Zerbino DD. Biopsy: its current history, management and future outlook. Lik Sprava Mar-Apr;(3-4):1-9
tumidity resulting from the presence of blood or other fluid in the tissues
Small Animal Diagnostic Ultrasound edited by Thomas G. Nyland, John S. Mattoon
Gliddon et al. Endoscope-Guided Biopsy of Mandibular Cyst. J Oral Maxillofac Surg 2005.
One can’t show this when it’s not there
One can’t hide this when it’s there.
TABLE OF CONTENTS
• Indications & Contraindications
• Various biopsy procedures
• Special considerations
• Essential biopsy principles
• Biopsy artifacts
• Healing of a biopsy wound
• The clinical presentation of any pathology can be
the mucosal surface change (i.e., change in texture,
ulceration, proliferation) or it can be submucosal
structural alterations (i.e., distortion or swelling
produced by a mass).
• The diagnosis of such pathology depends on the
history, examination, laboratory studies, biopsy and
other diagnostic techniques.
• Among all these investigations, obtaining an
accurate tissue diagnosis is the one needed in the
process of forming a definitive diagnosis and
treatment planning. This is referred to as the
• It is essential for the purpose of records and
research; to determine the prognosis in cases of
malignancy and also provides an indication towards
the response of the tumor to therapy (e.g., relative
resistance of malignant melanoma to
• There are many methods used to perform this
procedure. But whatever the procedure, the aim of
it should be to provide a suitably representative
sample for the pathologist to interpret, while
minimizing perioperative discomfort for the
• In the early 16th century, Sir Marcello Malphigi was
the one who formulated the basic microscopic
technique of utilizing the living tissues. He termed
it Bios-living, Opsis-visualizing.
• Later, Sir Georianni Morgahni in the early 17th
century popularized this method through his book
‘The site and causes of diseases’ which laid the
foundation for physiologic anatomy. From that date
of Malphigi’s formulation of the technique till now,
the method has spread wide with some additions
and deletions to the extent that its need has
become mandatory adjunct to complete history
and examination of the lesion in doubt.
• The term "biopsy" was introduced into medical
terminology in 1879 by Ernest Besnier.
• "... Not only skin changes (colloid degeneration of the
dermis) were studied in a definitive manner in a series of
necropsies, but we could enlighten the living many
points by examining fragments of integument or
diseased tissue fragments. There is in this latter mode of
investigation, true biopsy (a new word that we propose
to designate a new thing), regular clinical diagnostic
process whose importance is considerable.”
• In this quote from an article in the weekly Gazette
of Medicine and Surgery October 10, 1879 under
the Ernest Besnier signature, the word biopsy is
• The first diagnostic biopsy in Russia was performed
in 1875 by M. M. Rudnev.
It is possible to make out three stages in more than 100
year history of the method development:
• an occasional use of histologic procedure, involving
living organs and tissues accessible for observation and
study (approximately until the late 19th century);
• restricted application of biopsy (until the mid-20th
• present stage at which the method is widely
adopted and its use is general and total (with
respect to human organism) not only in oncology
but practically in all clinical specialties
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
• Biopsy is the removal and examination usually
microscopic of tissue from the living body,
performed to establish precise diagnosis.
-Chiles DG in 1987
• Biopsy is the removal of a tissue specimen either
totally or partially for microscopic examination and
-Pederson GW in 1988
• Biopsy is the removal of a piece of tissue from the
living body for diagnosis by microscopic
- Tomlins Christopher DC in 1998
• Biopsy is the removal of tissue from a living subject
for laboratory evaluation and analysis.
-Neelima AM in 2002.
INDICATIONS FOR BIOPSY
• To confirm a clinical impression of a lesion.
• 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 determine the nature of any intraosseous lesion
which cannot be identified clinically and
• To determine the nature of all abnormal tissue
removed from the oral cavity, including cysts and
• Persistent hyperkeratotic changes in surface tissues.
• Any persistent tumescence, either visible or palpable
beneath a relatively normal tissue.
• Lesions that interfere with local function (e.g.
• Any lesion that has the characteristics of a malignancy
1. Surface lesions:
• Color/texture change e.g., white, red or
• Ulcerated, fissured or both.
• Proliferate (fibroma, papilloma).
2. Subsurface lesions:
• Superficial, distort surface continuity, e.g.,
• Deep, detected by palpation, e.g., masses of
• Superficial, distort surface continuity
• Deep, detected by x-rays, e.g., radiopaque and
CONTRAINDICATIONS FOR BIOPSY
For ambiguous lesions
a) This should include inflammatory lesions of
allergic, viral, fungal or bacterial etiology.
b) Normal anatomical and racial variations, e.g., linea
alba, physiological racial pigmentation, leukedema,
c) Lesions caused by recent trauma.
• Compromised general health of the patient or a
history of coagulopathy or bleeding diathesis,
including patient on anticoagulant therapy.
• Proximity of lesion to vital anatomic, vascular,
neural or ductal structures and lesions in areas of
difficult surgical access.
• Pulsatile lesions or those suggestive of a vascular nature
should be referred for more in depth evaluation (e.g.,
• Intrabony radiolucent lesions should not be biopsied
without initial aspiration.
• Pigmented lesions generally should not be biopsied
incisionally (e.g. Spread of a melanoma, transformation
of premalignant pigmented lesions to malignant ones).
• Lesions that because of size or location present
technically difficult surgery e.g., posterior tongue and
oropharynx offer severe problems of access.
• Lesions that are clinically obviously malignant should be
biopsied only in the facility that will assure continual
VARIOUS BIOPSY TECHNIQUES
• The type of biopsy to be performed depends on the
location, size and clinical impression of the lesion.
Basic types include-
• Incisional, excisional, exploratory, punch biopsy,
needle biopsy, cytological smear, curettage biopsy,
Tissue may be obtained as:
• Tissue piece:
a. Incisional biopsy including geographical biopsies and
b. Excisional biopsy
d. Frozen section.
• Tissue core:
a. Fine needle cutting biopsy.
b. Tru-cut needle biopsy.
c. Vin Silverman needle biopsy.
• Cell aspirate:
Fine needle aspirate biopsy.
INCISIONAL BIOPSY / DIAGNOSTIC BIOPSY:
• It is a biopsy technique that samples only a
particular or representative part of the lesion.
• Using this technique multiple biopsy (that is, if the
lesion is large or has different characteristics at
different locations, more than one area of the
lesion need to be sampled) can be considered.
• This is often done by drawing a diagram of the
• It is chosen- 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.
• For central bony lesions (either cystic or solid) in
determining the nature and facilitate planning for
definitive removal/ reconstruction.
• Leaves noticeable scar.
• Risk of facial nerve damage.
