Pattern Of Direct Immunofluorescence (DIF) in the

diagnosis of PemphigusAzza M Abdel-Megaid1, Hisham D Gaber1, Amal Taha Abo-elghiet2,

Abeer Refaiy3 Aya Y Badran1

1. Departement of Dermatology, Veneareology and Andrology.2. Departement of Histology.3. Departement of Pathology.

Abstract:

Background: Immunofluorescence (IF) is a reliable biochemical staining

technique for the detection of antibodies, which are bound to antigen in the

tissue or circulating in body fluids. The relative simplicity and accuracy of the

technique has made immunofluorescence an unavoidably powerful technique in

the diagnosis of bullous diseases.

Objectives: To detect the pattern of direct immunofluorescence (DIF) in the

diagnosis of pemphigus.

Methods: Thirty-six patients with pemphigus disease were enrolled in our study.

The age of whom ranged from 21to 73 years with a mean age ± SD of

46.94±14.6 years Two skin biopsies (one from the bullous lesion and the other

one from adjacent area) were taken from each patient, lesional biopsy specimens

were used for histopathological examination after standard processing, while

perilesional skin samples were used for performing direct immunofluorescence.

Results: DIF was positive in all patients with pemphigus vulgaris (PV), showing

intraepidermal deposition of immunoglobulins and complement 3 (C3). IgG was

seen in all of them (100%), while C3 was also observed in 38.4% patients, and

IgM was positive in (3.8%) in a similar pattern.

Conclusion: Immunofluorescence (IF) studies are an important part of the

laboratory evaluation of immunologically mediated bullous dermatoses and have

become standard procedures for accurately diagnosing patients with these

disorders.

Key words: Direct Immunofluorescence, Autoimmune bullous disorders,

pemphigus.

Introduction:

Autoimmune bullous skin diseases are organ-specific autoimmune

diseases associated with pathogenic autoantibodies against structural proteins

that maintain cell-cell and cell-matrix adhesions in the skin and mucous

membranes (Sitaru and Zilliken, 2005). Antibodies may target the basement

membrane zone or interepidermal cell adhesion. Deposition of antibody can be

shown at the level of blister formation, at sites adjacent to blisters and in

uninvolved skin. The immunoreactant involved varies with the disease, but is

typically IgG or, less commonly, IgA or C3. The sites of antibody binding

(‘target antigen’) have been identified and characterized in most diseases

(Taghipour and Wojnarowsk, 2009).

The classification of autoimmune bullous dermatoses relies on histologic

criteria, primarily according to the localization of bullae; so, intraepidermal and

subepidermal bullous dermatoses are distinguished (Rados, 2011).

There is a great need for rapidly establishing the diagnosis of these

disorders since they may run a severe and potentially life-threatening course

(Kneisel A and Hertl M, 2011).

While histopathological examinations provide evidence for the level of

blister formation, immunofluorescence microscopy has been established to

identify tissue-bound and circulating autoantibodies. Direct

immunofluorescence microscopy represents the gold standard for detecting

tissue-bound autoantibodies. Indirect immunofluorescence microscopy with

defined tissue substrates is considered the first step in detecting circulating

autoantibodies. Confirmatory tests such as enzyme-linked-immunosorbent assay

(ELISA), immunoblot or immunoprecipitation analyses are performed utilizing

recombinant proteins or keratinocyte extract (Kneisel A and Hertl M, 2011).

Two types of Immunofluorescence are present, direct

immunofluorescence (DIF) and indirect immunofluorescence (IIF). DIF helps

detect molecules such as immunoglobulins and complement components within

biopsy specimens. The ideal site for the biopsy specimen depends on the type of

disorder. For bullous diseases, DIF is performed using perilesional skin, that is,

normal-appearing skin immediately adjacent to a lesion (vesicle, bulla, urticarial

plaque, or erythematous patch), as the immune deposits are partially or

completely degraded in inflamed or blistered skin, and DIF may be falsely

negative(Mutasim and Adams, 2001).

Aim of the study: To detect the pattern of direct immunofluorescence in the

diagnosis of pemphigus.

