![MI with contraction bands very high mag](https://upload.wikimedia.org/wikipedia/commons/6/6b/MI_with_contraction_bands_very_high_mag.jpg)
Histopathology (compound of three
Greek
Greek may refer to:
Greece
Anything of, from, or related to Greece, a country in Southern Europe:
*Greeks, an ethnic group.
*Greek language, a branch of the Indo-European language family.
**Proto-Greek language, the assumed last common ancestor ...
words: ''histos'' "tissue", πάθος ''pathos'' "suffering", and -λογία ''
-logia
''-logy'' is a suffix in the English language, used with words originally adapted from Ancient Greek ending in ('). The earliest English examples were anglicizations of the French '' -logie'', which was in turn inherited from the Latin '' -logi ...
'' "study of") refers to the
microscopic
The microscopic scale () is the scale of objects and events smaller than those that can easily be seen by the naked eye, requiring a lens or microscope to see them clearly. In physics, the microscopic scale is sometimes regarded as the scale be ...
examination of
tissue in order to study the manifestations of
disease
A disease is a particular abnormal condition that negatively affects the structure or function of all or part of an organism, and that is not immediately due to any external injury. Diseases are often known to be medical conditions that a ...
. Specifically, in clinical medicine, histopathology refers to the examination of a
biopsy
A biopsy is a medical test commonly performed by a surgeon, interventional radiologist, or an interventional cardiologist. The process involves extraction of sample cells or tissues for examination to determine the presence or extent of a dise ...
or surgical
specimen
Specimen may refer to:
Science and technology
* Sample (material), a limited quantity of something which is intended to be similar to and represent a larger amount
* Biological specimen or biospecimen, an organic specimen held by a biorepository ...
by a
pathologist
Pathology is the study of the causes and effects of disease or injury. The word ''pathology'' also refers to the study of disease in general, incorporating a wide range of biology research fields and medical practices. However, when used in t ...
, after the specimen has been processed and histological sections have been placed onto glass slides. In contrast,
cytopathology
Cytopathology (from Greek , ''kytos'', "a hollow"; , ''pathos'', "fate, harm"; and , '' -logia'') is a branch of pathology that studies and diagnoses diseases on the cellular level. The discipline was founded by George Nicolas Papanicolaou in ...
examines free cells or tissue micro-fragments (as "cell blocks").
Collection of tissues
Histopathological examination of tissues starts with
surgery,
biopsy
A biopsy is a medical test commonly performed by a surgeon, interventional radiologist, or an interventional cardiologist. The process involves extraction of sample cells or tissues for examination to determine the presence or extent of a dise ...
, or
autopsy. The tissue is removed from the
body
Body may refer to:
In science
* Physical body, an object in physics that represents a large amount, has mass or takes up space
* Body (biology), the physical material of an organism
* Body plan, the physical features shared by a group of anima ...
or
plant
Plants are predominantly photosynthetic eukaryotes of the kingdom Plantae. Historically, the plant kingdom encompassed all living things that were not animals, and included algae and fungi; however, all current definitions of Plantae exclu ...
, and then, often following expert dissection in the fresh state, placed in a
fixative which stabilizes the tissues to prevent
decay
Decay may refer to:
Science and technology
* Bit decay, in computing
* Software decay, in computing
* Distance decay, in geography
* Decay time (fall time), in electronics
Biology
* Decomposition of organic matter
* Tooth decay (dental caries ...
. The most common fixative is 10% neutral buffered
formalin
Formaldehyde ( , ) ( systematic name methanal) is a naturally occurring organic compound with the formula and structure . The pure compound is a pungent, colourless gas that polymerises spontaneously into paraformaldehyde (refer to section F ...
