CN105606823A - Detection method for PR genes of circulating tumor cells in peripheral blood of later-period breast cancer patient - Google Patents
Detection method for PR genes of circulating tumor cells in peripheral blood of later-period breast cancer patient Download PDFInfo
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Abstract
The invention discloses a detection method for PR genes of circulating tumor cells in peripheral blood of a later-period breast cancer patient. The detection method comprises the steps that the CTCs in the peripheral blood of the later-period breast cancer patient are separated and acquired through a film filtering device; the circulating tumor cells in the peripheral blood of the later-period breast cancer patient are identified through a cellular immunity fluorescent technique; thin sections are made through a cell wax block technique; the expression condition of the PR of the circulating tumor cells in the peripheral blood of the later-period breast cancer patient is detected through an immunologic tissue chemical technique. According to the detection method, the enrichment CTCs are separated with the help of an ISET technique, the CTCs are identified by depending on the cellular immunity fluorescent technique, and therefore the difficulties existing in CTC identification only through the ISET technique and the false negativity existing in CTC identification only through an immunological detection technique are overcome. The technology has the advantages that the requirement on equipment is not high, the method is easy to master, and real-time monitoring can be achieved. Through the technical method, the expression condition of the PR of the later-period breast cancer patient can be detected without needing to sample the breast cancer tissue. The technology belongs to a minimally invasive technology or even a non-invasive technology and can achieve real-time detection.
Description
Technical field
The present invention relates to a kind of new method of the PR of detection gene, especially detect mammary gland in late period by peripheral bloodThe method of cancer patient PR gene.
Background technology
ER as the progesterone receptor (ProgesteroneReceptor, PR) of prognostic factor at presentProduct, can cause and strengthen the reaction of estrogen to ER, play promote and collaborative effect, it isBecome most important index while formulating patient with breast cancer's endocrine therapy scheme, while only having ER positive, interior pointSecreting treated effect is 50%-60%, if when ER, PR are all positive, efficient can be up to 70%-80%,Be 10% and negative patient is efficient simultaneously. The detection of existing breast cancer PR need to be by performing the operation or puncturing workThe modes such as inspection obtain the tumor tissues sample of primary tumor or metastatic tumor, and right instead of each patient can obtainTumor tissues sample, and can not in therapeutic process, carry out continuous detection, having limited it becomes and hasThe curative effect monitoring index of effect. Can provide potential real-time tumor specimen but come off to us into the CTC of blood,Its mutation status can be used for guiding treatment equally.
Circulating tumor cell (Circulatingtumorcell, CTC) is come off and enter from entity tumorEnter the tumour cell of peripheral blood circulation, it is multiple that it is present in breast cancer, prostate cancer, colorectal cancer etc.In malignant tumour, it detects for especially prognosis and the selection of late tumor patient of assessment tumor patientSuitable individualized treatment has important clinical meaning. There is Wicresoft because CTC detects, the feature such as real-time,Be called as " liquid biopsy ".
The main method that CTCs detects at present comprises the immunology detection skill that relies on the survey of tumor related marker quality testingArt and the membrane filtration technique (Isolatingbysizeofepithelialtumor based on morphology enrichmentCells, ISET). The former is based on not synantigen mark of cell surface expression, will be for tumour cell tableSpecific antibody EpCAM, the CK etc. of face antigen are coated in magnetic bead surfaces to be known with the specificity that completes tumour cellNot, by externally-applied magnetic field screening enrichment CTCs. The advantage of this technology is that specificity is higher, existing commodityThe semi-automatic checkout equipment (CellSearch) of changing; But the disappearance that tumour specific antigen is expressed can cause vacationNegative findings. The latter is the difference according to cell size, utilizes the method for filtering by tumour larger volumeCell screening enrichment. The advantage of this technology is that method is simple, cheap, separates the CTCs tool obtainingThere is activity to can be used for follow-up study; But owing to lacking Morphologic Diagnosis goldstandard, this beneficiation technologies specificityRelatively poor.
