RU2619784C2 - System and kit for cytological specimens acquisition for research - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to microscopic examination of cytological specimens. The cytological specimen preparation system includes a retainer for cells, a cell surface modifier for cell surfaces modification, the first (103) and the second (105) supporting means for the specimen, each having at least two sides. Cytoplasmic or nuclear dye colorant is applied on at least one side of at least one of the supporting means. The first and second supporting means for a sample are attached to each other by a hinge. The process of cytological specimen preparation includes the stages of specimen cell fixation with a retainer, specimen cell surface modification by the cell surface modifier, provided with the first (103) and the second (105) supporting means for the specimen, having at least two sides, cells application on any of the first and second supporting means and cells coating by the first or the second supporting means, respectively. Application of the method or system for at least one target, selected from the group consisting of cancer screening, cancer diagnosis, prognosis with respect to the therapy, accompanying the observation of this cancer therapy.

EFFECT: invention provides simplification and automation of the study process of a cytologic specimen.

19 cl, 11 dwg

Description

FIELD OF THE INVENTION

The present invention relates to the field of microscopic examination of cytological samples.

BACKGROUND OF THE INVENTION

Cervical cancer is the second most common type of cancer in women in the world and the leading cause of cancer death among women in developing countries. About 30% of cancer cases in women are cervical cancer, with more than 100,000 new cases diagnosed each year, for example, in India. The estimated cumulative spread rate (CAGR) of cervical cancer is 2.56%, and at that rate, approximately 175,000 new cases of cervical cancer will be diagnosed in 2012.

One recommended cervical cancer screening tool is the detection of cytological cancer precursors in Papanicolaou analyzes (also called Pap smears, Pap tests, cervical swabs, or smear tests), which is a test used in gynecology to detect pre-tumor and malignant processes in the cervical canal, in particular in the transformation zone (overlapping of the cylindrical epithelium with stratified squamous epithelium).

When taking a Pap smear, a mirror is used that collects cells from the external mouth of the cervix and the mucous membrane of the cervical canal. These cells are examined under a microscope to observe anomalies. The objective of this analysis is to detect potentially precancerous changes that, among other reasons, cause the sexually transmitted human papillomavirus (HPV). This analysis is still an effective and widely used method for early detection of precancerous condition and cervical cancer. The analysis can also detect infections and abnormalities in the mucous membrane of the cervical canal and uterine mucosa (endometrium).

This procedure has effectively reduced the incidence of cervical cancer in developed countries. However, Pap analysis is now considered time-consuming and time-consuming. In addition, the required many stages and methodology make it very difficult to automate the entire process and its implementation at the medical care site, and this means that the samples cannot be analyzed at the collection site, but their delivery to the laboratory is required.

British Patent No. 1,555,507 A describes a plate made in such a way that a portion of a viscous solution of a transparent polymer in a mixed solvent that contains an organic compound and water is applied to a region of the plate whose area is less than the area of the cover plate, after which a cover plate with coating with a biological dye on the aforementioned portion of the viscous solution on a plate, in which the dye-coated side is in contact with the viscous solution, and ulation a coating plate to rest for some time.

British Patent No. 1557722 A describes a method for staining and sealing a biological sample, dried in air and fixed on a microscope slide, in which a viscous flowable transparent polymer composition and a biological dye are obtained in a mixed solvent containing water, a portion of this viscous flowable composition is applied to a fixed region biological sample, the area of which is less than the area of the cover plate, after which place the cover plate on the applied portion of viscous minutes flowable composition and leave a coating plate at rest as long as the dye is dissolved in the fluid composition does not come into contact with the sample and stain it.

US patent No. 3796594 describes dye-coated plates for differential staining of blood.

US Pat. No. 3,834,874 describes pre-stained microscope slides whose surface is coated with a dried mixture of methylene blue NN and cresyl violet acetate, used to detect malaria parasites in the blood.

European patent 0291153 A1 describes a set of plates for a microscope in which two rectangular plates face each other with their faces but are held at a certain distance from each other by means of their protruding parts, so that a capillary gap forms between the two plates.

US Pat. No. 4,224,277 describes a device for cover plates and pre-coated microscope slide plates made with the aforementioned device.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provided a system or kit for obtaining samples in cytological studies. The system or kit comprises a fixative for fixing the cells contained in the aforementioned sample, a cell surface modifier for modifying the surface of the cells contained in the aforementioned sample, a first sample support means having at least two sides and a second sample support means having at least two sides, with at least one side of at least one of the supporting means deposited cytoplasmic dye or nuclear dye.

As used herein, the term “cytological sample” is defined as any sample taken from an organism, preferably a mammal, in sufficient quantity for research and / or analysis. By way of non-limiting example, a cytological sample includes cell samples, skin samples, tissue samples, and mucosal samples.

As used herein, the term “fixative” means a composition that may or may not be present in liquid form and which fixes the cytological sample so that the cytological sample attaches to the plate for a period of time sufficient to examine and / or analyze the cytological sample.

As used herein, the term “cell surface modification” means that the cells are processed in such a way that they unfold and / or unfold, and / or that cell overlap is reduced. Reducing the degree of folding and coagulation of cells during the preparation of the sample leads to improved visibility of the cellular and morphological characteristics that are used for subsequent detection of abnormal cells in the sample, for example, in screening for cervical cancer.

The reduction in overlap leads to improved cell dispersion, which is an important factor, in particular in cell suspensions, where the cells tend to overlap, in particular polymorphic modifications, which are very small in size and tend to form clusters and precipitate into large cells. As a rule, both goals can be achieved by increasing the surface charge on the cells, which leads to increased repulsion between individual cells and, thus, improves dispersion.

