I have tried my best to focus on day to day problems in H&E staining and some troubleshooting and its probable solutions

2. CONTENTS
• Introduction of H&E stain
• Troubleshooting in H&E Stain
• Summary
• References

3. INTRODUCTION
• Waldeyer introduced hematoxylin and eosin
staining system in 1863.
• It is a most common and primary staining
technique in the histopathology today
• Today different types of hematoxylin (alum &
Iron) and eosin (Y, R, B) available.

4. TROUBLESHOOTING IN H&E STAIN

5. TROUBLESHOOTING IN H&E STAIN
White spots are seen in the section after deparaffinization step. If they are not
recognized at this point, spotty or irregular staining will be seen microscopically on
the stained section
The section not dried before xylene stage
properly
The slides must be dried and then
retreated with xylene to remove the
paraffin
The slide did not remain in xylene
long enough for complete
removal of the paraffin
The slides should be returned to
xylene for a longer time.

8. TROUBLESHOOTING IN H&E STAIN
The hematoxylin is too light (The nuclei are too pale).
The sections were
not stained long
enough in
hematoxylin.
The section
must be
restained.
The efficacy
of
hematoxylin
is lost/
reduced
Discard
hematoxylin
and replace
with fresh.
The
differenti-
ation step
was too
long.
Run back
and
restain
Pale nuclei in
bone sections
may be the
result of over
decalcification.
no
solution

9. The hematoxylin is too light

10. TROUBLESHOOTING IN H&E STAIN
The hematoxylin is too dark (The nuclei are overstained), or diffuse
hematoxylin staining of the cytoplasm
The sections were
stained too long in
hematoxylin.
Decolorize the section
and restain, making
appropriate adjustments
in the staining time of
hematoxylin.
The sections
are too thick.
Recut the
section.
The differentiation
step was too short.
Decolorize the section
and restain, making
appropriate adjustments
in the differentiation
times

12. TROUBLESHOOTING IN H&E STAIN
Red or red-brown nuclei.
The older
hematoxylin
Check the efficacy
of the
hematoxylin.
The sections were not
blued sufficiently
Allow a longer time for
bluing of the sections

13. TROUBLESHOOTING IN H&E STAIN
Pale staining with eosin.
The pH of the eosin solution
may be above 5.0, possibly
caused by carryover of the
bluing reagent.
Check the pH of the eosin solution, and
adjust it to a pH of 4 to 5 with acetic acid if
necessary.
Be sure the bluing reagent is completely
removed before transferring the slides to
the eosin.
The section is too thin.
Check the thickness of
the section and if
required recut

15. TROUBLESHOOTING IN H&E STAIN
Cytoplasm is overstained, and the differentiation is poor
The eosin solution
may be too
concentrated
Dilute the eosin
solution.
The section may
have been
stained for too
long.
Decrease the
staining time.
The sections may have
been passed through the
dehydrating alcohols too
rapidly
Allow more time in each
of the dehydrating
solutions for adequate
differentiation of the
eosin. (Also, check the
section thickness.)

18. Eosin blebs/ background
staining
May be due to egg albumin use for the coating of
the slide before taking section on the slide
Use as much egg albumin as required, or tried taking section on
slide without egg albumin (as it is not compulsory step)
TROUBLESHOOTING IN H&E STAIN

19. TROUBLESHOOTING IN H&E STAIN
Blue—black precipitate/crystals on top of the sections.
The metallic sheen that develops on most hematoxylin
solutions has been picked up on the slide.
Filter the hematoxylin solution daily before staining slides.

20. Hematoxylin crystals deposited within hepatocytes (arrows)
during staining with unfiltered hematoxylin staining solution.

21. TROUBLESHOOTING IN H&E STAIN
Water bubbles are seen microscopically in the stained sections
The sections were not completely dehydrated, and water is
present in the mounted section.
Remove the cover glass and mounting medium
with xylene. Return the slide to fresh absolute
alcohol (several changes). After the sections are
dehydrated, clear with fresh xylene and mount.

23. TROUBLESHOOTING IN H&E STAIN
Difficulty bringing some areas of the tissue in focus with light
microscopy.
Mounting medium may be present on top of the cover glass.
1. Wait  Let mounting medium dry  dressing of slide
2. Remove the cover glass and remount with a clean cover glass.
Review the method used for mounting sections, and modify if
needed.

24. The mounted stained sections do not show the usual
transparency and crispness when viewed by light
microscopy.
The mounting medium may be too thick, causing the
cover glass to be held too far above the tissue.
Remove the cover glass and mounting medium with
xylene. Remount the section with fresh mounting
medium.
TROUBLESHOOTING IN H&E STAIN

25. TROUBLESHOOTING IN H&E STAIN
The water and the slides turn milky when the slides are
placed in the water following the rehydrating alcohols.
Xylene has not been removed completely by the
alcohols.
Change the alcohols, back the slides up to absolute
alcohol, and rehydrate the sections..

26. TROUBLESHOOTING IN H&E STAIN
The mounting medium has retracted from the
edge of the cover glass.
The cover glass
is warped
Remove the
cover glass
and apply a
new cover
glass
The mounting medium has been
thinned too much with xylene
Apply a new cover glass with fresh mounting
medium. Keep the mounting medium
container tightly capped when not in use. Use
a small container for the mounting medium
and discard when it becomes too thick

27. SUMMARY
• The processing of a biopsy or specimen is subject to a
procedural protocol that results in a tissue fit for
diagnosis and interpretation.
• The staining procedures themselves are subject to
human and material errors and the result is an artifact
that in the least may interfere with adequate diagnosis
or at the most render the tissue so distorted as to be
undiagnosable.
• The need to recognize these artefacts and attempt to
overcome them is the single biggest challenge in the
pathological laboratory.

28. References
1. Bancroft JD. Theory and practice of histological techniques, 5th edition.
Philadelphia:Churchill Livingstone; 2005.
2. Brown RW (Ed.). Histologic Preparations: Common Problems and Their
Solutions. College of American Pathologists, Northfield IL, 2009.
3. Dumas T. ‘Going green’ in the lab. MLO. 2009;41(5):48- 49. Accessed November
1, 2009 at: http://www.mloonline.com/features/2009may/0509_education.pdf.
4. Kiernan JA. Histology FAQ Staining, Histochemistry and Histotechnology.
Accessed October 5, 2009 at: http:// www.ihcworld.com/_faq/histology-
faq/misc/m6.htm.
5. Luna LG. Histopathologic Methods and Color Atlas of Special Stains and Tissue
Artifacts, American Histolabs, Gaithersburg MD, 1992.
6. Thompson SW, Luna LG. An Atlas of Artifacts Encountered in the Preparation of
Microscopic Tissue Sections, Thomas, Springfield IL, 1978 (190 pages).
7. Wallington EA. Artifacts in Tissue Sections. Med Lab Sci. 1979;36(1):3-61.

29.  THANK YOU 