The Use of Kerosene/Olive Oil Proportioned Mixtures as Alternative Clearing Agent in Tissue Processing
Chapter One
AIMS & OBJECTIVES
AIM
To evaluate the efficiency of Olive oil and palm oil as a clearing agent for hematoxylin and eosin staining procedure and compare it with xylene.
OBJECTIVE
To determine whether tissues cleared and dewaxed with Olive oil and keroseneare same or superior with that of xylene treated tissues.
CHAPTER TWO
REVIEW OF LITERATURE
Xylene was first isolated and named by the French c emist Auguste Cahours in the year 1850, has been discovered as a component of wood tar.
Xylene (from Greek means “wood”), xylol or dimethylbenzene I any one of three isomers of dimethylbenzene, or in a combination form. It ha got a formula (CH3)2C6H4 with eac of these three compounds possess a central benzene ring with which two methyl groups are attached. These are colorless and flammable liquids with a great industrial value. The mixture is referred to as both xylene or xylenes. Xylene che ically exists in three isomeric forms. The isomers can be distinguished by the designations namely ortho (-o-), meta-(m-) and para(-p-), which specify to which carbon atoms (of the benzene ring) the two methyl groups are attached. Carbon atoms around the ring are counted starting from one of the ring carbons b nded to a methyl group, and counting towards the second methyl group, the o-isomer has the IUPAC name of 1,2-dimethylbenzene, the misomer is 1,3-dimethylbenzene and the p-isomer is 1,4-dimethylbenzene. Of the three isomers, the p-isomer is the most industrially used after since it can be oxidized to terephthalic acid11.
Based on respective isomers, the physical and chemical properties of xylene differs. The melting point for m-xylene is around 47.87 °C and for p xylene is roughly around 13.26 °C. The boiling point for each isomer is around 140 °C. The density of each isomer is around 0.87 g/ml. It is less denser than water. In air, xylene can be smelled at low concentrations between 0.08 to 3.6 ppm (parts per million).In water, xylene can be tasted at a level between 0.53 to 1.8 ppm4.
In laboratories xylene is used to prepare dry ice bath. It has also got its purpose in light microscopes, the synthetic immersion oil can be removed from the microscopic objective using xylene solvent. In histopathology laboratory the application of xylene are, it is used as a clearing agent, to remove the paraffin wax and in restaining of archival slides and prior to mounting.12
XYLENE AND ITS HEALTH HAZARDS
The conventional hematoxylin and eosin staining procedure is the gold standard technique and usage of xylene as a clearing agent in H/E procedure is valid, but its major demerits are cost containment, toxicity and polluted working environment13. Xylene is easily absorbed by the oro-respiratory mucosal tract following ingestion and inhalation4. The penetration of xylene through dermal and epidermal layers are enhanced when it is present in varied physical states. In humans, 64% of the intaken dose reaches the systemic circulation14. Xylene is rapidly distributed to the tissues once it is absorbed. On absorption by blood, it attaches to serum protein to form a complex. Of the total absorbed xylene, 95% is metabolized in the liver to methyl hippuric acid (MHA) and about 70–80% of metabolites are removed from body as urine within a time period of 24 hours. Since xylene is lipophilic, it is accumulated in adipose tissue. In murine studies, adipose tissues were examined to study the half-life for the elimination of xylene, which was estimated to be 7 hours, whereas in humans it is around 40 hours15. As per OSHA regulations, serum evaluation of MHA, which is the major metabolic product of xylene is mandatory among laboratory staffs and chemical factory employees. This is an accurate estimation of atmospheric exposure of xylene in the professional environment14.
