The document discusses various types of artefacts that can occur during tissue processing and slide preparation in histopathology. Artefacts are non-natural structures that are introduced and can compromise accurate diagnosis. They can occur during biopsy collection, fixation, processing, embedding, sectioning and staining. Examples include crush artefacts, thermal injury, surgical procedures, delayed fixation, contamination and diffusion of unfixed material. Care must be taken at each step and troubleshooting methods are provided to prevent or reduce artefacts.

1. NAMRATHA R
ARTEFACTS

2. DEFINITION
 An artefact (Latin ‘ars’- art + ‘factum’- made) in
histology means any non-natural feature or
structure accidentally introduced into
something being observed or studied
 According to Bernstein, Artefact refers to “An
artificial structure or tissue alteration on a
prepared microscopic slide caused by some
extraneous factors

3. RELEVANCE
 Confusion with normal structure or pathological
change
 Compromise accurate diagnosis
 Alteration of normal morphological and cytological
features and hiding structures
 May even lead to complete uselessness of the tissue,
thus creating serious errors and misdiagnosis of
correct histopathological impression
 Minor - involving only small portion of the specimen ,
do not interfere with an accurate diagnosis
 Excessive or may involve the entire specimen-
useless for diagnostic purposes

4.  From the time of biopsy to the final stage of
mounting.
 Surgical biopsy procedure
 Fixation
 Tissue processing
 Embedding
 Microtomy
 Mounting
 Staining
 Cover-slipping.

5. Pre fixation artefacts
 These are features and structures that have been
introduced prior to the collection of the tissues.
 Surgical procedure or handling prior to dissection
 Physical and chemical

6. BIOPSY PROCEDURE
 Pressure marks on the surface of the biopsied
specimen- Forceps with excessive force - crush or
compression artifacts
 Distorted tissue with scalloped serrations (produced
by beaks of the forceps)
 Crushed cells, appear as dark chromatin strands and
may give a false diagnosis of dysplastic lesions.
 Split artefacts occur on the surface and at the side of
the lesion due to scalpel or foceps which causes
multiple cuts in the tissue.
 This artefact may result in a split between epithelium
and connective tissue, giving a false impression of
vesiculo – bullous lesions.

7. CRUSH ARTEFACT
 Crush artifact is artificial elongation and distortion
in cells or tissue
 Tissue without supporting fibrous stroma
 Small cell carcinoma and lymphoma, thymoma,
olfactory neuroblastoma,
 Cells with high NC ratio. Elongated streaming
basophilic material, Nuclei pushed into each other
 Spillage of cytoplasmic contents into stroma
 Edges of sections and Small biopsies

8. Trouble shooting:
Use small atraumatic blunt forceps or Adson's
forceps without teeth
A suture should be placed in one edge of the specimen
(substitute for forceps for tissue immobilization)

9. AGONAL CHANGES
 The longer the agonal interval in fixation, the more
mitoses apparently progress to completion.
 Difficulty in recognizing mitoses
 Anoxia - release of hydrolytic enzymes from
cytoplasmic lysosomes
 The hydrolytic enzymes digest the cells, details are
lacking when they are seen under the microscope
 Autolyzed tissues - nuclear pyknosis, karyolysis
and karyorrhexis, cytoplasmic vacuolation and
disintegration of tissue structure.
 Storage of tissues at 40 C, but they can be
completely avoided by rapid fixation.