• Possible spread of malignant cells
(detection of cytokeratine 19 reverse transcriptase in
peripheral blood drained 15 mins after incision of
squamous cell carcinoma, not detected in excisional
biopsy group or in controls.
If at all Incisional biopsy is necessary, chemotherapy
should be administered before or after biopsy thus
effective in preventing secondary metastasis)
• 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.
• Deep narrow biopsy should be considered rather
than broad, shallow one, because superficial
changes may be different from those deeper in the
• Necrotic areas should be avoided because it may
GEOGRAPHICAL BIOPSY AND VITAL
• Lesions with areas of varying appearances and in
cases of widespread dysplastic disease or field
changes within the oral cavity affecting whole of
palate, tongue or floor of the mouth may require
several biopsies, in such situation vital staining
helps to choose the appropriate area for biopsy.
• 1% aqueous toludine blue dye is applied to the
affected lesion for approximately 30 seconds
followed by a rinse with water.
• Then 1% acetic acid stain is applied and rinsed with
• Toludine blue stains the epithelial surface blue and
with the application of acetic acid the blue stain
from normal epithelium is lost whereas the stain is
retained in premalignant and malignant
erythematous lesions and are not decolorized by
• Toluidine blue is not a specific stain for cancer cells
but since it is acidophilic metachromatic nuclear
dye that selectively stains the acid tissue
component (particularly nucleic acid DNA), the
dysplastic and anaplastic cells as they contain more
of DNA than normal stains, they retain toludine
• The technique is helpful in differentiating the small
dysplastic erythroplakia that requires biopsy from
erythematous lesions caused by infection,
inflammation or trauma.
• In dysplastic or malignant lesions with diffuse
marginal pattern preoperative toluidine blue
staining indicates the border of a lesion serving as a
guide for surgical excision than does the clinical
• The technique cannot show tumor that is present
beneath the normal epithelium.
• Electro-surgery refers to the cutting and coagulation of
tissue using very high-frequency, low-voltage electrical
• A blended current combines cutting and coagulation,
and is useful in producing a bloodless operative field.
• 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
The lesion is grasped
with forceps through
the loop electrode.
The electrode is
under the lesion,
• It is the removal of the lesion in toto 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
• 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
• Fibromas and papillomas.
• Sialoliths and smaller benign lesions of accessory
• Certain cicatrial lesions
• Entire lesion along with 2 to 3 mm of normal
appearing surrounding tissue is excised.
• For surface excision simple elliptical approach is
designed, like for the excision of pigmented and
hyperkeratotic lesions, fibroma, papillomas and
• For deep soft tissue lesions, modified elliptical that
is combined with deeper dissection is chosen.
• 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.
• 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.
• 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.
• Haemostasis is achieved with resorbable suture
inserted (to control bleeding and also assist in
healing). Firm pressure for few minutes aid in
• 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.
• CURRETTE is a French word ‘Curer’- meaning to
• It is indicated for 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
• Although the sample produced is usually soft tissue
but it may include bone fragment as well.
• 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.
• The technique most often is used for total removal
of small lesions but also finds applicability in the
partial removal of superficial abnormalities.
• It is extremely useful when used on fixed tissue
such as firmly attached palatal tissue, which heal by
secondary intention regardless of the technique.
• The technique is used for oral mucosal
malignancies such as squamous cell carcinoma as
well as leukoplakia and other mucosal
abnormalities that may require multiple biopsies.
• It is also helpful to diagnose oral manifestations of
mucocutaneous and other vesiculoulcerative
• As a definitive surgical excision procedure of
suspected malignant lesions and cases of vascular
• Technique is fast with low incidence of post surgical
• Suturing is usually not required as the surgical
wound heal readily by secondary intention with
minimal or no scar formation and with maximum
• The need for a post operative or suture removal
visit is uncommon.
• The technique can be used on any mucosal surface
accessible to the biopsy punch.
• Technique is designed primarily for use with
epithelial or superficial mesenchymal lesions.
• It is difficult to use biopsy punch to obtain
adequate representative tissue deeper than the
superficial lamina propria.
• Freely movable mucosa that cannot be well
supported as with the floor of the mouth and soft
palate may preclude the technique.
• 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.
• Various types of biopsy punches are available –
- Keye biopsy punch
• Even disposable biopsy punches are available.
• 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.
• 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
• Local pressure with sterile gauze is sufficient to
• Persistent hemorrhage can be treated with
chemical cautery such as silver nitrate, collagen
matrix, or by electrocautery.
• Post- operative instructions include avoidance of
inadvertent trauma to the area, either by diet or
through attempts at oral hygiene for 48 hours.
• Warm salt mater rinses recommended for
• Non-steroidal anti-inflammatory agents are
preferred for post-operative discomfort.
• In surgical oncology, treatment of malignant
oropharyngeal tumors involves the excision of
tumor with 1 cm margin of normal tissue around
the tumor- this is termed as clear margin. Failure to
achieve this reduces the chance of local control (for
radiotherapy) and recurrence can be expected.
• To overcome this problem, frozen section analysis is
undertaken from the mucosal and deep surfaces of
the defect intraoperatively; and if the tumor
remains then further resection is undertaken at the
time of primary resection.
• To make an immediate surgical therapeutic
• To determine whether a lesion is benign, malignant
• Establish the adequacy of clearance of margin after
• Ascertain metastatic involvement of regional lymph
• It reduces the time of processing from 18 hours to
Methods of frozen section:
• Freezing microtome using CO2 gas
• Refrigerated microtome(cryostat)
• 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
• 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.
• In this type of biopsy slides cannot be preserved for
Errors in diagnosis can be due to:
• Sampling by the surgeon or pathologist.
• Interpretation by the pathologist.
• Difference in communication between the two.
• There can be error in sampling and interpretation of
frozen tissue as compared to routinely processed
• Differentiation between reactive epithelial changes is
• It has the disadvantage that only 8-16 micron thick
section can be cut and finer details of tissue can not be
• 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.
• 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.
• Cutting needle biopsy that employed the Biopsy
gun was first devised in the early 1980s.
• Studies assessing the clinical utility of cutting
needle biopsy using Monopty biopsy instrument
(18G needle), which is a newly introduced simple
disposable tool for performing cutting needle
biopsy in head and neck lesions like lesions of
lymph nodes, salivary glands, palate and soft tissue
have shown a accuracy of 88% with no significant
rush artifacts or obscuring blood, both of which are
problems associated with manual biopsy
• Core biopsy specimen provides accurate
information about cell type and tissue
characteristics in head and neck lesions. It helps to
determine whether a lesion is malignant or benign
before performing a total resection.
• One of the major complications with this technique
is the possible spread of tumor cells along the
large-bore needle track.
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.
