H&E Staining Overview: A Guide to Best Practices

Hematoxylin and Eosin stains are used in many areas of the histology laboratory, including frozen sections, fine needle aspirates, and paraffin fixed embedded tissues. To better understand what makes a well-stained slide, it is important to understand the components of the stain.

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Hematoxylin is used to illustrate nuclear detail in cells. Depth of coloration is not only related to the amount of DNA in the nuclei but also to the length of time the sample spends in hematoxylin.

Hematoxylin is a reasonably simple dye to make. The dye itself is extracted from the tree Haematoxylum campechianum. Oxidation of the hematoxylin produces hematein, which is the actual dye used in an H&E stain. Addition of the mordant improves the ability of the hematein to attach to the anionic (negatively charged) components of the tissues.

Hematoxylins are typically classified by the mordant used before staining. Mordants strengthen the positive ionic charge of the hematin. This aids the bonding of the hematin to the anionic tissue component, which is most commonly chromatin. The type of mordant also influences the final color of the stained components. The most common mordant used in routine histology is aluminum ammonium sulfate (alum). This mordant causes the nuclei to be red in color, which is then changed to the more familiar blue color when the sample is later rinsed with a weakly basic solution.

Mayer's hematoxylin is an alum hematoxylin, a commonly used stain that may be employed for both progressive and regressive stains. It is often used as a nuclear counterstain for special stains and immunohistochemistry. For these applications, Mayer's is used to stain the nuclei and then blued without the use of a differentiator. Mayer's is a water-based stain.

Harris hematoxylin is another commonly used alum hematoxylin that may be used for progressive staining of cytology specimens but can also be used for either progressive or regressive staining in histology. The staining tends to provide clear nuclear detail. One challenge when using Harris is that it is best differentiated with a mild acid, as opposed to the more commonly used hydrochloric acid-based differentiators. Harris is an alcohol-based stain. Gill's hematoxylin is an alum hematoxylin. It may be used as a progressive or regressive stain and is available in different concentrations. Because it is made with water and ethylene glycol, autoxidation of the stain is typically prevented over months, making it more stable than Harris hematoxylin. However, the nature of Gills is such that extra-nuclear staining may occur. Mucin and even adhesives used on the slide may become heavily contaminated with Gills.

The hematoxylins that use iron salts as a mordant are typically used in special stains. This is because they can demonstrate more tissue structures than alum hematoxylins, such as myelin and elastin fibers. One of the best known is Weigert's, which is used in the Verhoeff-Van Gieson stain, shown in the image.

Many laboratories find ordering their stains to be the easiest way to ensure consistent and repeatable quality. A large variety of both hematoxylin and eosin stain combinations provide the ability to customize the desired results with very little hassle. As more techs retire and companies become leaner, staffing decreases make the use of commercially available reagents ideal because the techs are better able to focus on embedding and cutting, which are the areas of the laboratory that offer the least amount of automation.

Other eosin mixtures are sometimes used, such as EA50 and EA65. These stains are primarily used for cytology, and in addition to eosin Y, include light green, yellowish, and Bismarck brown. The addition of these two dyes provides for the variations in color from pale blue to pink cytoplasm, best noted in the squamous cells of a pap smear. The concentration of the mixture determines the designation of 50 or 65.

The differentiation of stains allows for the ability to selectively remove stain from tissues to the taste of the viewer. In the case of hematoxylin, hydrochloric acid (for rapid differentiation) and acetic acid (for slower, more controlled differentiation) are most commonly used. While hydrochloric acid (HCl) has historically been the standard, milder acids are being used to provide gentler dye removal. Part of this trend is due to the use of automated staining, which must accommodate the movement of the robotic arm in addition to the time spent in the reagent.

Bluing reagents, such as Scott's Tap Water, are used to change the hematoxylin from red to the traditional blue color we expect. These slightly basic solutions chemically alter the dye to produce this color change. In some locations, the tap water contains enough minerals so that the pH causes the water to be basic enough to allow for the bluing of nuclei without the need for a bluing specific reagent. In most cases, though, labs typically add this step to ensure appropriate bluing.

In combination, these components make up the standard stain most used in the histology laboratory.

The following table contains a protocol with a simple regressive stain that provides a nice balance of nuclear and cytoplasmic stains. This protocol is designed with a mild acid differentiator in mind.

Once the staining components have been selected, it is good to start with the baseline protocol. From there, edit either the hematoxylin in 30 second increments OR the eosin in 15 second increments. Remember, eosin will tend to penetrate much faster. Unless there is the need to significantly lighten or darken the eosin staining intensity, the shorter increments are best. It is also important that only one stain is changed at a time. It may appear that the hematoxylin is overstained, when the eosin just needs to be richer.

As laboratories continue to grow, the need for consistent results and continuous throughput is essential. Reproducibility is an important part of laboratory stain quality. When hand staining, human variables can make each stained slide rack look different from the last. The addition of automation not only removes the potential for inconsistency, but also frees technologists up to perform other tasks in the laboratory.

It is important that the proper balance of the dyes is achieved. Overstaining with hematoxylin can give the illusion of understained eosin, just as overstaining with eosin can cause the hematoxylin to appear lighter than it actually is. So, when optimizing the stain, make sure to only edit the time of one of the components. This technique will help eliminate the need to spend additional time adjusting the stain.

