TWI475229B - A reagent group for transfusion test and its test method - Google Patents

Description

Reagent group for test before blood transfusion and test method thereof

The invention relates to a reagent group and a test method thereof for pre-transfusion cooperation test, in particular to a reagent group capable of improving the sensitivity of the traditional manual condensed amine method and a test method thereof.

Blood type is a method of classifying blood and is a genetic feature. The clinically mentioned blood type refers to the difference in the structure of red blood cell surface antigen. Broadly speaking, blood types include antigenic systems with different blood components such as red blood cells, white blood cells, platelets, and plasma. The earliest known blood type system is the ABO blood group system. For example, the red blood cell contains the A antigen, which is called type A; this is Karl by the Institute of Pathology, University of Vienna, Austria. In 1900, Karl Landsteiner discovered that healthy human plasma has a cohesive effect on red blood cells of different human individuals, thus establishing an ABO blood group system. Later, the new blood type system was continuously discovered and discovered, and the International Blood Transfusion Society, established in 1935, was responsible for the identification and naming work. More than 600 antigens are currently recognized in 30 human blood group systems, but most of them are very rare.

Blood type system is of great significance for blood transfusion. Incompatibility of blood transfusion may lead to hemolysis reaction, resulting in hemolytic anemia, renal failure, shock and even death. For humans, although the "ABO blood group system" and the "Rh blood type system" are the two most important types in the 30 blood type systems recognized by the International Blood Transfusion Association, it is not only the difference between the two types of blood type systems. Blood type will have an incompatible reaction of blood transfusion. Therefore, patients must make a series of tests in the blood bank before blood transfusion to select the correct and safe blood to ensure the safety of blood transfusion.

In addition, the so-called cross-matching test is usually performed within a limited time before the blood transfusion. The blood plasma of the recipient is paired with the red blood cells of the donor to detect whether the blood of the recipient has an unknown antibody that reacts with the surface antigen of the blood donor's red blood cell surface antigen, or uses the red blood cells of the recipient and the plasma of the donor. Pairing is performed to detect the presence of an unknown surface antigen on the recipient's red blood cells that will agglutinate with the antibodies contained in the plasma of the donor. Therefore, looking at the above, the so-called "pre-transfusion test" includes three types: "ABO and Rh blood type identification", "antibody screening or identification" and "cross-matching blood test".

In the past, Asian countries generally use the so-called "Manual Polybrene" (MP method) to perform the pre-transfusion test. The main use of polyetherene (Polybrene) reagent is a polyvalent cationic amino group. A polymer that causes reversible agglutination of normal red blood cells. In the conventional manual condensed amine test procedure, the blood donor's red blood cells and the recipient's plasma are first combined with the red blood cell antigen in a low ionic medium (LIM), and then the condensed amine is added to make the red blood cell reversible. Sexual agglomeration, after centrifugation and precipitation, and then adding a citrate resuspension solution (negative citrate) to neutralize the positive charge of the condensed amine, the negative charge on the surface of the red blood cell can be restored. At this time, if the red blood cells are caused by the non-specific agglomeration caused by the condensed amine reagent, when the citrate resuspension solution is added, the surface of the red blood cells will return to a negative charge, and the red blood cells will be dispersed due to mutual repulsion, and the result is a negative reaction; The red blood cells are specifically agglomerated by the antigen-antibody, and are not scattered by the citrate resuspension solution, and the result is a positive reaction.

However, the traditional manual condensed amine method and reagent formulation are too sensitive to antibody detection, and are usually presented for antibodies that are not clinically meaningful (eg, cold antibodies, non-specific antibodies, etc.). Positive reaction, which will aggravate the workload of subsequent antibody identification, which is not only time consuming, but also may cause the patient to face no suitable The blood bag can be used in a dilemma, so this is a great risk when the patient is in urgent need of blood. Therefore, it is necessary to develop a reagent formulation or test method which has high detection sensitivity only for clinically significant antibodies, but has low detection sensitivity for antibodies which are not clinically significant.

