Cytogenetics
Cytogenetics is the science of studying the structure of chromosomes. In this science, chromosomes are analyzed using banding techniques or molecular cytogenetic methods. It is a branch of genetics that is related to the study of the structure and chromosomal composition of a cell, which includes the routine analysis of G-Band chromosomes and other banding techniques as well as molecular cytogenetics including FISH and CGH.
IHC immunohistochemistry
In immunohistochemistry (IHC) using monoclonal and polyclonal antibodies, it is used to detect and determine and distribute antigens. Tumor-specific antigens are either expressed in a new form or are increased in the body, and by detecting them, one can understand the health status of the cells in question.
In order to perform the immunohistochemical technique, sampling and biopsy are required. After performing the Zufaft process and preparing the sample in the form of a paraffin block, the samples are cut with a microtome and transferred to a slide. These samples are then incubated with an appropriate antibody. The binding site of the antibody to the antigen can be traced by a conventional or fluorescent microscope with a marker such as a fluorescent dye, enzyme, radioactive element or colloidal gold.
These markers are directly attached to primary antibody or secondary antibody.
FISH technique
The in situ hybridization technique was developed by Gall and Pardue as well as Jone et al. in 1969.This method has made it possible to locate genes or RNA or DNA sequences directly on chromosomes. In the FISH technique, single-stranded nucleic acids (usually DNA and sometimes RNA) interact with each other to form complexes or hybrids by creating molecules with high similarity or complementary sequences. After the hybridization of nucleic acids, the degree of homology of the sequences is determined. As a result, specific sequences are identified and located on specific chromosomes
In short, this technique is among the first histochemical techniques that includes the use of different categories of natural and synthetic dyes to stain cellular structures and intracellular communities. These compounds are generally non-specific and are used to classify specific molecules such as proteins, nucleic acids, lipids and carbohydrates. The first in situ hybridization was performed in the late 1960s, which did not use fluorescent materials and instead used probes labeled with radioisotopes.The FISH method is also used to detect nucleic acids and is an alternative to old methods that used radioactive probes.This method requires the hybridization of the nucleic acid probe with the desired nucleic acid in situ.
Flowcytometry technique
Cytometry means measuring and examining different cell components in a sample. Flow cytometry device determines multiple cell components and counts them simultaneously. Characteristics that can be measured by flow cytometry include cell size, cytoplasmic complexity, DNA and RNA content of the cell, and a wide range of intracellular or membrane-bound proteins.
In the flow cytometry method, stained cells are placed in a fluid flow either by means of monoclonal antibodies attached to fluorescent or fluorochromes attached to cellular components and pass individually in front of a light beam (laser) and then the light is scattered. and side fluorescence is collected by detectors. These detectors convert optical signals into electrical signals corresponding to the collected light. Light scattering at different angles can differentiate cells based on differences in size and internal complexity, while light emission from fluorescently labeled antibodies can differentiate cells based on differences in surface and cytoplasmic antigens. In this way, the cells can be distinguished based on characteristics such as the granulation volume and the amount of staining.
IHC immunohistochemistry
Immunohistochemistry is based on antigen-antibody detection by light microscopy to determine a specific antigen in a tissue, cell or a specific phase of the cell's life cycle.
Antigen is a protein related to the structure of different cells, including smooth muscle epithelial cells, striated muscles, lymphoid cells, or others.
Objectives of using immunohistochemistry
• In identifying the nature of cells and type of tissue
The use of immunohistochemistry in poorly differentiated or undifferentiated tumors plays a significant role in diagnosis.
Sometimes it is difficult to distinguish lymphoma from undifferentiated carcinomas or malignant melanoma or even sarcoma. In these cases, a final diagnosis can be reached by using a panel of relevant markers.
For example, it is possible to differentiate between lymphoma and undifferentiated carcinoma tumors using Pan cytokeratin and LCA (CD45) markers.
Or to differentiate epithelial tumors from malignant melanoma, pancytokeratin Melan A, HMB45 markers can be used and the final diagnosis can be made.
Also, for definitive diagnosis of neuroendocrine tumors, markers can be used that determine the nature and origin of the tumor, and this issue is considered important in terms of determining the type of treatment for the patient.
The markers used in the diagnosis of neuroendocrine tumors are NSE Chromogranin, Synaptophysin.
The opposite image is related to Synaptophysin staining in neuroendocrine tumor.
PCR method
This method is extremely simple and several processes can be performed one after the other by using temperature changes. This technique can be used as a powerful diagnostic method for the presence of mutations in the human genome, or to introduce specific mutations into the homologous gene.
PCR was performed manually by laboriously placing and moving test tubes between water baths of the required temperature. Today, devices are commercially available in which tube stations are fitted with heatable metal blocks and can be programmed to quickly change between the required temperatures. n PCR cycle amplifies the desired DNA 2n times.
