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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
A stain is an unwanted localized discoloration, often in fabrics or textiles.
Stain(s) or The Stain(s) may also refer to:
Color
* Stain (heraldry), a non-standard tincture
* Staining, in biology, a technique used to highlight contrast in samples
...
used in
histology
Histology,
also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures vis ...
.
It is the most widely used stain in
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 re ...
and is often the
gold standard
A gold standard is a monetary system in which the standard economic unit of account is based on a fixed quantity of gold. The gold standard was the basis for the international monetary system from the 1870s to the early 1920s, and from the la ...
.
For example, when a
pathologist
Pathology is the study of the causal, 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 us ...
looks at 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 ...
of a suspected
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
histological section
Histology,
also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures vis ...
is likely to be stained with H&E.
H&E is the combination of two histological stains:
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 ...
. The hematoxylin stains cell
nuclei a purplish blue, and eosin stains the
extracellular matrix
In biology, the extracellular matrix (ECM), also called intercellular matrix, is a three-dimensional network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide s ...
and
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. ...
pink, with other structures taking on different shades, hues, and combinations of these colors.
Hence a pathologist can easily differentiate between the nuclear and cytoplasmic parts of a cell, and additionally, the overall patterns of coloration from the stain show the general layout and distribution of cells and provides a general overview of a tissue sample's structure.
Thus, pattern recognition, both
by expert humans themselves and
by software that aids those experts (in
digital pathology
Digital pathology is a sub-field of pathology that focuses on data management based on information generated from digitized specimen slides. Through the use of computer-based technology, digital pathology utilizes virtual microscopy. Glass slide ...
), provides histologic information.
This stain combination was first introduced in 1876 by A. Wissowzky.
Uses
The H&E staining procedure is the principal stain in histology
in part because it can be done quickly,
is not expensive, and stains tissues in such a way that a considerable amount of
microscopic anatomy
Histology,
also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures vis ...
is revealed,
and can be used to diagnose a wide range of
histopathologic
Histopathology (compound of three Greek words: ''histos'' "tissue", πάθος ''pathos'' "suffering", and -λογία ''-logia'' "study of") refers to the microscopic examination of tissue in order to study the manifestations of disease. Spec ...
conditions.
The results from H&E staining are not overly dependent on the chemical used to
fix the tissue or slight inconsistencies in laboratory protocol,
and these factors contribute to its routine use in histology.
H&E staining does not always provide enough contrast to differentiate all tissues, cellular structures, or the distribution of chemical substances,
and in these cases more specific stains and methods are used.
Method of application
![Slide_rack_in_haematoxylin](https://upload.wikimedia.org/wikipedia/commons/6/63/Slide_rack_in_haematoxylin.jpg)
There are many ways to prepare the hematoxylin solutions (formulation) used in the H&E procedure,
in addition, there are many
laboratory protocols for producing H&E stained slides,
some of which may be specific to a certain laboratory.
Although there is no standard procedure,
the results by convention are reasonably consistent in that cell nuclei are stained blue and 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
extracellular matrix
In biology, the extracellular matrix (ECM), also called intercellular matrix, is a three-dimensional network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide s ...
are stained pink.
Histology laboratories may also adjust the amount or type of staining for a particular pathologist.
After tissues have been collected (often as
biopsies
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 ...
) and fixed, they are typically dehydrated and embedded in melted
paraffin wax
Paraffin wax (or petroleum wax) is a soft colorless solid derived from petroleum, coal, or oil shale that consists of a mixture of hydrocarbon molecules containing between 20 and 40 carbon atoms. It is solid at room temperature and begins to ...
, the resulting block is mounted on a
microtome and cut into thin slices.
The slices are affixed to microscope slides at which point the wax is removed with a solvent and the tissue slices attached to the slides are rehydrated and are ready for staining.
Alternatively, H&E stain is the most used stain in
Mohs surgery
Mohs surgery, developed in 1938 by a general surgeon, Frederic E. Mohs, is microscopically controlled surgery used to treat both common and rare types of skin cancer. During the surgery, after each removal of tissue and while the patient waits, ...
in which tissues are typically frozen, cut on a
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 ...
