DIF and IIF evaluates in vivo bound and circulating autoantibodies and are the preferred methods for diagnosing AIBDs. In pemphigus diseases and dermatitis herpetiformis, the titer of circulating autoantibodies reflects the disease activity. In patients with a classical clinical picture, the DIF confirms the diagnosis. Furthermore, this technique is essential in subtypes of AIBDs with atypical clinical manifestations (eg, no blisters or erosions) or clinically similar presenting manifestations, such as bullous pemphigoid, MMP, or EBA. A direct or indirect SSST is often crucial for the differential diagnosis between subtypes of these diseases, leading to proper treatment for severely affected patients.
Acquired autoimmune bullous diseases (AIBDs) are characterized by a pathogenic immune response against structural proteins of keratinocytes or of the dermoepidermal basement membrane zone (BMZ). Autoantibodies, both tissue-bound and circulating, account for the pathogenicity of this group of diseases known to be rare and also severe. The laboratory diagnosis includes histologic, immunohistochemical, and sometimes ultrastructural assessment. Immunofluorescence microscopy is a technique to rapidly show the reaction of an antigen (structural protein) and the pathogenic autoantibody in a patient suspected to have an AIBD. Direct immunofluorescence (DIF) is the method to show the deposition of immunoreactants (in vivo bound autoantibodies) in skin or mucous membranes. Indirect immunofluorescence (IIF) quantitates the respective circulating autoantibodies in the serum of the patient. Thus, immunofluorescence is the key instrument for diagnosing the three major groups of AIBDs: (1) pemphigus diseases, (2) BMZ diseases, including pemphigoid diseases such as bullous pemphigoid, mucous membrane pemphigoid (MMP), and epidermolysis bullosa acquisita (EBA) and (3) dermatitis herpetiformis, or Duhring disease. This article describes the method, practical application for the differential diagnosis of AIBDs, method to discern between subtypes within groups of those disorders (salt-split skin test [SSST]), and importance of the method to diagnose AIBDs representing with an atypical clinical picture.
Materials and methods
Biopsy and Blood Sample
If an AIBD is suspected, the biopsy for DIF should be taken when the routine lesional biopsy is taken within the edge of an active blister for routine pathology sent in formalin. A 4- to 6-mm punch or excision biopsy of patient skin or mucous membrane should be taken from an area of perilesional or clinically normal appearing skin or mucous membrane. For cosmetic reasons, the authors prefer to take the immunofluorescence biopsy from the (non–sun exposed) inner aspect of the upper arm if the blistering is widespread. If the immunofluorescence laboratory is nearby, such as within the same institution or hospital, the biopsy simply can be placed in a plastic tube (without any liquid) and transferred immediately to a freezer at −20°C. Alternatively, if the biopsy is to be sent to an immunofluorescence laboratory elsewhere, it can be placed in a small transport tube containing Michel’s medium. Samples can be stored in this medium for up to 28 days at room temperature or in the fridge (specimen should not be frozen in Michel’s medium) and sent worldwide to any specialized laboratory that performs diagnostic immunofluorescence.
A 2- to 5-mL sample of whole blood (without any additives) is centrifuged and the serum is used for IIF to discover circulating autoantibodies in AIBDs. The authors recommend always taking a biopsy and a blood sample from each patient.
Direct Immunofluorescence
For the DIF test, three to six cryocut sections (4- to 6-μ thick) from the patient’s skin or mucous membrane are placed on each of five microscope glass slides, air-dried for 10 minutes, and rinsed in phosphate buffered saline (PBS). For routine DIF, each slide is then covered with an appropriate dilution of one of the antisera against IgG, IgA, IgM, complement factor C3, and fibrinogen. The antibodies are conjugated to a fluorescent dye, most commonly fluorescein-iso-thio-cyanate (FITC). Incubation in a moist chamber (30 minutes, room temperature) is followed by two washings of 15 minutes each with PBS. The sections are then mounted with a drop of buffered glycerin or a special embedding medium containing antibleaching reagent (eg, EUROIMMUN Order no. ZF 1200–0130). For FITC conjugated antibodies, the results are read with a fluorescence ultraviolet microscope at 450 to 490 nm.
