RU2107911C1 - Differential diagnosis method for detecting the cases of hyperplastic processes and mammary gland cancer in histologic slices - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves dying cytoplasm of parenchymatous cells in histologic slices. Pregnancy-associated alpha2-glycoprotein detection is carried out by means of immune histochemical reaction with primary antibodies used against pregnancy-associated alpha2-glycoprotein in 8-10 mcg/l concentration. EFFECT: enhanced accuracy of prognosis.

Description

 The invention relates to medicine, namely to immunology and pathological anatomy.

 A known method for the differential diagnosis of hyperplastic processes and breast cancer in histological sections stained with the most popular staining method in histological practice is hematoxylin and zosin. The method is based on microscopic detection in the terminal sections of lobules, intralobular and extralobular ducts of cell proliferates. Differential diagnostic criteria in some cases include monomerphism, in others - cell polymorphism and hyperchromia of the nuclei, violation of the polarity of the cells and an increase in their mitotic activity (Ermilova V.D. Breast tumors. Guide to the pathological diagnosis of human tumors. / Ed. N.A. Kraevsky, A.V. Smolyannikov, D.S. Sarkisov .-- 3rd ed. -M .: Medicine, 1982, p. 210-232). With a sufficiently high degree of tumor differentiation, non-infiltrating breast cancers present significant difficulties for differential diagnosis with precancerous hyperplastic processes, as well as benign tumors. The most reliable sign is invasive growth (Golovin DI Errors and difficulties in the histological diagnosis of tumors: a guide for doctors. -L .: Medicine, 1982., p 304), which marks an already disseminated form of the disease. In this regard, it is necessary to resort to the study of numerous sections of material and advice. In some cases, all these techniques do not allow an affirmative answer to the question of the presence or absence of cancer, especially when examining urgent biopsy material.

 There is a method selected as a prototype for the diagnosis of cancer in histological sections, based on the immunological identification of individual substances in cells, which is currently one of the modern and rapidly developing methods for specifying histological and cytological diagnostics (Frank G.A., Pugachev K. K., Shabalova I.P., Shimbireva I.B. // Arch.pat. 1990.v. 52, N. 8, p. 70-74). Currently, a significant number of various types of biological substances are known that are classified as tumor markers: oncofetal antigens, isoenzymes, oncogen products, proteins specific for one or another cell, i.e. cell markers; mucimas and other glycoproteins; brakes and other connections (Romanenko A.M. // Arch. Pat. 1989, vol. 51, No. 4, pp. 58-62; Bobrow LA, Nartou AJ // Cancer Surv. - 1987. Vol.6, N 2 . - p. 209-225; Virji MA, Mercer DW, Herberman RB // CA. - 1988. - Vol.38, N 2.-p. 104-126.).

 Despite the abundance of studies that describe and use tumor markers, the latter do not distinguish between benign and malignant cells (Donat EE // Canad. S. wed. Technob-1987.-Vol. 49, N3. -P. 173-176; Wick MR // Arch, Patn. Lab. Med. -1986.-Vol. 110, N 3.-p. 180-181).

 The objective of the proposed method is to conduct an immunohistochemical reaction that allows using different strictly specific colors to show the differences in the intact, hyperplastic and atypical epithelium of the acini of the lobules, intralobular and extralobular ducts.

This object is achieved in that in a method based on an immunoperoxidase reaction, primary antibodies are used against one of the proteins of the macroglobulin family of pregnancy-associated alpha ₂ -glycoprotein (ABG), which is an inhibitor of proteinases. It should be emphasized that ABG is not a specific affiliation of the epithelium of the mammary gland. This protein is involved in the restriction of the activity of proteinases of various localization. Its biosynthesis is carried out mainly by lymphocytes (mainly B-lymphocytes) and monocytes. Thus, the normal breast structure does not contain ABH.

 It is known that malignant growth is accompanied by an increase in the activity of lysosomal proteins (Veremeenko KN, Goloborodko OP, Kizim AM Proteolysis in normal and pathological conditions. Kiev: Health, 1988, p.200).

 Simultaneously with this formation of complexes, proteins of the macroglobulin family - enzymes, activate a powerful immunosuppressive potential, which protects the malignant neoplasm from attacks of immunocompetent cells. Consequently, tumor growth increases its protective potential. The concentration of proteinase restrictors is well adjusted with the mass of the malignant tumor, which is associated with the local biosynthesis of macroglobulins by tumor cells (Zorin N.A., Zorina R.M. Gorin V.S. // Obstetrics and Gynecology. 1986. N 6, p. 6- 9).

 Epithelial lesions that underwent hyperplasia are in an intermediate position and, unlike normal cells, contain ABH, but in a much smaller amount than malignant cells. Thus, immunohistochemical detection of ABH will reflect the quantitative differences in the content of this protein in epithelial structures in different states corresponding to normal, hyperplasia and malignancy.

