Statistical modeling in pharmaceutical research and development.
12.biopsy
1. Biopsy
Presenter: Dr. Murari washani
Introduction
Many medical conditions, including all cases of cancer, must be diagnosed by
removing a sample of tissue from the patient and sending it to a pathologist
for examination. This procedure is
called a biopsy, a Greek-derived word that may be loosely translated as "view
of the living." Any organ in the body can be biopsied using a variety of
techniques, some of which require major surgery (e.g., staging splenectomy
for Hodgkin's disease), while others do not even
require local anesthesia (e.g., fine needle aspiration biopsy of thyroid, breast,
lung, liver, etc). After the biopsy specimen is obtained by the surgeon, it is
sent for examination to the pathologist, who prepares a written report with
information designed to help the management of patient's condition
properly.
Many types of biopsy exist.
• Aspiration or FNA Biopsy
• Cone Biopsy
• Core Needle Biopsy
• Suction Assisted Core Biopsy
• Endoscopic Biopsy
• Punch Biopsy
• Surface Biopsy
• Surgical Biopsy (or Excisional Biopsy)
• Incisional biospy
The surgeon will determine the most appropriate method of biopsy and guidance
based on various factors including:
• the tissue, organ or body part to be sampled
• how suspicious the abnormality appears
• the size, shape and other characteristics of the abnormality
• the location of the abnormality
• the number of abnormalities
• other medical conditions a patient may have
• the preference of the patient, and
• the imaging and biopsy systems available at a given hospital or healthcare
location
2. Biopsies are usually guided by the method that identifies the abnormality best.
Palpable lumps can be felt and therefore no additional guidance is needed in
most cases. Lesions discovered by an imaging test, for example, mammography
or CT, will often need biopsy that is guided by the modality that shows the lesion
the best. For example, CT is usually the method of choice for imaging the lungs,
so CT imaging is used to guide most lung biopsies. If an abnormality is seen well
on multiple imaging tests, the modality that provides the safest, most accurate,
fastest and/or least expensive route will be used to guide the biopsy. Lesions
discovered by a screening test such as PSA may require blind sampling biopsy
since there is often no focal visible or palpable abnormality to target.
Definition
There are oral lesions whose diagnosis can be made relying on data gathered
during the history and/or physical examination, but there are others where
histopathological studies are needed to confirm the presumed clinical diagnosis.
Biopsy is a surgical procedure to obtain tissue from a living organism for its
microscopical examination, usually to arrive at a diagnosis
Objectives
The aim of the biopsy is to:
• define a lesion on the basis of its histopathological aspect;
• to establish a prognosis in malignant or premalignant lesions;
• facilitate the prescription of specific treatment;
• contribute to the assessment of the efficacy of the treatment;
• act as a document with medical-legal value.
Examination
A thorough inspection of the oral cavity should be a part of any complete head
and neck examination. Close visual inspection should be performed with
adequate lighting, and each of the anatomic regions in the oral cavity should be
palpated. Approximately 10% of patients who are examined have some
abnormality of the oral mucosa. Some lesions can be diagnosed on the basis of
the history and clinical findings alone. For other lesions, additional information
may be required to properly guide any indicated therapy. Often, biopsy with
ample tissue for microscopic analysis is the definitive procedure.
Management of Mucosal Abnormalities
The development of a reasonable differential diagnosis is of prime importance in
determining if biopsy is indicated. Furthermore, the differential diagnosis aids
the clinician in selecting the appropriate technique if biopsy is necessary.
A waiting period of 2 weeks often helps in forming the differential diagnosis
because lesions that are related to infection, inflammation, or local trauma may
resolve during this time. Biopsy is strongly recommended for the evaluation of
most lesions that persist for 2 weeks or longer after the potential irritants are
removed.
3. Conventional cytologic examination of the oral cavity is associated with an
unacceptably high false-negative rate. However, for clinicians who are
uncomfortable with intraoral surgery, brush biopsy with sampling of the full
thickness of the mucosa may provide reliable information regarding the presence
of cellular atypia. A positive result requires referral for scalpel biopsy.
Indications
Biopsy is indicated for the assessment of any unexplained oral mucosal
abnormality that persists despite treatment or the removal of local irritants.
Malignancy is suspected when persistent oral mucosal lesions are red or red and
white or when they are ulcerated, indurated, or fixed to deeper tissues. Persistent
lesions that bleed easily or grow rapidly should also alert to the possibility of
malignancy. Unexplained pigmented oral lesions are of concern if they are new or
changed. Biopsy is recommended unless the pigmented area has been present
and unchanged for 5 years or longer.
Scalpel biopsy may be warranted even when the differential diagnosis includes
only benign entities. Lesions that interfere with function and those that have an
undesirable effect on esthetics should be excised.
Sometimes difficulty arise in distinguishing truly innocuous oral mucosal
changes and those that represent dysplasia or early invasive cancer. Therefore,
the decision to forego biopsy in an apparently benign lesion should be made with
great care and only when the patient understands the need for close follow-up
and agrees to comply.
Other lesions that should also be biopsed include:
• lesions that interfere with oral function, such as fibrous hyperplasias and
osseous lumps.
• lesions of unclear aetiology, particularly when associated with pain,
paraesthesia or anaesthesia
• interstitial lesions in lingual, buccal or labial muscles
• radiolucent or radio-opaque osseous lesions.
Contraindications
Oral mucosal biopsy has few contraindications. The standard biopsy techniques
may need to be modified in some patients, including those with conditions that
preclude the safe use of local anesthetic and those with severe bleeding diatheses
or coagulopathies. All invasive procedures in the oral cavity should be avoided in
patients who have used or are currently using injectable bisphosphonates.
Mucosal lesions in these individuals may be reflective of underlying
osteochemonecrosis, a condition that may be exacerbated by any manipulation.
