ORIGINAL ARTICLE
Year : 2021 | Volume
: 66 | Issue : 4 | Page : 360--365
Utility of Immunofluorescence Antigen Mapping in Hereditary Epidermolysis Bullosa
Raghavendra Rao, Varsha M Shetty
Department of Dermatology, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, India
Correspondence Address:
Varsha M Shetty
No. 21, Department of Dermatology, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal-576104, Karnataka
India
Abstract
Background: Epidermolysis bullosa (EB) is characterized by blisters and erosions on the trauma-prone areas of the body. It occurs as a result of mutation in the genes encoding structural proteins. Transmission electron microscopy (TEM) is considered the gold standard test in the laboratory diagnosis of EB. However, this test requires a lot of expertise and is not widely available. Immunofluorescence antigen mapping (IFM) is considered a suitable alternative with comparable sensitivity and specificity. However, there is paucity of studies analyzing the utility of IFM in the diagnosis of EB. Aims and Objectives: To study the utility of IFM in the laboratory diagnosis of EB. Materials and Methods: A cross-sectional study was conducted involving 179 biopsy specimens of patients with EB. IFM was carried out using a panel of monoclonal antibodies against K14, laminin 332, type IV collagen, and type VII collagen. Results: Diagnosis of EB simplex (EBS), junctional EB (JEB), and dystrophic EB (DEB) was made in 104, 28, and 26 biopsy specimens, respectively. The overall concordance rate was 41.3% with higher concordance rates in EBS. Conclusion: The present study is conducted to assess the efficacy of IFM in the diagnosis of EB with large sample size. Our study serves to establish IFM as an important tool in the diagnostic armamentarium of EB as the prognosis mainly rests on diagnosing the type of EB.
How to cite this article:
Rao R, Shetty VM. Utility of Immunofluorescence Antigen Mapping in Hereditary Epidermolysis Bullosa.Indian J Dermatol 2021;66:360-365
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How to cite this URL:
Rao R, Shetty VM. Utility of Immunofluorescence Antigen Mapping in Hereditary Epidermolysis Bullosa. Indian J Dermatol [serial online] 2021 [cited 2023 Dec 1 ];66:360-365
Available from: https://www.e-ijd.org/text.asp?2021/66/4/360/326108 |
Full Text
Introduction
Hereditary epidermolysis bullosa (EB) refers to a group of inherited blistering disorders affecting the skin and/or mucosa.[1] The blisters in EB develop predominantly on the trauma-prone areas of the body, hence these conditions are also termed "mechanobullous" diseases. Based on the ultrastructural level of split, four major types of EB have been recognized, namely, EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB), and Kindler syndrome (KS). The level of split in EBS is within the epidermis, whereas in JEB and DEB, the split occurs within the lamina lucida and sublamina densa regions, respectively.[1],[2] Patients with KS exhibit split at multiple planes.[2],[3] Based on the phenotype, protein mutated, and mode of transmission, the four major types have been further subclassified into 30 subtypes.[1] It is often difficult to distinguish various EB types based on the clinical phenotype alone, especially in the neonatal period. Clinical parameters such as nail changes, milia formation that are generally used to classify and subclassify patients with EB may not be evident in all patients in the neonatal period. Prognostication of EB, to a large extent, depends upon its types and subtypes. To offer proper counseling regarding the prognosis of the affected child to parents and other caregivers, the clinician must make a more precise diagnosis.[4]
Light microscopy has a limited role in the diagnosis of EB as the level of cleavage cannot be precisely ascertained.[4] Transmission electron microscopy (TEM) is considered the gold standard test for the laboratory diagnosis of EB. One of the main advantages of TEM is its ability to visualize the protein deficient in the epidermis or DEJ. However, TEM is expensive, time-consuming, and requires skill and expertise in interpretation. The results are to a certain extent operator-dependent. Hence, very few laboratories around the globe offer this facility.[4],[5],[6],[7] Immunofluorescence antigen mapping (IFM) is considered as an alternative tool in the diagnosis of EB; it is based on the principle of detecting structural proteins in the epidermis and or dermoepidermal junction (DEJ) using monoclonal antibodies.[1] There are very few studies describing the utility of IFM in the diagnosis of EB. In this study, we present the data of IFM done in our laboratory.
