CN111551719A - Kit and method for detecting expression of peripheral blood circulating tumor cells PD-L1 of pancreatic cancer patient - Google Patents
Kit and method for detecting expression of peripheral blood circulating tumor cells PD-L1 of pancreatic cancer patient Download PDFInfo
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- CN111551719A CN111551719A CN202010315133.3A CN202010315133A CN111551719A CN 111551719 A CN111551719 A CN 111551719A CN 202010315133 A CN202010315133 A CN 202010315133A CN 111551719 A CN111551719 A CN 111551719A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70532—B7 molecules, e.g. CD80, CD86
Abstract
The invention relates to a kit and a detection method for detecting the expression of peripheral blood circulating tumor cells PD-L1 of a pancreatic cancer patient, belonging to the technical field of molecular biology. The kit comprises 45mL of diluent, 1mL of destaining solution, 0.5mL of staining solution A, 1mL of staining solution B, 100 muL of anti-human PD-L1 primary antibody, 100 muL of goat anti-human IgG/HRP, 0.1% Triton X-100100 muL, and 0.3% H2O2100 mu L, 15ml of reagent A, 50ml of reagent B and 60ml of 6 × PBS buffer solution, separating and obtaining CTC in peripheral blood of a patient with advanced or recurrent pancreatic cancer, wherein a tissue specimen cannot be obtained by using a membrane filtration device, and further detecting the PD-L1 expression condition of the CTC by using an immunohistochemical technology.
Description
Technical Field
The invention relates to a kit and a detection method for detecting the expression of peripheral blood circulating tumor cells PD-L1 of a pancreatic cancer patient, belonging to the technical field of molecular biology.
Background
Pancreatic cancer is a common malignancy of the digestive system and one of the worst prognosis, among deaths caused by malignancies, pancreatic cancer ranks fourth, 90% of patients die within one year after diagnosis, and 5-year survival rate is less than 10%. Because early symptoms of pancreatic cancer are hidden, and the pancreatic cancer has no specificity, strong invasiveness, early metastasis and low surgical resection rate, the condition is mostly in middle and late stages when the pancreatic cancer is diagnosed, and the treatment is mainly systemic chemotherapy at present.
Circulating Tumor Cells (CTC) are tumor cells that have been shed from solid tumors into the peripheral blood circulation, and since their discovery in 1989, various methods have been used to detect Circulating tumor cells in the peripheral blood. Recent studies have shown that their detection is of great clinical significance for assessing the prognosis of patients with tumours, especially patients with advanced tumours, and for selecting appropriate individualized treatments. CTC detection is called "liquid biopsy" of tumors because of its characteristics of minimally invasive, real-time detection, etc.
The latest research shows that the curative effect of the combination of advanced pancreatic cancer chemotherapy and immunotherapy can be improved to a certain extent, but the expression of tumor cells PD-L1 with the curative effect of a PD-1 inhibitor is related; because the pancreas belongs to retroperitoneal viscera, the pancreatic tumor puncture biopsy is at risk of bleeding, pancreatic fistula, infection and the like, and the PD-L1 expression state can not be dynamically detected in real time, the detection based on the circulating tumor cell PD-L1 in the peripheral blood of a patient with advanced pancreatic cancer has important clinical guiding significance for the treatment of pancreatic cancer immune combined chemotherapy, and unnecessary operative trauma and puncture risk of part of patients are avoided.