4. RELATIVE CONTRAINDICATIONS
1: Allergic reactions
2: History of coagulopathy therapy
3: Proximity of lesions to vital anatomic, vascular, neural or ductal structures and
lesions
4: In areas of difficult surgical access demand at least a modicum of surgical
experience prior to biopsy
ABSOLUTE CONTRAINDICATIONS
• Pulsatile lesions or even those suggestive of a vascular nature should be
reffered .Vascular lesions should never be biopsied incisionally.
• intrabony radiolucent lesions should not be biopsied or removed without
prior investigational aspiration. Invasion of such lesions for biopsy
invariably results in sepsis of the lesion and surrounding tissues,and
surgical ablation of the lesion and or reconstruction is delayed or
compromised.
• Pigmented lesions should not be incisionally biopsied generally. It has
been suggested that the spread or seeding of malignant cells of melanoma
can occur through incisional biopsy, as can the transformation of
premalignant pigmented lesions to malignant ones.
Technique Selection
A differential diagnosis is formed by reviewing the features of the history and
physical findings in the context of the clinician's experiences and knowledge. The
result should be a group of possible diagnoses, beginning with the most likely
one. If the differential diagnosis includes malignancy, a tissue specimen must be
obtained. Incisional biopsy is indicated in this situation so that definitive
treatment of the potential malignancy is not compromised. If the differential
diagnosis does not include malignancy, lesions of reasonable size in manageable
locations can be completely excised at biopsy.
A variety of authors have proposed size limits for excisional biopsy. General
dentists, dermatologists, oral and maxillofacial surgeons, otolaryngologists, and
others undoubtedly have different comfort and skill levels; therefore, specific size
guidelines for incisional biopsy versus excisional biopsy have little value.
Similarly, clinicians who are uncomfortable with the regional anatomy should not
perform excisional biopsy of lesions near significant anatomic structures. When
excisional biopsy is being considered, should also be aware that it may produce
esthetic compromise as a result of scarring or residual deformity. The esthetic
5. outcome is of particular concern when a lesion on the lip is near the vermilion
border.
Numerous methods can be used to collect tissue samples from the oral mucosa
for histopathologic examination. Performing biopsy with a scalpel is the standard
and generally produces the most satisfactory specimen. Other techniques include
the use of a needle, biopsy punch, biopsy forceps, laser, or electrocautery device.
Needles may be appropriate in sampling cells from mass lesions, but they are of
no benefit in the evaluation of surface lesions. Electrocautery produces thermal
damage and artifact, which make evaluation of the specimen difficult; therefore,
electrosurgery should be avoided during oral mucosal biopsy. Electrosurgery may
be of benefit for wide local excisions of known intraoral malignancies after a
scalpel is used to atraumatically obtain marginal specimens for frozen sections.
A carbon dioxide or Nd:YAG laser produces a zone of thermal coagulation smaller
(approximately 500 µm) than that of electrocautery . If a laser is used for
incisional or excisional biopsy, a 0.5-mm margin should be maintained between
the cut and the representative area to be sampled. Although this technique may
result in good local hemostasis and minimal postoperative discomfort, it is
associated with potential shortcomings, including impingement on the specimen,
particularly at the deep margin, and the generation of excessive heat with
inadequate removal of the charred layer. The laser may be of great value in
managing the wound left by scalpel biopsy in areas of the mouth where closure is
difficult or inappropriate.
Biopsy forceps are long-handled instruments with biting ends that are cup
shaped to harvest an adequate amount of mucosa. They are particularly useful in
pharyngeal lesions in which the use of a scalpel is more challenging. The
specimen is not crushed in the cup and should be eminently assessable by the
pathologist.
The use of a biopsy punch in oral mucosal lesions is described and may be of
some value. Punch biopsy may be difficult on freely movable oral tissues and
probably offers no advantage compared with scalpel biopsy .The technique may
be appropriate in the hard palate and other sites with better support and tissue
that is bound down, and it is likely to produce a satisfactory specimen. The
wound heals by secondary intention, and discomfort may persist longer than
anticipated by the clinician and the patient.
When is biopsy not needed?
• There is no need to biopsy normal structures
• There is no need to biopsy irritative/traumatic lesions that respond to the
removal of a presumed local irritant
• There is no need to biopsy inflammatory or infectious lesions that respond to
specific local treatments, as pericoronitis, gingivitis or periodontal abscesses
• No incisional biopsies should be performed on suspected angiomatous
lesions.
6. Types of biopsy
According to the procedures applied, oral biopsies can be classified by:
a) Features of the lesion:
• Direct biopsy: when the lesion is located on the oral mucosa and can be
easily accessed with a scalpel from the mucosal surface.
• Indirect biopsy: when the lesion is covered by an apparently normal oral
mucosa
b) Area of surgical removal:
• Incisional biopsy: consists of the removal of a representative sample of
the lesion and normal adjacent tissue in order to make a definitive
diagnosis before treatment.
• Excisional biopsy: is aimed at the complete surgical removal of the lesion
for diagnostic and therapeutic purposes. This procedure is elective
when the size and location of the lesion allows for a complete removal
of the lesion and a wide margin of surrounding healthy tissue.
c) By the timing of the biopsy:
• Pre-operative
• Intra-operative
• Post-operative when aimed at checking the efficiency of a treatment.
General principles of biopsy:
Before the procedure is undertaken, the characteristics of the lesion (size, shape,
colour, texture, consistency, time of evolution, associated signs and symptoms,
regional nodes) should be described in the patient’s clinical records together with
a presumed diagnosis and possible differential diagnosis.
The patient should receive information on the technique that will be performed
and the reasons why it is performed, avoiding terms that may cause anxiety.
Informed consent is required.
