Effect of Fixation on Tissues
CHAPTER ONE
OBJECTIVES OF THE STUDY
- To compare the effects of formaldehyde based fixatives on tissues
- To determine the standard formaldehyde based fixative to be used on tissues
- To investigate the effect of the standard formaldehyde based fixative on tissues
CHAPTER TWO
LITERATURE REVIEW
HISTOLOGICAL TECHNIQUE
Histology is the study of the microscopic anatomy (microanatomy) of cells and tissues of plants and animals. It is commonly performed by examining cells and tissues under a light microscope or electron microscope, the specimen having been sectioned (cut into a thin cross section with a microtome), stained, and mounted on a microscope slide. In the 17th century, Italian Marcello Malpighi invented one of the first microscopes for studying tiny biological entities. Malpighi analysed several parts of the organs of bats, frogs and other animals under the microscope. Malpighi, while studying the structure of the lung, noticed its membranous alveoli and the hair-like connections between veins and arteries, which he named capillaries. His discovery established how the oxygen we breathe enters the blood stream and serves the body. In the 19th century, histology was an academic discipline in its own right. The French anatomist Bichat introduced the concept of tissue in anatomy in 1801, and the term “histology” first appeared in a book of Karl Meyer in 1819. The 1906 Nobel Prize in Physiology or Medicine was awarded to histologists Camillo Golgi and Santiago Ramon y Cajal. They had dueling interpretations of the neural structure of the brain based in differing interpretations of the same images. Cajal won the prize for his correct theory and Golgi for the staining technique he invented to make it possible (Lowe and Teffrey, 2000). Histological technique deals with the preparation of tissue for microscopic examination. The aim of good histological technique 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 (Titford and Bowman, 2012). The aim of Tissue Processing is to remove water from tissues and replace with a medium that solidifies to allow thin sections to be cut. Biological tissue must be supported in a hard matrix to allow sufficiently thin sections to be cut, typically 5 μm (micrometres; 1000 micrometres = 1 mm) thick for light microscopy and 80-100 nm (nanometre; 1,000,000 nanometres = 1 mm) thick for electron microscopy. For light microscopy, paraffin wax is most frequently used. Since it is immiscible with water, the main constituent of biological tissue, water must first be removed in the process of dehydration. Samples are transferred through baths of progressively more concentrated ethanol to remove the water. This is followed by a hydrophobic clearing agent (such as xylene) to remove the alcohol, and finally molten paraffin wax, the infiltration agent, which replaces the xylene. Paraffin wax does not provide a sufficiently hard matrix for cutting very thin sections for electron microscopy. Instead, resins are used. Epoxy resins are the most commonly employed embedding media, but acrylic resins are also used, particularly where immunohistochemistry is required. Thicker sections (0.35μm to 5μm) of resin-embedded tissue can also be cut for light microscopy. Again, the immiscibility of most epoxy and acrylic resins with water necessitates the use of dehydration, usually with ethanol. Processes involved in histological techniques includes:
Fixation:
This is the process by which the constituents of cells and tissue are fixed in a physical and partly also in a chemical state so that they will withstand subsequent treatment with various reagents with minimum loss of architecture. This is achieved by exposing the tissue to chemical compounds, call fixatives.
Dehydration:
Tissues are dehydrated by using increasing strength of alcohol; e.g. 50%, 70%, 90% and 100%. The duration for which tissues are kept in each strength of alcohol depends upon the size of tissue, fixative used and type of tissue; e.g. after fixation in aqueous fixative delicate tissue need to be dehydrated slowly starting in 50% ethyl alcohol directly whereas most tissue specimens may be put into 70% alcohol. Delicate tissue will get high degree of shrinkage by two great concentration of alcohol.
Clearing:
During dehydration water in tissue has been replaced by alcohol. The next step alcohol should be replaced by paraffin wax. As paraffin wax is not alcohol soluble, we replace alcohol with a substance in which wax is soluble. This step is call clearing.
Impregnation with Wax:
This is allowed to occur at melting point temperature of paraffin wax, which is 54-60oC. Volume of wax should be about 25-30 times the volume of tissues. The duration of impregnation depends on size and types of tissues and the clearing agents employed. Longer periods are required for larger pieces and also for harder tissue like bones and skin as compared to liver kidney, spleen, lung etc. Xylene is easiest way to remove. Total duration of 4 hours is sufficient for routine impregnation.
CHAPTER THREE
MARTERIALS AND METHOD
AREA OF STUDY
This study was carried out between January and March, 2017 at Imo state university Histopathology laboratory of the department of medical laboratory science, faculty of health science, Imo state university. Imo state university is located in Owerri Imo State Nigeria. Owerri lies within latitudes 5 o25o and 5 o 29 oN and longitudes 6 o59 o and 7o30 oE.
METHODOLOGY
Tissues meant for this study were harvested, cut out appropriately and washed in normal saline before fixing. The tissues were grouped according to the fixation process to be carried out on each group. Group 1 were tissues fixed in neutral buffered formalin, Group 2 were fixed in 10% formalin, Group 3 were fixed in 40% formaldehyde (formalin), while Group 4 were not fixed at all but started with dehydration (negative control). These prepared fixatives were in the ratio of 1:20 as fixation process was carried out for 48 hours After 48 hours, the fixed tissues were re-trimmed in to smaller bits of nearly equal sizes. Tissues were processed manually in the laboratory. After processing tissues were stained using haematoxylin and eosin staining technique.
MANUAL TISSUE PROCESSING PROCEDURES
Fixation: The tissues were fixed as follows for 48 hours:
Group 1 fixed in neutral buffered formalin
Group 2 were fixed in 10% formalin
Group 3 were fixed in 40% formaldehyde (formalin)
Group 4 were not fixed at all but started with dehydration (negative control).
Dehydration: all tissues were dehydrated by using increasing strength of alcohol:
CHAPTER FOUR
RESULTS
PHOTOMICROGRAPH OF TISSUE SECTION FIXED IN NEUTRAL BUFFERED FORMALIN (GROUP 1)
CHAPTER FIVE
DISCUSION, CONCLUSION AND RECOMMENDATION
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.
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.
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.
REFERENCES
- Akissane, M.J, (2000). Anderson’s pathology. ( 9th edition). CV Mosby company USA p. 1095-1099.
- Avwioro, O.G.(2010). principles and techniques. Histochemistry and tissue pathology. (2nd edition). Claverianum press, Nigeria. pp 10-50.
- Bonita, R., Beaglehole, R. Kjellestrom, T. (2006). Formaldehyde. (2nd edition).
- Goldstein, N.S., Ferkowicz, M.T., Odish, E., Mani, A., Hastah. F. (2003). Minimum formalin fixation time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma. Am J Clin Pathol 120(5):86-93.
- Hopwood, D. (2006).Host specificity in the Richelia-diatom symbioses revealed by hetR gene sequence analyses. Environ Microbiol 1(3): 431–438.
- Huang, B. Q. and Yeung, E. C. (2015). Chemical and Physical Fixation of Cells and Tissues: An Overview. (2nd edition).Springer publishers New York: 21-29.
- Janson, S., Wouter,s .J, Bergman, B., Carpenter, E.J. (2009). Fixation and fixatives. Theory and practice of histological techniques. Churchill Livingstone, New York: pp19-26.
- Leong, A.S. (2004). Fixation and fixatives. Laboratory histopathology. Churchill Livingstone, New York: 4.1-1 – 4.1-26.
