CN105510600B - The detection method of advanced breast cancer patient Peripheral Circulation tumour cell ER genes - Google Patents
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Abstract
The invention discloses a kind of detection method of advanced breast cancer patient Peripheral Circulation tumour cell ER genes：The CTC obtained in advanced breast cancer patient peripheral blood is separated using film filter；Utilize cellular immunofluorescence technical appraisement advanced breast cancer patient Peripheral Circulation tumour cell；Cut into slices with cell block fabrication techniques thin layer；And then the ER expressions of advanced breast cancer patient Peripheral Circulation tumour cell are detected by immunohistochemistry technology.By means of ISET technology separation and concentration CTCs, by cellular immunofluorescence technical appraisement CTCs, false negative existing for difficult and simple immunology detection technology existing for simple ISET technical appraisement CTCs is overcome.The technical equipment is less demanding, and method is easily grasped, and can monitor in real time.By the technical method, without the i.e. detectable advanced breast cancer patient ER expressions of the breast cancer tissue that draws materials.
Description
Technical field
The present invention relates to a kind of new method of detection ER genes, and advanced breast cancer patient ER is detected especially by peripheral blood
The method of gene.
Background technology
ERs (Estrogen receptor, ER) in treatment guidelines has been included currently as prognostic factor
As most important index when formulating patient with breast cancer's endocrine therapy scheme.The detection of existing expressions of ER needs to pass through hand
The mode such as art or aspiration biopsy obtains the tumor tissues sample of primary tumor or metastatic tumor, and so rather than each patient can obtain
Tumor tissues sample, and can not continuously be detected over the course for the treatment of, limit it and refer to as effective curative effect monitoring
Mark.But the circulating tumor cell into blood that comes off can provide potential real-time tumor specimen to us, its mutation status equally may be used
For guiding treatment.
Circulating tumor cell (Circulating tumor cell, CTC) is to be come off from entity tumor and enter peripheral blood
Liquid circulation tumour cell, it is present in the Several Kinds of Malignancy such as breast cancer, prostate cancer, colorectal cancer, its detect for
Assessing the prognosis of tumor patient especially late tumor patient and the suitable individualized treatment of selection has important clinical meaning
Justice.Because CTC detections have the characteristics that minimally invasive, real-time, it is referred to as " liquid biopsy ".
The main method of CTCs detections at present includes the immunology detection technology and base for relying on tumor related marker analyte detection
In the membrane filtration technique (Isolating by size of epithelial tumor cells, ISET) of morphology enrichment.Before
Person is to be based on cell surface expression difference antigen marker, by specific antibody EpCAM, CK for tumor cell surface antigen etc.
Magnetic bead surfaces are coated in complete the specific recognition of tumour cell, enrichment CTCs is screened by externally-applied magnetic field.The technology it is excellent
Gesture is that specificity is higher, the semi-automatic detection device (CellSearch) of existing commercialization；But tumour specific antigen is expressed
Missing can cause false negative result.The latter is the difference according to cell size, using the method for filtering by larger swollen of volume
Oncocyte screening enrichment.The advantage of the technology is that method is simple, cheap, and the CTCs for separating acquisition active can be used for
Follow-up study；But due to lacking Morphologic Diagnosis goldstandard, the beneficiation technologies specificity is relatively poor.
The content of the invention
In order to overcome conventional recycle tumour cell detection technique and advanced breast cancer patient to be not suitable for tissue specimen materials --
And then the deficiency of patient's ER states can not be assessed, it is thin that patent of the present invention provides a kind of advanced breast cancer patient Peripheral Circulation tumour
The detection method of born of the same parents' ER genes：The CTC obtained in advanced breast cancer patient peripheral blood is separated using film filter；Utilize cell
Immunofluorescence technique identifies advanced breast cancer patient Peripheral Circulation tumour cell；Cut with cell block fabrication techniques thin layer
Piece；And then the ER expressions of advanced breast cancer patient Peripheral Circulation tumour cell are detected by immunohistochemistry technology.
