Introduction of Histopathological Techniques

Histopathological Techniques and Cytology

Introduction Histology is the microscopic study of normal tissues; and histopathology is the study of abnormal or diseased tissues. This division of medical laboratory science was once referred to as morbid anatomy. At present, many workers prefer to call it 'cellular pathology'. 

Cytology, 

the study of cells, is an integral part of histopathology, and in most histological preparations, the components of individual cells are studied. Most of histopathological techniques are applied to killed tissues which have been fixed in such a way that they retain their structures and components as closely as possible to those of living tissues.

There are various techniques and methods designed and employed in histopathological investigations as it is impossible to study all normal and abnormal tissue components in a single preparation. 

For this reason, preparation of thin sections enables separate sections to be studied and stained in a variety of ways. However, a good knowledge of the structures of cells, organs and tissues is essential for the study to be worthwhile.

THE CELL

 

All living things are basically protoplasm. The protoplasm is contained within minute units, the cells. Aggregate of cells make up the tissues that form the human body. The name protoplasm is given to the main constituents together with water, proteins, carbohydrates, lipids, and inorganic substances. 

The cell which is a bundle of protoplasm enclosed within a plasma membrane has two main parts Cytoplasm and Nucleus

Cytoplasm The cytoplasm is surrounded by the cell membrane which allows selective flow of substances to and from the cell. 

Within the cytoplasm lies a variety of very fine structures known as organelles. These structures comprise the living material of the cell and each has its individual functions. In addition to the organelles, the cell contains some non-living substances which are collectively referred to as inclusions. 

Endoplasmic reticulum (ER) The reticulum is continuous with the outer layer of the nuclear membrane. Parts of the lining of the canals (tubules) are coated with granules of ribosomes (ribonucleoprotein) and this is referred to as rough or granular ER. The parts not coated are called smooth or agranular ER and are considered to synthesise fats and similar substances in some cells. 

Mitochondria These are small bodies involved in cell respiration. They are filamentous, measuring up to 7 nm in length and between 0.5 - 1.0 nm in diameter. They vary in numbers and may be evenly distributed in the whole cytoplasm or form aggregates in selected sites. They can be demon strated either in fresh unfixed specimens or in stained preparations. 

They are often referred to as the power house of the cell. They disappear quickly on the death of the cell due to autolysis. They are easily destroyed by acetic acid which therefore should not be part of cytoplasmic fixative.

Golgi apparatus The golgi apparatus is a specialised area of the smooth endoplasmic reticulum. It is associated with storage and synthesis of secretions prior to their being excreted from the cell. It has affinity for silver salts and osmium tetroxide. 

Lysosomes  Lysosomes are small spherical parcels of hydrolytic enzymes which break down large complex molecules into smaller molecules. When the lysosomes rupture, the enzymes are released and this causes death of the cell. This is known as autolysis. 

They are found in macrophages and leucocytes where they help in the phagocytosis of bacteria and the digestion of nutrient particles. 

Vacuoles The vacuoles are compartments present in the cytoplasm and are lined by membranes. They contain water, salts and nutrients.

Centrioles and centrosome The centrosome is the cell centre which is present in all cells but is only seen during cell division. It is seen in section as a clear area of cytoplasm just about 1.0 nm in diameter. The nucleus lies within the centrosome. 

Lying near the nucleus are pairs of small cylindrical bodies called the centrioles. They are associated with the organisation of chromosomes during cell division and in the formation of fibrillary material such as cilia.

Cytoplasmic inclusions Cytoplasmic inclusions are non-living substances that are seen in the cytoplasm. They are usually stored nutrients and particles produced or ingested by the cell. 

The common ones are:

Glycogen Liver cells act as store for accumulated glycogen when seen in sections. The glycogen is seen as fine granules or as larger amorphous masses. 

Fat Fat is normally stored in the fat cells though it may occur in other cells especially in pathological conditions. The accumulation of fat leads to the formation of minute globules which come together to form larger globules. 

Cytoplasmic granules Scattered throughout the cytoplasm are small globules which on fixation coagulate to form granules. They are involved in secretion hence they are also referred to as secretion granules. 

Pigments Pigments are frequently present in the cytoplasm of cells. They may be endogenous or exogenous in nature. Endogenous pigments like melanin and haemosiderin are produced within the body while exogenous pigments are particles of foreign matter which are phagocytosed and absorbed, for example, coal dust. Another group of pigments found in the cells is produced as a result of fixation or precipitation of the staining solutions. These pigments are easily identified and removed.

Nucleus

The nucleus controls the activities of the cell, especially those associated with reproduction. It is surrounded by two membranes which are similar to the cytoplasmic membranes. The nucleus contains the chromatin of the cell.

