CN115399314A

CN115399314A - Human ABO blood type anti-typing red blood cells capable of being freeze-dried and stored and storage method

Info

Publication number: CN115399314A
Application number: CN202211352847.7A
Authority: CN
Inventors: 商巧娜; 王伟权; 黄志刚; 王秀柱; 王丽; 郝进学
Original assignee: Tianjin Texiang Biotechnology Co ltd
Current assignee: Tianjin Texiang Biotechnology Co ltd
Priority date: 2022-11-01
Filing date: 2022-11-01
Publication date: 2022-11-29
Anticipated expiration: 2042-11-01
Also published as: CN115399314B

Abstract

The invention belongs to the field of human ABO blood type detection, and particularly relates to human ABO blood type reverse typing red blood cells capable of being freeze-dried and stored and a storage method. The invention adopts a method of fixing firstly and then freeze-drying, optimizes the freeze-drying process, and the freeze-dried red blood cells prepared by the method can be stored for one year at the temperature of 2-8 ℃ and can be directly used for cell reverse typing detection.

Description

Human ABO blood type reverse typing red blood cell capable of freeze-drying preservation and preservation method

Technical Field

The invention belongs to the field of human ABO blood type detection, and particularly relates to human ABO blood type reverse typing red blood cells capable of being freeze-dried and stored and a storage method.

Background

Blood grouping is the first task before clinical transfusion, because blood of different blood types can be subjected to hemolysis due to the coagulation of antigen and antibody when transfused mutually, and further the life safety of people can be endangered, so that correct blood grouping is a precondition for ensuring transfusion safety. Current methods of blood grouping include orthotyping and retrotyping. The positive typing method is used for detecting erythrocyte antigens, and the negative typing method is used for detecting antibodies in serum, wherein the detection of human blood group antigens/antibodies of A, B and O is the most important. The blood types can be divided into different types according to different antigens on the surface of erythrocyte membranes in human blood. The red blood cells of the blood group A carry blood group antigen A (hereinafter referred to as "A antigen"), and blood group antibody B (hereinafter referred to as "B antibody") is contained in the serum; the red blood cells of the B type blood are provided with blood group antigen B (hereinafter referred to as "B antigen"), blood group antibody A (hereinafter referred to as "A antibody") is contained in the serum, the red blood cells of the AB type blood are provided with the A antigen and the B antigen, and the serum is not provided with the A antibody and the B antibody; both A and B antigens are absent from erythrocytes in blood type O, and both A and B antibodies are present in the serum.

Blood group reverse typing tests the conventional method is to use red blood cell reagents of known blood groups to detect red blood cell antibodies in an unknown sample. However, the freshly prepared red blood cells are not easy to store, the antigenicity of the antigen on the surface of the red blood cell membrane is gradually reduced along with the prolonging of time, and the currently fresh human ABO blood group red blood cells can be stored for 3 months at most under the condition of 4 ℃, so that the blood group detection cost is greatly increased. Although there are many research institutes that disclose the use of lyophilization to prolong the storage time of red blood cells, intact human ABO blood group red blood cells are easily hemolyzed after lyophilization, and it is difficult to achieve neither hemolysis nor adverse effect on their use.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for preserving human ABO blood group red blood cells for a long time and an application thereof. The specific scheme is as follows.

A method for long-term preservation of human ABO blood group red blood cells, comprising the steps of:

step 1) washing of erythrocytes: washing packed red blood cells by using normal saline, then centrifuging at a low speed, and removing supernatant to obtain washed red blood cells;

step 2) erythrocyte fixing: adding the red blood cells washed in the step 1) into a fixing solution according to the volume ratio of 1 to 20, uniformly mixing, then carrying out low-speed centrifugation, removing supernatant, then adding 20 times of physiological saline, continuing the low-speed centrifugation, and repeating twice, wherein the fixing solution consists of 0.01-0.03% wt of glutaraldehyde, 0.4-0.8 g/L of sodium dihydrogen phosphate, 0.2-0.6 g/L of potassium chloride, 4-8 g/L of sodium chloride, 2-6 g/L of glucose and 2-6 g/L of sodium bicarbonate;

