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Immunohistochemistry for immunoglobulin G4 on paraffin sections for the diagnosis of pemphigus.

Pemphigus is a group of chronic, autoimmune, potentially fatal vesiculobullous diseases of the mucous membranes and skin. Pemphigus consists of 3 primary subsets (pemphigus vulgaris, pemphigus foliaceus, and paraneoplastic pemphigus) as well as other uncommon variants. (1,2) Pemphigus vulgaris is the most common subtype and accounts for approximately 70% of pemphigus cases. Although pemphigus is a relatively rare disease, with a reported incidence ranging from 0.1 to 3.2 cases per 100 000 person-years, (2) the incidence of pemphigus vulgaris has increased over time. (3) A population-based study demonstrated that the incidence of pemphigus vulgaris had an average yearly increase of 11% (incidence rate ratio, 1.1; 95% confidence interval, 1.0 to 1.2) from 1996 to 2006. (3) More importantly, pemphigus vulgaris is potentially fatal; the risk of death for patients with pemphigus vulgaris is 3 times greater than that for the general population (adjusted hazard ratio, 3.3; 95% confidence interval, 2.2 to 5.2). (3)

Pemphigus is characterized histologically by an intra-epidermal blister and immunopathologically by the finding of in vivo bound and circulating immunoglobulin G (IgG) antibodies directed against desmosomal adhesion proteins (desmoglein 1 and desmoglein 3) on the surface of keratinocytes. The binding of autoantibodies results in acantholysis and blistering, without a requirement for complement or inflammatory cells. Patients with active disease have circulating and tissue-bound autoantibodies of both the IgG1 and IgG4 subclasses, with IgG4 being predominant. (4-7)

The diagnosis of pemphigus is based on clinical history and presentation, histopathology from the edge of a blister, and direct immunofluorescence (DIF) on normal-appearing perilesional skin, as well as indirect immunofluorescence using the patient's serum. Direct immunofluorescence plays a crucial role in diagnosing pemphigus, especially when the clinical presentation and histology are not characteristic. Fresh-frozen tissue is required for DIF studies, which technically gives rise to a clinical challenge in the diagnosis of pemphigus. It is not infrequently encountered that, when paraffin sections of a biopsy specimen are histologically suspicious for pemphigus, frozen tissue is not available for DIF to confirm the diagnosis. Immunohistochemistry for total IgG performed on paraffin sections is of no diagnostic value because of the high background staining.

In the present study, we demonstrated that immunohistochemistry for IgG4 performed on paraffin sections could be a useful diagnostic test for pemphigus, especially when frozen tissue is not available for DIF.


Case Selection

This study was approved by the Institutional Review Board of the University of Chicago Medical Center. We searched our pathologic archive and retrieved 18 specimens of pemphigus from 15 patients, including 12 cases of pemphigus vulgaris and 6 of pemphigus foliaceus. In all cases, the diagnosis was confirmed by direct and/or indirect immunofluorescence studies. Thirty-six specimens served as controls, including 4 of normal skin and 32 of nonpemphigus vesiculobullous diseases. The study design is summarized in Table 1.

Immunohistochemistry for IgG4

Immunohistochemistry was performed on a Leica Bond-Max automated IHC/ISH plaftorm (Leica Microsystems Inc, Buffalo Grove, Illinois) (immunohistochemistry and in situ hybridization) according to the manufacturer's protocol with a slight modification. Four-micrometer paraffin sections were dewaxed in a Bond Dewax solution, rehydrated in alcohol and Bond Wash solution (Leica Microsystems). Antigen retrieval was performed using a high-pH (ER2) retrieval solution for 15 minutes followed by endogenous peroxidase blocking for 5 minutes on the machine. Anti-human IgG4 monoclonal antibody (clone HP6025, Invitrogen, Molecular Probes Inc, Eugene, Oregon) was applied at 1:50 dilution for 25 minutes at room temperature. Detection was performed using the biotin-free Bond Polymer Refine Red Detection system (Leica Microsystems) with a 15-minute postprimary step followed by 25 minutes' incubation with alkaline phosphatase-linked polymers. Detecting reaction was developed with red substrate provided in the Refine detection kit. Sections were then counterstained with hematoxylin on the machine, dehydrated in alcohols, and mounted with mounting medium (Sakura Finetek USA Inc, Torrance, California).

The IgG4 immunostained slides were independently evaluated in a masked manner by 3 pathologists (X.Z., V.P.-R., and C.R.S.), and the result was reported as either positive or negative. 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.


Three pathologists independently evaluated the IgG4 immunohistochemical stains using the same stringent criteria, and achieved a 100% interobserver agreement regarding positive versus negative results for all specimens.