In addition, the key to affecting the accuracy of traditional manual condensed amine method is that the ratio of plasma (or serum) to blood cells is greater than 50 times, and the detection sensitivity is less than 50 times; in general, the ratio of traditional manual condensed amine test tube method It is about 66.67 times (ie 100 μL: 1.5 μL). However, the above has mentioned that in addition to the "ABO blood group system" and "Rh blood type system" two blood types must be confirmed, there are other blood type systems will also have a blood transfusion incompatibility reaction, therefore, in order to further For more detailed screening or identification of antibodies, it is necessary to extract more blood samples from patients, which is undoubtedly worse for an ischemic patient. Therefore, if only a small amount of sample liquid is needed for the test, and the ratio of plasma (or serum) to blood cells is increased to more than 50 times, or even more than 100 times, a highly accurate test result can be obtained, which is nothing for the patient. It is a great gospel.

In the future, if combined with automated and parallel large-scale testing equipment and methods, it will save inspection time and manpower, reduce inspection costs, and at the same time achieve the goal of standardization of inspection operations.

The invention provides a reagent group for pre-transfusion cooperation test, which can improve the sensitivity of the traditional manual condensed amine method, and the reagent group comprises a blood cell cleaning solution, a low ionic medium solution, a condensed amine solution and a resuspension solution.

The blood cell cleaning solution of the reagent group is prepared by mixing Tween ^® 20 (polyoxyethylensorbitanmonolaurat) solution and a sodium chloride solution (Saline) having a concentration of 0.85 to 0.9% in an appropriate ratio, and the liquid volume ratio ranges from 1:5000. 20000, as a mild blood cell surface cleaner. Its main function is to clean and remove other substances such as other proteins on the surface of non-erythrocytes to avoid the sensitivity of the interference detection.

The low ionic medium solution of the above reagent group is firstly dissolved in a 5% Dextrose solution by an appropriate amount of ETDA sodium salt (Na ₂ EDTA. 2H ₂ O), and then with a concentration of 7-11% dextran solution (Dextran 40). It is prepared by mixing in an appropriate ratio, and the liquid volume ratio ranges from 7 to 12:1. Its main function is to provide a low-ion isotonic environment, which reduces the density of Na ⁺ and Cl ^- ion clouds around the red blood cells, allowing antibodies to attach to the blood cell antigen more quickly and in order to promote antigen-antibodies. The binding reaction.

The condensed amine solution of the reagent group is prepared from a polycondensate (Polybrene) powder in a sodium chloride solution having a concentration of 0.85 to 0.9% to prepare a condensed amine stock solution having a concentration of 0.05 to 0.5%, and a concentration of 18 to 25%. The polyethylene glycol solution is prepared by mixing in an appropriate ratio, and the liquid volume ratio ranges from 1:0.85 to 1.2. Its main function is to provide sufficient positively charged ions to neutralize the negatively charged ions on the surface of the blood cell, so that the blood cells produce non-specific reversible agglutination, and further promote the antigen-antibody binding reaction.

The resuspension solution of the reagent group is prepared by mixing a trisodium citrate solution having a concentration of 0.2 to 0.5 M and a Dextrose solution having a concentration of 5% in an appropriate ratio, and the liquid volume ratio ranges from 1.3 to 1.8: 1. Its main function is to neutralize the positive charge in the condensed amine solution, thereby restoring the negative charge on the surface of the red blood cell. At this time, if the red blood cells are agglutinated due to non-specificity, the red blood cells will be scattered due to the rejection of the negative charge on the surface of the red blood cells, and the result is a negative reaction; if the red blood cells are due to the antigen The specific agglutination caused by the antibody will not be spread by the influence of the citrate resuspension solution, and the result is a positive reaction.

