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H&E Staining

The document discusses common errors that can occur during hematoxylin and eosin staining and provides remedies. It defines staining terminology and describes the staining process. Common errors include inadequate drying before staining, over or under staining of hematoxylin or eosin, incomplete removal of reagents between steps, and issues with mounting. The remedies involve adjusting staining times, ensuring complete removal of water and reagents between steps, checking reagent quality, and remounting slides with clean materials. Proper technique and monitoring of the staining process are important to avoid errors.

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80 views31 pages

H&E Staining

The document discusses common errors that can occur during hematoxylin and eosin staining and provides remedies. It defines staining terminology and describes the staining process. Common errors include inadequate drying before staining, over or under staining of hematoxylin or eosin, incomplete removal of reagents between steps, and issues with mounting. The remedies involve adjusting staining times, ensuring complete removal of water and reagents between steps, checking reagent quality, and remounting slides with clean materials. Proper technique and monitoring of the staining process are important to avoid errors.

Uploaded by

sirahdemha
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPTX, PDF, TXT or read online on Scribd
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ERRORS IN H&E

STAINING
CAUSES AND REMEDIES
HEMATOXYLIN & EOSIN

The Hematoxylin and Eosin combination is the


most common staining technique used in
Histology. The diagnosis of most malignancies
is based on this staining procedure.
TERMINOLOGIES
Stain:
The dyes are used to facilitate examination of tissues, microorganisms, or other cells
under the microscope.
Mordant:
The chemical which promotes the binding/bridging between stain and the specific
tissue component.
Oxidizing agent:
A reagent that acts as an electron acceptor in a chemical oxidation-reduction reaction.
In other word, it enhance the dye to produce color.
Regressive:
The staining procedure needs over staining of one dye, then the addition of
differentiator to remove non specific stain and accomplish with counterstain.
Progressive:
The staining procedure needs optimal staining of one dye, Without use of
differentiator accomplished with counterstain.
TERMINOLOGIES
Differentiation:
The reagent use to decolorize the primary stain.
Blueing:
The reagent (alikaline) enhances the hematoxylin. e.g. Tap water, alkaline water, ammonia water,
lithium carbonate etc.
Counterstain:
The secondary stain use to accomplish the staining procedure and produces contrasting color
with the primary stain.
Basophilic:
pH > 7, Negative charged, An ionic i.e. Hematoxylin
Acidophilic:
pH < 7, Postively charged, Cat ionic i.e. Eosin
PRINCIPLE

The oxidation product of hematoxylin is hematin. The Hematin binds


with lysine residues of nucleus in the presence of metallic alum
(mordant). This oxidized product show blue color and less soluble in
basic condition and can convert to red color and more soluble in
acidic condition. The tissue is counterstain by acidophilic dye which
linked to the cytoplasm and connective tissues.
HEMATOXYLIN & EOSIN
Hematoxylin:
• Commonly used for nuclear staining.
• Specific Purple/blue coloration in Nucleus; Non specific (overstaining) cytoplasm,
connective tissues, mucins or in the background of tissue.
• The most common is Harris Hematoxylin (Regressive), other are Mayer’s
Hematoxylin and Gill I/II (Progressive)
• Harris hematoxylin is a strong nuclear stain and produces crisp nuclear staining.
As it also oxidises with air, so needs to be filter to remove precipitates.

Eosin:
• Negatively charge dye, stains cytoplasm & connective tissues.
• It produces optimal contrast with hematoxylin.
PROCEDURE (REGRESSIVE)
After microtomy
• Slide drying
• Deparaffinization
• Hydration
• Nuclear staining (Harris Hematoxylin)
• Rinse with water
• Differentiation
• Blueing
• Cytoplasmic staining (Eosin)
• Dehydration
• Clearing and mounting/coverslipping
The staining must be monitored properly. The reagents require filtration, must be
done on regular basis to avoid contamination with tissue or precipitation of reagent.
High Scoring H&E slides
Source RCPA web
High Scoring H&E slides
Source RCPA web
High Scoring H&E slides
Source RCPA web
ERROR # 1
White spots are seen in A. The section was not dried properly A. The slides must be treated
the section after de- before beginning deparaffinization. with absolute alcohol to
paraffinization step. If B. The slide did not remain in xylene remove the water and
they are not recognized long enough for complete removal then retreated with xylene
at this point, spotty or of the paraffin. to remove the paraffin. If
irregular staining will be incomplete drying is
seen microscop-ically on severe, the sections may
the stained section. loosen from the slides.
B. The slides should be
returned to xylene for a
longer time.
ERROR # 2
The nuclei are too pale (the hematoxylin is too light).

