This study was aimed at investigating the potentials of different proportioned mixtures of kerosene and olive oil as alternative clearing agents in place of xylene during tissue processing. It was carried out in the histopathology laboratory of the department of Medical Laboratory Science, Imo State University, Owerri between February and March (2017). Sample of tissues were harvested from a presumably healthy wistar rat. These tissues were fixed and grouped based on clearant to be used. Following all basic tissue processing protocols, tissues were processed manually with only alterations of the clearing process were necessary. Tissues were cleared by passing through two changes of xylene for (Group1control) and two changes of different proportions of mixtures of kerosene and olive oil for Group 2 (4:1), Group 3 (3:2), and Group 4 (2:3). Clearing was carried out in a hot air oven at 60o C based on Bernoulli’s principle. Following the stated criteria for evaluation, analysis were carried out and results were obtained showing that xylene was a colorless liquid with a strong pungent smell having hazardous properties unlike the kerosene and olive oil mixtures used which had distinct colors as well as smell and were less toxic. Group 2 (4:1)gave poor alternative results as tissues from this group were poorly sectioned and stained, Group 3 (3:2) gave excellent clearing and staining results comparable to the control. Group 4 (4:1) also gave good clearing and staining results as tissues were comparable to the control group. It was evident that xylene remained a very good clearing agent regardless of its hazardous properties. Although when used in the right ratio, a mixture of kerosene and olive oil served as good alternative clearing agent comparable to xylene. Toxic and costly chemicals used during tissue processing remains an issue which more researches should be carried on their alternatives.
Histology deals with the study of the microscopic anatomy of tissues. For these tissues to be studied microscopically, it has to be processed by procedures known as histological techniques. Tissue processing can be performed manually (hand processing), but where multiple specimens have to be dealt with it is more convenient and much more efficient to use an automated tissue processing machine (a “tissue processor”). Most Histological techniques have been developed to reserve the structural integrity of the specimens so that it can be viewed microscopically (Schwann, 2008).Tissue processing in histology is a physical process that involves chemical solutions reacting with biological specimens. Specimens from biopsies and autopsies require processing. These processes require different procedures which include: (a) Fixation- The purpose of fixation is to preserve tissues permanently in as life-like a state as possible. The most commonly used chemical solution is formalin. (b) Dehydration- Since the ultimate purpose of tissue specimen treatment is to infiltrate the tissue sample in paraffin, and since water and paraffin are not miscible, the sample must be dehydrated after the fixation step. This is usually achieved by subjecting the tissue sample to increasing concentrations of alcohols (c) Clearing- It is the process of replacing the dehydrant with a substance that will be miscible with the embedding medium (paraffin). The term “clearing” comes from the fact that the clearing agents often have the same refractive index as proteins. As a result, when the tissue is completely infiltrated with the clearing agent, it becomes translucent (Dapson, 2000). This change in appearance is often used as an indication of the effectiveness or completeness of the clearing process (d) Infiltration- This involves tissue sample treatment usually with paraffin wax. In this step the cleared tissue samples are placed into paraffin heated to a few degrees above its melting temperature. Several changes of paraffin may be required to remove the residual xylene so that the tissue is completely infiltrated with the molten paraffin. (e) Embedding- The aftermath of such processing is a sample that has been preserved, infiltrated and then embedded with paraffin. Embedding will give the tissue external support for microtomy. Once all these have been done, it is then sectioned on a microtome (Drury and Wallington, 2008).
Clearing means appearance of tissue after it has been treated by the fluid chosen to remove the dehydrating agent. It is otherwise referred to as dealcoholization since it involves the removal of the dehydrating agent which is normally alcohol although other dehydrating agents such as acetone and dioxane (diethylene dioxide) can also be used (An, Moreira, Kang, and Gruber., 2001). Not all dealcoholization agents can clear. When the dehydrating agent has been entirely replaced by the solvent, and the tissue has a translucent appearance afterwards, then it can be termed a clearing agent. The reason for this translucency is because most clearing agents have the same refractive index as proteins of tissue and the tissue becomes transparent. The end point of clearing can be noted by the transparent appearance of the tissue. Thus clearing serves two purposes which are
- Removes alcohol to make paraffin impregnation complete
- Acts as solvent for the mounting media which renders the tissues transparent and improves the refractive index, making microscopic examination easier.
