In AIBDs, the IFM aids in the differential diagnosis of disorders such as the pemphigoid diseases and various subtypes of epidermolysis bullosa acquisita (EBA) or bullous systemic lupus erythematosus (BSLE).

The technique allows to specify the level of blister formation in AIBDs with the common linear staining pattern with antibodies to immunoglobulins and/or complement components (immune deposits) along the basement membrane zone (BMZ) by the conventional immunofluorescence method. Two major types of the method are applied (see Table 18.1).

With AgM, the basal lamina (lamina densa) in a biopsy of an in vivo blister is visualized by staining with antibodies directed against, for example, type IV collagen. The level of tissue separation is then assessed with reference to the location of the basal lamina, i.e., either below or above the lesional split (junctional or dermolytic).

In the absence of in vivo blistering, AbM of a patient skin specimen (clinically normal-appearing skin of, for example, the inner aspect of the upper arm — non-sun exposed!) is the diagnostic method of choice. By overnight incubation in 1 M NaCl, an artificial split formation is induced within the lamina lucida. The sample is devoid of secondary changes which may interfere with regular IFM and thereby modify the diagnosis. In the subsequent DIF test (direct AbM), in vivo bound autoantibodies are visualized either on the epidermal, dermal, or both side(s) of the artificial blister (i.e., on the blister roof and/or blister floor), whereas the basal lamina, as a reference in this assay, is constantly found by AgM on the dermal side, i.e., blister floor.

In addition to studies on patient’s skin, the same principle is used modified with an IIF test performed with patient’s serum (indirect AbM) on cryostat sections of salt-separated normal human skin (obtained during surgical procedures with informed consent!). This technique demonstrates the location of binding of circulating autoantibodies either to the epidermal (i.e., bullous pemphigoid, BP) or dermal (i.e., EBA) side of the artificially created blister. This is particularly helpful for making the diagnosis in patients where a biopsy is not available. Indirect SSST is also markedly more sensitive than routine IIF.

The staining profile of immune deposits in SSST provides essential information for the differential diagnosis between those subtypes of AIBDs which are characterized by autoantibodies directed against BMZ proteins (Table 18.2). For instance, it allows to differentiate unequivocally between BP (epidermal fluorescence above the basal lamina = lamina densa) and EBA (exclusive dermal fluorescence below the basal lamina) which clinically may appear to be identical.

Epidermal fluorescence in SSST is a characteristic finding in several types of pemphigoid besides classical BP. Circulating autoantibodies binding to the dermal side (floor of the artificial blister) are seen in EBA, but also in BSLE, a subset of patients with mucous membrane pemphigoid and autoantibodies against laminin 332 within the lamina lucida, a bullous pemphigoid-like disease with autoantibodies to a 105 kDa lamina lucida glycoprotein unrelated to laminin 332, as well as anti-laminin-γ[gamma]1 pemphigoid [12–15]. Fluorescence on both – the epidermal and the dermal sides of the blister – is observed in mucous membrane pemphigoid and linear IgA dermatosis, partly reflecting the consequences of “epitope spreading.”

One major drawback of direct SSST on routinely obtained IF samples should be considered, i.e., that the induction of the artificial split means the original sample is lost for reinvestigation by routine DIF.

For a patient suspected to have one of the various subtypes of EBH, the immunofluorescence AgM technique supports to reach the primary diagnosis. It demonstrates (1) the level of split formation (intraepidermal, junctional, dermolytic) as well as (2) the expression of a specific structural protein in the skin (normal, reduced, or lacking; see Table 18.1).

The level of split formation can be determined on a cryosection of a lesional (blister) biopsy (see above). In addition, it is possible to gain information on the expression of specific structural proteins in the epidermis, the BMZ, or the dermis: in the latter situation, cryostat sections of a sample of normal-appearing skin (inner aspect of the upper arm) are incubated with different antibodies by IIF (Table 18.3). Taking the biopsy, attention has to be paid that the often minute and fragile epidermis is not kept in the punch instrument or gets lost during the final embedding procedure of the sample!