Patent Description:
In the medical testing and biological research, it is often necessary to perform smear preparation and staining on cell samples for convenient observation. The traditional smear preparation and staining process is carried out by hand, but the accuracy of the diagnosis is affected by a large number of cell accumulation and interference factors such as red blood cells, mucus, impurities and the like in the handmade cell smear, resulting in a false negative rate of <NUM>% to <NUM>%. In addition, the operator is likely to suffer eyestrain because the traditional preparation and staining process is time-consuming, laborious, and low-efficiency. In recent years, the application of liquid-based cytology technology has made a new breakthrough in the cell smear preparation and staining technology. In the liquid-based cytology technology, exfoliated cells are stored in a cell storage solution, which can effectively exclude interference factors such as red blood cells, mucus, impurities, and so on. Further, the liquid-based cytology technology can disperse and dissociate cell clusters to significantly improve the smear quality of pathological cytology, so that the cell structure and background are clear to facilitate diagnosis and significantly reduce the false negative rate.

By searching literature and patents, in the patents and literature related to cell smear preparation, the Chinese patent with the publication number of <CIT>, entitled "fully-automatic liquid-based thin-layer cell smear preparation and staining device" discloses a fully-automatic liquid-based thin-layer cell smear preparation and staining device. In the technical solution disclosed by this patent, the device can automatically realize the operation of sample placement, pipetting, smear preparation and staining. Although the device can prepare the smear automatically to some extent and replace a part of manual operations, the smear preparation and staining device will lead to different extents of staining under different temperatures without effective quality control. Moreover, the device can only realize sample placement and staining rather than the full automation of cleaning, glass slide receiving and other processes, so it still needs manual operations, thus increasing the labor cost and smear preparation time, which is not conducive to reducing the cost of the whole process. Other documents of the prior art are for example: <CIT> and <CIT>.

In view of the defects in the prior art, an objective of the present invention is to provide a full-automatic preparation method of a cast-off cell smear.

To achieve the above objective, the present invention provides a full-automatic preparation method of a cast-off cell smear, including the following steps:.

In S1, the pretreatment of the cast-off cell includes:.

In S12, the first buffer comprises a pH buffer, a surfactant, a mucus lysis agent, and a protein protectant; wherein <NUM>,<NUM> of the first buffer includes: <NUM> to <NUM> of Tris, <NUM> to <NUM> of Tween-<NUM>, <NUM> to <NUM> of dithiothreitol, <NUM> to <NUM> of glycine, <NUM> to <NUM> of citric acid, and the balance of water; and a pH of the first buffer is adjusted to <NUM> to <NUM>.

Preferably, in S11, the filtration is achieved through positive pressure filtration with a pressure of <NUM> Pa to <NUM>,<NUM> Pa.

Preferably, in S11, the structure of the cell cleaning chamber is arranged on an inclined plane, and an included angle is formed between the inclined plane and a horizontal plane.

Preferably, the included angle is <NUM>° to <NUM>°.

Preferably, in S2, the pretreated cast-off cell is transferred by a manipulator to the cyclic settling module.

Preferably, in S2, a constant temperature is controlled by a constant-temperature control system, and the constant-temperature control system includes a heating element and a temperature controller connected to the heating element; and the constant temperature is adjusted in a range of <NUM> to <NUM>.

Preferably, in S2, the moisturizing includes: adding <NUM> to <NUM> of a wetting agent, and removing the wetting agent after <NUM> to <NUM> to leave a wetting layer on a surface of a cell layer.

Preferably, in S3, the automatic liquid sealing is conducted with a novel polymer material liquid sealing agent, and the novel polymer material liquid sealing agent includes the following components in mass percentages:.

By reading the detailed description of the non-restrictive embodiments with reference to the following drawings, other features, objectives, and advantages of the present invention will become more apparent.

Reference numerals in the accompanying drawings:.

The present invention is described in detail below in conjunction with embodiments. The following embodiments are helpful for those skilled in the art to further understand the present invention but do not limit the present invention in any form.

Embodiment <NUM>, which is not part of the present invention.