United State Department of Health and Human services and Oak ridge National Laboratory reviewed the toxic effect of xylene and found out that xylene can cause health effects as a result of both acute (<14days) and chronic (>365 days) exposure. Accidental splash of xylene in the eye can result in eye damage. The effects can begin to occur with exposure to air levels of 100ppm. Following xylene ingestion, throat irritation can occur at approximately 200ppm within 3-5 minutes. Xylene exposure at 200ppm ≤ can cause lung irritation, causes chest pain and results in shortness of breath. Liver and kidney damage are confirmed as a result of high exposure to xylene. Extreme exposure can result in pulmonary oedema4. Depletion of adenosine triphosphate, which is a mitochondrial enzyme are confirmed in specifically affected cells. Renal injuries, hematologic discrepancies which are morbid and other immediate complications such as erythema, pruritis, exfoliation of the dermal and epidermal layers; all these changes predisposes the skin to acquire opportunistic infections which are considered to be noxious effects associated with the usage of xylene16.
CHAPTER THREE
RESEARCH METHODOLOGY
Preparation and mixture of solvents
- The two compounds, e., kerosene and xylene were mixed in the following proportion
- Kerosene (50%): Xylene (50%); Kerosene (70%):
Xylene (30%); Absolute Kerosene (100%); Absolute Xylene (100%).
Experimental design and setting
Preparation and mixture of solvents
A total of 120 soft tissue samples were selected measuring approximately 1.0–1.5 cm in size. The samples were randomly divided into 4 different groups as – A, B, C and D consisting of 30 each. A combination of kerosene and xylene is used in different combinations as a clearing agent.
Groups
- Group A – Kerosene (50%): Xylene (50%);
- Group B – Kerosene (70%): Xylene (30%);
Group C – Absolute Kerosene (100%); Group D – Absolute Xylene (control group) [Figure 1 a‑d].
Modified tissue processing
Tissues were randomly separated into 4 groups. Each tissue samples were kept for routine tissue processing till 5 changes of alcohol (70, 80, 90 and 2 changes of abs. Alcohol). During the process of clearing (except for Group D), instead of conventional xylene, 2 changes of mixture of kerosene and xylene were used in the ratio of Group A – 50:50; Group B – 70:30; and Group C – 100 kerosene for 2 h.
CHAPTER FOUR
RESULTS
Block tissue and sectioning
Block tissue and sectioning – the embedded blocks of tissue in Group A, B, C and D were carefully observed and analyzed. The tissues with Group A, B and D were properly embedded and in good shape without any form of shrinkage or depression in embedding paraffin wax. However, in Group C, the tissues were opaque in appearance, shrinked or depressed in the embedding paraffin wax and which also affected the proper sectioning of tissues and also there is observation of wrinkling or folding of tissue sections while cutting with microtome [Figure 1a‑d, Table 5 and Graph 1].
CHAPTER FIVE
CONCLUSION
By the present study, it can be suggested that kerosene and xylene in the ratio of 50:50 is a safest alternative to xylene when used alone without altering the tissue morphology and also the staining characteristic and most importantly without posing any health risk.
Xylene is an aromatic hydrocarbon which is extremely biohazardous. The exposure of xylene is maximum during dewaxing of sections and the histopathological laboratory technicians are routinely exposed to xylene during procedures. Effects to minimize the health hazards in the histopathology laboratory should be made to create a much more safer working environment by increasing the awareness among histopathology lab manager and assistants. Short educational programs targeting standard operating procedures, safety precautions and emergency management should be encouraged.
The xylene free method for paraffin sections was developed and in use at Vrinnevi hospital, Sweden since 19956. On account of the Occupational Safety and Health Administration regulations, various other xylene alternatives namely limonene reagents, aliphatic hydrocarbons, vegetable oils and mineral oils were tried in the past to avoid xylene usage in the laboratory.
This study is designed to establish whether the usage of Olive oil and palm oil at a maintained temperature as a clearing agent during tissue processing and as a dewaxing agent during staining procedure has any effect on transparency, rigidity, change after impregnation, ease in sectioning and quality of staining such as nuclear staining, cytoplasmic staining and clarity of staining as compared with the xylene treated counterparts. Our study results suggests that both the oils, especially Olive oil treated specimen showed better characteristic features than palm oil treated specimen with respect to both gross specimen evaluation and also with the quality of staining. Further studies with larger sample size and more parameters are required to validate the better results with the usage of biofriendly xylene alternatives.
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