Staining

Chapter 20 | H&E Staining

Gary W. Gill, CT(ASCP)

What is the difference between progressive vs. regressive hematoxylin staining?

progressive hematoxylin stains color primarily chromatin and to a much less extent cytoplasm to the desired optical density, regardless of the length of staining time. Regressive hematoxylin stains overstain chromatin and cytoplasm and require subsequent immersion in dilute acid to pull out the excess color from the chromatin and cytoplasm (table 1). if differentiation is omitted or incomplete, residual hematoxylin visually obscures fine chromatin detail and can prevent the uptake of eosin entirely. Gill hematoxylins no. 1 and 2 contain 2 and 4 gm hematoxylin per liter, respectively, and 25% ethylene glycol. they are progressive stains that can be applied for many minutes without overstaining and without differentiation in a dilute acid bath. harris hematoxylin contains 5 gm hematoxylin per liter of water. it overstains within minutes and requires differential extraction in dilute hcl to decolorize the cytoplasm (differentiation) and to remove excess hematoxylin from chromatin. Figure 1 illustrates the difference between the two approaches.

Are there reasons to prefer water or alcohol as the solvent for eosin formulations?

i prefer alcohol-based eosin formulations: 1) they are chemically more stable 2) they minimize, if not eliminate entirely, the unpredictable effects of various impurities such as water-soluble salts that in water may interfere with dye uptake, and 3) they tend to stain more slowly than water-based formulations (promotes a wider range of shades of eosin colors).

Is there a simple way to perform quality assurance (QA) on hematoxylin and eosin stains before using a batch for the first time?

Yes. Whether buying or making hematoxylin eosin solutions, one cannot be absolutely certain the product will perform. apart from unsound methods, limitations in ingredients, incorrect formulations (e.g., precipitated...