CN111551727A - Immunofluorescence kit and detection method for detecting ALK gene mutation of peripheral blood circulating tumor cells of non-small cell lung cancer patients - Google Patents

Abstract

The invention provides an immunofluorescence kit and a detection method for detecting ALK gene mutation of peripheral blood circulation tumor cells of non-small cell lung cancer patients, and belongs 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, 200 mu l of methanol, 200 mu l of 2% PFA, 100 mu l of 10% goat serum, 100 mu l of primary anti-working solution, 100 mu l of secondary antibody working solution and a DAPI blocking tablet. The detection method provided by the invention can detect the ALK expression condition of a patient with advanced or recurrent non-small cell lung cancer without taking a tissue sample by a puncture biopsy. The technology belongs to minimally invasive and can detect in real time. 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.

Description

Immunofluorescence kit and detection method for detecting ALK gene mutation of peripheral blood circulating tumor cells of non-small cell lung cancer patients

Technical Field

The invention provides an immunofluorescence kit and a detection method for detecting ALK gene mutation of peripheral blood circulation tumor cells of non-small cell lung cancer patients, and belongs to the technical field of molecular biology.

Background

Lung cancer is one of the main malignant tumors causing death of cancer patients, and both the morbidity and mortality of lung cancer are the first in China. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers, and more than 70% of these NSCLC patients have been advanced at the time of diagnosis. Despite the increasing treatment technologies such as surgery and chemoradiotherapy, the 5-year survival rate of NSCLC patients is still less than 20%, and the main causes of death include local recurrence and distant metastasis.

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 tumor because of its characteristics of minimal invasion, real-time detection, etc.

With the development of molecular biology, molecular targeted therapy targeting Anaplastic Lymphoma Kinase (ALK) has recently become a new treatment for NSCLC, especially in NSCLC patients who are advanced (losing the chance of surgery) or relapse after surgery. Other genes fused to the ALK gene in lung cancer include TFG, KIF5B, and the like. The small molecule inhibitor Crizotinib (Crizotinib) aiming at the ALK gene target is an ATP competitive Tyrosine Kinase Inhibitor (TKI), can specifically target and inhibit ALK, can also inhibit signal paths such as c-MET and ROSl, and has remarkable effect in the treatment of NSCLC. At present, the diagnosis and treatment guidelines for NSCLC at home and abroad are considered as follows: the time points for ALK mutation detection in NSCLC patients, including at the time of initial diagnosis and in the strive for re-detection as the disease progresses, should be reduced by repeated detection. In current clinical practice, the sample for detecting ALK of NSCLC patients is mainly tumor tissue, which is from operation or aspiration biopsy, and is difficult to detect for many 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 defects that a patient with advanced or recurrent non-small cell lung cancer cannot obtain a tissue sample by real-time or repeated puncture and further cannot evaluate the ALK state of the patient, the invention provides a detection method for the ALK gene mutation non-diagnosis purpose of peripheral blood circulation tumor cells of the patient with non-small cell lung cancer, which comprises the following steps: and (3) separating and obtaining the CTC in peripheral blood of a patient with advanced or recurrent non-small cell lung cancer, wherein the tissue specimen cannot be obtained, by using a membrane filtering device, and further detecting the ALK expression condition of the CTC by using an immunofluorescence technology.

The technical scheme adopted by the invention is as follows:

the invention provides an immunofluorescence kit for detecting ALK gene mutation of peripheral blood circulation tumor cells of non-small cell lung cancer patients, which comprises 45mL of diluent, 1mL of destaining solution, 0.5mL of staining solution A, 1mL of staining solution B, 200 mu l of methanol, 200 mu l of 2% PFA, 100 mu l of 10% goat serum, 100 mu l of primary antibody working solution, 100 mu l of secondary antibody working solution and a DAPI encapsulated tablet.

