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REVIEW ARTICLE
Year : 2012  |  Volume : 8  |  Issue : 2  |  Page : 192-198

Oral biopsy: Oral pathologist's perspective


1 Department of Oral Pathology and Microbiology, Farooquia Dental College, Mysore, India
2 Department of Oral Pathology, AB Shetty Memorial Institute of Dental Sciences, Nitte University, Mangalore, India
3 Department of Oral Medicine and Radiology, Yenepoya Dental College, Yenepoya University, Deralakatte, Karnataka, India

Date of Web Publication26-Jul-2012

Correspondence Address:
K L Kumaraswamy
Department of Oral Pathology and Microbiology, Farooquia Dental College, Mysore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.98969

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 > Abstract 

Many oral lesions may need to be diagnosed by removing a sample of tissue from the oral cavity. Biopsy is widely used in the medical field, but the practice is not quite widespread in dental practice. As oral pathologists, we have found many artifacts in the tissue specimen because of poor biopsy technique or handling, which has led to diagnostic pitfalls and misery to both the patient and the clinician. This article aims at alerting the clinicians about the clinical faults arising preoperatively, intraoperatively and postoperatively while dealing with oral biopsy that may affect the histological assessment of the tissue and, therefore, the diagnosis. It also reviews the different techniques, precautions and special considerations necessary for specific lesions.

Keywords: Biopsy, oral biopsy, surgical considerations in biopsy


How to cite this article:
Kumaraswamy K L, Vidhya M, Rao PK, Mukunda A. Oral biopsy: Oral pathologist's perspective. J Can Res Ther 2012;8:192-8

How to cite this URL:
Kumaraswamy K L, Vidhya M, Rao PK, Mukunda A. Oral biopsy: Oral pathologist's perspective. J Can Res Ther [serial online] 2012 [cited 2018 Nov 19];8:192-8. Available from: http://www.cancerjournal.net/text.asp?2012/8/2/192/98969


 > Introduction Top


Biopsy, a Greek-derived word (bio-life; opsia-to see) loosely translated as "view of the living," is defined as removal of tissue from the living organisms for the purpose of microscopic examination and diagnosis. The term "Biopsy" was introduced into medical terminology in 1879 by Ernest Besnier. [1] One of the earliest diagnostic biopsies was developed by the Arab physician Abulcasim (1103-1107AD). A needle was used to puncture a goiter and material was characterized. [2]


 > Indications Top


It is an accepted fact that microscopic analysis is the gold standard for the diagnosis of most lesions. According to the American Academy of Oral and Maxillofacial Pathology, any abnormal tissue removed from the oral and maxillofacial region should be submitted preferably to an oral and maxillofacial pathologist. The exceptions are in cases such as tori, exostosis, carious teeth lacking attached soft tissue, extirpated dental pulp and clinically normal tissues. [3] It is important for the clinician to decide whether a lesion needs to be biopsied or not before treating it. With regard to oral soft tissues, any lesion in question, if persisting for more than 2 weeks even after the removal of the irritating factor (if any), biopsy should be performed. Biopsy is also advisable in bony lesions that cannot be diagnosed radiographically and which are usually accompanied by pain, sensation alterations or other symptoms. [4] Any abnormal tissue removed from the oral cavity should be sent for histopathological analysis however confident the clinician may be with the diagnosis.


 > Contraindications Top


Although absolute contraindications are not present, there are some conditions where decision to proceed with biopsy should be done with caution. These are bleeding diathesis secondary to anticoagulation, lesion located near vital structures that could be injured by biopsy and in medical conditions that do not allow for the use of local anesthetics. The potential morbidity associated with a biopsy done in a previously irradiated region should be considered in deciding whether biopsy is advisable. [5] Biopsy is not advised in the case of multiple neurofibromas due to the risk of neurosarcomatous transformation, or in tumors of the greater salivary glands. Such biopsies must be performed by specialized surgeons in order to avoid damaging the nearby anatomical structures and causing the spread of tumor cells, as this would adversely affect the prognosis. [4]

Special considerations in specific lesions of the oral cavity [Table 1].
Table 1: Special considerations for specific clinical lesions

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 > Premalignant Lesions and Oral Squamous Cell Carcinoma Top


Oral premalignant lesions and early oral cancers are quite varied in appearance. Although clinical characteristics can raise the suspicion, biopsy of the lesion is required to establish a definitive diagnosis. Accurate diagnosis of malignant lesions depends on the quality of biopsy, adequate clinical information and correct interpretation of the biopsy. [6] Selection of the area to take a biopsy specimen of large red and white lesions may pose a problem because oral cancer originates at any location within the precancerous lesion, and it is often difficult to diagnose early cancerous transformation clinically. Therefore, multiple areas must be sampled and include both white and red lesions in speckled cases. Adjuvant aid such as using toluidine blue or direct fluorescence visualization can help a clinician highlight the most severe or significant site for biopsy. [7] Incisional, excisional or punch biopsy can be done based on the size of the lesion. Biopsies of the mucosa should be at least 4-5 mm in diameter and also in depth, as these lesions can have characteristic thickened epithelium with hyperkeratosis. [6] If the lesion is ulcerated, always including an area of adjacent intact epithelium is a good practice. For smaller, discrete lesions, an excisional biopsy may be more ideal.

Oral lichen planus

Diagnosis of oral mucosal lichen planus may be established clinically. [8] Biopsy of nonerosive lesional tissue offers definitive diagnosis. Erosive areas of lichen planus should be avoided as it may show nonspecific inflammatory changes. The history, typical oral lesions and skin or nail involvement are usually sufficient to make a clinical diagnosis of oral lichen planus. But, around 25% of the cases show only oral findings without skin manifestation. [9] Therefore, biopsy of lesional tissue offers definitive diagnosis. Also, biopsy is required to differentiate between oral lichen planus and other chronic white or ulcerative oral lesions. In case of gingival erosive lichen planus, direct immunofluorescence (DIF) is more suitable to differentiate from the other oral vesiculoulcerative conditions, as biopsy of this area would just suggest a nonspecific inflammatory process. [8] Adjacent perilesional tissue can be chosen if DIF is indicated. Positivity for oral lichen planus is considered when there is IgA, IgG, IgM or C3 deposition throughout the basement membrane zone and fibrinogen in the basement membrane in an irregular pattern. [8] Although adjacent perilesional tissue is indicated for immunofluorescence studies, Sano et al. in their study have proposed that no difference in sensitivity of DIF between biopsies performed in perilesional tissue (radius up to 1 cm from lesion) or distant tissue (radius greater than 1 cm) was seen. [10] Intraoral areas most sensitive to DIF are buccal floor, upper labial mucosa, hard palate and cheek mucosa. [8] While standard hematoxylin and eosin processing of tissue sample may be sufficient to rule out neoplasms, differentiation of lichen planus from other immunologic conditions such as benign mucous membrane pemphigoid and pemphigus may be improved by DIF analysis.

Oral vesiculobullous lesions

Oral mucosa may exhibit vesicles or bullae as a result of a variety of infections immunologically mediated, drug induced and hereditary diseases or mechanical injury. [11] In addition, there are diseases that mimic vesiculobullous diseases, which need to be critically differentiated. [11] Vesiculobullous lesions involving the oral cavity are common characteristics of a wide variety of diseases; the clinician attempting to diagnose intraoral ulcerative and vesiculobullous diseases is frequently confronted with several diseases having a similar if not identical clinical appearance. Histopathological evaluation as well as immunofluorescence provides critical information to facilitate a definitive diagnosis.

Biopsy of a fresh intact vesicle or bulla is difficult as it ruptures rapidly in the oral environment. Therefore, the site of biopsy for a vesiculobullous disease should be adjacent to bulla (perilesional) where epithelium is intact. [12],[13] Intact epithelium and connective tissue are critical in evaluating a specimen with vesiculobullous lesions. [12],[13] It is better to provide longer, broader shallower biopsy specimen than a deeper and narrow specimen in vesiculobullous lesions as it is a surface phenomenon. Gingival biopsy should be avoided as chronic inflammation of gingiva may confuse the histological aspects. [14] In most cases, oral lesions precede skin manifestations. Thus, earlier detection helps in controlling the disease at the initial stage. Biopsy and immunofluorescence are essential for making a diagnosis [Figure 1]. The histological analysis of the lesions that usually begin in the oral epithelium is essential as earlier diagnosis will allow proper treatment, delaying or even preventing the dissemination of the lesions. [14] It is important to note that if the patient is on topical steroids, biopsy should be taken only after discontinuing it for a period of 1 months, else it can alter the histopathologic and DIF findings. [12],[15]
Figure 1: Biopsy and blood for diagnosing vesiculobullous disease

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 > Immunofluorescence Techniques and Biopsy Top


When the choice is made to perform a diagnostic biopsy for any of the bullous diseases, the specimen must contain epithelium. A sample solely from ulcer or erosion will be of little diagnostic value. The reason is simple: the target antigens that will be exposed to immunofluorescence lie intraepithelial, or around the basement membrane for vesiculobullous lesions. Without the epithelium, the antigen cannot be identified using direct immunofluorescent techniques. In obtaining a biopsy in patients with vesiculobullous lesions, there are several important factors to be considered for immunofluorescence techniques [Table 2].
Table 2: Points to consider for sending biopsy for immunofluorescent studies[16]

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 > Salivary Gland Lesions Top


Salivary gland biopsy is usually performed to diagnose any autoimmune disorder such as Sjogren's syndrome or to confirm any tumor affecting the gland or therapeutic removal in case of mucocele. In diagnosing Sjogren's syndrome, labial salivary gland biopsy (LSG) is usually performed apart from other parameters as change in the minor salivary glands mirrors those in the major salivary glands. The LSG is performed on lower lip following administration of local anesthesia. Usually, a 1.5-2.0-cm horizontal incision is made on clinically normal labial mucosa, parallel to the vermillion border and lateral to the midline. Five or more accessory salivary gland lobules should be obtained for analysis. Care should be taken not to damage the muscle layer, arteries or the sensory nerve. [4],[13],[17]

Shane [18] has described that neonatal hemochromatosis (NH) can be safely and effectively diagnosed using minor salivary gland biopsy of the lower lip of neonates. In this study, lower lip biopsy for minor salivary gland was done similar to that done for Sjogren's syndrome, establishing the diagnosis of NH by demonstrating glandular iron deposition by a special stain, i.e. Perl's iron stain.

Other systemic disorders such as sarcoidosis and amyloid polyneuropathy can also be confirmed with lip biopsy. [19]

Mucoceles arise from blockage and subsequent rupture of minor salivary gland ducts. Excision of mucoceles must include few lobules of minor mucous glands that drain into the mucocele. This minimizes the recurrence potential of these lesions. [13]

Biopsy of the major salivary gland should be avoided as biopsy of parotid salivary gland can lead to scarring and increased vascularity, which makes it difficult to preserve the branches of facial nerve. Fine needle aspiration biopsy is done if abnormal lumps are found. [4] The biopsy needle removes a small core of gland tissue that is sent to the laboratory for analysis. For palatal swellings that are suspected salivary glands tumors, incisional biopsies should be as deep as possible, as the lesion can be present in considerable depth, beneath the mucosa; therefore, superficial biopsy may give a false-negative result. [13]


 > Candidal Infections Top


Oral candidiasis is an opportunistic infection of the oral cavity. Oral candidiasis is caused by an overgrowth or infection of the oral cavity by a yeast-like fungus, Candida. Oropharyngeal candidiasis manifests clinically as acute pseudomembranous, acute atrophic, chronic atrophic, chronic hyperplastic and angular cheilitis. Smears, swabs and oral rinse samples are the common specimens for diagnosing candidiasis.

