Remember me
Abstract
Background: There are several methods for the diagnosis of autoimmune bullous disease. Direct immunofluorescent (DIF) testing is an important diagnostic method in the diagnosis of immunobullous disease but requires skilled pathologist, fresh tissue and well-equipped laboratory to perform the procedure. The immunohistochemistry analysis of C4d and C3d is easily compared with other methods. This study was conducted to assess the value of immunohistochemistry (IHC) analysis for expressions of C3d and C4d in the diagnosis of bullous pemphigoid (BP). Aims and Objectives: This study was conducted to assess the value of immunohistochemistry (IHC) analysis for expressions of C3d and C4d in the diagnosis of bullous pemphigoid (BP). Materials and Method: We applied C4d and C3d immunohistochemistry on formalin-fixed, paraffin-embedded tissue on 30 cases of bullous pemphigoid that was confirmed by direct immunofluorescence (DIF) evaluation as well as 16 cases in control group (12 cases of herpetiform dermatitis, 3 cases of linear IgA dermatosis and 3 cases of bullous lichen planus). Results: Mean and SD of age were 68.13 ± 14.00, female to male ratio was 1:3. In cases where both C3d and C4d staining were positive, the intensity of C3d staining was higher than C4d. Twenty-two cases showed C4d-positive staining in IHC study, such that in seven cases focal staining and in 15 cases diffuse staining were observed. Also 26 cases showed C3d-positive staining in IHC study such that in four cases focal staining and in 22 cases diffuse staining were observed. In cases with C3d-positive staining, there were 21 cases of deposition only on the bullous floor, one case on the bullous roof and four cases on the bullous roof and floor. In cases with C4d-positive staining, there were 17 cases of deposition on the bullous floor, two cases only on the bullous roof and three cases on the roof and floor. All control cases were negative for C3d and C4d staining in the dermoepidermal junction. For C3d immunohistochemical staining, sensitivity, specificity, positive predictive value and negative predictive value were 86.66%, 100%, 100% and 80%, respectively, and for C4d immunohistochemical staining, respectively, were 73.3%, 100%, 100% and 66.66%. Conclusion: The immunohistochemical specificity of C4d and C3d on tissue blocks is the same as that of direct immunofluorescence test on fresh tissue, but it is less sensitive, so positive results for C3d and C4d immunohistochemical staining on paraffin blocks can be used to confirm the diagnosis of bullous pemphigoid.
Keywords: C3d, C4d, bullous pemphigoid, immunohistochemistry
Background 
Bullous pemphigoid (BP) is an autoimmune disorder characterised by intraepidermal detachment due to the autoimmune condition. Immune complex formation promotes disarray, resulting in flaccid blisters and erosions predominantly on the skin.[1]
Direct (DIF) and indirect (IIF) immunofluorescence allows localisation, titration and recognition of in situ and circulating autoantibodies. Immunofluorescence as a specific and sensitive technique is the best diagnostic approach in autoimmune bullous disorders.[2] Nevertheless, immunofluorescence typically requires other specimens.[3] Immediate tissue processing is needed if the fragment is kept in a saline solution. Preparation of frozen biopsies depends on a cryostat as a trained technician; in addition, a fluorescent microscope is necessary to analyze the slides.[4]
Immunohistochemistry (IHC) allows expression of antigen recognition and antibody bound to tissue, with advantage of using formalin-fixed paraffin-embedded (FFPE). New chromogens and antibodies, computerised analysis and automated processing increased sensitivity and specificity of IHC.[4] This method is widely used as a reliable method in diagnostic pathology.[2],[3]
However, there is scarce data about the use of IHC for the diagnosis of ABD, especially in comparison with IF assays. So this study was conducted to assess the value of immunohistochemical analysis for expressions of C3d and C4d in the diagnosis of bullous pemphigoid.
Patients and Methods Study setting
This article is a hospital-based study conducted in Ghaem and Imam Reza Hospitals.
Study population
The study population included patients with vesicular pemphigoid disease referred to Imam Reza and Ghaem hospitals whose diagnosis was confirmed by histology and direct immunofluorescence staining.
Inclusion criteria
Diagnosis of bullous pemphigoid confirmed by histopathology and direct immunofluorescence.
Sufficient data in the patient profile.
Exclusion criteria
Insufficient tissue for immunohistochemistry.
Uncertainty of diagnosis.
Incomplete profiles.
