CN109187978A - It is a kind of detect circulating tumor cell HER2, ER, PR immunofluorescent reagent box and its application - Google Patents

Abstract

The present invention relates to a kind of immunofluorescent reagent box for detecting circulating tumor cell HER2, ER, PR and its applications, the method of the invention, testing principle is as follows: enrichment cycles tumour cell and other rare cells first, then according to antigen-antibody reaction principle, using immunofluorescent detection method, expression of the destination protein in cell in the cell of enrichment is detected.This kit is by carrying out fluorescent marker to target cell and leukocyte common antigen CD45, positive, CD45 feminine gender the cell by screening destination protein, so that the circulating tumor cell to the specific protein positive in blood carries out interpretation and counting.

Description

It is a kind of detect circulating tumor cell HER2, ER, PR immunofluorescent reagent box and its Using

Technical field:

The present invention relates to a kind of medical detection methods, in particular to the detection of circulating tumor cell in blood, and in particular to Human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), progesterone receptor (PR) protein immunization luciferase assay reagent Box is suitable for different body fluid sample HER2, ER, PR antigens and detects, and further relates to use of the kit in detection HER2, ER, PR On the way.

Background technique:

Cancer morbidity is higher at present, seriously threatens human health.The more difficult early detection of some cancers is mostly evening when diagnosis Phase, such as lung cancer, colorectal cancer, oophoroma, cancer of pancreas etc..Suffer from correspondingly, 5 annual survival rates of advanced cancer are substantially less than early stage Person, precisely treatment can significantly extend the time-to-live of patient for the early early diagnosis of discovery.

It is human epidermal growth factor receptor 2 (Human epidermal growth factor receptor 2, HER2), female Hormone receptor (Estrogen receptor, ER), progesterone receptor (Progesterone receptor, PR) are important and exempt from One of epidemic disease group marker.In other Several Kinds of Malignancy for including breast cancer, gastric cancer, intestinal cancer, lung cancer, carcinoma of endometrium etc. The overexpression of HER2 albumen can be detected.Also often there are the high expression of ER, PR in including breast cancer, carcinoma of endometrium.It is clinical normal Reference is provided according to the expression status of HER2, ER, PR for the clinical treatment of patient.By taking breast cancer as an example, breast cancer be women most Common malignant tumour, year morbidity number account for global cancer neopathy number of cases 23%, and death toll accounts for 14%, occupies female tumor hair Raw the first (Jemal A, et al.CA Cancer J Clin, 2011).Clinical practice (OlivottoIA, etal.J ClinOncol, 2004) show: when ER, PR expression are the positive, endocrine therapeutic agents tamoxifen (Tamoxiefen, TAM) treated effect is 60%~75%, and (ER+PR- or ER-PR+) effective percentage drops to 35% when one of both is positive ~50%, show that ER, PR expression are closely related with endocrine therapy curative effect, testing result will directly determine the choosing of therapeutic scheme It selects.On the other hand, there are the amplification of HER2 gene and its overexpressions of coding albumen in 20%~30% patient with breast cancer (SlamonDJ, et al.Science, 1989), for these patients, the therapeutic effect of endocrine therapy and standard chemotherapeutic is simultaneously Undesirable, Anti-HER 2 drug can improve the prognosis of this part cancer patient.In same Endometrial Carcinomas, HER2, ER, The expression of PR shows similar result.Similar therapeutic scheme may be selected to treat patient.

Diagnosing tumor depends on three kinds of pathology, iconography and serology methods at present.Pathology are diagnosing tumors Goldstandard, but sample of tissue has certain traumatic, and materials position is limited to, simultaneously because tumor tissues is heterogeneous Property, single-point sample of tissue is not enough to react whole neoplastic state, and is difficult to repeat to sample dynamic monitoring.Imaging Method is diagnosing Act on wide in tumour, but generally there are radiation injuries for iconography.Moreover, iconography usually only reacts the letter such as tumor size Breath, it is difficult to judge the grade malignancy of tumour.More important point, iconography is typically only capable to the tumor tissues of detection 2mm or more, right Poor in lesser tumor tissues diagnostic sensitivity, there are hysteresis qualitys.Serology is now generally used for auxiliary judgment treatment of cancer The information such as curative effect, sampling is more convenient, but sensitivity and specificity are poor, and poor with pathologic, physiologic correlation.Likewise, Treatment aspect, clinically there is also certain limitations at present.For tumor patient, the patient especially shifted, at present Therapeutic scheme only usually is judged according to the information of primary tumor, is had ignored to the transfer stove even detection and treatment of micrometastasis stove. In therapeutic process, mainly judge to treat curative effect at present by iconography or serology, in terms of curative effect judges There are certain limitations.

