**3. Histopathology technique development**

Histopathology techniques refer to procedures that must be carried out to produce histological preparations of diseased tissue which will be analyzed visually. These techniques include 10% formalin buffer fixation, embedded paraffin, manual sectioning, dewaxed sectioning, routine staining with Hematoxylin**–**Eosin (HE), or special staining such as immunohistochemistry (IHC), in situ hybridization (ISH), or special staining for connecting tissue [3, 9].

Observations with histopathological techniques are observations done under a light microscope. Imaging evaluations are generated by comparing diseased tissue with control or healthy tissue [9]. The purpose of this evaluation is to help diagnose, plan therapy, and predict a disease's prognosis [10].

This histopathology technique, which has been practiced since 1970, can only be partially done automatically. The embedding and sectioning processes must be done manually [9]. Several new procedures have been developed to improve image quality from conventional histopathological techniques, such as fixation procedures to protect RNA from autolysis damage during the death process [10].

#### **3.1 Fixation**

Fixation is the initial procedure after a fresh specimen is taken during surgery. The purpose of fixation is to prevent the autolysis process and postmortem cell death, which can affect water, electrolytes, and enzyme activity dynamics. If the autolysis process and cell death are not prevented, then the cells or tissues in the specimen will become easily overgrown with microorganisms [9].

#### *Introduction of Histopathology DOI: http://dx.doi.org/10.5772/intechopen.110225*

Fixation methods are typically either physical or chemical. The physical method, for example, involves packing specimens cut during operations in vacuum plastic bags and storing them at 4°C. This method can protect RNA and prevent dehydration and enzyme autolysis. The chemical method is done by immersing the specimen in a chemical solution [3].

Chemical solutions that are routinely used are neutral buffered formalin 10% (NBF 10%) or Formalin-Fixed Paraffin-Embedded (FFPE), glutaraldehyde, mixed formaldehyde, and glutaraldehyde solution, and osmium tetraoxide. NBF 10% or FFPE is used routinely for specimens viewed under the light microscope. Meanwhile, glutaraldehyde, or a mixture of formaldehyde and glutaraldehyde and osmium tetraoxide is used routinely for specimens viewed under an electron microscope [3, 11]. Alcohol can also be used for fixation, but it is necessary to pay attention to its percentage and fixation time. This is because alcohol attracts water very quickly and denatures proteins. Research conducted by Arni et al. states that alcohol fixation can be done with ethanol with gradual concentrations, from 40% for 24 hours and up to 60% within the next 24 hours. The results of this study showed a good contour cell seen in the light microscope [11].

Neutral buffered formalin 10% (NBF 10%), paraformaldehyde 4%, and formalin are aldehyde groups. NBF 10%, paraformaldehyde 4% is a fixation medium that is routinely used by pathologists. Formaldehyde is a gas that dissolves in water to form methylene hydrate. Formalin is formed from 37 **to** 40% formaldehyde and 60**–**63% water. This process can take days if plain water is used to dissolve the formaldehyde. This is different if it is dissolved in a buffer solution at physiological pH. This fixation solution is known as neutral buffered formalin 10%. NBF 10% is enriched with 10% methanol to prevent precipitation to paraformaldehyde. Paraformaldehyde dissolved in water and containing 1% methanol is called 4% paraformaldehyde. NBF 10% is routinely used by pathologists to examine diseased tissue under a light microscope, while 4% paraformaldehyde is used in electron microscopes [12].

The aldehyde group can bond with nitrogen and several protein atoms or adjacent atoms to form a cross-link called a methylene bridge. Tissue reaction to formalin can occur within 24 hours. However, cross-links may weaken the longer the fixation process takes. The cross-links protect the proteins, carbohydrates, and lipids, which are trapped in and not chemically changed. However, if the fixation is carried out for several weeks, the cross-links will be reversible and break the carbohydrates, proteins, and fats' properties [3, 12].

Glutaraldehyde is a small molecule that has two aldehyde groups. Because of this, glutaraldehyde has greater potential to cross-link and is faster than formalin [12]. This protects the ultrastructural components. The cross-link reaction is irreversible. Due to the nature of glutaraldehyde, this fixation medium was used as the first fixation medium for electron microscopy specimen preparations. However, this fixation medium has a weakness when used for special immunohistochemical staining. Because the cross-link reaction is irreversible, staining antibodies cannot enter and allow background staining to occur [3].

Osmium tetraoxide was the first fixation medium for electron microscopy specimen preparation. This fixation medium protects cell and tissue structures that contain lipids and can react to form cross-links on hydrophilic or hydrophobic atoms. However, because of its expense and toxicity, osmium tetraoxide is occasionally used for the second medium fixation instead [3, 12].

#### **3.2 Tissue processing**

Tissue processing is a procedure for immersing tissue specimens into paraffin. This aims to make the tissue harder, making it easier to cut thinly. The tool for this procedure is a tissue processor [2, 3]. The tissue processor has a jar or tube consisting of glass or copper which contains 96% alcohol, absolute alcohol, chloroform, chloroform saturated with paraffin, paraffin bath, and water. The jar containing the liquid paraffin is made of heated copper. Tissue processing and embedding last about 26**–**100 hours or several days. The following is the time needed to produce network embedding with optimal results [2]:

Alcohol 96% 6**–**24 hours Alcohol absolute 6**–**24 hours Chloroform 6**–**24 hours Chloroform saturated with paraffin 6**–**24 hours Paraffin bath 2**–**4 hours Cool water (quickly).

