Preparation method and application of red blood cell lysis buffer for Rh double-group typing

Abstract

The invention provides a preparation method and application of a red blood cell lysis buffer for Rh double-group typing. The preparation method comprises the following steps: (1) preparing a stock solution 1; (2) preparing a stock solution 2; (3) mixing the stock solution 1 with H2O to obtain an application solution 1; (4) preparing application liquid 2; (5) adding 50 [mu] L of to-be-detected sample near-albuginea layer hematopoietic cells into 1 mL of the application liquid 1, and uniformly mixing; (6) centrifuging the mixed solution obtained in the step (5), and discarding the supernate; washing with normal saline under the condition of 1000g each time, and centrifuging; discarding supernate, dripping 2-3 drops of normal saline, and uniformly mixing; and (7) adding one drop of erythrocyte suspension and one drop of corresponding antibody into the blank card, and centrifugally observing the result. According to the preparation method of the red blood cell lysis buffer for Rh double-group typing, the detection rate is high, the preparation method is simple, the cost is low, and the red blood cell lysis buffer is very suitable for being popularized and popularized in primary medical institutions.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to a preparation method and application of a red blood cell lysate for Rh dual-group typing. Background Art

[0002] The Rh blood group system is the most complex red blood cell blood group system, and its importance in clinical blood transfusion is second only to the ABO blood group system. Currently, 56 Rh antigens have been discovered, including 15 polymorphic antigens, 17 high-frequency antigens, and 24 low-frequency antigens. Among them, the five antigens that are more closely related to clinical practice have different immunogenicities, from strong to weak, namely RhD, E, C, c, and e. Among the irregular antibodies detected clinically, Rh blood group system antibodies account for about 60wt%, which can cause fetal neonatal hemolytic disease, hemolytic transfusion reactions, etc. At present, most medical institutions have carried out Rh blood type compatible transfusions.

[0003] The scope of application of Rh-compatible blood transfusion includes: patients who have produced irregular antibodies of the Rh blood group system, even if the irregular antibody screening or identification of the transfusion specimen is negative. Patients whose irregular antibody specificity cannot be determined. Patients who are expected to need repeated blood transfusions, such as patients with thalassemia, blood system diseases, organ tissue and stem cell transplants, chronic gastrointestinal bleeding, kidney disease, tumors, etc. Patients whose repeated red blood cell transfusions are ineffective, including non-immune factors that cannot be ruled out. Patients with positive autoantibodies such as autoimmune anemia. Women of childbearing age, pregnant women, newborns, infants and children. Surgical patients who require large or multiple blood transfusions.

[0004] The frequency of haplotype expression of Rh antigen in the Han Chinese population shows a significant skewed distribution. The frequency of haplotype expression is the basis for producing irregular antibodies in the Rh blood type system and finding compatible blood. Rh incompatible blood transfusion will cause double groups and affect the judgment of Rh blood type. Therefore, accurate identification of Rh blood type, especially accurate judgment of Rh double group typing, has become the basis and prerequisite for carrying out Rh blood type compatible blood transfusion.

[0005] There are many conventional Rh typing detection methods at present, but when Rh typing shows double groups, conventional microcolumn method and test tube method cannot detect it. It needs to be detected by capillary centrifugation separation technology and molecular biology. However, this method has obvious limitations. Capillary centrifugation separation method requires the use of specific equipment and consumables. Data show that its detection rate is 64.63wt%. Molecular biology method requires genetic testing conditions and the detection is time-consuming and expensive, which makes it difficult to promote and use in grassroots hospitals.

[0006] Current studies have shown that erythrocytes continuously form vesicles in their cell membranes during maturation and aging. Continuous vesicle formation weakens the deformability of erythrocytes, reduces the elasticity of the cell membrane, and increases osmotic fragility. Young erythrocytes are not easily destroyed in erythrocyte lysates, while old erythrocytes are easily lysed and destroyed. Therefore, this patent proposes that a specific erythrocyte lysate can be prepared and the erythrocyte lysis method can be used to perform typing and identification of Rh typing dual-group samples.

[0007] Limitations of existing technologies: Technology, facilities and cost constraints: In order to accurately identify the patient's Rh typing from dual-group samples, laboratories may need to use more sophisticated techniques, such as molecular biology methods (such as PCR-DNA analysis). Although these methods can provide more accurate results, they place extremely high demands on hardware facilities and are usually time-consuming and costly. Balance between clinical needs and detection timeliness: In a clinical setting, it is very important to quickly obtain correct blood type information. However, for complex dual-group samples, it is not easy to achieve rapid and accurate judgment, which requires maximizing efficiency while ensuring accuracy. In summary, the difficulty in rapid judgment of Rh typing dual-group samples mainly stems from the complexity of the samples themselves and the limitations of existing technologies.