A. The sections were not stained long enough in hematoxylin.


B. The hematoxylin was overoxidized and should not have been used.

C. The differentiation step was too long.

D.
A. Pale section
The nuclei inmust
bonebesections mayThe
restained. be the
timeresult of over decalcification.
in the
hematoxylin may have to be increased, or a
method to increase tissue basophilia may be
needed.
B. Discard hematoxylin and replace with fresh.

C. Run back and restain.


D. No solution.
ERROR # 3
The nuclei are overstained (the hematoxylin is too dark), or diffuse hematoxylin staining of the
cytoplasm has occurred.

A. The sections were stained too long in hematoxylin.


B. The sections are too thick.
C. The differentiation step was too short.

A. Decolorize the section and restain, making appropriate adjustments in the staining time of
hematoxylin.
B. Recut the section.
C. Decolorize the section and restain, making appropriate adjustments in the differentiation
times.
ERROR # 4
Red or red-brown nuclei.

A. The hematoxylin is breaking down.


B. The sections were not blued sufficiently.

A. Check the oxidation status of the hematoxylin.


B. Allow a longer time for bluing of the sections; it is
impossible to over blue the sections.
ERROR # 5
Pale staining with eosin.

A. The pH of the eosin solution may be above 5.0, possibly caused by carryover of the
bluing reagent.
B. The sections may be too thin.
C. Slides may have been left too long in the dehydrating solutions.

A. Check the pH of the eosin solution, and adjust it to a pH of 4.6 to 5.0 with acetic
acid if necessary. Be sure the bluing reagent is completely removed before transfer-
ring the slides to the eosin.
B. Check the thickness of the section.
C. Restain with eosin and do not allow the stained slides to stand in the lower concen-
trations of alcohols.
ERROR # 6
Cytoplasm is overstained, and the differentiation is poor.

A. The eosin solution may be too concentrated, especially if phloxine is present.

B. The section may have been stained for too long.

C. The sections may have been passed through the dehydrating alcohols too rapidly
for good differentiation of the eosin to occur.
A. Dilute the eosin solution.
B. Decrease the staining time.
C. Allow more time in each of the dehydrating solutions for adequate differentiation of
the eosin. (Also, check the section thickness.)
ERROR # 7
Blue—black precipitate 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.


ERROR # 8
Water bubbles are seen microscopically in the stained sections. (Cornflakes artifact)

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
with synthetic resin. All dehydrating and clear-
ing solutions should be changed before stain-
ing any more sections.
ERROR # 9
Difficulty bringing some areas of the tissue in focus with light microscopy.

Mounting medium may be present on top of the cover glass.

Remove the cover glass and remount with a clean cover glass. Review the
method used for mounting sections, and modify if needed.
ERROR # 10
The mounting medium has retracted from the edge of the cover glass.
A. The cover glass is warped.
B. The mounting medium has been thinned too much with xylene.
A. Remove the cover glass and apply a new cover glass.
B. 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.
ERROR # 11
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.
ERROR # 12
The slides are hazy or milky in the last xylene rinse prior to cover slipping.

Water has not been completely removed from the sections before being
placed in the xylene

Change the alcohol solutions, especially the anhydrous or absolute reagents.


Redehydrate the sections and clear in fresh xylene.
ERROR # 13
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.
Random Errors

Incomplete dehydration/drying after microtomy Contamination from staining


Random Errors

Gelfoam artifact Calcium carbonate to neutralize formalin

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