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H&E Basics Part 1 - Introduction

By Cindy Sampias, JD CT(ASCP)HTL

For routine diagnosis, the use of Hematoxylin and Eosin (H&E) is by far preferred for viewing cellular and tissue structure detail by pathologists. The variation of stain intensity is often driven by the pathologist’s learning experience and personal preference. Because this stain demonstrates such a broad range of cytoplasmic, nuclear, and extracellular matrix features, nearly all teaching texts use H&E images. We continue to use this simple and essential stain today, which has remained unchanged for well over a century.

Fig 1: Skin H&E. Note the balanced coloration in this section of skin. The nuclei are stained purple, while the cytoplasmic components are pink.

The staining procedure for H&E follows a basic protocol:

  • Dewaxing
  • Dehydration
  • Hematoxylin
  • Differentiation
  • Bluing
  • Eosin
  • Dehydration
  • Clearing
  • Cover-slipping

The format is easily reproduced and the reagents resilient enough to allow for large numbers of slides to be stained consistently before reagents need to be changed.

Fig 2: Colon H&E. Note the balanced coloration in this section of colon. The cilia on the columnar epithelium are very crisp.
Photo credit: Stanley Hansen

In the past, stains and their components were routinely made by the laboratory. Commercially available, premade reagents were uncommon and expensive. So, as an economical way to perform H&Es, techs learned to make the dyes as needed. The challenge was to make sure the quality of the stain was consistent. Different techs follow recipes using their own individual approach, so making dyes was typically left to one person. That said, there were also more techs in the laboratory, so the time needed to prepare the reagents did not take away from the overall workload of the team.

Fig 3: Placenta H&E. The red blood cells in this placenta section represent the bright coloration that may be experienced with this simple stain

Many laboratories find ordering their stains to be the easiest way to ensure consistent and repeatable quality. A large variety of both hematoxylin and eosin stain combinations provide the ability to customize the desired results with very little hassle. 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.

Fig 4: Glomerulus (red arrow) and tubules (blue arrow) H&E. The nuclei are stained purple, while the cytoplasmic components are pink.

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.