Nuclear dye is used to stain the nuclei of cells contained in the sample when the sample is applied to the aforementioned first support agent for the sample, or it is covered by the aforementioned first support agent for the sample. The cytoplasmic dye serves to stain the cytoplasm of the cells contained in the sample when the sample is applied to the aforementioned second sample support, or covered by the aforementioned second sample support.

Also, the system or kit provides the opportunity to obtain a cytological sample in such a way that the cells in the sample are dispersed in such a way that they become suitable for observation using a microscope or for analysis using an automatic optical device for analyzing cells. Thus, one important distinguishing feature of the present invention is that it provides staining of the sample in place, i.e. at the place of medical care. In addition, it eliminates the need to prepare a coloring solution at the place of use, since dyes are preliminarily applied in an appropriate amount to the sample support. In addition, the need for a complex protocol and a multi-stage staining procedure is eliminated, as well as the problem of contamination, which occurs mainly due to dust and impurities during the staining process. In addition, deviations, such as excessive staining or insufficient staining, are minimized due to the dye being distributed at the exact dosage, which is required for optimal staining.

Another advantage is that you can easily adjust the pH level, which is important for cytoplasmic staining (where acidic pH is an advantage in some circumstances) and nuclear staining (where basic pH is an advantage in some circumstances).

The use of supportive means for the sample, on which dyes are previously applied, makes it possible to stain in a closed environment, i.e. in the cartridge, which additionally eliminates dust and impurities, and also increases the reproducibility of the staining process.

In one preferred embodiment, only one side of the first and / or second sample support means is coated with a suitable colorant. However, it may be advantageous to apply dye to both sides of the first and / or second sample support means; in this case, when using this system, it is not necessary to worry about which side of the support means is facing the sample. According to such an embodiment, errors are eliminated due to the use of an inappropriate side of the first and / or second sample support means.

According to a second aspect of the present invention, a method for obtaining samples for cytological studies, and this method includes the following stages:

fixation of cells contained in the aforementioned sample using a fixative,

surface modification of cells contained in the aforementioned sample using a cell surface modifier,

the manufacture of the first support means for the sample having at least two sides, and

a second supporting means for the sample having at least two sides, with at least one side of at least one of the supporting means applied cytoplasmic dye or nuclear dye,

applying cells to any of the first or second support means for the sample, and

coating the applied cells of the second or first supporting agent for the sample, respectively. It is important to understand that the steps of this method need not be carried out in the order presented above.

Thus, the present invention provides for the use of at least one type of prefabricated sample support means. A nuclear dye or cytoplasmic dye is applied to this prefabricated sample support. According to a preferred embodiment of the present invention, a nuclear dye is applied to at least one side of the first sample support means, and a cytoplasmic dye is applied to at least one side of the second sample support means. Alternatively, only one of the two sample support agents is coated with a dye (e.g., a nuclear dye), and the other type of dye (e.g., a cytoplasmic dye) is added to the cell suspension, for example, together with a fixative and / or cell surface modifier.

According to a preferred embodiment of the present invention, the first specimen support means and / or the second specimen support means are in the form of a plate and / or cover plate.

As used herein, the term “plate” means a transparent plate made of glass or plastic onto which samples, such as cells, can be applied for examination using an optical magnifying device such as a microscope.

As used herein, the term “cover plate” means a transparent plate made of glass or plastic that is used to coat samples, such as cells, before being examined with an optical magnifying device such as a microscope.

According to a common understanding, subject plates and cover plates differ from each other in size and thickness. In accordance with ISO 8255-2, microscope slides are 26 × 76 mm and 1 mm thick, while cover plates are typically 18 × 18 mm and a thickness of 100 to 200 μm. However, according to the context of the present invention, the terms “cover plate” and “subject plate” can be used interchangeably. In particular, in the preparation of smears, slides for the microscope are used to apply the sample and its coating after processing the smear.

According to the present invention, the first sample support means and the second sample support means are connected to each other by a hinge. This embodiment further simplifies the use of the system or kit according to the present invention. Such a hinge may, for example, consist of a tongue and groove joint made between two plastic frames holding the first and second sample support means. Other possibilities for creating such a hinge include the use of a piece of duct tape connecting the first and second sample support means. A person skilled in the art will be able to find other technical embodiments that allow the use of such a hinge without the use of inventive creativity.

According to another preferred embodiment of the present invention, the fixative and / or cell surface modifier are present in liquid form. Preferably, the fixative comprises at least one agent selected from the group consisting of:

• ethanol,

• isopropyl alcohol,

• acetic acid (preferably glacial acetic acid),

• formaldehyde and / or

• glutaraldehyde.

An even more preferred fixative is a mixture containing ethanol, isopropyl alcohol and acetic acid, preferably in a volume ratio of 7: 2: 1.

Alcohol-based fixators are extremely suitable for cytological smears, because they act quickly and provide good staining of the nuclei. Preferably, a mixture containing ethanol (70 vol.%), Isopropyl alcohol (70 vol.%) And glacial acetic acid (10 vol.%) Is used. This mixture has a low pH that can be maintained by using a buffer solution or by adjusting the concentration of acetic acid.

According to yet another preferred embodiment, the fixative comprises glutaraldehyde and phosphate buffered saline (PBS), and it preferably contains 2-20 wt.% Glutaraldehyde in 0.05-1 M phosphate buffered saline. Preferably, the pH of the fixative is from 5 to 6.5.

According to another preferred embodiment of the present invention, the cell surface modifier comprises at least one agent selected from the group consisting of:

• a tool that gives a positive charge to the cell surface,

• funds that impart a negative charge to the cell surface,

• chelating agent,

• anticoagulant,

• a means of preventing the formation of mucus,

• a means of supporting cell dispersion, and / or

• a tool that creates micropores on the cell surface.

