A method for detecting DNA ploidy based on flow cytometry

Abstract

The present invention relates to the technical field of DNA ploidy detection, and in particular, to a method for detecting DNA ploidy based on flow cytometry. The method comprises step 1, reagent preparation; step 2, urine concentration; step 3, cell washing; step 4, adding fixative; step 5, fixing cells; step 6, washing again; step 7, staining; step 8, on-machine detection; in the method for detecting DNA ploidy based on flow cytometry, the effectiveness and stability of the reagents are ensured by controlling the reagent dosage and storage conditions. During the experiment, accurate urine concentration, cell washing, fixation and staining steps improve the accuracy of detection, for example, targeted treatment methods for samples with different cell densities, and at the same time, the secondary combination of cell fixation and staining ensures the specific staining of cell DNA, thereby improving the accuracy of analyzing the properties of DNA ploidy.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA ploidy detection, and in particular to a method for detecting DNA ploidy based on flow cytometry. Background Art

[0002] Nucleic acid changes will occur long before cell morphology changes. The nucleic acid of cells that have a tendency to become cancerous or have already become cancerous will usually proliferate abnormally (increase in content), causing abnormal changes in the cell's DNA content. When normal human somatic cells are at rest, they have a total of 46 chromosomes, which are called diploid cells. However, if the cells become cancerous, tumor cells may proliferate out of control, causing abnormal changes in the cell's DNA content. For example, DNA tetraploid changes have been found in benign tumor cells; malignant tumor cells may have increased DNA aneuploidy; in chronic inflammation, due to continuous stimulation of cells, DNA cell fusion can also be found, followed by abnormal ploidy.

[0003] Although traditional flow cytometric analysis technology can objectively reflect the state of cell proliferation when used for urine cytology examination, it may be insufficient in terms of detection accuracy, stability and adaptability to different diseases. For example, there may be imperfections in the steps of reagent preparation, urine concentration, cell washing, staining and machine testing, resulting in unstable reliability of test results. There may be a lack of targeted processing methods for samples with different cell densities, which affects the accurate judgment of different disease stages, thereby affecting the cell fixation and staining effects, and further affecting the accurate analysis of DNA ploidy.

[0004] In view of this, there is an urgent need for a method for detecting DNA ploidy based on flow cytometry that can effectively improve detection accuracy and stability and ensure good cell fixation and staining effects to improve the shortcomings of existing technologies. Summary of the invention

[0005] The object of the present invention is to provide a method for detecting DNA ploidy based on flow cytometry to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides a method for detecting DNA ploidy based on flow cytometry, comprising the following steps:

[0007] S1. Reagent preparation: urine, nucleic acid dye, fixative, cleaning solution, wherein the main component of the nucleic acid dye is PI;

[0008] S2. Urine concentration: Pour the urine into a container and use a centrifugal concentration device to concentrate it. After the concentration is completed, the supernatant is sucked out and discarded, and the cells at the bottom are retained;

[0009] S3. Cell washing: Add washing solution to the container containing the cell sediment, centrifuge it, then aspirate and discard the supernatant, and repeat the cell washing step 1-2 times;

[0010] S4. Add fixative: Shake the container containing the cell pellet to disperse the cells evenly, then place the container on an oscillator and add fixative while shaking;

[0011] S5. Fix cells: Place the container with the fixative in a refrigerator for fixation;

[0012] S6. Clean again: take the container out of the refrigerator and repeat the cleaning steps in S3 3-4 times;

[0013] S7. Staining: After removing the supernatant, place the container on an oscillator, slowly add the nucleic acid dye while shaking the container, and then place the container at room temperature in a dark environment for 15 minutes;

[0014] S8. On-machine testing: Before on-machine testing, the container is shaken to make the cells evenly distributed in the solution, and then the cells are tested using a flow cytometer to reduce damage to the cells, and the data is analyzed.

[0015] As a further improvement of this technical solution, in S1,

[0016] The amount of urine is 42ml-56ml;

[0017] The dosage of the nucleic acid dye is 0.3ml-0.8ml;

[0018] The amount of the fixing solution is 0.8ml-1.6ml;

[0019] The amount of the cleaning solution used each time is 1.5ml-3ml.

[0020] As a further improvement of the present technical solution, in S3, the centrifugal speed of the centrifuge is 240g-260g.

[0021] As a further improvement of the technical solution, in S3, the centrifugation time of the centrifuge is 4 min-7 min.

[0022] As a further improvement of the technical solution, in S4, the oscillation frequency of the oscillator is 90-120r / min.

