Troubleshooting Guidance for Hematoxylin and Eosin (H&E) Stain

Author: Cynthia Sampias, JD, CT(ASCP)HTL
Reviewer: Michael D. Glenn, HT

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Continuing Education Credits


  • Review the basics of H&E staining and what optimal staining should look like.
  • Identify common problems encountered with staining techniques.
  • Consider solutions to common artifacts that influence stain results.
  • Discuss the advantages of staining platforms and the use of kits to standardize stain performance.
  • Address outside factors that can influence H&E stain success.
  • Identify strategies used for successful H&E staining.

Course Outline

  • H&E Basics
    • Why is H&E the standard?
      • Introduction
      • Preferred by Pathologists for Routine Diagnosis
      • Ease of Use and Reproducibility
    • Making reagents versus purchasing ready to use
      • Making Staining Reagents in the Laboratory
      • Purchasing Ready to Use Reagents
      • Author's Thoughts
    • Types of hematoxylin
      • Purpose of Hematoxylin
      • Components of Hematoxylin
      • What are Mordants?
      • Mayer's Hematoxylin
      • Harris' Hematoxylin
      • Gill's Hematoxylin
      • Iron Hematoxylins
    • Types of eosin
      • Purpose of Eosin
      • Eosin Y
      • Eosin-Phloxine
      • EA Family (50 and 65)
    • Differentiators
      • Purpose of Differentiating Solutions
    • Bluing
      • Bluing Solutions
    • Progressive versus regressive
      • Progressive and Modified Progressive Staining Methods
      • Regressive Staining Method
  • Protocol Selection
    • Selecting a protocol
      • Does One Staining Protocol Fit All?
      • A Good Baseline Staining Protocol
      • How Best to Optimize Staining Protocol
    • Automated Platform versus Hand Staining
      • Why Move from Hand Staining to an Automated Platform?
  • Understained Slides
    • Common causes
      • Poor Coloration: Is the Protocol Long Enough?
      • One Stain Component Overshadowing Another
      • Is the Differentiating Solution the Right Intensity?
    • The role of acidity in hematoxylin
      • The Importance of pH
    • Water and eosin
      • Eosin Differentiation
    • Lower cellularity of the sample can be misleading
      • Where is the Pink and Blue?
  • Overstained Slides
    • Common causes
      • Protocol is Too Long
      • One Stain Component Under Staining Compared to the Other
      • Is the Differentiator Too Strong or the Step Too Long?
    • The role of acidity in differentiation of hematoxylin
      • Acid Extends Hematoxylin Life: Why do I Need it for Differentiation?
    • Water and eosin
      • The Impact of Water on Eosin
    • Tissue considerations
      • Cellularity Makes a Difference
  • Uneven Staining
    • Common causes
      • Inadequate Deparaffinization
      • Suboptimal Section Quality
    • Water and eosin
      • Impact of Excess Water Following Eosin
      • Water in Xylene
    • Water quality
      • Tap Water versus Deionized Water
  • Staining Frozen Tissues
      • Challenges with Frozen Tissue Staining
  • Artifacts That Can Make Staining a Challenge
    • The impact of suboptimal tissue processing
      • Sample Collection
      • Sample Fixation
      • Choosing Processing Protocols that are Appropriate for your Tissues
    • Nuclear bubbling
      • Nuclear Bubbling: Created During Processing
      • Nuclear Bubbling: Created After Cutting
    • Floaters
      • Where are the Floaters with Respect to the Tissue?
      • Fungal and Bacterial Contamination
    • "Burnt" edges
      • Proper Specimen Collection and Handling
      • Related to Processing
    • Pigmentation
      • Causes of Pigmentation
  • Other Staining Considerations
    • Charged slides versus adding adhesives to waterbaths
      • Adhesives
      • Charged Slides
      • Charged Slides Used with Adhesives
    • Xylene substitutes
      • Xylene Versus Xylene Substitutes
      • Laboratory Safety
    • Are ethanol or reagent alcohols my only dehydrant options?
      • Methanol, Isopropyl Alcohol, and Flex
  • Conclusions
    • Common H&E challenges
      • Change Early, Change Often!
      • Maintenance and Good Quality Control
      • Ensure Training
    • A few favorite protocols
      • Well-Rounded, Standard H&E - Regressive Protocol
      • Well-Rounded, Standard H&E - Modified Progressive Protocol
      • Well-Rounded, Standard H&E - Progressive Protocol
      • Adjusting for Coloration
  • References
      • References

Additional Information

Level of Instruction: Basic to intermediate

Intended Audience: Clinical laboratory histotechnologists, technicians, and other medical laboratory personnel who have an interest in this subject matter. This course is also appropriate for histology and clinical laboratory science students, pathology residents, and practicing pathologists.

Author information: Cynthia Sampias, JD, CT(ASCP)HTL is currently a Senior Field Support Specialist with Leica Biosystems. Her primary focus is to assist histology teams on all core histology products/consumables and troubleshooting. During her career, she worked as a Histotechnologist and Global Project Manager for Covance Central Laboratory Services and as a Senior Cytotechnologist/Histotechnologist at Diagnostic Cytology Laboratories, both located in Indianapolis, Indiana. She holds a Doctor of Jurisprudence degree from Indiana University School of Law with a specialty in health care law, and a Masters of Public Affairs degree in Health Services Administration from Indiana University Northwest. She is certified as both a cytotechnologist and histotechnologist.

Reviewer information: Michael D. Glenn, HT is currently a Field Support Specialist at Leica Biosystems. He covers a twelve-state area within the United States, in which Michael supports, educates, and collaborates with doctors and histology professionals pertaining to the histology field. Michael received his BS from Indiana State University and Certificate in Histology from Indiana University School of Medicine. His professional background consists of research, clinical, and field support. Michael is a member of the National Society for Histotechnology.

Course description: This course is intended to examine common challenges with H&E staining and offer guidance for troubleshooting.

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Good Colon High mag
Another example of a well stained slide.  Note the color variation of the eosin stained components.
IMG 0848
IMG 2178
Appropriate staining with hematoxylin counterstain.
Automated staining instrument with interface capability