H&E stain

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Main types of staining seen on H&E stain. Eosinophilic, basophilic, chromophobic and amphophilic staining.png
Main types of staining seen on H&E stain.
Retina (part of the eye) stained with hematoxylin and eosin, cell nuclei stained blue-purple and extracellular material stained pink. Retina -- high mag.jpg
Retina (part of the eye) stained with hematoxylin and eosin, cell nuclei stained blue-purple and extracellular material stained pink.

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. [1] [2] [3] It is the most widely used stain in medical diagnosis [1] and is often the gold standard. [4] For example, when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E.

Contents

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. [5] [6] 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. [7] 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 introduced in 1877 by chemist Nicolaus Wissozky at the Kazan Imperial University in Russia. [8] [7]

Uses

The H&E staining procedure is the principal stain in histology [3] [7] [2] [5] in part because it can be done quickly, [7] is not expensive, and stains tissues in such a way that a considerable amount of microscopic anatomy [9] [10] is revealed, [7] [5] [4] and can be used to diagnose a wide range of histopathologic conditions. [8] The results from H&E staining are not overly dependent on the chemical used to fix the tissue or slight inconsistencies in laboratory protocol, [11] and these factors contribute to its routine use in histology. [7]

H&E staining does not always provide enough contrast to differentiate all tissues, cellular structures, or the distribution of chemical substances, [9] and in these cases more specific stains and methods are used. [10] [7]

Method of application

Rack of slides being removed from a bath of hematoxylin stain. Slide rack in haematoxylin.jpg
Rack of slides being removed from a bath of hematoxylin stain.

There are many ways to prepare the hematoxylin solutions (formulation) used in the H&E procedure, [11] [12] [6] in addition, there are many laboratory protocols for producing H&E stained slides, [9] some of which may be specific to a certain laboratory. [7] Although there is no standard procedure, [11] [9] the results by convention are reasonably consistent in that cell nuclei are stained blue and the cytoplasm and extracellular matrix are stained pink. [7] Histology laboratories may also adjust the amount or type of staining for a particular pathologist. [7]

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 and cut into thin slices. [6] 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. [6] 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. [9]

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. [13] [8] [14] After the application of haematoxylin, the tissue is counterstained with eosin (most commonly eosin Y). [6] [8] [7]

Results

Hematoxylin principally colors the nuclei of cells blue or dark-purple, [6] [15] [14] 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 [5] [7] [14] in up to five shades of pink. [8] The eosinophilic (substances that are stained by eosin) [5] structures are generally composed of intracellular or extracellular proteins. The Lewy bodies and Mallory bodies are examples of eosinophilic structures. Most of the cytoplasm is eosinophilic and is rendered pink. [10] [15] Red blood cells are stained intensely red.[ citation needed ]

Mode of action

Although hematein, an oxidized form of hematoxylin, [5] [16] [14] is the active colorant (when combined with a mordant), the stain is still referred to as hematoxylin. [8] [13] Hematoxylin, when combined with a mordant (most commonly aluminum alum) is often considered to "resemble" [10] a basic, positively charged, or cationic stain. [5] Eosin is an anionic (negatively charged) and acidic stain. [5] [10] The staining of nuclei by hemalum (a combination of aluminum ions and hematein) [14] is ordinarily due to binding of the dye-metal complex to DNA, but nuclear staining can be obtained after extraction of DNA [14] from tissue sections. The mechanism is different from that of nuclear staining by basic (cationic) dyes such as thionine or toluidine blue. [10] 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.[ citation needed ]

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 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. adipocytes, myelin around neuron axons, and Golgi apparatus membranes.[ citation needed ]

Examples of H&E stained tissues

Related Research Articles

<span class="mw-page-title-main">Histology</span> Study of the microscopic anatomy of cells and tissues of plants and animals

Histology, also known as microscopic anatomy or microanatomy, is the branch of biology that studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures visible without a microscope. Although one may divide microscopic anatomy into organology, the study of organs, histology, the study of tissues, and cytology, the study of cells, modern usage places all of these topics under the field of histology. In medicine, histopathology is the branch of histology that includes the microscopic identification and study of diseased tissue. In the field of paleontology, the term paleohistology refers to the histology of fossil organisms.

