Histotechnology- Fixation question & answers graded A+ 2023/2024

Histotechnology- FixationFixation: - correct answers Alteration of biological tissue by chemical or physical means which stabilizes proteins found in tissue, preventing further metabolic activity. Purpose of Fixation: - correct answers To kill tissue and preserve it by limiting the effects of decay, putrefaction, and autolysis. Fixation also hardens tissue which makes it easier to gross, and easier to section once it is processed and embedded. Physical Agents: - correct answers - Heat and Desiccation possess fixative properties but they are not very practical and do not result in the high level of structural preservation that can be achieved by using chemical fixative agents. Chemical Fixatives: - correct answers -Are grouped by the way they interact with tissues they are used on. -they can either be additive or nonadditive and coagulants or noncoagulants Additive Fixatives definition and examples - correct answers -Actually bond with and become part of the tissue proteins. -additive noncoagulants - formaldehyde, gluteraldehyde, and osmium tetroxide Nonadditive fixatives definition and examples - correct answers - Act on the structure of proteins but do not become part of the tissue -nonadditive coagulants- alcohol, acetone, acetic acid Coagulants definition and example: - correct answers - Work by precipitating proteins (can be additive or nonadditive) -nonadditive coagulants- alcohol, acetone, acetic acid Noncoagulants definition and example: - correct answers - Work by bonding with tissue proteins and altering their shape but do not directly precipitate the proteins. - additive noncoagulants- formaldehyde, gluteraldehyde, osmium tetroxide List the factors affecting Fixation - correct answers 1. Temperature increases 2. Size and thickness 3. Volume ratio of fixative to tissue 4. Tissue storage 5. Time elapsed before tissue is placed in fixative and time allowed for tissue to be thoroughly fixed 6. Tissues that have not been allowed adequate time for fixation will not do well in subsequent processing steps 7. Choice of Fixative 8. Penetration rates 9. the pH level of the fixative 10. Osmolality Temperature Increase: - correct answers -Speeds the rate of fixation but also increases the rate of autolysis which can negate the positive effects. -room temperature fixation in formaldehyde is the most commonly accepted procedure. Size and thickness: - correct answers - Plays a role in the rate of penetration of the fixative, making deeper regions more prone to autolysis. This is especially true with more delicate epithelial linings. - To remedy the problem, larger specimens should be cut into smaller pieces and regions of epithelia should be OPENED up to fixative solution. Volume ratio of fixative to tissue: - correct answers - If the concentration of the fixative becomes diluted with tissue liquids or the fixation solution volume is too low to allow for proper penetration- good fixation will not occur. - Rule of thumb- fix material in a solution volume greater than 20X the volume of the tissue itself Tissue Storage: - correct answers - Tissue storage of wet tissue is important should additional studies be required in the future. - Formalin fixed tissue can be stores in formalin indefinitely. However, since protein crosslinking is an ongoing process, tissue should be transferred to 70% ethanol if immunohistochemical stains may be required. Time elapsed before tissue is placed in fixative and time allowed for tissue to be thoroughly fixed - correct answers - The longer it takes for a sample to be immersed in fixative, there will be more negative effects of autolysis and other factors. - This is why biopsies and surgical samples produce much better quality microscopic studies than autopsy tissue. Tissues that have not been allowed adequate time for fixation will not do well in subsequent processing steps - correct answers - Various structural artifacts and poor staining may likely occur. - Ideally, formalin fixation should last 6-8 hours before further processing occurs. - smaller specimens are less susceptible to this need due to greatly improved penetration of solutions. Choice of fixative: - correct answers - This is a major concern depending on the type of tissue involved and the ultimate focus of the study. Penetration rates - correct answers - An important factor in how quickly the fixation process proceeds. In all cases, the smaller and/or thinner a specimen is (or can be cut to) will contribute to a good penetration rate. - Other issues can influence how the penetration rate affects the quality of fixation. Ex. Formaldehyde has a very rapid penetration rate but complete fixation can take many hours since protein crosslinking is an ongoing process. - Some coagulant fixatives penetrate much more slowly but the complete precipitation of protein occurs almost instantly. The pH level of the fixative - correct answers - concern for EM studies, should be kept at a pH between 7.2- 7.6 using buffers to preserve ultrastructural details - Not as important for LM, acceptible pH range 4-9 Osmolality: - correct answers - measure of the number of particles in solution. Physiologic osmolality = 0.3 Osm. - if the fixer osmolality is higher (hypertonic) water is drawn out of the cell causing shrinking.