Peripheral blood lymphocyte culture standardization process

Abstract

The invention discloses a peripheral blood lymphocyte culture standardization process, and relates to the technical field of cell culture, and the peripheral blood lymphocyte culture standardization process comprises the following steps: S1, blood sample planting; s2, cell culture; s3, harvesting the cells; s4, wafer dropping; s5, carrying out banding treatment; and S6, scanning and analyzing. The standardized process intends to standardize the whole peripheral blood chromosome preparation process, manual errors are reduced through instrument operation, experience judgment is replaced with specific numerical values, the standard operation process can avoid the slide preparation gap caused by human factors, then the peripheral blood chromosome karyotype analysis resolution is improved, a guarantee is provided for the reliability of clinical results, and the method is suitable for large-scale popularization and application. The step is the key of cytogenetics research, and detailed cell and chromosome data are provided; the standardized process aims to ensure that high-quality data output can be achieved in the culture, treatment and analysis process of the lymphocytes, so that the final analysis result is accurate and reliable.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a standardized process for culturing peripheral blood lymphocytes. Background Art

[0002] Peripheral blood lymphocytes are mainly lymphocytes in the blood circulation, which are composed of T cells and B cells. Peripheral blood lymphocyte culture is mainly used to study the function, cell proliferation and response of the immune system. By isolating lymphocytes from peripheral blood and proliferating them under specific culture conditions, we can gain a deeper understanding of cell behavior, immune response and related disease mechanisms.

[0003] Compared with molecular detection experiments with strict and standardized procedures, cytological experiments are more dependent on the operator's experience. In clinical practice, a series of experimental processes for peripheral blood lymphocyte culture, harvesting, and preparation lack standardization, and the experimental descriptions lack practicality. They are usually completed based on the experimenter's experience and feelings, resulting in uneven specimen preparation levels among clinical laboratories and significant differences in detection results.

[0004] Based on this, a standardized process for peripheral blood lymphocyte culture was designed with high resolution for peripheral blood chromosome karyotype analysis to ensure the reliability of clinical results. Summary of the invention

[0005] The present invention aims at the deficiencies of the prior art and provides the following technical solution: a standardized process for culturing peripheral blood lymphocytes, comprising the following steps:

[0006] S1, blood sample planting: used for sample processing;

[0007] S11, draw peripheral blood using a sodium heparin anticoagulant tube (3-5 ml), and mix by gently inverting up and down 10 times;

[0008] S12, under sterile conditions, use a 1000 μl pipette to draw 400 μl of blood, inoculate into human lymphocyte culture medium, and gently invert up and down 10 times to mix;

[0009] S2, cell culture: the inoculated peripheral blood was placed in a 37°C constant temperature incubator and cultured for 68-72 hours;

[0010] S21, culture medium improvement:

[0011] S211, Study new culture medium components or optimize existing culture medium to enhance lymphocyte proliferation and function;

[0012] S212, improve the gas concentrations of oxygen and carbon dioxide in the culture environment to better simulate the in vivo environment;

[0013] S213, explore the optimal culture temperature, humidity and fluctuation range to ensure the best growth conditions for cells;

[0014] S22, automated culture system:

[0015] S221, introduce automated equipment for cell culture, transfer, and separation to reduce human error and improve reproducibility;

[0016] S222, using high-throughput screening systems for large-scale cell screening and analysis to accelerate the experimental process;

[0017] S23, Cell Isolation and Enrichment:

[0018] S231, develop novel cell separation technologies, such as magnetic bead sorting or flow cytometry sorting, to improve the purity and quantity of lymphocytes;

[0019] S232, Application of microfluidics technology for cell separation and analysis to achieve more sophisticated cell manipulation and control;

[0020] S3, cell harvest:

[0021] S31, 2 hours before the completion of the culture, 30 μl of colchicine at a concentration of 20 μg / ml was added and the culture was continued for 2 hours;

[0022] S32, prepare hypotonic solution and fixative solution, the concentration of hypotonic solution is 0.075 mol / L KCl, select 2.8 g KCl and distilled water to make up to 500 ml, place it at 37°C for pre-warming, and the fixative solution includes methanol and glacial acetic acid in a ratio of 3:1;