At present, units such as Shandong province first medical university, Shandong province drug research institute combined with Shandong Qixin Biotechnology limited company, Shandong well-known Biotechnology limited company, Jinan Xin Biotechnology limited company, Shandong discovery biotechnology limited company and the like, carry out industrialized popularization on the key technology of detection and identification of circulating tumor cells, the project is a Shandong province major scientific and technological innovation project, the project takes the Shandong province drug research institute in Jinan school of Shandong first medical university as the core, realizes a registration system, relies on the core diagnostic technology of detection and identification of circulating tumor cells, and further registers, identifies and diagnoses a kit, and comprises PD1, PD-L1, ER, PR, Her-2, GPC-3, VEGF, P53, Vimentin, EGFR, RAS, CK, ALK-D5F3, CD20, ALK/EML4, Beta-catendin, E-Cacatenin, EP-CAM, HPV, IDH-1, PSA, PSMA, VEGF, GFAP, cytokeratin, AE1/AE3, estrogen receptor, progestin receptor, BCA-225, CA 125, CEA, EMA, ERCC1, HPV, Ki-67, P53, TOP2A and the like are used as tracers for expression of CTCs, and the identification and diagnosis kit is registered to be an ultrasensitive, ultrafast, high-coverage, low-cost, accurate and specific kit, and industrially popularized by cooperating with Shandong Qicheng Xin Biotech Co., Ltd, Shandong well-known Biotech Co., Ltd, Jinan En Biotech Co., Ltd, Shandong discovery Biotech Co., Ltd registered in Jinan.
Disclosure of Invention
In order to solve the problem of real-time dynamic detection of PD-L1 expression and avoid the risk of bleeding, pancreatic fistula, infection and the like in the process of biopsy, the invention provides a detection method for non-diagnosis purpose of peripheral blood circulating tumor cell PD-L1 expression of pancreatic cancer patients, wherein CTC in peripheral blood of advanced stage or recurrent pancreatic cancer patients incapable of obtaining tissue specimens is obtained by separation through a membrane filtration device, and the PD-L1 expression condition of the CTC is further detected by using an immunohistochemical technology.
The technical scheme adopted by the invention is as follows:
a kit for detecting the expression of peripheral blood circulating tumor cells PD-L1 of a pancreatic cancer patient comprises 45mL of diluent, 1mL of destaining solution, 0.5mL of staining solution A, 1mL of staining solution B, 100 muL of anti-human PD-L1 primary antibody, 100 muL of goat anti-human IgG/HRP, 0.1% Triton X-100100 muL, 0.3% H2O2100. mu.L, 15mL of reagent A, 50mL of reagent B, and 60mL of 6 × PBS buffer.
The diluent comprises: ethylenediaminetetraacetic acid with the content of 0.5g/L and sodium sulfate with the content of 6 g/L; 4g/L of sodium chloride, 2g/L of dihydroxymethylurea and 6g/L of hydroxyethyl piperazine ethanethiosulfonic acid; dodecyl sulphobetaine with the content of 0.5g/L, sodium azide with the content of 0.5g/L, polyoxyethylene polyoxypropylene ether block copolymer with the content of 0.3g/L and the balance of water.
The destaining solution is composed of ethanol and acetic acid according to the volume ratio of 1: 2.
The staining solution A is a DAB staining solution; the staining solution B is hematoxylin staining solution.
The reagent A is formaldehyde-xylene, and the volume ratio of the formaldehyde-xylene is 1: 3, preparing a composition; the reagent B is xylene.
The method for detecting the expression of the peripheral blood circulating tumor cells PD-L1 of the pancreatic cancer patient by using the kit in a non-diagnostic purpose comprises the following steps:
(1) separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent pancreatic cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device: collecting peripheral blood of patients with advanced or recurrent pancreatic cancer who cannot obtain tissue specimens: 5ml of peripheral blood of the median cubital vein;
(2) peripheral blood sample pretreatment: diluting the collected peripheral blood sample by using 45ml of diluent, and adding polyformaldehyde to fix the peripheral blood sample for 10 minutes after dilution, wherein the fixed final concentration is 0.25%;
(3) and (3) filtering the peripheral blood sample by using a membrane filtration tumor cell separation device, and separating to obtain peripheral blood CTC: adding the pretreated peripheral blood sample into a blood sample container of a membrane filtration tumor cell separation device, and naturally filtering the blood sample by means of gravity;
(4) after the filtration is finished, taking the filter out of the membrane filtration tumor cell separation device, adding 0.5ml of circulating tumor cell staining solution A into the filter, staining for 3min, and washing with PBS buffer solution; adding 1ml of staining solution B after completely filtering the filtrate, staining for 2min, washing for 2 times by using 1ml of pure water, taking down the filter membrane, placing on a glass slide, drying, and observing under a microscope to determine whether CTC exists;
(5) detecting the expression of PD-L1 of CTC by using immunohistochemical technology.