The detection of body fluid tumour cell based on circulating tumor cell (Circulating Tumor Cell, CTC) can Some solutions are provided for above-mentioned limitation.CTC refers to de- from primary tumor or transfer stove in tumour formation or progression It drops into sanguimotor tumour cell, the detection of CTC prompts presence or the intraepithelial neoplasia cells of internal primary tumor or transfer stove Presence, be that metastatic lesion forms " seed " cell, be the positive evidence of tumor development.It constantly shows on evidence in recent years Show: early stage tumor development, the pre-neoplastic of the even visible lesion formation of iconography is just sent out into blood, this to send out It is likely to be present in during the entire process of tumor development.So carrying out detection to CTC is conducive to early detection tumour, auxiliary diagnosis Or reference is provided for selection therapeutic scheme.Meanwhile dynamic detection is carried out to CTC and can be appreciated that the sensibility of patient for treatment's drug, Drug resistance signal is found early.

Currently, mainly being detected by immunohistochemical method to HER2, ER, PR expression.It is swollen due to existing Tumor heterogeneity and the sightless reason of micro metastasis, the detection of HER2, ER, PR can not direct antimers in cancerous tissue in situ The expression of index of correlation in liquid and transfer stove.State of the studies have shown that HER2 in breast cancer tissue and CTC is not consistent, symbol Conjunction rate about 48%~97% (Lee JS, et al.Breast Cancer Res Treat, 2016)；There are about 21% breast cancer Patient, the expression of ER, PR are different from primary tumor (MacFarlane R, et al.J ClinOncol, 2008) in transfer stove. The detection of HER2, ER, PR are to carry out on tissue sections in tissue, individual piece can not reflect patient's entirety HER2, ER, PR expression and the result for lacking multiple protein detection simultaneously.Patient's HER2, ER, PR expression is examined based on CTC It surveys, effect of the patient to particular medication can be predicted, the selection for disease treatment scheme provides reference, and preferably guidance is clinical Using.Goal of the invention:

Kit in the present invention can detect the table of HER2, ER, PR in the tumor patients CTC such as breast cancer, carcinoma of endometrium Informative opinion is provided to the medication of patient up to horizontal.

HER2, ER, PR detection of tumor patient at present, mainly organizationally carries out Immunohistochemical detection.It is certain Transfer stove is not easy to find when very little, and there are it is invasive, cannot sample repeatedly this detected, be unable to dynamic monitoring, Single tissue samples are not enough to the shortcomings that reflecting whole tumor load.By immunofluorescence technique in patient CTC in the present invention The protein expression level of HER2, ER, PR are detected, and can more accurately reflect the disease burden of patient.And it can be repeatedly Materials, the dynamic monitoring state of an illness, thus to take therapeutic scheme to provide more foundations.

Aktas B etc. (Aktas B.BMC Cancer, 2016) etc. have detected 96 patient with breast cancer CTC EpCAM, The concordance rate of HER2, ER, PR expression is 59% (p=in the equimolecular expression of HER2, ER, PR, discovery primary tumor and CTC 0.262) concordance rate of HER2, ER, PR expression is 67% in, 39% (p=0.51) and 44% (p=0.62), transfer stove and CTC (p=0.04), 43% (p=0.16) and 46% (p=0.6).The research detects HER2, ER, PR with reverse transcription PCR, RNA is easy degradation, and can not identify the expression status of unicellular middle HER2, ER, PR.

Summary of the invention:

In view of the deficiencies of the prior art, the present invention provides a kind of method and its kit for detecting HER2, ER, PR antigen.

The prior art there is no using immunofluorescence method while detect the report that HER2, ER, PR are expressed in CTC.

