Red blood cell preservation reagent, red blood cell preservation pretreatment method of reagent and application of red blood cell preservation reagent

Abstract

The invention relates to a red blood cell preservation reagent, the red blood cell preservation reagent comprises a single aldehyde, the single aldehyde is glutaraldehyde, and the mass fraction of the glutaraldehyde in a solvent is 0.00125%-0.04%. The invention also provides a method for preserving red blood cells and application of the red blood cell preservation reagent in blood group reverse typing detection, blood group antibody titer determination or red blood cell antibody screening and identification, and the application is not used for disease diagnosis and treatment. Experiments are carried out aiming at reagent types, concentration, treatment time and reaction temperature variables, and a reagent red blood cell treatment method which is good in maintenance of cell membrane stability, low in antigen damage degree and stable in antigenicity within 3 months of storage life is established by controlling hydroformylation treatment conditions and using a single glutaraldehyde fixing formula.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biotechnology, and more specifically, to red blood cell preservation reagents, red blood cell pretreatment methods, and their applications. Background Technology

[0002] Transfusion medicine is a crucial method and support for modern medicine, and transfusion immunoassay is a key step in assessing transfusion safety. Reagent red blood cells, as an indispensable core tool in modern transfusion immunoassay practice, can qualitatively / semi-quantitatively detect unknown blood type antibodies in patient serum using known antigens. This provides significant assistance for prenatal blood type antibody monitoring in pregnant women, detection of antibodies related to hemolytic disease of the newborn, and avoidance of antibody-related hemolytic transfusion reactions. To ensure the stability and comparability of test results to a certain extent, manufacturers typically use reagent red blood cell preservation solutions to preserve reagent red blood cells. However, research has found that with prolonged storage, changes in the spatial structure of blood type substances can lead to deviations in qualitative and semi-quantitative blood type experiments, affecting the accuracy of results and threatening patient lives. Therefore, improving the antigen stability of reagent red blood cells is of great significance for related testing and clinical transfusion.

[0003] The use of aldehyde reagents for long-term preservation of biological samples is a common laboratory method and technique. The principle behind this is that aldehydes enhance the mechanical strength of cell membranes through cross-linking of proteins. However, when treating erythrocytes with reagents using traditional tissue fixation conditions, the treatment can be too harsh, leading to the destruction of the spatial structure of erythrocyte membrane proteins and hindering effective detection of target antibodies. Furthermore, the type and concentration of reagents, treatment time, and reaction temperature all affect the fixation effect and membrane stability during erythrocyte aldehyde treatment.

[0004] Chinese patent document CN202210959869.3 only addresses how to fix and preserve cells using fixatives. However, experiments have shown that the concentration range of such solutions can severely damage red blood cell antigens.

[0005] Chinese patent document CN2022113913652 only detected the carbohydrate blood group antigens (ABO blood group antigens) highly expressed on red blood cells, but did not detect or verify the effect of the long-term presence of aldehydes on the protein blood group antigens expressed on numerous red blood cells. In this invention, experiments have demonstrated that the contact time between aldehydes and red blood cells has a destructive effect on red blood cell blood group antigens. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a pretreatment method for erythrocyte reagents that can both ensure the stability of erythrocyte blood group antigens and increase the stability of erythrocyte reagents by controlling the concentration of aldehydes and cell concentrations. Furthermore, by controlling the processing time and temperature, a method for determining the optimal scheme for pretreatment of erythrocyte reagents using aldehydes before preservation is proposed.

[0007] In a first aspect, the present invention provides a red blood cell preservation reagent comprising a single aldehyde, wherein the single aldehyde is glutaraldehyde, and the mass fraction of the glutaraldehyde in the solvent is 0.125 mM (0.00125%) to 4.0 mM (0.04%).

[0008] As a preferred example, the mass fraction of glutaraldehyde in the solvent is 0.125 mM (0.00125%) to 3.0 mM (0.03%).

[0009] As a preferred example, the mass fraction of glutaraldehyde in the solvent is 0.03 mM / mL (0.003%) to 1 mM / mL (0.01%).

[0010] Secondly, the present invention provides a method for preserving red blood cells, wherein the method involves adding red blood cells to a red blood cell preservation reagent to preserve the red blood cells.

[0011] As a preferred example, the glutaraldehyde concentration in erythrocytes is in the range of 0.004 mM / 10⁻¹⁰. 10 Red blood cells -0.3mM / 10 10 Red blood cells.

