1. Presented by
Dr.Vimi Jain
Oral And Maxillofacial Surgery

2. OUTLINE
• Introduction
• Definitions
• Historical perspective
• Indications & Contraindications
• Objectives
• Classification
• Various biopsy procedures
• Special considerations
• Essential biopsy principles

3. • Stains
• Errors
• Biopsy artefacts
• Complications
• Biopsy results : WHAT IF?
• Situation in Sub-region
• Conclusion
• References

4. INTRODUCTION
The diagnosis of any pathology depends
• History
• Examination
• Laboratory studies
• Biopsy
• Other diagnostic technique

5. Biopsy is essential for:
• The definitive diagnosis and treatment planning
• To determine the prognosis in cases of malignancy
• Provides an indication towards the response of the
tumor to therapy (e.g., relative resistance of malignant
melanoma to radiotherapy)
• The purpose of record and research work.

6. • Aim of Biopsy should be to provide a suitably
representative sample for the pathologist to
interpret, while minimizing perioperative discomfort
for the patient.

7. DEFINITION OF BIOPSY
• Biopsy is the removal of tissue for
examination, microscopic analysis, chemical
analysis, and bacterial analysis or a
combination of all four. The term is used most
frequently to indicate removal of tissue from a
living subject for analysis.
-Tiecke RW in 1965

8. HISTORICAL PERSPECTIVE
• 1870, Ruge and Joham Vert in Berlin introduced surgical
biopsy as an essential tool for diagnosis.
• 1889, Emarch put forward an argument that
confirmations should be made before surgeries for
malignancies.
• Williams halsted 1st introduced this principle in United
States.

9. • This was adapted to study cells from other body
systems
• Along with this were innovations in various kinds
of tissue preparations and staining techniques
• 1941, study of exfoliated cells from female genital
tract by Papanicolaou

10. INDICATIONS FOR BIOPSY
• When an inflammatory lesion is not responding to
conservative therapy after 10 to 14 days.
• For the determination of the more definitive
treatment of the lesion.
• To confirm a clinical impression of a lesion.

11. • To determine the nature of any intraosseous lesion
which cannot be identified clinically and
radiographically.
• To determine the nature of all abnormal tissue removed
from the oral cavity, including cysts and granulomas.
• Persistent hyperkeratotic changes in surface tissue.

12. • Any persistent tumescence, either visible or
palpable beneath a relatively normal tissue.
• Any lesion that has the characteristics of a
malignancy (e.g. Erythroplakia).
• Lesion that interfere with local function(e.g
fibroma)

13. CONTRAINDICATION
 RELATIVE CONTRAINDICATION
• This should include inflammatory lesions of allergic,
viral, fungal or bacterial etiology.
• Normal anatomical and racial variations, e.g., linea
alba, physiological racial pigmentation, leukedema,
exostoses etc.

14. • Compromised general health of the patient or a history
of coagulopathy or bleeding disorder, including patient
on anticoagulant therapy.
• Proximity of lesion to vital anatomic, vascular, neural
or ductal structures and lesions in areas of difficult
surgical access.
• Lesions caused by recent trauma.

15. ABSOLUTE CONTRAINDICATION:
• Pulsatile lesions or those suggestive of a vascular
nature should be referred for more in depth evaluation
(e.g., hemangioma).
• Pigmented lesions generally should not be biopsied
incisionally (e.g. Spread of a melanoma,
transformation of premalignant pigmented lesions to
malignant ones).

16. • Lesions that because of size or location present
technically difficult surgery e.g., posterior tongue and
oropharynx offer severe problems of access.
• Intrabony radiolucent lesions should not be biopsied
without initial aspiration.

17. • Lesions that are clinically obviously malignant should
be biopsied only in the facility that will assure
continual care.

18. OBJECTIVE OF BIOPSY
• To confirm a diagnosis made on clinical
findings.
• To determine the treatment plan
• Valuable self teaching diagnostic aid.
• As a medical record

19. • Grade tumors
• To detect receptors
• For screening purposes
• Detecting enzymes and antigens
• Monitoring, recurrence and prognosis
• Research purposes

20. • Microbiology
• Medicolegal

21. CLASSIFICATION OF BIOPSY

22. SURGICAL
NON-SURGICAL
SOFT TISSUE
BONE
DIAGNOSTIC
CURATIVE •EXCISIONAL
DIAGNOSTIC
CURATIVE
•CURETTAGE
•TREPHINE
•ASPIRATION
•FROZEN
•ENUCLEATION
•RESECTION
•CURETTAGE
ASPIRATION CYTOLOGY
•SCALPEL
•CAUTERY
•EXFOLIATIVE
•FNAC
•INCISIONAL
22

23. According to the procedures applied, oral biopsies can
also be classified by:
Features of the lesion:
• Direct biopsy: when the lesion is located on the oral
mucosa and can be easily accessed with a scalpel
from the mucosal surface.
• Indirect biopsy: when the lesion is covered by an
apparently normal oral mucosa.

24.  By the timing of the biopsy/ Clinical timing of
sampling:
• Pre-operative
• Intra-operative
• Post-operative

25.  Purpose of the biopsy.
• Diagnostic Biopsy
• Experimental Biopsy

26. VARIOUS BIOPSY TECHNIQUE
i. Incisional Biopsy
ii. Excisional Biopsy
iii. Electro-surgery Biopsy
iv. Curettage Biopsy
v. Punch Biopsy
vi. Frozen-section Biopsy
vii. Fine needle aspiration biopsy (FNAC)

27. viii. Aspiration Biopsy
ix. Trephine Biopsy
x. Exfoliative cytology
xi. Fine needle Biopsy
xii. Trucut needle Biopsy
xiii. Vim Silverman needle Biopsy

28. xiv. Sissor Biopsy
xv. Shave Biopsy
Xvi Ultra sound guided Biopsy
xvii. CT guided Biopsy
xviii. MRI guided Biopsy
xix. ENDOSCOPE guided Biopsy

29. xx. VELOSCOPE guided Biopsy
xxi. Exploration Biopsy
xxii. Unexplained/unplanned Biopsy
xxiii.Touch impression or imprint cytology

30. INCISIONAL BIOPSY / DIAGNOSTIC
BIOPSY:
• An incisional biopsy is a
biopsy that samples only a
particular portion or
representative part of a
lesion.

