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4biopsy 150328072327 Conversion Gate01 PDF
4biopsy 150328072327 Conversion Gate01 PDF
1
BIOPSY
3
• 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,
ulceration, proliferation) or it can be submucosal
structural alterations (i.e., distortion or swelling
produced by a mass).
5
• 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.
6
• 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
HISTORY
8
• 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.
9
• The term "biopsy" was introduced into medical
terminology in 1879 by Ernest Besnier.
11
It is possible to make out three stages in more than 100
year history of the method development:
12
• 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
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
• Biopsy is the removal and examination usually
microscopic of tissue from the living body,
performed to establish precise diagnosis.
-Chiles DG in 1987
15
• Biopsy is the removal of a piece of tissue from the
living body for diagnosis by microscopic
examination.
- Tomlins Christopher DC in 1998
16
INDICATIONS FOR BIOPSY
• To confirm a clinical impression of a lesion.
17
• To determine the nature of any intraosseous lesion
which cannot be identified clinically and
radiographically.
18
• Persistent hyperkeratotic changes in surface tissues.
19
1. Surface lesions:
20
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
CONTRAINDICATIONS FOR BIOPSY
RELATIVE
For ambiguous lesions
22
• Compromised general health of the patient or a
history of coagulopathy or bleeding diathesis,
including patient on anticoagulant therapy.
23
ABSOLUTE
• Pulsatile lesions or those suggestive of a vascular nature
should be referred for more in depth evaluation (e.g.,
hemangioma).
24
• Lesions that because of size or location present
technically difficult surgery e.g., posterior tongue and
oropharynx offer severe problems of access.
25
VARIOUS BIOPSY TECHNIQUES
• The type of biopsy to be performed depends on the
location, size and clinical impression of the lesion.
Basic types include-
26
•PUNCH
•ELECTROCAUTERY
•SOFT TISSUE
DIAGNOSTIC •INCISIONAL CURETTAGE
•FROZEN SECTION
SOFT TISSUE
•SCALPEL
CURATIVE •EXCISIONAL
•CAUTERY
SURGICAL
•CURETTAGE
DIAGNOSTIC •TREPHINE
•ASPIRATION
BONE •FROZEN
•ENUCLEATION
CURATIVE
•RESECTION
•CURETTAGE
NON-SURGICAL
•EXFOLIATIVE
ASPIRATION CYTOLOGY
•FNAC
27
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
• 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
TISSUE PIECE:
INCISIONAL BIOPSY / DIAGNOSTIC BIOPSY:
• It is a biopsy technique that samples only a
particular or representative part of the lesion.
34
Other indication includes:
• Chronic non-healing ulcer or squamous cell
carcinoma.
• Leukoplakia/ erythroplakia.
• Lichen planus.
36
Disadvantages:
• Leaves noticeable scar.
40
Technique:
41
• 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.
42
Advantages:
• The technique is helpful in differentiating the small
dysplastic erythroplakia that requires biopsy from
erythematous lesions caused by infection,
inflammation or trauma.
43
Disadvantage:
44
ELECTRO-SURGERY BIOPSY
• Electro-surgery refers to the cutting and coagulation of
tissue using very high-frequency, low-voltage electrical
currents.
47
Indications:
• It is the usual approach for smaller lesions (less
than 1 cm in diameter).
48
• Mucoceles
• Myoblastoma
• Keratoacanthoma
49
Principle:
50
51
Technique:
• For surface excision simple elliptical approach is
designed, like for the excision of pigmented and
hyperkeratotic lesions, fibroma, papillomas and
superficial pathology.
52
elliptical incision vertical deep
53
• For most Incisional and excisional biopsies, ellipse
technique is employed.
54
• 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.
55
• Haemostasis is achieved with resorbable suture
inserted (to control bleeding and also assist in
healing). Firm pressure for few minutes aid in
haemostasis.
58
59
PUNCH BIOPSY:
60
61
Indications:
62
• 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.
63
Contraindication:
64
Advantages:
• Technique is fast with low incidence of post surgical
morbidity.
66
• 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
Technique:
68
69
• After the biopsy site has been anesthetized, the site
is gently blotted with sterile gauze.
70
• 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.
71
• Persistent hemorrhage can be treated with
chemical cautery such as silver nitrate, collagen
matrix, or by electrocautery.
72
• Post- operative instructions include avoidance of
inadvertent trauma to the area, either by diet or
through attempts at oral hygiene for 48 hours.
73
74
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.
77
Technique:
• Biopsy tissue is frozen in a mixture of isopentene
and solid carbon dioxide at -70o.
78
• 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.
79
Errors in diagnosis can be due to:
• Sampling by the surgeon or pathologist.
80
Disadvantages:
81
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.
82
• Secondly the technique is simpler, easier and faster
than the traditional suction method.
83
• Cutting needle biopsy that employed the Biopsy
gun was first devised in the early 1980s.
84
• 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.
85
TRU-CUT NEEDLE BIOPSY:
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
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
Advantage:
• They are easy to interpret then aspiration cytology to the
pathologist
90
CELL ASPIRATE
FINE NEEDLE ASPIRATION CYTOLOGY:
(FNAC)
• Kun in 1847, gave the first description of the
technique for aspiration biopsy.