The most commonly used clearing agent is xylene. Unfortunately, xylene is considered to be toxic although most histological processing laboratories use xylene on a daily basis. (Whalen, Dufrense and Wilkel, 2005; Taskinen, Anttila, Lindbohm, Sallmen and Hemminki 2009).Other Commonly used clearing agents includes Toluene and Benzene, Chloroform, Carbon tetrachloride, Cedar wood oil (Histological)(Bancroft and Stevens, 2009).All of these clearing agents have their specific advantages and disadvantages. Xylene is a type of clearing agent that reasonably works well for short-term clearing of small tissue blocks. Long-term immersion of tissue in xylene results in tissue distortions. Toluene works well, and is more tolerant of small amounts of water left in the tissues, but is three times more expensive than xylene. Chloroform was formally used, but its hazardous and slow. Methyl salicylate is rarely used because it is expensive, but it smells nice (it is oil of wintergreen). There are newer clearing agents available for use. Many of them are based on limolene, a volatile oil found in citrus peels. Another uses long chain aliphatic hydrocarbons (Clearite) are used also. Although they represent less of a health hazard, they are less forgiving with poorly fixed, dehydrated, or sectioned tissues (Beusa, 2000; Savoleinen and Pfaffli, 2008). When the tissue is to be cleared directly from water as in frozen section, no dealcoholization process is involved in this case glycerin and gum syrup are used. A good clearing agent should be miscible with both alcohol and paraffin wax, should not produce excessive shrinkage, hardening and damage of tissues, should not dissolve aniline dyes, should make the tissue transparent and so on (Bruce, 2013).
Xylene has been the most used clearing agent due to its properties that fits in as a good clearing agent. Xylene is an alkane which is an aromatic hydrocarbon widely used in industry and medical technology as a solvent. It is a colorless, sweet-smelling liquid or gas occurring naturally in petroleum, coal and wood tar, and is so named because it is found in crude wood spirit (Gr. xy`lon- wood). It has a chemical formula of C6 H4 (CH 3)2 and is referred to as “dimethyl benzene” because it consists of a six-carbon ring to which two methyl groups are bound. It exists in three isomeric forms: ortho-, meta- and para-xylene (Dapson and Dapson, 2005). Xylene is used as a solvent in the printing, rubber, paint and leather industries. It is found in small amounts in airplane fuel, gasoline and cigarette smoke. Xylene is used in histological laboratories for tissue processing, staining and cover slipping (Kandyala, Raghavendra, Rajasekharam, 2010). Its high solvency factor allows maximum displacement of alcohol and renders the tissue transparent, enhancing paraffin infiltration. In staining procedures, its excellent dewaxing and clearing capabilities contribute to brilliantly stained slides. Laboratory-grade xylene is composed of m-xylene (40-65%), p-xylene (20%), o-xylene (20%) and ethyl benzene (6-20%) and traces of toluene, trimethyl benzene, phenol, thiophene, pyridine and hydrogen sulfide. Histopathological technicians who routinely come in contact with xylene-contaminated solvents in the workplace are the population most likely to be exposed to high levels of xylene. The current Occupational Safety and Health Administration permissible exposure limit for xylene is 100 parts per million as an 8-h time-weighted average (TWA) concentration.Besides occupational exposure, the principal pathway of human contact is via soil contamination from leaking underground storage tanks containing petroleum products. Xylene can leak into the soil, surface water or ground water where it may remain for months or more before it breaks down into other chemicals. However, as it evaporates easily, most of it goes into the air and gets broken down by sunlight into other less-harmful chemicals. Most people begin to smell xylene in air at 0.08-3.7 ppm (parts per million) and begin to taste it in water at 0.53-1.8 ppm. The main effect of inhaling xylene vapor is depression of the central nervous system, with symptoms such as headache, dizziness, nausea and vomiting. (Savoleinen and Pfaffli, 2008).