This method comprises the following steps that：
First, separated using film filter and obtain advanced breast cancer patient Peripheral Circulation tumour cell：
1st, advanced breast cancer disease peripheral blood in patients is gathered：Median basilic vein 10ml, it is divided into after 2 pipes write identical numbering,
It is respectively designated as 2 groups of 1 group of experiment and experiment；
2nd, 2 pipe peripheral blood samples are pre-processed respectively：By 10 times of dilutions of peripheral blood sample of collection, diluent ingredient：
1mmol/l EDTA+0.1%BSA, peripheral blood sample is fixed 10 minutes with 4% paraformaldehyde after dilution；
3rd, peripheral blood sample is separated using film filter, is enriched with Peripheral Circulation tumour cell：By the peripheral blood of pretreatment
Sample is added in the blood sample container of film filter, it is relied on gravity natural filtration；
4th, after filtering terminates, filter is removed from film filter, PBS is rinsed 2-3 times, Peripheral Circulation tumour cell quilt
It is trapped on filter membrane.
2nd, cellular immunofluorescence technical appraisement advanced breast cancer patient Peripheral Circulation tumour cell is utilized：
1st, obtained to 1 group of separation of experiment in the filter of Peripheral Circulation tumour cell and add Cytoperm/Cytofix reagents
300 μ l, fixed, perforation 15min；
2nd, PBS is rinsed 3 times, 5 minutes every time, is added the μ l of wash buffer 300 afterwards, is closed 30min；
3rd, the μ l of CK8/18/19, CD45 primary antibody suspension 300 are added, two kinds of primary antibodies are diluted with wash buffer, are mixed
Afterwards final concentration of 1:250,45min is incubated at room temperature；
4th, Wash buffer wash 3 times, 5 minutes every time, add the μ l of secondary antibody suspension 300 afterwards, two kinds of secondary antibodies with
Wash buffer dilute, and final concentration of 1 after mixing:500, at room temperature lucifuge be incubated 30min；
5th, Wash buffer are washed 3 times, 5 minutes every time, add the μ l of Hoechst 300 afterwards, wash dye nucleus 5min；
6th, PBS is rinsed 2 times, 3 minutes every time, is taken out filter membrane afterwards and is placed on slide, and the anti-fluorescent quenching envelopes of 10 μ l are added dropwise
Close fluid-tight piece；
7th, result is observed under fluorescence microscope in 2h, takes pictures, record CTC situations, it is determined whether CTC be present；As observed
CTC then continues to detect in next step, otherwise stops detection.
3rd, with cell block fabrication techniques CTC cell blocks：
1st, the filter of 2 groups of identical blood samples numberings of experiment is removed from filter, opens and to remove filter suitable for reading, will circulate
Tumour cell dyeing liquor is added in filter, dyes 2min, and PBS is rinsed well, and filter membrane is removed with ophthalmology tweezers, is placed
On slide, observed under the microscope after suitably drying, it was demonstrated that CTC be present；
2nd, CTC cell blocks are made；
A, decolourize：Filter membrane on above-mentioned slide is removed, is placed in immersion 4-6 hours in destainer, sloughs CTC dyeing
Liquid, the destainer are：95% alcohol and 100% dimethylbenzene by volume 1:1 mixes；
B, wrap up：Immersion takes out filter membrane after terminating, and is wrapped up with blotting paper；
C, fixation：Wrappage is placed in 10% neutral formalin, fixed 4-6h.
D, waxdip embeds：Fix after terminating and take out wrappage, be placed in after dehydration in FFPE box, waxdip embedding makes thin
Born of the same parents' paraffin mass.
E, thin layer is cut into slices：With slicer by cell paraffin mass serial section, the thin layer that thickness is 2-4 μm is made and cuts into slices.