32. INDICATION
• when the lesion is extensive (larger than 1 cm in diameter)
or potentially malignant (requiring wide excision) or to avoid
an adjacent structure, e.g., nerve or artery.
• Chronic non-healing ulcer or squamous cell carcinoma.
• Premalignant lesion
• Bullous lesions (pemphigus, pemphigoid etc).

33. • Granulomatous diseases (crohn’s disease, orofacial
granulomatosis, ulcerative colitis, tuberculosis).
• Minor salivary gland tumour commonly seen on
palate.

34. CONTRAINDICATION
• Pulsatile/ vascular lesions.
• Pigmented lesions.

35. DISADVANTAGES
• Leaves noticeable scar.
• Possible spread of malignant cells
• Crush, splits and haemorrhage are the
artefacts most frequently found in incisional
oral biopsies

36. PRINCIPLE
• Representative areas of the lesion (the area that shows
complete tissue changes) should be biopsied in wedge
fashion from the edge of the lesion including some of
the normal tissue

37. • Deep narrow biopsy should be considered rather
than broad, shallow one, because superficial
changes may be different from those deeper in the
tissues.
• Necrotic areas should be avoided because it may
be nondiagnostic.

39. EXICISIONAL BIOPSY
• It is the removal of the lesion in
total at the time the surgical
diagnostic procedure is
performed.
• Not only the entire lesion is made
available for pathological
examination, but also complete
excision may constitute definitive
treatment for few lesions.

40. INDICATION
• It is the usual approach for smaller lesions (less than 1
cm in diameter).
• It is used for clinically benign lesions, be they
superficial or deep, soft or hard tissue.
• Includes the excision of pigmented and hyperkeratotic
lesions.
• Fibromas and papillomas.

41. • Mucoceles
• Myoblastoma
• Keratoacanthoma
• Sialoliths and smaller benign lesions of accessory
salivary glands
• Certain cicatrial lesions

42. PRINCIPLE
• Entire lesion along with 2 to 3 mm of normal
appearing surrounding tissue is excised.

43. TECHNIQUE
• For surface excision simple elliptical approach is
designed, like for the excision of pigmented and
hyperkeratotic lesions, fibroma, papillomas and superficial
pathology.
• For deep soft tissue lesions, modified elliptical that is
combined with deeper dissection is chosen.
continue…

44. • For most Incisional and excisional biopsies, ellipse
technique is employed.
• After obtaining the anesthesia i.e., local infiltration (LA
with vasoconstrictor) around the periphery of the
lesion, the lesion is isolated and immobilized with a
traction suture, hook or forceps.
continue…

45. • Care should be taken not to mutilate the specimen
when grasping it with forceps
• An elliptical area at the surface is outlined using
no.15 sharp scalpel blade (to avoid tearing the
tissue), incision is taken down to the connective
tissue layer to form a ‘V’ at the base of the lesion,
this provides a good specimen and also leaves a
wound that is easy to close.
continue…

46. • The length of the incision should be three times its
width to allow for tension-free primary closure.
• High volume suction devices should be avoided as
they can aspirate small surgical specimen.
• Hemostasis is achieved with resorbable suture
inserted (to control bleeding and also assist in
healing). Firm pressure for few minutes aid in
hemostasis.
continue…

47. • The tissue is fixed immediately upon removal in 10%
formalin / 70% alcohol, 20 times the volume of the
surgical specimen.
• If the specimen is thin, then place it upon a piece of
glazed paper and drop into fixative so that it prevents
curling of the tissue.
• The biopsy specimen should be marked with a silk
suture to orient the specimen to the pathologist.

49. ELECTRO-SURGERY BIOPSY
• Electro-surgery refers to the cutting and coagulation
of tissue using very high-frequency, low-voltage
electrical currents.

50. INDICATION
• Lesion excisions on the face are usually performed
with only a cutting current to limit scarring at the
wound base, which can be produced by the effects
of thermal coagulation

51. Electro-surgical Technique
The lesion is grasped with forceps through the loop
electrode. The electrode is activated going under the
lesion, removing the growth.

52. CURRETAGE BIOPSY
• CURRETTE is a French word ‘Curer’- meaning to clean.

53. INDICATION
• Intraosseous lesions that lie in cavities such as
maxillary antrum and cystic lesions within the jaws.
• Also used in very friable cellular lesions like sinuses
and fistulae within the soft tissues when only small
amounts of surface material are necessary for
evaluation.

54. TECHNIQUE
• These extremely small segments of tissue after
fixation are centrifuged and then the sediment is
placed in medium such as agar, they are then
sectioned as a cellblock.

55. PUNCH BIOPSY
• It is an alternative technique of tissue removal
applicable to both incisional and excisional biopsy.

56. INDICATION
• For total removal of small lesions
• Fixed tissue such as firmly attached palatal tissue,
which heal by secondary intention regardless of the
technique.

57. • For oral mucosal malignancies such as squamous
cell carcinoma as well as leukoplakia and other
mucosal abnormalities that may require multiple
biopsies.
• To diagnose oral manifestations of
mucocutaneous and other vesiculoulcerative
diseases.

58. CONTRAINDICATION
• As a definitive surgical excision procedure of
suspected malignant lesions and cases of vascular
lesions.

59. ADVANTAGES
• Technique is fast with low incidence of post surgical
morbidity.
• Suturing is usually not required as the surgical wound
heal readily by secondary intention with minimal or no
scar formation and with maximum esthetic results.
• The need for a post operative or suture removal visit is
uncommon.

60. • The technique can be used on any mucosal surface
accessible to the biopsy punch.

61. DISADVANTAGES
• Freely movable mucosa that cannot be well supported as
with the floor of the mouth and soft palate may preclude
the technique.
• It is difficult to use biopsy punch to obtain adequate
representative tissue deeper than the superficial lamina
propria

62. • Punch biopsy should be used with caution when the
lesion overlie significant submucosal structures such as
mental foramen or nasopalatine foramen and occurs in
inaccessible areas such as the maxillary posterior buccal
alveolar ridge and anterior lingual aspect of the
mandible.