Further, the primary anti-working solution consists of mouse anti-CK, rat anti-CD 45 and rabbit anti-ALK, and is diluted and mixed uniformly by adopting BD wash buffer according to the ratio of 1:100&1:500&1: 400; the secondary antibody working solution consists of a mouse anti-sheep with fluorescence labeling, a rat anti-sheep with fluorescence labeling and a rabbit anti-sheep with fluorescence labeling, and is diluted and mixed uniformly according to a ratio of 1:500 by adopting BD wash buffer.

Further, the diluent is composed of 1mmol/L EDTA +0.1% BSA +0.1% trehalose +0.2% polyoxyethylene polyoxypropylene ether block copolymer, and the base solution is Tris-HCl buffer.

Further, the decoloring solution is composed of 95% alcohol and 100% xylene according to a volume ratio of 1: 1.

Further, the staining solution A is a DAB staining solution; the staining solution B is hematoxylin staining solution.

The invention also provides a method for detecting ALK gene mutation of peripheral blood circulation tumor cells of non-small cell lung cancer patients by using the immunofluorescence kit in a non-diagnosis purpose manner, which comprises the following steps:

(1) separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent non-small cell lung cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device: collecting peripheral blood of patients with advanced or recurrent non-small cell lung cancer who can not obtain tissue samples: 5ml of peripheral blood of the median cubital vein;

(2) peripheral blood sample pretreatment: diluting the collected peripheral blood sample by 10 times by using a 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; filtering the filtrate completely, adding 1ml of staining solution B, staining for 2min, washing with 1ml of pure water for 2 times, adding 200 μ l of 2% PFA into the filter, fixing at room temperature for 5min, and rinsing with 0.5ml PBS for 3 times, each time for 2 min; adding 200 μ l of precooled methanol into the filter, and fixing at 4 deg.C for 15 min; taking down the filter membrane, flatly attaching the lower surface of the filter membrane to one side of a glass slide, drying the glass slide at room temperature for 4-5min, quickly taking off the filter membrane from the surface of the glass slide at one time by using a pair of tweezers, transferring the filter membrane to the center of the glass slide, dropwise adding 2ul of patch tablets, drying, observing under a microscope, and determining whether CTC exists;

(5) and detecting ALK expression of CTC by using an immunofluorescence method.

The specific method for detecting ALK expression of CTC by adopting an immunofluorescence method comprises the following steps:

(1) and (3) decoloring: taking down the filter membrane with CTC from the glass slide, soaking in a decolorizing solution for 4-6 hours, removing the CTC staining solution, and washing with PBS for 2min × 3 times;

(2) and (3) sealing: dripping 100 μ l 10% goat serum onto the filter membrane, standing at room temperature for 30min, and sucking off excessive serum;

(3) primary antibody incubation: discarding the blocking solution on the slide, immediately dropping 100 μ l of primary-antibody dilution solution, and incubating at 37 deg.C for 60min (or incubating overnight at 4 deg.C, and rewarming at 37 deg.C for 30min the next day);

(4) after completion, rinsing with PBS for 3 times, each time for 3 min;

(5) and (3) secondary antibody incubation: dripping 100 mul of second antibody diluent, and incubating for 30min at room temperature;

(6) after completion, rinsing with PBS for 3 times, each time for 2 min;

(7) sealing the piece by using a sealing agent containing DAPI, reading the piece and collecting a drawing;

(8) after the light collection was completed, the sections were removed and then stained with giemsa renbergii, which was compared with the IF results.

Drawing standard (drawing positive cells).

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 ALK expression condition of a patient with advanced or recurrent non-small cell lung 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 immunofluorescence image of ALK circulating tumor cells in peripheral blood of a patient with advanced non-small cell lung cancer.

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 cases of non-small cell lung cancer patients (and detecting 10 cases of normal human samples as negative controls).