<FIG> schematically shows a cyclic settling module used by the full-automatic preparation method of a cast-off cell smear of the present invention in an embodiment.

As shown in <FIG>, the device for preparing a cast-off cell smear of the present invention includes a cyclic settling module, a heating temperature control system, and an UV sealing device. The cyclic settling module includes: rotary settling disc <NUM>, glass slide storage and pushing mechanism <NUM>, a sample addition pipette, pipetting mechanism <NUM>, and cleaning assembly <NUM>. The settling disc <NUM> includes a constant-temperature turntable provided with a plurality of spaced glass slide clamping slots <NUM> along an outer circumference, a thermal insulation housing, and a settling smear preparation assembly <NUM>. The glass slide storage and pushing mechanism <NUM> includes glass slide case <NUM>, glass slide feeding fork <NUM>, and a glass slide discharging fork. The pipetting mechanism <NUM> includes reagent arm Z shaft <NUM>, transverse reagent arm R shaft <NUM>, and reagent pipette assembly <NUM> located at an end of the transverse reagent arm R shaft <NUM>. The reagent pipette assembly <NUM> forms an arc and is located directly above the settling cup <NUM>.

It should be noted that a constant-temperature control system is integrated inside the rotary settling disc <NUM> to control a temperature in the rotary settling disc, such that the temperature in the rotary settling disc <NUM> is always kept at <NUM> to <NUM>. 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 rotary settling disc is lower than <NUM>, the heating element is controlled to heat the rotary settling disc; and when the temperature controller detects that the temperature in the rotary settling disc is higher than <NUM>, the heating element is controlled to stop heating. In addition, an outer layer of the rotary settling disc <NUM> is provided with a thermal insulation housing, and the thermal insulation housing is made from a thermal insulation material and wraps a periphery and a lower part of the rotary settling disc <NUM> to keep the temperature in the rotary settling disc <NUM> stable, which provides a stable external environment for a staining reaction and avoids a sudden temperature change to affect a quality of a cell smear. The settling smear preparation assembly <NUM> includes a glass slide clamping slot, lifting mechanism <NUM>, and settling cup <NUM>, and the lifting mechanism <NUM> controls the settling cup <NUM> to move up and down along a central axis of the constant-temperature turntable. The lifting mechanism <NUM> controls the settling cup <NUM> to rise, fork motor <NUM> drives glass slide feeding fork <NUM> to push a glass slide in the glass slide case <NUM> into glass slide clamping slot <NUM> through glass slide inlet <NUM>, and then the lifting mechanism <NUM> controls the settling cup <NUM> to lower down, such that a bottom of the settling cup <NUM> is tightly engaged with the glass slide. The reagent pipette assembly <NUM> includes vertically arranged arc bracket <NUM>, an integrated reagent pipette, and an integrated cleaning pipette, where the integrated reagent pipette and the integrated cleaning pipette each are connected to an external container through an internal suction tube. The lifting drive motor <NUM> drives the reagent arm Z shaft <NUM> to move up and down to complete the addition of a reagent and the extraction of a waste liquid. The cleaning assembly <NUM> includes a cleaning board and cleaning board pushing mechanism <NUM>. After the constant-temperature turntable completes Papanicolaou staining or settling cup cleaning once, the constant-temperature turntable rotates at least once to transfer the next settling cup <NUM> to be operated to a low below the reagent pipette assembly <NUM>. The integrated reagent pipette works in cooperation with the integrated cleaning pipette to clean a settling cup to be cleaned while subjecting a sample in another settling cup to Papanicolaou staining.

