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GOOD MORNING
1
BIOPSY
One can’t show this when it’s not there
One can’t hide this when it’s there.
TABLE OF CONTENTS
• Introduction
• History
• Definitions
• Indications & Contraindications
• Classification
• Various biop...
• Errors
• Biopsy artifacts
• Healing of a biopsy wound
• Complications
• Conclusion
• References
4
Introduction
• The clinical presentation of any pathology can be
the mucosal surface change (i.e., change in texture,
ulce...
• Among all these investigations, obtaining an
accurate tissue diagnosis is the one needed in the
process of forming a def...
• There are many methods used to perform this
procedure. But whatever the procedure, the aim of
it should be to provide a ...
HISTORY
8
• In the early 16th century, Sir Marcello Malphigi was
the one who formulated the basic microscopic
technique of utilizing...
• The term "biopsy" was introduced into medical
terminology in 1879 by Ernest Besnier.
• "... Not only skin changes (collo...
• In this quote from an article in the weekly Gazette
of Medicine and Surgery October 10, 1879 under
the Ernest Besnier si...
It is possible to make out three stages in more than 100
year history of the method development:
• an occasional use of hi...
• present stage at which the method is widely
adopted and its use is general and total (with
respect to human organism) no...
DEFINITION OF BIOPSY
• Biopsy is the removal of tissue for examination,
microscopic analysis, chemical analysis, and
bacte...
• Biopsy is the removal and examination usually
microscopic of tissue from the living body,
performed to establish precise...
• Biopsy is the removal of a piece of tissue from the
living body for diagnosis by microscopic
examination.
- Tomlins Chri...
INDICATIONS FOR BIOPSY
• To confirm a clinical impression of a lesion.
• When an inflammatory lesion is not responding to
...
• To determine the nature of any intraosseous lesion
which cannot be identified clinically and
radiographically.
• To dete...
• Persistent hyperkeratotic changes in surface tissues.
• Any persistent tumescence, either visible or palpable
beneath a ...
1. Surface lesions:
• Color/texture change e.g., white, red or
pigmented.
• Ulcerated, fissured or both.
• Proliferate (fi...
2. Subsurface lesions:
Soft tissue-
• Superficial, distort surface continuity, e.g.,
Mucocele.
• Deep, detected by palpati...
CONTRAINDICATIONS FOR BIOPSY
RELATIVE
For ambiguous lesions
a) This should include inflammatory lesions of
allergic, viral...
• Compromised general health of the patient or a
history of coagulopathy or bleeding diathesis,
including patient on antic...
ABSOLUTE
• Pulsatile lesions or those suggestive of a vascular nature
should be referred for more in depth evaluation (e.g...
• Lesions that because of size or location present
technically difficult surgery e.g., posterior tongue and
oropharynx off...
VARIOUS BIOPSY TECHNIQUES
• The type of biopsy to be performed depends on the
location, size and clinical impression of th...
SURGICAL
NON-SURGICAL
SOFT TISSUE
BONE
DIAGNOSTIC
CURATIVE
•PUNCH
•ELECTROCAUTERY
•SOFT TISSUE
CURETTAGE
•FROZEN SECTION
•...
Tissue may be obtained as:
• Tissue piece:
a. Incisional biopsy including geographical biopsies and
vital staining
b. Exci...
• Tissue core:
a. Fine needle cutting biopsy.
b. Tru-cut needle biopsy.
c. Vin Silverman needle biopsy.
• Cell aspirate:
F...
TISSUE PIECE:
INCISIONAL BIOPSY / DIAGNOSTIC BIOPSY:
• It is a biopsy technique that samples only a
particular or represen...
31
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Indication:
• It is chosen- when the lesion is extensive (larger
than 1 cm in diameter) or potentially malignant
(requirin...
Other indication includes:
• Chronic non-healing ulcer or squamous cell
carcinoma.
• Leukoplakia/ erythroplakia.
• Lichen ...
Contraindications:
• Pulsatile/ vascular lesions.
• Pigmented lesions.
36
Disadvantages:
• Leaves noticeable scar.
• Risk of facial nerve damage.
• Possible spread of malignant cells
(detection of...
Principle:
• Representative areas of the lesion (the area that
shows complete tissue changes) should be biopsied
in wedge ...
39
GEOGRAPHICAL BIOPSY AND VITAL
STAINING
• Lesions with areas of varying appearances and in
cases of widespread dysplastic d...
Technique:
• 1% aqueous toludine blue dye is applied to the
affected lesion for approximately 30 seconds
followed by a rin...
• Toludine blue stains the epithelial surface blue and
with the application of acetic acid the blue stain
from normal epit...
Advantages:
• The technique is helpful in differentiating the small
dysplastic erythroplakia that requires biopsy from
ery...
Disadvantage:
• The technique cannot show tumor that is present
beneath the normal epithelium.
44
ELECTRO-SURGERY BIOPSY
• Electro-surgery refers to the cutting and coagulation of
tissue using very high-frequency, low-vo...
Electro-surgical
technique
The lesion is grasped
with forceps through
the loop electrode.
The electrode is
activated going...
EXCISIONAL BIOPSY
• It is the removal of the lesion in toto at the time the
surgical diagnostic procedure is performed.
• ...
Indications:
• It is the usual approach for smaller lesions (less
than 1 cm in diameter).
• It is used for clinically beni...
• Mucoceles
• Myoblastoma
• Keratoacanthoma
• Sialoliths and smaller benign lesions of accessory
salivary glands
• Certain...
Principle:
• Entire lesion along with 2 to 3 mm of normal
appearing surrounding tissue is excised.
50
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Technique:
• For surface excision simple elliptical approach is
designed, like for the excision of pigmented and
hyperkera...
elliptical incision vertical deep
53
• For most Incisional and excisional biopsies, ellipse
technique is employed.
• After obtaining the anesthesia i.e., local...
• An elliptical area at the surface is outlined using
no.15 sharp scalpel blade (to avoid tearing the
tissue), incision is...
• Haemostasis is achieved with resorbable suture
inserted (to control bleeding and also assist in
healing). Firm pressure ...
CURRETTAGE BIOPSY
• CURRETTE is a French word ‘Curer’- meaning to
clean.
• It is indicated for intraosseous lesions that l...
• Although the sample produced is usually soft tissue
but it may include bone fragment as well.
• These extremely small se...
59
PUNCH BIOPSY:
• It is an alternative technique of tissue removal
applicable to both incisional and excisional biopsy.
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Indications:
• The technique most often is used for total removal
of small lesions but also finds applicability in the
par...
• The technique is used for oral mucosal
malignancies such as squamous cell carcinoma as
well as leukoplakia and other muc...
Contraindication:
• As a definitive surgical excision procedure of
suspected malignant lesions and cases of vascular
lesio...
Advantages:
• Technique is fast with low incidence of post surgical
morbidity.
• Suturing is usually not required as the s...
Disadvantage:
• Technique is designed primarily for use with
epithelial or superficial mesenchymal lesions.
• It is diffic...
• Punch biopsy should be used with caution when
the lesion overlie significant submucosal structures
such as mental forame...
Technique:
• Various types of biopsy punches are available –
- Keye biopsy punch
- Belt-driven.
• Even disposable biopsy p...
69
• After the biopsy site has been anesthetized, the site
is gently blotted with sterile gauze.
• The edge of the blade of t...
• The tissue is then grasped with an atraumatic
forceps and the base of the tissue core is released
using a scalpel blade ...
• 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 ...
74
FROZEN SECTION
• In surgical oncology, treatment of malignant
oropharyngeal tumors involves the excision of
tumor with 1 c...
Indication:
• To make an immediate surgical therapeutic
decision.
• To determine whether a lesion is benign, malignant
or ...
• It reduces the time of processing from 18 hours to
5 minutes
Methods of frozen section:
• Freezing microtome using CO2 g...