The invention provides a pre-transfusion cooperation test method which can improve the traditional manual condensed amine method, and the test method combined with the above reagent group can reduce the detection sensitivity of detecting antibodies without clinical significance, but at the same time has clinical significance for detection. The sensitivity of detection of antibodies is increased.

The invention provides a pre-transfusion test method with high detection sensitivity and the reagent set of the invention, which can reduce the interference of non-surface antigen substances attached to the red blood cells, thereby improving the accuracy and sensitivity of the test for clinically meaningful antibodies. Reduce the amount of reagents and samples used.

The invention provides a pre-transfusion test method with high detection sensitivity and the reagent set, which can be used for detecting the identification of ABO blood type and Rh blood type, and the standard plasma reagent and the red blood cell sample to be tested are used, or used. The standard red blood cell reagent and the plasma sample to be tested, the method steps and the reagent group are the same as those disclosed in the present specification.

The invention provides a pre-transfusion test method with high detection sensitivity and the reagent set, which can be used for detecting antibodies contained in plasma (or serum) to be tested, and at this time, a red blood cell reagent of known blood cell surface antigen is used. The test, method steps and reagent sets are the same as those disclosed in the present specification.

The invention provides a pre-transfusion test method with high detection sensitivity and the reagent set, which can be used for performing cross-matching blood test of blood recipients and blood donors, and using blood donors when performing large cross-matching blood test Plasma (or serum) and blood donors' blood cells; while performing small cross-matching tests, blood cells and blood donors of the recipients are used Plasma. The method steps and reagent sets are the same as those disclosed in the present specification.

The invention provides a pre-transfusion cooperation test method which can improve the traditional manual condensed amine method, and the test method can prepare a large amount of blood cell solution in advance before the test, in order to respond to a large number of subsequent tests. Especially when the test requires the use of standard red blood cell reagents, such as: ABO blood type and Rh blood type identification, antibody screening or identification.

The invention provides a pre-transfusion test method with high detection sensitivity and the reagent set, which can reduce the use amount of the detection reagent and the patient sample, and thus can be used for developing an automated and parallel large-scale detection device and method. It can save inspection time and manpower, reduce inspection cost, and at the same time achieve the goal of standardization of inspection operation process.

The embodiments of the present invention are further described in the following description, and the embodiments of the present invention are set forth to illustrate the present invention, and are not intended to limit the scope of the present invention. In the scope of the invention, the scope of protection of the invention is defined by the scope of the appended claims.

definition

The "about" recited in the specification of the present invention represents a range of plus or minus five percent of the value.

The flow of the pre-transfusion test method using the reagent set of the present invention is shown in Figure 1:

1. As shown in step 120, the appropriate amount of the blood cell solution is added to a container, which is a container for holding a liquid, such as a test tube, a microplate, etc., and the container is made of glass or plastic. ...Wait.

2. As shown in step 122, an appropriate amount of the blood cell cleaning solution is added to the container, and the liquid in the container is uniformly mixed to wash away other substances on the blood cell other than surface antigen.

3. As shown in step 124, the container is placed in a centrifuge and centrifuged at a centrifugal force of 100 to 1200 G to precipitate the blood cell at the bottom of the container, and then the supernatant can be removed.

4. According to the actual situation of the test, the above steps 122 and 124 can be repeatedly performed to ensure that the surface impurities of the blood cell are removed, and the detection sensitivity can be improved.

5. As shown in step 126, an appropriate amount of sodium chloride solution (concentration 0.85 to 0.9%) is added to the container, and the liquid in the container is uniformly mixed to prepare a blood cell suspension having an appropriate concentration of 2.5 to 3.0%.

6. As shown in step 128, add an appropriate amount of plasma solution to the container and mix the liquid in the container evenly.

7. As shown in step 130, an appropriate amount of the low ionic medium solution is added to the vessel and the liquid in the vessel is uniformly mixed.