It can be further seen from <FIG> that the glass slide case <NUM> is loaded with glass slides, snapped by the glass slide case clasp <NUM>, and then inserted into the glass slide case clamping slot <NUM>, thereby completing fixation. The lifting mechanism <NUM> controls the settling cup <NUM> to rise, the glass slide feeding fork <NUM> pushes a glass slide out, and the rotary settling disc <NUM> rotates to make the glass slide enter a corresponding glass slide clamping slot through the glass slide inlet. The lifting mechanism <NUM> makes the settling cup <NUM> to lower down, such that a bottom of the settling cup <NUM> is hermetically attached to the glass slide. A sample is injected by the sample addition pipette into the settling cup <NUM>, the constant-temperature turntable makes the settling cup rotate to a position below the integrated reagent pipette, and the lifting drive motor <NUM> drives the reagent arm Z shaft <NUM> to lower down, thereby allowing Papanicolaou staining. After the Papanicolaou staining is completed, the glass slide clamping slot rotates to a discharging position, the glass slide discharging fork lowers down first, the glass slide enters the constant-temperature turntable through a glass slide outlet, the glass slide discharging fork rises to a position below the glass slide, the glass slide discharging fork exits, and the glass slide is pulled out. The glass slide clamping slot rotates to a cleaning position, the lifting mechanism controls the settling cup to rise, and the cleaning board motor <NUM> drives the cleaning board pushing mechanism <NUM> to push the cleaning board into the glass slide clamping slot through a cleaning board inlet; the lifting mechanism controls the settling cup <NUM> to lower down, the bottom of the settling cup <NUM> is hermetically attached to the cleaning board <NUM> to form a sealed structure, and the integrated cleaning pipette leads the cleaning reagent into the settling cup <NUM>; after the cleaning is completed, the integrated cleaning pipette extracts a waste liquid; and the lifting mechanism <NUM> controls the settling cup <NUM> to rise, the cleaning board motor <NUM> drives the cleaning board pushing mechanism to pull the cleaning board out, and the lifting mechanism controls the settling cup to lower down.

<FIG> schematically shows a UV sealing device used by the full-automatic preparation method of a cast-off cell smear of the present invention in an embodiment.

As shown in <FIG>, the UV sealing device includes frame <NUM> and fork <NUM>, adhesive addition arm <NUM>, adhesive addition pipette <NUM>, curing cup <NUM>, curing box <NUM>, UV lamp <NUM>, adhesive addition cleaning tank <NUM>, adhesive addition arm control mechanism <NUM>, and sample sliding rail <NUM> that are arranged on the frame <NUM>. The fork <NUM> is arranged at an end of the sample sliding rail <NUM>, and can slide within the sample sliding rail <NUM>. The curing box <NUM> and the UV lamp <NUM> are arranged above the sample sliding rail <NUM> from one side provided with the fork <NUM> to the other side, the curing cup <NUM> is arranged on the curing box <NUM>, the adhesive addition arm <NUM> is arranged above the curing cup <NUM>, and the adhesive addition pipette <NUM> is arranged on the adhesive addition arm <NUM>. The adhesive addition arm control mechanism <NUM> controls the adhesive addition arm <NUM> to move in a vertical direction and a horizontal direction. For example, the adhesive addition arm control mechanism <NUM> is connected to the adhesive addition arm <NUM> through a gear or belt transmission mechanism, and can drive the adhesive addition arm <NUM> to rotate in a horizontal direction; or the adhesive addition arm control mechanism is connected to the adhesive addition arm <NUM> through a gear-rack mechanism or a gear-worm mechanism, and can drive the adhesive addition arm <NUM> to move up and down in a vertical direction.

It can be further seen from <FIG> that a lamp cover is provided outside the UV lamp <NUM>, the lamp cover is located above the sample sliding rail, and a sample glass slide with an adhesive is cured under the UV lamp inside the lamp cover.

It should be noted that a structure of the adhesive addition arm <NUM> is provided with a novel polymer material liquid sealing adhesive receiving device, an injection system, a suction system, and a waste adhesive receiving device, where the adhesive addition pipette <NUM> includes a adhesive addition pipette and a adhesive suction pipette; the novel polymer material liquid sealing adhesive receiving device is connected to the injection system; the injection system is connected to the adhesive addition pipette; the adhesive suction pipette is connected to the suction system; and the suction system is connected to the waste adhesive receiving device.

It may be conceived that, in some other embodiments, one or more adhesive suction pipettes are provided, and when there are a plurality of adhesive suction pipettes, the plurality of adhesive suction pipettes are uniformly arranged along a circumference of the adhesive addition pipette; and the injection system is provided with a measurement device.

