2 English reference flow cytometry
Flow cytometry (FCM) is a high-tech developed in 1970s. It integrates computer technology, laser technology, fluid mechanics, cytochemistry and cytoimmunology, and has the function of analyzing and sorting cells. It can not only measure the size of cells and the characteristics of internal particles, but also detect antigens on cell surface and cytoplasm, DNA and RNA content in cells, and is widely used in hematology, immunology, oncology, pharmacology, molecular biology and other disciplines.
3 A brief history of the development of flow cytometry Flow cytometry (FCM) is a new analysis technology and sorting technology, which can quickly measure the structure of cells or subcellulars. Its characteristics are: ① the measurement speed is fast, and tens of thousands of cells can be measured within 1 second at the earliest; (2) Multi-parameter measurement can be carried out, and the physical and chemical characteristics of the same cell can be measured, which has obvious statistical significance; ③ It is a comprehensive high-tech method, which combines the knowledge and achievements of laser technology, computer technology, fluid mechanics, cytochemistry, image technology and other fields. ④ It is not only a cell analysis technique, but also an accurate sorting technique.
To sum up, flow cytometry mainly includes the techniques of sample flow, cell sorting and counting, data collection and analysis. The current development level of FCM embodies people's efforts and achievements in this field for half a century.
1934, Moldavan 1 first put forward the idea of letting suspended single red blood cells flow through a glass capillary, counting with a microscope under bright field of vision and measuring with a photoelectric recording device. Before that, people used to measure static cells, because if a single cell flows through a narrow pipe in sequence, it is easy to cause a large number of cells to deposit and agglomerate. From 65438 to 0953, crossland-Taylor realized from Reynolds' research on the flow law of Newtonian fluid in a circular tube that the faster the sheath fluid flows through the central axis of the tube, the stronger its ability to carry objects and the stronger its hydrodynamic aggregation. Therefore, the flow chamber is designed so that the cell suspension to be analyzed flows around the axis of the circular tube, and the outer layer surrounds the sheath fluid. Both cell suspension and sheath fluid are used as layer fluid. This laid a foundation for the liquid flow technology in modern flow cytometry.
From 65438 to 0956, based on years of research, Kurt produced Kurt counter by using Kurt effect. The basic principle is that when a cell passes through a pore, only the conductivity difference between the cell and the suspension medium will affect the resistance characteristics of the pore, thus forming an electric pulse signal. By measuring the intensity and number of electric pulses, information about the size and number of cells can be obtained. In 1967, Holm et al. designed a device to excite fluorescent stained cells with mercury arc lamp, and then counted it with photoelectric detection equipment. In 1973, Steinkamp designed a device, which can not only analyze and count the cells labeled with two-color fluorescent pigments by laser, but also sort the cells. In this way, the main course of modern FCM counting technology is basically completed.
Modern FCM data acquisition and analysis technology originated from histochemistry, and its pioneer was Kamentsky. In 1965, Kamentsky put forward two new viewpoints on the basis of histochemistry: (1) Cell components can be quantitatively determined by spectrophotometry, that is, spectrophotometry can quantitatively obtain important information of cell histochemistry. (2) Different components of cells can be measured by multiple parameters at the same time, so that cells can be classified. In other words, for the same cell, all kinds of information about different components can be obtained at the same time, which can be used as the basis for identifying cells. Kamentsky is not only quick-thinking but also pragmatic. He was the first person to connect the computer interface to the instrument and record and analyze multi-parameter data, and also the first person to display and analyze multi-parameters by using two-dimensional histogram.
The pioneers of flow cytometry in cytochemistry are Van Dilla and Los Alamos in the United States. In 1967, they developed a flow cytometer in which the liquid beam, the illumination optical axis and the detection system optical axis are orthogonal to each other. They stained DNA with fluorescence Feulgen reaction for the first time to show that there is a linear relationship between DNA activity and fluorescence, and clearly show the various stages of cell cycle on the histogram of DNA. Gohde and Dittrich then put this technology into practice, and they measured the cell cycle by flow cytometry to study the pharmacokinetics of cells. The key of FCM in immunohistochemistry is immunofluorescence staining of cells, which is not much different from cytochemistry.
In recent 20 years, a lot of research and application work has been done on FCM at home and abroad, and many achievements have been made. Especially with the improvement of instruments and methods, people are more and more committed to sample preparation, cell labeling, software development and other aspects to expand the application field and use effect of FCM. The application of FCM in immunohistochemistry is similar, emphasizing the popularization of clinical application.