92
• 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.
93
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.
95
•Absence of reactive fibrosis to interfere with subsequent
surgery
•Readily repeatable
96
Disadvantages:
• Success of FNA depends on obtaining a
representative sample (if the specimen is small
with few or no cells).
97
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).
100
• Ideally the aspiration should have a creamy
consistency with a high cell and low fluid content.
102
GOOD MORNING
103
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
ASPIRATION CYTOLOGY GUN
105
FRANZEN’S HANDEL WITH SYRINGE & NEEDLE FITTED ON IT
FOR PERFORMING FNAC
106
Causes of unsatisfactory yield with fine needle
aspiration include:
• Needle missing a lesion and picking up
inflammatory cells.
107
COMPLICATION & HAZARDS OF FNAC
109
USE OF FNA:
110
• 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.
112
• Aspirated blood often indicates a vascular lesion
e.g., hemangioma or aneurysmal bone cyst.
113
• 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
Salivary gland swelling –
117
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
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.
120
Aspiration biopsy of radiolucent lesions
123
• 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
124
Osseous window
•Lesions within the jaw (i.e., central lesion) require the
use of a cortical window.
125
• 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.
126
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.
127
•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.
128
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
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
Available in a range of sizes,
from 7 guage to 12 guage.
135
• 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
• Caution should be exercised in patients with soft
bones secondary to radiation therapy, multiple
myeloma, or osteoporosis.
137
138
• In some patients, the sternum may be the first
choice for aspiration because of positioning
limitations or obesity.
139
Equipment
1. Sterile gloves 7. Heparinized 10 cc syringe
2. Sterile drape 8. 3 cc and 10 cc syringes
3. Povidone-iodine 9. 1% lidocaine
4. Bone marrow 10. Glass slides
aspiration needle or 11- 11. Specimen bottle with
or 13-gauge Jamshidi formalin
needle
12. 4“ x 4“ gauze
5. 25- and 22-gauge 13. Pressure dressing and
needles
tape
6. No. 11 scalpel blade
140
141
Tests on Bone Marrow Aspirate
and Biopsy Specimens
142
TEST PURPOSE
145
Microbiologic cultures Detect bacterial, viral,
and fungal infections in
patients with fever of
unknown origin and
immunodeficiency
syndromes
146
Clinical Exam
• 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
SCISSORS BIOPSY
• Is one of the ways to remove skin tissue for a
biopsy specimen.
148
• Small forceps with teeth and a pair of sharp curved
or straight iris scissors are the only surgical
instruments required.
149
150
SHAVE BIOPSY
• A scalpel or razor blade is used to scrape lesion,
performed superficially or deeply.
156
157
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.
158
Indications
159
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
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
• 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.
163
Endoscope guided biopsy
• Endoscopy is defined as “the examination of the
interior of a canal or hollow viscous by means of an
endoscope.”
164
• 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.
165
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
168
169
170
171
172
EXPLORATION BIOPSY
• Used for inrtaosseous lesions of maxilla and
mandible
• Instruments: Chisel, Bone burs, Periosteal Elevator
173
TISSUE SCRAPINGS
EXFOLIATIVE CYTOLOGY:
• Cyto – Cell -- Logos – Study
• Study of cells.
174
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 cervix
cancer
176
• Normal oral squamous epithelium continuously
sheds the most superficial cells.
177
• 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
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
Indications:
• For quick laboratory evaluation of suspected
malignant and premalignant oral lesions and
multiple premalignant and extensive lesion and
lesions leading to field cancerization.
180
• To identify the presence of certain specific cells in
non-malignant red patches or ulcerative lesions.
181
• Certain benign hereditary skin lesions having their
representative oral manifestations.
182
Contradictions:
• Deep seated lesions (both soft and hard tissue).
• Fibrous lesions.
• Polypoid growth.
• Non-ulcerative lesions.
183
• Atrophic lesions.
184
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.
186
Demerits:
• Relatively limited information provided with exfoliated
material when compared to a histological preparation.
188
Target for cytological Prediction:
189
Requisite for diagnostic cytology:
• Diagnosis must be based on the synthesis of entire
evidence available rather than the changes in
individual cell.
190
Technique:
199
Class I (Normal) indicates only normal cells.
Advantage:
• Feasible and economical
• Detect the malignancy at the tumor margin
• Less time consumable
201
• 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.
202
This is particularly useful in -
• In the diagnosis of certain neoplastic lesions which
can simulate inflammatory lesions on frozen
section
203
• 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.
206
ARMAMENTARIUM:
207
• Wide mouthed bottle with 10% formalin (at least
20 times the volume of the specimen should be
used)
208
Site selection and handing of the specimen-
209
• 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.
211
TISSUE STABILIZATION
213
• 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.
216
• Manipulation and grasping of any lesion suspected of
being a tumor should be minimal. Vigorous
manipulation of malignant tumors can cause tumour
cell emboli.
218
• Surgeons can place sutures in the specimen to
assist in orientation and provide a written
description of the specimen in relation to suture.
