CN108575987A - Red blood cell processing method and its application - Google Patents

Abstract

The invention provides a method for treating red blood cells, which comprises collecting red blood cells and sequentially contacting the red blood cells with a saline solution containing bromelain and a saline solution containing glutaraldehyde. The invention also provides a soluble membrane containing the red blood cells, a reagent card and a method for identifying blood types by using the red blood cells. The erythrocytes treated by the method of the present invention have a long shelf life and are easy to transport, and can be pre-stored in the reaction chamber of the reagent card directly or in the form of a soluble film, and the test result can be obtained conveniently after adding the serum or plasma to be tested. Anti-determined test pre-added operation of anti-determined cells.

Description

Red blood cell processing method and its application

technical field

The invention relates to a method for processing red blood cells and its application, belonging to the fields of biotechnology and medicine.

Background technique

The ABO blood type is determined by both cell surface antigens and antibodies in the blood. Only by testing both the positive and negative typing can the blood type be correctly determined. In order to increase the reliability of ABO positive typing, it is necessary to perform a reverse typing test at the same time as the positive typing test, that is, to use known blood type standard red blood cells as reagents to detect blood group antibodies in the serum or plasma to be tested. This involves the preservation of red blood cell reagents. Usually the erythrocyte reagent is in the erythrocyte preservation solution, maintained at 2-8°C, and requires a full cold chain during transportation. Red blood cell reagents are susceptible to hemolysis due to the influence of external temperature. For example, during transportation, if the temperature is too high, the lifespan of the cells will be significantly reduced, and the direct contact of the cells with ice cubes used for temperature control will cause cell hemolysis. In addition, excessive turbulence during transportation will also cause cell hemolysis and reduce cell life, so the validity period of the reagent red blood cells is usually only 2 to 3 months.

In addition, regarding the reverse determination of blood type testing, there is currently no method in the industry to add red blood cells to the reaction system in advance, and it is necessary to temporarily take the stored red blood cell reagents before the test, which increases the complexity of the operation.

Contents of the invention

In order to overcome the above problems, in one aspect, the present invention provides a method of processing red blood cells, which comprises collecting red blood cells and sequentially contacting the red blood cells with a bromelain-containing physiological saline solution and a glutaraldehyde-containing physiological saline solution.

In one embodiment, the content of bromelain in the physiological saline solution containing bromelain is 0.1 mg/ml to 10 mg/ml.

In one embodiment, said contacting comprises incubating said erythrocytes with said bromelain-containing saline solution at 37° C. for 30 min.

In one embodiment, the content of glutaraldehyde in the glutaraldehyde-containing physiological saline solution is 0.03% to 1% by weight.

In one embodiment, said contacting comprises incubating said erythrocytes with said glutaraldehyde-containing saline solution for 1 hour at room temperature or overnight at 4°C.

In one embodiment, the erythrocytes are human type A erythrocytes, human type B erythrocytes, human type AB erythrocytes or human type O erythrocytes.

In another aspect, the present invention provides erythrocytes obtained by the above method.

In another aspect, the present invention provides a soluble membrane containing red blood cells prepared by the following steps:

1) adding the above-mentioned treated red blood cells into the solution of water-soluble polymer film-forming material, and mixing; and

2) Allow the mixture obtained in step 1) to form a solid film.

In some embodiments, the water-soluble polymer film-forming material is selected from PVA, PVP/PVA composite, PAA, and chitosan. In preferred embodiments, the weight ratio of PVP to PVA in the PVP/PVA composite may be 7:3, 6:4, 5:5 or 4:6.

In some embodiments, step 2) forms the mixture into a solid film by using blow molding, casting, or electrospinning.

In some embodiments, the dissolvable film has a thickness of 10 μm to 1 mm.

On the other hand, the present invention provides a method for reverse ABO blood typing, comprising contacting the serum or plasma to be tested with red blood cells treated by the method of the present invention or with the soluble membrane.

On the other hand, the present invention provides a reagent card for reverse ABO blood typing, wherein one or more reaction chambers of the reagent card contain red blood cells or the soluble membrane treated by the method of the present invention.

The erythrocytes processed by the method of the present invention have a long shelf life and are easy to transport, and can be stored in the reaction chamber of the reagent card directly or in the form of a soluble film, and the detection results can be obtained conveniently after adding the serum or plasma to be tested, reducing the The operation of the negative test.

Description of drawings

Figure 1 shows the photos of the reverse quantitative performance comparison of untreated A cells and membrane-forming reconstituted A cells.

Fig. 2 shows the photos of the test results of untreated A cells, formylated A cells, and membrane-forming reconstituted A cells on plasma samples of different blood types.

Detailed ways

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

As used herein, "bromelain" refers to the polypeptide hydrolase derived from pineapple, which belongs to sulfhydryl protease, also known as bromelain (bromelain), with a molecular weight of about 33,000, which has been widely used in the food and pharmaceutical industries.

