Biopsy seminar

BIOPSY

Presented byDr.Vimi JainOral And Maxillofacial Surgery

OUTLINE

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

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

INTRODUCTIONThe diagnosis of any pathology depends• History• Examination• Laboratory studies• Biopsy • Other diagnostic technique

Biopsy is essential for:

• The definitive diagnosis and treatment planning

• To determine the prognosis in cases of malignancy • Provides an indication towards the response of the

tumor to therapy (e.g., relative resistance of malignant melanoma to radiotherapy)

• The purpose of record and research work.

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

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

HISTORICAL PERSPECTIVE• 1870, Ruge and Joham Vert in Berlin introduced surgical

biopsy as an essential tool for diagnosis.

• 1889, Emarch put forward an argument that confirmations should be made before surgeries for malignancies.

• Williams halsted 1st introduced this principle in United States.

• This was adapted to study cells from other body systems

• Along with this were innovations in various kinds of tissue preparations and staining techniques

• 1941, study of exfoliated cells from female genital tract by Papanicolaou

INDICATIONS FOR BIOPSY

• When an inflammatory lesion is not responding to conservative therapy after 10 to 14 days.

• For the determination of the more definitive treatment of the lesion.

• To confirm a clinical impression of a lesion.

• To determine the nature of any intraosseous lesion which cannot be identified clinically and radiographically.

• To determine the nature of all abnormal tissue removed from the oral cavity, including cysts and granulomas.

• Persistent hyperkeratotic changes in surface tissue.

• Any persistent tumescence, either visible or palpable beneath a relatively normal tissue.

• Any lesion that has the characteristics of a malignancy (e.g. Erythroplakia).

• Lesion that interfere with local function(e.g fibroma)

CONTRAINDICATION RELATIVE CONTRAINDICATION

• This should include inflammatory lesions of allergic, viral, fungal or bacterial etiology.

• Normal anatomical and racial variations, e.g., linea

alba, physiological racial pigmentation, leukedema, exostoses etc.

• Compromised general health of the patient or a history of coagulopathy or bleeding disorder, including patient on anticoagulant therapy.

• Proximity of lesion to vital anatomic, vascular, neural or ductal structures and lesions in areas of difficult surgical access.

• Lesions caused by recent trauma.

ABSOLUTE CONTRAINDICATION:

• Pulsatile lesions or those suggestive of a vascular nature should be referred for more in depth evaluation (e.g., hemangioma).

• Pigmented lesions generally should not be biopsied incisionally (e.g. Spread of a melanoma, transformation of premalignant pigmented lesions to malignant ones).

• Lesions that because of size or location present technically difficult surgery e.g., posterior tongue and oropharynx offer severe problems of access.

• Intrabony radiolucent lesions should not be biopsied without initial aspiration.

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

OBJECTIVE OF BIOPSY

• To confirm a diagnosis made on clinical findings.

• To determine the treatment plan

• Valuable self teaching diagnostic aid.

• As a medical record

• Grade tumors

• To detect receptors

• For screening purposes

• Detecting enzymes and antigens

• Monitoring, recurrence and prognosis

• Research purposes

• Microbiology

• Medicolegal

CLASSIFICATION OF BIOPSY

SURGICAL

NON-SURGICAL

SOFT TISSUE

BONE

DIAGNOSTIC

CURATIVE •EXCISIONAL

DIAGNOSTIC

CURATIVE

•CURETTAGE•TREPHINE•ASPIRATION•FROZEN

•ENUCLEATION •RESECTION•CURETTAGE

ASPIRATION CYTOLOGY

•SCALPEL •CAUTERY

•EXFOLIATIVE•FNAC

•INCISIONAL

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According to the procedures applied, oral biopsies can also be classified by:

Features of the lesion:

• Direct biopsy: when the lesion is located on the oral mucosa and can be easily accessed with a scalpel from the mucosal surface.

• Indirect biopsy: when the lesion is covered by an apparently normal oral mucosa.

By the timing of the biopsy/ Clinical timing of

sampling:

• Pre-operative

• Intra-operative

• Post-operative

Purpose of the biopsy.

