Facts in artifacts

INTRODUCTION
It is a fact that artifact is not a fact but
misinterpreted as a fact. It is derived from
the two words art and factum. Artifacts are a
common phenomenon encountered in a
variety of diagnostic procedures in
medicine. It is defined as being any structure
or feature that has been produced by the
processing of a tissue.[1] Artifacts may be
produced at any stage beginning from the
time of biopsy to the final stage of
mounting. These artifacts result in alteration
of normal morphologic or cytologic features,
[2] thus interfering or obscuring the
interpretation of histopathological diagnosis.
According to the stage at which they are
formed artifacts can be classified into
different categories as artifacts produced
during:

Surgical biopsy procedure

Fixation

Tissue processing

Embedding

Microtomy

Mounting

Staining

Cover-slipping.

BIOPSY PROCEDURE
A few of the artifacts encountered in
microscopic sections are caused by factors
related to surgical procedures. Intralesional
injection produces epithelial vacuolation,
connective
tissue
separation
and
extravasation of red blood cells.[3] This is

best avoided by administering anesthetics to

the area adjacent to the biopsy site.
Hemorrhage is a common complication
occurring during surgery. An inexperienced
histopathologist may interpret this for a
pathologic change [Figure 1a]. Pressure
marks on the surface of the biopsied
specimen can be produced when held by
forceps with excessive force prior to
fixation, referred to as crush or compression
artifacts.[4,5] These are usually seen at the
periphery of the lesion. Split artifacts are
produced by penetration of forceps into the
tissue, leaving gaps and compression zones
around tissues[4] [Figure 1b]. Crush/split
artifacts can be avoided by use of blunt
forceps rather than toothed forceps. In punch
biopsy, fragmentation artifacts are common
which may be attributed to the use of
scissors at the base of tissue for releasing it.
According to Meghna and Ahmed Mujib,
punch biopsy procedure produced fewer
artifacts when compared to biopsy by
scalpel.[6]
(a)
Hemorrhage
artifact.
(b)
Histopathological section showing split
artifact due to usage of sharp forceps during
biopsy procedure. (c) Coagulation of
proteins within the tissues as a result of
biopsy using laser. (d) Venetian blind artifact
produced vibration of tissue block/knife
Coagulation of proteins results from
dehydration of the tissues during biopsy
which may be caused either by cauterization
or by chemicals used in sterilization of
surgical instruments. Electrocautery in
parotid surgery causes oncocytoid changes
in the acinar cells.[7] Laser or electrosurgery
produces tissue distortion by inducing
carbonization, nuclear elongation and
vacuolar degeneration
Another commonly observed artifact in
microscopic tissue sections is entrapped

suture material. Presence of suture material

is of less significance in pathology but in
turn can damage microtome knives leading
to tears in sections.[8] Removal of suture
material prior to fixation is required. Gel
foam or surgical sponge is used to control
bleeding in various surgical procedures. The
presence of gel foam on a histological
section produces a characteristic appearance
of distorted spaces on the surface which may
be filled with blood surrounded by slightly
basophilic gelatin walls.[8]

FIXATION
Fixation is a process which attempts to
preserve the tissues in a life like condition
by preventing autolysis and putrefaction.
The volume of fixative should be 20 times
that of the specimen with thickness not
exceeding 6 mm.[9] Autolytic changes may
occur due to fresh tissues sticking together,
adherence of specimen to the inner surface
of the fixative container, inadequate amount
of fixative, thick tissue specimens, or
insufficient time spent in fixative.[1]
Inaccurate fixation produces shrinkage or
crenation with hypertonic saline and
swelling/bursting of cells with hypotonic
saline. Usage of a normal phosphate
buffered saline (PBS) based fixative corrects
such problems. Alcohol fixatives tend to
make tissue sections brittle resulting in
microtome
sectioning
artifacts
with
chattering and a venetian blind appearance
[Figure 1d].
Intraepithelial
cleft
formation
and
acantholysis occurs as a result of formation
of calcium carbonate residue due to formalin
evaporation from unsealed bottles.[1]
Acid formalin hematin, a dark brown
microcrystalline pigment is produced by the
reaction of formic acid (unbuffered
formalin) and heme molecule from
hemoglobin in an acidic pH. It is usually
found adjacent to erythrocytes in tissue
sections and may simulate microorganisms.
This can be confirmed by polarized light

