Histopathology

Histopathology

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Histopathology refers to the microscopic examination of tissue in order to study the manifestations of disease
Disease
A disease is an abnormal condition affecting the body of an organism. It is often construed to be a medical condition associated with specific symptoms and signs. It may be caused by external factors, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune...

. Specifically, in clinical medicine, histopathology refers to the examination of a biopsy
Biopsy
A biopsy is a medical test involving sampling of cells or tissues for examination. It is the medical removal of tissue from a living subject to determine the presence or extent of a disease. The tissue is generally examined under a microscope by a pathologist, and can also be analyzed chemically...

 or surgical specimen
Specimen
A specimen is a portion/quantity of material for use in testing, examination, or study.BiologyA laboratory specimen is an individual animal, part of an animal, a plant, part of a plant, or a microorganism, used as a representative to study the properties of the whole population of that species or...

 by a pathologist
Pathology
Pathology is the precise study and diagnosis of disease. The word pathology is from Ancient Greek , pathos, "feeling, suffering"; and , -logia, "the study of". Pathologization, to pathologize, refers to the process of defining a condition or behavior as pathological, e.g. pathological gambling....

, after the specimen has been processed and histological section
Histological section
Histological section refers to thin slices of tissue applied to a microscopic slide, usually around 5 to 10 micrometres thick, which are viewed under a microscope...

s have been placed onto glass slides. In contrast, cytopathology
Cytopathology
Cytopathology is a branch of pathology that studies and diagnoses diseases on the cellular level. The discipline was founded by Rudolf Virchow in 1858. A common application of cytopathology is the Pap smear, used as a screening tool, to detect precancerous cervical lesions and prevent cervical...

 examines free cells or tissue fragments.

Collection of tissues


Histopathological examination of tissues starts with surgery
Surgery
Surgery is an ancient medical specialty that uses operative manual and instrumental techniques on a patient to investigate and/or treat a pathological condition such as disease or injury, or to help improve bodily function or appearance.An act of performing surgery may be called a surgical...

, biopsy
Biopsy
A biopsy is a medical test involving sampling of cells or tissues for examination. It is the medical removal of tissue from a living subject to determine the presence or extent of a disease. The tissue is generally examined under a microscope by a pathologist, and can also be analyzed chemically...

, or autopsy
Autopsy
An autopsy—also known as a post-mortem examination, necropsy , autopsia cadaverum, or obduction—is a highly specialized surgical procedure that consists of a thorough examination of a corpse to determine the cause and manner of death and to evaluate any disease or injury that may be present...

. The tissue is removed from the body
Body
With regard to living things, a body is the physical body of an individual. "Body" often is used in connection with appearance, health issues and death...

 or plant
Plant
Plants are living organisms belonging to the kingdom Plantae. Precise definitions of the kingdom vary, but as the term is used here, plants include familiar organisms such as trees, flowers, herbs, bushes, grasses, vines, ferns, mosses, and green algae. The group is also called green plants or...

, and then placed in a fixative
Fixation (histology)
In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay, thereby preventing autolysis or putrefaction...

 which stabilizes the tissues to prevent decay
Decomposition
Decomposition is the process by which organic material is broken down into simpler forms of matter. The process is essential for recycling the finite matter that occupies physical space in the biome. Bodies of living organisms begin to decompose shortly after death...

. The most common fixative is formalin (10% formaldehyde
Formaldehyde
Formaldehyde is an organic compound with the formula CH2O. It is the simplest aldehyde, hence its systematic name methanal.Formaldehyde is a colorless gas with a characteristic pungent odor. It is an important precursor to many other chemical compounds, especially for polymers...

 in water).

Preparation for histology


The tissue is then prepared for viewing under a microscope
Microscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...

 using either chemical fixation or frozen section.

Chemical fixation


In chemical fixation
Fixation (histology)
In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay, thereby preventing autolysis or putrefaction...

, the samples are transferred to a cassette, a container designed to allow reagents to freely act on the tissue inside. This cassette is immersed in multiple baths of progressively more concentrated ethanol
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...

, to dehydrate the tissue, followed by toluene
Toluene
Toluene, formerly known as toluol, is a clear, water-insoluble liquid with the typical smell of paint thinners. It is a mono-substituted benzene derivative, i.e., one in which a single hydrogen atom from the benzene molecule has been replaced by a univalent group, in this case CH3.It is an aromatic...

 or xylene, and finally extremely hot liquid (usually paraffin
Paraffin
In chemistry, paraffin is a term that can be used synonymously with "alkane", indicating hydrocarbons with the general formula CnH2n+2. Paraffin wax refers to a mixture of alkanes that falls within the 20 ≤ n ≤ 40 range; they are found in the solid state at room temperature and begin to enter the...