The specific method for detecting the expression of PD-L1 of CTC in the step (5) is as follows:
(1) and (3) decoloring: taking down the filter membrane with CTC from the glass slide, soaking in a decolorizing solution for 4-6 hours, and removing the CTC staining solution;
(2) dropping 100 μ l of 0.1% Triton X-100, incubating at room temperature for 15min, and washing with DI water for 2min × 3 times;
(3) 100 μ l of 0.3% H was added dropwise2O2Incubating at room temperature for 10min, washing with PBS for 2min × 3 times, (4) adding 100 μ lAnti-human PD-L1 primary antibody, incubating at room temperature for 2h or overnight at 4 ℃, washing with PBS for 2min × 3 times;
(5) dropping 100 mul goat anti-human IgG/HRP, incubating for 20min at 18-26 ℃, washing for 2min x 3 times with PBS;
(6) dripping 100 mul of DAB color development solution, incubating at 18-26 ℃, and observing the color development condition under a microscope at any time, wherein the observation time is 3-10 min;
(7) after the color development is finished, discarding DAB color development liquid, flushing with running water for 5min, and dyeing with hematoxylin for 5 min;
(8) the hydrochloric acid alcohol is differentiated for 8 seconds, and tap water is rewound for 5 min;
(9) dehydrating the rewound CTC by using 75% ethanol, 95% ethanol and 100% ethanol in a gradient manner for 10min respectively, then adding 15mL of reagent A, oscillating uniformly and filtering; adding reagent B, decolorizing for 30 min, drying, and sealing with neutral resin;
(10) and (5) performing microscopic examination by using an optical microscope.
The device for separating tumor cells by membrane filtration comprises a filter, a blood sample container, a waste liquid tank and an iron stand, wherein the iron stand is provided with a base, a vertical frame and a support, the blood sample container is arranged at the upper part of the iron stand through the support, the filter is arranged below the blood sample container, the filter is communicated to the waste liquid tank through an infusion apparatus, and the waste liquid tank is arranged on the base.
The filter comprises a filter upper opening, a filter membrane carrying platform and a filter lower opening, and the filter membrane is arranged on the filter membrane carrying platform; the upper port of the filter is connected with a blood sample container, and the lower port of the filter is connected with a waste liquid cylinder through an infusion apparatus.
The filter membrane is made of hydrophobic materials, and filter holes with the caliber of 8 microns are uniformly distributed on the filter membrane.
Immunosuppression is closely related to immune escape and over-expression of tumor cells PD-L1, tumor cells can be combined with PD-1 on the surface of T cells of immune cells through PD-L1 on the surface of the tumor cells to conduct inhibitory signals, so that the T cells cannot identify the tumor cells and send attack signals to the tumor cells, and the immune escape of the tumor cells is caused, therefore, the detection of the expression condition of Circulating Tumor Cells (CTC) PD-L1 of pancreatic cancer has important value for the curative effect evaluation of the pancreatic cancer chemotherapy combined immunotherapy.
The invention has the beneficial effects that:
(1) the detection method provided by the invention can detect the PD-L1 expression condition of a patient with advanced or recurrent pancreatic cancer without taking a tissue sample by a puncture biopsy. The technology belongs to minimally invasive and can detect in real time.
(2) The method provided by the invention can avoid false positive results caused by edge effect possibly generated in the dyeing process, has good stability, reduces the loss of cells and improves the detection accuracy.
