A method and application for fetal red blood cell flow cytometry detection
By combining flow cytometry and nanoflow cytometry with centrifugation and fluorescent labeling, the limitations of existing technologies that can only detect fetal red blood cell counts have been overcome, enabling the detection of hemoglobin levels and improving the accuracy and comprehensiveness of the detection.
Patent Information
- Application Number
- CN202211623094.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-16
- Publication Date
- 2025-12-02
- Estimated Expiration
- 2042-12-16
AI Technical Summary
Existing fetal red blood cell testing methods can only detect the number of fetal red blood cells, but cannot detect the number of hemoglobin cells, resulting in reduced accuracy and an inability to fully understand the fetal health status.
The flow cytometry technique is used to separate fetal blood by centrifugation, label hemoglobin in red blood cells with specific mouse monoclonal antibodies and fluorescent dyes, and then use a nanoflow cytometer to excite fluorescence and scattered light to detect the amount of hemoglobin.
It improves the accuracy of fetal red blood cell detection, and can simultaneously detect the number of red blood cells and hemoglobin, providing a comprehensive understanding of the fetus's health status.
Abstract
Description
Technical Field
[0001] This invention relates to the field of fetal red blood cell detection technology, specifically a method and application for fetal red blood cell flow cytometry detection. Background Technology
[0002] Red blood cells, often abbreviated as RBC in routine laboratory tests, are the most numerous type of blood cell and the primary medium for transporting oxygen in vertebrates. They also play an immune role. Mature red blood cells in mammals are anucleate, meaning they have lost their DNA. Red blood cells primarily release energy through anaerobic glycolysis, and glucose-6-phosphate dehydrogenase and pyruvate kinase are essential enzymes for this process. If the activity of these enzymes decreases or is absent, red blood cells cannot perform anaerobic glycolysis. The quantity and quality of red blood cells in fetal blood can directly reflect the fetal health. To ensure fetal safety, regular testing of fetal red blood cells is necessary. Currently, methods for testing fetal red blood cells generally only detect the number of red blood cells, determining fetal health solely based on the number. The amount of hemoglobin in fetal red blood cells cannot be detected, reducing the accuracy of the test and hindering the understanding of fetal health. Summary of the Invention
[0003] The purpose of this invention is to provide a method and application for fetal red blood cell flow cytometry detection, which has the advantage of being able to detect the number of hemoglobin in red blood cells. This solves the problem that current methods for detecting fetal red blood cells can only detect the number of fetal red blood cells, and the health of the fetus is determined solely by the number of fetal red blood cells. The number of hemoglobin in fetal red blood cells cannot be detected, which reduces the accuracy of the detection and is not conducive to understanding the health status of the fetus.
[0004] To achieve the above objectives, the present invention provides the following technical solution: a method for detecting fetal erythrocytes by flow cytometry, comprising the following steps:
[0005] S1: Fetal blood is collected and placed in a test tube. The test tube containing fetal blood is then added to an anticoagulant tube. An anticoagulant is added to the blood in the anticoagulant tube. The anticoagulant tube containing blood and anticoagulant is placed in a blood centrifuge. The blood centrifuge centrifuges the fetal blood in the anticoagulant tube. After centrifugation, the fetal blood in the anticoagulant tube separates into layers. The proportion of each layer is recorded. Red blood cells are at the bottom layer. The proportion of red blood cells in the blood is calculated based on the proportion of red blood cells in the layers. The bottom layer of red blood cells is removed and placed in a new test tube. The model of the blood centrifuge is TGL-12B-C. The speed of the blood centrifuge is 3000 rpm. The centrifugation time of the blood is 30 minutes. The anticoagulant is sodium citrate. After adding sodium citrate to the anticoagulant tube, it needs to be inverted and mixed 5 times. The ratio of sodium citrate to blood is 1:4.