Technique:
• Biopsy tissue is frozen in a mixture of isopentene
and solid carbon dioxide at -70o.
• Sections of 5-7μm are ...
• The remainder of tissue is stored in 10% buffered
formaldehyde and routinely processed; embedded
in paraffin, sectioned ...
Errors in diagnosis can be due to:
• Sampling by the surgeon or pathologist.
• Interpretation by the pathologist.
• Differ...
Disadvantages:
• There can be error in sampling and interpretation of
frozen tissue as compared to routinely processed
tis...
TISSUE CORE
• Although FNAB is widely used technique in the
diagnosis of head and neck lesions, the diagnostic
accuracy is...
• Secondly the technique is simpler, easier and faster
than the traditional suction method.
• It also eliminates the possi...
• Cutting needle biopsy that employed the Biopsy
gun was first devised in the early 1980s.
• Studies assessing the clinica...
• Core biopsy specimen provides accurate
information about cell type and tissue
characteristics in head and neck lesions. ...
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...
87
VIM SILVERMAN NEEDLE BIOPSY:1938
It consists of:
1)Outer canula 16 G in size.
2)Inner trocar.
3)Inner split longitudinal n...
89
Advantage:
• They are easy to interpret then aspiration cytology to the
pathologist
• To distinguish between reactive chan...
CELL ASPIRATE
FINE NEEDLE ASPIRATION CYTOLOGY:
(FNAC)
• Kun in 1847, gave the first description of the
technique for aspir...
Patient selection/Indication:
1)The disease must be localized and clearly defined
by clinical examination or by any availa...
• Aspiration of soft tissue pathology is employed only when
fluid is detected or suspected and in of little value in the
d...
Other advantages include –
• The cost savings as compared to conventional open
biopsy, rapid and effective aid in diagnosi...
•Treatment options including preoperative counseling
can be investigated earlier without the need for
further diagnostic s...
•Absence of reactive fibrosis to interfere with subsequent
surgery
•No problems with wound healing, e.g. after radiotherap...
Disadvantages:
• Success of FNA depends on obtaining a
representative sample (if the specimen is small
with few or no cell...
Technique:
• Standard disposable needles (21 – 23G) are used for all
palpable lesions (for children and where the eyelids ...
• The needle is inserted into the lesion using no suction and
once the needle is within the lesion the change in
resilienc...
• Usually 2-3 slides are prepared on each mass.
• One slide is immediately fixed in 95% ethanol
solution and subsequently ...
• Ideally the aspiration should have a creamy
consistency with a high cell and low fluid content.
• The number of needle i...
• After any needle biopsy, direct pressure should be
applied over the site to reduce the incidence of
hematoma formation.
...
GOOD MORNING
103
A- needle is introduced into the mass.
B – plunger is retracted after needle enters the mass
C – suction is maintained whi...
ASPIRATION CYTOLOGY GUN
105
FRANZEN’S HANDEL WITH SYRINGE & NEEDLE FITTED ON IT
FOR PERFORMING FNAC
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Causes of unsatisfactory yield with fine needle
aspiration include:
• Needle missing a lesion and picking up
inflammatory ...
COMPLICATION & HAZARDS OF FNAC
•Haematoma: Bleeding from puncture site &
haematoma formation are commonest complications o...
LIMITATIONS OF FNAC
•Only a small population of cells is sampled, thus the
reliability of test depends on adequacy of samp...
USE OF FNA:
• Commonest indication is to distinguish between
both benign and malignant neoplasia as well as
non-neoplastic...
• Lymph nodes – FNAB is an excellent initial
diagnostic modality in the evaluation of
lymphadenopathy. Many infectious pro...
Bone and Cartilage
• Aspiration using 21 or 23G needles can be
undertaken if the cortical bone is thin or absent.
• A larg...
• Aspirated blood often indicates a vascular lesion
e.g., hemangioma or aneurysmal bone cyst.
• Withdrawn air suggests lik...
• Skin and soft tissue - It is usually possible to obtain
tissue by incisional at excisional biopsy, but FNA is
possible o...
Salivary gland swelling –
• Incisional or cutting biopsies are contraindicated owing to
risks of tumor seeding or fistula ...
Terminology:
• Regarding the confusion over the use of the terms
aspiration biopsy and aspiration cytology.
• If an aspira...
ASPIRATION BIOPSY
• Aspiration biopsy is the use of a needle & syringe to
penetrate a lesion for aspiration of its content...
Used to rule out &/or differentiate
• Fluid filled cavities
• Vascular lesions
• Hematomas
• Empty cavities
• Cyst (when l...
119
INTRAOSSEOUS OR HARD TISSUE BIOPSY
TECHNIQUE AND SURGICAL PRINCIPLES
•A lesion either on or within the osseous tissues...
120
•Before performing hard tissue biopsy, the dentist
should carefully palpate the area of the jaws where the
suspect les...
121
Aspiration biopsy of radiolucent lesions
•Any radiolucent lesion that requires biopsy should
undergo aspiration before...
Mucoperiosteal flaps
•Because of their location within or proximal to the
jaws, most lesions of hard tissue must be approa...
123
•The location of the lesion dictates where the flap
incisions are to be made. It is important to avoid major
neurovasc...
124
• All mucoperiosteal flaps for biopsies in or on the jaws
should be full thickness and incised through mucosa,
submuco...
125
Osseous window
•Lesions within the jaw (i.e., central lesion) require the
use of a cortical window.
•If expansion of a...
• The size of the window depends on the size of the
lesion and the proximity of the window to normal
anatomic structures s...
127
Removal of the specimen
•The technique for removal of the biopsy specimen
depends on the nature of the biopsy (excisio...
128
•The convex surface of the instrument is the portion
that actually separates the specimen from
surrounding bone. This ...
Bone marrow aspiration
• Site: Sternum, Posterior iliac crest
• SALAH BM aspiration needle is used (strong wide
bore needl...
130
TREPHINE BIOPSY
• Jamshidi Trephine needle is used
• H&E staining
• Time in 7 days
• Better marrow architectural pattern b...
Available in a range of sizes,
from 7 guage to 12 guage.
Needles in the market, with
lengths from 27mm to
44mm.
All Needle...
133
A biopsy needle retrieves a sample of bone and it is
sent for examination.
• Bone marrow biopsy is a diagnostic procedure
...
• Other indications for bone marrow examination
include evaluation of immunodeficiency
syndromes, confirmation of unusual ...
• Unexplained anemia in peripheral blood
• Unexplained thrombocytopenia in peripheral blood
• Pancytopenias in peripheral ...
• Caution should be exercised in patients with soft
bones secondary to radiation therapy, multiple
myeloma, or osteoporosi...
138
• In some patients, the sternum may be the first
choice for aspiration because of positioning
limitations or obesity.
139
Equipment
1. Sterile gloves
2. Sterile drape
3. Povidone-iodine
4. Bone marrow
aspiration needle or 11-
or 13-gauge Jamshi...
141
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Tests on Bone Marrow Aspirate
and Biopsy Specimens
TEST PURPOSE
Bone marrow
morphology
Performed on stained smears of all aspirate
and biopsy specimens. Determine cell linea...
Cytochemistry,
histochemistry
Differentiate acute
myelogenous leukemia
(AML) from acute
lymphatic leukemia
Flow cytometry,...
Iron stains Perform on all aspirate
and biopsy specimens to
assess iron stores,
anemia, and iron
accumulation
Molecular st...
Microbiologic cultures Detect bacterial, viral,
and fungal infections in
patients with fever of
unknown origin and
immunod...
• Palpation of the area of the lesion with comparison to
the opposite side.
• Any radiolucent lesion should have an aspira...
SCISSORS BIOPSY
• Is one of the ways to remove skin tissue for a
biopsy specimen.
• This procedure entails snipping off a ...
• Small forceps with teeth and a pair of sharp curved
or straight iris scissors are the only surgical
instruments required...
