Histology is the study of tissues. For these tissues to be studied properly, they are prepared via various processes. These procedures are known as histological techniques. Tissue processing can be performed manually or by the use of an automated tissue processing machine (a “tissue processor”). Tissue processing schedules for enclosed, automatic processors are devised according to species and size / type of tissue. Most Histological techniques have been developed to reserve the structural integrity of the specimens so that it can be viewed microscopically (Schwann, 2008). The aim of good histological technique is to preserve microscopic anatomy of tissue and make them hard, so that very thin section (4 to 5 micron) can be made. After staining, the section should represent the anatomy of the tissue as close to as possible to their structure in life. This is achieved by passing the total as selected part of the tissue through a series of process. For light microscopy, three techniques can be used: the paraffin technique, frozen sections, and semi thin sections. The paraffin technique is the most commonly used (Preece, 2008). Histological techniques involved in preparation of tissues for light microscopy includes: Fixation, Dehydration, Cleaning, Embedding, Cutting, Staining.
Fixation as one of the most important steps in tissue processing involves fixing tissues to keep the cellular components as “lifelike” as possible, it is essential that all biochemical and proteolytic processes are inactivated and structures are immobilized and locked in space by a “fixation” step (Mayer, Franz, and Karl, 2009). Two approaches are normally used to “fix” biological samples: chemical and physical fixation methods. Chemical fixation is the most common approach used in specimen preservation. The tissues are immersed in a fixative that kills and stabilizes the cell contents. Physical fixation is accomplished by microwaving and cryopreserving the samples, procedures that rapidly inactivate cellular activities using microwave (MV) energy and low temperatures, respectively(Huang and Yeung, 2015). Most fixatives act by denaturing or precipitating proteins which then form a sponge or meshwork, tending to hold the other constituents. Good fixative is most important factors in the production of satisfactory results in histopathology. It is important to realize that a fixative will initially produce a number of changes to the tissues in what is usually an aqueous environment. These will include shrinkage, swelling and hardening of various components. Despite these initial effects tissues will undergo further changes during processing when they are placed in a non-aqueous environment. For example fixation in 10% buffered formalin initially causes slight swelling of tissue specimens. During processing however the specimen may shrink 20% – 30% of its volume. 3
Fixatives are divided into three main groups which are:
Among the various available fixatives, formaldehyde is the most popular in great part because of its low cost, ease of preparation, and because it preserves morphologic detail with few artifacts. Formaldehyde is a gas but is soluble in water to the extent of 37-40% w/v. This solution of formaldehyde in water is called formalin or full strength formalin. Formalin is one of the commonly used fixative in all laboratories since it is cheap penetrates rapidly and does not over harden the tissues. Since it oxidizes to formic acid if kept standing for long period so it should be neutralized by phosphates or calcium carbonate otherwise it tends to form artifact; a brown pigment in tissues. To remove this pigment picric alcohol or saturated alcoholic sodium hydroxide may be used (Shi et al., 2004).The commercial formalin becomes cloudy on standing especially when stored in a cool place due to formation of precipitate of paraformaldehyde which can be filtered. Formalin on prolonged exposure can cause either dermatitis its vapour may damage the nasal mucosa and cause sinusitis Most laboratories use neutral-buffered formalin (10%) for tissue fixation which introduces cross-links, whereas coagulative fixatives are less popular. Problems with formalin fixation comprise delay of fixation and variations in the duration of the fixation mainly. Solutions to these problems could be to start fixation soon (<30 min) after surgical removal of the tissue and to avoid overfixation (>24–48 hrs). For tissue processing, the most important problem is inadequate tissue dehydration prior to paraffin embedding. This can be prevented by preparing all fixatives to be used freshly every week, depending on the volume of tissue processed. If consistently applied, these procedures could eliminate some of the sources of variation in immunohistochemical stains (Werner, Chott, Fabiano and Battifora, 2000).
1.1 JUSTIFICATION OF THE STUDY
Fixation is an important stage of tissue processing that keeps the cellular components as “lifelike” as possible. Various fixation agents are used and Formalin is one of the commonly used fixative in all laboratories since it is cheap penetrates rapidly and does not over harden the tissues. For tissue processing, the most important problem is inadequate tissue dehydration prior to paraffin embedding (Werner et al., 2000). Good fixative is most important factor in the production of satisfactory results in histopathology (Talukder, 2011). It is important to realize that a fixative will initially produce a number of changes to the tissues in what is usually an aqueous environment. These will include shrinkage, swelling and hardening of various components. Despite these initial effects tissues will undergo further changes during processing when they are placed in a non-aqueous environment. For example fixation in 10% buffered formalin initially causes slight swelling of tissue specimens. During processing however the specimen may shrink 20% – 30% of its volume. The particular fixative employed will also influence the degree to which individual elements will stain with various histochemical and immuno-histochemical reagents. Thus the total effect on tissues of a particular fixative should be assessed after a tissue has been processed, sectioned and stained to demonstrate the required elements (Hopwood, 2006; Mayer et al., 2009). Thus this study is important to accesses the total effect of fixative on a tissue as well as to determine the standard fixative reduce cellular changes to the minimum and provide satisfactory results in tissue processing since there is no ideal fixative that can be used. Hence the study on the effect of fixation on tissues.
1.2 SPECIFIC AIM
1.3 OBJECTIVES
To investigate the effect of the standard formaldehyde based fixative on tissues