Cellular and Molecular Pathology - Essay Example

Cellular and molecular pathology The most suitable method of removing adequate tissue for histopathological examination and the most reliable way of making sure you get the correct tissue needed Materials to be used in histological preparations are removed from anaesthetized animals. Since this case involves a human being and thus human materials, which is a scarce situation. Surgical materials or rather tissues are the best source of human tissue due to the ability to remove the normal tissue together with the affected/diseased tissues. In obtaining the right tissue for the examination, there are two distinct methods that can be employed. That is the physical examination technique and the liver biopsy technique. During physical examination, the medical history of the suspected patient is checked and more attention is paid to the patient’s abdomen. Presence of lumps in the liver can be felt as a patient lies flat with the liver being swollen. Other cases present an enlarged spleen and ability to hear a unique sound/noise when a stethoscope is used to listen to the blood vessels. The noise is usually caused by tumor pressure on the vessels. Liver biopsy is the appropriate method that can be used in obtaining materials for the study. In this case, a definite diagnosis is provided. It is appropriate since it deals with the actual tissues and fluids from the liver thus giving appropriate results rather than the suspect results as provided by the physical method of testing. Liver biopsy is done through obtaining a sample of the liver or rather a tissue fluid using a fine needle. The obtained tissue or fluid is prepared and checked under the observation microscope for the presence of cancer cells. In many incidences, about 70% biopsy shows a positive result for cancer (Brown, 2010). However, in fewer situations, there are risks involved whereby about 0.4% of the cases, some patients develop severe blood loss since a number of tumors are supplied with major and numerous blood vessels thus the heavy bleeding. 2. Methods of fixation and processing that are most suitable for the studies to be performed a) Tissue fixation Fixation is the process by which obtained tissue samples are preserved in a life –like state preventing damage and distortions and is always carried out sooner after tissue removal through surgeries and immediately after death in the case of autopsies. There are several fixatives such as alcohols, mercurials, oxidizing agents, picrates and aldehydes. In this case I would prefer the use of formaldehyde which is also regarded as a combination of formalin and glutaraldehyde. The choice is coherently based on the neutral nature of the formaldehyde solution and the ability to penetrate the tissues cells thus encouraging visibility during the observation time. Formaldehyde has a standard solution hydrogen potential at 10% buffered formalin. The buffer is significant in prevention of acidity that would in turn cause precipitation of the formol-heme tint in the tissues. Formalin has an osmotic pressure that is equal to that of the mammalian cells thus preventing the tissue structure changes due to its reaction. The ratio of the fixative to be used stands at 10:1 to the fixative tissue. The fixative specimen would also boost the fixation process. The process of fixation is also increased by altering the temperature which when increased increases all chemical reactions. In the event where the hematoxylin and esion tissues are to be obtained for a pathology test, formalin is a recommended fixative to be used in the preservation process of the tissues since it is more tolerant and harmless to the tissues (Pathol, 2010). Formalin and alcohol are considered as the best fixatives that penetrate the tissues. Penetration is faster in thin sections compared to thick sections of tissues. However, formalin is only recommended for shirt time fixation and cannot be used in the preservation of skeletal specimens since after a long duration it softens the bones and changes the color of tissues thus making it ineffective. b) Tissue processing Tissues obtained for disease process diagnosis are processed in histology laboratories in order to produce microscopic slides for viewing under the microscopes. Once fixation of tissues is completed, the tissue processing procedure is carried in order to convert to a thin microscopic form. The most common and reliable method is the use of paraffin which has been considered as quite automated for large scale tissue processing. The tissues entrenched in paraffin are able to be sectioned at any point ranging from three through to ten microns at a routine of between six to eight sections. Tissue processing here therefore refers to the dipping of fixed tissues into paraffin. Processing of tissues involves a lot of stages since the wet fixed tissues are unable to be dipped into paraffin. The moisture present in the tissues must at the first stage be eliminated through dehydration using various alcohol brands at 70%, 90% or rather 100% at distinct times. This can even involve the use of formalin and alcohol. Other dehydration agents can also be applied but they have greater risks like acetone which is highly inflammable. Dioxane is also suited for use but is consisting of toxic fumes thus a health hazard (Pathol, 2010). Clearing becomes the next step after dehydration whereby the dehydrator is eliminated through the use of a substance that is miscible with paraffin. Xylene is commonly used in the clearing process because of its efficiency and cheapness in acquisition. Other substances such as Toluene can also be used due to its improved tolerance to