Drawings
FIG. 1 is a schematic structural view of a membrane filtration apparatus according to the present invention;
FIG. 2 is a schematic sectional view showing the structure of a filter of the membrane filtration apparatus of the present invention;
FIG. 3 is a schematic view showing the structure of a filter membrane of the membrane filtration apparatus of the present invention;
FIG. 4 is an image of circulating tumor cells isolated from peripheral blood of a pancreatic cancer patient.
In the figure: 1 iron stand, 2 blood sample containers, 3 filters, 4 transfusion devices, 5 waste liquid jars, 6 filter upper ports, 7 filter membranes, 8 filter membrane platforms, 9 filter lower ports, 10 filter holes, 11 bases, 12 vertical frames and 13 supports.
Detailed Description
The invention is elucidated below with reference to the figures and embodiments.
The specific specification of the kit used in the invention is shown in table 1:
TABLE 1
The method is applied to the embodiment of separating, obtaining and identifying 10 pancreatic cancer patients (detecting 10 normal human samples as negative controls at the same time) peripheral blood circulating tumor cells.
Preparing the diluent: 0.5g of ethylenediamine tetraacetate, 6g of sodium sulfate; 4g of sodium chloride, 2g of dihydroxymethylurea, 6g of hydroxyethylpiperazine ethanethiosulfonic acid; 0.5g of dodecyl sulphobetaine, 0.5g of sodium azide, 0.3g of polyoxyethylene polyoxypropylene ether block copolymer and 980.5g of water.
Example 1
Firstly, separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent pancreatic cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device, and determining whether the CTCs exist:
collecting 5ml of fasting 8-12 hours fasting blood from the median cubital vein, diluting peripheral blood with 45ml of diluent, and then adding 3ml of 4% paraformaldehyde to fix the diluted blood sample for 10 minutes;
at fixed intervals, a membrane filtration device was assembled: as shown in fig. 1, 2 and 3, the filter device comprises a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5 and an iron stand 1;
wetting the filter 3 with 10ml of PBS, then adding the fixed peripheral blood sample into the blood sample container 2 of the membrane filtration device, allowing it to naturally filter by gravity, and the CTC being trapped on the filter membrane 7;
the tumor cells are typically larger than 15 microns in diameter, while the blood cells (including red blood cells, white blood cells) are typically smaller than 8 microns in diameter, so that when peripheral blood containing CTCs is filtered, the blood cells can be filtered by being smaller than filter pores 10, while the CTCs are retained on filter membrane 7 by being larger than filter pores 10.
After the filtration is finished, taking the filter 3 from the filter device, opening and removing the upper opening 6 of the filter, adding 0.5ml of circulating tumor cell staining solution A into the filter, staining for 3min, and washing with PBS buffer solution; filtering the filtrate completely, adding solution B, 1ml, staining for 2min, and pure water 1ml, washing filter 3 with PBS buffer solution, taking down filter membrane 7 with ophthalmic forceps with cell surface facing upwards, and placing on glass slide;
the filters were dried and observed under a microscope to determine the presence of CTCs.
FIG. 4 is an image of a circulating tumor cell isolated from peripheral blood of a patient with pancreatic cancer, wherein it can be seen that the cell nuclei are abnormal in shape, large in size, irregular in shape, and larger than 15um in cell diameter (long end); high nuclear-to-cytoplasmic ratio, the nuclear-to-cytoplasmic ratio is more than 0.8; nuclear chromatin border shift, large nucleoli, abnormal nuclear division.
By observation, no CTCs were detected in 10 healthy volunteers; CTCs were detected in 4 patients with recurrent pancreatic cancer and 3 patients with advanced pancreatic cancer (Table 2), except that CTCs were not detected in 2 patients with recurrent pancreatic cancer and 1 patient with advanced pancreatic cancer, and the positive rate of this detection was 70%.
It is worth noting that when the hydroxyethyl piperazine ethiosulfonic acid or polyoxyethylene polyoxypropylene ether block copolymer is not added into the diluent, the prepared blood sample has poor stability, a part of the blood sample can form delamination, blood cells are easy to aggregate and adhere, and the final detection effect is influenced.