[0023] S33, harvested using an automatic harvester, set the centrifugation parameters to 1500 rpm, centrifuged for 7 minutes, removed the supernatant, and retained 0.5 ml of cell pellet;

[0024] S34, add 5 ml of hypotonic solution, mix with an oscillator for 30 seconds, and place in a 37°C water bath for hypotonic treatment for 30 minutes;

[0025] S35, add 2 ml of freshly prepared fixative, mix gently 5 times, and place in a 37°C water bath for 10 minutes;

[0026] S36, centrifuge at 1500 rpm for 7 min, remove the supernatant, and retain 0.5 ml of the cell pellet;

[0027] S37, add 8 ml of freshly prepared fixative solution, mix gently 5 times, let stand for 5 minutes, centrifuge at 1500 rpm × 7 min, repeat 3 times, and finally retain 0.5 ml of cell suspension;

[0028] S4, drop tablets:

[0029] S41, controlling the humidity and temperature of the dripping room to 50%-55% and 23-25°C;

[0030] S42, placing a clean glass slide in the glass slide slot at the center mark position of the airing board;

[0031] S43, use a shaker to mix each tube of cell suspension for 1 min, and use a 100 μl pipette to draw 35 μl of cell suspension;

[0032] S44, with the elbow at a 60° angle on the table, place the pipette perpendicular to the surface of the slide and quickly drop the suspension at a height of 1 cm to form a complete large circle that does not exceed the edge of the slide;

[0033] S45, after the liquid surface is dry, the slide is placed in a 75°C electric oven and baked for 3 hours;

[0034] S5, banding processing:

[0035] S51, improved banding technology:

[0036] S511, cooling the baked slides, and placing them in a 0.025% trypsin solution at 37°C for digestion for 60 seconds, wherein the pH of the trypsin solution is between 7.2 and 7.4, and the trypsin time can be adjusted according to the banding effect;

[0037] S512, rinse once in 0.85% NaCl solution at 37°C;

[0038] S513, use Giemsa stock solution diluted 5 times as working solution, stain at 37°C for 30 seconds, then rinse with tap water and dry;

[0039] S52, Data Analysis and Artificial Intelligence:

[0040] S521, Application of machine learning and data analysis techniques to optimize chromosome banding conditions;

[0041] S522, develop an intelligent decision-making system to automatically adjust banding conditions to achieve the best staining effect;

[0042] S6, Scanning and Analysis:

[0043] S61, scan the karyotype using a film scanner, set to capture 120 mitotic phases;

[0044] S62, cleavage phases with more than 400 bands were selected for analysis and detailed karyotype analysis was performed.

[0045] As an improvement of the above technical solution, in step S513, the method for preparing the Giemsa mother solution includes the following steps:

[0046] A1, weigh the raw materials: weigh 3.5 g of Giemsa powder;

[0047] A2, prepare the stock solution: add 3.5 g Giemsa powder into a mixed solvent of 500 ml methanol and 500 ml glycerol, use a measuring cylinder to mix, and mix upside down until the powder is completely dissolved;

[0048] A3, storage and stirring: the prepared mother solution was divided into light-proof glass bottles, the glass bottles were placed in a 37°C water bath, and stirred for 24 hours to ensure that the reagents were fully dissolved;

[0049] A4, heating treatment: transfer the mother liquor to a 75°C water bath for heating treatment for 72 hours. During this process, mix the mother liquor more than ten times a day to ensure the uniformity of the reagent;

[0050] A5, prepare the working solution: take 45 ml of buffer solution with a pH value in the range of 6.4 to 6.8, add 3 ml of the mother solution, and dilute it into the working solution; after heating the working solution at 37°C for 30 minutes, it can be used for staining experiments.