microscopy and prevented by using neutral

buffered formalin or fixation in phenol
formalin.[9] This pigment can be removed
using saturated alcoholic picric acid
solution. Deposition of chrome oxide
pigments within tissues occurs with the use
of chrome fixatives.
Fall out of sections from slides during
staining procedures can occur as an after
effect of inadequate fixation. Over fixation
causes bleaching artifacts. Unfixed areas
within a tissue move and localize in some
place other than the original location
producing streaming artifact. This change
is seen associated with glycogen in
gluteraldehyde fixation, where there is
considerable loss and diffusion of glycogen
in tissues.[10] Generally fixation at room
temperature is sufficient to maintain
excellent morphological detail, but a rise in
temperature can increase the rate of fixation
(microwave oven with an optimum
temperature of 45-55C).[11] Under heating
produce poor sectioning quality and
overheating causes vacuolation, over-stained
cytoplasm and pyknotic nuclei.

GROSSING
PROCESSING

AND

Floaters or cross-contamination artifacts are

small pieces of tissue that appear on a slide
which do not belong to that particular area
and have floated in during grossing,
processing or floatation of cut-sections.
They may also arise from sloppy procedures
on the cutting bench such as dirty towels,
instruments or gloves that have remnants of
tissue that is carried over to the next case.
Therefore, it is essential to do only one
specimen at a time and clean thoroughly
before opening the container of the next
case. Thin and narrow tissue specimens tend
to curl during processing. This will cause
difficulty in orienting the tissue while
embedding leading to formation of
tangential sections

(a) Tangential section of epithelium caused

by improper orientation. (b) Thick and thin
section formed due to loosely attached
microtome knife. (c) Knife scoring appeared
in the section due to a small nick in the knife
edge. (d) Folds and wrinkles within the
histological section produced by a
blunt microtome knife

According to Panja et al., tissue processing

by microwave method produced the least
amount of tissue shrinkage. The usage of
noxious chemicals like formalin and xylene
is eliminated by this technique.[11,12] If the
automatic tissue processor is improperly
adjusted or there is a power failure, the
basket containing cassettes may remain
elevated resulting in dehydration of tissue
specimens by exposure to air. Excessive
dehydration will give the tissue a dry
homogenous appearance. This might also
cause cracking and excessive staining of
tissue sections.[1,13] Biopsy foam pads used
in embedding cassettes may produce gridlike/triangular-shaped artifacts resembling
vascular channels.[14]
Incomplete dehydration leads to water
entrapment within the tissues and cause
inadequate staining or opacity within the
section. This may be prevented by frequent
changing of processing solutions and
covering their containers to avoid moisture
contamination[1] Inadequate infiltration of
tissue with paraffin results in wrinkles that
run in all directions.[1] This occurs due to
faulty fixation, dehydration, clearing and
insufficient time in molten wax. Properly
fixed small tissues when processed with
long schedules, becomes excessively
shrunken, dry, brittle and difficult to cut.
They appear as overstained, fragmented or
crushed sections. Prevention is by using
short schedule of processing.[13]

EMBEDDING
Entrapment of air around the tissue is a
common finding during embedding. This
causes the tissue to fall out or vibrate during

the cutting procedure leading to venetian

blind artifact which appears as zones of
compressed tissue separated by open spaces.
[8] Embedding of multiple tissues having
variable consistencies in the same block can
produce artifacts.[1] Hydrophilic processing
fluids may be retained within the embedded
block of tissue and result in wrinkled tissue
sections. If the hardness of the embedding
medium is greater than the infiltrated tissue,
wrinkles or cracks appear in the tissue
sections. Use of soft wax or hard embedding
medium,
rapid
cooling
of
wax,
contamination
with
clearing
agent,
denaturation of wax and insufficient
dehydration results in tear artifacts.[13]

