Blood cell detection and separation method based on flow cytometry

Abstract

The invention relates to a blood cell detection and separation method based on flow cytometry, which relates to the technical field of cell detection, overcomes the limitation of the traditional method and the existing flow cytometry, and provides an excellent cell analysis tool for clinical medicine and life science research. First, the method selects one or more novel blood cell markers, which have high selectivity and sensitivity to blood cells. The markers can clearly distinguish the blood cells from other cell types, and lay a foundation for accurate identification of the blood cells. Secondly, by optimizing parameter setting of the flow cytometer, including laser power, a fluorescence detection channel and the like, efficient analysis of blood cells is realized. Through multi-parameter analysis, fluorescence signals and other related parameters of the cells can be obtained, and the accuracy and sensitivity of blood cell detection are further improved. In addition, the method is also combined with a cell sorting function of a flow cytometer, and an efficient cell separation strategy is provided. According to the analysis result of the flow cytometry, the blood cells can be independently separated and collected, so that a blood cell subset with relatively high purity is obtained. Therefore, a reliable basis is provided for subsequent blood cell research and application.

Description

Technical Field

[0001] The invention belongs to the technical field of cell detection, and in particular relates to a blood cell detection and separation method based on flow cytometry. Background Art

[0002] Blood cell testing is a critical process in clinical medicine and research to assess patient health and diagnose diseases. Traditional blood cell testing methods usually rely on microscopic observation and manual counting, which have many limitations, including cumbersome operation, time-consuming, high subjectivity, and the inability to provide real-time data.

[0003] With the advancement of technology, flow cytometry has become a powerful tool for analyzing and separating blood cells. Flow cytometry uses the principle of flowing cells through an optical detection system to detect specific markers of different types of cells, such as fluorescent markers, to analyze millions of cells at high speed and high throughput, providing accurate and reliable results.

[0004] However, despite the advantages of flow cytometry, there are still some challenges. For example, for some special types of blood cells, accurate separation and detection is still a challenge, which requires more advanced technologies and methods to achieve. In addition, the degree of automation of flow cytometry also needs to be further improved to reduce the dependence of operators and reduce potential operating errors.

[0005] Therefore, the present invention aims to overcome the limitations of traditional blood cell detection and flow cytometry, and provide an efficient, accurate and automated blood cell detection and separation method to meet the needs of clinical and research fields for rapid and precise cell analysis. By combining flow cytometer technology and special labeling methods, the present invention makes the classification and separation of blood cells more accurate and reliable, providing a more powerful tool for medical diagnosis and disease research. Summary of the invention

[0006] The purpose of the present invention is to solve the above technical problems and provide a blood cell detection and separation method based on flow cytometry.

[0007] The present invention uses a new cell labeling method called "Ultra-Precision Fluorescent Labeling" (UPFL). The UPFL method is based on a combination of highly specific antibodies and highly affinity fluorescent labeling agents. These antibodies have extremely low cross-reaction rates, ensuring that each cell type can be accurately labeled. In addition, the fluorescence signal intensity of the UPFL labeling agent is optimized to ensure high sensitivity of the detection results.

[0008] The flow cytometer we use is the latest model XYZ-2000, which has 16-channel multi-parameter detection capabilities. The instrument can simultaneously measure multiple parameters of each cell, such as cell size, cell shape, color and intensity of fluorescent markers, etc. The simultaneous measurement of these parameters not only improves the detail of cell characteristics, but also shortens the detection time, making it suitable for high-throughput analysis.

[0009] The method of the present invention realizes a highly automated cell detection and separation process. Sample preparation includes automatic collection and pre-treatment of blood samples without manual intervention. The loading and data collection of cell samples are also fully automated, reducing the operator's operation workload and operation errors.

[0010] We have developed a set of advanced cell separation and identification algorithms based on machine learning and deep learning technologies. These algorithms can accurately separate and identify different types of cells based on multi-parameter characteristics of cells, such as size, shape, fluorescence signals, etc. The innovation of the algorithm lies in its high adaptability and ability to cope with the differences between different samples.

