CN106124266A - A kind of liquid-based cell sample manufacturing method - Google Patents

A kind of liquid-based cell sample manufacturing method Download PDF

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Publication number
CN106124266A
CN106124266A CN201610458798.3A CN201610458798A CN106124266A CN 106124266 A CN106124266 A CN 106124266A CN 201610458798 A CN201610458798 A CN 201610458798A CN 106124266 A CN106124266 A CN 106124266A
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Prior art keywords
filter membrane
liquid
preparation bin
cell
chamber
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Granted
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CN201610458798.3A
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CN106124266B (en
Inventor
毛立新
陈志俊
王云皓
项伟平
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Hangzhou Healthsky Biotechnology Co Ltd
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Hangzhou Healthsky Biotechnology Co Ltd
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Priority to CN201610458798.3A priority Critical patent/CN106124266B/en
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Abstract

The invention discloses a kind of liquid-based cell sample manufacturing method, it comprises the steps: step 1, take cell smearing device, and this cell smearing device include pallet, the preparation bin being fixed on pallet, be positioned at preparation bin lower surface and the microscope slide of closure preparation bin lower end, removable be located at preparation bin and along the circumferentially disposed filter membrane of preparation bin, be placed in preparation bin and be positioned at the filter chamber above filter membrane and be placed in preparation bin and be positioned at the cleaning chamber below filter membrane;Adding cleanout fluid in preparation bin, cleanout fluid fully enters cleaning chamber and its liquid level flushes with filter membrane.Step 2, sampling originally adds in filter chamber with the mixture preserving liquid, and mixture enters through filter membrane and cleans intracavity, bottom cell settlement extremely cleaning chamber;Step 3, removes the impurity on filter membrane and filter membrane, and natural subsidence removes supernatant;Add cleanout fluid, centrifugal, abandon supernatant;Step 4, extracts microscope slide, fixing, dyeing, mounting, has the effect reducing loss cell.

Description

A kind of liquid-based cell sample manufacturing method
Technical field
The present invention relates to cell detection field, particularly to a kind of liquid-based cell sample manufacturing method.
Background technology
Liquid-based cytology, is to use liquid-based thin-layer cell detection system detection cervix cells and carry out cytology specification Diagnosis.Compared with traditional cervical scraping smear Pap smear inspection, liquid-based cytology significantly improve sample satisfaction and Abnormal cervical cell recall rate, liquid-based cytology is that the most more advanced a kind of cervical cancer cell checks skill Art.
Application publication number is CN102680290A, Shen Qing Publication day is that in JIUYUE, 2012 Chinese patent of 19 days discloses one Planting the flaking method automatically making liquid basal cell slide in production facility, this flaking method includes: a, separation cell, uses cell Cell sap is extracted and discharges by destroyer repeatedly, and pockets of for adhesion cell is broken up separation;B, filtration sedimentation, carry cell Take liquid to be centrifuged, settle after making cell filtration;C, extraction Cell sap, the Cell sap after settling extracts, and instills in preparation bin Pathology slide on;D, centrifugal film-making, recentrifuge, make the cell in preparation bin be deposited on pathology slide, form cellular layer, It is liquid basal cell slide.The principle of this flaking method is to utilize various separation means to be isolated by the Cell sap in sample Come, and this Cell sap is dropped on the pathology slide in preparation bin, by making cell be deposited to disease preparation bin centrifugal treating On reason slide, obtain liquid basal cell slide.
It is in place of the deficiencies in the prior art, in the process utilizing various separation means that the Cell sap in sample is separated In, inevitably there is the situation that Cell sap repeatedly shifts in it, and there is the phenomenon of loss cell in this transfer process, and Loss cell amount by the impact such as technical merit of operator, this may by cause final be deposited on pathology slide thin Born of the same parents lack representativeness, it is also possible to affect repeatability and check result.
Summary of the invention
It is an object of the invention to provide a kind of liquid-based cell sample manufacturing method, which solve asking of loss cell in separation process Topic, has the effect reducing loss cell.