The injection system can add a new polymer material liquid sealing adhesive in the novel polymer material liquid sealing adhesive receiving device dropwise to a surface of a sample glass slide through the adhesive addition pipette. The volume of the adhesive injected by the injection system is measured by the measurement device, and the volume of the adhesive dripped is controlled by the measurement device. The suction system can suck the excess new polymer material liquid sealing adhesive to the waste adhesive receiving device through the adhesive suction pipette.

It can be further seen from <FIG> that the adhesive addition cleaning tank <NUM> is arranged on the frame <NUM> and is located on a movement path of the adhesive addition arm <NUM>. The adhesive addition pipette <NUM> on the adhesive addition arm <NUM> is cleaned by the adhesive addition cleaning tank <NUM> after adhesive addition and suction.

Sealing is conducted with the sealing device <NUM>. <NUM>µL to <NUM>µL of the adhesive is added dropwise, and the UV lamp <NUM> has a wavelength of <NUM> to <NUM>. Standing is conducted for <NUM> to <NUM>, and then adhesive suction is conducted by the adhesive addition pipette <NUM> for <NUM> to <NUM> to remove the excess new polymer material liquid sealing adhesive, such that a liquid thin layer is formed on a surface of the sample layer, and the liquid thin layer wraps an internal moisturizer and has a thickness of <NUM> to <NUM>.

With reference to <FIG> and <FIG>, the full-automatic preparation method of a cast-off cell smear in the present invention is further explained, including the following steps:.

A structure of the cyclic settling module in this embodiment may refer to Embodiment <NUM>, and the automatic liquid sealing device in S3 may refer to the sealing device in Embodiment <NUM>.

It should be noted that, in this embodiment, a glass slide can also be laser-labeled in the cyclic settling module to ensure the traceability of each sample, thereby avoiding wrong sample identification. In particular, in the existing techniques, a smear preparation technician needs to check samples one by one when receiving a test sheet and the samples and then manually attach labels to sample bottles and glass slides in a one-to-one correspondence manner. The above process is easy to cause false detection due to human negligence, and thus it usually takes a lot of time for verification. This embodiment provides a solution of laser labeling on a glass slide, which directly solves the problem of false detection caused by human error. A QR code preset in a sample cup body is scanned such that a central control computer connected to the cyclic settling module determines the information of a sample, then the central control computer rotates a turntable to make a corresponding glass slide rotate to a laser labeling machine, and the glass slide is laser-labeled to complete the information docking between the sample and the glass slide, thereby ensuring the traceability of the sample.

Embodiment <NUM>, which corresponds to the present invention.

A full-automatic preparation method of a cast-off cell smear is provided in this embodiment, and a device used by the method may refer to Embodiment <NUM>. The method in this embodiment is different from the method in Embodiment <NUM> in that, in S1, the pretreatment of the cast-off cell includes:.

It should be noted that the cast-off cells intercepted on the surface of the filter membrane can be resuspended in the first buffer, which is attributed to hydrophilicity of the surface of the membrane and characteristics of the first buffer.

In this embodiment, the filter membrane may be prepared by allowing a heavy ion beam to penetrate through a polymer membrane, and the polymer membrane may be, for example, PE, PC, PET, PP, or PI. During the preparation of the filter membrane, the heavy ion beam causes a radiation damage along a penetration path on the polymer membrane to leave corresponding traces, and then these traces are chemically etched to form through holes, where a time of the chemical etching can be controlled to allow a through-hole diameter of <NUM> to <NUM> and a uniform through-hole size, such that a filter membrane obtained has advantages such as stable properties, strong structure, low adsorption, and no shedding, and can intercept <NUM>% of particles with a size larger than the pore size. Therefore, the filter membrane selectively allows RBCs, WBCs, lymphocytes, and a cell storage solution to flow through the filter membrane while intercepting squamous epithelial cells and glandular cells on a surface of the filter membrane.

In addition, in some preferred embodiments, in S11, the filtration is achieved through positive pressure filtration with a pressure of <NUM> Pa to <NUM>,<NUM> Pa.