#### **3.3 Sectioning**

Sectioning is a procedure for cutting tissue that has been embedded with paraffin (paraffin block) into several ribbons. Cutting is done using a tool called the microtome. Thickness is generally 3**–**5 micrometers, except for neural networks, for which optimal results (not too thick) are around 7 micrometers [3, 9].

The ribbon that is formed is put in warm water at a temperature of about 45°C. This pulls and thins the ribbon, allowing it to stretch without bending. It can then be easily positioned on the glass slide. Previously, the glass slides should have been labeled and given glue made from egg whites. After the ribbon is attached, the glass slide is dried at room temperature or heated at room temperature at 37°C [3, 9].

#### **3.4 Staining**

A staining procedure is required to be able to evaluate the resulting image under a light microscope. This is because the prepared specimens produced from paraffinization have low contrast. This makes it difficult to distinguish structures [9].

Sectioning, followed by staining, previously had to go through a rehydration stage with xylene as well as absolute alcohol with up to 95% alcohol. This rehydration stage aims to pull out the paraffin from the intracell and the extracellular and replace it with water. This is done because routine dye stains are water-soluble [9].

The following are some of the dyes that are often used by pathologists: Hematoxylin**–**eosin (HE) stain, osmic acid stain, periodic-acid Schiff (PAS) stain, Masson-Mallory trichrome, and Giemsa [13]. He is the oldest routine stain and is still used as a routine stain by pathologists. There has been an emergence of histopathological dye development because, since 1903, several researchers considered HE staining inadequate. These researchers are M. Heidenhain (1903), Masson (1923), Langeron (1925), and Gabe (1969). They assumed this because no new structures were found when only relying on HE staining. Currently, in addition to staining for specific cell parts, staining techniques have been developed molecularly with immunohistochemistry, immunofluorescence, and in situ hybridization [3, 13].

#### *Introduction of Histopathology DOI: http://dx.doi.org/10.5772/intechopen.110225*

Osmic acid is used to color fat produced by cells. An example is myelin, which is a glial cell produced by Schwan cells in the peripheral nervous system. PAS staining is used for cells that produce glucose, causing a pink granule to appear in the cytoplasm. The Masson Mallory trichome stain is used to differentiate collagen fibers in several connective tissues. Giemsa is used to stain blood cells for the cytoplasmic granules to be differentiated [13].

Hematoxylin can be found in the inner part of the logwood tree, which was originally found in Central America. Presently, it is grown in the Caribbean islands, Australia, India, Malaysia, and West Africa [14]. When dissolved, hematoxylin contains one or more aluminum-hematein complex cations known as hemalum. Hemalum has a bond with acids, producing a blue or purple color in the nucleus and nuclei. Cations will react with DNA and rRNA in the nucleus [9, 13, 15]. Meanwhile, eosin is a solution that binds to structures other than acids in the cytoplasm and gives it a red color [15].

Hematoxylin is currently scarce, which is why preventive measures are being taken by using substitutes that have the same properties. Substitutes have to be cationic and able to bind to acids. Some of the substitute materials studied are the thiazine, anthocyanin, and anthocyanidins groups. Toluidine blue is a member of the thiazine group. It is used by dissolving it in water until the pH reaches 3**–**4 or unbuffered. This toluidine blue solution can stain the cell nucleus and sulfated glycosaminoglycans. Sulfated glycosaminoglycans include bony matrix, mast cell granules, and mucousproducing cells. Meanwhile, anthocyanins and anthocyanidins are dyes produced by flowers and fruits, such as Rosella extract and some others that contain flavonoids. The extract is taken by heating it in water with an acidic pH to remove carbohydrates or sugars. The addition of metal salts such as aluminum potassium sulfate (alum) can increase the stain's intensity (**Figure 6**) [14].

#### **3.5 Mounting**

Mounting is the last procedure after the coloring process. The staining process is followed by blocking the specimen, making it unable to accept water or other solutions and thereby last a long time. This process begins with cleaning the remaining paint with running water, adding graded alcohol from absolute alcohol to 95% alcohol, then cleaning with xylene [3, 9, 15].

#### **Figure 6.**

*Villi intestinalis with 400 magnification. A = HE staining intestinalis. The goblet cell cytoplasm looks empty (long arrow). B=PAS staining. The goblet cell cytoplasm looks pink or purple (small arrow).*

#### **Figure 7.**

*Peripheral nervous system with 400 magnification. Blue circle shows artifact shadow because of difference of thickness section.*

Mounting is done immediately after cleaning with xylene. The specimen preparation is covered with a cover glass with a waterproof adhesive. After that, the evaluation process is carried out under a microscope. Visible cell structure and contour, artifacts, and any color ingredients that did not enter are evaluated [3, 9, 15].

#### **3.6 Artifacts**

Artifacts are errors in performing histopathological procedures or techniques. Several types of artifacts can be found. Broken tissue can be caused by tissue being too hard, or the microtome blade needing replacement. Shadow images in the microscope can be caused by different cutting thicknesses. Folded tissue can arise after sectioning if the tissue is not stretched in warm water. Inconsistent staining thickness or outof-place paint drops can be caused by mishandling the process of cleaning paint with running water (**Figure 7**) [3, 9, 15].

## **4. Conclusions**

Histopathology is basic knowledge for a pathologist. Making a definite diagnosis, planning therapy, and predicting the prognosis of patients' diseases are very dependent on changes in their macroscopic and microscopic structures. These two structural changes are sometimes incompatible because macroscopic changes are preceded by microscopic changes, either due to internal or external stressors. Histopathological evaluation can be done if the imaging obtained is good enough. This is related to the histopathological technique and the type of microscope used.
