The hematoxylins and eosin

Introduction

Hematoxylin and eosin (H&E) is the most widely used histological stain. It is simple to use, easy to automate and demonstrates different tissue structures clearly. Hematoxylin stains the cell nuclei blue-black, showing clear intranuclear detail, whilst eosin stains cell cytoplasm and most connective tissue fibers in varying shades and intensities of pink, orange and red. Automated staining machines and commercially prepared hematoxylin and eosin solutions are commonly used in today’s laboratories for routine staining. Many laboratories now have to adhere to strict ISO standards to gain accreditation from regulating authorities. These standards cover validation of chemicals and stains used within the laboratory and variability between batches of ‘in-house’ stains. These regulations and the underpinning paperwork have resulted in many laboratories using commercially produced reagents where validation is performed by the supplier (see Chapter 1 ). Students of histological techniques should have a basic knowledge of the dyes and their preparation techniques in order to troubleshoot and/or modify procedures for specialized use. Hematoxylin may also be used as a stain without eosin to demonstrate connective tissues such as elastic fibers, muscle striations and mucins.

Eosin

Eosin is a fluorescent, xanthene dye which binds to salts with eosinophilic compounds containing positive charges. It is the most suitable stain to combine with an alum hematoxylin to demonstrate the general histological architecture of tissues. Eosin has the ability, with correct differentiation, to distinguish between the cytoplasm of different types of cell, connective tissue fibers and matrices, by staining these differing shades of red and pink.

There are several types of eosin available commercially but Eosin Y is the most widely used and is soluble in water and alcohol. As a cytoplasmic stain, it is usually used as a 0.5 or 1.0% solution in distilled water, with a crystal of thymol added to inhibit the growth of fungi. The addition of a little acetic acid (0.5 ml to 1000 ml stain) is said to sharpen the staining. The majority of the differentiation of eosin staining occurs in the subsequent tap water wash, but a little further occurs during dehydration through the alcohols. The intensity of eosin staining and the degree of differentiation is a matter of individual and laboratory taste. Good film-based photomicrographs of H&E stained tissues are obtained when the eosin staining is relatively intense and the differentiation slight. This is achieved by doubling the routine staining time. However, modern digital cameras can be used with the full range of eosin staining. Alternative red dyes, e.g. phloxine or Biebrich scarlet can be used as an eosin substitute but give a more intense red color to the tissues, and are rarely as amenable to subtle differentiation, making them less effective.

Eosin staining may be intense after mercuric fixation and difficulty may be experienced in obtaining adequate differentiation. Over-differentiation of the eosin, until only the red blood cells and granules of eosinophil polymorphs are stained red, facilitates the location and identification of this type of cell. Combining 10 ml 1% phloxine B, 100 ml 1% eosin Y, 780 ml 95% alcohol and 4 ml glacial acetic acid produces a cytoplasmic stain where muscle is clearly differentiated from collagen and red cells stain bright red.

Hematoxylin

Hematoxylin is extracted from the heartwood (‘logwood’) of the tree Hematoxylon campechianum which originated in the Mexican State of Campeche, but is now mainly cultivated in the West Indies. It is extracted from the logwood with hot water and then precipitated out from the aqueous solution using urea (see prior editions of this text). Hematoxylin itself is not a stain, but hematein, the major oxidization product, is a natural dye responsible for the color properties.

Hematein can be produced from hematoxylin in two ways:

• 1.

  Natural oxidation or ‘ripening’ by exposure to light and air. This slow process can take up to 3–4 months, but the resultant solution retains its staining ability for a long time. Ehrlich’s and Delafield’s hematoxylin solutions are examples of naturally ripened hematoxylins.

• 2.

  Chemical oxidation. Chemical oxidizing agents convert the hematoxylin to hematein rapidly, so these solutions are ready for use immediately after preparation. Sodium iodate used in Mayer’s hematoxylin and mercuric oxide used in Harris’s hematoxylin are examples of these agents. These solutions have a shorter useful life than the naturally oxidized hematoxylins because the oxidation process continues in air and light, converting the hematein to a colorless compound.

Hematein is anionic with a poor affinity for tissue and is inadequate as a nuclear stain without the presence of a mordant. The metal cation in the mordant confers a net positive charge to the dye-mordant complex and enables it to bind to anionic tissue sites, e.g. nuclear chromatin. The type of mordant used influences the type of tissue components stained and their final color. Aluminum, iron and tungsten salts are the most useful mordants for hematoxylin, but solutions using lead are used for the demonstration of argyrophil cells.

Most mordants are incorporated into the staining solution, but certain hematoxylins such as Heidenhain’s iron require the tissue section to be pre-treated with the mordant before staining.

Hematoxylin solutions are classified according to the mordant used:

• Alum hematoxylins

• Iron hematoxylins

• Tungsten hematoxylins

• Molybdenum hematoxylins

• Lead hematoxylins

• Hematoxylins without mordant.