Oral tissue biopsy may be necessary for lesions that cannot be diagnosed on the basis of the history and clinical findings alone. A thorough inspection of the oral cavity should be a part of any complete head and neck examination. Approximately 10% of patients who are examined will have some abnormality of the oral mucosa. Biopsy is often the definitive procedure that provides tissue for microscopic analysis when additional information is required to guide any indicated therapy. [1, 2]
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; 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.
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 the clinician 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.
Even experienced clinicians have difficulty in distinguishing between 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.
Oral mucosal biopsy has few contraindications. The standard biopsy techniques may require modification in some patients, including those with conditions that preclude the safe use of local anesthetic and those with severe bleeding diatheses or coagulopathies. Invasive procedures that may stimulate bone should be avoided when possible, or conducted with great caution in patients who have used or are currently using injectable bisphosphonates. Mucosal lesions in these individuals may be reflective of underlying medication-related osteonecrosis of the jaws (MRONJ), a condition that may be exacerbated by any manipulation. Caution should also be taken with lesions that appear vascular, especially when there is a palpable pulsation or bruit.
The oral cavity (see the image below) is oval shaped and is separated into the oral vestibule and the oral cavity proper. It is bound by the lips anteriorly, the cheeks laterally, the floor of the mouth inferiorly, the oropharynx posteriorly, and the palate superiorly. The oropharynx begins superiorly at the junction between the hard palate and the soft palate, and inferiorly behind the circumvallate papillae of the tongue. The bony base of the oral cavity is represented by the maxillary and mandibular bones. The oral cavity includes the lips, gingivae, retromolar trigone, teeth, hard palate, cheek mucosa, mobile tongue, and floor of the mouth.
The tongue is basically a mass of muscle that is almost completely covered by a mucous membrane. From anterior to posterior, the tongue has 3 surfaces: tip, body, and base. The tip is the highly mobile, pointed anterior portion of the tongue. Posterior to the tip lies the body of the tongue, which has dorsal (superior) and ventral (inferior) surfaces.
The lips surround the entrance to the oral cavity. The upper lip extends from the base of the nose superiorly to the nasolabial folds laterally and to the free edge of the vermilion border inferiorly. The lower lip extends from the superior free vermilion edge superiorly, to the commissures laterally, and to the mandible inferiorly. Around the circumferential vermilion-skin border, a fine line of pale skin accentuates the color difference between the vermilion and normal skin.
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 aesthetics 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. To ensure correct approximation of the vermilion border, it should be marked prior to excision.
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, as shown in the image below.
This clamping occludes the labial artery and its local branches while immobilizing and tensing 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 clamp (shown below) 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 (see the image below).
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 that are remote from teeth may be safely excised as long as attention is paid to the underlying vascular anatomy. Since 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. Some clinicians use dressings, packings, or even surgical stents to assist in managing palatal wounds such as those created by biopsy.
Most biopsies in the oral cavity are performed for the evaluation of mucosal changes. When pathologic entities arise from deeper tissues, techniques must be adapted and appropriate instrumentation selected. Such situations may include soft tissue masses deep to the surface with normal-appearing overlying mucosa, lesions within the jaws, and tumors of salivary gland origin.
Intraosseus lesions generally require biopsy to establish a tissue diagnosis. Most of these lesions are cysts and benign tumors; however, primary malignancies, metastatic disease, and jaw lesions associated with systemic disease should be considered. Due to behavioral differences and therefore varying treatment recommendations among jaw cysts and tumors, a tissue diagnosis is recommended to provide direction.
The standard approach to intrabony lesions of the jaws involves reflection of a mucoperiosteal flap using a crestal incision and releasing incisions as needed. These should be placed far enough from the planned entry into bone to provide good access and to ensure that incision lines lie over sound bone. Aspiration with an 18-gauge needle is recommended prior to flap reflection and entry into bone as some of these lesions may be vascular. If the lesion has not eroded and perforated bone, a window is made with a rotary instrument. The opening must be large enough to permit the sampling of an adequate amount of pathologic tissue so that a histopathologic diagnosis can be made.
Patients with deeper soft tissue masses might benefit from fine-needle aspiration biopsy (FNAB) to check for the presence of cells that suggest malignancy. FNAB is rarely used in the oral cavity; however, some authors do describe using FNAB in the diagnosis of oral cavity and oropharyngeal lesions. FNAB is further described in Technique.
Additional techniques have been published, including an approach described for lesions of the tongue that may be difficult to access. One such report describes a translingual biopsy involving the blind passage of an instrument through the tongue to a lesion that is not amenable to traditional instrumentation because of its location. Further research into the success and safety of this technique is necessary before it gains acceptance. 
A differential diagnosis is formed by reviewing the history and physical findings and assessing the 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.
For both incisional and excisional biopsies, the physician's armamentarium 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)
Suture for closure (if indicated)
Laser or electrocautery device for fulguration (if indicated)
Specimen bottle containing formalin and biopsy data sheet
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.
Allis forceps (see the image below) are optional. They have sharp teeth to grasp heavy tissue.
Finer instruments, such as small-toothed tissue forceps should be used in the handling of more delicate biopsy specimens. Grasping instruments without teeth may crush tissue and impact negatively on the pahtologist's ability to read and interpret biopsy material.