150
SHAVE BIOPSY
• A scalpel or razor blade is used to scrape lesion,
performed superficially or deeply.
• Shave excision usua...
The blade is held horizontal to
the skin surface and
brought below the lesion.
The other hand is used to
stretch and stabi...
153
Assisted guidance:
• Most tumours that are visible or palpable can be
examined without the aid of radiologic imaging,
wher...
Ultrasound guided biopsy
• This technique has the advantages of being
noninvasive, quick, and easy, and it can be
performe...
• However, Ultrasound guided biopsy is not possible
when gas/bone prevents the visualization of the
biopsy region.
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CT guided biopsy
• A computed tomography guided biopsy, uses real-
time CT images to help the doctor guide a needle to
the...
Indications
1. Lymph nodes or masses that are not completely
identifiable using ultrasound.
2. Lesions near the skull base...
Disadvantages of CT include
• radiation exposure,
• limited possible scan plane orientations,
• low soft tissue contrast, ...
MRI guided
• Interventional MRI is a method for procedure
guidance that combines the imaging benefits of
magnetic resonanc...
162
• Scientific interest has also focused on MRI-guided,
minimally invasive thermal tumour ablation using
the unique temperat...
Endoscope guided biopsy
• Endoscopy is defined as “the examination of the
interior of a canal or hollow viscous by means o...
• Endoscopy may prove to be an important tool for
the internal examination of large jaw cysts that may
contain regional ne...
Endoscope positioned into the lesion.
166
Endoscopic view showing areas of thickened lining
containing exophytic protrusions measuring up to 10
mm in diameter.
167
Velscope
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EXPLORATION BIOPSY
• Used for inrtaosseous lesions of maxilla and
mandible
• Instruments: Chisel, Bone burs, Periosteal El...
TISSUE SCRAPINGS
EXFOLIATIVE CYTOLOGY:
• Cyto – Cell -- Logos – Study
• Study of cells.
• Rudolf Virchow (1955) stated “Ev...
Rudolf Virchow (1821-1905)
“ THE FATHER OF CELLULAR PATHOLOGY” 175
Dr. George N.
Papanicolaou
(1883-1962)
1920s “ father of
exfoliative cytology”
Developed PAP test
for diagnosis of
uterine...
• Normal oral squamous epithelium continuously
sheds the most superficial cells.
• If malignant or other disease processes...
• Exfoliative cytology is the study of superficial cells
which have been either exfoliated or shed actually
from mucous me...
The lesion is repeatedly scraped with a moistened
tongue depressor or spatula or cytobrush type
instrument. The cells obta...
Indications:
• For quick laboratory evaluation of suspected
malignant and premalignant oral lesions and
multiple premalign...
• To identify the presence of certain specific cells in
non-malignant red patches or ulcerative lesions.
• To see malignan...
• Certain benign hereditary skin lesions having their
representative oral manifestations.
• For the study of the change of...
Contradictions:
• Deep seated lesions (both soft and hard tissue).
• Fibrous lesions.
• Polypoid growth.
• Non-ulcerative ...
• Atrophic lesions.
• Densely keratinized lesions.
• Smooth surface lesions.
• Lesions giving inadequate specimen sampling...
Merits:
• Exfoliative cytology implicates its importance in the
field of diagnosis with the principle that any change
in t...
• It helps to follow up recurrent carcinomas.
• It is valuable for screening of clinically non-
suspected lesions.
• Easy ...
Demerits:
• 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 les...
Target for cytological Prediction:
• Squamous cell Carcinoma remains the chief target
for cytological investigation.
• She...
Requisite for diagnostic cytology:
• Diagnosis must be based on the synthesis of entire
evidence available rather than the...
Technique:
• Instruments for removing the cells should have a
90-degree angle or straight blade.
• The cement spatula used...
• Scraping of the lesion should be done while the
tissue is stretched or taut. A single stroke is
preferred over many smal...
• Fixation must be immediate. Air dried smears are
inconsistent in their staining properties. A safe and
adequate fixation...
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Interpretation and Reporting:
• The evaluation of slide is based on the
morphological features and staining quality of the...
Class I (Normal) indicates only normal cells.
Class II (Atypical) indicates the presence of mild atypia but no
evidence of...
Touch impression or Imprint
cytology
• It is a method in which gentle grazing or sliding of
glass slide over the cut surfa...
• A direct imprint is prepared by pressing a glass slide
gently on to the freshly cut surface of the
specimen, avoiding a ...
This is particularly useful in -
• In the diagnosis of certain neoplastic lesions which
can simulate inflammatory lesions ...
• In the diagnosis of malignancy confined to one
small area of a large specimen
The amount of tissue that can be frozen fo...
Imprint of a massively necrotic carcinoma showing a
cluster of viable cancer cells on a necrotic background.
Diagnosis was...
Imprint of a lymph node showing malignant cells.
206
ARMAMENTARIUM:
207
• Wide mouthed bottle with 10% formalin (at least
20 times the volume of the specimen should be
used)
• For exploratory bi...
Site selection and handing of the specimen-
• By carefully selecting the area or areas, which will
produce a good diagnost...
• Deep sections are preferred over shallow
specimens. If possible, biopsies from the mucosa
should include the epithelium,...
• Sterile technique should be utilized both to prevent
infection, which complicates the diagnostic procedure,
and to avoid...
TISSUE STABILIZATION
•Soft tissue biopsies in the oral cavity are frequently
performed on movable structures, such as the ...
•This method also aids in hemostasis by compressing
the labial arteries. Instruments are available to
perform this same fu...
• The chalazion clamp, used by ophthalmologists, is a
helpful tool for oral biopsies on the oral lips,
anterior buccal muc...
215
HEMOSTASIS
•The use of suction device for aspiration of surgical
hemorrhage during biopsy should be avoided. This is
espec...
• Manipulation and grasping of any lesion suspected of
being a tumor should be minimal. Vigorous
manipulation of malignant...
• Orientation of mucosal biopsies (particularly
superficial lesion) is important, especially because
they are small and ha...
• Surgeons can place sutures in the specimen to
assist in orientation and provide a written
description of the specimen in...
220
• Fixation and preservation of tissue are essential to avoid
distortion of histologic and cytologic details.
• 10% formali...
• Tissue should never be placed on cotton, gauze, or
paper prior to fixation. There is rapid absorption of
fluid from the ...
• If culture is desired, then the tissue should not be
placed in fixative until the material for
bacteriological study is ...
However, fresh material is needed for the following
purpose:
1. Frozen section
2. Immunocytochemistry
3. Cytological exami...
• No fixative will penetrate a piece of tissue thicker
than 1 cm. For dealing with specimen thicker than
this, following m...
Temperature:
• The fixation can be carried out at room temperature.
Tissue should not be frozen once it has been placed in...
• Factor affecting fixation:
1. Size and thickness of piece of tissue.
2. Tissue covered by large amount of mucous fix
slo...
Labeling of the specimen:
• The patient’s name, the location of the specimen, and
the date of the surgical procedure are a...
Details required in pathology form
• Patient data
• Clinical details of lesion
• Any medical history with details of medic...
230
Follow-up and Reporting of Biopsy Result to the
Patient
• Patients should be seen 1 to 2 weeks
postoperatively to ensure h...
General Principle of Gross
Examination
1. Proper identification and orientation of the
specimen.
2. Unlabelled specimen sh...
5. Surgeon should be instructed to submit all the
material that they have removed, not the selected
portion from it.
6. Pl...
7. Measurements are usually given in centimeter
unless the specimen is very small in which mm can
be used.
8. Tissues shou...
Processing
• The fixed tissue is dehydrated by immersion in a
series of solvent and impregnated with paraffin
wax.
• The b...
236
• The paraffin block containing the tissue is mounted on a
cutting machine microtome and tissue section of 3-6 mm
thicknes...
