Method of formation of risk groups of neoplastic disorders in cervical epithelium

FIELD: medicine.

SUBSTANCE: formation of a risk group of neoplastic disorders in a cervical epithelium is carried out by IF staining of a cervical epithelium smear with using a monoclonal antibody reacting with a sialylated conformational glycopeptide antigen determinant as a part of MUC1 followed by with cell finish staining with chromogenic nuclear stain. The sample is examined by alternated fluorescent light and transmission light. Each cell with a depolarised stained surface membrane detected by fluorescent light is examined in transmission light. If the cells with the depolarised stained surface membrane detected by fluorescent light show cytological symptoms of dyscariosis, and/or proliferative parabasal cell complexes with no symptoms of dyscariosis show depolarised stained surface membranes or granular stained cytoplasm, a patient is subsumed to the risk group of the presence of neoplastic disorders in the cervical epithelium.

EFFECT: higher analysis accuracy.

5 dwg

 

The invention relates to medicine, in particular to methods of physical analysis of biological material in vitro. The invention can be used for the cytological diagnosis of dysplasia of the cervical epithelium and cervical cancer (cervical cancer).

The development of cervical cancer passes through the stages of dysplasia and intraepithelial cancer. The difficulty in diagnosing the disease in the early stages is that the initial forms of neoplasia can be the focal character, leaking clinically asymptomatic, camouflage background diseases.

The "gold standard" in the primary diagnosis of cervical cancer (BL) is cytological examination. Cytological studies of the determination of the nature of violations and the severity of the pathological changes based on the characteristics of the nuclei and cell maturity. In most cases, the limitations of the method associated with the quality of sampling and preparation of material, low information content of the material at small sizes pathological lesions. In particular, as a separate issue experts identify the difficulties in identification and interpretation of cytologic picture when dysplastic disorders mainly represented by small population discretions cells. Thus, it is important to search for more IU the W, which would allow to increase the efficiency of cytological diagnosis of precancerous abnormalities and malignant pathology in the cervical epithelium.

Currently as additional methods used immunocytochemical (ISH) staining of cells of the cervical epithelium. To identify cells using monoclonal antibodies to various biological markers, which are produced by epithelial cells, the expression of which depends on the differentiation and proliferative activity of the cells and an increase in neoplastic changes.

A known method for the diagnosis of dysplasia and cervical cancer in the lining of the BL (US 7.455.973), based on ICH determining in a sample of cervical epithelial several markers that are expressed in the cell nucleus: protein pl4ARF, p21.sup.waf1 protein, topoisomerase Toroa, cycline E, proteins belonging to the group of MSM. The expression of these markers increased in severe pathological cervical disorders. Due to the variability of their expression the best specificity and sensitivity of the test in relation to the diagnosis of intraepithelial violations, according to the authors, can be obtained by using combinations of antibodies to three different markers. The main criteria for cytologic diagnosis are features of the structure of the nucleus and the degree of SP is lost cells. As ICH staining of nuclear proteins escapes the structure of the kernel method is also used in combination with Cytology of smears stained by traditional methods for verification of existing pathological disorders.

There is also known a method of determining the presence or absence of cervical cancer using a set of antibodies (EP 1586903), comprising at least one antibody from each of the following groups:

- 1 - antibodies that interact with cells of the glandular epithelium: anti-MUC1, anti-cytokeratin 7, anti-cytokeratin 18;

- 2 - antibodies that interact with cell adenocarcinoma: anti - cytokeratin 8 and anti-NK;

- 3 - antibodies that interact with the atypical squamous cells: anti-NMP179, anti-P16INK4Aanti-Ki-67, anti-p53, anti-P21, anti-EMA, anti-CEA and anti-MIB-1.

