Histology is the microscopic study of tissues of living organisms. The Histology Department of the Central Laboratory at Candos is mainly concerned with the examination of diseased tissues and the diagnosis of cancer in- patients from all hospitals and clinics of Mauritius and Rodrigues. The procedures adopted for the preparation of materials for microscopic examinations are known as histological techniques and it is with these techniques that the Medical Laboratory Technicians in the Histology Department are primarily concerned.
The various ways of preserving and processing tissues, cutting and staining sections and the ability to recognize whether or not the procedures have been performed correctly constitute the skill of the histology technician. For the work to be performed competently, knowledge of the complex histological structure of tissues to be examined is essential.
The Histologist / Histopathologist examines the preparation and issues a report. The report based on years of study and experience is in fact an opinion on the case under scrutiny and would decide whether a leg should be amputated or a breast removed. Opinions differ from pathologist to pathologist: one may think that a tumour is malignant, another may think that it benign. A final diagnosis that happens to be wrong can lead to disastrous consequences to the patient and the Histology department.
Histology –the study of microscopic anatomy-really got started after the development of the compound microscope in the nineteenth century. It has travelled a long way from the first time a cell was seen, when Robert Hooke observed cork cells in 1665. Although the compound microscope is still the most widely used type of microscope in research and medicine, variations of the compound microscope have been invented for use in special investigations. One uses polarised light to detect birefringent materials and another uses ultraviolet light with fluorescent dyes. The most important recent invention is the Electron microscope, which has opened the new field of ultra-structure research.
The object of histological technique is the treatment of a piece of tissue for microscopic examination. Fixation is the first step in preparing a piece of tissue for study. The surgeon upon removing the tissue from the body puts it immediately into a jar containing a fixative fluid such as 15% formol saline. Fixatives are usually a mixture of chemicals which act as preservatives, preventing autolysis and bacterial decay. Autolysis is the "self digestion" of the tissue caused by enzymes found in the cell. This happens within seconds of the cutting off of the blood supply causing alteration in the architecture of the tissue. Putrefaction caused by bacteria or fungi also accelerates the degeneration of the cells. Quick fixation minimises these changes. Protein is the principal component of most animal tissues and most fixatives such as alcohol, acetic acid, and mercuric chloride work on the proteins of the cell rather than the carbohydrate or fat constituents.
After fixation the tissue is infiltrated with a substance such as paraffin wax, celloidin, resin, or gelatin which will act as support while the material is being sectioned. Paraffin wax is the best support medium and is used routinely in the histology laboratory. It is, however, insoluble in water and in order to infiltrate the tissue thoroughly, all the water must be removed (dehydration) by passing through ascending grades of alcohol. The alcohol being immiscible with wax is replaced by a wax solvent such as xylene (clearing or de-alcoholisation). The tissue is then impregnated for two hours with molten wax placed in a thermostatically controlled oven and finally embedded in a mould containing molten wax. After cooling and solidifying, the piece of tissue, now encased in a block of wax, is ready for sectioning.
Sections are cut with a machine called a Microtome which produces a series of sections at a constant, though, adjustable thickness. The choice of thickness depends on the material and the purpose of the investigation. Sections are routinely cut at 5 microns which is about one cell layer thick. The skill of the Histology technician is reflected most in the cutting of sections. Nowadays disposable knives are available which facilitates the work of the microtomist.
A few years ago, microtome knives had to be sharpened regularly on a hone. A good microtome knife was valued beyond riches by its owner and it was said that the chastity of his womenfolk was far less important to the microtomist than the edge of his knife! Good knives were rarely left unguarded for a moment, and special precautions (chains and padlocks) had been used in many laboratories to repel the casual borrower. Now disposable knives are available in pack of 100, and section cutting has become comparatively very easy. The sections still embedded in wax are floated on the surface of a waterbath heated to 50 °C. to remove all wrinkles and then mounted on glass slides. The sections are dried on a hot plate. Before staining, the sections are de-waxed in xylene.
An entirely different approach is to freeze the tissue solid by liquid Nitrogen or Carbon dioxide and then sections cut using a freezing microtome or cryostat. The frozen sections produced in this way are important when time does not permit the production of wax sections as the patient is on the operating table and the nature of the lesion must be determined urgently. They are also needed to demonstrate the presence of materials such as fats and enzymes, which might be destroyed in wax techniques.
Most unstained tissues are almost transparent and the recognition of specific structures is difficult as all the various components of a tissue have more or less the same refractive index. Hence, there is no contrast. The purpose of staining is to render prominent the different tissue elements so that they may be easily recognized and studied.
The tendency nowadays is towards selective or differential staining by which tissue can recognise elements morphologically similar. The staining technique most commonly used in histology is " Haematoxylin and Eosin" which stains nuclei blue and other tissue elements shades of pink.
Histology has contributed a great deal to the advancement of science. It has become one of the most widely used research and diagnostic technique available to biology and medicine. It has found widespread use in medical research whenever direct observation of normal or pathological disease is required. It is very important for the diagnosis of cancer. About 8000 specimens are processed annually at the Central Lab, out of which about 12% turns out to be malignant.
Histology is widely used during the prolonged tests of drugs and cosmetics in order to determine their effects in the user. It is being used by the food processing industries to investigate changes within the tissues of stored food.
In the coming years, automation will increase the usefulness of Histology by making the many steps involved in processing a specimen cheaper and faster. Already processing and staining machines are in use. Staining methods will become more precise as more is learned about the chemistry of the cell. The expanding field of histochemistry and immunocytochemistry could become more diagnostic as we learn more about the chemical constituents of normal and diseased cells.
Author : Mr. A K DREEPAUL , PMLT , Histology & Cytology Dept. Central Lab , Candos