[0051] Beneficial effects of the present invention: This standardized process intends to standardize the entire process of peripheral blood chromosome preparation, reduce manual errors by instrument operation, and replace empirical judgments with specific values. The standard operating process can avoid the production gap caused by human factors, thereby improving the resolution of peripheral blood chromosome karyotype analysis and providing a guarantee for the reliability of clinical results; by transferring the peripheral blood sample to the culture medium, the cell culture process begins. This step ensures that the lymphocytes can begin to proliferate and expand in a suitable environment, providing a sufficient number of cells for subsequent analysis; in the incubator, the cells grow under suitable temperature, humidity and gas conditions. This stage allows the lymphocytes to proliferate and may stimulate the cells to enter the division cycle during the culture process to prepare for subsequent analysis; by harvesting the cells, the cells can be separated and concentrated from the culture bottle. This step is for subsequent processing and Preparation for analysis ensures that cells can be effectively processed and concentrated before analysis; the cell suspension is dropped onto a glass slide through a dropper technique to form a cell monolayer. This step is to prepare for banding processing so that the cells can clearly display their structural characteristics during the banding process; through banding processing such as using staining reagents, the chromosomes of the cells are stained to make them easier to observe under a microscope. This process can help analyze the structure and number of chromosomes and conduct genetic research; scanning and analyzing cells under a microscope can observe the morphology of cells, chromosomal abnormalities and other genetic characteristics. This step is the key to cytogenetics research and provides detailed cell and chromosome data; this standardized process is designed to ensure that lymphocytes can achieve high-quality data output during culture, processing and analysis, so that the final analysis results are accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It is a flow chart of the present invention. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0054] See also Figure 1 The present invention provides a technical solution: a standardized process for culturing peripheral blood lymphocytes, comprising the following steps:

[0055] S1, blood sample planting: used for sample processing; the processing of blood samples ensures that lymphocytes in peripheral blood can grow and proliferate smoothly in the culture medium.

[0056] S11, use 3-5ml sodium heparin anticoagulant tube to draw peripheral blood, and gently invert it upside down 10 times to mix; sodium heparin as an anticoagulant can effectively prevent blood from coagulation during the extraction process, thereby ensuring that the lymphocytes in the blood sample are not damaged. Gently inverting the blood to mix it helps to fully distribute the anticoagulant in the blood and prevent blood stratification or clot formation. This can maintain the consistency and uniformity of the blood sample and ensure the representativeness and reliability of the cells in subsequent processing.

[0057] S12, under sterile conditions, use a 1000μl pipette to draw 400μl of blood, inoculate it into human lymphocyte culture medium, and gently invert it 10 times to mix; operating under sterile conditions can prevent contamination by exogenous microorganisms and ensure the purity and safety of cell culture; using a pipette to accurately draw blood and inoculate it into the culture medium can effectively control the amount of blood added, so that the concentration of cells in the culture medium is moderate, which is conducive to cell growth and proliferation. Gentle mixing can fully mix the blood and culture medium, ensure that lymphocytes can be evenly distributed in the culture medium, and avoid data deviation caused by uneven distribution of cells during the culture process.

[0058] S2, cell culture: the inoculated peripheral blood was placed in a 37°C constant temperature incubator and cultured for 68-72 hours;

[0059] S21, culture medium improvement:

[0060] S211, research new culture medium components or optimize existing culture medium to enhance lymphocyte proliferation and function; optimizing culture medium components can enhance lymphocyte proliferation and function and ensure that cells receive optimal nutrition and growth support during culture.

[0061] S212, improve the gas concentrations of oxygen and carbon dioxide in the culture environment to better simulate the in vivo environment; adjusting the oxygen and carbon dioxide concentrations to simulate the in vivo environment helps maintain the physiological state of cells and avoids the decline of cell function due to inappropriate gas concentrations.

[0062] S213, explore the optimal culture temperature and fluctuation range to ensure the best growth conditions for cells; determining the optimal culture temperature and fluctuation range can ensure that cells grow under optimal conditions and reduce cell stress and slow proliferation caused by environmental instability.

[0063] S22, automated culture system:

[0064] S221, introduce automated equipment for cell culture, transfer and separation to reduce human errors and improve repeatability; automated equipment reduces human errors, increases experimental consistency and repeatability, and improves operational efficiency and data reliability.

