Immunofluorescence Patterns in Selected Dermatoses, Including Blistering Skin Diseases Utilizing Multiple Fluorochromes

Background: Autoimmune vesiculobullous disorders represent a heterogeneous group of dermatoses whose diagnosis is made based on clinical history, histologic features, and immunopathologic features. The most commonly used techniques for the diagnosis of these diseases are direct and indirect immunofluorescence (DIF and IIF), including salt-split processing. NaCl split skin is used to determine the level of blister formation, and the localization of autoantibodies relative to the split. Classically, immunofluorescence has been performed with one fluorochrome in the diagnosis of autoimmune bullous skin diseases. Aims: To compare DIF and IIF of the skin, using a single fluorochrome versus multiple fluorochromes. Materials and Methods: We studied 20 autoimmune skin disease cases using fluorescein isothiocyanate (FITC) alone, in comparison to multiple fluorochromes (with or without DNA counterstaining). Results: The use of multiple fluorochromes helped to simultaneously visualize reactivity in multiple skin areas, in contrast to using FITC alone. Conclusions: Using multiple fluorochromes allows simultaneous labeling of two or more antigens within the same cell/or tissue section, assists in colocalization of unknown antigens with known molecules, and helps in ruling out "background" staining.