Regarding the surgical technique:
• Regional block local analgesia rather than infiltrative techniques is
preferred;
• Elliptical incisions should be attempted in order to ease suture;
• Incisions parallel to nerves and vases are preferred;
• If the lesion is smaller than 1 cm, excisional biopsy should be performed. If
larger, an incisional technique including representative areas of the lesion with
healthy margins should be chosen;
• When a malignant lesion is suspected, incisional technique is mandatory.
Samples must be oriented with a suture or a piece of paper, and introduced in a
container with a fixing solution (10% formalin)
7. The number and location of the biopsies will be decided on the basis of the
clinical appearance of the lesion. If a lesion shows several areas where biopsy
would be indicated, more than one sample should be taken. In these cases with
precancerous or suspicious lesions, toluidine blue staining could be useful to
choose the area most relevant to biopsy.
The biopsy should be large enough to include normal and suspicious tissue and
for the pathologist to give a diagnosis without further specimens (small samples
are difficult to orientate and handle and certain processes as sample fixation may
end in a reduction of the size of the specimen).
There are different procedures for undertaking oral biopsies. However, the
selection of both technique and surgical instruments to use to avoid artifacts is
controversial. The use of CO2 laser for the procurement of diagnostic biopsy
specimens is compromised by thermal cytological artifacts. Problems of this
nature are also witnessed with electrocautery. Punch biopsy has been suggested
to reduce artifacts, although this has not been confirmed under controlled
experimental conditions. Punch biopsy may tear the tissue in vesiculobullous
conditions. Scalpel biopsy is the most widely accepted technique and the one that
shows fewer limitations for obtaining samples from the oral cavity.
Scalpel technique for biopsy taking:
In order to obtain good visibility, good illumination is needed. A Farabeuf-type
separator or similar instrument to retract the lips and cheeks, and moderate-
volume surgical aspiration are required.
The instruments suggested are:
- local anaesthetic syringe
- Fine, single use, two-sided needles
- Small and short scalpel blades (no. 15, 11, 12 or even 5)
- Mosquito forceps
- Allis tweezers
- 2/0 to 5/0 non-traumatic suture material
- Gauze
- Container with fixing solution
A biopsy technique can be reduced to six steps: selection of the area to biopsy,
preparation of the surgical field, local anaesthesia, incision, handling of the
specimen and suture of the resulting wound.
1. Selection of the area to biopsy
When dealing with small-sized lesion, an excisional biopsy will be performed,
whereas incisional biopsy performed in the most representative area of the lesion
is used for large lesions (long axis larger than 1 cm). If there is any doubt about
the malignant character of the lesion, vital staining with toluidine blue can be use
as an adjunct to select representative areas. Toluidine blue is a basic dye that
fixes to nucleic acids and stains the nuclear content of malignant cells; in these
cases samples should be taken from areas with deep blue patches, as light blue
areas are not significant. Toluidine blue is used in three steps:
• wash the area with 1% acetic acid
8. • apply a 1% toluidine blue water solution for 1 minute
• Mouthwash with 1% acetic acid
The sample must include healthy tissue at the margin of the lesion.
2. Preparation of the surgical field.
The surgical area is disinfected with a quaternary ammonium compound. Iodine-
containing surface antiseptics should not be used, as they may stain the tissues. A
0.12- 0.20 % chlorhexidine solution is preferred.
3. Local anaesthesia:
An local anaesthetic solution with vasoconstrictor should be used and infiltrated
away from the lesion are to avoid introducing artifacts in the sample.
4. The incision:
Oral tissues should be immobilized far from the area to biopsy with non-toothed
tweezers. A clean and defined incision is performed to obtain a slice of tissue
when aiming at incisional biopsy. Soft tissues incisions should be elliptical in
shape producing a “V” wedge that includes both the lesion and healthy margins.
If various lesions are present, multiple biopsies should be taken.
5. Tissue handling
The specimen is handled gently to avoid crush artifacts and introduced in the
fixing solution. The role of the fixing agent is to preserve the cellular architecture
of the tissues. There are authors that suggest the placement of the specimen on a
sterile paper with the mucous surface facing upwards to avoid distortion and
curling of the sample margins.
The best fixing agent is a 10% formalin solution, as it induces less ultrastructural
alterations in the samples. 70% ethanol can also be used. The samples should
never be put in isopropyl or methyl alcohol, saline or distilled water - as severe
alterations may be provoked. The volume of the fixing agent should exceed 10 to
20-fold the volume of the sample. When immunofluorescence or immunostaining
are needed, specimens should not be fixed, but sent as soon as possible to the
laboratory for freezing or put in Michel’s solution.
When the material is sent to the pathologist, it should be accompanied with a
detailed report that includes identification of the patient, clinical records, clinical
signs and a probable diagnosis as well as the orientation of the sample. An
explanatory diagram of the biopsy area may be useful for this purpose.
6. Suture
The suture should achieve good haemostasis, facilitate healing and should be
removed after 6-8 days
What are the most frequent errors that should be avoided when
taking oral biopsies?
In order to obtain a quality, artifact-free oral biopsy that permits the pathologist
establish a histological diagnosis, the clinician should avoid:
9. • pressing the sample with the tweezers, particularly if toothed, as may
produce tissue tears and “pseudomicrocysts”
• infiltrating anaesthetic solution within the lesion, as it can cause sample
alterations
• applying products to the lesion that induce tissue modifications
• using an insufficient volume of fixing solution
• Inclusion of undesired material in the sample: glove powder, calculus,
restorative materials, etc.
• taking insufficient amount of tissue in extension and depth.