63. TECHNIQUE
• Various types of biopsy punches are available –
- Keye biopsy punch
- Belt-driven.
• Even disposable biopsy punches are available.
continue…

64. • After the biopsy site has been anesthetized, the site is
gently blotted with sterile gauze.
• The edge of the blade of the biopsy punch is placed on
the site and rotated back and forth using moderate
pressure to an appropriate depth until the external
bevel is not visible and creates a clearly defined
surgical margin.
continue…

65. • The tissue is then grasped with an atraumatic forceps
and the base of the tissue core is released using a
scalpel blade or fine curved scissors.
• Punch size varies from 2-6 mm in diameter.
• Suture is rarely needed, as the hemorrhage is
minimal.
continue…

66. • Local pressure with sterile gauze is sufficient to induce
haemostasis
• Persistent hemorrhage can be treated with chemical
cautery such as silver nitrate, collagen matrix, or by
electrocautery.
continue…
• Post- operative instructions include avoidance of
inadvertent trauma to the area, either by diet or
through attempts at oral hygiene for 48 hours.

67. • Warm salt water rinses recommended for palliation.
• Non-steroidal anti-inflammatory agents are preferred
for post-operative discomfort.

69. FROZEN SECTION
• The frozen section procedure is a pathological
laboratory procedure to perform rapid microscopic
analysis of a specimen.
• It is used most often in oncological surgery.
• The technical name for this procedure is
cryosection.

70. INDICATION
• To make an immediate surgical therapeutic decision.
• To determine whether a lesion is benign, malignant or
non-neoplastic.
• Establish the adequacy of clearance of margin after
resection.

71. • Ascertain metastatic involvement of regional lymph
nodes.

72. ADVANTAGES
• If more tissue is needed to make an accurate diagnosis,
the surgeon is able to obtain an additional sample,
avoiding a second operation.
• If the tissue is determined to be cancerous and is
amenable to surgery, the mass can be removed at that
time.

73. • If the tissue is determined to be benign (not
cancerous), then the mass may not always need to
be removed and the
surgery can end.
• The frozen section biopsy can help ensure that the
mass being removed is the intended tissue for
removal.

74. • It can help ensure that the entire mass and its
surrounding borders are removed.
• It allows for the collection of proper tissue samples for
further scientific research.
• The surgeon and pathologist are able to collaborate to
care for the patient

75. Disadvantages
• There can be error in sampling and interpretation
of frozen tissue as compared to routinely processed
tissue.
• Differentiation between reactive epithelial changes
is difficult.

76. • It has the disadvantage that only 8-16 micron thick
section can be cut and finer details of tissue can not
be examined.

77. Technique
• Biopsy tissue is frozen in a mixture of isopentene and
solid carbon dioxide at -70o.
• Sections of 5-7μm are made on a refrigerated
microtome adhered to a glass slide at room
temperature, fixed with formal acetic alcohol (50ml
formalin, 450ml 90% alcohol and 25ml of glacial
acetic acid) and stained with haematoxylin and eosin.
continue…

78. • The procedure is completed within 5-10 mins from
the time of receiving specimen till it is stained.
• The remainder of tissue is stored in 10% buffered
formaldehyde and routinely processed; embedded in
paraffin, sectioned to 3 μm and stained with
haematoxylin and eosin.
continue…

79. • In this type of biopsy slides cannot be preserved for
future reference.

80. Fine needle aspiration cytology
(FNAC)
• Fine Needle Aspiration Cytology (FNAC) is a simple,
quick and inexpensive method that is used to sample
superficial masses like those found in the neck and is
usually performed in the outpatient clinic

81. INDICATION
• Lymph nodes - Reactive changes, lymphoma,
metastatic cancer
• Thyroid gland - Solitary or dominant nodule, suspected
malignancy, nontoxic goiter versus autoimmune
thyroiditis

82. • Salivary glands - Benign and malignant neoplasms,
inflammatory lesions, and cysts
• Cystic lesions of the neck - Branchial cleft and
thyroglossal duct cysts
• Miscellaneous - Parathyroid neoplasms, dermoid
cysts, and teratomas

83. CONTRAINDICATION
• No absolute contraindications exist to performing fine
needle biopsy of neck masses.
• However, a mass in the area of the carotid bifurcation
may represent a carotid body tumor, and serious
complications and one death have been reported
following fine needle biopsy of carotid body tumors.

84. • Patients with bleeding disorders or those on
anticoagulant therapy should receive appropriate
medical consultation prior to fine needle biopsy.

85. ADVANTAGES
• Small size of the needle avoids damage to vital
structure in the head and neck.
• Cells can be fixed, stained and examined within
minutes.
• In cases of deep lesions ultrasound or radiological
guidance may be used to ensure that the needle
enters the lesion.

86. •Treatment options including preoperative counseling
can be investigated earlier without the need for
further diagnostic surgery
•Requires little equipment
•Painless, no anesthesia is required
•Outpatient or bedside procedure

87. •Absence of reactive fibrosis to interfere with subsequent
surgery
•No problems with wound healing, e.g. after radiotherapy
•Readily repeatable
•Cost effective

88. Disadvantages
• Success of FNA depends on obtaining a
representative sample (if the specimen is small with
few or no cells).
• Experience is required for interpretation.
• Definitive diagnosis not always possible.
• False negative and false positive results are possible.

89. ASPIRATION TECHNIQUE
• Fix the target mass between 2 fingers of the
nondominant hand.
• Advance the needle(21-23G) attached to the syringe,
into the mass. Do not apply suction during the
advancement.
continue…

90. • Once the needle tip is within the mass, apply
suction by pulling back on the plunger of the
syringe.
• While maintaining suction, move the needle rapidly
back and forth with short strokes within the mass.
• Release negative pressure after sampling is
completed and before withdrawing the needle from
the mass.
continue…

91. • Withdraw the needle.
• The cytologic specimen is within the needle and,
perhaps, the hub of the needle.
• Once withdrawn, detach the syringe from the needle.
Fill the syringe with air and then reattach it to the
specimen-containing needle.
• Expel the specimen onto a glass microscope slide

92. A- needle is introduced into the mass.
B – plunger is retracted after needle enters the mass
C – suction is maintained while needle is moved back
and forth within the mass
D – suction is released and plunger returned to original
position before needle is withdrawn

93. NON ASPIRATION(ZAJDELA)
TECHNIQUE
• Fix the target mass between 2 fingers of the
nondominant hand
• Hold the needle by the hub between the thumb and
index finger.
• Advance the needle into the target mass.
continue…

94. • Attach an air-filled syringe, with the plunger already
retracted, to the specimen-containing needle.
• Expel the specimen onto a glass microscope slide
• Move the needle back and forth within the mass with
short, rapid strokes. Simultaneously, move the needle
in a rotary clockwise-counterclockwise motion.
• Withdraw the needle.