The above-mentioned technical purpose of the present invention has the technical scheme that
A kind of liquid-based cell sample manufacturing method, comprises the steps:
Step 1, takes cell smearing device, and this cell smearing device includes pallet, the preparation bin being fixed on pallet, is positioned at preparation bin Lower surface and the microscope slide of closure preparation bin lower end, removable be located in preparation bin and along the circumferentially disposed filter membrane of preparation bin, put In preparation bin and be positioned at filter chamber above filter membrane and be placed in preparation bin and be positioned at the cleaning chamber below filter membrane;In system Adding cleanout fluid in sheet storehouse, cleanout fluid fully enters cleaning intracavity;
Step 2, samples this and preserves the mixture of liquid, being added in the filter chamber of the cell smearing device after step 1 processes, The mixture of sample and preservation liquid enters through filter membrane and cleans intracavity, and the cell settlement in sample is to cleaning bottom chamber;
Step 3, removes the filter membrane after step 2 processes and the impurity on filter membrane, removes the supernatant after natural subsidence;
Step 4, the cell smearing device that step 3 of learning from else's experience processes, extracts microscope slide thereon, fixing, dyeing, mounting and get final product.
The flaking method of prior art utilizes various separation means to separate cell and impurity, during this, Cell sap There is the situation repeatedly shifting and being centrifuged, the situation that Cell sap repeatedly shifts and is centrifuged has the disadvantage that
1, in Cell sap repeatedly transfer process, the cell of its loss is the most, and this loss amount is grasped to a certain extent Making the technical merit impact of personnel, this, by causing the cell being finally deposited on pathology slide to lack representativeness, also affects reproduction Property check result;
2, in general, Cell sap transfer needs by external tool (such as liquid-transfering gun etc.), is utilizing external tool (such as liquid-transfering gun Deng) during transfer Cell sap, it inevitably exists draws and the step of tapping;Drawing and in tapping step, Cell may be repeatedly by coming from the interior rifle of external tool (such as liquid-transfering gun etc.) under external tool (such as liquid-transfering gun etc.) acts on The extruding of wall, impulse force impact when coming from absorption and tapping, so, this situation will cause cell to be destroyed, and impact is made Sheet effect and Checking on effect;
3, the centrifugally operated of Cell sap is loaded down with trivial details.
To sum up, flaking method of the prior art will affect the reliability of liquid-based cytology result;
The application is by being directly provided with filter membrane on cell smearing device so that sample and preservation liquid can directly be entered by filter membrane Row filters, and filter membrane can not be passed through more than the impurity in filter membrane aperture in aperture, and wherein the aperture of filter membrane can be according to actual need Ask and select;Little granule foreign, cell and preservation liquid are entered by filter membrane and clean intracavity, and little granule foreign and cell are clearly Under washing liquid effect, sedimentation velocity is different, general cell settlement speed faster, so (selecting cell several in standing sedimentation a period of time Sedimentation and time point that little granule foreign major part does not settles completely, current real work finds under normal temperature and pressure with 0.5 ~ 1hr is preferred) after, remove the impurity on filter membrane and filter membrane, remove the supernatant after natural subsidence and i.e. can get cell with few The system of amount impurity;After the most cleaned liquid of this system separates, can further cell be purified;Purify in this process Process is carried out on same cell smearing device, and it does not exist the situation repeatedly shifting cell, thin by the flaking method of the application Born of the same parents' loss amount is few and the method is affected by the technical merit of operator and almost negligible disregards, and improves and is finally deposited to pathology glass The representativeness of the cell on sheet, also improves repeatability and checks result;By removable for filter membrane be located at preparation bin in and along preparation bin week To arranging and being fixed on sleeve bottom, it is simple to change, and it is easy to select during the course to install or remove, the most flexibly, with film-making Process compatible;It addition, the flaking method of the application by action of gravity reach filter separate with natural subsidence, during cell several It is not affected by coming from the extruding of interior rifle wall of external tool (such as liquid-transfering gun etc.), impulse force impact etc., cell damage can be reduced, carry The reliability of high liquid-based cytology result.
More preferably: in described preparation bin, be provided with sleeve, the upper end of described sleeve be provided with for preparation bin The collar flange that end face abuts;Described filter membrane is fixed on the lower end of sleeve.
Use technique scheme, realize the releasable connection between filter membrane and preparation bin by sleeve, easy disassembly, And sleeve can adapt with sample introduction equipment, in order to realize automation mechanized operation.
More preferably: between described sleeve outer wall and preparation bin inwall, leave gap.
Use technique scheme, first, after entering cleaning chamber by the mixture of the sample of filter membrane and preservation liquid When its liquid level is higher than filter membrane, owing to filter membrane has certain blocking, then clean the cell of intracavity and between solution preferentially enters Gap (for comparing filter chamber), can reduce cell as far as possible and again be back to the probability of filter chamber, increase the cell concentration gathered;Its Secondary, when inserting sleeve, without friction between sleeve and centrifuge tube, convenient, and sleeve outer wall will not be caused because of friction and be centrifuged There is material chips to enter cleaning intracavity between inside pipe wall and affect Checking on effect.
