WO2022073335A1 - 一种全自动的脱落细胞制片方法 - Google Patents

一种全自动的脱落细胞制片方法 Download PDF

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WO2022073335A1
WO2022073335A1 PCT/CN2021/088915 CN2021088915W WO2022073335A1 WO 2022073335 A1 WO2022073335 A1 WO 2022073335A1 CN 2021088915 W CN2021088915 W CN 2021088915W WO 2022073335 A1 WO2022073335 A1 WO 2022073335A1
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cells
exfoliated cells
exfoliated
cell
constant temperature
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PCT/CN2021/088915
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English (en)
French (fr)
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陈石磊
曾斌
陆龙飞
朱海龟
汪钰青
聂垒
胡付余
王扩业
李一鸣
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嘉兴晶铸生物科技有限公司
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Priority to EP21876858.8A priority Critical patent/EP4206642A4/en
Priority to US18/029,674 priority patent/US20230366790A1/en
Priority to JP2023546374A priority patent/JP7550996B2/ja
Publication of WO2022073335A1 publication Critical patent/WO2022073335A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G01N1/312—Apparatus therefor for samples mounted on planar substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G01N2001/4088—Concentrating samples by other techniques involving separation of suspended solids filtration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N2035/00039—Transport arrangements specific to flat sample substrates, e.g. pusher blade
    • G01N2035/00049—Transport arrangements specific to flat sample substrates, e.g. pusher blade for loading/unloading a carousel
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N2035/00089—Magazines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N2035/00099—Characterised by type of test elements
    • G01N2035/00138—Slides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G01N2035/0401—Sample carriers, cuvettes or reaction vessels
    • G01N2035/0403—Sample carriers with closing or sealing means
    • G01N2035/0405—Sample carriers with closing or sealing means manipulating closing or opening means, e.g. stoppers, screw caps, lids or covers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G01N2035/0401—Sample carriers, cuvettes or reaction vessels
    • G01N2035/0437—Cleaning cuvettes or reaction vessels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G01N2035/0439—Rotary sample carriers, i.e. carousels
    • G01N2035/0441—Rotary sample carriers, i.e. carousels for samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1002—Reagent dispensers

Definitions

  • the invention relates to the technical field of instruments for pathological cell, diagnostics and pathological biomolecular diagnostics, in particular to a cell sheet method, in particular to a fully automatic exfoliated cell sheet preparation method.
  • the purpose of the present invention is to provide a fully automatic method for preparing exfoliated cells.
  • the present invention proposes a fully automatic method for preparing exfoliated cells, which comprises the following steps:
  • Step S1 Pretreatment of exfoliated cells: automated pretreatment to remove interfering substances for exfoliated cells that need to be prepared;
  • Step S2 automatically transfer the exfoliated cells pretreated in step S1 to a circulating sedimentation module for constant temperature sedimentation, and dye and moisturise them;
  • Step S3 automatic liquid sealing of the exfoliated cells after moisturizing in step S2.
  • the inventor of this case is the first to combine the circulating sedimentation module with automatic sealing, so that the fully automatic production of exfoliated cells in this case can be fully automated.
  • the type settling module can be produced and dyed together, and the silo itself can be used repeatedly instead of one-time use. Therefore, this case has an excellent recycling rate.
  • the circulating sedimentation module has the following advantages: First, cells can be allowed to settle in a specific area of the glass slide. Second, cells can be Papanicolaou stained in a cyclic sedimentation module. Third, moisturizing the cells after staining for the purpose of mounting.
  • the operation of laser labeling the glass slides can also be performed in the circulating sedimentation module to ensure the traceability of each specimen and avoid sample identification errors.
  • the production technician needs to check one by one, and manually affix a one-to-one corresponding label on the specimen bottle and glass slide. This process is prone to false detection due to human negligence, so it usually takes a lot of time to check.
  • the present invention provides a solution for laser coding on glass slides, which directly solves the problem of false detection caused by human error. Then the central control computer rotates the turntable and rotates the corresponding glass slide to the laser marking machine to carry out laser marking on the glass slide to obtain the information of the specimen. Complete the information docking between the specimen and the glass slide to ensure the traceability of the specimen.
  • circulating sedimentation module in this case is that the slides are automatically loaded and pushed into their respective workstations, and can be automatically pushed into the sealing chamber after the steps of dyeing and moisturizing.
  • the method of the present application can cooperate with the device to realize automatic sealing.
  • the method for preparing exfoliated cells of the present invention cell sedimentation and staining can be carried out continuously without any supervision.
  • the cell sheet technology is adopted, so that step S3 and step S2 are connected, thereby realizing automatic production, which greatly improves production efficiency, realizes high-throughput output when unattended, and produces high-quality cell sheet samples. , the effect is good.
  • the fully automatic production method of exfoliated cells of the present invention automates production, staining and sealing, and realizes unattended high-throughput production, high-quality production and sealing. piece.
  • the exfoliated cell pretreatment includes:
  • Step S11 Use a cell washing chamber to clean and filter the cells, wherein the bottom of the cell washing chamber includes a filter membrane, the diameter of the through holes is 5-20 ⁇ m, and the size of the through holes is uniform.
  • White blood cells, lymphocytes and cell preservation solution flow through, while squamous epithelial cells and glandular cells are trapped on the surface of the filter membrane;
  • Step S12 The squamous epithelial cells and glandular cells trapped in the filter membrane are resuspended by washing with the first buffer to obtain the squamous epithelial cells and glandular cells trapped in the filter membrane.
  • the exfoliated cells trapped on the surface of the filter membrane can be resuspended with the first buffer. This is due to the hydrophilicity of the membrane surface and the characteristics of the first buffer.
  • the filter membrane used can be prepared by penetrating a polymer film by a heavy ion beam, and the polymer film can be, for example, PE, PC, PET, PP or PI.
  • the heavy ion beam forms radiation damage on the polymer film in its penetration path, and leaves corresponding traces. These traces are then chemically etched to form through holes.
  • the diameter of the through hole is controlled between 5-20um, and the size of the through hole is uniform.
  • the obtained filter membrane has the advantages of stable properties, strong structure, low adsorption and no shedding, and can retain 100% of the particles larger than the pore size.
  • the membrane selectively allows red blood cells, white blood cells, lymphocytes and cell preservation fluid to pass through, while squamous epithelial cells and glandular cells are trapped on the surface of the filter membrane.
  • step S11 positive pressure filtration is used during filtration, and the pressure of the positive pressure filtration used is 50-1000 Pa.
  • the positive pressure filtration method used in this case can simplify the mechanical structure and avoid the risk of cross-contamination of cell samples.