Antibodies interact with nuclear, membrane and cytoplasmic proteins. Antibodies each group conjugated with different fluorescent labels, which allows us to differentiate the object binding with the antibody of the respective group. The main objective of the known method is the differential diagnosis of pathological disorders in the glandular epithelium from those in the flat epithelium. However, the known method does not allow one to determine the nature of the violations in the squamous cells. is ntitle to MUCl-anti-MUCl and anti-EMA - used as markers of different cell types. Perhaps, the authors suggest the use of different clones of antibodies differing in reactivity, but their specificity against antigenic determinants MUCl not specified. The method is designed for automated screening, first and foremost, for selection of patients at risk. The method does not exclude the need for further verification of pathological disorders traditional cytological methods. Furthermore, the method requires the use of special equipment and software, and makes it possible to perform research only in large medical centers.

As a prototype of the claimed invention, the set of features adopted method of assessment of the cervical epithelium in relation to the presence of precancerous and neoplastic diseases of the BL, which uses a panel of two or more antibodies that interact with membrane and cytoplasmic proteins of the cells of the cervical epithelium (US 6.869.801). In the method prototype used a panel of two or more antibodies that interact with membrane and cytoplasmic proteins of the cells of the cervical epithelium. This panel includes at least one antibody that communicates with the columnar epithelial cells and at least one the antibody, communicating with the cells of the squamous epithelium. Antibodies bind to cells of the squamous epithelium, differ in the interaction with cells of different degrees of maturity. Use the following antibodies:

- antibody V, which detects surface glycoprotein, dimeric protein with a molecular weight (MB) of about 180 kDa and interacts with the basal and parabasal cells, as well as cross-reacts with cylindrical cells and stromal elements.

- antibody S, which detects high molecular weight glycoprotein with MB more than 500 kDa (not identified mucin) and communicates with a cylindrical cells;

- antibody 9G5, which detects protein with MB 40 kDa and interacts with the superficial and intermediate squamous cells;

- antibody HG3, which detects protein with MB 180 kDa and interacts with the superficial and intermediate squamous cells;

- antibody WS, which detects protein with MB 200-210 kDa and interacts with parabasal and intermediate cells of the squamous epithelium.

The method is based on registration in cervical smears ratio of cells stained with antibodies of different specificity. The increase in the relative number of Mature and undifferentiated, immature cells parabasal layers of the epithelium, rascenivaesh a sign of possible pathological disorders in the mucous BL. Evaluation of the characteristics of the cellular composition of the stroke is performed either qualitatively, on the basis of visual assessment of the relative number of cells stained with antibodies of different specificity, or quantitatively, the results of the computation of the relevant cells. The disadvantage of this method is the need to analyze multiple samples, painted by various antibodies. Quantitative evaluation can lead to a false-negative result with a small number of atypical cells in the sample. In addition, the appearance of cells with insufficient differentiation in the flat epithelium may occur in atrophic and reactive changes that are not associated with neoplasia, which may be the reason for the low specificity of the test. The result of the test allows you to select the cases with potentially high risk of the presence of pathological disorders, but does not allow to verify their character.

The claimed invention is directed to solving the problem of increasing the effectiveness of cytological diagnosis of precancerous abnormalities and cervical cancer.

Clinical applications of the proposed method achieves the following technical and economic results:

to simplify the process of research by combining in one study, the effective detection of atypical cells and verification of the nature of the violations on the specifics ISH staining;

- reduce the cost of technical research procedures;

to reduce the number of false-negative results of cytological examination, due to the uninformative strokes, the complexity of interpreting cytological picture with small cell variant heavy dyskaryosis or inferiority of the material;

- effectively visualize cells with insufficient differentiation in ICH colouring and spend a targeted assessment of pathological disorders in accordance with the morphological criteria;

- to increase the efficiency of detection of neoplastic disorders of the small size of the nidus;

- provides the possibility of clarifying the diagnosis of latent forms of severe cervical neoplasia due to the anomalous nature ISH staining in proliferating complexes parabasal cells that do not have sufficient morphological signs of atypia;

- available for research in laboratories equipped with a fluorescent microscope.

These technical results by carrying out the invention are achieved due to the fact that as in the known method the formation of groups at risk of neoplastic disorders in epithelium performed by immunofluorescent staining smear cervical epithelium with PR the application of monoclonal antibodies which communicates with the high-molecular glikoproteinom MUCl, followed by counterstaining cells chromogenic nuclear dye.