(a microtome that cuts frozen tissue), fixed in alcohol, and then stained.
The H&E staining method involves application of
haematoxylin
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 ...
mixed with a metallic salt, or
mordant
A mordant or dye fixative is a substance used to set (i.e. bind) dyes on fabrics by forming a coordination complex with the dye, which then attaches to the fabric (or tissue). It may be used for dyeing fabrics or for intensifying stains in ...
, often followed by a rinse in a weak acid solution to remove excess staining (''differentiation''), followed by ''bluing'' in mildly
alkaline water.
After the application of haematoxylin, the tissue is
counterstain
A counterstain is a stain with colour contrasting to the principal stain, making the stained structure easily visible using a microscope.
Examples include the malachite green counterstain to the fuchsine stain in the Gimenez staining technique ...
ed with eosin (most commonly
eosin Y
Eosin Y, also called C.I. 45380 or C.I. Acid Red 87, is a member of the triarylmethane dyes. It is produced from fluorescein by bromination.
Use
Eosin Y is commonly used as the red dye in red inks.
It is commonly used in histology, most nota ...
).
Results
Hematoxylin principally colors the
nuclei of
cells blue or dark-purple,
along with a few other tissues, such as
keratohyalin
Keratohyalin is a protein structure found in cytoplasmic granules of the keratinocytes in the stratum granulosum of the epidermis. Keratohyalin granules (KHG) mainly consist of keratin, profilaggrin, loricrin and trichohyalin proteins which con ...
granules and
calcified
Calcification is the accumulation of calcium salts in a body tissue. It normally occurs in the formation of bone, but calcium can be deposited abnormally in soft tissue,Miller, J. D. Cardiovascular calcification: Orbicular origins. ''Nature Ma ...
material. 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 some other structures including
extracellular matrix
In biology, the extracellular matrix (ECM), also called intercellular matrix, is a three-dimensional network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide s ...
such as
collagen in up to five shades of pink.
The
eosinophilic
Eosinophilic (Greek suffix -phil-, meaning ''loves eosin'') is the staining of tissues, cells, or organelles after they have been washed with eosin, a dye.
Eosin is an acidic dye for staining cell cytoplasm, collagen, and muscle fibers. ''E ...
(substances that are stained by eosin)
structures are generally composed of intracellular or extracellular
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, respo ...
s. The
Lewy bodies
Lewy bodies are the inclusion bodies – abnormal aggregations of protein – that develop inside nerve cells affected by Parkinson's disease (PD), the Lewy body dementias ( Parkinson's disease dementia and dementia with Lewy bodies (DLB)), and ...
and
Mallory bodies are examples of eosinophilic structures. Most of 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. ...
is eosinophilic and is rendered pink.
Red blood cell
Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "holl ...
s are stained intensely red.
Mode of action
Although
hematein, an
oxidized
Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is the gain of electrons or a ...
form of hematoxylin,
is the active colorant (when combined with a mordant), the stain is still referred to as ''hematoxylin''.
Hematoxylin, when combined with a mordant (most commonly aluminum
alum) is often considered to "resemble"
a basic, positively charged, or
cationic
An ion () is an atom or molecule with a net electrical charge.
The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by convent ...
stain.
Eosin is an
anionic
An ion () is an atom or molecule with a net electrical charge.
The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by conven ...
(negatively charged) and acidic stain.
The staining of nuclei by hemalum (a combination of aluminum ions and hematein)
is ordinarily due to binding of the dye-metal complex to DNA, but nuclear staining can be obtained after extraction of DNA
from tissue sections. The mechanism is different from that of nuclear staining by basic (cationic) dyes such as
thionine
Thionine, also known as Lauth's violet, is the salt of a heterocyclic compound. It was firstly synthesised by Charles Lauth. A variety of salts are known including the chloride and acetate, called respectively thionine chloride and thionine ac ...
or
toluidine blue
Toluidine blue, also known as TBO or tolonium chloride ( INN) is a blue cationic (basic) dye used in histology (as the toluidine blue stain) and sometimes clinically.
