A Guide to Immunofluorescence and its Applications in Dermatopathology
By Ing Wei Khor
Immunofluorescence (IF) is a method for detecting a specific protein in tissue or cells using an antibody that binds to that protein. Because the antibody is attached to a fluorescent dye, it (and by extension its protein partner) can be detected using a fluorescence microscope. IF is commonly used in dermatopathology to aid in the diagnosis and management of a wide range of skin diseases. These diseases include vasculitides; psoriasis; as well as bullous and connective tissue disorders. IF is also useful in diagnosing and managing diseases that involve the skin and other organs, such as systemic lupus erythematosus.
How Does Immunofluorescence in Dermatopathology Work?
Two types of IF are commonly used in dermatopathology—direct IF and indirect IF.
Direct IF
This form of IF involves applying fluorescent antibodies that recognize the protein of interest directly to tissue sections on glass slides. If the specific protein is present in the tissue, the antibodies will bind to the tissue and are then detected using fluorescent microscopy. Any antibody that does not bind to the tissue is removed by washing the tissue section with saline. Pathologists most commonly use sections of fresh frozen tissues, eg, skin biopsies, in direct IF. For the diagnosis of some diseases, such as autoimmune skin diseases, pathologists may use tissues that have been fixed in formalin and embedded in paraffin (formalin-fixed paraffin-embedded or FFPE tissue) when frozen tissue is not available.
A successful direct IF requires an effective antibody that is specific for the protein of interest. Important qualities include the “tightness” of the binding between the antibody and the protein, as well as the specificity of the binding (referring to the ability of the antibody to recognize the protein, while not recognizing other proteins).
Indirect IF
In this type of IF, the proteins being detected are usually antibodies in the patient’s blood. Several dilutions of serum (blood without the cells) are placed on a layer of tissue, often monkey esophagus tissue. The antibodies of interest, typically antibodies against the body’s own proteins, bind to the tissue layer. After this step, the tissue layer is washed in saline. The antibodies are then detected by applying another set of antibodies that are specific for human antibodies and that are bound to fluorescent dyes. Pathologists detect this second set of antibodies using a fluorescence microscope.
One advantage of indirect IF is that it does not require the production of specific fluorescent antibodies against the protein of interest, as is required in direct IF. Instead, indirect IF can use commercially available anti-human antibodies that are produced in mice or rats.
Fluorescent Dyes and Detection
The most common fluorescent dye used in IF is fluorescein isothiocyanate (FITC), which fluoresces green when light is shone on it. Other fluorescent dyes commonly used in IF are rhodamine and TRITC, a modified form of rhodamine.
If the protein (or antibody) of interest is present, pathologists can visualize the detecting antibody that is linked to FITC or another fluorescent dye by using a fluorescence microscope. Depending on the particular fluorescent dye, the microscope will detect the fluorescence at a certain wavelength. With IF, pathologists are also able to see where in the cell the protein of interest is located and determine the amount of protein present.
How Immunofluorescence Is Used in Dermatopathology
IF is used, together with clinical and histological evaluation, for diagnosing skin diseases characterized by abnormal proteins or antibodies against the body’s own proteins (autoantibodies). These diseases include autoimmune bullous diseases, vasculitides, and psoriasis. For some diseases, such as pemphigus diseases, pathologists may also use the amount of autoantibodies present (as determined by IF) to determine the severity of the disease.
In some skin disease cases, patients do not show typical clinical signs. In other cases, two different skin diseases may show similar clinical signs. IF is especially useful for establishing the diagnosis in these difficult situations through identifying disease-specific proteins or autoantibodies.
Ing Wei Khor is a trained scientist and medical writer, who is passionate about communicating science and medicine clearly and simply to a wide audience.
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Villani AP, Chouvet B, Kanitakis J. Application of C4d immunohistochemistry on routinely processed tissue sections for the diagnosis of autoimmune bullous dermatoses. Am J Dermatopathol. 2016;38:186-188. https://pubmed.ncbi.nlm.nih.gov/25793311/. Accessed November 20, 2020.
Pohla-Gubo G, Hintner H. Direct and indirect immunofluorescence for the diagnosis of bullous autoimmune diseases. Dermatol Clin. 2011;29:365-372. https://pubmed.ncbi.nlm.nih.gov/21605801/. Accessed November 20, 2020