• Stab incision with a scalpel
• Canula is inserted with the trocar fully retracted until
the specimen notch is with in the tissue to be
VIM SILVERMAN NEEDLE BIOPSY:1938
It consists of:
1)Outer canula 16 G in size.
3)Inner split longitudinal needle.
• Small incision made with the scalpel before the canula
and trocar are inserted up to the tissue to be
• The trocar is then completely removed and replaced
by the split cutting needle.
• They are easy to interpret then aspiration cytology to the
• To distinguish between reactive changes and recurrent
malignancy in possible cervical metastasis.
FINE NEEDLE ASPIRATION CYTOLOGY:
• Kun in 1847, gave the first description of the
technique for aspiration biopsy.
• Greg and Gray in 1904 used needle aspiration to
demonstrate the organisms in the lymph nodes.
• Franzem et al 1956 gave the technique of fine
needle aspiration biopsy which is used today
(needles of 21G or smaller).
1)The disease must be localized and clearly defined
by clinical examination or by any available
radiological investigating technique.
2)The most commonly diagnosed malignant lesion,
with this method is squamous cell carcinoma &
benign lesions are Pleomorphic adenoma and
relative lymph node hyperplasia.
• Aspiration of soft tissue pathology is employed only when
fluid is detected or suspected and in of little value in the
diagnosis of solid oral lesions.
• Although it is not a substitute for conventional biopsy but it
is valuable in producing immediate results and free of
complications and even helpful in distinguishing between
benign from malignant neoplasms, to initiate treatment, or
even to indicate the need for further investigation.
Other advantages include –
• The cost savings as compared to conventional open
biopsy, rapid and effective aid in diagnosis of swelling in
lymph nodes and parotid tumors.
• 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
•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
•Cuts down on bed occupancy for diagnostic investigations
•Absence of reactive fibrosis to interfere with subsequent
•No problems with wound healing, e.g. after radiotherapy
• 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
• Standard disposable needles (21 – 23G) are used for all
palpable lesions (for children and where the eyelids are
involved smaller 25 G needles are used).
• Thicker needles tend to cause more bleeding and are
prone to blockage.
• The needle is attached to a standard 10-20 ml disposable
syringe capable of producing good suction.
• The barrel of the syringe is supported with free hand and
the lesion is approached, as vertically as possible
• The needle is inserted into the lesion using no suction and
once the needle is within the lesion the change in
resilience confirms the entry of needle into the mass.
• Then suction or negative pressure is applied and is moved
back and forth in the lesion for 10 to 15 times at different
angulations making sure that the needle is within the
• When the mass has been adequately sampled, the
negative pressure is released from the syringe and the
needle is withdrawn.
• Then air is drawn into the syringe and the aspirate is
deposited on a clean-labeled microscopic slide.
• Usually 2-3 slides are prepared on each mass.
• One slide is immediately fixed in 95% ethanol
solution and subsequently stained with
Papanicolaou’s or haematoxylin and eosin stain.
• Another slide is allowed to dry for staining with a
May-Graunwald or Wright stain.
• Ideally the aspiration should have a creamy
consistency with a high cell and low fluid content.
• The number of needle insertion necessary for a
good aspirate depends on the consistency of the
• Even it is possible to perform FNAC using a 25 G
needle alone in cases of small lesions that is just
relaying on the capillary pressure which keeps the
cells within the needle lumen.
• This technique gives a better feel for the
consistency of the tissue and produces less blood in
• After any needle biopsy, direct pressure should be
applied over the site to reduce the incidence of
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
FRANZEN’S HANDEL WITH SYRINGE & NEEDLE FITTED ON IT
FOR PERFORMING FNAC
Causes of unsatisfactory yield with fine needle
• Needle missing a lesion and picking up
• Lack of cells in central area of necrosis,
hemorrhage, cystic change.
• Small malignant tumour being masked by larger
• Lack of cells in dense fibro sclerotic tissue.
COMPLICATION & HAZARDS OF FNAC
•Haematoma: Bleeding from puncture site &
haematoma formation are commonest complications of
the procedure. Firm finger pressure for 2-3 minutes
immediately after procedure reduces the frequency of
•Infection: Introduction of infection is not a significant
•Dissemination of tumour: Local dissemination by
seeding of malignant cells along the needle tract is a
rare complication & reported in cancers of lung,
prostate & pancreas. 108
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
•Requires clinical information or relevant investigation
(e.g. x-ray finding), which further limit utility of FNAC.
USE OF FNA:
• Commonest indication is to distinguish between
both benign and malignant neoplasia as well as
• Secondly to differentiate between a local
recurrence or nodal metastasis
• Lymph nodes – FNAB is an excellent initial
diagnostic modality in the evaluation of
lymphadenopathy. Many infectious processes can
be diagnosed because cultures may be obtained
from bacteria and fungus.
Aspirates from enlarged lymph nodes can
• Reactive hyperplasia or inflammation.
• Malignant disease.
• It is also used to confirm the cervical lymph node
metastasis from previously treated local neoplasms.
Bone and Cartilage
• Aspiration using 21 or 23G needles can be
undertaken if the cortical bone is thin or absent.
• A larger bore 18G needle can be used to gain
access to the lesion prior to inserting a smaller 25G
needle for the actual aspiration, or access may be
gained by penetrating the cortical wall with a small
• Aspirated blood often indicates a vascular lesion
e.g., hemangioma or aneurysmal bone cyst.
• Withdrawn air suggests likely entry into the
maxillary sinus or a traumatic cyst.
• Aspirated serous fluid sometimes glistering with
cholesterol crystal is indicative of a cyst.
• If aspiration of a central bony defect is
nonproductive, the probability of a solid lesion, i.e.
neoplasia or tumor, exists
• Skin and soft tissue - It is usually possible to obtain
tissue by incisional at excisional biopsy, but FNA is
possible on all lesions larger them 5 mm.
Salivary gland swelling –
• Incisional or cutting biopsies are contraindicated owing to
risks of tumor seeding or fistula formation.
• Thus FNAB is most widely used in the assessment of salivary
• The primary indication for FNAB of salivary gland is to
distinguish among benign, malignant and inflammatory
• The accuracy percentage is higher for benign as compared
to malignant tumors with a sensitivity and specificity of 80%
• Regarding the confusion over the use of the terms
aspiration biopsy and aspiration cytology.
• If an aspirate of cells is obtained using fine needles
(21-25G) the technique is called “fine needle
aspiration cytology” (FNAC) or fine needle
• Whereas, if a core of tissue is produced using larger
bore needles (14-18G), the procedure is best
referred to as fine needle cutting biopsy (FNCB) or
true cut biopsy
• Aspiration biopsy is the use of a needle & syringe to
penetrate a lesion for aspiration of its contents for
purpose of analysis.
