Hematoxylin and Eosin Stain (H&E)

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Hematoxylin and Eosin Stain (H&E)

Hematoxylin and Eosin (H&E) is the standard stain in histology. It is the main and general diagnostic method in histopathology laboratories and morphological evaluation. The stain has been the preferred choice for many years because it is compatible with various fixatives and clearly displays cell features. Features such as cytoplasm, extracellular matrix, and nucleus are clearly elaborated by the technique. Abnormal cells and tissues can be identified by only staining frozen or paraffin sections with perfectly executed H&E staining. In addition, most malignant cases are detected using this stain by determining morphologically changed cells that provide useful information for cancer diagnosis (Anderson, 2011).

Böhmer introduced hematoxylin stain in 1865 while Fischer introduced eosin in 1875. Schwarz came up with the idea of double staining technique by use of consecutive solutions of carmine and picric acid. In 1867, Wissowzky implemented the idea and proposed the combination of two independent dyes to carry out staining. He was the first to use H&E stain in laboratory experiments, in 1867. Hematoxylin is blue-purple in color and stains the chromatin in the nucleus deep purplish-blue making it easily visible. Eosin, by contrast, is a non-specific pink dye that stains the cytoplasm and extracellular matrix giving them an orange-pink color. Eosin is the counterstain that contrasts the purplish-blue nucleus to make other cell components such as erythrocytes, fluid, and cell membrane visible (Anderson, 2011). The red blood cells stain pink while the other components such as the cytoplasm assume different shades of orange-pink. Most cells and tissues are squishy, transparent, and colorless, and their features cannot easily be recognized without enhancement; thus, the need to stain them for clear identification.

Eosin is a negatively charged and acidic stain, which binds to the positively charged proteins in cells and tissue components. The stain has a pH of about 3.5, and the intensity of staining depends on the pH in that the higher the pH, the lower the intensity. Eosin is synthetic while hematoxylin is a natural dye obtained from Haematoxylon campechianum Linnaeus a logwood tree found in Central America (Brown, n.d.). It is not naturally basic and does not have staining ability, but acts together with a mordant that gives it a basic characteristic. Mordants are aluminum salts that bind to the tissue because the stain binds to the mordant. It is first oxidized to hematein, which when bound to aluminum, gains a positive charge. The hematein-aluminum compound binds negatively charged molecules in tissue components and cells such as the nucleus. The staining capacity depends on pH and the higher the pH, the higher the intensity.

In preparing 1000 ml of hematoxylin stain, the ingredients include 10 g commercially obtained hematoxylin dark crystals and 500 ml hot tap water at about 70-80°C. The crystals are thoroughly mixed with the hot water. In another vessel, 10 g potassium or aluminum alum is thoroughly mixed with 500ml of hot water. The two mixtures are mixed together, and thymol crystals added to prevent fungal growth. The mixture is then put in a translucent vase, covered with paper towel for air circulation, and placed at room temperature for a week. The solution is then transferred to an airtight dark bottle after a week, and placed in a dark room to mature for 3 weeks.

Preparation of 1000 ml of eosin stain involves the following procedure. 10 g of eosin Y crystals is added to 1000 ml of hot water, diluted and mixed thoroughly, then placed in a dark, airtight container. Neither maturation nor thymol is required for this stain. Alum solution, which is the mordant, is prepared according to the aforementioned steps. The two solutions are then mixed together to form the eosin stain. The alum solution can continually be added to replace the consumed hematoxylin stain in the dish. However, caution should be taken in order to maintain the staining qualities of the nucleus (Gill, 2010).

To prepare a tissue for staining, it is first embedded in paraffin, sectioned, and mounted on a slide before staining with H&E. It is then placed in a series of water, alcohol and xylene to give the cells affinity for the stain. Water is used for hydration, alcohol for dehydration, and xylene for clearing. Two methods are mainly used of perform the H&E staining technique. The common method is progressive staining where the prepared tissue slide is first placed in the hematoxylin for about 3 minutes. Secondly, it is rinsed in the first and second tap water baths, and then transferred to the eosin stain bath for about 5 seconds. It is then rinsed successively in tap water baths to remove the excess stain. Finally, the slide is dried in an oven and then mounted in mounting media for examination under a microscope.

The regressive method involves putting the tissues in a strong hematoxylin type called Harris hematoxylin. The hematoxylin is then removed from other cell components except the nucleus using an acid alcohol. It is stained with eosin dye, rinsed, dehydrated, cleared and cover slipped in a mounting media (August & Kaufmann, 1997). In some cases, hematoxylin dye can be used solely to perform immunohistochemical procedures as a counterstain. However, the drawback of this single staining is its incompatibility with immunofluorescence, and it is essential to use an immunofluorescence-compatible tissue when staining.

H&E stain has various advantages over other stains used in histopathology. The technique yields outstanding results that can easily be utilized for differentiation of cell and tissue structures. The stained slides can be examined under low microscopic power because of the detailed and easily viewed sections. Another benefit is the ready-to-use reagents that are readily available. The staining procedure is also user friendly because it involves direct, simple steps, and low-cost equipment such as a microtome and light microscope. H&E stain is also certified and tested according to DIN EN ISO 13485 standards. In addition, the stain is also economical and timesaving because it takes only a few minutes to execute the procedure.

A main drawback of H&E stain is the fact that conventionally, it is prepared using alcohol and xylene solvents, which are flammable, toxic and hazardous. However, there is an alternative way of preparing xylene and alcohol free stains using hot tap water, which has quality staining characteristics for use in cytological procedures, in the laboratory. Xylene and alcohol free stains have advantages such as being nonflammable, nontoxic, and easily disposable. Another disadvantage of H&E stain is that components grazing the edges of the nuclei may be mistaken for nuclear fragments giving false results (August & Kaufmann, 1997).

In most pathology laboratories, H&E stain is the most common in differentiating cells and tissue components as either pathological, normal, degeneratively altered, or inflamed. The stain is popular because it yields excellent results, easy to use, and time effective. The benefits of this stain greatly outweigh its drawbacks, explaining its popularity in histopathology laboratories.

H&E stained sectioned biopsy of uterine cervix with marked dysplasia (precancerous changes) ×100 (Gill, 2010).

A cross-section of the medulla showing various structures; H-Nucleus, C-Cytoplasm, T-Connective tissue (Brown, 2002).

References

Anderson, J. (2011). An introduction to routine and special staining. Leica Biosystems. Retrieved on 18 Oct. from http://www.leicabiosystems.com/pathologyleaders/an-introduction-to-routine-and-special-staining/

August, J. T. & Kaufmann, S. H.E. (1997). Apoptotis: Pharmacological implications and therapeutic opportunities. San Diego, CA: Academic Press.

Brown, H. S. (2002). Hematoxylin & Eosin. Sigma-Aldrich Cooperation. Retrieved on 18 Oct. from http://www.sigmaaldrich.com/img/assets/7361/Primer-H&Emay04.pdf

Brown, H. S. (n.d.).The science and application of Hematoxylin and Eosin staining. Robert H. Lurie Comprehensive Cancer Center Northwestern University. Retrieved on 18 Oct. from http://www.feinberg.northwestern.edu/research/docs/cores/mhpl/HandE_troubleshooting.pdf

Gill, G. W. (2010). H&E staining: Oversight and insights. Connection (Chapter 13). Retrieved on 18 Oct. from http://www.dako.com/08066_12may10_webchapter13.pdf