The feature of the proposed method lies in the fact that as monoclonal antibodies that interact with high molecular glikoproteinom MUCl, use of a monoclonal antibody that interacts with Valerevna conformational glycopeptides antigenic determinant in the composition of MUCl. The sample examined by alternating fluorescent lighting and illumination in transmitted light. Each cell depolarized by staining the surface of the membrane revealed by fluorescent lighting, are examined in transmitted light. When detecting the transmitted light in the cells with depolarized staining of surface membrane cytological signs of dyskaryosis and/or the detection of the complexes proliferating parabasal cells without signs of dikaiosu depolarized staining of the surface of the membrane or granular staining of the cytoplasm, the patient is referred to the group at risk for neoplastic disorders in the cervical epithelium.

The invention consists in the following.

Mucous BL is represented by two types of epithelium: squamous epithelium covering the epithelium, and a cylindrical lining the inner surface Kahn is La. Under the action of damaging factors columnar epithelium may undergo metaplasia can be transformed into the flat.

Dysplasia is a pathological process in which disturbed proliferation and cell differentiation. Morphologically this is reflected in the increase in the thickness of parabasal layers of the epithelium and the appearance of atypical cells. Dysplastic disorders may progress to cancer and intraepithelial often accompany invasive cancer. In the mucous BL dysplasia in the majority of cases found in stratified squamous and metaproterenol epithelium in the transformation zone, in areas of squamous metaplasia in the cervical canal.

When dysplasia and primary cervical cancer in the material scraping the mucous BL an increase in the number of atypical cells with dyskaryosis. Signs of dyskaryosis are the variability of the size and shape of nuclei, high nuclear-cytoplasmic ratio, disruption of chromatin structure, hyperchromia, multicore, abnormal number of nucleoli. With increasing severity of violations increases the relative number of parabasal cells of the type and severity of them dyskaryosis.

It is known that in normal epithelium MUCl is produced by columnar epithelial cells and is not reproduced by the cells of Mature squamous epithelium. Express the ia antigen persists in metaproterenol flat epithelium.

Pathological disturbances in a flat epithelium atypical cells in cervical smears are almost always painted ICH reaction with anti-MUCl antibody. However, due to the fact that the expression of MUC1 can be observed both in normal and atypical cells, and anti-MUCl antibody for the diagnosis of pathological disorders in the mucous BL up to the present time was considered inappropriate.

Molecule MUCl characterized by high polymorphism. Due to the long, polymorphic on the size of the polypeptide chain and variable composition of the carbohydrate side chains of the molecular structure MUCl creates a lot of potential antigenic determinants. As a result, the majority of monoclonal antibodies raised to the MUCl, unique in its ability to interact with carbohydrate and peptide epitopes in the molecule. The structure of MUCl, which is produced by normal cells of different organs, different. The experience of numerous studies and comparative analysis of published data shows that the results of the MUCl in tissues significantly depend on the specificity of monoclonal antibodies that are used for analysis.

The inventors have found that the determination of the expression of MUCl in the cells of the cervical epithelium with the use of monoclonal antibodies to the / establishment, which is produced by a strain of hybrid cultivated cells Mus.Musculus L No. wscc/P/D (ICO) and interacts with Valerevna glycopeptides conformational antigenic determinant in the composition of MUCl, can be used to detect pathological disorders in the mucous BL.

So, normally superficial and intermediate cells of the squamous epithelium in most cases, not colored by reaction with ICO, or they are experiencing a relatively weak uniform color throughout the surface of the membrane. When atrophy or reparative changes in the parabasal squamous cells observed polarized homogeneous staining of the cytoplasm or weak uniform staining throughout the cytoplasm.

In undifferentiated cells parabasal layer flat and metaproterenol epithelium the expression of the antigen recognized ICO (MUC1/HKO25), increased in comparison with the cells of the overlying layers. In the cells of immature squamous metaplasia staining of the surface of the membrane is polarized. Cells Mature and maturing squamous metaplasia lose polarization, they observed variable intensity uniform color throughout the surface of the membrane and/or cytoplasm.