In the above solution, unlike the fact that positive pressure filtration is adopted in the prior art, positive pressure filtration is adopted in the present invention, which can make a mechanical structure simple and eliminate the risk of cross-contamination of a cell sample.

In addition, in S12, the first buffer includes a pH buffer, a surfactant, a mucus lysis agent, and a protein protectant, wherein <NUM>,<NUM> of the first buffer includes: <NUM> to <NUM> of Tris, <NUM> to <NUM> of Tween-<NUM>, <NUM> to <NUM> of DTT, <NUM> to <NUM> of glycine, <NUM> to <NUM> of citric acid, and the balance of water; and a pH of the first buffer is adjusted to <NUM> to <NUM>.

In some other embodiments, in order to improve the recovery efficiency of a cell suspension, a cell cleaning chamber may be arranged on an inclined plane, that is, the structure of the cell cleaning chamber is arranged on the inclined plane, and an included angle of <NUM>° to <NUM>° is formed between the inclined plane and a horizontal plane.

In some other embodiments, in S2, the moisturizing includes: <NUM> to <NUM> of a wetting agent was added, and the wetting agent was removed after <NUM> to <NUM> to leave a wetting layer on a surface of a cell layer.

A full-automatic preparation method of a cast-off cell smear is provided in this embodiment, and a device used by the method may refer to Embodiment <NUM>. The method in this embodiment is different from the method in Embodiment <NUM> in that, a composition of the first buffer is different, which is shown in Table <NUM>:.

When the components of the first buffer are not as follows: Tris: <NUM> to <NUM>, Tween-<NUM>: <NUM> to <NUM>, DTT: <NUM> to <NUM>, glycine: <NUM> to <NUM>, citric acid: <NUM> to <NUM>, and water: the balance, and when the pH of the first buffer is adjusted to <NUM> to <NUM>, it will cause the imbalance of the first buffer, the cleaning effect will also be affected, and excessive impurities will affect the adsorption of a glass slide, such that a number of adsorbed cells is lower than a standard value, resulting in an unqualified smear.

A full-automatic preparation method of a cast-off cell smear is provided in this embodiment, and a device used by the method may refer to Embodiment <NUM>. The method in this embodiment is different from the method in Embodiment <NUM> in that, a composition of the polymer material liquid sealing adhesive is different, which is shown in Table <NUM>:.

Claim 1:
A full-automatic preparation method of a cast-off cell smear, comprising the following steps:
S1: pretreatment of a cast-off cell: automatically pretreating the cast-off cell to be prepared to remove interfering substances to obtain a pretreated cast-off cell;
S2: automatically transferring the pretreated cast-off cell obtained in S1 to a cyclic settling module to allow constant-temperature settling, and staining and moisturizing the pretreated cast-off cell to obtain a moisturized cast-off cell; and
S3: subjecting the moisturized cast-off cell obtained in S2 to automatic liquid sealing;
wherein in S1, the pretreatment of the cast-off cell comprises:
S11: using a cell cleaning chamber to clean and filter the cast-off cell, wherein a bottom of the cell cleaning chamber comprises a filter membrane with a through-hole diameter of <NUM> to <NUM> and a uniform through-hole size; and the filter membrane selectively allows red blood cells (RBCs), white blood cells (WBCs), lymphocytes, and a cell storage solution to flow through the filter membrane while intercepting squamous epithelial cells and glandular cells on a surface of the filter membrane; and
S12: using a first buffer to rinse the filter membrane with the squamous epithelial cells and the glandular cells intercepted to obtain a suspension of the squamous epithelial cells and the glandular cells;
characterized in that in S12, the first buffer comprises a pH buffer, a surfactant, a mucus lysis agent, and a protein protectant; wherein <NUM>,<NUM> of the first buffer includes: <NUM> to <NUM> of Tris, <NUM> to <NUM> of Tween-<NUM>, <NUM> to <NUM> of dithiothreitol, <NUM> to <NUM> of glycine, <NUM> to <NUM> of citric acid, and the balance of water; and a pH of the first buffer is adjusted to <NUM> to <NUM>.