 The essence of the proposed method for immunohistochemical detection of ABH in histological sections of the breast, its proliferates and cancer are as follows. The reaction involves a number of steps.

 1. Rehydration of sections and removal of paraffin residues from them. For this, sections are carried out through three portions of o-xylene, absolute ethanol, alcohol 70 and 56%, as well as buffered saline solution (PBS) - for three minutes in each portion of the liquid.

 2. Blocking endogenous peroxidase is carried out by introducing into the cut several drops of 3% hydrogen peroxide for 10 minutes

 3. Blocking non-specific sorption of immunoglobulins is achieved by applying horse serum to a slice at a dilution of 1:20 for 30 minutes.

4. The stage of processing histological sections with primary antibodies at a concentration of 8-10 μg / ml, diluted in PBS with the addition of 3% albumin and 0.01% sodium azide. Incubation of sections is carried out for 30 min at 37 ^o C.

 5. Incubation of histological sections with secondary antibodies at a dilution of 10 μg / ml PBS for 30 minutes.

 6. Processing sections with a complex of avidin-peroxidase at a concentration of 25 μg / ml for 30 minutes

 7. The manifestation of peroxidase activity of 3,3-diaminobenzidine tetrachloride (DAB) and PBS in the ratio of 500 μg DAB per 1 ml of PBS. 0.03% hydrogen peroxide is added to the freshly prepared solution and the sections are incubated with the substrate for 10 minutes. The reaction is stopped by immersion of the slices in distilled water. Subsequently, the sections are carried out with up-concentration alcohols and three portions of o-xylene, enclosed in a balm.

 When conducting an immunohistochemical reaction, a "positive control" is used, that is, sections of a similar tissue under investigation with a known positive reaction. In each case, apply a "negative control"; those. sections of a similar test tissue, in which during the immunoperoxidase reaction, 4 or 5 steps are omitted to detect possible background staining. In other words, an endogenous peroxidase clogging test is used.

 As a result of the reaction, a clear bright coloration of the cytoplasm of the cells of the malignant neoplasm, reaching a brown color, is obtained. The epithelial structures of the normal gland are not stained. Parenchymal formations of hyperplasia sites have an uneven golden yellow color.

 The proposed method allows simultaneous recognition in the histological section of epithelial cells of normal tissue, areas of hyperplasia and cancer. This allows you to increase the accuracy of diagnosis as individual of these states of the body, and to carry out differential diagnosis between them.

 Example 1. Immunohistochemical detection of ABH in breast tissue with the presence of foci of the correct typical proliferation of epithelium of ducts and lobules.

Fragments of the surgical material are fixed with a 15% aqueous neutral formalin solution for 24 hours at room temperature. Subsequently, the material is subjected to paraffin wiring, which consists of conducting it on a battery, including a number of liquids: o-xylene for 30 minutes, two portions of 70% and 80% ethanol for 6 hours each, three portions of 96% ethanol for 6 hours in each, absolute alcohol for 30 minutes, absolute alcohol - xylene in equal proportions for 2 hours, xylene paraffin in equal proportions at a temperature of 37 ^° C for 2 hours, three portions of paraffin at a temperature of 58 ^° C for one hour each. The paraffin-impregnated pieces are cooled in air and glued onto wooden blocks. Histological sections 5-8 μm thick are prepared using a microtome.

Histological sections are dewaxed by treating them in three portions of o-xylene for 5 minutes each and then heating the sections in an thermostat at 37 ^° C for three hours. Dehydration of the histological sections is achieved by treating them in four portions of ethanol (absolute, 96, 70 and 56%) for three minutes each. Next, the sections are transferred for 5 min in a 0.05 M solution of PBS pH 7.6. Blocking endogenous peroxidase (this and subsequent reaction steps is carried out in Petri dishes) is carried out with a fresh 3% hydrogen peroxide solution. The duration of the stage is 10 min. PBS 5 min.

Next, the sections are kept under a drop of 5% solution of equine serum albumin (50 mg of albumin in 1 ml of PBS). After that, excess albumin is shaken off and the glass slides carefully wiped around the slice. A drop of affinity-purified rabbit anti-ABH antibody is applied to the latter at a concentration of 8 μg / ml PBS. The antibody solution includes 3% albumin and 0.01% sodium azide. Incubation of sections was carried out for 30 min at 37 ^o C in Petri dishes with the creation of the conditions of a wet chamber (filter paper moistened with PBS was placed on the bottom of the cup). Two portions of PBS for 5 minutes each, after which the PBS solution is changed to a new one. Histological sections were incubated in secondary antibodies at a dilution of 10 μg per 1 ml of PBS for 30 minutes. SFR 5 min. Slices are treated with avidin-peroxidase complex for 30 minutes. Immediately before use, the complex is diluted 200 times in PBS. SFR 5 min. Immunological manifestation is carried out in a 0.03% solution of the substrate for 10 minutes The solution is prepared by mixing immediately before use 1 mg of DAB with 2 ml of PBS and 0.2 ml of 0.3% hydrogen peroxide. The reaction is stopped by rinsing the sections in two portions of distilled water.

