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Use of Immunofluorescence in Oral Pathology by Robert M. Law, M.D.
Many dermatologic diseases manifest themselves in the oral cavity and offer dentists an opportunity to identify systemic illnesses that may go unrecognized. Although routine histologic evaluation of mucosal biopsies will provide pathologists with useful diagnostic information, many scenarios require the use of ancillary techniques. Erosive lesions, blistering disorders, and lichen planus are situations in which immunofluorescence (IF) examination plays a vital role in establishing a definitive diagnosis. This technique requires fresh tissue, specialized equipment, and a pathologist with experience in immunofluorescent microscopy.
Oral lichen planus (LP): This chronic inflammatory condition of the mouth exhibits a female predilection, and occurs in roughly 2% of the population. Typically, there are three main clinical categories of disease. Reticular lichen planus displays the classic Wickham's striae, which are lacy white keratotic striations. Erosive or erythematous LP exhibits reddened mucosa, and ulcerative LP is a painful process presenting with an overlying yellow fibrinous exudate. Biopsy for immunofluorescence will demonstrate fibrillar deposition of fibrinogen as well as IgM or C3 positive cytoid bodies. This distinguishes LP from other causes of lichenoid mucositis such as lupus, plasma cell gingivitis, or apthous stomatitis.
This includes the pemphigus family of immunobullous disorders. In pemphigus vulgaris, patients tend to have large flaccid bullae on the skin with oral involvement often preceding cutaneous manifestations. In up to 50% of cases, oral involvement is the sole manifestation of disease. This is in contrast to pemphigus foliaceus, where erosions are the rule, and oral involvement is not typically seen. IF studies will highlight the intercellular 'fish-net' deposition of IgG within the epidermis in both diseases.
Many of the immunobullous diseases show subepidermal clefting, and IF is critical in distinguishing among them. Bullous pemphigoid (BP), cicatricial pemphigoid, epidermolysis bullosa acquisita (EBA), bullous lupus, and linear IgA dermatosis all exhibit separation beneath the basal cell layer. BP typically presents as tense bullae on an erythematous base over the trunk and extremities, with oral involvement in up to one-third of cases. A close relative of BP is cicatricial pemphigoid (aka mucous membrane pemphigoid, MMP), which typically involves only mucosal surfaces and can heal with significant scarring. Both BP and MMP exhibit linear IgG staining along the basement membrane zone, but MMP may also involve antibodies to laminin 5 in addition to the BPAG1/BPAG2 antibodies seen in BP.
Technical aspects of direct immunofluorescence:
Punch biopsies (3-4 mm) as well as shave biopsies including submucosa are submitted to the laboratory in Zeus' or Michel's medium. The anatomic location of the biopsy is guided by the specific clinical scenario (see Table 1). In bullous disorders, a perilesional biopsy typically produces the most useful information, as false negatives may occur in ulcerated or eroded skin. In the laboratory, samples are washed to remove excess salts from the transport medium, and the biopsies are carefully mounted and frozen in liquid nitrogen prior to sectioning. Using a cryostat, 5 micron sections are cut and mounted onto charged glass slides. The tissue sections are incubated with fluorescence tagged antibodies (fluorescene isothiocyante, FITC) directed against human IgA, IgG, IgM, C3, and fibrinogen. Each slide is then carefully examined for fluorescence using a microscope with an ultraviolet light source and equipped with filters to detect the FITC signal. The location, pattern, and intensity of each immune reactant is then reported with the final impression. In some biopsies, it may then be necessary to induce an artefactual cleft within the lamina lucida of the basement membrane zone using 1M NaCl. This salt-splitting technique allows for distinction between such diseases as bullous pemphigoid, EBA, and bullous lupus.