TABLE 2 results of CTC assay in examples
Secondly, detecting the expression condition of PD-L1 of CTC by using an immunohistochemical technology:
taking down the filter membrane 7 carrying CTC on the glass slide from the glass slide, soaking in a destaining solution of ethanol and acetic acid mixed uniformly according to a volume ratio of 1:2 for 4-6 hours, removing CTC staining solution, dripping 100 mul of 0.1% Triton X-100, incubating at room temperature for 15min, washing with DI water for 2min × 3 times, dripping 100 mul of 0.3% H2O2Incubating for 10min at room temperature, washing for 2min × 3 times by PBS, dripping 100 mul PD-L1 (human) primary antibody, incubating for 2h (or overnight at 4 ℃), washing for 2min × 3 times by PBS, dripping 100 mul goat anti-human IgG/HRP, incubating for 20min at room temperature (18-26 ℃), washing for 2min × 3 times by PBS, dripping 100 mul DAB developing solution, incubating at room temperature (18-26 ℃) and observing the developing condition under a microscope at any time (generally 3-10 min, the time can not exceed 10 min), discarding the DAB developing solution after the developing is finished, flushing for 5min by running water, staining for 5min by hematoxylin, differentiating for 8 seconds by hydrochloric acid alcohol, returning blue by running water for 5min, 75% ethanol (1 min), 95% ethanol (1 min), dehydrating by 100% ethanol (1 min), adding formaldehyde and xylene with the volume ratio of 1: 3, oscillating uniformly, filtering, decolorizing, drying xylene, sealing by neutral resin, inspecting under an optical microscope, reading cell membranes and judging the cell membranes according to the coloring degree of PD-L1 expressed by a specialist.
The decoloring effect of the decoloring solution composed of ethanol and acetic acid is better than that of a decoloring solution composed of 95% ethanol and 100% dimethylbenzene according to the volume ratio of 1: 1.
When the reagent A is not adopted and only the dimethylbenzene is used for decolorization, the accuracy of the detection result is 85%, and the reagent A and the reagent B are adopted, namely, the cell concentration of the invention is realized, the form is clear, the dispersion uniformity is good, and the accuracy can reach 99%.
The detected circulating tumor cells are applied to immunohistochemical detection of expression of PD-L1 and are compared with expression results of PD-L1 of pancreatic cancer operation or puncture tissue specimens, differences of the expression results are observed, the potential of CTCs serving as noninvasive biopsy for real-time evaluation of pancreatic cancer PD-L1 expression is verified, and an important basis is provided for evaluation of pancreatic cancer chemotherapy and immunotherapy combined immunotherapy prognosis.
Claims (7)
1. A kit for detecting the expression of peripheral blood circulating tumor cells PD-L1 of a pancreatic cancer patient is characterized by comprising 45mL of diluent, 1mL of destaining solution, 0.5mL of staining solution A, 1mL of staining solution B, 100 muL of anti-human PD-L1 primary antibody, 100 muL of goat anti-human IgG/HRP, 0.1% Triton X-100100 muL and 0.3% H2O2100. mu.L, 15mL of reagent A, 50mL of reagent B, and 60mL of 6 × PBS buffer.
2. The kit of claim 1, wherein the diluent comprises: ethylenediaminetetraacetic acid with the content of 0.5g/L and sodium sulfate with the content of 6 g/L; 4g/L of sodium chloride, 2g/L of dihydroxymethylurea and 6g/L of hydroxyethyl piperazine ethanethiosulfonic acid; dodecyl sulphobetaine with the content of 0.5g/L, sodium azide with the content of 0.5g/L, polyoxyethylene polyoxypropylene ether block copolymer with the content of 0.3g/L and the balance of water.
