KR20210002212A - Production of Glutaraldehyde-treated red blood cells with weakened expression of ABO and RhD antigens for use as a quality control material in tube technique - Google Patents

Abstract

The present invention relates to a method and composition for preparing red blood cells for quality control of in vitro blood type testing, in which ABO and RhD blood type phenotypes are simultaneously attenuated by treating red blood cells with glutaraldehyde. The present invention is to provide red blood cells for quality control of in vitro blood type testing by treating glutaraldehyde to red blood cells to attenuate ABO and RhD blood type phenotypes.

Description

Production of Glutaraldehyde-treated red blood cells with weakened expression of ABO and RhD antigens for use as a quality control material in tube technique, with weakened ABO and RhD blood type phenotypes }

The present invention relates to a method for producing red blood cells and a composition for producing red blood cells for quality control in an in vitro blood type test in which glutaraldehyde is treated on red blood cells to weaken the ABO and RhD blood type phenotypes.

ABO and RhD blood type tests are very important tests that require the accuracy of the tests as they are directly related to the success of the patient's transplantation or the life of the patient. In particular, if the ABO subtype of a weakly expressed blood donor is regarded as type O or the patient's RhD variant is incorrectly determined as positive for normal RhD, it is possible to induce antibodies to the patient during transfusion or to cause side effects of hemolytic transfusion.

The transfusion medicine program of the Korean Association for Clinical Quality Management includes a subtype ABO test twice a year and a drug D test that detects RhD variants through a special antigen test program since 2016. However, the rate of correct answer for the 2016-2018 ABO subtype blood group was 80.7% to 96.0% of the participating institutions, and 34.8% to 80.9% of the institutions reporting the RhD variant intended for the drug D test, depending on the rounds. Compared to the sport, the percentage of correct answers was very low. In particular, the drug D test samples sent in 2017 were all weak compared to normal RhD positive in the RhD blood type test, but 47.6% and 21.3% of the institutions that reported it as RhD positive even though they were RhD mutant samples that showed aggregation. In addition, 63.8% of organizations incorrectly reported RhD mutant samples with weakly expressed RhD antigens sent out in the second round in 2018 as RhD negative. These data show weak aggregation seen in ABO subtypes and RhD variants in addition to aggregation of normal red blood cells with 4+ aggregation grade, which is used as an internal quality control substance for ABO and RhD blood group testing in all laboratories (e.g., 2+ or less. ) This suggests that visible red blood cells need to be used as quality control materials in actual laboratory.

After the report that glutaraldehyde reduces erythrocyte ABO antigen, attempts to treat red blood cells with glutaraldehyde were reported for the purpose of preparing pluripotent blood for transfusion or extending the shelf life of erythrocyte reagents, and the preparation method varied for each report. I did. In particular, although there have been reports of ABO blood type and RhD blood type antigen modification on the possibility of producing red blood cells for the purpose of pluripotent donating blood using glutaraldehyde in Korea, this was only applied as a laboratory study of antigen modification for the purpose of pluripotent donating blood. Regarding the lowering of the titer of the type test reagent, a high glutaraldehyde solution of 2 to 3% was used, but the weakened hemagglutination level was not shown.

In addition, KODE technology and a method of modifying red blood cell H antigen using a glycosyltransferase enzyme have been reported as a method of expressing a weak ABO blood type to prepare a weak phenotypic quality control substance for ABO blood type testing, but reports on red blood cells treated with glutaraldehyde There was no. In addition, these chemical or enzyme-based methods are limited to ABO blood type antigens, where carbohydrates are the main antigens, so there are hardly any attempts as a weak phenotypic quality control substance for RhD blood type testing, where protein is the main antigen.

Under this background, the present inventors have made intensive research efforts to produce red blood cells for in vitro blood type testing, and as a result, glutaraldehyde at a specific concentration exhibits a weak phenotype for both ABO and RhD blood type tests, thereby producing red blood cells suitable for use as quality control substances. It confirmed that it can be used to, and completed the present invention.

One object of the present invention is to provide a method for producing red blood cells for quality control of an in vitro blood type test, comprising the step of reducing the ABO and RhD blood type phenotypes by treating red blood cells with glutaraldehyde.

