If a patient is suspected of having a tumor-like formation, the doctor prescribes a series of standard examinations.

The diagnosis of a malignant lesion can only be made on the basis of an assessment of changes in the biopsy specimen - immunohistochemical study (IHC) is considered one of the most reliable ways to detect tumor cells.

What is an immunohistochemical study?

The essence of immunohistochemical research is the study under a microscope of samples of biological material, that is, tissues obtained by biopsy.

Tissues are pretreated with certain specific antibodies.

And cancer cells themselves have been the objects of comprehensive research for several decades.

In the course of these studies, scientists were able to establish that tumor cells produce specific proteins or, as they are also called, antigens.

These proteins have the ability to bind to antibodies. It is on this principle that the IHC is built - the tissue taken from the patient for research is processed with a number of standard antibodies and a study is carried out under a microscope.

If antibodies interact with cancer cells, then they acquire the property of fluorescence, that is, a glow occurs that differs in different wavelengths. That is, if such a change in the biopsy specimen under study is detected, then it is already possible to expose a malignant formation with a greater degree of probability.

To date, antibodies to most of the most common neoplasms have been developed and are actively used.

Immunohistochemical study allows:

• Determine the type of neoplasm and its subspecies.
• Establish how common the primary cancer focus is throughout the body.
• Determine the source of metastases if the biopsy is from secondary cancers.
• Assess how effective the treatment of patients with cancer is.
• Determine the stage of the malignant neoplasm.
• To find out the proliferation of tumors, that is, to establish the growth rate of neoplasms.

IHC is considered to be a more informative method compared to the more familiar histological one. But in some cases, it is histology that is needed, so it is advisable to use both analyzes.

Indications

The IHC method can be used to study almost any tissue in the human body. This type of study is prescribed mainly if there is a suspicion of the development of a tumor process.

Immunohistochemical study is used:

• To determine the type of primary, mostly solitary, neoplasms.
• to detect metastases.
• When it is necessary to determine the prognosis of the development and course of tumors.
• As a method for studying receptors for a number of hormones.
• To determine the type of lymphoproliferative conditions.
• For the detection of microorganisms.

There are no contraindications to IHC. This analysis cannot be performed only if there are insurmountable difficulties in taking samples of damaged tissue.

How is the analysis carried out?

IHC is carried out in several stages, the very first is pre-laboratory, that is, tissue sampling for analysis.

The sample is obtained by biopsy, in some cases it is possible to pinch off a piece of tissue or remove it during an endoscopic or surgical operation.

How biological material will be obtained depends on the location of the neoplasm and on its type. The collected material is placed in formalin and only after that it is sent to the laboratory center.

In the laboratory, a tissue sample is subjected to several changes:

• The material is degreased and filled with paraffin. Thus, histological blocks are obtained, they can be stored forever, therefore, IHC can be repeated if necessary.
• The next stage of microtomy is obtaining the thinnest sections with a width of up to 1.0 microns from paraffin blocks. Sections are distributed on special glasses.
• The resulting sections are stained with immunohistochemical preparations, that is, with antibody solutions in a certain concentration. A small panel can be used for research - it includes 5 types of antibodies. A large panel contains from 6 to dozens of types of markers. Which antibodies will interact with tumor cells depends on the proposed type of neoplasm.

The results of the conducted IHC become known within 7-15 days.

IHC in breast cancer

Certain immunohistochemical markers have been deduced for determination. If a malignancy is suspected, the number of receptors such as estrogen and progesterone is mandatory.

An excess of these hormones provokes the growth of a malignant formation and affects the appearance of metastases. IHC for the expression of progesterones and estrogens allows you to more accurately determine the stage of the disease and find out whether hormonal treatment is indicated in this case.

Tumors with a high concentration of hormones are mostly indistinguishable increased activity and successfully treated with antihormonal drugs.

When conducting IHC, such an indicator as Ki-67 is necessarily determined, it indicates the malignancy of the process. If in breast cancer Ki-67 reaches only 15%, then the outcome of the disease is considered favorable.

At a level of 30%, they talk about the rapid rate of tumor development, the cessation of its growth occurs under the influence of chemotherapy. If the indicator is less than 30%, then the treatment of patients is carried out with hormonal agents.

Examination of patients made it possible to establish that if Ki-67 is less than 10%, then the survival rate approaches 95%. At a level of this indicator of 90%, death is observed in almost one hundred percent.