). During this 12 to 16 hour process, paraffin will replace the water in the tissue, turning soft, moist tissues into a sample miscible with paraffin, a type of wax. This process is known as tissue processing.

The processed tissue is then taken out of the cassette and set in a mold. Through this process of embedding, additional paraffin is added to create a paraffin block which is attached to the outside of the cassette.

The process of embedding then allows the sectioning of tissues into very thin (2 - 7 micrometer) sections using a microtome
Microtome
A microtome is a sectioning instrument that allows for the cutting of extremely thin slices of material, known as sections. Microtomes are an important device in microscopy preparation, allowing for the preparation of samples for observation under transmitted light or electron radiation...

. The microtome slices the tissue ready for microscopic examination. The slices are thinner than the average cell, and are layered on a glass slide for staining.

Frozen section processing


The second method of histology processing is called frozen section processing. In this method, the tissue is frozen and sliced thinly using a microtome
Microtome
A microtome is a sectioning instrument that allows for the cutting of extremely thin slices of material, known as sections. Microtomes are an important device in microscopy preparation, allowing for the preparation of samples for observation under transmitted light or electron radiation...

 mounted in a below-freezing refrigeration device called the cryostat
Cryostat
A cryostat is a device used to maintain cold cryogenic temperatures. Low temperatures may be maintained within a cryostat by using various refrigeration methods, most commonly using cryogenic fluid bath such as liquid helium. Hence it is usually assembled into a vessel, similar in construction...

. The thin frozen sections are mounted on a glass slide, fixed immediately & briefly in liquid fixative, and stained using the similar staining techniques as traditional wax embedded sections. The advantages of this method is rapid processing time, less equipment requirement, and less need for ventilation in the laboratory. The disadvantage is the poor quality of the final slide. It is used in intra-operative pathology for determinations that might help in choosing the next step in surgery during that surgical session (for example, to preliminarily determine clearness of the resection margin of a tumor during surgery).

Staining of the Processed Histology Slides


This can be done to slides processed by the chemical fixation or frozen section slides. To see the tissue under a microscope, the sections are stained with one or more pigments. The aim of staining is to reveal cellular components; counterstains are used to provide contrast.

The most commonly used stain in histopathology is a combination of hematoxylin and eosin
Eosin
Eosin is a fluorescent red dye resulting from the action of bromine on fluorescein. It can be used to stain cytoplasm, collagen and muscle fibers for examination under the microscope. Structures that stain readily with eosin are termed eosinophilic....

 (often abbreviated H&E). Hematoxylin is used to stain nuclei
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...

 blue, while eosin stains cytoplasm
Cytoplasm
The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...

 and the extracellular connective tissue matrix pink. There are hundreds of various other techniques which have been used to selectively stain cells. Other compounds used to color tissue sections include safranin
Safranin
Safranin is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring all cell nuclei red. This is the classic counterstain in a Gram stain...

, Oil Red O
Oil Red O
Oil Red O is a lysochrome diazo dye used for staining of neutral triglycerides and lipids on frozen sections and some lipoproteins on paraffin sections...

, congo red
Congo red
Congo red is the sodium salt of 3,3'-bis. It is a secondary diazo dye...

, silver salts and artificial dyes. Histochemistry refers to the science of using chemical reactions between laboratory chemicals and components within tissue. A commonly performed histochemical technique is the Perls Prussian Blue reaction, used to demonstrate iron deposits in diseases like Hemochromatosis.

Recently, antibodies
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...

 have been used to stain particular protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s, lipid
Lipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...

s and carbohydrate
Carbohydrate
A carbohydrate is an organic compound with the empirical formula ; that is, consists only of carbon, hydrogen, and oxygen, with a hydrogen:oxygen atom ratio of 2:1 . However, there are exceptions to this. One common example would be deoxyribose, a component of DNA, which has the empirical...

s. Called immunohistochemistry
Immunohistochemistry
Immunohistochemistry or IHC refers to the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and...

, this technique has greatly increased the ability to specifically identify categories of cells under a microscope. Other advanced techniques include in situ hybridization to identify specific DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 or RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....

 molecules. These antibody staining methods often require the use of frozen section histology.
Digital cameras are increasingly used to capture histopathological images.

Interpretation


The histological slides are examined under a microscope by a pathologist
Pathology
Pathology is the precise study and diagnosis of disease. The word pathology is from Ancient Greek , pathos, "feeling, suffering"; and , -logia, "the study of". Pathologization, to pathologize, refers to the process of defining a condition or behavior as pathological, e.g. pathological gambling....