95. ASPIRATION
CYTOLOGY GUN

96. FRANZEN’S HANDEL WITH SYRINGE
& NEEDLE FITTED ON IT FOR
PERFORMING FNAC

97. Causes of unsatisfactory yield with
FNAC
• Needle missing a lesion and picking up inflammatory
cells.
• Lack of cells in central area of necrosis, hemorrhage,
cystic change.
• Small malignant tumour being masked by larger
benign tumour.
• Lack of cells in dense fibro sclerotic tissue.

98. COMPLICATION
• Fine needle biopsy (FNB) of the neck has a very low
morbidity rate
• Hematoma is the most common significant complication.
Most hematomas are small and resolve without
treatment.
• Infection following fine needle biopsy of the neck is rare.

99. • Recurrent laryngeal nerve paralysis following fine
needle biopsy of the thyroid gland is noticed .
• Puncture of the trachea during fine needle biopsy
of the thyroid gland can cause coughing and mild
hemoptysis
• fine needle biopsy has been associated with
infarction and subsequent necrosis of the biopsied
neck mass.

100. • One fatality resulting from a fine needle biopsy of a
neck mass has been reported. The death followed fine
needle biopsy of a carotid body tumor. The immediate
cause was thrombosis of the adjacent internal carotid
artery.

101. LIMITATIONS OF FNAC
•Only a small population of cells is sampled, thus the
reliability of test depends on adequacy of sample & its
representative character. An inadequate sample, which
is not representative of true lesion, results in false
negative report.
•Requires clinical information or relevant investigation
(e.g. x-ray finding), which further limit utility of FNAC.

102. TREPHINE BIOPSY
• Bone marrow examination refers to the
pathologic analysis of samples of bone marrow
obtained by bone marrow biopsy (often called a
trephine biopsy) and bone marrow aspiration.

104. INDICATIONS
• Detect and stage malignancy.
• Differentiate benign hematologic diseases (e.g.,
aplastic anemia, macroglobulinemia).
• Evaluate progression of human immunodeficiency virus
• classify an anemia as hypoproliferative, a maturation
disorder, or resulting from hemorrhage or hemolysis

105. • Unexplained anemia in peripheral blood
• Unexplained thrombocytopenia in peripheral blood
• Pancytopenias in peripheral blood
• Lymphoproliferative disorders
• Abnormal cells in peripheral blood
• Diagnosis and stage of lymphomas and leukemias

106. • Metastatic disease
• Minimal residual disease in lymphomas and leukemias
posttreatment
• Chromosomal abnormality
• Immunodeficiency syndromes
• Fever of unknown origin

107. PROCEDURE

109. Equipment
1. Sterile gloves
2. Sterile drape
3. Povidone-iodine
4. Bone marrow
aspiration needle or
11- or 13-gauge
Jamshidi needle
5. 25- and 22-gauge
needles
6. No. 11 scalpel
blade
7. Heparinized 10 cc syringe
8. 3 cc and 10 cc syringes
9. 1% lidocaine
10. Glass slides
11. Specimen bottle with
formalin
12. 4“ x 4“ gauze
13. Pressure dressing and
tape

110. SITES FOR BONE MARROW COLLECTION
• Posterior iliac crest
• Sternum

111. Caution should be exercised in patients with
soft bones secondary to radiation therapy,
multiple myeloma, or osteoporosis.

112. TISSUE CORE
• Although FNAB is widely used technique in the
diagnosis of head and neck lesions, the diagnostic
accuracy is not as good as histological analysis.
• Many pathologists consider core tissue for the
histologic study to be useful because core biopsy
specimen is larger in size and thus suitable for
conventional histopathological analysis than the
cytologic material obtained from FNB.

113. • Secondly the technique is simpler, easier and faster
than the traditional suction method.
• It also eliminates the possibility of inadvertent
suction of a specimen through a biopsy needle into
the syringe and the fragmentation of the specimen
due to the aspiration step.

114. TRU-CUT NEEDLE BIOPSY:
• It consists of wide bore 14G and consists of a long
15.2cm )canula and trocar with a 2cm notch at the tip
of the trocar.
Technique
• L.A.
• Stab incision with a scalpel
• Canula is inserted with the trocar fully retracted until
the specimen notch is with in the tissue to be biopsied.

116. VIM SILVERMAN NEEDLE BIOPSY:1938
It consists of:
1)Outer canula 16 G in size.
2)Inner trocar.
3)Inner split longitudinal needle.
Technique:
L.A.
Small incision made with the scalpel before the canula
and trocar are inserted up to the tissue to be biopsied.
The trocar is then completely removed and replaced by
the split cutting needle.

118. ADVANTAGE
• They are easy to interpret then aspiration cytology to
the pathologist
• To distinguish between reactive changes and recurrent
malignancy in possible cervical metastasis.

119. EXFOLIATIVE CYTOLOGY
• It is the study of superficial cells which have been
either exfoliated or shed actually from mucous
membrane, renal tubules etc. and also includes the
study of those cells which have been collected being
scraped or pulled off by tissue surface and may also
be found in body fluids such as sputum, saliva etc

120. The lesion is repeatedly
scraped with a
moistened tongue
depressor or spatula or
cytobrush type
instrument
The cells obtained are
smeared on a glass slide and
immediately fixed with a
fixative spray or solution.

121. Indications
• For quick laboratory evaluation of suspected
malignant and premalignant oral lesions and multiple
premalignant and extensive lesion and lesions leading
to field cancerization.
• For sequential laboratory evaluation of post-operative
or post-irradiated malignant lesions.
• Recurrent oral cancers after treatment.