Immunohistochemical examination is prescribed not only for malignant lesions of the mammary gland, this study is informative for:

• infertility.
• Pathological changes in reproductive function.

endometrial IHC

Immunohistochemical examination of endometrial tissues is prescribed:

• With infertility.
• With frequent miscarriage.
• Patients with multiple unsuccessful attempts ECO.
• When diagnosing chronic form endometrium.

IHC allows you to determine whether there are cells that prevent conception. This analysis simultaneously determines how the uterine tissue receptors react to hormonal stimulation, if pathology is detected, then this indicates changes in the endometrium - endometritis, hyperplasia, desynchronization of the process of endometrial differentiation.

It is these violations in two out of three cases that become the main causes of problems with conception and gestation.

The collection of endometrial tissue, depending on the detected pathology, is carried out in different days cycle, the day of the biopsy should be appointed by the doctor. With the help of IHC, you can set cancer diseases and HPV infection of several subspecies.

Deciphering the results

The pathologist examines the prepared samples, he must have a certificate of special preparation for conducting analyzes using the IHC method.

In conclusion, indicators of antibodies are taken out, to which the tropism of the material under study is determined.

Be sure to indicate the morphological structure of the tissue, that is, the type of atypical cells and their number.

The detection of certain antigens indicates the type of cancer pathology. But it must be borne in mind that the diagnosis is made only on the totality of the data of all diagnostic procedures. An oncologist can interpret the IHC results.

Research price

The cost of an immunohistochemical study depends on how many antibodies are used in the analysis.

A standard study (from 2 to 5 antibodies) in most clinics costs between 4-5 thousand rubles. If up to a dozen antigens are detected at once, then the cost can be much higher - from 15 thousand rubles.

Video on how immunohistochemistry helps to diagnose a malignant tumor:

Immunohistochemical (IHC) study is a method for identifying specific antigenic properties of malignant tumors. are used to detect the localization of a particular cellular or tissue component (antigen) in situ by binding it to labeled antibodies and are an integral part of modern diagnostics cancer, providing detection of localization in tissues of various cells, hormones and their receptors, enzymes, immunoglobulins, cell components and individual genes.

Goals of the IHC study

IHC studies allow:

1) to carry out histogenetic diagnosis of tumors;

2) determine the nosological variant of the neoplasm;

3) identify the primary tumor by metastasis with an unknown primary focus;

4) determine the prognosis of a tumor disease;

5) determine the malignant transformation of cells;

6) identify opportunities;

7) identify both resistance and sensitivity of tumor cells to chemotherapeutic drugs;

8) determine the sensitivity of tumor cells to radiation therapy.

How is an IHC study performed?

The IHC study begins with the collection of material. To do this, it is carried out, in which a tissue column is taken from the tumor and nearby tissues, or the material comes from the operation. Then the material is fixed. After fixing, the material is sent to the wiring, which allows you to prepare it for work (degrease and additionally fix it). After wiring, all samples are embedded in paraffin, receiving histological blocks. Paraffin blocks are stored forever, so it is possible to conduct an IHC study in the presence of paraffin blocks made earlier.

The next stage of the IHC study is microtomy - the laboratory assistant makes sections from paraffin blocks up to 1.0 microns thick and places them on special histological glasses.

Then sequentially routine staining and immunohistochemical examination are performed, allowing at each stage to more and more differentiate the phenotype and nosology of the tumor.

As you can see, an IHC study is a complex multi-stage process, and therefore, for an IHC study, you should choose the most modern laboratory with highly qualified specialists and a high degree of automation - this way you will negate the risks of receiving poor-quality diagnostics. Such a laboratory today is UNIM.

Separately, it should be said about the timing of the this study. On average in Russia, IHC research is carried out in terms of 10 days to several weeks. When contacting UNIM, you can do an IHC test in just 3 days! Also, the advantage of conducting IHC research at UNIM is your materials for research from any city in Russia. If necessary, specify the cost by submitting an application for research, or call the hotline (free of charge in Russia): 8 800 555 92 67.

Alternative names: IHC, English: Immunohistochemistry or IHC.

Immunohistochemical examination is a special method for diagnosing tumor diseases. The essence of the method is to study under a microscope tissue samples previously treated with specific antibodies.

At present, antibodies have been created to almost all tumors.

With the help of immunohistochemical studies, the following is performed:

Indications for immunohistochemistry

With this method, you can examine any tissue. The main indication for carrying out is the suspicion of a tumor process.