<span class="mw-page-title-main">Haematoxylin</span> Natural stain derived from hearthwood and used in histology

Haematoxylin or hematoxylin, also called natural black 1 or C.I. 75290, is a compound extracted from heartwood of the logwood tree with a chemical formula of C
16H
14O
6. This naturally derived dye has been used as a histologic stain, as an ink and as a dye in the textile and leather industry. As a dye, haematoxylin has been called palo de Campeche, logwood extract, bluewood and blackwood. In histology, haematoxylin staining is commonly followed by counterstaining with eosin. When paired, this staining procedure is known as H&E staining and is one of the most commonly used combinations in histology. In addition to its use in the H&E stain, haematoxylin is also a component of the Papanicolaou stain which is widely used in the study of cytology specimens.

<span class="mw-page-title-main">Eosin</span> Group of bromo derivatives of fluorescein used as red dye

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 result of the actions of bromine on eosin. In addition to staining proteins in the cytoplasm, it can be used to stain collagen and muscle fibers for examination under the microscope. Structures that stain readily with eosin are termed eosinophilic. In the field of histology, Eosin Y is the form of eosin used most often as a histologic stain.

<span class="mw-page-title-main">Mordant</span> Substance used for binding dyes to fabrics

A mordant or dye fixative is a substance used to set dyes on fabrics. It does this by forming a coordination complex with the dye, which then attaches to the fabric. It may be used for dyeing fabrics or for intensifying stains in cell or tissue preparations. Although mordants are still used, especially by small batch dyers, they have been largely displaced in industry by directs.

<span class="mw-page-title-main">Staining</span> Technique used to enhance visual contrast of specimens observed under a microscope

Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology, in cytology, and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses of diseases at the microscopic level. Stains may be used to define biological tissues, cell populations, or organelles within individual cells.

<span class="mw-page-title-main">Eosinophilic</span> Quality of being colored by eosin

Eosinophilic is the staining of tissues, cells, or organelles after they have been washed with eosin, a dye.

<span class="mw-page-title-main">Basophilic</span> Microscopic appearance of cells stained with a basic dye

Basophilic is a technical term used by pathologists. It describes the appearance of cells, tissues and cellular structures as seen through the microscope after a histological section has been stained with a basic dye. The most common such dye is haematoxylin.

<span class="mw-page-title-main">Eosin Y</span> Chemical compound

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.

<span class="mw-page-title-main">Hematein</span> Chemical compound

Hematein 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 Colour Index, haematein is called haematine.

<span class="mw-page-title-main">Masson's trichrome stain</span> Biological staining procedure used in study of tissues

Masson's trichrome is a three-colour staining procedure used in histology. The recipes emerged from Claude L. Pierre Masson's (1880–1959) original formulation have different specific applications, but all are suited for distinguishing cells from surrounding connective tissue.

<span class="mw-page-title-main">Papanicolaou stain</span> Histological staining method

Papanicolaou stain is a multichromatic (multicolored) cytological staining technique developed by George Papanicolaou in 1942. The Papanicolaou stain is one of the most widely used stains in cytology, where it is used to aid pathologists in making a diagnosis. Although most notable for its use in the detection of cervical cancer in the Pap test or Pap smear, it is also used to stain non-gynecological specimen preparations from a variety of bodily secretions and from small needle biopsies of organs and tissues. Papanicolaou published three formulations of this stain in 1942, 1954, and 1960.

<span class="mw-page-title-main">Phosphotungstic acid-haematoxylin stain</span> Biological stain used for staining of tissues

Phosphotungstic acid haematoxylin (PTAH) is a mix of haematoxylin with phosphotungstic acid, used in histology for staining.