[0023] As a further improvement of the technical solution, in S4, the speed of adding the fixing solution is 1-2 drops per second.

[0024] As a further improvement of the technical solution, in S5, the temperature of the refrigerator is controlled at 3-5°C, and the fixed time in the refrigerator is 4h-10h.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. In this method for detecting DNA ploidy based on flow cytometry, the effectiveness and stability of the reagents are ensured by controlling the reagent dosage and storage conditions. During the experiment, precise urine concentration, cell washing, fixation and staining steps improve the accuracy of detection. For example, targeted treatment methods are used for samples with different cell densities. At the same time, the secondary combination of cell fixation and staining ensures the specific staining of cell DNA, thereby improving the accuracy of analyzing the properties of DNA ploidy.

[0027] 2. This method of detecting DNA ploidy based on flow cytometry has good adaptability and practicality. On the one hand, by processing and testing urine, it avoids the pain and rejection caused by invasive examinations for patients; on the other hand, through the secondary combination of cell fixation and staining, it can accurately analyze the DNA ploidy properties of cells, thereby providing accurate information for the detection of urinary tract tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a flowchart of the method for detecting DNA ploidy based on flow cytometry of the present invention;

[0029] Figure 2 A line graph showing the certainty, stability and adaptability of the present invention;

[0030] Figure 3 It is a line graph of the reliability, repeatability and specificity of the present invention. DETAILED DESCRIPTION

[0031] The following will be combined with the accompanying drawings in the present invention to clearly and completely describe the technical solutions in 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 those skilled in the art without creative work are within the scope of protection of the present invention.

[0032] according to Figure 1 As shown, the embodiment of the present invention provides a method for detecting DNA ploidy based on flow cytometry, and the specific steps are as follows:

[0033] Step 1. Reagent preparation: Urine, the amount of urine is 42ml-56ml; Nucleic acid dye, the main component is PI, the amount of nucleic acid dye is 0.3ml-0.8ml, in order to ensure that the dye is within the validity period and storage, it needs to be refrigerated at 2℃-8℃ and avoid light; Fixative, the amount of fixative is 0.8ml-1.6ml, in order to effectively fix the cells, at the same time, not affecting subsequent staining and detection, the fixative needs to be refrigerated at 2℃-8℃; Cleaning solution, the amount of cleaning solution each time is 1.5ml-3ml to remove impurities and unbound substances;

[0034] Step 2: Urine concentration: Pour urine into a container and use a centrifugal concentration device to concentrate it. During the concentration process, pay attention to the volume change of urine to ensure that the concentration effect meets the requirements. After the concentration is completed, the supernatant is sucked out and discarded to avoid disturbing the cell sediment at the bottom and retain the cells at the bottom;

[0035] Step 3, cell washing: Add washing solution to the container containing cell sediment, use centrifuge for centrifugation, the centrifugal speed of the centrifuge is 240g-260g, the centrifugal time of the centrifuge is 4min-7min, the centrifuge should be balanced during the centrifugation process to avoid excessive vibration, then aspirate and discard the supernatant, remove it cleanly, and repeat the cell washing step 1-2 times;

[0036] Step 4: Add fixative: Shake the container containing the cell sediment to disperse the cells evenly, then place the container on an oscillator with an oscillation frequency of 90-120r / min. Add fixative while oscillating at a speed of 1-2 drops per second to avoid bubbles.

[0037] Step 5. Fix the cells: Put the container with the fixative solution into the refrigerator for fixation. The temperature of the refrigerator should be controlled at 3-5℃. The fixation time in the refrigerator is 4h-10h. If long-term storage is required, the container with the fixative solution can be placed in a -20℃ refrigerator for one year. During the storage process, ensure that the sample is well sealed to avoid contamination.

[0038] Step 6: Wash again: Take the container out of the refrigerator and repeat the washing steps in step 3 3-4 times, that is, add washing solution to the container, put it into the centrifuge for centrifugation, ensure that the centrifuge is balanced and the centrifugal speed is moderate, absorb the supernatant after centrifugation and discard it, try to remove impurities, and repeat the operation to achieve the purpose of sufficient washing;

[0039] Step 7: Staining: After removing the supernatant, place the container on an oscillator, slowly add nucleic acid dye while shaking the container to ensure that the cells are fully in contact with the staining solution, and then place the container at room temperature in a light-proof environment for 15 minutes. During the staining process, cover the container with a shading material to prevent light from affecting the staining effect. The shading material can be fabric, such as velvet or cotton with good shading performance. This type of material is soft in texture, can cover the container tightly and is not easy to let light through; it can also be aluminum foil, which has strong shading properties, can effectively block light, and is easy to shape and can fit tightly to the surface of the container;

[0040] Step 8. Machine testing: Before the machine testing, shake the container to make the cells evenly distributed in the solution, then use the flow cytometer to test the cells to reduce damage to the cells, and analyze the data, carefully observe and eliminate debris and adhesions (FSC-A / FSC-H, SSC-A / SSC-H, PI-A / PI-H), accurately determine the position of the G0 / G1 peak, when the cell density is relatively consistent, refer to the adjacent normal samples for comparative analysis; if the density difference is large, make another tube of sample mixed with normal whole blood to confirm the G0 / G1 peak position.