Summary of the invention
Be not suitable for tissue specimen in order to overcome traditional circulating tumor cell detection technique and advanced breast cancer patientDraw materials--and then deficiency that cannot assess patient PR state, the invention provides a kind of advanced breast cancer patientThe detection method of Peripheral Circulation tumour cell PR gene: utilize film filter to separate and obtain mammary gland in late periodCTC in cancer peripheral blood in patients; Utilize cellular immunofluorescence technical appraisement advanced breast cancer peripheral blood in patients to followRing tumour cell; Use the section of cell block fabrication techniques thin layer; And then pass through immunohistochemistry technologyDetect the PR expression of advanced breast cancer peripheral blood in patients circulating tumor cell.
The method concrete steps are as follows:
One, utilize film filter to separate and obtain advanced breast cancer peripheral blood in patients circulating tumor cell:
1, gather advanced breast cancer disease peripheral blood in patients: median basilic vein 10ml, is divided into 2 pipes and writes phaseWith numbering after, respectively called after test 1 group and experiment 2 groups;
2,2 pipe peripheral blood sample are carried out respectively to pretreatment: by the 10 times of dilutions of peripheral blood sample that gather, rareRelease liquid composition: 1mmol/lEDTA+0.1%BSA, after dilution, fix 10 points of peripheral blood sample with 4% paraformaldehydeClock;
3, utilize film filter to separate peripheral blood sample, enrichment Peripheral Circulation tumour cell: by pretreatmentPeripheral blood sample join in the blood sample container of film filter, make it rely on gravity natural filtration;
4, after filtration finishes, take off filter from film filter, PBS rinses 2-3 time, and peripheral blood followsRing tumour cell is trapped within on filter membrane.
Two, utilize cellular immunofluorescence technical appraisement advanced breast cancer peripheral blood in patients circulating tumor cell:
1, add from obtaining in the filter of Peripheral Circulation tumour cell to experiment 1 componentCytoperm/Cytofix reagent 300 μ l, fixing, perforation 15min;
2, PBS rinsing 3 times, each 5 minutes, adds washbuffer300 μ l, sealing 30min afterwards;
3, add CK8/18/19, CD45 primary antibodie suspension 300 μ l, two kinds of primary antibodies are all with washbufferDilution, the final concentration after mixing is 1:250, under room temperature, hatches 45min;
4, Washbuffer washing 3 times, each 5 minutes, add afterwards two anti-suspension 300 μ l,Two kind two anti-, and all with washbuffer dilution, the final concentration after mixing is 1:500, and under room temperature, lucifuge is hatched30min；
5, Washbuffer washing 3 times, each 5 minutes, add afterwards Hoechst300 μ l, washTransfect cell core 5min;
6, PBS rinsing 2 times, each 3 minutes, take out afterwards filter membrane and be placed on slide, drip 10 μThe anti-fluorescent quenching confining liquid of l mounting;
7, the interior observed result of 2h under fluorescence microscope, takes pictures, records CTC situation, determines whether to existCTC; As observe CTC and proceed next step detection, otherwise stop detecting.
Three, use cell block fabrication techniques CTC cell block:
1, from filter, take off the filter of 2 groups of identical blood sample numberings of experiment, open and remove on filterMouthful, circulating tumor cell dyeing liquor is joined in filter, dyeing 2min, PBS buffer solution is rinsed well,Take off filter membrane with ophthalmology tweezers, be placed on slide, examine under a microscope after suitably dry, confirm to depositAt CTC;
2, make CTC cell block;
A, decolouring: the filter membrane on above-mentioned slide is taken off, be placed in destainer and soak 4-6 hour, sloughCTC dyeing liquor, described destainer is: 95% alcohol and 100% dimethylbenzene by volume 1:1 mix;
B, parcel: soak and finish rear taking-up filter membrane, wrap up with blotting paper;
C, fixing: wrappage is placed in to 10% neutral formalin, fixing 4-6h;
D, waxdip embedding: after fixing end, take out wrappage, dehydration is placed on FFPE boxIn, cell paraffin mass is made in waxdip embedding;
E, thin layer section: with slicer, by cell paraffin mass serial section, making thickness is 2-4The thin layer section of μ m.