Incisional biopsy
When incisional biopsy is contemplated, the biopsy site should be carefully
considered. Much has been written about selecting a site at the periphery of a
lesion to ensure the inclusion of healthy tissue. This site selection is certainly
important in an ulcerated oral lesion. Selecting only the center of an ulcer results
in an inadequate specimen devoid of mucosa. Some authors generalize this
principle to nonulcerated lesions as well. Advocates of this approach might argue
that the edge of many lesions is an area of activity and interest; however, the
concept that the specimen must contain healthy tissue for pathologic diagnosis
probably has little merit. The overriding principle that guides site selection for
incisional biopsy should be the acquisition of the most representative sample of
the suspected pathologic condition. An overzealous attempt to include the
periphery of a lesion with clinically healthy tissue may inadvertently lead to a
missed diagnosis or an under diagnosis.
Persistent diffuse color and texture changes on oral mucosal surfaces may be the
clinical expression of oral epithelial dysplasia. Specimens of similarly affected
mucosa may yield adjacent zones of mild-to-severe dysplasia, carcinoma in situ,
and microinvasive squamous cell carcinoma. This result raises the question of
how a clinician knows whether incisional biopsy samples are sufficient for the
most important histologic diagnosis of a diffuse area of mucosal change.
Incisional biopsy can lead to a diagnosis of mild or moderate dysplasia despite
the presence of invasive cancer within millimeters of the biopsy site. Therefore, a
diagnostic adjunct may be used to guide the clinician to the biopsy site that is
most likely to be associated with carcinoma in situ or invasive cancer.
One such adjunct is staining with toluidine blue (tolonium chloride), a dye that
predictably stains affected mucosa and not unaffected areas. Numerous findings
support the effectiveness of this vital staining technique as a tool to enhance the
diagnostic abilities of even experienced clinicians. A recommended protocol
begins with careful assessment of the lesion in question on the first day. Potential
irritants that may provoke an inflammatory response are removed for 2 weeks.
Loose dentures, sharp spots on tooth cusps or dental prostheses, and
parafunctional habits may all result in mucosal inflammation that can be
clinically indistinguishable from an early oral cancer.
10. On the return visit in 14 days, the area is reevaluated. Persistent mucosal
abnormalities, particularly those with red components, are stained by using an
application technique. A few drops of toluidine blue are applied to the lesion and
surrounding mucosa. Patients then rinse their mouths several times with water or
a mild acetic acid solution. The dorsum of the tongue retains some stain because
of its rough papillary contour.
On the remaining surfaces of the oral mucosa, any stain that persists and cannot
be wiped off indicates the need for incisional biopsy. Biopsy should be focused on
the area of greatest staining. Toluidine blue does not interfere with routine
histopathologic examination nor does it hamper computer-assisted cytologic
screenings of brush biopsy samples. Following this protocol, the sensitivity and
specificity of toluidine blue staining are more than 90%. When properly applied,
toluidine blue staining is a highly sensitive and specific test for carcinoma in situ
and invasive oral cancer.
Other diagnostic adjuncts involving chemiluminescence or tissue fluorescence
are now available and may augment the ability of the clinician to visualize areas
of suspicious mucosal change.
Large diffuse zones of persistent mucosal change require multiple incisional
biopsies. Close observation and/or repeat biopsy are indicated for areas
diagnosed as mild or moderate dysplasia. Severe dysplasia, carcinoma in situ,
and invasive cancers should be treated by using the principles of oncologic
surgery.
The administration of local anesthetic for incisional biopsy is generally simple
and straightforward. A small amount of local anesthetic infiltrated in an area
peripheral to the lesion provides adequate anesthesia in nearly every situation.
To avoid distorting the lesion, the anesthetic should not be injected directly into
it. Theoretically, direct injection can also result in an inadvertent seeding of cells
deeper into the tissues. The judicious use of a vasoconstrictive agent in the
anesthetic solution improves local hemostasis and can be helpful when indicated.
The minimal requirements for an adequate specimen vary somewhat with the
nature of the pathologic entity. As a general principle, including tissue subjacent
to the epithelium and removing a wedge of manageable size is desirable.
Therefore, a minimal depth of 3 mm, minimal length of 3-6 mm, and minimal
width of 1-2 mm are advised
As with excisional biopsy, suction devices should be used with caution or
completely avoided to prevent inadvertent loss of the specimen. If necessary, a
suction tip covered with gauze safely keeps the field clear.
The armamentarium for biopsy includes the following:
• Blade handle with a No. 15 blade
• Fine tissue forceps with teeth
11. • Local anesthetic solution and syringe
• Retractor appropriate for the site
• Suture for traction, if needed
• Needle holder
• Suture for closure, if indicated
• Fine-tipped scissors
• Laser or electrocautery device for fulguration, if indicated
• Specimen bottle containing formalin and biopsy data sheet
• Gauze sponges
Small wedge-shaped incisional biopsy sites can usually be closed with a single
suture. Small wounds in the floor of the mouth or on the tonsillar pillars heal well
without primary closure. Hemostasis may be achieved by using a laser or
electrocautery unit if necessary.
Excisional biopsy
Given a differential diagnosis that includes only benign entities, the clinician may
elect to remove a lesion in its entirety. As indicated previously, the size of the
lesion is only one of several factors that may affect the decision to perform
excisional biopsy. The location of the lesion, the nature of its attachment to the
underlying tissue, the accessibility of the lesion, and the regional anatomy all
contribute to the decision. Small, pedunculated, exophytic masses in accessible
areas are excellent candidates for excisional biopsy.
The preferred methods for the administration of local anesthetic are regional
blocks or field blocks, which are accomplished by means of infiltration peripheral
to the lesion. Much of the oral mucosa is easily movable, and an assistant may
need to stabilize the area by using an instrument or his or her fingers.
Stabilization and traction techniques specific to the various anatomic area within
the oral cavity are discussed in Special Considerations below.