In differentiated columnar epithelial cells in the normal colored apical surface of the cells. In contrast, undifferentiated proliferating reserve cells are located layers and groups, or not painted, or they are experiencing a relatively weak staining cytoplasm is s, evenly represented in all cells. Inflammatory changes in the nature of the staining columnar epithelium does not differ from the norm.

In neoplastic disorders high expression of MUCl/ICO observed in the basal and parabasal cells flat and metaproterenol epithelium. Intensive depolarized staining of surface membrane distinguishes insufficiently differentiated cells from more differentiated cellular elements.

In low proliferative dysplasia parabasal cells occupy not more than one-third of the epithelial layer and seldom get to smear when scraping with the mucosa. Picture ISH staining with UA ICO with weak dysplasia practically does not differ from that of the norm. Signs of weak dyskaryosis detected mainly in unpainted, negative ITH reaction, the cells of the intermediate type.

In moderate and severe dysplasia, intraepithelial cancer (expressed cervical interepithelial violations, CIN) or invasive cervical cancer among colored cellular elements in the majority of cases are detected painted by reaction with MCA ICA atypical cells with dyskaryosis varying degrees of severity. With the exception of cases in which a majority of the cells expressed dystrophic changes in the cytograms is dominated by bare atypical nuclei. Indirect signs of presence of atypia in the study in fluorescent lighting stroke, painted ICH reaction with MCA ICA is the high intensity of this color is variable in size and shape of the cells, the uneven distribution of staining on the surface membrane, the presence of colored intracellular inclusions.

In severe dysplasia and primary cervical cancer in the cervical epithelium, along with atypical cells, in some cases in smears attended painted ICH reaction with ICO massive complexes of proliferating cells parabasal type, enlarged, sometimes with hyperchromic nuclei, slightly pronounced polymorphism. Intensive depolarized staining of the surface of the membrane or granular staining of the cytoplasm of cells in these complexes distinguishes them from proliferating reserve cells, proliferating glandular epithelium and stratum cells immature squamous metaplasia. The presence of groups of cells with similar staining closely associated with pathological disorders as they are not observed in normal and reactive changes of the epithelium.

The inventive method is based on ICH staining of cervical smears with anti-MUCl monoclonal antibody product hybrid strain of cultured cells Mus.Musculus L No. wscc/P/D (ICO), the analysis of paintings fluoresc nnogo coloring and finding cells with depolarized intracellular distribution of staining, the analysis of the morphology of the stained cells by alternating fluorescent lighting and illumination in transmitted light.

Features ISH staining cells of the cervical epithelium with MCA ICA used to identify the pool of undifferentiated cells with pathological changes. The use of fluorescent variant coloring allows you to alternate fluorescent lighting with illumination in transmitted light and to analyze the morphology of cell nuclei. This approach allows us to determine the presence of dyskaryosis in cells, morphological changes which are the main criterion for assessing the presence and severity of pathological disorders in the cervical epithelium.

Intense immunofluorescent staining effectively renders even a single parabasal cells among unpainted or homogeneous colored component of the smears of blood cells, mucus, debris, massive layers of proliferating epithelium. Sighting evaluation of the morphology of the nucleus in the cell elements with depolarized by staining the surface of the membrane greatly increases the probability of detecting cells with dyskaryosis, and also gives the opportunity to verify the extent of the violations in accordance with conventional cytological criteria.

This approach is especially important in case the s, when the strokes aren't helpful for cytological analysis because of the small number of atypical cells, promiscuity material due to pronounced inflammatory changes, a large number of blood cells, and also in cases where pathological changes are mainly small population discretions cells.

Thus, using the technique of immunofluorescence staining in the case of using anti-MUCl monoclonal antibodies ICA allows you to combine high sensitivity ICH detection and high specificity of cytological analysis method.

The method is as follows.