The positive cell-surface charge agent is preferably at least one water-soluble compound selected from the group consisting of poly-L-lysine hydrochloride and polyvinylpyrrolidone (PVP).

A negative cell surface charge, preferably at least one water-soluble compound selected from the group consisting of dextransulfate, sodium poly-4-styrene sulfonate, polymethacrylic acid, carboxymethyl cellulose and / or sodium polyacrylate.

It is important to understand that you can also use any other tool, the molecules of which produce a positive / negative charge in solution, according to the context of the present invention. However, it is useful if the aforementioned agent is water soluble.

The aforementioned polymers having different molecular weights and / or different concentrations can be used. It turns out that high molecular weight polymers give cells a high charge. Similarly, a high concentration (for example, a concentration of 5 to 10 mg / ml PVP in an alcohol fixative works well) gives an increased charge to the cell surface (see FIG. 5).

The chelating agent and / or anticoagulant is preferably at least one compound selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), hydroxyethylenediamine triacetic acid (HEDTA), nitrilotriacetic acid (NTA), sodium citrate, and / or disodium or dioxate other citrates or oxalates.

The mucus preventing agent is preferably at least one compound selected from the group consisting of sodium hydroxide, N-acetyl-L-cysteine and / or sodium hypochlorite. Preferably, the cell surface modifier contains from 2 to 5% w / v NaOH and 0.25 g / 50 ml N-acetyl-L-cysteine, or 0.5 to 6% w / v NaOCl.

The cell dispersion maintaining agent and / or the micropore creating agent on the cell surface is preferably a surfactant, more preferably at least one compound selected from the group consisting of dithiothreitol and / or tert-octylphenoxypolyoxyethanol (Triton X-100) . The presence of these agents creates regulated pores in the cell membranes, providing quick access of the dye to the cytoplasm and nucleus.

The aforementioned compounds provide proper fixation, inhibit coagulation and promote good dispersion of cell samples. They do not affect the staining of the cells, which is carried out by methylene blue, eosin azure (EA) and orange G (see Fig.6), and contribute to the fact that the cells remained stable for approximately one week (see Fig.7).

It is particularly preferred that a mixture for a cell preparation is provided that includes at least a fixative and a cell surface modifier. Alternatively, it is preferred that in the method of the present invention, the steps of fixing the cells contained in the aforementioned sample and modifying the surface of the cells contained in the aforementioned sample are carried out simultaneously.

According to these embodiments, the cell preparation mixture serves to simultaneously fix the cells and modify the surface of the cells in the sample.

According to another preferred embodiment, the fixative and / or cell surface modifier may further include a dye.

In addition, it is preferable that the nuclear dye contains at least one agent selected from the group consisting of:

• carmine,

• methylene blue,

• neutral / toluene red,

• hematoxylin,

• safranin,

• Nile blue.

Similarly, it appears preferable that the cytoplasmic dye contains at least one agent selected from the group consisting of:

• eosin,

• Alcian blue,

• xylidine crimson,

• the scarlet dye Biebrich (Biebrich),

• tartrazine,

• Van Gieson dye (picric acid and acid fuchsin)

• Wright's dye.

Preferably, you can use a combination of dyes, which, for example, contains the so-called coloring mixture Papanicolaou, where this combination includes hematoxylin, orange G, eosin Y, light green SF yellowish and sometimes brown Bismarck Y.

Another suitable combination of dyes is used in the Masson tri-color dye, which is composed of Weigert hematoxylin, acid fuchsin, crimson crimson, phosphomolybdenum acid and light green SF yellowish, or, alternatively, green persistent FCF, methyl blue, blue water or aniline blue.

Another suitable combination of dyes is used in the tricolor dye Lilly (Lillie), which is similar to the three-color dye Masson, but it contains the scarlet dye Bibrich, replacing acid fuchsin and / or xylidine crimson.

In addition, fluorescent dyes such as 4 ', 6-diamidino-2-phenylindole (DAPI) can also be used.

In addition, certain combinations of commercially available dyes can also be used, such as cell staining kits, such as the HCS CellMask ™ kit, supplied by Invitrogen.

Nuclear dye is used to stain the nuclei of cells contained in the sample when the sample is applied to the aforementioned first support agent for the sample, or it is covered by the aforementioned first support agent for the sample. The cytoplasmic dye serves to stain the cytoplasm of the cells contained in the sample when the sample is applied to the aforementioned second sample support means or covered by the aforementioned second sample support means.

In another preferred embodiment of the present invention, the first and second sample support means are provided for placement in an automatic sample staining device.

The method according to the present invention preferably may further include at least one step selected from the group consisting of:

a) a detailed examination of the cervix by colposcopy;

b) performing a human papillomavirus (HPV) DNA analysis in the aforementioned biological sample or in a new sample having comparable properties;

c) performing a biomarker analysis in the aforementioned biological sample or in a new sample having comparable properties; and / or

d) visual examination of the aforementioned biological sample or a new sample having comparable properties by a medical expert.

According to another aspect of the present invention, the use of a system, kit or method corresponds to at least one purpose selected from the group consisting of:

• cancer screening,

• cancer diagnosis,

• prognosis in relation to this therapy,

• concomitant observation of this cancer therapy.

Preferably, the cytological sample is a human cell sample. Preferably, the cytological sample is a sample of cervical cells. However, since the principles of cancer and cell transformation are universal, this method can also be used for samples of other body tissues that are subject to check for abnormalities, such as breast samples, prostate samples, liver samples, lung samples, blood samples, etc. .d.