The results for IgG4 immunohistochemical stains as well as previously performed immunofluorescence studies are summarized in Table 2. In pemphigus vulgaris cases, the immunoreactivity for IgG4 was most condensed to the intercellular junctions of suprabasal keratinocytes, consistent with the known location of desmoglein 3 (Figure 1, A). Positive IgG4 immunoreactivity could be observed on both sides of the acantholytic split if the detached portion of epidermis was present in the tissue section (Figure 1, B). Of the 12 pemphigus vulgaris cases, 9 cases were immunohistochemically positive for IgG4, resulting in a sensitivity of 75.0%. In contrast, pemphigus foliaceus cases showed immunoreactivity for IgG4 in the intercellular junctions of superficial keratinocytes, reflecting the known location of desmoglein 1 (Figure 1, C). Four of the 6 pemphigus foliaceus cases exhibited immunore-activity for IgG4 (sensitivity 66.7%). The overall sensitivity of IgG4 immunostain was 72.2%.

Out of 4 normal skin and 32 nonpemphigus vesiculobullous disease controls, 1 bullous pemphigoid case showed IgG4 positivity (Figure 1, D), resulting in a specificity of 97.2%. However, although this case was regarded as positive based on immunohistochemical findings, the morphologic features (subbasal instead of suprabasal blister, as well as inflammation and exudate in the blister, Figure 1, E) were different from those seen in pemphigus, and would lead to the accurate diagnosis. None of the cases with acantholysis mimicking pemphigus, such as Grover disease and staphylococcal scalded skin syndrome, exhibited IgG4 immunoreactivity. Nonspecific staining was present in some control cases; these were associated with debris and exudate, or stained in a homogeneous, mostly cytoplasmic pattern (Figure 1, F through H), instead of the distinct, condensed, continuous immunoreactivity localized to the intercellular junctions of keratinocytes, as seen in pemphigus.


Immunohistochemistry for IgG4 exhibited a higher sensitivity in pemphigus cases with active acantholysis. Of the 12 pemphigus vulgaris specimens, 10 displayed acantholytic lesions, in which 8 specimens showed positive immunostains for IgG4 (sensitivity 80.0%). Four of the 6 pemphigus foliaceus specimens exhibited acantholytic lesions, and all of them were positive for IgG4 immunostain (sensitivity 100.0%). It is worth mentioning that in one patient, both the pemphigus foliaceus lesion (with acantholysis) and perilesional skin were biopsied at the same time. The lesional biopsy showed positive IgG4 immunoreactivity, whereas the perilesional biopsy was negative (Figure 2, A and B). In another patient with pemphigus foliaceus, the biopsy was taken from the edge of the blister, and IgG4 immunostain showed strong positivity in the acantholytic lesion, and was negative in the adjacent intact epidermis (Figure 2, C). The overall sensitivity for specimens with acantholytic lesions was 85.7%.


The classic clinical presentation and histopathology of pemphigus usually lead to a straightforward diagnosis; however, in equivocal cases, the diagnosis might be hindered because of the unavailability of frozen tissue for DIF. In this study, we used immunohistochemistry performed on paraffin sections as a diagnostic tool for pemphigus. The rationale is 2-fold: first, IgG4 constitutes only 5% of total IgG. (8) In contrast to immunohistochemistry for total IgG, the background level of routine IgG4 immunostain on paraffin sections is usually of very low intensity, and false positivity is rarely seen. Secondly, IgG4 is the predominant subclass of IgG in active pemphigus, as demonstrated by ELISA and indirect immunofluorescence. (4-7) Therefore, intraepidermal IgG4 deposits should be detectable via immunohistochemical techniques.

IgG4 is the rarest subclass of IgG, and is associated with limited categories of disease, including IgE-related allergy, IgG4-related systemic sclerosing disease, and pemphigus. (8) The mechanistic basis for the association of IgG4 with IgG4-related systemic sclerosing disease is still enigmatic; it remains unknown whether IgG4 is pathogenic or an epiphenomenon in this setting. (8,9) In contrast, the pathogenic role of IgG4 in pemphigus has been extensively demonstrated. Autoantibody production in pemphigus vulgaris and pemphigus foliaceus is polyclonal, and most circulating autoantibodies against desmogleins are of the IgG4 subclass. Circulating IgG4 antibodies are present in more than 70% of patients with active disease, and the titer of serum IgG4 is closely related to the active disease status. (5,6) IgG1 autoantibodies against desmogleins can be detected in patients with pemphigus in remission, as well as healthy relatives of pemphigus vulgaris patients and healthy carriers of pemphigus vulgaris-prevalent human leukocyte antigen class II alleles; however, the IgG4 autoantibodies are rarely present in these populations, but almost exclusively present in patients with active disease. (5,6) The pathogenic capacity of IgG4 autoantibody was further demonstrated by tissue culture and animal experiments. IgG4 antibodies purified from patients with pemphigus vulgaris or pemphigus foliaceus reproduce the disease clinically and histologically in mice and cultured human skin. (10,11) Furthermore, newborn infants of mothers with active disease temporarily exhibit pemphigus-like lesions, because of the placental transfer of autoantibodies. (12,13)