The following indications for IHC are defined:

Contraindications

There are no contraindications to this research method. Its implementation is impossible only if it is not possible to obtain a biopsy material.

How is an immunohistochemical study performed?

The study itself consists of four stages:

The results of the study become known after 7 days (with a standard study - "small panel") or after 15 (extended study - "large panel").

Interpretation of results

The study of samples is carried out by a pathologist who has undergone special training in IHC. In conclusion, the doctor notes to which antibodies the tropism (affinity) of the tissue is determined. Additionally, the morphological structure of the sample is described - which cells and in what quantity are present.

The detection of tissue affinity for certain standard tumor antibodies indicates a specific type of cancer.

Additional Information

Immunohistochemical study is currently the most accurate method for diagnosing tumor diseases. It allows you to make a final diagnosis with an accuracy of up to 99%, determine the type of tumor, and identify its primary localization.

Literature:

Sometimes the results of histology are not enough. If it is not possible to obtain a definitive answer and make a diagnosis, or it is necessary to clarify the molecular parameters of the tumor formation, an immunohistochemical study is prescribed.

What it is

The method of immunohistochemistry is used as an additional method. It helps to identify specific proteins in the biological material that are characteristic of a particular type of cell and, as a result, to distinguish one type of tumor formation from another. The study also helps to identify:

1. Immunohistochemistry markers that are responsible for tumor sensitivity to exposure medicines, radiation therapy.
2. Metastatic nature of the origin of the formation and determine the primary source of the lesion.
3. Hormones and their receptors.
4. cell malignancy.

IHC as a diagnostic method is widely used in oncology. This type of tissue study involves the use of certain reagents. They contain antibodies or protein compounds that are labeled with certain substances.

IMPORTANT! Antibodies (immunoglobulins, specific proteins) are produced by various cells of the body and provide immunity when an antigen is introduced. They are often produced when viruses, bacteria enter, during oncology, vaccination, tissue transplantation, and sometimes against their own cells, which leads to autoimmune pathologies. They are obtained from various animals. [>

Upon contact with antigens that may be contained in the studied biopsy, a specific reaction occurs (antigens are intercellular substances of tissue, components of cellular structures). As a result, it is possible to judge the presence or absence of a particular substance in the tissues.

IMPORTANT! Antigens - a genetically foreign body that, when ingested, causes an immune response, namely the production of antibodies to suppress this foreign agent. In the case of IHC analysis, antigens are molecules that are produced by pathologically altered tumor cells.

Structural specificity of the study - fundamentally distinguishes IHC from other methods of immunological diagnostics that use the antigen-antibody reaction. That is, experts evaluate, in addition to the presence of certain substances in tissues (with or without staining) and their quantitative composition (by staining intensity), also the spatial distribution of various structural elements (cytoplasm, nucleus, cell membrane, etc.) in a thin colored section.

Research Methods

The reaction can be obtained in several ways. A direct approach is often used, in which one type of labeled antibody is used. The latter must bind to the detected substance or target molecule. If the latter is present in the biopsy, then the tissue under study is stained.

The indirect method is considered more informative and sensitive. Its essence lies in the phased introduction of 2 different antibodies. Unmarked primary antibodies react with the tumor antigen in the biopsy, forming an antibody-antigen complex. Then, secondary already labeled antibodies (reagent) are injected, which bind to a certain part of the primary antibody molecule. That is, primary antibodies are antigens for secondary ones.

When is the study scheduled?

IHC is prescribed when the usual histological examination is not enough. For example, when a breast tumor has a non-specific microscopic structure that does not allow an accurate final diagnosis.

What shows

Biopsy with immunohistochemistry is an important part of the verification of the diagnosis in cancer of the breast, prostate, soft tissues, etc. The study is especially important for the detection of lymphomas, since it is often impossible to diagnose this disease without IHC. The IHC study plays an important role in determining the histological type of undifferentiated tumor formations, as well as metastases that have an unexplained primary focus.

Immunohistochemistry of breast cancer is useful in predicting the course of the tumor process and the possibility of targeted therapy. It helps to evaluate:

• functional state of formation cells;
• malignant potential and tumor growth rate;
• tendency to metastasize.

How is the analysis done?

To conduct a study, samples of the tumor itself and nearby tissues are needed. They can be obtained by biopsy or after surgical intervention. The material must go through several stages of preparation before the study: fixation, washing, dehydration, immersion in paraffin or filling with another substance.