Four, detect advanced breast cancer peripheral blood in patients circulating tumor cell by immunohistochemistry technologyPR expression:
1. roasting sheet: section is baked sheet 2 hours in 60 DEG C of insulating boxs;
2. dewaxing: cut into slices and place respectively 5 minutes in two bottles of dimethylbenzene;
3. aquation: section is placed respectively 1 minute successively in absolute ethyl alcohol, 95% ethanol, 85% ethanol(slightly adjusting depending on indoor temperature situation): running water, distilled water flushing;
4. antigen retrieval: the reparation liquid EDTA in pressure cooker is boiled, put into section to completely submerging and repairMultiple liquid covers high-pressure pot cover and pressure valve, and 800W is heated to pressure cooker jet latter 1.5 minutes, from fire, and willThe slightly cooling pot cover of opening of pressure cooker, section is naturally cooling in reparation liquid;
5. distilled water flushing;
6. remove endogenous peroxydase: with 3%H2O2 effect section 10 minutes;
7. distilled water flushing;
8.PBS embathe 5 times each 1.5 minutes;
9. drip primary antibodie, 4 DEG C of refrigerator overnight, then put it into PBS and embathe 5 times, embathe at every turn1.5 minute;
10. dripping two resists;
In 11.37 DEG C of water-baths, hatch 20 minutes;
12.PBS embathe 5 times each 1.5 minutes;
Under 13. microscopes, control DAB colour developing;
14. running water rinse, and haematine dye liquor is redyed 2 minutes, hydrochloride alcohol differentiation, and ammoniacal liquor returns indigo plant;
15. running water rinse 5 minutes;
16.75%-95%-100% gradient alcohol dehydration, dimethylbenzene is transparent, neutral Instant cement mounting;
17. cell pathology experts read sheet, interpretation PR expression.
Utilize the membrane filtration dress described in cellular immunofluorescence technical appraisement Peripheral Circulation tumour cell stepPut: comprise filter, blood sample container, waste liquid cylinder and iron stand, described iron stand be provided with base, stand andSupport, described blood sample container is arranged at iron stand top by support, and the below of blood sample container is filter,Filter is by transfusion device UNICOM to waste liquid cylinder, and waste liquid cylinder is arranged on base.
Described filter comprises that filter is suitable for reading, filter membrane, carry filter membrane platform and filter end opening, and filter membrane is placed in and carries filterOn film platform; The filter blood sample container that connects suitable for reading, filter end opening connects waste liquid cylinder by transfusion device.
Described filter membrane is that hydrophobic material is made, and is evenly covered with bore and is the filter opening of 10 microns on it.
Beneficial effect: by means of ISET technology separation and concentration CTCs, rely on cellular immunofluorescence technical appraisementCTCs, not only can overcome the difficulty that simple ISET technical appraisement CTCs exists, and also can catch epithelium and resistThe tumour cell that former expression weakens or lacks, overcomes the false negative that simple immunology detection technology exists.Meanwhile, this technical equipment is less demanding, and method is easy to grasp, and energy Real-Time Monitoring, has clinical expansionApplication may. By this technical method, the breast cancer tissue that need not draw materials can detect advanced breast cancer troublePerson PR expression. This technology belongs to Wicresoft even without wound, and can detect in real time.
Brief description of the drawings
Fig. 1 is film filter structural representation of the present invention;
Fig. 2 is the structural representation cutaway view of the filter of film filter of the present invention;
Fig. 3 is the structural representation of the filter filter membrane of film filter of the present invention;
Fig. 4 is that advanced breast cancer cancer peripheral blood in patients separates the circulating tumor cell striograph obtaining;
Fig. 5 is advanced breast cancer peripheral blood in patients circulating tumor cell SABC PR colored graph picture.
1. iron stand 2. blood sample container 3. filter 4. transfusion device 5. waste liquid cylinder 6. filters suitable for reading 7. in figure8. years filter membrane platforms of filter membrane, 9. filter end opening, 10. filter opening, 11. bases, 12. stands, 13 supports.
Detailed description of the invention
Below in conjunction with drawings and Examples, the present invention is further described.