  • the first buffer solution includes pH buffer, surfactant, mucus lysing agent and protein protectant, for example: every 1000ml of the first buffer solution includes Tris: 0.5-2g, Tween- 20: 2-10 g, dithiothreitol: 0.1-0.5 g, glycine: 0.5-5 g, citric acid: 0.2-0.5 g, the balance is water, and the pH of the first buffer is adjusted to 7.5-8.
  • the first buffer prepared by using the above-mentioned components has an excellent effect on improving the cleaning effect. Impurities such as red blood cells and white blood cells, and the first buffer prepared with components other than those in this case will also affect the morphology of the prepared cells, thereby affecting the interpretation of the results.
  • the cell washing bin structure is arranged on an inclined plane, and there is an inclined angle between the inclined plane and the horizontal plane.
  • the inclination angle is 5-30°.
  • setting the cell washing chamber on an inclined plane can help to improve the recovery efficiency of the cell suspension.
  • the pretreated exfoliated cells are transferred to the circulatory sedimentation module via a robotic arm.
  • the constant temperature is controlled by a constant temperature control system
  • the constant temperature control system includes a heating element and a temperature controller connected to the heating element, and the adjustment range of the constant temperature is 25-45°C.
  • the sample can be kept at a constant temperature during the dyeing process, thereby ensuring the repeatability of the dyeing.
  • the reaction temperature is lower than 25°C or higher than 45°C, unsuitable temperature, firstly, will affect the number of settled cells, resulting in insufficient number of settled cells, and secondly, unsuitable temperature will affect the number of settled cells.
  • too low or too high temperature will cause the dye to be too light or too dark, and the depth of coloring will affect the interpretation of the result.
  • the constant temperature control system controls the temperature in the circulating sedimentation module to be maintained at a constant temperature
  • the temperature accuracy error range is within ⁇ 2°C.
  • step S2 when performing the moisturizing step, it includes: adding 0.2-0.3 ml of a wetting agent, and sucking it off after 3-8 seconds, leaving a wetting layer on the surface of the cell layer.
  • the sealing sheet adopts a new type of polymer material liquid sealing sheet
  • the new type of polymer material liquid sealing sheet includes the following components:
  • the mass percentage of photoinitiator is 2%-8%
  • the wetting agent used in step S2 and step S3 is a solution comprising acrylate, for example: using trimethylolpropane triacrylate, tripropylene glycol diacrylate, hexanediol diacrylate wherein one or a combination of them.
  • the traditional sealing method uses a neutral resin (which includes xylene solvent, which is a carcinogen) to drip on the surface of the cell smear, and then needs to be ventilated in a fume hood for several hours due to xylene. Only then can it be used for interpretation.
  • a novel non-xylene polymer material is used for liquid sealing.
  • the new polymer material After being sprayed on the surface of the cell layer and irradiated by ultraviolet light, the new polymer material will be rapidly cured, and the cured layer formed after curing is completely transparent, which can better show the morphology of the prepared cell sample, and the polymer The material is harmless to the human body, and the curing process only takes a few seconds.
  • the present invention has the following advantages and beneficial effects:
  • the inventor of this case is the first to combine the cyclic sedimentation module with automatic sealing, so that the fully automatic production of exfoliated cells in this case can be fully automated. Working together, the bin itself can be used repeatedly instead of one-time use. Therefore, this case has an excellent recycling rate.
  • the fully automatic exfoliated cell production method of the present invention realizes automatic production, staining and sealing, and realizes unattended high-throughput production and high-quality production and sealing.
  • a new type of non-xylene polymer material is used for liquid sealing. After being sprayed on the surface of the cell layer and irradiated by ultraviolet light, the new polymer material will be rapidly cured, and the cured layer formed after curing is completely transparent, which can better show the morphology of the prepared cell sample, and the polymer The material is harmless to the human body, and the curing process only takes a few seconds.
  • Fig. 1 schematically shows the structure of the circulating sedimentation module adopted in an embodiment of the fully automatic exfoliated cell preparation method of the present invention
  • Fig. 2 schematically shows an ultraviolet sealing device used in an embodiment of the fully automatic exfoliated cell preparation method of the present invention.
  • Fig. 1 schematically shows the structure of a circulating sedimentation module used in an embodiment of the fully automatic exfoliated cell preparation method of the present invention.
  • the exfoliated cell tableting device of the present invention includes a circulating sedimentation module, a heating temperature control system and an ultraviolet sealing device.
  • the circulating sedimentation module includes: a rotary sedimentation plate 12 , a slide storage and push mechanism 25 , a sample adding needle, a liquid pipetting mechanism 26 and a cleaning component 29 .
  • the settling plate 12 includes a constant temperature turntable with a plurality of spaced glass clip slots 23 arranged in the outer circumference, a heat preservation shell, and a settling tablet assembly 30 .
  • the slide storage and push mechanism 25 includes a slide cassette 2 , a slide-in fork 8 and a slide-out fork.
  • the pipetting mechanism 26 includes a reagent arm Z axis 10 , a lateral reagent arm R axis 9 and a reagent needle assembly 27 located at the end of the lateral reagent arm R axis 9 .
  • the reagent needle assembly 27 forms an arc and is located just above the sedimentation cup 5 .
  • a constant temperature control system is integrated inside the rotary settling pan 12 to control its temperature, so that the temperature inside the rotary settling pan 12 is always maintained within 25-45°C.
  • the constant temperature control system includes a heating element and a temperature controller connected to the heating element. When the temperature controller detects that the temperature in the disk is lower than 25°C, it controls the heating element to heat the rotating turntable. When the temperature is higher than 45°C, the heating element is controlled to stop heating.
  • the outer layer of the rotating turntable 12 is provided with a thermal insulation shell, and the thermal insulation shell is made of thermal insulation material, which wraps the outer periphery and the lower part of the rotary settling disc 12 to keep the internal temperature of the rotary settling disc 12 stable and provide stable reflection for dyeing. external environment to avoid sudden temperature changes that affect the quality of cell preparations.
  • the sedimentation tablet assembly 30 includes a slide slot, a jacking mechanism 7 and a sedimentation cup 5 .
  • the jacking mechanism 7 controls the sedimentation cup 5 to move up and down along the central axis of the constant temperature turntable 17 .
  • the jacking mechanism 07 controls the settling cup 05 to lift up
  • the shifting fork motor 18 drives the slide fork 8 to push the slides in the slide cassette 02 into the slide slot 23 from the slide inlet 24, and the jacking mechanism 7 controls the settling cup 5 Descend to seal the bottom of the sinker cup 05 with the glass slide.