As with incisional biopsy, suction devices should be used with caution or
completely avoided during excisional biopsy to prevent inadvertent loss of the
specimen. If necessary, a suction tip covered with gauze safely keeps the field
clear. When excisional biopsy is planned, the lines of excision should ensure that
the entire lesion is removed.
Two incisions forming an ellipse are made around the lesion with the blade
angled toward the lesion. These incisions produce a wedge-shaped specimen that
is deepest under the center of the lesion and leaves a wound that is simple to
close .Closure is facilitated by developing an ellipse that is 3 times longer than it
is wide .In general, properly designed elliptical wounds can be closed easily;
however, depending on the location of the biopsy site and the size of the wound,
mucosal undermining may help in producing a tension-free closure.
Aspiration or FNA Biopsy is performed with a fine needle attached to a
syringe. Aspiration biopsy is often referred to as Fine Needle Aspiration (FNA).
12. FNA biopsy is a percutaneous (through the skin) biopsy. FNA biopsy is typically
accomplished with a fine gauge needle (22 gauge or 25 gauge). The FNA
procedure is often performed, for example, to diagnose nonpalpable breast
abnormalities .FNA may be performed under image guidance such as ultrasound.
The area is first cleansed and then usually numbed with a local anesthetic. The
needle is placed into the region of the abnormality such as a cyst or tumor. Once
the needle is placed, a vacuum is created with the syringe and multiple in and out
needle motions are performed. The cells to be sampled are sucked into the
syringe through the fine needle. Three or four samples are usually taken.
Before microscopic examination is made, the sample of fluid and cells is
sometimes spun at high speed in a centrifuge (a device for separating substances
in a liquid by spinning the mixture at high speeds) then a small amount is placed
on a slide and covered with a plastic slip. A smear is prepared by spreading
samples of fluid and cells onto glass slides. The specimens are then fixed
(preserved) and stained to improve viewing. The preservation (fixing) is often
performed by heating the slide with a Bunsen burner or by using a methanol
solution. A cytologist (pathologist who examines cells) then uses a microscope to
examine individual cells for abnormalities, paying particular attention to the size,
shape and structure of the cell and cell nucleus.
Tumors of deep, hard-to-get-to structures such as the pancreas, lung, and liver
are good candidates for FNA. Such FNA procedures are typically done by a
radiologist under guidance by ultrasound or computed tomography (CT) imaging
and usually require no anesthesia or only local anesthesia. Thyroid abnormalities
are also excellent candidates for FNA.
FNAC
• Standard disposable 21-25G needle with 10ml / 20ml syringe
Advantages (over tru-cut biopsy):
• Excellent patient compliance
• Can be readily repeated
• Minimal / no complications such as pain or bleeding
Disadvantages:
• Inadequate sample with little or no cells
• False +ve/–ve results.
Uses of fine needle aspiration
Lymph nodes: Reactive hyperplasia/inflammation, Malignant disease,
Lymphoma.
13. • Salivary gland swellings
• Thyroid gland
• Skin and soft tissue,Bone &cartilage.
Failures in aspiration biopsy – due to:
• Needle missing the lesion / picking up inflammatory cells
• Lack of cells in central area - necrosis/cystic change / hemorrhage
• Lack of cells in dense fibro-sclerotic tissue
• Small malignant lesion masked by large benign mass
Drawbacks of aspiration cytology:
• Inadequate sample with few or no cells
• A false negative result lesion diagnosed as benign when it is malignant
• A false positive result lesion diagnosed as malignant when it is benign
Guided FNAC
Ultrasound, computed tomography (CT) & magnetic resonance imaging
(MRI) used in percutaneous biopsy procedures.
• Previously inaccessible lesions can now be routinely biopsied
• Ultrasound shows the exact location of the needle tip during the actual
procedure
• Fine needles are seen more easily than larger bore needles
• Ultrasound and fine needle aspiration together can investigate non-
palpable disease and to differentiate tissues within a lesion
• MRI & CT offer enhanced definition over ultrasound imaging
TRU-CUT BIOPSY
• It consists of wide bore 14G needle and consists of a long 15.2cm (6in)
canula and trocar with a 2cm notch at the tip of the trocar.
Technique:
• Local analgesia
• Stab incision with a scalpel
• Canula is inserted with the trocar fully retracted until the specimen notch
is within the tissue to be biopsied.
14. • The trocar is stabilised and the canula is withdrawn to expose the
specimen notch
• Tissue prolapses in to the notch and is then cut by pushing the canula back
over the trocar again, before the whole assembly is withdrawn.
• The specimen is recovered by pushing the trocar out of the canula and
placing it in formal saline for routine pathology.
VIM SILVERMAN BIOPSY
• It consists of:
Outer canula 16 G in size.
Inner trocar.
Inner split longitudinal needle.
• Technique:
Local analgesia
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 splitcutting needle. This is pushed in to the lesion by
about 2cm; firm stability of the canula is maintained by other hand. The
specimen is cut by holding the hub of the cutting needle and pushing the
canula downwards in rapid spiral motion to cover the cutting needle. The
whole instrument is removed and the specimen placed in formal saline.
• Advantage:
1. They are easy to interpret then aspiration cytology to the pathologist
2. To distinguish between reactive changes and recurrent malignancy in
possible cervical metastasis.
Cone Biopsy removes a piece of tissue which is cylindrical or cone shaped. Cone
biopsy is performed to diagnose cervical cancer. Cone biopsy is often done
following a pap smear, colposcopy (examination of the cervix under illuminated
magnification), and a punch biopsy.
After the tissue is removed, it is analyzed in the pathology laboratory to
determine whether cancer is present. Cone biopsy may also be performed as a
treatment if a cancer is small enough to be completely removed during biopsy.