(corresponding to 3.7% w/v
formaldehyde
Formaldehyde ( , ) (systematic name methanal) is a naturally occurring organic compound with the formula and structure . The pure compound is a pungent, colourless gas that polymerises spontaneously into paraformaldehyde (refer to section ...
in neutral buffered water, such as
phosphate buffered saline
Phosphate-buffered saline (PBS) is a buffer solution (pH ~ 7.4) commonly used in biological research. It is a water-based salt solution containing disodium hydrogen phosphate, sodium chloride and, in some formulations, potassium chloride and po ...
).
Preparation for histology
The tissue is then prepared for viewing under a
microscope
A microscope () is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisi ...
using either chemical fixation or frozen section.
If a large sample is provided e.g. from a surgical procedure then a pathologist looks at the tissue sample and selects the part most likely to yield a useful and accurate diagnosis - this part is removed for examination in a process commonly known as grossing or cut up. Larger samples are cut to correctly situate their anatomical structures in the cassette. Certain specimens (especially biopsies) can undergo
agar pre-embedding to assure correct tissue orientation in cassette & then in the block & then on the diagnostic microscopy slide. This is then placed into a plastic cassette for most of the rest of the process.
Chemical fixation
In addition to formalin, other chemical fixatives have been used. But, with the advent of
immunohistochemistry (IHC) staining and diagnostic
molecular pathology testing on these specimen samples, formalin has become the standard chemical fixative in human diagnostic histopathology. Fixation times for very small specimens are shorter, and standards exist in human diagnostic histopathology.
Processing
Water is removed from the sample in successive stages by the use of increasing concentrations of
alcohol.
Xylene is used in the last dehydration phase instead of alcohol - this is because the wax used in the next stage is soluble in
xylene
In organic chemistry, xylene or xylol (; IUPAC name: dimethylbenzene) are any of three organic compounds with the formula . They are derived from the substitution of two hydrogen atoms with methyl groups in a benzene ring; which hydrogens are s ...
where it is not in alcohol allowing wax to permeate (infiltrate) the specimen.
This process is generally automated and done overnight. The wax infiltrated specimen is then transferred to an individual specimen embedding (usually metal) container. Finally, molten wax is introduced around the specimen in the container and cooled to solidification so as to embed it in the wax block.
This process is needed to provide a properly oriented sample sturdy enough for obtaining a thin
microtome section(s) for the slide.
Once the wax embedded block is finished, sections will be cut from it and usually placed to float on a waterbath surface which spreads the section out. This is usually done by hand and is a skilled job (histotechnologist) with the lab personnel making choices about which parts of the specimen microtome wax ribbon to place on slides. A number of slides will usually be prepared from different levels throughout the block. After this the thin section mounted slide is stained and a protective cover slip is mounted on it. For common stains, an automatic process is normally used; but rarely used stains are often done by hand.
Frozen section processing
![Touch prep on a lymph node](https://upload.wikimedia.org/wikipedia/commons/d/db/Touch_prep_on_a_lymph_node.jpg)
An initial evaluation of a suspected lymphoma is to make a "touch prep" wherein a glass slide is lightly pressed against excised lymphoid tissue, and subsequently stained (usually
H&E stain
Hematoxylin and eosin stain ( or haematoxylin and eosin stain or hematoxylin-eosin stain; often abbreviated as H&E stain or HE stain) is one of the principal tissue stains used in histology. It is the most widely used stain in medical diagnos ...
) for evaluation under
light microscopy.
The second method of histology processing is called
frozen section processing. This is a highly technical scientific method performed by a trained histoscientist. In this method, the tissue is frozen and sliced thinly using a
microtome mounted in a below-freezing refrigeration device called the
cryostat
A cryostat (from ''cryo'' meaning cold and ''stat'' meaning stable) is a device used to maintain low cryogenic temperatures of samples or devices mounted within the cryostat. Low temperatures may be maintained within a cryostat by using various r ...
. 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
A resection margin or surgical margin is the margin of apparently non-tumorous tissue around a tumor that has been surgically removed, called " resected", in surgical oncology. The resection is an attempt to remove a cancer tumor so that no por ...
of a tumor during surgery).