Working principle of flow cytometry
Dye the cells to be tested and make single cell suspension. The sample to be tested is pressed into the flow chamber at a certain pressure, and the phosphate buffer solution without cells is ejected from the sheath fluid tube at a high pressure. The inlet direction of the sheath fluid tube forms a certain angle with the flow direction of the sample to be tested, so that the sheath fluid can flow around the sample at a high speed to form a circular flow, and the cells to be tested are lined up under the sheath fluid and pass through the detection area in turn.
Flow cytometry usually uses laser as light source. The focused and shaped beam is vertically irradiated on the sample flow, and the cells dyed by fluorescence generate scattered light and excited fluorescence under the irradiation of the laser beam. These two signals are simultaneously received by forward photodiode and photomultiplier tube in 90 direction. The light scattering signal is detected at a small forward angle, which basically reflects the size of the cell. The receiving direction of the fluorescence signal is perpendicular to the laser beam, and after being separated by a series of dichroic mirrors and bandpass filters, a plurality of fluorescence signals with different wavelengths are formed.
The intensity of these fluorescent signals represents the intensity of antigens on the cell membrane surface or the concentration of substances in its nucleus. After being received by the photomultiplier tube, it can be converted into an electrical signal, and then the continuous electrical signal is converted into a digital signal that can be recognized by the computer through the A/D converter. The computer processes the measured signals and displays the analysis results on the computer screen. The liquid can be printed out and stored in the hard disk as a data file for future query or further analysis.
Depending on different measurement parameters, the display of test data can be selected in various forms. Single-parameter data is expressed in the form of histogram, and its X axis is measured intensity and Y axis is cell number. Generally speaking, the resolution of flow cytometry coordinate axis is 5 12 or 1024 channels, which depends on the resolution of its analog-to-digital converter. For two-parameter or multi-parameter data, the histogram of each parameter can be displayed separately, and two-dimensional three-point map, contour map, gray scale map or three-dimensional stereo map can also be selected.
Cell sorting is achieved by separating droplets containing a single cell. The nozzle of the flow chamber is equipped with an UHF transistor, which vibrates after charging, so that the ejected liquid flow is broken into uniform droplets, and the cells to be detected are dispersed in these droplets. These droplets have different positive and negative charges. When the droplets flow through the deflection plate of several thousand volts, they are deflected under the action of high-voltage electric field and fall into their respective collection containers, and the uncharged droplets fall into the middle waste container, thus realizing cell separation.
5 detection range 1. Flow cytometry can detect cell structure, including cell size, cell granularity, cell surface area, nuclear plasma ratio, DNA content and cell cycle, R NA content and protein content.
2. Flow cytometry can detect cell functions, including cell surface/cytoplasm/nucleus specific antigen, cell activity, intracellular cytokines, enzyme activity, hormone binding sites and cell receptors.
Clinical application of 6 flow cytometry 1. Application of flow cytometry in oncology: Flow cytometry can detect tumor cell proliferation cycle, tumor cell surface markers, oncogene expression products, multidrug resistance analysis and apoptosis;
2. Application of flow cytometry in hematology: leukemia and lymphoma cell detection, activated platelets, hematopoietic stem cell (CD34+) count, leukemia and lymphoma immunophenotyping, reticulocyte count, cell transplantation cross matching and immune status monitoring;
3. Application of flow cytometry in immunology: analysis of lymphocytes and their subsets, immunophenotyping of lymphocytes and detection of cytokines can be carried out.
Scientific research and application mainly include cell dynamics function research, environmental microorganism analysis, flow cytometry and molecular biology research.
8. Preparation of samples for routine detection 8. 1 Take a certain amount of cells (about 1× 106 cells /ml) by direct immunofluorescence technique, directly add antibodies connected with fluorescein for immunolabeling reaction (for example, double-label or multi-label staining, several different fluorescein labeled antibodies can be added at the same time), and incubate for 20 ~ 60 minutes. This method is simple, accurate and easy to analyze, and is suitable for simultaneous determination of multiple parameters in the same cell population. Although the cost of direct labeling antibody reagent is high, it reduces the interference of strong nonspecific fluorescence in indirect labeling method, so it is more suitable for the detection of clinical specimens.
8.2 Indirect immunofluorescence method Take a certain amount of cell suspension (about 1X 106 cells /ml), first add the specific first antibody, wash off the unbound antibody after the reaction is complete, then add the fluorescently labeled second antibody to generate antigen-antibody anti-antibody complex, and detect the fluorescence emitted by fluorescein labeled on it after being excited by FCM. This method is low in cost and widely used in the detection of scientific research specimens. However, because the secondary antibody is generally a polyclonal antibody, its specificity is poor and its non-specific fluorescence background is strong, which easily affects the experimental results. Therefore, negative or positive controls should be added when preparing specimens. In addition, the internal standard method is not suitable for the determination of samples with few cells because of its many steps and increased cell loss.