[0006] S2: A specific murine monoclonal antibody is added to red blood cells. The murine monoclonal antibody binds to the corresponding protein inside the red blood cells. A flow cytometry fluorescent agent is poured into the detection antigen. The flow cytometry fluorescent agent labels the detection antigen. The labeled antigen is added to the extracted red blood cells. The antigen binds to the murine monoclonal antibody. The bound red blood cells are placed in a nanoflow cytometer. The nanoflow cytometer emits a laser to irradiate the red blood cells. The laser activates the fluorescent agent labeled in the antigen. The fluorescent agent emits excitation fluorescence and scattered light. The fluorescence ratio is observed to understand the proportion of the detected substance in the red blood cell fluid, and the positive ratio of the fluorescent substance is detected.
[0007] Preferably, the blood separates into three layers after centrifugation: plasma, red blood cells, and hemoglobin. Plasma, with its lower density, is on the top layer, followed by red blood cells, which are in the middle layer. Hemoglobin, with its highest content, is on the bottom layer. Plasma is pale yellow and transparent, white blood cells are white and opaque, and red blood cells are dark red. The blood layers differ at different stages of fetal development. During the last 4-6 weeks of pregnancy, adult hemoglobin in the fetus gradually increases, accounting for 75%. Granulocytes appear in the fetal blood circulation after 8 weeks of pregnancy. By 32 weeks of pregnancy, the fetal thymus and spleen produce lymphocytes, which in turn produce white blood cells.
[0008] Preferably, the red blood cell tubes extracted by layering need to be stored in a low-temperature chamber, the temperature of which needs to be maintained at around 4 degrees Celsius, and the storage time should not exceed 24 hours.
[0009] Preferably, the murine monoclonal antibody needs to be stored in a cold storage at a temperature of -20 degrees Celsius or -80 degrees Celsius, and antimicrobial drugs need to be added during storage.
[0010] Preferably, the nanoflow cytometer contains a forward photodiode and a 90° photomultiplier tube. Excitation fluorescence and scattered light are received by the forward photodiode and the 90° photomultiplier tube. The light scattering signal is detected at a small forward angle. This signal reflects the size of the cell volume. The direction of fluorescence signal reception is perpendicular to the laser beam. After being separated by a series of dichroic mirrors and bandpass filters, multiple fluorescence signals of different wavelengths are formed.
[0011] An application of fetal erythrocyte flow cytometry detection includes the following steps:
[0012] A: When it is necessary to test the number of red blood cells in whole blood, the whole blood sample is taken out and placed in a test tube, and stored in a suitable environment. The storage time should not be too long. A murine monoclonal antibody corresponding to the red blood cell surface antigen is added to the whole blood. The murine monoclonal antibody will bind to the corresponding antigen on the surface of red blood cells. During this period, it is necessary to stain the specific antigen corresponding to the murine monoclonal antibody. A special fluorescent staining agent is added to the specific antigen to stain it. After staining, the specific antigen is added to the mixture of murine monoclonal antibody and whole blood to obtain a new mixture. Then, the new mixture is placed in a nanoflow cytometer. The nanoflow cytometer emits a laser to activate the fluorescent staining agent in the specific antigen, causing it to emit excitation fluorescence and scattered light. The excitation fluorescence and scattered light are collected and detected. After the test, the proportion of positive cells in the whole blood needs to be checked. The red blood cell count and red blood cell size are determined by the test results.
[0013] B: When it is necessary to detect the amount of hemoglobin inside red blood cells, the whole blood sample is centrifuged. The red blood cells separated by centrifugation are removed and stored in a suitable environment. Then, the red blood cells are broken down to allow hemoglobin to flow out. A murine monoclonal antibody corresponding to hemoglobin is added to the red blood cell solution. The murine monoclonal antibody will bind to the hemoglobin. During this process, a specific antigen corresponding to the murine monoclonal antibody needs to be stained with a special staining agent. The stained specific antigen is added to the mixture of murine monoclonal antibody and red blood cell solution to obtain a new mixture. The new mixture is then placed in a nanoflow cytometer. The nanoflow cytometer emits a laser to activate the fluorescent staining agent in the specific antigen, causing it to emit excitation fluorescence and scattered light. The excitation fluorescence and scattered light are collected and detected. After the detection, the proportion of positive hemoglobin in the red blood cell solution needs to be checked. The amount and size of hemoglobin are determined based on the above detection results.
