Histopathology (compound of three

Greek Greek may refer to: 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 of all kno ...

words: 'tissue', 'suffering', and ''

-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 '' -lo ...

'' 'study of') is 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 betwe ...

examination of tissue in order to study the manifestations of

disease A disease is a particular abnormal condition that adversely affects the structure or function (biology), function of all or part of an organism and is not immediately due to any external injury. Diseases are often known to be medical condi ...

. 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, an interventional radiologist, or an interventional cardiology, interventional cardiologist. The process involves the extraction of sampling (medicine), sample ...

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 f ...

by a

pathologist Pathology is the study of disease. 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 the context of modern medical treatme ...

, 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 Surgery is a medical specialty that uses manual and instrumental techniques to diagnose or treat pathological conditions (e.g., trauma, disease, injury, malignancy), to alter bodily functions (e.g., malabsorption created by bariatric surgery s ...

, or

autopsy An autopsy (also referred to as post-mortem examination, obduction, necropsy, or autopsia cadaverum) is a surgical procedure that consists of a thorough examination of a corpse by dissection to determine the cause, mode, and manner of deat ...

. 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 anim ...

plant Plants are the eukaryotes that form the Kingdom (biology), kingdom Plantae; they are predominantly Photosynthesis, photosynthetic. This means that they obtain their energy from sunlight, using chloroplasts derived from endosymbiosis with c ...

, and then, often following expert dissection in the fresh state, placed in a fixative which stabilizes the tissues to prevent decay. The most common fixative is 10% neutral buffered

formalin Formaldehyde ( , ) (systematic name methanal) is an organic compound with the chemical formula and structure , more precisely . The compound is a pungent, colourless gas that polymerises spontaneously into paraformaldehyde. It is stored as ...

(corresponding to 3.7% w/v

formaldehyde Formaldehyde ( , ) (systematic name methanal) is an organic compound with the chemical formula and structure , more precisely . The compound is a pungent, colourless gas that polymerises spontaneously into paraformaldehyde. It is stored as ...

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 pota ...

Preparation for histology

The tissue is then prepared for viewing under a

microscope A microscope () is a laboratory equipment, 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 ...

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 Agar ( or ), or agar-agar, is a jelly-like substance consisting of polysaccharides obtained from the cell walls of some species of red algae, primarily from " ogonori" and " tengusa". As found in nature, agar is a mixture of two components, t ...

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 Immunohistochemistry is a form of immunostaining. It involves the process of selectively identifying antigens in cells and tissue, by exploiting the principle of Antibody, antibodies binding specifically to antigens in biological tissues. Alber ...

(IHC) staining and diagnostic

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. Molecular pathology shares some aspects of practice ...

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 Alcohol may refer to: Common uses * Alcohol (chemistry), a class of compounds * Ethanol, one of several alcohols, commonly known as alcohol in everyday life ** Alcohol (drug), intoxicant found in alcoholic beverages ** Alcoholic beverage, an alco ...

. 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 su ...

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 A microtome (from the Greek ''mikros'', meaning "small", and ''temnein'', meaning "to cut") is a cutting tool used to produce extremely thin slices of material known as ''sections'', with the process being termed microsectioning. Important in sc ...

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 water bath 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

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 diag ...

) for evaluation under

light microscopy Microscopy is the technical field of using microscopes to view subjects too small to be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of microscopy: optical, el ...

. The second method of histology processing is called

frozen section 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 tha ...

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

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 ...

. 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 edge or "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 th ...

of a tumor during surgery).

Staining of 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 A pigment is a powder used to add or alter color or change visual appearance. Pigments are completely or nearly solubility, insoluble and reactivity (chemistry), chemically unreactive in water or another medium; in contrast, dyes are colored sub ...

. 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 American and British English spelling differences#ae and oe, or hematoxylin (), also called natural black 1 or Colour Index International, C.I. 75290, is a chemical compound, compound extracted from wood#Heartwood and sapwood, heart ...

and

eosin Eosin is the name of several fluorescent acidic compounds which bind to and from salts with basic, or eosinophilic, compounds like proteins containing basic amino acid residues such as histidine, arginine and lysine, and stains them dark red ...

(often abbreviated H&E). Hematoxylin is used to stain nuclei blue, while eosin stains the

cytoplasm The cytoplasm describes all the material within a eukaryotic or prokaryotic cell, enclosed by the cell membrane, including the organelles and excluding the nucleus in eukaryotic cells. The material inside the nucleus of a eukaryotic cell a ...

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 endo ...

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, 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 Iron overload is the abnormal and increased accumulation of total iron in the body, leading to organ damage. The primary mechanism of organ damage is oxidative stress, as elevated intracellular iron levels increase free radical formation via the ...

. Recently,

antibodies An antibody (Ab) or immunoglobulin (Ig) is a large, Y-shaped protein belonging to the immunoglobulin superfamily which is used by the immune system to identify and neutralize antigens such as bacteria and viruses, including those that caus ...

have been used to stain particular

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

lipid Lipids are a broad group of organic compounds which include fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids include storing ...

s and

carbohydrate A carbohydrate () is a biomolecule composed of carbon (C), hydrogen (H), and oxygen (O) atoms. The typical hydrogen-to-oxygen atomic ratio is 2:1, analogous to that of water, and is represented by the empirical formula (where ''m'' and ''n'' ...

s. Called

, 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 Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

RNA Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...