Subjects and Methods: The study was conducted at the department of Dermatology, Andrology

and Venereology, Assiut University Hospital during the period of 2013-2015 on

36 patients with pemphigus, after exclusion of patients on corticosteroids and/or

any immunosuppressive agent at, or one month before the time of the study.

Age, sex, type of lesions, site, mucous membrane affection, duration,

associated symptoms, previous treatment, other system affection and a

written informed consent were signed by every patient.

Two skin biopsies (one from the bullous lesion and the other one from

adjacent area) were taken from each patient after local anesthesia using a

6mm punch.

Lesional biopsy specimens were collected in 10% formalin solution and

used for histopathological examination after standard processing. The

sections were stained with hematoxylin and eosin and examined under the

light microscope. Perilesional skin samples were used for performing

direct immunofluorescence using a 6 mm punch from perilesional

skin,samples quickly frozen in liquid nitrogen at (-196 C), processesd

and stained using the following antisera: anti-IgG, anti-IgA, anti-IgM and

anti-C3(manufactured by Thermo Fisher Scientific Company). Then

imaged by Olympus camera attached to Olympus epifluoerescence

microscope (Olympus, Japan). Evaluation of biopsy sections was made by

positive and negative marks only.

Statistical analysis:

All analyses were performed with the SPSS 20.0 software.Categorical

variables were described by number and percent (N, %), where continuous

variables described by mean and standard deviation (Mean, SD). Continuous

variables were tested for normal distribution using Kolmogorove Smirnov

test.

Results

Among patients with pemphigus, pemphigus vulgaris (PV) was the most

frequent disease 26/36 (72.7%), followed by pemphigus foliaceous (PF) 7/36

(19.4%), and lastly pemphigus herpetiformis (PH) 3/36 (8.3%). The age of this

group ranged from 21to 73 years with a mean age ± SD of 46.94±14.6 years. In

this group males were more frequent among patients having PV and PF, while

all PH patients were females (Table 1).

Table (1): Age and sex distribution in patients with pemphigus

DiseaseSex, n (%) Age

mean+SD (range)Male Female

Pemphigus (n=36)20(55.6

%)16(44.4%

)46.94±14.6(21-73)

Pemphigus Vulgaris (26)16(61.5%

) 10(38.5%)43.88±15.06(21-73)

Pemphigus Foliaceous (7) 4(57.1%) 3(42.9%) 57.57±10.67(45-72) Pemphigus

Herpetiformis(3) 0(0%) 3(100%) 46.67±7.64(40-55)

Histopathological examination of all patients clinically diagnosed as PV

(n=26) confirmed the diagnosis histopathologically. Their biopsies showed

supra-basal intra-epidermal bullae, acantholytic cells and tombstones appearance

(figure1).

Fig.1. Histopathological examination of lesional skin in PV (H&E x100).

Patients with PF, all patients clinically diagnosed as PF (n=7) had

confirmed the diagnosis histopathologically. Their biopsies showed subcorneal

intra-epidermal bullae with mixture of inflammatory cells (figure 2).

Fig.2. Histopathological examination of lesional skin in PF (H&Ex100).

We had 3 female patients presented with itchy grouped vesicular lesions,

and were clinically diagnosed as dermatitis herpetiformis (DH) or pemphigus

herpetiformis (PH), on examination of their biopsies with H &E, all patients

showed subcorneal blister with polymorphs and esinophils, so the diagnosis of

DH was excluded. And we describe them as having PH (Figure 3).

Fig.3. Histopathological examination of lesional skin in PH (H&E,x100).

DIF was positive in all patients with pemphigus (100%), showing

intraepidermal deposition of immunoglobulins and complement 3 (C3).

Table (4):DIF in patients with pemphigus

 Disease IgG IgM IgA C3

Pemphigus group: 36(100%) 1(2.8%) 0(0%) 14(38.9%) Pemphigus Vulgaris

(26) 26(100%) 1(3.8%) 0(0%) 10(38.4%)

Pemphigus Foliaceous

(7) 7(100%) 0(0%) 0(0%) 4(57.1%)

Pemphigus

Herpetiformis (3) 3(100%) 0(0%) 0(0%) 0(0%)

DIF of patients with PV, showing intraepidermal deposition of

immunoglobulins. IgG in all patients (100%), IgM in only one patient (3.8%),

and C3 was observed in 10 patients (38.4%) (Figure 6-7). IgA was not detected

in these patients.

Fig. 6. Direct immunofluorescence findings in PV showing deposition of IgG

in the intercellular spaces of the epidermis (x100).

Fig.7. Direct immunofluorescence findings in PV showing deposition of C3

in the intercellular spaces of the epidermis (x100).

DIF in patients with PF showed intraepidermal deposition of

immunoglobulins, mainly in the upper part of the epidermis, but this pattern

cannot be seen in all patients, and the diagnosis was helped by clinical,

pathological and ELISA result. IgG was seen in all patients (100%), while

C3 was also observed in 4 (71.4%) patients.

Fig.8.:Direct immunofluorescence findings in PF showing deposition of

IgG in the intercellular spaces of the epidermis (x100).

DIF was positive in all patients with PH showed intraepidermal deposition

of immunoglobulins, mainly in the upper part of the epidermis , but this pattern

cannot be seen in all patients, and the diagnosis was helped by clinical,

pathological and ELISA result. IgG was seen in all of them (100%) (Figure 9).

Fig.9. Direct immunofluorescence findings in PH showing deposition of IgG

in the intercellular spaces of the epidermis (x100).

Discussion

Autoimmune bullous disorders are a group of disorders characterized by

autoantibodies directed against various skin autoantigens resulting in blister

formation. These disorders may be further classified as intraepidermal and

subepidermal depending on the level of bulla. In cases where clinical and

histopathological diagnosis could be very challenging, direct

immunofluorescence will help at arriving with an accurate diagnosis.

Immunofluorescence techniques help in better understanding and classification

of the various immunobullous disorders (Khannan et al., 2015).

Regarding DIF in PV patients, in our study, we found that IgG was

detected in 100% of patients, IgM was deposited in (3.8%)of patients, while C3

was also observed in 38.4% of patients. These results were in the agreement

with Kabir et al., 2009, who found that 87.5% of PV patients showed

intercellular lace like deposition of IgG in the whole thickness of the epidermis.

They stated that the diagnosis of blistering diseases can often made on the basis

of clinical features but in some cases it may be possible to produce only

differential diagnosis, they also stated that less than 50% of clinical diagnosis

showed concordance with the final diagnosis in their study. DIF study has a

definitive role in distinguishing immune mediated blistering diseases from

others. They concluded that DIF technique combined with routine histology is a

useful method in distinguishing most of immunobullous diseases, if not all.

A similar study done by Monsef et al., 2012 found that all cases of PV

had positive DIF in intercellular region; IgG was detected in all (100%) of

patients, IgM in 11.1% of patients and C3 in 33.3% of patients. They concluded

that immunofluorescence tests are sensitive diagnostic methods for autoimmune

bullous diseases.

Another study done by Khannan et al., 2015 found that 100% of cases

with PV showed deposition of IgG in intercellular region, while C3 was also

observed in 84% of patients.

In patients with pemphigus foliaceus (n=7), DIF was positive in all

patients in the present study showing intraepidermal deposition of

immunoglobulins, mainly in the upper part of the epidermis, but this pattern

cannot be seen in all patients. IgG was seen in all of them (100%), while C 3 was

observed in (57.1%) of patients. Similar results were obtained by Kabir et al.,

2009 who found that IgG was positive in all patients with pemphigus foliaceus

(100%) in the intercellular substance (ICS) in the full thickness of epidermis.

Also, Khannan et al., 2015 found that 100% of cases with PF showed ICS

deposition of IgG.

Huang et al., 2007, stated that in some cases of pemphigus, the results

of immunofluorescence staining may be identical in PV and PF. Similarly,

Khandpur et al., 2010 stated that in a majority of the cases, the diagnosis of PV

and PF, the two major variants of pemphigus, rests upon clinical, histological

and immunofluorescence features, however, at times, differentiation between

these two variants, and between pemphigus and other vesicobullous disorders is

a diagnostic challenge as the DIF shows a similar fluorescence pattern in PV and

PF. In these cases ELISA may have a role in the differentiation between the two

variants.

Conclusions

Immunofluorescence (IF) studies are an important part of the laboratory

evaluation of immunologically mediated bullous dermatoses and have

become standard procedures for accurate diagnosis of patients with these

disorders.