Kerosene widely used in kerosene lamps andlanterns, is a thin, clear liquid formed from hydrocarbons,with density of 0.78-0.81g/cm3. Kerosene is obtained fromthe fractional distillation of petroleum between 150 ° C and275 ° C, resulting in a mixture of carbon chains containing12 – 15 carbon atoms. It is sometimes spelt as kerosine in scientific and industrial usage. In Nigeria, kerosene can be readily bought at any filling stations. Its use as a cooking fuel is mostly restricted to some portable stoves. Kerosene, a flammable hydrocarbon liquid is widely used in Nigeria and India as fuel for domestic activities. In countries such as Nigeria, kerosene is the main fuel used for cooking, especially by the poor. Kerosene stoves have replaced the traditional wood-based cooking appliances that are unhealthy and inefficient. Kerosene has been used to treat pools of standing water to prevent mosquitoes from breeding. It is used as a solvent like xylene and in conjunction with cutting oil as a thread cutting and reaming lubricant. Kerosene is not considered to be carcinogenic orteratogenic as observed in xylene (Kunhua et al., 2011). The acute health risks involved in handling and using kerosene are minimal, provided that the product(s) are used in accordance with current safety practices. The most common health effect associated with chronic or repeated kerosene exposure is dermatitis (Ritchie et al., 2003).
Various xylene substitutes were tried in the past such as limonene reagents, aliphatic hydrocarbons, vegetable oils and mineral oils to avoid xylene in the laboratory. However, some of these substitutes were found to be less effective as clearing agents and more expensive than xylene. (Beusa, 2000; Whalen et al., 2005; Kandyala et al., 2010).One of this vegetable oil substitutes is olive oil. Olive oil is obtained from the olive (the fruit of Oleaeuropaea; family Oleaceae), a traditional tree crop of the Mediterranean Basin. The oil is produced by pressing whole olives. It is commonly used in cooking, whether for frying or as a salad dressing. It is also used in cosmetics, pharmaceuticals, and soaps, and as a fuel for traditional oil lamps, and finds uses in some religions. Olive trees have been grown around the Mediterranean since the 8th millennium BC. Spain is by far the largest producer of olive oil, followed by Italy and Greece. Per capita consumption is however highest in Greece, followed by Spain, Italy, and Morocco. Consumption in North America and northern Europe is far less, In 2013, world production of virgin olive oil was 2.8 million tonnes (table), a 20% decrease from the 2012 world production of 3.5 million tonnes (Vivante, 2011).Although further research in the use of olive oil as a clearing agent is expected, where olive oil–treated specimen can be subjected to all stains and advanced histological procedures (like immunohistochemistry), in order to consider this agent as a better and safer substitute for xylene studies shows that Olive oil remains a very good possible substitute of xylene in clearing tissues.(Sugunakar, Surapaneni , Pavan, Thokala andPavan, 2015).
1.1 JUSTIFICATION OF STUDY
Xylene is considered to be toxic although most histological processing laboratories use xylene on a daily basis (Whalen et al., 2005; Taskinen, et al., 2009). The main effect of inhaling xylene vapor is depression of the central nervous system, with symptoms such as headache, dizziness, nausea and vomiting. (Savoleinen and Pfaffli, 2008). Histopathological technicians who routinely come in contact with xylene-contaminated solvents in the workplace are the population most likely to be exposed to high levels of xylene. Thus there is a need to find substitutes that are less toxic, easily accessible, cheaper and with the same potency of xylene as a good clearing agent, to save all especially histopathological technicians who routinely come in contact with xylene during tissue processing from its hazardous implications on their health. Kerosene and olive oil are not only easily accessible, cheap and less toxic; they are as well environmentally friendly and have been shown each by various researches to stand a chance of substituting xylene in clearing. A mixture of both might give even better results if used in the right proportion. Hence the study on the use of kerosene/olive oil mixtures as alternative clearing agent in tissue processing.
1.2 SPECIFIC AIM
- To investigate the potentials of different proportioned mixtures of kerosene and olive oil as alternative clearing agents in place of xylene during tissue processing.
- To determine the best proportion of mixture of kerosene and olive oil that can clear tissues ideally.
- To compare the clearing potentials of mixture of kerosene and olive oil to that of xylene in tissue processing.