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ORIGINAL ARTICLE
Year : 2017  |  Volume : 62  |  Issue : 6  |  Page : 649-653
Detection of anti-basement membrane zone antibodies in the blister fluid in subepidermal autoimmune bullous diseases


1 Department of Dermatology, Kasturba Medical College, Manipal University, Manipal, Karnataka, India
2 Department of Community Medicine, Kasturba Medical College, Manipal University, Manipal, Karnataka, India

Date of Web Publication24-Nov-2017

Correspondence Address:
Raghavendra Rao
Department of Dermatology, Kasturba Medical College, Manipal University, Manipal - 576 104, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijd.IJD_403_17

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   Abstract 


Background: Subepidermal autoimmune bullous diseases (sAIBD) are diverse of conditions with clinicopathological overlap. Circulating autoantibodies in the serum can be demonstrated using indirect immunofluorescence (IIF) microscopy. Artificially split normal human skin is considered as an optimum substrate for the demonstration of anti-basement membrane zone (BMZ) antibodies using IIF in sAIBD; it not only helps to detect the presence of circulating antibodies in the serum but also helps to subclassify these conditions into “roof” and “floor” binding disorders. Aim: In this study, we evaluated the utility of IIF to detect anti-BMZ antibodies in the blister fluid of patients with sAIBD. Materials and Methods: Twenty-two patients with a clinical diagnosis of sAIBD were enrolled in the study. IIF of serum and blister fluid were done simultaneously using salt-split skin as a substrate. Results: Anti-BMZ antibodies could be detected in the blister fluid using IIF in all patients in the study group. Limitation: We could not do enzyme-linked immunosorbent assay of blister fluid. This would have given us the quantitative data of circulating antibodies in the blister fluid. Conclusion: Blister fluid offers an alternate source for the detection of autoantibodies in patients with sAIBD. It may be of particular help in children and in elderly with poor venous access.


Keywords: Blister fluid, indirect immunofluorescence, subepidermal autoimmune bullous diseases


How to cite this article:
Surya R, Tejasvi B, Shenoi SD, Pai S, Rao C, Rao R. Detection of anti-basement membrane zone antibodies in the blister fluid in subepidermal autoimmune bullous diseases. Indian J Dermatol 2017;62:649-53

How to cite this URL:
Surya R, Tejasvi B, Shenoi SD, Pai S, Rao C, Rao R. Detection of anti-basement membrane zone antibodies in the blister fluid in subepidermal autoimmune bullous diseases. Indian J Dermatol [serial online] 2017 [cited 2022 Aug 17];62:649-53. Available from: https://www.e-ijd.org/text.asp?2017/62/6/649/219217

What was known?

  • Subepidermal autoimmune bullous diseases are heterogeneous group of condition with clinical and histopathological overlap
  • Indirect immunofluorescence microscopy of serum using salt-split skin is a simple technique, which will enable subclassification of sAIBD into “roof” and “floor” binding conditions.



   Introduction Top


Subepidermal autoimmune bullous diseases (sAIBD) are heterogeneous group of diseases characterized clinically by tense blisters on the skin with or without mucous membrane involvement. These include bullous pemphigoid (BP), epidermolysis bullosa acquisita (EBA), linear IgA disease (LAD), pemphigoid gestationis (PG), mucous membrane pemphigoid (MMP) and recently described anti-p200 pemphigoid.[1],[2],[3] Autoantibodies in these conditions target the antigens present in the dermoepidermal junction triggering inflammation and blister formation.[4] Histopathology in these conditions reveals subepidermal blisters with granulocytes-rich inflammatory infiltrates.[3] The gold standard test to demonstrate the tissue-bound antibodies is direct immunofluorescence (DIF) microscopy of biopsy specimen obtained from the perilesional skin; it characteristically reveals linear staining IgG, IgA, and C3 classes of immunoreactants along the basement membrane zone (BMZ).[5] Circulating antibodies in patient's serum can be demonstrated by indirect immunofluorescence (IIF). Sensitivity of IIF microscopy depends on the substrate used; salt-split skin (SSS) is considered as the optimum substrate for IIF in patients with sAIBDs.[6],[7] This study was undertaken to identify the presence of anti-BMZ antibodies in the blister fluid of patients with sAIBDs using IIF microscopy and to compare it with that of the serum.