Chromatin This is the material from which chromosomes are formed when the cell divides. Chromatin granules are scattered all over the nucleus and have affinity for basic dyes. 

The chromatinis mainly made up of deoxyribonucleic acid (DNA), the molecules of which store the genetic information of the cell. Lying within the nucleus of most cells is a small spheroidal body called the nucleolus. It is very rich in ribonucleic acid (RNA).

Chromosomes Small thread like bodies which are seen in the nucleus during cell division are the chromosomes. A chromosome has a bifid structure formed by two chromatids lying side by side and joined together at the centromere. There are 23 pairs of chromosomes in the normal body cells: one of each pair being derived from the father and the other from the mother. 

They are referred to as diploid sets and are made up of one pair of sex chromosomes and 22 pairs of somatic (body) chromosomes also known as autosomes. In the female, the sex chromosomes are similar and are symbolised as XX while the male ones are dissimilar and are designated as XY.

The female ovum and the male spermatozoan contain the haploid set which is a single set of 23 chromosomes. One half of the spermatozoa contains an X chromosome and the other half contains a Y chromosome. The chromosomes of the spermatozoan therefore consist of 22 autosomes and I X or Y. That of the ovum consists of 22 autosomes and I X. When the ovum and the spermatozoa fuse together (fertilization), the sex of the new embryo is determined by the sex chromosome carried by the spermatozoa.

METABOLISM OF THE CELL

Metabolism (the chemical reactions which occur in the body) consists of:

1. Anabolic reactions during which the essential constituents of the cells are synthesized. For example, the synthesis of protein from amino acid. Adenosine triphosphate (ATP) supplies the energy necessary for anabolic reaction.

2. Catabolic reactions are those during which complex substances are broken down to yield simple molecules that produce the energy rich compound ATP. These reactions involve the oxidation of nutrients such as carbohydrates, lipids and proteins by glycolysis, glycogenolysis, fatty acid oxidation and proteolysis.

CELL DIVISION

To replace damaged and worn out cells, and essentially for growth, multiplication of cells takes place. The orderly process by which cell divides is called mitosis. In mitosis, two new cells (daughter cells) are produced with each having 46 chromosomes which carry genes identical to those of the original cell. Mitosis is a continuous process even though four distinct stages (phases) are recognised. 

Just before the cell division begins, there is a resting phase during which there is an increase in DNA, RNA and proteins in preparation for the cell division. This phase is known as the interphase. When the nuclear material in the cell has doubled, cell division can begin in the following sequence. 

Prophase The nuclear chromatin becomes concentrated to form a tangled mass of chromosomes. Each pair of chromosomes consists of two identical chromatids attached at a point called centromere. 

The two centrioles separate and move towards opposite poles of the cell joined by protein fibres known as mitotic or achromatic spindle. Without this spindle, cell division cannot take place. At the end of the prophase, the nucleolus and nuclear membrane disappear to appear again when cell division is complete. 

Metaphase At this stage, the chromosomes move to the centre of the cell and arrange themselves in a line along the mitotic spindle. Each chromosome divides longitudinally into two chromatids. 

Anaphase The centromeres separate and the two chromatids are pulled apart towards opposite ends of the cell by the spindle fibres. The cell now contains two sets of identical chromosomes, and the cytoplasm begins to constrict. 

Telophase During this final stage of cell division, the chromosomes become thread like, and then become a mass of chromatin. A nucleolus appears and a nuclear membrane forms around each set of chromosomes.

The cytoplasmic membrane continues to constrict and finally divides to give rise to two identical daughter cells which are exact replicas of the parent cell. Figure shows the different stages of mitosis.

TISSUE FORMATION

Tissues are formed by the binding together of living cells with non-living substances called intercellular substances. In some cases, tissues are made up of one type of cell which performs the particular function of that tissue. The non-living substances include tissue fluids and fibres such as collagen, elastic and reticular fibres. These substances are involved in the support and strengthening of the tissues, and in the maintenance and nourishment of the cells.

Collagen Collagen is a tough, thick (about 140 nm) fibre of proteinous nature commonly found in the connective tissues of bones and cartilages. Collectively the fibres are referred to as white fibres. An enzyme, collagenase, is capable of dissolving the collagen

Elastic fibers These are made up of the fibrous protein, elastin. They are long, thread-like fibers which impart yellow color to tissues when present in large numbers. For this reason they are also referred to as yellow fibres. They are present in abundance in the walls of blood vessels, trachea, lungs and dermis. Their function is to provide these tissues the power of elastic recoil.

Reticular fibres These are often connected with collagen fibres and structurally they are similar to each other. They form a delicate network of fibres and provide support for the cells, capillaries and nerve fibres. They can also be found at the junction of connective tissues and other types of tissues.