10. CURLING ARTEFACT- Incisional
biopsies
 Small delicate strip of oral
mucosa, the shrinking process
caused by formalin fixation
 Curling and bending of the tissue,
thus making its correct orientation
during difficult .
 TS: Specimen placed with its
mucosal surface up on a piece
of the sterile paper (usually that
which held the suture material)
and allowed to remain unfixed
for some time while the incision
is being sutured
 Adequate depth of the
specimens can prevent this
artefact

11.  Pseudomicrocysts- lined with surface epithelium,
forced inward by the teeth of the instrument,
along with compressed surrounding connective
tissue, thus making evaluation impossible
 Intralesional injection of anaesthetic solution -
haemorrhage with extravasation and separation
of connective tissue bands with vacuolization

13. THERMAL INJURY
 Iatrogenic- Electric cautery
 Laser Beam
 Infiltration with over heated wax
 Embedding using over heated forceps
 Drying section mounted slides on hot plate or
incubator
 LEEP- Large thermal artefact zone-
uninterpretable margins

14.  Dehydration and denaturation of protein – coagulation
and condensation
 Increased acidophilia and amorphous appearance to
the epithelium and connective tissue.
 The epithelium cells appear detached
 nuclei assume a spindled, palisading configuration.
 separation of the epithelium from the basement
membrane
 Loss of nuclear and cytoplasmic detail
 Alternating zones of coagulation - "Herring bone"
appearance.
 Connective tissue- coagulated
 Glandular tissue- vacuolated
Electrocautery in parotid surgery causes
oncocytoid changes in the acinar cells

16. TROUBLE SHOOTING
 This can be prevented by using the cutting and
not the coagulation electrodes when obtaining
the specimen, so that low milliampere current is
produced, that will allow cutting and liberation of
the specimen
 Use of electrocautery is contra-indicated as
biopsy technique
 The incision margin should be adequaltely away
from the interface of the lesion

17. SURGICAL SUCTION
 Negative pressure of suction pulls air and
mobilises tissue fluids
 Gross- high density tissue with visible air
bubbles
 Micro- Extracellular round spaces with fluid
 Oral tissue- Basophilic material with acid
mucopolysaccharides- Myxoid material around
dental follicles
 Nuclei may line up around spaces
 DDX- Vascular and glandular spaces
 Soft tissue emphysema, Pneumotosis intestinalis,
oedema

18. RADIATION
 Dosage dependant
 High dosages- Cell death
 Small dosages- Acute
 Intermediate
 Chronic

19. RADIATION ARTEFACT
 Acute
1. Tissue oedema
2. Endothelial proliferation
3. Swelling of cells
4. Proliferation of collagen
fibres
5. Ischemic effects
 Intermediate-
Regeneration
1. Fibrosis and
pleomorphic fibroblasts
 Chronic
1. Thin and depigmented
epithelium
2. Dense fibrosis with
pleomorphic fibroblasts
3. Prominent vascularity
4. Multinucleate giant cells
5. Pleomorphic epithelial
cells
Hyperchromasia,
irregular nuclear con
tours,
6. Macronucleoli
7. Vaculoisation of
cytoplasm
8. Normal N/C ration

20. CHEMICAL ARTEFACTS
Chemotherapy
 Lung- Interstitial fibrosis, type II pneumocyte
hyperplasia, Hyaline membrane formation
 Liver- Necrosis, fibrosis, Cholestasis,
granuloas, vascular changes, GIT and Skin
 shrunken and separated from each other, Loss of
cellular and nuclear detail, Hypereosinophila and
vacuolation, Nuclear pyknosis

21. MONSEL’S SOLUTION
 Hemostatic agent to
control bleeding –
skin/ mucosal biopsy
 Coagulation and
necrosis upto a
depth of 0.6mm
 Ferric sulphate –
macrophages with
granular material
 General basophilia,
partial
desquamation,
cracking of
epithelium and
necrosis
 Restitution stain(0.5%
HCL and 70/5 Ethanol
at 60 degrees
improves image
quality

22. Steroids
 Intra articular,soft
tissue,nasal
 Steroid granulomas
 Bubbly basophilic
material surrounded
by histiocytes
 Birefringent
crystalline particles
 DDx - Rhematoid
granuloma
 Infectious granulomas

23. MYOSPHERULOSIS
 Sac like structures
containing
endobodies- Nasal
specimens
 Hemostatic packing with
petroleum based
ointment
 30-100micromm
 Foreign body-
type granulomatous
reaction to lipid-
containing material
and blood
 DDx - Protothecosis

