|Year : 1992 | Volume
| Issue : 6 | Page : 379-383
Direct immunofluorescence as a diagnostic and prognostic marker in pemphigus
Kaur Jagjit Sethi, AJ Kanwar, S Kaur, S Sehgal
Kaur Jagjit Sethi
Source of Support: None, Conflict of Interest: None
Direct immunoflurescence was carried out in 20 patients with pemphigus vulgaris and repeated at intervals of 3 months in each patient on 3 occasions or earlier in patients who had a relapse. No correlation was observed between the disease activity and positive DIF. Complement deposition showed an increase or reappeared in patients who had a relapse. It is thus a better indication of an imminent relapse.
Keywords: Direct immunofluorescence, Pemphigus vulgaris
|How to cite this article:|
Sethi KJ, Kanwar A J, Kaur S, Sehgal S. Direct immunofluorescence as a diagnostic and prognostic marker in pemphigus. Indian J Dermatol Venereol Leprol 1992;58:379-83
|How to cite this URL:|
Sethi KJ, Kanwar A J, Kaur S, Sehgal S. Direct immunofluorescence as a diagnostic and prognostic marker in pemphigus. Indian J Dermatol Venereol Leprol [serial online] 1992 [cited 2020 May 26];58:379-83. Available from: http://www.ijdvl.com/text.asp?1992/58/6/379/3857
| Introduction|| |
Pemphigus vulgaris is an autoimmune bullous disorder in which IgG antibodies are detected in the intercellular substance and in the serum of the patients by direct and indirect immunofluorescence tests. 
The direct immunofluorescence test (DIF), which detects the antibody deposition in the tissues, is a very reliable diagnostic test for pemphigus and it can remain positive for several years after regression of the disease.  sub The levels of antibody as detected by indirect immunofluorescense tests (IIF) usually show a direct correlation with the disease activity.  However there are many exceptions to this and, therefore, the titre of antibodies cannot be regarded as a reliable index for assessing the disease severity  and thus cannot be used as a guide for monitoring the therapy.  The purpose of the present study was to elucidate the role of DIF as a diagnostic and prognostic marker in patients of pemphigus.
| Material and Methods|| |
Patients : In this prospective study 20 patients (16 men, 4 women; age 18 to 55 years) of pemphigus vulgaris were included. The patients were divided into 2 groups of 10 patients each - group A and group B. Group A comprised of recently diagnosed patients and group B of patients who had earlier been treated with dexamethasone-cylophosphamide pulse therapy or oral corticosteroids or oral corticosteroids along with immunosuppressives (conventional treatment) and were in various phases of remission. The duration of the disease ranged from 1 '/ 2 to 12 months in Group A and in group B from 1 '/ 2 to 6 years. The diagnosis of pemphigus vulgaris was confirmed by histopathology and immunofluorescence in all patients. Patients were followed up regularly every month for 1 year. DIF test was performed at intervals of 3 months in each patient on 3 occasions. Biopsies were taken from a- site which was close to previous biopsy site.
Immunofluorescence Studies : For DIF biopsy specimens were taken from the lesional and perilesional skin in patients with active lesions and from upper back in clinically inactive patients. Fluorescin labelled monospecific antisera raised against IgA, IgG, IgM and complement component C3 were used (Chemopol Ltd). The conjugates were checked for monospecificity before use. Four frozen sections from each biopsy were incubated with fluorescein isothiocyanate (FITC) labelled antihuman immunoglobulin and complement for a period of 30 minutes at 30°C. These slides were washed gently in buffer and then mounted in buffer glycerin mixtures. The slides were screened in an incident light under Zeiss 16 research microscope fitted with an HBO 50 ultraviolet lamp. The immunofluorescence pattern was studied and the positivity was graded arbitrarily as strongly positive (+++), moderately positive (++), and weakly positive (+).
Therapeutic Regimens: Eight patients in Group A, received monthly dexamethasone cyclophosphamide pulses, the other 2 patients having mild cutaneous involvement were treated initially with oral corticosteroids 60-120 mg/day and later were maintained on oral dapsone 100 mg daily. Five patients out of the 8 receiving monthly pulses were maintained on intervening oral corticosteroids 20-40 mg/ day and oral corticosteroids 30-40 mg/ day. Patients in group B were not on any medication and the period of remission was from 2 to 4 years.
| Results|| |
Group A patients : The first biopsy in patients in group A had positive DIF with deposits of IgG, IgM and complement varying from + to +++ [Table - 1]. In 2 patients, the IgG deposition was strongly positive (+++) at the initial biopsy; it became less (i.e.+) in one patient at 3 months and disappeared completely at 6 months. In the other patient, it became negative at 3 months but again became positive (++) at 6 months. This was associated with a clinical relapse. In 1 patient, the initial IgG deposition was ++ at first biopsy; it remained ++ at 3 months and became negative at 6 months. In 7 patients, IgG deposition was (+) at initial biopsy; in 6 remained + at 3 months while in 1 it became +++ (This was associated with a clinical relapse). At 6 months, in 4 of these patients, IgG deposition remained +, in 1 it increased to ++ and in 2, it was negative.
The IgM complement deposition showed a gradual reduction in nearly all patients except in one in whom the complement increased from mild (+) to moderate ( ++) at 6 months.
