European Journal of Cardiovascular Medicine

AIBD (Autoimmune blistering disorders) present a disease group having characteristic features of forming auto-antibodies against adhesion proteins of dermis/sub-epithelium or epidermis/epithelium. The disease clinically present itself as erosions and/or blisters on epithelial surfaces as skin and mucosa adjoining the skin. Two major groups of AIBDs are pemphigus and pemphigoid with various variants. It can be challenging to differentiate AIND from other disorders clinically owing to significant clinical overlapping and clinical variations.¹ 

First investigation done generally in resource-limited settings include histopathological assessment which can help in diagnosis. For establishment of immunological disease mechanism, DIF or direct immunofluorescence is usually a first immunological assessment. However, it is not readily available in India. Most common AIBD seen in India is pemphigus and is diagnosed with certainty from intercellular immune deposits in the epidermis assessed from DIF, histopathology, Tzanck smear, and clinical features. DIF assess immune deposits and does not reveal target antigens against the formed autoantibodies. A hinderance is seen for diagnosis, especially in sub-epidermal AIBDs. Semi-quantitative multiparametric or multivariant ELISAs target multiple and limited antigens and Quantitative ELISA for specific antigens have various commercially available kits that helps to identify antigens against which antibodies are formed.^2,3   

IIF (indirect immunofluorescence) has a configuration similar to multivariant ELISA which helps in identification of antigenic target of pathogenic antibodies. However, IIF is costly and need IF (immunofluorescence) microscope along with dark room need and trained laboratory personnel. Quantitative ELISA need acquisition of various samples before running the test which might delay in reporting, especially in low limited resource settings. On the other hand, multi-parametric/multi-variant ELISAs can be done with ELISA plate reader known as traditional spectrophotometer which is available at all the facilities in single sample with lesser time and cost.⁴ The present study was aimed to assess the concordance rate between ELISA and autoimmune bullous disorders assessed using DIF at an Indian healthcare Center.

The present retrospective study was aimed to assess the concordance rate between ELISA and autoimmune bullous disorders assessed using DIF at an Indian healthcare Center. The study subjects were from Department of Dermtology of the Institute. Verbal and written informed consent were taken from guardians/parents of all the subjects before study participation.

The study retrospectively assessed retrieved reports for multivariant ELISA done during the defined study period. DIF and clinical data were retrieved from histopathology reports of the Institute. Among total 676 samples assessed during study period. The study excluded subjects with unavailable DIF or inadequate clinical data or mucosa/skin biopsy samples, or with improper sample processing. After exclusion, 170 samples were finally included and analysis was done from data of 506 subjects.

For all the subjects, final diagnosis was made using combined DIF findings, histopathological data, and clinical presentation which was considered gold-standard as done in resource limited setting. To diagnose pemphigus foliaceous, histopathological finding of sub-corneal split or bullae, clinical picture, and positive DIF showing intercellular deposit of IgG in the epidermis was taken. For pemphigus vulgaris, histopathological evidence of intraepidermal split/bullae, clinical picture, and DIF showing intercellular deposition of IgG, was taken. For bullous pemphigoid, histopathological evidence of inflammatory infiltrate in the upper dermis with/without subepidermal bullae/split, clinical picture, and positive DIF showing linear IgG deposits along dermo-epidermal junction (DEJ) with/without IgM and C3 were considered.⁵

Subjects that had predominant mucosa involvement and evident mucosa scarring in mucosa of either conjunctiva, esophagus, larynx, trachea, genitals, nasal cavity, and oral cavity with DIF showing linear IgG deposits (DEJ) with/without IgM and C3 were diagnosed as mucous membrane pemphigoid.⁶ EBA (Epidermolysis bullosa acquisata) was considered with presence of scarring and/ or atrophy with/without milia formation, clinical presentation, and tense bullae mostly over the trauma-prone areas. The diagnosis was confirmed using DIF depicting linear IgG deposition with or without deposition of u-serration pattern and C3/IgA/IgM.