step 3) freeze-drying pretreatment: adding the erythrocytes fixed in the step 2) into the freeze-drying preservation solution according to the volume ratio of 1 to 20, uniformly mixing, then carrying out low-speed centrifugation, discarding the supernatant, then adding physiological saline with the volume 20 times that of the erythrocytes, continuously carrying out low-speed centrifugation, repeating twice, discarding the supernatant, then adding the freeze-drying preservation solution according to the volume ratio of 1 to 20, and uniformly mixing, wherein the freeze-drying preservation solution consists of 0.4 to 0.8 g/L of sodium dihydrogen phosphate, 0.2 to 0.6 g/L of potassium chloride, 4 to 8 g/L of sodium chloride, 8 to 12 g/L of trehalose and 3 to 7 g/L of bovine serum albumin;

step 4) red blood cell freeze-drying: freeze-drying the red blood cells treated in the step 3), and then storing at 2-8 ℃.

Further, the fixing solution consists of 0.03 wt% of glutaraldehyde, 0.68 g/L of sodium dihydrogen phosphate, 0.4 g/L of potassium chloride, 6 g/L of sodium chloride, 2 g/L of glucose and 2 g/L of sodium bicarbonate.

Glutaraldehyde in the fixing solution is used to fix hemoglobin and proteins on the erythrocyte membrane. The purpose of fixing the erythrocytes is to allow the erythrocytes to be lyophilized without hemolysis. In the prior art, glutaraldehyde is more used for observing cell morphology. If the erythrocytes are not fixed or insufficiently fixed before freeze-drying, the erythrocytes are hemolyzed to some extent; however, if the erythrocytes are fixed too sufficiently before lyophilization (e.g., too high a concentration of fixative or too long a time for fixation), the sensitivity of the antigen on the erythrocyte membrane may be affected, and even the antigen may be inactivated.

Further, the freeze-drying preservation solution consists of 0.68 g/L sodium dihydrogen phosphate, 0.4 g/L potassium chloride, 6 g/L sodium chloride, 10 g/L trehalose and 5 g/L bovine serum albumin.

The trehalose and the bovine serum albumin in the freeze-drying preservation solution are mainly used for protecting antigens on the surfaces of the erythrocytes, so that the antigens are not easy to fall off, and the sensitivity of the antigens on the surfaces of the erythrocytes after freeze-drying can be unchanged.

Further, the volume ratio of packed red blood cells washed with physiological saline in the step 1) is 10.

Further, the low-speed centrifugation operation in the step 1) is centrifugation for 3-6min at 600-800g, and the centrifugation is repeated for 3 times.

Further, the low-speed centrifugation operation in the step 2) and the step 3) is centrifugation for 3-6min at 600-800 g.

The centrifugal washing is performed for multiple times, and the effect of the centrifugal washing is to wash out residual components such as blood plasma, stationary liquid and the like so as to avoid interference on the freeze-drying process of the red blood cells.

Further, the freeze-drying operation of step 4) comprises: 1) Freezing: 4 ℃, 2h, 20 ℃ below zero, 2h,40 ℃ below zero and 2h; 2) Sublimation drying: setting the vacuum degree to be 5, and drying for 12 hours; 3) And (3) resolving and drying: the vacuum degree is set to 10, and the drying is carried out for 12h.

In the freezing step of the freeze-drying operation, a programmed cooling method is adopted, namely a gradient cooling method of 4 ℃ freezing for 2h, -20 ℃ freezing for 2h and-40 ℃ freezing for 2h is set, which is different from the traditional freeze-drying method that the temperature is reduced by the degree of every minute, the temperature is directly reduced to the preset temperature and then the preset temperature is maintained. In fact, the freeze-drying process is very harmful to cells, and if the freeze-drying operation is not proper, the freeze-dried cells are subjected to hemolysis at the moment of re-dissolving. After long-term research, the research team of the invention finds that the hemolysis can be minimized by adopting the programmed cooling method of the invention.

The freeze-dried human ABO blood group red blood cells prepared by the method.

The application of the freeze-dried human ABO blood group red blood cells in blood group reverse typing detection.

Further, the freeze-dried human ABO blood group red blood cells can be directly used for detecting antibodies in serum.