   Materials and Methods Top


This study was conducted in a tertiary care teaching hospital over 1 year. This study was approved by the Institutional Ethical Committee Review Board. Twenty-two patients with a clinical diagnosis of subepidermal autoimmune blistering diseases (sAIBD) were enrolled in this prospective study. A perilesional biopsy was undertaken, and the clinical diagnosis was confirmed in each case by DIF microscopy. Tense, freshly erupted blister of 0.5 cm or more in size was chosen; using a 22-gauge needle, about 0.5 ml of blister fluid was aspirated and collected in an aliquot. Two milliliter of serum was collected simultaneously, and both serum and the blister fluid were stored in deep freezer until further use. SSS was prepared by overnight incubation of normal human skin (NHS) in 1 molar sodium chloride for 8 h in a rotator at 4°C in a refrigerator. This was then gently teased using a forceps to separate epidermis from the dermis. Specimen of SSS and NHS were then placed side by side and blocked together in optimum cutting temperature compound. Frozen sections of this block were obtained using a cryostat set at -26°C in a special glass slide with inbuilt adhesive properties (obtained from Hendley's UK); frozen sections thus yielded both NHS and SSS sections side by side [Figure 1] and [Figure 2].
Figure 1: Line diagram showing the method adopted in taking frozen section in the present study Abbreviations: NHS: normal human skin, SS: Salt-split skin, SE: serum, BF: blister fluid

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Figure 2: Normal human skin and salt-split skin sections stained with IgG showing linear staining of BMZ; band localizing to the dermal side in the split skin (red circle represents the level of split) fluorescein isothiocyanate X100

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Serum and blister fluid were serially diluted (in 1:10 and 1:100 concentration) using phosphate buffer solution (PBS). This was then incubated with frozen sections (six sections per patient-three each for serum and blister fluid) of NHS/SSS for 60 min as shown in [Figure 1]. This was washed in PBS, and the secondary incubation was done for 60 min with fluorescein isothiocyanate conjugated IgG and IgA (DAKO, Denmark). The slides were again washed in PBS, mounted in buffered glycerol, and examined under fluorescence microscope. The intensity of staining was graded subjectively as strong positive (+++), moderately strong (++), weak (+), and negative (-); all the slides were read by the same investigator. Data were entered and analyzed using Statistical Package for the Social Sciences 15.0 (Chicago, SPSS Inc). The intensity of staining with IgG and IgA in blister fluid and serum was compared using Wilcoxon signed rank test; P < 0.05 was considered to be statistically significant.


   Results Top


The mean age of patients in the study group was 57.5 years (Mean ± standard deviation 57.5 ± 13); sex distribution was equal among the study participants. Disease-wise distribution is described in [Table 1]. BP accounted for majority of the cases in this study (n = 13), followed by EBA (n = 7). Out of seven patients with EBA, six patients presented with inflammatory phenotype and were diagnosed initially as BP. This provisional diagnosis was revised to EBA after IIF of patient's serum using salt-split study showed dermal staining pattern in these six patients.
Table 1: Disease-wise distribution and mean age at presentation

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Indirect immunofluorescence

In the present study, IIF on SSS detected the presence of anti-BMZ antibodies in all patients, both in the serum and blister fluid. However, IIF using NHS substrate was negative in five patients (5/22) in the serum group and nine patients (9/22) in blister fluid group making it an inferior substrate when compared to SSS, for the detection of anti-BMZ antibodies in patients with subepidermal autoimmune blistering diseases (sAIBDs).