It turns out that the cytological sample is selected from the group consisting of:

• smear sample,

• tissue section,

• liquid sample and / or

• other cytological samples.

A smear sample, for example, is similar or identical to the samples used in the Pap tests (also called Pap smear, Pap test, cervical smear, or smear test). A tissue section is obtained, for example, using a microtome. The liquid sample may preferably consist of a suspension of cells, for example, obtained by smear.

Other suitable samples include, but are not limited to, samples obtained by fine needle aspiration biopsy for cytological examination (FNAC), abrasive cytological samples and / or exfoliated samples.

In many cases, in order to make a sample, for example, a tissue section, or a smear available for examination, it is actually placed on a subject plate. However, other devices can also be used to apply the sample, for example, such as a small cuvette, when the sample is a liquid sample, or a cartridge, in the case where the sample is a brush sample. The term "slice", which is used in flowcharts, thus, does not in any way limit the scope of the present invention.

Example: Rotational application of nuclear dyes and cytoplasmic dyes on cover plates and subject plates

Subject plates and cover plates were cleaned according to the standard cleanroom protocol.

1. Getting dyes:

a) azure eosin Y (EA): eosin Y 0.23% w / v, green persistent F 0.08% w / v, brown bismarck 0.05% w / v, phosphofungsten acid 0, 2% w / v, solution in denatured alcohol;

b) orange G (OG): OG 0.3% w / v%, phosphofungsten acid 0.01% w / v%, solution in denatured alcohol;

c) methylene blue (MB) / cresyl violet (CV): 80 g / l MB in methanol, 40 g / l CV in methanol.

2. Rotational coating on a rotary coating device from Holmarc (India):

a) MB / CV slide: 1000 rpm for 20 seconds (200 μl of dye);

b) EA / OG Coating Plate: 3000 rpm for 20 seconds (70 μl of dye).

3. The concentration of the applied product and the rotation speed used for coating, take the following values:

i) Cover plates

The code Speed (r / min) Time (s) Volume (μl) OE1 2000 twenty one hundred OE2 2000 10 one hundred OE3 1000 twenty one hundred OE4 1000 10 one hundred OE5 500 10 one hundred OE6 500 5 one hundred

OE7 500 10 one hundred OE8 500 5 one hundred OE9 500 10 one hundred OE10 500 5 one hundred

ii) Subject plates

The code Speed (r / min) Time (s) Volume (μl) OE11 1000 10 150 OE12 1000 5 150 OE13 1000 10 200 OE14 1000 5 200 OE15 500 10 200 OE16 500 5 200 OE17 1000 10 200

MB / CV coverage (1: 1)

i) Subject plates

The code Speed (r / min) Time (s) Volume (μl) Dilution MS1 1000 10 150 5x MS2 1000 10 150 5x MS3 1000 5 150 5x MS4 1000 5 150 5x MS5 1000 10 200 5x MS6 1000 10 200 5x MS7 1000 5 200 5x MS8 1000 5 200 5x

Brief Description of the Drawings

These and other aspects of the present invention become apparent and are explained in relation to the options for implementation described later in this document. In the drawings:

1 schematically represents an exemplary system or kit according to an embodiment of the present invention.

Figure 2 represents an exemplary method of manufacturing a cytological plate according to the present invention.

Figure 3 presents a variety of problems that arise in the process of obtaining samples according to the prior art.

4 represents an exemplary process flow and functionality of each of the components according to the present invention.

Figure 5 shows how the zeta potential (i.e., cell membrane potential) of cervical cells increases with increasing concentration of polyvinylpyrrolidone (PVP).

6 represents cells in which cell nuclei (FIG. 6A) or cell cytoplasm (FIG. 6B) are stained using the protocols of the present invention.

Fig.7 represents the gradual destruction of cells over time in the main fixative. This degradation is enhanced when the pH of the fixative is chosen closer to neutral, without any change in staining pattern.

Fig. 8 represents an exemplary process of staining cervical cells on subject plates.

Fig.9 is an exemplary flowchart illustrating the preparation of a dye and an exemplary staining process.

10 represents first and second sample support means according to an exemplary embodiment.

11 represents the results of preliminary spot application of the dye by the method according to the present invention.

Detailed Description of Embodiments

Although the present invention is illustrated and described in detail in the drawings and in the above description, these illustrations and description should be considered as illustrative or exemplary, but not restrictive; the present invention is not limited to the described embodiments. Other varieties of the described embodiments may be understood and practiced by those skilled in the art in practicing the claimed invention as a result of studying the drawings, description and appended claims. In the claims, the word “including” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude the plural. The fact that certain measured values are presented in mutually different dependent claims does not in itself show that a combination of these measured values cannot be used as an advantage. No symbols in the claims should not be construed as limiting its scope.

Although embodiments of the described system and embodiments of the described method are presented in detail in the description with reference to the drawings, these descriptions and drawings should be considered as exemplary and non-limiting; the present invention is not limited to the described embodiments.

For example, the present invention may be practiced in a configuration in which a cell preparation mixture is delivered to one or more containers and mixed in place before the sample is dispersed therein. It may be advantageous to pre-apply a nuclear dye to a plate and a cytoplasmic dye to a coating plate without deviating from the present invention. All such modifications are considered as embodiments of the present invention. Additional options and combinations should be obvious to a person skilled in the art, and all such options are considered to be included in the scope of the described methods.

Figure 1 schematically represents the described system 100, according to a variant implementation. The container 101 contains a mixture for the cell preparation in liquid form. The presented container 101 has a lid 109, which is airtight when closed and therefore protects the liquid 107 contained in the container 101.