Specifically, the freeze-dried human ABO blood group red blood cells are redissolved by purified water with the same volume as that of freeze-dried liquid before freeze-drying without washing, and then are directly used for human ABO blood group reverse typing detection to detect the antibody in unknown serum.

Has the beneficial technical effects.

1. The invention innovatively uses a method of fixing the red blood cells firstly and then freeze-drying the red blood cells, the freeze-dried red blood cells are not easy to cause hemolysis, and the sensitivity of the antigen on the red blood cell membrane is equivalent to that of the red blood cells prepared freshly. Furthermore, the performance of the erythrocytes prepared by this method is consistent with that of commercially available countercommitted erythrocytes.

2. In the process of freeze-drying the erythrocytes, the programmed cooling operation is used, so that the activity of the erythrocytes can be effectively protected, and the erythrocytes obtained by subsequent freeze-drying are not easy to be hemolyzed.

3. The complete human ABO blood group red blood cells prepared by the method of fixing firstly and then freeze-drying can be stored for at least one year at the temperature of 2-8 ℃.

4. The complete freeze-dried human ABO blood group red blood cells prepared by the method can be directly used for blood group reverse typing detection, and washing is not needed before use. The general freeze-dried red blood cells need to be washed before use, firstly, the influence of freeze-drying liquid is eliminated, and secondly, red blood cell fragments and hemoglobin are washed away, and the freeze-dried red blood cells prepared by the method have complete shapes and do not undergo hemolysis, so that no extra red blood cell fragments and free hemoglobin exist. In addition, the freeze-drying protective solution provided by the invention does not have adverse effect on detection, so that the freeze-drying red blood cells can be directly used for blood type reverse typing detection.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrases "comprising a component of' 8230; \8230;" does not exclude the presence of another like element in a process, method, article, or apparatus that comprises the element.

As used in this specification, the term "about" typically means +/-5% of the stated value, more typically +/-4% of the stated value, more typically +/-3% of the stated value, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, and even more typically +/-0.5% of the stated value.

In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description of "in a certain range" is merely for convenience and brevity and should not be construed as an inflexible limitation on the disclosed range. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, the description of range 1-6 should be taken to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, e.g., 1,2,3,4,5 and 6. The above rules apply regardless of the breadth of the range.

Example one

This example provides a specific method for long-term preservation of human ABO blood group red blood cells.

Step 1) washing red blood cells: washing packed red blood cells with physiological saline, wherein the volume ratio of the packed red blood cells is physiological saline: red blood cells =10, then placed in a low-speed centrifuge for centrifugation for 3-6min at 600-800g, repeated for 3 times, and supernatant fluid is discarded to obtain washed red blood cells; the centrifugation operation comprises centrifugation at 610g for 6min, centrifugation at 716g for 5min or centrifugation at 795g for 3min.

Step 2) fixing red blood cells: fixing the red blood cells by using a fixing solution, and mixing the red blood cells washed in the step 1) in a volume ratio of 1: the ratio of the stationary liquid =1 = 20) is added into the stationary liquid, the mixture is mixed for 1min, then the mixture is put into a low-speed centrifuge for centrifugation for 3-6min at 600-800g, the supernatant is discarded, then 20 times of physiological saline is added for continuous centrifugation for 3-6min at 600-800g, and the centrifugation is repeated twice, wherein the stationary liquid consists of 0.03% wt of glutaraldehyde, 0.68 g/L of sodium dihydrogen phosphate, 0.4 g/L of potassium chloride, 6 g/L of sodium chloride, 2 g/L of glucose and 2 g/L of sodium bicarbonate; the centrifugation operation comprises centrifugation at 610g for 6min, centrifugation at 716g for 5min or centrifugation at 795g for 3min.

Step 3), freeze-drying pretreatment: adding 250 mu L of packed erythrocyte into 5mL of freeze-dried erythrocyte liquid, uniformly mixing for 1min, centrifuging for 3-6min at 600-800g, discarding the supernatant, adding 5mL of physiological saline, continuously centrifuging for 3-6min at 600-800g, repeating twice, discarding the supernatant, adding 5mL of freeze-dried preservation liquid, and uniformly mixing, wherein the freeze-dried preservation liquid consists of 0.68 g/L of sodium dihydrogen phosphate, 0.4 g/L of potassium chloride, 6 g/L of sodium chloride, 10 g/L of trehalose and 5 g/L of bovine serum albumin; the centrifugation operation comprises centrifugation at 610g for 6min, centrifugation at 716g for 5min or centrifugation at 795g for 3min.