The method of the invention, testing principle are as follows:

Enrichment cycles tumour cell and other rare cells by known method are adopted first, wherein the enrichment method can be with Using method described in Chinese patent 200810097889.4,201310057307.0,2015104411229.Then basis The principle of antigen-antibody reaction detects the expression of destination protein in the cell of enrichment using known immunofluorescent detection method. This kit by carrying out fluorescent marker to target cell and leukocyte common antigen CD45, it is positive by screening destination protein, The cell of CD45 feminine gender, so that the CTC to the specific protein positive in body fluid carries out interpretation and counting.On this basis, by purpose Protein positive, CD45 feminine gender candidate GTC it is classified according to high, medium and low expression according to destination protein fluorescence intensity.But Immunofluorescent detection method is used to detect HER2, ER, PR expression in GTC and encounters many difficulties in practice, comprising: cell holds It easily falls off from slide, fluorescence is weaker, non-specific dyeing, substrate background height etc..

The present invention passes through multiple screening study, finally obtains a kind of suitable method, solves above-mentioned difficulties, while in reality It during trampling, is found surprisingly that, compared to the prior art detection accuracy and accuracy rate greatly improve testing result of the invention.

Detection method of the invention, steps are as follows:

1) enrichment of CTC

Blood and other humoral samples are taken, CTC therein can be enriched with, wherein the enrichment, method, which can be selected, appoints A kind of what method for separating and concentrating of CTC, is selected from: red blood cell removal, negative enrichment, positive enrichment, density gradient separation, film mistake Filter method, microfluidic method, Flow Cytometry etc.；Enriched blood specimen can not also be applied to multiple glass slides.

Wherein the CTC (circulating tumor cell) refers to de- from primary tumor or transfer stove in tumour formation or progression It drops into sanguimotor tumour cell, is enriched with to obtain usually using selected by flow cytometry apoptosis, magnetic bead positive method for separating, preferably Obtained using negative enrichment method, the best approach of the present invention is as follows:

The abnormal cell of various hypotypes is obtained using the negative enrichment method of removal blood plasma, red blood cell, leucocyte；Use room Temperature or 20% constant humidity condition of room temperature spontaneously dry slide；Use the fixed cell of fixative and antigen.

Sample after enrichment is fixed using fixer, and the fixer is selected from: ethyl alcohol, methanol, formalin, acetone, ice (periodates-lysine-paraformaldehyde is fixed for acetic acid, formaldehyde, paraformaldehyde, glutaraldehyde, methacrylaldehyde, picric acid, PLP liquid Liquid), ECD-G liquid (carbon two sub- amide-glutaraldehyde) etc..It is solid after can also being carried out using these reagents in following detection step It is fixed.

2) CTC of enrichment is subjected to Immunofluorescence test, steps are as follows for immunofluorescent detection method: 2.1CYP1 washes load glass Piece 3min × 3 time, every time 100~150 μ L, it is ensured that cover full entire sample area；

2.2 suck surplus liquid on glass slide, and CYPP is added and acts on 5min.3min × 1 time of developing a film is same as above with CYP1；It sucks Surplus liquid, adds 200 μ l ice acetone: methanol (7: 3) acts on 5min, and CYP1 develops a film 3min × 3 time, sucks excessive moisture；

2.3 plus 100~150 μ l confining liquid room temperatures closing, 20~30min.Extra confining liquid is sucked, adds 100~150 μ l dilute ER antibody, PR antibody and the CD45 antibody released are protected from light 1~2h of incubation in room temperature wet box；(direct labelled antibody can also be 2.5 It is added).

2.4 are protected from light, and take the μ L/ piece of CYP2100~150 to develop a film 3min × 3 time, suck excessive moisture；

The fluorescence secondary antibodies that have diluted of 2.5 plus 100~150 μ L, be protected from light in wet box incubation (37 DEG C, 25~30min or room temperature 30 ~60min).CYP2 washes 3min × 4 time, sucks excessive moisture.

2.6 redye: taking 10 μ L DAPI dye liquors, add to sample area.

2.7 covereds, and suck peripheral liquid.Under the microscope or it is placed under 2~8 DEG C or -20 DEG C of environment and is protected from light guarantor It deposits.

3) microscopy observation is carried out to sample

Sample is placed under fluorescence microscope, entire sample area is scanned.If it was found that being switched to when sheet or round signal It is further identified under high power lens.The coordinate of each positive cell is recorded, and 40 × object lens are taken pictures under different channels respectively.It must Oil mirror observation can be used when wanting.