Indirect Immunofluorescence
Different commercially available animal tissues, such as the frequently used monkey or guinea pig esophagus, serve as antigenic substrates. For special purposes, such as for the SSST, normal human skin (NHS) is used as a substrate. The source for the NHS samples is excess cutaneous material from the Dermatosurgery Unit of the authors’ Department of Dermatology, provided that the respective patient has given informed consent for use, but other places use donated abdominopexy skin. In the first step, circulating autoantibodies bind to their respective antigens (mostly keratinocyte or BMZ structural proteins) in the tissue. For this purpose the substrate is covered with a series of dilutions of patient serum in PBS (mostly starting with 1:10), incubated in a moist chamber for 30 minutes at room temperature, and rinsed then twice for 15 minutes in PBS. In the next step the tissue sample is incubated for 30 minutes with a second antibody directed against various immunoglobulin classes of the autoantibody that has bound in vitro in the first step. Typically these second antibodies are raised in mice (usually mouse anti-human IgG1), swine, or rat and are conjugated to a fluorescent dye like FITC. Again the slides are washed twice, mounted, and viewed under the microscope.
SSST
Salt-split skin is the substrate of choice for the differential diagnosis between subtypes of AIBDs with autoantibodies directed against proteins of the dermoepidermal BMZ (for example bullous pemphigoid vs epidermolysis bullosa acquisita). Ray Gammon at the University of North Carolina, Chapel Hill first described using incubation in 1M sodium chloride (NaCl) to separate the skin between the epidermis and dermis in the lamina lucida ( Fig. 1 ). This technique can be performed on the patient’s skin (direct SSST) and on separated NHS using patient serum (indirect SSST).
Direct SSST
Usually a biopsy that has already been investigated with routine DIF is prepared for the direct SSST. If two biopsies (normal and perilesional) were obtained, the authors prefer the one from normal-appearing skin. If only a perilesional biopsy from the patient is available, the authors also use this one. When the biopsy is prepared for the SSST, the sample is lost for a reinvestigation using routine DIF because it is placed overnight in 1M NaCl. This technique produces an artificial blister within the lamina lucida (see Fig. 1 ). The DIF is then repeated on cryostat sections of the salt-split sample, as described earlier. The distribution of in vivo bound autoantibodies can then be seen on either the epidermal or the dermal side of the artificial blister (ie, on the blister roof or blister floor; Fig. 2 F, G). This method allows one to distinguish, for example, between bullous pemphigoid (epidermal staining) and the BP-variant of EBA (dermal staining), which clinically may look identical (see Fig. 2 A–C).
Indirect SSST
For indirect SSST, NHS is incubated for 1 night in 1M NaCl. An IIF with the serum of the patient is then performed on cryostat sections of that substrate as described. The test, which is more sensitive than routine IIF, shows circulating autoantibodies binding to either the epidermal or the dermal or both sides of the artificial blister. This technique again allows, for example, differentiation between bullous pemphigoid and EBA (see Fig. 2 H).
Patterns of staining
Pemphigus Diseases
Pemphigus diseases are characterized by intraepithelial blister formation from acantholysis as the result of autoantibody reactivity directed against (desmosomal) intercellular adhesion proteins of keratinocytes such as desmoglein 1 or 3. On a biopsy, a typical net-like (chicken wire–like) intercellular substance pattern (ICS) ( Fig. 3 A) is seen in the DIF with anti-IgG and anti-C3, and rarely also with anti-IgA (in IgA pemphigus) ( Box 1 ). IIF on monkey esophagus shows circulating IgG autoantibodies in the serum of the patients (see Fig. 3 B). The amount of autoantibodies is reflected in the ICS titer, and correlates with the disease activity in pemphigus diseases. Patient follow-up is performed through monitoring the ICS titer with IIF. To detect technical pitfalls in the immunofluorescence technique (eg, decrease in brightness because of the time span of the microscope lamp), the authors study the actual serum together with a former sample on one slide containing two cuts of the substrate to see an increase or decrease of the titer. In addition to DIF and IIF, enzyme-linked immunosorbent assays (ELISA) or immunoblot techniques are used to specify the autoantibodies against different antigens, such as desmoglein 3 and 1.
Related posts:
Diagnosis and Clinical Features of Pemphigus Foliaceus
Pathogenesis of Endemic Pemphigus Foliaceus
Pemphigoid Gestationis: Pathogenesis and Clinical Features
Pathophysiology of Dermatitis Herpetiformis: A Model for Cutaneous Manifestations of Gastrointestinal Inflammation
Pathogenesis of Epidermolysis Bullosa Acquisita
Nail Involvement in Autoimmune Bullous Disorders
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