Step 4), red blood cell freeze-drying: and (3) treating the red blood cells treated in the step 3): 1) Freezing: 4 ℃, 2h, 20 ℃ below zero, 40 ℃ and 2h; 2) Sublimation drying: setting the vacuum degree to be 5, and drying for 12 hours; 3) And (3) resolving and drying: setting the vacuum degree to 10, and drying for 12h.

And finally, storing the human ABO blood group red blood cells obtained by freeze-drying at 2-8 ℃.

Example two

Freeze-dried red blood cell hemolytic assay

1. Principle of detection

The erythrocyte is mainly composed of erythrocyte membrane and a large amount of hemoglobin, when the erythrocyte membrane is broken, the surface antigen of the membrane is destroyed, the hemoglobin is released, and the phenomenon of hemolysis occurs. Therefore, the concentration of free hemoglobin in the erythrocyte suspension with different storage periods can be used as one of the quality indexes of the erythrocyte in vitro storage effect (namely whether hemolysis occurs).

2. Detection method

1) Dissolving 0.1g of o-toluidine with 30mL of glacial acetic acid, adding distilled water to 50mL to prepare a 0.2% o-toluidine solution, and storing at 2-8 ℃; 3.67mL of 3% hydrogen peroxide is absorbed, distilled water is added to 100mL, 0.1% hydrogen peroxide solution is prepared, and the hydrogen peroxide solution is prepared fresh in each experiment; absorbing 50mL of glacial acetic acid solution, adding distilled water to 500mL, and preparing into 10% glacial acetic acid solution; 0.01g of bovine hemoglobin is weighed and dissolved in 1mL of physiological saline to prepare 10mg/mL of hemoglobin stock solution, and the hemoglobin stock solution is diluted to 100mg/L of hemoglobin standard solution when in use. 20 mu L of the supernatant to be detected is added into the measuring tube, 20 mu L of double distilled water is added into the blank tube, and 20 mu L of 100mg/L of hemoglobin standard solution is added into the standard tube. And then adding 1mL of 0.2% o-toluidine solution into each tube, then adding 1mL of 0.1% hydrogen peroxide solution, uniformly mixing and standing for 10min, adding 10mL of 10% glacial acetic acid solution into each tube, uniformly mixing and standing for 10min, and then measuring the absorbance by an ultraviolet-visible spectrophotometer. During the measurement, a blank tube is used for zero setting, and the detection wavelength is 435nm. The free hemoglobin concentration was determined by the following formula:

free hemoglobin concentration (mg/L) = measurement tube absorbance/standard tube absorbance × 100 (mg/L).

2) mu.L of the lyophilized erythrocytes stored according to the method of the present invention (20. Mu.L of the lyophilized erythrocytes of the present invention were reconstituted with purified water and the concentration was identical to that of commercially available erythrocytes) were added to the test tube, and 20. Mu.L of commercially available anti-fixed erythrocytes was added to the control tube. The results are shown in the following table. Data for commercial red blood cells at day 0 are not detectable, and since it is difficult to obtain commercial red blood cells produced that day, data for day 0 are missing from the table below.

TABLE 1 free hemoglobin concentration Change (mg/L) of type A erythrocyte suspension

TABLE 2 change in free hemoglobin concentration (mg/L) of type B erythrocyte suspensions

Remarking: the freeze-dried red blood cells and the red blood cells sold in the market are preserved at the temperature of 2-8 ℃.

The experimental results are as follows:

according to the national standard GB18469-2012 'quality requirement of whole blood and component blood', the hemolysis rate at the end of the storage of the washed red blood cells is less than 0.8 percent of the total amount of the red blood cells, namely the concentration of free hemoglobin is less than 880mg/L.

Along with the prolonging of the preservation time, the freeze-dried red blood cells are preserved for 420 days at the temperature of 2-8 ℃, and the concentration of free hemoglobin of each red blood cell suspension is basically unchanged; commercial cells rapidly increase free hemoglobin after 90 days, suggesting increased hemolysis, which does not meet national GB standards. However, after 240 days of the experiment, the concentration of free hemoglobin is decreased due to decomposition of hemoglobin by bacteria.