  • the reagent needle assembly 27 includes a vertically arranged arc support 28 , an integrated reagent needle and an integrated cleaning needle, wherein the integrated reagent needle and the integrated cleaning needle are connected to the external container through a suction pipe.
  • the lift drive motor 19 drives the reagent arm Z axis 10 to move up and down to complete reagent filling and waste liquid extraction.
  • the cleaning assembly 29 includes a cleaning plate and a cleaning plate pushing mechanism 11 .
  • the thermostatic turntable rotates at least once every time the pasteurization or sedimentation cup cleaning is completed, and the next sedimentation cup 5 to be operated is transferred to the bottom of the reagent needle assembly 27 .
  • the integrated reagent needle and the integrated cleaning needle work together, and when the sample in one sedimentation cup is pasteurized, the other sedimentation cup in a washable state can be cleaned.
  • the slide cassette 2 with the slides installed is fastened with the slide cassette buckle 22 and inserted into the slide cassette catch slot 21 to complete the fixing.
  • the jack-up mechanism 7 controls the sinker cup 5 to rise, the slide-in fork 8 pushes the glass slide out, and through the rotation of the rotary sinker plate 12 , the glass slide inlet enters the corresponding glass slide slot.
  • the lifting mechanism 7 puts down the sedimentation cup 5, and the bottom of the sedimentation cup 5 is closed with the glass slide.
  • the sample injection needle injects the sample into the sedimentation cup 5
  • the thermostatic turntable rotates the sedimentation cup to the lower part of the integrated reagent needle
  • the elevating drive motor 19 drives the Z-axis 10 of the reagent arm to descend to perform the Pasteur staining process.
  • the slide slot is rotated to the slide-out position
  • the slide-out fork 13 descends first
  • the slide enters the thermostatic turntable 17 from the slide outlet
  • the slide-out fork 13 rises to the bottom of the slide
  • the slide-out fork 13Exit pull the slide out.
  • the slide card slot rotates to the cleaning position, the jacking mechanism controls the sedimentation cup to rise, the cleaning plate motor 20 drives the cleaning plate pushing mechanism 11 to push the cleaning plate from the inlet of the cleaning plate into the glass card slot, the jacking mechanism controls the sedimentation cup 05 to descend, The bottom of the sedimentation cup 05 is closed with the cleaning plate 31 to form an airtight structure.
  • the integrated cleaning needle 03 passes the cleaning reagent into the sedimentation cup 05. After the cleaning is completed, the integrated cleaning needle 03 draws out the waste liquid, and the jacking mechanism 7 controls the sedimentation cup 5 to rise and clean.
  • the plate motor 20 drives the cleaning plate pushing mechanism to pull out the cleaning plate, and the jacking mechanism controls the descending of the settling cup.
  • Fig. 2 schematically shows an ultraviolet sealing device used in an embodiment of the fully automatic exfoliated cell preparation method of the present invention.
  • the UV sealing device includes a frame 18 and a fork 23 , a glue application arm 14 , a glue sample needle 16 , a curing cup 4 , a curing box 13 , an ultraviolet lamp 3 , The glue cleaning tank 17 , the glue adding arm control mechanism 15 and the sample slide rail 24 provided on the rack 18 .
  • the fork 23 is disposed at one end of the sample slide rail 24 and can slide in the sample slide rail 24 .
  • the top of the sample slide rail 24 is sequentially provided with the curing box 13 , the ultraviolet light Lamp 3, a curing cup 4 is provided on the curing box 13, a gluing arm 14 is disposed above the curing cup 4, and a gluing sample needle 16 is set on the gluing arm 14;
  • the gluing arm control mechanism 15 controls the gluing arm 14 Move in its vertical and horizontal directions, for example: the gluing arm control mechanism 15 is connected to the gluing arm 14 through a gear or belt transmission mechanism, and can drive the gluing arm 14 to rotate in the horizontal direction; or through a gear rack A mechanism or a worm gear mechanism is connected to the glue adding arm 14 and can drive the glue adding arm 14 to move up and down along the vertical direction.
  • the ultraviolet lamp 3 is provided with a lampshade on the outside, the lampshade is located above the sample slide rail, and the sample slide after dispensing is cured by the ultraviolet lamp in the lampshade.
  • the structure of the glue adding arm 14 is provided with a new polymer material liquid sealing glue accommodating device, an injection system, a suction system and a waste glue accommodating device, wherein the glue adding sample needle 16 includes a glue adding needle and a suction system. glue needle; the novel polymer material liquid sealing glue accommodating device is connected to the injection system, the injection system is connected to the glue needle, the glue suction needle is connected to the suction system, and the suction system is connected to the waste glue accommodating device.
  • glue suction needles there are one or more glue suction needles, and the plurality of glue suction needles are evenly arranged on the circumference of the glue filling needle; a metering device is provided on the injection system .
  • the injection system can drop the new polymer material liquid sealing glue in the new polymer material liquid sealing glue accommodating device to the surface of the sample slide through the glue needle, and the volume of the glue injected by the injection system is measured by the metering device. , and the volume of the glue drop is controlled by the metering device; the suction system can suck the excess new polymer material liquid sealing glue to the waste glue accommodating device through the glue suction needle.
  • the gluing cleaning tank 17 is provided on the frame 18 and is located on the path of the gluing arm 14 movement.
  • the glue-adding sample needle 16 on the glue-adding arm 14 is cleaned by the glue-adding cleaning tank 17 after adding glue and sucking glue.
  • the sealing device 1 is used for sealing, the volume of the glue added is 50-500 ⁇ L, and the wavelength of the ultraviolet lamp 6 is 200-400 nm. Let it stand for 3s-10s, and suck the glue through the glue sample needle 16 to absorb the excess liquid sealing glue of the new polymer material.
  • the glue sucking time is 5s-20s, so that a liquid thin layer is formed on the surface of the sample layer.
  • the liquid thin layer Wrap the inner moisturizing agent, the thickness of the thin layer is 0.02mm-0.08mm.
  • Step S1 Pretreatment of exfoliated cells: automated pretreatment to remove interfering substances for exfoliated cells that need to be prepared;
  • Step S2 automatically transfer the exfoliated cells pretreated in step S1 to a circulating sedimentation module for constant temperature sedimentation, and dye and moisturise them;
  • Step S3 automatic liquid sealing of the exfoliated cells after moisturizing in step S2.
  • Embodiment 1 For the structure of the circulating sedimentation module in this embodiment, reference may be made to Embodiment 1, and for the device for automatic liquid sealing in step S3, reference may be made to the sealing device of Embodiment 1.
  • the operation of laser labeling the glass slides can also be performed in the circulating sedimentation module to ensure the traceability of each specimen and avoid sample identification errors.