Staining of processed histology slides
![Eosinophilic, basophilic, chromophobic and amphophilic staining](https://upload.wikimedia.org/wikipedia/commons/b/bb/Eosinophilic%2C_basophilic%2C_chromophobic_and_amphophilic_staining.png)
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
A pigment is a colored material that is completely or nearly insoluble in water. In contrast, dyes are typically soluble, at least at some stage in their use. Generally dyes are often organic compounds whereas pigments are often inorganic compoun ...
. The aim of staining is to reveal cellular components; counterstains are used to provide contrast.
The most commonly used stain in histology is a combination of
hematoxylin
Haematoxylin or hematoxylin (), also called natural black 1 or C.I. 75290, is a compound extracted from heartwood of the logwood tree ('' Haematoxylum campechianum'') with a chemical formula of . This naturally derived dye has been used as a ...
and
eosin
Eosin is the name of several fluorescent acidic compounds which bind to and form salts with basic, or eosinophilic, compounds like proteins containing amino acid residues such as arginine and lysine, and stains them dark red or pink as a resul ...
(often abbreviated H&E). Hematoxylin is used to stain
nuclei blue, while eosin stains the
cytoplasm
In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. ...
and the extracellular connective tissue matrix of most cells 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 O or basic red 2) is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring cell nuclei red. This is the classic counterstain in both Gram stains and endos ...
,
Oil Red O
Oil Red O (Solvent Red 27, Sudan Red 5B, C.I. 26125, C26H24N4O) is a lysochrome (fat-soluble dye) diazo dye used for staining of neutral triglycerides and lipids on frozen sections and some lipoproteins on paraffin sections. It has the appearance ...
,
congo red
Congo red is an organic compound, the sodium salt of 3,3′-( ,1′-biphenyl4,4′-diyl)bis(4-aminonaphthalene-1-sulfonic acid). It is an azo dye. Congo red is water-soluble, yielding a red colloidal solution; its solubility is greater in organic ...
,
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
In histology, histopathology, and clinical pathology, Perls Prussian blue is a commonly used method to detect the presence of iron in tissue or cell samples. Perls Prussian Blue derives its name from the German pathologist Max Perls (1843–188 ...
reaction, used to demonstrate iron deposits in diseases like
Hemochromatosis
Iron overload or hemochromatosis (also spelled ''haemochromatosis'' in British English) indicates increased total accumulation of iron in the body from any cause and resulting organ damage. The most important causes are hereditary haemochromatos ...
.
Recently,
antibodies have been used to stain particular
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
s,
lipid
Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids includ ...
s and
carbohydrate
In organic chemistry, a carbohydrate () is a biomolecule consisting of carbon (C), hydrogen (H) and oxygen (O) atoms, usually with a hydrogen–oxygen atom ratio of 2:1 (as in water) and thus with the empirical formula (where ''m'' may or m ...
s. Called
immunohistochemistry, 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 or
RNA molecules. These antibody staining methods often require the use of frozen section histology. These procedures above are also carried out in the laboratory under scrutiny and precision by a trained specialist medical laboratory scientist (a histoscientist). Digital cameras are increasingly used to capture histopathological images.
Interpretation
The histological slides are examined under a microscope by a
pathologist
Pathology is the study of the causes and effects of disease or injury. The word ''pathology'' also refers to the study of disease in general, incorporating a wide range of biology research fields and medical practices. However, when used in t ...
, a medically qualified specialist who has completed a recognised training program. This
medical diagnosis
Medical diagnosis (abbreviated Dx, Dx, or Ds) is the process of determining which disease or condition explains a person's symptoms and signs. It is most often referred to as diagnosis with the medical context being implicit. The information r ...
is formulated as a pathology report describing the histological findings and the opinion of the pathologist. In the case of
cancer
Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
, this represents the tissue diagnosis required for most treatment protocols. In the removal of
cancer
Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
, the pathologist will indicate whether the
surgical margin
A resection margin or surgical margin is the margin of apparently non-tumorous tissue around a tumor that has been surgically removed, called " resected", in surgical oncology. The resection is an attempt to remove a cancer tumor so that no portio ...
is cleared, or is involved (residual cancer is left behind). This is done using either the
bread loafing
Bread loafing is a common method of processing surgical specimens 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 t ...
or
CCPDMA method of processing.