In keeping with the concept that IgG4 autoantibody is pathogenic and associated with active pemphigus, our results showed that immunohistochemistry for IgG4 had a higher sensitivity in specimens with active acantholysis, or active disease status, and the positive IgG4 immunoreactivity often concentrated to the acantholytic site. Of the 3 pemphigus vulgaris specimens that were negative for IgG4 immunostain, 1 specimen did not have acantholytic lesions. Neither of the 2 pemphigus foliaceus specimens that were immunohistochemically negative for IgG4 showed acantholysis, although the lesional biopsy from 1 of these patients exhibited positive IgG4 immunolabeling. Apparently the positive DIF results seen in perilesional biopsy specimens are due to the presence of IgG1 autoantibodies against desmogleins, which is the predominant subclass of autoantibodies in inactive pemphigus, whereas IgG4 anti-desmoglein autoantibodies are commonly present in acantholytic/preacantholytic sites, and are less likely to be detected by immunohistochemistry in intact perilesional skin. These findings are also clinically significant and relevant. When pemphigus is clinically suspected, usually perilesional biopsies will be taken and tissue will be frozen for DIF studies, in addition to obtaining lesional skin for routine histopathology. Exclusively lesional biopsies, in contrast, are usually performed when pemphigus is not strongly considered in the clinical differential diagnosis. When histopathology shows acantholysis suspicious for pemphigus, often no frozen tissue is available to confirm the diagnosis in such cases. Certainly we initiated the present study because of these clinical settings, and we further entertain the fact that immunohistochemistry for IgG4 performed on paraffin sections are more useful in such scenarios, given the higher sensitivity of this assay in specimens with acantholysis.


Immunohistochemistry for IgG4 also demonstrated excellent specificity (97.2%) for pemphigus. However, nonspecific immunoreactivity does occur, and caution needs to be exercised when interpreting the IgG4 immunostains. As in all immunohistochemical stains, necrotic debris and serum exudate tend to nonspecifically bind colorimetric substrate, but such a nonspecific staining pattern is not difficult to recognize given the close association with debris and exudate instead of intact cells. Nonspecific cytoplasmic staining was also observed in some cases, which may be partially because of the higher antibody concentration we used for immunohistochemistry. None of the observed nonspecific immunoreactivity meet the criteria that defined positive immunoreactivity. Both the condensed, continuous staining pattern and the intercellular junctional location of immunoreactivity are of great importance. By using such stringent criteria, a high interobserver agreement rate can be achieved. In the present study, only one control case (bullous pemphigoid) was considered as positive for IgG4 immunostain. Although immunohistochemical findings in this case largely met our criteria for IgG4 positivity, there were subtle differences. The positivity was located right above the subbasal blister adjacent to nonspecifically stained exudate, and the intercellular stain was strong at the basal and lateral cellular borders, with gradual attenuation towards the apex. Based on the constellation of findings, we speculate that the intercellular staining observed in this bullous pemphigoid case is not real IgG4 immunoreactivity, but resulted from nonspecifically stained exudate permeating into the spongiotic cellular junction, mimicking intercellular junctional immunostain. Furthermore, the subbasal location of the blister helps to distinguish bullous pemphigoid from pemphigus. None of the cases with acantholysis mimicking pemphigus exhibited IgG4 immunoreactivity. In cases such as staphylococcal scalded skin syndrome, the morphology is indistinguishable from pemphigus foliaceus, and IgG4 immunostains provide a valuable tool to confirm a diagnosis.

In conclusion, 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 to validate the clinical value of this test.


(1.) Groves RW. Pemphigus: a brief review. Clin Med. 2009; 9(4):371-375.

(2.) loannides D, Lazaridou E, Rigopoulos D. Pemphigus. J Eur Acad Dermatol Venereol. 2008; 22(12):1478-1496.

(3.) Langan SM, Smeeth L, Hubbard R, Fleming KM, Smith CJ, West J. Bullous pemphigoid and pemphigus vulgaris--incidence and mortality in the UK: population based cohort study. BMJ. 2008; 337:a180.

(4.) Futei Y, Amagai M, Ishii K, Kuroda-Kinoshita K, Ohya K, Nishikawa T. Predominant IgG4 subclass in autoantibodies of pemphigus vulgaris and foliaceus. J Dermatol Sci. 2001; 26(1):55-61.