9 Quality control and precautions Flow cytometry is not a fully automatic instrument, and accurate experimental results need accurate manual technical cooperation, so sample preparation needs standardization, and the instrument itself needs quality control.
9. 1 Influencing factors and quality control of immunological detection by flow cytometry Flow cytometry is widely used in immunology, and sample preparation for immunofluorescence staining is very important, which often affects the detection results due to artificial nonspecific fluorescence interference (especially indirect immunofluorescence staining) or low cell concentration. The solutions to these factors are as follows:
(1) Ensure that the concentration of the sample before computer detection is 1X 106/ml. Too low cell concentration will directly affect the detection results.
(2) Using protein blocking agents to block nonspecific binding sites, especially in indirect immunofluorescence technology. Commonly used protein blocking agents are 0.5% bovine serum albumin and 1% fetal bovine serum.
(3) After fluorescent antibody staining, thoroughly clean, pay attention to the stirring and centrifugation speed, and reduce overlapping cells and cell fragments.
(4) Setting control samples, using irrelevant control matched with antibody source and background control of fluorescent antibody.
(5) When judging the results, attention should be paid to subtracting the background fluorescence. In order to make the quantitative analysis of immunofluorescence more accurate, the curve peak of the control group was subtracted from the curve peak of the experimental group by computer program software, so as to obtain more accurate quantitative results of immunofluorescence.
(6) Avoid light after staining to ensure the stability of cell immunofluorescence.
9.2 Quality control of DNA ploidy analysis There is still no uniform standard for the quality control of DNA ploidy analysis, and the experimental results reported in various literatures are quite different. 1993, 10 In June, the American Organization for Research on Cancer formulated a unified standard for the determination of FCMDNA. According to these standards and combined with many years' practice of experienced experts in China, we expounded the quality control and matters needing attention of FCM DNA analysis technology.
(1) When collecting fresh samples or aspirating samples with biopsy needles, bleeding and necrotic tissues should be avoided.
(2) Specimens should be fixed in time or stored at low temperature after collection, so as to avoid tissue autolysis and DNA degradation, leading to errors in test results.
(3) The concentration of fixative should be able to penetrate tissues and cells, and 70% ethanol is the best fixative.
(4) During the preparation of single cell suspension, pay attention to separate the components of cells to be tested, reduce the interference of other components, and be careful not to damage this group of cells.
(5) Collection of cell samples shall ensure sufficient cell concentration, namely 1X 106/ml, and impurities, fragments, lumps and overlapping cells shall
(6) Attention should be paid to the preparation of paraffin-embedded tissue single cells: tissue without autolysis and necrosis should be selected when taking materials, and tumor tissue samples should be selected in areas rich in tumor cells; The thickness of paraffin tissue section should be appropriate, preferably 40 ~ 50 μ m, too thin or too thick sections will affect the detection results; Thoroughly dewaxing, so as to avoid residual paraffin affecting the digestive activity of the enzyme. The way to verify whether dewaxing is complete is to discard xylene and add 100% ethanol. If no floc floats, the wax has been removed. Hydration should be enough to restore the tissue to a state similar to fresh tissue; Pay attention to the digestion time and the activity of digestive enzymes. Routine use of 0.5% pepsin, pH 1.5.
9.3 Operation (1) Flow Cytometer is in the best condition in the whole working process, which can ensure the accuracy and precision of quantitative detection. Using standard samples to adjust the coefficient of variation of the instrument to the minimum range and the resolution to the best state can avoid the detection error caused by the change of instrument conditions in the measurement process.
(2) Coefficient of variation (CV) is an important index to evaluate the accuracy of the instrument. For calibration samples, the smaller the CV value, the better. The smaller the CV value, the higher the accuracy of instrument calibration. Calibration samples include abiotic samples (fluorescent microspheres) and biological cell samples (human lymphocytes, chicken red blood cells, etc.). ). At present, there are commercial reagents for abiotic fluorescent microspheres, and the CV is generally
9.4 Data analysis (1) When there are too many fragments, impurities or lumps in the sample, the number of cells measured is below 20%, and the baseline of the square graph is increased, the analysis should be abandoned.
(2) When conducting cell cycle analysis, the number of sample cells should be 65,438+00,000, excluding fragments, impurities and lumps. When the proportion of aneuploid cells to the total number of cells is less than 65,438+00%, other diagnostic indexes should be combined and conclusions should not be drawn blindly. At least aneuploid cells account for more than 20% of the total number of cells, so the existence of aneuploid can be confirmed.
(3) CV value of normal diploid cells in 3)DNA analysis >: 8%, but the CV value of tumor cells is >; 8%, which is related to the heterogeneity of tumor cells. In addition, during DNA ploidy analysis, 10% drift occurs in different individuals of homologous tissues.