8. As shown in step 132, the vessel is warmed to 35-39 °C.

9. As shown in step 134, add an appropriate amount of the polycondensed amine solution to the container and mix the liquid in the container evenly.

10. As shown in step 136, place the container in a centrifuge to 100~1200G. The centrifugal force was centrifuged to precipitate the blood cells at the bottom of the vessel, followed by removal of the supernatant.

11. As shown in step 138, add an appropriate amount of the resuspension solution and mix the liquid in the container evenly. At this time, if the red blood cells are agglutinated due to non-specificity, the red blood cells will be scattered due to the rejection of the negative charge on the surface of the red blood cells; if the red blood cells are specifically agglutinated due to the antigen and antibody, they will not spread.

12. As shown in step 140, the valence of hemagglutination is determined based on the condition of aggregation or dispersion of the blood cells.

The following is exemplified by an embodiment, using a 96-well micro-plate as the container described above, in combination with the pre-transfusion test method with high detection sensitivity of the present invention, the reagent group and three groups of red blood cells. Reagents (SI, SII, SIII reagent blood cells provided by Formosa Biotechnology), for the screening of 429 patient samples for antibody screening, supplemented by traditional manual condensed amine method, traditional European and American national standard anti-human ball The protein antibody LIAT method (LISS additive indirect antiglobin test) and the automated instrument method Gel Test method used in Europe and the United States as a comparison to verify the accuracy of the invention in clinical trials, and compare the time spent on the four detection methods to clear The innovation and progress of the present invention are disclosed.

Embodiment 1

The distribution of the reagent set proposed according to the present invention is as follows:

1. The blood cell cleaning solution: a Tween® 20 solution and a 0.9% sodium chloride solution are uniformly mixed at a liquid volume ratio of 1:10000.

2. The low ionic medium solution: firstly dissolve 0.05 gram of ETDA sodium salt (Na ₂ EDTA. 2H ₂ O) in 90 mL of a 5% Dextrose solution, and then a liquid volume ratio of 10% to glucomannan. :1 is evenly mixed.

3. The condensed amine solution: firstly, a condensed amine solution having a concentration of 0.4% is prepared by condensing amine powder and sodium chloride solution, and then uniformly mixed with a polyethylene glycol solution having a concentration of 20% at a liquid volume ratio of 1:1.

4. The resuspension solution is prepared by uniformly mixing a liquid volume ratio of 3:2 from a trisodium citrate solution having a molar concentration of 0.4 M and a Dextrose solution having a concentration of 5%.

The antibody screening test of 428 samples was performed in sequence according to the steps described in the pre-transfusion fitting test method proposed by the present invention. The detailed steps are as follows, and refer to the first figure at the same time:

1. As shown in step 120, 12 μL of the reagent hematocrit solution was added to a well of a 96-well microplate.

2. As shown in step 122, 300 μL of the blood cell washing solution is added to the well, and the liquid in the well is uniformly mixed.

3. As shown in step 124, the test tube is placed in a centrifuge and centrifuged at 700 G centrifugal force to precipitate the blood cell at the bottom of the well, and then the supernatant can be removed.

4. As shown in step 126, 12 μL of a 0.9% sodium chloride solution was added to the well, and the liquid in the well was uniformly mixed to prepare a blood cell suspension having a concentration of 2.5%.

5. As shown in step 128, add 50 μL of plasma solution to the well and mix the liquid in the well.

6. Add 150 μL of the low ionic medium solution to the well as shown in step 130. In the tank, and mix the liquid in the tank.

7. Place the 96 microwell plate into the warmer as shown in step 132 and warm to 37 ° C for 10 minutes.

8. As shown in step 134, 30 μL of the concentrated amine solution having a concentration of 0.4% was added to the well, and the liquid in the well was uniformly mixed.

9. As shown in step 136, the 96-well microplate is placed in a centrifuge and centrifuged at 114 G centrifugation to precipitate the blood cells at the bottom of the well, and then the supernatant can be removed.