25. Spironolactone bodies
 Adrenal gland
following therapy
 In cytoplasm of cells
of Z. glomerulosa or
fasciculata
 Rounded laminated
eosinophilic bodies
 2-25µm
 Derivatives of ER

26. MEDICAL BIOMATERIALS
 Biological substances- Cellulose, collagen,
fibrin, silk
 Synthetic polymers- Oriented molecular structure-
Birefringent,orange to red with Sudan stain
 Dyes
 Minerals and metals
 Phagocytosis, proteolysis and hydrolysis-
Absorbtion
 Fragmentation- Foreign body reaction,
Calcification, infective nidus, embolus

27. SUTURES
 Isolated fragments or
complete fibre
bundles- transverse/
oblique/ longtitudinal
 Can damage the
microtome knife-
tears and knife lines
in sections

28.  Silk sutures- strong
birefringence under
polarised light
 Silk sutures around
which has formed a
granulomatous
reaction, the so
called 'stitch
granuloma'.

29. Cellulose
contamination
 Luminal surface of
GIT
 Shredding of sections
 Plant cells with
strongly staing walls
and square shape
 Can be mechanically
implanted during
dissection

30. Gelfilm/Gelfoam- absorbable gelatin
 Thin film/sponge- used
to control bleeding
 Sections adhering to
specimen surface
 Slightly basophilic
walls of varying
thickness surrounding
distorted spaces
containing blood or
cells
 No tissue reaction
 Surround distorted
spaces with blood

31. Starch
- A powder in surgical gloves , contaminate body
tissues during an operation.
- Difficult to see in sections under normal bright
field microscopy.
 Spherical to slightly angular (hexagonal), very pale
in H&E with small central dark spot.
 Refractile, glassy, polygonal, PAS +ve bodies,
generally 5-20 mm in diameter.
 Light blue on H and E staining and deep liliac-red with
PAS.
 Maltose cross birefringence under polarized
 Starch artefacts can be prevented by the alternate
use of rubber gloves

32. STARCH
CONTAMINATION

33.  Foam pads to prevent escape of small specimens
 A fine pattern of local pattern or molding- pore
structure of foam
 Troubleshooting- Lens or gauze paper by
allowing the tissue to fix before placing it into the
sponge pad because after fixation the surface is
firmer and less prone to indentation.

34. 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.
 Fixation Artefacts: During fixation, tissues
commonly change in volume.
 Inhibition of respiration,
 Changes in membrane permeability
 Changes in ion transport through the
membranes

35. PROLONGED FIXATION
 Prolonged fixation in formalin - secondary
shrinkage.
 Inaccurate fixation- Shrinkage or crenation with
hypertonic saline and swelling/bursting of cells with
hypotonic saline.
 Normal phosphate buffered saline (PBS) based
fixative corrects such problems.
 Alcohol fixatives - tissue sections brittle resulting
in microtome sectioning artifacts with chattering
and a “venetian blind” appearance
 Intraepithelial cleft formation and acantholysis
occurs as a result of formation of calcium carbonate
residue due to formalin evaporation from
unsealed bottles.

36. TROUBLE SHOOTING
 Dewax the section
↓
 Transfer to absolute alcohol
↓
 Place the slide in Coplin jar containing picric acid
solution for 48 hrs
↓
 Wash in 90% alcohol
↓
 Wash in 70% alcohol
↓
 Wash in water

37. TISSUE PROCESSING
ARTEFACT
 Inadequate or incomplete fixation
Vessel shrinkage in CNS.
 Perivascular shrinkage in nerve tissues
 Perfusion fixation and gentle prolonged
processing
 Chloroform as a clearing agent
 Celloidin paraffin double embedding

38. DELAYED FIXATION
 Fixation in formalin and embedded in paraffin
shrink by 33%.
 Delayed fixation- Cell shrinkage and cytoplasmic
clustering.
 The nuclear chromatin cannot be distinguished
and the nucleoli are sometimes not visualized.
 Vascular structures, nerves and glands show a
loss of detail and an impression of scarring or
loss of cellularity is seen