More preferably: under normal temperature and pressure, the density of described cleanout fluid is 0.9 ~ 1.2g/cm3
Use technique scheme, be conducive to holding an optimum between cell settlement and sedimentation rate of impurities.
More preferably: in step 1, after cleanout fluid fully enters cleaning intracavity, its liquid level flushes with filter membrane.
Using technique scheme, in filter process, aperture is less than the impurity of cleanout fluid less than filter membrane 7 aperture but density Float over upper surface and be predominantly located in filter chamber, separation purity can be improved.
In sum, the method have the advantages that
The mixture of sample and preservation liquid enters through filter membrane and cleans intracavity, during, aperture is more than the impurity in filter membrane aperture Can not pass through filter membrane, aperture floats over upper surface and big less than filter membrane aperture but density less than the impurity of the cleanout fluid of embodiment Part is positioned at filter chamber, and the cell in sample is settled down to clean part bottom chamber under the effect of cleanout fluid and passes through filter membrane When failing to timely enter bottom directly next step (the standing sedimentation time is 0.5 ~ 1hr).
Accompanying drawing explanation
Fig. 1 is the cross-sectional view of embodiment 10;
Fig. 2 is the cross-sectional view of embodiment 11.
In figure, 1, pallet;2, preparation bin;3, microscope slide;4, sealing ring;5, sleeve;6, collar flange;7, filter membrane;8、 Filter chamber;9, chamber is cleaned;10, gap.
Detailed description of the invention
Below in conjunction with accompanying drawing, the present invention is described in further detail.
This specific embodiment is only explanation of the invention, and it is not limitation of the present invention, people in the art The present embodiment can be made after reading this specification by member as required does not has the amendment of creative contribution, but as long as at this All protected by Patent Law in the right of invention.
As indicated without special, normal temperature and pressure described herein, refer to 20 ~ 30 DEG C/81 ~ 122kPa.
Embodiment 1: cleanout fluid, it prepares by the following method:
Take 3g NaCl, 5g glycerol, 3g dehydrated alcohol and 89g distilled water, mixing, stir, filter, take filtrate and get final product.Room temperature Under normal pressure, the density of this cleanout fluid is 1.1 ~ 1.2g/cm3
Embodiment 2: cleanout fluid, is with the difference of embodiment 1, and its formula is: 4g NaCl, 8g glycerol, 4g are anhydrous Ethanol and 84g distilled water.
Embodiment 3: cleanout fluid, is with the difference of embodiment 1, and its formula is: 5g NaCl, 10g glycerol, 5g without Water-ethanol and 80g distilled water.
Embodiment 4: preserve liquid, it prepares by the following method:
Step A: take 1g KCl, 1g KH2PO4、0.1g Na2HPO4With 77.79g distilled water, mixing, stir;
Step B: take 5g ethylene glycol and 15g isopropanol, mixing, stir;
Step C: ethylene glycol/isopropanol that addition 0.01g EDTA-2Na and step B obtain in the mixed solution that step A obtains Mixture, mixing, stir;
Step D: add 0.1g formaldehyde, mixing in the mixed solution that step C obtains, stir, to obtain final product.
Embodiment 5: preserving liquid, be with the difference of embodiment 4, its formula is: 2g KCl, 2g KH2PO4、0.5g Na2HPO4, 68.95g water, 8g ethylene glycol, 18g isopropanol, 0.05g EDTA-2Na and 0.5g formaldehyde.
Embodiment 6: preserving liquid, be with the difference of embodiment 4, its formula is: 3g KCl, 3g KH2PO4、1g Na2HPO4, 61.9g water, 10g ethylene glycol, 20g isopropanol, 0.1g EDTA-2Na and 1g formaldehyde.
Embodiment 7: sample collection (i.e. the preparation of the mixture of sample and preservation liquid), comprises the steps:
Step i: fetching has the specimen cup preserving liquid of 10ml embodiment 4 to take out, bowl cover of outwarding winding, preparation for acquiring cell sample;
Step ii: using each position of sampling brush swipe cervix uteri to carry out cell sample collection, sample takes out the guarantor i.e. washing step i Liquid storage preserves whole cell sample, to obtain final product.
Embodiment 8: sample collection (i.e. the preparation of the mixture of sample and preservation liquid), exists with the difference of embodiment 7 In, it uses the preservation liquid of embodiment 5.