[0065] S222 uses a high-throughput screening system for large-scale cell screening and analysis to accelerate the experimental process; the high-throughput screening system can quickly process a large number of samples, accelerate the cell screening and analysis process, and improve the overall efficiency of the experiment.

[0066] S23, Cell Isolation and Enrichment:

[0067] S231, Develop novel cell separation technologies, such as magnetic bead sorting or flow cytometry sorting, to improve the purity and quantity of lymphocytes; novel cell separation technologies such as magnetic bead sorting and flow cytometry sorting can improve the purity and quantity of cells and ensure high-quality cell materials required for experiments.

[0068] S232, Apply microfluidics technology for cell separation and analysis to achieve more precise cell manipulation and control; Microfluidics technology enables precise cell manipulation and control, improves the accuracy of cell separation and analysis, and helps to obtain more accurate experimental data.

[0069] S3, cell harvest:

[0070] S31, 2 hours before the completion of the culture, add 30 μl of colchicine at a concentration of 20 μg / ml and continue to culture for 2 hours; colchicine can inhibit the formation of spindles, thereby preventing chromosome separation during cell division. This helps to "freeze" chromosomes at a specific stage of cell division (usually metaphase or anaphase), facilitating subsequent cytogenetic analysis, such as chromosome karyotype analysis.

[0071] S32, prepare hypotonic solution and fixative. The concentration of hypotonic solution is 0.075 mol / L KCl. Select 2.8 g of KCl and mix it with distilled water to 500 ml. Place it at 37°C for preheating. The fixative includes methanol and glacial acetic acid in a ratio of 3:1. The hypotonic solution 0.075 mol / L KCl is used to swell the cells, which is convenient for observing the chromosomes in the cells. In a hypotonic environment, the liquid concentration in the cells is high, which makes the cells swell and the separated chromosomes are more spaced, thereby improving the visibility of the chromosomes. The fixative methanol and glacial acetic acid, 3:1, are used to fix the cells to prevent the cells from changing during the treatment process. The mixture of methanol and glacial acetic acid can quickly fix the structure in the cells and keep the chromosomes and cell membranes stable for subsequent staining and microscopic observation.

[0072] S33, harvested using a fully automatic harvester, set the centrifugation parameters to 1500rpm, centrifuged for 7 minutes, removed the supernatant, and retained 0.5ml of cell pellet; the use of a fully automatic harvester and centrifugation process can efficiently collect cell pellets and remove supernatant, ensuring a high concentration of cell pellets for further processing.

[0073] S34, add 5 ml of hypotonic solution, mix with an oscillator for 30 seconds, and place in a 37°C water bath for hypotonic treatment for 30 minutes; hypotonic treatment causes the cells to swell and further separate the chromosomes, which helps to improve the resolution and observability of chromosomes under a microscope.

[0074] S35, add 2 ml of freshly prepared fixative, mix gently 5 times, and place in a 37°C water bath for 10 minutes; after mixing the cells with the fixative, the fixative can quickly fix the structure of the chromosomes to avoid changes during subsequent processing and staining. This step is crucial to preserve the morphology and structure of the chromosomes.

[0075] S36, centrifuge at 1500 rpm for 7 min, remove the supernatant, and retain 0.5 ml of the cell pellet;

[0076] S37, add 8 ml of freshly prepared fixative solution, mix gently 5 times, let stand for 5 minutes, centrifuge at 1500 rpm × 7 min, repeat 3 times, and finally retain 0.5 ml of cell suspension;

[0077] The supernatant is removed by centrifugation and the fixative is added repeatedly to ensure adequate fixation of the cell sample and further remove residual hypotonic fluid and other impurities. The final retained cell suspension will be used for observation and analysis under a microscope after fixation.

[0078] S4, drop tablets:

[0079] S41, control the humidity and temperature of the drop slide room at 50%-55% and 23-25℃; ensuring moderate humidity and temperature will help form uniform drop slides and prevent the slides from drying too quickly, resulting in distortion of chromosome samples or uneven sample dispersion.