[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0015] This invention has the advantage of being able to detect the amount of hemoglobin in red blood cells, which solves the problem that current methods for detecting fetal red blood cells can only detect the number of fetal red blood cells and determine the health of the fetus solely based on the number of fetal red blood cells. The amount of hemoglobin in fetal red blood cells cannot be detected, which reduces the accuracy of the detection and is not conducive to understanding the health status of the fetus. Detailed Implementation
[0016] A method for detecting fetal red blood cells by flow cytometry includes the following steps:
[0017] S1: Fetal blood is collected and placed in a test tube. The test tube containing fetal blood is then added to an anticoagulant tube. Anticoagulant is added to the blood in the anticoagulant tube. The anticoagulant tube containing blood and anticoagulant is placed in a blood centrifuge. The blood centrifuge centrifuges the fetal blood in the anticoagulant tube. After centrifugation, the fetal blood in the anticoagulant tube separates into layers. The proportion of each layer is recorded. Red blood cells are at the bottom layer. The proportion of red blood cells in the blood is calculated based on the proportion of red blood cells in the layers. The bottom layer of red blood cells is removed and placed in a new test tube. The model of the blood centrifuge is TGL-12B-C. The speed of the blood centrifuge is 3000 rpm. The centrifugation time of the blood is 30 minutes. The anticoagulant is sodium citrate. After adding sodium citrate to the anticoagulant tube, it needs to be inverted and mixed 5 times. The ratio of sodium citrate to blood is 1:4.
[0018] S2: Specific murine monoclonal antibodies are added to red blood cells. The murine monoclonal antibodies bind to corresponding proteins inside the red blood cells. Flow cytometry fluorescent agents are added to the detection antigen, labeling it. The labeled antigen is then added to the extracted red blood cells, where it binds to the murine monoclonal antibodies. The bound red blood cells are placed in a nanoflow cytometer, which emits a laser to irradiate the red blood cells. The laser activates the fluorescent agent labeled in the antigen, causing it to emit excitation fluorescence and scattered light. The fluorescence percentage is observed to determine the proportion of the detected substance in the red blood cell fluid, and the positive percentage of the fluorescent substance is detected. After centrifugation, blood separates into three layers: plasma, red blood cells, and hemoglobin. Plasma has the lowest density and is on top, followed by red blood cells, and hemoglobin has the highest content and is on the bottom. Plasma is pale yellow and transparent, white blood cells are white and opaque, and red blood cells are dark red. The blood layering varies at different stages of fetal development, especially during the last 4-6 months of pregnancy. During the first week of gestation, the amount of adult hemoglobin in the fetus gradually increases, accounting for 75%. Granulocytes appear in the fetal blood circulation after 8 weeks of gestation. At 32 weeks of gestation, the fetal thymus and spleen produce lymphocytes, which become antigens in the body, thus producing white blood cells. The red blood cell test tubes extracted by stratification need to be placed in a low-temperature incubator for preservation. The temperature of the low-temperature incubator needs to be maintained at about 4 degrees Celsius, and the preservation time should not exceed 24 hours. Mouse monoclonal antibodies need to be stored in a cold storage. The storage location for mouse monoclonal antibodies is -20 degrees Celsius or -80 degrees Celsius. Antimicrobial drugs need to be added to the storage of mouse monoclonal antibodies. The nanoflow cytometer contains a forward photodiode and a 90° photomultiplier tube. The excitation fluorescence and scattered light are received by the forward photodiode and the 90° photomultiplier tube. The light scattering signal is detected at a small forward angle. This signal reflects the size of the cell volume. The direction of fluorescence signal reception is perpendicular to the laser beam. After separation by a series of dichroic mirrors and bandpass filters, multiple fluorescence signals of different wavelengths are formed.