The fluid 107 has such a composition that it simultaneously performs a variety of functions. It can be provided for performing two main groups of functions, namely, cell fixation and cell preparation. The term “fixation”, as used herein, has the meaning of killing, preservation and curing (tissue, cells, etc.) for subsequent microscopic examination. Cell preparation is a function by which cells are prepared for placement on a plate. The corresponding process will be described in detail below.

The plate 103 resembles a rectangular glass plate, which is usually used as a cytological plate, except that, among other things, a nuclear dye is applied to it. The subject plate is intended to cover with a cover plate, which resembles a cover plate, usually used as a cytological plate, i.e. representing a rectangular glass plate, except that it has a cytoplasmic dye. After presenting the main parts of the described system, a detailed description will now be given with respect to these parts and the intended method of use.

A cytological sample is obtained from patients in a conventional manner. As an example, a sample can be collected from the cervix of a patient being examined for cervical cancer. The sample is obtained using a special wooden spatula or cotton swab, or brush. The sample obtained in this way is immersed in the mixture 107 for the cell preparation, preferably located inside the container 109, and mixed or shaken so that the constituent components of the sample, in particular the cells, are uniformly suspended in the mixture for the cell preparation.

According to one embodiment, the mixture for the cell preparation necessarily contains a fixative, the task of which is to fix the cells for subsequent placement on the subject plate. The main component of the fixative is a mixture of ethanol, isopropyl alcohol and glacial acetic acid, in a ratio of 7: 2: 1, respectively. Although other ratios can be used, the aforementioned ratio provides an appropriate pH to keep the cells without destruction for a period of time. If long-term storage is not provided, in the interests of other functions of the latch, this ratio can be changed through some experimentation. Other functions may be, for example, the rate of staining of cells in subsequent stages.

According to an alternative embodiment, the main component of the fixative is a mixture containing 2-4 vol.% Glutaraldehyde in 0.1 M phosphate-buffered saline (PBS). This solution may contain sodium salts or potassium salts.

However, in order to obtain high-quality subject plates, i.e. platelets, which contain mainly a single layer of cells that do not overlap and are evenly distributed on the surface of the plate, surface, other compounds are added to the fixative, and all of them are collectively referred to as the term “cell preparation”. It was found that cells do not stick together if they contain a charge on their surface. In order for the cells to acquire a surface charge, other components are added to the fixative. Poly-L-lysine hydrochloride (PLL) or polyvinylpyrrolidone (PVP), when mixed in appropriate amounts, gives a positive charge to the cells and, therefore, they repel each other, which contributes to the formation of a cell monolayer. Alternatively, compounds that impart a negative charge can also be used. Some of the compounds that can be used are dextran sulfate, sodium poly-4-styrene sulfonate, polymethacrylic acid, carboxymethyl cellulose and / or sodium polyacrylate. This list is by no means exhaustive. Knowing the corresponding principle, you can use other various compounds.

In addition, blood coagulation and thickening of other cells in the sample should be prevented. Thus, anticoagulants are added to the cell preparation mixture. One or more of the following is also added to the cell preparation mixture: ethylenediaminetetraacetic acid (EDTA), hydroxyethylenediaminetriacetic acid (HEDTA), nitrilotriacetic acid (NTA), sodium citrate, disodium trixalate (Na ⁺ ₂ C ₂ O ₄ ^2- ) or dimethyl oxalate (CH ₃ ) ₂ C ₂ O ₄ ). These compounds can be used in various concentrations, which are for EDTA from 1 to 1.5 mg per 1 ml of a mixture for a cell preparation; for sodium citrate, 3.8% with respect to the volume of the mixture for the cell preparation, for oxalate 1% with respect to the volume of the mixture for the cell preparation, etc. As a result of some experimentation, it is also possible to determine the appropriate amounts for the other aforementioned compounds.

In addition, mucus inhibiting agents, such as 1% NaOH, 2% NaOH + 0.25 g N-acetyl-L-cysteine or 3% w / v NaOCl, are added to the cell preparation mixture.

In addition, surfactants are added to the cell preparation mixture to improve the dispersion of the cells in the cell preparation mixture and to form a limited number of pores in the membrane that promote staining of the cells. A suitable surfactant is dithiothreitol (DTT). About 1% w / v% with respect to the mixture for the cell preparation is a suitable concentration of surfactant in the mixture for the cell preparation. Using the above concentration, the sample can be stored without significant cell disruption for approximately one week. If this is not necessary, the surfactant can be used in a concentration of 1 to 2% w / v.

Thus, this only component, namely the mixture for the cell preparation, is used for the preparation of cells in the manufacture of on-site application plates for cytological examination.

The plate 103 is similar to a conventional cytological plate, but contains a pre-applied dye. According to one embodiment, the cytoplasmic dye is pre-coated on a plate. As the dye, choose methylene blue (MB) or cresyl violet (CV), or a mixture of these two dyes. For this purpose, you can use other equivalent nuclear dyes, which are hydrophobic in nature. The dye is applied to this plate by the rotational coating method, using the appropriate technology, as a result of which a uniform dye layer is formed on the plate. A coating thickness of 10 nm to 10 μm provides a sufficient amount of dye.

However, good cytoplasmic staining is achieved when it is carried out in an environment in which the pH is from 4.5 to 6.5. To achieve this, according to one embodiment, the cytoplasmic dye that is previously applied to the plate has a neutral pH and the mixture for the cell preparation has a pH of 4.5 to 6.5, i.e. the mixture for the cell preparation contains acetic acid, and the pH of the mixture for the cell preparation is adjusted so that it is in a predetermined range during the preparation. According to another embodiment, in which the cell preparation mixture contains glutaraldehyde and PBS, the colorant precoated on the plate contains suitable acid mixed with it before it is applied to the plate. A suitable acid is hydrochloric acid (HCl). The amount of HCl for mixing with the dye is controlled so that when the sample is dispersed in a predetermined amount of the mixture for the cell preparation and placed on a plate, the pH value is in the optimal range, namely from 4.5 to 6.5.