[0080] S42, mark the center of the slide slot on the drying board and place a clean slide in the slot; ensuring that the droplet is positioned accurately will help spread the cell suspension evenly and avoid sample deviation or uneven distribution.

[0081] S43, use an oscillator to mix each tube of cell suspension for 1 minute, and use a 100μl pipette to aspirate 35μl of cell suspension; ensure that the cell suspension is uniform to avoid affecting the final chromosome observation effect due to uneven distribution of cells.

[0082] S44, support your elbow on the table at a 60° angle, hold the pipette perpendicular to the surface of the slide, and quickly drip the suspension from a height of 1 cm to form a complete large circle without exceeding the edge of the slide; drip the cell suspension at an appropriate angle and height to form a complete large circle, ensure that the sample evenly covers the slide, and reduce bubbles or edge effects.

[0083] S45, after the liquid surface is dry, place the glass slide in a 75°C electric oven and bake for 3 hours; by heating in the electric oven, the sample is further dried so that the cells are fixed on the glass slide, which is convenient for subsequent staining and microscopic analysis.

[0084] S5, banding processing:

[0085] S51, improved banding technology:

[0086] S511, cool the baked slides and place them in a 0.025% trypsin solution at 37°C for 60 seconds. The pH of the trypsin solution is between 7.2-7.4. The trypsin time can be adjusted according to the banding effect. The use of trypsin and pH control can effectively remove proteins on the cell surface and optimize the clarity and contrast of cell staining.

[0087] S512, rinse once in 0.85% NaCl solution at 37°C; rinsing with 0.85% NaCl solution can remove unbound enzymes and other impurities and ensure uniform staining.

[0088] S513, dilute Giemsa stock solution 5 times as working solution, stain at 37°C for 30 seconds, then rinse with tap water and dry; dilution and time control of Giemsa staining help to obtain the best banding effect, making the chromosome structure clearer and easier to observe and analyze.

[0089] S52, Data Analysis and Artificial Intelligence:

[0090] S521, Apply machine learning and data analysis techniques to optimize chromosome banding conditions; Use machine learning and data analysis techniques to automatically identify and adjust optimal staining conditions to improve the consistency and accuracy of staining effects.

[0091] S522, develop an intelligent decision-making system to automatically adjust the banding conditions to achieve the best staining effect; develop an intelligent system that can automatically adjust the banding conditions according to real-time data, so as to achieve the best banding effect under different experimental conditions and improve the overall experimental efficiency and result quality.

[0092] S6, Scanning and Analysis:

[0093] S61, use the scanner to scan the karyotype and set it to shoot 120 mitotic phases; Scanning the karyotype with the scanner and setting it to shoot 120 mitotic phases can ensure that enough cell division phases are obtained to provide a comprehensive view of the sample. This helps to increase the coverage of the cell karyotype and ensure that no key information is missed.

[0094] S62, select mitotic phases with more than 400 bands for analysis and conduct detailed karyotype analysis; photographing multiple mitotic phases helps capture the chromosome distribution at different stages of the cell cycle and enhances the accuracy and representativeness of karyotype analysis.

[0095] The cell culture process begins by transferring the peripheral blood sample into the culture medium. This step ensures that the lymphocytes can begin to proliferate and expand in a suitable environment to provide a sufficient number of cells for subsequent analysis; in the incubator, the cells grow under suitable temperature, humidity and gas conditions. This stage allows the lymphocytes to proliferate and may stimulate the cells to enter the division cycle during the culture process, preparing for subsequent analysis; by harvesting the cells, the cells can be separated and concentrated from the culture flask. This step prepares for subsequent processing and analysis, ensuring that the cells can be effectively processed and concentrated before analysis; the cell suspension is dropped on the slide by the drop technique to form a cell monolayer. This step is to prepare for banding, so that the cells can clearly show their structural characteristics during the banding process; through banding, such as using staining reagents, the chromosomes of the cells are stained to make them easier to observe under a microscope. This process can help analyze the structure and number of chromosomes and conduct genetic research; scanning and analyzing cells under a microscope can observe the morphology of cells, chromosomal abnormalities and other genetic characteristics. This step is key to cytogenetic research and provides detailed cell and chromosome data; this standardized process is designed to ensure high-quality data output during the culture, processing and analysis of lymphocytes, making the final analysis results accurate and reliable.