 The result is read under different magnifications of the light microscope by the presence of cytoplasm staining in experimental micropreparations and the "positive control", in the absence of color - in the "negative control". Using the method showed that the cytoplasm of the epithelium of the ducts and lobules of normal breast tissue is not stained. In the foci of hyperplasia, the cytoplasm of epithelial structures is colored in golden yellow. Different cells of this zone have an uneven color, which is explained by the varying degree of their hyperplasia. The data obtained make it possible to more objectively distinguish between areas of intact and hyperplasticized epithelium, which increases the accuracy of diagnosis of hyperplastic processes in the mammary gland.

 Example 2. Immunohistochemical detection of ABH in breast cancer tissue.

A fragment of the operating material is fixed in an aqueous neutral solution of formalin of 15% concentration during the day at room temperature. Subsequently, the material is subjected to paraffin wiring, which consists in conducting it through a battery, including a number of liquids: o-xylene for 30 minutes, three portions of 70% and 80% ethanol for 6 hours each, three portions of 96% ethanol for 6 hours in each, absolute alcohol for 30 minutes, absolute alcohol-xylene in equal proportions for 2 hours, xylene-paraffin in equal proportions at a temperature of 37 ^° C for 2 hours, three portions of paraffin at a temperature of 58 ^° C for one hour each. The paraffin-impregnated pieces are cooled in air and glued onto wooden blocks. Histological sections 5-8 μm thick are prepared using a microtome. Histological sections are dewaxed by treating them in three portions of o-xylene for 5 minutes each and then heating the sections in an thermostat at 37 ^° C for 3 hours. Histological sections are dehydrated by treating them in four portions of ethanol (absolute, 96, 70 and 56 %) for three minutes each.

Next, the sections are transferred for 5 min in a 0.05 M solution of PBS with a pH of 7.6. Blocking of endogonal peroxidase (this and subsequent reaction steps is carried out in Petri dishes) is carried out with a fresh solution of 3% hydrogen peroxide. The duration of the stage is 10 minutes. SFR 5 min. Next, the sections are kept under a drop of 5% solution of equine serum albumin (50 mg of albumin in 1 ml of PBS). After that, excess albumin is shaken off and the specimen slides are carefully taken around the sections. A drop of affinity-purified rabbit primary anti-ABH antibody is applied to the latter at a concentration of 8 μg / ml PBS. The antibody solution includes 3% albumin and 0.01% sodium azide. Incubation of sections was carried out for 30 minutes at 37 ^° C in Petri dishes with the creation of a wet chamber (filter paper moistened with PBS was placed on the bottom of the cup). Two portions of PBS for 5 minutes each, after which the PBS solution is changed to a new one. Histological sections were incubated in secondary antibodies at a dilution of 10 μg per 1 ml of PBS for 30 minutes. SFR 5 min. Slices are treated with avidin-peroxidase complex for 30 minutes. Immediately before use, the complex is diluted 200 times in PBS. SFR 5 min. Immunological manifestation is carried out in a 0.03% solution of the substrate for 10 minutes The solution is prepared by mixing immediately before use 1 mg of DAB with 2 ml of PBS 0.2 ml of 0.3% hydrogen peroxide. The reaction is stopped by rinsing the sections in two portions of distilled water.

 Next, the sections are carried out through fresh ascending concentrations (56, 70, 96% and absolute alcohol) and three portions of xylene by thorough rinsing in each liquid. Then the slices are enclosed in a balm.

 Result: the cytoplasm of tumor cells is distinctly and uniformly stained yellow-brown, which is explained, given the absolute specificity of the method, by the presence of ABH in it.

 Output. The proposed method allows with high specificity and relative simplicity to conduct differential diagnosis of hyperplastic processes and breast cancer in histological sections. The pathomorphological diagnosis of malignant neoplasms, as you know, is by far the most informative. The method has a certain applied value, as it allows to facilitate, improve and objectify, i.e. to increase the accuracy of the process of histological diagnosis of breast cancer, especially its non-infiltrating forms, the diagnosis of which still presents significant difficulties in the work of practical pathologists.

Claims (1)

 A method for the differential diagnosis of hyperplastic processes and breast cancer, including an immunohistochemical study of parenchymal cells of their tissues on histological stained sections, characterized in that when immunoperoxidase staining of the histological structures of the micropreparation is used, primary antibodies against pregnancy-associated alpha-2 glycoprot are used in the processing of histological sections concentration of 8 - 10 μg / ml and in the presence of uneven staining of the cytoplasm of golden yellow cells the colors diagnose the hyperplastic process in the mammary gland, and in the presence of a uniform brown color - cancer.