Nuclear dyes are preliminarily applied to the cover plate 105. Orange G (OG) and eosin azure (EA) are mixed and preliminarily applied to the cover plate 105. In addition, the combination of nuclear dyes is also preliminarily applied to the cover plate by a rotational coating method, the thickness of which is from 10 nm to 10 μm.

In addition, nuclear staining is most effective when the pH of the medium is basic and ranges from 7 to 10. Nuclear staining can be carried out at a pH of 7 or even in the range of 6 to 7, but then this staining is weak or slow. Thus, a nuclear dye is preliminarily applied to the cover plate together with a base such as sodium hydroxide (NaOH) or ammonium hydroxide (NH ₄ OH). Thus, when the mixture for the cell preparation itself is acidic and applied to a plate that is neutral, or when the mixture for the cell preparation is neutral (pH 7) and its pH changes on the plate to 4.5-6.5 in the case of cytoplasmic staining, this pH level again returns to the range from 7 to 10 due to the base mixed with a nuclear dye, previously applied to the cover plate.

Although several embodiments of the described system are presented above, numerous alternative embodiments can be developed using the principles on which the described system is based. For example, cytoplasmic dyes can be mixed with a mixture for a cell preparation so that the cytoplasm of the cells stains when the sample is dispersed in it. When the predetermined time elapses after the sample is introduced into the cell preparation mixture, a small amount of the obtained sample is applied to a plate coated with a nuclear dye and base, so that the pH of the resulting sample changes in place and the cell nuclei are stained. According to such an embodiment, there is no coating on the cover plate, and therefore, it is a cover plate, commonly used as a cytological plate. Another embodiment that can be developed is to stain the nuclei of the cells, using first a mixture for the cell preparation, in which the pH exceeds 7, and which contains a nuclear dye, and then change the pH in place on the subject plate to 5- 6.5 using a plate on which the cytoplasmic dye and acid are preliminarily applied. All such other embodiments are considered to be included in the present invention.

Figure 2 represents a method of manufacturing a cytological slide. The first stage in the procedure described below is a stage not of the described process, but of the process usually carried out to obtain a cytological sample. However, the operation of obtaining a cytological sample is represented as step 211 in the block by a dashed line and a dashed arrow to show that it essentially does not form part of the described method.

However, when the sample is obtained in a known manner using a spatula or stick, or a brush, or any other suitable tool, the sample is dispersed in a specially prepared mixture along the length of the cell preparation described above, and the dispersion is carried out at the appropriate step 213. The mixture for the cell preparation is used to prepare cells in the sample to place them on the prepared plate. Dispersion can be accomplished by mixing the sample in a mixture for a cell preparation using a tool with which to obtain a sample, such as a spatula. Alternatively, the container containing the cell preparation mixture is shaken gently with a sample containing instrument immersed in it. It should be understood that part of the instrument containing the sample is immersed in the mixture for the cell preparation. This dispersion of the sample in the mixture for the cell preparation may require a minimum time. This may depend on the exact composition of the mixture for the cell preparation.

At application step 215, the amount of sample dispersed in a mixture for a cell preparation called a “fabricated sample” is applied to a plate on which a cytoplasmic stain has been previously applied and left to spread uniformly on the plate. The amount of the mixture produced, applied to the plate, should be determined, since the pH of the mixture must be changed in place at this stage or in the subsequent stage, and it can be fewer or more drops in different places on the plate. When the manufactured sample comes into contact with the pre-applied dye, and the dye is dissolved in the mixture for the cell preparation, it begins staining of the cytoplasm of the cells in the manufactured sample.

After incubation for an appropriate period of time to stain the cytoplasm of the cells in the fabricated sample, a coating plate on which at least a nuclear dye has been pre-applied is used to coat the coated fabricated sample in the coating step 217 so that the surface of the coating plate on which a nuclear dye is preliminarily applied; it comes into contact with the manufactured sample coated with it. The period of time that must expire before placing the cover plate may depend on the exact composition of the mixture for the cell preparation according to the various embodiments described above. This period can be established experimentally and defined as the minimum period of time that must elapse before the cover plate can be placed on the sample.

When a cover plate pre-coated with a nuclear dye comes into contact with a manufactured sample deposited on a plate, the nuclear dye dissolves in the mixture for the cell preparation and begins staining of the cell nuclei. As in the case of the time required for staining after applying the manufactured sample to the plate, keeping for a minimum period of time for staining the cells with nuclear dye is required before the fabricated plate can be dried in an accelerated mode, if necessary, for example, by heating.

In this way, a research plate is made by means of a microscope or an automatic optical device for analyzing cells. As can be seen from the above description, the manufacture of a cytological plate is aimed at the production of a plate in place using fewer stages than the number of stages in the methods known to date. It should be understood that although the manufacture of a single plate is described in this description, the same system can be used for the simultaneous production of more than one plate with the same manufactured sample, which may be required in accordance with normal laboratory standards or guidelines. The volume of the mixture for the cell preparation and the amount of sample obtained from the patients can be standardized so that it is possible to produce the number of slides required at one time.

It can be seen that the kit used in practice on the basis of the described system and method may contain a predetermined amount of the mixture for the cell preparation in order to produce the number of slides required according to the relevant laboratory practice, as well as standardize the amount of the obtained sample, the preferred tool for its preparation, dispersion method the sample to obtain the sample preparation, as well as the minimum period of time required at various stages of the method, so as to require The required number of high-quality subject plates could be produced locally for further research.