  • the production technician needs to check one by one, and manually affix a one-to-one corresponding label on the specimen bottle and glass slide. This process is prone to false detection due to human negligence, so it usually takes a lot of time to check.
  • a solution of laser coding on the glass slide is provided, which directly solves the problem of false detection caused by human error.
  • the preset two-dimensional code enables the central control computer connected to the circulating sedimentation chamber to determine the specimen information, and then the central control computer rotates the turntable to rotate the corresponding glass slide to the laser marking machine to carry out laser marking on the glass slide. In order to complete the information docking between the specimen and the slide, the traceability of the specimen is ensured.
  • a fully automatic method for preparing exfoliated cells is provided in the examples, and the device used can refer to Example 1.
  • the difference is that in the step S1, the pretreatment of exfoliated cells includes:
  • Step S11 Use a cell washing chamber to clean and filter the cells, wherein the bottom of the cell washing chamber includes a filter membrane, the diameter of the through holes is 5-20 ⁇ m, and the size of the through holes is uniform.
  • White blood cells, lymphocytes and cell preservation solution flow through, while squamous epithelial cells and glandular cells are trapped on the surface of the filter membrane;
  • Step S12 The squamous epithelial cells and glandular cells trapped in the filter membrane are resuspended by washing with the first buffer to obtain the squamous epithelial cells and glandular cells trapped in the filter membrane.
  • exfoliated cells trapped on the surface of the filter membrane can be resuspended with the first buffer. This is due to the hydrophilicity of the membrane surface and the characteristics of the first buffer.
  • the filter membrane used can be prepared by the heavy ion beam penetrating a polymer film, for example, the polymer film can be PE, PC, PET, PP or PI.
  • the heavy ions The beam forms radiation damage on the polymer film in its penetrating path and leaves corresponding traces. These traces are then chemically etched to form through holes.
  • the diameter of the through holes can be controlled to 5 Between -20um, the size of the through holes is uniform, and the obtained filter membrane has the advantages of stable properties, strong structure, low adsorption and no shedding, and can retain 100% of the particles larger than the pore size, thereby realizing the membrane selectively makes red blood cells. , leukocytes, lymphocytes and cell preservation solution flow through, while the squamous epithelial cells and glandular cells are trapped on the surface of the filter membrane.
  • step S11 positive pressure filtration is used during filtration, and the pressure of the positive pressure filtration used is 50-1000 Pa.
  • the positive pressure filtration method used in this case can simplify the mechanical structure and avoid the risk of cross-contamination of cell samples.