• Applicable to both intra osseous as well as soft
Used to rule out &/or differentiate
• Fluid filled cavities
• Vascular lesions
• Empty cavities
• Cyst (when lesion is cystic fluid is yellow in color
occasionally red due to presence of blood)
INTRAOSSEOUS OR HARD TISSUE BIOPSY
TECHNIQUE AND SURGICAL PRINCIPLES
•A lesion either on or within the osseous tissues of the
jaws requires investigation. The most common
intraosseous lesions we encounter are periapical
granulomas and cysts of the jaws. Because these have a
characteristic radiographic appearance and are usually
asymptomatic, a presumptive diagnosis is frequently
•Treatment may involve surgical removal of the cyst in
the form of an Excisional biopsy. When a lesion is large,
perforating into soft tissues, or suspected of
malignancy, incisional biopsy is indicated.
•Before performing hard tissue biopsy, the dentist
should carefully palpate the area of the jaws where the
suspect lesion is located. Comparison with the
opposite side is helpful. Bone that feels smooth and
firm to palpation usually indicates that the lesion has
not expanded or eroded the cortical plate.
•A spongy feel to the jaw when compressed indicates
erosion or thinning of the cortical plates.
Aspiration biopsy of radiolucent lesions
•Any radiolucent lesion that requires biopsy should
undergo aspiration before surgical exploration. This
provides valuable diagnostic information regarding
the nature of the lesion.
•For e.g, brisk, pulsating blood may indicate a
vascular lesion, which should not undergo surgical
exploration by the general dentist. The return of
straw colored fluid would corroborate presumptive
diagnosis of a cyst, and surgical removal can then
be undertaken without hesitation. The aspiration of
air may indicate that then needle tip is within the
maxillary sinus or a traumatic bone cavity.
•Because of their location within or proximal to the
jaws, most lesions of hard tissue must be approached
through a mucoperiosteal flap. The choice of flap
depends chiefly on the size and location of the lesion.
•The size of the lesion dictates how much access is
necessary when Excisional biopsy is indicated. Access
may necessitate extension of the mucoperiosteal flap.
•The location of the lesion dictates where the flap
incisions are to be made. It is important to avoid major
neurovascular structures when possible and to keep
incisions over sound bone for closure. Optimally flap
design should provide 4 to 5 mm of sound bone around
the anticipated surgical margins.
•A central lesion that may have eroded the cortical
plate of the jaw is always approached with flap
elevation in an area away from the lesion and over
sound bone. This technique allows establishment of the
proper tissue plane for dissection.
• All mucoperiosteal flaps for biopsies in or on the jaws
should be full thickness and incised through mucosa,
submucosa, and periosteum. The dissection to expose
bone is always performed subperiosteally
•Lesions within the jaw (i.e., central lesion) require the
use of a cortical window.
•If expansion of a central lesion has eroded the cortical
plate to the point that an osseous void is seen once the
flap has been elevated, this window can be enlarged
with rongeurs or burs.
• If the cortical plate is intact, a rotating bur should be
used to remove an osseous window.
• The size of the window depends on the size of the
lesion and the proximity of the window to normal
anatomic structures such as roots and
• Once the window has been created, it can be
enlarged with a rongeur. The osseous window
specimens should always be submitted for
histopathologic examination with the primary
Removal of the specimen
•The technique for removal of the biopsy specimen
depends on the nature of the biopsy (excisional versus
incisional) and the consistency of the tissue
•Most small lesions that have a connective tissue
capsule (e.g., cysts) can be removed in their entirely. A
dental curette is used to peel the c.t. wall of the
specimen from surrounding bone. The concave
surface of the instrument should always be kept in
contact with the osseous surface of the bone cavity.
•The convex surface of the instrument is the portion
that actually separates the specimen from
surrounding bone. This technique is used until the
specimen is free and removed.
•The bony cavity is inspected after irrigation with
sterile saline. Any residual fragments of soft tissue
within the cavity should be removed with curettes.
•Once the cavity is devoid of residual pathologic
tissue, it is irrigated and flap is replace and sutured in
its proper location.
Bone marrow aspiration
• Site: Sternum, Posterior iliac crest
• SALAH BM aspiration needle is used (strong wide
bore needle with stylet)
• Stains with Romanowskys stain pearls reaction for
iron on smears
• Indication: anemias, leukaemias, granulomatous
• Time in 1-2 hrs
• Jamshidi Trephine needle is used
• H&E staining
• Time in 7 days
• Better marrow architectural pattern but cell
• Indications: aplastic anemia
Available in a range of sizes,
from 7 guage to 12 guage.
Needles in the market, with
lengths from 27mm to
All Needles in this range have
a mm scale, providing more
precise penetration, and a
beveled tip cannula which
makes penetration much
The needle also features a
rapid, safe, locking knob. 132
A biopsy needle retrieves a sample of bone and it is
sent for examination.
• Bone marrow biopsy is a diagnostic procedure
commonly used to
(a) detect and stage malignancy,
(b) differentiate benign hematologic diseases (e.g.,
aplastic anemia, macroglobulinemia), and
(c) evaluate progression of human immunodeficiency
• Bone marrow evaluation can classify an anemia as
hypoproliferative, a maturation disorder, or resulting
from hemorrhage or hemolysis
• Other indications for bone marrow examination
include evaluation of immunodeficiency
syndromes, confirmation of unusual infections in
the marrow (e.g., miliary tuberculosis, fungi), and
sampling of marrow for chromosomal analysis
• 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
• Metastatic disease
• Minimal residual disease in lymphomas and
• Chromosomal abnormality
• Immunodeficiency syndromes
• Fever of unknown origin
• Caution should be exercised in patients with soft
bones secondary to radiation therapy, multiple
myeloma, or osteoporosis.
Tests on Bone Marrow Aspirate
and Biopsy Specimens
Performed on stained smears of all aspirate
and biopsy specimens. Determine cell lineages,
relative abundance of hematopoietic precursor
cells. Biopsy gives more information
on marrow cellularity, presence of aplasia, and
Stains used to enhance cell characteristics:
H&E, periodic acid-Schiff, Prussian blue
for iron stores, Wright-Giemsa for details of
nuclei and cytoplasm, trichrome for collagen
tissue, silver for myelofibrosis or tumor
metastases, peroxidase and esterase for acute
(AML) from acute
Analyze B-cell and T-cell
surface markers in
lymphoid neoplasms and
Determine lineage and
stage of differentiation.
Cytogenetic analysis Detect chromosomal
changes in leukemias,
Iron stains Perform on all aspirate
and biopsy specimens to
assess iron stores,
anemia, and iron
reaction, fluorescence in
Establish clonality of a
determine cell lineage,
rearrange B-cell and Tcell
receptor genes, and
detect minimal residual
disease in leukemias.