Immunofluorescence studies are considered the ‘gold standard’ for the

diagnosis of autoimmune blistering diseases.

Limitations

Small sample size of the study.

The rarity of some types of pemphigus (eg. Pemphigus herpetiformis).

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كلية الطب األمراض الجلدية و قسم

التناسلية

نمط الوميض الفلوريسيني المناعي المباشر في تشخيص مرض الفقاعالطبيبة/ آيه يوسف محمد بدران

مدرس مساعد بقسم األمراض الجلدية والتناسليةوأمراض الذكورة

تحت إشراف أ.د/ عزه محفوظ عبد المجيد

أستاذ األمراض الجلدية والتناسلية وأمراض الذكورةكلية الطب ــ جامعة أسـيوط

أ.د/ أمل طـــه أبوالغيطأستاذ الهستولوجيا

كليه الطب – جامعه أسيوطد/هشام دياب جابر

مدرس بقسم األمراض الجلدية والتناسلية وأمراض الذكورةكلية الطب ــ جامعة أسـيوط

2016

:المقدمة تضم األمراض الفقاعية المناعية العديــد من األنــواع ، تشــترك جميعهــا في-

وجود أجسام مضادة للبروتينات التى تحافــظ على التصــاق الخاليــا ببعضــهاالبعض في الجلد وكذلك في األغشية المخاطية.

حيث تقوم هذه االجسام المضاده بفصل خاليــا الجلــد واالغشــيه المخاطيــه- عن بعضــها البعض ممــا يــؤدي الي ظهــور الفقاعــات الجلديــه والتقرحــات

بالجلد واالغشيه المخاطيه. وتختلف االعراض الظاهره في المريض حسب مكــان االنفصــال, حيث انــه-

من الممكن ان يكون االنفصال في طبقه االدمه او في طبقه البشره. و تعتمد هــذه األمــراض في تشخيصــها على : الفحص اإلكليــنيكي ، الفحص-

المجهري والفحص المجهري الفلوريسيني المناعي وكذلك اســتخدام تقنيــة إليزا وغيرها من الفحوصات التي تعتمد على وجــود األجســام المضــادة في

الدم. ــةحيث يقوم الفحص الم- جهري بتحديد ما إذا كان اإلنفصال موجود في طبق

األدمة أم بطبقــة البشــره , ولكن ال يمكن االعتمــاد علي الفحص المجهــري وحـــده في التشـــخيص ، حيث البـــد من اســـتخدام الفحص المجهـــري الفلوريسيني المناعي بطريقتيــة المباشــرة )حيث تؤخــذ العينــة من الجلــد(والغير مباشرة ) حيث تؤخذ العينة من الدم( وكذلك استخدام تقنية إ ليزا.

أهـداف البحث : تحديد طريقة ترسب األجسام المضادة في المرضــى المصــابين بــاألمراض

الفقاعية المناعية بالجلد وتحديد نوعها ومكانها.

: وتشــتمل هــذه المجموعــة على المرضــى م""ريض36 تضــمنت هــذه الدراسة الفقاع الشائع " الفقاع الورقيالمصابون بمرض الفقاع بأنواعه المختلفة )

(" الفقاع الحلئي . مرضى7 مريض يعانون من مرض الفقاع الشائع و 26وقد وجد أن

3يعانون من مرض الفقاع الورقي و مريض يعانون من مرض الفقاع.الحلئي

عام73 إلى 21وقد كان متوسط أعمارهم يتراوح من ولقد وجد بالفحص المجهري الفلوريسيني المناعي في مرض الفقاع

(الجلوبولين المناعي جالشائع أن ) (IgG) في100موجود بنسبة % (الجلوبولين المناعي مالمرضى، أما ) (IgM) 3.8وجد بنسبة %،

%، وجميعها موجودة في طبقة38.4وجد بنسبة C3 وكذلك المتمم الثالث .البشرة بالجلد بين الخاليا وبعضها

:وعن مرض الفقاع الحلئي وجد بالفحص المجهري الفلوريسيني المناعي في مرض الفقاع الشائع أن

الجلوبولين المناعي ج) (IgG) في المرضى100موجود بنسبة % .