Microscopic visual artifacts can potentially cause misdiagnosis of samples.
Following are examples of general features of suspicious findings that can be appreciated from low to high magnification on histopathology:
File:Systematic microscopy 2 - Orientation.jpg, Orientation (lowest magnification): In this case oriented by the skin surface (green). A lesion is seen (red) and its demarcation can be discerned (diffuse in this case)
File:Systematic microscopy 3 - Architectural pattern.jpg, Architectural pattern of any suspicious cells, in this case nests of cells, as well as components of the intervening stroma.
File:Systematic microscopy 4 - Cellular arrangement.jpg, Cellular arrangement, including crowding and cell polarity (common tendencies among cells at the border, such as elongation or " palisading" in this case). Amount of mitoses
In cell biology, mitosis () is a part of the cell cycle in which replicated chromosomes are separated into two new nuclei. Cell division by mitosis gives rise to genetically identical cells in which the total number of chromosomes is maintai ...
can also be appreciated at this level.
File:Systematic microscopy 5 - Subcellular features.jpg, Subcellular features (may need highest magnification)
In myocardial infarction
After a
myocardial infarction
A myocardial infarction (MI), commonly known as a heart attack, occurs when blood flow decreases or stops to the coronary artery of the heart, causing damage to the heart muscle. The most common symptom is chest pain or discomfort which may ...
(heart attack), 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
Pyknosis, or karyopyknosis, is the irreversible condensation of chromatin in the nucleus of a cell undergoing necrosis or apoptosis. It is followed by karyorrhexis, or fragmentation of the nucleus.
Pyknosis (from Ancient Greek meaning "thick, clo ...
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
Granulation tissue is new connective tissue and microscopic blood vessels that form on the surfaces of a wound during the healing process. Granulation tissue typically grows from the base of a wound and is able to fill wounds of almost any siz ...
formation at margins, which matures during the following month, and gets increased collagen deposition and decreased cellularity until the
myocardial scarring
Myocardial scarring is the accumulation of fibrous tissue resulting after some form of trauma to the cardiac tissue. Fibrosis is the formation of excess tissue in replacement of necrotic or extensively damaged tissue. Fibrosis in the heart is often ...
is fully mature at approximately 2 months after infarction.
[Chapter 11 in: 8th edition.]
See also
*
Anatomical pathology
Anatomical pathology (''Commonwealth'') or Anatomic pathology (''U.S.'') is a medical specialty that is concerned with the diagnosis of disease based on the macroscopic, microscopic, biochemical, immunologic and molecular examination ...
*
Molecular pathology
*
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. The microtome device that ...
*
Medical technologist
A medical laboratory scientist (MLS) or clinical laboratory scientist (CLS) or medical technologist (MT) performs diagnostic testing of blood and body fluids in clinical laboratories. The scope of a medical laboratory scientist's work begins wit ...
*
Laser capture microdissection
Laser capture microdissection (LCM), also called microdissection, laser microdissection (LMD), or laser-assisted microdissection (LMD or LAM), is a method for isolating specific cells of interest from microscopic regions of tissue/cells/organisms ...
*
List of pathologists
A list of people notable in the field of pathology.
A
* John Abercrombie, Scottish physician, neuropathologist and philosopher.
* Maude Abbott (1869–1940), Canadian pathologist, one of the earliest women graduated in medicine, expert in co ...
References
External links
Virtual Histology Course - University of Zurich(German, English version in preparation)
Histopathology of the uterine cervix - digital atlas (IARC Screening Group)- University of Iowa
{{pathology
Anatomical pathology
Pathology