Immunofluorescent techniques are extremely useful in the diagnosis of several dermatologic diseases. University medical centers and most private dermatopathology laboratories have the facilities to carry out these tests. Transport solutions, such as Michel's solution, are easily available so that specimens of skin to be tested for direct immunofluorescence can be mailed if necessary. Two types of immunofluorescent testing are available: the direct technique and the indirect technique.
Direct immunofluorescent testing is carried out on skin specimens. With this technique, immunoglobulin(s) or complement components that have been deposited in the skin can be identified. The skin specimen for this test is taken by standard biopsy technique, but instead of placing it in formalin, the specimen either is immediately frozen in liquid nitrogen or is placed in transport solution. In the laboratory, fluoresceinlabeled antibodies to immunoglobulins G (IgG), M (lgM), and A (IgA), complement components, and fibrin are layered over the tissue. If anyone of these proteins is present, the antibodies will adhere to the tissue and will be visible on fluorescent microscopy.
Positive direct immunofluorescent tests are expected in six diseases described in this book: dermatitis herpetiformis, pemphigoid, pemphigus, lupus erythematosus, lichen planus, and some forms of vasculitis. In dermatitis herpetiformis, perilesional skin will contain globules of IgA (and sometimes C3) located in the superficial papillary dermis. In pemphigoid, IgG and C3 are found at the dermal-epidermal junction of both lesional and perilesional skin. In pemphigus, IgG and, occasionally, C3 neatly outline the epidermal cell membranes in a net-like fashion.
IgG and C3 are present at the dermal-epidermal junction in the lesional skin of patients with all types of lupus erythematosus; about 70% of patients with systemic lupus erythematosus will have similar deposits in sun-exposed but nonlesional skin. In lichen planus, globular deposits of fibrin are often found in the papillary dermis; additionally, IgM may coat the cytoid bodies (apoptotic keratinocytes) present in the papillary dermis. In the neutrophilic types of vasculitis, IgG, C3, and fibrin are generally found in and around the affected vessels.
Indirect immunofluorescent studies identify specific antibodies circulating within the patient's plasma. These antibodies are directed against individual antigens found within the patient's skin. When indirect studies are performed, the patient's serum is layered over an appropriate substrate such as slices of epithelium (for the bullous diseases), certain tumor cells (for the antinuclear factors of lupus erythematosus), or specific organisms (such as T. pallidum for syphilis). If the patient's serum contains antibodies, they will adhere to the antigen present in the substrate. These attached antibodies are visualized when fluorescein-tagged antibodies to human immunoglobulin are applied and the specimen is examined under fluorescence microscopy.
Almost all patients with pemphigus will have circulating antibodies directed toward desmosomal-related antigens on the outer surface of epithelial cell membranes. Approximately 80% of patients with pemphigoid will have circulating antibody directed against hemidesmosomal-related antigens present within the basement membrane zone of the dermal-epidermal junction. Most patients with primary syphilis and all patients with secondary syphilis will have circulating antibodies that will bind to T. pallidum fixed on microscopic slides (the FTA-Abs test). More than 95% of patients with systemic lupus erythematosus and a varying proportion of those with other types of lupus erythematosus will have antibodies that bind to one or more antigens present within cell nuclei. This latter test is known as the fluorescent antinuclear antibody (FANA) test. All of the above indirect immunofluorescent tests can be titered in order to estimate the amount of antibody that is present. Antibody titer is very roughly correlated with the severity of the disease, and in some instances, the clinician can judge the results of therapy by following the decline in antibody titer.
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