4) result judges

Positive cell: destination protein fluorescence signal is positive and has core thin without haematogenous Swine lymphocyte antigen fluorescence signal Born of the same parents are denoted as a positive cell.

Negative cells: cell has haematogenous Swine lymphocyte antigen fluorescence signal, no matter whether destination protein has fluorescence equal It is denoted as feminine gender.

In above step, need to be marked directly on using to fluorescein on antibody or by fluorescent marker Secondary antibody is detected；The fluorescein of labelled antibody has different emission spectrum, preferably green fluorescence (such as Alexa Fluor 488), orange fluorescence (such as Alexa Fluor 555), red (such as Alexa Fluor 594), crimson fluorescent (such as Alexa Fluor 647) combination；The fluorescein of labelled antibody is not limited to Alexa Fluor line fluorescent element, can equally adopt With Dylight, China green dyestuff, fluorescein isothiocynate (Fluorescein isothiocynate, FITC), biotin (biotin), the series such as quantum dot, Texas Red, phycoerythrin (PE), phycocyanin (APC), rhodamine (rhodamine) Fluorescein labelled antibody or different series fluorescein labelled antibody combination.

CD45 antibody is can also to be selected from following for the antibody of the general antigen of leucocyte for excluding blood borne cell The antibody of cell surface antigen: CD3, CD11b, CD14, CD16, CD19, CD34, CD35, CD36, CD38, CD41, CD58, CD61,CD66b,CD235a；Immunofluorescence test is carried out on tissue or cell line can be omitted CD45 antibody.Of the invention Detection method includes the operation with automation equipment by hand.

Detection method of the invention can be combined with fluorescence in situ hybridization (FISH) technology, and step is following any: (i): first carrying out fluorescence in situ hybridization again with Immunofluorescence test；(ii): first carrying out immunofluorescence inspection again with fluorescence in situ hybridization It surveys；(iii): while carrying out Immunofluorescence test and fluorescence in situ hybridization.

Contain surfactant and protein protective agent in buffer CYP1, CYP2, CYPP that this detection process uses, these Surfactant can use 0~10% Triton X-100, saponin, Tween, NP-40, SDS, polyethylene glycol；Albumen 0~10% BSA, trehalose (Trehalose), sucrose can be used in protective agent.

As well known to a person skilled in the art the confining liquid that this detection process uses can also be selected: animal blood serum, 0.5%~10%BSA, Fc receptor antibody.

As well known to a person skilled in the art restorative procedure can be used to handle sample for this detection process, include Enzymic digestion reparation, EDTA reparation, Pressure method, Microwave method etc..

The core dyestuff that this detection process uses is not limited to DAPI, equally can be using Hoechst, propidium iodide etc..

As well known to a person skilled in the art the incubation process of specific antibody and cell can be divided into Immunofluorescence test Liquid dyeing and solid dyeing.Liquid dyeing is that cell is prepared into cell suspension, carries out cell permeabilization in suspension, antibody is incubated The operation such as educate, then by cell be transferred to glass slide fix, mounting；And solid dyeing is that cell is first transferred to glass slide to consolidate It is fixed, then the operation such as carry out cell permeabilization, antibody incubation.

The present invention is to carry out above-mentioned Immunofluorescence test, devises a kind of kit convenient for operation, in the kit It include the main agents in above-mentioned Immunofluorescence test step, such as following reagent:

Buffer CYP1, CYP2, CYPP, confining liquid detect the specific antibody at least one and phase of HER2, ER, PR The fluorescence secondary antibody answered, the CD45 antibody of fluorescent marker, antibody diluent, nuclear targeting liquid are right involved in operating process Photo cell may include or be not included in kit.

The preferred following components of kit of the present invention:

The explanation of title term is carried out to reagent of the present invention below:

The HER2 sequence that HER2 antibody is directed in this kit belongs to known array, is published in Yamamoto T, et al.Similarity of protein encoded by the human c-erb-B-2 gene to epidermal Growth factor receptor.Nature, 1986,319 (6050): entitled c-erb-B-2 in 230-4. document.

The FR sequence that FR antibody is directed in this kit belongs to known array, is published in Green S, et al.Human Oestrogen receptor cDNA:sequence, expression and homology to v-erb-A.Nature, 1986,320 (6058): entitled oestrogen receptor (ER) in 134-9. document

The PR sequence that PR antibody is directed in this kit belongs to known array, is published in Kastner P, et al.Two distinct estrogen-regulated promoters generate transcripts encoding the two Functionally different human progesterone receptor forms A and B.EMBOJ, 1990,9 (5): entitled human progesterone receptor (hPR) in 1603-14. document.