Embodiment 9: sample collection (i.e. the preparation of the mixture of sample and preservation liquid), exists with the difference of embodiment 7 In, it uses the preservation liquid of embodiment 6.
Embodiment 10: cell smearing device, as it is shown in figure 1, this cell smearing device includes pallet 1, is fixed on above pallet 1 Preparation bin 2, be positioned at preparation bin 2 lower surface and closure preparation bin 2 lower end microscope slide 3, microscope slide 3 is plugged in the lower end of pallet 1 In face.It is provided with sealing ring 4 at corresponding microscope slide 3 on preparation bin 2.
With reference to Fig. 1, being provided with sleeve 5 in preparation bin 2, the upper end of sleeve 5 is provided with for abutting with the upper surface of preparation bin 2 Collar flange 6.The lower end of sleeve 5 is fixed with filter membrane 7, and the aperture of filter membrane 7 can select according to demand, filter membrane 7 edge Preparation bin 2 circumferentially disposed.
With reference to Fig. 1, the space in preparation bin 2 is separated into and is positioned at cleaning chamber 9 below filter membrane 7, is placed in set by filter membrane 7 In the cylinder 5 and filter chamber 8 above filter membrane 7, the gap 10 between the outer wall and the inwall of preparation bin 2 of sleeve 5.
Embodiment 11: cell smearing device, is with the difference of embodiment 10, as in figure 2 it is shown, the outer wall of sleeve 5 and Abut between the inwall of centrifuge tube 2, i.e. there is not gap 10(between outer wall and the inwall of centrifuge tube 2 of sleeve 5 with reference to Fig. 1).
Embodiment 12: a kind of liquid-based cell sample manufacturing method, comprises the steps:
Step 1, the cell smearing device of Example 10, in preparation bin 2, add the cleanout fluid of embodiment 1, cleanout fluid all enters Entering to clean in chamber 9, its liquid level highly flushes with filter membrane 7;
Step 2, the sample that Example 4 prepares and the mixture of preservation liquid, added to the cell after step 1 processes In the filter chamber 8 of smear machine, the mixture of sample and preservation liquid enters through filter membrane 7 and cleans in chamber 9, during, aperture is more than The impurity in filter membrane 7 aperture can not pass through filter membrane 7, and aperture is less than the cleanout fluid of embodiment less than filter membrane 7 aperture but density Impurity float over upper surface and be predominantly located in filter chamber 8, the cell in sample be settled down under the effect of cleanout fluid clean Bottom chamber 9 part by filter membrane 7 when failing to timely enter bottom directly next step (the standing sedimentation time is 0.5 ~ 1hr);
Step 3, removes the filter membrane 7 after step 2 processes and the impurity on filter membrane 7 (the most directly removes sleeve 5 and is fixed on Filter membrane 7 on sleeve 5), remove the supernatant after natural subsidence;
Step 4, the cell smearing device that step 3 of learning from else's experience processes, extract microscope slide 3 thereon, fixing, dyeing, mounting, met The sample of liquid-based cytology, and identical to 10 its results of the method parallel test.
Embodiment 13: be with the difference of embodiment 12, its cleanout fluid using embodiment 2 and the system of embodiment 5 The standby sample obtained and the mixture of preservation liquid.Obtain meeting the sample of liquid-based cytology, and to the method parallel test 10 times its result is identical.
Embodiment 14: be with the difference of embodiment 12, its cleanout fluid using embodiment 3 and the system of embodiment 6 The standby sample obtained and the mixture of preservation liquid.Obtain meeting the sample of liquid-based cytology, and to the method parallel test 10 times its result is identical.
Embodiment 15: be with the difference of embodiment 12, its cleanout fluid uses Beijing Suo Laibao Science and Technology Ltd. The Precoll cell separation liquid provided.Obtain meeting the sample of liquid-based cytology, and to the method parallel test 10 times its Result is identical.
Embodiment 16: be with the difference of embodiment 12, its preservation liquid uses Wuhan cause sincerity to reach scientific and technological development to be had The liquid based cytology that limit company provides preserves liquid.Obtain meeting the sample of liquid-based cytology, and to the method parallel test 10 times its result is identical.