[0041] In the present invention, the reagents are first prepared, and the dosage and storage conditions of urine, nucleic acid dye, fixative and cleaning solution are determined, which can not only ensure that each reagent works within the validity period, but also provide an accurate material basis for subsequent experiments. For example, the main component of nucleic acid dye is PI, which can be stored at 2°C-8°C and avoided from light to ensure its stability and effectiveness. It can also effectively improve the accuracy and reliability of the experiment.

[0042] In addition, by combining cell fixation with nucleic acid dye staining for a secondary time, the cell DNA is specifically stained, which is convenient for accurate detection on the flow cytometer. The reaction mechanism of the secondary binding is that the fixative first fixes the cells to keep the cell structure stable for subsequent operations, and then the nucleic acid dye combines with the DNA in the cells under specific conditions. Since PI is a nuclear staining reagent that stains DNA, it releases red fluorescence after embedding into double-stranded DNA, and thus the DNA ploidy properties of the cells can be analyzed by detecting the fluorescence signal by flow cytometry.

[0043] The effectiveness and stability of the reagents are ensured by controlling the reagent dosage and storage conditions. During the experiment, precise urine concentration, cell washing, fixation and staining steps improve the accuracy of the test. For example, targeted treatment methods are used for samples with different cell densities. At the same time, the secondary combination of cell fixation and staining ensures the specific staining of cell DNA, thereby improving the accuracy of analyzing the ploidy properties of DNA.

[0044] This method has good adaptability and practicality. On the one hand, by processing and testing urine, it avoids the pain and rejection caused by invasive cystoscopy for patients, thereby improving patient acceptance; on the other hand, through the secondary combination of cell fixation and staining, it can accurately analyze the DNA ploidy properties of cells, thereby providing accurate information for the detection of urinary tract tumors.

[0045] According to different raw material dosages, the DNA ploidy detection provided by the present invention is further illustrated through the following specific examples.

[0046] Example 1

[0047] Reagent preparation: Take 45 ml of urine, prepare 0.5 ml of nucleic acid dye, 1 ml of fixative, and 2 ml of cleaning solution each time;

[0048] Urine concentration: Pour urine into a container and concentrate it using a centrifugal concentration device. Closely observe the volume change. After concentration, discard the supernatant and retain the cell pellet.

[0049] Cell washing: add washing solution to the cell pellet, centrifuge at 250g for 5 min, ensure the centrifuge is balanced, discard the supernatant, and repeat twice;

[0050] Add fixative: shake the container to disperse the cells, place it on an oscillator at 100 r / min, and add 1 drop of fixative per second to avoid bubbles;

[0051] Fix cells: Place in a 4°C refrigerator for 8 hours to ensure that the sample is well sealed;

[0052] Wash again: Take out the cells from the refrigerator and repeat the cell washing steps three times;

[0053] Staining: After removing the supernatant, add nucleic acid dye while shaking, incubate at room temperature in the dark for 15 minutes, and shade with velvet cloth;

[0054] On-machine detection: Oscillate to evenly distribute the cells, detect using a flow cytometer, and analyze the data to determine the G0 / G1 peak position.

[0055] Example 2

[0056] Reagent preparation: Take 50 ml urine, 0.6 ml nucleic acid dye, 1.2 ml fixative, and 2.5 ml cleaning solution each time;

[0057] Urine concentration: the same operation as in Example 1;

[0058] Cell washing: centrifugation at 245g for 6 min, repeated twice;

[0059] Add fixative: Oscillate at 110 r / min and add 2 drops of fixative per second;

[0060] Fixed cells: fixed in 3℃ refrigerator for 10h;

[0061] Wash again: Repeat the washing steps four times;

[0062] Dyeing: The same operation as in Example 1, but using cotton cloth with good light-shielding performance for light-shielding;

[0063] Machine testing: the same as in Example 1.