Using this technical method to separate obtains and identifies that 8 routine advanced breast cancer cancer patients (detect 8 examples simultaneouslyNormal person's sample does negative control) Peripheral Circulation tumour cell, detects its PR expression.
One, utilize film filter to separate and obtain the advanced breast cancer patient periphery that cannot obtain tissue specimenCTC in blood:
Gather advanced breast cancer peripheral blood in patients 10ml, be divided into 2 pipes, same blood sample of patient is write identicalNumbering, respectively called after test 1 group and experiment 2 groups; Then 2 pipe peripheral blood sample are located respectively in advanceReason: by the 10 times of dilutions of peripheral blood sample that gather, diluent ingredient: 1mmol/lEDTA+0.1%BSA),After dilution, fix peripheral blood sample 10 minutes with 4% paraformaldehyde; In fixing interval, assembling film isolated by filtrationTumour cell (ISET) easy device (as shown in Figure 1, 2, 3); This filter is by filter 3, filterFilm 7, blood sample container 2, waste liquid cylinder 5, iron stand 1 form, and utilize film filter to separate peripheral blood sample,Enrichment Peripheral Circulation tumour cell: with the wetting filter 3 of 10mlPBS, then by pretreated peripheral blood sampleJoin in the blood sample container 2 of film filter, make it rely on gravity natural filtration; After filtration finishes,From film filter, take off filter 3, PBS rinses 2-3 time, and peripheral blood CTC is trapped within on filter membrane 7.
Tumour cell diameter is generally greater than 15 microns, and haemocyte (comprising red blood cell, leucocyte) diameterBe generally less than 10 microns, therefore ought contain the peripheral blood of CTC after filtering, haemocyte is less than filter because of diameterHole 10 can be filtered across, and CTC is trapped within on filter membrane 7 because diameter is greater than filter opening 10.
Two, utilize cellular immunofluorescence technical appraisement advanced breast cancer peripheral blood in patients circulating tumor cell:
Obtain (experimental group 1) in the filter of Peripheral Circulation tumour cell adds to separationCytoperm/Cytofix reagent 300 μ l, fixing, perforation 15min; PBS rinsing 3 times, each 5Minute, add afterwards washbuffer300 μ l, sealing 30min; Add CK8/18/19, CD45Primary antibodie suspension 300 μ l, two kinds of primary antibodies are all with washbuffer dilution, and the final concentration after mixing is1:250, hatches 45min under room temperature; Washbuffer washing 3 times, each 5 minutes, adds afterwardsTwo anti-suspension 300 μ l, two kind two is anti-all with washbuffer dilution, and the final concentration after mixing is1:500, under room temperature, lucifuge is hatched 30min; Washbuffer washing 3 times, each 5 minutes, afterwardsAdd Hoechst300 μ l, wash transfect cell core 5min; PBS rinsing 2 times, each 3 minutes, afterwardsTake out filter membrane and be placed on slide, drip the anti-fluorescent quenching confining liquid of 10 μ l mounting; Under fluorescence microscopeObserved result in 2h, takes pictures, records CTC situation (as experimental group 1 is observed application experiment group of CTC2 carry out next step detects).
Testing result: 8 routine healthy volunteers all do not find circulating tumor cell; Suffer from 8 routine advanced breast cancersIn person's peripheral blood, there are 5 examples to detect that CTC exists.
Three, use the section of cell block fabrication techniques thin layer:
From filter, take off the filter 3 that CTC numbering detected in testing 2 groups, open and remove filterSuitable for reading 6, circulating tumor cell dyeing liquor is joined in filter 3, dyeing 2min, PBS buffer solution rinsesTotally, take off filter membrane 7 with ophthalmology tweezers, be placed on slide, examine under a microscope after suitably dry,Alleged occurrence CTC;
Filter membrane on above-mentioned slide 7 is taken off, be placed in destainer soak 4 ?6 hours, slough CTCDyeing liquor, described destainer is: 95% alcohol and 100% dimethylbenzene by volume 1:1 mix; Soak knotAfter bundle, take out filter membrane 7, wrap up with blotting paper; Wrappage is placed in to 10% neutral formalin, fixing 4-6h;After fixing end, take out wrappage, dehydration is placed in FFPE box, and cell paraffin is made in waxdip embeddingPiece; With slicer, by cell paraffin mass serial section, making thickness is the thin layer section of 2-4 μ m.
