A Review of

Artifacts in
Histopathology
Yoomin Sabig (074153)
Jaaisha Amjad (075628)
Fathmath Looseen (072239)
 A review of artifacts in
histopathology
Journal of Oral and Maxillofacial pathology
May-August 2018

Syed Ahmed Taqi

Syed Abdus Sami
Lateef Begum Sami
Syed Ahmed Zaki
 Contents
What are artifactss?
Classification of artifacts
Prefixation artifacts
Fixation artifacts
Artifacts related to bone tissue
Tissue-processing artifacts
Artifacts related to microtomy
Artifacts related to floatation and mounting
Staining artifacts
Mounting artifacts
Conclusion
References
What are artifacts?
An artificial structure or tissue alteration on a prepared
microscopic slide as a result of an extraneous factor

Aim:
To review the various causes of artifacts and how to identify
and prevent them from interfering in the accurate diagnosis of
lesions
Classification of artifacts
Prefixation artifacts
Fixation artifacts
Artifacts related to bone tissue
Tissue-processing artifacts
Artifacts related to microtomy
Artifacts related to floatation and mounting
Staining artifacts
Mounting artifacts
Prefixation Artifacts
Prefixation artifacts
Injection artifacts
intralesional injection of anaesthetic solution cause
- bleeding with extravasation
- separation of connective tissue bands with
This image shows separation of connective tissue
vacuolization bands due to intralesional injection of anesthetic solution

Squeeze artifacts
Form of tissue distortion from minimal compression
Includes crush, hemorrhage, splits, fragmentation and
pseudo-cysts
prevented by placing a suture within the mucosa that
is to be removed and hold its end by an artery forcep
This image shows hemorrhage occurring
during surgery
Prefixation artifacts
Fulguration artifacts
Occurs during electrosurgical or laser cutting of tissue –
results in a zone of thermal necrosis and tissue distortion
Prevented by using the cutting and not the coagulation
electrodes when obtaining the specimen This image shows fulguration artifacts

Starch artifact
A lubricant for surgical gloves
Appear as numerous blue, small, spherical structures
(hematoxylin and eosin stain)

This image shows starch artifacts in a tissue

Prefixation artifacts
Autolysis artifacts
show increased eosinophilia
increased binding of eosin to denatured
intracytoplasmic proteins
vacuoles in cytoplasm
nuclear changes - pyknosis

Improper Fixation
Solutions such as normal saline do not fix tissue
Show features of autolysis artifact
separation of epithelium from connective tissue
Fixation Artifacts
Fixation Artifacts
Formalin pigments
Heme and formalin bind to from formalin-heme
complex
Appears as brown-black amorphous to
microcrystalline granules
Removed by immersion in saturated picric acid
This image shows formalin pigments in tissue

Mercury pigments
Dark brown granular deposits
Removed by treatment in alcoholic iodine

Microwave fixation artifact

Underheating results in poor sectioning quality
Overheating produces vacuolation, overstained This image shows mercury pigments in tissue

cytoplasm and pyknotic nuclei

Fixation Artifacts
Osmolality of the fixative solution
Hypertonic fixatives - cellular shrinkage and
increased extracellular spaces
Isotonic and hypotonic fixatives - results in cell
swelling
Slightly hypertonic solutions are recommended

Freezing artifact
Appear as Swiss cheese holes in epithelium This image shows freezing artifacts in muscle tissue
(interstitial vacuoles along with vacuoles inside
the cell cytoplasm) and tissue spaces
Represent areas where ice crystals rupture the
tissue.
 Fixation artifacts
Streaming artifact
Diffusion of unfixed material to give false localization by
coming to rest in a place other than its original location
Mostly seen in formalin fixed tissue.
Prevented by using glycogen fixatives or by freeze drying
This image shows streaming artifacts in tissue
Ice-crystals artifacts
Results from:
1. slow freezing of tissue
2. inappropriate quenching techniques
3. tissue samples too large
Appears as intercellular clefts in highly cellular tissue and
intracellular clefts and vacuoles in skeletal muscle.
Carbondioxide expansion freezers are recommended for This image shows ice crystals in tissue

prevention
Artifacts related to bone
tissue
Artifacts related to bone tissue
Bone dust artifacts

Issue Remedy

Dust produced when ensure saw blades and drills are

undecalcified resin sections are sharp
cut trim blocks with a knife or razor
blade after decalcification

fragments deposited on cut

gently brush surface of blocks with
surfaces while others may be
soft brush after sawing to remove
implanted more deeply in
debris
specimen.
Artifacts related to bone tissue
Effect of incomplete decalcification
Damage to microtome knife
Damage to soft tissue surrounding calcified area when trimming paraffin block

This image shows bone trabeculae stained strongly with hematoxylin due incomplete decalcification
Artifacts related to bone tissue
Decalcification
Acid decalcifying fluids that hydrolyze the digestion of cellular and other
tissue components more rapidly when tissue is fixed or partially fixed

Issue Remedy

Impairment or loss of
basophilia
Inactivation of enzymes
Loss of iron and ribonucleic By fixation of specimen
acid
Destruction of tooth enamel
Artifacts related to bone tissue
Overdecalcification

Issue Remedy

Stains strongly with

Fixation of specimen
eosin
before decalcification
Show marked loss of
Radiographic check of
nuclear basophilia
progress and ensure
Nuclear and This image shows over decalcification in
complete decalcification a section of bone
cytoplasmic detail

poorly preserved
Fixation Artifacts
Tissue-processing Artifacts
Improper dehydration