[0012] As a preferred example, the glutaraldehyde concentration in erythrocytes is in the range of 0.008 mM / 10⁻¹⁰. 10 Red blood cells -0.24mM / 10 10 Red blood cells.

[0013] As a preferred example, the cell treatment temperature is 4°C.

[0014] Thirdly, the present invention provides a reagent red blood cell, which is prepared by a method for preserving red blood cells.

[0015] Fourthly, the present invention provides the application of red blood cell preservation reagents in blood typing, blood type antibody titer determination, or red blood cell antibody screening and identification, wherein the application is not for the purpose of disease diagnosis and treatment.

[0016] Fifthly, the present invention provides the application of reagent red blood cells in blood typing, blood type antibody titer determination, or red blood cell antibody screening and identification, wherein the application is not for the purpose of disease diagnosis and treatment.

[0017] The advantages of this invention are as follows: This invention conducts experiments on the variables of reagent type, concentration, treatment time and reaction temperature. By controlling the conditions of aldehyde treatment and using a single glutaraldehyde (GA) fixed formulation, a reagent-based red blood cell treatment method is established that maintains good cell membrane stability, has low antigenic damage, and maintains antigenicity within a 3-month storage period.

[0018] This invention controls the conditions of aldehyde treatment, using a single glutaraldehyde fixation formulation to ensure the functionality of reagent red blood cells in routine blood type-related serological tests and cellular immunological tests (such as flow cytometry). While maintaining the natural conformation required for the antigenicity of red blood cell blood type antigens, the aldehyde treatment improves the stability of red blood cell antigens, achieving the goal of maintaining antigenic stability during the storage period of reagent red blood cells. This further enhances the accuracy and uniformity of transfusion immunological test results, providing a guarantee for transfusion safety. Attached Figure Description

[0019] Figure 1 Preservation of the appearance of red blood cells treated with different glutaraldehyde at the terminal stage.

[0020] Figure 2 Preservation of morphology of red blood cells treated with different glutaraldehyde at the terminal stage.

[0021] Figures A to F show the microscopic cell morphology of red blood cells pretreated with different concentrations of glutaraldehyde in group B at the end of the preservation experiment (90 days). The corresponding aldehyde treatment concentrations are as follows: A: control group; B: 0.01%; C: 0.02%; D: 0.03%; E: 0.035%; F: 0.04%.

[0022] Figure 3 : To preserve the differences in membrane PS between different aldehyde treatment groups at the end of the preservation period.

[0023] The vertical axis represents Annexin V, indicating the extent of PS flaring in the membrane, while the horizontal axis represents different glutaraldehyde treatment concentrations. Groups A, B, and C are three parallel treatments. *: p < 0.05. Figure 4 The AF study statistically analyzed the differences in membrane PS eversion between the experimental and control groups with different glutaraldehyde treatment concentrations. The vertical axis represents Annexin V, indicating the extent of membrane PS eversion, and the horizontal axis represents different glutaraldehyde treatment concentrations. Groups A, B, and C were three parallel treatment groups. *: p < 0.05. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0025] Example 1

[0026] Materials and Methods

[0027] 1. Sample Source

[0028] The red blood cell samples used in the system construction and validation were all taken from the research blood used in the station. Among them, type O blood samples (n=7) were used to confirm the early aldehyde treatment conditions, and type O blood samples (n=4) and type AB blood samples (n=3) were used for subsequent red blood cell preservation stability validation.

[0029] 2. Reagents and Instruments

[0030] In this study, the erythrocytes prepared using aldehyde-treated reagents designed for blood typing immunoassays were all fixed using a single glutaraldehyde (Sigma, 1002381005) fixation method and preserved using a laboratory-made 4k erythrocyte preservation solution (Patent Application No. 202010613045.1, Invention Title: A Reagent Erythrocyte Preservation System and its Preparation Method). During the preservation period of the aldehyde-treated erythrocytes, an apoptosis cell detection kit (Invitrogen, V13241) and a hemoglobin quantitative detection kit (BioAssaySystems) were used to conduct validation experiments related to the preservation performance of the reagent erythrocytes under different treatment conditions.

[0031] The blood group monoclonal antibodies used in this study are mainly as follows: monoclonal antibodies

[0032] -A / -B / -H / -M / -C / -c / -E / -e (Shanghai Blood Biotechnology), monoclonal antibody -Jkb / -Fya / -k (Sanquin). The main instruments involved include: a serological centrifuge (KA2200, KUBOTA), a BIO-RAD gel card incubator, and a card centrifuge (Bio-RAD).