Anti-BMZ antibodies belonged exclusively to IgG class in 19 patients (12 patients with BP, 6 patients with EBA, and 1 patient with lichen planus pemphigoides). One patient with LAD showed BMZ staining exclusively with IgA class. A combined IgG and IgA class of autoantibodies were detected in two patients (one each of BP and EBA). All seven patients with EBA showed BMZ staining on the dermal side of the split (“floor” pattern), whereas rest of the patients exhibited staining on the epidermal side of the split (“roof” pattern). There was no difference in the type of immunoreactants deposited and the pattern of staining between serum and blister fluid group [Figure 3] and [Figure 4].
Figure 3: Linear staining of IgG on the epidermal side of the split in blister fluid (a) and in the serum (b) in a patient with bullous pemphigoid (red circle represents the level of split) fluorescein isothiocyanate X200

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Figure 4: Linear staining of IgG on the dermal side of the split in blister fluid (a) and in the serum (b) in a patient with epidermolysis bullosa acquisita (red circle represents the level of split) fluorescein isothiocyanate X200

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In the BP subgroup [Table 2], intensity of staining with IgG class of antibodies was similar in both serum and blister fluid groups but for one patient in whom antibodies could not be detected in the blister fluid in 1:100 titer, that is, at higher dilution. On the other hand, in EBA group [Table 3], IIF was negative in blister fluid in 1:100 titer in two patients (both these patients showed weak staining in serum) and one patient showed less intense staining in 1:100 in blister fluid compared to serum (3+ vs. 2+). While other four EBA patients, as well as a case of lichen planus pemphigoides, showed no difference in the staining intensity between blister fluid and serum groups. A patient of LAD revealed moderately strong reaction with IgA in serum in contrast to blister fluid which showed weak reaction. Wilcoxon signed rank test indicated the asymptotic significance (two-tailed) of anti-IgG antibodies in 1:10 dilution between serum and blister fluid to be statistically significant (Z = 2.236, P = 0.025). No statistically significant association was noted with respect to anti-IgG antibodies in 1:100 dilution between serum and blister fluid (Z = 1.394, P = 0.163); and anti-IgA antibodies in 1:10 dilution between serum and blister fluid (Z = 0.000, P = 1.000).
Table 2: Intensity of staining with immunoglobulin G on salt-split skin with immunoglobulin G in bullous pemphigoid

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Table 3: Intensity of staining with immunoglobulin G on salt-split skin with immunoglobulin G in epidermolysis bullosa acquisita

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   Discussion Top


BP is the most common sAIBDs in clinical practice; it is characterized by tense blisters on urticated background with or without mucosal lesions. These clinical criteria, however, are not very specific to BP as these findings also can be seen in certain other sAIBDs, especially EBA. In the clinical setting, this distinction is important as BP has got a favorable prognosis in contrast to EBA which is known to run a protracted course often necessitating prolonged course of immunosuppressive therapy.[7] Given this scenario, we need a simple tool to distinguish these two conditions. Gammon et al. in 1984 described IIF performed on SSS as an ideal test to differentiate anti-lamina lucida antibodies (e.g., BP) from anti-sublamina densa antibodies (e.g., EBA).[8] They incubated the NHS in 1 molar sodium chloride for 72 h at 4° centigrade; subsequently, the epidermis was dislodged from the dermis by applying lateral traction. We tried a modification of the original technique; we could obtain the dermoepidermal separation by overnight incubation of NHS in 1 molar sodium chloride. In this study, we blocked NHS and SSS side by side; frozen section thus taken from this block contained both NHS and SSS. This is the deviation from our previous practice of taking separate sections of NHS and SSS in two different glass slides. Both these techniques have helped us to reduce the time required to perform IIF using two substrates.