Core of the invention technology is that the CTC of enrichment is carried out Immunofluorescence test, smear methods therein by step 2) It is that the present inventor obtains by many experiments and data screening, the screening process in relation to condition is as follows:

In step 2.2, surplus liquid on glass slide is sucked, CYPP is added and acts on 5min, 100~150 μ l is added to close liquid chamber 20~30min of temperature closing；

Screening process is as follows: test different types and various concentration gradient penetrate reagent, different rear fixating reagents, envelope Closing the time finally screens suitable reaction condition.

In step 2.3, HER, ER, PR antibody and CD45 antibody for adding 100~150 μ l to dilute are protected from light in room temperature wet box It is incubated for 1~2h；

Screening process is as follows: test different antibodies concentration, incubation temperature, incubation time, liquid of developing a film finally screen and are suitble to Reaction condition.

In step 2.5 plus the fluorescence secondary antibody that has diluted of 100~150 μ L, be protected from light in wet box incubation (37 DEG C, 25~30min or 30~60min of room temperature).

Screening process is as follows:

By above-mentioned screening, smear condition and method of the invention is obtained, acquired results detection accuracy and accuracy rate are significantly It improves.

Kit of the invention is mainly for detection of the CTC in blood, it can also be used to detect chest/seroperitoneum, irrigating solution, Tumour cell in hydropericardium, cerebrospinal fluid, urine, cell line, drainage-fluid, sputum etc..Can also be applied to cell sheet such as smear/ Creep plate, Immunofluorescence test of tissue etc..

Kit of the invention can be adapted for kinds cancer cell.Cancer may include following at least one: breast cancer, The cancer of the esophagus, gastric cancer, colon cancer, lung cancer, carcinoma of endometrium, prostate cancer, carcinoma of testis, the cancer of the brain, cutaneum carcinoma, the carcinoma of the rectum, sarcoma, gas Pipe cancer, head and neck cancer, cancer of pancreas, liver cancer, oophoroma, lymph cancer, cervical carcinoma, carcinoma of vulva, melanoma, celiothelioma, kidney, bladder Cancer, thyroid cancer, osteocarcinoma, cancer, sarcoma and soft tissue cancer.

Beneficial effects of the present invention are further illustrated below by way of experimental data:

Beneficial effects of the present invention:

1. with the prior art according to protein expression situation in tumor tissues come direction of medication usage compared with, testing result of the invention Real-time instruction can be carried out to medication, can not only reflect whole tumour (including carcinoma in situ and that may be present sightless micro- Small transfer stove) load, it equally can reflect the expression of HER2, ER, PR in the tumour cell easily shifted in body fluid, from And more accurately it is used for medication guide.

2. thin to HER2, ER, PR in CTC compared with the prior art carries out immunohistochemistry detection to HER2 in tumor tissues Born of the same parents carry out counting the differentiation with expression quantity, can more accurately reflect that the correlation of HER2, ER, PR and clinical parameter and guidance are used Medicine.The expression of HER2, ER, PR can be detected on same cell simultaneously, be conducive to the selection of patient treatment protocol.

3. compared with prior art, sample used by this research is the cell in the body fluid such as blood source, in materials It is more more convenient than the detection technique based on tumor tissues.

4. compared with the prior art detects protein expression in tumor tissues, using immunofluorescence to HER2, ER in CTC, The expression of PR, which is detected, can carry out phenotypic analysis to these cells.

Detailed description of the invention:

Fig. 1 be same sample fluorescence display figure, respectively the general antigen CD4 5 of target protein HER2, ER, PR, leucocyte, Nucleus dyestuff DAPI, figure is closed.

Specific embodiment:

Embodiment 1

Material: the blood sample smear of feminine gender enrichment, control cell can use BT-474 cell smear.

