Flow cytometric DNA analysis

Flow cytometric DNA analysis is an examination method that can study interphase cells and is not affected by the state of cell proliferation, and is important for detecting malignant cells in pleural effusion. Flow cytometry detection range: 1. Flow cytometry can detect cell structure, including cell size, cell size, cell surface area, nucleoplasmic ratio, DNA content and cell cycle, RNA content, protein content. 2. Flow cytometry can detect cellular functions, including cell surface/cytoplasmic/nuclear specific antigens, cellular activities, intracellular cytokines, enzyme activities, hormone binding sites, and cellular receptors. Basic Information Specialist classification: cardiovascular examination classification: blood examination Applicable gender: whether men and women apply fasting: not fasting Tips: Keep a normal mindset. Normal value The body is in a dynamic balance with no disease. Clinical significance 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, conduct multidrug resistance analysis, and detect apoptosis; 2. Application of flow cytometry in hematology to detect leukemia and lymphoma cells, activated platelets, hematopoietic stem cell (CD34+) counts, immunophenotyping of leukemias and lymphomas, reticulocyte counts, cross-matching of cell transplantation, and Immune status monitoring; 3. Flow cytometry in immunology can be used for lymphocyte and its subgroup analysis, lymphocyte immunophenotyping, detection of cytokines. Abnormal results There were data on flow cytometry analysis of 71 cases of pleural effusion. The results showed that the sensitivity of malignant pleural effusion was 52% and the specificity was 100%. If used in conjunction with routine testing, the sensitivity of the diagnosis can be as high as 94%. DNA analysis of cancerous pleural effusion cells showed an increase in the proportion of aneuploid and S phase, G2/M phase cells. People who need to be examined are suspected of having cancerous pleural effusion and other related diseases. Precautions Flow cytometry is not a fully automated instrument. Accurate experimental results require accurate manual techniques. Therefore, specimen preparation requires specification and the instrument itself requires quality control. (1) Influencing factors and quality control of flow cytometry immunological detection Flow cytometry has a wide range of applications in immunology. The preparation of immunofluorescent staining specimens is very important, often due to the occurrence of anthropogenic non-specific fluorescence interference (especially in indirect immunofluorescence staining) or low cell concentration during specimen preparation. Test results. The solutions to these influencing factors are as follows: (1) Ensure that the concentration of the specimen before the machine is detected is 1X106 cells/ml, and the cell concentration is too low, which directly affects the detection result. (2) 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. (3) Fluorescent antibody is washed thoroughly after staining, pay attention to mixing and centrifugation speed, and reduce overlapping cells and cell debris. (4) A control sample was set up using a background control of unrelated control and fluorescent antibodies that matched the same source of antibody. (5) When judging the result, attention should be paid to subtracting the background fluorescence. In order 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 can be obtained. (6) Pay attention to avoiding light after dyeing to ensure the stability of cellular immunofluorescence. (2) There is still no uniform standard for the quality control of DNA ploidy analysis. The experimental results reported in various literatures are quite different. In October 1993, the American Cancer Research Organization developed a unified standard for the determination of FCM DNA, which we combined according to these standards. Experienced experts have practiced for many years to explain the quality control and precautions of FCM's DNA analysis technology. (1) When fresh specimens for surgical resection or biopsy needles are taken, the hemorrhagic necrotic tissue should be avoided. (2) Specimens should be fixed or cryopreserved in time after collection to avoid autolysis and DNA degradation, resulting in errors in test results. (3) The fixative should adopt a concentration that is highly penetrating to the tissue cells, and 70% of the ethanol is better. (4) During the preparation of single cell suspension, take care to separate the components of the cells to be tested, reduce the interference of other components, and take care not to damage the cells. (5) The collection of cell samples should ensure sufficient cell concentration, ie 1×10 6 cells/ml, impurities, debris, clumps and overlapping cells should