Another object of the present invention is to provide a composition for producing red blood cells for quality control of in vitro blood type testing, comprising glutaraldehyde as an active ingredient.

One aspect of the present invention for achieving the above object is to provide a method for producing red blood cells for quality control of an in vitro blood type test, comprising the step of simultaneously weakening the ABO and RhD blood type phenotypes by treating red blood cells with glutaraldehyde.

In addition, another aspect of the present invention provides a composition for producing red blood cells for quality control of an in vitro blood type test, comprising the step of reducing ABO and RhD blood type phenotypes by treating red blood cells with glutaraldehyde.

Hereinafter, the present invention will be described in detail.

The term "glutaraldehyde" of the present invention means a compound having the structure of the following formula (I). Glutaraldehyde is a material widely used in laboratory as a preservative and fixative. It is composed of 8 hydrogen atoms, 5 carbon atoms and 2 oxygen atoms to form a total of 15 atoms, and there are 14 chemical bonds in the molecule, which are 6 non-hydrogen bonds, 2 multiple bonds, 4 single bonds, 2 It consists of two double bonds and two aldehydes (aliphatic).

[Formula 1]

As used herein, the term "red blood cells" refers to cells in the shape of a disc with a hollow in the center, and is one of the blood cells present in the blood. Specifically, red blood cells are donut-shaped blood cells specialized for transporting oxygen from the lungs to all the cells of the body, and contain about 280 million pieces of a protein called hemoglobin that has a high oxygen retention capacity. In this way, a single red blood cell can carry about 1.12 billion oxygen molecules. Red blood cells are attached to the outer wall of the cell membrane as a mixed structure of carbohydrates, fats, and proteins, and the structures act as antigens to determine blood types.

The red blood cells of the present invention exhibit a phenotype similar to the ABO subtype or RhD variant in which the blood type antigen is weakened, because glutaraldehyde can mask all kinds of antigens in the red blood cell membrane, and the higher the concentration, the weaker the blood type antigen appears. Because. Specifically, the term "ABO subtype or RhD variant" of the present invention appears when the number of antigens in the red blood cell membrane is less than normal or the antigen is qualitatively modified, and most of them can be caused by inherited factors such as genetics or diseases. Is known.

The term “ABO blood type” in the present invention is a blood type classification method discovered by Karl Landsteiner of Austria in 1901. The types of human blood types classified according to the ABO formula are A, B, AB, and O. There are, and the AB type can be divided into A _₁ B and A _₂ B type. Basically, it is classified by whether or not hemagglutination is caused by the reaction of blood type antigen and antibody.

The term "RhD blood type" of the present invention is a classification method according to the presence or absence of D antigen in red blood cells, and it is classified as Rh positive if D antigen is present and Rh negative if there is no D antigen.

The term "in vitro blood type test" of the present invention is one of ABO blood type determination test or RhD blood type test technique. Specifically, for the ABO blood type test, both a hemocytosis test for antigens in red blood cells and a serotype test for antibodies in serum should be performed, and only hemocytotype test for RhD blood type is performed. More specifically, the blood type test is known to be safer because the manual method using slides or test tubes is used in many institutions, and the test tube method is more accurate than the slide method and there is no risk of exposure to the sample to examiners. More specifically, all of the above tests are based on hemagglutination, and the aggregation level is divided into 0, 0.5+ (trace), 1+, 2+, 3+ and 4+, and the stronger the blood type antigen, the better the relationship with the blood type reagent. In the reaction, hemagglutination is strong and hemagglutination grade is also increased, and normal type A, B, AB, and RhD positives show strong hemagglutination of 3+ or more in most of the blood type reagents anti-A, anti-B and anti-D. . The red blood cells for quality control of the in vitro blood type test of the present invention can be used for both the slide method and the test tube method, but cannot be used in the recently introduced columnar aggregation method.

The term "quality control red blood cells" of the present invention is red blood cells that are already aware of the result value that performs the same test as the patient's sample during the ABO blood type or RhD blood type determination test, and serves to confirm the reliability of the blood type test performed in the laboratory every day. In charge of Specifically, when ABO and RhD blood type tests were performed with red blood cells known to show an aggregation grade of 2+ in anti-A, anti-B and anti-D reagents, if the anti-D reagent was not 2+, but 0.5+ If the agglutination grade was shown, the RhD blood type test performed with anti-D reagents on the same day is unreliable, so other patient samples cannot be tested until the cause is resolved.