122. • To identify the presence of certain specific cells in non-
malignant red patches or ulcerative lesions.
• To see malignancy associated change in buccal
squamous cells in patients with malnutrition.
• For evaluation of vesicular lesion.
• For detection of sex chromosomes.

123. • Certain benign hereditary skin lesions having their
representative oral manifestations.
• For the study of the change of the oral epithelial cells
followed by chemotherapy.
• For the study of buccal mucosa in various anemia.
• Mass screening of oral cancer.

124. Contradictions
• Deep seated lesions (both soft and hard tissue).
• Fibrous lesions.
• Polypoid growth.
• Non-ulcerative lesions.
• Lesions do not show positive changes in the cells of
the superficial layers.

125. • Atrophic lesions.
• Densely keratinized lesions.
• Smooth surface lesions.
• Lesions giving inadequate specimen sampling
through the adopted technique.

126. ADVANTAGES
• Exfoliative cytology implicates its importance in the
field of diagnosis with the principle that any change in
the superficial cells can be the reflection of the change
in the immediate underlying tissue.
• Cytology is an adjunct to but not a substitute for the
surgical biopsy.

127. • It is quick, simple, painless, bloodless, non- invasive
procedure.
• If guards against false negative biopsies.

128. DISADVANTAGES
• Relatively limited information provided with exfoliated
material when compared to a histological
preparation.
• Exfoliative cytology is limited to tissue, which
exfoliate cells into reasonably accessible sites.
• Majority of the lesions occur in the oral cavity are
benign which do not lend themselves to cytologic
smear.

129. • Positive results gives definite value whereas
negative results is of considerably less value.
• Cytology positive with biopsy positive - definite value.
• Cytology positive with biopsy negative - Repeat biopsy.
• Cytology negative with biopsy negative - negative.
• Cytology negative with biopsy positive – Repeat
cytology.

130. Technique:
• Instruments for removing the cells should have a 90-
degree angle or straight blade.
• The cement spatula used in mixing crown and bridge
cement or wax carver can be used.
• A tongue blade slit longitudinally is a second choice,
but it should be moistened to avoid dehydration
damage to the cells.
continue…

131. • The cotton swab has been recommended in selected
cases.
• Scraping of the lesion should be done while the
tissue is stretched or taut. A single stroke is
preferred over many small strokes. Excess saliva
should be removed by an air blast prior to removal
of the cell.
continue…

132. • Smearing should be done on a standard glass slide
(1x3 inches) with an etched label area at one end. The
glass slide should be clean and dry.
• The cells should be evenly disturbed over the central
one third of the slide. The slide must be labeled
continue…

133. • Fixation must be immediate. Air dried smears are
inconsistent in their staining properties. A safe and
adequate fixation is 95% isopropyl alcohol, there is
little danger of fire with this solution, and it is easily
obtained, inexpensive, available and easy to store.
continue…

134. • The cells should be fixed for at least 1 hr before
staining. If the slide is to be mailed the fixative can
be discarded after fixation and the mailing
container tightly sealed to prevent drying.
• History should be supplied to the pathologist as in
done with a biopsy.

140. Interpretation and Reporting
• The evaluation of slide is based on the morphological
features and staining quality of the cells especially
the nuclei.
• The ratio of nucleus to cytoplasm is also a critical
factor papanicolaou’s classification is often used in
reporting diagnostic equation. The cytologic smear is
usually reported into one of the V classes: -

141. Class I (Normal) indicates only normal cells.
Class II (Atypical) indicates the presence of mild atypia but no
evidence of malignant change.
Class III (Intermediate) the cells display wider atypia, may be
suggestive of cancer, but they are not clear-cut and may
represent precancerous lesions, carcinoma in situ. Biopsy
is recommended.
Class IV (Suggestive of cancer) A few malignant cells with many
cells having borderline characteristics. Biopsy is mandatory.
Class V (Positive for cancer) obvious malignant cells. Biopsy is
mandatory

142. Touch impression or
Imprint cytology
• It is a method in which gentle grazing or sliding of glass
slide over the cut surface of a resected tumor immediately
after the surgery.

143. INDICATION
• For diagnosis of certain neoplastic lesions which can
simulate inflammatory lesions on frozen section
• For diagnosis of certain benign inflammatory lesions
which can simulate malignancy on frozen section

144. Advantage
• Feasible and economical
• Detect the malignancy at the tumor margin
• Less time consumable

145. TECHNIQUE
• A direct imprint is prepared by pressing a glass slide
gently on to the freshly cut surface of the specimen,
avoiding a gliding movement, which will distort the
shape of the cells.
• The imprint slide is immediately fixed in 95 % ethyl
alcohol for 5-6 seconds and then stained (rapid
haematoxylin and eosin).

146. Imprint of a massively necrotic carcinoma showing a
cluster of viable cancer cells on a necrotic background.
Diagnosis was not possible on frozen section.

147. Imprint of a lymph node showing malignant cells.

149. • Is one of the ways to remove skin tissue for a
biopsy specimen.
• This procedure entails snipping off a growth that is
attached to the skin with a stalk.
• Scissors biopsy is indicated for pedunculated and
very superficial growth.

150. • Small forceps with teeth and a pair of sharp curved
or straight iris scissors are the only surgical
instruments required.
• Depending on lesion size and morphology,
anesthesia may or may not be necessary.

151. • Bleeding after this procedure is usually minimal and
can be easily controlled by application of 35%
aluminium chloride solution
• The lesion to be removed is lightly grasped with
forceps by gently pulling upward, traction
provides a firm cutting surface and allows clear
visualization of the lesion base.

152. SHAVE BIOPSY

153. • A scalpel or razor blade is used to scrape lesion,
performed superficially or deeply.
• Shave excision usually extends to the level of the
middle dermis, with the subcutaneous tissue left
undisturbed.
• Skin lesions with a minimal dermal component, such
as seborrheic keratoses or fibrous papules are
excellent candidates for shave excision technique.

155. ULTRASOUND GUIDED BIOPSY

156. • This technique has the advantages of being
noninvasive, quick, and easy, and it can be
performed with the patient under local anesthesia.
• It has an advantage over blind percutaneous biopsy
because the needle can be visualized in the organ
and the organ scanned after biopsy for possible
complications.