Microbiologic cultures Detect bacterial, viral,
and fungal infections in
patients with fever of
unknown origin and
• Palpation of the area of the lesion with comparison to
the opposite side.
• Any radiolucent lesion should have an aspiration biopsy
performed prior to surgical exploration.
• Information from the aspiration will provide
valuable information about the lesion.
• Fluid Filled
• Without Contents
• Is one of the ways to remove skin tissue for a
• 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.
• Depending on lesion size and morphology,
anesthesia may or may not be necessary.
• Small forceps with teeth and a pair of sharp curved
or straight iris scissors are the only surgical
• 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.
• Bleeding after this procedure is usually minimal and
can be easily controlled by application of 35%
aluminium chloride solution
• 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
• Skin lesions with a minimal dermal component,
such as seborrheic keratoses or fibrous papules are
excellent candidates for shave excision technique.
The blade is held horizontal to
the skin surface and
brought below the lesion.
The other hand is used to
stretch and stabilize the skin
surrounding the lesion
during the shave biopsy.
Smooth, unidirectional cutting
with the blade separates
the lesion above from the
deep (reticular) dermis
• Most tumours that are visible or palpable can be
examined without the aid of radiologic imaging,
whereas for deep lesions that cannot be palpated,
as well as for small, deep mobile lesions that are
difficult to palpate require radiologic control to
ensure that target tissue sample is obtained and
secondly this guidance reduces the number of
aspirates and helpful in differentiating the tissues
within a lesion.
• Ultrasound, computed tomography and MRI are
used in percutaneous biopsy procedures
Ultrasound guided biopsy
• 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
• Another advantage is that, unlike other
radiographic biopsy procedures, ionizing radiation
is not used for imaging.
• However, Ultrasound guided biopsy is not possible
when gas/bone prevents the visualization of the
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.
1. Lymph nodes or masses that are not completely
identifiable using ultrasound.
2. Lesions near the skull base: CT is optimal for
localizing these lesions.
Disadvantages of CT include
• radiation exposure,
• limited possible scan plane orientations,
• low soft tissue contrast, and,
• poor vessel conspicuity without administration of
intravenous contrast medium.
• 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
• 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.
• The mean procedure time for MRI-guided needle
insertion per pass is less than 10 minutes for
aspiration as well as core biopsy.
Endoscope guided biopsy
• Endoscopy is defined as “the examination of the
interior of a canal or hollow viscous by means of an
• 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.
• Endoscopy may prove to be an important tool for
the internal examination of large jaw cysts that may
contain regional neoplastic processes within the
• Especially in the case in large cysts that extend into
areas that are difficult to inspect and sample
through a standard “bony window” technique
• Used for inrtaosseous lesions of maxilla and
• Instruments: Chisel, Bone burs, Periosteal Elevator
UNEXPECTED/ UNPLANNED BIOPSY
• When as a result of surgical procedure (Tooth
extraction) some suspicious tissue is obtained
• Cyto – Cell -- Logos – Study
• Study of cells.
• Rudolf Virchow (1955) stated “Every cell is derived
from a cell & that human disease processes were
essentially disease of the cells.”
Rudolf Virchow (1821-1905)
“ THE FATHER OF CELLULAR PATHOLOGY” 175
Dr. George N.
1920s “ father of
Developed PAP test
for diagnosis of
• Normal oral squamous epithelium continuously
sheds the most superficial cells.
• If malignant or other disease processes affect the
area, the deeper cells lose their cohesiveness and
are exfoliated at the same time as the superficial
• Exfoliative cytology 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
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.
• 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.
• Mass screening of oral cancer.
• 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.
• For the study of buccal mucosa in various anemia.
• Certain benign hereditary skin lesions having their
representative oral manifestations.
• For the study of the change of the oral epithelial
cells followed by chemotherapy.
• 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.
• Atrophic lesions.
• Densely keratinized lesions.
• Smooth surface lesions.
• Lesions giving inadequate specimen sampling
through the adopted technique.
• Patients with underlying blood dyscrasias.
• 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.
• It is quick, simple, painless, bloodless, non-
• If guards against false negative biopsies.
• It helps to follow up recurrent carcinomas.
• It is valuable for screening of clinically non-
• Easy and a most feasible method for detection of
malignancies in oral epidemiological survey
• It gains to evaluate the emergent concepts of
proclaimed cancer, ca-in-situ, and related lessons of
the oral cavity.
• Relatively limited information provided with exfoliated
material when compared to a histological preparation.
• 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
• 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
Target for cytological Prediction:
• Squamous cell Carcinoma remains the chief target
for cytological investigation.
• Shed cells with less cohesion or poor adhesiveness
and red oral lesions usually considered as benign
are the prime targets for cytological smearing.
Requisite for diagnostic cytology:
• Diagnosis must be based on the synthesis of entire
evidence available rather than the changes in
• Adequate clinical data.
• Uniformity of the technical method employed.
• Thorough knowledge of normal cells and extensive
physiological variation of the sites to be diagnosed.
• Instruments for removing the cells should have a
90-degree angle or straight blade.
• The cement spatula used in mixing crown and
bridge cement at wax carver is ideal.
• A tongue blade slit longitudinally is a second
choice, but it should be moistened to avoid
dehydration damage to the cells.
• The cotton swab has been recommended in
• 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.
• Smearing should be done on a standard glass slide
(1x3 inches) with an etched label area atone 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
• 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.
• 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.
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: -
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
Class V (Positive for cancer) obvious malignant cells. Biopsy is
Touch impression or Imprint
• 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.
• Feasible and economical
• Detect the malignancy at the tumor margin
• Less time consumable
• 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).
This is particularly useful in -
• In the diagnosis of certain neoplastic lesions which
can simulate inflammatory lesions on frozen
• In the diagnosis of certain benign inflammatory
lesions which can simulate malignancy on frozen
• In the diagnosis of malignancy confined to one
small area of a large specimen
The amount of tissue that can be frozen for rapid
intraoperative diagnosis is limited. On the other
hand, the imprint technique can easily cover a larger
portion of the specimen, thus reducing errors due to
• In the diagnosis of malignancy when the submitted
specimen is limited in quantity
Small fragments of tissue, which may prove to be
difficult for frozen-section interpretation, are often
large enough to provide sufficient cells for imprint
Imprint of a massively necrotic carcinoma showing a
cluster of viable cancer cells on a necrotic background.
Diagnosis was not possible on frozen section.
Imprint of a lymph node showing malignant cells.
• Wide mouthed bottle with 10% formalin (at least
20 times the volume of the specimen should be
• For exploratory biopsy – bone burs, chisel,
periosteal elevator and curette are included.
• Electric cautery should not be used for removal of
tissue because the surgical margins get coagulated.
Cautery can however be used on a postsurgical site
in order to control bleeding.
