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 of a suspected
cancer, the
histological section is likely to be stained with H&E.
H&E is the combination of two histological stains:
hematoxylin and
eosin. 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
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 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
Fix or FIX may refer to:
People with the name
* Fix (surname)
Arts, entertainment, and media Films
* ''Fix'' (film), a feature film by Tao Ruspoli Music
* ''Fix'' (album), 2015 album by Chris Lane
* "Fix" (Blackstreet song), 1997 song by Black ...
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, the resulting block is mounted on a
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''. Important in science, microtomes are used in microscopy, allow ...
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
In chemistry, an alkali (; from ar, القلوي, al-qaly, lit=ashes of the saltwort) is a base (chemistry), basic, ionic compound, ionic salt (chemistry), salt of an alkali metal or an alkaline earth metal. An alkali can also be defined as ...
water.
After the application of haematoxylin, the tissue is
counterstained with eosin (most commonly
eosin Y).
Results
Hematoxylin principally colors the
nuclei of
cells
Cell most often refers to:
* Cell (biology), the functional basic unit of life
Cell may also refer to:
Locations
* Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery w ...
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
Collagen () is the main structural protein in the extracellular matrix found in the body's various connective tissues. As the main component of connective tissue, it is the most abundant protein in mammals, making up from 25% to 35% of the whole ...
in up to five shades of pink.
The
eosinophilic (substances that are stained by eosin)
structures are generally composed of intracellular or extracellular
proteins. 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
In histopathology, a Mallory body, Mallory-Denk body, and Mallory's hyaline, is an inclusion found in the cytoplasm of liver cells. Mallory bodies are damaged intermediate filaments within the liver cells.
Associated conditions
Mallory bodies ar ...
are examples of eosinophilic structures. Most of the
cytoplasm is eosinophilic and is rendered pink.
Red blood cells are stained intensely red.
Mode of action
Although
hematein
Hematein (US spelling) or haematein is an oxidized derivative of haematoxylin, used in staining. Haematein should not be confused with haematin, which is a brown to black iron-containing pigment formed by decomposition of haemoglobin. In the Col ...
, 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
An alum () is a type of chemical compound, usually a hydrated double salt, double sulfate salt (chemistry), salt of aluminium with the general chemical formula, formula , where is a valence (chemistry), monovalent cation such as potassium or a ...
) is often considered to "resemble"
a basic, positively charged, or
cationic stain.
Eosin is an
anionic (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.
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 lamina
The basal lamina is a layer of extracellular matrix secreted by the epithelial cells, on which the epithelium sits. It is often incorrectly referred to as the basement membrane, though it does constitute a portion of the basement membrane. The ba ...
e need to be stained by
PAS stain or some
silver stains, if they have to be well visible.
Reticular fibers 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 around neuron
axons, 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
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Histopathology
Histotechnology
Staining dyes
Staining