10. As shown in step 138, add 45 μL of the resuspension solution at a concentration of 0.4 M to the well, and mix the liquid in the well. At this time, if the red blood cells are agglutinated due to non-specificity, the red blood cells will be scattered due to the rejection of the negative charge on the surface of the red blood cells; if the red blood cells are specifically agglutinated due to the antigen and antibody, they will not spread.

11. As shown in step 140, the valence of hemagglutination is determined based on the condition of aggregation or dispersion of the blood cells. If the agglutination phenomenon is not obvious, it can be further viewed with an imaging amplifying device such as a magnifying glass or a microscope to obtain a more accurate interpretation result.

Please refer to the second figure, which is an image of the hemagglutination reaction after performing antibody screening in this example. If there is agglutination reaction like D hole, the result is positive, if there is no agglutination reaction such as A~C, E , F, the result is negative.

Of the 429 samples, 175 were positive, 214 were negative, 40 were false negative, and no false positives were produced. 174 positive antibody results and 40 false negative undetected antibodies, see Table 3.

In terms of detection time, 32 test samples can be simultaneously detected using the detection method and the reagent set of the present invention, and it takes about 25 minutes from the start of the test to the result, and 429 samples take a total of 84 minutes.

[Comparative] - Traditional manual condensed amine method

The reagent groups required for the traditional manual condensed amine method are as follows:

1. Low ionic medium solution: 0.2 gram of ETDA sodium salt (Na ₂ EDTA. 2H ₂ O) was added and uniformly mixed in 100 mL of a 5% Dextrose solution.

2. Condensed amine solution: 0.05% of a 0.05% polyamine solution and 99.5 mL of a 0.9% sodium chloride solution are uniformly mixed.

3. Resuspension solution: Trisodium citrate with a molar concentration of 0.2 M and a 5% Dextrose solution were uniformly mixed at a liquid to volume ratio of 3:2.

In the traditional manual condensed amine method, the 428 test antibody screening experiments were performed in sequence. The detailed steps are as follows:

1. Add 100 μL of plasma and 50 μL of reagent 2.5% hemoglobin solution to a test tube and mix them evenly.

2. Add 600 μL of the low ionic medium solution to the test tube, mix well, and let stand for 1 minute.

3. Add 100 μL of a 0.05% concentrated polyamine solution to the test tube and mix for 15 seconds.

4. Place the test tube in a centrifuge and perform a centrifugal sedimentation treatment for 15 seconds to precipitate the blood cells. At the bottom of the tube, remove the supernatant.

5. Add 100 μL of the resuspension solution to the test tube and mix well.

6. According to the condition of blood cell aggregation or dispersion, the valence of hemagglutination is read within 1 minute.

Among the results of 429 specimens detected by traditional manual condensed amine method, 215 specimens were positive, 214 specimens were negative, and 6 specimens were pseudo-negative, with no false positive results. 209 positive antibody results and 6 false negative undetected antibodies, see Table 3.

In terms of detection time, a sample is in accordance with the traditional manual condensed amine test tube method, and it takes about 3 minutes from the start to the result, so it takes about 321 minutes to detect 429 samples.

[Comparative] - Anti-human globulin antibody LIAT method for standard European and American national standards

1. Add 100 μL of plasma and 50 μL of reagent 2.5% hemoglobin solution to a test tube and mix them evenly.

2. Add 100 μL of N-HANCE (LISS additive) to the tube.

3. Leave at 37 ° C for 10 minutes, wash the red blood cells 3 to 4 times with 3 mL of 0.9% sodium chloride solution, and the supernatant should be drained for the last time.

4. Add 2 drops of anti-human globulin antibody (AHG) and centrifuge to observe the presence or absence of agglutination.

5. If there is no agglutination, add 1 drop of check cells, centrifuge for 15 seconds, observe the agglutination reaction, and have a reaction of 2+ or more, indicating that the negative result is truly negative. If no agglutination occurs, it may be a false negative reaction and the steps must be redone.