Four, detect advanced breast cancer peripheral blood in patients circulating tumor cell by immunohistochemistry technologyPR expression:
The paraffin section application row immunohistochemical method of obtaining is detected to the expression of PR gene, will cutSheet is baked sheet 2 hours in 60 DEG C of insulating boxs, cuts into slices and places respectively 5 minutes in two bottles of dimethylbenzene, will cutSheet is placed respectively 1 minute (depending on indoor temperature situation successively in absolute ethyl alcohol, 95% ethanol, 85% ethanolSlightly adjust): running water, distilled water flushing;
Antigen retrieval: according to first antibody description and specific experiment operating experience, experiment section is usedDiverse ways carries out antigen retrieval, and this experiment is used the reparation of EDTA (PH=9.0) hot high pressure: at electromagnetic ovenUpper reparation liquid (EDTA) in pressure cooker is boiled, putting into section and repairing liquid to submerging completely and cover high pressurePot cover and pressure valve, 800W is heated to pressure cooker jet latter 1.5 minutes, from fire, pressure cooker is slightly coolingOpen pot cover, section is naturally cooling in reparation liquid, then uses distilled water flushing, removes endogenous after rinsingProperty peroxidase: (adopt the detection system of peroxidase, must carry out endogenous peroxydase envelopeClose processing, if do not processed, the red blood cell in tissue, the judgement that granulocyte can disturb coloration result)3%H2O2 effect section 10 minutes for this experiment, preserves and does not all affect coloration result collection antigen, envelopeClose effect more remarkable, distilled water flushing, PBS embathes 5 times each 1.5 minutes, drips PR primary antibodie, 4DEG C refrigerator overnight, then puts it into PBS and embathes 5 times, embathes 1.5 minutes at every turn; Drip two and resist,In 37 DEG C of water-baths, hatch 20 minutes, PBS embathes 5 times each 1.5 minutes, controls DAB under microscopeColour developing, running water rinses, and haematine dye liquor is redyed 2 minutes, hydrochloride alcohol differentiation, ammoniacal liquor returns indigo plant, fromWater is rinsed 5 minutes, 75%-95%-100% gradient alcohol dehydration, and dimethylbenzene is transparent, neutral quick-drying rubber sealSheet, 2-3 name cell pathology expert reads sheet, interpretation PR expression.
Testing result: 8 routine healthy volunteers all do not find circulating tumor cell; Suffer from 8 routine advanced breast cancersThe CTC that person's peripheral blood detects, wherein 5 examples detect that through SABC it has PR to express, positive rate is62.5% (table 1).
Fig. 4 is that Peripheral Blood In Patients With Breast Cancer separates the circulating tumor cell striograph obtaining, and its nucleusGreatly, nucleus out-of-shape; High nucleocytoplasmic ratio.
Fig. 5 is Peripheral Blood In Patients With Breast Cancer circulating tumor cell PR immunohistochemical staining image, cell membrane andCytoplasm xanthochromia. Judge positive degree according to its dye distribution and intensity, the painted cell that accounts for of cell is less than 10%For (-), painted 10%-25% is (+), and painted 25%-50% is (++), is paintedly greater than 50% for (+++).Table 1 embodiment testing result (PR)
Claims (8)
1. a detection method for advanced breast cancer peripheral blood in patients circulating tumor cell PR gene, its feature existsIn, utilize film filter to separate the CTC obtaining in advanced breast cancer peripheral blood in patients; Identify late periodPeripheral Blood In Patients With Breast Cancer circulating tumor cell; Use the section of cell block fabrication techniques thin layer; DetectThe PR expression of advanced breast cancer peripheral blood in patients circulating tumor cell.