There are two main methods used to perform cone biopsy. The LEEP (also called
LLETZ) method, short for loop electrosurgical excision procedure, removes the
tissue by using a wire that is heated by an electrical current. Patients are given
local anesthesia and the procedure can be performed quickly. Another method of
cone biopsy involves using a surgical scalpel or laser to remove the tissue. This
procedure typically requires general anesthesia and may be performed in a
hospital or outpatient facility. However, an overnight hospital stay is not usually
required.
15. The most common side effects of cone biopsy include cramping/discomfort and
moderate or mild bleeding for a few weeks after the procedure. Patients should
avoid sexual intercourse, tampons, and douching until the incision is completely
healed, which may take several weeks. Patients should also discuss other possible
side effects of cone biopsy prior to the procedure.
The advantages of cone biopsy are that it provides a large sample of tissue for
analysis and it can sometimes completely remove the cancer so the patient does
not need additional surgery. If a cone biopsy is recommended after abnormal
Pap smear results, a patient may wish to ask if a colposcopy (looking at the cervix
with magnification) or cervical biopsy would be an appropriate alternative for her
(if they have not already been performed), based on her individual case.
Core Needle Biopsy (or core biopsy) is performed by inserting a small hollow
needle through the skin and into the organ or abnormality to be investigated. The
needle is then advanced within the cell layers to remove a sample or core. Needle
biopsy is also a type of percutaneous (through the skin) biopsy. The needle may
be designed with a cutting tip to help remove the sample of tissue. Core biopsy is
often performed with the use of spring loaded gun to help remove the tissue
sample.
Core biopsy is typically performed under image guidance such as CT imaging,
ultrasound or mammography. The needle is either placed by hand or with the
assistance of a sampling device. Multiple insertions are often made to obtain
sufficient tissue, usually multiple samples are taken. Patients may experience a
slight pressure, but usually do not experience significant pain. As tissue samples
are taken, a click may be heard from the sampling instrument. The core tissue
samples will be sent to the pathology laboratory for diagnosis. In some cases, the
pathologist can attend the biopsy to examine imprints of the samples with a
microscope. This can allows the pathologist to determine the adequacy of the
sample and perhaps offer preliminary results.
Vacuum Assisted Biopsy: Core biopsy is sometimes suction assisted with a
vacuum device. This method enables to removal of multiple samples with only
one needle insertion. Vacuum assisted core biopsy is being used more and more
in breast biopsy procedures and is guided via stereotactic mammography or
ultrasound imaging. However, unlike core biopsy, the vacuum assisted biopsy
probe is inserted just once into the tissue through a tiny skin nick. Multiple
samples are then taken using a rotation of the sampling needle aperture
(opening) and with the assistance of suction.
Endoscopic Biopsy is a very common type of biopsy. Endoscopic biopsy is
done through an endoscope (a fiber optic cable for viewing inside the body)
which is inserted into the body along with sampling instruments. The endoscope
allows the physician to visualize the abnormality and guide the sampling.
Endoscopic biopsy may be performed of the gastrointestinal tract (alimentary
tract endoscopy), urinary bladder (cystoscopy), abdominal cavity (laparoscopy),
16. joint cavity (arthroscopy), mid-portion of the chest (mediastinoscopy), or trachea
and bronchial system (laryngoscopy and bronchoscopy), either through a natural
body orifice or a small surgical incision. The endoscopist can directly visualize an
abnormal area on the lining of the organ in question and pinch off tiny bits of
tissue with forceps attached to a long cable that runs inside the endoscope.
Punch Biopsy is typically used by dermatologists to sample skin rashes, moles
and other small masses. After a local anesthetic is injected, a biopsy punch, which
is similar in function to a small (3 mm to 4 mm or 0.15 inch in diameter) version
of a cookie cutter, is used to cut out a cylindrical piece of skin. The opening is
typically closed with a suture (small stitches) and heals with minimal scarring.
Punch biopsy may also be performed when removing small tissue samples from
the cervix.
Surface Biopsy involves sampling or scraping the surface of a sore or tumor to
remove cells for pathologic testing. Surface biopsy is often performed by
dermatologists to remove a small piece of skin to test for carcinoma (cancerous
tissue).
BRUSH BIOPSY
Brush biopsy is a noninvasive method of evaluating oral mucosal lesions for
cellular atypia. It is a three-layer transepithelial exfoliative cytology technique.
Computerized brush biopsy analysis (Oral CDx®) became commercially available
in 1999.
Brush Bx Kit
1. Brush Bx instrument.
2. Pre-coded glass slide and test form.
3. Alcohol/carbowax fixative pouch.
4. Pre-addressed submission container.
Brush Bx Procedure (three-layer exfoliative computer-assisted cytology)
1. Open fixative.
2. Wet brush with water or saliva.
3. Use mild (flat ulcerated) to firm (thick keratinized) pressure for 5 (flat)-10
(thick) rotations.
4. See pink micro-bleeding; bend brush handle and bristles.
5. Spread immediately over entire glass slide.
6. Squeeze onto slide entire contents of fixative to saturate specimen.
7. Dry alcohol for 15-20 minutes.
8. Complete submission form.
Evaluation of Results 1. Negative: no cellular abnormalities, normal spectral
analysis of keratin, normal nuc/cyto ratio; F/U. 2. Positive: atypical epithelial
changes indicate scalpel Bx; cancer or dysplasia requires referral.
17. Indications
• For precancerous / cancerous oral mucosal lesions
Advantages
• Easy to perform; requires less time
• Well tolerated by the patient
CURETTAGE
• Curette – French word curer, meaning to clean out
• Applies to anatomical & pathological cavities
• Designed for scraping out cavities (diagnostic/therapeutic
purposes)
• Used primarily for intra-osseous lesions
• Samples produced are usually soft tissues
• Done on the surface of tumors or on small epidermal lesions with minimal
to no topical anesthetic using a round curette blade.
