Introduction of Histopathology

Abstract

Histopathology is the science that studies disease pathology through morphological changes that can be seen microscopically. The word histopathology was first known to be introduced in a book entitled “On the Nature and Structure Characteristic of Cancer” by Johannes Muller in 1838. Before histopathology, doctors distinguished diseases based on macroscopically visible morphology organ changes (anatomy) in the operating room or during forensic autopsies. However, this expertise requires another diagnosis method to ensure that several differential diagnoses indicate the same anatomical morphological changes. Definitive diagnosis is obtained by taking a small organ tissue sample during surgery (biopsy), which is then viewed microscopically. This diagnosis method is called histopathological diagnosis. For example, a person with hearing loss has received a differential diagnosis of conduction or sensorineural hearing loss. An external macroscopic physical examination for this patient is as necessary as internal imaging. The condition’s pathological cause can be ascertained by additional histopathology biopsies. However, invasive histopathology procedures can be uncomfortable and cause harm. Histopathology for diagnostic or research purposes requires two fundamental things: the microscope as a tool and the development of histopathological techniques.

1. Introduction

Histopathology is very widely used, not only as a diagnostic tool but also as an examination tool, monitoring the effects of therapy or estimating a disease’s prognosis. Microscopes are necessary equipment for carrying out histopathological functions. Histopathological preparations rely on microscopes, as does result examination [1].

A microscope is a tool for viewing cell and tissue structures that cannot be seen by the naked eye because of their micron or smaller size. The cell is the smallest unit of life. Several cells that come together and perform the same function are referred to as tissue. Organs are several tissues that come together and form the same function. The microscope was first introduced by Zacharias Jansen and his father in 1591 [2], and it was then perfected by Leeuwenhoek in 1673. Furthermore, along with the need to see certain tissue structures, microscopes evolved from light to fluorescence microscopes [2].

Histopathology is a technique for making histological preparations for diseased tissues. These techniques include fixation techniques, tissue processing, tissue sectioning, and tissue staining. Fixation techniques are needed for hardening, keeping the cell death process from continuing, and keeping structural molecules from being damaged. Tissue processing aims to remove water from the intracell and replace it with a medium that can harden the cell structure to make it easy to cut. Tissue sectioning refers to cutting the prepared specimen into several slices. Finally, tissue staining aims to color the cell and tissue structures with specific staining according to the examination’s purpose [3].

Microscopy and histopathology technique development are fundamental things to know when making examination preparations and reading tissue preparations from appropriate controls to ensure good examination quality [4].

2. Microscope development

A microscope is a tool used to view tissues and organs’ microscopic structures. Microscopes work the same way eyes work. Light, which is an electromagnetic spectrum, is transmitted to the cornea and through the eye’s diaphragm (pupil) until it reaches the retina. Whereas in light microscopes, the light that hits the specimen preparation is transmitted by the microscope diaphragm and then captured by the objective and ocular lens for the eye. The resulting image’s magnification depends on the objective and ocular lenses’ sizes [2]. The following are the microscopes commonly used for a histopathology examination.

2.1 Light microscopy

Early in the sixteenth century, the first microscope was assembled by Jansen and his father. It used the light from the sun (Figure 1). The scientists at that time did not feel comfortable using this microscope because the light source did not fall on one point, thus interfering with the view. Sun or white light consists of several color spectrums. When it falls on a simple lens like Jansen’s work, it will make the colors scatter according to their wavelength. This is known as chromatic aberration. This became the basis for Leeuwenhoek in 1673 to make two lenses with different mediums. Colors that degraded due to certain wavelengths could be absorbed by the lens medium. The achromatic is a lens medium that can correct two spectrums of blue and red, while an apochromat medium is a lens medium that corrects two spectrums of green and yellow [2, 3]. Leeuwenhoek’s microscope can magnify a maximum of 300 times the resulting image. At the time, he could identify bacteria, muscles, teeth, and blood cells [3]. The microscope’s components were then developed to correct chromatic aberration with the discovery of lens mediums that fit examination needs. Leeuwenhoek’s principal work was the basic development of the light microscope (Figure 2).

Figure 1.

Jansen’s microscope with the single lens source [2].

Figure 2.

Light microscopy model in general source [2].

Light microscopes’ components and models evolve with their needs and uses. Currently, apart from being a diagnostic tool, light microscopes are also used for teaching and learning purposes. Several types of microscopes have been established with their respective advantages for teaching purposes. The double or triple ocular lens and light microscope models that are connected to the computer with or without application are widely used. Figure 3 shows the resulting image with a light microscope.

Figure 3.

Image of the spinal cord by light microscope with 400 times magnification.

2.2 Electron microscopy

The electron microscope was first assembled by Ernst Ruska in 1920. This microscope’s purpose is to view biological ultrastructures and identify diseases that are difficult to explain. Its working principle is to use light with short wavelengths to produce better resolution and magnification. Resolution is the ratio between the wavelength and the width of the diaphragm opening [3, 5].