2. the inspection of advanced breast cancer peripheral blood in patients circulating tumor cell PR gene according to claim 1Survey method, is characterized in that, described film filter comprise filter (3), blood sample container (2),Waste liquid cylinder (5) and iron stand (1), described iron stand (1) is provided with base (11), stand (12)And support (13), described blood sample container (2) is arranged on iron stand (1) by support (13)Portion, the below of blood sample container (2) is filter (3), filter (3) is by transfusion device (4) UNICOMTo waste liquid cylinder (5), waste liquid cylinder (5) is arranged on base (11).
3. the inspection of advanced breast cancer peripheral blood in patients circulating tumor cell PR gene according to claim 2Survey method, is characterized in that: described filter (3) comprises filter (6) suitable for reading, filter membrane (7), carriesFilter membrane platform (8) and filter end opening (9), filter membrane (7) is placed in and carries on filter membrane platform 8; On filterMouth (6) connects blood sample container (2), and filter end opening (9) connects waste liquid cylinder (5) by transfusion device (4).
4. the inspection of advanced breast cancer peripheral blood in patients circulating tumor cell PR gene according to claim 3Survey method, is characterized in that: described filter membrane (7) is made for hydrophobic material, and on it, even cloth profuselyFootpath is the filter opening (10) of 10 microns.
5. the inspection of advanced breast cancer peripheral blood in patients circulating tumor cell PR gene according to claim 1Survey method, is characterized in that, the described film filter separation that utilizes is obtained outside advanced breast cancer patientCTC in all blood, step is as follows:
1) gather patient with advanced cancer peripheral blood: median basilic vein 5ml;
2) peripheral blood sample is carried out respectively to pretreatment: by the 10 times of dilutions of peripheral blood sample that gather, rareRelease liquid composition: 1mmol/lEDTA+0.1%BSA, after dilution, fix peripheral blood sample with 4% paraformaldehyde10 minutes;
3) utilize film filter to separate peripheral blood sample, enrichment Peripheral Circulation tumour cell: will be pre-The peripheral blood sample of processing joins in the blood sample container (2) of film filter, makes it rely on gravity certainlySo filter;
4) after filtration finishes, take off filter (3) from film filter, PBS rinses 2-3 time,Peripheral Circulation tumour cell is trapped within on filter membrane (7).
6. the inspection of advanced breast cancer peripheral blood in patients circulating tumor cell PR gene according to claim 1Survey method, is characterized in that, described qualification advanced breast cancer peripheral blood in patients circulating tumor cell,Step is as follows:
1) in obtaining the filter (3) of Peripheral Circulation tumour cell, separation addsCytoperm/Cytofix reagent 300 μ l, fixing, perforation 15min;
2) PBS rinsing 3 times, each 5 minutes, adds washbuffer300 μ l, envelope afterwardsClose 30min;
3) add CK8/18/19, CD45 primary antibodie suspension 300 μ l, two kinds of primary antibodies are all with washBuffer dilution, the final concentration after mixing is 1:250, under room temperature, hatches 45min;
4) Washbuffer washing 3 times, each 5 minutes, adds two anti-suspensions 300 afterwardsμ l, three kind two is anti-all with washbuffer dilution, and the final concentration after mixing is 1:500, room temperatureLower lucifuge is hatched 30min;
5) Washbuffer washing 3 times, each 5 minutes, add afterwards Hoechst300 μ l,Wash transfect cell core 5min;
6) PBS rinsing 2 times, each 3 minutes, take out afterwards filter membrane (7) and be placed on slide,Drip the anti-fluorescent quenching confining liquid of 10 μ l mounting;
7) the interior observed result of 2h under fluorescence microscope, takes pictures, records CTC situation, determines whetherThere is CTC.
7. the inspection of advanced breast cancer peripheral blood in patients circulating tumor cell PR gene according to claim 1Survey method, is characterized in that, described utilization cell block fabrication techniques thin layer section, and step is as follows:
1) after peripheral blood filters, from filter, take off filter (3), open and remove on filterMouth (6), joins circulating tumor cell dyeing liquor in filter (3), dyeing 2min, and PBS is slowRush liquid and rinse well, take off filter membrane (7) with ophthalmology tweezers, be placed on slide, suitably dry afterExamine under a microscope alleged occurrence CTC;
2) make thin layer section:
A, decolouring: the above-mentioned filter membrane with CTC (7) is taken off from slide, be placed in 95%In alcohol and the 100% dimethylbenzene destainer that 1:1 mixes by volume, soak 4-6 hour, slough CTCDyeing liquor;
B, parcel: soak and finish rear taking-up filter membrane, wrap up with blotting paper;
C, fixing: wrappage is placed in to 10% neutral formalin, fixing 4-6h;
D, waxdip embedding: after fixing end, take out wrappage, dehydration is placed in FFPE box,Cell paraffin mass is made in waxdip embedding;
E, thin layer section: with slicer, by cell paraffin mass serial section, making thickness is 2-4 μ mThin layer section.