be <2%, and at least 20% of the samples of tumor cell DNA aneuploids should be analyzed. Tumor cells are present. (6) When preparing the single cell of paraffin-embedded tissue, attention should be paid to the selection of the tissue without autolysis and necrosis. For the tumor tissue specimen, the area containing the tumor cells is selected; the thickness of the paraffin tissue sheet should be suitable, preferably 40 to 50 μm. Excessively thin or too thick slices will affect the test results; thoroughly dewaxed to prevent residual paraffin from affecting the digestive activity of the enzyme. Verify that the dewaxing is complete by discarding xylene and adding 100% ethanol. Floating, indicating that the wax has been removed; hydration is sufficient to restore the tissue to a state similar to fresh tissue; pay attention to the time of digestion and the activity of digestive enzymes. 0.5% pepsin was used routinely, pH 1.5. (3) Operational aspects (1) The flow cytometer is in an optimal state throughout the working process, which can ensure the accuracy and accuracy of quantitative detection. Using the standard sample to adjust the instrument's coefficient of variation to the minimum range, the resolution is in the best state, to avoid detection errors caused by changes in instrument conditions during the measurement process. (2) The important index for evaluating the accuracy of the instrument is the coefficient of variation (CV) of the instrument. For the calibration sample, the smaller the CV value is, the smaller the CV value is, 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.). At present, non-bioluminescent microspheres have commercial reagents, and CV is generally <2% to 3%. (4) Data analysis (1) When there are too many debris or agglomerates in the sample, the number of cells measured is below 20%, and the baseline of the histogram should be abandoned. (2) When performing cell cycle analysis, the number of sample cells should be 10,000, excluding debris, impurities and agglomerates. When the number of aneuploid cells accounts for less than 10% of the total number of cells, it is necessary to combine other diagnostic indicators. In conclusion, at least aneuploid cells account for more than 20% of the total number of cells, and the presence of aneuploids can be determined. (3) In the DNA analysis, the analysis was abandoned when the CV value of the normal diploid cell group was >8%, but the CV value of the tumor cells was >8%, which was related to the heterogeneity of the tumor cells. In addition, in DNA ploidy analysis, 10% of the individuals in the homologous tissue will drift. (4) Quality control of DNA ploidy standards, using 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, normal diploid tissue as Internal standard. Inspection process Flow cytometry analysis: cells in pleural fluid were directly stained with fluorescent dyes (such as propidium iodide), and DNA content, cell cycle distribution, and DNA ploidy of pleural fluid cells were analyzed by flow cytometry. Sample preparation for routine testing by flow cytometry: (1) Direct immunofluorescence labeling Take a certain amount of cells (about 1X106 cells/ml), directly add the fluorescein-conjugated antibody for immunolabeling reaction (such as double-labeled or multi-labeled staining, add several antibodies labeled with different fluorescein at the same time), incubate After 20 to 60 minutes, wash with PBS (pH 7.2 to 7.4) for 1 or 2 times, resuspend in buffer, and test on the machine. The method has the advantages of simple operation, accurate result and easy analysis, and is suitable for simultaneous determination of multiple parameters of the same cell group. Although the cost of the direct-labeled antibody reagent is high, it reduces the interference of strong non-specific fluorescence in the indirect labeling method, and thus is more suitable for the detection of clinical specimens. (two) indirect immunofluorescence labeling Take a certain amount of cell suspension (about 1X106 cells / ml), first add a specific first antibody, wash the unbound antibody after the reaction is complete, and then add a fluorescently labeled secondary antibody to form an antigen-antibody-antibody complex The FCM detects the fluorescence emitted by the labeled fluorescein after it is excited. The method is low in cost, and the secondary antibody is widely used, and is mostly used for the detection of scientific specimens. However, since 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, a negative or positive control should be added to the preparation of the specimen. In addition, due to the large number of steps in the standard labeling method, the loss of cells is increased, and it is not suitable to measure specimens with a small number of cells. Not suitable for the crowd no.