This document discusses various artifacts that can occur during biopsy and tissue processing. It defines an artifact as a defect or distortion resulting from how the tissue was handled from biopsy to fixation. Artifacts can occur during tissue manipulation, transport, fixation, processing, embedding and sectioning. Specific artifacts discussed include squeeze artifacts from improper forceps use, heat artifacts from electrosurgery, autolysis artifacts from delayed fixation, curling artifacts from thin specimens, and orientation artifacts from unlabeled specimens. Proper techniques such as gentle tissue handling, rapid fixation in adequate formalin volume, and specimen orientation labeling can prevent many artifacts.

1. Akhil s
Post graduate student
SMIDS
1

2. INTRODUCTION
Biopsy of the tissue helps in formulating diagnosis
by permitting study of both the cells and their
relationship to the surrounding tissues.
Biopsy provides the information regarding the benign
and malignant nature of the tumor.
2

3. The specimen removed from the oral cavity
are often small, the chances of producing
artifacts are greater.
Sometimes the state of the tissue is so
severe that the tissue is useless for the
histopathological diagnosis.
3

4. These includes errors by the surgeon or
assistant in handling the tissue at the time of
biopsy
problems in the transport of the tissue to
laboratory & faulty tissue processing.
4

5. • Some artefacts are easily distinguished from
normal or pathological tissue components, and
some are difficult to distinguish from such
entities.
5

6. CONTENTS
DEFINITION
ARTIFACTS DURING TISSUE MANIPULATION
INJECTION
FORCEPS
FULGuRATION
IMPROPER SURGICAL REMOVAL
ARTIFACTS DUE TO IMPROPER SPECIMEN TRANSPORT
ARTIFACT DUE TO IMPROPER FIXATION
FREEZE ARTIFACTS
CURLING ARTIFACTS 6

7.  ARTIFACTS DUE TO IMPROPER
ORIENTATION
ARTIFACTS DUE TO
IMPROPER EMBEDDING
ARTEFACTS DUE TO
UNDECALCIFIED BODIES
ARTEFACTS DUE TO SECTIONING
FAULT
ARTEFACTS DURING
HISTOCHEMICAL PROCEDURE
ARTIFICIAL DEPOSITS
FOREIGN BODY ARTEFACTS
7

8. Latin. Ars- art or skill, factum- made
(any artificial product)
DEFINITION
is a defect or distortion that occurs as a result
of the way the tissue was handled right from the
time of the biopsy taking to the fixation process
including problems in the transportation of the
tissue to the lab and the histotechnical procedure
used for embedding and staining.
8

9. Artefacts occur at each of the following stages in
the
• During handling of the tissue
• during the fixation of tissues,
• during processing, paraffin embedding and
microtomy,
• during the mounting of tissue sections onto glass
slides,
• staining procedures and coverslipping.
9

10. Use of gloves: Starch powder is used as a lubricant
of surgical gloves
• Oral cytological smear & tissue sections
• Refractile, glassy, polygonal bodies (5-20microns in
diameter)
• Exhibit central dot/ Y shaped structure
• H&E – light blue
• Lugol solution – blue-black
• PAS +ve – Lilac red & diastase resistant
Artifacts by surgeons
10

11. Starch granules resembles atypical epithelial cells.
 Spore like structure with dark central area can resemble a
pyknotic nuclei
Polarised microscope –”Maltese cross*” configuration
EM – spherical, faceted balls (2.5 – 30microns)
*- cross where arms look like arrowheads pointing inwards/
Garden flower having brilliant red 5 parted flower. 11

12. • Starch also produces clinically significant
diseases (granulomatous reaction)
• It produces acute postsurgical reaction possibly
mediated by HyperSensitivity mechanisms.
12

13. INJECTION ARTEFACTS:
Local anesthesia when injected
into the areas to be biopsied can
produce 2 major tissue changes.
Needle insertion may produce
hemorrhage with extravasation of
the blood which will mask the
normal cellular architecture.
ARTEFACTS DURING MANIPULATION OFTHETISSUES
13

