method 1 was weaker than that of method 2. The cells were screened with double sample size. Method 1 was negative, and method 2 had a weaker agglutination band on the upper end of the gel column.

Table 2 Agglutination strength of each well of clinical sample 1

Note: 3+/2+ is recorded as 4/3, 2+ is recorded as 3, 2+/1+ is recorded as 3/2, 1+ is recorded as 2, ± is recorded as 1, - is recorded as 0.

2.3 The agglutination strength of each well of the clinical sample 2 is shown in Table 3, and the photos of the experimental results are shown in Figure 2. The agglutination strength of the positive wells of method one was weaker than that of method two.

Table 3 Agglutination strength of each well of clinical sample 2

Note: 4+ is recorded as 5, 4+/3+ is recorded as 5/4, 3+ is recorded as 4, 2+ is recorded as 3, 2+/1+ is recorded as 3/2, 1+ is recorded as 2, ± is recorded is 1, - is recorded as 0.

2.4 At the end of 10 minutes, obvious sedimentation of erythrocytes can be seen by the naked eye, and the sedimentation of erythrocytes is more obvious at the end of 15 minutes.

3. Conclusion

Pre-transfusion blood type reexamination, erythrocyte irregular antibody screening and cross-matching test are important technical means for compatible blood transfusion, which are of great significance to blood transfusion safety. The study found that the ineffective transfusion of red blood cells is closely related to the low titer of irregular antibodies. Therefore, it is of great significance to improve the detection rate of low-titer irregular antibodies and reduce the occurrence of missed detection to improve the efficacy of red blood cell transfusion and ensure the safety of blood transfusion. Some studies have used multiple methods for simultaneous detection, which can reduce the incidence of missed detection of low-titer irregular antibodies.

Indirect anti-human globulin tests are widely used in the diagnosis of immune hemolytic diseases, blood transfusion compatibility testing (including irregular antibody screening and cross-matching tests). The experiment consists of two parts, the first step of alloantibody sensitization of erythrocytes (erythrocyte sensitization) and the second step of agglutination of sensitized erythrocytes with the second antibody (anti-human globulin antibody) (agglutination). reaction). Among them, whether the red blood cell sensitization reaction is sufficient or not is an important factor affecting the sensitivity of the experiment. Studies have shown that preventing the sensitizing reaction mixture from contacting the gel column reagent, ensuring sufficient reaction time, and increasing the amount of serum to be measured can improve the sensitivity of the indirect anti-human globulin test.

The above-mentioned method 1 adopts the method recommended in the "National Clinical Testing Operation Regulations" and the reagent instructions. The red blood cell sensitization reaction is mixed and reacted in the column reaction chamber on the upper part of the micro-column, and the sensitivity is lower than that of the method 2. The red blood cell sensitization reaction of method two is completed in the test tube, and the antibody to be tested is not neutralized by anti-human globulin antibody (secondary antibody) during the reaction process; when the red blood cell sensitization reaction of method one is carried out in the microcolumn reaction chamber, the reaction During the process, the anti-human globulin antibody (secondary antibody) in the gel reagent neutralizes part of the free antibody to be tested. The effect of the antibody to be tested is not obvious at high concentrations, and the agglutination strength decreases or even false negatives occur at low concentrations. The second is that the volume of the micro-column reaction chamber is small, and it is difficult to mix the reaction solution fully. In addition, the erythrocytes in the reaction solution slowly settle under the action of gravity, the antigen-antibody in the reaction chamber of the first method has a relatively short contact time due to the sedimentation of the erythrocytes, and the reaction is not sufficient. Mixing times makes the antigen-antibody fully contacted and reacted for a relatively long time, and the reaction is more sufficient. Therefore, in clinical testing, the second method can be used in experiments such as erythrocyte irregular antibody screening and cross-matching test, that is, the erythrocyte sensitization reaction is completed in a test tube, and increasing the mixing times during the reaction process can improve the detection of alloantibodies. rate, and ensure the safety of blood transfusion.

In this application, clinical sample 1, magnified sample screening cell 1, method 2, there is a weak agglutination band on the upper end of the gel column, which is not easy to observe when the light is weak, and it is easy to be missed. You should develop the habit of reading the card under strong light. .

In the clinical sample detection of this application, the same type of serum diluted sample is used to make it closer to the characteristics of clinical weakly positive samples and present more realistic clinical test results.

In short, the red blood cell sensitization reaction is completed in the test tube, the experiment is easy to mix well, and it can effectively prevent the anti-human globulin antibody from neutralizing the free test antibody. Sensitivity of the indirect antiglobulin assay by microcolumn agglutination.

To sum up, according to the indirect anti-human globulin test method provided by the microcolumn agglutination method, the red blood cell sensitization reaction is completed in the test tube, without contact with the second antibody, without being neutralized, and the mixing is easy and can be increased in the experiment. The number of times of mixing can increase the contact and reaction of the antigen and antibody, so the red blood cell sensitization reaction is more sufficient and the reaction sensitivity is improved; after the red blood cell sensitization reaction is completed, the microcolumn reagent is added to perform the agglutination reaction, and there is no need to worry about the neutralization of the primary antibody, which is very difficult to operate. reduce.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the patent of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (5)

1. A method for testing indirect anti-human globulin by a microcolumn agglutination method, comprising:

adding red blood cells and a first antibody into a test tube to perform red blood cell sensitization reaction, wherein the red blood cells are donor/receiver red blood cells or reagent red blood cells in a matching test, and the first antibody is receiver/donor serum in the matching test, or an antibody to be detected or an autoantibody;

after the erythrocyte sensitization reaction is finished, the product after the reaction is added into an anti-human globulin microcolumn gel tube for agglutination reaction.

2. The method of claim 1, wherein the anti-human globulin reagent is contained in the gel tube.

3. The method of claim 2, wherein the anti-human globulin reagent comprises a solid phase gel molecular sieve and a liquid phase secondary antibody, and the secondary antibody is an anti-human globulin antibody.

4. The indirect anti-human globulin test method according to claim 3, wherein said second antibody fills the pores of the molecular sieve and is higher than the microcolumn by a predetermined distance.

5. The indirect anti-human globulin test method according to claim 3, wherein said method comprises:

taking 5 test tubes, adding sample as reaction liquid according to the ratio of antigen cells/to-be-detected samples of 2/1, uniformly mixing, putting in a water bath box, incubating in water bath at 37 ℃ for 15 minutes, and uniformly mixing for 1 time at intervals of 5 minutes;

each tube absorbs 75ul of reaction solution and adds the reaction solution into a micro-column gel tube of an anti-human globulin card, and the result is immediately read centrifugally.

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