Diagnosis of basal cell cancer can be made with some limitation, as morphology
of the tumor is often disrupted.
Bone marrow biopsy
In cases of abnormal blood counts, such as unexplained anemia, high white cell
count, and low
platelet count, it is necessary to examine the cells of the bone marrow. In adults,
the sample is usually taken from the pelvic bone, typically from the posterior
superior iliac spine. This is the prominence of bone on either side of the pelvis
underlying the "bikini dimples" on the lower
back/upper buttocks.
With the patient lying on his/her stomach, the skin over the biopsy site is
deadened with a local anesthetic. The needle is then inserted deeper to deaden
the surface membrane covering the bone (the periosteum). A larger rigid needle
with a very sharp point is then introduced into the marrow space. A syringe is
attached to the needle and suction is applied. The marrow cells are then drawn
into the syringe. This suction step is occasionally uncomfortable, since it is
impossible to deaden the inside of the bone. The contents of the syringe, which to
18. the naked eye looks like blood with tiny chunks of fat floating around in it, is
dropped onto a glass slide and smeared out. After staining, the cells are visible to
the examining pathologist or hematologist.
This part of procedure, the aspiration, is usually followed by the core biopsy, in
which a slightly larger needle is used to extract core of bone. The calcium is
removed from the bone to make it soft, the tissue is processed and tissue sections
are made. Even though the core biopsy procedure involves a bigger needle, it is
usually less painful than the aspiration.
SENTINAL NODE BIOPSY
The sentinel lymph node (SLN) is the first lymph node to drain a metastatic
tumor cell that drains via the lymphatic route. The concept of the SLN is based on
the orderly progression of tumor cells within the lymphatic system. Mapping of
the lymph flow from the tumor site to the regional lymphatic drainage area can
be used to identify the primary draining lymph node (ie, SLN). If the SLN can be
identified and examined for the presence of tumor metastases, an elective lymph
node dissection for staging does not need to be performed
Although CT scanning and MRI are commonly used to classify tumors of the
neck, their overall accuracy is limited. The only highly accurate means of
identifying lymph node disease is to perform a staging lymph node dissection.
For disease in its early stages, clinicians are reluctant to perform an elective
lymph node dissection because of the associated morbidity and lack of beneficial
effects.
Pathophysiology
SCC arises from basal keratinocytes of the skin. It typically manifests as a firm
nodule on an erythematous base with elevated borders and insidious margins.
Central ulceration or crusting may be present. Irregular nests of epidermal cells
invading the dermis in varying degrees characterize SCC. Grading is based on the
degree of cell differentiation. The most common route of spread for metastatic
SCC is lymphatic in nature.
INDICATIONS
Elective dissection of a clinically negative (ie, N0) neck causes overtreatment for
most patients, while no equivocal advantage in survival has been demonstrated
when compared with a delayed dissection for patients with metastases in the
neck. SLN biopsy can help determine the presence of lymph node metastases in
patients with T1-T2, N0 oral and oropharyngeal SCCs
After the tissue is removed from the patient, it is processed in one or both of two
major ways:
19. Histologic sections
This involves preparation of stained, thin (less than 5 micrometers, or 0.005
millimeters) slices mounted on a glass slide, under a very thin pane of glass called
a coverslip. There are two major techniques for preparation of histologic sections:
Permanent Sections
This technique gives the best quality of specimen for examination, at the expense
of time. The fresh specimen is immersed in a fluid called a fixative for several
hours (the necessary time dependent on the size of the specimen). The fixative,
typically formalin (a 10% solution of formaldehyde gas in buffered water), causes
the proteins in the cells to denature and become hard and "fixed." Adequate
fixation is probably the most important technical aspect of biopsy processing.
The fixed specimen is then placed in a machine that automatically goes through
an elaborate overnight cycle that removes all the water from the specimen and
replaces it with paraffin wax. The next morning, a technical professional, called a
histologic technician, or "histotech," removes the paraffin-impregnated specimen
and "embeds" it in a larger bloc of molten paraffin. This is allowed to solidify by
chilling and is set in a cutting machine, called a microtome. The histotech uses
the microtome to cut thin sections of the paraffin block containing the biopsy
specimen. These delicate sections are floated out on a water bath and picked up
on a glass slide.
The paraffin is dissolved from the tissue on the slide. With a series of solvents,
water is restored to the sections, and they are stained in a mixture of dyes. The
most common dyes used are hematoxylin a natural product of the heartwood of
the logwood tree, Haematoxylon campechianum, which is native to Central
America, and eosin, an artifcial aniline dye. The stain combination, casually
referred to by pathologists as "H and E" yields pink, blue sections that make it
easier for us to distinguish different parts of cells. Typically, the nucleus of cells
stains dark blue, while the cytoplasm stains pink.
Frozen sections
This technique allows one to examine histologic sections within a few minutes of
removing the specimen from the patient, but the price paid is that the quality of
the sections is not nearly as good as those of the permanent section. Still, a skilled
pathologist and a knowledgeable surgeon can work together to use the frozen
section's rapid availability to the patient's great benefit.
Smears
The specimen is a liquid, or small solid chunks suspended in liquid. This material
is smeared on a microscope slide and is either allowed to dry in air or is "fixed" by
spraying or immersion in a liquid. The fixed smears are then stained,
coverslipped, and examined under the microscope.
Like the frozen section, smear preparations can be examined within a few
minutes of the time the biopsy was obtained. This is especially useful in FNA
20. procedures, in which a radiologist is using ultrasound or CT scan to find the area
to be biopsied. He or she can make one "pass" with the needle and immediately
give the specimen to the pathologist, who can within a few minutes determine if a
diagnostic specimen was obtained. The procedure can be terminated at that
point, sparing the patient the discomfort and inconvenience of repeated sticks.