238
• When the mounting media dries, the specimen is ready
for microscopic examination and storage.
• It takes 24-48 hours to ...
• For specimens containing bone and teeth - they
need to be softened by decalcifying in acid to
enable a thin section to b...
• Pathologists and clinicians sometimes request slide
consultations.
• For difficult specimens general and oral pathologis...
• Sometimes, there can be discrepancies between
the diagnosis or interpretation and the clinical
finding – in such cases f...
Good Morning
243
Stains
• Generally the stains are classified as:
A. Acid stains
B. Basic stains
C. Neutral stains
• All dyes are composed ...
• Acid Dyes:
In an acid dye the basic component is coloured and
the acid component is colourless. Acid dyes stain
basic co...
• Neutral Dyes:
When an acid dye is combined with a basic dye a
neutral dye is formed. As it contains both coloured
radica...
Special Stains:
1. PAS (Periodic Acid Schiff) stain: This stain
demonstrates glycogen and neutral mucous substances,
outli...
c. PAS stain: It is used for fungi, amoeba and Tricomonas.
d. Modified Giemsa (2% Giemsa in water): Detects
Helicobacter p...
In large lesions
Accessible area
Characteristic portion.
For multiple lesions
Most representative site.
Material curetted ...
For red & white lesions include both red & white
area 250
ULCERS
Include margin,
deep part of
ulcer and site of
maximal clinical
activity.
AVOID
Superficial
ulcers &
necrotic tissu...
For Polypoid lesions include base
252
For Vesiculobullous lesions
Fluid is more representative. Intact vesicle or bulla
should be biopsied.
253
For LICHEN PLANUS – representative area should
be biopsied
254
For LEUKOPLAKIA – Most dysplastic
area should be biopsied
255
For MUCOCELE lesions – careful excisional biopsy
256
For GRANULOMATOUS LESIONS –
deep incisional biopsy + fresh sample to
microbiology if infective agent suspected
257
Do not cut into pigmented and vascular lesions
258
259
ERRORS DURING SURGICAL
REMOVAL OF BIOPSY
SPECIMEN
260
INJECTION
261
HANDLING THE TISSUE
Handle tissue with minimum force
USE OF FORCEPS
Avoid use of forceps on the surface of biopsy.
USE OF ...
SELECTION OF THE SPECIMEN
Tissue should be representative of whole lesion.
Adequate amount of tissue at least 1 x 0.5 cm i...
Biopsy of skin or mucosa representative sample of
both epithelium and connective tissue has to be
taken
264
PRECAUTIONS AT FIXATION
265
SELECTION OF FIXATIVE
Depends on purpose and method of tissue processing
10% neutral buffered formalin
Request for 10% for...
Do not add tap water to dilute the fixative
267
PURPOSE OF FIXATION
To retain tissue as much as possible in living condition
268
CONTAINER OF FIXATIVE
Wide mouth clear bottle
INTRODUCING INTO FIXATIVE
Avoid curling of tissue.
DELAY IN FIXATION
Leads t...
VOLUME OF FIXATIVE
270
• Disadvantage of formalin is that it makes the
specimen unsuitable for immunofluorescent
techniques as it forms protein c...
• Because rapid freezing of specimens require special
supplies and keeping them frozen during
transportation is a packagin...
273
Lost specimen:
• The value of entire hospital admission for diagnosis
biopsy is all too frequently negated by the loss of
...
Mislabeling of specimen:
• Another error which can easily occur is for the
specimens to be improperly labeled, so that
mat...
Failure to note orientation of the specimen:
• Failure to identify the orientation of the mass of
tissue removed in enbloc...
Tissue Artifacts
"Alterations in tissue morphology that result from
various forms of mechanical, chemical or thermal
insul...
CAUSES OF TISSUE ARTIFACTS
• Clinical application of chemicals.
• Local injection of anaesthetics.
• Surgical sectioning.
...
1.DURING SURGERY:
• Injection Artifacts
• Forceps Artifacts
• Fulguration Artifacts
• Laser Artifacts
• Suction Artifacts
...
2.DURING FIXATION AND TRANSPORT:
• Fixation Artifacts
• Freezing Artifacts
• Artifacts during transportation
3.TISSUE PROC...
INJECTION ARTIFACTS
• Injection of large amounts of anesthetic solution
into the area to be biopsied can produce following...
HOW TO AVOID
Infiltration of an anaesthetic agent
• Injections directly into the lesion should be avoided.
• If hemeostasi...
FORCEPS ARTIFACTS
• When the teeth of the instrument penetrates the
specimen, it resulted in voids or tears and
compressio...
TO AVOID
• Using small atraumatic or Adson forceps with or
without teeth. These will produce little mechanical
distortion ...
CRUSH ARTIFACT
• It’s a form of tissue distortion resulting from even
the most minimal compression of the tissue.
• Crushi...
CAUSES
• Mutilation of the tissues with forceps.
• Dull scalpel blades.
HOW TO AVOID
• Delicate handling of specimen.
• A ...
FULGURATION ARTIFACTS
• The use of cautery for biopsies.
• Heat produces marked alterations in both the
epithelium and con...
• The fibrous connective tissue, fat and muscle gain
an amorphous appearance when subjected to such
procedures.
• Its effe...
TO AVOID
• Only the cutting and not the coagulation electrode
should be used while obtaining a biopsy specimen.
• The inci...
• This technique involves use of the scalpel for initial
incision in and around the lesion and electrosurgery
to complete ...
ONCOCYTOID ARTIFACT IN SALIVARY GLAND
TISSUES
• This unusual tissue artifact occur in the serous acini
of parotid gland ti...
• The two cells can be distinguished by the fact that
the altered acinar cells possess uniform round
nuclei in the basal h...
LASER ARTIFACTS
• Lasers are being used for excision and ablation of
oral mucosal lesions with the chief advantage being
m...
• The CO2, Nd: YAG systems lends to cytological
artifacts in keratinocytes, with the keratinocytes
showing atypical change...
ARTIFACTS INDUCED BY SUCTION TIPS
• Induced by the vacuum effect of the surgical
suction tips.
• These are seen in various...
• In highly vascularised tissue such as tissue in
periapical granulomas and in inflamed odontogenic
cysts.
• Suctioning in...
FIXATION ARTIFACTS
• Although fixation is used to prevent the occurrence
of various artifacts, it may itself be the cause ...
Volume changes
• Various factors have been suggested such as
inhibition of respiration, changes in membrane
permeability a...
Artifactual pigments
These include:
1) Formalin pigment.
2) Mercury pigment.
3) Dichromate deposits.
299
FORMALIN PIGMENT
• This pigment appears as a brown/ brownish black
deposit in tissues fixed in any formalin fixative with
...
MERCURY PIGMENT
• Tissues fixed in fixative mixtures containing
mercury have been found to develop a uniform
granular blac...
DIFFUSION ARTIFACTS
• This important group of artifacts is related to the
diffusion of unfixed material to give a false
lo...
• Materials may also diffuse out the tissue and may
lead to errors where localization of these materials
is important for ...
IMPROPER FIXATION
• Delay in fixation and inadequate fixation alters the
staining quality of the cells.
• Cells appears sh...
HOW TO AVOID
• For optimal fixation the amount of fixative should
be approximately 20 times the volume of the
specimen.
• ...
FREEZING ARTIFACTS
• These are characterised by the formation of
interstitial vacuoles within the cell cytoplasm
resulting...
HOW TO AVOID
• As formalin freezes at 12.2°F, containers should
have adequate insulation from extreme cold and
should cont...
ARTIFACTS DUE TO TISSUE PROCESSING.
• Small biopsy specimen, often a narrow strip of
delicate mucosa, will bend and twist ...
Sponge artifacts
• Utilization of sponges has been advocated as an
efficient means of holding specimen during
processing.
...