• Diagnostic Biopsy

• Experimental Biopsy

VARIOUS BIOPSY TECHNIQUE

i. Incisional Biopsyii. Excisional Biopsyiii. Electro-surgery Biopsyiv. Curettage Biopsyv. Punch Biopsyvi. Frozen-section Biopsyvii. Fine needle aspiration biopsy (FNAC)

viii. Aspiration Biopsy

ix. Trephine Biopsy

x. Exfoliative cytology

xi. Fine needle Biopsy

xii. Trucut needle Biopsy

xiii. Vim Silverman needle Biopsy

xiv. Sissor Biopsy

xv. Shave Biopsy

Xvi Ultra sound guided Biopsy

xvii. CT guided Biopsy

xviii. MRI guided Biopsy

xix. ENDOSCOPE guided Biopsy

xx. VELOSCOPE guided Biopsy

xxi. Exploration Biopsy

xxii. Unexplained/unplanned Biopsy

xxiii.Touch impression or imprint cytology

INCISIONAL BIOPSY / DIAGNOSTIC BIOPSY:

• An incisional biopsy is a biopsy that samples only a particular portion or representative part of a lesion.

INDICATION• when the lesion is extensive (larger than 1 cm in diameter)

or potentially malignant (requiring wide excision) or to avoid an adjacent structure, e.g., nerve or artery.

• Chronic non-healing ulcer or squamous cell carcinoma.

• Premalignant lesion

• Bullous lesions (pemphigus, pemphigoid etc).

• Granulomatous diseases (crohn’s disease, orofacial granulomatosis, ulcerative colitis, tuberculosis).

• Minor salivary gland tumour commonly seen on palate.

CONTRAINDICATION

• Pulsatile/ vascular lesions.

• Pigmented lesions.

DISADVANTAGES

• Leaves noticeable scar.

• Possible spread of malignant cells

• Crush, splits and haemorrhage are the artefacts most frequently found in incisional oral biopsies

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

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• 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.

EXICISIONAL BIOPSY• It is the removal of the lesion in

total at the time the surgical diagnostic procedure is performed.

• Not only the entire lesion is made available for pathological examination, but also complete excision may constitute definitive treatment for few lesions.

INDICATION• It is the usual approach for smaller lesions (less than 1

cm in diameter).

• It is used for clinically benign lesions, be they superficial or deep, soft or hard tissue.

• Includes the excision of pigmented and hyperkeratotic lesions.

• Fibromas and papillomas.

• Mucoceles

• Myoblastoma

• Keratoacanthoma

• Sialoliths and smaller benign lesions of accessory salivary glands

• Certain cicatrial lesions

PRINCIPLE

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

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.

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• 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.

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• Care should be taken not to mutilate the specimen when grasping it with forceps

• An elliptical area at the surface is outlined using no.15 sharp scalpel blade (to avoid tearing the tissue), incision is taken down to the connective tissue layer to form a ‘V’ at the base of the lesion, this provides a good specimen and also leaves a wound that is easy to close.

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• The length of the incision should be three times its width to allow for tension-free primary closure.

• High volume suction devices should be avoided as they can aspirate small surgical specimen.

• Hemostasis is achieved with resorbable suture inserted (to control bleeding and also assist in healing). Firm pressure for few minutes aid in hemostasis.

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• 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.

ELECTRO-SURGERY BIOPSY

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

INDICATION

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

Electro-surgical Technique The lesion is grasped with forceps through the loop

electrode. The electrode is activated going under the lesion, removing the growth.

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

INDICATION

• Intraosseous lesions that lie in cavities such as maxillary antrum and cystic lesions within the jaws.

• Also used in very friable cellular lesions like sinuses and fistulae within the soft tissues when only small amounts of surface material are necessary for evaluation.

TECHNIQUE

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

PUNCH BIOPSY• It is an alternative technique of tissue removal

applicable to both incisional and excisional biopsy.

INDICATION

• For total removal of small lesions

• Fixed tissue such as firmly attached palatal tissue, which heal by secondary intention regardless of the technique.

• For oral mucosal malignancies such as squamous cell carcinoma as well as leukoplakia and other mucosal abnormalities that may require multiple biopsies.

• To diagnose oral manifestations of mucocutaneous and other vesiculoulcerative diseases.

CONTRAINDICATION

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

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.

DISADVANTAGES

• Freely movable mucosa that cannot be well supported as with the floor of the mouth and soft palate may preclude the technique.