small amounts left in the tissues but becomes more expensive in acquisition as it is three times more expensive compared to xylene. The final stage in processing involves infiltration of the tissue with the paraffin. Paraffin has different melting points and hardness and can either be warm or cold paraffin but largely dependent on the decision of the user at this point. Paraplast which is a composition of plasticizers can be used since it makes the paraffin blocks softer for penetration. There are other existing alternatives for the use of paraffin as an embedding agent which are inclusive of the use of plastics that allow for the acquisition of finer and thinner sections. Methyl methacrylate, glycol methacrylate and epon among others fall in the alternative category of agents of embedment. However, the alternative agents though having been accredited with efficiency and widespread use have limitations since they are very expensive to acquire and are complex in their application. The plastics also require very special dehydration and clearing agents which are rather expensive (Pathol, 2010). 3. a) Possible techniques performed on the sample of tissues obtained to help in the microscopal examination of the tissue Before the samples are observed under the microscope they have to be stained, sectioning, cover slipped and decalcified and mounted in order to give a better and clearer visual impression. According to Dragovich (2011), carcinoembryonic antigen (CEA) is a glycoprotein that manifests its presence in the normal mucosal cells but improves its levels in association with colorectal cancer thus making it poses a role in tumor creations. The samples that have been obtained from the surgery are taken to the lab where a specialist doctor on cancer treatment is charged with the responsibility of examining the obtained, fixed and processed samples under a microscope. The samples are stained in order to make them visible through the microscope. The common staining method is the hematoxylin and oesion technique. Hematoxylin is an oxidized product of logs commonly known as hematin, and does not directly stain tissues but rather gives a link to the tissues which is facilitated by metal cations as aluminum and tungsten. Eosin is an acidic dye with cytoplasmic affinity and is less problematic in the laboratory (Burn & Jaros, 2011). Cover slipping is a technique involving the covering of stained slides with thin glass for tissue protection against scratches thus providing better visual quality. Clearing is performed on the stained slide to remove water particles that might be present in the slide. The covered sample slides are then subjected to decalcification in order to remove persistent calcium deposits which would not have been removed during the fixation, clearing and processing process then put under the microscope for examination and observations. Under other circumstances, the biopsy specimen may be subjected to several tests to ascertain the category and give a better classification of the cancer so as to ascertain the presence or absence of the carcinoembryonic antigen in the colon cancer (Visokai and Lipsika, 2012). The search for designated and specific changes in genes in the cancer cells are significant in devising the better method of treatment to be accorded. The levels of CEA are significant in the assessment and detection of recurrent colorectal cancer. Patients with increased levels of CEA usually have diseases that have been greatly confined in the colon but the sensitivity is set to increase with the advancement of tumor stages. A tumor tissue is tested to check for changes such as microsatellite instability which is present in most colorectal cancers. This helps for further screening or rather identify the early stages of the disease and recommend the better treatment methods. The test is significant in checking for the presence or absence of the proteins related to the microsatellite instability (Pathol, 2010). 3. b) Other staining methods The process of embedment must be put to a reverse with the aim of removing the paraffin that was used as an embedment agent thus allowing soluble dyes and water to pierce the sections. Clearing is done since no stain is possible on tissue samples containing paraffin. Staining involves the use of varied dyes chosen based on their abilities. The common staining method is the hematoxylin and eosin while other special stains are also used based on the situations. The other staining methods used would include which involve the use of compounds such as Oil Red, the Congo red compound, use of silver salts, safranin and the use of artificial dyes. Of recent times, certain antibodies have also been used in staining specific proteins, carbohydrates and even lipids through immunohistochemistry which has largely assisted in the identification of different categories of cells under the microscopes. The antibody technique in staining requires. Another staining technique is in situ hybridization which is significant in the process of DNA and RNA molecule identification. References Brown, R., 2010. Histological preparations:Common problems and their solutions. Chicago: ASCP Press. Burn, D. and Jaros, E. (2011) Multiple system atrophy:cellular and molecular pathology. Journal of clinical pathology, 54(6), p.419-426. Dragovich, T. (2011) Colon Adenocarcinoma. Drugs, Diseases, and Procedures, p.1-7. Pathol, S. (2010) Effect of formalin tissue fixation and processing on Immunohistochemistry. The American Journal of Surgical Pathology, 24(7), p.1016-1019. Visokai, L. and Lipsika, L. (2012) Tumor markers in staging and prognosis of colorectal carcinoma. Neoplasma, 55(2), p.138-42. Read More