SECTIONING ARTIFACTS
• These artifacts can be caused as a result of:
1) Improper processing.
2) Improper sectioning.
310
IMPROPER SURGICAL REMOVAL
311
WRONG ORIENTATION OF SPECIMEN
312
CURLING AND CRUSH ARTEFACT
313
HEMORRHAGE ARTEFACT
314
SPLIT ARTEFACT
315
FRAGMENTATION ARTEFACT
316
HEALING OF BIOPSY WOUNDS
• The healing of a biopsy wound of the oral cavity is
identical with the healing of a similar wou...
Primary healing
• Primary healing, healing by primary intention or
healing by first intention is that type of healing
whic...
• When the edges of the wound are brought into contact
and held in place by sutures, the blood clots, and in a
matter of h...
• In time, these cells form thin, delicate collagen
fibrils which intertwine and coalesce in a general
direction parallel ...
• When there is a close apposition of the edges of the
wound, the surface epithelium proliferates rapidly
across the line ...
Secondary healing:
• Healing by second intention, healing by granulation
or healing of an open wound occurs when there is
...
• After the removal of the lesion, the blood filling the
defect clots and the repair process begins.
• It is basically ide...
• Cellular proliferation begins around the periphery of
the wound, and the fibroblasts and endothelial cells
grow into the...
• As the granulation tissue matures, it becomes more
fibrous through condensation of collagen bundles,
and the surface of ...
Complications
326
1. Haemorrahage –
• Immediate or subsequent haemorrhage can be
serious problem following biopsy.
• Highly vascular tissue ...
2. Infection –
• When tumors on the various skin or mucosal
surfaces are biopsied, there is always a possibility
that alre...
3. Poor wound Healing
Unsatisfactory healing of the incision may be due to
• Ischemia of the skin overlying a tumor mass (...
4. Spread of tumor cells
• Prominent reasons for local tumor cell contamination
are the following:
-Lack of awareness that...
5. Injury to adjacent organs
• Injury may occur to surrounding structure (blood
vessels).
• Structures through which the b...
Other complications
• Post operative pain.
• Paraesthesia - in the lips or the tongue,
• Swelling and bruising - in the to...
CONCLUSION
• For entities of uncertain significance or etiology, a
biopsy provides the simplest and most speedy
means of o...
• Careful handling of the tissue and prompt
appropriate fixation will enable a confident
histological diagnosis to be reac...
•R. Rajendran and B. Sivapathasundharam: Shafer’s
textbook of oral pathology, 5th edition (2006), Elsevier.
•Neville Brad ...
BIOPSY
BIOPSY
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BIOPSY

  1. 1. GOOD MORNING 1
  2. 2. BIOPSY One can’t show this when it’s not there One can’t hide this when it’s there.
  3. 3. TABLE OF CONTENTS • Introduction • History • Definitions • Indications & Contraindications • Classification • Various biopsy procedures • Special considerations • Essential biopsy principles 3
  4. 4. • Errors • Biopsy artifacts • Healing of a biopsy wound • Complications • Conclusion • References 4
  5. 5. Introduction • 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. 5
  6. 6. • 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 biopsy. • 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 radiotherapy). 6
  7. 7. • 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 patient. 7
  8. 8. HISTORY 8
  9. 9. • 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. 9
  10. 10. • 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.” 10
  11. 11. • 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 first delivered. • The first diagnostic biopsy in Russia was performed in 1875 by M. M. Rudnev. 11
  12. 12. 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 century); 12
  13. 13. • 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 13
  14. 14. 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 14
  15. 15. • 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 diagnosis. -Pederson GW in 1988 15
  16. 16. • Biopsy is the removal of a piece of tissue from the living body for diagnosis by microscopic examination. - Tomlins Christopher DC in 1998 • Biopsy is the removal of tissue from a living subject for laboratory evaluation and analysis. -Neelima AM in 2002. 16
  17. 17. 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. 17
  18. 18. • 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. 18
  19. 19. • 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. Fibroma). • Any lesion that has the characteristics of a malignancy (e.g. Erythroplakia). 19
  20. 20. 1. Surface lesions: • Color/texture change e.g., white, red or pigmented. • Ulcerated, fissured or both. • Proliferate (fibroma, papilloma). 20
  21. 21. 2. Subsurface lesions: Soft tissue- • Superficial, distort surface continuity, e.g., Mucocele. • Deep, detected by palpation, e.g., masses of various consistency. Hard tissue- • Superficial, distort surface continuity • Deep, detected by x-rays, e.g., radiopaque and radiolucent changes. 21
  22. 22. CONTRAINDICATIONS FOR BIOPSY RELATIVE 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, exostoses etc. c) Lesions caused by recent trauma. 22
  23. 23. • 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. 23
  24. 24. ABSOLUTE • Pulsatile lesions or those suggestive of a vascular nature should be referred for more in depth evaluation (e.g., hemangioma). • 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). 24
  25. 25. • 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 care. 25
  26. 26. 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, unplanned biopsy. 26
  27. 27. SURGICAL NON-SURGICAL SOFT TISSUE BONE DIAGNOSTIC CURATIVE •PUNCH •ELECTROCAUTERY •SOFT TISSUE CURETTAGE •FROZEN SECTION •EXCISIONAL DIAGNOSTIC CURATIVE •CURETTAGE •TREPHINE •ASPIRATION •FROZEN •ENUCLEATION •RESECTION •CURETTAGE ASPIRATION CYTOLOGY •SCALPEL •CAUTERY •EXFOLIATIVE •FNAC •INCISIONAL 27
  28. 28. Tissue may be obtained as: • Tissue piece: a. Incisional biopsy including geographical biopsies and vital staining b. Excisional biopsy c. Curetting d. Frozen section. 28
  29. 29. • Tissue core: a. Fine needle cutting biopsy. b. Tru-cut needle biopsy. c. Vin Silverman needle biopsy. • Cell aspirate: Fine needle aspirate biopsy. • Scrapings: Exfoliative cytology. 29
  30. 30. TISSUE PIECE: 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 lesion. 30
  31. 31. 31
  32. 32. 32
  33. 33. 33
  34. 34. Indication: • 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. 34
  35. 35. Other indication includes: • Chronic non-healing ulcer or squamous cell carcinoma. • Leukoplakia/ erythroplakia. • Lichen planus. • Bullous lesions (pemphigus, pemphigoid etc). • Granulomatous diseases (crohn’s disease, orofacial granulomatosis, ulcerative colitis, tuberculosis). • Minor salivary gland tumor (in palate). 35
  36. 36. Contraindications: • Pulsatile/ vascular lesions. • Pigmented lesions. 36
  37. 37. Disadvantages: • 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) 37
  38. 38. 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. • 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. 38
  39. 39. 39
  40. 40. GEOGRAPHICAL BIOPSY AND VITAL STAINING • 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. 40
  41. 41. Technique: • 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 water. 41
  42. 42. • 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 acetic acid. • 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 blue stain. 42
  43. 43. Advantages: • 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 examination alone. 43
  44. 44. Disadvantage: • The technique cannot show tumor that is present beneath the normal epithelium. 44
  45. 45. ELECTRO-SURGERY BIOPSY • Electro-surgery refers to the cutting and coagulation of tissue using very high-frequency, low-voltage electrical currents. • 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 thermal coagulation. 45
  46. 46. Electro-surgical technique The lesion is grasped with forceps through the loop electrode. The electrode is activated going under the lesion, removing the growth. 46
  47. 47. EXCISIONAL BIOPSY • 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 lesions. 47
  48. 48. Indications: • 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. 48
  49. 49. • Mucoceles • Myoblastoma • Keratoacanthoma • Sialoliths and smaller benign lesions of accessory salivary glands • Certain cicatrial lesions 49
