Effects Of Fixation On Tissues

Medical and Health Science Project and Seminar Material

Effects Of Fixation On Tissues


Abstract


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.


Chapter One


1.0 Introduction

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 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 (Bonita, Beaglehole and Kjellestrom, 2006; Hopwood, 2006; Mayer et al., 2009). Specimen should be transported in glass, plastic or metal container or in a plastic bag in 10% formalin. If formalin is not available at hand, place the specimen in refrigerator at 4oC to slow down autolysis. The container should have an opening larger enough so that the tissue can be removed easily after it has hardened by fixation. Chemical fixation is the most common approach used in the fixation of biological specimens for light microscopy (LM) and electron microscopy (EM). Relatively large specimens can be fixed when compared to the physical methods. Chemical fixation requires a fixative which is composed of fixing agent(s) and vehicle(s). Fixing agents are traditionally classified as coagulants and non-coagulants, but they can also be classified as additive and no additive, and acidic or basic. Coagulant fixing agents such as ethanol, methanol, and acetone can cause protein denaturation ((Talukder, 2011). These fixing agents precipitate proteins by replacing water, resulting in conformation change of protein molecules and their solubility. The non-coagulant fixing agents such as aldehydes and osmium tetroxide (OsO4) react with proteins and other components, forming intermolecular and intramolecular cross-links resulting in a better retention of the cellular organization. A vehicle is usually a buffer solution with additional compounds such as inorganic salts added to optimize fixation of targeted organelles such as cytoskeletal elements (Goldstein, Ferkowicz, Odish, Mani and Hastah, 2003). A buffer is used to maintain the desired pH regardless of the chemical nature of the fixing agent(s) selected. The buffer chosen has to be compatible with other components of the fixative and stains. The fixative also needs to have a suitable osmotic concentration compatible with cells and tissues, so that biological materials will neither swell nor shrink during fixation. A great variety of chemical compounds can be used as fixatives. In recent years, the most commonly used fixatives for botanical specimens are ethanol, methanol, acetone, formaldehyde, glutaraldehyde, and osmium tetroxide (OsO4). Ethanol and methanol are coagulant fixing agents. They denature proteins by replacing water in the tissue environment and are usually combined with other fixing agent(s) as a fixative for light microscopy. The alcohols, especially ethanol, are also used as dehydrating solvents for both light microscopy and electron microscopy (Shi, Key and Kalra, 2004).Fixing agents can be used alone or combined with others in a fixative. For high resolution light microscopy and transmission electron microscopy (TEM), the common design is to combine formaldehyde with glutaraldehyde as the primary fixative With the advantage of the rapid penetration and stabilization of tissue by formaldehyde molecules and the stable cross-links generated by glutaraldehyde that come later. Unfortunately, there is no ideal fixative. Not all substances and macromolecules can be “fixed” by the fixing agents. Components such as nucleic acids are indirectly stabilized through protein fixation. Furthermore, the vacuole compartments, different types of cell walls, and ergastic substances can pose challenges to a fixation protocol. Compromises have to be made in selecting a protocol that is best suited for the objective of the experiment. In order to achieve good quality fixation, it is important to consider some variables which includes: Rate of Penetration, Concentration of Fixing Agents and Additives, Length of Fixation, Temperature and so on.

Fixatives are divided into three main groups which are:

  1. Microanatomical fixatives – such fixatives preserves the anatomy of the tissue.
  2. Cytological fixatives – such fixation are used to preserve intracellular structures or inclusion.
  3. Histochemical fixatives: Fixative used to preserve the chemical nature of the tissue for it to be demonstrated further. Freeze drying technique is best suited for this purpose.

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

  • To determine the effect of fixation on tissues

1.3 Objectives

  1. To compare the effects of formaldehyde based fixatives on tissues
  2. To determine the standard formaldehyde based fixative to be used on tissues
  3. To investigate the effect of the standard formaldehyde based fixative on tissues

Chapter Five


5.0 Discussion, Conclusion and Recommendation

5.1 Discussion

From this study, neutral buffered formalin gave the best results as tissue section fixed in it gave excellent staining as well as differentiation. There was no artifact seen unlike the group 4 that was not fixed at all. This study was carried out in line with a research by Hopwoods,(2006), who concluded that 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). From this study, the actual effect of a fixative can be determined rightly after staining as not much could be deducted before staining. The non fixed group showed artifacts present in tissue section. This result obtained was similarly noted by Hopwood, (2006) who noted that even the most careful fixation does alter the sample and introduce artifacts that can interfere with interpretation of cellular ultrastructure. There had to be variation in timing to achieve proper fixation this was just in concordance with what Werner, et al., (2000) noted, that the problems with formalin fixation comprise delay of fixation and variations in the duration of the fixation mainly (Werner, et al., 2000). As against a research by O’Leary, (2011), although neutral buffered formalin fixative was not used in demonstrating nucleic acid particles, but from this study neutral buffered formalin fixative stands tall as a standard fixative for tissues of any kind unlike what he concluded that the Progress toward clinical application of potentially useful markers is hampered by the established routine fixation methods that fail to conserve the structure of nucleic acids and proteins in tissues and the limited ability to extract sufficient high-quality RNA or protein from fixed tissue (O’Leary, 2011). The reason for the different result obtained was probably due to the routine fixative used which was formalin of which this study has shown neutral buffered formalin to be better off. As against a research by Lewis et al., 2001, which showed that Formaldehyde fixation at room temperature results in poor preservation of high-molecular weight DNA, the size of the extracted DNA being directly related to the fixation temperature (Lewis et al., 2001) this research shows that formaldehyde fixation gives good results but not as excellent as neutral buffered fixed tissues. Another research by leong, (2004), showed that formaldehyde has a greater chance for oxidation in this concentration of tissue fixative and eventually the solution will start to drop in pH, in spite of the buffer. It is that 10% buffered formalin solutions be used no longer than 3 months after they were initially mixed (leong, 2004). As similarly noted by leong, (2004), one of the reason for the attributed failure of formaldehyde fixatives might be as a result of its oxidation property as this was conquered by the neutral buffered formalin fixative used as against others in this study. In a research Shi, et al., (2004), they suggests that tissues fixed in formalin at 4°C exhibit the least amount of degradation of the nucleic acids (Shi, et al., 2004). From this study formalin fixed tissues gave good results as with the research above although the temperature at which fixation was carried out might have contributed to the better results obtained when compared to this study.


5.2 Conclusion

From this study, neutral buffered formalin gave the best results with no artifacts or shrinkage as well as formalin and formaldehyde fixatives which gave good results also. Buffered formalin remains a standard fixative for use in tissue processing protocols. Non- fixation gave the poorest of all results this shows that non-fixation should be largely discouraged in tissue processing procedures in the laboratory.


5.3 Recommendation

From this study, it is recommended that neutral buffered formalin be widely used in the laboratory and this fixative be applied to even more research also fixation should be considered an essential step in tissue processing thus should not be skipped at all as this will help improve results obtained during tissue processing generally.


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