[0008] Therefore, there is an urgent need for a preparation method and application of a red blood cell lysate for Rh dual-group typing. Summary of the invention

[0009] The purpose of the present invention is to solve the problem that it is difficult to quickly determine the Rh typing of Rh typing dual-group samples. By preparing a specific red blood cell lysate and using a red blood cell lysis method, the rapid identification of Rh dual-group typing is achieved through simple experimental operations, thereby providing a basis and guarantee for conducting Rh blood type compatibility transfusions and finding compatible blood.

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] A method for preparing a red blood cell lysate for Rh dual-group typing comprises the following steps:

[0012] (1) Stock solution 1: prepare 300 mL of 1.4 wt% potassium bicarbonate solution, add 300 mL of normal saline and 300 μL of 0.5 wt% surfactant to obtain stock solution 1;

[0013] (2) Stock solution 2: Prepare 300 mL of 1.4 wt % potassium bicarbonate solution, add 300 mL of normal saline and 900 μL of 0.5 wt % surfactant to obtain stock solution 2;

[0014] (3) Mix the stock solution 1 with H 2 O mixed to obtain application liquid 1;

[0015] (4) Mix the stock solution 2 with H 2 O to obtain a mixed solution, and at the same time, 0.5 μL of 0.5 wt % surfactant was added to each mL of the mixed solution to obtain application solution 2;

[0016] (5) Add 50 μL of the packed red blood cells near the buffy coat layer of the sample to be tested to 1 mL of application solution 1 and mix well;

[0017] (6) Centrifuge the mixture of step (5) and discard the supernatant; wash with physiological saline 3-4 times, each centrifugation condition is 1000g, centrifugation for 1-2 minutes; discard the supernatant, add 2-3 drops of physiological saline, and mix well;

[0018] (7) Add 1 drop of red blood cell suspension and 1 drop of corresponding antibody to the blank card. For Rh typing, for the four antigen tests of RhCcEe, add anti-C, anti-c, anti-E, and anti-e antibodies according to the actual situation of the test sample. Centrifuge and observe the results; if the test result is still double group, use application solution 2 to perform a second operation according to the above method.

[0019] The method of the present invention significantly improves the detection rate of the Rh dual-group sample typing results through a carefully designed lysate formula and operating steps. The method utilizes the characteristics that the cell membrane of red blood cells continuously forms vesicles during maturation and aging, resulting in weakened deformability, decreased cell membrane elasticity, and increased osmotic fragility. Young red blood cells are relatively difficult to be destroyed, while old red blood cells are more easily lysed. By adjusting the concentration of a surfactant (such as Triton X-100), selective lysis of red blood cells of different age groups can be achieved, thereby effectively separating the patient's own red blood cells for Rh typing detection.

[0020] Application Solution 1 and Application Solution 2 are designed to correspond to different lysis intensities. Application Solution 1 is used to initially lyse the more fragile old red blood cells, while Application Solution 2 further processes the remaining red blood cells, ensuring that even the most difficult to lyse relatively young red blood cells can be effectively separated. This step-by-step lysis strategy improves the discrimination of different red blood cell types in Rh dual group samples, thereby improving the detection rate.

[0021] Furthermore, the physiological saline solution is a sodium chloride solution with a concentration of 0.9 wt%.

[0022] Furthermore, the surfactant is Triton X-100.

[0023] Furthermore, the centrifugation method in step (6) is: first centrifuge at 1000g for 15-20s, and use a dropper to blow away the red blood cell buckle; then centrifuge at 1000g for 1-2min, and discard the supernatant.

[0024] Furthermore, in step (3), the stock solution 1 and H 2 O is mixed in a volume ratio of 3:(6-8).

[0025] Furthermore, in step (4), the stock solution 2 and H 2 O is mixed in a volume ratio of 2:(7-9).

[0026] Furthermore, in step (1), the concentration of the potassium bicarbonate solution is 1.3-1.5 wt %.

[0027] Furthermore, the concentration of the potassium bicarbonate solution in step (2) is 1.3-1.5 wt %.