  50. 50. Principle: • Entire lesion along with 2 to 3 mm of normal appearing surrounding tissue is excised. 50
  51. 51. 51
  52. 52. 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. 52
  53. 53. elliptical incision vertical deep 53
  54. 54. • 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 54
  55. 55. • 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. 55
  56. 56. • Haemostasis is achieved with resorbable suture inserted (to control bleeding and also assist in healing). Firm pressure for few minutes aid in haemostasis. • 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. 56
  57. 57. CURRETTAGE BIOPSY • CURRETTE is a French word ‘Curer’- meaning to clean. • 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 evaluation. 57
  58. 58. • 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. 58
  59. 59. 59
  60. 60. PUNCH BIOPSY: • It is an alternative technique of tissue removal applicable to both incisional and excisional biopsy. 60
  61. 61. 61
  62. 62. Indications: • 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. 62
  63. 63. • 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 diseases. 63
  64. 64. Contraindication: • As a definitive surgical excision procedure of suspected malignant lesions and cases of vascular lesions. 64
  65. 65. 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. • The technique can be used on any mucosal surface accessible to the biopsy punch. 65
  66. 66. Disadvantage: • 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. 66
  67. 67. • 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. 67
  68. 68. Technique: • Various types of biopsy punches are available – - Keye biopsy punch - Belt-driven. • Even disposable biopsy punches are available. 68
  69. 69. 69
  70. 70. • 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. 70
  71. 71. • 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. • Local pressure with sterile gauze is sufficient to induce haemostasis 71
  72. 72. • Persistent hemorrhage can be treated with chemical cautery such as silver nitrate, collagen matrix, or by electrocautery. 72
  73. 73. • 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 palliation. • Non-steroidal anti-inflammatory agents are preferred for post-operative discomfort. 73
  74. 74. 74
  75. 75. FROZEN SECTION • 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. 75
  76. 76. 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. • Ascertain metastatic involvement of regional lymph nodes. 76
  77. 77. • It reduces the time of processing from 18 hours to 5 minutes Methods of frozen section: • Freezing microtome using CO2 gas • Refrigerated microtome(cryostat) 77
  78. 78. 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. • The procedure is completed within 5-10 mins from the time of receiving specimen till it is stained. 78
  79. 79. • 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 future reference. 79
  80. 80. Errors in diagnosis can be due to: • Sampling by the surgeon or pathologist. • Interpretation by the pathologist. • Difference in communication between the two. 80
  81. 81. 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. • It has the disadvantage that only 8-16 micron thick section can be cut and finer details of tissue can not be examined. 81
  82. 82. 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. 82
  83. 83. • 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. 83
  84. 84. • 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 techniques. 84
  85. 85. • 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. 85
  86. 86. 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. 86
  87. 87. 87
  88. 88. 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. 88
  89. 89. 89
  90. 90. Advantage: • They are easy to interpret then aspiration cytology to the pathologist • To distinguish between reactive changes and recurrent malignancy in possible cervical metastasis. 90
  91. 91. CELL ASPIRATE FINE NEEDLE ASPIRATION CYTOLOGY: (FNAC) • 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). 91
  92. 92. Patient selection/Indication: 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. 92
  93. 93. • 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. 93
  94. 94. 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 the lesion. 94
  95. 95. •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 95
  96. 96. •Absence of reactive fibrosis to interfere with subsequent surgery •No problems with wound healing, e.g. after radiotherapy •Readily repeatable 96
  97. 97. 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. 97
  98. 98. Technique: • 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 98
  99. 99. • 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 lesion. • 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. 99
  100. 100. • 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. 100
  101. 101. • 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 lesion. • 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 the aspiration. 101
  102. 102. • After any needle biopsy, direct pressure should be applied over the site to reduce the incidence of hematoma formation. 102
  103. 103. GOOD MORNING 103
  104. 104. 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 104
  105. 105. ASPIRATION CYTOLOGY GUN 105
  106. 106. FRANZEN’S HANDEL WITH SYRINGE & NEEDLE FITTED ON IT FOR PERFORMING FNAC 106
  107. 107. Causes of unsatisfactory yield with fine needle aspiration include: • 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. 107
  108. 108. 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 complications. •Infection: Introduction of infection is not a significant hazard. •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
  109. 109. 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. 109
  110. 110. USE OF FNA: • Commonest indication is to distinguish between both benign and malignant neoplasia as well as non-neoplastic conditions. • Secondly to differentiate between a local recurrence or nodal metastasis 110
  111. 111. • 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 differentiate between- • Reactive hyperplasia or inflammation. • Malignant disease. • Lymphoma. • It is also used to confirm the cervical lymph node metastasis from previously treated local neoplasms. 111
  112. 112. 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 bur. 112
  113. 113. • 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 113
  114. 114. • 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. 114
  115. 115. 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 gland masses. • The primary indication for FNAB of salivary gland is to distinguish among benign, malignant and inflammatory lesions. • The accuracy percentage is higher for benign as compared to malignant tumors with a sensitivity and specificity of 80% and 98%. 115
  116. 116. Terminology: • 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 aspiration (FNA). • 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 116
  117. 117. ASPIRATION 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 tissue masses. 117
  118. 118. Used to rule out &/or differentiate • Fluid filled cavities • Vascular lesions • Hematomas • Empty cavities • Cyst (when lesion is cystic fluid is yellow in color occasionally red due to presence of blood) 118
  119. 119. 119 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 possible. •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.
  120. 120. 120 •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.
  121. 121. 121 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.
  122. 122. Mucoperiosteal flaps •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.
  123. 123. 123 •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.
  124. 124. 124 • 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
  125. 125. 125 Osseous window •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.
  126. 126. • 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 neurovascular bundles. • 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 specimen. 126
  127. 127. 127 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 encountered. •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.
  128. 128. 128 •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.
