Serological diagnosis of autoimmune bullous skin diseases: prospective comparison of the BIOCHIP mosaic-based indirect immunofluorescence technique with the conventional multi-step single test strategy

Abstract

Background: Various antigen-specific immunoassays are available for the serological diagnosis of autoimmune bullous diseases. However, a spectrum of different tissue-based and monovalent antigen-specific assays is required to establish the diagnosis. BIOCHIP mosaics consisting of different antigen substrates allow polyvalent immunofluorescence (IF) tests and provide antibody profiles in a single incubation.

Methods: Slides for indirect IF were prepared, containing BIOCHIPS with the following test substrates in each reaction field: monkey esophagus, primate salt-split skin, antigen dots of tetrameric BP180-NC16A as well as desmoglein 1-, desmoglein 3-, and BP230gC-expressing human HEK293 cells. This BIOCHIP mosaic was probed using a large panel of sera from patients with pemphigus vulgaris (PV, n=65), pemphigus foliaceus (PF, n=50), bullous pemphigoid (BP, n=42), and non-inflammatory skin diseases (n=97) as well as from healthy blood donors (n=100). Furthermore, to evaluate the usability in routine diagnostics, 454 consecutive sera from patients with suspected immunobullous disorders were prospectively analyzed in parallel using a) the IF BIOCHIP mosaic and b) a panel of single antibody assays as commonly used by specialized centers.

Results: Using the BIOCHIP mosaic, sensitivities of the desmoglein 1-, desmoglein 3-, and NC16A-specific substrates were 90%, 98.5% and 100%, respectively. BP230 was recognized by 54% of the BP sera. Specificities ranged from 98.2% to 100% for all substrates. In the prospective study, a high agreement was found between the results obtained by the BIOCHIP mosaic and the single test panel for the diagnosis of BP, PV, PF, and sera without serum autoantibodies (Cohen's κ between 0.88 and 0.97).

Conclusions: The BIOCHIP mosaic contains sensitive and specific substrates for the indirect IF diagnosis of BP, PF, and PV. Its diagnostic accuracy is comparable with the conventional multi-step approach. The highly standardized and practical BIOCHIP mosaic will facilitate the serological diagnosis of autoimmune blistering diseases.

Figure 1

BIOCHIP mosaic for immunobullous disorders. (A) On a standard-sized slide, there are ten incubation fields each with six different BIOCHIPs. (B, C) Representative stainings after incubation with a bullous pemphigoid (B) and pemphigus vulgaris (C) serum. Desmoglein (Dsg) 1, Dsg3, and BP230gC (C-terminal globular domain of BP230) are expressed in human HEK293 cells. BP180 NC16A is directly coated on the BIOCHIP. HEK293 cells transfected with pTriEx-1 serve as negative control.

Figure 2

Correlation of serum autoantibody levels with disease activity. Values by conventional ELISA (Dsg3 (A), Dsg1 (C), BP180 (E)) paralleled indirect immunofluorescence titers by the BIOCHIP mosaic (Dsg3 (B), Dsg1 (D), BP180 (E)) obtained by testing sera from patients with pemphigus vulgaris (PV1-3) (A,B), pemphigus foliaceus (PF1-3) (C,D), and bullous pemphigoid (BP1-3) (E,F). Both ELISA values and indirect immunofluorescence titers appeared to correlate with disease activity determined by a clinical score (4, more than 10 lesions; 3, 4–10 lesions; 2, 1–3 lesions; 1, clinical remission on immunosuppressive therapy; 0 clinical remission without immunosuppressive therapy). Dsg, desmoglein; IF, immunofluorescence.

Figure 3

Evaluation of the BIOCHIP mosaic for the routine diagnosis of autoimmune bullous diseases. A large panel of consecutive sera (n = 453) from patients with suspected autoimmune bullous disease was subjected to both the BIOCHIP mosaic (black columns) and the routine multi-step procedures of the autoimmune laboratory of the department of dermatology, Luebeck (grey columns; detailed in [38]). Obtained diagnoses were grouped into BP (bullous pemphigoid), “pemphigoid” diseases (including linear IgA disease, p200-pemphigoid, mucous membrane pemphigoid and dermatitis herpetiformis), PV/PNP (pemphigus vulgaris/ paraneoplastic pemphigus), PF (pemphigus foliaceus), and PG (pemphigoid gestationis). PNP and “pemphigoid” sera are further specified in Table 4.