40. TROUBLE SHOOT
 Fix specimen immediately in 10% formalin solution as soon as
the tissue is removed.
 Dewax the section
↓
 Transfer to 70% absolute alcohol
↓
 Immerse in iodine solution for 3 min with continous
agitation
↓
 Wash in water
↓
 Immerse in sodium thiosulfate solution until the yellow color
of iodine is completely removed
↓
 Wash in water

41. ZONAL FIXATION
 Large tissues surrounded by a capsule and
rapidly degenerating tissue(glandular tissue)
 Fixative penetrates slowly- different degrees of
fixation at different levels within tissue
 Insufficient time for fixation, too large a specimen
and reagent with poor penetration rate

42. Diffusion of unfixed material
 Rest in places other than
their original locations and
are most commonly seen
in glycogen.
 This can be prevented by
using smaller blocks (Reale
and Luciano 1970) or
stronger fixatives for larger
bits.
 Fixation of tissue for
glycogen should be
prompt, as there is an
initial sharp loss of
glycogen postmortem and
it should be carried out at
40 C in 80% alcohol or in
Rossman’s solution.
 Troubleshooting- Glycogen
fixatives- formal alcohol and
Bouin

43. DIFFUSION ARTEFACTS
 Small molecules like inorganic ions and biogenic
amines can be lost from tissues
 chromogranin, in case of adrenaline and nor
adrenaline.
 This can be demonstrated by placing the
adrenals in iodate.
 The catecholamines can be seen leaving the
tissue as a red cloud of aminochromes.
 Generated can only be retained by precipitation
 Can be prevented by proper fixation for accurate
localization and also by preventing the leaching of
ions from the tissue

44. Formalin pigment artefact
 As solutions age, formic acid developes from the
formaldehyde content- lowers the pH.
 Causes crystals of formalin pigment, or acid
formaldehyde hematin tissues.
 Acid formalin+ Haemoglobin=Acid
formaldehyde haematin
 Black to brown finely granular birefringent
deposit in the vicinty of RBCs
 Tissues richly vascular- Spleen, lymph node
 Occur in nearly all tissues eventually, especially
when they are stored in those solutions for
extended periods.

45.  The pigment is birefringent in polarized light
and will appear as numerous bright white motes
on the slide.
 Troubleshooting:
 Bring sections to water via xylene and ethanol.
 Place into saturated picric acid in absolute
ethanol for 1 hour.
 Optionally, treat with saturated aqueous lithium
carbonate to remove picric acid discolouration.
 Wash well with water.
 Continue with the staining method.

46. Trouble shooting
 Prevented by using 10% neutral buffered
formalin (NBF) or phenol formalin as the
fixative.
 The NBF should be changed every six months at
a minimm
 If NBF is not available and either 10% formalin or
10% formal saline are being used, they should be
stored over marble chips or crushed oyster
shells to neutralise any formic acid as it is
produced.

47. Mercuric chloride artefact
 Seen with fixatives containing mercuric
chloride.
 Crystalline or amorphous greenish-brown
artefact pigment of mercury is randomly
deposited in tissues.
 Treatment of specimens with iodine
(Lugol’s iodine) during processing or
sections prior to staining, will produce
mercuric iodide which can be washed out of
the tissues.
 A subsequent treatment with sodium
thiosulphate then removes residual iodine.