Materials and Methods
In this cross-sectional study, we analyzed skin biopsy specimens of 179 patients with EB who were subjected to IFM over 5 years from 2014 to 2019. This included skin biopsy specimens of both in-house patients (n = 45) as well the biopsy specimens sent to our laboratory by the trained dermatologists from outside centers (n = 134). All biopsy specimens were washed overnight in a rotator at 4°C. They were then embedded in an optimum cutting temperature (OCT) compound and 6 μm frozen sections were taken on special adhesive slides. The slides were washed in phosphate buffer solution (PBS) and stained with monoclonal antibodies (anti-human immunoglobulin derived from mouse) against type VII collagen (type VII col), type IV collagen (type IV col), laminin 332 (lam 332), and keratin 14 protein (K14) for 1 h [Table 1]. The sections were again washed in PBS and then incubated with secondary antibody for an hour (anti-IgG mouse-specific antibody raised in rabbit and tagged to a fluorescent dye at 1:200 dilution). Frozen sections of normal human skin (NHS) were used as a control. The slides were then mounted in buffered glycerol and examined under a fluorescent microscope by a trained dermatologist.{Table 1}
Results
Patient demographics
Out of 179 biopsy specimens, 105 belonged to male (58.6%) and 74 to female patients (41.3%); the average age of the patient in the study group was 31.5 years (range: 1 day–63 years). The majority of the biopsy specimens belonged to neonates (59.78%), infants (9.49%), and postinfantile age group (30.7%) accounted for the rest of the cases. A simple algorithm as shown in [Figure 1] was used to categorize patients with EB based on IFM.{Figure 1}
Immunofluorescence characteristics in the group showing split on frozen section (n = 125)
Frozen sections of 125 biopsy specimens showed a sub-epidermal split on IFM; among these, a diagnosis of EBS was offered in 76 specimens as the basement membrane zone (BMZ) staining with lam 332, type IV col, and type VII col were located on the floor of the split. In addition, 18 specimens showed clumping of K14 staining, whereas another eight sections showed the absence of K14 staining of basal keratinocytes.
A diagnosis of JEB was made in 27 specimens; BMZ staining with type IV col and type VII col were seen on the dermal side of the split whereas the staining with lam 332 was either absent (n = 8) or reduced (n = 19).
Twenty-two cases were diagnosed as DEB; BMZ staining with type IV col and lam 332 were seen on the roof of the split ("toward the epidermal side"). Out of these, 10 specimens showed complete absence of type VII col staining at BMZ, whereas 12 cases showed reduced staining of type VII col when compared with NHS, which was used as a control.
Immunofluorescence characteristics in the group showing no split on frozen section (n = 54)
There was no split either at the DEJ or within the epidermis in 54 specimens. Among them, 28 specimens showed clumping of K14 staining, thus a diagnosis of EBS was offered in these patients. Four specimens revealed reduced staining of type VII col, whereas one specimen showed a complete absence of staining at BMZ with lam 332. In another 14 specimens, BMZ staining with lam 332 and type VII col were comparable with that of NHS, thus ruling out the possibility of severe generalized JEB and DEB in these patients.
In the remaining seven specimens, a conclusive diagnosis could not be ascertained. The main reasons for inconclusive reporting being poor specimen (absence of epidermis) in four samples and formalin contamination of the three specimens.
Correlation of clinical data and IFM findings
Using IFM, a precise diagnosis of EBS could be established in 47 out of 62 clinically suspected cases of EBS (concordance rate of 76%). The concordance rate in JEB and DEB were 43% and 30%, respectively. Concordance rate was 43.2% (54 out of 125 cases) in the group, which showed a split compared with the group without split (35%). In addition, IFM could establish the diagnosis of EB subtype in 60 out of 71 cases (85%) where the subclassification of EB was not possible clinically [Table 2].{Table 2}
Discussion
In 1981, Hintner et al. described the utility of IFM in the diagnosis of EB; besides revealing the level of the split it also helps in assessing whether a particular protein is expressed normally, reduced, or absent. IFM is a type of indirect immunofluorescence test wherein initially, a primary antibody is added to the frozen sections of the patient's skin. The primary antibody belongs to the IgG subclass raised in mice against the different structural proteins that are likely to be mutated in EB. Later, a secondary antibody is added, which is tagged to a fluorochrome to identify the antigen-antibody complex. The secondary antibody is an anti-mouse antibody of the IgG subclass. Fluorescein is one of the commonly used fluorochromes that emits lime-green color fluorescence, the intensity of which corresponds to the abundance of the protein.[4],[8] A previous study by Yiasemides et al. has shown that IFM has a higher sensitivity and specificity than TEM (97% vs. 71% and 100% vs. 81%).[9] Presently, IFM is considered the primary modality in the diagnosis and classification of EB with the advantage of being relatively cheap, simple, and quick to perform and interpret.[1]
In this study, we have analyzed the biopsy specimens of patients with EB who were subjected to IFM using a limited panel of monoclonal antibodies against type VII col, type IV col, lam 332, and K14. Though type IV col is not affected in EB, it provides a useful clue to distinguish DEB from JEB, especially when there is cleavage at the DEJ. In DEB, the staining with type IV col is seen on the roof whereas, in the case of JEB, the staining is seen on the floor.