Measurements
Using the information available in the computer system and archives of the Pathology Department of Ghaem and Imam Reza Hospitals, patients with related conditions who have been diagnosed with bullous pemphigoid histopathology have been selected and using the examination files of direct immunofluorescence results, we isolated specimens whose diagnosis of bullous pemphigoid was confirmed by direct immunofluorescence staining of basal membranes with C3 or IgG, then we removed the formalin-fixed paraffin blocks of these patients from the pathology archive of Ghaem and Imam Reza hospitals. And blocks that had insufficient tissue were removed and finally 3–4 μ sections were prepared from the remaining 30 tissue blocks of these patients and the immunohistochemical tests were performed on the blocks using C4d and C3d monoclonal antibodies (abd/clone 10-11, abcam/ab 136916) and the results of C3d and C4d deposits. Deposition staining along the dermoepidermal junction was scored as (zero: no reactivity, +1: weak reactivity, +2: moderate reactivity, +3: strong reactivity) and cases with a score of +1, +2 and +3 were considered positive cases. To determine the specificity and sensitivity of this method, 16 skin samples from patients with autoimmune bullous diseases other than bullous pemphigoid diagnosed with direct immunofluorescence were used as a control group, which included 12 cases of herpetiform dermatitis, three cases of linear IgA dermatosis and one case was bullous lichen planus. Finally, the results of immunohistochemistry in the case group were compared with the results of their direct immunofluorescence and also with the results of IHC in the control group, and the sensitivity and specificity of immunohistochemistry in the diagnosis of this disease were measured.
Study tools
In this study, a data checklist was used to collect information. In addition, data collection was based on laboratory tests.
Ethical considerations
The study group adheres to the principles of medical ethics introduced by the Health Ministry and the Declaration of Helsinki and legislation in the medical ethics committee of Mashhad University of Medical Sciences. In addition, ethical committee of Mashhad University of Medical Sciences approved protocol of study with ethical code as 3302.
Statistical analysis
Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) 16 software (Chicago, IL). Fisher's Exact test, t-test, Mann–Whitney and Kruskal–Wallis tests were used to determine the relationship between C4d and C3d expression and each of the clinical-pathological parameters, and then the sensitivity and specificity of immunohistochemistry were determined. P-value less than 0.05 was considered statistically significant.
Results Demographic data
In the study, 13 patients (43.3%) were male and 17 patients (56.7%) were female and according to Fisher's Exact Test, there was no significant relationship between gender and C4d and C3d staining results (P = 1.0), also the mean and standard deviation of age in total was 68.13 ± 14.00 years and there was no significant relationship between C4d and C3d expression and age (P = 0.07) [Table 1].
Table 1: Frequency distribution of gender and age based on C3d and C4d testsClinical data
In the study performed on 30 bullous pemphigoid blocks, 22 cases (73.3%) with C4d marker, 26 cases (86.7%) with C3d marker and in total staining with two markers, 26 cases (86.7%) had a positive staining in the dermoepidermal junction [Table 2]. In addition, according to C4d marker, 15 cases (68.2%) had a diffuse staining and seven cases (31.8%) had a focal staining, and according to C3d marker, 22 cases (84.6%) had a diffuse staining and four cases (15.4%) had a focal staining [Table 3]. In C3d positive cases, 27% had an intensity of +1, 42% had an intensity of +2 and 31% had an intensity of +3 and in C4d positive cases, 46% had an intensity of +1, 36% had an intensity of +2 and 18% had an intensity of +3.
In cases with C3d-positive staining, there were 21 cases of deposition only on the blister floor (80.8%), one case on the blister roof (3.8%) and four cases on the blister roof and floor (15.4%) [Figure 1]a. In total, in 25 cases, staining on the floor of the blister (96%) and in 5 cases, staining on the roof of the blister (19%) was observed. In cases with C4d-positive staining, there were 17 cases of deposition on the blister floor (77.3%), two cases only on the blister roof (9.1%) and three cases on the roof and floor (13.6%) [Figure 1]b. In total, in 20 cases, staining on the floor of blister (90%) and in five cases staining on the roof of the blister (22%) was observed [Table 4].