14. LA injected rapidly may results in separation of the
epithelium & CT or there may be separation of CT
bands with vacuolization.
Hence LA should be injected slowly away from the
area to be biopsied.
14

15. 15

16. FORCEP / SQUEEZE / CRUSH ARTEFACTS
Most frequently encountered.
Occurs when the tissue have
been removed by using
toothed forceps or hemostat.
During surgical procedure the
tissue may be grasped with
excessive force during
removal.
16

17. When the teeth of the instruments penetrate the
tissue, results in voids/tears of the tissue along
with compression of the surrounding tissue.
The surface epithelium, may be forced through the
CT producing small pseudocyst/microcyst, which
distort the normal relationship of the tissue.
Compression of the tissue results in loss of
cytological detail with rupture of the nuclear and
cell membrane.
17

18. Excessive force with forceps – voids 18

19. Squeeze artifacts - pseudo cyst 19

20. Incorrect use of forceps -
pseudocysts 20

21. Tissue grasped with hemostat21

22. COMPRESION BY IMPROPER GRASP
22

23. Folded artefact
Cracked tissue artefact
Knife mark + folded arterfact
23

24. Crush artifact
At one margin the section shows darkly staining,
distorted cell nuclei, some obviously stretched out and
flattened, but maintaining an intense basophilia.
 Some are almost fiber-like. 24

25. Generally these squeeze artifacts are easily
recognized and there is no problem in,
arriving at proper diagnosis but in the
biopsies of the epithelial neoplasm there may
be problem in accurate diagnosis.
The problems can be avoided by handling the
tissue with a suture during incision.
25

26. The tissue forceps should be used gently for
handling the specimen.
If the use of the tissue forceps can’t be
avoided, Care must be taken to provide
adequate retraction and visualization of the
area
26

27. FULGURATION / HEAT ARTEFACTS
Electrosurgery has an advantage of producing
hemostasis at the biopsy site by coagulating
the severed blood vessel, this leads to a
profound artifactual alteration.
The heat produced may alter both the
epithelium and the connective tissue.
Microscopically: A broad band of basophilic
coagulum along the surgical margins giving
amorphous appearance.
27

28. The epithelium may have an appearance of the
detached surface and the nuclei assume a spindle
palisading configuration.
Separation of the epithelium from the basement
membrane.
Hard tissue biopsy should be removed with chisel
and not with surgical bur to avoid generation of
the heat which will damage the biopsy specimen.
28

29. Elongated
hyperchromatic
epithelium
29

30. Small biopsy – distorted by heat
– non- diagnostic
30

31. 31

32. Hence the use of the electrosurgical procedure should not be
encouraged.
 If at all done it should be limited to the larger specimen as the
artifacts can obscure the details in the smaller specimen.
Care should be, taken to use the cutting and not the coagulation
electrode, so that the low current will be produced that will
allow cutting & liberation of the specimen.
32

33. The margin of the incision should, be far
away from the interface of the lesion and
normal tissue to prevent thermal changes in
that area.
Accidental contact of cutting tip with the
metal instrument used to hold the specimen
should be avoided as this will also cause
tissue changes.
33

34. Electrosurgery, when used in combination with the
scalpel procedures better results were seen.
The scalpel is used for the initial incision
around the lesion to be biopsied and the
electrosurgery is used to complete the removal of
the specimen.
This method has an advantage of producing superior
hemostasis and also reduces the amount of the
heat, to which the specimen is exposed.
34

35. Ideally single electrosurgery incision should
be made with a thin electrode at a speed of
7mm/sec and the successive incision should be
separated by cooling intervals of 8- 10 sec
avoid generation of the heat sufficient to
initiate an adverse affect.
35

