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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. It is the most widely used stain in medical diagnosis and is often the gold standard. For example, when a pathologist 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 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 and cytoplasm 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), 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 is revealed, and can be used to diagnose a wide range of histopathologic 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

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 and extracellular matrix 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) 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 in which tissues are typically frozen, cut on a cryostat (a microtome that cuts frozen tissue), fixed in alcohol, and then stained. The H&E staining method involves application of haematoxylin mixed with a metallic salt, or mordant, 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 counterstained with eosin (most commonly eosin Y).


Results

Hematoxylin principally colors the nuclei of cells blue or dark-purple, along with a few other tissues, such as keratohyalin granules and calcified material. Eosin stains the cytoplasm and some other structures including extracellular matrix 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, res ...
s. The Lewy bodies and Mallory bodies are examples of eosinophilic structures. Most of the cytoplasm 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 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 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. 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. Basal laminae need to be stained by PAS stain 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. adipocytes, myelin 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 p ...
s, and Golgi apparatus 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