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Immunofluorescence Examination of Skin Biopsies - Robert M. Law, M.D.
IMMUNOFLUORESCENCE EXAMINATION OF SKIN BIOPSIES by Robert M. Law, M.D.
Routine histologic evaluation of skin biopsies provides dermatopathologists with useful diagnostic information, but many clinical scenarios require the use of ancillary techniques. Blistering disorders, connective tissue disease, and vasculitis 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 dermatopathologist with experience in immunofluorescent microscopy.
Intraepidermal blisters: This includes the pemphigus family of immunobullous disorders (pemphigus vulgaris, pemphigus foliaceus, and IgA pemphigus). In pemphigus vulgaris, patients tend to have large flaccid bullae with oral involvement. This is in contrast to pemphigus foliaceus, where intact bullae are rare, and oral involvement is not seen. IF studies will highlight the intercellular 'fish-net' deposition of IgG within the epidermis. In IgA pemphigus, patients present with pruritic vesiculopustules on the trunk, extremities, or axillae. Similar to other variants of pemphigus, intercellular deposition of immunoglobulins (IgA, in this case) is seen on direct immunofluorescence.
Subepidermal blisters: 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 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.
EBA and bullous lupus are subepidermal blisters which target type VII collagen, but can look identical to BP on direct immunofluorescence examination. Salt-splitting samples will distinguish between these entities, with BP antibodies localizing to the epidermal side of the basement membrane zone and EBA/bullous lupus localizing to the dermal side.
Linear IgA dermatosis classically presents with vesicles of the periphery of annular erythematous lesions. Chronic bullous disease of childhood is a synonomous entity which presents in the first 5-6 years of life. Linear IgA disease has been associated with certain drug use (e.g., vancomycin, captopril) and oral involvement may occur in up to 70% of patients. IF demonstrates a linear band of IgA along the basement membrane zone, and antibodies are directed to a fragment of BPAG2.
Connective tissue diseases: Immunofluorescence is also helpful in evaluating for the presence of connective tissue diseases such as lupus erythematosus or dermatomyositis. Granular deposition of immunoglobulins (typically IgM) and complement (C3) is seen along the basement membrane zone in lesional skin, but may also be seen in normal skin during periods of active disease. Lesions present longer than six weeks generally have a greater sensitivity of detection using IF, while the more evanescent lesions of subacute cutaneous lupus erythematosus (SCLE) may exhibit false negatives on immunofluorescence. In all cases of suspected connective tissue disease, serologic studies are a vital adjunctive diagnostic test.
Vasculitis: The patient presenting with palpable purpura can be a diagnostic challenge. In some cases of vasculitis, immunoreactants may deposit within vessel walls. These immune complexes are highlighted using direct immunofluorescence, typically as punctate intramural granular deposits. Henoch-Schoenlein purpura is a specific situation in which the demonstration of IgA deposits by IF is essential for establishing the diagnosis.
Technical aspects of direct immunofluorescence
The optimal specimens for thorough dermatopathologic evaluation are two 3-4 mm punch biopsies: one submitted in formalin for routine hematoxylin/eosin staining, and a second biopsy placed fresh in a tightly sealed container of Michel's transport medium for direct immunofluorescent examination. Splitting of a single punch biopsy is suboptimal, as the lesion may be missed when the specimen is divided. 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 (fluorescein 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 (see Figure 1). 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.