ERRORS IN TISSUE

PROCESSING

Dr. SHASWAT BHATTARAI

1st year JR, Pathology
- Specimen receive and registration
- Grossing
- Fixation
- Processing
- E mbedding
- Staining
 Specimen receive and registration

• The specimen name is identical to that

written on the request form.
• All the patient details are properly
written mentioned.
• Date of biopsy/procedure
• Time of specimen receive
• Register the specimen with a lab
number
 Grossing
• The specimen should be completely
covered with the fixation fluid.
• If no fluid/water, change with 10 % formalin
solution or other fixative according to your
lab protocol.
 Fixation
• The major problems related to fixation are delayed or
incomplete fixation, autolysis is caused by delayed fixation.
• In H & E section, the tissue may show loss or total
disappearance of nuclear chromatin. Some cells may
disappear (intestinal mucosa), shrlnk and leave artefactual
space around.
• Prevention: Add fixative to the specimen as soon as possible
• Open uterus for endometrial fixation Open GIT specimen for
mucosal fixation.
• Slice solid organ e.g breast, kidney, thyroid.
• Bisect lymph nodes
 Fixation
Incomplete fixation
• The cells characterized by smudge nuclei
(indistinct nuclear pattern), nuclear bubbling
Prevention:
• Prolong fixation time
• Thin gross section
• Fresh formalin solution
• Cassete should not be tightly backed
• Agitation of cassettes in the fixative
 Processing
• Most problem encountered in processing is
related to either over processing or under
processing.
Overdehyration:
• Due to excessive dehydration, which results in
microchatter around the edges of the tissue
on H & E Stain.
Prevention:
• Small biopsies processed separately
• Shorten the dehydration time
 Processing
Poor processing
• Due to improper dehydration (water in tissue),
impaired clearing, clearing agent in paraffin,
too much heat during processing.
Prevention:
• No cassettes condensation
• Absolute alcohol is fresh, free of Water
• Fluid are changed according to the schedule
• Heat is used only for paraffin wax.
 Embedding and specimen orientation
It refers to casting or blocking
(paraffin blocking) "
Hints:
Specimen orientation
• Proper pressure force
applied to the entire
specimen during
orientation and initial
solidification to obtain
flat tissue.
 Embedding and specimen
orientation
• Hard tissue such as bone
will section more easily if
they are embedded
diagonally.

• Tissue with wall such as

cyst, gall bladder and GIT
must be embedded on
the edge.
 Troubleshooting in Embedding and
specimen orientation
Soft Mushy Tissue
Incorrect orientation
The most common cause is
thick sections at gross Section may be incorrectly
examination and have been oriented at the embedding
compressed between the top section if the correct
and bottom of the cassette. method wasn't indicated.
Prevention:
• The tissue section should be Prevention:
thin • Marking the side of tissue
• Adequate time for fixation to be embedded facing
up with ink.
• Fluid change according to
schedule.
 Troubleshooting in Embedding and
specimen orientation
Tissue not embedded at the
Tissue carryover same level
Small pieces or fragments of If the tissue is not properly
tissue may be carried from 1 flattened by pressing it down
tissue to the next at the uniformly when it is placed in the
embedding table, resulting embedding mold or multiple
tissues to be palced in the same
in cross-contamination. mold are embedded at different
Prevention: levels.
• Carefully clean forceps used Prevention:
• Press the tissue uniformly.
at the embedding table • Keep the paraffin molten enough to
between specimens. get all pieces embedded at the same
• Open only the cassette with level.
the tissue to be embedded. • Work very fast when embedding
multiple pieces.
 Troubleshooting in Decalcification
Underdecalcification
Bone dust . The tissue still hard and
sectioning is difficult
When obtaining sections of
bone with a saw commonly Over decalcification
used for the process, bone dust Occur when the end point
is pressed into the surface of of decalcification is not
the bone. carefully checked, result in
poorly stained sections.
Prevention: Prevention
• Use a saw with diamond • Choose decalcificatian
blade agent that fits the need of
the lab
• Trim the bone surfaces • Develop good method for
after decaIcification detecting the end point of
decalcification.
Troubleshooting in frozen section
Freezing artifact

If ice crystals were formed due

to improper freezing of tissue.

Prevention:
• Snap freezing
• Make sure the tissue wasn’t
immersed in saline before
freezing.
 Troubleshooting in frozen section
Tissue not embedded flat Block loosens from the
chuck while sectioning
If the tissue is not embedded
flat on the chuck, then sectioning Occur if the check was too
will have to be deeper into the cold
block and some important parts when the embedding medium
of the tissue may be wasted. was applied.
Prevention: Prevention
• Place the tissue on a slide,
• Reattach the tissue block to a
surround it with the
clean chuck with additional
medium, when the medium
begin to turn white, coat embedding medium
the chuck with embedding
medium and invert over
the tissue. Then remove the
slide.
 Troubleshooting in microtomy
Crooked ribbons

Result when the horizontal edges

(to and bottom) of the
block are
not parallel.
If the lower block edge is not
parallel to the knife edge.
Prevention:
• The upper and lower edge
should be parallel.