4th, the ER that advanced breast cancer patient Peripheral Circulation tumour cell is detected by immunohistochemistry technology expresses feelings
Condition：
1. roasting piece：The section of CTC wax stones thin layer bakes piece 2 hours in 60 DEG C of insulating boxs；
2. dewaxing：Cut into slices and placed respectively in two bottles of dimethylbenzene 5 minutes；
3. aquation：Section is placed 1 minute (depending on Indoor Temperature respectively in absolute ethyl alcohol, 95% ethanol, 85% ethanol successively
Degree situation slightly adjusts)：Running water, distilled water flushing；
4. antigen retrieval：Reparation liquid EDTA in pressure cooker is boiled, section is put into reparation liquid is submerged completely and covers height
Pot cover and pressure valve are pressed, is heated to after pressure cooker jet 1.5 minutes, from fire, pressure cooker is slightly cooled down to opening pot cover, repairing of cutting into slices
Natural cooling in multiple liquid；
5. distilled water flushing；
6. remove endogenous peroxydase：With 3%H2O2 effects section 10 minutes；
7. distilled water flushing；
8.PBS embathe 5 times it is each 1.5 minutes；
9. ER primary antibodies are added dropwise, 4 DEG C of refrigerator overnights, then put it into PBS and embathe 5 times, embathe every time 1.5 minutes；
10. secondary antibody is added dropwise；
It is incubated 20 minutes in 11.37 DEG C of water-baths；
12.PBS embathe 5 times it is each 1.5 minutes；
13. controlled under microscope, DAB colour developings；
14. running water rinses, haematine dye liquor is redyed 2 minutes, and hydrochloride alcohol differentiation, ammoniacal liquor returns indigo plant；
15. running water rinses 5 minutes；
16.75%-95%-100% gradient alcohol dehydrations, dimethylbenzene is transparent, neutral Instant cement mounting；
17. cell pathology expert's diagosis, interpretation ER expressions.
Utilize the film filter described in cellular immunofluorescence technical appraisement Peripheral Circulation tumour cell step：Including
Filter, blood sample container, waste liquid cylinder and iron stand, the iron stand are provided with base, stand and support, and the blood sample container passes through branch
Erection is placed in iron stand top, and the lower section of blood sample container is filter, and filter is set by transfusion device UNICOM to waste liquid cylinder, waste liquid cylinder
In on base.
The filter includes filter suitable for reading, filter membrane, carries mouth under filter membrane platform and filter, and filter membrane, which is placed in, to be carried on filter membrane platform；
Filter is suitable for reading to connect blood sample container, and mouth connects waste liquid cylinder by transfusion device under filter.
The filter membrane is made up of hydrophobic material, is uniformly covered with the filter opening that bore is 10 microns thereon.
Beneficial effect：By means of ISET technology separation and concentration CTCs, by cellular immunofluorescence technical appraisement CTCs, not only
Difficulty existing for simple ISET technical appraisement CTCs can be overcome, it is thin also to capture the tumour that epithelium antigen expression weakens or lacked
Born of the same parents, that is, overcome false negative existing for simple immunology detection technology.Meanwhile the technical equipment is less demanding, method is easy to grasp,
And can monitor in real time, have clinical application may.It is i.e. detectable without the breast cancer tissue that draws materials by the technical method
Advanced breast cancer patient ER expressions.The technology belongs to minimally invasive or even noninvasive, and can detect in real time.
Brief description of the drawings
Fig. 1 is the film filter structural representation of the present invention；
Fig. 2 is the structural representation sectional view of the filter of inventive film filter；
Fig. 3 is the structural representation of the filter filter membrane of inventive film filter；
Fig. 4 is the circulating tumor cell striograph that the separation of advanced breast cancer cancer peripheral blood in patients obtains；
Fig. 5 is advanced breast cancer patient Peripheral Circulation tumour cell SABC ER dye images.