As used herein, "physiological saline solution" refers to a sodium chloride solution having an osmotic pressure substantially equal to that of human blood plasma, usually with a sodium chloride concentration of 0.85-0.9%. Or it can also be replaced by a physiologically compatible buffer solution, such as a phosphate buffer solution. At this time, "physiological saline solution" can also include these buffer solutions in a broad sense.

As used herein, "type A red blood cells," "type B red blood cells," "type AB red blood cells," or "type O red blood cells" designate red blood cells by their surface antigens: a red blood cell with an A antigen is a type A red blood cell, and a red blood cell with a B Red blood cells with antigens are type B red blood cells; red blood cells with both A and B antigens are type AB red blood cells; red blood cells with neither A nor B antigens are type O red blood cells. Herein, type A erythrocytes are also sometimes simply referred to as "A cells".

As used herein, "solid film" or "membrane" refers to a sheet-like structure formed by film-forming polymers, the thickness of which is generally much smaller than the length and width. In the selection of film-forming polymers, water-soluble polymer materials are generally selected. The polymer material should be relatively stable in terms of chemical properties, and will not chemically react with red blood cells or the substance to be detected. In addition, it is hoped that the solution of the polymer material has better film-forming properties at different concentrations. The polymer material of aqueous solution state can adopt multiple suitable methods to form film-like thing, for example, flow-casting method film formation (for example, liquid is spread on horizontal stainless steel plate or glass plate with the scraper of certain size, then dry to form film ). When forming a film, a certain amount of red blood cells treated with pineapple enzyme and glutaraldehyde can be added to the polymer material solution in solution state, so that red blood cells are entrained in the formed film. The formed film can be stored for a long time after being dried. The content of erythrocytes in the membrane can be controlled by controlling the mixing ratio of erythrocytes and membrane-forming polymers before membrane formation. For example, 2-15% (wt) film-forming polymer solution and red blood cell suspension can be mixed at a volume ratio of 1:1 to form a film. A film formed of a soluble polymer material can be completely or partially dissolved when exposed to a solvent (such as water, or aqueous serum, plasma), and is referred to as a "dissolvable film" herein. These film-forming polymer materials include, for example but not limited to, polyvinyl alcohol (PVA), polyvinylpyrrolidone/polyvinyl alcohol (PVP/PVA) composites, polyacrylic acid (PAA), and chitosan, etc., or other A substance that forms a membrane with red blood cells, for example, that dissolves rapidly in serum, allowing the red blood cells to react with the serum.

As used herein, "formylated cells" refer to erythrocytes that have been treated with bromelain and glutaraldehyde without undergoing the membrane-forming process. "Membrane-forming reconstituted cells" or "reconstituted cells" refer to erythrocytes that have been treated with bromelain and glutaraldehyde to form a film with a film-forming polymer, and then reconstituted with saline solution (for example, erythrocytes The concentration can be 1-10%).

As used herein, "reagent card" and "test card" are used interchangeably and refer to a card-shaped carrier on which wells, grooves, chambers, etc. for carrying out chemical or biological reactions are formed. For example, a blood type reagent card is shown in Figure 1, in which a plurality of microcolumns arranged in parallel are formed in the card body. Red blood cells, used for blood typing.

The present invention is further illustrated below through specific examples.

Example 1. Red blood cell processing and film formation and redissolution

Red blood cell treatment: take 2ml of 3% (vol) fresh A cells, centrifuge at 2000rpm (380g) for 3min, remove the supernatant, wash with 2ml of normal saline for three times, discard the supernatant, and resuspend with 2ml of normal saline containing 1mg/ml pineapple enzyme red blood cells. The resuspended erythrocytes were incubated at 37° C. for 30 min, then centrifuged and washed 3 times with 2 ml of normal saline, and the supernatant was discarded after the last washing, and the erythrocytes were resuspended with 2 ml of normal saline containing 0.1% glutaraldehyde. The resuspended erythrocytes were incubated overnight at 4°C on a rotator mixer. After incubation, wash 4 times with 2 ml of normal saline, and finally resuspend red blood cells with 2 ml of normal saline to obtain red blood cell suspension, which is stored at room temperature.

Erythrocyte film formation: mix 10% (wt) PVA aqueous solution with the above-prepared erythrocyte suspension 1:1 (vol), and dry to form a film by casting method with a thickness of 20 μm. Store the membrane at room temperature for future use.

Red blood cell reconstitution: Dissolve the membrane with an appropriate amount of normal saline before use until the red blood cell concentration is 3%.

Example 2. Reverse titer analysis of type A erythrocytes preserved in different ways

Take 15 μl of 3% A cells with different storage time: untreated A cells in the red blood cell preservation solution and the film-forming and reconstituted A cells of Example 1, and add them to the reverse blood group test card. Next, take 15 μl of the anti-A working solution diluted twice with the antibody diluent, add it to the above test card, centrifuge at 55 g for 3 min, and centrifuge at 200 g for 2 min. The observation results are shown in Figure 1. The figure shows that the detection performance of untreated A cells decreased by about 2 times after storage at 4°C for 2 months, while the untreated A cells failed to perform the reverse assay due to severe hemolysis at 6 months. The A cells preserved by the dissolvable membrane did not show any hemolysis phenomenon and the anti-assay performance did not decrease significantly when they were preserved at room temperature for 2 months and 6 months.