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.
• 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.
• 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
• Sterile technique should be utilized both to prevent
infection, which complicates the diagnostic procedure,
and to avoid contamination of the specimen.
• 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
• The anesthesia should be subjected slowly enough to
provide blanching but not rapid swelling.
•Soft tissue biopsies in the oral cavity are frequently
performed on movable structures, such as the lips,
soft palate, and tongue.
•Accurate surgical incisions are easiest to perform on
tissues that are properly stabilized.
•Several methods are available to achieve tissue
stabilization. An assistant’s fingers pinching the lip on
the both sides of the biopsy area can immobilize the
•This method also aids in hemostasis by compressing
the labial arteries. Instruments are available to
perform this same function.
•Heavy retraction sutures or towel clips can be used
to aid immobilization of the tongue or soft palate.
When used, the sutures should be placed deeply into
the substance of the tissue, away from the proposed
biopsy site. They will be useful for secure
stabilization without pulling through the tissue
• The chalazion clamp, used by ophthalmologists, is a
helpful tool for oral biopsies on the oral lips,
anterior buccal mucosa, or tongue. This clamp, with
a solid metal back and ring like opening anteriorly,
is tightened in place around the lesion to be
• It performs the two important functions of
providing a firm surface to work and yields nearly
• Sutures can be placed in the center of the ringed
opening before the clamp is loosened.
•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.
•Gauze wrapped over the tip of a low-volume suction
device or simple gauze compresses are adequate in
most cases, unless severe haemorrhage is
• Manipulation and grasping of any lesion suspected of
being a tumor should be minimal. Vigorous
manipulation of malignant tumors can cause tumour
• Suturing is not often necessary when small incisional
biopsies are taken, but most excisions require
suturing. Black silk suture material in preferable.
• Orientation of the specimen is extremely important to
the pathologist Multiple lesions must be adequately
labeled and often oriented with a diagram of the
• A black silk suture placed through each end,
preferably in normal tissue will often serve to orient
• Orientation of mucosal biopsies (particularly
superficial lesion) is important, especially because
they are small and have limited morphologic
characteristics after being immersed in formalin.
• Proper orientation of the surface lesion specimen
assists the oral pathologist in sectioning the
specimen to avoid tangential cuts.
• Improper orientation will lead to the sectioning of
either epithelium or the connective tissue alone,
but not both.
• Surgeons can place sutures in the specimen to
assist in orientation and provide a written
description of the specimen in relation to suture.
• At least two adjoining margins must be clearly
identified to ensure correct orientation, with the
help of short suture and a long suture
• Fixation and preservation of tissue are 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
• Other fixatives, which are used with satisfactory results,
include parachlorphenol (camphorated), sodium
hypochlorite, and Zephesin.
• 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. 221
• 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.
• Container used for fixation should have a wide
mouth, so that after fixation the specimen can be
• Containers that have caps that will rust should be
avoided since the iron oxide will often interfere
markedly with the staining reaction in the
• If culture is desired, then the tissue should not be
placed in fixative until the material for
bacteriological study is obtained, either by
smearing the tissue on a plate or by placing it for a
short time in a nutrient medium.
• If formalin is not available at hand, place the
specimen in refrigerator at 4oC to slow down
autolysis. The container should have an opening
larger enough so that the tissue can be removed
easily after it has hardened by fixation.
However, fresh material is needed for the following
1. Frozen section
3. Cytological examination
4. Microbiological sampling before histopathology
5. Chromosome analysis
6. Research purpose
7. Museum display
• No fixative will penetrate a piece of tissue thicker
than 1 cm. For dealing with specimen thicker than
this, following methods are recommended:
1. Solid organ: Cut slices as necessary as but not
thicker than 5 mm.
2. Hollow organ: Either open or fill with fixative or
pack lightly with wool soaked in fixative.
3. Large specimen, which require dissection: Inject
fixative along the vessels or bronchi as in case of lung
so that it reaches all parts of the organs.
• 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
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
• Factor affecting fixation:
1. Size and thickness of piece of tissue.
2. Tissue covered by large amount of mucous fix
3. The same applies to tissue covered by blood or
organ containing very large amount of
4. Fatty and lipomatous tissue fix slowly.
5. Fixation is accelerated by agitation.
6. Fixation is accelerated by maintaining temperature
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.
• 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.
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
• Investigations done, if any
• Site and biopsy type
• Clinical diagnosis with differential diagnosis
• Previous biopsy done, if any, with details.
Follow-up and Reporting of Biopsy Result to the
• 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.
• If the microscopic diagnosis is inconsistent with the
clinical impression, the clinician is strongly advised
to discuss any concerns directly with the
General Principle of Gross
1. Proper identification and orientation of the
2. Unlabelled specimen should never be processed.
3. A properly completed histopathology requisition
form containing patient’s name, age, sex, relevant
clinical data, surgical findings, nature of operation
and name of tissue submitted.
4. Careful search and examination of all the tissue
submitted in order.
5. Surgeon should be instructed to submit all the
material that they have removed, not the selected
portion from it.
6. Place the specimen on cutting board in an anatomic
position and record the following information:
a. Types of specimen
b. Structure included.
h. Surgical margin, whether included or not involved by
7. Measurements are usually given in centimeter
unless the specimen is very small in which mm can
8. Tissues should be measured by aggregate pieces in
9. Endoscopic biopsies should be numbered.
• The fixed tissue is dehydrated by immersion in a
series of solvent and impregnated with paraffin
• The block should not be more than 3 mm thick and
not larger than 20 mm on the sides. Otherwise the
chemicals will not adequately infiltrate them during
processing (automatic / manually hand processed)
• The paraffin block containing the tissue is mounted on a
cutting machine microtome and tissue section of 3-6 mm
thickness are cut.
• The sections are then placed on glass slides with an
• The paraffin is dissolved out of the section by immersing
in xylene followed by rehydration of tissue by passing
through graded series of alcohol solutions to water.
• The tissue is then stained with haematoxylin & eosin -
most commonly used stain, this stained section is then
dehydrated in alcohol, immersed in xylene and mounted
with a cover slip using a mounting media
• When the mounting media dries, the specimen is ready
for microscopic examination and storage.
• It takes 24-48 hours to fix, process, section and stain a
specimen before the pathologist can report on it.
• Thus a typed diagnostic report should normally be ready
by third day evening in most of the cases where
diagnosis is straight forward.