MICROTOMY/SECTIONI
NG ARTIFACTS
Thick and thin sections and chatter/venetian
blind artifact are formed as a result of
loosely attached microtome knife or tissue
block, steep angle of the cutting knife, hard
tissue or wax, and presence of calcification
in tissues[15] [Figure 2b]. Scratch lines
appear in the sections due to small nicks in
the knife edge, large knife clearance angle,
hard material embedded in the wax or hard
material in the tissue[15] [Figure 2c].
Crumbling of sections occur on cutting if the
knife is blunt or the wax is too soft or due to
contamination of wax with clearing agent or
water or due to slow cooling of wax[15]
[Figure 2d]. Loss of bevel on the knife edge
produces compression of the block which in
turn leads to formation of creases in the cut
sections.[15] Displacement of tissue
components
especially
bone
during
microtomy is a common finding in
association with the use of dull knife, soft
embedding medium, rough sectioning,
incorrect blotting and poor adhesion of
sections to the glass slide [Figure 3a].
(a) Displacement of bone during microtomy
in association with the use of dull knife. (b)
Microscopic section showing folding. (c)

Residual wax within the stained section. (d)

Stain deposits within salivary gland tissue

FLOATATION
MOUNTING

AND

Artifacts that appear in this stage include

contamination by microorganisms (fungi),
air borne fibers, hair, cellulose fibers,
floaters or bubbles beneath the sections.
Contamination by exfoliated squamous cells
is another common artifact caused by fingers
or sneezes/coughs.[13] Special care has to
be taken during processing and floatation in
order to avoid folding of microscopic tissue
sections [Figure 3b]. As the tissue sections
are flattened in the water bath, bubbles of air
may become trapped beneath them.
Collapsed bubble artifact occur due to
collapsing of air bubbles entrapped beneath
the sections leaving cracked areas when dry,
which fail to adhere to the glass slide
properly and show altered staining.[16]

STAINING
Failure to remove wax from sections
completely result in impairment of staining
known as residual wax artifact [Figure 3c].
Stain deposits may appear in sections if the
dye solutions are old or unfiltered [Figure
3d]. Eosin flakes, seen above the focal plane
of the tissue section, are precipitated dye
derived from an unfiltered stock solution.[1]
Drying up of sections between the last
xylene and cover slipping result in
entrapment of minute bubbles over the
nuclei leading to dark nuclei lacking visible
detail (corn flake artifact).[8] The presence
of water in the sections masks microscopic
detail and causes leaching of stains [Figure
4a and andb].b]. Washing eosin-stained
sections in tap water with an acidic pH leads
to leaching of the stain into mounting media.
This is more common where there is high
humidity and is due to atmospheric moisture
being absorbed by alcohols and particularly
xylene substitutes. If there is presence of
moisture still in the section after cover

slipping due to moisture in alcohols and

clearing agents, albeit in small amounts,
eosin will bleed from the section.[13]
(a and b) Section showing eosin leaching.
(c) Thin mounting media resulting in the
formation of air bubbles. (d) Section with
cracking of DPX (H&E stain, 100)

COVER SLIPPING
Bubbles may form under the cover slips if
the mounting media is too thin [Figure 4c].
Incorrectly prepared resin based mountants
tend to decay over time causing
crystallization and cracking of mounting
media [Figure 4d]. Bleaching of stain is an
unwanted outcome of prolonged exposure of
the sections to light. Hence, stained sections
should be stored in dark storing cabinets.
Presence of fingerprints can be avoided by
using slide holders. If mounting bench is
kept neat and tidy, unwanted elements like
debris, fibers or even fungi may be
prevented from contaminating the tissue
sections.[1]

ARTIFACTS
DIAGNOSIS

IN

Artifacts are well-known for their diagnostic

misinterpretation but not always. Few
artifacts have been proven as diagnostic
clues to histopathology.
Cholesterol clefts in radicular cysts or
periapical granuloma are produced as a
result of dissolution of lipids during
processing that leave behind needle like
spaces [Figure 5a]. Lacunar cells, the
diagnostic clue to nodular sclerosis, a
variant of Hodgkin's lymphoma is an artifact
induced by formalin fixation and absent with
other fixatives. These cells are formed by
retraction of cytoplasm towards the nuclear
membrane thus giving the appearance of
cells enclosed within lacunae.[17] Max
Joseph Space (Caspary Joseph Space)
associated with lichen planus is an

artifactual space in the subepithelial region

caused during processing and is attributed to
basal cell degeneration[18] [Figure 5b].
Formalin induced fluorescence can detect
melanin pigment in amelanotic melanoma
where melanin is not demonstrable in

routine hematoxylin and eosin (H and E)

section.[19]
(a) Clefts formed as a result of dissolution
of cholesterol crystals. (b) Max Joseph space
formed as a result of basilar degeneration
(H&E stain, 100)