  • the first buffer solution includes a pH buffer, a surfactant, a mucus lysing agent and a protein protectant, for example, every 1000ml of the first buffer solution includes Tris: 0.5-2g, Tween-20 : 2-10g, dithiothreitol: 0.1-0.5g, glycine: 0.5-5g, citric acid: 0.2-0.5g, the balance is water, and the pH of the first buffer is adjusted to 7.5-8.
  • the cell cleaning chamber in order to improve the recovery efficiency of the cell suspension, can be arranged on an inclined plane, that is, the cell cleaning chamber structure is arranged on an inclined plane, and the inclined plane is connected to the horizontal plane. There is an inclined angle between them, and the inclined angle is 5-30°.
  • step S2 when performing the moisturizing step, it includes: adding 0.2-0.3 ml of wetting agent, sucking it off after 3-8 seconds, leaving a layer of moisturizing on the surface of the cell layer layer.
  • Example 1 a fully automatic method for producing exfoliated cells is provided, and the device used can refer to Example 1.
  • the difference is that the composition of the first buffer is different, and its specific composition is shown in Table 1. :
  • the balance is water, and adjusting the pH of the first buffer to 7.5-8 will lead to the imbalance of the first buffer, the cleaning effect will also be affected, and too much impurities will affect the adsorption of the glass slide , so that the number of adsorbed cells is lower than the standard, resulting in unqualified production.
  • Example 2 a fully automatic method for producing exfoliated cells is provided, and the device used can refer to Example 1.
  • the difference is that the components of the polymer material liquid seal are different, and the specific components are shown in Table 2. shown:
  • each of the above-mentioned components can be purchased from Guangzhou Wuxing Materials or Shanghai Boyue New Materials, where the leveling agent can be one of water-based leveling agent BYK 333, TEGO410 leveling agent, TEGO300 leveling agent or In various combinations thereof, the wetting agent can be one of trimethylolpropane triacrylate, tripropylene glycol diacrylate, hexanediol diacrylate or a combination of multiple thereof.

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Abstract

一种全自动的脱落细胞制片方法，其包括以下步骤：步骤S1：脱落细胞的预处理，对需要制片的脱落细胞进行自动化预处理去除干扰物质；步骤S2：将预处理后的脱落细胞自动转移至循环式沉降模组内进行恒温沉降,染色和保湿；步骤S3：将保湿后的脱落细胞进行自动液态封片。该全自动的脱落细胞制片方法实现了自动化制片、染色以及封片，实现了无人值守的高通量的产出，高质量的制片和封片。

Description

一种全自动的脱落细胞制片方法 技术领域
本发明涉及病理细胞、诊断学、病理生物分子诊断学用仪器技术领域，具体涉及一种细胞片方法，特别涉及一种全自动的脱落细胞制片方法。
背景技术
在医学检测和生物学研究中，常需要通过对细胞样本进行制片与染色，以便更好地观察。传统的制片染色过程均采用手工操作，但是由于手工涂片存在着大量的细胞堆积以及红细胞、黏液、杂质等干扰因素而影响正确诊断，会出现2％～50％的假阴性率，并且容易使诊断人员眼工作疲劳，且费时费力，效率低等问题。近年来，液基细胞学技术的应用使细胞的制片与染色技术有了新的突破，液基细胞学技术是一种将脱落细胞保存在细胞保存液中，能有效祛除红细胞、黏液、杂质等干扰诊断因素并将细胞团分散离解，显著改变病理细胞学制片质量，细胞结构和背景清晰，利于鉴别诊断，明显降低了假阴性率。
通过对文献和专利的检索，与细胞制片相关的专利及文献，例如：公开号为CN106018033A，公开日为2016年10月12日，名称为“全自动液基薄层细胞制片染色装置”的中国专利文献公开了一种全自动液基薄层细胞制片染色装置。在该专利文献所公开的技术方案中，装置可以全自动实现放样，移液，制片，染色的机构。其虽然在一定程序上能够自动制片，部分取代人工操作，但是该制片染色装置将在不同的温度将会导致不同的染色深浅，缺少有效的质量控制，同时其仅能实现放样与染色，而不能实现清洗，收片等全流程的自动化，因此还是需要人工的参与，从而提高了人力成本与制片时间，不利于降低整个过程的成本。
发明内容
针对现有技术中的缺陷，本发明的目的在于提供一种全自动的脱落细胞制片方法。
为了实现上述目的，本发明提出一种全自动的脱落细胞制片方法，其包括以下步骤：
步骤S1：脱落细胞的预处理：对需要制片的脱落细胞进行自动化预处理去除干扰物质；
步骤S2：将步骤S1预处理后的脱落细胞自动转移至循环式沉降模组内进行恒温沉降,并对其进行染色和保湿；
