The Study Of Application Of C3d And C4d Immunohistochemistry In Bullous Pemphigoid

Background: Bullous pemphigoid(BP)is the most frequently encountered acquired auto-immune blistering disease especially in the elder beyond the age of 60.Clinical features of the disease are tense pruritic fluid filled blisters and bulla measuring up to several centimeters in diameter.BP180 and BP230 are recognized as BP antigens mainly,both of which are part of the hemipontin.Most antibodies in BP specifically bind to the NC16 A region of BP180,which is a non-collagenous extracellular antigenic site.Separation in BP occurs at the level of the lamina lucida.A skin biopsy from an established blister revealed subepidermal blisters,often associated with superficial dermal lymphocytes,eosinophils,granulocytes,mast cells,and monocytes/macrophages.These inflammatory cells are also present in the blister fluid of BP.Direct immunofluorescence(DIF)showed specific Ig G and/or C3 deposition were linear along the basement membrane zone.Complement components and activation fragments including C1 q,C3,C3 c,C3d,C4,C4 d,C5,C5b-9,FB,FH have been proved at the basement membrane zone and blister fluid in BP.These findings suggest that both the classical and alternative pathways involved in the pathogenesis of BP.As the final products of complement,C3 d and C4 d were stable in tissues,while the rest of complement split products were decomposed by enzymes.Combining with basement membrane by covalent bond,C4 d can exist for a long time.Therefore,C4 d showed the existence of humoral immune response especially in organ transplantation.In recent years,some important discoveries have been made in the application of C4 d in pathology,such as the application of C4 d in the diagnosis of lymphoma,autoimmune diseases,and gestational nephropathy.C3 d is one of the smallest fragments of complement C3 molecule cleaved by protease,which always maintains a covalent connection with the antigen.In recent years,complement split products C3 d and C4 have been proved to be deposited along the basement membrane zone in formalin fixed paraffin embedded tissue,which are expected to be potential substitutes for DIF in the near future.DIF using frozen section material from a fresh/preserved perilesional biopsy is the gold standard for the immunopathologic diagnosis of BP.DIF in BP shows linear dermoepidermal junction(DEJ)staining for C3,with or without staining for Ig G.But DIF depends on the availability of fresh tissue and specialized laboratories.Currently,there are few researches on the diagnosis of BP by C3 d,C4d immunohistochemical staining in formalin-fixed paraffin-embedded tissue,and it is controversial that immunohistochemistry(IHC)can replace DIF in the diagnosis of skin bullous diseases.Clinically,DIF is not easy to be obtained,so it is of great significance to evaluate the diagnostic value of IHC by this research.Objective: Whether IHC can be used as a sensitive method to assist the diagnosis of BP.To compare the degree of complement deposition in anti-BP180 Ig G+-type BP and anti-BP180 Ig G--type BP.Methods: We examined 36 patients with confirmed BP between 2015 and 2018 by IHC for C3 d,C4d,and 10 healthy controls were selected as research object.Result: By IHC diagnostic deposits of immunoreactants were found in 88.8% of all tested cases.Deposits of C3 d,C4d were found in 83.3%,33% of cases respectively.Conclusion: The deposition of C3,IgG by DIF was nondistinctive of that of C3 d by IHC.However,the result of C4 d is the opposite of C3 d.The deposition of C3 d had no correlation with the existence of anti-BP 180 Ig G.C4 d is correlated with the existence of anti-BP180 Ig G.Our findings demonstrate that paraffin section C3 d with but not C4 d immunohistochemical staining has been proved to be diagnostically useful,especially when DIF is unavailable.Negative findings,however,do not exclude a possible diagnosis of BP and should prompt an additional biopsy for DIF.