  129. 129. 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 conditions, myelomas • Time in 1-2 hrs 129
  130. 130. 130
  131. 131. TREPHINE BIOPSY • Jamshidi Trephine needle is used • H&E staining • Time in 7 days • Better marrow architectural pattern but cell morphology indistinct • Indications: aplastic anemia 131
  132. 132. Available in a range of sizes, from 7 guage to 12 guage. Needles in the market, with lengths from 27mm to 44mm. All Needles in this range have a mm scale, providing more precise penetration, and a beveled tip cannula which makes penetration much smoother. The needle also features a rapid, safe, locking knob. 132
  133. 133. 133
  134. 134. 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 virus • Bone marrow evaluation can classify an anemia as hypoproliferative, a maturation disorder, or resulting from hemorrhage or hemolysis 134
  135. 135. • 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 135
  136. 136. • 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 leukemias posttreatment • Chromosomal abnormality • Immunodeficiency syndromes • Fever of unknown origin 136
  137. 137. • Caution should be exercised in patients with soft bones secondary to radiation therapy, multiple myeloma, or osteoporosis. 137
  138. 138. 138
  139. 139. • In some patients, the sternum may be the first choice for aspiration because of positioning limitations or obesity. 139
  140. 140. 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 140 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
  141. 141. 141
  142. 142. 142 Tests on Bone Marrow Aspirate and Biopsy Specimens
  143. 143. TEST PURPOSE Bone marrow morphology 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 granulomas. 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 leukemias. 143
  144. 144. Cytochemistry, histochemistry Differentiate acute myelogenous leukemia (AML) from acute lymphatic leukemia Flow cytometry, immunochemistry Analyze B-cell and T-cell surface markers in lymphoid neoplasms and myeloid leukemias. Determine lineage and stage of differentiation. Cytogenetic analysis Detect chromosomal changes in leukemias, lymphomas, and myelodysplastic syndromes 144
  145. 145. Iron stains Perform on all aspirate and biopsy specimens to assess iron stores, anemia, and iron accumulation Molecular studies (polymerase chain reaction, fluorescence in situ hybridization, restriction fragment length polymorphism) Establish clonality of a cell population, determine cell lineage, rearrange B-cell and Tcell receptor genes, and detect minimal residual disease in leukemias. 145
  146. 146. Microbiologic cultures Detect bacterial, viral, and fungal infections in patients with fever of unknown origin and immunodeficiency syndromes 146
  147. 147. • 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. • Solid • Fluid Filled • Vascular • Without Contents Clinical Exam
  148. 148. SCISSORS BIOPSY • 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. • Depending on lesion size and morphology, anesthesia may or may not be necessary. 148
  149. 149. • Small forceps with teeth and a pair of sharp curved or straight iris scissors are the only surgical instruments required. • 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 149
  150. 150. 150
  151. 151. SHAVE BIOPSY • 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. 151
  152. 152. 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 below. 152
  153. 153. 153
  154. 154. Assisted guidance: • 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 154
  155. 155. 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 complications. • Another advantage is that, unlike other radiographic biopsy procedures, ionizing radiation is not used for imaging. 155
  156. 156. • However, Ultrasound guided biopsy is not possible when gas/bone prevents the visualization of the biopsy region. 156
  157. 157. 157
  158. 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. 158
  159. 159. Indications 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. 159
  160. 160. 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. 160
  161. 161. MRI guided • 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. 161
  162. 162. 162
  163. 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. • The mean procedure time for MRI-guided needle insertion per pass is less than 10 minutes for aspiration as well as core biopsy. 163
  164. 164. 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. 164
  165. 165. • 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 165
  166. 166. Endoscope positioned into the lesion. 166
  167. 167. Endoscopic view showing areas of thickened lining containing exophytic protrusions measuring up to 10 mm in diameter. 167
  168. 168. Velscope 168
  169. 169. 169
  170. 170. 170
  171. 171. 171
  172. 172. 172
  173. 173. EXPLORATION BIOPSY • Used for inrtaosseous lesions of maxilla and mandible • Instruments: Chisel, Bone burs, Periosteal Elevator UNEXPECTED/ UNPLANNED BIOPSY • When as a result of surgical procedure (Tooth extraction) some suspicious tissue is obtained unexpectedly 173
  174. 174. TISSUE SCRAPINGS EXFOLIATIVE CYTOLOGY: • 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.” 174
  175. 175. Rudolf Virchow (1821-1905) “ THE FATHER OF CELLULAR PATHOLOGY” 175
  176. 176. Dr. George N. Papanicolaou (1883-1962) 1920s “ father of exfoliative cytology” Developed PAP test for diagnosis of uterine cervix cancer 176
  177. 177. • 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 cells. 177
  178. 178. • 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 178
  179. 179. 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. 179
  180. 180. 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. • Mass screening of oral cancer. 180
  181. 181. • 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. 181
  182. 182. • Certain benign hereditary skin lesions having their representative oral manifestations. • For the study of the change of the oral epithelial cells followed by chemotherapy. 182
  183. 183. 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. 183
  184. 184. • Atrophic lesions. • Densely keratinized lesions. • Smooth surface lesions. • Lesions giving inadequate specimen sampling through the adopted technique. • Patients with underlying blood dyscrasias. 184
  185. 185. Merits: • 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- invasive procedure. • If guards against false negative biopsies. 185
  186. 186. • It helps to follow up recurrent carcinomas. • It is valuable for screening of clinically non- suspected lesions. • Easy and a most feasible method for detection of malignancies in oral epidemiological survey programme. • It gains to evaluate the emergent concepts of proclaimed cancer, ca-in-situ, and related lessons of the oral cavity. 186
  187. 187. Demerits: • 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 cytology. 187
  188. 188. • 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. 188
  189. 189. 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. 189
  190. 190. Requisite for diagnostic cytology: • Diagnosis must be based on the synthesis of entire evidence available rather than the changes in individual cell. • Adequate clinical data. • Uniformity of the technical method employed. • Thorough knowledge of normal cells and extensive physiological variation of the sites to be diagnosed. 190
  191. 191. 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 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 selected cases. 191
  192. 192. • 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 labeled 192
  193. 193. • 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. 193
  194. 194. 194
  195. 195. 195
  196. 196. 196
  197. 197. 197
  198. 198. 198
  199. 199. 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: - 199
  200. 200. 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 200
  201. 201. 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. Advantage: • Feasible and economical • Detect the malignancy at the tumor margin • Less time consumable 201
  202. 202. • 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). 202
  203. 203. This is particularly useful in - • In the diagnosis of certain neoplastic lesions which can simulate inflammatory lesions on frozen section • In the diagnosis of certain benign inflammatory lesions which can simulate malignancy on frozen section 203
  204. 204. • 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 inadequate sampling. • 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 interpretation. 204
  205. 205. Imprint of a massively necrotic carcinoma showing a cluster of viable cancer cells on a necrotic background. Diagnosis was not possible on frozen section. 205
  206. 206. Imprint of a lymph node showing malignant cells. 206
  207. 207. ARMAMENTARIUM: 207
  208. 208. • Wide mouthed bottle with 10% formalin (at least 20 times the volume of the specimen should be used) • 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. 208
  209. 209. 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. 209
  210. 210. • 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 infections. 210
  211. 211. • 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 tissue. • The anesthesia should be subjected slowly enough to provide blanching but not rapid swelling. 211
  212. 212. TISSUE STABILIZATION •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 lips. 212
  213. 213. •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 213
  214. 214. • 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 biopsied. • It performs the two important functions of providing a firm surface to work and yields nearly complete hemostasis. • Sutures can be placed in the center of the ringed opening before the clamp is loosened. 214
  215. 215. 215
  216. 216. 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. •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. 216
  217. 217. • 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. • 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. 217
  218. 218. • 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. 218
  219. 219. • 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 219
  220. 220. 220
  221. 221. • 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 diagnostic specimens. • 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
  222. 222. • 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 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. 222
  223. 223. • 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. 223
  224. 224. However, fresh material is needed for the following purpose: 1. Frozen section 2. Immunocytochemistry 3. Cytological examination 4. Microbiological sampling before histopathology 5. Chromosome analysis 6. Research purpose 7. Museum display 224
  225. 225. • 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. 225
  226. 226. 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. 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. 226
  227. 227. • Factor affecting fixation: 1. Size and thickness of piece of tissue. 2. Tissue covered by large amount of mucous fix slowly. 3. The same applies to tissue covered by blood or organ containing very large amount of blood. 4. Fatty and lipomatous tissue fix slowly. 5. Fixation is accelerated by agitation. 6. Fixation is accelerated by maintaining temperature around 60oc. 227
  228. 228. 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. 228
  229. 229. 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. 229
  230. 230. 230