8. the inspection of advanced breast cancer peripheral blood in patients circulating tumor cell PR gene according to claim 1Survey method, is characterized in that, described detection advanced breast cancer peripheral blood in patients circulating tumor cellPR expression, step is as follows:
1) roasting sheet: the section of CTC wax stone thin layer is baked sheet 2 hours in 60 DEG C of insulating boxs;
2) dewaxing: cut into slices and place respectively 5 minutes in two bottles of dimethylbenzene;
3) aquation: section is placed respectively to 1 point successively in absolute ethyl alcohol, 95% ethanol, 85% ethanolClock (slightly adjusting depending on indoor temperature situation): running water, distilled water flushing;
4) antigen retrieval: the reparation liquid EDTA in pressure cooker is boiled, put into section to submerging completelyRepair liquid and cover high-pressure pot cover and pressure valve, 800W is heated to pressure cooker jet latter 1.5 minutes, fromFire, by the slightly cooling pressure cooker pot cover of opening, section is naturally cooling in reparation liquid;
5) distilled water flushing;
6) remove endogenous peroxydase: with 3%H2O2 effect section 10 minutes;
7) distilled water flushing;
8) PBS embathe 5 times each 1.5 minutes;
9) drip primary antibodie, 4 DEG C of refrigerator overnight, then put it into PBS and embathe 5 times, soak at every turnWash 1.5 minutes;
10) dripping two resists;
11) in 37 DEG C of water-baths, hatch 20 minutes;
12) PBS embathe 5 times each 1.5 minutes;
13) under microscope, control DAB colour developing;
14) running water rinses, and haematine dye liquor is redyed 2 minutes, hydrochloride alcohol differentiation, and ammoniacal liquor returns indigo plant;
15) running water rinses 5 minutes;
16) 75%-95%-100% gradient alcohol dehydration, dimethylbenzene is transparent, neutral Instant cement mounting;
17) cell pathology expert reads sheet, interpretation PR expression.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610058588.5A CN105606823B (en) | 2016-01-28 | 2016-01-28 | The detection method of advanced breast cancer patient Peripheral Circulation tumour cell PR genes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610058588.5A CN105606823B (en) | 2016-01-28 | 2016-01-28 | The detection method of advanced breast cancer patient Peripheral Circulation tumour cell PR genes |
Publications (2)
| Publication Number | Publication Date |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109239030A (en) * | 2018-08-10 | 2019-01-18 | 北京莱尔生物医药科技有限公司 | A kind of kit and application detecting circulating tumor cell HER2 different loci and ER, PR |
| CN111534586A (en) * | 2020-04-21 | 2020-08-14 | 山东省肿瘤防治研究院（山东省肿瘤医院） | Kit and method for detecting CEA gene mutation of peripheral blood circulating tumor cells of non-small cell lung cancer patients |
| CN112485426A (en) * | 2020-12-01 | 2021-03-12 | 山东省药物研究院 | Circulating tumor cell IHC antigen repairing method based on ISET principle |
| WO2021213257A1 (en) * | 2020-04-21 | 2021-10-28 | 山东第一医科大学(山东省医学科学院) | Detection and identification method for circulating tumor cells (ctcs) |
| WO2021213311A1 (en) * | 2020-04-22 | 2021-10-28 | 山东第一医科大学(山东省医学科学院) | Immunofluorescence kit for detecting pd-l1 gene expression of patient with colorectal cancer by means of peripheral blood circulating tumor cells |