36. ARTEFACTS DURING IMPROPER
SURGICAL REMOVAL
Biopsy specimen if shallow and minute, correct evaluation of
the epithelium & its interrelationship with the underlying
connective tissue is impossible.
36

37. 37

38. Prevention:
Deeper elliptical incisions should be used
involving adjacent normal mucosa.
The clinician should know the history and
appearance, the basic type of the lesion with
which he is dealing and how the dimensions of
the specimen should be modified for each
clinical situation.
38

39. Example:
1. Pemphigus : A longer, broader and
shallow surface specimen should be taken
rather than deep, narrow and short specimen
2. SCC: A deeper incision should be taken
39

40. • Artifacts due to fixation occurs mainly due to:
Improper fixation
Delay in fixation
Use of inadequate volume of fixative
Use of inappropriate solution
Inadequate fixation time
ARTEFACTS DURING FIXATION
40

41. Adequate Fixation
41

42. Delay in fixation & inadequate
fixation time produces similar
changes
The specimen will lack the detail and loss of
cellularity due to autolysis.
The staining quality of the cells is altered
and the cell cytoplasm and nuclear structure
appears completely indistinct.
42

43. Fixation delayed 30 min – false hyperchromasia
Poor detail of fibroblast nuclei
Excellent indication - loss of detail of RBC
43

44. The cell appears shrunken showing clumping of the cytoplasm with
vacuole formation and there may be lysis of the cell membrane.
The nucleoli may not be seen.
SG interstitium – opaque & acellular
nuclei of individual units is not visible
44

45. Vascular, nervous & glandular structure shows loss of the
details which may give an impression of the scar formation.
In severe cases, the entire tissue undergoes autolysis and
appears fibrillar, eosinophilic and necrotic
Generation of fibrous component
Amorphous collagen bundles (scar formation)
45

46. Alcohol use results in poor staining of the
epithelium and improper fixation of the
connective tissue
 Water/saline fixative will alter the tissue
morphology and may result in an artifacts
simulating acantholytic disease (pemphigus)
making evaluation difficult.
Alcohol fixation –Acantholysis, hyperchromatic, disintegration of fibrous tissue
46

47. This slide shows areas which crystals once occupied but which
dissolved during fixation and processing.
The characteristic shape suggests cholesterol which is often
deposited as tapering needle-crystals. 47

48. 48

49. Autolysis Of Nuclei
(Inadequate Fixation)
49

50. Effect Of Osmolality Of
Fixative
Crenation of tissue due
to hypertonic fixative
Normally fixed tissue
50

51. • To avoid fixation artifacts, specimen should
be immediately fixed after removal in 10%
formalin and the volume should be
approximately 20 times the volume of the
specimen & should be kept overnight.
51

52. Tissue In Fixative
Fixative is only 5 times the
volume of the specimen.
Fixative is 20 times the
volume of the specimen. 52

53. ARTEFACTS DUE TO IMPROPER
SPECIMEN TRANSPORT
53

54. Leakage or evaporation of the
formalin can results if the container
is not sealed properly.
 If such tissue is again rehydrated
and placed in other container results
in an artifacts resembling
acantholysis.
54

55. FREEZE ARTIFACTS
Occurs if the tissue is frozen prior to the
fixation.
Specimen received through mail during the month
of winter results in freeze artifacts.
Freeze temp, of the aqueous formalin is 12.2o F &
hence the temp in the winter season will be
sufficiently cool to freeze the formalin.
55

56. Biopsy which are left overnight in the mail
box.
The water within the tissue may be freezed and
form ice crystals.
The processed freeze artifacts is characterized
by the formation of interstitial vacuoles and
also within the cytoplasm mainly in the areas
of the ice crystals. 56

57. This slide shows ice-crystal artifact which appears as
intercellular clefts in highly cellular tissues and as
intracellular clefts and vacuoles in skeletal muscle.
57

58. So during the transportation of the tissue the freezing
should be avoided
Use the container which provides insulation from the
extreme cold.
58