[0096] Specifically, in step S513, the method for preparing the Giemsa mother solution includes the following steps:

[0097] A1, weigh the raw materials: weigh 3.5 g of Giemsa powder;

[0098] A2, prepare the stock solution: add 3.5 g Giemsa powder into a mixed solvent of 500 ml methanol and 500 ml glycerol, use a measuring cylinder to mix, and mix upside down until the powder is completely dissolved;

[0099] A3, storage and stirring: the prepared mother solution was divided into light-proof glass bottles, the glass bottles were placed in a 37°C water bath, and stirred for 24 hours to ensure that the reagents were fully dissolved;

[0100] A4, heating treatment: transfer the mother liquor to a 75°C water bath for heating treatment for 72 hours. During this process, mix the mother liquor more than ten times a day to ensure the uniformity of the reagent;

[0101] A5, prepare the working solution: take 45 ml of buffer solution with a pH value in the range of 6.4 to 6.8, add 3 ml of the mother solution, and dilute it into the working solution; after heating the working solution at 37°C for 30 minutes, it can be used for staining experiments.

[0102] By dissolving Giemsa powder in methanol and propylene glycol, all chemical components are ensured to be evenly mixed to obtain high-quality mother liquor; stirring in a 37°C water bath and heating at 75°C can ensure that the Giemsa powder is completely dissolved and evenly distributed, prevent precipitation, and improve the uniformity and accuracy of dyeing; adjusting the working solution to an appropriate pH range helps to improve the dyeing effect and ensure the reliability of the experimental results.

[0103] Experimental data example

[0104] Experimental data set 1

[0105] Blood sample volume in step S11: 4.5 ml;

[0106] Cell culture time in step S2: 72 h;

[0107] Colchicine concentration in step S31: 20 μg / ml; incubation time: 1 hour;

[0108] Centrifugation parameters in step S33: 1500 rpm, 7 minutes;

[0109] The droplet drying time in step S45 is: 75°C for 3 hours;

[0110] Band staining time in step S51: 30 seconds;

[0111] The number of scanned split phases in step S61 is: 120;

[0112] The number of analysis bands is selected in step S62: 500 bands;

[0113] Technical effects of experimental data set 1: Cell growth was stable, and the number of division phases and analyzed bands was relatively balanced, indicating that the current parameter settings could effectively maintain cell stability and high band resolution.

[0114] Experimental data set 2

[0115] Blood sample volume in step S11: 3.8 ml;

[0116] Cell culture time in step S2: 66 h;

[0117] Colchicine concentration in step S31: 20 μg / ml; incubation time: 1.5 hours;

[0118] Centrifugation parameters in step S33: 1500 rpm, 7 minutes;

[0119] The droplet drying time in step S45 is: 90°C for 1 hour;

[0120] Band staining time in step S51: 35 seconds;

[0121] The number of scanned split phases in step S61 is: 120;

[0122] The number of analysis bands is selected in step S62: 400 bands;

[0123] Technical effect of experimental data set 2: Shorter droplet drying time and slightly longer band staining time resulted in a slightly lower number of analyzed bands, which may have affected the comprehensiveness of the data, but the overall number of cell division phases was stable.

[0124] Experimental data set 3

[0125] Blood sample volume in step S11: 4.0 ml;

[0126] Cell culture time in step S2: 72 h;

[0127] Colchicine concentration in step S31: 25 μg / ml; incubation time: 2 hours;

[0128] Centrifugation parameters in step S33: 1500 rpm, 7 minutes;

[0129] The droplet drying time in step S45 is: 75°C for 3 hours;

[0130] Band staining time in step S51: 25 seconds;

[0131] The number of scanned split phases in step S61 is: 120;

[0132] The number of analysis bands is selected in step S62: 400 bands;

[0133] Technical results of experimental data set 3: Higher colchicine concentration and longer exposure time resulted in lower number of analytical bands, but cell division arrest was more effective and the number of mitotic phases was stable, but the droplet drying time and staining time needed to be optimized to avoid potential interference.