Among the 429 specimens tested by the anti-human globulin antibody LIAT method operating in the traditional European and American standards, 114 specimens were positive, 214 specimens were negative, 101 specimens were pseudo-negative, and no false positives. The result is produced. 114 positive antibody results and 101 false negative undetected antibodies, see Table 3.

In terms of detection time, a sample is in accordance with the detection process of the LIAT method, and it takes about 17 minutes from the start to the result. Therefore, it takes about 1819 minutes to detect 429 samples, which takes about 30.5 hours.

[Comparative] - Gel Test method used in automated instruments

The Gel Test method's detection reagents and cassettes use the Cellbind product from Sanquin Blood Supply The Netherlands. The procedure is as follows:

1. The reagent hemoglobin solution was diluted to 0.5% with Cellbind LISS solution.

2. Peel the tin foil on the desired card string.

3. Add 50 μL of reagent blood cells at a concentration of 0.5%.

4. Add 50 μL of the plasma solution to mix.

5. Leave at 37 ° C for 15 minutes.

6. Place the card in a Cellbind centrifuge and centrifuge for 10 minutes to interpret the results.

Among the 429 specimens tested by the Gel Test method using automated instruments in Europe and the United States, 128 specimens were positive, 214 specimens were negative, and 87 specimens were pseudo-negative, with no false positive results. These 128 positive antibody results and 87 false negative samples were not detected, see Table 3.

In terms of detection time, a specimen is opened according to the detection procedure of the Gel Test method. It takes about 30 minutes to get the results, so it takes about 270 minutes to test 429 samples.

[Comprehensive comparison]

Table 1 shows the results of the above four test methods for the pre-transfusion test.

Table 2 compares the detection efficiencies of the above four pre-transfusion test methods.

Table 3 shows the results of the above four test methods for the pre-transfusion test for various antibodies.

As shown in Table 3, (Note 1) Cold represents a cold type antibody, (Note 2) I represents a non-specific antibody, which is a non-clinical antibody, and can be treated by the detection method and the reagent group proposed by the present invention. The test results were negative. However, in the traditional manual condensed amine method, the test result is a positive reaction.

From the test results of the present embodiment, it is seen that the pre-transfusion test method and reagent set proposed by the present invention have the shortest detection time, the fastest speed, and the highest accuracy, and the number of positive antibodies detected in Table 1 can be It is seen that the present invention has a high degree of accuracy (present invention: Gel Test = 153: 128).

From the test results of the present embodiment, it is seen that the pre-transfusion test method proposed by the present invention has a plasma to blood cell ratio of 166.7 times, that is, 50 μL / (12 μL × 2.5%) = 166.7 times, that is, from the conventional A total of about 850 μL of total liquid required for the manual condensing amine method, which is reduced to 250 μL in this example, also maintains the accuracy of the test.

122~140‧‧‧ steps in the process

The first figure is a schematic flow chart of the pre-transfusion fitting test method proposed in the present invention.

The second figure is the image of the blood cell agglutination reaction after the test according to the flow of the blood pre-matching test method proposed in the 96-microplate. In the figure, A~C, E, F are non-agglutination, D is There is an agglutination reaction.

122~140‧‧‧ steps in the process

Claims (17)