The recovery rate of the red blood cells after freeze-drying is in a descending trend along with the prolonging of the preservation time, and the hemolysis rate is not statistically different (P > 0.05) in different preservation time periods.

EXAMPLE III

Freeze-dried erythrocyte antigen titer detection

The titer is determined by a test tube method, which refers to the clinical blood transfusion detection operating protocol. The antigen titer of the lyophilized erythrocytes prepared in the first example of the present invention was measured and compared with those of the A-type and B-type in the commercial ABO reverse typing kit (human erythrocytes).

The detection method comprises the following steps:

taking out A type or B type lyophilized erythrocytes from refrigerator, re-dissolving with appropriate amount of purified water, transferring the re-dissolved erythrocytes to centrifuge tube with appropriate specification, centrifuging at 3000r for 5min, and collecting packed erythrocytes and making into 2% erythrocyte suspension with physiological saline. The commercially available erythrocytes were also prepared in the same manner as a 2% erythrocyte suspension and ready for use. Taking 12 test tubes, and sequentially marking as: 1. 100 μ l of a 2% suspension of erythrocytes of the corresponding blood type previously prepared is then added to each tube of the dilution, mixed and incubated for 15min at room temperature, centrifuged at 3000r for 10s, in order to obtain the titer of the antigen (e.g. 32, instead of 1/32 or 1. If agglutination still exists in the test tube with the highest dilution degree, indicating that the reaction end point is not reached, and continuing to dilute and detect.

TABLE 3 agglutination strength interpretation criteria

The lyophilized erythrocytes obtained by the method of example were reconstituted with purified water and compared with the antigen titer of commercially available erythrocyte suspensions according to the above measurement protocol. Data for commercial red blood cells at day 0 are not detectable, and since it is difficult to obtain commercial red blood cells produced that day, data for day 0 are missing from the table below.

TABLE 4 type A erythrocyte suspension antigen potency

TABLE 5 type B erythrocyte suspension antigen potency

The experimental results are as follows:

the A-type red blood cells prepared by the method of the first embodiment of the invention have no change when being stored for 390 days at 2-8 ℃, and have a decrease when being detected at 420 days. The antigen titer of the commercial A-type erythrocyte reagent can only be maintained for 90 days when being stored at 2-8 ℃, and gradually decreases from the detection of 120 days. Similar results were also obtained on type B red blood cell experiments.

Example four

Comparative test example 1

According to the principles and methods of the hemolysis detection/antigen titer detection of the lyophilized erythrocytes of the second and third examples, the hemolysis and antigen titer detection is carried out by fixing and protecting the erythrocytes of human type A and B with the fixing solution and the lyophilization protection solution of different test groups in the following tables, and then lyophilizing according to the method of the first example.

Comparative test example 2

According to the principles and methods of hemolysis test/antigen titer test of lyophilized erythrocytes in examples two and three, erythrocytes of human type A and B were fixed and protected by the fixing solution and lyophilization protection solution of group # 2 in comparative experiment example 1, and subjected to lyophilization by the different lyophilization methods in the following table for hemolysis test.

Results of the experiment

TABLE 6 change in free hemoglobin concentration (mg/L) of type A erythrocyte suspensions

TABLE 7 change in free hemoglobin concentration (mg/L) of type B erythrocyte suspensions

From the results in tables 6 and 7, the hemolysis rates of the 1# group, 2# group, 3# group and 1-2# group meet the requirements of the national standard GB 18469-2012. The 1-3# group and the 1-4# group use the traditional freeze-drying method, so the cell hemolysis is serious, the national standard requirement is exceeded, and the protection effect is not achieved. Therefore, the fixing link before the freeze-drying of the erythrocytes and the freeze-drying process are the key links for preventing the erythrocytes from being easily hemolyzed after the freeze-drying of the erythrocytes.