• It is difficult to use biopsy punch to obtain adequate representative tissue deeper than the superficial lamina propria

• 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.

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

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• 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.

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• 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.

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• Local pressure with sterile gauze is sufficient to induce

haemostasis

• Persistent hemorrhage can be treated with chemical cautery such as silver nitrate, collagen matrix, or by electrocautery.

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• 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 water rinses recommended for palliation.

• Non-steroidal anti-inflammatory agents are preferred for post-operative discomfort.

FROZEN SECTION

• The frozen section procedure is a pathological laboratory procedure to perform rapid microscopic analysis of a specimen.

• It is used most often in oncological surgery.

• The technical name for this procedure is cryosection.

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.

ADVANTAGES• If more tissue is needed to make an accurate diagnosis,

the surgeon is able to obtain an additional sample, avoiding a second operation.

• If the tissue is determined to be cancerous and is amenable to surgery, the mass can be removed at that time.

• If the tissue is determined to be benign (not cancerous), then the mass may not always need to be removed and the

surgery can end.

• The frozen section biopsy can help ensure that the mass being removed is the intended tissue for removal.

• It can help ensure that the entire mass and its surrounding borders are removed.

• It allows for the collection of proper tissue samples for further scientific research.

• The surgeon and pathologist are able to collaborate to care for the patient

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.

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.

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• The procedure is completed within 5-10 mins from the time of receiving specimen till it is stained.

• The remainder of tissue is stored in 10% buffered formaldehyde and routinely processed; embedded in paraffin, sectioned to 3 μm and stained with haematoxylin and eosin.

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

Fine needle aspiration cytology (FNAC)

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

INDICATION

• Lymph nodes - Reactive changes, lymphoma, metastatic cancer

• Thyroid gland - Solitary or dominant nodule, suspected malignancy, nontoxic goiter versus autoimmune thyroiditis

• Salivary glands - Benign and malignant neoplasms, inflammatory lesions, and cysts

• Cystic lesions of the neck - Branchial cleft and thyroglossal duct cysts

• Miscellaneous - Parathyroid neoplasms, dermoid cysts, and teratomas

CONTRAINDICATION

• No absolute contraindications exist to performing fine needle biopsy of neck masses.

• However, a mass in the area of the carotid bifurcation may represent a carotid body tumor, and serious complications and one death have been reported following fine needle biopsy of carotid body tumors.

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

ADVANTAGES • Small size of the needle avoids damage to vital

structure in the head and neck.

• Cells can be fixed, stained and examined within minutes.

• In cases of deep lesions ultrasound or radiological guidance may be used to ensure that the needle enters the lesion.

•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

•Absence of reactive fibrosis to interfere with subsequent surgery

•No problems with wound healing, e.g. after radiotherapy •Readily repeatable

•Cost effective

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.

ASPIRATION TECHNIQUE

• Fix the target mass between 2 fingers of the nondominant hand.

• Advance the needle(21-23G) attached to the syringe, into the mass. Do not apply suction during the advancement.

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• Once the needle tip is within the mass, apply suction by pulling back on the plunger of the syringe.

• While maintaining suction, move the needle rapidly back and forth with short strokes within the mass.

• Release negative pressure after sampling is completed and before withdrawing the needle from the mass.

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• Withdraw the needle.

• The cytologic specimen is within the needle and, perhaps, the hub of the needle.

• Once withdrawn, detach the syringe from the needle. Fill the syringe with air and then reattach it to the specimen-containing needle.

• Expel the specimen onto a glass microscope slide

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

NON ASPIRATION(ZAJDELA) TECHNIQUE

• Fix the target mass between 2 fingers of the nondominant hand

• Hold the needle by the hub between the thumb and index finger.

• Advance the needle into the target mass.

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• Attach an air-filled syringe, with the plunger already retracted, to the specimen-containing needle.

• Expel the specimen onto a glass microscope slide

• Move the needle back and forth within the mass with short, rapid strokes. Simultaneously, move the needle in a rotary clockwise-counterclockwise motion.

• Withdraw the needle.

ASPIRATION CYTOLOGY GUN

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

Causes of unsatisfactory yield with FNAC

• Needle missing a lesion and picking up inflammatory cells.

