1. Tissue processing- Trimming and Sectioning
2. Tissue processing- Staining and Quality study of the slide
Tissue processing is an routine but essential component in histopathology department and it do contribute a lot of information to the physician in diagnose the cancer state or condition of any other life threatening disease. Firstly, the surgeon will be remove part of the organ in the surgery theater room and send to histopathology department by immerse the removed part of the organ in a fixative solution. The purpose of this fixation is to avoid the cell being digested by own cytoplasmic enzyme or by bacteria beside preserve the structure and molecular composition (Mescher, 2010; Hewitson and Darby, 2010). For routine histological investigation, the tissue will be immersed in the 37 % formaldehyde (Mescher, 2010), in addition, neutral buffered formalin and 4 % paraformaldehyde could be employed for routine fixation as well (Hewitson and Darby, 2010). In fact, there are two ways to perform fixation, first of all, the chemical fixation (immerse the whole removed organ part into a fixative solution and whole tissue will be fixed via diffusion manner) while the other one will be intravenous perfusion of fixatives (infuse the fixative solution via blood stream by cannulation of the aorta) (Hewitson and Darby, 2010). Both ways have their own risk and advantage but this scope is beyond the objective of this experiment.
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Once the tissue have been received, then it will be embedded into either paraffin (for light microscopy) or plastic resin (for both light and electron microscopy). In this step, dehydration and clearing the tissue is the two main objectives before sectioning. The dehydration will be achieved by immersing the tissue into a series of ethanol solution with different concentration (50 % to 70 %). To remove the ethanol from the tissue, the tissue will be immersed in a solvent that able to dissolve both ethanol and embedding materials. As a result, the tissue will become transparent and the clearing of this tissue is achieved. Then, the tissue will be placed into a melted paraffin in order to replace the solvent. Once all the space of the tissue have been replaced by the paraffin, then the tissue coated with paraffin will be solidified and ready for sectioning (Mescher, 2010). If the tissue was prepared for Immunohistochemical staining purpose, then it will undergo antigen retrieval step, which is using the enzyme digestion or heat-induced antigen retrieval. This step is mainly to recover back the cellular protein or plasmic component that have been cross-linked in the fixation step (Hewitson and Darby, 2010).
For sectioning part, a specialized instrument will be used and called as microtome. This instrument able to cut the embedded tissue into a very thin film (4 µm to 5 µm), hence, the pathologist able to observed any alternation on the structure of the tissue and identify the etiology of a certain disease. Beside this approach, as claimed by Mescher (2010), rapid freeing also could be employed but the principle of this approach is beyond the objective of this experiment. After the embedded tissue have been trimmed and sectioned, the thin paraffin film will be place on a warm water bath in order to expand the paraffin. The details on how to use a microtome and the precaution in expansion of the paraffin film have been described in the procedure of this report.
Once the slide have been dried out, then it will be stained to visualize and differentiate the border between each cell type. The most common stained to be used is the Hematoxylin and Eosin staining (H&E) and this staining technique also is the staining that employed in this experiment. H&E staining stain up the cell sorely based on the attraction between the acidic component and basic compound. Hematoxylin is an basic dye, thus, it will stain up the DNA, RNA, ribosome and nuclei in a blue colour while the eosin is an acidic dye and stain up all the cytoplasmic protein and keratin in a pink colour. The basophilic and acidophilic are the terms to categories the component that stained up by basic dye and acidic dye respectively. Beside the attraction force between the negative charge and positive charge that carried by the cellular component, there are some stain also is rely on chemical reaction between the cellular compound and the dye component. For instance, the Periodic acid and Schiff reagent is one of the staining techniques that will react with DNA and stain it in purple colour (Mescher, 2010; Young, Lowe, Steven and Heath, 2006).
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- To learn the whole process for tissue processing (from fixation to staining).
- To identify the any artifacts on the slide and propose some suggestions for improvement.
Trimming and Sectioning Part:
- The safety lock of microtome handle should be locked before and trimming started.
- The chilled block was place on the block holder of microtome.
- The safety guard on the blade was removed and the position of it was adjusted until the blade was almost touch the block.
- The angle of the block holder was adjusted to an optimal angel against the blade.
- The thickness of the film was first adjusted to 8 or 10 µm for trimming process.
- The handle was unlocked and turned in a clock-wise direction and this step was repeated until the block was trimmed properly (normally until the surface of the block become smooth and minimal crack).
- Once the block was trimmed, the thickness of the film will be adjusted to 4 or 5 µm for sectioning.
- A tooth stick was prepared and adhered at the end of the film that have been sectioned out.
- Without break the adherent of the subsequent film, the sectioned film was pulled slowly and gently.
- The chain of sectioned film was placed on the water bath (600C).
- The handle of the microtome was locked and the safety guard of the blade was placed.
- A clean slide was prepared and film was fished and adhered on the middle of slide.
- The slide was dried by oriented vertically in a slide holder before heated with hot plate.
- Step 8 onward were repeated for the second slide preparation.
Staining and Quality study Part:
- The dried slides were placed in a slide holder.
- The details of subsequent steps for the staining part could refer to the Appendix 1as shown in the appendices.
- All slides were observed under light microscope and the result was captured.