[0028] The present invention ensures the controllability and consistency of the lysis process by strictly controlling the ratio of potassium bicarbonate solution, physiological saline and surfactant in the stock solution, as well as the surfactant concentration in the subsequent mixed solution. This helps to avoid excessive lysis or insufficient lysis, and ensures the reliability of the experimental results. The reagents used in this method are all common chemical reagents and physiological saline, the preparation process is simple, the cost is low, and it is suitable for promotion and popularization in primary medical institutions. The entire experimental process is easy to operate, no special equipment and consumables are required, the centrifugation condition is a conventional 1000g, and the centrifugation time is short (1-2 minutes), which greatly shortens the detection time and improves work efficiency. All operations can be completed under ordinary laboratory conditions, without the need for special instruments or complex techniques, and are easy to implement in resource-limited environments. Using physiological saline for 3-4 washings ensures that the red blood cells after lysis are fully cleaned, removes residual lysis solution and other impurities, and improves the accuracy of subsequent detection. If the initial detection result shows a double group, a secondary operation can be performed by using liquid 2 to further confirm the result. This double verification mechanism increases the reliability and accuracy of the detection. This method is not only applicable to the typing of Rh bigroup samples, but can also be extended to other application scenarios that require selective lysis of red blood cells, such as hematology research, transfusion medicine, etc. The reagents used are conventional chemical reagents, harmless to the human body, safe to operate, and in line with laboratory safety standards.

[0029] The present invention also provides application of the method for preparing the red blood cell lysate for Rh dual-group typing in performing Rh dual-group typing.

[0030] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0031] 1. In order to cope with the challenges of rapid blood type detection of Rh bi-group patients in primary medical institutions, the present invention has developed a method for preparing a red blood cell lysate. The lysate has a simple configuration and low cost, which greatly reduces the operating threshold and is convenient for wide promotion and popularization. The experimental method is simple and fast to operate, and does not require special equipment and consumables, so that it can be efficiently implemented even in resource-limited environments. The present invention not only provides new ideas and methods for rapid blood type detection of Rh bi-group patients, but also provides a solid foundation and guarantee for the selection of Rh compatible blood transfusions, ensuring the accuracy and safety of blood selection, thereby improving the safety and effectiveness of clinical blood transfusions. This method is expected to significantly improve the ability of primary medical units to deal with complex blood type situations, promote wider clinical applications, and improve the ability to ensure the safety of patient blood transfusions.

[0032] 2. The present invention provides a method for preparing a red blood cell lysate for Rh dual group typing, which has a simple preparation method and low cost, and is very suitable for promotion and popularization in primary medical institutions. The experimental methodology established based on this lysate is simple and fast to operate, does not require special equipment and consumables, greatly reduces the difficulty of implementation, and enables efficient execution even in resource-limited environments. The scientific basis of this method is that during the maturation and aging of red blood cells, their cell membranes continuously form vesicles, resulting in weakened deformability, decreased cell membrane elasticity, and increased osmotic fragility. Therefore, young red blood cells are relatively difficult to be destroyed in the lysate, while old red blood cells are easily lysed. This feature enables the method of the present invention to effectively separate and identify the Rh typing of the patient's own red blood cells in the Rh dual group sample, and provides a new idea and new method for rapid detection of blood type in Rh dual group patients. At the same time, this method also provides a solid foundation and guarantee for the selection of Rh compatible blood transfusions, ensuring the accuracy and safety of blood selection, thereby improving the safety and effectiveness of clinical blood transfusions.

[0033] 3. This method utilizes the characteristics that during the maturation and aging process of red blood cells, the cell membrane continuously forms vesicles, resulting in weakened deformability, decreased cell membrane elasticity, and increased osmotic fragility. Young red blood cells are relatively difficult to be destroyed, while old red blood cells are more easily lysed. By adjusting the concentration of the surfactant, selective lysis of red blood cells of different ages can be achieved, thereby effectively separating young red blood cells from old red blood cells. Application liquid 1 and application liquid 2 are designed to correspond to different lysis intensities. Application liquid 1 is used for the initial lysis of relatively fragile old red blood cells, while application liquid 2 further processes the remaining red blood cells to ensure that even the relatively young red blood cells that are most difficult to lyse can be effectively separated.