157. • However, Ultrasound guided biopsy is not possible
when gas/bone prevents the visualization of the
biopsy region.
• Another advantage is that, unlike other
radiographic biopsy procedures, ionizing
radiation is not used for imaging

158. CT GUIDED BIOPSY
• A computed tomography guided biopsy, uses real-
time CT images to help the doctor guide a needle to
the suspect lesion to obtain a tissue sample.
• Occasionally, intravenous (IV) contrast is needed to
help the radiologist identify and target the lesion
prior to the biopsy.
• The CT image is immediately available on a monitor,
allowing the radiologist to view the biopsy target.

159. Indications
• Lymph nodes or masses that are not completely
identifiable using ultrasound.
• Lesions near the skull base: CT is optimal for
localizing these lesions.

160. Disadvantages
• radiation exposure.
• limited possible scan plane orientations.
• low soft tissue contrast.
• poor vessel conspicuity without administration of
intravenous contrast medium.

161. MRI GUIDED BIOPSY
• Interventional MRI is a method for procedure
guidance that combines the imaging benefits of
magnetic resonance, including excellent tissue
contrast and multiplanar imaging capability, and
good vessel depiction of MRI with the increased
patient access that is possible with newer magnet
designs.

163. • Scientific interest has also focused on MRI-guided,
minimally invasive thermal tumour ablation using the
unique temperature sensitivity of MRI or its capability
to demonstrate changes in tissue relaxation
parameters (T1 and T2) that occur in the process of
necrosis.

164. • The mean procedure time for MRI-guided needle
insertion per pass is less than 10 minutes for
aspiration as well as core biopsy.
• The mean procedure time for MRI-guided needle
insertion per pass is less than 10 minutes for
aspiration as well as core biopsy.

165. ENDOSCOPE GUIDED BIOPSY
• Endoscopy is defined as “the examination of the
interior of a canal or hollow viscous by means of an
endoscope.”
• Endoscopic technique may prove to be particularly
important when dealing with large jaw cystic lesions
that may contain neoplastic processes such as
ameloblastomas or carcinomatous entities within
certain regions of their lining.

166. • Endoscopy may prove to be an important tool for the
internal examination of large jaw cysts that may
contain regional neoplastic processes within the cyst
lining.
• Especially in the case in large cysts that extend into
areas that are difficult to inspect and sample
through a standard “bony window” technique

167. Endoscope positioned into the lesion.

168. Endoscopic view showing areas of thickened lining
containing exophytic protrusions measuring up to 10
mm in diameter.

169. VELSCOPE

174. EXPLORATION BIOPSY
Used for inrtaosseous lesions of maxilla and mandible
Instruments: Chisel, Bone burs, Periosteal Elevator.

175. UNEXPECTED/ UNPLANNED BIOPSY
When as a result of surgical procedure (Tooth
extraction) some suspicious tissue is obtained
unexpectedly

176. ESSENTIAL BIOPSY PRINCIPLE

177. ARMAMENTARIUM

178.  Site selection and handing of the
specimen-
• By carefully selecting the area or areas, which will
produce a good diagnostic specimen, the clinician
can avoid having repeat biopsy.
• The extent of biopsy is determined by the size and
location of the lesion. Pathologists prefer the entire
lesion as the most desirable specimen.

179. • Deep sections are preferred over shallow
specimens. If possible, biopsies from the mucosa
should include the epithelium, lamina propria,
submucosa, and a portion of any deeper tissue, such
as muscle and fat, which may be affected by the
clinical lesion.

180. • Sections taken from the surface or center of a
lesion are often necrotic and do not represent the
most typical characteristics of the lesion.
• The periosteum should be avoided whenever
possible as it is often a barrier to growth and
infections.

181. Sterile technique should be utilized both to
prevent infection, which complicates the diagnostic
procedure, and to avoid contamination of the
specimen.

182. Anesthesia in the majority of cases is local,
with infiltration used more commonly than
block.
• Infiltration must be slow and far enough from
the lesion to prevent explosion of the tissue
elements, and particularly the separation of
epithelium from connective tissue.

183. HEMOSTASIS
•The use of suction device for aspiration of surgical
hemorrhage during biopsy should be avoided. This is
especially true of the high-volume evacuators available
in most dental offices.
•Small surgical specimens can be easily aspirated into
these devices and lost.

184. •Gauze wrapped over the tip of a low-volume suction
device or simple gauze compresses are adequate in
most cases, unless severe haemorrhage is
encountered.

185.  Manipulation and grasping of any lesion suspected of
being a tumor should be minimal. Vigorous
manipulation of malignant tumors can cause tumour
cell emboli.
 Suturing is not often necessary when small incisional
biopsies are taken, but most excisions require
suturing. Black silk suture material in preferable.

186. Orientation of the specimen is
extremely important to the pathologist Multiple
lesions must be adequately labeled and often
oriented with a diagram of the original lesion.
• A black silk suture placed through each end,
preferably in normal tissue will often serve to orient
the lesson.

187.  Fixation and preservation
• It is essential to avoid distortion of histologic and
cytologic details.
• 10% formalin (4% formaldehyde) has a relatively long
shelf life, and it is the more commonly used fixative for
diagnostic specimens.
• Other fixatives, which are used with satisfactory results,
include parachlorphenol (camphorated), sodium
hypochlorite, and Zephesin.

188. • When there is doubt about the action of some
chemical on tissue, it is preferable to place the
specimen first in cool or cold water (preferably an
isotonic solution) and then in formalin or soon as
possible.
• Tissue should never be placed on cotton, gauze, or
paper prior to fixation. There is rapid absorption of
fluid from the cells, often causing extensive
distortion that would not occur if the specimen is
left on glass or metallic surface for several hours.

189. • Container used for fixation should have a wide
mouth, so that after fixation the specimen can be
easily removed.
• Containers that have caps that will rust should be
avoided since the iron oxide will often interfere
markedly with the staining reaction in the laboratory.

190. • Temperature:
The fixation can be carried out at room temperature.
Tissue should not be frozen once it has been placed in
the fixative solution, for a peculiar ice crystals distortion
will result.

191. • Speed of fixation:
The speed of fixation of most fixative is almost 1
mm/hour. Therefore, a fixation time of several hours
is needed for most specimens.
• Amount of fixative fluid:
This should be approximately 10-20 times the volume
of the specimen. Fixative should surround the
specimen on all sides.