The types of electron microscopes are the scanning electron microscope (SEM), the transmission electron microscope (TEM), and the cryo-electron microscope (Cryo-EM) [5, 6, 7]. SEM is a type of electron microscope that can only take ultrasound images on surfaces, such as the skin’s epidermal layer. TEM is a type of electron microscope that can take ultra-cell images of cytoplasmic organelles such as mitochondria and rough endoplasmic reticulum (RER). Cryo-EM is a type of electron microscope that can produce ultra-cell images up to atoms in 3D for single particle analysis, such as on certain proteins, with better resolution [5, 6, 7].

The difference in preparation between a light microscope and SEM or TEM is that the specimen’s maximum size before processing is cut at 1 mm³ as well as its fixative medium, which will be discussed in detail in topic 3 [3]. The differences in preparation between the light microscope and Cryo-EM are specimen screening and preparation, data acquisition, image processing, and structure validation [6]. Figure 4 shows an image result from EM.

Figure 4.

Image of nucleus and RER neuron cell by TEM.

2.3 Phase contrast microscopy/interference microscopy

The working principle of a phase-contrast microscope or interference microscope is that the light is blocked by the specimen, thus producing a “halo” in the resulting image. A halo pattern will form on the bright and dark bands when light passes through the specimen. Besides the halo, the resulting image is also a color gradation and a quantitative index of refraction. This microscope shows biological structures in larger pieces than other types of microscopes [3].

2.4 Fluorescence microscopy

Fluorescence is light that is reflected by objects or specimens that are lit at a certain wavelength. Objects or specimens that can emit fluorescence alone are called fluorophores, also called primary fluorescence or autofluorescence. Some examples are porphyria, elastic fiber, collagen, vitamin A, and lipofuscin [3].

Specimen fluorescence can also be induced by adding a substance, such as a certain antibody painted with a fluorescence agent like blue, green, or ultraviolet fluorochrome [2]. This was first introduced by Coons, Creech, and Jones in 1941.

Fluorescence microscopy’s development from two dimensions (2D) to three dimensions (3D) makes pathological models in biological tissues clearer. In 3D fluorescence microscopy, axial scanning is required for several specimen pieces that make up the volume. Data is transferred to a computer with certain applications. The required variables can be calculated quantitatively or only qualitatively [8]. This microscope’s weakness is that the entire field of view will show fluorochrome, including areas that are not the focus of research [3]. This weakness became the basis for confocal microscopy development.

2.5 Confocal microscopy

Confocal microscopy enhances the image results from fluorescence microscopy. Confocal microscopes have a pinhole component to only focus the image on the part being examined. Microscopic imaging results can be seen at one time [3].

Histopathology has an important role in the diagnosis. However, ordinary histological techniques after a biopsy are not able to describe details such as the boundaries of cancer cell development that must be taken during the operation at the same time (during surgery). To overcome this, the confocal microscope technique was developed into confocal laser endomicroscopy (CLE). The CLE technique greatly improves the surgical outcome and prognosis of most cancers [8].

The CLE technique requires intravenous injection of fluorosphere contrast enhancer before surgery begins. Sodium fluorescence (SF) is a type of fluorosphere that has been studied. It has a composition of 5 mL in 10% saline solution and must be injected just before surgery. The second type is 5-aminolevulinic acid (5-ALA) with a composition of 20 mg/kg, which is injected 3 hours before surgery. The time of contrast enhancer administration from seven studies gave different results in terms of cell contour [8].

Pathological feature observation results using the CLE technique were better than those with ordinary histological techniques. The contour cells could be differentiated in terms of vascular proliferation, cellular density, and irregular cell phenotype between high-grade gliomas, low-grade gliomas, and normal glia at the same time during surgery. Based on research, the imaging results were better when using SF to show the cells’ contours (Figure 5) [8].

Figure 5.

A. Confocal imaging of CLE during operation. B. Convivo ex vivo CLE image that show low cellular density (red circle). Source [8].

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].

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].

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 mucous-producing 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].

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).

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].

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 out-of-place paint drops can be caused by mishandling the process of cleaning paint with running water (Figure 7) [3, 9, 15].

Figure 7.

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

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.

Acknowledgments

Thank you to the head of the Faculty of Vocational Studies, Universitas Airlangga, who provided support in receiving the invitation to write for IntechOpen. Gratitude is also extended to the head of the Ph.D. program of the Faculty of Medicine, Universitas Airlangga, who gave his appreciation to those involved in writing these chapters and allowed them to be used for assessment points.

Conflict of interest

The authors declare there was no conflict of interest.

Appendices and nomenclature

5-ALA

5-aminolevulinic acid

CLE

confocal laser endomicroscopy

Cryo-EM

cryo electron microscope

DNA

deoxyribonucleic acid

FFPE

formalin fixed paraffin embedded

HE

hematoxylin – eosin

IHC

immunohistochemistry

ISH

in situ hybridization

NBF

normal buffered formalin

PAS

periodic-acid Schiff

RER

rough endoplasmic reticulum

RNA

ribonucleic acid

SEM

scanning electron microscope

SF

sodium fluorescence

TEM

transmission electron microscope