Bullous pemphigoid: analysis of C4d by immunohistochemistry in suspected cases

Abstract

Background

Bullous pemphigoid (BP) is the most common type of subepidermal bullous disease. The detection of linear deposition of immunoglobulin G (IgG) and/or C3 at the dermo-epidermal junction using direct immunofluorescence (DIF) is the gold standard for diagnosis; however, it requires a fresh tissue specimen. Since the immunohistochemistry (IHC) of complement component C4d is easy and more accessible, this technique can also serve as an efficient method. Our objective is to compare the IHC results for C4d with the DIF results in cases suspected of BP.

Methods

Seventeen cases of bullous skin lesions and the dermatitis mimicking bullous diseases were evaluated through DIF and IHC. Clinical and demographic data were obtained from the laboratory database.

Results

Out of 17 cases, seven were diagnosed with BP, while the other seven cases were diagnosed with other bullous diseases or dermatitis mimicking bullous diseases, including pharmacodermia (n = 4), contact dermatitis (n = 1), dermatitis herpetiformis (n = 3) and non-specific bullous disease (n = 2). In the DIF analysis, IgG and C3 were positive in 57.1% of the BP cases, while all other diseases were negative. C4d immunoexpression was positive in 85.7% of the BP, whereas it was negative in almost all other cases.

Conclusion

The C4d IHC study is a valuable diagnostic tool for BP and is especially helpful in cases where clinical suspicion and histology strongly suggest BP, but the frozen samples are negative for DIF.

Introduction

Bullous pemphigoid (BP) is the most common autoimmune subepidermal bullous disease involving the skin and mucosa, occurring predominantly in elderly patients (Oh, Kim, Lee, 2022; Oryani et al., 2017). It usually presents as multiple tense blisters with mixed superficial inflammation that begins as pruritic urticarial plaques with progression to vesicles, blisters, and crusted erosions (Oh, Kim, Lee, 2022; Oryani et al., 2017). The diagnosis of BP is made by a skin biopsy taken from the edge of a recent blister or the perilesional area (Oh, Kim, Lee, 2022; Mohammed et al., 2021; Villani et a., 2016). The classic histological features of well-developed BP include unilocular non-acantholytic subepidermal blisters with conspicuous eosinophilic infiltration in the blister cavity and dermis (Oh, Kim, Lee, 2022; Ates, 2018). Although these histological features are important in identifying the disease, it is impossible to obtain an accurate diagnosis based on histological features alone (Oh, Kim, Lee, 2022; Villani et a., 2016; Mohammed et al., 2021).

The primary event in the pathogenesis of BP is an autoantibody response to hemidesmosomal antigens specific to basal keratinocytes (BP180 and BP230). The binding of autoantibodies to the target antigens leads to the activation of the complement cascade (classical and alternative pathways and their factors), the recruitment of inflammatory cells, and the release of proteolytic enzymes that weaken the dermo-epidermal junction (DEJ). It is also recognized that autoantibodies have a direct interaction with the function of adhesion molecules in the DEJ for the formation of blisters (Oh, Kim, Lee, 2022; Mohammed et al., 2021; Kamyab et al. 2019).

The gold standard test for diagnosing BP is direct immunofluorescence (DIF) of the perilesional skin, which shows linear deposits of IgG and/or C3 along the DEJ. This analysis is considered the most sensitive diagnostic test for BP (sensitivity of 82–90.5% and specificity of 98%). Although direct immunofluorescence (DIF) remains essential in the diagnosis of BP, suitable skin samples, frozen or fixed in Michel’s medium, are not always available to the pathologist, once the professional usually receives samples that have already been fixed in formalin. This is either because the referring doctor did not suspect of BP (clinically atypical presentations), or because of the difficulty of handling and sending frozen skin samples or samples in suitable media to the laboratory. In addition, there is a need for qualified laboratory equipment and pathologists, which are not always available in all health services. For this reason, DIF is expensive and, consequently, not accessible to all patients (Oh, Kim, Lee, 2022; Mohammed et al., 2021; Kamyab et al. 2019; Villani et al. 2016).

Stable components of the complement cascade, such as C4d, can be detected using immunohistochemical analysis (IHC), and their diagnostic value for BP has been widely studied. C4d is a stable component of classical complement activation and represents a degradation product of complement factor C4b. C4d binds covalently to the tissue via a thioester bond, which is resistant to elimination, making it possible to evaluate it using the IHC technique, which offers several advantages over DIF. Among them, it does not require an additional biopsy, it does not need frozen samples, and it is a widely used technique; therefore, it is easy to apply in clinical practice, and because it is carried out on paraffin-fixed tissue samples, the histological examination and interpretation of the immunopathological results can be carried out simultaneously (Oh, Kim, Lee, 2022; Mohammed et al., 2021; Kamyab et al. 2019; Magro, Dyrsen, 2008).

