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.
For new professionals entering the field of histology, the days of making reagents are quickly becoming a thing of the past. I find this shift may be problematic, primarily because some of the art that is characteristically histology is lost.
One of my personal challenges when working with students is helping them to troubleshoot staining anomalies. When stains are made by the laboratory, the team learns from hands on experience what happens when a component is missing or inappropriately added to the mixture. These changes can make subtle alterations in the stained slides, which the histology team is ultimately expected to fix. Ultimately, newer histology teams can struggle when troubleshooting the subtle changes that can be easily fixed, without having the experience of making reagents and the lessons learned in that process.
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 Haematoxylum campechianum. 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. 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. So for those who want to see richer looking reds, phloxine may be added. Other eosin mixtures are sometimes used, such as EA50 and EA 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 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. 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 noncellular material, such as mucin, becomes stained with the hematoxylin.
This extracellular staining can be an indicator of well differentiated tumors. 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.
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. Eosin also stains red blood cells intensely red. Progressive staining — When tissue is left in the stain just long enough to reach the proper end point.
The slides have to be examined at different interval to find out when the staining is optimum. Regressive staining — In this method the tissue is overstained and then destained differentiate until the proper endpoint is reached. About Overview. Two types of staining methods Progressive staining — When tissue is left in the stain just long enough to reach the proper end point.
Thus, the stain discloses abundant structural information, with specific functional implications. A limitation of hematoxylin staining is that it is incompatible with immunofluorescence. It is useful, however, to stain one serial paraffin section from a tissue in which immunofluorescence will be performed.
Hematoxylin, generally without eosin, is useful as a counterstain for many immunohistochemical or hybridization procedures that use colorimetric substrates such as alkaline phosphatase or peroxidase.
0コメント