Example 3. Contrastive testing of A cells preserved in different ways with plasma samples

Take 15 μl of 3% A cells with different storage times: untreated A cells (stored in erythrocyte preservation solution), formaldehyde A cells of Example 1 (stored in physiological saline) and membrane-forming reconstituted A cells (preserved in a dissolvable film, reconstituted with physiological saline before use), and added to the reverse blood type test card. Next, take 15 μl of type A, type B, type AB and type O plasma respectively, add them to the above test card, centrifuge at 55g for 3min, and 200g for 2min, the results are shown in Figure 2. The figure shows that untreated type A red blood cells have a slight decline in assay performance after 2 months of storage at 4°C, while untreated A cells cannot be tested at 6 months due to severe hemolysis. The A cells preserved by formaldehyde or film-forming were stored at room temperature for 6 months without any hemolysis and no significant decline in detection performance.

In addition, similar results were obtained by repeating the above example with B-type erythrocytes, AB-type erythrocytes and O-type erythrocytes (results not shown). In the invention, after the red blood cells are treated with bromelain and glutaraldehyde, the red blood cells are no longer hemolyzed, and at the same time, the activities of A and B antigens are retained, and can be used for ABO reverse typing. The erythrocytes after hydroformylation and film-forming treatment have better stability, have the advantages of long shelf life, and can be transported at room temperature. The anti-fixed erythrocyte reagent prepared by the invention has a shelf life of at least 6 months at 2-25°C, and greatly reduces the requirements for transportation. In this way, the transportation cost can be saved, and the product stability and validity period can be improved.

In addition, in the above Examples 2 and 3, in order to compare with untreated A cells, the hydroformylated or film-forming and reconstituted A cells of the present invention were added to the test card only during the experiment, but those skilled in the art It can be understood that the hydroformylated or membrane-forming reconstituted A cells of the present invention can be directly stored in the test card. For example, after the erythrocytes processed by the method of the present invention form a film, they can be conveniently pre-distributed to each test hole of the test card. When the user performs a reverse test with the test card, the sample to be tested (such as serum or Plasma) is added to the sample hole, and after the serum or plasma enters the reaction chamber of the test card, the dissolvable membrane is quickly dissolved, so that the released red blood cells can quickly participate in the reaction. In this way, the shelf life of red blood cells can be extended, and the operation process of reverse determination test can be reduced.

Therefore, the embodiments described above are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments provided herein, all other embodiments obtained by those skilled in the art without making creative efforts fall within the protection scope of the present invention.

Claims (13)

1. a kind of method of processing red blood cell, including collect red blood cell and successively with containing bromelain normal saline solution and containing penta The normal saline solution of dialdehyde contacts the red blood cell.

2. the method as described in claim 1, wherein the content of bromelain is in the normal saline solution containing bromelain 0.1mg/ml to 10mg/ml.

3. method as claimed in claim 1 or 2, wherein the contact includes allowing the red blood cell and the life containing bromelain Reason saline solution one arises from 37 DEG C of incubation 30min.

4. the method as described in claim 1, wherein the content of glutaraldehyde is with weight in the normal saline solution containing glutaraldehyde Amount is calculated as 0.03% to 1%.

5. method as described in claim 1 or 4, wherein the contact includes allowing the red blood cell and the life containing glutaraldehyde Reason saline solution is incubated at room temperature 1 hour or 4 DEG C and is incubated overnight together.

6. the method as described in claim 1, wherein the red blood cell behaviour red blood cells of type A, human B-type red blood cell, people's AB types are red Cell or the O-shaped red blood cell of people.

7. the red blood cell for handling and obtaining through method according to any one of claims 1 to 6.

8. a kind of dissolvable film containing red blood cell, is made by the steps：

1) red blood cell of claim 7 is added in the solution of water soluble polymer filmogen, mixing；And

2) mixture that step 1) obtains is allowed to form solid film-shaped object.

9. dissolvable film as claimed in claim 8, wherein the water soluble polymer filmogen is multiple selected from PVA, PVP/PVA Close object, PAA and chitosan.

10. dissolvable film as claimed in claim 8, wherein step 2) allow the mixture to form solid-state by using the tape casting Membranoid substance.

11. dissolvable film as claimed in claim 8, thickness is 10 μm to 1mm.

12. a kind of method carrying out abo blood group reverse type, including serum or blood plasma to be checked and the red blood cell of claim 7 is allowed to connect It touches or is contacted with the dissolvable film described in any one of claim 8 to 11.

13. a kind of abo blood group reverse type reagent card, wherein including claim in one or more reaction chambers of the reagent card The dissolvable film of any one of 7 red blood cell or claim 8 to 11.