192.  Labeling of the specimen:
• The patient’s name, the location of the specimen,
and the date of the surgical procedure are all
essential.
• If multiple biopsies are taken from the same patient,
the specimens should be placed in individual
containers or wrapped and labeled in individual
gauze, which has been saturated in 10% formalin.

193. • If the specimen is to be mailed, it is better to
place it in gauge within the specimen container
so that if a rupture with loss of fluid occurs, the
gauze will maintain the tissue in a moist fixed
state.

194.  Details required in pathology form
• Patient data
• Clinical details of lesion
• Any medical history with details of medication
• Oral habits - all forms of tobacco and alcohol
consumption
• Investigations done, if any
• Site and biopsy type
• Clinical diagnosis with differential diagnosis
• Previous biopsy done, if any, with details.

196.  Follow-up and Reporting of Biopsy Result to the
Patient
• Patients should be seen 1 to 2 weeks postoperatively
to ensure healing and to discuss the results of the
biopsy.
• It is the responsibility of the clinician (not the
assistant or secretary) to explain the diagnosis and
any further management if necessary.

197. • If the microscopic diagnosis is inconsistent with
the clinical impression, the clinician is strongly
advised to discuss any concerns directly with the
pathologist

198. SPECIAL CONSIDERATIONS
In large lesions
Accessible area
Characteristic portion.
For multiple lesions
Most representative site.
Material curetted from interior of the lesion .

199. For red & white lesions include both red & white
area

200. Include margin,
deep part of ulcer
and site of maximal
clinical activity.
AVOID Superficial
ulcers & necrotic
tissue
ULCERS

201. For Polypoid lesions include base

202. For Vesiculobullous lesions
Fluid is more representative. Intact vesicle or
bulla should be biopsied.

203. LICHEN PLANUS – representative area should be
biopsied

204. LEUKOPLAKIA – Most dysplastic area should be
biopsied

205. MUCOCELE lesions – careful excisional biopsy

206. GRANULOMATOUS LESIONS – deep incisional
biopsy + fresh sample to microbiology if infective
agent suspected

207. Do not cut into pigmented and vascular lesions

208. 208

210. • PAS stain: It is used for fungi, amoeba and
Tricomonas.
• Modified Giemsa (2% Giemsa in water): Detects
Helicobacter pylori.
• Congo-red: It is used for identification of amyloid.

211. • Sudan-Black: It is used for fat staining.
• Masson’s Trichrome: It is used for
differentiation of connective tissue elements.
• Papanicolaou’s stain: It is used to stain cells in
cervical and sputum smear for cytology.

212. ERRORS DURING
SURGICAL REMOVAL OF
BIOPSY SPECIMEN

213. INJECTION

214. • HANDLING THE TISSUE:
Tissue should be handled with minimum force.
• USE OF FORCEP:
Avoid use of forcep on the use of biopsy
• USE OF ELECTRO-CAUTERY:
Different view for its use.however combination of
cautery and scarpel should be used

215. SELECTION OF THE SPECIMEN
Tissue should be representative of whole lesion.
Adequate amount of tissue at least 1 x 0.5 cm in
size.

216. Biopsy of skin or mucosa representative sample of both
epithelium and connective tissue has to be taken

217. Tissue Artifacts
• Alterations in tissue morphology that result from
various forms of mechanical, chemical or thermal
insult to the tissue removed for diagnostic
purposes are termed as tissue artifacts.

218. CAUSES OF TISSUE ARTIFACTS
• Clinical application of chemicals.
• Local injection of anaesthetics.
• Surgical sectioning.
• Excessive heat.
• Freezing.
• Surgical mishandling of the specimen.
• Inadequate fixation.
• Improper fixation medium.
• Faulty tissue processing.
• Improper staining.

219. 1.DURING SURGERY:
• Injection Artifacts
• Forceps Artifacts
• Fulguration Artifacts
• Laser Artifacts
• Suction Artifacts

220. 2.DURING FIXATION AND TRANSPORT
• Fixation Artifacts
• Freezing Artifacts
• Artifacts during transportation
3.TISSUE PROCESSING ARTIFACTS
• During Embedding
• During Sectioning

221. INJECTION ARTIFACTS
Injection of large amounts of anesthetic solution into the
area to be biopsied can produce following tissue
changes:
• The needle insertion may produce hemorrhage with
extravasation, which in turn masks the cellular
architecture.
• The separation of connective tissue bands with
vacoulation may occur.
• Tissue edema or distortion.

222. HOW TO AVOID
Infiltration of an anaesthetic agent
• Injections directly into the lesion should be avoided.
• If hemeostasis is a consideration, injections deep
to the lesion, or immediately after the biopsy has been
performed, is effective.

223. FORCEPS ARTIFACTS
• When the teeth of the instrument penetrates the
specimen, it resulted in voids or tears and compression
of the surrounding tissue.
• The surface epithelium may be forced through the
connective tissue producing small "pseudocysts".
• Compression of tissues results in loss of cytological
details with nuclear dimensions and relationships
being especially affected.

224. TO AVOID
• Using small atraumatic or Adson forceps with or
without teeth. These will produce little mechanical
distortion of the tissue.
• The area of the lesion should never be grasped with
forceps.
• If the use of forceps is mandatory, only the peripheral
aspect should be used for holding and delivery of
the specimen.

225. CRUSH ARTIFACT
• It’s a form of tissue distortion resulting from even the
most minimal compression of the tissue.
• Crushing produces a destructive and dangerous type of
artifact by rearranging tissue morphology and
squeezes the chromatin out of the cell nuclei.
Inflammatory and tumors cells are most susceptible.

226. CAUSES
• Mutilation of the tissues with forceps.
• Dull scalpel blades.
HOW TO AVOID
• Delicate handling of specimen.
• A suture placed to the edge of the specimen to
substitute for forceps.

227. FULGURATION ARTIFACTS
• The use of cautery for biopsies.
• Heat produces marked alterations in both the
epithelium and connective tissue.
• The epithelial cells appear detached and the nuclei
assume a palisading configuration.
• Separation of the epithelium from the basement
membrane has also been reported.