48.  Dewax the section
↓
 Transfer to 70% absolute
alcohol
↓
 Immerse in iodine solution
for 3 min with continous
agitation
↓
 Wash in water
↓
 Immerse in sodium
thiosulfate solution until the
yellow color of iodine is
completely removed
↓
 Wash in water

49. FREEZING DAMAGE
 Freezing during transport before fixation also
causes cytoplasmic condensation and it
occurs secondary to cell dehydration
 After frozen section- thaw the block in fixative and
process to paraffin
 Ice crystal damage and freeze thaw changes
 Nuclei surrounded by a free space
 Smaller size with darker chromatin
 Nuclear and cytoplasmic detail not well
defined

50.  Ice crystal artefact
 Clefts and vacuoles in
the central region of
the specimen
 Frozen section chatter

51. TROUBLE SHOOTING
 Due to slow freezing of tissue
● Solution: Freeze fast (flash/snap); the faster
the freeze, the smaller the ice crystals, the less
tissue damage (best freezing method is arguably
liquid nitrogen)
● Smaller tissues yield less artifact - optimally
tissue should be 0.5 x 0.5 x 0.3 cm or less
● Never freeze fragments larger than the
diameter of the chuck
● Avoid freezing fat around tissue
● Blot the outer surface of the tissue dry with
gauze before making your block

52. UNDER AND OVER HEATING
 Optimum temperature for microwave fixation is
45- 550C.
 Underheating results in poor sectioning quality,
whereas overheating above 650C produces
vacuolization,
 Overstained cytoplasm and pyknotic nuclei .
 The mechanism whereby microwaves bring
about tissue stabilization involves protein
denaturation.
 The time taken for IHC and in - situ
hybridization can be significantly decreased

53.  Chemical changes can also lead to artefacts.
 Gluteraldehyde used to fix tissues will add
carbonyl groups to tissues in which they were not
present and these groups will react with
Schiff’s reagent
 This can be overcome by using Bouin’s fixation
medium for the storage of specimens.

54. GROSSING
 Floaters or cross-contamination artifacts - 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.
 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.
 One cassette to another during processing
 During section embedding via contaminated
forceps,molds, hot plates,casette covers
 Via section flotation baths
 During staining – cells are shed from section and
smear onto another slide

55. Troubleshoot
 Floater artefact may be suspected if
 A tissue fragment looks different from others by
virtue of section thickness and/or staining intensity
 A tissue fragment is on a slightly different plane
from others, especially if superimposed
 A tissue fragment showing pathologic changes
totally different from others and of a type that one
would not have expected at all under the clinical
circumstances of the particular case.
 Thorough rinsing of board and instruments
between specimens
 Flotation baths skimmed samples

56. PROCESSING ARTEFACTS
Poor processing
 Dehydration is the first step in processing - removal of
aqueous fixative fluids from the tissue by using
compounds like alcohol
 Clearing is replacing the dehydrating agent with fluid
that is miscible with dehydrating fluid and embedding
medium
 Tissues immersed in too great a concentration of
alcohol will usually show a high degree of shrinkage
due to rapid removal of water. These are referred to
as shrinkage artefacts.
 Troubleshoot: After fixation, tissue needs to be
dehydrated slowly. Starting with 50% alcohol can
prevent this artefact

57. PROCESSING ARTEFACTS
 Tissue placed in acetone – a dehydrating agent, for a
prolonged period of time tissue – brittle affecting
subsequent sectioning.
 Prolonged immersion in clearing agent also renders
the tissues brittle - crumbling and crystallization of
tissues during cutting.
 Use the proper amount of clearing agent and no
clearing agent should be left behind to contaminate
the wax.
 Incomplete dehydration - water entrapment within the
tissues and cause inadequate staining or opacity
within the section.
 Frequent changing of processing solutions and
covering their containers to avoid moisture
contamination
 Inadequate infiltration of tissue with paraffin results in
wrinkles that run in all directions.

58. Artifacts during impregnation
 The function of wax impregnation is to remove
clearing agent (wax solvent) from the tissue and for
them to be completely permeated by the paraffin wax
which is subsequently allowed to harden to produce a
block from which sections may be cut.
 The artifact produced during this procedure is
Crystallization:
 Thicker the tissue the more clearing agent it will carry
and hence it requires more change of wax to be
removed it.
 Even if a small amount of clearing agents
contaminates the wax it will lead to crumbling and
crystallization of tissue during cutting.

