Keratinocyte footprint assay discriminates antilaminin‐332 pemphigoid from all other forms of pemphigoid diseases

Abstract

Summary Background Antilaminin‐332 mucous membrane pemphigoid is a chronic severe pemphigoid disease characterized by autoantibodies to laminin‐332. At present no commercial assay is available to demonstrate antilaminin‐332 antibodies, and diagnosis relies on in‐house techniques with limited sensitivities. Objectives In order to move, keratinocytes cultured in vitro secrete laminin‐332 to attach to the culture dish. In that way, they leave behind a unique footprint trail of laminin‐332. We aimed to develop a sensitive and specific laboratory assay to determine antilaminin‐332 autoantibodies in patient serum based on binding of patient IgG to these unique footprints. Methods Normal human keratinocytes were grown on glass coverslips and incubated with patient or control serum for 1 h. The binding of IgG was then investigated by immunofluorescence. After validating the test for its ability to identify antilaminin‐332 autoantibodies it was converted into a daily available test based on binding of IgG to dried coverslips that can be stored frozen. The staining patterns of sera from patients with antilaminin‐332 pemphigoid were then compared with those of sera from patients with other autoimmune bullous diseases and normal human sera. Results IgG of all antilaminin‐332 pemphigoid sera (n = 16) bound to laminin‐332 footprints, while all normal human controls (n = 55) were negative. From the sera of patients with other diseases (n = 72) four sera tested positive. The footprint assay was also positive for sera that were negative by salt‐split skin analysis, demonstrating that it is a very sensitive technique. Conclusions The keratinocyte footprint assay is a fast and specific assay to confirm or rule out the presence of antilaminin‐332 autoantibodies. What's already known about this topic? Antilaminin‐332 mucous membrane pemphigoid is a severe form of pemphigoid, and patients may have an increased risk of malignancies. The diagnosis of antilaminin‐332 mucous membrane pemphigoid is complicated by the lack of specific commercial tests for antilaminin‐332 antibodies and can be confirmed only in specialized laboratories. Keratinocytes in culture need laminin‐332 for adhesion and migration and therefore deposit it on the bottom of the culture dish. What does this study add? The keratinocyte footprint assay detects antilaminin‐332 autoantibodies in patient serum using the native laminin‐332 produced by cultured keratinocytes. What is the translational message? The keratinocyte footprint assay is a fast and specific assay to confirm or rule out the presence of antilaminin‐332 autoantibodies.

Comparison of Two Diagnostic Assays for Anti-Laminin 332 Mucous Membrane Pemphigoid

“…In the biochip, untransfected cells that do not express laminin 332 serve as internal controls; however, they may show some autofluorescence. These observations also reflect the reported higher sensitivity of the footprint assay compared with the biochip (10,24). a Sixty-one sera; in seven sera of patients originally classified as non-anti-laminin 332 mucous membrane pemphigoid, an additional anti-laminin 332 IgG was detected in this study.…”

“…We included 54 anti-laminin 332 MMP patients (21 females, 33 males) in our study (Table S1). Sixteen of these sera were already described by Giurdanella et al (24). Most of the other sera were used for the establishment of the indirect IF test using recombinant laminin 332 (10).…”

European Guidelines (S3) on diagnosis and management of mucous membrane pemphigoid, initiated by the European Academy of Dermatology and Venereology – Part II

“…In a large group of unselected MMP patients, Bernard and coworkers detected laminin 332 antibodies in 20% of sera, with a specificity of 91% (3/32 of healthy controls) 55 . Further, a sensitive (100%, n = 16) and specific assay (96.9%, n = 127), based on detection by IIF of IgG binding to laminin 332 secreted by human keratinocytes, named the keratinocyte footprint assay, has been reported 84 . Moreover, a sensitive and specific assay based on IIF on HEK293 cells expressing the laminin 332 heterotrimer on the cell surface (biochip mosaic), has recently been developed.…”

“… 55 Further, a sensitive (100%, n = 16) and specific assay (96.9%, n = 127), based on detection by IIF of IgG binding to laminin 332 secreted by human keratinocytes, named the keratinocyte footprint assay, has been reported. 84 Moreover, a sensitive and specific assay based on IIF on HEK293 cells expressing the laminin 332 heterotrimer on the cell surface (biochip mosaic), has recently been developed. When a large cohort of 93 laminin 332 positive MMP patients was assayed, a sensitivity of 84% and specificity of 99.6% were obtained.…”

Novel diagnostic method to differentiate antilaminin‐332 pemphigoid from other forms of pemphigoid

“…In this issue of the BJD , Hendri Pas’ group presents an excellent innovative diagnostic test that has been developed in Groningen, the Netherlands . Their paper describes the keratinocyte footprint assay, a specific diagnostic method to detect the presence of antilaminin‐332 autoantibodies.…”