Example 1

Firstly, separating and acquiring CTCs in peripheral blood of a patient with advanced or recurrent non-small cell lung cancer, wherein a tissue specimen cannot be obtained, by using a membrane filtering device, and determining whether the CTCs exist:

collecting 5ml of fasting 8-12 hr fasting blood from the median cubital vein, diluting peripheral blood with 45ml of diluent (component: 1mmol/L EDTA +0.1% BSA +0.1% trehalose +0.2% polyoxyethylene polyoxypropylene ether block copolymer), and fixing the diluted blood sample with 3ml of 4% paraformaldehyde for 10 min;

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.

By observation, no CTCs were detected in 10 healthy volunteers; except that no CTC was detected in 2 patients with recurrent non-small cell lung cancer and 1 patient with advanced non-small cell lung cancer, CTC was detected in 7 patients (table 2), the positive rate of the detection was 70%, and it is noted that when 0.1% trehalose or 0.2% polyoxypropylene ether block copolymer was not added to the diluent, 0.3% trehalose or 0.3% polyoxypropylene ether block copolymer was singly used to prepare a blood sample with poor stability, and some blood samples were also stratified, so that aggregation and adhesion of blood cells easily occur, which affects the final detection effect.

TABLE 2 results of CTC assay in examples

Secondly, detecting ALK expression condition of CTC by using immunofluorescence technology:

(1) and (3) decoloring: taking down the filter membrane with CTC from the glass slide, soaking in a decolorizing solution for 4-6 hours, removing the CTC staining solution, and washing with PBS for 2min × 3 times;

(2) and (3) sealing: dropping 100 μ l 10% goat serum onto the filter membrane, standing at room temperature for 30min, and removing excess serum (note: goat serum is diluted with PBS buffer solution with pH of 7.4);

(3) primary antibody incubation: discarding the blocking solution on the glass slide, immediately dropwise adding 100 μ l of primary anti-dilution solution, wherein the primary anti-working solution is composed of mouse anti-CK, rat anti-CD 45 and rabbit anti-ALK, respectively diluting and uniformly mixing by adopting BD wash buffer according to the ratio of 1:100&1:500&1:400, and incubating at 37 ℃ in a wet box for 60min (or incubating overnight at 4 ℃, and re-warming at 37 ℃ for 30min the next day);

(4) after completion, rinsing with PBS for 3 times, each time for 3 min;

(5) and (3) secondary antibody incubation: dripping 100 μ l of secondary antibody diluent, wherein the secondary antibody working solution is composed of fluorescence labeled mouse anti-sheep, fluorescence labeled rat anti-sheep and fluorescence labeled rabbit anti-sheep, respectively diluting and mixing uniformly by BD wash buffer according to a ratio of 1:500, and incubating for 30min at room temperature;

(6) after completion, rinsing with PBS for 3 times, each time for 2 min;

(7) sealing the piece by using a sealing agent containing DAPI, reading the piece and collecting a drawing;

(8) after the light collection was completed, the sections were removed and then stained with giemsa renbergii, which was compared with the IF results.

FIG. 4 is an immunofluorescence staining image of circulating tumor cells in peripheral blood of a patient with advanced non-small cell lung cancer, wherein the tumor cells are found to be large in cell size, abnormal in nucleoplasmic ratio and typical in immunological expression of CTCs according to immunological and morphological expression, wherein A is merge; b is DAPI; c is CD 45; d is ALK; e is CK.

The detected circulating tumor cells are applied to immunohistochemistry to confirm the ALK expression and are compared with the ALK result of a non-small cell lung cancer gross sample, the difference is observed, the targeted therapy of the non-small cell lung cancer is guided mainly aiming at the patients with negative ALK expression of the gross sample and positive circulating tumor cell expression, and a new thought is provided for the targeted therapy of the non-small cell lung cancer.