[0033] 3 Experimental Methods

[0034] 3.1 Confirmation of the working concentration range for aldehyde treatment of erythrocytes

[0035] Because the erythrocyte concentration (aldehyde:erythrocyte) ratio is not fixed in various immunological assays for different purposes, the (glutaraldehyde:erythrocyte) concentration range used in this assay is described in the form of "n aldehydes / erythrocytes", with a minimum value of 0.004 mM / 10⁻¹⁰. 10 Red blood cells (0.004 mM / mL) (effectively protects blood group antigen stability, but the concentration is slightly lower in maintaining membrane stability), with a maximum value of 0.3 mM / 10. 10 Red blood cells (not conducive to the long-term preservation of protein-based blood group antigens, but can effectively maintain the stability of the membrane itself). Within this range, the concentration should not exceed 0.24 mM / 102 10 Red blood cells are beneficial for the long-term preservation of blood group antigens, with a concentration not lower than 0.008 mM / 10⁻⁶. 10 [Red blood cells are beneficial for long-term preservation due to membrane stability]. To clarify that aldehyde treatment of red blood cells does not affect the effective working concentration range for blood group antigen detection, this study tested low GA concentrations of 0.03 mM / mLRBC and high concentrations of 1 mM / mLRBC in preliminary experiments [“ThermoFisher, HFH01, MAN0003641Rev3.02”], and detected some blood group antigens for various blood group systems on the treated red blood cells. In the initial experiment, the aldehyde treatment of red blood cells was carried out at room temperature, and the main operating procedures are as follows:

[0036] Prepare 10 mL each of GA working solutions with final concentrations of 5 mM and 1 mM using 1xPBS;

[0037] Take 10 μL of 50% HCT red blood cell suspension and add 10 mL of 5 M mGA working solution to make the high concentration group;

[0038] Take 60 μL of 50% HCT red blood cell suspension and add 10 mL of 1 mM GA working solution to make the low concentration group;

[0039] After thoroughly mixing by inverting 10 times, allow the mixture to stand for 10 minutes. Centrifuge to completely remove the supernatant, then wash three times with PBS containing 0.1% BSA and resuspend. After processing, store the red blood cells at a final concentration of 3% using 0.1% BSA as a diluent, and detect some antigens of the H / MNS / RHD / RHCE / KIDD / KELL / Duffy blood group system.

[0040] 3.2 Selecting the optimal aldehyde treatment conditions for erythrocyte reaction based on the weakening trend of blood type antigens.

[0041] Based on preliminary experimental results, red blood cells were treated with working concentrations of 0.03 / 0.06 / 0.1 / 0.12 / 0.13 / 0.15 mM / mLRBCGA (corresponding to final concentrations of 1 / 2 / 3 / 3.5 / 4 / 4.5 mM) (final red blood cell concentration at treatment was 3%). The reaction temperatures were 37℃ / 25℃ / 4℃. The B antigen and RhD antigen of the reagent-treated red blood cells were detected after 10 min / 20 min / 30 min / 1 hr of treatment. Except for the above variables, the experimental procedure remained unchanged from 3.1. Agglutination results were referenced to column agglutination intensity. Anti-B gel cards were incubated at room temperature, while anti-D gel cards were incubated at 37℃.

[0042] 3.3 Aldehyde Treatment Reagent Red Blood Cell Preservation Test

[0043] Pre-cooled GA working solution was used to treat erythrocytes (type O n=4, type AB n=3) with GA at final concentrations of 0.125mM / 0.25mM / 0.5mM / 1mM / 2mM / 3mM / 3.5mM / 4mM, and the reaction was carried out at 4℃ for 10 min. After removing the supernatant and washing three times, the cells were resuspended in 4k erythrocyte preservation solution at 3% and 10% cell concentrations and stored at 4℃. Antigen stability (including A, B, D, C, c, E, e, M, Jkb, Fya) was monitored in erythrocyte samples treated with different GA concentrations at storage periods (days 0, 30, 60, and 90). Preservation appearance, erythrocyte morphology, erythrocyte hemolysis rate, and PS eversion of the cell membrane were also monitored.

[0044] result

[0045] 1. Effects of different glutaraldehyde treatment concentrations on serological detection of erythrocyte blood group antigens

[0046] Traditional aldehyde fixation of cells severely damages erythrocyte blood group antigens, making it unsuitable for processing erythrocyte-based reagents. Considering that the cell concentration during aldehyde treatment of erythrocytes is closely related to the yield and homogeneity of the formulation, a reliable reference range has been lacking in previous blood group immunology studies. See Table 1 for the effect of GA treatment concentration on serological detection of erythrocyte blood group antigens.