[0019] Mouse hybridoma monoclonal antibodies are produced by fusing B cells from immunized mice with myeloma cells, then screening for mouse hybrid fusion cells that can proliferate indefinitely and secrete antibodies, followed by screening, antibody preparation, and antibody purification. Antibodies are immunoglobulins, which are modified globulin molecules produced by stimulation by specific antigens. Antibody production is due to the interaction of various immune cells caused by antigens entering the human body, which leads to the differentiation and proliferation of B cells in lymphocytes to form plasma cells, which can produce and secrete antibodies.
[0020] Nanoflow cytometry is an advanced, high-throughput quantitative characterization of nanoparticles based on sheath flow single-molecule fluorescence detection technology. It covers the blind spot of traditional flow cytometry for particle size detection below 200 nm, and encompasses the characterization of nanoparticles as well as subcellular structures, bacteria, viruses, extracellular vesicles (exosomes), and other natural biological nanoparticles. It opens a window to the nanoworld for flow cytometry technology. Nanoflow cytometry can realize the particle size and distribution of single nanoparticles (7-1000 nm) and particle concentration.
[0021] Sodium citrate, also known as sodium citric acid, has the chemical formula C6H5Na3O7 and a molecular weight of 258.07. It is an organic compound that appears as colorless orthorhombic prismatic crystals. It is stable in air, soluble in water and glycerol, slightly soluble in ethanol, and its aqueous solution is slightly alkaline. It has a cooling sensation when tasted. When heated to 100°C, it becomes a dihydrate. It is commonly used as a buffer, complexing agent, and bacterial culture medium. In medicine, it is used for diuresis, expectoration, sweating, and preventing blood clotting. It is also used in food, beverages, electroplating, and photography.
[0022] Fluorescent dyes are substances that, after absorbing a certain wavelength of light, emit another wavelength of light with a longer wavelength than the absorbed light. They are mostly compounds containing benzene rings or heterocycles and conjugated double bonds. Fluorescent dyes can be used alone or in combination to form composite fluorescent dyes. Composite fluorescent dyes are synthesized using fluorescence resonance energy transfer (FRET) technology. They consist of a donor molecule and an acceptor fluorophore molecules that are very close together and can transfer energy between each other. The composite dye is excited at the excitation wavelength of the acceptor molecule and emits light at the emission wavelength of the donor molecule. The development of fluorescent dyes has been very rapid. Fluorescent dyes developed for scientific research and clinical applications have basically covered the entire spectral range from ultraviolet to visible and infrared light. Due to their high sensitivity and ease of operation, fluorescent dyes have gradually replaced radioactive isotopes as detection markers. They are widely used in fluorescence immunoassay, fluorescent probes, cell staining, etc., including specific DNA staining for chromosome analysis, cell cycle, apoptosis and other related studies. In addition, many nucleic acid dyes are very useful counterstains in multicolor staining systems. They can be used as background controls and to label cell nuclei to make the spatial relationship of intracellular structures clear at a glance.
[0023] An application of fetal erythrocyte flow cytometry detection includes the following steps:
[0024] A: When it is necessary to test the number of red blood cells in whole blood, the whole blood sample is taken out and placed in a test tube, and stored in a suitable environment. The storage time should not be too long. A murine monoclonal antibody corresponding to the red blood cell surface antigen is added to the whole blood. The murine monoclonal antibody will bind to the corresponding antigen on the surface of red blood cells. During this period, it is necessary to stain the specific antigen corresponding to the murine monoclonal antibody. A special fluorescent staining agent is added to the specific antigen to stain it. After staining, the specific antigen is added to the mixture of murine monoclonal antibody and whole blood to obtain a new mixture. Then, the new mixture is placed in a nanoflow cytometer. The nanoflow cytometer emits a laser to activate the fluorescent staining agent in the specific antigen, causing it to emit excitation fluorescence and scattered light. The excitation fluorescence and scattered light are collected and detected. After the test, the proportion of positive cells in the whole blood needs to be checked. The red blood cell count and red blood cell size are determined by the test results.