The previous studies have shown that blister fluid in BP contains various inflammatory mediators, including interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor-α, and interferon-γ.[8],[9],[10],[11] Wakugawa et al. have shown elevated levels of eotaxin and IL-5 in blister fluid of BP patients. The eotaxin levels in blister fluid significantly correlated with the number of eosinophils in the dermis.[12] In fact, levels of cytokines were higher in blister fluid when compared to the serum; this could be related to the localized production of these cytokines by the keratinocytes during the inflammatory process.[11] However, only a few studies have investigated the presence of antibodies in blister fluid.[4],[13],[14] In 1970, Bean detected BMZ antibodies in the serum and blister fluid of three BP patients.[13] Zhou et al. studied IIF of serum and blister fluid in 35 patients with sAIBDs. This included BP (30 patients), PG (2), LAD (2), and one patient with MMP. Anti-BMZ antibodies were detected in the SSS in 24 patients (twenty-one cases of BP, two cases with LAD, and one patient with MMP) by IIF in their series. The antibody titer in blister fluid has been found to be the same (16 patients) or one dilution less (five patients) than that in serum in BP. They concluded that, unlike cytokines, antibodies (mainly IgG1 and IgG4) in the blister fluid are derived from the vascular compartment through the process of diffusion.[4] Daneshpazhooh et al., in their series of 35 patients with sAIBDs detected anti-BMZ antibodies in the blister fluid in 28 patients, using IIF. Interestingly, in one of their BP case, IIF was positive in blister fluid but was negative in serum; this patient had limited disease on the scalp. The authors concluded that in patients who have recently been affected by BP or suffer from limited BP, IIF on blister fluid might be more useful than that on serum. They also showed that the use of both serum and blister fluid samples in BP patients increased the sensitivity of IIF in detecting IgG anti-BMZ antibodies from 92% to 96%.[14] Patsatsi et al. performed BP enzyme-linked immunosorbent assay (ELISA) (BP 180 and 230) of blister fluid in thirteen newly diagnosed BP patients, before starting treatment.[15] Antibodies could be detected by ELISA in nine patients (69.2%) in blister fluid in contrast to serum where it could be found in eleven patients (84.6%). Sensitivity of IIF in this group of patients with BP was 61.5% (positive in 8 of 13 patients). They concluded that the sensitivity of detecting anti-BMZ antibodies in blister fluid using traditional diagnostic techniques such as IIF was comparable with that of the ELISA technique.

We could detect anti-BMZ antibodies in the blister fluid in all patients in the study group. Autoantibodies belonged predominantly to IgG class; anti-IgA antibodies were detected only in three cases. The previous studies have reported autoantibodies of IgA class in 35%–40% patients with BP;[4],[14] in contrast, it was detected in only one patient of BP in the present study. It is not clear yet whether the presence of antibodies of IgA class has any effect on the clinical course and/or response to treatment in BP patients. The cohort in the present study included two children (one each of LAD and EBA); IIF using blister fluid was positive on SSS in both these patients.


   Conclusion Top


Blister fluid as an alternative source for the detection of anti-BMZ antibodies using IIF. It is less traumatic than venipuncture and is particularly useful in children and elderly, from whom it may often be difficult to obtain the blood samples. The aspiration of blister fluid is also therapeutic and is often used to relieve discomfort of multiple tense fluid-filled lesions and can also provide large amount of materials for the diagnosis and investigations.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
De A, Rao R, Balachandran C. Salt split technique: A useful tool in the diagnosis of subepidermal bullous disorders. Indian J Dermatol 2010;55:334-6.  Back to cited text no. 1
[PUBMED]  [Full text]  
2.
Zillikens D, Kawahara Y, Ishiko A, Shimizu H, Mayer J, Rank CV, et al. A novel subepidermal blistering disease with autoantibodies to a 200-kDa antigen of the basement membrane zone. J Invest Dermatol 1996;106:1333-8.  Back to cited text no. 2
    