Test for lignin
Toluidine blue solution is used in testing for lignin, a comp ...
.
Staining by basic dyes occurs only from solutions that are less acidic than hemalum, and it is prevented by prior chemical or enzymatic extraction of nucleic acids. There is evidence to indicate that co-ordinate bonds, similar to those that hold aluminium and hematein together, bind the hemalum complex to DNA and to carboxy groups of proteins in the nuclear
chromatin
Chromatin is a complex of DNA and protein found in eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in r ...
.
The structures do not have to be acidic or basic to be called basophilic and eosinophilic; the terminology is based on the affinity of cellular components for the dyes. Other colors, e.g. yellow and brown, can be present in the sample; they are caused by intrinsic pigments such as
melanin
Melanin (; from el, μέλας, melas, black, dark) is a broad term for a group of natural pigments found in most organisms. Eumelanin is produced through a multistage chemical process known as melanogenesis, where the oxidation of the amino ...
.
Basal laminae need to be stained by
PAS stain
PAS or Pas may refer to:
Companies and organizations
* Pakistan Academy of Sciences
* Pakistan Administrative Service
* Pan Am Southern, a freight railroad owned by Norfolk Southern and Pan Am Railways
* Pan American Silver, a mining company i ...
or some
silver stain In pathology, silver staining is the use of silver to selectively alter the appearance of a target in microscopy of histological sections; in temperature gradient gel electrophoresis; and in polyacrylamide gels.
In traditional stained glass, silv ...
s, if they have to be well visible.
Reticular fiber
Reticular fibers, reticular fibres or reticulin is a type of fiber in connective tissue composed of type III collagen secreted by reticular cells. Reticular fibers crosslink to form a fine meshwork (reticulin). This network acts as a supportin ...
s also require silver stain. Hydrophobic structures also tend to remain clear; these are usually rich in fats, e.g.
adipocyte
Adipocytes, also known as lipocytes and fat cells, are the cells that primarily compose adipose tissue, specialized in storing energy as fat. Adipocytes are derived from mesenchymal stem cells which give rise to adipocytes through adipogenesis. I ...
s,
myelin
Myelin is a lipid-rich material that surrounds nerve cell axons (the nervous system's "wires") to insulate them and increase the rate at which electrical impulses (called action potentials) are passed along the axon. The myelinated axon can be ...
around neuron
axon
An axon (from Greek ἄξων ''áxōn'', axis), or nerve fiber (or nerve fibre: see spelling differences), is a long, slender projection of a nerve cell, or neuron, in vertebrates, that typically conducts electrical impulses known as action po ...
s, and
Golgi apparatus
The Golgi apparatus (), also known as the Golgi complex, Golgi body, or simply the Golgi, is an organelle found in most eukaryotic cells. Part of the endomembrane system in the cytoplasm, it packages proteins into membrane-bound vesicles ins ...
membranes.
Examples of H&E stained tissues
References
Further reading
* Kiernan JA (2008) Histological and Histochemical Methods: Theory and Practice. 4th ed. Bloxham, UK: Scion.
* Lillie RD, Pizzolato P, Donaldson PT (1976) Nuclear stains with soluble metachrome mordant lake dyes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues. Histochemistry 49: 23–35.
* Llewellyn BD (2009) Nuclear staining with alum-hematoxylin. Biotech. Histochem. 84: 159–177.
* Puchtler H, Meloan SN, Waldrop FS (1986) Application of current chemical concepts to metal-haematein and -brazilein stains. Histochemistry 85: 353–364.
External links
SIGMA-ALDRICH H&E Informational Primer
Protocol
Routine Mayer's Hematoxylin and Eosin Stain (H&E)Rosen Lab, Department of Molecular and Cellular Biology, Baylor College of Medicine) Step by step protocol
{{DEFAULTSORT:HandE stain
Microscopy
Microbiology techniques
Laboratory techniques
Histopathology
Histotechnology
Staining dyes
Staining