The subjects that had suspicion of AIBD and were DIF negative were considered non-AIBD. Six recombinant antigens used with multivariant ELISA were envoplakin, collagen VII, bullous pemphigoid antigen 1 and 2, and desmoglein 1 and 3 which can help to diagnose IgG mediated AIBDs as epidermolysis bullosa acquisita (EBA), mucous membrane pemphigoid (MMP), bullous pemphigoid (BP), paraneoplastic pemphigus (PNP), pemphigus foliaceous (PF), and pemphigus vulgaris (PV). ELISA was performed in the serum of the subjects simultaneously with DIF on first day of presentation, not considering the previous treatment.

Autoantibodies bound with antigens were assessed by incubation of wells using secondary enzyme-labelled anti-human IgG which subsequently catalyzed the color reaction. Optical density was assessed at 450nm with 620nm as reference wavelength using ELISA reader. A positive EISA was considered for value of >1.7. with multiple positivity, highest value was taken for diagnosis. For envoplakin, test was considered positive only when it was associated by desmoglein reactivity. Reactivity in all wells was considered negative.

The data gathered was subjected to statistical evaluation using using the chi-square test, Fisher’s exact test, Mann Whitney U test, and SPSS (Statistical Package for the Social Sciences) software version 24.0 (IBM Corp., Armonk. NY, USA) using ANOVA, chi-square test, and student's t-test. The significance level was considered at a p-value of <0.05

The present retrospective study was aimed to assess the concordance rate between ELISA and autoimmune bullous disorders assessed using DIF at an Indian healthcare Center. The study assessed data of 676 subjects after screening where 19.5% (n=132) subjects were excluded owing to incomplete clinical and/or DIF data and 10% (n=34) subjects had improper or inadequate tissue. There were 506 subjects in the study including 58.1% (n=118) subjects with AIBD and 76.6% (n=388) subjects had AIBD. Non-AIBD subjects having IgA mediated dermatosis as linear IgA disease, dermatitis herpetiformis, and other diseases as erosive lichen planus, lupus erythematosus, vasculitis, and diabetic bullae.

It was seen that among 388 subjects with AIBD, 62.88% (n=244) subjects had pemphigus aged mean 43.63±14.78 years. There were 37.11% (n=144) subjects in pemphigoid group in the mean age of 60.14±15.71 years. The study results had higher number of subjects with pemphigoid as multivariant ELISA is primarily used for diagnosis of pemphigoid group of disorders. for 388 subjects with AIBD, positive multivariant ELISA was seen for 82.32% (n=326) subjects. For 110 subjects without AIBD, ELISA was positive in 29.09% (n=32) subjects. In pemphigus group, a positivity of 89.2% was seen for pemphigus vulgaris, 97.9% for pemphigus foliaceous, 90.7% for pemphigus vulgaris, and 63.2% for pemphigus foliaceous using multivariant ELISA.

The study results showed that there was statistically good agreement seen in results of multivariant ELISA and final diagnosis of subjects made following DIF results, histopathology, and clinical results with p<0.001. In pemphigus vulgaris group, highest agreement was seen in all group of subjects with p<0.001. The groups with non AIBDs, bullous pemphigoid, and pemphigus foliaceous had significant moderate level of agreement with p<0.001. A fair agreement was seen for mucous membrane pemphigoid group (p<0.001). Number of subjects having epidermolysis bullosa acquisata and paraneoplastic pemphigus was very low and no agreement was seen between multivariant ELISA and diagnosis by composite criteria.

It was also seen that NPV (negative predictive value) and PPV (positive predictive value) for pemphigus vulgaris was 92.4% and 86.5% respectively. A lower PPV was seen for pemphigus foliaceous group compared to pemphigus vulgaris group with 66.5% and had higher NPV of 95%. The specificity and sensitivity for multivariant ELISA for bullous pemphigoid were 95.7% and 64.1% respectively. The NPV and PPV was 90.2% and 81.6% respectively. In mucous membrane pemphigoid group, higher specificity and lower sensitivity was seen as 99% and 21.2% respectively. The PPV for mucous membrane pemphigoid was lowest in all the groups with 60% PPV and 95.4% NPV (Table 1).