[0034] 4. The present invention ensures the controllability and consistency of the lysis process by strictly controlling the ratio of potassium bicarbonate solution, physiological saline and surfactant in the stock solution, as well as the concentration of surfactant in the subsequent mixed solution. This helps to avoid excessive lysis or insufficient lysis, and ensures the reliability of the experimental results. The reagents used in this method are all common chemical reagents and physiological saline, the preparation process is simple, the cost is low, and it is suitable for promotion and popularization in primary medical institutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the Rh dual-group sample microcolumn gel card typing results before and after red blood cell lysis treatment, where A is before red blood cell lysis treatment; B is after red blood cell lysis treatment. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1

[0038] This embodiment provides a method for preparing a red blood cell lysate for Rh dual-group typing, comprising two sample tubes with different concentrations, and the specific composition is formulated as follows:

[0039] (1) Stock solution 1: prepare 300 mL of 1.4 wt% potassium bicarbonate solution, add 300 mL of normal saline (0.9 wt% sodium chloride solution) and 300 μL of 0.5 wt% surfactant Triton X-100 to obtain stock solution 1;

[0040] (2) Stock solution 2: Prepare 300 mL of 1.4 wt% potassium bicarbonate solution, add 300 mL of physiological saline (0.9 wt% sodium chloride solution) and 900 μL of 0.5 wt% surfactant Triton X-100 to obtain stock solution 2;

[0041] (3) Mix the stock solution 1 with H 2 O are mixed in a volume ratio of 3:7 to obtain application liquid 1;

[0042] (4) Mix the stock solution 2 with H 2 O were mixed in a volume ratio of 2:8 to obtain a mixed solution, and 0.5 μL of 0.5 wt% surfactant Triton X-100 was added to each mL of the mixed solution to obtain application solution 2;

[0043] (5) Add 50 μL of the packed red blood cells near the buffy coat layer of the sample to be tested to 1 mL of application solution 1 and mix well;

[0044] (6) Centrifuge at 1000 g for 15 s, and use a dropper to blow away the red blood cell buckle; centrifuge at 1000 g for 1 min, and discard the supernatant; wash with saline three times, each time at 1000 g for 1 min; discard the supernatant, add 2 drops of saline, and mix well;

[0045] (7) Add 1 drop of red blood cell suspension and 1 drop of corresponding antibody to the blank card, centrifuge and observe the results; if the test result is still double population, use application solution 2 to perform a second operation according to the above method.

[0046] According to the above steps, the experiment was performed on 48 samples of Rh typing double groups in the initial microcolumn agglutination method, and Rh antigen typing was further tested using red blood cell lysate. Figure 1 , Table 1.

[0047] Table 1 Comparison of the results of Rh dual group sample typing by two methods

[0048] Experimental methods Number of cases Number of detected cases Detection rate Example 1 48 31 64.58wt%* Improved capillary centrifugation method 82 53 64.63wt%

[0049] Note: Compared with the modified capillary centrifugation method, χ2=0.000, *P>0.05.

[0050] After testing 48 samples and comparing them with the patient's historical results before blood transfusion, 31 samples were accurately typed, with an accurate detection rate of 64.58wt% (31 / 48). The modified capillary centrifugation method (Literature data: Wu Changlin, Shao Chaopeng, Zhang Yinze, et al. Application of capillary centrifugation in RhCcEe blood typing in patients with thalassemia undergoing regular blood transfusion) showed that the modified capillary centrifugation method is currently a commonly used method for detecting Rh dual groups, with an accurate detection rate of 64.63wt% (53 / 82). The chi-square test analysis showed that there was no statistically significant difference in the detection rate between the two methods (P>0.05).

[0051] The high detection rate of Rh dual group sample typing results has important clinical and practical significance, which is specifically reflected in the following aspects: 1. Improve transfusion safety: Accurate matching: The Rh blood group system is one of the most important blood group systems in transfusion medicine. For Rh dual group patients, accurate typing (RhCcEe) is crucial. The high detection rate ensures that the patient's blood type can be correctly identified, so that the corresponding blood can be selected for transfusion, avoiding transfusion reactions caused by blood type incompatibility. Reduce immune response: Avoid multiple Rh incompatible transfusions, which may trigger an immune response and produce Rh blood group system antibodies, which may cause severe hemolytic reactions in future transfusions or pregnancies. Accurate Rh typing can achieve Rh compatible transfusions, which can effectively avoid this situation and protect patients from unnecessary immune risks. 2. Improve treatment effects and auxiliary diagnosis: Effective treatment: For certain blood diseases or hereditary diseases, such as autoimmune hemolytic anemia, thalassemia, etc., accurate Rh typing helps to develop personalized treatment plans. Improving the detection rate of Rh typing can improve the effect of transfusion therapy. Assisted prenatal examination: In prenatal examination, Rh typing is very important for assessing the risk of Rh incompatibility between mother and baby. Accurate Rh typing can help doctors detect potential risks earlier and take preventive measures to prevent the occurrence of hemolytic disease of the newborn. 3. Improve the quality of medical services: Optimize resource allocation: A high detection rate means that more Rh double group patients can get accurate blood type identification, reducing the possibility of misdiagnosis and repeated testing. This not only improves the efficiency of medical services, but also saves valuable medical resources. Enhance patient trust: Accurate test results can enhance patients' trust in medical institutions and improve patient satisfaction. This is of great significance for establishing a good doctor-patient relationship and promoting the continuous improvement of medical service quality. 4. Promote scientific research progress: Basic research: Detection methods with high detection rates provide reliable data support for scientific research, which helps to deeply understand the molecular mechanisms and biological characteristics of the Rh blood type system. This plays an important role in promoting the development of new diagnostic tools and treatments. Clinical research: In clinical trials and clinical treatments, accurate Rh typing data is indispensable. A high detection rate can ensure the reliability and reproducibility of research results, thereby promoting scientific progress in related fields. 5. Rapid response to emergencies: In emergency situations, such as traffic accidents, natural disasters, etc., rapid and accurate blood typing is essential for timely provision of appropriate blood products. High detection rate methods can complete complex blood typing analysis in a short time, buying precious time to save lives.