The biological object under study should be dense, but not fragile. First, the material is washed, dehydrated and compacted in alcohols of increasing strength. It is then clarified by immersion in a 1:1 mixture of absolute alcohol and O-xylene. After that, they are placed in several portions of pure O-xylene. And only then the biological object is impregnated in paraffin. The last stage helps to conserve the tissue and preserve its structural features in the paraffin block for a long time.

Then the laboratory assistant makes sections from these blocks, using a special device - a microtome, up to 1 micron thick. The resulting sections are glued to a glass slide, which is lubricated with glycerin and a mixture of protein. Dry.

In order for the test material to react with the antibody, it must be degreased and paraffin removed. The procedure is called deparaffinization. For its implementation, 3 portions of O-xylene are used, as well as alcohols of descending strength.

The last step is coloring. It is carried out in order to clearly see the structure of tissues under a microscope. Laboratory assistants can use a huge amount of dyes. Conventionally, they can be classified according to:

• origin, for example, animals - carmine, vegetable - hematoxylin, synthetic - eosin;
• chemical properties, for example, neutral, basic, acidic.

Most often, eosin, hematoxylin are used for this procedure. The biological material is placed in a hematoxylin solution for several minutes, then washed and the staining results are differentiated. After that, staining in eosin, washing and dehydration in alcohols of ascending strength are foreseen. Sequential multi-stage staining allows at each stage to more and more determine the nosology and phenotype of the tumor formation. The pathologist examines the material under a microscope after each staining. And only after assessing the changes and interpreting the information received, appoints a panel of antibodies.

To obtain accurate data on the pathological process and verify the disease, the laboratory assistant starts processing the prepared sample with special reagents containing labeled antibodies. The result is a reaction between the antibody and antigen. This is accompanied by a characteristic visualization that depends on the substances used for labelling.

What do the results mean?

Immunohistochemistry of the breast is a complex multi-stage analysis, the result of which is influenced by many factors, for example, poor quality of reagents, insufficient fixation of the biological object under study, or an incorrectly selected antibody panel. The number of desired target cells is in the hundreds, so the specialist must understand what he is looking for and use the necessary antibodies for this.

Histologists evaluate the staining intensity of tumor cells after interaction with reagents using a special scale:

• 0-1+ - no oncology;
• 2+ - oncological formation is negative;
• 3+ - oncological education is positive.

The duration of IHC in breast cancer largely depends on the method of its implementation. There are manual and automatic. The latter is considered more perfect, since the probability of a technical error is minimized, and it is carried out much faster.

Before determining the tactics of treatment, the oncologist asks various questions: what is the likelihood of a recurrence of the disease in a patient or his death, what result of treatment can be expected. To do this, the specialist studies the features and characteristics of the tumor and, depending on various predictive and prognostic factors, prescribes treatment tactics.

Prognostic factors

These factors include fundamental signs that determine the peculiarity of the course of the disease and its outcome. With operable forms of breast oncology, such factors include: the morphological structure and its size, the state of the lymph nodes. That is, such characteristics determine the chronology of the disease.

Features of prognostic factors:

1. Based on the biological characteristics of the tumor formation.
2. Interrelated with the patient's life expectancy, regardless of the tactics of therapy.
3. Divide patients according to the degree of risk of death, relapse.
4. Examples: index of mitotic activity, proteolytic enzymes, antigenesis, etc.

The mitotic activity index (MAI) is an independent prognostic factor that characterizes the life expectancy of patients. In patients with an index less than 10 during the follow-up, the lethal outcome was 6%, in patients with an index greater than 10 - 28%.

Proteolytic enzymes play a role in the process of metastasis. In transformed malignant cells, more of these enzymes are produced than in healthy ones, therefore, in malignant tumors, these substances are released into the intercellular space and increase in activity. That is, enzymes are involved in the process of oncogenesis. That. patients with low levels of these enzymes have a better prognosis for disease-free survival.

The ability of a neoplasm to grow and metastasize largely depends on angiogenesis - the formation of new blood vessels. During the observation of patients for 10 years, it was found that in women with a high value of microvessel density in the tumor, the prognosis is worse.

Predictive factors

These factors predict the effectiveness of treatment. They help to identify a group of patients with a more or less significant response to therapy. For example, to determine the efficiency hormonal drugs, the level of steroid hormone receptors.

