INTRODUCTION TO HISTOPATHOLOGY & CYTOPATHOLOGY
Cells are the simplest unit of living matter that can maintain life and reproduce within themselves. Groups of these cells unite to perform a specific function, called tissues. The tissues of the body consist of a large number of cells and they are classified according to the shape size and functions of the cells.
Histology is the study of typical tissues particularly involving the investigations of microscopic structures of the more specialized tissues including the detailed knowledge of structures of individual cells. Histology and Cytology, i.e. the study of normal cells, are very closely interrelated and in most histological preparations, the components of the individual cells are also studied.
Ideally, microscopic examination of the living tissues is carried out so as to obtain a true picture of the cell and its components or any abnormalities, but only small primitive living organisms can be examined in this ways as most tissues are too thick and inaccessible for direct inspection. Thus, the majority of the histological techniques are applied to preserve the tissues in such a way so as to retain the structures as closely as possible to that of living tissues. It must be appreciable that the completed Histological operations from all methods show certain alterations of the cells and tissues as a whole and these changes are called artifacts. These changes may occur during the preparation of specimens, the specific methods are used for appropriate purposes causing least tissue damage.
Histopathology is the study of diseased or atypical tissues present in the body. Histopathology laboratory prepares tissue sections for establishing the histopathologic diagnosis. Because of its convincing Physical evidence, histotechnology has proved to be one of the most effective tools for diagnosing tissue abnormality and cancerous conditions. In the recent years with the advent of the freezing microtome, the teamwork of the surgeons and histopathologists has greatly contributed to the progress of Medical Science.
The specimen submitted to the histopathology laboratory comes mostly from the operation theatre in the form of small pieces of tissues (biopsies) or organ. The specimens are submitted either fresh (unfixed) or immersed in a fixative fluid. The basic steps of specimen processing include fixation, embedding, microtomy, staining, mounting and microscopic examination.
Histopathology specimens – Specimens received for histopathology examination ranges from small biopsies to complete organ specimen. They are removed out of body using following techniques at the time of surgery:-
Incision biopsy – In this, only a portion of the lesion is sampled. This procedure is strictly used for diagnostic purposes.
Excision biopsy – In this, entire lesion is removed usually with the rim of normal tissues. This procedure serves both as diagnostic and therapeutic purpose. These specimens are received for examination in the laboratory by biopsy. Similarly, already processed specimens are received in the form of slides and paraffin blocks are also received for ancillary tests.
Cytopathology is the study of diseased or abnormal cells. Exfoliative Cytology, concerned with the early detection of tumors or cancer. The malignant cells diffuse into the body fluids and secretions. The exfoliative cytological technique deals with the study of body fluids and techniques deal with the study of body fluids and secretions by direct smear method and also by the study of the concentrated material.
The pathologist evaluates the smears for exfoliative cells as well as the tissue sections, the Characteristics of not only cancerous and precancerous conditions but also a variety of other alteration produced by inflammatory and degenerative processes. There are some inherent problems faced by the lab in preparing specimens for histological studies, as soon as the tissue is removed from the body for histological examination, it is cut off from its blood supply and begins to decompose (autolysis). To preserve as nearly as possible the natural state of the tissue or cell it is essential to check the autolysis with a minimum of delay which is accomplished by placing the tissue in certain chemicals which prevents the decomposition of tissues.
These tissues are either too soft or too hard and calcified which makes them difficult to cut into microscopic sections. Thus the procedure of decalcification, dehydration, and embedding precede microtomy and staining.
As the specimen reaches the histopathology laboratory, first of all, the morphological description of the tissue is noted by the pathologist and following Gross examination and internal examination of the tissue, a portion of the tissue is trimmed and handed over to the Histotechnologist for laboratory processing. The specimen processing includes the following basic steps:
- Fixation – when a tissue is removed from the body it undergoes self-destruction. To preserve the natural state as nearly as possible the tissue is immediately placed into a solution called fixative and the process is called fixation.
- Dehydration – After Fixing the tissues in proper fixatives, the water content of the tissue is necessary to remove out to prepare it for the embedding as mostly used embedding media are immiscible with water.
DEHYDRATION OF TISSUES – TECHNIQUE OF REMOVING WATER FROM THE TISSUES
- Clearing – Clearing of tissues is done to make the tissues more transparent and to remove the alcohol content of the tissues that are commonly used in dehydration process because the embedding medium is still not miscible with alcohol. so the alcohol is replaced with a medium which is miscible with both alcohol and embedding medium and also makes the tissue more transparent.
- Embedding – To provide necessary hardness to cut sections, infiltration of paraffin or other embedding media is carried out followed by the impregnation of the same embedding media into the tissue which makes the tissue hard for easy sectioning into the thin slices.
- Microtomy – The Thin sections or slices of the tissue are cut by using equipment called microtome, which is performed after the tissue get impregnated by paraffin and solidified followed by molding it into the block. The thin sections of the block containing tissue are cut and placed over the slide for the next step i.e. staining.
- Staining – The paraffin from the sections on the slide is removed by drying and then by dewaxing. During dewaxing, the sections on the slide are treated first with Xylene and then the sections are rehydrated with Ethanol in decreasing concentrations. The rehydrated sections are stained by using appropriate staining methods.
- Mounting – This process is done to preserve the stained tissues on the slide. After the appropriate staining procedure, the sections on the slides are dehydrated by treating with Ethanol in increasing concentration and finally with Xylene. After this the mounting media such as DPX or Canada balsam and then a coverslip is placed over the stained sections on the slide, the tissue section is said to be mounted.
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