59. The tissue sometimes may twist by its own and get fixed in
this unnatural position, after being excised.
Particularly seen in the smaller or the thin specimen such
as in case of delicate strip of the oral mucosa or the
gingival specimen.
 Formalin fixation of the tissue causes shrinkage and
curling which results in difficultly in the orientation of
the tissue during the process of embedding.
CURLINGARTEFACTS
59

60. 60

61. 61

62. HOW TO PREVENT?
The thin and small specimen, place on the cardboard/
gauze to maintain its form before immersing it into the
formalin.
Non corrosive pins used to secure the tissue in the
position over the cardboard. This results in the proper
orientation of the tissue.
62

63. Curling is less problematic when the lesion have
relatively thick keratotic surface.
The keratin layer serve as a back bone enabling the
specimen to retain the shape during the fixation
63

64. Avoid:
Use of dry gauze to place unfixed tissue, because
this may cause dehydration of the tissue due to water
absorption from tissue.
64

65. Flat lesion with ulcerated, bloody or thin
epithelium.
 The specimen which has similar dimension without
any labelling can result in difficult orientation.
ARTEFACTS DUETO IMPROPERORIENTATION
65

66. Once the specimen
placed in the
formalin the entire
surface will be
darkened, it becomes
difficult to identify
the surface specially
when it is ulcerated.
66

67. This leads to improper
orientation.
Subsequently incorrect
sectioning i.e. instead
of cutting the section
perpendicularly to the
epithelial surface it may
be cut parallel to the
epithelial surface.
67

68. PREVENTION
Identify the sides with
label or suture tags.
Suture tags placed either on
the top, bottom or the side
of the specimen.
This should be accompanied
with diagrammatic
representation which shows
the pathologist the proper
surface. 68

69. Over Dehydration
Cracking of
tissue
69

70. Incomplete Dehydration
Bubbles in cells
70

71. ARTEFACTS DUE TO IMPROPER
EMBEDDING
71

72. Embedding
A. Orient tissue
1. cross section
2. longitudinal section
B. Dissection orientation
Plane of section effects
72

73. Procedure
1. Place tissue cassette
in melted paraffin
2. Fill mold with paraffin
3. Place tissue in mold
4. Allow to cool
73

74. Tissue distortation seen especially
in the hard tissue like bone/
teeth, due to improper orientation
of specimen to the microtome blade.
This will obviate the jumping that
may occur if the blade had to meet
the entire hard tissue.
In case of the tough epithelial
surface as in palms; the blade
should first cut the softer
connective tissue then epithelial
surface
74

75. Hard tissue within the specimen can alter the
quality of the section.
Undecalcified tissue may result in the tears
and rips while sectioning which may cause
problem in proper interpretation.
ARTEFACTS DUETO UNDECALCIFIED BODIES
75

76. Undecalcified bone left in the soft tissue
The rip in the tissue is unavoidable
76

77. Indication of the possibility of calcification in
the soft tissue should be made depending on the
case history form of the patient that accompanies
the specimen so that decalcification can be
carried out prior to the processing of the
specimen.
77

78. ARTEFACTS DUE TO FAULTY SECTIONING (Knife
marks)
Block
Knife
78

79. Sections may show tears or rips in the
vertical direction due to damaged microtome
knife.
Knife may show small nicks along the
cutting edge and it require sharpening
79

80. Poor sectioning
1. knife marks (scratches perpendicular to knife
edge)
80

81.  Sections may have thin/thick
zones i.e. the section with no
uniform thickness which may be
due to:
Presence of the hard tissue
which needs treatment with
softening agents (Blocks soaked
in 4% phenol)
Adjust the screws of the
microtome which may be not
tight.
81

82.  Sections may be compressed
due to:
1.Blunt knife
2.Section cut at a fast speed
3.Too soft wax
compression (waves parallel to knife edge) 82