| WO2022001826A1 (en) * | 2020-07-01 | 2022-01-06 | 天津市肿瘤医院（天津医科大学肿瘤医院） | Immunofluorescence kit for detecting e-cadherin expression of peripheral blood circulating tumor cells of patient with pancreatic cancer |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1737526A (en) * | 2005-07-20 | 2006-02-22 | 徐华 | Making method of body fluid cell lump paraffin section |
| CN102980995A (en) * | 2012-12-04 | 2013-03-20 | 南京市妇幼保健院 | Method for detecting protective effect of estrogen receptor on penis vascular endothelial cell |
| CN103820390A (en) * | 2014-03-04 | 2014-05-28 | 李胜 | Method and device for obtaining circulating tumor microembolus |
| CN104471402A (en) * | 2012-04-13 | 2015-03-25 | 鹿特丹伊拉斯谟大学医疗中心 | Biomarkers for triple negative breast cancer |
-
2016
- 2016-01-28 CN CN201610058588.5A patent/CN105606823B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1737526A (en) * | 2005-07-20 | 2006-02-22 | 徐华 | Making method of body fluid cell lump paraffin section |
| CN104471402A (en) * | 2012-04-13 | 2015-03-25 | 鹿特丹伊拉斯谟大学医疗中心 | Biomarkers for triple negative breast cancer |
| CN102980995A (en) * | 2012-12-04 | 2013-03-20 | 南京市妇幼保健院 | Method for detecting protective effect of estrogen receptor on penis vascular endothelial cell |
| CN103820390A (en) * | 2014-03-04 | 2014-05-28 | 李胜 | Method and device for obtaining circulating tumor microembolus |
Non-Patent Citations (2)
| Title |
|---|
| BAHRIYE AKTAS ET AL.: "Comparison of estrogen and progesterone receptor status of circulating tumor cells and the primary tumor in metastatic breast cancer patients", 《GYNECOLOGIC ONCOLOGY》 * |
| MATTHEW G. KREBS ET AL.: "Analysis of Circulating Tumor Cells in Patients with Non-small Cell Lung Cancer Using Epithelial Marker-Dependent and -Independent Approaches", 《JOURNAL OF THORACIC ONCOLOGY》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109239030A (en) * | 2018-08-10 | 2019-01-18 | 北京莱尔生物医药科技有限公司 | A kind of kit and application detecting circulating tumor cell HER2 different loci and ER, PR |
| CN111534586A (en) * | 2020-04-21 | 2020-08-14 | 山东省肿瘤防治研究院（山东省肿瘤医院） | Kit and method for detecting CEA gene mutation of peripheral blood circulating tumor cells of non-small cell lung cancer patients |
| WO2021213257A1 (en) * | 2020-04-21 | 2021-10-28 | 山东第一医科大学(山东省医学科学院) | Detection and identification method for circulating tumor cells (ctcs) |
| CN111534586B (en) * | 2020-04-21 | 2022-05-13 | 山东省肿瘤防治研究院（山东省肿瘤医院） | Kit and method for detecting CEA gene mutation of peripheral blood circulating tumor cells of non-small cell lung cancer patients |
| WO2021213311A1 (en) * | 2020-04-22 | 2021-10-28 | 山东第一医科大学(山东省医学科学院) | Immunofluorescence kit for detecting pd-l1 gene expression of patient with colorectal cancer by means of peripheral blood circulating tumor cells |
| WO2022001826A1 (en) * | 2020-07-01 | 2022-01-06 | 天津市肿瘤医院（天津医科大学肿瘤医院） | Immunofluorescence kit for detecting e-cadherin expression of peripheral blood circulating tumor cells of patient with pancreatic cancer |
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Effective date of registration: 20180425 Address after: 250200 Zhangqiu District, Ji'nan, Shandong, 3 south of the Shandong Academy of pharmacy, 6866 south of ten East Road and 6866 west of Ming Bu road. Patentee after: Shandong discovery Biotechnology Co., Ltd. Address before: 250117 Ji Yankuang Road, Huaiyin District, Ji'nan, Shandong Province, No. 440 Patentee before: Shandong Inst. of Tumor Prevention and Cure |