228. • The fibrous connective tissue, fat and muscle gain an
amorphous appearance when subjected to such
procedures.
• Its effect is to boil tissue fluid and precipitate
protein. Microscopically such tissue shows a
coagulated and torn appearance that makes
histological evaluation impossible in the cauterised
areas.

229. • Alternating areas of coagulation impart a
"Herring Bone" appearance to the tissue if the
procedure is not done properly

230. TO AVOID
• Only the cutting and not the coagulation electrode
should be used while obtaining a biopsy specimen.
• The incision margins should be sufficiently far away
from the lesion - normal tissue interface to prevent
thermal changes in that significant area.
• Combination of electro surgery and a scalpel should
always be considered

231. • This technique involves use of the scalpel for initial
incision in and around the lesion and electrosurgery
to complete the removal of the specimen.
• This ensures superior hemostasis while reducing the
amount of heat to which the tissue is exposed.

232. LASER ARTIFACTS
• Lasers are being used for excision and ablation of oral
mucosal lesions with the chief advantage being
minimization of intraoperative haemorrhage.
DISADVANTAGE
• Produces the thermal artifactual changes.

233. • The CO2, Nd: YAG systems lends to cytological
artifacts in keratinocytes, with the keratinocytes
showing atypical changes consisting of
hyperchromatism, pleomorphism and nuclear
elongation along with a wide margin of coagulative
thermal changes.

234. ARTIFACTS INDUCED BY SUCTION TIPS
• Induced by the vacuum effect of the surgical
suction tips.
• These are seen in various types of surgical
specimen, most notably in the connective tissue
around odontogenic cysts and dental follicles.
• Manifest by the formation of large often
pleomorphic connective tissue vacuoles resembling
traumatized adipose tissue.

235. • In highly vascularised tissue such as tissue in
periapical granulomas and in inflamed odontogenic
cysts.
• Suctioning induces extravasation of blood and focal
accumulation of erythrocytes within the connective
tissue vacuoles.

236. Complications

237. Haemorrahage
Immediate or subsequent haemorrhage can be serious
problem following biopsy.
• Highly vascular tissue (hemangioma)
• In a region where venous pressure is markedly
increased (e.g. a supraclavicular lymph node biopsy
in the presence of superior vena caval obstruction)

238. • From a large friable tumor mass
• Where an adjacent blood vessel of moderate
size may be severed and allowed to retract.
• When the wound becomes infected and late
secondary haemorrhage occurs.

239. Infection
• When tumors on the various skin or mucosal surfaces
are biopsied, there is always a possibility that
already present bacteria, may thus gain access to the
depths of the tumor and to the adjacent normal
tissue.
• Aseptic technique must always be observed

240. Poor wound Healing
Unsatisfactory healing of the incision may be due to
• Ischemia of the skin overlying a tumor mass
(tension)
• Implantation of atumor cells
• Previous X-ray therapy

241. Spread of tumor cells
Prominent reasons for local tumor cell contamination
are the following:
• -Lack of awareness that the spread of tumor cells
commonly occurs and is increase by prolonged and
unnecessary manipulation of tissue.
• -Careless protection of the tissue during the
Incision or Excision of malignant neoplasm.
• -Failure to change contaminated drapes,
instruments or material when indicated

242. Injury to adjacent organs
• Injury may occur to surrounding structure (blood
vessels).
• Structures through which the biopsy is accomplished
may be injured. Certain of the vital structures must be
avoided in needle biopsy (blind methods) and the
adequate surgical exposure is essential when the
biopsy is taken with a scalpel.

243. Other complications
• Post operative pain.
• Paraesthesia - in the lips or the tongue,
• Swelling and bruising - in the tongue, lips and buccal
mucosa
• Procedures in the floor of the mouth can lead to
submandibular or sublingual duct damage.
• Removal of mucocoeles from the lip carries the risk of
further gland damage and ‘recurrence’.

244. Biopsy Results: What If ?
They don’t corroborate your clinical impression
• Repeat the biopsy!!!
• Determine if the tissue was looked at by a
Pathologist
• The results show malignancy

245. Situation In Our Sub-region
• Inadequate facilities
• Few number of experienced Pathologist
• Patient associated factors e.g poverty, ignorance,
religious beliefs

246. CONCLUSION

247. • For entities of uncertain significance or etiology, a
biopsy provides the simplest and most speedy means
of obtaining the perfect diagnosis. In the concern of
patient’s welfare, correct diagnosis is of extreme
importance.
• A carefully selected, performed and interpreted
biopsy is critical in rendering an accurate diagnosis.
• When considering biopsy, a little forward planning
and thought can greatly improve the diagnostic
value obtained.

248. • Careful handling of the tissue and prompt
appropriate fixation will enable a confident
histological diagnosis to be reached. Inadequate
care at any stage could result in a nondiagnostic
biopsy and may necessitate the patient having a
repeat procedure with its ensuing physical and
psychological morbidity.

249. References
•R. Rajendran and B. Sivapathasundharam: Shafer’s
textbook of oral pathology, 5th edition (2006), Elsevier.
•Neville Brad W., Damm Douglas D., Allen Carl M. and
Bouquet Jerry E.: Oral and Maxillofacial Pathology, 2nd
Edition (2004) Saunders.
•Martin S. Greenberg and Michael Glick: Burket’s Oral
Medicine Diagnosis and Treatment, 10th Edition (2003);
BC Decker Inc.

250. •Peterson, Ellis, Hupp and Tucker: contemporary oral
and maxillofacial surgery, 4th edition.
•S M Balaji: Text book of oral and maxillofacial surgery,
1st edition.
•Theory & practice of Histological techniques , 2nd & 3rd
ed. , Bancroft

251. Journal of Cancer Research and Therapeutics - April-
June 2012 - Volume 8 - Issue 2
S I Talukder. www.talukderbd.com. Histopathology
Techniques: Tissue Processing and Staining
Sylvie-Louise Avon. Oral Soft-Tissue Biopsy: An
Overview. J Can Dent Assoc 2012;78:c75
K. L. Kumaraswamy, M. Vidhya. Oral biopsy: Oral
pathologist’s perspective. Journal of Cancer Research
and Therapeutics - April-June 2012 - Volume 8 - Issue 2

252. THANK YOU
FOR
YOUR TIME