Application Of Immunofluorescence On The Diagnosis Of Autoimmune Bullous Diseases And Congenital Epidermolysis Bullosa

Immunofluorescence is an important diagnostic technology, it is widely used through using fiuorescein-labeled secondary antibody to recognize primary antibody, which can interact with antigen to detect whether there is corresponding antibody or antigen in the blood or in the tissue, then through the localization we can mapping it and give diagnosis. This method plays an important role in the diagnosis of autoimmune bullous diseases, it also extended to the diagnosis of congenital epidermolysis bullosa in recent years. In this study, we investigated the application of immunofluorescence technique in autoimmune bullous diseases and congenital epidermolysis bullosa.Part 1 Application of indirect immunofluorescence on the diagnosis of autoimmune intraepidermal bullous diseaseObjective To evaluate the diagnostic value of indirect immunofluorescence on three different kinds of substrates including normal skin, monkey esophagus and salt-split skin for the diagnosis of autoimmune intraepidermal bullous disease.Methods Clinical data of 71 patients with pemphigus and 56 cases of control including 26 patients with autoimmune subepidermal bullous diseases,15 patients with chronic eczema and 15 healthy people were collected. Indirect immunofluorescence on normal skin, monkey esophagus and salt-split skin three different kinds of substrates were performed respectively, observing patterns of fluorescein deposition and comparing its difference in different kinds of autoimmune intraepidermal bullous diseases and its sensitivities and specificities on different substrates.Results Intercellular deposition of IgG was visible when indirect immunofluorescence on normal skin, monkey esophagus and salt-split skin three different kinds of substrates was positive in some pemphigus patients, there sensitivities were 31.0%,83.1%,70.4% respectively and specificities were 96.4%,96.4%,94.6% respectively. The sensitivities between normal skin and monkey esophagus, normal skin and salt-split skin indirect immunofluorescence showed statistical significance (P< 0.001), the specificities among three different kinds of substrates had no statistical significance (P> 0.05). In both Dsgl and Dsg3 positive and only Dsgl positive pemphigus patients, sensitivities difference between normal skin and salt-split skin indirect immunofluorescence was statistical significance (P< 0.01). The sensitivity of only Dsg3 positive pemphigus patients using monkey esophagus as substrate was significantly higher than only Dsgl positive pemphigus patients, and the sensitivity difference was statistically significant (P< 0.05). Compare anti-Dsgl ELISA level between monkey esophagus indirect immunofluorescence positive and negative patients among only Dsgl positive pemphigus patients, there was no significant difference (P> 0.05), so did salt-split skin indirect immunofluorescence positive and negative patients.Conclusion Using monkey esophagus or salt-split skin as substrate indirect immunofluorescence is better than normal skin with higher sensitivity in pemphigus patients, salt-split skin can be used instead of normal skin when monkey esophagus is not availiable. Its sensitivity is higher in patients with only existing Dsg3 antibody than in patients with only Dsgl antibody when using monkey esophagus as substrate. The result of monkey esophagus and salt-split skin indirect immunofluorescence is not related with anti-Dsgl ELISA index level.Part 2 Application of indirect immunofluorescence on the diagnosis of autoimmune subepidermal bullous diseaseObjective To evaluate the diagnostic value of indirect immunofluorescence on three different kinds of substrates including normal skin, monkey esophagus and salt-split skin for the diagnosis of autoimmune subepidermal bullous diseases.Methods A total of 26 patients with autoimmune subepidermal bullous diseases, control including 71 patients with pemphigus,15 patients with chronic eczema and 15 healthy people were enrolled into this study. Indirect immunofluorescence on normal skin, monkey esophagus, salt-split skin three different kinds of substrates were performed respectively, observing patterns of fluorescein deposition and comparing its sensitivities and specificities on different substrates.Results Fluorescein linear deposition along the basement membrane zone was visible when indirect immunofluorescence on normal skin, monkey esophagus, salt-split skin three different kinds of substrates was positive in some pemphigoid patients. The sensitivities of subepidermal bullous diseases on the three substrates were 42.3%,69.2%, 96.1% respectively, the specificities were 98.0%,100.0%,97.0% respectively, and the sensitivities of bullous pemphigoid were 40.0%,75.0%,90.0% respectively. For pemphigoid patients, there existed statistical significance between normal-skin and salt-split skin indirect immunofluorescence sensitivity (P< 0.001), so did monkey esophagus and salt-split skin indirect immunofluorescence sensitivity (P< 0.05), specificities among the three different substrates had no statistical significance (P> 0.05). For bullous pemphigoid patients, there existed statistical significance between normal skin and salt-split skin indirect immunofluorescence sensitivity (P< 0.01). Compare anti-BP180 ELISA level between monkey esophagus indirect immunofluorescence positive and negative bullous pemphigoid patients, there was no significant difference (P> 0.05)Conclusion For pemphigoids, using salt-split skin as substrate is better than using normal skin and monkey esophagus with higher sensitivity and the result of monkey esophagus indirect immunofluorescence is not related with anti-BP180 ELISA index level.Part 3 Application of salt-split skin indirect immunofluorescence on the diagnosis of autoimmune subepidermal bullous diseasesObjective To assist diagnose three cases of special autoimmune subepidermal bullous diseases using salt-split skin indirect immunofluorescence method.Methods Salt-split skin indirect immunofluorescence was used to screen out special autoimmune subepidermal bullous diseases, enzyme-linked immunosorbent assay and western blot were used to establish definitive diagnosis.Results Clarify a definitive diagnosis of one case bullous pemphigoid, one epidermolysis bullosa acquisita and one P200 pemphigoid.Conclusion Salt-split skin indirect immunofluorescence shows fluorescein linear deposition along the epidermal side of the split in bullous pemphigoid, while linear deposition along the dermal side of the split in epidermolysis bullosa acquisita and P200 pemphigoid. Salt-split skin indirect immunofluorescence dermal side linear deposition hints further implement ELISA and western blot to detect corresponding antibodies to clarify the diagnosis.Part 4 Application of immunofluorescence on the classification diagnosis of congenital epidermolysis bullosaObjective To describe patterns of immunofluorescence mapping staining in different forms of EB and to classification diagnosis EB patients using different antibodies immunofluorescence mapping or immunohistochemistry mapping.Methods Tissue specimens were obtained from patients with congenital epidermolysis bullosa in outpatients and were made into frozen sections and paraffin sections, using immunofluorescence and immunohistochemistry methods staining keratin 14, type-IVcollagen and type-Ⅶ collagen respectively, at the same time using normal skin and salt-split skin as controls. Results were observed to make clear which parts of the blister the corresponding antibody labeled, then classifying EB patients.Results The blisters in epidermolysis bullosa simplex patients lay in the K14 labeled layer or below, and above the type-IV collagen labeled layer, patients with dystrophic epidermolysis bullosa blisters were below type-Ⅳ collagen, at the same time the label of type-Ⅶ collagen decreased significantly than control or even disappear. The cleft of salt-split skin lay below K14 labeled layer and above the type-Ⅳ collagen, type-Ⅶ collagen labeled layer.Conclusion Immunofluorescence mapping is an important method for EB diagnosis and subtyping EB patients, and it is superior to immunohistochemistry.