[0011] The present invention can be widely used in clinical medicine and life science research. In clinical practice, it can be used for rapid and accurate leukemia diagnosis, disease monitoring and drug efficacy evaluation. In life science research, it provides a high-throughput cell analysis tool that can be used for phenotypic research, drug screening and disease mechanism research.

[0012] The present invention provides a user-friendly data analysis interface that can convert the massive data generated by the flow cytometer into easy-to-understand graphics and reports. Users can customize the content and format of the report to meet their specific needs.

[0013] We conducted a series of experimental verifications, including the automation of sample processing, the accurate separation and identification of different types of blood cells, and the reliability of data processing. The experimental results show that the method of the present invention has high consistency and repeatability.

[0014] The UPFL labeling method and adaptive algorithm of the present invention have brought innovative breakthroughs to the field of blood cell detection and separation. By combining high-precision cell labeling and high-throughput flow cytometry, the present invention realizes highly automated cell analysis with the advantages of high precision, high efficiency and wide application.

[0015] This example provides a more detailed refinement of the invention content to more clearly convey the core features and advantages of the invention. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0017] Example:

[0018] A blood cell detection and separation method based on flow cytometry, the blood cell detection and separation method using flow cytometry comprising the following steps:

[0019] S1: Use an appropriate blood collection needle and blood collection tube to collect a sufficient amount of blood sample, usually 2 to 5 ml;

[0020] S2: Use anticoagulants (e.g. EDTA) to prevent blood clotting;

[0021] S3: Add ammonium chloride, a red blood cell lysis buffer, to achieve this. Mix the sample to lyse the red blood cells;

[0022] S4: Wash the cell suspension to remove the cell culture medium, enzymes and cell debris in the suspension. This can be achieved by adding PBS and centrifuging. Repeat the washing steps to ensure that the blood is washed clean;

[0023] S5: Use fluorescently labeled CD4 antibodies;

[0024] S6: Mix the cell suspension with the fluorescent antibody and incubate it so that the antibody can bind to the target molecule. Incubate at 4°C for 30 minutes.

[0025] S7: Turn on the flow cytometer and preheat it. Before use, perform instrument calibration, including laser calibration and detection channel calibration, and inject the labeled cell suspension into the sample device of the flow cytometer. Make sure the number of injected cells is within the linear range of the instrument, start the flow cytometer, and start data acquisition. Excite the laser to illuminate the cells in the sample, and then measure the fluorescence signals of different wavelengths and the scattered light signals.

[0026] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A blood cell detection and separation method based on flow cytometry, characterized in that: The blood cell detection and separation method using flow cytometry comprises the following steps: S1: Use an appropriate blood collection needle and blood collection tube to collect a sufficient amount of blood sample, usually 2 to 5 ml; S2: Use anticoagulants (e.g. EDTA) to prevent blood clotting; S3: Add ammonium chloride, a red blood cell lysis buffer, to achieve this. Mix the sample to lyse the red blood cells; S4: Wash the cell suspension to remove the cell culture medium, enzymes and cell debris in the suspension. This can be achieved by adding PBS and centrifuging. Repeat the washing steps to ensure that the blood is washed clean; S5: Use fluorescently labeled CD4 antibodies; S6: Mix the cell suspension with the fluorescent antibody and incubate it so that the antibody can bind to the target molecule. Incubate at 4°C for 30 minutes. S7: Turn on the flow cytometer and warm it up. Before use, perform instrument calibration, including laser calibration and detection channel calibration, and inject the labeled cell suspension into the sample device of the flow cytometer. Make sure the number of injected cells is within the linear range of the instrument, start the flow cytometer, and start data acquisition. Excite the laser to illuminate the cells in the sample, and then measure the fluorescence signals of different wavelengths and scattered light signals.