(5.) Kricheli D, David M, Frusic-Zlotkin M, 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(2): 337-342.

(6.) Ayatollahi M, Joubeh S, Mortazavi H, Jefferis R, Ghaderi A. IgG4 as the predominant autoantibody in sera from patients with active state of pemphigus vulgaris. J Eur Acad Dermatol Venereol. 2004; 18(2):241-242.

(7.) 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):1-8.

(8.) Aalberse RC, Stapel SO, Schuurman J, Rispens T. Immunoglobulin G4: an odd antibody. Clin Exp Allergy. 2009; 39(4):469-477.

(9.) Pickartz T, Mayerle J, Lerch MM. Autoimmune pancreatitis. Nat Clin Pract Gastroenterol Hepatol. 2007; 4(6):314-323.

(10.) Rock B, Martins CR, Theofilopoulos AN, et al. The pathogenic effect of IgG4 autoantibodies in endemic pemphigus foliaceus (fogo selvagem). N Engl J Med. 1989; 320(22):1463-1469.

(11.) Yeh SW, Cavacini LA, Bhol KC, et al. Pathogenic human monoclonal antibody against desmoglein 3. Clin Immunol. 2006; 120(1):68-75.

(12.) Wasserstrum N, Laros RK Jr. Transplacental transmission of pemphigus. JAMA. 1983; 249(11):1480-1482.

(13.) Hup JM, Bruinsma RA, Boersma ER, de Jong MC. Neonatal pemphigus vulgaris: transplacental transmission of antibodies. Pediatr Dermatol. 1986; 3(6): 468-472.

Xuefeng Zhang, MD, PhD; Elizabeth Hyjek, MD, PhD; Keyoumars Soltani, MD; Vesna Petronic-Rosic, MD, MSc; Christopher R. Shea, MD

Accepted for publication January 16, 2012.

From the Departments of Pathology (Drs Zhang and Hyjek) and Medicine (Dermatology) (Drs Soltani, Petronic-Rosic, and Shea), University of Chicago Medical Center, Chicago, Illinois.

The authors have no relevant financial interest in the products or companies described in this article.

Presented at the United States & Canada Academy of Pathology Annual Meeting; February 28, 2011; San Antonio, Texas.

Reprints: Christopher R. Shea, MD, Department of Medicine (Dermatology), University of Chicago Medical Center, 5841 S Maryland Ave, MC5067, Chicago, IL 60637 (e-mail: cshea@medicine.bsd.
Table 1. Summary of Cases Enrolled in the Study

Pemphigus (IF+) No. Control No.

Vulgaris (a) 12 Normal skin 4
Foliaceus (b) 6 Grover disease 8
 Hailey-Hailey 4
 Bullous pemphigoid 6
 Staphylococcus scalded skin 3
 Dermatitis herpetiformis 3
 Herpes simplex virus 4
 Erythema multiforme 1
 Bullous impetigo 1
 Epidermolysis bullosa 1
 Porphyria cutanea tarda 1
Total 18 36

Abbreviation: IF, immunofluorescence.

(a) From 10 patients (2 patients each with 2 specimens).

(b) From 5 patients (1 patient with 2 specimens).

Table 2. Summary of Results
of IgG4 Immunohistochemistry

Specimen Acantholytic
No. DIF (a) IIF (a) Lesion (b) IgG4

Pemphigus Vulgaris
1 N/A >1:160 2 2
2 + 1:160 2 +
3 + >1:160 + 2
4 + 1:40 + 2
5 + Suggestive + +
6 + Trace + +
7 + 1:80 + +
8 N/A >1:160 + +
9 (c) N/A 1:160 + +
10 (c) N/A 1:160 + +
11 (d) + 1:160 + +
12 (d) + 1:160 + +
Pemphigus Foliaceus
13 N/A >1:160 2 2
14 (e) + 1:160 2 2
15 (e) + 1:160 + +
16 + >1:160 + +
17 + >1:160 + +
18 + >1:160 + +

Abbreviations: DIF, direct immunofluorescence; Ig, immunoglobulin;
IIF, indirect immunofluorescence; N/A, not available.

(a) +/2 is defined as positive/negative.

(b) +/2 is defined as present/absent in the biopsied specimen.

(c,d,e) Specimen number followed by the same superscript letter
represents the same patient.
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Author:Zhang, Xuefeng; Hyjek, Elizabeth; Soltani, Keyoumars; Petronic-Rosic, Vesna; Shea, Christopher R.
Publication:Archives of Pathology & Laboratory Medicine
Date:Nov 1, 2012
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