What Is DNA Flow Cytometry?

Flow cytometry DNA analysis is an inspection method that can study interstitial cells and is not affected by the cell proliferation status and is important for detecting malignant cells in pleural effusion. Detection range of flow cytometer: (1) Flow cytometer can detect cell structure, including: cell size, cell size, cell surface area, nuclear plasma ratio, DNA content and cell cycle, RNA content, protein content. (2) Flow cytometry can detect cell functions, including: cell surface / cytoplasmic / nuclear specific antigens, cell activity, intracellular cytokines, enzyme activity, hormone binding sites, and cell receptors.

Flow cytometry DNA analysis

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Flow cytometry DNA analysis is an inspection method that can study interstitial cells and is not affected by the cell proliferation status and is important for detecting malignant cells in pleural effusion. Detection range of flow cytometer: (1) Flow cytometer can detect cell structure, including: cell size, cell size, cell surface area, nuclear plasma ratio, DNA content and cell cycle, RNA content, protein content. (2) Flow cytometry can detect cell functions, including: cell surface / cytoplasmic / nuclear specific antigens, cell activity, intracellular cytokines, enzyme activity, hormone binding sites, and cell receptors.

Name

Flow cytometry DNA analysis

category

Special inspection

The body is in homeostasis and free of disease.

Clinical application of flow cytometry: (1) Application of flow cytometry in oncology: Flow cytometry can detect tumor cell proliferation cycle, detect tumor cell surface markers, oncogene expression products, and perform multidrug resistance Analysis and detection of apoptosis Cross-matching and immune status monitoring of cell transplantation (3) Application of flow cytometry in immunology: analysis of lymphocytes and its subpopulations, immunotyping of lymphocytes, detection of cytokines Abnormal results: Data indicate that 71 cases of pleural effusion were analyzed by flow cytometry DNA. The results showed that the sensitivity to the diagnosis of malignant pleural effusion was 52% and the specificity was 100%. When combined with routine tests, the diagnostic sensitivity can reach 94%. DNA analysis of cancerous pleural effusion cells showed an increase in the proportion of aneuploidy, S-phase, and G2 / M-phase cells. People to be checked: Suspected of cancer-related pleural effusion and other related diseases

