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E-IJD ORIGINAL ARTICLE
Year : 2019  |  Volume : 64  |  Issue : 4  |  Page : 338
Immunohistochemistry for immunoglobulin G4 in the diagnosis of pemphigus


1 Department of Pathology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
2 Faculty of Medicine, Medical Students' Research Committee, Isfahan University of Medical Sciences, Isfahan, Iran

Date of Web Publication5-Jul-2019

Correspondence Address:
Elnaz Babaei Pour
Department of Pathology, School of Medicine, Isfahan University of Medical Sciences, Isfahan
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijd.IJD_87_18

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   Abstract 


Introduction: Pemphigus comprises of a group of autoimmune bullous disorders with intraepithelial lesions involving the skin and mucous membranes. Pemphigus is characterized histologically by an intraepidermal blister and immunopathologically by the finding of in vivo bound immunoglobulin G (IgG) antibodies against desmosomal adhesion proteins on the surface of keratinocytes. Indirect immunofluorescence for IgG is considered as a gold standard method for diagnosis of this group of bullous disorders on the condition that fresh frozen tissue is accessible. Aim: We designed a new diagnostic method by immunohistochemistry (IHC) for IgG4 on paraffin sections instead of fresh frozen tissue and evaluated sensitivity, specificity, and positive and negative predictive values of this method. Materials and Methods: We searched our pathologic archive of pemphigus of 35 patients, including 29 cases of pemphigus vulgaris (PV) and 6 patients with pemphigus foliaceus (PF). In all cases, the diagnosis was confirmed by indirect immunofluorescence studies. Thirty-five specimens served as controls, including 31 specimens of safe margins of basal cell carcinoma and 4 specimens of normal skin. Sections with condensed and continuous immunoreactivity localized to the intercellular junctions of keratinocytes were considered as positive. Results: Sensitivity of IgG4 was estimated to be 72.4% in PV group and 83.3% in PF group. The overall sensitivity and specificity of IgG4 IHC for diagnosis of pemphigus were 74.2% and 82.8%, respectively, in PV and PF groups. Furthermore, positive predictive value and negative predictive value were 81.2% and 76.3%, respectively. Conclusion: Immunohistochemical labeling for IgG4 on paraffin-embedded tissue provides a sensitive and specific test for diagnosing pemphigus in a situation when fresh frozen tissue is unavailable.


Keywords: Immunoglobulin G, immunohistochemistry, pemphigus


How to cite this article:
Heidarpour M, Rajabi P, Pour EB, Fayyazi E. Immunohistochemistry for immunoglobulin G4 in the diagnosis of pemphigus. Indian J Dermatol 2019;64:338

How to cite this URL:
Heidarpour M, Rajabi P, Pour EB, Fayyazi E. Immunohistochemistry for immunoglobulin G4 in the diagnosis of pemphigus. Indian J Dermatol [serial online] 2019 [cited 2019 Jul 17];64:338. Available from: http://www.e-ijd.org/text.asp?2019/64/4/338/262186





   Introduction Top


Pemphigus comprises of a group of autoimmune bullous disorders with intraepithelial lesions involving the skin and mucous membranes. There are several types of pemphigus which vary in severity and blister location: pemphigus foliaceus (PF), pemphigus vulgaris (PV), and paraneoplastic pemphigus.

In PV which is the most common form of this spectrum, patients show flaccid blisters and erosions involving both the skin and oral mucosa and occur when antibodies attack desmoglein 3.[1] Actually, circulating desmoglein-reactive autoantibodies directed against keratinocyte cell surfaces lead to a prototypical organ-specific human autoimmune disorder with a poor prognosis and a potentially fatal condition in the absence of medical treatment.

PF characterized by superficial bullae and erosion, particularly on the sebaceous areas, the least severe of pemphigus group,[2] and desmoglein 1, the protein that is destroyed by the autoantibody, is found just in the top dry layer of the skin.

Although pemphigus is a relatively rare disease, with a reported incidence ranging from 0.1 to 3.2 cases/100,000 person-years, the incidence of PV has increased over time.[3]

Pemphigus is characterized histologically by an intraepidermal blister and immunopathologically by the finding of in vivo bound and circulating immunoglobulin G (IgG) antibodies directed against desmosomal adhesion proteins on the surface of keratinocytes.