Experimental procedure:

1. extracting 3.5ml peripheral blood in ACD (sodium citrate) anticoagulant tube.WithHuman peripheral leucocytes go Except kit feminine gender enriched tumor cell, and it is fixed on glass slide；

2.CYP1 washes glass slide 3min × 3 time, every time 100~150 μ L, it is ensured that covers full entire sample area；

3. sucking surplus liquid on glass slide, CYPP is added and acts on 5min, CYP1 is same as above 3min × 1 time of developing a film；It sucks more Extraction raffinate body, adds 200 μ l ice acetone: methanol (7: 3) acts on 5min, and CYP1 develops a film 3min × 3 time, sucks excessive moisture；

4. 100~150 μ l confining liquid room temperatures is added to close 25~30min.Extra confining liquid is sucked, 100 μ l is added to dilute HER2 antibody, ER antibody, PR antibody and CD45 antibody are protected from light 1.5~2h of incubation in room temperature wet box；

5. be protected from light, take CYP2100-150 μ L/ piece to develop a film 3min × 3 time, suck excessive moisture；

6. adding the secondary antibody that has diluted of 100 μ l, incubation (37 DEG C, 25~30min) are protected from light in wet box.CYP2 washes 3min × 4 time, Suck excessive moisture.

7. redying: after DAPI pipe brief centrifugation, 10 μ L DAPI dye liquors are taken at liquid level, add to sample area.

8. cover plate: covered, under the microscope.HER2, ER, PR are partly or entirely expressed to positive, CD45 antibody yin Property cell be considered as positive cell (Fig. 1, this experiment PR AlexaThe fluorescence secondary antibody of 700 labels, unused kit AlexaThe secondary antibody of 594 labels), and record the quantity of positive cell.

As a result illustrate:

Embodiment 2

Material: appropriate anticoagulated blood 1 is managed, and carries out Protein Detection to it after being enriched with using membrane filtering method

Experimental procedure:

1. extracting appropriate peripheral blood to be put into containing in anti-coagulants heparin tube, slight concussion is mixed.

2. suspension addition film is separated by filtration in tumour cell technique device, filter and filter membrane are slow transitted through.

3. after to be filtered, 50mI0.01MPBS is added in continuation in film filter, thin by what is adhered to around tube wall Born of the same parents' suspension pours in film filter, it is allowed to pass through filter and filter membrane；

4. the cell on fixed filter membrane；

5. operating detection albumen with embodiment 1.

Embodiment 3

Material: appropriate anticoagulated blood 1 is managed, and carries out Protein Detection to it after being enriched with using microfluidic methods

Experimental procedure:

1. the appropriate blood extracted is enriched with using the micro-fluidic chip of various principles.

2. sample carries out protein immunization fluorescence detection after enrichment.

Embodiment 4

Material: frozen tissue section

Experimental procedure:

1. being loaded with the glass slide of slice by taking out in freezing microtome, it is put into wet box.

2. when glass slide reach room temperature and it is not dry when, add CYP1 in washing on slice glass slide 3-5 times；

3. sucking surplus liquid, CYPP is added to be incubated for, CYP1 develops a film；

4. sucking surplus liquid, seals up and close the closing of liquid chamber temperature；

5. suck extra confining liquid, add the HER2 antibody, ER antibody, PR antibody diluted, room temperature is protected from light when being incubated for appropriate Between；

6. being protected from light, CYP2 develops a film, and sucks excessive moisture；

7. adding the secondary antibody diluted, it is protected from light incubation appropriate time.

8.CYP2 developing a film, excessive moisture is sucked.

9. adding 10 μ lDAPI, mounting microscopy.Positive cell is judged whether it is according to cellular morphology and HER2, ER, PR expression.

Claims (10)

1. a kind of method of HER2, ER, PR expression in detection circulating tumor cell, the method, steps are as follows:

1) enrichment of circulating tumor cell

Blood and other humoral samples are taken, circulating tumor cell therein (Circulating Tumor Cell, CTC) is carried out Enrichment, wherein the enrichment, the method for separating and concentrating of any rare karyocyte is can be selected in method, and be selected from: red blood cell is gone It removes, negative enrichment, positive enrichment, density gradient separation, membrane filter method, microfluidic method, Flow Cytometry etc.；It can not also It is enriched that blood specimen is applied to multiple glass slides,

Sample after enrichment is fixed using fixer, and the fixer is selected from: ethyl alcohol, methanol, formalin, acetone, glacial acetic acid, Formaldehyde, paraformaldehyde, glutaraldehyde, methacrylaldehyde, picric acid, PLP liquid, ECD-G liquid,