  231. 231. 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. • If the microscopic diagnosis is inconsistent with the clinical impression, the clinician is strongly advised to discuss any concerns directly with the pathologist. 231
  232. 232. General Principle of Gross Examination 1. Proper identification and orientation of the specimen. 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. 232
  233. 233. 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. c. Dimensions d. Weight e. Shape f. Colour g. Consistency h. Surgical margin, whether included or not involved by tumour. 233
  234. 234. 7. Measurements are usually given in centimeter unless the specimen is very small in which mm can be used. 8. Tissues should be measured by aggregate pieces in volume. 9. Endoscopic biopsies should be numbered. 234
  235. 235. Processing • The fixed tissue is dehydrated by immersion in a series of solvent and impregnated with paraffin wax. • 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) 235
  236. 236. 236
  237. 237. • 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 adhesive. • 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 237
  238. 238. 238
  239. 239. • 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 reached. 239
  240. 240. • 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 the specimen. • 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 histopathological examination. 240
  241. 241. • Pathologists and clinicians sometimes request slide consultations. • 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. 241
  242. 242. • 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 histology features. • And even pathologist can have a look in the lesion before biopsy if the location or clinical features of the lesson are unusual. 242
  243. 243. Good Morning 243
  244. 244. Stains • 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. 244
  245. 245. • 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. 245
  246. 246. • 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 stain. 246
  247. 247. Special Stains: 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. 247
  248. 248. c. PAS stain: It is used for fungi, amoeba and Tricomonas. d. Modified Giemsa (2% Giemsa in water): Detects Helicobacter pylori. 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. 248
  249. 249. In large lesions Accessible area Characteristic portion. For multiple lesions Most representative site. Material curetted from interior of the lesion . SPECIAL CONSIDERATIONS 249
  250. 250. For red & white lesions include both red & white area 250
  251. 251. ULCERS Include margin, deep part of ulcer and site of maximal clinical activity. AVOID Superficial ulcers & necrotic tissue 251
  252. 252. For Polypoid lesions include base 252
  253. 253. For Vesiculobullous lesions Fluid is more representative. Intact vesicle or bulla should be biopsied. 253
  254. 254. For LICHEN PLANUS – representative area should be biopsied 254
  255. 255. For LEUKOPLAKIA – Most dysplastic area should be biopsied 255
  256. 256. For MUCOCELE lesions – careful excisional biopsy 256
  257. 257. For GRANULOMATOUS LESIONS – deep incisional biopsy + fresh sample to microbiology if infective agent suspected 257
  258. 258. Do not cut into pigmented and vascular lesions 258
  259. 259. 259
  260. 260. ERRORS DURING SURGICAL REMOVAL OF BIOPSY SPECIMEN 260
  261. 261. INJECTION 261
  262. 262. 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. 262
  263. 263. SELECTION OF THE SPECIMEN Tissue should be representative of whole lesion. Adequate amount of tissue at least 1 x 0.5 cm in size. 263
  264. 264. Biopsy of skin or mucosa representative sample of both epithelium and connective tissue has to be taken 264
  265. 265. PRECAUTIONS AT FIXATION 265
  266. 266. SELECTION OF FIXATIVE Depends on purpose and method of tissue processing 10% neutral buffered formalin Request for 10% formalin From the Pathology Lab 266
  267. 267. Do not add tap water to dilute the fixative 267
  268. 268. PURPOSE OF FIXATION To retain tissue as much as possible in living condition 268
  269. 269. CONTAINER OF FIXATIVE Wide mouth clear bottle INTRODUCING INTO FIXATIVE Avoid curling of tissue. DELAY IN FIXATION Leads to artefacts. FRESH FIXATIVE Formalin kept for longer will not fix tissue properly LABELLING THE BOTTLE Patients NAME , AGE , SEX , HOSPITAL NUMBER 269
  270. 270. VOLUME OF FIXATIVE 270
  271. 271. • 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 studies. • 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 bath. 271
  272. 272. • 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 processing. • At present, immunofluorescence can be performed with biopsy specimens handled for several days at ambient temperature in a preservative liquid medium, Michel’s medium. 272
  273. 273. 273
  274. 274. Lost specimen: • The value of entire hospital admission for diagnosis biopsy is all too frequently negated by the loss of specimen. • This can be prevented only by a definite and formed routine which is rigidly enforced by the pathology department. 274
  275. 275. 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. 275
  276. 276. 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. 276
  277. 277. 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." 277
  278. 278. 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. 278
  279. 279. 1.DURING SURGERY: • Injection Artifacts • Forceps Artifacts • Fulguration Artifacts • Laser Artifacts • Suction Artifacts 279
  280. 280. 2.DURING FIXATION AND TRANSPORT: • Fixation Artifacts • Freezing Artifacts • Artifacts during transportation 3.TISSUE PROCESSING ARTIFACTS: • During Embedding • During Sectioning 280
  281. 281. INJECTION ARTIFACTS • Injection of large amounts of anesthetic solution into the area to be biopsied can produce following tissue changes: 1) The needle insertion may produce hemorrhage with extravasation, which in turn masks the cellular architecture. 2) The separation of connective tissue bands with vacoulation may occur. 3) Tissue edema or distortion. 281
  282. 282. 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. 282
  283. 283. 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. 283
  284. 284. 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. 284
  285. 285. 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. 285
  286. 286. 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. 286
  287. 287. 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. 287
  288. 288. • 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. • Alternating areas of coagulation impart a "Herring Bone" appearance to the tissue if the procedure is not done properly 288
  289. 289. 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 289
  290. 290. • 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. 290
  291. 291. ONCOCYTOID ARTIFACT IN SALIVARY GLAND TISSUES • 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. 291
  292. 292. • 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. 292
  293. 293. 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. 293
  294. 294. • 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. 294
  295. 295. 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. 295
  296. 296. • 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. 296
  297. 297. FIXATION ARTIFACTS • Although fixation is used to prevent the occurrence of various artifacts, it may itself be the cause of various artifacts. • The most common of these are: • Volume changes. • Artifactual pigments. • Diffusion artifacts 297
  298. 298. Volume changes • Various factors have been suggested such as inhibition of respiration, changes in membrane permeability and changes in ion transport through the membranes etc. 298
  299. 299. Artifactual pigments These include: 1) Formalin pigment. 2) Mercury pigment. 3) Dichromate deposits. 299
  300. 300. FORMALIN PIGMENT • 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. 300
  301. 301. MERCURY PIGMENT • Tissues fixed in fixative mixtures containing mercury have been found to develop a uniform granular black deposit known as mercury pigment. DICHROMATE DEPOSIT • Tissues fixed in potassium dichromate containing fixatives show an insoluble yellow-brown oxide precipitated if transferred directly to alcohol without proper washing in water. 301
  302. 302. DIFFUSION ARTIFACTS • 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 artifact. 302
  303. 303. • 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 histochemistry. 303
  304. 304. IMPROPER FIXATION • Delay in fixation and inadequate fixation alters the staining quality of the cells. • Cells appears shrunken and shows clumping of cytoplasm. • 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. 304
  305. 305. HOW TO AVOID • For optimal fixation the amount of fixative should be approximately 20 times the volume of the specimen. • The morphology of a tissue specimen will be altered by use of different fixatives. 305
  306. 306. FREEZING ARTIFACTS • 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 during transport. • Swiss cheese holes in the epithelium and tissue spaces are seen where forming ice crystals rupture. 306
  307. 307. 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) 307
  308. 308. 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 & through suture 308
  309. 309. Sponge artifacts • Utilization of sponges has been advocated as an efficient means of holding specimen during processing. • 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 superficial bristles. 309
  310. 310. SECTIONING ARTIFACTS • These artifacts can be caused as a result of: 1) Improper processing. 2) Improper sectioning. 310
  311. 311. IMPROPER SURGICAL REMOVAL 311
  312. 312. WRONG ORIENTATION OF SPECIMEN 312
  313. 313. CURLING AND CRUSH ARTEFACT 313
  314. 314. HEMORRHAGE ARTEFACT 314
  315. 315. SPLIT ARTEFACT 315
  316. 316. FRAGMENTATION ARTEFACT 316
  317. 317. 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. 317
  318. 318. Primary healing • 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. 318
  319. 319. • 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 the area. • 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 incision. 319
  320. 320. • 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. 320
  321. 321. • 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. 321
  322. 322. Secondary healing: • 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 approximated. • 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. 322
  323. 323. • 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. 323
  324. 324. • 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 and tissues. 324
  325. 325. • As the granulation tissue matures, it becomes more fibrous through condensation of collagen bundles, and the surface of the granulation tissue becomes epithelized. • The lesion becomes less vascular, the only evidence of the wound may be a small depressed area of the mucosa. 325
  326. 326. Complications 326
  327. 327. 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. 327
  328. 328. 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 normal tissue. • Aseptic technique must always be observed 328
  329. 329. 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 329
  330. 330. 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 330
  331. 331. 5. 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. 331
  332. 332. 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’. 332
  333. 333. CONCLUSION • 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. 333
  334. 334. • 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. 334
  335. 335. •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. References 335

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