[0064] Example 3

[0065] Reagent preparation: Take 48 ml urine, 0.4 ml nucleic acid dye, 0.9 ml fixative, and 1.8 ml cleaning solution each time;

[0066] Urine concentration: the same operation as in Example 1;

[0067] Cell washing: centrifugation at 255g for 4 min, repeated once;

[0068] Add fixative: shake at 95 r / min, add 1.5 drops of fixative per second;

[0069] Fixed cells: fixed in 5℃ refrigerator for 6h;

[0070] Wash again: Repeat the washing three times;

[0071] Dyeing: The same operation as in Example 1, but using aluminum foil to shield light;

[0072] Machine testing: the same as in Example 1.

[0073] Example 4

[0074] Reagent preparation: Take 52 ml urine, 0.7 ml nucleic acid dye, 1.5 ml fixative, and 2.8 ml cleaning solution each time;

[0075] Urine concentration: the same operation as in Example 1;

[0076] Cell washing: centrifugation at 240 g for 7 min, repeated twice;

[0077] Add fixative: shake at 120 r / min, add 1.8 drops of fixative per second;

[0078] Fixed cells: fixed in 4℃ refrigerator for 9h;

[0079] Wash again: Repeat the wash four times;

[0080] Dyeing: same as in Example 1;

[0081] Machine testing: the same as in Example 1.

[0082] Table 1 Amounts of raw materials used in Examples 1-4

[0083]

[0084] In order to verify that the method for detecting DNA ploidy based on flow cytometry prepared in the embodiment of the present invention has the effects of accuracy, stability, strong adaptability, etc., the detection method provided in the embodiment of the present invention is described through the following test examples.

[0085] Test example

[0086] The purpose of this test group is to explore the effects of different ingredient ratios on the detection method.

[0087] Experimental objective: To detect the accuracy, stability, adaptability, reliability, repeatability and specificity of the cytometry method of the present invention for detecting DNA ploidy.

[0088] Test method: Test group A, test group B, test group C and test group D respectively use the component ratios of DNA ploidy detection provided in Examples 1-4; the control examples use control group A, control group B, control group C, control group D, control group E, control group F, control group G and control group H, wherein:

[0089] Control group A

[0090] Reagent preparation and subsequent steps were performed according to Example 1, but the urine concentration step was omitted, and cell washing was performed directly. Furthermore, no light-shielding material was used during staining.

[0091] Control group B

[0092] Reagent preparation: Take 35 ml urine, 0.2 ml nucleic acid dye, 0.6 ml fixative, and 1 ml cleaning solution each time;

[0093] The subsequent steps were carried out according to Example 1.

[0094] Control group C

[0095] Reagent preparation and subsequent steps were performed as in Example 2, except that the centrifugation speed was increased to 300 g and the centrifugation time was 4 min during cell washing, and repeated washing was not performed.

[0096] Control group D

[0097] Reagent preparation: Take 60ml urine, 1ml nucleic acid dye, 2ml fixative, and 4ml cleaning solution each time;

[0098] The subsequent steps were carried out according to Example 2.

[0099] Control group E

[0100] Reagent preparation and subsequent steps were performed as in Example 3, except that the refrigerator temperature was 1° C. and the fixation time was 12 h when the cells were fixed.

[0101] Control group F

[0102] Reagent preparation: Take 50 ml urine, 0.2 ml nucleic acid dye, 0.7 ml fixative, and 1.2 ml cleaning solution each time;

[0103] The subsequent steps were carried out according to Example 3.

[0104] Control group G

[0105] The reagent preparation and subsequent steps were carried out according to Example 3, but the fixed cells were not washed again.

[0106] Control group H

[0107] Reagent preparation: Take 45 ml urine, 1.2 ml nucleic acid dye, 1.8 ml fixative, and 3.5 ml cleaning solution each time;

[0108] The subsequent steps were carried out according to Example 4.

[0109] Specific detection indicators are shown in Table 2.

[0110] Table 2 Test indicators of each sample

[0111]

[0112] according to Figure 2 , Figure 3 As shown in Table 2, the above comparative data show that the accuracy, stability, adaptability, reliability, repeatability and specificity of the test group are generally higher, among which the various indicators of test group B are relatively the highest, followed by test groups A and D, and test group C is slightly lower;

[0113] The indicators of the control group were significantly lower than those of the experimental group. The indicators of control group A were the lowest because urine was not concentrated and the staining was not shielded from light. Control group B reduced the amount of urine and reagents, but the effect was poor. Control group C increased the centrifugation speed and reduced the number of washing times, with average results. Control group D increased the amount of urine and reagents, and the effect was slightly better than some control groups but still lower than the experimental group. Control group E lowered the refrigerator temperature and extended the fixation time, but the effect was not good. Control group F reduced the amount of nucleic acid dye and fixative, but the effect was poor. Control group G did not perform re-washing, and the effect was not good. Control group H increased the amount of some reagents, but the effect was still lower than the experimental group.

