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H&E Basics Part 2: What’s in it?

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By Cindy Sampias, JD CT(ASCP)HTL

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.

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 Hematoxylon 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.

Fig. 1: In this image of large cell anaplastic medublastoma, the hematoxylin plays a key role in the diagnosis. Note the mitotic figures, indicating a rapidly growing tumor. Nuclear irregularity and chromatin clumping is also noted in the cells. Photo credit: Frontalcortex.com

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, auto-oxidation 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 know is Weigert's, which is used in the Verhoff-Van Gieson stain, shown in the image.

Fig. 2: Note the elastin fibers in aorta stained with Verhoff-Van Gieson, aka Elastic stain. Changes in these fibers suggest disease processes that may not otherwise be detected Photo credit: Stanley Hansen

Having reviewed the nuclear stains, let’s talk about the cytoplasmic component dyes. Eosin is the most commonly used counterstain that distinguishes between the cytoplasm and nuclei of cells. It is typically pink, with different shades of pink for different types of connective tissue fibers.

Eosin Y is the most commonly used form of eosin and may be used in both water and alcohol. The addition of a small amount of acetic acid will also sharpen the staining of the eosin. Eosin with phloxine added will enhance the reds seen with H&E staining. So, for those who want to see richer looking reds, phloxine may be added.

Fig. 3: The nuclei in this image have a nice contrast with the cytoplasm and red blood cells (RBC) within the blood vessel. Note the intensity of the RBCs as compared to the pink of the cytoplasm of the surrounding cells.

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.

Fig. 4: In this image from a pap smear with a patient exhibiting squamous cell carcinoma, the beauty of the varied cytoplasmic coloration can be appreciated. The red arrow indicates a malignant nucleus with very little cytoplasm. The black arrow illustrates a normal zed acute inflammatory cell, similar in size to the RBCs on the previous image. Note how much larger the malignant cell is compared to the normal nuclei on the previous image.

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 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.

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