Overtreament in high conc of alcohol - high degree of

shrinkage of tissue (shrinkage artifacts)
Overtreatment in lower dilution of alcohol - macerates the
tissue (vacuolization)
Paraffin unble to infiltrate properly - block too difficult to
cut results in tearing artifacts and holes
This image shows gastrointestinal
Remedy : rehydrate tissue section and repeat processing biopsy with incomplete dehydration

Improper clearing

Prolonged treatment in xylene - make tissue brittle,

crumble and crystalize during cutting
Not cleared properly in xylene - paraffin does not
impregnate, tissue becomes distorted during sectioning
.
Tissue-processing Artifacts
Improper embedding
Too long exposure during embedding - excessive hardening
which makes tissue friable and sectioning gives rise to cracks

Orientation artifact
This image shows tangential section of
Improper orientation - disorderly arranged histological
epithelium caused by improper orientation
features

Loss of soluble substances

preparation of paraffin wax, cellulose nitrate uses fat
solvents
fat dissolves from fat cells during processing of adipose
tissue and appear as ovoid spaces surrounded by a rim of
cytoplasm This image shows cholesterol clefts in
odontogenic cysts

Fixation Artifacts
 Artifacts related to microtomy
Scores and tearing in sections

Issue Remedy

using different part of

nick or blemish in the
knife or resharpening it
knife edge

This image shows scoring and tearing of

section due to nick in knife edge
Floater artifact

Pieces of tissue that appear on slides which do not belong there

- Tissue floated during processing
- From sloppy procedures on cutting bench using dirty towel,knife,gloves
- Improper cleaning of water bath
Artifacts related to microtomy
Moth eaten effect (holes from roughing)

Issue Remedy

Excessive rough
trimming of paraffin
blocks with greater Knife sharpened
thickness  regularly  This image shows tangential cut artifact
Void spaces or holes showing a hole

in thin sections

Artifacts related to microtomy

Alternate thick and thin sections

Issue Remedy

Wax too soft for

Cool block with ice 
tissue 

Block or blade is Tighten the block and

loose  blade  This image shows curling artifact due to
folding of tissue due to blunt microtome
knife
Artifacts related to microtomy
Chatters
Thick and thin zones that are parallel to the knife edge

Issue Remedy

Ensure knife is securely

Tiny vibrations in
clamped into its holder and
knife edge
the holder to microtome
This image shows venetian blind artifact due
to vibration of knife edge
Excessive steep knife Reduce clearance angle to
angle  minimum
Artifacts related to microtomy
Compression artifact

Issue Remedy

Blunt knife Sharpen the knife 

Wax too soft for Cool block with ice 

tissue of sectioning Use a higher melting
condition point wax
This image shows displacement of bone during
microtomy due to use of dull knife
 Artifacts related to
floatation and mounting
Artifacts related to floatation and mounting
Prolonged floating of sections on the water bath
May cause expansion of tissue beyond the
original size.
Give epithelium an acantholytic appearance
mimicking edema.

Folds and wrinkles in section

Occurs when extremely thin paraffin sections are
forced to stretch unevenly around other
structures with varying consistencies.
Appear as darker-stained strands. This image shows wrinkles and folds due to uneven
Can be removed by stretching or gently tapping stretching of tissue sections

with forcep.
Artifacts related to floatation and mounting

Contamination
Sometimes water bath may get contaminated
Can be reduced or prevented by regular cleaning of water surface and using
distilled water rather than tap water.

Air bubbles
Due collapsing of the air bubbles trapped beneath the sections
Use freshly boiled water in floatation bath for prevention.
Staining artifacts

Residual wax
Failed to remove the wax results in inadequate
staining of the section
Result in Pink disease aertifact

Artifacts related to addition of acetic acid to eosin

Undifferentiated completely homogenous and
flat deep pink‑stained
This image shows inadequate staining due to
residual wax
Staining artifacts
Artifact due to mordent of hematoxylin
Appearance of a black pigment
throughout the section.

Artifacts due to fluorescent sheen of of

hematoxylin
If the fluorescent sheen is not
properly removed;
In the finished slide, sheets or clumps
This image shows stain deposit due to formation of
of dye particles will be seen. fluorescent sheen in hematoxylin solution
Mounting artifacts
Residual water and air bubbles
formed under the coverslip when the mounting
medium is too thin.
use adequate thickness of mounting medium and
removal of air bubbles from under the slide.

Artifact related to excessive use of mounting media

Excessive use of mounting media
Gives a foggy appearance to the mounting media. This image shows air bubbles formed during
prevented by using adequate amount of mounting mounting procedure

media with proper consistency.

Mounting artifacts

Dry mounting artifact

Three distinct features of artifacts

a. Section may exhibit brown stippling which resembles pigment
b. Highly refractile lines outlining cells and tissue
c. Trapped air may be seen in the nuclei leading to dark lacking details.
 Classification of artifacts
Different types of artifacts

Conclusion found during different steps

in histopathology
Why artifacts occurs and how
we can minimize or prevent
them.
References
Taqi, S. A., Sami, S. A., Sami, L. B., & Zaki, S. A. (2018). A review of artifacts in
histopathology. Journal of Oral and Maxillofacial Pathology, 8.
Thank
You!