<span class="mw-page-title-main">Phosphotungstic acid</span> Chemical compound

Phosphotungstic acid (PTA) or tungstophosphoric acid (TPA), is a heteropoly acid with the chemical formula H3PW12O40]. It forms hydrates H3[PW12O40]·nH2O. It is normally isolated as the n = 24 hydrate but can be desiccated to the hexahydrate (n = 6). EPTA is the name of ethanolic phosphotungstic acid, its alcohol solution used in biology. It has the appearance of small, colorless-grayish or slightly yellow-green crystals, with melting point 89 °C (24 H2O hydrate). It is odorless and soluble in water (200 g/100 ml). It is not especially toxic, but is a mild acidic irritant. The compound is known by a variety of names and acronyms (see 'other names' section of infobox).

<span class="mw-page-title-main">Acidophile (histology)</span>

Acidophile is a term used by histologists to describe a particular staining pattern of cells and tissues when using haematoxylin and eosin stains. Specifically, the name refers to structures which "love" acid, and take it up readily. More specifically, acidophilia can be described by cationic groups of most often proteins in the cell readily reacting with acidic stains.

<span class="mw-page-title-main">Brazilin</span> Chemical compound

Brazilin is a naturally occurring, a homoisoflavonoid, red dye obtained from the wood of Paubrasilia echinata, Biancaea sappan, Caesalpinia violacea, and Haematoxylum brasiletto. Brazilin has been used since at least the Middle Ages to dye fabric, and has been used to make paints and inks as well. The specific color produced by the pigment depends on its manner of preparation: in an acidic solution brazilin will appear yellow, but in an alkaline preparation it will appear red. Brazilin is closely related to the blue-black dye precursor hematoxylin, having one fewer hydroxyl group. Brazilein, the active dye agent, is an oxidized form of brazilin.

Infantile digital fibromatosis (IDF), also termed inclusion body fibromatosis, Reye tumor, or Reye's tumor, usually occurs as a single, small, asymptomatic, nodule in the dermis on a finger or toe of infants and young children. IMF is a rare disorder with approximately 200 cases reported in the medical literature as of 2021. The World Health Organization in 2020 classified these nodules as a specific benign tumor type in the category of fibroblastic and myofibroblastic tumors. IDF was first described by the Australian pathologist, Douglas Reye, in 1965.

<span class="mw-page-title-main">Perls Prussian blue</span> Histologic method to stain for iron

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 (1843–1881), who described the technique in 1867. The method does not involve the application of a dye, but rather causes the pigment Prussian blue to form directly within the tissue. The method stains mostly iron in the ferric state which includes ferritin and hemosiderin, rather than iron in the ferrous state.

<span class="mw-page-title-main">Luxol fast blue stain</span>

Luxol fast blue stain, abbreviated LFB stain or simply LFB, is a commonly used stain to observe myelin under light microscopy, created by Heinrich Klüver and Elizabeth Barrera in 1953. LFB is commonly used to detect demyelination in the central nervous system (CNS), but cannot discern myelination in the peripheral nervous system.

Verhoeff's stain, also known as Verhoeff's elastic stain (VEG) or Verhoeff–Van Gieson stain (VVG), is a staining protocol used in histology, developed by American ophthalmic surgeon and pathologist Frederick Herman Verhoeff (1874–1968) in 1908. The formulation is used to demonstrate normal or pathologic elastic fibers.

<span class="mw-page-title-main">Dmitri Leonidovich Romanowsky</span> Russian physician

Dmitri Leonidovich Romanowsky was a Russian physician who is best known for his invention of an eponymous histological stain called Romanowsky stain. It paved the way for the discovery and diagnosis of microscopic pathogens, such as malarial parasites, and later developments of new histological stains that became fundamental to microbiology and physiology.

References

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