The scraping of the cervical epithelium receive as well as for routine Cytology, spatula from the mucous membrane of the external OS of the cervix in the area of its transition into the cervical canal, with a spatula or brush from the cervical canal. Prepare a smear on a glass slide and fix it in 95% ethanol or other alcohol cytological fixative.

Spend ISH staining using the technique of immunofluorescence analysis. Swabs incubated in buffered physiological solution rn,6 (SFR) for 5 min, then on the glass is applied in 200 μl of a solution of monoclonal antibodies ICO at a concentration of 10 μg/ml, prepared with 1% solution of bovine sivaraman the th albumin (BSA) in SFR. Glass incubated in a humid chamber for 1 hour at room temperature (20-24°C). After incubation, the glass is rinsed three times in SFR and put them in a 200 μl solution antivitamin antibodies labeled with fluorescent label, prepared with 1% solution of BSA in SFR. Incubate in moist chamber for 1 hour, washed three times in SFR.

After completing ISH staining the drug Domracheva with hematoxylin, conclude in a mixture of glycerine and SFR (rn,4, v/v 1:1) and analyzed by fluorescent microscope with built-in illuminator light of day.

Examine sequentially the entire area of the smear.

1) In transmitted light at low magnification (lens ×10) assess the existence and General nature of the morphology of the cellular elements in a, including the obvious signs of atypia that may be sufficient to assess the nature of pathological changes. In the absence of obvious signs of atypia pass to smear with fluorescent light.

2) With fluorescent light at higher magnification (lens ×20), assess the presence of cells stained by immunofluorescence reaction, their shape and size, the nature of intracellular staining.

3) assessment Criteria nature immunofluorescent staining of cells are the distribution of staining relative to the boundaries and the nucleus of the cell. The WPPT is erencereu staining:

- polarized - staining limited portion of the surface of the membrane and/or cytoplasm;

- depolarized - uniform staining of the entire surface of the membrane and/or cytoplasm;

membrane - intensity staining of the boundaries of the cells is higher than the intensity of the colouring of its internal parts; fluorescent staining fully or partially screens the nucleus;

- cytoplasmic - staining intensity of the cell boundary is not higher than the intensity of staining its internal part; the dark area in the cytoplasm, corresponding to the shape and position of the nucleus.

4) Register detached and spaced groups of cells with depolarized by staining the surface of the membrane. Alternating between fluorescent lighting and illumination in transmitted light, aiming to evaluate the morphology of the nuclei of cells at higher magnification (lens ×40).

5) In accordance with conventional cytological criteria make a conclusion about the presence or absence of pathological disorders. The extent of pathological disorders set in accordance with cytological criteria.

In cases when the research is not detected atypical cells with unambiguously interpret the signs of dyskaryosis, they proliferating cells parabasal type with intensive is a diversified granular staining of the cytoplasm or intensive depolarized the membrane staining indicates a high probability of hidden or small pathological disorders. In such cases it is advisable to re-survey and regular monitoring.

After analyzing the fluorescence strokes can be zakrasheny with hematoxylin and eosin and subjected to the control of cytological research.

Examples of the performance of the proposed method is demonstrated on the accompanying micrographs of drugs, processed in accordance with the described method.

Figure 1 Patient X., clinical diagnosis of severe dysplasia of the epithelium of the BL. Intensive depolarized staining of the surface membrane with MCA ICA in parabasal cells of the type with a pronounced dyskaryosis, normal cells of the squamous epithelium is not painted. And image in fluorescent light, B is the image of the same section in transmitted light, staining with hematoxylin (HC.×400).

Figure 2. Patient W., clinical diagnosis of severe dysplasia of the epithelium of the BL. Intensive depolarized staining of the surface membrane and cytoplasm with MCA ICA in parabasal cells of the type with a pronounced dyskaryosis, erythrocytes are not painted. And image in fluorescent light, B is the image of the same section in transmitted light, staining with hematoxylin (HC.×400).

Figure 3. Patient M., clinical diagnosis of microinvasive cervical cancer. Intensive depolarized staining with UA ICO surface membrane petty dyskaryotic the ski cells. And image in fluorescent lighting; B - image of the same section in transmitted light, staining with hematoxylin (HC.×400).