[0052] Improving the detection rate of Rh bi-group sample typing results is not only a technological breakthrough, but also an important guarantee in clinical practice. It not only improves the safety of blood transfusion and the accuracy of diagnosis, but also optimizes the allocation of medical resources, enhances patient trust, promotes scientific research progress, and plays an important role in emergency and public health fields. This method, which is simple to operate, does not require special equipment and consumables, and has a reasonable detection rate, provides a solid foundation for the management and treatment of Rh bi-group patients and significantly improves the overall level of medical services.

[0053] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a red blood cell lysate for Rh dual-group typing, characterized in that: The following steps are involved: (1) Stock solution 1: prepare 300 mL of potassium bicarbonate solution, add 300 mL of normal saline and 300 μL of a 0.5 wt % surfactant to obtain stock solution 1; (2) Stock solution 2: Prepare 300 mL of potassium bicarbonate solution, add 300 mL of normal saline and 900 μL of a 0.5 wt % surfactant to obtain stock solution 2; (3) mixing the stock solution 1 with H2O to obtain the application solution 1; (4) mixing stock solution 2 with H2O to obtain a mixed solution, and adding 0.5 μL of 0.5 wt% surfactant per mL of the mixed solution to obtain application solution 2; (5) Add 50 μL of the packed red blood cells near the buffy coat layer of the sample to be tested to 1 mL of application solution 1 and mix well; (6) centrifuging the mixed solution of step (5) and discarding the supernatant; Wash with saline 3-4 times, centrifuging at 1000g for 1-2 min each time; discard the supernatant, add 2-3 drops of saline, and mix well; (7) Add 1 drop of red blood cell suspension and 1 drop of corresponding antibody to the blank card, centrifuge and observe the results; if the test result is still double population, use application solution 2 to perform a second operation according to the above method.

2. According to the method for preparing a red blood cell lysate for Rh dual-group typing according to claim 1, the physiological saline is a sodium chloride solution with a concentration of 0.9wt%.

3. The method for preparing a red blood cell lysate for Rh dual-group typing according to claim 1, wherein the surfactant is Triton X-100.

4. According to the method for preparing red blood cell lysate for Rh dual-group typing as described in claim 1, the centrifugation method in step (6) is: first, centrifuge at a centrifugal force of 1000g for 15-20s, and use a dropper to blow away the red blood cell buckle; then centrifuge at a centrifugal force of 1000g for 1-2min, and discard the supernatant.

5. The method for preparing a red blood cell lysate for Rh dual-group typing according to claim 1, wherein in step (3), the stock solution 1 and H2O are mixed in a volume ratio of 3:(6-8).

6. The method for preparing a red blood cell lysate for Rh dual-group typing according to claim 1, wherein in step (4), the stock solution 2 and H2O are mixed in a volume ratio of 2:(7-9).

7. The method for preparing a red blood cell lysate for Rh dual-group typing according to claim 1, wherein the concentration of the potassium bicarbonate solution in step (1) is 1.3-1.5 wt%.

8. The method for preparing a red blood cell lysate for Rh dual-group typing according to claim 1, wherein the concentration of the potassium bicarbonate solution in step (2) is 1.3-1.5 wt%.

9. Use of the method for preparing a red blood cell lysate for Rh dual-group typing according to any one of claims 1 to 8 in performing Rh dual-group typing.

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