TABLE 8 type A erythrocyte suspension antigen potency

TABLE 9 antigenic potency of type B erythrocyte suspensions

As shown in the results of tables 8 and 9, the lyophilized erythrocytes obtained from group # 1, group # 2, group # 3 and group # 1-1 had the antigen titer of the surface A antigen and B antigen preserved at 2-8 ℃ for at least one year. Therefore, the erythrocyte freeze-drying preservation solution and the freeze-drying process can be proved to be the key links for maintaining the unchanged sensitivity of the erythrocyte surface antigen. However, the antigen titer of 1-2# was significantly lower than that of the other control groups from the 0 day detection, which indicates that too much fixation affects the sensitivity of the antigen on the erythrocyte membrane; 1-1# although the good antigen sensitivity was maintained by the effective lyophilization process protection, hemolysis was severe because it was not immobilized. Therefore, it is not simple to lyophilize human erythrocytes with neither hemolysis nor to maintain the sensitivity of surface antigens.

As is clear from the results shown in tables 6, 7, 8 and 9, the lyophilized erythrocytes obtained from group # 1 to group # 3 were preserved at 2-8 ℃ for at least 1 year.

The foregoing detailed description is to be construed as illustrative only and not limiting, and many forms can be made by those skilled in the art without departing from the spirit and scope of the invention as set forth in the appended claims.

Claims

1. A method for long-term preservation of human ABO blood group red blood cells, comprising the steps of:

step 2) erythrocyte fixing: adding the red blood cells washed in the step 1) into a fixing solution according to the volume ratio of 1 to 20, uniformly mixing, centrifuging at a low speed, removing supernatant, adding 20 times of physiological saline, continuously centrifuging at a low speed, and repeating twice, wherein the fixing solution consists of 0.01-0.03% of wt glutaraldehyde, 0.4-0.8 g/L of sodium dihydrogen phosphate, 0.2-0.6 g/L of potassium chloride, 4-8 g/L of sodium chloride, 2-6 g/L of glucose and 2-6 g/L of sodium bicarbonate;

step 3) freeze-drying pretreatment: adding the erythrocytes fixed in the step 2) into a freeze-drying preservation solution according to the volume ratio of 1 to 20, uniformly mixing, centrifuging at a low speed, discarding the supernatant, adding 20 times of physiological saline, continuously centrifuging at a low speed, repeating twice, discarding the supernatant, adding the freeze-drying preservation solution according to the volume ratio of 1 to 20, and uniformly mixing, wherein the freeze-drying preservation solution consists of 0.4 to 0.8 g/L of sodium dihydrogen phosphate, 0.2 to 0.6 g/L of potassium chloride, 4 to 8 g/L of sodium chloride, 8 to 12 g/L of trehalose and 3 to 7 g/L of bovine serum albumin;

2. The method of claim 1, wherein the fixative solution consists of 0.03% wt glutaraldehyde, 0.68 g/L sodium dihydrogen phosphate, 0.4 g/L potassium chloride, 6 g/L sodium chloride, 2 g/L glucose, and 2 g/L sodium bicarbonate.

3. The method of claim 1, wherein the lyophilized preservation solution consists of 0.68 g/L sodium dihydrogen phosphate, 0.4 g/L potassium chloride, 6 g/L sodium chloride, 10 g/L trehalose, and 5 g/L bovine serum albumin.

4. The method according to claim 1, wherein the volume ratio of packed red blood cells washed with physiological saline in step 1) is 10.

5. The method as claimed in claim 1, wherein the low speed centrifugation operation in step 1) is 600-800g centrifugation for 3-6min, which is repeated 3 times.

6. The method as claimed in claim 1, wherein the low speed centrifugation operation in step 2) and step 3) is 600-800g centrifugation for 3-6min.

7. The method of claim 1, wherein the lyophilizing of step 4) comprises: 1) Freezing: 4 ℃ for 2h, 20 ℃ below zero for 2h,40 ℃ below zero for 2h; 2) Sublimation drying: setting the vacuum degree to be 5, and drying for 12 hours; 3) And (3) resolving and drying: setting the vacuum degree to 10, and drying for 12h.

8. Lyophilized human ABO blood group red blood cells obtained by the method of any one of claims 1-7.

9. Use of the lyophilized human ABO blood group red blood cells of claim 8 in a blood group reverse typing assay.

10. The use of claim 9, wherein said lyophilized human ABO blood group red blood cells are used directly for the detection of antibodies in serum.