Claims (7)

1. An immunofluorescence kit for detecting ALK gene mutation of peripheral blood circulation tumor cells of a patient with non-small cell lung cancer is characterized by comprising 45mL of diluent, 1mL of destaining solution, 0.5mL of staining solution A, 1mL of staining solution B, 200 mu l of methanol, 200 mu l of 2% PFA, 100 mu l of 10% goat serum, 100 mu l of primary antibody working solution, 100 mu l of secondary antibody working solution and a DAPI blocking tablet.

2. The immunofluorescence kit according to claim 1, wherein the primary antibody working solution is composed of mouse anti-CK, rat anti-CD 45 and rabbit anti-ALK, and is diluted and mixed uniformly according to a ratio of 1:100&1:500&1:400 by adopting BD wash buffer; the secondary antibody working solution consists of a mouse anti-sheep with fluorescence labeling, a rat anti-sheep with fluorescence labeling and a rabbit anti-sheep with fluorescence labeling, and is diluted and mixed uniformly according to a ratio of 1:500 by adopting BD wash buffer.

3. The immunofluorescence kit of claim 1, wherein the diluent is composed of 1mmol/L EDTA +0.1% BSA +0.1% trehalose +0.2% polyoxyethylene polyoxypropylene ether block copolymer and the base is Tris-HCl buffer.

4. The immunofluorescence kit according to claim 1, wherein the destaining solution consists of 95% alcohol and 100% xylene in a volume ratio of 1: 1.

5. The immunofluorescence kit according to claim 1, wherein the staining solution a is DAB staining solution; the staining solution B is hematoxylin staining solution.

6. A method for detecting ALK gene mutation of peripheral blood circulation tumor cells of a patient with non-small cell lung cancer by using the immunofluorescence kit of any one of claims 1-5 for non-diagnostic purposes, which is characterized by comprising the following steps:

(1) separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent non-small cell lung cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device: collecting peripheral blood of patients with advanced or recurrent non-small cell lung cancer who can not obtain tissue samples: 5ml of peripheral blood of the median cubital vein;

(2) peripheral blood sample pretreatment: diluting the collected peripheral blood sample by 10 times by using a 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; filtering the filtrate completely, adding 1ml of staining solution B, staining for 2min, washing with 1ml of pure water for 2 times, adding 200 μ l of 2% PFA into the filter, fixing at room temperature for 5min, and rinsing with 0.5ml PBS for 3 times, each time for 2 min; adding 200 μ l of precooled methanol into the filter, and fixing at 4 deg.C for 15 min; taking down the filter membrane, flatly attaching the lower surface of the filter membrane to one side of a glass slide, drying the glass slide at room temperature for 4-5min, quickly taking off the filter membrane from the surface of the glass slide at one time by using a pair of tweezers, transferring the filter membrane to the center of the glass slide, dropwise adding 2ul of patch tablets, drying, observing under a microscope, and determining whether CTC exists;

(5) and detecting ALK expression of CTC by using an immunofluorescence method.

7. The method of claim 6, wherein the specific method for detecting ALK expression in CTCs 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, removing the CTC staining solution, and washing with PBS for 2min × 3 times;

(2) and (3) sealing: dripping 100 μ l 10% goat serum onto the filter membrane, standing at room temperature for 30min, and sucking off excessive serum;

(3) primary antibody incubation: discarding the blocking solution on the slide, immediately dropping 100 μ l of primary-antibody dilution solution, and incubating at 37 deg.C for 60min (or incubating overnight at 4 deg.C, and rewarming at 37 deg.C for 30min the next day);

(4) after completion, rinsing with PBS for 3 times, each time for 3 min;

(5) and (3) secondary antibody incubation: dripping 100 mul of second antibody diluent, and incubating for 30min at room temperature;

(6) after completion, rinsing with PBS for 3 times, each time for 2 min;

(7) sealing the piece by using a sealing agent containing DAPI, reading the piece and collecting a drawing;

(8) after the light collection was completed, the sections were removed and then stained with giemsa renbergii, which was compared with the IF results.

Images