1. iron stand, 2. blood sample container, 3. filter, 4. transfusion device, 5. waste liquid cylinder, 6. filters, 7. filter membrane 8. suitable for reading in figure
Carry mouth, 10. filter openings, 11. bases, 12. stands, 13 supports under filter membrane platform, 9. filters.
Embodiment
The present invention is further described with reference to the accompanying drawings and examples.
With this technical method separate obtain and identify 8 advanced breast cancer cancer patients (while detect 8 normal person's samples
Do negative control) Peripheral Circulation tumour cell, detects its ER expression.
First, obtained using film filter separation in the advanced breast cancer patient peripheral blood that can not obtain tissue specimen
CTC：
Advanced breast cancer patient peripheral blood 10ml is gathered, is divided into 2 pipes, same blood sample of patient writes identical numbering, respectively
It is named as 2 groups of 1 group of experiment and experiment；Then 2 pipe peripheral blood samples are pre-processed respectively：10 times of the peripheral blood sample of collection is dilute
Release, diluent ingredient：1mmol/l EDTA+0.1%BSA), fix peripheral blood sample 10 minutes with 4% paraformaldehyde after dilution；
Phase between fixed, assembling film are separated by filtration tumour cell (ISET) easy device (as shown in Figure 1, 2, 3)：The filter is by filtering
Device 3, filter membrane 7, blood sample container 2, waste liquid cylinder 5, iron stand 1 are formed, and are separated peripheral blood sample using film filter, are enriched with peripheral blood
Circulating tumor cell：Filter 3 is soaked with 10mlPBS, then the peripheral blood sample of pretreatment is added to the blood sample of film filter
In container 2, it is set to rely on gravity natural filtration；After filtering terminates, filter 3 is removed from film filter, PBS is rinsed 2-3 times,
Peripheral blood CTC is trapped within filter membrane 7.
Tumor cell diameter is generally higher than 15 microns, and haemocyte (including red blood cell, leucocyte) diameter is generally less than 10
Micron, therefore work as the peripheral blood containing CTC after filtering, haemocyte can be filtered across because diameter is less than filter opening 10, and CTC is because straight
Footpath is trapped within filter membrane 7 more than filter opening 10.
2nd, cellular immunofluorescence technical appraisement advanced breast cancer patient Peripheral Circulation tumour cell is utilized：
(1 group of experiment) adds Cytoperm/Cytofix examinations in the filter 3 for obtaining Peripheral Circulation tumour cell to separation
The μ l of agent 300, fixed, perforation 15min；PBS is rinsed 3 times, 5 minutes every time, adds the μ l of wash buffer 300, closing afterwards
30min；The μ l of CK8/18/19, CD45 primary antibody suspension 300 are added, two kinds of primary antibodies are diluted with wash buffer, after mixing
Final concentration of 1:250,45min is incubated at room temperature；Wash buffer are washed 3 times, 5 minutes every time, add secondary antibody suspension afterwards
300 μ l, two kinds of secondary antibodies are diluted with wash buffer, and final concentration of 1 after mixing:500, at room temperature lucifuge be incubated 30min；
Wash buffer are washed 3 times, 5 minutes every time, add the μ l of Hoechst 300 afterwards, wash dye nucleus 5min；PBS is rinsed 2 times,
3 minutes every time, filter membrane 7 was taken out afterwards and is placed on slide, the anti-fluorescent quenching confining liquid mountings of 10 μ l are added dropwise；Under fluorescence microscope
Observation result in 2h, take pictures, record CTC situations (application experiment group 2 carries out next step detection if experimental group 1 observes CTC).
Testing result：8 healthy volunteers do not find circulating tumor cell；In 8 advanced breast cancer patient peripheral bloods
In with the presence of 6 detect CTC.