[0025] B: When it is necessary to detect the amount of hemoglobin inside red blood cells, the whole blood sample is centrifuged. The red blood cells separated by centrifugation are removed and stored in a suitable environment. Then, the red blood cells are broken down to allow hemoglobin to flow out. A murine monoclonal antibody corresponding to hemoglobin is added to the red blood cell solution. The murine monoclonal antibody will bind to the hemoglobin. During this process, a specific antigen corresponding to the murine monoclonal antibody needs to be stained with a special staining agent. The stained specific antigen is added to the mixture of murine monoclonal antibody and red blood cell solution to obtain a new mixture. The new mixture is then placed in a nanoflow cytometer. The nanoflow cytometer emits a laser to activate the fluorescent staining agent in the specific antigen, causing it to emit excitation fluorescence and scattered light. The excitation fluorescence and scattered light are collected and detected. After the detection, the proportion of positive hemoglobin in the red blood cell solution needs to be checked. The amount and size of hemoglobin are determined based on the above detection results.
[0026] In summary, this method and application of fetal red blood cell flow cytometry detection solves the problem that current methods for detecting fetal red blood cells can only detect the number of fetal red blood cells, and the health of the fetus can only be determined by the number of fetal red blood cells. However, the number of hemoglobin in fetal red blood cells cannot be detected, which reduces the accuracy of the detection and is not conducive to understanding the health status of the fetus.
Claims
1. A method for detecting fetal erythrocytes by flow cytometry, characterized in that, Includes the following steps: S1: Fetal blood is collected and placed in a test tube. The test tube containing fetal blood is then added to an anticoagulant tube. An anticoagulant is added to the blood in the anticoagulant tube. The anticoagulant tube containing blood and anticoagulant is placed in a blood centrifuge. The blood centrifuge centrifuges the fetal blood in the anticoagulant tube. After centrifugation, the fetal blood in the anticoagulant tube separates into layers. The proportion of each layer is recorded. Red blood cells are at the bottom layer. The proportion of red blood cells in the blood is calculated based on the proportion of red blood cells in the layers. The bottom layer of red blood cells is removed and placed in a new test tube. The model of the blood centrifuge is TGL-12B-C. The speed of the blood centrifuge is 3000 rpm. The centrifugation time of the blood is 30 minutes. The anticoagulant is sodium citrate. After adding sodium citrate to the anticoagulant tube, it needs to be inverted and mixed 5 times. The ratio of sodium citrate to blood is 1:
4. S2: A specific murine monoclonal antibody is added to red blood cells. The murine monoclonal antibody binds to the corresponding protein inside the red blood cells. A flow cytometry fluorescent agent is poured into the detection antigen. The flow cytometry fluorescent agent labels the detection antigen. The labeled antigen is added to the extracted red blood cells. The antigen binds to the murine monoclonal antibody. The bound red blood cells are placed in a nanoflow cytometer. The nanoflow cytometer emits a laser to irradiate the red blood cells. The laser activates the fluorescent agent labeled in the antigen. The fluorescent agent emits excitation fluorescence and scattered light. The fluorescence ratio is observed to understand the proportion of the detected substance in the red blood cell fluid, and the positive ratio of the fluorescent substance is detected.
