Dermatopathology
Comparison of C3d immunohistochemical staining to enzyme-linked immunosorbent assay and immunofluorescence for diagnosis of bullous pemphigoid

https://doi.org/10.1016/j.jaad.2020.02.020Get rights and content

Background

Bullous pemphigoid (BP), the most common autoimmune blistering disease, may be diagnostically challenging. Direct immunofluorescence (DIF), indirect immunofluorescence (IIF), enzyme-linked immunosorbent assay (ELISA), and recently, C3d immunohistochemistry (IHC), are used as adjuncts to diagnosis.

Objective

To compare C3d IHC to DIF, IIF, and ELISA testing in BP diagnosis.

Methods

C3d IHC was performed on skin biopsy specimens from 51 patients (27 with BP and 24 with other blistering diseases) and compared to DIF and IIF, with anti-BP180 or anti-BP230 ELISA results used as the gold standard.

Results

We found C3d IHC, DIF, and IIF had similar sensitivity (74.1%, 63.1%, and 70.4%), specificity (95.8%, 100%, and 100%), positive predictive value (95.2%, 100%, and 100%), and negative predictive value (76.7%, 70.6%, and 75%) for BP. Cases with positive C3d IHC, DIF, and IIF had significantly higher anti-BP180 and anti-BP230 by ELISA than cases with negative testing (P < .0001). False-negative IIF results were associated with lower BP230 compared with true-positive results (P = .03).

Limitations

This was a single-center, retrospective study.

Conclusion

Our study compared C3d IHC to DIF and IIF in BP diagnosis, demonstrating C3d IHC on fixed tissue provides similar diagnostic utility to immunofluorescence and ELISA.

Section snippets

Case selection

Use of specimens in this study was provided through a protocol approved by University of Pennsylvania Institutional Review Board (#820338). Patients with at least 1 positive BP180 or BP230 ELISA, or both, in the setting of clinical concern for BP (based on clinical documentation) and a skin biopsy specimen within 1 year of ELISA testing, between 2010 and 2014, were identified from our dermatopathology database NewPath. This search yielded 110 patients. We considered positive ELISA testing in

Results

We identified 110 patients in our dermatopathology database for whom ELISA testing was performed in addition to a skin biopsy for routine histopathology with C3d. Of the 110 patients identified, 51 also had DIF and IIF testing. Representative interpretations of positive and negative C3d staining results are provided for reference (Fig 1). No significant differences in age or sex were identified between the 24 patients who tested BP negative by ELISA compared with the 27 who tested BP positive (

Discussion

In this study, we found that C3d IHC, DIF, and IIF had similar test characteristics for identifying BP using ELISA for anti-BP180 or anti-BP230 as the diagnostic gold standard. Notably, the areas under the receiver operating characteristic curve for each test were not significantly different, suggesting similar discriminative abilities for BP diagnosis. Together, these data suggest that C3d may be used to support a diagnosis of pemphigoid, particularly when fluorescence microscopy may be

Conclusion

This study is unique in that it examines a cohort of patients who were diagnosed with the aid of ELISA testing but corroborates previous reports that have suggested that C3d IHC may be helpful in diagnosing BP. The ability to use IHC on fixed tissue adds to the diagnostic toolkit for BP, potentially reducing the need for additional biopsy specimens or ancillary tests if positive. At institutions where immunofluorescence or ELISA may not be readily available, C3d IHC may be incorporated into a

References (23)

  • C. Muglia et al.

    Questioning the specificity and sensitivity of ELISA for bullous pemphigoid diagnosis

    Cutis

    (2017)

    • Cited by (12)

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      Nowadays, the diagnosis relies on clinical presentation, histopathology, and combined immunologic tests, including direct immunofluorescence (DIF), indirect immunofluorescence (IIF), enzyme-linked immunosorbent assay (ELISA), and immunoblotting techniques [6, 7]. Although evidence has suggested sensitivities and specificities of DIF, IIF, and immunoblotting [8, 9, 10], ELISA was thought to be a novel gold standard in BP diagnosis because of its high accuracy and ability to track disease activity[11, 12, 13, 14, 15, 16]. However, these testing methods have many limitations in clinical practice.

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      Fc-domain site-directed mutagenesis indicated that complement-dependent cytotoxicity was necessary for disease development in this model, whereas antibody-dependent cellular cytotoxicity played a minor role. Studies of pemphigoid skin biopsies have shown that C3d staining has similar sensitivity (74.1%) and specificity (95.8%) to those of direct or indirect immunofluorescence for BP diagnosis, using BP180/230 ELISA as the gold standard (Wang et al., 2020). Complement activation at the BMZ subsequently induces disease pathology in pemphigoid by C3/C5-mediated chemotaxis of mast cells, neutrophils, and/or eosinophils (Bieber et al., 2021; Edwards et al., 2019) and can promote autoantibody production by engagement of B-cell complement receptors by C3d immune complexes (Nikitin et al., 2019).

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    Funding sources: Dr Simpson is supported by a Dermatology Foundation Physician-Scientist Career Development Award. Dr Takeshita is supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases grant K23-AR-068433. This study was supported by a Dermatology Foundation Dermatopathology Career Development Award to Dr Chu.

    Conflicts of interest: Dr Novoa has served as consultant for Enspectra Health and received speaker's honoraria from Novartis Argentina and HealthCert for work unrelated to this manuscript. Dr Takeshita receives a research grant to the Trustees of the University of Pennsylvania from Pfizer Inc for work that is unrelated to this manuscript and received payment for continuing medical education work related to psoriasis that was supported indirectly by Eli Lilly. Dr Payne has served as a consultant for SyntImmune Inc, holds equity in Cabaletta Bio, Inc, and has patents licensed by Novartis and Cabaletta Bio, Inc, focused on cellular therapies for autoimmune diseases. Drs Wang, Moshiri, Simpson, and Chu have no conflicts of interest to declare.

    IRB approval status: Use of human specimens in this study was provided through University of Pennsylvania Institutional Review Board-approved protocol #820338.

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