83. This slide shows displacement of
a trabeculae of cancellous bone
which is lying over the marrow.
Displacement can be caused by:
A dull knife,
Excessively rough sectioning,
By using an embedding media
which is too soft for the
density of the tissue being cut,
By careless flotation technique.
83

84. All the artefacts can be avoided by using
thorough knowledge of all the equipments used,
its maintenance and use.
84

85. A. Artifacts deposits
1.Formalin pigment
2.Mercury pigment
3.Chromate deposits
4.Paraffin wax
5.Dust contamination
6.Rust contamination
B. Foreign body artifacts
ARTEFACTS OCCURING DURING HISTOCHEMICAL PROCEDURE
85

86. Artifacts deposits
The deposits due to some reagents used the
processing of the tissue or section with tissue
components.
86

87. Formalin pigment
Formation of the acid formaldehyde, haematin within the
tissue due to action of acid formaldehyde solution on the
hemoglobin
Seen as brown/brownish black deposit
Morphology may vary but commonly seen as microcrystalline
deposits i.e. anisotropic (birefringent)
Found in association:
Blood within the tissue
Most commonly in spleen, liver, lung, bone marrow
Areas of the hemorrhage
Along with blood vessel
87

88. Formalin Pigment
88

89. Avoided by:
• Treating the unstained tissue sections with
saturated alcoholic picric acid
• Alcoholic solution of both Na/K hydroxide (but
deleterious effect on staining)
• Treat with 10% ammonium hydroxide in 70%
alcohol for 5-15 minutes
• Change fixative on regular basis.
Prevention: By fixing the tissue in non acid
formaldehyde (buffered formalin),
89

90. Mercury pigment
When the tissue fixed in the solution
containing mercuric chloride.
Appears as dark brown or grey granules or
irregular masses which are distributed
throughout the tissue.
Removed by oxidation with iodine to mercuric
iodide with subsequently removed with sodium
thiosulphate.
90

91. Chromate deposits
Tissue fixed in chromate containing solution yield
an yellowish-brown to black precipitate with in
the tissue.
 To prevent this tissue should be thoroughly
washed in water after fixation to prevent reaction
between chrome salts with dehydrating alcohol.
Removed by treating sections with 1% HCL in 70%
alcohol for 30 min 91

92. Paraffin wax
 Paraffin wax sometimes are retained within the
tissue, particularly in the nuclei
This can be recognized by
Its position
Its refractile appearance
Its birefringence
Removed by treating in xylene 92

93. A section of skin where the superficial
epidermis and keratin have failed to
stain with both H&E because of
residual wax.
The final clearing of the section prior
to cover slipping has removed all
traces of the wax leaving no evidence
of the cause of the patchy staining.
93

94. Dust contamination: Seen in H &E section
Rust contamination: Can occur from the metal caps
94

95. Foreign body artifacts
Presence of foreign bodies makes interpretation difficult
Examples:
1. Cotton in the section resemble eosinophilic amyloid like substance
95

96. Silk sutures around which has formed a granulomatous reaction,
the so called 'stitch granuloma'. The sutures of course are
birefringent.
96

97. The section shows cellulose fibers which are
birefringent.
• Same section under polarised
light
97

98. ARTEFACTS DURING SECTIONING
98

99. RIBBON & CONSECUTIVE SECTIONS ARE CURVED
CAUSES Solutions
Leading & trailing edges of block
not parallel.
Trim until they are parallel
Knife blunt in one area Sharpen/ use different part of knife.
Surplus area of wax at one side Trim excess wax.
Tissue of varying consistency Reorient the block by turning block
through 90o & Cool block with ice
regularly.
99

100. ALTERNATE THIN AND THICK
SECTION
Problem Solution
Wax too soft for that tissue Cool wax with ice/ re-embed in
high- melting point of wax.
Block/knife loose Tighten
Insufficient clearance angle Increase angle
100