Embodiment 17: a kind of liquid-based cell sample manufacturing method, comprises the steps:
Step 1, the cell smearing device of Example 11, in preparation bin 2, add the cleanout fluid of embodiment 1, cleanout fluid all enters Entering to clean in chamber 9, its liquid level flushes with filter membrane 7;
Step 2, the sample that Example 4 prepares and the mixture of preservation liquid, added to the cell after step 1 processes In the filter chamber 8 of smear machine, the mixture of sample and preservation liquid enters through filter membrane 7 and cleans in chamber 9, during, aperture is more than The impurity in filter membrane 7 aperture can not pass through filter membrane 7, and aperture is less than the cleanout fluid of embodiment less than filter membrane 7 aperture but density Impurity float over upper surface and be predominantly located in filter chamber 8, the cell in sample be settled down under the effect of cleanout fluid clean Bottom chamber 9 part by filter membrane 7 when failing to timely enter bottom directly next step (the standing sedimentation time is 0.5 ~ 1hr);
Step 3, removes the filter membrane 7 after step 2 processes and the impurity on filter membrane 7 (the most directly removes sleeve 5 and is fixed on Filter membrane 7 on sleeve 5), remove the supernatant after natural subsidence;
Step 4, the cell smearing device that step 3 of learning from else's experience processes, extract microscope slide 3 thereon, fixing, dyeing, mounting, met The sample of liquid-based cytology, and identical to 10 its results of the method parallel test.

Claims (5)

1. a liquid-based cell sample manufacturing method, it is characterised in that comprise the steps:
Step 1, takes cell smearing device, and this cell smearing device includes pallet (1), the preparation bin (2) that is fixed on pallet (1), It is positioned at preparation bin (2) lower surface and the microscope slide (3) of closure preparation bin (2) lower end, removable is located at preparation bin (2) and along film-making The circumferentially disposed filter membrane (7) in storehouse (2), be placed in preparation bin (2) in and be positioned at the filter chamber (8) of filter membrane (7) top and be placed in Preparation bin (2) is interior and is positioned at the cleaning chamber (9) below filter membrane (7);Adding cleanout fluid in preparation bin (2), cleanout fluid is whole Enter and clean in chamber (9);
Step 2, samples this and preserves the mixture of liquid, being added to the filter chamber (8) of the cell smearing device after step 1 processes In, the mixture of sample and preservation liquid enters through filter membrane (7) and cleans in chamber (9), and the cell settlement in sample is to cleaning chamber (9) Bottom;
Step 3, removes the filter membrane (7) after step 2 processes and the impurity on filter membrane (7), after natural subsidence (or centrifugal) Remove the supernatant;
Step 4, the cell smearing device that step 3 of learning from else's experience processes, extract microscope slide thereon (3), fixing, dyeing, mounting, to obtain final product.
A kind of liquid-based cell sample manufacturing method the most according to claim 1, it is characterised in that described preparation bin is provided with in (2) Sleeve (5), the upper end of described sleeve (5) is provided with the collar flange (6) for abutting with the upper surface of preparation bin (2);Described mistake Filter membrane (7) is fixed on the lower end of sleeve (5).
A kind of liquid-based cell sample manufacturing method the most according to claim 2, it is characterised in that described sleeve (5) outer wall and system Gap (10) is left between sheet storehouse (2) inwall.
A kind of liquid-based cell sample manufacturing method the most according to claim 1, it is characterised in that under normal temperature and pressure, described cleaning The density of liquid is 0.9 ~ 1.2g/cm3
A kind of liquid-based cell sample manufacturing method the most according to claim 4, it is characterised in that in step 1, cleanout fluid all enters In entering to clean chamber (9), its liquid level rear highly flushes in filter membrane (7).
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CN108687087A (en) * 2017-04-06 2018-10-23 希森美康株式会社 Smear the cleaning method and smear slide preparing apparatus and smear slide preparing of component
CN109001017A (en) * 2018-10-24 2018-12-14 清华大学 The planktonic organism settler for avoiding bubble from interfering
CN109030151A (en) * 2018-07-27 2018-12-18 上海皓信生物科技有限公司 Liquid-based flaking method
CN109507004A (en) * 2018-11-21 2019-03-22 杭州海世嘉生物科技有限公司 A kind of natural subsidence sectioning cells dyeing work station control method and system
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CN108687087A (en) * 2017-04-06 2018-10-23 希森美康株式会社 Smear the cleaning method and smear slide preparing apparatus and smear slide preparing of component
CN108687087B (en) * 2017-04-06 2022-05-13 希森美康株式会社 Method for cleaning smear member and smear sample producing device
CN111051847A (en) * 2017-07-20 2020-04-21 贝克顿·迪金森公司 Manual method of depositing samples directly onto slides for liquid-based cytology
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