[0047] Table 1

[0048]

[0049] *Anti-D and anti-Fya reagents are IgG antibodies, and experimental results are expressed as agglutination intensity in classical anti-human sphere media;

[0050] #The results of all other antibody experiments are expressed as agglutination intensity using the saline method.

[0051] The results suggest that under the above reaction conditions, a high GA concentration of 1 mM / mLRBC exceeds the tolerance range of erythrocytes, while treatment with a low GA concentration of 0.03 mM / mLRBC leads to a certain degree of reduction in blood group antigens.

[0052] 2. Effects of different glutaraldehyde treatment conditions on serological detection of erythrocyte blood group antigens

[0053] Red blood cells with a concentration of 3% were treated with glutaraldehyde at concentrations of 0.01%, 0.02%, 0.03%, 0.035%, and 0.05%, respectively, while other conditions remained unchanged. The B and RHD antigens of the red blood cells were then detected. The experimental results are shown in Table 2. The effect of different glutaraldehyde treatment conditions on the detection of ABO blood group antigens in red blood cells.

[0054] Table 2

[0055]

[0056] The effects of different glutaraldehyde treatment conditions on the detection of erythrocyte RHD blood group antigens are shown in Table 3.

[0057] Table 3

[0058]

[0059] Based on the above test results, reaction time is the most significant factor affecting the weakening of erythrocyte antigens after aldehyde treatment; the longer the treatment time, the weaker the antigen reactivity. Simultaneously, glutaraldehyde concentration is inversely proportional to the antigen reaction intensity. Low-temperature treatment demonstrated a better antigen protection effect in this experiment.

[0060] 3. Comparison of Red Blood Cell Antigen Stability

[0061] Antigen monitoring (including A, B, D, C, c, E, e, M, Jkb, and Fya) was performed on red blood cell samples stored for 0, 30, 60, and 90 days. Statistical differences were found in some antigens in the aldehyde-treated group compared to the control group. The specific results are shown in Table 4, which compares the antigen stability of red blood cells stored at different concentrations of aldehyde.

[0062] Table 4

[0063]

[0064] *Median score of erythrocyte agglutination intensity (P25, P75)

[0065] #Frideman multivariate nonparametric analysis was performed. Results are expressed as Z-statistics and p-values ​​for paired comparisons. Within the same column (a, b), the aldehyde treatment concentration groups labeled a and b were statistically significant compared to the corresponding Ctl. groups; no significant differences were found between the unlabeled groups and the Ctl. groups.

[0066] 4. Appearance and morphology of red blood cells preserved with different concentrations of glutaraldehyde during shelf life:

[0067] The appearance of red blood cells preserved at different glutaraldehyde treatments during the final stage of preservation is shown in the figure. Figure 1 The morphology of erythrocytes treated with different glutaraldehyde at the end of the preservation period is shown in the figure. Figure 2 , Figure 3 .

[0068] 5. Comparison of hemolysis rate of erythrocytes treated with different concentrations of glutaraldehyde during the shelf life

[0069] To explore the optimal concentration of glutaraldehyde for fixing erythrocytes and the changes in erythrocytes during storage, the team further reduced the concentration of glutaraldehyde in the pretreatment to the ranges of 0.00125% (0.125 mM), 0.0025% (0.25 mM), 0.005% (0.5 mM), 0.01% (1.0 mM), 0.02% (2.0 mM), and 0.03% (3.0 mM), and monitored hemolysis. Table 3 shows the comparison of hemolysis rates of erythrocytes treated with different concentrations of glutaraldehyde.

[0070] Table 3

[0071]

[0072] By monitoring the changes in free hemoglobin in the supernatant of reagent red blood cells during the storage period, the team found that there was no significant difference between red blood cells treated with different concentrations of glutaraldehyde and those in the control group.

[0073] 6. Monitoring of PS eversion of erythrocyte membranes treated with different concentrations of glutaraldehyde during the storage period

[0074] Membrane stability is an important reference for evaluating the preservation effect of erythrocyte reagents. The team used the Annexin V detection kit to monitor the eversion of phosphatidylserine (PS) in the cell membrane of erythrocyte samples stored on day 0, day 30, day 60 and day 90. Figure 4 To preserve the differences between the different glutaraldehyde treatment groups and the control group at the terminal stage (90 days).