In pemphigus, IgG4 autoantibodies that dominate the autoimmune response are obviously pathogenic. However, IgG1 autoantibodies also probably have blister-inducing potential that needs further investigation.[4] The diagnosis of pemphigus is based on the clinical findings, histopathology data from the edge of a blister and direct immunofluorescence (DIF) of IgG on normal-appearing perilesional skin. DIF plays a crucial role in diagnosing pemphigus, especially when the clinical presentation and histology are not characteristic. Fresh frozen tissue is needed for DIF studies, which technically gives rise to a clinical challenge in the diagnosis of pemphigus. Immunohistochemistry (IHC) for total IgG performed on paraffin sections is of no diagnostic value because of the high background staining.

In the present study, we evaluated the sensitivity and specificity of IHC for IgG4 in the diagnosis of pemphigus. Findings of DIF were considered as the gold standard.


   Materials and Methods Top


Case selection

This study was approved by the Ethics Committee of Isfahan University of Medical Sciences (Approval code: 395418). We searched our pathologic archive of pemphigus of 35 patients, including 29 cases of PV and 6 patients with PF. In all patients, the diagnosis was confirmed by DIF studies. Thirty-five specimens served as controls, including 31 specimens of safe margins of basal cell carcinoma and 4 specimens of normal skin.

Study design

This study was done from August 2014 to August 2016 in Alzahra Hospital, Isfahan, Iran. Written informed consent was taken from all patients. DIF for IgG4 was performed on fresh frozen tissue for the diagnosis of pemphigus. All the patients had acantholytic lesions. IgG4 has been related to the pathogenesis of many diseases, many of which have been known and studied for decades. However, in the recent years, new systemic IgG4-related diseases have been described, and new concepts regarding the implication of IgG4 in many inflammatory and tumoral situations have of appeared. Some diseases involve the skin, either primarily or as apart of their systemic manifestations. Therefore, due to the presence of such diseases, the diagnostic method mentioned in this article is a corroborate diagnostic method and the presence of another diagnostic method such as clinical manifestation of the disease and hematoxylin and eosin slide are necessary. The long-term sustainability of IgG4 kit (stored for 1 year at −20°C), monoclonality and no cross-reaction with other IgG4 subclasses, the presence of positive control tissue available in almost all pathology laboratories (spleen and tonsillary tissue), and no need of fluorescent microscope are some of the issues that suggest easy and inexpensive access to this method in comparison with the DIF method.

Immunohistochemistry for immunoglobulin G4

IHC was performed on formalin-fixed, paraffin-embedded, 4-μm-thick tissue sections. Briefly, the sections were deparaffinized three times for 5 min in xylene and then dehydrated using a graded ethanol series. Endogenous peroxidase activity was halted by the administration of 3% hydrogen peroxide and methanol for 5 min. For antigen retrieval, the sections were treated with phosphate-buffered saline (PBS; pH: 9) at 95°C for 15 min in a microwave oven and allowed to cool for 20 min at room temperature. Subsequently, the sections were incubated at 37°C for about 1 h in a humidified chamber with anti-IgG4 antibody (Abcam, ab109493) as the primary antibody, 1/100 dilution. The sections were incubated for 10 min at 45°C with secondary antibody (DAKO real envision). Subsequently, the sections were rinsed three times in PBS and incubated with horseradish peroxidase (DAB + choromogen DAKO) for 10 min for visible light microscopy.

Study measurements and outcomes

Positivity was defined as distinct, condensed, continuous immunoreactivity localized to the intercellular junctions of keratinocytes, in essentially the same pattern as that seen in immunofluorescence studies. Reactivity that did not meet all these criteria was considered nonspecific. Sensitivity and specificity were calculated with standard tables.

Statistical analysis

All statistical analyses were performed using SPSS (Statistical Package for the Social Sciences, Chicago, IL, USA) version 21. Differences between groups were analyzed using Student's t-test for continuous variables and Chi-square test for proportions. P < 0.05 was considered statistically significant.


