University Medical Center Groningen
The diagnosis of autoimmune blistering diseases
Autoimmune blistering diseases can be divided into two main groups according to the level of blister formation, i.e. intraepidermal blistering in pemphigus and subepidermal blistering in pemphigoid. The combination of direct and indirect immunofluorescence together with immunoblotting and ELISA is essential for a complete diagnosis of autoimmune blistering diseases.
Direct immunofluorescence
The biopsy of patient's skin or mucosa is an important part in the diagnosis of autoimmune blistering diseases. The biopsy should be taken perilesional, i.e. from the redness next to the blister. Since immunofluorescence does not work on formalin-fixed material, it is necessary to store a biopsy preferably in normal saline. Normal saline ensures that undesired background is reduced and enhances specific immunofluorescent staining (see Figure 1). If it will take longer than 24 hours to transport the biopsy to our laboratory, it can be stored on dry ice or in Michel's fixative.
The biopsy is stained for the presence of immunoglobulins and complement. In pemphigus the antibodies are deposited on the surface of keratinocytes; a so-called honeycomb pattern (Figure 2). Pemphigoid shows a linear pattern along the basal membrane. A distinction can be made here between a linear n-serration pattern (figure 3), compatible with bullous pemphigoid and most other forms of pemphigoid, or a u-serration pattern, that can be seen in epidermolysis bullosa acquisita (figure 4). Finally, a granular pattern of IgA along the basal membrane can also be found, which is seen in dermatitis herpetiformis (Figure 5).
Together with the findings of the serological examination, we can further (sub)classify autoimmune blistering diseases. This is important because every variant of a blistering disease has a different prognosis and requires different treatment
Serology of bullous dermatoses
Serological testing is needed to determine whether circulating antibodies to proteins (antigens) involved in autoimmune blister diseases are present in patient's blood. This requires a tube of clotted blood from the patient. The combination of serology and biopsy increases the sensitivity and specificity of the diagnosis of autoimmune blistering diseases.
Two substrates are used. In the first place monkey esophagus, where two patterns can be distinguished. The antibodies in pemphigus serum will bind to the epithelial cells, resulting in a chicken wire pattern (Figure 6). In the case of pemphigoid, the antibodies will bind at the site of the basal membrane, giving a linear pattern (Figure 7).
The other substrate is human salt split skin. Incubating the skin of healthy persons with high molar sodium chloride for approximately 24 hours creates a split at the site of the basal membrane zone. Certain proteins involved in pemphigoid will be in the roof of the blister (Figure 8), including BP180 and BP230, while other proteins are at the dermal site of the blister, such as collagen 7 and laminin 332 (Figure 9). Incubation with serum from pemphigoid patients can give an epidermal or dermal staining, depending on the antigen to which the antibodies are directed.
With both pemphigus and pemphigoid, it is necessary to perform additional tests, such as ELISA and immunoblotting, in order to determine the antigen involved in more detail.
Additional serology of bullous dermatoses
Enzyme-Linked Immune Sorbent Assay (ELISA) is a laboratory test that can measure the amount of antibodies in the blood against a certain autoantigen. The advantage of this is that the disease activity can be monitored to see if the medication is effective or if there is a risk of relapse. The laboratory has anti-desmoglein 1 and anti-desmoglein 3 ELISAs (pemphigus) and anti-NC16A and anti-BP230 ELISAs (pemphigoid).
All sera with a differential diagnosis of pemphigus or pemphigoid are routinely screened with ELISA to see if antibodies to an autoantigen are indeed present. The antibody titer of patients under treatment at the UMCG is checked at each visit.
Immunoblot is a test in which a patient's serum is placed on a nitrocellulose membrane on which skin specific proteins are present. If the serum contains antibodies that are directed against one of these proteins, they will bind to it. This can be made visible with a color method and a diagnosis can be made based on the position where the IgG is bound (Figure 10). With pemphigoid, the technique is particularly suitable for detecting antibodies against BP180 (the antigen that most pemphigoid patients make antibodies to). In addition, it is a very adequate test to detect paraneoplastic pemphigus because it can make periplakine and envoplakine antibodies visible. The initial diagnosis in the event of suspected paraneoplastic pemphigus is immunoblot and indirect immunofluorescence on rat bladder. If the outcome is not clear, the serum is tested by immunoprecipitation. Immunoprecipitation is an extremely sensitive but also laborious test that can detect very low concentrations of antibodies in the blood. Three autoantigens specific to paraneoplastic pemphigus are the combination envoplakine and periplakine and / or the protein A2ML1 (Figure 11). The antibody response may differ per patient.
Special serology tests
Additional tests can be carried out if the results from the above techniques are not clear. The immunodermatology laboratory has a number of tests developed by the laboratory itself. The In Vitro Binding Assay (ivba) is a test for pemphigus that detects antibodies directed against desmosomes. Patient serum is added to cultured keratinocytes. If anti-desmosomal antibodies are present, they will bind to the desmosomes which can be visualized with immunofluorescence (Figure 12). This test also shows the presence of antibodies if IIF on apenoesofagus and ELISA are negative. Patients who have negative ELISA values can be monitored with this test for the presence of antibodies.
The Keratinocyte Footprint Assay (KFA) was most recently developed by us. With this test, antibodies can be detected in the blood against laminin-332. Keratinocytes in culture crawl over the glass on which they are grown. Just like a snail leaves a slime trail, keratinocytes do that too. This trace contains laminin-332. When serum from a patient with anti-laminin-332 antibodies is placed on such a slide, the auto-antibodies will bind to this track and this can be made visible with immunofluorescence (Figure 13). As the trails do not contain any other autoantigens, the test is 100% specific for the diagnosis of anti-laminin-332 mucosal pemphigoïd. Compared to other anti-laminin 332 tests, this test is also the most sensitive.