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

, 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 a diagnosis with the medical context being implicit. The information ...

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 Cell growth#Disorders, abnormal cell growth with the potential to Invasion (cancer), invade or Metastasis, spread to other parts of the body. These contrast with benign tumors, which do not spread. Po ...

, this represents the tissue diagnosis required for most treatment protocols. In the removal of

, the pathologist will indicate whether the

surgical margin A resection margin or surgical margin is the edge or "margin" of apparently non-tumorous tissue around a tumor that has been surgically removed, called "Resection (surgery), resected", in surgical oncology. The resection is an attempt to remove ...

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 ...

or CCPDMA method of processing. Microscopic visual artifacts can potentially cause misdiagnosis of samples. Scanning of slides allows for various methods of digital pathology, including the application of

artificial intelligence Artificial intelligence (AI) is the capability of computer, computational systems to perform tasks typically associated with human intelligence, such as learning, reasoning, problem-solving, perception, and decision-making. It is a field of re ...

for interpretation. 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 Cell polarity refers to spatial differences in shape, structure, and function within a cell. Almost all cell types exhibit some form of polarity, which enables them to carry out specialized functions. Classical examples of polarized cells are de ...

(common tendencies among cells at the border, such as elongation or " palisading" in this case). Amount of

mitoses Mitosis () is a part of the cell cycle in eukaryotic cells in which replicated chromosomes are separated into two new nuclei. Cell division by mitosis is an equational division which gives rise to genetically identical cells in which the ...

can also be appreciated at this level. File:Systematic microscopy 5 - Subcellular features.jpg, Subcellular features (may need highest magnification)

Architectural patterns

Major histopathologic architectural patterns include: File:High-magnification micrograph of basal-cell carcinoma.jpg, Nests: islands of cells of similar type. File:Prostate adenocarcinoma - acinar pattern.jpg, Acinar or tubular: Each acinus consists of cells that surround a lumen. File:Typical carcinoid tumor of the lung, trabecular pattern.jpg,

Trabecula A trabecula (: trabeculae, from Latin for 'small beam') is a small, often microscopic, biological tissue, tissue element in the form of a small Beam (structure), beam, strut or rod that supports or anchors a framework of parts within a body or ...

r, elongated (rod-shaped) groups of cells. File:Encapsulated Papillary Carcinoma of the Breast, H&E (15768688957).jpg, Papillary: Protuberances of epithelioid cells around fibrovascular cores. File:Micropapillary urothelial carcinoma, very high mag.jpg, Micropapillary: Papillary tufts without fibrovascular cores File:Histopathology of fascicular growth in a leiomyoma.jpg, Fascicular: Generally the same cell type throughout, but some form band-like groups that are aligned in the same direction. File:Histopathology of woven or storiform pattern.jpg, Woven or storiform: Elongated cells or nuclei wherein small bundles are aligned in an otherwise haphazard pattern. File:Micrograph of prostate cancer with Gleason score 10 (5+5) with solid sheets of cells (crop).jpg, Solid: More or less the same cell type throughout, with no spaces between, and no other particular pattern. File:Papillary urothelial carcinoma with cribriform morphology, very high mag.jpg, Cribriform: Solid with multiple clear spaces. File:Bovine Bone Sample and 430 times Magnification.jpg,

Whorl A whorl ( or ) is an individual circle, oval, volution or equivalent in a whorled pattern, which consists of a spiral or multiple concentric objects (including circles, ovals and arcs). In nature File:Photograph and axial plane floral diagra ...

ed: Multiple

concentric objects In geometry, two or more objects are said to be ''concentric'' when they share the same center. Any pair of (possibly unalike) objects with well-defined centers can be concentric, including circles, spheres, regular polygons, regular polyh ...

, or spiral-shaped File:Histopathology of cartwheel pattern in dermatofibrosarcoma protuberans, annotated.jpg, Cartwheel pattern: Center points that radiate cells or connective tissue outward

Nuclear patterns

Major nuclear patterns include: File:Histopathology of clear cell renal cell carcinoma, grade 1, high magnification.jpg, Monomorphic when having relatively similar sizes and shapes. File:Pleomorphic nuclei.jpg, Pleomorphic when having different sizes and shapes. This often correlates with an increased nucleus to cytoplasm ratio. These features generally indicate

malignancy Malignancy () is the tendency of a medical condition to become progressively worse; the term is most familiar as a characterization of cancer. A ''malignant'' tumor contrasts with a non-cancerous ''benign'' tumor in that a malignancy is not ...

. File:Fine versus coarse chromatin.jpg, ''Fine chromatin'' when inconspicuous (essentially only nucleoli seen in the nuclei), versus ''coarse'' chromatin. File:Heterochromatic versus euchromatic nuclei.jpg, Sometimes " heterochromatic" versus " euchromatic" nuclei are used for visual appearance, but this strictly refers to the molecular structure of DNA. File:Well-differentiated neuroendocrine tumor with salt-and-pepper chromatin.png, Granular "salt-and-pepper" chromatin.

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 {{Authority control Anatomical pathology Pathology