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H&E Basics Part 3: Getting down to business-Protocol Selection

By Cindy Sampias, JD CT(ASCP)HTL

The next step to having a good stain, is determining what type of H&E stain is desired. There are typically 3 types of H&E stains: progressive, modified progressive, and regressive.

Progressive staining occurs when the hematoxylin is added to the tissue without being followed by a differentiator to remove excess dye. Because there is no differentiation step, background staining can occur-especially with charged or treated slides. Pathologists sometimes prefer this type of stain, because the non-cellular material, such as mucin, becomes stained with the hematoxylin. This extracellular staining can be an indicator of well differentiated tumors.

Fig. 1: In this section of colon, the mucin is still visible with the goblet cells.

Table: This protocol is 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 under stained 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.

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 minute
Xylene 2 minutes
Fig.2 In this image, the nuclei in this sample of colon seem to overwhelm the other cellular components. Even the red blood cells appear muted. Less time in hematoxylin OR additional time in eosin can provide a better balance.
Fig. 3: The nuclei in this section of kidney are lost in the pink of the eosin. Additional time in hematoxylin would make these nuclei much easier to see.

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 under stain 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 (eg, 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.

Fig. 4: Lymph node
Fig. 5: Kidney - Note the differences between this section of lymph node and kidney, both sectioned at 4 um. These samples, stained with the same protocol, are very different in appearance due to cellularity.

The use of clean and fresh dewaxing reagents is essential for the removal of paraffin from the slide prior to the addition of the dyes. While xylene is the most commonly used solvent, xylene substitutes are gaining in popularity because they are considered less hazardous and more ecofriendly. Water in solvents, whether from reagent contamination or a high humidity environment, reduces the ability of the solvent to remove the paraffin. Remaining paraffin prevents the dyes from penetrating the tissues, thus giving an uneven appearance.

The simplest way to prevent this from occurring is to change reagents more frequently. Adding a small of amount of desiccant pellets (about a tablespoon per reagent vessel) will also reduce water contamination within solvents. These measures are especially important when using a xylene substitute, as these reagents tend to be far less tolerant of any water contamination than xylene.