2) CTC of enrichment is subjected to Immunofluorescence test, steps are as follows for immunofluorescent detection method: 2.1) CYP1 washes glass slide 3min × 3 time, every time 100~150 μ L, it is ensured that cover full entire sample area；

2.2) surplus liquid on glass slide is sucked, CYPP is added and acts on 5min, is same as above 3min × 1 time of developing a film with CYP1；It sucks more Extraction raffinate body, adds 200 μ l ice acetone: methanol (7:3) acts on 5min, and CYP1 develops a film 3min × 3 time, sucks excessive moisture；

2.3) plus 100~150 μ l confining liquid room temperatures close 20~30min, suck extra confining liquid, 100~150 μ l is added to dilute ER antibody, PR antibody and CD45 antibody, be protected from light 1~2h of incubation in room temperature wet box；

2.4) it is protected from light, takes 100~150 μ L/ piece of CYP2 to develop a film 3min × 3 time, suck excessive moisture；

2.5) it plus the fluorescence secondary antibody that has diluted of 100~150 μ L, is protected from light incubation in wet box, CYP2 washes 3min × 4 time, and it is extra to suck Moisture,

2.6) it redyes: taking 10 μ L DAPI dye liquors, add to sample area,

2.7) covered, and suck peripheral liquid under the microscope or is placed under 2~8 DEG C or -20 DEG C of environment and is kept in dark place,

3) microscopy observation is carried out to sample

Sample is placed under fluorescence microscope, entire sample area is scanned, if switching to high power when discovery sheet or round signal It is further identified under mirror, records the coordinate of each positive cell, and 40 × object lens are taken pictures under different channels respectively, when necessary Oil mirror observation can be used,

4) result judges

Positive cell: the destination protein fluorescence signal positive and the karyocyte without haematogenous Swine lymphocyte antigen fluorescence signal, It is denoted as a positive cell,

Negative cells: cell has haematogenous Swine lymphocyte antigen fluorescence signal, no matter whether destination protein has fluorescence to be denoted as It is negative.

2. the method according to claim 1, wherein being needed in detection process using to fluorescein, the fluorescence The plain combination for preferentially using green fluorescence, orange fluorescence, red fluorescence, crimson fluorescent, is selected from: Alexa Fluor, Dylight, China green dyestuff, fluorescein isothiocynate, biotin, quantum dot, Texas Red, phycoerythrin, phycocyanin, sieve It is red bright.

3. the method according to claim 1, wherein CD45 antibody used in detection process is for leucocyte The antibody of general antigen can also be selected from following antibody for excluding blood borne cell: CD3, CD11b, CD14, CD16, CD19、CD34、CD35、CD36、CD38、CD41、CD58、CD61、CD66b、CD235a。

4. the method according to claim 1, wherein the method can be with fluorescence in situ hybridization (FISH) technology Combination, step are following any: (i): first carrying out fluorescence in situ hybridization again with Immunofluorescence test；(ii): first former with fluorescence Position hybridization carries out Immunofluorescence test again；(iii): while carrying out Immunofluorescence test and fluorescence in situ hybridization.

5. the method according to claim 1, wherein in buffer CYP1, CYP2, CYPP that detection process uses Containing surfactant and protein protective agent, surfactant is selected from: 0~10% Triton X-100, saponin, Tween, NP-40, SDS, polyethylene glycol；Protein protective agent is selected from: 0~10% BSA, trehalose, sucrose.

6. the method according to claim 1, wherein the confining liquid that detection process uses is selected from: animal blood serum, 0.5%~10%BSA, Fc receptor antibody.

7. the method according to claim 1, wherein the restorative procedure that detection process uses is selected from: enzymic digestion is repaired Multiple, EDTA reparation, Pressure method, Microwave method.

8. the method according to claim 1, wherein the core dyestuff that uses of detection process is selected from DAPI, Hoechst, propidium iodide.

It include the main agents in claim 1 detection method step: buffer 9. a kind of kit, in the kit CYP1, CYP2, CYPP, confining liquid detect at least one and corresponding fluorescence secondary antibody of specific antibody of HER2, ER, PR, glimmering The CD45 antibody of signal, antibody diluent, nuclear targeting liquid, optional related control cell include or do not include In kit.

10. kit according to claim 9, contains following components: