|Year : 2014 | Volume
| Issue : 3 | Page : 164-168
Direct immunofluorescence in autoimmune vesiculobullous disorders: A study of 59 cases
Kamal Ahmed1, T Narayana Rao2, G Swarnalatha3, Sameera Amreen4, Bhagyalaxmi5, AS Kumar1
1 Department of Dermatology, Deccan College of Medical Sciences, Hyderabad, Andhra Pradesh, India
2 Department of Dermatology, Andhra Medical College, Visakhapatnam, Andhra Pradesh, India
3 Department of Pathology, Apollo Hospitals, Hyderabad, Andhra Pradesh, India
4 Department of Pathology, Osmania Medical College, Hyderabad, Andhra Pradesh, India
5 Department of Pathology, Andhra Medical College, Visakhapatnam, Andhra Pradesh, India
|Date of Web Publication||17-Sep-2014|
H. No. 12-2-709/C/191, Padmanabha Nagar, Hyderabad - 500 028, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Background: The autoimmune bullous diseases are characterized by pathogenic autoantibodies directed at target antigens whose function is either cell-cell adhesion within the epidermis or adhesion of stratified squamous epithelium to dermis or mesenchyme.
Aim: To assess the correlation between clinical, histopathological and direct immunofluorescence patterns in autoimmune vesiculobullous diseases and to assess the diagnostic value of direct immunofluorescence in various autoimmune vesiculobullous diseases.
Materials and Methods: The study was conducted for a period of 24 months from August 2006 to August 2008. Total of 59 patients aged 3-80 years with vesiculobullous lesions of both sexes attending the department of dermatology were selected for the study. These samples were tested and analyzed under light microscope and direct immunofluorescence (DIF) testing.
Results: Among the patients of vesiculobullous disorders studied most common disorder was found to be pemphigus vulgaris, constituting 40.6% cases. About 43.5% of patients presented with flaccid vesicles and bullae. Around 26.4% of the patients presented with tense bullae. Mucosal involvement was found in 57.6% of cases. Tzanck smear was positive for acantholytic cells in 54.2% of cases. Bullae were located sub epidermally in 30.5% of the patients, for 54.2% of patients they were located intra epidermally and no bullae were seen in 15.25% of cases. DIF was positive in 93.2% cases of autoimmune vesiculobullous disorders. Out of 59 cases of autoimmune vesiculobullous disorders, only 41 (69.4%) cases correlated clinically and histopathologically with direct immunofluorescence patterns.
Conclusion: Our study proves DIF is not only diagnostic but also confirmatory for all autoimmune bullous disorders.
Keywords: Direct immunofluorescence, pemphigus vulgaris, bullous pemphigoid
|How to cite this article:|
Ahmed K, Rao T N, Swarnalatha G, Amreen S, Bhagyalaxmi, Kumar A S. Direct immunofluorescence in autoimmune vesiculobullous disorders: A study of 59 cases. J NTR Univ Health Sci 2014;3:164-8
|How to cite this URL:|
Ahmed K, Rao T N, Swarnalatha G, Amreen S, Bhagyalaxmi, Kumar A S. Direct immunofluorescence in autoimmune vesiculobullous disorders: A study of 59 cases. J NTR Univ Health Sci [serial online] 2014 [cited 2020 Apr 4];3:164-8. Available from: http://www.jdrntruhs.org/text.asp?2014/3/3/164/140935
| Introduction|| |
The pathogenesis of autoimmune bullous diseases (ABD) is yet to be completely deciphered. Consequently, there has been enormous development in the fields of immunopathology, diagnostics and treatment of ABDs and there is still a long way to go. The last century saw the management of these disorders improve by leaps and bounds due to the demonstration of acantholysis as the primary pathology in a majority of intraepidermal bullous diseases, identification of various autoantibodies with their pathogenic significance and a great reduction in mortality with the discovery and widespread use of corticosteroids. However, these disorders are still associated with significant morbidity, considerable mortality and impaired quality of life. 
Immunofluorescence is a histochemical laboratory staining technique used for demonstrating the presence of antibodies bound to antigens in tissues or circulating body fluids. These techniques are essential to supplement clinical findings and histopathology in the diagnosis of immunobullous disorders. They permit early diagnosis, treatment, and subsequent monitoring of disease activity in patients with these potentially life-threatening disorders.