Essentially, the method can be described as comprising three stages. The first stage is the dispersion of a cytological sample in a mixture for a cell preparation, and at the same time many functions are performed, such as dispersing cells in a mixture for a cell preparation, modifying the cell surface, imparting an electric charge to the cell surface so that they are distributed evenly and do not connect to each other another, and preparing the cell surface for staining. In addition, the mixture for the cell preparation may also have a pH that is optimal for staining. The second stage is the staining of the cytoplasm of cells. The third stage is a change in the pH of the fabricated sample in place and staining of the nuclei of cells in the fabricated sample. Despite the fact that only this embodiment of the described method is presented above, many embodiments of this method can be developed on its basis. For example, a mixture for a cell preparation may also contain a cytoplasmic dye, so that, in addition to the functions already described for the first stage, the cytoplasm can also be stained. Suitable changes can be made to the mixture for the cell preparation, so that the nuclei of the cells are stained first, and then the cytoplasm. All such options are considered as embodiments of the described method, and therefore, the present invention extends to them.

Figure 3 presents a variety of problems that arise in the process of obtaining samples according to the prior art. On figa polymorphic modification overlaps with the cell, which complicates the subsequent visual examination of the aforementioned cells or introduces distortions in the automatic image analysis. FIG. 3B represents cells that are folded, resulting in problems similar to those discussed with respect to FIG. 3A. Fig. 3C represents cells that partially overlap, again complicating subsequent visual examination and introducing distortions into the automatic image analysis. 3D represents cells that must be present in order to provide appropriate image analysis, including visual analysis or automatic image analysis, i.e. these cells are expanded and the cells are properly dispersed without any overlap.

Figure 4 represents an exemplary process flow as well as the functionality of each of the components according to the present invention. It should be noted that this example is by no means restrictive. In addition, it should be noted that components I and II (i.e., fixative and cell surface modifier) can be combined.

Figure 5 shows how the zeta potential (i.e., cell membrane potential) of cervical cells increases with increasing concentration of polyvinylpyrrolidone (PVP). Polyvinylpyrrolidone has a positive effect on the charge of cell membranes. In sample 1, only an alcohol-based fixative is used. Samples 2-4 use the same alcohol-based fixative that contains 2, 4, or 5 mg / ml PVP.

6 represents stained cells. In FIG. 6A, the cell nucleus is stained with methylene blue, and in FIG. 6B, the cytoplasm of the cells is stained with azure azure (EA) and orange G (OG). It is important to note that this staining is not affected by the previous steps carried out according to the present invention, i.e. the use of chelating agents and anticoagulating agents (to prevent cell thickening), such as ethylenediaminetetraacetic acid (EDTA), hydroxyethylenediaminetriacetic acid (HEDTA), nitrilotriacetic acid (NTA), sodium citrate, oxalates; the use of mucus-preventing agents, such as 1% NaOH, 2% NaOH + 0.25 g / 50 ml N-acetyl-L-cysteine, 3% NaOCl; the use of surfactants (to improve cell dispersion and the formation of a limited number of pores in the membrane), such as dithiothreitol (DTT), Triton X-100, etc.

7 represents the gradual destruction of cells over time in an alcohol-based fixative. This degradation slows down when the pH of the fixative approaches neutral, for example, from 4.5 to 6.5, by adding a base or an appropriate buffer solution.

Fig. 8 is an exemplary process for making dyes in combination with a surfactant and a base (for changing the pH at the place of use). A surfactant such as Triton X-100 or DTT is mixed directly with a fixative, such as an alcohol or glutaraldehyde based fixative, in which samples are then collected. Optionally, the aforementioned solution may also contain a dye, for example, a cytoplasmic dye, including eosin azure (EA) and / or orange G (OG). The pH of the solution is preferably maintained in the range from 4.5 to 6.5. Another dye (nuclear dye, such as hematoxylin, methylene blue, etc.) is applied to the plate together with some basic compound such as NaOH, NaCl, NH ₄ OH, etc. The concentration of the base should be chosen so that the pH level of the desired volume of fixative (having an acidic nature) can quickly change to the main range from 7.5 to 9.5. It should be noted that a nuclear dye can also be added to the cell suspension, and a cytoplasmic dye can be applied to the plate.

Fig.9 is an exemplary flowchart illustrating the manufacture of a dye, as well as an exemplary staining process. A mixture of methylene blue (MB) and cresyl violet (CV) is used as a nuclear dye. Other nuclear dyes that are hydrophilic can also be used for this purpose. The dye is applied to the plate by rotational coating using the appropriate protocol to ensure the formation of a uniform dye layer with a thickness of 10 nm to 10 μm. To stain the cytoplasm, orange G (OG) and eosin azure (EA) are mixed together using a single solvent, and then applied to the cover plate. This ensures that the nuclear dye and cytoplasmic dye are pre-applied, and the corresponding types of staining can be simultaneously carried out in a single device for staining. The subject plate and the cover plate (i.e., the first and second supporting means for the sample) can be combined so that they are attached to each other by a hinge, as shown in FIG. 10. A spacer between 2 and 100 μm thick can be provided between these surfaces, so that when the cells are placed between two supporting means for the sample, the cells are protected against compression.

FIG. 10 represents an exemplary embodiment including a first specimen support means (plate 1001) and a second specimen support means (cover plate 1002), both specimen support means being connected by a hinge 1005. In addition, a spacer 1003 is provided, thickness which can range from 2 to 100 microns to prevent cell compression. The spacer may have shock absorbing and / or self-adhesive properties. 10, a cytoplasmic dye is preliminarily deposited on a plate, and a nuclear dye is preliminarily deposited on a cover plate. However, the reverse configuration can also be used (i.e., the cytoplasmic dye can be applied to the integumentary plate, and the nuclear dye can be applied to the subject plate). In addition, the cover plate and the plate may be close or even the same size, or differ from each other even more than shown in Fig.10. As discussed above, a nuclear dye or cytoplasmic dye can also be introduced into the cell suspension in advance.