• In rest of the cases, there may be need of additional
sampling of tissue blocks, study of deeper level section
of existing blocks, or special stains including immuno
histochemical procedures before a firm diagnosis can be
• For specimens containing bone and teeth - they
need to be softened by decalcifying in acid to
enable a thin section to be cut. They delay the
diagnosis by days or weeks according to the size of
• Furthermore, tissue may also be wasted on the part
of the pathologist in attempting to gather adequate
clinical information in cases where such
information is not provided in the request form for
• Pathologists and clinicians sometimes request slide
• For difficult specimens general and oral pathologist
consult with one another – if variation in opinion
as to the exact nature of the lesion exerts then
clinical should be extremely caution in deciding on
the final management of patients.
• Sometimes, there can be discrepancies between
the diagnosis or interpretation and the clinical
finding – in such cases features should be
reevaluated with consultation between pathologist
and clinician, and there should always be a frank
discussion of terminology and the clinical and
• And even pathologist can have a look in the lesion
before biopsy if the location or clinical features of
the lesson are unusual.
• Generally the stains are classified as:
A. Acid stains
B. Basic stains
C. Neutral stains
• All dyes are composed of acid and basic
components. Dye is a compound which can colour
fibres and tissue constituents.
• Acid Dyes:
In an acid dye the basic component is coloured and
the acid component is colourless. Acid dyes stain
basic components e.g. eosin stains cytoplasm. The
colour imparted is shade of red.
• Basic Dyes:
In a basic dye the acid component is coloured and
the basic component is colourless. Basic dyes stain
acidic components e.g. basic fuchsin stains nucleus.
The colour imparted is shade of blue.
• Neutral Dyes:
When an acid dye is combined with a basic dye a
neutral dye is formed. As it contains both coloured
radicals, it gives different colours to cytoplasm and
nucleus simultaneously. This is the basis of Leishman
1. PAS (Periodic Acid Schiff) stain: This stain
demonstrates glycogen and neutral mucous substances,
outlines basement membranes and reticulin and makes
evident most types of fungi and parasites.
2. Stains for micro-organism:
a. Gram-stain: Gram stain allows the separation of
bacteria those that retain the crystal-violet-iodine
complex (gram-positive) and those that are decolorized
by alcohol treatment and counterstained by eosin,
safranin or fuchsin.
b. Ziehl_Neelsen stain: This stain detect acid fast bacilli.
c. PAS stain: It is used for fungi, amoeba and Tricomonas.
d. Modified Giemsa (2% Giemsa in water): Detects
3. Congo-red: It is used for identification of amyloid.
4. Sudan-Black: It is used for fat staining.
5. Masson’s Trichrome: It is used for differentiation of
connective tissue elements.
6. Papanicolaou’s stain: It is used to stain cells in cervical
and sputum smear for cytology.
In large lesions
For multiple lesions
Most representative site.
Material curetted from interior of the lesion .
For red & white lesions include both red & white
deep part of
ulcer and site of
HANDLING THE TISSUE
Handle tissue with minimum force
USE OF FORCEPS
Avoid use of forceps on the surface of biopsy.
USE OF ELECTROCAUTERY
Topic of debate
Combination of cautery and scalpel should be considered.
SELECTION OF THE SPECIMEN
Tissue should be representative of whole lesion.
Adequate amount of tissue at least 1 x 0.5 cm in size.
Biopsy of skin or mucosa representative sample of
both epithelium and connective tissue has to be
SELECTION OF FIXATIVE
Depends on purpose and method of tissue processing
10% neutral buffered formalin
Request for 10% formalin
From the Pathology Lab
Do not add tap water to dilute the fixative
PURPOSE OF FIXATION
To retain tissue as much as possible in living condition
CONTAINER OF FIXATIVE
Wide mouth clear bottle
INTRODUCING INTO FIXATIVE
Avoid curling of tissue.
DELAY IN FIXATION
Leads to artefacts.
Formalin kept for longer will not fix tissue properly
LABELLING THE BOTTLE
Patients NAME , AGE , SEX , HOSPITAL NUMBER
• Disadvantage of formalin is that it makes the
specimen unsuitable for immunofluorescent
techniques as it forms protein cross- linkings.
• Perilesional skin is used for immunofluorescence.
• Quick freezing is the most widely used method for
handling biopsy specimens for immunofluorescent
• This can be performed by immersing the biopsy
specimen immediately after biopsy either in liquid
nitrogen or cold solid carbon dioxide or in a hexane
• Because rapid freezing of specimens require special
supplies and keeping them frozen during
transportation is a packaging challenge, an
excellent alternative is to place the specimen in a
room temperature transportation medium that
permits convenient transport to the laboratory for
• At present, immunofluorescence can be performed
with biopsy specimens handled for several days at
ambient temperature in a preservative liquid
medium, Michel’s medium.
• The value of entire hospital admission for diagnosis
biopsy is all too frequently negated by the loss of
• This can be prevented only by a definite and
formed routine which is rigidly enforced by the
Mislabeling of specimen:
• Another error which can easily occur is for the
specimens to be improperly labeled, so that
material from one patient becomes confused with
that from another, or the biopsy from one site is
confused with that from another.
• A meticulous routine of immediate labeling of all
material will minimize such error.
Failure to note orientation of the specimen:
• Failure to identify the orientation of the mass of
tissue removed in enbloc dissection will result in
confusion, if the pathologist reports that the tumor
was inadequately excised at one point.
"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."
CAUSES OF TISSUE ARTIFACTS
• Clinical application of chemicals.
• Local injection of anaesthetics.
• Surgical sectioning.
• Excessive heat.
• Surgical mishandling of the specimen.
• Inadequate fixation.
• Improper fixation medium.
• Faulty tissue processing.
• Improper staining.
2.DURING FIXATION AND TRANSPORT:
• Fixation Artifacts
• Freezing Artifacts
• Artifacts during transportation
3.TISSUE PROCESSING ARTIFACTS:
• During Embedding
• During Sectioning
• Injection of large amounts of anesthetic solution
into the area to be biopsied can produce following
1) The needle insertion may produce hemorrhage
with extravasation, which in turn masks the
2) The separation of connective tissue bands with
vacoulation may occur.
3) Tissue edema or distortion.
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.
• 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.
• 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
• If the use of forceps is mandatory, only the
peripheral aspect should be used for holding and
delivery of the specimen.
• 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.
• 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.
• 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.
• The fibrous connective tissue, fat and muscle gain
an amorphous appearance when subjected to such
• 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
• Alternating areas of coagulation impart a "Herring
Bone" appearance to the tissue if the procedure is
not done properly
• 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
• 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.
ONCOCYTOID ARTIFACT IN SALIVARY GLAND
• This unusual tissue artifact occur in the serous acini
of parotid gland tissue.
• Many of these have been misinterpreted as
oncocytosis, oncocytoma and both. Serous acinar
cells resemble oncocytes.
• The two cells can be distinguished by the fact that
the altered acinar cells possess uniform round
nuclei in the basal half of the cells like their
unaffected counterparts. In contrast the oncocytes
have a centrally placed nucleus.
• It is believed that the oncocytosis artifact of salivary
gland tissue subjected to electrocautery is a
consequence of electrothermal discharge.
• Lasers are being used for excision and ablation of
oral mucosal lesions with the chief advantage being
minimization of intraoperative haemorrhage.
• Produces the thermal artifactual changes.
• 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
ARTIFACTS INDUCED BY SUCTION TIPS
• Induced by the vacuum effect of the surgical
• 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.
• In highly vascularised tissue such as tissue in
periapical granulomas and in inflamed odontogenic
• Suctioning induces extravasation of blood and focal
accumulation of erythrocytes within the connective
• Although fixation is used to prevent the occurrence
of various artifacts, it may itself be the cause of
• The most common of these are:
• Volume changes.
• Artifactual pigments.
• Diffusion artifacts
• Various factors have been suggested such as
inhibition of respiration, changes in membrane
permeability and changes in ion transport through
the membranes etc.
• This pigment appears as a brown/ brownish black
deposit in tissues fixed in any formalin fixative with
an acid pH and is more likely to occur in blood rich
• Tissues fixed in fixative mixtures containing
mercury have been found to develop a uniform
granular black deposit known as mercury pigment.
• Tissues fixed in potassium dichromate containing
fixatives show an insoluble yellow-brown oxide
precipitated if transferred directly to alcohol
without proper washing in water.
• This important group of artifacts is related to the
diffusion of unfixed material to give a false
localization by coming to rest in some place other
than it's original location.
• Because of relatively slow penetration of the
fixatives, the large molecules tend to diffuse during
the period of fixation. Well-known examples of
such localization occur with glycogen i.e. streaming
• Materials may also diffuse out the tissue and may
lead to errors where localization of these materials
is important for diagnosis e.g. Enzymes in
• Delay in fixation and inadequate fixation alters the
staining quality of the cells.
• Cells appears shrunken and shows clumping of
• The nuclear chromatin was indistinct and nucleoli
were sometimes not seen.
• Vascular structures, nerve and glands exhibited loss
of detail and the impression of scar formation and
loss of cellularity was created.
HOW TO AVOID
• For optimal fixation the amount of fixative should
be approximately 20 times the volume of the
• The morphology of a tissue specimen will be
altered by use of different fixatives.
• These are characterised by the formation of
interstitial vacuoles within the cell cytoplasm
resulting from ice crystal formation.
• Freezing artifacts are an inadvertent occurrence
that may destroy tissue.
• This may result from freezing prior to fixation or
• Swiss cheese holes in the epithelium and tissue
spaces are seen where forming ice crystals rupture.
HOW TO AVOID
• As formalin freezes at 12.2°F, containers should
have adequate insulation from extreme cold and
should contain sufficient amounts of fixative.
• Freezing artifacts can also be avoided by using
Lillie's AAF (Acetic Alcohol Formalin)
ARTIFACTS DUE TO TISSUE PROCESSING.
• Small biopsy specimen, often a narrow strip of
delicate mucosa, will bend and twist on fixation.
• When this occurs, the orientation between
connective tissue and the epithelium is lost,
especially if the tissue does not possess an
identifiable submucosa or muscle base.
• This problem may be avoided if the surgeon would
label the top & bottom or the side with a through &
• Utilization of sponges has been advocated as an
efficient means of holding specimen during
• The degree of sponge penetration is related to the
consistence of the tissue and dependant on quality
and quantity of stroma, matrix and vasculature.
• With increased sponge thickness or multiple
sponges there is an increased penetration of the
• These artifacts can be caused as a result of:
1) Improper processing.
2) Improper sectioning.
HEALING OF BIOPSY WOUNDS
• The healing of a biopsy wound of the oral cavity is
identical with the healing of a similar wound in any
other part of the body and thus may be classified as
either primary healing or secondary healing.
• The nature of the healing process depends upon
whether the edges of the wound can be brought into
apposition, often by suturing, or whether the lesion
must fill in gradually with granulation tissue.
• Primary healing, healing by primary intention or
healing by first intention is that type of healing
which occurs after the excision of a piece of tissue
with the close apposition of the edges of the wound.
• This is the form of healing one might expect after
the excision of a lesion in an area of the oral cavity
where the pliability of the tissues is such that the
wound may be drawn together and sutured.
• When the edges of the wound are brought into contact
and held in place by sutures, the blood clots, and in a
matter of hours numerous leukocytes are mobilized to
• Connective tissue cells in the immediate
vicinity undergo transformation into fibroblasts which
in turn undergo mitotic division, and the new
fibroblasts begin to migrate into and across the line of
• In time, these cells form thin, delicate collagen
fibrils which intertwine and coalesce in a general
direction parallel to the surface of the wound.
• At the same time, endothelial cells of the capillaries
begin to proliferate, and small capillary buds grow
out and across the wound and form new capillaries
which fill with blood, and a rich network of young
capillaries and capillary loops are formed.
• When there is a close apposition of the edges of the
wound, the surface epithelium proliferates rapidly
across the line of incision and reestablishes the
integrity of the surface.
• The delicate connective tissue fibrils eventually
coalesce into denser bundles and usually contract.
• Shows a small linear scar which may be depressed
below the surface.
• Wound heals rapidly.
• Healing by second intention, healing by granulation
or healing of an open wound occurs when there is
loss of tissue and the edges of wound cannot be
• Removal of a lesion of the palate or a large lesion of
the alveolar ridge is usually followed by healing by
second intention, since the edges of the wound
cannot be coapted.
• After the removal of the lesion, the blood filling the
defect clots and the repair process begins.
• It is basically identical with healing by primary
intention except that the fibroblast and capillaries have
a greater distance to migrate; more granulation tissue
must form, and the healing is slower.
• Cellular proliferation begins around the periphery of
the wound, and the fibroblasts and endothelial cells
grow into the clot along fibrin strands.
• Polymorph nuclear leukocytes and, later,
lymphocytes, and mononuclear phagocytes migrate
into the granulation tissue from the adjacent vessels
• As the granulation tissue matures, it becomes more
fibrous through condensation of collagen bundles,
and the surface of the granulation tissue becomes
• The lesion becomes less vascular, the only evidence
of the wound may be a small depressed area of the
1. 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)
• 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.
2. 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
• Aseptic technique must always be observed
3. Poor wound Healing
Unsatisfactory healing of the incision may be due to
• Ischemia of the skin overlying a tumor mass (tension)
• Implantation of tumor cells
• Previous X-ray therapy
4. 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
5. Injury to adjacent organs
• Injury may occur to surrounding structure (blood
• 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.
• Post operative pain.
• Paraesthesia - in the lips or the tongue,
• Swelling and bruising - in the tongue, lips and
• 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’.
• 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
• 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
• 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
•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