• Lack of cells in central area of necrosis, hemorrhage, cystic change.

• Small malignant tumour being masked by larger benign tumour.

• Lack of cells in dense fibro sclerotic tissue.

COMPLICATION

• Fine needle biopsy (FNB) of the neck has a very low morbidity rate

• Hematoma is the most common significant complication.

Most hematomas are small and resolve without treatment.

• Infection following fine needle biopsy of the neck is rare.

• Recurrent laryngeal nerve paralysis following fine needle biopsy of the thyroid gland is noticed .

• Puncture of the trachea during fine needle biopsy of the thyroid gland can cause coughing and mild hemoptysis

• fine needle biopsy has been associated with infarction and subsequent necrosis of the biopsied neck mass.

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

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.

TREPHINE BIOPSY• Bone marrow examination refers to the

pathologic analysis of samples of bone marrow obtained by bone marrow biopsy (often called a trephine biopsy) and bone marrow aspiration.

INDICATIONS

• Detect and stage malignancy.

• Differentiate benign hematologic diseases (e.g., aplastic anemia, macroglobulinemia).

• Evaluate progression of human immunodeficiency virus

• classify an anemia as hypoproliferative, a maturation disorder, or resulting from hemorrhage or hemolysis

• 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

PROCEDURE

Equipment

1. Sterile gloves2. Sterile drape3. Povidone-iodine4. Bone marrow aspiration needle or 11- or 13-gauge Jamshidi needle5. 25- and 22-gauge needles6. No. 11 scalpel blade

7. Heparinized 10 cc syringe8. 3 cc and 10 cc syringes9. 1% lidocaine10. Glass slides 11. Specimen bottle with formalin12. 4“ x 4“ gauze13. Pressure dressing and tape

SITES FOR BONE MARROW COLLECTION• Posterior iliac crest• Sternum

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

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.

• 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.

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.

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.

ADVANTAGE

• They are easy to interpret then aspiration cytology to the pathologist

• To distinguish between reactive changes and recurrent malignancy in possible cervical metastasis.

EXFOLIATIVE CYTOLOGY

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

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.

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.

• 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.

• Certain benign hereditary skin lesions having their representative oral manifestations.

• For the study of the change of the oral epithelial cells followed by chemotherapy.

• For the study of buccal mucosa in various anemia.

• Mass screening of oral cancer.

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.

• Atrophic lesions.

• Densely keratinized lesions.

• Smooth surface lesions.

• Lesions giving inadequate specimen sampling through the adopted technique.

ADVANTAGES• Exfoliative cytology implicates its importance in the

field of diagnosis with the principle that any change in the superficial cells can be the reflection of the change in the immediate underlying tissue.

• Cytology is an adjunct to but not a substitute for the surgical biopsy.

• It is quick, simple, painless, bloodless, non- invasive procedure.

• If guards against false negative biopsies.

DISADVANTAGES• Relatively limited information provided with exfoliated

material when compared to a histological preparation.

• Exfoliative cytology is limited to tissue, which exfoliate cells into reasonably accessible sites.

• Majority of the lesions occur in the oral cavity are benign which do not lend themselves to cytologic smear.

• 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.

Technique:

• Instruments for removing the cells should have a 90-degree angle or straight blade.

• The cement spatula used in mixing crown and bridge cement or wax carver can be used.

• A tongue blade slit longitudinally is a second choice, but it should be moistened to avoid dehydration damage to the cells.

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• The cotton swab has been recommended in selected cases.

• Scraping of the lesion should be done while the tissue is stretched or taut. A single stroke is preferred over many small strokes. Excess saliva should be removed by an air blast prior to removal of the cell.

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• Smearing should be done on a standard glass slide (1x3 inches) with an etched label area at one end. The glass slide should be clean and dry.

• The cells should be evenly disturbed over the central one third of the slide. The slide must be labeled

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• 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.

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• The cells should be fixed for at least 1 hr before staining. If the slide is to be mailed the fixative can be discarded after fixation and the mailing container tightly sealed to prevent drying.

• History should be supplied to the pathologist as in done with a biopsy.

Interpretation and Reporting

• The evaluation of slide is based on the morphological features and staining quality of the cells especially the nuclei.

