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

(based on 138 customer ratings)

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

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Continuing Education Credits

P.A.C.E.® Contact Hours (acceptable for AMT, ASCP, and state recertification): 1 hour(s)
Course number 578-031-17, approved through 12/31/2019
Course number 20-603459, approved through 12/31/2019


  • 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.

Customer Ratings

(based on 138 customer ratings)

Course Outline

Click on the links below to preview selected pages from this course.
  • 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
    • Types of hematoxylin
    • Types of eosin
    • Differentiators
      • Purpose of Differentiators
    • Bluing
    • Progressive versus regressive
      • Progressive and Modified Progressive Staining
      • Regressive Staining
  • Protocol Selection
  • Understained Slides
  • 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
    • Tissue considerations
      • Cellularity Makes a Difference
  • Uneven Staining
    • Common causes
      • Inadequate Deparaffinization
      • Suboptimal Section Quality
    • Water and eosin
    • 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
    • "Burnt" edges
    • Pigmentation
  • Other Staining Considerations
    • Charged slides versus adding adhesives to waterbaths
      • Adhesives
      • Charged Slides
      • Charged Slides Used with Adhesives
    • Xylene substitutes
    • Are ethanol or reagent alcohols my only dehydrant options?
  • 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.

Another example of a well stained slide.  Note the color variation of the eosin stained components.
Automated staining instrument with interface capability
Good Colon High mag
IMG 0848
IMG 2178
Appropriate staining with hematoxylin counterstain.
How to Subscribe
Histology CE Package$65 Add to cart
Individual course$20 Add to cart

Another example of a well stained slide.  Note the color variation of the eosin stained components.

Automated staining instrument with interface capability

Good Colon High mag

IMG 0848

IMG 2178

Appropriate staining with hematoxylin counterstain.