In the ABO blood type test and RhD test, only normal red blood cells showing an erythrocyte aggregation grade of 3+ or higher are used as quality control substances in the actual laboratory. At this time, the red blood cells for quality control of the in vitro blood type test of the present invention are used instead or additionally. It can be used as a substance or as an educational substance for laboratory personnel to detect weak phenotypes.

The term “quality control” of the present invention refers to all means of managing an analysis value within a certain confidence range through statistical processing in order to ensure the accuracy and precision of the analysis value of measurement and examination in work environment measurement and health examination.

The method of treating glutaraldehyde to red blood cells of the present invention may include reacting glutaraldehyde with red blood cells for 30 minutes in a refrigerated state, and the concentration of adding glutaraldehyde may be 0.1 to 0.35% by weight, It is not limited thereto.

In a specific embodiment of the present invention, a red blood cell concentrate of A _₁ B type RhD-positive blood type is prepared and reacted with glutaraldehyde diluted to 0.25% to weaken the AB type antigen and the RhD antigen at the same time. It was confirmed that aggregation of 2+ grades occurred in reactions of anti-A, a blood type reagent, and anti-B, and anti-D, a RhD blood type reagent (Figs. 1 to 2), and glutar diluted to 0.25% Refrigerated storage of red blood cells whose phenotype was weakened by treatment with aldehydes, and even if these red blood cells reacted with ABO blood type reagent and RhD blood type after one week, the aggregation grade of about 2+ was maintained, and it was confirmed that storage for one week was possible (Figure 3). .

Another aspect of the present invention comprises the steps of: (a) washing red blood cells with saline; (b) treating the washed red blood cells with glutaraldehyde; (c) separating red blood cells; And (d) preparing a blood cell suspension from the separated red blood cells, providing a method for producing red blood cells for quality control of a test tube method blood type test.

The terms of the present invention, "red blood cells", "glutaraldehyde", "in vitro blood type test" and "quality control red blood cells", are as described above.

More specifically, more specifically, the red blood cells in step (a) may be concentrated red blood cells of the A _₁ type B RhD-positive EDTA residual sample, and the amount is not limited thereto because it is proportional to the amount of red blood cells to be prepared. The saline solution may have a pH of 6 to 8, specifically 7.3 to 7.4, and may be washed with a known method under conditions of 2500 to 3500 rpm, specifically 3000 rpm. In addition, the washing time may be 1 to 10 minutes, specifically 2 to 5 minutes, and 1 to 5 washing times, but is not limited thereto.

The step (b) is a step of treating glutaraldehyde to the red blood cells washed in step (a), wherein the glutaraldehyde is 0.1 to 5%, specifically 0.1 to 0.5%, more specifically 0.1 to 0.35 It may be glutaraldehyde diluted in %, and the diluted glutaraldehyde may be reacted with the washed red blood cells in a refrigerated state of preferably 1 to 8 degrees Celsius.

The step (c) is a step of separating red blood cells from the reaction product of step (b). Specifically, the glutaraldehyde-treated red blood cells cultured in a refrigerated solution are used as a supernatant at 2500 to 4000 rpm, specifically 3000 to 3500 rpm. It may be a removing step, and the condition may be 5 to 20 seconds, specifically 10 to 18 seconds, washing 1 to 5 times to remove the supernatant, but is not limited thereto.

The step (d) is a step of preparing a blood cell suspension from the red blood cells separated in step (c), specifically, 1 to 5% blood cell suspension, more specifically 2 to 5% blood cell suspension using saline It may be to prepare, but is not limited thereto, and a method of preparing a blood cell suspension may be prepared by a generally well-known method.

The present invention is to provide red blood cells for quality control of in vitro blood type tests by treating glutaraldehyde to red blood cells to weaken the ABO and RhD blood type phenotypes.