For smear preparation, lesion should be scraped with spatula or tongue blade and smeared gently on labeled glass slide and fixed immediately in 95% ethyl alcohol or with spray fixative. To diagnose Candida, two to three sequentially numbered glass slides should be made and labeled. Care should be taken that when they are fixed they do not stick together. In cases of denture stomatitis, which is a common but usually undiagnosed condition among the elderly, a smear of the fitting surface of the denture should be taken. [20]

In cases where culture is indicated, material should be collected with swabs. Samples are obtained by rubbing a sterile cotton-tipped swab over the lesional tissues. The swabs should be transported to the laboratory as quickly as possible to prevent desiccation. Candida albicans can survive at least 24 h on a moist swab without loss of viability, but immersion of the swabs in buffered charcoal (Amies transport medium) may be done to prevent desiccation or in an enrichment medium to increase the isolation sensitivity. [21]

In the oral rinse technique, the patient is requested to rinse the mouth for 1 min with 10 mL of phosphate-buffered saline supplied in a universal container (denture has to be removed if the person is a denture wearer) and then expel it back to the universal container and sent to the laboratory. [21] Oral rinse method is used for culturing and differentiating oral yeast carriage and candidal infection.

In cases of chronic hyperplastic candidosis, biopsy specimen is usually advisable.


 > Tooth Top


If there is a need to study the histopathology of the dental pulp, once the tooth is removed, the clinician should drill away the crown or apical third of the root to facilitate the penetration of the fixative into the pulp chamber. When the tooth specimen is sent for biopsy, one should inform the tissue of interest. This is because in case of enamel, the ground sections have to be prepared whereas for the study of the dental pulp decalcification sections are required.


 > Surgical Considerations Top


Apart from the representative site selection, there are many issues to be kept in mind while performing biopsy.

Remove sufficient tissue

Generally, the larger the sample, the greater the chance of an accurate diagnosis. Fixation causes shrinkage, color changes and firming of the tissue. The greater size of tissue biopsy allows for that shrinkage and permits the pathologist to better orient and cut the specimen avoiding tangential sectioning. [7] Tiny samples may inhibit the histology technician from producing a quality slide and may also impair the pathologist's ability to provide an unequivocal diagnosis.

Avoid the use of solutions that stain the surface

Toluidine blue staining can be used to select the most representative portion of the precancerous lesion and malignant oral lesions. But, the preparation of the area of biopsy with iodine tincture or other colored solutions is not recommended as it can interfere with tissue processing and staining procedures. [22]

For the surgical procedure of biopsy, local anesthesia should be given away from the lesion to avoid artifacts in the sample. If block anesthesia is not possible, infiltration should be given at least 3-4 mm away from the lesion. Four-point anesthesia technique [Figure 2] can be used as cardinal reference (top, bottom, left, right). [4] Intralesional injection of anesthetic solution produces hemorrhage with extravasation and separation of connective tissue bands with vacuolization. [4]
Figure 2: Four-point anesthesia technique

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Inclusion of undesired material in the sample

Foreign body inclusion into the sample such as the cotton, glove starch, calculus or restorative material can make the interpretation of the specimen quite difficult. Cotton in the section can mimic amyloid-like substance, and presence of glove starch leads to starch artifacts that can appear as atypical epithelial cells in histopathological sections. [23],[24]

If it is a hard tissue lesion, it is important to take a radiograph of the specimen so that it might also help in assessing whether the lesion is been removed to its entirety.

Handling tissues gently

Using sharp instruments is a minimum requirement for better biopsy technique. Crushing of tissue with tissue forceps during the procedure or rough handling of tissue can destroy the histological features, rendering accurate microscopic assessment difficult. Tissue distortion or artifacts is also caused when using electrosurgery or laser. Although electrosurgery or laser has the advantage of producing hemostasis, it also induces profound artefactual alterations by producing heat, leading to tissue protein coagulation, resulting in an amorphous epithelial and connective tissue appearance. In such situations, epithelial cells appear detached, fusiform and hyperchromatic, making it useless for diagnosis, especially if the specimen is small. Hence, use of electrosurgery to obtain routine biopsy specimens should be discouraged. If used, it should be limited to relatively large specimen, as the artifact could obscure all details of a smaller specimen. [4],[24],[25]


 > Different Techniques to Minimize the Artifacts Top


The specimen obtained with oral biopsy techniques are typically small, and chance for artifacts is considerable. Most of the artifacts caused because of improper handling goes unnoticed clinically but might pose potential diagnostic problems to the pathologist during histopathological examination. There are many techniques available to reduce the artifacts in the specimen.

Traditional method of using the toothed tissue forceps to grasp the specimen should be avoided. Alternatively, blunt forceps instead of toothed forceps can be used to grasp the tissue and to grasp the tissue away from the main site of interest. Inadvertent use of toothed forceps leads to perforation, leaving gaps and compression zones around the tissue. [4] A better option to handle the tissue is to pass the suture and hold it with the artery forceps, which provides the traction and controls the specimen, aiding biopsy. The same suture can also be used to orient the biopsy sample for sectioning. In the traditional scalpel biopsy, incisions are placed on either side in the shape of an ellipse that converges in a "V" to join in deeper sublesional tissue. In this method, the length should be approximately three-times more than the width.

Alternately, punch biopsy is becoming popular these days to the traditional scalpel biopsy. The oral mucosal punch is a rapid and simple tool for obtaining the representative sample. The instrument consists of a cylindrical cutting blade attached to a plastic handle. Diameter from 2 to 8 mm with stepwise increments of 0.25-0.50 mm is available. [4] This removes a core of tissue (usually around 4 mm), the base of which can be released using curved scissors or a scalpel. Previous studies have indicated that oral mucosal punch induces fewer artifacts compared with the conventional incisional scalpel biopsy. But, this oral mucosal punch may be difficult to biopsy freely movable tissues (e.g., soft palate, floor of mouth)

Other instruments such as B forceps (B standing for biopsy) can also be used to obtain a biopsy. This forceps is based on the chalazion forceps, which consists of autopressure forceps with two elongated rectangular plates at the operator ends. These forceps are placed and the handle is released, which exerts spontaneous pressure on the tissue. The compressive effect makes the tissue to be raised within the window and the pressure induces ischemia in the work zone. Biopsy with a scalpel or punch is then carried out. The so-called B forceps designers propose several advantages, including speed, because the ischemia produced by the clamp stabilizes the tissue and increases visibility, facilitating dissection and also reducing artifacts. [26],[27]

Lasers and electrosurgical knives are also often used to perform oral biopsies. These techniques have the advantage of producing a completely bloodless surgical area, but it can induce thermal artifacts. Hence, it is best to excise with a scalpel and use electrosurgery to control hemorrhage at the biopsy site.


 > Orientation Top


Orientation is important for all surgical specimens submitted for microscopic examinations. Orientation of mucosal biopsies (particularly superficial lesion) is important, especially because they are small and have limited morphologic characteristics after being immersed in formalin. Proper orientation of the surface lesion specimen assists the oral pathologist in sectioning the specimen to avoid tangential cuts. Improper orientation will lead to the sectioning of either epithelium or the connective tissue alone, but not both. Surgeons can place sutures in the specimen to assist in orientation and provide a written description of the specimen in relation to suture. At least two adjoining margins must be clearly identified to ensure correct orientation, with the help of short suture and a long suture [12] [Figure 3]. Illustrations are also very helpful and should be included. By providing this information to the pathologist, it is possible to assess anterior, posterior, superior and inferior margins, allowing for the identification of areas that might require further excision.
Figure 3: An excisional biopsy specimen showing orientation margins with long- and short-length sutures

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 > Specimen Preparation and Fixation Top


The specimen obtained with oral biopsy procedures are typically small and mucosal biopsies, which do not include underlying muscle tissue, tend to curl and distort. This creates difficulty in orientation of the specimen for tissue processing. This problem can be overcome by placing it with the mucosal surface up on a piece of stiff sterile paper immediately after biopsy and then dropping into the formalin fixative slowly. [12],[24] Also, this curling artifact can be avoided if sufficient depth of the specimen is included.

When more than one specimen is removed from a patient, each specimen should be placed in separate containers with appropriate descriptions for each labeled container. Once the specimen is placed in the container, it should be labeled with patient name, date of biopsy, site of biopsy and hospital number.

The clinician should make sure that the specimen is placed in a wide mouth container. If a narrow-mouthed container is used, the specimen may have to be handled roughly or the container needs to be broken to get the fixed specimen out. Therefore, a suitable wide-mouthed inert, clean and clear (not brown) container must always be used. The dark-colored bottles should be avoided as we cannot see whether the tissue is into the fixative or it is adhering to the wall of the bottle. Most histopathology laboratories will supply suitable containers containing appropriate volume of 10% neutral buffered formalin for fixation. Fixation is mandatory to inhibit autolysis of tissue once they are removed from the patient. Sometimes, formalin is further diluted with water by ancillary staff or specimens are placed in alternative solutions such as saline or tap water, which results in poor fixation and artefactual changes. [13] The volume of fixative should exceed 10-15-times the volume of the specimen for proper fixation. Formalin fixes specimens by forming intermolecular bridges between proteins and cross-links between protein end groups. Disadvantage of this protein cross-linking produced by formalin is that it makes unsuitable for immunofluorescent techniques. [13] Specimens to be submitted for immunofluorescence testing (in cases of autoimmune or vesiculobullous disorders) need some special consideration. Perilesional skin is used for immunofluorescence for DIF patients. Monkey or guinea pig esophagus rat bladder epithelium for paraneoplastic pemphigus or blister fluid can be used for DIF. Quick freezing is the most widely used method for handling biopsy specimens for immunofluorescent studies. This can be performed by immersing the biopsy specimen immediately after biopsy either in liquid nitrogen or cold solid carbon dioxide or in a hexane bath. The quick frozen biopsy is then mounted in tissue embedding compound and sectioned in a cryostat. Tissue substrates for DIF techniques are processed similarly. Because rapid freezing of specimens require special supplies and keeping them frozen during transportation is a packaging challenge, an excellent alternative is to place the specimen in a room temperature transportation medium that permits convenient transport to the laboratory for processing. At present, immunofluorescence can be performed with biopsy specimens handled for several days at ambient temperature in a preservative liquid medium, as described by Michel et al. Michel's medium is now commercially available. [28]

Fresh frozen tissue [Table 3] is also required where cases are intended for genetic testing and flow cytometry [5] (e.g., in cases of lymphoma). Also, in suspected cases of fungal, mycobacterial, bacterial or viral infections, a small portion of fresh specimen should be sent separately without fixation for culture. [5] The other main situation where fresh tissue is processed is when frozen sections are used to examine surgical margins perioperatively. In cases of electron microscopic studies, specimens should be fixed in gluteraldehyde. [13]
Table 3: Indications for fresh tissue

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 > Information to Pathologist Top


To diagnose a specimen properly, the oral pathologist may need to review the clinical history and the setting in which the patient's condition manifested. Therefore, the clinician should submit a biopsy data sheet with pertinent thorough history and radiograph as appropriate. Adequate clinical history and description should be provided so that it enables the pathologist to provide a useful and meaningful diagnosis. A labeled diagram on the pathology form may be very useful, showing the area biopsied and size of the lesion indicating where the specimen was taken from. Most of the labs have their own prescribed requisition form that can be collected from the laboratory [Table 4].
Table 4: Details required in pathology form

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 > Conclusion Top


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 Top

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[PUBMED]  Medknow Journal  


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]


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