[0134] Experimental data set 4

[0135] Blood sample volume in step S11: 4.2 ml;

[0136] Cell culture time in step S2: 66 h;

[0137] Colchicine concentration in step S31: 20 μg / ml; incubation time: 0.75 hours;

[0138] Centrifugation parameters in step S33: 1500 rpm, 7 minutes;

[0139] The droplet drying time in step S45 is: 75°C for 12 hours;

[0140] Band staining time in step S51: 30 seconds;

[0141] The number of scanned split phases in step S61 is: 120;

[0142] The number of analysis bands is selected in step S62: 500 bands;

[0143] Technical effects of experimental data group 4: The number of split phases and bands similar to those of group 1 were maintained, the display parameter settings were stable, and changes in droplet drying time and band staining time had little effect on the experimental results.

[0144] Experimental data set 5

[0145] Blood sample volume in step S11: 4.1 ml;

[0146] Cell culture time in step S2: 72 h;

[0147] Colchicine concentration in step S31: 20 μg / ml; incubation time: 1.25 hours;

[0148] Centrifugation parameters in step S33: 1500 rpm, 7 minutes;

[0149] The droplet drying time in step S45 is: 75°C for 1 hour;

[0150] Band staining time in step S51: 30 seconds;

[0151] The number of scanned split phases in step S61 is: 120;

[0152] The number of analysis bands is selected in step S62: 400 bands;

[0153] Technical effect of experimental data set 5: The experimental results show that the cell culture time is relatively consistent with the number of bands, but a slightly reduced droplet drying time may lead to uneven staining and affect the accuracy of the analysis results. The above examples are only used to illustrate the technical solution of the present invention, but not to limit it.

Claims

1. A standardized process for culturing peripheral blood lymphocytes, characterized in that: The following steps are involved: S1, blood sample planting; S2, cell culture; S3, cell harvest; S4, drop sheet; S5, banding treatment; S6, scanning and analysis.

2. The standardized process for culturing peripheral blood lymphocytes according to claim 1, characterized in that: In step S1, a blood sample is planted for sample processing; step S1 includes the following steps: S11, draw peripheral blood using a sodium heparin anticoagulant tube (3-5 ml), and mix by gently inverting up and down 10 times; S12. Under sterile conditions, use a 1000 μl pipette to draw up 400 μl of blood, inoculate into human lymphocyte culture medium, and gently mix by inverting up and down 10 times.

3. The standardized process for culturing peripheral blood lymphocytes according to claim 2, characterized in that: In step S2, cell culture: the inoculated peripheral blood is placed in a 37°C constant temperature incubator for 68-72 hours. Step S2 includes the following steps: S21, culture medium improvement: S211, Study new culture medium components or optimize existing culture medium to enhance lymphocyte proliferation and function; S212, improve the gas concentrations of oxygen and carbon dioxide in the culture environment to better simulate the in vivo environment; S213, explore the optimal culture temperature and fluctuation range to ensure the best growth conditions for cells; S22, automated culture system: S221, introduce automated equipment for cell culture, transfer, and separation to reduce human error and improve reproducibility; S222, using high-throughput screening systems for large-scale cell screening and analysis to accelerate the experimental process; S23, Cell Isolation and Enrichment: S231, develop novel cell separation technologies, such as magnetic bead sorting or flow cytometry sorting, to improve the purity and quantity of lymphocytes; S232, Apply microfluidics technology for cell separation and analysis to achieve more sophisticated cell manipulation and control.