59. LOSS OF SOLUBLE
SUBSTANCES
 Cholesterol- tapering needle like crystals in
vessel wall- atheroma and old haemorrhage
 Dissolve – characteristic space
 Neutral lipid from adipose cells- ovoid spaces

60. ARTEFACTS OF 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. .
 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.

61. ARTEFACTS OF MICROTOMY
 Microtomy, the means by which tissues are
sectioned, so that microscopic examination is
possible, involves some artefacts that can get
incorporated if proper technique is not followed

62. Venetian blind effect
 Fine parallel cracks in section
 Tiny vibrations in the knife as it passses hard
brittle blocks.
 Disposable bades are not supported in the holder.
 Steep angle of the cutting knife, hard tissue or
wax, and presence of calcification in tissues
 TS: Cutting thinner sections
 Sharpening the knife before cutting.

64. Knife lines- Scores in
sections in the
direction of cutting due
to a damaged knife
edge.
If the tissue is cut
tangentially, the connective
tissue cores may become
entrapped within the
epithelium, giving a false
impression of invasive
squamous cell carcinoma

65.  Alternate thick and
thin sections - the wax
is too soft for tissue,
block or blade is loose,
clearance angle is
insufficient or
mechanism of
microtome is faulty.
 TS: Cooling the block,
tightening the block or
blade and increasing
the clearance angle.
 Wrinkles and folding
of tissue sections -
very thin paraffin
sections are forced to
stretch unevenly around
other structures which
have different
consistencies.
 TS: Removed by gentle
teasing with forceps
or by transferring the
sections from the
slide to another water
bath at high

66.  Scratch lines - small nicks in the knife edge,
large knife clearance angle, hard material
embedded in the wax or hard material in the
tissue
 Crumbling of sections - 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
 Displacement of tissue components -
especially bone - use of dull knife, soft
embedding medium, rough sectioning, incorrect
blotting and poor adhesion of sections to the
glass slide

67.  COARSE CHATTER
 Cutting dense
tissue(uterus/ cervix in
large blocks)
 Movement of holder or
specimen or knife-
Vibration in the section.
 Poorly designed
microtome
 Chatters or chaffers
are thick or thin zones
parallel to knife edge
as it cuts the tissue
 narrow parallel bands,
usually evenly spaced
across the tissue
specimen
 TS: Suitable
microtome
 Blocks of a sensible
size
 , changing the
orientation and soaking
the block face with
detergent or water.
HOLES FROM
ROUGHING
 Lymph nodes, very
cellular organs
 Excessively rough
trimming

69. (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

70.  Artifacts during lifting of tissue sections:
 Artifacts such as Tissue Folds produce when
tissue adheres to the under surface of the blade,
seen most commonly with fatty tissues and
mainly seen due to dull blade
 TS: transferring the sections to a new water bath
or by passing light of Bunsen burner over the
section and
 adding small amount of detergent may be helpful.
Tissue

71. ARTEFACTS OF FLOTATIONAND
MOUNTING
 After sectioning and
mounting.
 Section dropped rather
than pulled across the
water.
 Freshly boiled water less
likely to produce artefact.
 Air bubbles trapped in a
section after flotation and
mounting can collapse on
drying ,leaving zones
which cracks and fails to
adhere properly to the
slide
 Increase temperature of
water bath results into
expansion of tissue
beyond its limit and
“Parched Earth (crackes)”
 Contamination by
microorganisms (fungi),
air borne fibers, hair,
cellulose fibers, floaters or
bubbles bene
 Contamination by
exfoliated squamous cells
is another common
artifact caused by fingers
or sneezes/coughs.