FIG. 1 is a photograph showing a blood cell aggregation level by reacting A _₁ B type RhD-positive red blood cells treated with various concentrations of glutaraldehyde and ABO type and RhD blood type reagents. It can be seen that red blood cells treated with glutaraldehyde diluted to 0.25% showed a 2+ degree of hemagglutination, which is commonly seen in the ABO subtype or suspected of the RhD variant in the ABO and RhD blood group reagents.
Figure 2 is a graph confirming the aggregation grade from 0 to 4+ by reacting red blood cells of 10 A _₁ B RhD-positive samples treated with various concentrations of glutaraldehyde with various blood type reagents. The numbers in (parentheses) mean the average hemagglutination level of 10 samples, and the stronger the glutaraldehyde concentration, the lower the hemagglutination level.
3 is a graph showing the change in aggregation level over time by refrigerating the red blood cells of 10 A _₁ B RhD positive samples treated with 0.25% diluted glutaraldehyde for 7 days and then reacting with various blood type reagents. . The numbers in (brackets) refer to the average hemagglutination strength of 10 samples.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

Example 1. Preparation of red blood cell concentrate

In order to prepare red blood cells to be used as a specimen, the following experiment was performed.

Specifically, 1 mL of concentrated red blood cells from a fresh A _₁ B-type RhD-positive EDTA residual sample was put into a tube using a pipette, and then pH 7.3 to 7.4 phosphate buffer solution was used for 3 minutes at 3000 rpm for 3 minutes. It was washed twice to prepare a red blood cell concentrate.

Example 2. Preparation of diluted glutaraldehyde

In order to confirm an appropriate concentration of glutaraldehyde for attenuating the red blood cell phenotype, the following experiment was performed.

Specifically, 25% glutaraldehyde may be diluted to a desired concentration by diluting it with sterile saline, and more specifically, 0%, 0.5%, 0.375%, 0.3125%, 0.25% and 0.125% in Example 1 The prepared _A₁ B type RhD positive red blood cell concentrate was treated.

As a result, as shown in FIGS. 1 and 2, when red blood cells treated with 0% glutaraldehyde were reacted with anti-A, anti-B, and anti-D blood type reagents, a strong aggregation reaction of normal 4+ grade This occurred, and the red blood cells treated with glutaraldehyde diluted to 0.5% did not aggregate. On the other hand, in the red blood cells treated with glutaraldehyde diluted to 0.25%, it was confirmed that 2+ grade aggregation occurred in all of the blood type reagents tested (FIGS. 1 and 2).

Through this, it was considered that glutaraldehyde diluted to 0.25% was a suitable concentration for preparing red blood cell subtypes, and the following experiment was conducted with glutaraldehyde at the concentration.

Example 3. Preparation of red blood cells for quality control of blood type test

The following experiment was performed to prepare red blood cells for quality control of blood type tests with modified phenotypes.

Example 1 1 mL of the prepared red blood cell concentrate and the glutaraldehyde diluted in Example 2 were reacted in a refrigerated state for 30 minutes, and then the glutaraldehyde-treated red blood cells cultured in the refrigerator were subjected to phosphate buffered saline at 3400 rpm for 15 seconds, 3 times. After washing and removing the supernatant, a 3 to 5% blood cell suspension was prepared using phosphate buffered saline. Even after 7 days of glutaraldehyde-treated red blood cells prepared in this way were tested for in vitro blood typing, it was confirmed that the aggregation level of about 2+ was maintained, and it was confirmed that refrigeration for at least one week was possible (FIG. 3).

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

Claims (6)

A method for producing red blood cells for quality control of an in vitro blood type test, comprising the step of simultaneously weakening the ABO and RhD blood type phenotypes by treating glutaraldehyde on red blood cells.
The method of claim 1, wherein the glutaraldehyde is represented by the following formula (I).
[Formula 1]

The method of claim 1, wherein the concentration of glutaraldehyde is 0.1% to 5% by weight.
The method of claim 1, wherein the concentration of glutaraldehyde is 0.1% to 0.35% by weight.
A composition for producing red blood cells for quality control of in vitro blood type testing, comprising glutaraldehyde as an active ingredient.
The composition of claim 5, wherein the concentration of glutaraldehyde is 0.1% to 0.35% by weight.

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