[0075] It should be noted that the present invention differs from 2022113913652 (a red blood cell preservation reagent and its application) in that;

[0076] 1. Reagents used: This invention uses a single aldehyde reagent, namely glutaraldehyde. The prior art uses formaldehyde and glutaraldehyde, while this invention uses only a single aldehyde reagent, making it simpler. Using glutaraldehyde as a single aldehyde overcomes the risk of instability associated with paraformaldehyde in the prior art due to its inherent chemical properties.

[0077] 2; Concentration range: 0.125 mM (0.00125%) to 3.0 mM (0.03%) in this invention. Comparative documents show glutaraldehyde at 0.004% to 0.030% and paraformaldehyde at 0.04% to 0.14%. The aldehyde reagents used in this invention have precise concentrations, far less than the aldehyde concentration ranges mentioned in the comparative documents.

[0078] 3. Processing conditions: This invention specifies the use of 4°C. Comparative literature uses 18-25°C. Experiments have demonstrated that processing cells at 4°C results in less damage to the antigen compared to room temperature.

[0079] 4. Membrane stability: This invention used Annexin V to detect the eversion of PS, a marker of cell membrane stability. The prior art did not provide a description of this detection. PS eversion as a marker of membrane stability is widely accepted, and this invention demonstrates that the pretreatment method involved in this invention can effectively prevent PS eversion, indicating increased membrane stability.

[0080] 5. Protein Antigen Stability Testing: This invention tested and evaluated carbohydrate blood group antigens (ABO blood group system) and protein blood group antigens with different structures (Rh, MN blood group system, Duffy, Kidd, etc.). The prior art only tested carbohydrate blood group antigens (ABO blood group system) and did not test protein antigens. Histological knowledge clearly shows that high aldehyde concentrations cause changes in protein structure, which is the main reason why high-concentration aldehyde reagents cannot be used as red blood cell pretreatment methods. This invention tested blood group system antigens, which are the most common sources of accidental antibodies in humans, ensuring the effectiveness of the reagent cells. The prior art only focuses on the ABO system and can only prove the applicability of ABO reagent cells. For other types of blood group reagent cells used for accidental antibody detection (such as antibody screening and antibody identification cells), the prior art does not provide testing and verification of the effect on protein antigens, therefore its effectiveness cannot be fully demonstrated.

[0081] 6. Application Scenarios and Scope: As a reagent for red blood cell pretreatment. The prior art requires the use of a specific diluent. This invention, as a pretreatment method, does not impose specific requirements on subsequent preservation reagents, making it more applicable. In contrast, the prior art requires the use of a diluent, limiting its scope of use.

[0082] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A red blood cell preservation reagent, characterized in that, The red blood cell preservation reagent contains a single aldehyde, namely glutaraldehyde, and the mass fraction of glutaraldehyde in the solvent is 0.00125%-0.04%.

2. The red blood cell preservation reagent according to claim 1, characterized in that, The glutaraldehyde has a mass fraction of 0.00125%-0.03% in the solvent.

3. The red blood cell preservation reagent according to claim 2, characterized in that, The glutaraldehyde has a mass fraction of 0.003%-0.01% in the solvent.

4. A method for preserving red blood cells, characterized in that, The method involves adding red blood cells to the red blood cell preservation reagent according to any one of claims 1-3 to preserve the red blood cells.

5. The method for preserving red blood cells according to claim 4, characterized in that, Glutaraldehyde: Red blood cell concentration range is 0.004 mM / 10 10 Red blood cells -0.3mM / 10 10 Red blood cells.

6. The method for preserving red blood cells according to claim 5, characterized in that, The glutaraldehyde concentration range for erythrocytes is 0.008 mM / 10⁻⁶. 10 Red blood cells -0.24mM / 10 10 Red blood cells.

7. The method for preserving red blood cells according to any one of claims 4-6, characterized in that, The cell treatment temperature was 4℃.

8. A reagent of red blood cells, characterized in that, The reagent red blood cells are prepared by the method described in any one of claims 4-7.

9. The application of the red blood cell preservation reagent according to any one of claims 1-3 in blood typing, blood type antibody titer determination, or red blood cell antibody screening and identification, wherein the application is not for the purpose of disease diagnosis and treatment.

10. The application of the reagent red blood cells according to claim 8 in blood type reverse typing, blood type antibody titer determination or red blood cell antibody screening and identification, wherein the application is not for the purpose of disease diagnosis and treatment.

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