3.
Goyal N, Rao R, Shenoi SD, Pai S, Kumar P, Bhogal BS, et al. Epidermolysis bullosa acquisita and anti-p200 pemphigoid as major subepidermal autoimmune bullous diseases diagnosed by floor binding on indirect immunofluorescence microscopy using human salt-split skin. Indian J Dermatol Venereol Leprol 2017;83:550-5.  Back to cited text no. 3
[PUBMED]  [Full text]  
4.
Zhou S, Wakelin SH, Allen J, Wojnarowska F. Blister fluid for the diagnosis of subepidermal immunobullous diseases: A comparative study of basement membrane zone autoantibodies detected in blister fluid and serum. Br J Dermatol 1998;139:27-32.  Back to cited text no. 4
    
5.
Schmidt E, Zillikens D. Modern diagnosis of autoimmune blistering skin diseases. Autoimmun Rev 2010;10:84-9.  Back to cited text no. 5
    
6.
Woodley DT. Immunofluorescence on salt-split skin for the diagnosis of epidermolysis bullosa acquisita. Arch Dermatol 1990;126:229-31.  Back to cited text no. 6
    
7.
Yang B, Wang C, Chen S, Chen X, Zhou G, Tian H, et al. Accuracy of indirect immunofluorescence on sodium chloride-split skin in the differential diagnosis of bullous pemphigoid and epidermolysis bullosa acquisita. Indian J Dermatol Venereol Leprol 2011;77:677-82.  Back to cited text no. 7
[PUBMED]  [Full text]  
8.
Gammon WR, Briggaman RA, Inman AO 3rd, Queen LL, Wheeler CE. Differentiating anti-lamina lucida and anti-sublamina densa anti-BMZ antibodies by indirect immunofluorescence on 1.0 M sodium chloride-separated skin. J Invest Dermatol 1984;82:139-44.  Back to cited text no. 8
    
9.
Ameglio F, D'Auria L, Bonifati C, Ferraro C, Mastroianni A, Giacalone B, et al. Cytokine pattern in blister fluid and serum of patients with bullous pemphigoid: Relationships with disease intensity. Br J Dermatol 1998;138:611-4.  Back to cited text no. 9
    
10.
Bhol KC, Rojas AI, Khan IU, Ahmed AR. Presence of interleukin 10 in the serum and blister fluid of patients with pemphigus vulgaris and pemphigoid. Cytokine 2000;12:1076-83.  Back to cited text no. 10
    
11.
Sun CC, Wu J, Wong TT, Wang LF, Chuan MT. High levels of interleukin-8, soluble CD4 and soluble CD8 in bullous pemphigoid blister fluid. The relationship between local cytokine production and lesional T-cell activities. Br J Dermatol 2000;143:1235-40.  Back to cited text no. 11
    
12.
Wakugawa M, Nakamura K, Hino H, Toyama K, Hattori N, Okochi H, et al. Elevated levels of eotaxin and interleukin-5 in blister fluid of bullous pemphigoid: Correlation with tissue eosinophilia. Br J Dermatol 2000;143:112-6.  Back to cited text no. 12
    
13.
Bean SF. Blister fluid in bullous pemphigoid. Acta Derm Venereol 1970;50:95-6.  Back to cited text no. 13
    
14.
Daneshpazhooh M, Shahdi M, Aghaeepoor M, Hasiri G, Chams C. A comparative study of antibody titers of blister fluid and serum in patients with subepidermal immunobullous diseases. Int J Dermatol 2004;43:348-51.  Back to cited text no. 14
    
15.
Patsatsi A, Vyzantiadis TA, Devliotou-Panagiotidou D, Chrysomallis F, Sotiriadis D. Detection of anti-BP180NC16a and anti-BP230 autoantibodies in blister fluid of patients with bullous pemphigoid: The first survey in Greece. Clin Exp Dermatol 2008;33:183-5.  Back to cited text no. 15
    

What is new?

  • Blister fluid in sAIBD contains autoantibodies and other cytokines and may be used instead of serum for IIF microscopy
  • This may be particularly useful technique in patients with poor venous access and children.


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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