, a medically qualified specialist who has completeted a recognised training programme (5 - 5.5 years in the United Kingdom). This medical diagnosis
Medical diagnosis
Medical diagnosis refers both to the process of attempting to determine or identify a possible disease or disorder , and to the opinion reached by this process...

 is formulated as a pathology report describing the histological findings and the opinion of the pathologist. In the case of cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...

, this represents the tissue diagnosis required for most treatment protocols. In the removal of cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...

, the pathologist will indicate whether the surgical margin
Surgical margin
Surgical margin, also known as tumor free margin, free margin, normal skin margin, and normal tissue margin, usually refers to the visible normal tissue or skin margin that is removed with the surgical excision of a tumor, growth, or malignancy.-Definition:Surgical margin in a surgery reports...

 is cleared, or is involved (residual cancer is left behind). This is done using either the bread loafing
Bread loafing
Bread loafing is a common method of processing surgical specimen for histopathology. The process involves cutting the specimen into 3 or more sections. The cut sections are mounted by embedding in paraffin or frozen medium. The cut edge is then thinly sliced with a microtome or a cryostat...

 or CCPDMA
CCPDMA
CCPDMA is the acronym for "complete circumferential peripheral and deep margin assessment". It is the preferred method for the removal of certain cancers, especially skin cancers.A classical example of CCPDMA is Mohs surgery...

 method of processing.

In myocardial infarction



After a myocardial infarction
Myocardial infarction
Myocardial infarction or acute myocardial infarction , commonly known as a heart attack, results from the interruption of blood supply to a part of the heart, causing heart cells to die...

, no histopathology is seen the first ~30 minutes. The only possible sign the first 4 hours is waviness of fibres at border. Later, however, a coagulation necrosis is initiated, with edema and hemorrhage. After 12 hours, there can be seen karyopyknosis and hypereosinophilia of myocytes with contraction band necrosis in margins, as well as beginning of neutrophil infiltration. At 1 – 3 days there is continued coagulation necrosis with loss of nuclei and striations and an increased infiltration of neutrophils to interstitium. Until the end of the first week after infarction there is beginning of disintegration of dead muscle fibres, necrosis of neutrophils and beginning of macrophage removal of dead cells at border, which increases the succeeding days. After a week there is also beginning of granulation tissue formation at margins, which matures during the following month, and gets increased collagen deposition and decreased cellularity until the myocardial scarring
Myocardial scarring
Myocardial scarring is fibrous tissue that replaces normal tissue destroyed by injury or disease pertaining to the muscular tissue of the heart....

 is fully mature at approximately 2 months after infarction.

See also

  • Histology
    Histology
    Histology is the study of the microscopic anatomy of cells and tissues of plants and animals. It is performed by examining cells and tissues commonly by sectioning and staining; followed by examination under a light microscope or electron microscope...

  • Pathology
    Pathology
    Pathology is the precise study and diagnosis of disease. The word pathology is from Ancient Greek , pathos, "feeling, suffering"; and , -logia, "the study of". Pathologization, to pathologize, refers to the process of defining a condition or behavior as pathological, e.g. pathological gambling....

  • Anatomical pathology
    Anatomical pathology
    Anatomical pathology or Anatomic pathology is a medical specialty that is concerned with the diagnosis of disease based on the gross, microscopic, chemical, immunologic and molecular examination of organs, tissues, and whole bodies...

  • Molecular pathology
    Molecular pathology
    Molecular pathology is an emerging discipline within pathology which is focused in the study and diagnosis of disease through the examination of molecules within organs, tissues or bodily fluids....

  • Biopsy
    Biopsy
    A biopsy is a medical test involving sampling of cells or tissues for examination. It is the medical removal of tissue from a living subject to determine the presence or extent of a disease. The tissue is generally examined under a microscope by a pathologist, and can also be analyzed chemically...

  • Frozen section procedure
    Frozen section procedure
    The frozen section procedure is a pathological laboratory procedure to perform rapid microscopic analysis of a specimen. It is used most often in oncological surgery. The technical name for this procedure is cryosection....

  • Medical technologist
    Medical technologist
    A Medical Laboratory Scientist is a healthcare professional who performs chemical, hematological, immunologic, microscopic, and bacteriological diagnostic analyses on body fluids such as blood, urine, sputum, stool, cerebrospinal fluid , peritoneal fluid, pericardial fluid, and synovial...

  • Laser capture microdissection
    Laser capture microdissection
    Laser capture microdissection , also called Microdissection, Laser MicroDissection , or Laser-assisted microdissection is a method for isolating specific cells of interest from microscopic regions of tissue/cells/organisms....

  • List of pathologists

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