步骤S3：将步骤S2保湿后的脱落细胞进行自动液态封片。
在本发明所述的技术方案中，本案发明人首创地将循环式沉降模组与自动封片联合应用，使得本案的全自动的脱落细胞制片可以实现全自动化生产，尤其是，本案的循环式沉降模组可以制片染色共同进行，仓体本身可以循环反复使用，而非一次性使用。因此，本案具有极好地循环利用率。
此外，在本发明所述的技术方案中，循环式沉降模组具有如下所述的优点：其一,可以让细胞沉降在玻片的特定区域。其二,可以在循环式沉降模组内对细胞进行巴氏染色。其三,染色完毕后对细胞进行保湿处理,以备封片之用。
另外，在本发明所述的技术方案中，循环式沉降模组内还可以通过对玻片进行激光打标签的操作，以保证各个标本的可追溯性,避免了样品标识错误。尤其是，在现有的技术中，制片技师在收到检测单以及标本后，需要一一核对，并在标本瓶、玻片上人工贴上一一对应的标签。这个过程易因人为疏忽造成误检，因此通常花费大量时间核对，而本发明中提供了在玻片上激光打码的解决方案，直接解决了人为错误导致误检的问题，通过扫描标本杯身预置的二维码，以使得与循环沉降仓连接的中控计算机确定标本信息，随后中控计算机旋转转盘，将对应的玻片转动至激光打标机处，对玻片进行激光打码，以完成标本与玻片的信息对接，确保了标本的可追溯性。
再者，本案中的循环式沉降模组的另一特征是玻片是自动装载推入各自的工位,在经过染色、保湿步骤后可以自动推入封片仓。
又如，不同于现有技术中封片需要手动完成，采用本案的方法可以配合装置实现自动封片。采用本发明所述的脱落细胞制片方法可以在无人值守下连续进行细胞沉降和染色工作,为了能衔接该模块高通量的产出细胞涂片的功能,本案发明人创造性地采用液态封片技术，以使得步骤S3与步骤S2进行衔接，由此实现了自动化生产，极好地提高了生产效率，实现了在无人值守时的高通量产出，并且制得的细胞片样本质量高、效果佳。
综上所述可以看出，本发明所述的全自动的脱落细胞制片方法自动化制片、染色以及封片，实现了无人值守的高通量的产出，高质量的制片和封片。
优选地，在所述步骤S1中，脱落细胞预处理包括：
步骤S11：使用细胞清洗仓对细胞进行清洗过滤，其中，所述细胞清洗仓的底部包括过滤膜，其通孔直径为5-20μm，通孔大小均一，所述过滤膜选择性地使得红细胞、白细胞、淋巴细胞以及细胞保存液穿流通过，同时将鳞状上皮细胞及腺细胞截留在过滤膜的表面；
步骤S12：通过第一缓冲液洗使得截留在过滤膜的鳞状上皮细胞及腺细胞重新悬浮，以获得截留在过滤膜的鳞状上皮细胞及腺细胞。
在本发明所述的脱落细胞制片方法中，截留在过滤膜表面的脱落细胞可以用第一缓冲液使其重新悬浮起来。这是由于膜表面的亲水性和第一缓冲液特点决定的。
在本发明所述的脱落细胞制片方法中，所采用的过滤膜可以通过重离子束流穿透高分子薄膜制备获得，该高分子薄膜例如可以为：PE、PC、PET、PP或是PI，制备过程中，重离子束流在其穿透路径，在高分子薄膜上形成辐射损伤，并留下相应痕迹，随后对这些痕迹进行化学蚀刻，蚀刻形成通孔，通过控制化学蚀刻的时间可将通孔直径控制在5-20um之间，通孔大小均一，由此获得的过滤膜具有性质稳定，结构强韧，低吸附无脱落的优点，并且可以100％截留大于孔径的微粒，由此实现膜选择性地使得红细胞、白细胞、淋巴细胞以及细胞保存液穿流通过，同时将鳞状上皮细胞及腺细胞截留在过滤膜的表面的效果。
优选地，在所述步骤S11中，过滤时采用正压过滤，所采用的正压过滤的压强为50-1000Pa。
上述方案中，不同于现有技术采用正压过滤法，本案中采用正压过滤，其可以让机械结构简单，并且免除了细胞样品交叉污染的风险。
优选地，在所述步骤S12中，第一缓冲液包括pH缓冲剂、表面活性剂、粘液裂解剂以及蛋白保护剂，例如：每1000ml第一缓冲液中，包括Tris：0.5-2g、Tween-20：2-10g、二硫苏糖醇：0.1-0.5g、甘氨酸：0.5-5g、柠檬酸：0.2-0.5g，余量为水，并调节第一缓冲液的pH至7.5-8。
在本案中，优选地采用上述的组分配制获得的第一缓冲液对于清洗效果有着极好的提高作用，若不采用本案的组分配制或是采用常规的缓冲液，会不利于有效去除诸如红细胞以及白细胞等的杂质，并且采用非本案的组分配制的第一缓冲液，对于制备得到的细胞形态也会因此受到影响，进而影响结果的判读。
优选地，在所述步骤S11中，所述细胞清洗仓结构设于倾斜平面上，所述倾斜平面 与水平平面间具有倾斜夹角。
优选地，所述倾斜夹角为5-30°。
上述方案中，将细胞清洗仓设于倾斜平面上，可以有利于提高细胞悬浮液的回收效率。
优选地，在所述步骤S2中,所述预处理后的脱落细胞经机械臂转移至循环式沉降模组。
优选地，在所述步骤S2中，恒温通过恒温控制系统进行控制，所述恒温控制系统包括加热元件以及与加热元件连接的温度控制器，恒温温度的调节范围为25-45℃。
上述反应时考虑到，由于温度对染色有很大影响，因此，通过设置加热元件以及温度控制系统可以保证在染色过程中样品一直保持恒温，进而保证了染色的重复性。尤其是，当反应温度低于25℃或是高于45℃时，不适宜的温度，第一，会影响沉降细胞的数目，导致没有足够数目的沉降细胞，第二，不适宜的温度会影响巴氏染色的效果，温度过低或者过高均会导致染料着色过浅或者过深，着色的深浅影响结果的判读。
需要指出的是，在本发明所述的技术方案中，当温度达到预设定的恒温温度时，例如30℃，则恒温控制系统控制循环式沉降模组内的温度恒定维持在恒温温度下，其温度精度误差范围在±2℃以内。
优选地，在所述步骤S2中，进行保湿步骤时，其包括：加入润湿剂0.2-0.3ml，3-8秒后吸走，在细胞层表面留下一层润湿层。
优选地，在所述步骤S3中，封片采用新型高分子材料液态封片，所述的新型高分子材料液态封片包括以下组分：
质量百分比为50％-80％的UVLED光固化丙烯酸酯树脂、
质量百分比为5％-40％的光固化丙烯酸酯单体、
质量百分比为0.5％-8％的反应活性胺丙烯酸树脂、
质量百分比为2％-8％的光引发剂、
质量百分比为0.05％-0.5％的流平剂、
以及质量百分比为0.1％-0.5％润湿剂。
上述方案中，步骤S2以及步骤S3所采用的润湿剂为包括丙烯酸酯的溶液，例如：采用三羟甲基丙烷三丙烯酸酯、二缩三丙二醇二丙烯酸酯、己二醇二丙烯酸酯的其中之一或其多种组合。
需要说明的是，传统封片采用的是中性树脂(其包括二甲苯溶剂，二甲苯溶剂为致癌物)点滴在细胞涂片表面，随后由于二甲苯的原因，需要在通风柜中通风数小时后才可以进行判读使用。但是，需要指出的是，在本发明所述的技术方案中，本案采用的是非二甲苯的新型高分子材料进行液态封片。该新型高分子材料在喷涂于细胞层表面后经过紫外光照射后，其会迅速固化，固化后所形成的固化层完全透明，可以较好地展现所制得地细胞样品形态，并且该高分子材料对人体无害，固化过程仅需要数秒。
与现有技术相比，本发明具有如下所述的优点以及有益效果：
1、本案发明人首创地将循环式沉降模组与自动封片联合应用，使得本案的全自动的脱落细胞制片可以实现全自动化生产，尤其是，本案的循环式沉降模组可以制片染色共同进行，仓体本身可以循环反复使用，而非一次性使用。因此，本案具有极好地循环利用率。
2、本发明所述的全自动的脱落细胞制片方法实现了自动化制片、染色以及封片，实现了无人值守的高通量的产出，高质量的制片和封片。