11 represents the results of preliminary application of dyes at the place of use by the method according to the present invention. Dyestuffs are applied to the platelets and integumentary plates using a rotational coating as described in Example 1. The pre-coated platelets were then used in cervical cell staining experiments. For this purpose, a sample of cervical cells collected in the medium was applied to a subject plate, and then a cover plate was placed on top. Subject plates were examined under a microscope 3 minutes after staining and images were obtained. Fig. 11A represents staining with orange G. Fig. 11B represents staining with eosin azure, and Fig. 11C represents staining with methylene blue (MB) and cresyl violet (CV). 11D is a cervical cell cytoplasm image of a combination of orange G (OG) and azure eosin (EA) 3 minutes after staining.

Claims (66)

1. System (100) for obtaining a cytological sample for research, and this system contains: fixative for fixing the cells contained in the above sample, a cell surface modifier for modifying the surface of cells contained in the aforementioned sample, first support means (103) for the sample having at least two sides, and second sample support means (105) having at least two sides, moreover, at least one side of at least one of the supporting means is applied cytoplasmic dye or nuclear dye, and the first supporting means for the sample and the second supporting means for the sample are connected to each other by hinge. 2. A method for obtaining a cytological sample for research, and this method includes the steps of: fixing the cells contained in the above sample using a fixative, modifying the surface of cells contained in the above sample using a cell surface modifier, providing a first supporting agent (103) for a sample having at least two sides, and a second supporting agent (105) for a sample having at least two sides, with a cytoplasmic applied to at least one side of at least one of the supporting agents a dye or a nuclear dye, wherein the first specimen support means and the second specimen support means are connected to each other by a hinge, applying cells to any of the first or second supporting means for the sample and coating the applied cells with a second or first supporting agent for the sample, respectively. 3. The system of claim 1 or the method of claim 2, wherein the nuclear dye is applied to at least one side of the first sample support means and the cytoplasmic dye is applied to at least one side of the second sample support means. 4. The system of claim 1 or the method of claim 2, wherein the first sample support means and / or second sample support means are in the form of a plate and / or cover plate. 5. The system of claim 1 or the method of claim 2, wherein the fixative and / or cell surface modifier are present in liquid form. 6. The system according to p. 1 or the method according to p. 2, in which the latch contains at least one tool selected from the group consisting of: • ethanol, • isopropyl alcohol, • acetic acid (preferably glacial acetic acid), • formaldehyde, • glutaraldehyde. 7. The system of claim 1 or the method of claim 2, wherein the fixative comprises a mixture comprising ethanol, isopropyl alcohol and glacial acetic acid, preferably in a volume ratio of 7: 2: 1. 8. The system of claim 1 or the method of claim 2, wherein the fixative comprises glutaraldehyde and a phosphate-buffered saline solution, wherein the 0.1-1 M phosphate-buffered saline solution preferably contains from 2 to 20 wt.% Glutaraldehyde. 9. The system according to claim 1 or the method according to claim 2, wherein the fixative has a pH value of from 5 to 6.5. 10. The system of claim 1 or the method of claim 2, wherein the cell surface modifier comprises at least one agent selected from the group consisting of: • a tool that gives a positive charge to the cell surface, • funds that impart a negative charge to the cell surface, • chelating agent, • anticoagulant, • a means of preventing the formation of mucus, • a means of supporting cell dispersion, and / or • a tool that creates micropores on the cell surface. 11. The system according to p. 1 or the method according to p. 2, which provides for the manufacture of a mixture for a cell preparation, which contains at least a fixative and a modifier of the cell surface. 12. The method of claim 2, wherein the steps of fixing the cells contained in the aforementioned sample and modifying the surface of the cells contained in the aforementioned sample are carried out simultaneously. 13. The system according to p. 1 or the method according to p. 2, in which the nuclear dye contains at least one agent selected from the group consisting of: • carmine, • methylene blue, • neutral / toluene red, • hematoxylin, • safranin, • Nile blue. 14. The system of claim 1 or the method of claim 2, wherein the cytoplasmic dye contains at least one dye selected from the group consisting of: • eosin, • Alcian blue, • xylidine crimson, • scarlet dye Biebrich, • tartrazine, • dye Van Gieson, • Wright's dye. 15. The system of claim 1, or the method of claim 2, wherein the first and second sample support means are configured to be placed in an automatic sample staining device. 16. The method according to p. 2, further comprising at least one stage selected from the group consisting of: a) a detailed examination of the cervix by colposcopy; b) performing an HPV DNA analysis of the aforementioned biological sample or a new sample having comparable properties; c) performing a biomarker analysis of the aforementioned biological sample or a new sample having comparable properties; and / or d) visual examination of the aforementioned biological sample or a new sample having comparable properties by a qualified pathologist. 17. The use of the system according to any one of paragraphs. 1, 3-11, 13-15 or the method according to any one of paragraphs. 2-16, for at least one purpose selected from the group consisting of: • cancer screening, • cancer diagnosis, • prognosis in relation to this therapy, • concomitant observation of this cancer therapy. 18. The method according to p. 2 or the system according to p. 1, in which the cytological sample is a sample of the cervix. 19. The method according to p. 2 or the system according to p. 1, in which the cytological sample is selected from the group consisting of: • smear sample, • tissue section, • liquid sample and / or • other cytological samples.

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