IFM could ascertain the major type of EB in 88.26% of biopsy specimens in our study (158/179). This was higher than the rates reported by Hiremagalore et al. (77%)[2] but less than that reported by Yiasemides et al. (97%) and Barzegar et al.(95%).[9],[10]
EBS was the most common type of EB encountered in our study (n = 104, 58%) similar to Hiremagalore et al.(37%).[2] In contrast, Barzegar et al. reported DEB (65%) as the commonest variety in their study with EBS (14%) being the least common type.[10] This could be attributed to the racial and ethnic variations in different populations. Concordance rate between the clinical and IFM diagnosis for EBS in the present study was 76%; EBS was diagnosed based on the staining of all the antigens (type IV col, type VII col, and lam 332) to the floor of the split and or abnormal K14 staining in the form of reduced, absent staining, or clumping as compared with NHS [Figure 2] and [Figure 3]. There was a reduction in the intensity or total absence of staining with K14 in eight specimens raising the possibility of an autosomal recessive type of EBS. In addition, 46 specimens exhibited clumping of K14 indicating mutations of keratin protein. However, genetic analysis could not be done in any of our cohorts due to the lack of facilities.{Figure 2}{Figure 3}
A diagnosis of JEB and DEB was made in 28 (15.64%) and 26 (14.5%) biopsy specimens, respectively, in the present study. A previous study from India (n = 86) also observed these two subtypes to be less common than EBS (15 cases of JEB, 17 cases of DEB, and 2 cases of KS).[2] This is in contrast to the Iranian study (n = 95), wherein DEB (62 cases) was found to be the predominant type of EB (14 cases of JEB, 13 cases of EBS, and 1 case of KS).[10]
IFM could confirm the diagnosis of JEB in 28 biopsy specimens; nine specimens showed complete absence of staining with lam 332 thus confirming the diagnosis of the severe, generalized type of JEB (formerly known as Herlitz type). In the remaining 19 specimens, there was a marked reduction in the staining of BMZ with lam 332 when compared with that of NHS thus suggesting the diagnosis of generalized intermediate type (formerly known as a non-Herlitz type of JEB). The subepidermal split was seen in 27 specimens and staining with type IV col was seen localizing to the floor of the split, which is a consistent feature of JEB [Figure 4] and [Figure 5]. In seven cases, where a provisional clinical diagnosis of JEB was considered, one showed features of EBS and three each showed features of JEB and DEB on IFM. The target proteins in the generalized intermediate type of JEB include collagen type XVII or lam 332. JEB associated with pyloric atresia shows reduced or absent staining of α6β4 integrin and plectin.[5] However, monoclonal antibodies against these proteins could not be used in our study.{Figure 4}{Figure 5}
A diagnosis of DEB was offered in 26 specimens; 10 specimens showed complete absence of BMZ staining with type VII col, which is a consistent feature of recessive DEB (RDEB) [Figure 6] and [Figure 7]. The remaining 16 specimens showed a marked reduction of BMZ staining with type VII col suggesting the diagnosis of dominant DEB (DDEB). IFM offered a diagnosis of DEB in 11 out of 37 clinically suspected cases thus amounting to a concordance rate of 30%.{Figure 6}{Figure 7}
Two cases that were clinically diagnosed as Kindler syndrome (KS); however, on IFM showed features of EBS thus amounting to a discordance. Both these cases did not display a split on IFM and a diagnosis of EBS was made by clearly ruling out JEB and DEB. In addition, a broad reticulated staining pattern of type IV col, type VII col, and lam 332 was not seen in our patients. It is important to note that a definitive diagnosis of KS is impossible without gene sequencing.[10]
Overall, the concordance between clinical features and IFM was 41.3% in contrast to Hiremagalore et al. with a reported concordance of 57%. The difference in the rate of concordance could be due to interobserver variation in clinical evaluation and diagnosis, incomplete phenotypic expression of the disease at the time of biopsy, and variability in biopsy techniques. Majority of the cases (134 out of 179) had their biopsy samples sent to us for IFM reporting, which could have resulted in variations in biopsy technique as well as clinical evaluation. Ideally, shave/punch biopsy from an artificially induced blister is recommended in patients with EB;[4] however, the type, as well as the site of biopsy, was not mentioned in some of the samples that were sent to us for reporting. Moreover, the present study sought concordance between IFM and clinical features and not with other diagnostic modalities such as TEM or gene sequencing, which could have probably yielded higher concordance rates and also established accuracy. Nonetheless, IFM could establish the diagnosis in 60 out of 71 cases (85%) where the subclassification of EB was not possible clinically.
Yiasemides et al. reported a higher rate of sensitivity, specificity, and predictive value of IFM compared with TEM though it was not statistically significant. Out of a total of 33 cases of EB, IFM could classify EB in 97% of cases compared with TEM, which could classify in 80% of cases. In seven cases where the results of TEM and IFM did not match, the IFM results agreed with the genetic results in six of the seven cases. These discordant cases mainly were JEB and DEB where IFM was more accurate than TEM in clinching the diagnosis.[9] Moreover, the present study revealed that it is easier to identify the type of EB in those specimens that display a split owing to the localization of the antibodies to the floor or roof of the split, thus helping in easy interpretation.
Conclusion
To conclude, EB continues to be a challenging scenario in the Indian setup as there are not many dedicated diagnostic and referral centers. Given TEM and DNA-based mutational analysis is out of reach for most of the practicing dermatologists in India, it is vital to increase awareness regarding IFM as a useful diagnostic tool. The diagnostic accuracy of IFM can be improved with a larger panel of monoclonal antibodies and an improvement in biopsy techniques.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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