219
220
• Fixation and preservation of tissue are essential to avoid
distortion of histologic and cytologic details.
222
• 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.
223
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
• No fixative will penetrate a piece of tissue thicker
than 1 cm. For dealing with specimen thicker than
this, following methods are recommended:
225
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.
227
Labeling of the specimen:
• The patient’s name, the location of the specimen, and
the date of the surgical procedure are all essential.
229
230
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
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
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
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
Processing
235
236
• The paraffin block containing the tissue is mounted on a
cutting machine microtome and tissue section of 3-6 mm
thickness are cut.
240
• Pathologists and clinicians sometimes request slide
consultations.
241
• 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.
242
Good Morning
243
Stains
• Generally the stains are classified as:
A. Acid stains
B. Basic stains
C. Neutral stains
244
• 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
• 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
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.
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 ELECTROCAUTERY
Topic of debate
Combination of cautery and scalpel should be considered.
262
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
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
266
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 to artefacts.
FRESH FIXATIVE
Formalin kept for longer will not fix tissue properly
LABELLING THE BOTTLE
Patients NAME , AGE , SEX , HOSPITAL NUMBER
269
VOLUME OF FIXATIVE
270
• Disadvantage of formalin is that it makes the
specimen unsuitable for immunofluorescent
techniques as it forms protein cross- linkings.
272
273
Lost specimen:
274
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.
275
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
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
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
1.DURING SURGERY:
• Injection Artifacts
• Forceps Artifacts
• Fulguration Artifacts
• Laser Artifacts
• Suction Artifacts
279
2.DURING FIXATION AND TRANSPORT:
• Fixation Artifacts
• Freezing Artifacts
• Artifacts during transportation
280
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.
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HOW TO AVOID
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FORCEPS ARTIFACTS
• When the teeth of the instrument penetrates the
specimen, it resulted in voids or tears and
compression of the surrounding tissue.
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TO AVOID
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CRUSH ARTIFACT
• It’s a form of tissue distortion resulting from even
the most minimal compression of the tissue.
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CAUSES
• Mutilation of the tissues with forceps.
HOW TO AVOID
• Delicate handling of specimen.
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• The fibrous connective tissue, fat and muscle gain
an amorphous appearance when subjected to such
procedures.
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TO AVOID
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• This technique involves use of the scalpel for initial
incision in and around the lesion and electrosurgery
to complete the removal of the specimen.
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ONCOCYTOID ARTIFACT IN SALIVARY GLAND
TISSUES
• This unusual tissue artifact occur in the serous acini
of parotid gland tissue.
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• 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.
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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.
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• 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.
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ARTIFACTS INDUCED BY SUCTION TIPS
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• In highly vascularised tissue such as tissue in
periapical granulomas and in inflamed odontogenic
cysts.
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FIXATION ARTIFACTS
297
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.
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Artifactual pigments
These include:
1) Formalin pigment.
2) Mercury pigment.
3) Dichromate deposits.
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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.
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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.
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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.
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• 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.
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IMPROPER FIXATION
• Delay in fixation and inadequate fixation alters the
staining quality of the cells.
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HOW TO AVOID
• For optimal fixation the amount of fixative should
be approximately 20 times the volume of the
specimen.
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FREEZING ARTIFACTS
307
ARTIFACTS DUE TO TISSUE PROCESSING.
• Small biopsy specimen, often a narrow strip of
delicate mucosa, will bend and twist on fixation.
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SECTIONING ARTIFACTS
310
IMPROPER SURGICAL REMOVAL
311
WRONG ORIENTATION OF SPECIMEN
312
CURLING AND CRUSH ARTEFACT
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HEMORRHAGE ARTEFACT
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SPLIT ARTEFACT
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FRAGMENTATION ARTEFACT
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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.
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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.
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• 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.
319
• In time, these cells form thin, delicate collagen
fibrils which intertwine and coalesce in a general
direction parallel to the surface of the wound.
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• 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.
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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.
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• After the removal of the lesion, the blood filling the
defect clots and the repair process begins.
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• Cellular proliferation begins around the periphery of
the wound, and the fibroblasts and endothelial cells
grow into the clot along fibrin strands.
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• As the granulation tissue matures, it becomes more
fibrous through condensation of collagen bundles,
and the surface of the granulation tissue becomes
epithelized.
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Complications
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1. Haemorrahage –
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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.
328
3. Poor wound Healing
Unsatisfactory healing of the incision may be due to
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4. Spread of tumor cells
330
5. Injury to adjacent organs
• Injury may occur to surrounding structure (blood
vessels).
331
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’.
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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.
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References
•R. Rajendran and B. Sivapathasundharam: Shafer’s
textbook of oral pathology, 5th edition (2006), Elsevier.
335
• Marx RE. Oral and Maxillofacial Pathology. A
rationale for diagnosis and treatment. 2003.
Quintessence publishing co, Inc. Chicago.
337
• Oral Brush Biopsy Technique Instruction Outcomes for
Senior Dental Students David L. Hall, D.D.S. Journal of
Dental Education Volume 70, Number 8.
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THANK YOU
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