CN111562377A - Kit and method for detecting expression of peripheral blood circulating tumor cells CA199 of pancreatic cancer patient - Google Patents

Abstract

The invention relates to a kit and a detection method for detecting the expression of peripheral blood circulating tumor cells CA199 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 goat anti-CA 199 primary antibody, 100 muL of rabbit anti-goat IgG/HRP, 0.1% Triton X-100100 muL and 0.3% H₂O₂100 mu L of the sample, 15ml of the reagent A, 50ml of the reagent B and 60ml of 6 × PBS buffer solution, the detection method of the invention is to separate and obtain CTC in peripheral blood of a patient with advanced or recurrent pancreatic cancer, wherein a tissue sample cannot be obtained by utilizing a membrane filtration device, and further detect the CA199 expression condition of the CTC by utilizing an immunohistochemical technology.

Description

Kit and method for detecting expression of peripheral blood circulating tumor cells CA199 of pancreatic cancer patient

Technical Field

The invention relates to a kit and a detection method for detecting the expression of peripheral blood circulating tumor cells CA199 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 5%. As early symptoms of pancreatic cancer are hidden, the pancreatic cancer is not specific, the pancreatic cancer is highly invasive, the pancreatic cancer is early in metastasis, the pancreatic cancer is mostly in the middle and late stages when the pancreatic cancer is diagnosed, the surgical resection rate is low, and the pancreatic cancer is diagnosed only by 15%, the selection of the treatment method for pancreatic cancer patients is particularly important, and unnecessary surgical trauma and risks can be avoided for part of patients.

CAl99(carbohydrate antigen 99), also known as tumor associated 199 antigen, gastrointestinal cancer associated antigen or glycoantigen, is a mucin tumor marker that is glycolipids on the cell membrane and exists as mucin in the blood. CAl99 is an antigenic substance highly associated with adenocarcinoma, produced by adenocarcinoma cells, and is introduced into the blood stream via the thoracic duct. The normal reference value of the serum CAl99 is 39 kU/L, the CAl99 concentration in the malignant tumor serum is obviously increased, especially for patients with pancreatic cancer and colon cancer, the increase is several times to dozens of times different, even can reach 1000U/mL, so the method is mainly used for early diagnosis of pancreatic cancer, and in the current clinical practice, the sample detected by the CA199 of the pancreatic cancer patient is mainly tumor tissue, is from operation or puncture biopsy, and is difficult to detect for a plurality of times or in real time.

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 overcome the problem of multiple or real-time detection and simultaneously avoid the risks of bleeding, pancreatic fistula, infection and the like in the process of puncture biopsy, the invention provides a kit and a detection method for detecting the CA199 expression of peripheral blood circulating tumor cells of a pancreatic cancer patient, wherein CTC in peripheral blood of a pancreatic cancer patient in a late stage or a recurrent stage, which cannot obtain a tissue specimen, is obtained by separation through a membrane filtration device, and the CA199 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 CA199 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 goat anti-CA 199 primary antibody, 100 muL of rabbit anti-goat 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.

The diluent is 19g/L of potassium chloride, 4.8 g/L of ethylene diamine tetraacetic acid dipotassium, 2.6 g/L of pyridinium sodium, 17g/L of monopotassium phosphate, 9g/L of sodium hydroxide, 0.2g/L of hydroxyethyl piperazine ethanethiosulfonic acid, 2g/L of dihydroxymethyl urea and the balance of purified water.

The destaining solution consists of 0.5 percent hydrochloric acid-70 percent ethanol solution.

The staining solution A is a DAB staining solution; the staining solution B is hematoxylin staining solution.

The reagent A is formaldehyde-methanol according to a volume ratio of 2: 1, preparing a composition; the reagent B is xylene.

The method for detecting the CA199 expression of the peripheral blood circulation tumor cells 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) and detecting the CA199 expression of the CTC by using an immunohistochemical technology.

The specific method for detecting the CA199 expression of the CTC in the step (5) is as follows:

s1 decolorization: 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;

s2 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;

s3 was added dropwise with 100. mu.l of 0.3% H₂O₂Incubating for 10min at room temperature, washing for 2min × 3 times with PBS, (4) dripping 100 mul goat anti-CA 199 primary antibody, incubating for 2h at room temperature or overnight at 4 ℃, and washing for 2min × 3 times with PBS;

s4, 100 mul of rabbit anti-sheep IgG/HRP is dripped, the incubation is carried out for 20min at the temperature of 18-26 ℃, and the washing is carried out for 2min multiplied by 3 times by PBS;

s5, 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;

s6, after the color development is finished, discarding the DAB color development solution, flushing with running water for 5min, and dyeing with hematoxylin for 5 min;

s7, carrying out alcohol differentiation for 8 seconds by hydrochloric acid, and bluing tap water for 5 min;

s8, 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;

s9 microscopic examination under 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.

The invention has the beneficial effects that:

(1) the detection method provided by the invention can detect the CA199 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 kit Specifications

Components	Content (wt.)
		6 × PBS buffer	60 mL
Diluent liquid	50 mL
		Decolorizing liquid	1mL
Staining solution A	0.5 mL
		Dyeing liquid B	1mL
Goat anti-CA 199 primary antibody	100μL
		Rabbit anti-sheep IgG/HRP	100μL
0.1% Triton X-100	100μL
		0.3% H2O2	100μL
Reagent A	15mL
		Reagent B	50 mL

The technical method is used for separating, obtaining and identifying 15 pancreatic cancer patients (5 normal human samples are simultaneously detected as negative controls) and the peripheral blood circulating tumor cells.