• The ratio of nucleus to cytoplasm is also a critical factor papanicolaou’s classification is often used in reporting diagnostic equation. The cytologic smear is usually reported into one of the V classes: -

Class I (Normal) indicates only normal cells.

Class II (Atypical) indicates the presence of mild atypia but no evidence of malignant change.

Class III (Intermediate) the cells display wider atypia, may be suggestive of cancer, but they are not clear-cut and may represent precancerous lesions, carcinoma in situ. Biopsy is recommended.

Class IV (Suggestive of cancer) A few malignant cells with many cells having borderline characteristics. Biopsy is mandatory.

Class V (Positive for cancer) obvious malignant cells. Biopsy is mandatory

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.

INDICATION

• For diagnosis of certain neoplastic lesions which can simulate inflammatory lesions on frozen section

• For diagnosis of certain benign inflammatory lesions which can simulate malignancy on frozen section

Advantage• Feasible and economical

• Detect the malignancy at the tumor margin

• Less time consumable

TECHNIQUE

• A direct imprint is prepared by pressing a glass slide gently on to the freshly cut surface of the specimen, avoiding a gliding movement, which will distort the shape of the cells.

• The imprint slide is immediately fixed in 95 % ethyl alcohol for 5-6 seconds and then stained (rapid haematoxylin and eosin).

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

Imprint of a lymph node showing malignant cells.

• 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.

• Small forceps with teeth and a pair of sharp curved or straight iris scissors are the only surgical instruments required.

• Depending on lesion size and morphology, anesthesia may or may not be necessary.

• Bleeding after this procedure is usually minimal and can be easily controlled by application of 35% aluminium chloride solution

• The lesion to be removed is lightly grasped with forceps by gently pulling upward, traction provides a firm cutting surface and allows clear visualization of the lesion base.

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.

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.

• However, Ultrasound guided biopsy is not possible when gas/bone prevents the visualization of the biopsy region.

• Another advantage is that, unlike other radiographic biopsy procedures, ionizing radiation is not used for imaging

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.

Indications • Lymph nodes or masses that are not completely

identifiable using ultrasound.

• Lesions near the skull base: CT is optimal for localizing these lesions.

Disadvantages • radiation exposure.

• limited possible scan plane orientations.

• low soft tissue contrast.

• poor vessel conspicuity without administration of intravenous contrast medium.

MRI GUIDED BIOPSY

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

• 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.

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

ENDOSCOPE GUIDED BIOPSY• Endoscopy is defined as “the examination of the

interior of a canal or hollow viscous by means of an 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.

• 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

Endoscope positioned into the lesion.

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

VELSCOPE

EXPLORATION BIOPSY

Used for inrtaosseous lesions of maxilla and mandibleInstruments: Chisel, Bone burs, Periosteal Elevator.

UNEXPECTED/ UNPLANNED BIOPSY

When as a result of surgical procedure (Tooth extraction) some suspicious tissue is obtained unexpectedly

ESSENTIAL BIOPSY PRINCIPLE

ARMAMENTARIUM

Site selection and handing of the specimen-

• By carefully selecting the area or areas, which will produce a good diagnostic specimen, the clinician can avoid having repeat biopsy.

• The extent of biopsy is determined by the size and location of the lesion. Pathologists prefer the entire lesion as the most desirable specimen.

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

• Sections taken from the surface or center of a lesion are often necrotic and do not represent the most typical characteristics of the lesion.

• The periosteum should be avoided whenever possible as it is often a barrier to growth and infections.

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.

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.

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.

Fixation and preservation

• It is essential to avoid distortion of histologic and cytologic details.

• 10% formalin (4% formaldehyde) has a relatively long shelf life, and it is the more commonly used fixative for diagnostic specimens.

• Other fixatives, which are used with satisfactory results, include parachlorphenol (camphorated), sodium hypochlorite, and Zephesin.

• When there is doubt about the action of some chemical on tissue, it is preferable to place the specimen first in cool or cold water (preferably an isotonic solution) and then in formalin or soon as possible.

• Tissue should never be placed on cotton, gauze, or paper prior to fixation. There is rapid absorption of fluid from the cells, often causing extensive distortion that would not occur if the specimen is left on glass or metallic surface for several hours.

• 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.

• 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.