Table 1: Agreement values, NPV, PPV, specificity, and sensitivity in multivariant ELISA to diagnose AIBDs

The present study assessed data of 676 subjects after screening where 19.5% (n=132) subjects were excluded owing to incomplete clinical and/or DIF data and 10% (n=34) subjects had improper or inadequate tissue. There were 506 subjects in the study including 58.1% (n=118) subjects with AIBD and 76.6% (n=388) subjects had AIBD. Non-AIBD subjects having IgA mediated dermatosis as linear IgA disease, dermatitis herpetiformis, and other diseases as erosive lichen planus, lupus erythematosus, vasculitis, and diabetic bullae. These data were comparable to the previous studies of Schmidt E et al⁷ in 2021 and Gornowicz-Porowska J et al⁸ in 2018 where authors assessed subjects with demographic and disease data comparable to the present study in their respective studies.

The study results showed that among 388 subjects with AIBD, 62.88% (n=244) subjects had pemphigus aged mean 43.63±14.78 years. There were 37.11% (n=144) subjects in pemphigoid group in the mean age of 60.14±15.71 years. The study results had higher number of subjects with pemphigoid as multivariant ELISA is primarily used for diagnosis of pemphigoid group of disorders. for 388 subjects with AIBD, positive multivariant ELISA was seen for 82.32% (n=326) subjects. For 110 subjects without AIBD, ELISA was positive in 29.09% (n=32) subjects. In pemphigus group, a positivity of 89.2% was seen for pemphigus vulgaris, 97.9% for pemphigus foliaceous, 90.7% for pemphigus vulgaris, and 63.2% for pemphigus foliaceous using multivariant ELISA. These results were consistent with the findings of van Beek N et al⁹ in 2017 and Kumar V et al¹⁰ in 2025 where results reported by the authors in their studies were comparable to the results of the present study.

It was seen that there was statistically good agreement seen in results of multivariant ELISA and final diagnosis of subjects made following DIF results, histopathology, and clinical results with p<0.001. In pemphigus vulgaris group, highest agreement was seen in all group of subjects with p<0.001. The groups with non AIBDs, bullous pemphigoid, and pemphigus foliaceous had significant moderate level of agreement with p<0.001. A fair agreement was seen for mucous membrane pemphigoid group (p<0.001). Number of subjects having epidermolysis bullosa acquisata and paraneoplastic pemphigus was very low and no agreement was seen between multivariant ELISA and diagnosis by composite criteria. These findings were in agreement with the results of Ravi D et al¹¹ in 2017 and Kasperkiewicz M et al¹² in 2017 where agreement results for ELISA and DIF in AIBDs comparable to the present study were also reported by the authors in their studies.

The study results also showed that NPV (negative predictive value) and PPV (positive predictive value) for pemphigus vulgaris was 92.4% and 86.5% respectively. A lower PPV was seen for pemphigus foliaceous group compared to pemphigus vulgaris group with 66.5% and had higher NPV of 95%. The specificity and sensitivity for multivariant ELISA for bullous pemphigoid were 95.7% and 64.1% respectively. The NPV and PPV was 90.2% and 81.6% respectively. In mucous membrane pemphigoid group, higher specificity and lower sensitivity was seen as 99% and 21.2% respectively. The PPV for mucous membrane pemphigoid was lowest in all the groups with 60% PPV and 95.4% NPV. These results correlated with the findings of Mahajan et al¹³ 2005 and Martin LK et al¹⁴ in 2011 where NPV, PPV, specificity, and sensitivity of ELISA and DIF reported by the authors for AIBDs was comparable to the results of the present study.

The present study, considering its limitations concludes that there is a good agreement between gold standard diagnosis using histopathology, DIF, and clinical findings to the multivariant ELISA, hence, multivariant ELISA can be used to diagnose Autoimmune blistering disorders at places with limited resources where DIF is not available. Also, multivariant ELISA can help in improving etiological diagnosis for various AIBDs where target antigens are seen in multivariant panel.

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