4. The standardized process for culturing peripheral blood lymphocytes according to claim 3, characterized in that: In step S3, cell harvesting includes the following steps: S31, 2 hours before the completion of the culture, 30 μl of colchicine at a concentration of 20 μg / ml was added and the culture was continued for 2 hours; S32, prepare hypotonic solution and fixative solution, the concentration of hypotonic solution is 0.075 mol / L KCl, select 2.8 g KCl and distilled water to make up to 500 ml, place it at 37°C for pre-warming, and the fixative solution includes methanol and glacial acetic acid in a ratio of 3:1; S33, harvested using an automatic harvester, set the centrifugation parameters to 1500 rpm, centrifuged for 7 minutes, removed the supernatant, and retained 0.5 ml of cell pellet; S34, add 5 ml of hypotonic solution, mix with an oscillator for 30 seconds, and place in a 37°C water bath for 30 minutes of hypotonic treatment; S35, add 2 ml of freshly prepared fixative, mix gently 5 times, and place in a 37°C water bath for 10 minutes; S36, centrifuge at 1500 rpm for 7 min, remove the supernatant, and retain 0.5 ml of the cell pellet; S37, add 8 ml of freshly prepared fixative, mix gently 5 times, let stand for 5 minutes, centrifuge at 1500 rpm × 7 min, repeat 3 times, and finally retain 0.5 ml of cell suspension.

5. The standardized process for culturing peripheral blood lymphocytes according to claim 4, characterized in that: In step S4, the film dropping process includes the following steps: S41, controlling the humidity and temperature of the dripping room to 50%-55% and 23-25°C; S42, placing a clean glass slide in the glass slide slot at the center mark position of the airing board; S43, use a shaker to mix each tube of cell suspension for 1 min, and use a 100 μl pipette to draw 35 μl of cell suspension; S44, with the elbow at a 60° angle on the table, place the pipette perpendicular to the surface of the slide and quickly drop the suspension at a height of 1 cm to form a complete large circle that does not exceed the edge of the slide; S45, after the liquid surface is dry, place the glass slide in a 75°C electric oven and bake for 3 hours.

6. The standardized process for culturing peripheral blood lymphocytes according to claim 5, characterized in that: The band display process in step S5 includes the following steps: S51, improved banding technology: S511, cooling the baked slides, and placing them in a 0.025% trypsin solution at 37°C for digestion for 60 seconds, wherein the pH of the trypsin solution is between 7.2 and 7.4, and the trypsin time can be adjusted according to the banding effect; S512, rinse once in 0.85% NaCl solution at 37°C; S513, use Giemsa stock solution diluted 5 times as working solution, stain at 37°C for 30 seconds, then rinse with tap water and dry; S52, Data Analysis and Artificial Intelligence: S521, Application of machine learning and data analysis techniques to optimize chromosome banding conditions; S522, develop an intelligent decision-making system to automatically adjust the banding conditions to achieve the best staining effect.

7. The standardized process for culturing peripheral blood lymphocytes according to claim 6, characterized in that: In step S6, scanning and analyzing includes the following steps: S61, scan the karyotype using a film scanner, set to capture 120 mitotic phases; S62, cleavage phases with more than 400 bands were selected for analysis and detailed karyotype analysis was performed.

8. The standardized process for culturing peripheral blood lymphocytes according to claim 6, characterized in that: In step S513, the Giemsa mother solution preparation method The following steps are involved: A1, weigh the raw materials: weigh 3.5 g of Giemsa powder; A2, prepare the stock solution: add 3.5 g Giemsa powder into a mixed solvent of 500 ml methanol and 500 ml glycerol, use a measuring cylinder to mix, and mix upside down until the powder is completely dissolved; A3, storage and stirring: the prepared mother solution was divided into light-proof glass bottles, the glass bottles were placed in a 37°C water bath, and stirred for 24 hours to ensure that the reagents were fully dissolved; A4, heating treatment: transfer the mother liquor to a 75°C water bath for heating for 72 hours. During this process, mix the mother liquor more than ten times a day to ensure the uniformity of the reagent; A5, prepare the working solution: take 45 ml of buffer solution with a pH value in the range of 6.4 to 6.8, add 3 ml of the mother solution, and dilute it into the working solution; after heating the working solution at 37°C for 30 minutes, it can be used for staining experiments.

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