3、本案采用的是非二甲苯的新型高分子材料进行液态封片。该新型高分子材料在喷涂于细胞层表面后经过紫外光照射后，其会迅速固化，固化后所形成的固化层完全透明，可以较好地展现所制得地细胞样品形态，并且该高分子材料对人体无害，固化过程仅需要数秒。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1示意性地显示了本发明所述的全自动的脱落细胞制片方法在一种实施方式中所采用的循环式沉降模组结构；
图2示意性地显示了本发明所述的全自动的脱落细胞制片方法在一种实施方式中所采用的紫外封片装置。
附图标记如下：
Figure PCTCN2021088915-appb-000001
Figure PCTCN2021088915-appb-000002
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变化和改进。这些都属于本发明的保护范围。
实施例1
图1示意性地显示了本发明所述的全自动的脱落细胞制片方法在一种实施方式中所采用的循环式沉降模组结构。
如图1所述，本发明所述的脱落细胞制片装置包括了循环式沉降模块、加热温控系统以及紫外封片装置。其中，循环式沉降模组包括：旋转沉降盘12、玻片存储与推送机构25、加样针、移液机构26以及清洗组件29。其中：沉降盘12包括外周向设置有若干间隔玻片卡槽23的恒温转盘、保温外壳以及沉降制片组件30。其中：玻片存储与推送机构25包括玻片匣2、入片拨叉8以及出片拨叉。移液机构26包括试剂臂Z轴10、横向试剂臂R轴9以及位于横向试剂臂R轴9末端的试剂针组件27。试剂针组件27形成弧形位于沉降杯5正上方。
需要说明的是，旋转沉降盘12内部集成有恒温控制系统，以对其温度进行控制，使得旋转沉降盘12内部的温度始终保持在25-45℃内。该恒温控制系统包括加热元件以及与加热元件连接的温度控制器，当温度控制器检测到盘内温度低于25℃时，其控制加热元件对旋转转盘进行加热，当温度控制器检测到盘内温度高于45℃时，则控制加热元件停止加热。此外，在旋转转盘12的外层设有保温外壳，该保温外壳由保温材料构成，其将旋转沉降盘12的外周和下部包裹，以保持旋转沉降盘12内部温度稳定，为染色反映提供稳定的外环境，避免发生突然的温度骤变而影响细胞制片质量。沉降制片组件30包括玻片卡槽、顶升机构7以及沉降杯5，顶升机构7控制沉降杯5 沿恒温转盘17的中心轴方向上下运动。顶升机构07控制沉降杯05抬起，拨叉电机18驱动入片玻叉8将玻片匣02内玻片从玻片入口24推入玻片卡槽23，顶升机构7控制沉降杯5下降，使沉降杯05底部与玻片密闭接合。试剂针组件27包括竖向设置的弧形支架28、集成试剂针以及集成清洗针，其中，集成试剂针和集成清洗针通过内吸管道与外部容器连接。升降驱动电机19驱动试剂臂Z轴10上下运动，完成试剂加注和废液抽取。清洗组件29包括清洗板以及清洗板推送机构11。恒温转盘每完成一次巴氏染色或者沉降杯清洗，恒温转盘转动至少一次，将下一个需要操作的沉降杯5转移至试剂针组件27下方。集成试剂针与集成清洗针协同工作，对一个沉降杯内样本进行巴氏染色时，可对另一个处于可清洗状态的沉降杯进行清洗。
而进一步参考图1可以看出，装好玻片的玻片匣2扣好玻片匣卡扣22，插入玻片匣卡槽21内，完成固定。顶升机构7控制沉降杯5上升，入片拨叉8将玻片推出，通过旋转沉降盘12的旋转，从玻片入口进入相应的玻片卡槽。顶升机构7将沉降杯5放下，沉降杯5底部与玻片闭合。加样针将样本注入沉降杯5，恒温转盘将沉降杯转至集成试剂针下方，升降驱动电机19驱动试剂臂Z轴10下降，进行巴氏染色流程。巴氏染色完成后，玻片卡槽旋转至出片位置，出片拨叉13先下降，玻片从玻片出口进入恒温转盘17，出片拨叉13上升至玻片下方，出片拨叉13退出，将玻片拉出。玻片卡槽旋转至清洗位，顶升机构控制沉降杯上升，清洗板电机20驱动清洗板推送机构11将清洗板由清洗板入口推入玻片卡槽，顶升机构控制沉降杯05下降，沉降杯05底部与清洗板31闭合形成密闭结构，集成清洗针03将清洗试剂通入沉降杯05，清洗完成后，集成清洗针03将废液抽出，顶升机构7控制沉降杯5上升，清洗板电机20驱动清洗板推送机构把清洗板拉出，顶升机构控制沉降杯下降。
图2示意性地显示了本发明所述的全自动的脱落细胞制片方法在一种实施方式中所采用的紫外封片装置。
如图2所示，紫外封片装置包括机架18以及设于机架18上的拨叉23、加胶臂14、加胶样针16、固化杯4、固化盒13、紫外灯3、加胶清洗槽17、加胶臂控制机构15以及机架18上设置的样本滑轨24。其中，拨叉23设置在样本滑轨24的一端，并能够在样本滑轨24内滑动，样本滑轨24的上方从设置拨叉23的一侧到另一侧依次设置有固化盒13、紫外灯3，固化盒13上设置有固化杯4，固化杯4上方设置 有加胶臂14，加胶臂14上设置有加胶样针16；所述加胶臂控制机构15控制加胶臂14在其竖直方向以及水平方向上运动，例如：加胶臂控制机构15通过齿轮或皮带传输机构连接加胶臂14，并能够驱动加胶臂14在水平方向内转动；或是通过齿轮齿条机构或者涡轮蜗杆机构连接加胶臂14，并能够驱动加胶臂14沿着竖直方向上下运动。
进一步参考图2可以看出，紫外灯3的外部设置有灯罩，所述灯罩位于样本滑轨上方，滴胶后的样本载玻片在灯罩内接受紫外灯固化。
需要说明的是，加胶臂14的结构内设置有新型高分子材料液态封片胶容纳装置、注射系统、抽吸系统以及废胶容纳装置，其中，加胶样针16包括加胶针以及吸胶针；所述新型高分子材料液态封片胶容纳装置连接注射系统，注射系统连接加胶针，所述吸胶针连接抽吸系统，抽吸系统连接废胶容纳装置。
可以想到的是，在一些其他的实施方式中，所述吸胶针为一个或多个，所述多个吸胶针均匀设置在加胶针的周向上；所述注射系统上设置有计量装置。
所述注射系统能够将新型高分子材料液态封片胶容纳装置内的新型高分子材料液态封片胶通过加胶针滴加到样本载玻片表面，注射系统注射加胶的体积通过计量装置计量，并通过计量装置控制滴胶的体积；所述抽吸系统能够将多余的新型高分子材料液态封片胶通过吸胶针抽吸到废胶容纳装置。
此外，进一步参考图2可以看出，加胶清洗槽17设置在机架18上并位于加胶臂14运动的路径上。加胶臂14上的加胶样针16加胶吸胶后通过加胶清洗槽17清洗。
采用封片装置1进行封片，滴加的胶体积为50-500μL，所述紫外灯6的波长为200-400nm。静置3s-10s，通过加胶样针16进行吸胶，以吸取多余的新型高分子材料液态封片胶，吸胶时间5s-20s，使在样本层表面形成液态薄层，该液态薄层包裹内部保湿剂，该薄层厚度在0.02mm-0.08mm。
结合图1至图2对本案中的全自动的脱落细胞制片方法进行进一步说明，其包括如下步骤：
步骤S1：脱落细胞的预处理：对需要制片的脱落细胞进行自动化预处理去除干扰物质；
步骤S2：将步骤S1预处理后的脱落细胞自动转移至循环式沉降模组内进行恒温沉降,并对其进行染色和保湿；
步骤S3：将步骤S2保湿后的脱落细胞进行自动液态封片。
关于本实施例中的循环式沉降模组的结构可以参考实施例1，而步骤S3进行自动液态封片的装置可以参考实施例1的封片装置。
需要说明的是，在本实施例中，循环式沉降模组内还可以通过对玻片进行激光打标签的操作，以保证各个标本的可追溯性,避免了样品标识错误。尤其是，在现有的技术中，制片技师在收到检测单以及标本后，需要一一核对，并在标本瓶、玻片上人工贴上一一对应的标签。这个过程易因人为疏忽造成误检，因此通常花费大量时间核对，而本实施例中提供了在玻片上激光打码的解决方案，直接解决了人为错误导致误检的问题，通过扫描标本杯身预置的二维码，以使得与循环沉降仓连接的中控计算机确定标本信息，随后中控计算机旋转转盘，将对应的玻片转动至激光打标机处，对玻片进行激光打码，以完成标本与玻片的信息对接，确保了标本的可追溯性。