3rd, cut into slices with cell block fabrication techniques thin layer：
The filter 3 that CTC numberings are detected in 2 groups of experiment is removed from filter, opens and removes filter suitable for reading 6, will
Circulating tumor cell dyeing liquor is added in filter 3, dyes 2min, and PBS is rinsed well, and filter membrane is removed with ophthalmology tweezers
7, it is placed on slide, is observed under the microscope after suitably drying, it was demonstrated that CTC be present；
Filter membrane 7 on above-mentioned slide is removed, immersion 4-6 hours in destainer is placed in, sloughs CTC dyeing liquors, it is described
Destainer is：95% alcohol and 100% dimethylbenzene by volume 1:1 mixes；Immersion takes out filter membrane 7 after terminating, with water suction paper bag
Wrap up in；Wrappage is placed in 10% neutral formalin, fixed 4-6h；Fix after terminating and take out wrappage, stone is placed in after dehydration
In wax embedded box, waxdip embedding makes cell paraffin mass；With slicer by cell paraffin mass serial section, it is 2-4 μ that thickness, which is made,
M thin layer section.
4th, the ER that advanced breast cancer patient Peripheral Circulation tumour cell is detected by immunohistochemistry technology expresses feelings
Condition：
The paraffin section application row immunohistochemical method of acquisition is detected to the expression of ER genes, will be cut into slices at 60 DEG C
Piece is baked in insulating box 2 hours, cut into slices and placed respectively in two bottles of dimethylbenzene 5 minutes, will cut into slices successively in absolute ethyl alcohol, 95% second
Placed respectively in alcohol, 85% ethanol 1 minute (slightly being adjusted depending on indoor temperature situation)：Running water, distilled water flushing；
Antigen retrieval：According to first antibody specification and specific experiment operating experience, different sides is used to experimental slides
Method carries out antigen retrieval, and this experiment uses EDTA (PH=9.0) hot high pressure reparation：By the reparation liquid in pressure cooker on electromagnetic oven
(EDTA) boil, be put into section to reparation liquid is submerged completely and cover high-pressure pot cover and pressure valve, 800W is heated to pressure cooker jet
1.5 minutes afterwards, from fire, pressure cooker is slightly cooled down to opening pot cover, the natural cooling in liquid is repaired of cutting into slices, then rushed with distilled water
Wash, endogenous peroxydase is removed after flushing：(use the detecting system of peroxidase, it is necessary to carry out endogenous peroxidating
Thing enzyme Seal treatment, if without handling, red blood cell, granulocyte in tissue can disturb the judgement of coloration result) this experiment
With 3%H2O2 effects section 10 minutes, coloration result collection antigen, which is preserved, all not to be influenceed, and sealing effect is than more significant, distillation
Water rinses, PBS embathe 5 times it is each 1.5 minutes, ER primary antibodies are added dropwise, 4 DEG C of refrigerator overnights, then puts it into PBS and embathes 5 times, often
It is secondary to embathe 1.5 minutes；Secondary antibody is added dropwise, is incubated 20 minutes in 37 DEG C of water-baths, PBS embathe 5 times it is each 1.5 minutes, controlled under microscope
System, DAB colour developings, running water are rinsed, and haematine dye liquor is redyed 2 minutes, and hydrochloride alcohol differentiation, ammoniacal liquor returns indigo plant, and running water rinses 5 points
Clock, 75%-95%-100% gradient alcohol dehydrations, dimethylbenzene is transparent, neutral Instant cement mounting, 2-3 name cell pathology experts
Diagosis, interpretation ER expressions.
Testing result：8 healthy volunteers do not find circulating tumor cell；In 8 advanced breast cancer patient peripheral bloods
The CTC detected, wherein 6 detect that it has ER expression through SABC, positive rate is 75.0% (table 1).
Fig. 4 is the circulating tumor cell striograph that Peripheral Blood In Patients With Breast Cancer separation obtains, and its nucleus is larger, nucleus
It is in irregular shape；High nucleocytoplasmic ratio.