Flow cytometry is not a fully automated instrument. Accurate experimental results also require accurate manual techniques. Therefore, specimen preparation needs to be standardized, and the instrument itself needs quality control. (1) Influencing factors and quality control of flow cytometry immunoassay Flow cytometry has a wide range of applications in immunology. The preparation of specimens for immunofluorescence staining is very important, often due to artificial non-specific fluorescence during the preparation of specimens. Interference (especially in indirect immunofluorescence staining) or low cell concentration can affect test results. The methods to solve these influencing factors are as follows: Make sure that the concentration of the specimen before the test is 1X106 cells / ml, and the low cell concentration directly affects the test results. The use of protein blocking agents to block non-specific binding sites is especially necessary for indirect immunofluorescence labeling. Commonly used protein blocking agents are 0.5% bovine serum albumin and 1% fetal bovine serum. Wash the fluorescent antibody well after staining, pay attention to the mixing and centrifugation speed, and reduce overlapping cells and cell debris. Set up the control sample, and use the irrelevant control and the background control of the fluorescent antibody that match the isotype of the antibody source. When judging the results, the background fluorescence should be subtracted. To make the quantitative analysis of immunofluorescence more accurate, the computer program software is used to subtract the curve peak of the control group from the curve peak of the experimental group by the fitting curve method. More accurate immunofluorescence quantitative results. Avoid light after staining to ensure the stability of cell immunofluorescence. (2) There is still no unified standard for the quality control of DNA ploidy analysis. The experimental results reported in different literatures are quite different. In October 1993, the American Cancer Research Organization formulated a unified standard for FCMDNA determination. Experienced experts with many years of practice explain the quality control and precautions of FCM's DNA analysis technology. When removing fresh specimens or biopsy needles for surgical resection, avoid bleeding and necrosis. After the specimens are collected, they should be fixed or stored at low temperature in time to avoid autolysis and DNA degradation of the tissues, which will cause errors in the test results. The fixative should be used at a concentration that is highly permeable to tissue cells. 70% ethanol fixation effect is better. During the preparation of the single cell suspension, pay attention to separate the components of the cells to be tested, reduce the interference of other components, and be careful not to damage the group of cells. The collection of cell samples should ensure a sufficient cell concentration, ie 1X106 cells / ml, impurities, debris, clumps and overlapping cells should be <2%. For analysis of tumor cell DNA aneuploid, at least 20% of the tumor cells presence. Pay attention to the preparation of paraffin-embedded tissue single cells: when collecting materials, select non-autolytic and necrotic tissues. For tumor tissue samples, select areas containing tumor cells. The thickness of the paraffin tissue pieces should be appropriate, preferably 40- 50 m. Too thin or too thick sections will affect the test results. Thoroughly dewax to prevent the residual paraffin from affecting the digestive activity of the enzyme. The method to verify the complete dewaxing is to discard xylene and add 100% ethanol. If there is no floc Floating indicates that the wax has been removed; hydration should be sufficient to reduce the tissue to a state similar to fresh tissue; pay attention to the time of digestion and the activity of digestive enzymes. Routinely use 0.5% pepsin, pH 1.5. (3) Operation The flow cytometer is in the best state during the entire working process, which can ensure the accuracy and precision of quantitative detection. Use the standard sample to adjust the variation coefficient of the instrument in the smallest range and the best resolution, which can avoid detection errors caused by changes in instrument conditions during the measurement process. The important index for evaluating the accuracy of the instrument is the coefficient of variation (CV) of the instrument. For calibration samples, the smaller the CV value, the better, and the smaller the CV value, indicating that the accuracy of the instrument calibration is higher. Calibration samples include non-biological samples (fluorescent microspheres) and biological cell samples (human lymphocytes, chicken red blood cells, etc.). Currently, non-biofluorescent microspheres have commercial reagents, and the CV is generally <2% -3%. (4) Analysis of data When there are too many debris impurities or clumps in the sample, the number of measured cells is below 20%, and the baseline of the histogram is raised, the analysis should be abandoned. When performing cell cycle analysis, the number of sample cells should be 10,000, and debris, impurities and clumps should be excluded. When the number of aneuploid cells accounts for less than 10% of the total number of cells, other diagnostic indicators should be combined, and conclusions should not be made blindly. The presence of aneuploidy can be confirmed by at least 20% of the total number of aneuploid cells. During DNA analysis, the analysis of the normal diploid cell group CV value> 8% was abandoned, but the CV value of the tumor cells> 8% was related to the heterogeneity of the tumor cells. In addition, during DNA ploidy analysis, different individuals of homologous tissues will drift by 10%. The quality control of the DNA ploidy standard uses the same individual homologous normal tissue, the same fixation method, the same sample processing method, the same staining method, simultaneous staining, the same instrument detection conditions, and normal diploid tissue as the internal standard .

Flow cytometry DNA analysis: Stain cells in pleural fluid directly with a fluorescent nucleic acid dye (such as propidium iodide). Flow cytometry analyzes the DNA content, cell cycle distribution, and DNA ploidy number of pleural fluid. Sample preparation for routine detection by flow cytometry: (1) Take a certain amount of cells (approximately 1X106 cells / ml) by direct immunofluorescence labeling, and directly add antibodies linked to fluorescein for immunolabeling reactions (such as double standard or multi-label For standard staining, several antibodies labeled with different fluorescein can be added at the same time). After incubation for 20-60 minutes, wash with PBS (pH7.2-7.4) 1-2 times, add buffer to resuspend, and test on the machine. The method is simple, accurate, and easy to analyze. It is suitable for simultaneous determination of multiple parameters in the same cell population. Although direct-labeled antibody reagents are more expensive, they reduce the interference of strong non-specific fluorescence in indirect labeling methods, so they are more suitable for the detection of clinical specimens. (2) Take a certain amount of cell suspension (about 1 × 106 cells / ml) by indirect immunofluorescence labeling, first add a specific primary antibody, wash the unbound antibody after the reaction is complete, and then add a fluorescently labeled second antibody. An antigen-antibody-antibody complex is generated, and the fluorescence emitted by the labeled fluorescein is excited by FCM. This method has low cost, and the secondary antibody is widely used, and it is mostly used for the detection of scientific specimens. However, because the secondary antibody is generally a polyclonal antibody, the specificity is poor, and the non-specific fluorescent background is strong, which easily affects the experimental results. Therefore, negative or positive controls should be added when preparing specimens. In addition, due to the many steps of the inter-standard method, the loss of cells is increased, so it is not suitable for measuring samples with a small number of cells.

DNA, localized pleural mesothelioma, pneumonia-like pleural effusion, tuberculous pleurisy, phenylketonuria in children, etc.

Pleural effusion, lung pleural effusion, malignant pleural effusion, bloody pleural effusion, exudative pleural effusion, etc.

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