3. The kit of claim 1, wherein the destaining solution consists of ethanol and acetic acid in a volume ratio of 1: 2.
4. The kit according to claim 1, wherein the staining solution A is DAB staining solution; the staining solution B is hematoxylin staining solution.
5. The kit according to claim 1, wherein the reagent A is formaldehyde-xylene in a volume ratio of 1: 3, preparing a composition; the reagent B is xylene.
6. A method for the non-diagnostic purpose of detecting the expression of PD-L1 in peripheral blood circulating tumor cells of a pancreatic cancer patient using the kit of any one of claims 1-5, comprising the steps of:
(1) separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent pancreatic cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device: collecting peripheral blood of patients with advanced or recurrent pancreatic cancer who cannot obtain tissue specimens: 5ml of peripheral blood of the median cubital vein;
(2) peripheral blood sample pretreatment: diluting the collected peripheral blood sample by using 45ml of diluent, and adding polyformaldehyde to fix the peripheral blood sample for 10 minutes after dilution, wherein the fixed final concentration is 0.25%;
(3) and (3) filtering the peripheral blood sample by using a membrane filtration tumor cell separation device, and separating to obtain peripheral blood CTC: adding the pretreated peripheral blood sample into a blood sample container of a membrane filtration tumor cell separation device, and naturally filtering the blood sample by means of gravity;
(4) after the filtration is finished, taking the filter out of the membrane filtration tumor cell separation device, adding 0.5ml of circulating tumor cell staining solution A into the filter, staining for 3min, and washing with PBS buffer solution; adding 1ml of staining solution B after completely filtering the filtrate, staining for 2min, washing for 2 times by using 1ml of pure water, taking down the filter membrane, placing on a glass slide, drying, and observing under a microscope to determine whether CTC exists;
(5) detecting the expression of PD-L1 of CTC by using immunohistochemical technology.
7. The method of claim 6, wherein the specific method for detecting PD-L1 expression in CTC in step (5) is as follows:
(1) and (3) decoloring: taking down the filter membrane with CTC from the glass slide, soaking in a decolorizing solution for 4-6 hours, and removing the CTC staining solution;
(2) dropping 100 μ l of 0.1% Triton X-100, incubating at room temperature for 15min, and washing with DI water for 2min × 3 times;
(3) 100 μ l of 0.3% H was added dropwise2O2Incubating for 10min at room temperature, washing for 2min with PBS for × 3 times, (4) dripping 100 μ L of anti-human PD-L1 primary antibody, incubating for 2h at room temperature or overnight at 4 ℃, and washing for 2min with PBS for × 3 times;
(5) dropping 100 mul goat anti-human IgG/HRP, incubating for 20min at 18-26 ℃, washing for 2min x 3 times with PBS;
(6) dripping 100 mul of DAB color development solution, incubating at 18-26 ℃, and observing the color development condition under a microscope at any time, wherein the observation time is 3-10 min;
(7) after the color development is finished, discarding DAB color development liquid, flushing with running water for 5min, and dyeing with hematoxylin for 5 min;
(8) the hydrochloric acid alcohol is differentiated for 8 seconds, and tap water is rewound for 5 min;
(9) dehydrating the rewound CTC by using 75% ethanol, 95% ethanol and 100% ethanol in a gradient manner for 10min respectively, then adding 15mL of reagent A, oscillating uniformly and filtering; adding reagent B, decolorizing for 30 min, drying, and sealing with neutral resin;
(10) and (5) performing microscopic examination by using an optical microscope.
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| WO2022001824A1 (en) * | 2020-07-01 | 2022-01-06 | 山东第一医科大学第二附属医院 | Kit and method for detecting pd-l1 gene mutations in circulating tumor cells in peripheral blood of patient with small cell lung cancer |
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| WO2022001824A1 (en) * | 2020-07-01 | 2022-01-06 | 山东第一医科大学第二附属医院 | Kit and method for detecting pd-l1 gene mutations in circulating tumor cells in peripheral blood of patient with small cell lung cancer |
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Application publication date: 20200818 |