Fig. 5 is Peripheral Blood In Patients With Breast Cancer circulating tumor cell ER immunohistochemistries, and cell membrane and cytoplasm are yellow
Dye.Positive degree is judged according to its dye distribution and intensity, it is (-) that cell color, which accounts for cell less than 10%, colours 10%-25%
For (+), coloring 25%-50% is (++), and coloring is (+++) more than 50%.
The embodiment testing result (ER) of table 1
Claims (4)
1. a kind of detection method of the non-diagnostic purpose of advanced breast cancer patient Peripheral Circulation tumour cell ER genes, its feature exist
In, utilize film filter separation obtain advanced breast cancer patient peripheral blood in CTC；Identify advanced breast cancer patient peripheral blood
Circulating tumor cell；Cut into slices with cell block fabrication techniques thin layer；And then detect advanced breast cancer patient Peripheral Circulation and swell
The ER expressions of oncocyte；
Described obtains advanced breast cancer patient Peripheral Circulation tumour cell using film filter separation, and step is as follows：
1) advanced breast cancer disease peripheral blood in patients is gathered：Median basilic vein 5ml；
2) peripheral blood sample is pre-processed：By 10 times of dilutions of peripheral blood sample of collection, diluent ingredient：1mmol/l EDTA+
0.1%BSA, peripheral blood sample is fixed 10 minutes with 4% paraformaldehyde after dilution；
3) using film filter separation peripheral blood sample, it is enriched with Peripheral Circulation tumour cell：The peripheral blood sample of pretreatment is added
Enter into the blood sample container (2) of film filter, it is relied on gravity natural filtration；
4) after filtering terminates, filter (3) is removed from film filter, PBS is rinsed 2-3 times, Peripheral Circulation tumour cell quilt
It is trapped on filter membrane (7)；
Described identification advanced breast cancer patient Peripheral Circulation tumour cell, step are as follows：
1) obtained to separation and the μ l of Cytoperm/Cytofix reagents 300 are added in the filter (3) of Peripheral Circulation tumour cell, Gu
Fixed, perforation 15min；
2) PBS is rinsed 3 times, 5 minutes every time, is added the μ l of wash buffer 300 afterwards, is closed 30min；
3) the μ l of CK8/18/19, CD45 primary antibody suspension 300 are added, three kinds of primary antibodies are diluted with wash buffer, after mixing
Final concentration of 1:250,45min is incubated at room temperature；
4) Wash buffer are washed 3 times, 5 minutes every time, add the μ l of secondary antibody suspension 300 afterwards, three kinds of secondary antibodies are with wash
Buffer dilutes, and final concentration of 1 after mixing:500, at room temperature lucifuge be incubated 30min；
5) Wash buffer are washed 3 times, 5 minutes every time, add the μ l of Hoechst 300 afterwards, wash dye nucleus 5min；
6) PBS is rinsed 2 times, 3 minutes every time, is taken out filter membrane (7) afterwards and is placed on slide, and the anti-fluorescent quenching closings of 10 μ l are added dropwise
Fluid-tight piece；
7) result is observed under fluorescence microscope in 2h, takes pictures, record CTC situations, it is determined whether CTC be present；
Described is as follows with the section of cell block fabrication techniques thin layer, step：
1) after peripheral blood filtering, filter (3) is removed from filter, opens and to remove filter (6) suitable for reading, circulating tumor is thin
Born of the same parents' dyeing liquor is added in filter (3), dyes 2min, and PBS is rinsed well, and filter membrane (7) is removed with ophthalmology tweezers, is placed
On slide, observed under the microscope after suitably drying, it was demonstrated that CTC be present；
2) thin layer section is made：
A, decolourize：The above-mentioned filter membrane (7) with CTC is removed from slide, is placed in 95% alcohol and 100% dimethylbenzene by body
Product ratio 1:4-6 hours are soaked in 1 destainer mixed, slough CTC dyeing liquors；
B, wrap up：Immersion takes out filter membrane (7) after terminating, and is wrapped up with blotting paper；