• The lower block edge is

parallel to the knife edge.
• No problem in the blade
 Troubleshooting in microtomy
Block face unevenly sectioned

Occur when the block holder is not

parallel to the blade. One side of
the block is exhausted while
attempting to get a complete
section of the block face.
Prevention:
• Ensure at the beginning of the
sectioning that the block holder
is adjusted so that the block
face and the blade are perfectly
parallel.
 Troubleshooting in microtomy
T h i c k section

Prevention:
• Adjust the thickness
 Troubleshooting in microtomy
Vertical scratches

Caused by defect in the blade

edge, calcium, bone or hard
material in the specimen.
Prevention:
• Ensure at the beginning of
the sectioning that the
block holder is adjusted so
that the block face and the
blade are perfectly parallel.
 Troubleshooting in microtomy
Holes in the section
Occur when block is faced too
aggressively.
The specimen is either
excessively dehydrated or
improperly processed.
Prevention:
• Ensure to chill the block with ice
before cutting and discard
ribbons until the hole disappear.
• Facing the block less
aggressively, with smaller
micrometer advances of the
block for each section removed.
 Troubleshooting in microtomy
Failure of ribbon to form
Commonly caused by dull
blade. Could result from too
hard paraffin, too much blade
tilt.
Prevention:
• Paraffin with lower melting
point.
• Decrease blade tilt.
• Change room temperature ”
 Troubleshooting in microtomy
Washboarding or Undulation in the
section

Commonly occurs in very hard tissue

such as uterus or in over fixed tissue.
It is the macroscopic type of chatter
commonly caused by loose
clamping of blade or block.
Prevention:
• Proper clamping of blade and
block.
• Ensure the block holder shaft is
not over extended.
• Ensure the microtome is in good
working order.
• Decrease the blade tilt.
 Troubleshooting in microtomy
Chatter or microscopic Vibration

Commonly caused by over dehydration or

lack of moisture in the tissue. It could also
result from dull blade or too much blade
tilt which cause the section to be scrabed
rather than cut or cutting too rapidly.
Prevention:
• Proper processing.
• Restore moisture by facing the block
down on an ice tray.
• Decrease the blade tilt.
• Decrease cutting speed: one wheel per
second is considered reasonable speed.
 Troubleshooting in E staining
H
Incomplete Deparaffinization
White spots may be seen in tissue sections
after the deparaffinization step. Usually
caused by water left in the tissue, incomplete
drying or not leaving the slides in xylene long
enough.
Prevention:
• Dry section properly before beginning
deparaffinization.
• Allow sufficient time in xylene for complete
deparaffinization.
• Avoid contaminated xylene, change fluids
according to the schedule.
• If the slides have been stained, decolorize
and restain.
 Troubleshooting in H & E staining
Pale nuclear staining
The Nuclei is too Iight due to:
• Slide not exposed to the
hematoxylin long enough
exhausted (over oxidized or
depleted) hematoxylin.
• Over differentiation.

Prevention:
• Leave the slide longer.
• Use fresh hematoxylin.
• Time the differentiation.
 Troubleshooting in H & E staining
Dark nuclear staining

The nuclei is too dark due to :

• Slide exposed to the

hematoxylin too long.
• Section are too thick.
• Differentiation step is too short .
Prevention:
• Thin sections.
• Decrease hematoxylin
exposure.
• Increase time for
Troubleshooting in H & E staining
Red or red-brown Nuclei

If the nuclei is stained red or

reddish brown instead of blue,
either the hematoxylin is
breaking down or the blueing
step was not properly done.

Prevention:
• New hematoxylin
• Proper blueing
 Troubleshooting in E staining
H black precipitate
Blue

• Hematoxylin precipetate

Prevention:
• Filter the hematoxylin.
 Troubleshooting in E staining
H Cytoplasmic staining
Pale
Eosin pH is over 5, high PH may result from carryover of
the blueing agent. The section may be too thin or left
long in the dehydration.

Prevention:
• Check Eosin pH
• Completely remove blueing agent before
transferring the slides to eosin.
• To allow stained slides to stand in the lower
concentration of alcohols after the eosin. The more
water in the alcohol, more eosin will be removed.
 Troubleshooting in H& E
Darkstaining
Cytoplasmic staining

If the cytoplasm is overstained and

the differentiation is poor, the contrast
between the nucleus and the
cytoplasm is lost.
Prevention:
• Avoid over concentration of eosin,
dilute eosin solution.
• Do not leave sections in eosin for
long and allow sufficient time in
dehydration solution, specially 70%
alcohol to allow good eosin
differentiation.
• Check section for proper thickness.
 Troubleshooting in H& E staining
Dark basophilic staining of
nuclei and cytoplasm,
especially around tissue
edges

Laser and electrocautry

techniques denature
macromolecules and produce
heat artifact, generally marked
by dark basophilic staining
in nuclei and cytoplasm.

Prevention:
Nothing to be done.
THANK YOU