In this experiment, ileum was the organ been employed to achieve the objectives of this experiment. As shows in the result, there are 3 main artifacts could be identified which are the fine line formation due to using a blunt blade during section, the tore off of part of the tissue and the hole formation due to overexpansion of the paraffin film.
In Figure 1 and Figure 2, there was a series of fine line could be observed at the bottom of this field. This is due to the blunt blade was used. Once the blade blunt, it was no longer perpendicular to the position of the block, eventually, the surface of the film will not as smooth as sharp end of the blade. To overcome this, the position of the blade should be adjusted to the sharper end or replace a brand new blade before section and trimming. Further enhance the quality of the film, the block should be trimmed and sectioned slowly. Beside this reason, another reasons could be proposed that poor processing and debris in unfiltered wax (Leica Microsystems, 2008; Leica Microsystems, 2010).
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In Figure 1 again, the tissue obviously have been tore off and the complete morphology of the tissue could not be study. After revise all the step in staining, the possible reason lead to this situation could be the stacked slides in the slide holder. During the staining step, the slide was found stacked with another slide. In order to separate it, it slides against the stacked slide and cause some part of the tissue was sloughed off from the slide. The similar artifact due to physical trauma also was shown in the catalogue done by Leic Microsystems (2008). To prove this, the same block was sectioned and stained again without stacked to any other slide. As a result, as shown in Figure 2, there is no any part of tissue is lost and shows complete morphology of the tissue. Beside this reason, as claimed by Leica Microsystems (2008), the tissue is not fully fixed on the slide due to shorter drying process. If there is any water droplet present beneath the film and did not be removed completely, then the film will not fix on the slide and easily to slough off. This situation will be exacerbated when the sectioned film contain any wrinkles or crack.
In Figure 3, there are some holes could be observed at the right side of the field. Compared to the Leica Microsystems instruction manual, the hole formed could be due to the high temperature of the water bath have been used in the fishing step. Besides that, the time for expanding the film too longer also may cause the similar artifacts. The paraffin was highly heat-sensitive, the temperature of the water bath should reduced to 58 0C whereas the melting point of the paraffin wax is 58 0C to 62 0C (ChemicalBook, 2010; Leica Microsystems, 2008; Leica Microsystems, 2010).
Besides these three artifacts, there no other artifact can be identified. The slide do not show any effect from the chemical reaction, contaminant by other types of tissues, bubble beneath the section and so on. However, there still have several precaution steps or maintenance steps should be carried out. First of all, the staining techniques should be identified and appropriate slide should be chosen. Different type of slide was designed differently from each other in order to enhance specific staining process. For instance, for the Immunohistochemical staining should employed the aminoalkylsilane (AAS) slide instead of normal glass slide because aminoalkylsilane (AAS) slide could provide better adhesion of the section and prevent it from lifting. The block should be ensured is cold and hard enough before trimming or sectioning. It is because the warm paraffin wax no longer able to provide strong support to the tissue and make the block not amenable for cutting. The section will plenty of wrinkles will be the result of sectioning from a warm block (Leica Microsystems, 2010).
The whole process of the tissue processing was studied and there are few artifacts was identified and proposed with rationales which are fine line due to the blunt end of the blade was use, part of the tissue was lost due to physical trauma and short drying time and lastly, hole formation due to high temperature was used.
ChemicalBook.(2010). Paraffin wax. [Online]. Available at: http://www.chemicalbook.com/ChemicalProductProperty_EN_CB2854418.htm [ Accessed by 8 February 2014]
Hewitson, T. D. and Darby, I.A., 2010. Histology Protocols. USA: Human Press.
Leica Microsystems, 2008.101 Steps To Better Histology
Leica Microsystems, 2010. Microtomy and Paraffin Section Preparation.
Mescher, A.L., 2010. Junqueira's Basic Histology: Texts and Atlas. 12th ed. USA: The McGraw-Hill Education (Asia).
Young, B., Lowe, J.S., Stevens, A. and Heath, J.W., 2006.Wheater's Functional Histology: A Text and Colour Atlas. 5th ed. USA: Elsevier Limited.
- Name the glass slide used for IHC staining.
aminoalkylsilane (AAS) slide
- Briefly describe the principle behind IHC.
IHC (Immunohistochemical) techniques is employing the specificity of the antibodies that target on the specific cell antigen. After the binding, a particular cell morphology could be enhanced. Beside the cell antigen, protein or any enzyme that produced by the cell also could be stained by using this technique. The antibodies used here must totally different species from the tissue that are examining.
First of all, the tissue was covered a layer of solution that contain specific antibodies. After few minutes, the solution is washed off with an intention to wash away all unbound and excess antibodies. To make the antibodies visible, there are 2 ways but not limited to, the antibodies was pre-linked with fluorescent substances or conjugated with an enzyme. If the antibodies was linked with fluorescent materials, the position of the antibodies could be observed under an ordinary fluorescent microscope while the enzyme-conjugated antibodies will be only visible after the substrate have been added and catalyzed by the enzyme. Despite any method was employed, the region of the target cell could be sure that be emphasized and shows a high contrast to the other region of the tissue.
State the component s stained by hematoxylin and eosin
- Hematoxylin: Nuclei, ribosome, rough endoplasmic reticulum, DNA and RNA
- Eosin: cytoplasmic protein and collagen