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Xylene 2 minutes Xylene 2 minutes 100% ethanol 2 minutes 100% ethanol 2 minutes 95% ethanol 2 minutes Water wash 2 minutes Hematoxylin 3 minutes Water wash 1 minute Differentiator (mild acid) 1 minute Water wash 1 minute Bluing 1 minute Water wash 1 minute 95% ethanol 1 minute Eosin 45 seconds 95% ethanol 1 minute 100% ethanol 1 minute 100% ethanol 1 minute Xylene 2 minutes Xylene 2 minutes Coverslip

With regressive and modified progressive staining, a differentiator is used. If the differentiator is made in-house, there is the potential for it to be either too weak or too strong. Both scenarios will impact staining. If the differentiator is stronger than intended, it will remove more hematoxylin and will make the nuclei pale. Time is also important. Too much time in a properly prepared differentiator will also remove more hematoxylin and will ultimately understain the nuclei.

Mild acidity is critical to the shelf life of hematoxylin. Without it, the alkalinity of the tap water rinse will raise the pH such that the dye lake can precipitate, and the color will change from cherry red to purple red. Adding small amounts of acetic acid to the hematoxylin periodically will aid in maintaining appropriate pH and can extend the life of the stain.

Water is used as a differentiator for eosin. It is common to follow the eosin step with 95% ethanol. The ethanol aids with rinsing the slide, while water pulls excess eosin from the tissue. This step can help with coloration control but extending the time provides for lighter stains, while shortening the time maintains brighter coloration. However, excess water in xylene can continue the differentiation process and can be seen after coverslipping as a pink haze on the slide.

Not all tissues are created equally. Cysts and fatty samples, even when processed correctly, may be very difficult to see grossly once the slide has been stained. These samples often have open spaces where fluids or fat were in the cell, and the thinness of the cell walls may give the appearance of being light when the coloration is simply an artifact of the tissue type.

Highly cellular samples (e.g., tonsil, lymph node) can be very concerning. Remember that lymphocytes have little cytoplasm, and there is not nearly the cellular material between cells as with other tissues. For this reason, the hematoxylin does not have to compete with the eosin. The compact nature of the cells also concentrates the DNA, giving these highly cellular tissues the appearance of being overstained, when in reality, they may simply need to be sectioned thinner.

The H&E stain provides a comprehensive picture of the microanatomy of organs and tissues. Hematoxylin precisely stains nuclear components, including heterochromatin and nucleoli, while eosin stains cytoplasmic components including collagen and elastic fibers, muscle fibers and red blood cells. In a high-quality H&E stain, there are subtle differences in the shades of color produced by the stains, particularly eosin, and this aids in the detection and interpretation of morphological changes associated with disease.

It is important that people performing and assessing H&E stains for quality are aware of the subtleties of the stain, know what can be achieved when the stain is properly performed with high-quality reagents, and know what to look for microscopically. The maintenance of consistent, high-quality H&E stains is a fundamental requirement in histopathology laboratories.

In the following sections, the basic steps in performing an H&E stain are outlined.

Remove the Wax

Following the preparation of a paraffin section, all the elements are infiltrated with and surrounded by paraffin wax which is hydrophobic and impervious to aqueous reagents. The majority of cell and tissue components have no natural color and are not visible. The first step in performing an H&E stain is to dissolve all the wax away with xylene (a hydrocarbon solvent).

Hydrate the Section

After thorough de-waxing, the slide is passed through several changes of alcohol to remove the xylene, then thoroughly rinsed in water. The section is now hydrated so that aqueous reagents will readily penetrate the cells and tissue elements.

Apply the Hematoxylin Nuclear Stain

The slide is now stained with a nuclear stain such as Harris hematoxylin, which consists of a dye (oxidized hematoxylin or hematein) and a mordant or binding agent (an aluminum salt) in the solution. Initially this stains the nuclei and some other elements a reddish-purple color.

Complete the Nuclear Stain by “Blueing”

After rinsing in tap water, the section is “blued” by treatment with a weakly alkaline solution. This step converts the hematoxylin to a dark blue color. The section can now be rinsed and checked to see if the nuclei are properly stained, showing adequate contrast and to assess the level of background stain.

Remove Excess Background Stain (Differentiate)

On most occasions when Harris hematoxylin is employed, a differentiation (destaining) step is required to remove non-specific background staining and to improve contrast. A weak acid alcohol is used. After this treatment, blueing and thorough rinsing is again required. Staining methods that include a destaining or differentiation step are referred to as “regressive” stains.

Apply the Eosin Counterstain

The section is now stained with an aqueous or alcoholic solution of eosin (depending on personal preference). This colors many nonnuclear elements in different shades of pink.

Rinse, Dehydrate, Clear and Mount (Apply Cover Glass)

Following the eosin stain, the slide is passed through several changes of alcohol to remove all traces of water, then rinsed in several baths of xylene which “clears” the tissue and renders it completely transparent. A thin layer of polystyrene mountant is applied, followed by a glass coverslip. If the stain and all the subsequent steps have been properly performed, the slide will reveal all the important microscopic components and be stable for many years.

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