Figure 4. Patient S., the clinical diagnosis of microinvasive cervical cancer. Intense granular cytoplasmic staining with UA ICO proliferating cells parabasal type. And image in fluorescent lighting; B - image of the same section in transmitted light, staining with hematoxylin (HC.×400).

Figure 5. Patient K., clinical diagnosis of microinvasive cervical cancer. Examples of visualization of single cells with heavy dyskaryosis in uninformative and illegible smears cervical epithelium ISH staining with UA ICO. And the image in transmitted light, staining with hematoxylin; B, G - image of the same area in fluorescent lighting (HC.×200).

Thus, using the proposed method of forming risk neoplasia FB allows to detect pathological changes in the epithelium in the early stages of their development. Early treatment of precancerous disorders provides an opportunity to prevent the development of malignant neoplasms. Early detection of cervical cancer will allow for a more efficient, safe and organ-sparing treatment, which is especially important for patients young, reproductive age.

The method of forming the group R is ska neoplastic disorders in the cervical epithelium, including immunofluorescent staining smear cervical epithelium using a monoclonal antibody that interacts with high molecular glikoproteinom MUC1, followed by counterstaining cells chromogenic nuclear dye, characterized in that as monoclonal antibodies that interact with high molecular glikoproteinom MUC1, using monoclonal antibody IC, the product of a hybrid strain of cultured cells Mus.Musculus L No. wscc/P/D, the sample examined by alternating fluorescent lighting and illumination in transmitted light, with each cell depolarized by staining the surface of the membrane revealed by fluorescent lighting, are examined in transmitted light; detecting the transmitted light in the cells with depolarized staining of surface membrane cytological signs of dyskaryosis and/or the detection of the complexes proliferating parabasal cells without signs of dyskaryosis depolarized staining of the surface of the membrane or granular staining of the cytoplasm of a patient is referred to the group at risk for neoplastic disorders in the cervical epithelium.



 

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20 cl, 9 dwg

FIELD: medicine.

SUBSTANCE: one should register the values of pH and Redox potential (Red) of liquid means in bio-objects simultaneously. It is necessary to plot a graph ▵pH/▵Red according to fluctuations of variation values in measured parameters at their temporal diagram to transfer it into a columnar one where the height of every column is proportional to the area of this graph between neighboring measurements, correspondingly. According to the ratio of area sums of positive columns to that of negative ones during a certain period of time ( from a minute- to a year-long ones) one should conclude upon a state in a bio-object. The value ranged 0.5-2 is considered to be a standard. The method enables to provide efficient control for a two-phase systemic process.

EFFECT: higher efficiency and accuracy of diagnostics.

1 cl, 9 dwg, 5 ex, 1 tbl

FIELD: medicine, clinical neurology, neurosurgery.

SUBSTANCE: one should perform crystallographic study if cerebrospinal fluid and at protein concentration in liquor samples being from 0.50 g/l and higher at predominance of crystals as suppressed dendrites upon a crystallographic picture one should diagnose a malignant cerebral tumor, and at protein concentration being below 0.50 g/l and predominance of crystals as branched dendrites - a benign cerebral tumor. The innovation enables to fulfill not only due diagnostics of cerebral neoplasms, but detect the degree of tumor process malignancy at the early stages of its development.

EFFECT: higher accuracy of differential diagnostics.

3 dwg, 2 ex, 1 tbl

FIELD: biology, medicine, in particular histological investigation of shells with natural surface by using light-optical microscope.

SUBSTANCE: claimed method includes surface relief investigation by using plane field microscope in reflected light at one-side falling shadow-forming lighting. Light-reflecting ability of specimen surface is provided by silver impregnation. Said impregnation is carried out by specimen hold in 10 % silver nitrogen solution for 10 min followed by reducing thereof with 1 % ascorbic acid solution for 1 min.

EFFECT: method for investigation of natural shell surface relief of improved quality due to visual representation of surface three-dimension image on tissue level.

3 cl, 4 dwg

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