Direct immunofluorescence (DIF) is a one-step histological staining procedure for identifying in vivo antibodies that are bound to tissue antigens. ,
| Materials and methods|| |
This study was conducted during the period of 24 months from August 2006 to August 2008. A total of 59 patients aged 3-80 years with vesiculobullous lesions of both sexes attending the Department of Dermatology were selected for the study. All patients were subjected to a detailed history taking and clinical examination. Their particulars regarding age, gender, occupation, personal and family history, presenting complaints, duration, general condition and findings on clinical examination were recorded in the proforma. Routine hematological and biochemical investigations were done and reports recorded. Tzanck smear was done in all cases and findings were noted. Two 4 mm punch biopsies were done in all cases, one from the vesicle (fixed in 10% buffered formalin) and another from the perilesional area (fixed in Michel's medium) were sent to the Department of Histopathology. Salt spilt DIF was done in all patients with subepidermal blisters.
Direct immunofluorescence is a one-step procedure used to detect and localize immunoreactants deposited in vivo in the patient's skin or mucosa. The immunoreactants include antibodies, complement components, and fibrinogen. , Frozen sections 5 μm in thickness are cut with the cryotome and placed on slides. These are dried for 10 min with an electric fan. Inadequate drying of sections between processing steps may lead to their detachment during washing. The sections are then washed in phosphate-buffered saline (PBS) at a pH of 7.4 for 10 min to remove surrounding optimal cutting temperature compound. The sections are fan-dried once more and incubated with monospecific fluorescein isothiocyanate-labeled antisera for 30 min at 37°C. Antisera to immunoglobulin G (IgG), IgA, IgM, fibrinogen, and the C3 component of complement should be routinely employed. Antisera to particular subclasses of immunoglobulins and other components of complement are also available, but are less commonly used. Sensitivity and specificity of staining may be maximized by the use of the optimal dilution of the labeled antisera. This is determined by a chess-board titration procedure utilizing a known positive tissue specimen.  The sections are washed in PBS to remove unbound antisera, fan-dried, and mounted in a drop of buffered glycerol. They are then viewed with the fluorescence microscope. 
The distribution and type of immunoreactant deposition is recorded. The class and subclass of immunoglobulins and the presence or absence of complement is noted. Excessive fibrin deposition indicates that immunoreactants have been present for more than 24-48 h. Immunoreactants are deposited in two main patterns: In the epidermal intercellular space (ICS) and along the basement membrane zone (BMZ) [Figure 1], [Figure 2], [Figure 3]. ICS immunoreactants may be found throughout the epidermis or restricted to certain layers. BMZ deposits may be smooth and linear, granular and discontinuous or a combination of the two  [Table 1].
|Figure 1: Intercellular deposits of immunoglobulin G and C3c|
(Fish net pattern)
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|Figure 2: Linear deposits of immunoglobulin G and C3c along the basement membrane zone (shore line fluorescence pattern)|
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|Figure 3: Granular deposits of immunoglobulin A at the tips of the papillary dermis and along the basement membrane zone|
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A number of artefacts must be differentiated from significant findings. , Autofluorescence by proteins such as keratin and elastic fibers may be a significant problem.
| Results|| |
Autoimmune vesiculobullous disorders were found to be more common in females with male to female ratio was 0.73:1. Around 25.4% of patients belonged to 31-40 age group, the mean age being 39.88 years.
Among the patients studied with vesiculobullous disorders most common disorder was found to be pemphigus vulgaris, constituting 40.6% cases [Table 2].
About 43.5% of patients presented with flaccid vesicles and bullae. Around 26.4% of the patients presented with tense bullae. Mucosal involvement was found in 57.6% of cases. Tzanck smear was positive for acantholytic cells in 54.2% of cases. Bullae were located sub epidermally in 30.5% of patients, for 54.2% of patients they were located intraepidermally and no bullae were seen in 15.25% of cases. DIF was positive in 93.2% cases of autoimmune vesiculobullous disorders. Of 59 cases of autoimmune vesiculobullous disorders, only 41 (69.4%) cases correlated clinically and histopathologically with DIF patterns. Of the 55 patients who were found to have positive DIF study, IgG was present in 46 (83.6%) patients, C3 was present in 53 (96.36%) cases, IgM was present in 3 (5.45%) patients [Table 3]. DIF was inconclusive in 6 (10.1%) of cases.
|Table 2: Distribution of Different Types Of Autoimmune Vesiculobullous Disorders|
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| Discussion|| |
Immunological disorders are classified largely by the clinical and immunofluorescence pattern. Even after the advent of systemic corticosteroids some of them may prove fatal. It is therefore necessary to diagnose and treat the condition early.