2. The method for detecting fetal erythrocytes by flow cytometry according to claim 1, characterized in that: After centrifugation, the blood separates into three layers: plasma, red blood cells, and hemoglobin. Plasma, with its lower density, is on top, followed by red blood cells, which are in the middle layer. Hemoglobin, with its highest content, is on the bottom layer. Plasma is pale yellow and transparent, white blood cells are white and opaque, and red blood cells are dark red. The blood layers differ at different stages of fetal development. In the last 4-6 weeks of pregnancy, adult hemoglobin in the fetus gradually increases, accounting for 75%. Granulocytes appear in the fetal blood circulation after 8 weeks of pregnancy. By 32 weeks of pregnancy, the fetal thymus and spleen produce lymphocytes, which become antigens in the body, thus producing white blood cells.
3. The method for detecting fetal erythrocytes by flow cytometry according to claim 1, characterized in that: The red blood cell tubes extracted by layering need to be stored in a low-temperature incubator. The temperature of the incubator needs to be maintained at around 4 degrees Celsius, and the storage time should not exceed 24 hours.
4. The method for detecting fetal erythrocytes by flow cytometry according to claim 1, characterized in that: The murine monoclonal antibody needs to be stored in a cold storage at a temperature of -20 degrees Celsius or -80 degrees Celsius. Antimicrobial drugs need to be added during the storage of the murine monoclonal antibody.
5. The method for detecting fetal erythrocytes by flow cytometry according to claim 1, characterized in that: The nanoflow cytometer contains a forward photodiode and a 90° photomultiplier tube. Excitation fluorescence and scattered light are received by the forward photodiode and the 90° photomultiplier tube. The light scattering signal is detected at a small forward angle, and this signal reflects the size of the cell volume. The direction of fluorescence signal reception is perpendicular to the laser beam. After being separated by a series of dichroic mirrors and bandpass filters, multiple fluorescence signals of different wavelengths are formed.
6. The application of fetal erythrocyte flow cytometry detection according to any one of claims 1-5, characterized in that: Includes the following steps: A: When it is necessary to test the number of red blood cells in whole blood, the whole blood sample is taken out and placed in a test tube, and stored in a suitable environment. The storage time should not be too long. A murine monoclonal antibody corresponding to the red blood cell surface antigen is added to the whole blood. The murine monoclonal antibody will bind to the corresponding antigen on the surface of red blood cells. During this period, it is necessary to stain the specific antigen corresponding to the murine monoclonal antibody. A special fluorescent staining agent is added to the specific antigen to stain it. After staining, the specific antigen is added to the mixture of murine monoclonal antibody and whole blood to obtain a new mixture. Then, the new mixture is placed in a nanoflow cytometer. The nanoflow cytometer emits a laser to activate the fluorescent staining agent in the specific antigen, causing it to emit excitation fluorescence and scattered light. The excitation fluorescence and scattered light are collected and detected. After the test, the proportion of positive cells in the whole blood needs to be checked. The red blood cell count and red blood cell size are determined by the test results. B: When it is necessary to detect the amount of hemoglobin inside red blood cells, the whole blood sample is centrifuged. The red blood cells separated by centrifugation are removed and stored in a suitable environment. Then, the red blood cells are broken down to allow hemoglobin to flow out. A murine monoclonal antibody corresponding to hemoglobin is added to the red blood cell solution. The murine monoclonal antibody will bind to the hemoglobin. During this process, a specific antigen corresponding to the murine monoclonal antibody needs to be stained with a special staining agent. The stained specific antigen is added to the mixture of murine monoclonal antibody and red blood cell solution to obtain a new mixture. The new mixture is then placed in a nanoflow cytometer. The nanoflow cytometer emits a laser to activate the fluorescent staining agent in the specific antigen, causing it to emit excitation fluorescence and scattered light. The excitation fluorescence and scattered light are collected and detected. After the detection, the proportion of positive hemoglobin in the red blood cell solution needs to be checked. The amount and size of hemoglobin are determined based on the above detection results.