   Results Top


Thirty-five pemphigus cases (29 PV and 6 PF) were enrolled in this study. The mean age was 37.4 and 57.6 years in case and control group, respectively. All of the pemphigus cases had acantholytic lesions and DIF was positive for all of the samples. Moreover, 35 specimens were used as controls.

Two pathologists independently assessed IgG4 immunohistochemical stain slides using the same criteria and achieved a 100% interobserver agreement regarding positive versus negative results for all the specimens.

Of the 29 PV cases, 21 samples were immunoreactive for IgG4. Most of the positive specimens were condensed to the intercellular junctions of suprabasal keratinocytes [Figure 1]. Five samples from six PF specimens were immunoreactive for IgG4 [Figure 2]. IHC studies revealed that among 35 control samples, 29 were negative for IgG4. However, six specimens of control group were positive for IgG4 [Figure 3]. There was not a significant difference between IHC results of PV and PF (P = 0.51). However, a significant difference was seen among IHC results of PV (P = 0.001) and PF (P = 0.003) in comparison to control group.
Figure 1: Pemphigus vulgaris shows extensive acantholytic cells in the bullous cavity (a; H and E, ×400). Immunoglobulin G4 immunoreactivity with distinct continuous pattern localized to intercellular junction of the keratinocytes in both sides of the blister is obvious (b; immunohistochemistry, ×400). Immunohistochemistry findings are compatible direct immunofluorescence by immunoglobulin G4 (c; direct immunofluorescence, ×400). Pemphigus vulgaris shows in low power (d; H and E, ×100). Lace-like pattern immunoreactivity for immunoglobulin G4 is very distinct and comparable with direct immunofluorescence (e; immunohistochemistry, ×400 and f; direct immunofluorescence, ×400)

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Figure 2: Pemphigus foliaceus with sub granular layer blister (a; H and E, ×400) and immunoglobulin G4 immunoreactivity in intercellular junction (b; immunohistochemistry, ×400)

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Figure 3: Positive staining in pemphigus foliaceus (a; immunohistochemistry, ×400], nuclear staining in pemphigus foliaceus that was considered negative because of nuclear staining instead of intercellular junction staining (b; immunohistochemistry, ×400), and normal skin without any staining (c; immunohistochemistry, ×400)

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Sensitivity of IgG4 was estimated to be 72.4% and 83.3% in PV and PF groups, respectively. The overall sensitivity and specificity of IgG4-IHC for diagnosis of pemphigus were 74.2% and 82.8%, respectively. Furthermore, positive predictive value and negative predictive value were 81.2% and 76.3%, respectively.


   Discussion Top


Pemphigus is a group of autoimmune vesiculobullous diseases characterized by IgG antibodies directed against desmosomal adhesion proteins, with IgG4 being the most important subclass in active disease. DIF for IgG conducted on fresh frozen tissue plays a crucial role in diagnosing pemphigus, but the diagnosis might be hindered when frozen tissue is not available.[5],[6]

In this study, we used IHC performed on paraffin sections as a diagnostic tool for pemphigus. The existence of the IgG4 subclass, its upregulation by anti-inflammatory agents, and its own anti-inflammatory characteristics may help the immune system to dampen inappropriate inflammatory reactions. IgG4 constitutes only 5% of total IgG and is known to have poor complement and leukocyte activating properties. It is predominant in PF and PV, while several investigations suggest a pathogenic role of IgG4 in pemphigus.

IHC for total IgG is usually of very low intensity and false positivity is seen.[7] Therefore, intraepidermal IgG4 deposits should be detectable via immunohistochemical techniques. In our study, IgG4 antibodies were present in 74.2% of patients with active disease. Autoantibody production in PV and PF is polyclonal, and most circulating autoantibodies against desmogleins are of the IgG4 subgroup. Circulating IgG4 antibodies are present in more than 70% of patients with active disease, and the titer of serum IgG4 closely relates to the activity of the disease.[4] Another study in 2000 on 27 PV cases showed that PV-IgG4 was found in 62% of the patients and was absent in the controls.[8] In our study, IgG4 antibodies were present in 17.14% of control group. IgG4 has formerly been described to be a blocking or protective antibody because it has poor effector functions in vitro, as compared with the other IgG subclasses. It is the pathogenic autoantibody in PF and it may also be important in other autoimmune diseases.[9]