73. Residual wax artefact-
prevent penetration of
both aqueous and
alcoholic dye solutions -
area totally devoid of
stain, nuclei - muddy and
lack detail
Xylene treatment
and re-staining
Stain deposits may appear
in sections if the dye
solutions are old or
unfiltered, undisolved stain,
open racks
Replace staining
solutions, stain in
closed vertical jars
Eosin flakes- Above the focal plane of the tissue
section- precipitated dye derived from an unfiltered
stock solution.
Drying up of sections between the last xylene and
cover slipping - entrapment of minute bubbles over
the nuclei leading to dark nuclei lacking visible

75.  Incomplete staining- Automated staining
machines
 Leaching of substances from tissues into the
dye -- weak staining of calcium by alizarin red S,
resulting from loss of calcium ions into aqueous
fixative
 Leaching of the stain into mounting media
Washing eosin-stained sections in tap water with
an acidic pH-
 Eosin bleed - If there is presence of moisture still
in the section after cover slipping due to moisture
in alcohols and clearing agents.

76. Section showing eosin
leaching.

77.  Contamination by microorganisms- Regular
replacement
 TS: Preservative- Thymol or merthiolate
 Mucous contamination- Saliva- Failure to
adequately wash after incubation with saliva-
strong PAS staining with residual mucus
 TS: Diluting with water or saline, comercial
lyophilised diastase preparations

78. ARTEFACTS OF COVER
SLIPPING
 Stained sections are protected from damage by the
application of cover-glasses with appropriate
mounting media.
 Bubbles are formed under the cover- slip when the
mounting medium is too thick.
 TS: A clearing agent is compatible with the
mounting medium to be used because some
solvent may be carried over to the mounting
stage.
 Mounting medium of adequate thickness
 Use adequate amount of mounting media
 If the mounting media has attained greater
viscosity, xylene can be used as a thinning agent

79. COVER SLIPPING
 Bleaching of stain is an unwanted outcome of
prolonged exposure of the sections to light.
 TS: Stained sections should be stored in dark
storing cabinets

80. ARTEFACTS OF BONE
 Reprecipitation
artefacts are caused by
lack of agitation and
inadequate volume of
decalcifying fluid.
 They are seen as round
granules or crystalline
masses which lie mainly
in the soft tissues and
bone marrow.,
secondarily calcium
phosphate and it
stains strongly with
alum hematoxylin
 Overdecalcification
may hamper cutting
qualities, affects
staining properties
and histological
details are destroyed.
 Neutralizing the bone
section by 1%
aqueous solution of
lithium carbonate
 Surface
decalcification of the
paraffin block can rectify
incomplete
decalcification

81. DIAGNOSTIC
ARTEFACTS
 Crush artefact in small
cell carcinoma-
Nuclear molding and
azzopardi effect
 Ground glass orphan
annie nuclei
 An artefact of paraffin
embedded tissue

82.  Fried egg artefact-
Oligodendroglioma,
hairy cell leukemia
and chromophobe
RCC- Autolytic
imbibation of water-
Delayed fixation. Not
seen in FS.
 Lacunar cell of NS
HL- Formalin
fixation artefact

83.  Peritumoural
retraction artefact in
BCC
 Loss of bullous
pemphigoid antigen.
 Differentiates from
SCC,
Trichoepithelioma
 Tigroid backround
FNA of seminoma-
Fraagile cytoplas.
Lace like background.

84.  Formalin induced
flouresence-
Amelanotic
melanoma-biogenic
amines, including
DOPA and dopamine,
to show fluorescence
after exposure to
formaldehyde
(formalin-induced
fluorescence).
 Max Joseph space-
basal cell

85. summary
 Pre fixation artefacts- Crush, split, agonal
changes and pseudomicrocyst
 Thermal
 Suction
 Chemical- Chemo,Monsels solution,
myospherulosis, sutures, gelfoam, starch

86. FIXATION
 Delayed, inadequate and prolonged fixation
 Zonal fixation and diffusion/streaming
 Pigment artefacts
 Freezing and heating artefacts
 Processing artefacts
 Emedding artefacts
 Artefacts of microtomy- Venetian blind,chatter, folds,
rough holing
 Staining artefacts- Wax artefact, Staind deposits,
Eosin flakes, Cornflake artefact
 Mounting and coverslippind artefacts- Bubbles,
Bleaching
 Artefacts of bone
 Diagnostic artefacts

87. THANK YOU