Labeling of the specimen:

• The patient’s name, the location of the specimen, and the date of the surgical procedure are all essential.

• If multiple biopsies are taken from the same patient, the specimens should be placed in individual containers or wrapped and labeled in individual gauze, which has been saturated in 10% formalin.

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

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

Follow-up and Reporting of Biopsy Result to the 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

SPECIAL CONSIDERATIONS

In large lesions

Accessible area

Characteristic portion.

For multiple lesions

Most representative site.

Material curetted from interior of the lesion .

For red & white lesions include both red & white area

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

ULCERS

For Polypoid lesions include base

For Vesiculobullous lesions

Fluid is more representative. Intact vesicle or bulla should be biopsied.

LICHEN PLANUS – representative area should be biopsied

LEUKOPLAKIA – Most dysplastic area should be biopsied

MUCOCELE lesions – careful excisional biopsy

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

Do not cut into pigmented and vascular lesions

208

• PAS stain: It is used for fungi, amoeba and Tricomonas.

• Modified Giemsa (2% Giemsa in water): Detects Helicobacter pylori.

• Congo-red: It is used for identification of amyloid.

• Sudan-Black: It is used for fat staining.

• Masson’s Trichrome: It is used for differentiation of connective tissue elements.

• Papanicolaou’s stain: It is used to stain cells in cervical and sputum smear for cytology.

ERRORS DURING SURGICAL REMOVAL OF BIOPSY SPECIMEN

INJECTION

• HANDLING THE TISSUE: Tissue should be handled with minimum force.

• USE OF FORCEP: Avoid use of forcep on the use of biopsy

• USE OF ELECTRO-CAUTERY: Different view for its use.however combination of cautery and scarpel should be used

SELECTION OF THE SPECIMEN

Tissue should be representative of whole lesion.

Adequate amount of tissue at least 1 x 0.5 cm in size.

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

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.

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.

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 PROCESSING ARTIFACTS

• During Embedding • During Sectioning

INJECTION ARTIFACTSInjection of large amounts of anesthetic solution into the area to be biopsied can produce following tissue changes:• The needle insertion may produce hemorrhage with

extravasation, which in turn masks the cellular architecture.

• The separation of connective tissue bands with vacoulation may occur.

• Tissue edema or distortion.

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.

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.

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.

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.

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.

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.

• 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

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

• 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.

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.

• 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.

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.

• 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.

Complications

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.

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

Poor wound Healing

Unsatisfactory healing of the incision may be due to • Ischemia of the skin overlying a tumor mass

(tension)• Implantation of atumor cells • Previous X-ray therapy

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

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.

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’.

Biopsy Results: What If ?

They don’t corroborate your clinical impression• Repeat the biopsy!!!• Determine if the tissue was looked at by a

Pathologist• The results show malignancy

Situation In Our Sub-region

• Inadequate facilities• Few number of experienced Pathologist• Patient associated factors e.g poverty, ignorance,

religious beliefs

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.

• 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.

References •R. Rajendran and B. Sivapathasundharam: Shafer’s textbook of oral pathology, 5th edition (2006), Elsevier.

•Neville Brad W., Damm Douglas D., Allen Carl M. and Bouquet Jerry E.: Oral and Maxillofacial Pathology, 2nd Edition (2004) Saunders.

•Martin S. Greenberg and Michael Glick: Burket’s Oral Medicine Diagnosis and Treatment, 10th Edition (2003); BC Decker Inc.

•Peterson, Ellis, Hupp and Tucker: contemporary oral and maxillofacial surgery, 4th edition.

•S M Balaji: Text book of oral and maxillofacial surgery, 1st edition.

•Theory & practice of Histological techniques , 2nd & 3rd ed. , Bancroft

Journal of Cancer Research and Therapeutics - April-June 2012 - Volume 8 - Issue 2

S I Talukder. www.talukderbd.com. Histopathology Techniques: Tissue Processing and Staining

Sylvie-Louise Avon. Oral Soft-Tissue Biopsy: An Overview. J Can Dent Assoc 2012;78:c75

K. L. Kumaraswamy, M. Vidhya. Oral biopsy: Oral pathologist’s perspective. Journal of Cancer Research and Therapeutics - April-June 2012 - Volume 8 - Issue 2

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