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 for 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 (see the image below).
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 (see the image below). 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. 
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.
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, the physician 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 (see the image below).
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 thermal artifact that may have an adverse affect on the histologic interpretation of the specimen. 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.
When incisional biopsy is contemplated, the biopsy site should be carefully considered. The primary principle directing site selection is the attainment of the most representative sample of the lesion. Many texts direct clinicians to the periphery of the lesion to include surrounding tissue. That is certainly an appropriate approach for incisional biopsy of an ulcerated oral lesion as the ulcer itself has no mucosal covering. When sampling such lesions, an incisional biopsy must extend into the surrounding tissue to ensure that specimen includes intact mucosa from the active area at the periphery. For any lesion covered with intact mucosa, the incisional biopsy site of choice is the one judged to best represent the abnormality. In such cases, peripheral sampling with inclusion of healthy tissue is not indicated, provides no advantage to the pathologist, and may lead to a missed diagnosis or under-diagnosis of the lesion. For more extensive lesions and/or those with varied appearances, multiplebiopsies may beconsidered.
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. A significant body of evidence supports the effectiveness of this vital staining technique as a tool to enhance the diagnostic abilities of even experienced clinicians. [6, 7, 8] 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 dark staining 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.
In the past decade, other adjunctive techniques have emerged that may facilitate the early detection of potentially malignant lesions.  These new detection modalities use chemiluminescence or tissue autofluorescence to augment the clinician's ability to visualize areas of suspicious mucosal change. [8, 10, 11, 12] These methods are intended to serve as diagnostic aids and are adjunctive to the standard visual and tactile oral examination. They are predicated on the concept that mucosal tissues with dysplastic changes have different absorbance and reflectance profiles when exposed to various forms of light energy. 
Chemiluminescent light based systems (ViziLite Plus, MicroLux DL, Orascoptic DK) use the application of a diffuse chemiluminescent light source to visualize abnormal oral mucosa not visible under normal incandescent light. After a 1% acetic acid oral rinse to remove surface debris and slightly desiccate the oral mucosa, cells with abnormal nuclear structure are purported to preferentially reflect the low-energy blue-white light emitted.  Under illumination, normal epithelium absorbs the light, appearing light blue, while abnormal tissue reflects the light and appears white, with sharper, more distinct margins. Toluidine blue, discussed above, is used as a stain to aid in further lesion assessment (see the image below).
Tissue autofluorescence–based systems (VELscope) rely on the exposure of oral epithelium to specific wavelengths of light in order to produce an emission of energy in the form of fluorescence. The proposed mechanism of this fluorescence is related to reflective and absorptive patterns due to naturally occurring fluorophores in the oral mucosal tissue. This fluorescence is variable and it is affected by changes in tissue metabolism and structure. Further, the presence of hemoglobin, vessel dilation, and inflammation in the examined tissue can affect its appearance. Illuminating oral tissues with the VELscope device (see below) causes normal mucosa to emit a pale green light, whereas abnormal mucosa appears dark in presentation.
While these modalities may be useful adjuncts in the detection of oral lesions, the most definitive evaluation method for potentially malignant tissue is still a biopsy that provides the pathologist with an adequate sample of tissue in which the architecture has been preserved. 
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, as shown below, 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.
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 Technical Considerations.
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. See the image below.
Closure is facilitated by developing an ellipse that is 3 times longer than it is wide, as shown below.
The relative merits of perpendicular and parallel excisions on the lip should be considered prior to picking up a blade. Among the factors that impact on this choice are the size and position of the lesion in relation to the vermilion border and the need to create a wedge three times longer than its width.
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.
Fine-Needle Aspiration Biopsy
Patients with deeper soft tissue masses might benefit from fine-needle aspiration biopsy (FNAB) to check for the presence of cells that suggest malignancy. FNAB is rarely used in the oral cavity; however, some authors do describe using FNAB in the diagnosis of oral cavity and oropharyngeal lesions. 
In this technique, a thin, hollow needle is inserted into the mass to extract cells that, after being stained, will be examined under a microscope. Fine-needle aspiration is accomplished by first rinsing the oral cavity with either dilute hydrogen peroxide or chlorhexidine for about 60 seconds. The tissue or cavity to be sampled is then prepared with topical anesthetic. Local anesthetic with a vasoconstrictor is then injected around the base of the lesion at its periphery. Multiple passes into, but not beyond, the lesion are then performed with a 22-gauge, 1.5-inch needle, and a specimen is obtained by negative pressure. The material within the syringe is spread onto a slide and fixed.
Advantages of FNAB over open biopsy may include avoiding unnecessary damage to vital structures, low risk of infection, and patient comfort. Disadvantages of FNAB may include high false-negative rates, insufficient room in the oral cavity to properly perform the movements necessary to aspirate material, and difficulty fixing the lesion. A review of the literature reveals a wide range of false-negative rates for FNAB.
FNAB has been used in both soft and hard tissue lesions in the oral cavity. It may play a role in the evaluation of deeper lesions, which have a high index of suspicion for malignancy. In such situations, a positive FNAB result confirms the presence of malignant cells and may preclude the need for more invasive tissue sampling. Clearly, a negative FNAB result in that context would mandate additional diagnostic procedures.
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 (see the image below).
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.