Previous studies have already shown that C4d immunolabeling by IHC is useful in the diagnosis of BP. Although these studies have shown different sensitivities, IHC seems to be a possible substitute for DIF (Oh, Kim, Lee, 2022; Kamyab et al. 2019). We hypothesize that C4d detected by IHC could also be a promising diagnostic marker for BP, particularly in cases that are IgG and/or C3 negative by the DIF method. In this study, we aim to evaluate the correlation between the IHC study for C4d and the DIF results in cases with suspected BP and to compare our results with the literature.

Methods

This is a retrospective study based on 44 patient cases, accumulated between January 2018 and May 2023, in which the clinical suspicion and/or histological evaluation had BP disease as the differential diagnosis. The study was carried out in a pathology reference laboratory accredited by the College of American Pathologists and the National Accreditation Organization. Only cases in which there were concomitant paraffin-embedded biopsy samples and DIF reports were selected (n = 17). Positive and negative DIF cases without paraffin-fixed skin samples were excluded. Clinical and demographic data, including gender and age, were obtained from patient records in the laboratory database.

The hematoxylin and eosin (HE) stained slides were reviewed and their respective paraffin-embedded tissue blocks were cut into 4 μm thick sections. The sections were incubated at 70 °C for 1 h. Antigen retrieval was carried out using “EnVisionTM FLEX Target Retrieval Solution, High pH” (Dako Omnis), in PT Link equipment (Dako Omnis), heated to 96 °C for 20 min. The container was cooled to 65 °C and then the sections were washed with “EnVisionTM FLEX Wash Buffer” (Dako) for 5 min. Murine anti-human C4d antibody (1:300, clone ZM78, Zeta Corporation) was used and incubated for 20 min at 20 °C. The assays were carried out on the Autostainer Link 48 automated platform (Dako), using “EnVisionTM FLEX HRP DAB + Chromogen” (Dako). Tonsil tissue was used as a positive control. A positive result was considered when linear marking was identified in the DEJ, cytoplasmic, and membranous in the basal cells of the epidermis.

The results were presented using descriptive statistics to compare the DIF and IHC. The study protocol was approved by the Research Ethics Committee (CAAE: 76,301,423.0.0000.5363).

Results

Seventeen cases of bullous skin lesions with biopsy and DIF reports were selected. The average age of the patients was 58 ± 16.1 years, with 8 (47%) men and 9 (53%) women. The lesions were located on the lower extremities (n = 6; 35.3%), trunk (n = 6; 35.3%), upper extremities (n = 4; 23.5%), and limbs, without further specification (n = 1; 5.9%). The selected cases first underwent histopathological analysis via HE and DIF staining. Of these, 7 cases were diagnosed as BP and 10 cases as other bullous diseases or dermatitis mimicking bullous diseases, including pharmacodermia (n = 4), contact dermatitis (n = 1), dermatitis herpetiformis (n = 3) and non-specific bullous disease (n = 2).

In the DIF analysis, 4 cases were diagnosed as BP according to clinical and anatomopathological exam associated with positive DIF for IgG and C3; 1 case was diagnosed as BP according to clinical examination and the anatomopathological result associated with positive DIF only for C3; and 2 cases were diagnosed as BP according to clinical examination and very suggestive anatomopathological result, but with negative DIF for IgG and C3. DIF analysis revealed linear deposition of IgG along the DEJ in 4 cases and linear deposition of C3 along the DEJ in 5 cases (Fig. 1). All the cases that were diagnosed as other bullous diseases or dermatitis mimicking bullous diseases had negative DIF for IgG and C3. These results are shown in Table 1.

Fig. 1

Subepidermal blistering with inflammatory cells (HE, A × 400) and linear C3 deposition (direct immunofluorescence, B × 200)

Table 1 Results of direct immunofluorescence and immunohistochemistry staining

A paraffin-fixed sample from each patient with bullous lesions or dermatitis mimicking bullous diseases was submitted for immunohistochemical examination for C4d. Of these, 7 cases were positive, with 6 diagnosed as BP based on clinical history and histopathological results and 1 case diagnosed as unspecified subepidermal bullous disease. IHC revealed linear deposition of C4d along the DEJ, cytoplasmic, and membranous in the basal cells of the epidermis (Fig. 2). Of the other 10 cases that were negative in the IHC, 9 were diagnosed as other bullous diseases or dermatitis mimicking bullous diseases and 1 was diagnosed as BP. These results are shown in Table 1.

Fig. 2

Linear deposition of C4d at the dermo-epidermal junction and in basal keratinocytes (A, B) in bullous pemphigoid samples, with negative staining observed in control samples (C, D) (X200)."

Of the 7 cases diagnosed as BP via IHC for C4d, 4 were positive on DIF for IgG and/or C3, 2 were positive only on IHC and 1 was positive only on DIF for IgG and C3. Of the remaining 10 cases diagnosed as other bullous diseases or dermatitis mimicking bullous diseases, all samples were negative on DIF for IgG and C3 and on IHC for C4d, with the exception of a single case, which was positive only on IHC.

This case that tested positive for C4d on the IHC and negative for IgG and C3 on the DIF was diagnosed as unspecified subepidermal bullous disease. However, his biopsy was sent to the laboratory in saline solution and not in Michel’s medium, which may have led to a false-negative DIF result.

Discussion

Studies published in recent years have raised the hypothesis that the analysis of C4d by IHC in cases of BP can elucidate this diagnosis and thus allow immunopathological confirmation of the bullous process without the inherent disadvantages of DIF (Tables 2 and 3).

Table 2 General information of the included literature

Table 3 Main findings of included literature

Kassaby et al. (2017) reported the case of an 80-year-old woman with a clinical history and classic histological findings of BP, in whom conventional DIF for IgG/C3 was negative and DIF for C4d was positive. This result was important for laboratory confirmation of the diagnosis, clearly demonstrating that DIF applied to a perilesional frozen section for C4d can be positive and useful in the diagnosis of BP, even when conventional DIF for IgG and C3 is negative.

Subsequently, Harvey and Wood also reported a case of BP in which the DIF for IgG and C3 had been negative, while positive for C4d in the IHC. Taken together, these cases demonstrate that the investigation of C4d in formalin-fixed tissue can be useful as an adjuvant test for the diagnosis of BP, particularly in cases with a negative DIF result (Harvey, Wood, 2019).

The study by Chandler, Zone, and Florell showed that C4d IHC is a sensitive method for confirming the deposition of immunoreagents in paraffin-embedded tissue. It was observed that, in 70% of BP cases, the immunopathological process could be confirmed with the C4d IHC study. In agreement, Mohammed Soliman et al. concluded that C4d IHC proved to be a reliable method for detecting autoimmune bullous diseases. Specifically, in the case of BP, five of the six cases studied were positive for C4d via IHC.

In the study by Kamyab et al. (2019), the analysis of C4d by IHC showed a sensitivity of 90% and a specificity of 86.7% in the diagnosis of BP, being useful, especially in cases where frozen samples are not available or paraffin-fixed tissue samples are the only material available for analysis.

On the other hand, the study by Magro e Dyrsen presented contradictory results regarding the diagnostic value of C4d by IHC. They studied 17 cases of BP with positive C3d and C4d via IHC and found invariable agreement between C3d via IHC and classic DIF, while positivity for C4d via IHC was found in only 4 DIF-positive cases. As a result, this study revealed that the investigation of C4d has a lower sensitivity compared to C3d in cases of BP. In agreement, the study by Thakur et al. showed that, overall, C3d is significantly more useful than C4d in the diagnosis of BP via IHC (Thakur et al., 2023).

C3d and C4d are stable components of Complement activation. One difference between them is that C4d represents the stable component of classical Complement activation, while C3d can be formed from classical or alternative activation. According to Nelson et al., Complement activation, via the classical and alternative pathways, has a well-established role in BP. It is believed that the reason why the frequencies of C3d and C4d positivity differ in BP patients is due to the fact that C3d and C4d represent different pathways in Complement activation (Nelson et al. 2006).

An immunological explanation for a negative C4d laboratory result is that the specific IgG autoantibody that is deposited on the basement membrane in negative cases is not an efficient activator of the classical Complement pathway. Therefore, little C4d is generated. Several studies have shown that IgG4, which does not activate the classical Complement pathway, is the IgG subclass most consistently deposited on the basement membrane in BP. One or more complement-fixing IgG subclasses (IgG1, IgG2, and IgG3) are deposited on the basement membrane in most cases of BP, but not all. Perilesional skin biopsies, with significant IgG4 deposition, may have linear C3 deposition in the basement membrane without IgG identifiable by standard DIF. The absence of IgG by DIF, despite the consistent presence of IgG4 by more sensitive techniques, is explained by the lack of sensitivity of commercial anti-human IgG preparations for IgG4. The presence of C3 may be caused by IgG1-3 deposits below the detection threshold of standard DIF or may be further evidence of alternative pathway activity in BP (Oh, Kim, Lee, 2022; Chandler et al., 2022).

In addition, several other reasons could explain a negative DIF result in BP patients, including loss of immunoglobulins and complement in biopsies of damaged skin, inadequate transportation, delays in freezing the samples, and immunological mechanisms of the BP itself that are independent of Complement (Damman et al. 2021).

Our study has some limitations that should be considered, such as the unavailability of data on serum antigens from BP patients, as well as possible technical difficulties in accurate interpretation. As this was a single-center study, the number of cases included was limited and, in addition, the samples used for DIF and IHC analysis differed, despite corresponding to the same cases.

Conclusion

Detecting the linear deposition of autoantibodies and/or Complement fragments along the DEJ using DIF is considered the gold standard for diagnosing BP. However, the need for frozen samples in DIF is an operational disadvantage. In routine examinations, paraffin-embedded tissue slides stained with HE are examined to identify histological features, requiring additional skin biopsy to obtain a frozen sample for DIF testing in suspected cases of BP. In addition, although rare, false-negative DIF results are possible, which can lead to an inaccurate diagnosis. In this study, we hypothesized that IHC for the investigation of C4d could be used in the diagnosis of BP, thus allowing immunopathological confirmation of the bullous process without the disadvantages of DIF.