Clinical factors that help interpret response to therapy in metastatic cancer include:

1. Localization and quantitative indicator of metastases. For example, if the bone is damaged, the prognosis is relatively favorable; damage to the bone, 2 organs, lungs - less favorable; with damage to more than 2 organs, the presence of metastases in the liver, brain, etc. - unfavorable.
2. The time interval between the treatment of the primary neoplasm and the appearance of metastases. The longer it lasts, the better the prognosis.
3. The general health of the patient.
4. Age category. Postmenopausal women are more likely to have a favorable response to therapy.
5. Steroid hormone receptors in the tumor (hormone-independent neoplasms do not respond to hormonal treatment).

Metastatic breast cancer is considered a chronic pathology. After a complete regression obtained as a result of chemotherapy, a life expectancy of 5-10 years is quite rare, in about 5-20% of cases. Therefore, treatment is considered the best option- consistent application of therapeutic effects with as little toxicity as possible in order to obtain the maximum palliative effect and delay the progression of pathology, death.

The reliability of breast IHC results reaches 99%. The technique is sensitive and highly specific. It helps to verify pathological process and establish the stage of the disease. For its implementation, a minimum of biopsy and reagents is required, while it will be informative even after a long time after taking a biopsy.

Method of determination Histological examination of the biopsy according to the histological classification of the World Health Organization (WHO) with hematoxylin-eosin staining. IHC study using a spectrum of tissue-specific and prognostic antibodies (up to five antibodies) (peroxidase and avidin-biotin methods).

Material under study See in the description

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A comprehensive study of biopsy specimens of metastatic formations of various localizations, including a morphological description and assessment of the expression spectrum of tissue markers, to determine the histogenesis of the primary tumor focus.

According to statistics, in 3-15% of cases, malignant tumors manifest as metastases. Metastases without an identified primary focus are characterized by random, atypical localization and rapid progression of the process. The average life expectancy of patients with three or more metastatic lesions is three months. At the same time, the localization of the primary focus in 60-70% of patients is detected only at autopsy. Timely IHC diagnostics determines the tactics of treatment before the identification of the primary focus.

Pathological diagnosis of metastases of undetected tumors involves determining their morphological type, clarifying the likely source of metastasis, and assessing the malignant potential. The first task is solved with routine histological examination biopsy material (staining with hematoxylin-eosin), the second - using special research methods: histochemical, immunohistochemical (IHC) or molecular genetic method FISH (Fluorescence In Situ Hybridization).

In routine morphological diagnosis, metastatic tumors, according to the recommendations of the European Society for Medical Oncology ESMO (European Society for Medical Oncology) (2004), are divided into five major categories: adenocarcinoma, squamous cell carcinoma, neuroendocrine cancer, undifferentiated cancer, undifferentiated tumor. These morphological categories, along with data on the prevalence of the process, in many cases make it possible to determine an adequate examination (and treatment) plan. So, for example, when detecting metastasis of squamous cell carcinoma in the cervical lymph nodes endoscopic examination of the organs of the upper respiratory and digestive tract is necessary. IHC study, depending on the morphological type of neoplasm, allows to clarify the histogenesis of the tumor and/or to determine the probable localization of the primary focus.

For IHC studies of primary tumors and their metastases, a wide range of tissue-specific markers is used: - cytospecific (clusters of leukocyte differentiation (CD - Clusters of Differentiation), smooth muscle actin, myoglobin, thyroglobulin); - proliferation markers (Ki67, PCNA - Proliferating Cell Nuclear Antigen); - tumor markers– oncofetal antigens (fetoprotein, carcinoembryonic antigen); hormones (estrogen, progesterone); -enzymes, protein products of cellular oncogenes, etc.

Uniform algorithms for IHC study of tumor metastases without a specified primary focus have not been developed. Correct determination of the direction of differentiation of tumor cells and a number of biological parameters of the tumor is in itself an indication for prescribing certain therapy regimens. For example, the detection of metastases of adenocarcinoma in the lymph nodes of the axillary region may be an indication for therapy similar to that of breast cancer of the corresponding stage. Detection of the expression of estrogen and progesterone receptors in such adenocarcinoma may be an indication for the appointment of antihormonal therapy, regardless of the presence of a detectable tumor node in the mammary gland. An example of an algorithm recommended for use in differential diagnosis.

Material for research: tumor biopsy fixed in paraffin block.

Literature

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