The diagnosis of these diseases is facilitated by the use of a battery of tests that include histopathology, immunofluorescence microscopy, electron microscopy and immunosorbent assays. The common immunoglobulin deposit patterns of DIF of autoimmune bullous diseases are shown in [Table 3].
Keeping in view of these facts, it was thought that it would be worthwhile to study vesiculobullous disorders clinically, histopathologically and to correlate with immunofluorescence studies in some cases.
Among the patients studied most common disorder was found to be pemphigus vulgaris, constituting 40.6% cases. Our series is in accordance with other Indian series each of which has maximum cases of pemphigus vulgaris. ,
Around 25.4% of patients belonged to 31-40 age group, but in some Indian studies maximum number of patients belong to 21-60 years. ,
Pemphigus vulgaris affects males and females equally.  Although, in the present study, there was a slight male predominance, out of 59 cases, 24 cases diagnosed as pemphigus vulgaris with a distribution among males were 13 (54.2%) and 11 (45.8%) were females, which probably reflects the higher male attendance in the outpatient department. Male dominance has been reported in the same ratio.  However, there was overall female predominance in our study that is, out of 59 cases of autoimmune vesiculobullous dermatoses studied 25 patients (42.4%) were males and 34 patients (57.6%) were females with a male to female ratio of 0.73:1.
In this study, maximum number of patients presented with flaccid bullae accounting for 43.5% of cases (mostly pemphigus vulgaris).
There is 26.4% present with tense bullae with hemorrhagic fluid, which is seen in subepidermal blistering disorders.
In this study, about 57.6% patients had mucosal involvement and the lesions were mostly distributed in the oral, buccal, palatal, gingival, conjunctiva, and genital mucosa. Mucosal lesions were most commonly seen in pemphigus vulgaris accounting for 40.6% of cases. In this study conducted by Arya et al. pemphigus vulgaris showed initial lesions involving mucous membranes in 37.2% cases and lesions including both skin and mucosae in 13.9% cases. 
Nature and distribution of lesions as well as mucosal involvement in different types of pemphigus in our series has followed the pattern seen in earlier studies. ,,,,
Of 59 patients of autoimmune vesiculobullous dermatoses, 32 patients (54.2%) showed acantholytic cells that is, positive Tzanck smear and 27 patients (45.76%), showed no acantholytic cells that is, negative Tzanck smear.
In our study, bullae are located subepidermally in 18 patients that is, 30.5% according to histopathological examination. The bullae are formed intraepidermally in 32 patients contributing 54.2% and no bullae in 9 (15.25%) cases.
Analysis of our study showed that out of 59 cases, 41 (69.4%) cases correlated clinically and histopathologically with DIF patterns.
Of the remaining 17 (28.8) patients, 12 (20.3%) which did not correlate with clinical, histopathological findings, gave diagnostic findings with DIF, 5 (8.5%) patients had only oral lesions, which were not conclusive by clinical, histopathological examination, gave positive DIF finding suggestive of pemphigus vulgaris. In six patients even DIF was inconclusive.
| Conclusion|| |
Our study further proves that DIF is not only diagnostic, but also confirmatory for all autoimmune vesiculobullous disorders. DIF is most useful in oral lesions in which clinical findings and histopathology may be inconclusive. Salt split DIF is very useful in differentiating bullous pemphigoid from epidermolysis bullosa acquisita which have the same histopathological picture [Figure 4] and [Figure 5]. Since most of the autoimmune vesiculobullous disorders require prolonged and aggressive therapy, the diagnosis may not be confirmatory only with mere histopathology; hence, DIF studies are a must for proper management.
|Figure 4: Salt split immunofl uorescence - Deposition of IgG & C3c over the roof the Blister (Bullous Pemphigoid)|
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|Figure 5: Salt split immunofl uorescence -Deposition of IgG over the base the Blister (Epidermolysis Bullosa Acquisita)|
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]