In keeping with the concept that IgG4 autoantibody is pathogenic and associated with active pemphigus, our results showed that IHC for IgG4 had a higher sensitivity in specimens with PF. When pemphigus is clinically suspected, usually perilesional biopsies are taken and tissue is frozen for DIF studies, in addition to obtaining lesional skin for routine histopathology. In contrast, exclusively lesional biopsies are usually done when pemphigus is not considered in the clinical differential diagnosis. When histopathology demonstrates acantholysis suspicious for pemphigus, often no frozen tissue is accessible for diagnosis in such cases. We initiated the present study because of these clinical settings and our results demonstrated that IHC for IgG4 performed on paraffin sections was more useful in such scenario, given the higher sensitivity of this assay in specimens with acantholysis. IHC for IgG4 also demonstrated good specificity (82.8%) for pemphigus. However, nonspecific immunoreactivity occurs.

The main limitation of our study was that we did not compare the findings in other immunobullous disorders, e.g., bullous pemphigoid. Also the number of specimens were small to come to a concrete decision.


   Conclusion Top


Immunohistochemical labeling for IgG4 provides a sensitive and specific test for diagnosing pemphigus; it is likely to be particularly valuable in cases where frozen tissue is not available for DIF, and especially when active acantholytic lesions are examined. Our results warrant further studies with a larger sample size with samples from other immunobullous disorders to validate the clinical importance of this test.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initial would not be published and due efforts would be given to conceal their identity, but anonymity could not be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Zhang X, Hyjek E, Soltani K, Petronic-Rosic V, Shea CR. Immunohistochemistry for immunoglobulin G4 on paraffin sections for the diagnosis of pemphigus. Arch Pathol Lab Med 2012;136:1402-7.  Back to cited text no. 1
    
2.
Kusudo E, Endo Y, Kitayama N, Ishida Y, Fujisawa A, Dainichi T, et al. Pemphigus foliaceus developed in a patient with long-term erosion on the genital skin. J Dermatol 2017;44:e126-7.  Back to cited text no. 2
    
3.
Langan SM, Smeeth L, Hubbard R, Fleming KM, Smith CJ, West J, et al. Bullous pemphigoid and pemphigus vulgaris – Incidence and mortality in the UK: Population based cohort study. BMJ 2008;337:a180.  Back to cited text no. 3
    
4.
Sitaru C, Mihai S, Zillikens D. The relevance of the IgG subclass of autoantibodies for blister induction in autoimmune bullous skin diseases. Arch Dermatol Res 2007;299:1-8.  Back to cited text no. 4
    
5.
Qian Y, Jeong JS, Ye J, Dang B, Abdeladhim M, Aoki V, et al. Overlapping IgG4 responses to self- and environmental antigens in endemic pemphigus foliaceus. J Immunol 2016;196:2041-50.  Back to cited text no. 5
    
6.
Azimi H, Majidi J, Estakhri R, Goldust M. IgG antibodies in patients with pemphigus vulgaris before and after diagnosing with immunofluorescence. Pak J Biol Sci 2013;16:589-92.  Back to cited text no. 6
    
7.
Kwon EJ, Yamagami J, Nishikawa T, Amagai M. Anti-desmoglein IgG autoantibodies in patients with pemphigus in remission. J Eur Acad Dermatol Venereol 2008;22:1070-5.  Back to cited text no. 7
    
8.
Kricheli D, David M, Frusic-Zlotkin M, Goldsmith D, Rabinov M, Sulkes J, et al. The distribution of pemphigus vulgaris-IgG subclasses and their reactivity with desmoglein 3 and 1 in pemphigus patients and their first-degree relatives. Br J Dermatol 2000;143:337-42.  Back to cited text no. 8
    
9.
Rock B, Martins CR, Theofilopoulos AN, Balderas RS, Anhalt GJ, Labib RS, et al. The pathogenic effect of IgG4 autoantibodies in endemic pemphigus foliaceus (fogo selvagem). N Engl J Med 1989;320:1463-9.  Back to cited text no. 9
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

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