C, fixation：Wrappage is placed in 10% neutral formalin, fixed 4-6h；
D, waxdip embeds：Fix after terminating and take out wrappage, be placed in after dehydration in FFPE box, waxdip embedding makes cell stone
Wax stone；
E, thin layer is cut into slices：With slicer by cell paraffin mass serial section, the thin layer that thickness is 2-4 μm is made and cuts into slices；
The ER expressions of described detection advanced breast cancer patient Peripheral Circulation tumour cell, step are as follows：
1) piece is baked：The section of CTC wax stones thin layer bakes piece 2 hours in 60 DEG C of insulating boxs；
2) dewax：Cut into slices and placed respectively in two bottles of dimethylbenzene 5 minutes；
3) aquation：Section is placed 1 minute respectively in absolute ethyl alcohol, 95% ethanol, 85% ethanol successively, running water, distillation
Water rinses；
4) antigen retrieval：Reparation liquid EDTA in pressure cooker is boiled, section is put into reparation liquid is submerged completely and covers pressure cooker
Lid and pressure valve, are heated to after pressure cooker jet 1.5 minutes, from fire, pressure cooker are slightly cooled down to opening pot cover, cut into slices and are repairing liquid
Middle natural cooling；
5) distilled water flushing；
6) endogenous peroxydase is removed：With 3%H2O2 effects section 10 minutes；
7) distilled water flushing；
8) PBS embathe 5 times it is each 1.5 minutes；
9) ER primary antibodies are added dropwise, 4 DEG C of refrigerator overnights, then puts it into PBS and embathes 5 times, embathe every time 1.5 minutes；
10) secondary antibody is added dropwise；
11) it is incubated 20 minutes in 37 DEG C of water-baths；
12) PBS embathe 5 times it is each 1.5 minutes；
13) controlled under microscope, DAB colour developings；
14) running water is rinsed, and haematine dye liquor is redyed 2 minutes, and hydrochloride alcohol differentiation, ammoniacal liquor returns indigo plant；
15) running water rinses 5 minutes；
16) 75%-95%-100% gradient alcohol dehydrations, dimethylbenzene is transparent, neutral Instant cement mounting；
17) cell pathology expert diagosis, interpretation ER expressions.
2. the inspection of the non-diagnostic purpose of advanced breast cancer patient Peripheral Circulation tumour cell ER genes according to claim 1
Survey method, it is characterised in that described film filter includes filter (3), blood sample container (2), waste liquid cylinder (5) and iron stand
(1), the iron stand (1) is provided with base (11), stand (12) and support (13), and the blood sample container (2) passes through support (13)
Iron stand (1) top is arranged at, the lower section of blood sample container (2) is filter (3), and filter (3) passes through transfusion device (4) UNICOM to waste liquid
Cylinder (5), waste liquid cylinder (5) are arranged on base (11).
3. the inspection of the non-diagnostic purpose of advanced breast cancer patient Peripheral Circulation tumour cell ER genes according to claim 2
Survey method, it is characterised in that：The filter (3) includes filter (6) suitable for reading, filter membrane (7), carries mouth under filter membrane platform (8) and filter
(9), filter membrane (7), which is placed in, carries on filter membrane platform 8；Filter (6) suitable for reading connects blood sample container (2), and mouth (9) passes through transfusion device under filter
(4) waste liquid cylinder (5) is connect.
4. the inspection of the non-diagnostic purpose of advanced breast cancer patient Peripheral Circulation tumour cell ER genes according to claim 3
Survey method, it is characterised in that：The filter membrane (7) is made up of hydrophobic material, is uniformly covered with the filter opening that bore is 10 microns thereon
(10)。
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|---|---|---|---|---|
| CN105954246B (en) * | 2016-04-29 | 2020-10-30 | 上海乾翼生物科技有限公司 | Method and kit for detecting free rare tumor cells in human biological fluid sample |
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