Application Of Direct Immunofluorescence In Autoimmune Bullous Dermatoses

[Research Background] : The Autoimmune bullous dermatoses(AIBD)is the autoimmune disease manifested as the autoimmune disease with erythema,blisters and bullosa as the basic damage on the skin and/or mucosa.Its clinical manifestations are in the normal skin or erythema on the basis of blisters,bullosa,often accompanied by erosion,including Pemphigoid,Bullous Pemphigoid(BP),Linear Ig A bullous dermatosis(LABD),etc.Bullous pemphigoid(BP)is the most common.It is characterized by the presence of autoantibodies in the body,which bind to specific structural proteins and mediate skin and/or mucosal damage.Therefore,the detection of patients’ autoantibodies is of great significance for diagnosis and clinical decision-making.Monitoring the level of autoantibodies helps to assess the activity of the disease.With the development of science and technology,many new techniques and methods have appeared in clinical examination,which help to further clarify clinical diagnosis and reduce the probability of misdiagnosis and missed diagnosis.Immunofluorescence technique plays an important role in the diagnosis of common autoimmune bullous skin diseases and is also the gold standard for the diagnosis of some bullous skin diseases.IF is to label the antigen or antibody with fluorescent pigment,combine it with the corresponding antibody or antigen,and test it through fluorescence microscope,so as to obtain the test result,which can be used to detect various antigens in tissues or suspended on cells.It has the characteristics of high precision,high sensitivity,high accuracy and anti-interference.Direct immunofluorescence(DIF),by detecting the immune reactants formed by autoantibodies and antigens,can well diagnose autoimmune bullous bullous skin disease(AIBD),and can also be used in the diagnosis of other autoimmune skin diseases,such as lupus erythematosus,vasculitis.The diagnosis of DIF provides the basis for the next development of treatment plan,and the change of DIF results before and after treatment is considered to be one of the main indicators of therapeutic efficacy.The deposition of immunoreactants in tissues was detected by labeling frozen sections with fluorescent antibodies,mainly located in epithelial tissue,basal membrane zone(BMZ),and around blood vessels.The immunoreactants measured by DIF include immunoglobulin antibodies,such as Ig A,Ig G,Ig M,fibrin,complement C3,C4,etc.The staining intensity grades range from + to ++++,and the deposition patterns range from granular,linear,and reticular.DIF results of AIBD were different.BP mainly showed linear deposition of Ig G and(or)C3 in the basement membrane zone(BMZ),and a few patients had linear deposition of Ig A,Ig E,Ig G and Ig M granular deposition.In pemphigus DIF test,Ig G,C3,Ig A and Ig M were deposited between spinous cells,which were reticular,linear and granular.Labds produce linear or banded and uniform Ig A deposits on the BMZ.Occasionally,Ig G,Ig M,and C3 are found.The diagnosis of various types of AIBD is mainly based on the comprehensive assessment of clinical manifestations,histopathological findings,DIF or IIF,so as to obtain the most accurate clinical diagnosis.The lack of any one of them may increase the rate of missed diagnosis and/or misdiagnosis,and the correct diagnosis is the basis of treatment.DIF plays an important role in the diagnosis of AIBD.According to its deposition site,composition,intensity and pattern,DIF provides clues for clinical diagnosis and is the cornerstone of subsequent treatment and disease remission.[Objective(s)]:The application value of DIF in AIBD was analyzed retrospectively,and the characteristics of fluorescent deposition of DIF in different types of AIBD were analyzed,and the results of DIF detection were compared with the results of histopathology and antibody detection by ELISA.[Methods]: The clinical data of all patients who underwent histopathologic examination and direct immunofluorescence in the outpatient/inpatient department of dermatology of our hospital from January 2016 to December 2022 were collected.The subjects were divided into AIBD group and control group according to clinical manifestations,histopathological examination,DIF and serum antibody detection results.The clinical data of the two groups were retrospectively analyzed to compare the difference of the positive rate of DIF between and within the groups.[Results]: The positive rate of DIF in AIBD group and control group was significantly different(P<0.001),and the positive rate of DIF in AIBD group was significantly higher than that in control group.In AIBD group,the positive rate of DIF in BP group and pemphigus group was slightly higher than that detected by histopathology and ELISA,but there was no significant difference among the three detection methods(P>0.05).DIF was positive in 90 of 94 BP patients(95.7%)and negative in 4 cases(4.3%),mainly with continuous linear deposition of Ig G and/or C3 in the basal membrane zone.Among 136 patients with pemphigus,134(98.5%)were positive for DIF,and 2(1.5%)were negative for DIF,mainly composed of Ig G and C3 interspinous cell reticular deposition.DIF was positive in 1 of 2 DH patients(50%),mainly Ig A and Ig G granules deposited in the basement membrane.DIF of 2patients with LABD was positive,showing linear continuous deposition of Ig A in the basement membrane zone.There were 75 cases(79.79%)of Ig G deposition in BP patients,82 cases(87.23%)of C3 deposition,Ig A,Ig M,C4,Fib and C1 q deposition were rare components,129 cases(94.85%)of Ig G deposition in pemphigus patients,111 cases(81.62%)of C3 deposition.Different sedimentary components can be found in each subtype of pemphigus.There was no significant difference in the distribution of immunofluorescence markers in different AIBD severity groups(P>0.05).[Conclusion]: DIF has an important role in the clinical diagnosis of autoimmune bullous dermatoses(AIBD)and has a high sensitivity(97%),especially for pemphigus vulgaris,dermatitis herpetiformis,and linear Ig A bullous dermatosis,making it the "gold standard" for diagnosis.The results of this study,as well as other domestic studies,show that the positivity rate of DIF testing in AIBD patients is significantly higher than that in other control groups.The specific deposition sites,deposition types,and deposition patterns of DIF have important implications for the classification of AIBD diseases,particularly Ig G,C3,Ig A,and Ig M.The positivity rate of DIF in this study is slightly higher than that of histopathology and ELISA,but there is no statistical difference between the three,which is consistent with other research results.However,DIF also has its limitations,and the positivity rate cannot reach 100%.In this study,3% of the patients were DIF negative and for these patients,further immunological examinations such as serum antibody detection are needed.