101. SPLIITING OF SECTIONS AT RIGHT
ANGLE TO KNIFE
Problem Solution
Nick in the knife edge Sharpen
Hard particles in tissue Calcium deposits
remove with
sharp scalpel
Hard particles in wax Re-embed in fresh
filtered wax
101

102. AREAS OF TISSUE NOT PRESENT IN
SECTION
Incomplete impregnation of
tissue
Return tissue to vacuum
impregnation bath for few
hours
102

103. SECTION ROLL INTO A TIGHT COIL
INSTEAD OF REMAINING FLAT ON
KNIFE
Knife blunt
Sharpen
103

104. SECTION EXPAND AND DISINTEGRATE
ON WATER SUFACE
1. Poor impregnation of tissue
2. Water temperature too high
Return tissue to vacuum impregnation
bath for few hours
Cool
104

105. Micrograph shows a circular deposit adjacent to a section and
which contained recognizable squamous cells, cell debris,
some bacteria and an amorphous background which stains
weakly with hematoxylin 105

106.  This deposit resulted from someone sneezing close to the section while it was
drying on a hotplate.
 Squamous cell contamination on sections is usually arises from the fingers or scalp
of the microtomist during flotation and less commonly from sneezes or coughs.
106

107. Excess albumin (stain) 107

108. Improper staining
Over staining,
Use proper concentration of staining .
solutions with proper timing
Under staining,
108

109. Staining
1. Inadequate rehydration (uneven staining)
2. Too dark or too light
3. Inadequate agitation
109

110. Mounting sections Folds & Tears
110

111. Improper mounting
Air bubbles, dust,
racked cover slip
Use fresh DPX with clean un-cracked cover slip
111

112. Improper Cover slip
Bubbles
112

113. Improper Cover slipping
2. Excess Permount
3. Two cover slips
113

114. If the mountant is not of
satisfactory quality it may break
down in time.
 This effect is usually evident
after about six months.
 This has occurred with the
mountant on this section which
has crystallized.
114

115. Guidelines to avoid
artifacts
1. Good clinical judgment in selecting best area for biopsy.
2. Submit sufficient tissue or whole specimen.
3. Handle tissue with care, without pulling/crushing the
specimen. Minute specimen should be placed on the
cardboard prior to fixation.
4. Not to contaminate tissue with foreign material
115

116. 5. Place specimen in the fixative immediately
after excision.
6. Label specimen properly with sufficient
clinical data to permit pathologist to return
with most definitive diagnosis
116

117. More than proper surgical technique is required to
facilitate the proper diagnosis of an oral biopsy specimen.
The proper preparation of the tissue for microscopic
analysis depend on steps taken by the surgeon, assistant
and histotechnician to reduce the inclusion of artifacts.
There are many ways that exact interpretation of tissue
specimen can be compromised.
117

118. REFERENCES
Cellular pathology technique by cullings. 4th ed
Theory and practice of histological techniques: Sheehan 2nd ed
Joseph E. “Artifacts in oral biopsy specimen”. J oral maxillofac surg: 1985. 43:
163-172:
Giuseppe Ficarra. “Artifacts created during oral biopsy procedure”. J cranio-max
surg. 1987. 15 : 35-38:
G.L. Lovas. “Starch artifacts in oral cytologic specimen” Oral Surg: August , 1985.
Elizabeth McInnes “Artefacts in histopathology” Comp Clin Path (2005) 13: 100–
108
118

119. • Chatterjee S. Artefacts in histopathology. J OralMaxillofac
Pathol 2014;18:111-6.
• Bindhu PR, Krishnapillai R, Thomas P, Jayanthi P. Facts
in artifacts. J Oral Maxillofac Pathol 2013;17:397-401.
• Kumar K,Shetty DC,Dua M.Biopsy and tissue processing
artifacts in oral mucosal tissues.Int J Head Neck Surg
2012:3(2):92-8.
119

120. 120