A reagent group for pre-transfusion cooperation test, the reagent group comprises a blood cell cleaning solution, a low-ionic medium solution, a polycondensation amine solution, and a resuspension solution; wherein the low-ion medium solution is from 0.05 to 0.2 g The ETDA sodium salt is first dissolved in a 5% Dextrose solution, and then uniformly mixed with a concentration of 7-11% dextran at a liquid volume ratio of 7 to 12:1; the condensed amine solution is firstly condensed by the condensed amine powder. Dissolved in 0.85~0.9% sodium chloride solution to prepare a concentration of 0.05~0.5% condensed amine solution, and then uniformly mixed with a concentration of 18~25% polyethylene glycol solution at a liquid volume ratio of 1:0.85~1.2. . The scope of the patent application item of the first set of reagent, wherein the blood cell-based cleaning liquid and a solution of Tween ^® 20 concentration of 0.85% to 0.9% sodium chloride solution at an appropriate percentage of the preparation. The scope of the patent application item of the reagent set 2, wherein the concentration of Tween ^® 20 solution and 0.85 to 0.9% sodium chloride solution as a liquid volume ratio of 1: 5,000 to 20,000 uniformly mixed together. The reagent set according to claim 1, wherein the resuspension solution is prepared from a trisodium citrate solution and a Dextrose solution in an appropriate ratio. The reagent group according to claim 1 or 4, wherein the resuspension solution is a trisodium citrate solution having a molar concentration of 0.2 to 0.5 M and a Dextrose solution having a concentration of 5%, and the liquid volume ratio is 1.3~. 1.8:1 is evenly mixed. The reagent group according to any one of the claims 1 to 4 is used for the pre-transfusion combined test, which is ABO blood type identification, Rh blood type identification, antibody screening and identification or cross-matching test. The reagent group according to Item 6 of the patent application, wherein the blood volume (or serum) and the volume of blood cells in the test before the blood transfusion test are more than 100 times. A pre-transfusion fitting test method using the reagent set according to any one of claims 1 to 7, comprising the steps of: (a) adding an appropriate amount of a hematocrit solution to a container capable of containing a liquid; Adding an appropriate amount of blood cell cleaning solution to the container and mixing the liquid in the container; (c) placing the container in a centrifuge for centrifugation to precipitate the blood cell at the bottom of the container, and then The supernatant can be removed; (d) an appropriate amount of sodium chloride solution is added to the container, and the liquid in the container is uniformly mixed to prepare a blood cell suspension of an appropriate concentration; (e) an appropriate amount of plasma solution is added to the container. And mixing the liquid in the container uniformly; (f) adding an appropriate amount of the low ionic medium solution to the container, and mixing the liquid in the container; (g) heating the container to a suitable temperature; Adding an appropriate amount of a suitable concentration of the polycondensed amine solution to the container and mixing the liquid in the container uniformly; (i) placing the container in a centrifuge for centrifugation to precipitate the blood cell at the bottom of the container Then the supernatant can be removed (J) adding an appropriate amount of resuspension solution, the liquid in the vessel and mixed; and (k) based on blood cell aggregation or dispersed condition, interpretation of the valence of the blood cell agglutination. The pre-transfusion test method according to claim 8, wherein the container for holding the liquid is a test tube or a microplate. The pre-transfusion test method according to claim 8 or 9, wherein the liquid-filled container is made of glass, ceramic, plastic or rubber. The pre-transfusion test method according to item 8 of the patent application scope, wherein the appropriate temperature of the step (g) is 35 to 39 °C. The pre-transfusion test method according to item 8 of the patent application scope, wherein the centrifugal sedimentation treatment in the step (c) is performed by centrifugal precipitation treatment with a centrifugal force of 100 to 1200 G. The pre-transfusion test method as described in claim 8 wherein step (k) is further viewed using a magnifying imaging device when the valence of hemagglutination is interpreted. The pre-transfusion fitting test method according to claim 13, wherein the magnifying imaging device is a magnifying glass or a microscope. For example, the pre-transfusion test method described in item 8 of the patent application is for ABO blood type identification, Rh blood type identification, antibody screening and identification or cross-matching test. The pre-transfusion test method according to item 8 of the patent application scope, wherein the plasma (or serum) and the volume of the blood cell at the time of the test are more than 100 times. The pre-transfusion test method according to item 8 of the patent application scope, wherein the steps (a) to (d) can be prepared before the test, in order to be used for subsequent large-scale detection.