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ROUTINE STAINING

High-quality staining is central to the successful interpretation of pathology slides. In fact, it is probably not advisable to consider doing margin control surgery (MCS) without a well-equipped laboratory and trusted staff. Most laboratories use the hemotoxylin and eosin (HE) stain to colorize slides. This picks up the various tissue and cellular components in different ways, resulting in a highly reproducible artifact allowing assessment of the cellular and tissue morphology. Pathologists use these staining characteristics to make determinations about the features of disease they see microscopically. In a sense, it is medicine’s version of physiognomy, the study of how someone’s personality can be determined just by looking at their facial characteristics. Though it may seem superficial and far less sophisticated than an involved genetic analysis, the centuries-old method of HE staining continues to be the gold standard in diagnostic pathology.

An alternative to HE is the toluidine blue stain, a metachromatic stain which stains the mucopolysaccharides around basal cell carcinoma (BCC) a distinctive magenta color. This can be helpful in difficult and subtle cases of BCC but due to several factors—the perceived inferior cellular detail being the most pertinent—it fell out of favor and its use has largely been abandoned.

HE is composed of hematoxylin and eosin. Hematoxylin is an extract derived from the Haematoxylum campechianum logwood, a tree native to Mexico. This stains acidic cell components such as nucleic acids, glycosaminoglycans, and acid glycoproteins into shades of blue or purple. Eosin is an acidic dye and serves as an excellent counterstain to hematoxylin as it targets the cytoplasm of cells, specifically mitochondria, secretory granules, and collagen. It gives differing shades of pink to the cytoplasm of different types of cells and connective tissues.

There is a range of protocols for performing the HE stain but they all have the same key steps. The first step is always slide fixation, in which chemicals or heat is used to adhere the tissue sections to the slide before staining. Frozen section laboratories typically use 10% neutral buffered formalin, alcoholic formalin, alcohol, or acetone as a fixative. If the slides are removed too quickly from the fixative, they will be inadequately dehydrated, which profoundly alters the staining quality.

Following fixation, the key steps are nuclear staining (hematoxylin), cytoplasm staining (eosin), dehydration, clearing, and coverslipping. Each one of these steps is a potential cause for morphologic distortion (Table 7.1).

TABLE 7.1Sample Rapid Frozen Section HE Staining Method

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