Preparing the diluent: 19g of potassium chloride, 4.8g of ethylene diamine tetraacetic acid dipotassium, 2.6g of pyridinium sodium, 17g of potassium dihydrogen phosphate, 9g of sodium hydroxide, 0.2g of hydroxyethyl piperazine ethanethiosulfonic acid, 2g of dihydroxymethyl urea and 945.4g of purified 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); nuclear chromatin border shift, large nucleoli, abnormal nuclear division.

By observation, no CTCs were detected in 5 healthy volunteers; CTCs were detected in 6 patients with recurrent pancreatic cancer and 5 patients with advanced pancreatic cancer (Table 2), except that CTCs were not detected in 3 patients with recurrent pancreatic cancer and 1 patient with advanced pancreatic cancer, and the positive rate of this detection was 73.3%.

It is worth noting that when the diluent is not added with dihydroxymethylurea or hydroxyethylpiperazine ethanethiosulfonic acid, the prepared blood sample has poor stability, a part of the blood sample can be layered, 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 CA199 expression condition of the 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 decolorizing solution of 0.5% hydrochloric acid-70% ethanol solution for 4-6 hr, removing CTC staining solution, dropping 100 μ l of 0.1% Triton X-100, incubating at room temperature for 15min, DI water washing for 2min × 3 times, dropping 100 μ l of 0.3% H₂O₂Incubating for 10min at room temperature, washing for 2min × 3 times by PBS, dripping 100 mul of goat anti-CA 199 primary antibody, incubating for 2h at room temperature (or overnight at 4 ℃), washing for 2min × 3 times by PBS, dripping 100 mul of rabbit anti-goat IgG/HRP, incubating for 20min at room temperature (18-26 ℃), washing for 2min × 3 times by PBS, dripping 100 mul of 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), after the developing is finished, discarding the DAB developing solution, flushing with running water for 5min, dyeing with hematoxylin for 5min, differentiating with hydrochloric acid alcohol for 8 s, returning blue with running water for 5min, 75% ethanol (1 min), 95% ethanol (1 min), dehydrating with 100% ethanol (1 min) in a gradient manner,then adding a mixture of 2: 1, uniformly oscillating formaldehyde and methanol, filtering, decoloring dimethylbenzene, airing, and sealing with neutral resin; and (4) microscopic examination is carried out under an optical microscope, and a cytopathologist reads the cell membrane, and the CA199 expression condition is judged according to the staining degree of the cell membrane and the cytoplasm.

The decolorizing solution prepared from 0.5 percent hydrochloric acid-70 percent ethanol solution has better decolorizing effect than the decolorizing solution prepared from 95 percent ethanol and 100 percent 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 immunohistochemistry to confirm the expression of CA199 and are compared with the expression result of CA199 in a pancreatic cancer operation or puncture tissue specimen, the difference of the expression result is observed, the potential of CTCs used as noninvasive biopsy for evaluating the pancreatic cancer CA199 expression in real time is verified, and an important basis is provided for evaluating the pancreatic cancer chemotherapy and immunotherapy combined immunotherapy prognosis.

Claims (7)

1. A kit for detecting the expression of peripheral blood circulating tumor cells CA199 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 goat anti-CA 199 primary antibody, 100 muL of rabbit anti-goat 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 according to claim 1, wherein the diluent is 19g/L of potassium chloride, 4.8 g/L of dipotassium ethylenediamine tetraacetic acid, 2.6 g/L of pyridinium sodium, 17g/L of potassium dihydrogen phosphate, 9g/L of sodium hydroxide, 0.2g/L of hydroxyethylpiperazine ethanethiosulfonic acid, 2g/L of dihydroxymethylurea and the balance of purified water.

3. The kit of claim 1, wherein the destaining solution consists of a 0.5% hydrochloric acid-70% ethanol solution.

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-methanol in a volume ratio of 2: 1, preparing a composition; the reagent B is xylene.

6. A method for non-diagnostic detection of CA199 expression in peripheral blood circulating tumor cells of a pancreatic cancer patient using the kit of any of claims 1 to 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) and detecting the CA199 expression of the CTC by using an immunohistochemical technology.

7. The assay of claim 6, wherein the specific method for detecting CA199 expression of CTCs in step (5) is as follows:

s1 decolorization: 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;

s2 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;

s3 was added dropwise with 100. mu.l of 0.3% H₂O₂Incubating at room temperature for 10min, washing with PBS for 2min × 3 times, dripping 100 μ l goat anti-CA 199 primary antibody, incubating at room temperature for 2h or overnight at 4 deg.C, and washing with PBS for 2min × 3 times;

s4, 100 mul of rabbit anti-sheep IgG/HRP is dripped, the incubation is carried out for 20min at the temperature of 18-26 ℃, and the washing is carried out for 2min multiplied by 3 times by PBS;

s5, 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;

s6, after the color development is finished, discarding the DAB color development solution, flushing with running water for 5min, and dyeing with hematoxylin for 5 min;

s7, carrying out alcohol differentiation for 8 seconds by hydrochloric acid, and bluing tap water for 5 min;

s8, 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;

s9 microscopic examination under optical microscope.

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