Group B patients : Five patients in this group did not show any relapse during the period of study. Only 1 patient had IgG deposition (++) at the time of initial biopsy. It became less i.e. + at 3 months and negative at 6 months. In 4, though IgG was negative initially, it became + at 3 months. At 6 months, in 2 it became ++; in 1 it became negative while in 1, it was +. IgM though initially absent in all 5, became positive i.e. + in 2 at 3 months; it remained + in one while in the other, it disappeared at 6 months. IgM appeared for the first time (i.e.+) at 6 months in one patient. The complement deposition was less consistent: it was + in one patient initially, disappeared at 3 months and reappeared at 6 months. In one patient, it appeared only at 3 months (+) and became ++ at 6 months. In 2 patients, it appeared for the first time at 6 months.
In the other 5 patients in group B who relapsed, IgG deposition was positive in 3 patients (+++ in 1, + in 2) initially. It was present in all 5 at 3 months (+++ in 1, ++ in 2 and + in 2) while at 6 months, it was +++ in 1, ++ in 2 and + in 1. There was however, no significant correlation between extent of cutaneous involvement and IgG deposition. IgM deposition was again inconsistent; however, it appeared at 6 months in 3 patients. The increase in complement deposition was however, striking. At 6 months, it was present in 4 out of 5 patients and the intensity of deposition was greater compared to initial levels.
| Comments|| |
Demonstration of immunoglobulins especially IgG and complement in the intercellular space by DIF is a very reliable diagnostic test for pemphigus, it becomes positive early at the onset and remains positive for a long period after clinical remission.
The role of DIF as a prognostic marker and indicator of disease activity has, however, not been evaluated fully. Only a few studies are available highlighting the importance of DIF as a marker of disease activity and prognosis. 
The results of our study indicate that the intensity of DIF coincided fairly well with clinical activity but occasionally in an individual patient a perfect correlation was not consistently observed. However, the persistence of positive DIF in patients whose lesions had cleared clinically signified a persisting immunologic activity and possibility of relapse. The baseline IgG, IgM and complement were higher in the patients who relapsed.
The role of complement in the pathogenesis of pemphigus is controversial, since antibody alone can induce acantholysis in vitro. , Although intercellular complement is reported to be mostly present in the involved skin,  it may also be demonstrated in the uninvolved skin. Its presence is thus a marker of the disease activity, the skin becomes negative for complement during remission. 
However, our study did not reveal so. One patient in group A and 5 patients in Group B showed presence of complement even when the disease was in remission. The interesting observation was that 3 out of 5 patients who relapsed in group B showed increasing complement deposits 2-3 months before an oncoming relapse. Among the other 5 patients also, who did not have any relapse for at least 1 year i.e., during the period of the study, reappearance of complement was observed in 4 (+ in 2 and ++ in 2). Three of these 4 patients showed a clinical relapse during a further follow-up of 15 months to 2 years. Similarly findings were observed by David et al . 
Our study shows that the patients continue to have positive DIF despite clinical inactivity of pemphigus. In patients who relapse, DIF tends to remain positive and the complement deposition may show an increase or may reappear. It is, thus, a better indicator of an imminent relapse. The study highlights that a positive DIF is perhaps not a good prognostic marker for disease activity as it continued to remain positive even in patients who were clinically inactive. A longer follow up is required to assess the exact significance of positive DIF and its relevance to relapse. At present the clinical activity of the disease is the only parameter taken into consideration for deciding the duration of treatment or its withdraw]. In the light of the findings of the DIF and complement brought out in the present study, the recommendations about the duration of therapy with corticosteroids and immunosuppressives may have to be looked into afresh.
| References|| |
|1.||Beutner EH, Lever WF, Witebspy E, et al. Autoantibodies in pemphigus vulgaris. JAMA 1965; 192: 682-6. |
|2.||Judd KP, Lever WF. Correlation of antibodies in skin and serum with disease severity in pemphigus. Arch Dermatol 1979; 115: 428-31. [PUBMED] |
|3.||Weissman V, Feuerman EJ, Joshua H, et al. The correlation between the antibody in sera of patients with pemphigus vulgaris and their clinical state. J Invest Dermatol 1978; 71: 107-11. [PUBMED] |
|4.||Cressvell SN, Black MM, Bhogal B, et al. Correlation of circulating intercellular antibody titres with disease activity. Clin Exp Dermatol 1981; 6: 477-80. |
|5.||Fitzpatrick RE, Newccomer VD. The correlation of disease acivity and antibody titres in pemphigus. Arch Dermatol 1980; 116: 285-9. |
|6.||David M, Weissman V, Ben A, et al. The usefulness of immunofluorescent tests in pemphigus patients in clinical remission Br J Dermatol 1989; 120: 391-4. |
|7.||Bhogal B, Wojnarowoska E, Black MM, et al. The distribution of immunoglobulins and C3 component of complement in multiple biopsies from the uninvolved and perilesional skin in pemphigus. Clin Exp Dermatol 1986; 11: 49-52. |
|8.||Jordon RE, Schroeter AL, Rogers RS, et al. Classical and alternative pathway activation of complement in pemphigus vulgaris lesions. J Invest Dermatol 1974; 63: 256-61. |
[Table - 1], [Table - 2], [Table - 3]
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