BiopsyPresenter: 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 iscalled 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 evenrequire 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

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.

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.

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 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.

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) 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 • 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:

• 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.

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 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). 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.

• 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. • 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 analgesiaSmall 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.

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), 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.

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 lowplatelet 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 lowerback/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 isimpossible to deaden the inside of the bone. The contents of the syringe, which to 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:

Histologic sectionsThis 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 SectionsThis 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.

SmearsThe 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 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 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 InfectionPoor wound healing Spread of tumor cells Injury to adjacent organsHypersensitivity to local anesthesiaHypertrophy 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.