[0114] It can be seen that the urine concentration step plays an important role in improving the accuracy and stability of the test. The indicators of the control group A without urine concentration were significantly lower than those of the test group, indicating that this step can effectively retain cell precipitation, remove impurities, and provide a better sample basis for subsequent testing;

[0115] The appropriate dosage of reagents is crucial to the detection effect. If the dosage is too small, the indicators of control group B and control group F are low; if the dosage is too large, control group H also fails to achieve the best effect. This shows that the appropriate dosage of reagents can ensure good cell fixation and staining effects and improve the accuracy and reliability of the detection.

[0116] The use of shading materials during staining can improve the detection effect. The control group A did not use shading materials, and the indicators were low, indicating that shading materials can avoid the influence of light on the staining effect and ensure the accuracy and specificity of the detection;

[0117] The centrifugation speed, number of repetitions and re-washing in the cell washing step have an impact on the detection effect. Too high or too low centrifugation speed, reduced number of washing times, and no re-washing will reduce the accuracy and stability of the test. For example, the indicators of control group C and control group G are relatively low;

[0118] The refrigerator temperature and time for fixing cells must also be within the appropriate range. If the temperature is too low or the time is too long, as in the control group E, the indicators are not good. This indicates that appropriate fixation conditions can ensure the stability of the cell structure and facilitate subsequent detection.

[0119] On the whole, the best experimental method is Example 2, which performs more prominently in terms of accuracy, stability, adaptability, reliability, repeatability and specificity, and is at a higher level than other examples and the control group in all indicators. At the same time, the various operating steps of Example 2 not only meet the requirements of the invention, but also have good stability and repeatability, and can provide a more reliable method for detecting DNA ploidy based on flow cytometry.

[0120] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for detecting DNA ploidy based on flow cytometry, characterized in that: It consists of the following steps: S1. Reagent preparation: urine, nucleic acid dye, fixative, cleaning solution, wherein the main component of the nucleic acid dye is PI; wherein the amount of urine is 50 mL; the amount of nucleic acid dye is 0.6 mL, and the nucleic acid dye is stored at 2°C-8°C and protected from light; the amount of fixative is 1.2 mL, and the fixative is stored at 2°C-8°C; the amount of cleaning solution each time is 2.5 mL; S2. Urine concentration: Pour urine into a container and use a centrifugal concentration device to concentrate it. During the concentration process, pay attention to the volume change of urine to ensure that the concentration effect meets the requirements. After the concentration is completed, the supernatant is sucked out and discarded to avoid disturbing the cell sediment at the bottom and retain the cells at the bottom; S3. Cell washing: Add washing solution to the container containing cell sediment, use centrifuge for centrifugation, the centrifugal speed of the centrifuge is 245g, the centrifugal time of the centrifuge is 6min, ensure the balance of the centrifuge during the centrifugation process, then absorb and discard the supernatant, remove it cleanly, and repeat the above steps 1-2 times; S4. Add fixative: shake the container containing the cell sediment to disperse the cells evenly, then place the container on an oscillator with an oscillation frequency of 110 r / min, add fixative while oscillating, and add 2 drops of fixative per second to avoid bubbles; S5. Fix cells: Place the container with the fixative in a refrigerator for fixation. The temperature of the refrigerator should be controlled at 3°C ​​and the fixation time should be 10 hours. If long-term storage is required, the container with the fixative can be placed in a -20°C refrigerator. It can be stored for at least one year. During the storage process, ensure that the sample is well sealed to avoid contamination. S6. Clean again: take the container out of the refrigerator and repeat the cleaning steps in S3 3-4 times; S7. Staining: After removing the supernatant, place the container on an oscillator and slowly add the nucleic acid dye while shaking the container to ensure that the cells are fully in contact with the staining solution. Then place the container at room temperature in a dark environment for 15 minutes. Use a light-shielding material to cover the container during the staining process. S8. On-machine testing: Before on-machine testing, oscillate the container to make the cells evenly distributed in the solution, then use flow cytometry to test the cells to reduce damage to the cells, and analyze the data, carefully observe and eliminate debris and adhesions, and accurately determine the position of the G0 / G1 peak. When the cell density is relatively consistent, you can refer to the adjacent normal samples for comparative analysis; if the density difference is large, you need to make another tube of sample mixed with normal whole blood to confirm the G0 / G1 peak position.

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