Special Considerations
The excision of lip lesions requires planning and tissue stabilization. In general,
excision lines should be parallel to the nerves and vessels; however, a substantial
excision from the lip parallel to its long axis may result in a decrease of visible
vermilion after healing. The white roll of the lip may be pulled in because of the
loss of tissue and contracture during healing. Although this excision is unlikely to
produce a major deformity, the esthetics may be compromised. Excisions made
perpendicular to the long axis of the lip are far less likely to produce this type of
distortion; however, with the 3:1 rule (ie, the specimen should be 3 times longer
that it is wide), the excision of a lesion near the vermilion border requires the
clinician to cross onto the skin to produce a wound that can be closed properly.
This incision may result in a scar that is more visible and more troublesome to
the patient than one confined to only the labial mucosa
Excisional biopsy of the lip may be difficult to perform without assistance.
Bleeding can obscure the field, and the mobility of the labial tissues further
complicates what should be a simple procedure. Local hemostasis and tissue
stabilization are managed better if the clinician and an assistant each firmly grip
the lip by placing their thumb and index finger on either side of the lesion .This
clamping occludes the labial artery and its local branches and immobilizes and
tenses the tissue. Another approach is to use a chalazion clamp, which reduces
bleeding, tenses and stabilizes the tissue, and provides the operator with a
convenient handle so that control can be maintained throughout the procedure.
After the specimen is removed from the field, the chalazion is loosened slowly
and carefully, and any bleeding sites are addressed before the wound is closed.
Tongue lesions present similar difficulties. An assistant can stabilize the tongue
by wrapping the tip in a gauze sponge and grasping it firmly. Alternatively, heavy
sutures or towel clamps can be used to control the tongue after appropriate local
anesthesia is achieved .When tongue wounds are closed, deep sutures should be
placed when indicated, and mucosal sutures should be placed fairly close to each
other. Inadequately closed wounds on this movable and highly vascularized organ
tend to re-open, with resultant oozing and/or delayed healing.
A different set of issues arises when excision is planned for a lesion that appears
to emanate from the keratinized attached gingiva. A periodontist or oral and
maxillofacial surgeon should evaluate and manage these lesions. A number of
pathologic entities that appear to arise from the gingiva are actually derived from
the periodontal ligament. Recurrence is more likely in these lesions if their true
21. origin is not addressed. In addition, even the appropriate excisions of small
amounts of attached gingiva may result in significant periodontal defects.
Lesions on the hard palate and remote from teeth may be safely excised as long as
attention is paid to the underlying vascular anatomy. Because keratinized oral
tissues are tightly bound to the skeleton, denuded bone may remain after this
type of excisional biopsy is performed. Inform the patient that denuded bone in
the oral cavity can cause discomfort for several weeks. Even if bone is not
exposed, resultant wounds that involve the attached gingiva and palate are
generally not closed primarily.
Specimen Handling
Surgical specimens obtained with any of the biopsy techniques discussed above
should be handled appropriately. During the biopsy procedure, the lesion is
grasped with an Allis forceps or secured with a traction suture .The use of any
instrument that crushes the specimen makes the pathologist's work more
difficult, if not impossible. The specimen should be removed from the field and
placed into a solution of 10% formalin. The volume of formalin should be at least
20 times the volume of the specimen.
Special tests may require that a second specimen be submitted in a different
solution. For example, in the diagnosis of lesions possibly related to an
autoimmune process, immunofluorescent studies may be of value, in addition to
standard hematoxylin and eosin staining. Specimens for direct
immunofluorescence testing must be submitted in Michel solution.
Communication
Thorough documentation is essential, and the patient's record should include a
description of the lesion and its location, as well as a diagram to illustrate the
chart entry. Any lesion that is being followed up should be photographed. Data
gathering may involve radiography, vital staining, and other modalities, the result
of which should be appropriately documented. Any information that may assist
the pathologist in making a diagnosis should be included on the biopsy report.
The surgeon should be encouraged to review the histopathology slides,
particularly when malignancy is diagnosed or when the microscopic and clinical
assessments differ.
COMPLICATIONS OF BIOPSY:
Haemorrhage
Infection
Poor wound healing
Spread of tumor cells
Injury to adjacent organs
Hypersensitivity to local anesthesia
22. Hypertrophy scar or keloid
Repeat Biopsy
• Not taken from representative areas
• Inadequate size
• Improper procedure
• Mishandling of tissues
• Incorrect orientation
• Misinterpretation
Recent advances:
• Automation of cytologic screening
• Use of powerful computers
• Cyto-analyzer – based on measurements various cellular parameters
• Image analysis – quantitative analysis of various cell components
• Flow cytometry – can measure multiple physical characteristics of cells
suspended in a solute at a rate of 3000 – 5000 cells per second
The purpose of the fixative is to stabilize the protein in the tissue. Once the
tissue is removed from the body it will go through a process of self-destruction.
This process is known as Autolysis – which starts soon after the cell death
creating an enzyme attack, which in place causes the breakdown of protein and
eventual liquefaction of the cell. The objective of fixation is to preserve cells and
tissue constituents in as close a life-like state as possible and to allow them to
undergo further preparative procedures without change. Fixation arrests
autolysis and bacterial decomposition and stabilizes the cellular and tissue
constituents so that they withstand the subsequent stages of tissue processing.
Fixation should also provide for the preservation of tissue substances and
proteins, therefore, it is the first step and the foundation in a sequence of events
that culminates in the final examination of a tissue section.
Fixatives Help Maintain a proper relationship between cells and extracellular
substances. They also Render cell constituent’s insoluble, with tissue proteins
serving as the primary target for stabilization. Factors affecting fixation are
temperature, volume ratio, size, time penetration and tissue storage.