实施例2：
在实施例中提供了一种全自动的脱落细胞制片方法，其所采用的装置可以参考实施例1，其区别在于，所述步骤S1中，脱落细胞预处理包括：
步骤S11：使用细胞清洗仓对细胞进行清洗过滤，其中，所述细胞清洗仓的底部包括过滤膜，其通孔直径为5-20μm，通孔大小均一，所述过滤膜选择性地使得红细胞、白细胞、淋巴细胞以及细胞保存液穿流通过，同时将鳞状上皮细胞及腺细胞截留在过滤膜的表面；
步骤S12：通过第一缓冲液洗使得截留在过滤膜的鳞状上皮细胞及腺细胞重新悬浮，以获得截留在过滤膜的鳞状上皮细胞及腺细胞。
需要说明的是，截留在过滤膜表面的脱落细胞可以用第一缓冲液使其重新悬浮起来。这是由于膜表面的亲水性和第一缓冲液特点决定的。
在本实施例中，所采用的过滤膜可以通过重离子束流穿透高分子薄膜制备获得，该高分子薄膜例如可以为：PE、PC、PET、PP或是PI，制备过程中，重离子束流在其穿透路径，在高分子薄膜上形成辐射损伤，并留下相应痕迹，随后对这些痕迹进行化学蚀刻，蚀刻形成通孔，通过控制化学蚀刻的时间可将通孔直径控制在5-20um之间，通孔大小均一，由此获得的过滤膜具有性质稳定，结构强韧，低吸附无脱落的优点，并且可以100％截留大于孔径的微粒，由此实现膜选择性地使得红细胞、白细胞、淋巴细胞以及细胞保存液穿流通过，同时将鳞状上皮细胞及腺细胞截留在过滤膜的表面的效果。
此外，在一些优选的实施方式中，在所述步骤S11中，过滤时采用正压过滤，所采用的正压过滤的压强为50-1000Pa。
上述方案中，不同于现有技术采用正压过滤法，本案中采用正压过滤，其可以让机械结构简单，并且免除了细胞样品交叉污染的风险。
此外，在所述步骤S12中，第一缓冲液包括pH缓冲剂、表面活性剂、粘液裂解剂以及蛋白保护剂，例如：每1000ml第一缓冲液中，包括Tris：0.5-2g、Tween-20：2-10g、二硫苏糖醇：0.1-0.5g、甘氨酸：0.5-5g、柠檬酸：0.2-0.5g，余量为水，并调节第一缓冲液的pH至7.5-8。
而在一些其他的实施例中，为了提高细胞悬浮液的回收效率，可以将细胞清洗仓设于倾斜平面上，也就是说，将细胞清洗仓结构设于倾斜平面上，而倾斜平面与水平平面间具有倾斜夹角，该倾斜夹角为5-30°。
在一些其他的实施例中，在所述步骤S2中，进行保湿步骤时，其包括：加入润湿剂0.2-0.3ml，3-8秒后吸走，在细胞层表面留下一层润湿层。
实施例3-6：
在本实施例中提供了一种全自动的脱落细胞制片方法，其所采用的装置可以参考实施例1，其区别在于第一缓冲液的成分有所不同，其具体成分如表1所示：
表1.(wt％,余量为水)
Figure PCTCN2021088915-appb-000003
当第一缓冲液的组分不采用以下组分：Tris：0.5-2g、Tween-20：2-10g、二硫苏糖醇：0.1-0.5g、甘氨酸：0.5-5g、柠檬酸：0.2-0.5g，余量为水，并调节第一缓冲液的pH至7.5-8时，会导致第一缓冲液的失衡，清洗效果也会受到影响，并且，过多的杂质会影响玻片的吸附，以致吸附细胞数目低于标准，导致制片不合格。
实施例7-10
在本实施例中提供了一种全自动的脱落细胞制片方法，其所采用的装置可以参考实施例1，其区别在于高分子材料液态封片的成分有所不同，其具体成分如表2所示：
表2.(wt％)
Figure PCTCN2021088915-appb-000004
上述各个成分可以从广州五行材料或是上海博觉新材料处购得相应的产品，其中，流平剂可以采用水性流平剂BYK 333、TEGO410流平剂、TEGO300流平剂的其中之一或其多种组合，润湿剂可以采用三羟甲基丙烷三丙烯酸酯、二缩三丙二醇二丙烯酸酯、己二醇二丙烯酸酯的其中之一或其多种组合。
需要说明的是，本发明的保护范围中现有技术部分并不局限于本申请文件所给出的实施例，所有不与本发明的方案相矛盾的现有技术，包括但不局限于在先专利文献、在先公开出版物，在先公开使用等等，都可纳入本发明的保护范围。
此外，本案中各技术特征的组合方式并不限本案权利要求中所记载的组合方式或是具体实施例所记载的组合方式，本案记载的所有技术特征可以以任何方式进行自由组合或结合，除非相互之间产生矛盾。
还需要注意的是，以上所列举的实施例仅为本发明的具体实施例。显然本发明不局限于以上实施例，随之做出的类似变化或变形是本领域技术人员能从本发明公开的内容直接得出或者很容易便联想到的，均应属于本发明的保护范围。
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变化或修改，这并不影响本发明的实质内容。在不冲突的情况下，本申请的实施例和实施例中的特征可以任意相互组合。

Claims (10)

  1. 一种全自动的脱落细胞制片方法，其特征在于：包括以下步骤：
    步骤S1：脱落细胞的预处理：对需要制片的脱落细胞进行自动化预处理去除干扰物质；
    步骤S2：将步骤S1预处理后的脱落细胞自动转移至循环式沉降模组内进行恒温沉降,并对其进行染色和保湿；
    步骤S3：将步骤S2保湿后的脱落细胞进行自动液态封片。
  2. 根据权利要求1所述的脱落细胞制片方法，其特征在于：在所述步骤S1中，脱落细胞预处理包括：
    步骤S11：使用细胞清洗仓对细胞进行清洗过滤，其中，所述细胞清洗仓的底部包括过滤膜，其通孔直径为5-20μm，通孔大小均一，所述过滤膜选择性地使得红细胞、白细胞、淋巴细胞以及细胞保存液穿流通过，同时将鳞状上皮细胞及腺细胞截留在过滤膜的表面；
    步骤S12：通过第一缓冲液洗使得截留在过滤膜的鳞状上皮细胞及腺细胞重新悬浮，以获得截留在过滤膜的鳞状上皮细胞及腺细胞。
  3. 根据权利要求2所述的脱落细胞制片方法，其特征在于：在所述步骤S11中，过滤时采用正压过滤，所采用的正压过滤的压强为50-1000Pa。
  4. 根据权利要求2所述的脱落细胞制片方法，其特征在于，在所述步骤S12中，第一缓冲液包括pH缓冲剂、表面活性剂、粘液裂解剂以及蛋白保护剂。
  5. 根据权利要求2所述的脱落细胞制片方法，其特征在于，在所述步骤S11中，所述细胞清洗仓结构设于倾斜平面上，所述倾斜平面与水平平面间具有倾斜夹角。
  6. 根据权利要求5所述的脱落细胞制片方法，其特征在于：所述倾斜夹角为5-30°。
  7. 根据权利要求1所述的脱落细胞制片方法,其特征在于，在所述步骤S2中,所述预处理后的脱落细胞经机械臂转移至循环式沉降模组。
  8. 根据权利要求1所述脱落细胞制片方法，其特征在于，在所述步骤S2中，恒温通过恒温控制系统进行控制，所述恒温控制系统包括加热元件以及与加热元件连接的温度控制器，恒温温度的调节范围为25-45℃。
  9. 根据权利要求1所述的细胞制片方法，其特征在于，在所述步骤S2中，进行保湿步骤时，其包括：加入润湿剂0.2-0.3ml，3-8秒后吸走，在细胞层表面留下一层润 湿层。
  10. 根据权利要求1所述的细胞制片方法，其特征在于：在所述步骤S3中，封片采用高分子材料液态封片，所述的高分子材料液态封片包括以下组分：
    质量百分比为50％-80％的UVLED光固化丙烯酸酯树脂、
    质量百分比为5％-40％的光固化丙烯酸酯单体、
    质量百分比为0.5％-8％的反应活性胺丙烯酸树脂、
    质量百分比为2％-8％的光引发剂、
    质量百分比为0.05％-0.5％的流平剂、
    以及质量百分比为0.1％-0.5％润湿剂。
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