Figure 1: Linear C3d staining along the dermoepidermal junction (roof and floor) on formalin-fixed paraffin-embedded tissue from a case of bullous pemphigoid (×400)(a) Linear C4d staining along the dermoepidermal junction (roof and floor) on formalin-fixed paraffin-embedded tissue from a case of bullous pemphigoid, (×400) (b)All control cases were negative for C3d and C4d staining in the dermoepidermal junction. Based on the evaluation performed for C4d marker, specificity, sensitivity, positive predictive value, negative predictive value and accuracy were 100%, 73.33%, 100%, 66.66% and 82.6%, respectively. Also, in the study performed with C3d marker, specificity, sensitivity, positive predictive value, negative predictive value and accuracy were 100%, 86.66%, 100%, 80% and 91.3%, respectively. Also, in total, C3d and C4d staining specificity, sensitivity, positive and negative predictive value and accuracy were 100%, 86.66%, 100%, 80% and 91.3%, respectively, which were similar to C3d alone [Table 5].
Table 5: Sensitivity, specificity, negative and positive predictive value of C4d and C3d Discussion Cutaneous autoimmune diseases are characterised by the presence of antibodies against specific adhesion antigens in the epidermis or dermoepidermal junction.[5] Binding of these antibodies to the target antigens induces the keratinocytes of the epidermis or the basement membrane region to lose their connections, leading to blistering. Diagnosis of autoimmune bullous diseases is based on clinical findings, histopathology and immunofluorescence.[6],[7]
Complement C3 protein plays an important role in activating the complement system in the classical and secondary pathways. C3b reacts with other components of the complement cascade and eventually leads to the formation of a membrane attack complex.[8] The membrane attack complex leads to lysis of target cells. C4d is also a complementary classic track product. C4b and C3b inactivation is caused by destructive factors, C4d and C3d.[9] C3 and other components of the complement system disappear after the cell damage has resolved, but C4d and C3d remain attached to the target cell and can therefore be used as markers to activate complement.[10] Recently, few immunohistochemical studies have been performed on paraffin-embedded tissue to show C3d and C4d deposits in the skin, so in this study, we examined the immunohistochemical expression of C3d and C4d in paraffin-embedded tissue as a substitute for DIF. In our study, it was observed that out of 30 bullous pemphigoids examined, 26 showed linear C3d deposition along the dermoepidermal junction (86.7%) and in four cases no deposition was observed along the dermoepidermal junction (13.3%). These results were 100% in the Magro et al. study and 97% in the Pfaltz et al. study, which was slightly higher than our study,[11],[12] also 22 out of 30 bullous pemphigoids showed linear C4d deposition along the dermoepidermal junction (73%), which is almost identical to studies by Chandler et al. (77%), Kwon et al. (83%) and Villani et al. (86.2%),[13],[14],[15] but in the study by Magro et al., was 23%, which is less than our study and other studies, which could be due to differences in the handling of samples (such as duration of fixation) and the IHC protocol.[11] In our study, there was a significant relationship between the C3d and C4d staining (P = 0.00). A total of 68.2% of C4d positive cases, had diffuse staining and 31.8% had focal staining, and for C3d positive cases, 84.6% of cases had diffuse staining and 15.4% had focal staining. In C3d positive cases, 27% had an intensity of +1, 42% had an intensity of +2 and 31% had an intensity of +3 which was almost similar to the Pfaltz et al. study[12] and in C4d positive cases, 46% had an intensity of +1, 36% had an intensity of +2 and 18% had an intensity of +3, which was similar to the study by Magro et al.[11], the number of cases with an intensity of +1 and +2 was more than +3. There was a significant relationship between the staining intensity of two markers (P = 0.001) and the staining intensity of C3d was higher than C4d, which was similar to the Magro et al. study.[11] Deposition on the blister floor was observed in 77% of C4d positive cases and 81% of C3d positive cases. The deposition in the floor of the blister was higher than the roof, which showed more sensitivity for staining on the blister floor than the blister roof, which was similar to the studies by Pfaltz et al. and Chandler et al.[12],[13]
Finally, specificity, sensitivity, positive predictive value, negative predictive value and accuracy of C4d immunohistochemical staining were 100%, 73.33%, 100%, 66.66% and 82.6%, also, specificity, sensitivity, positive predictive value, negative predictive value and accuracy of C3d immunohistochemical staining were 100%, 86.66%, 100%, 80% and 91.3% respectively. Accordingly, most studies in this field have been consistent with the results of our study,[11],[12],[13],[14],[15] which indicates the diagnostic efficiency of immunohistochemistry study in bullous pemphigoid.
Conclusion From our study and its comparison with direct immunofluorescence results as the diagnostic gold standard method, it was found that the specificity of immunohistochemical staining of C4d and C3d on tissue blocks is the same as direct immunofluorescence test on fresh tissue, but the sensitivity of staining with C3d and C4d is lower. In addition immunohistochemical staining of C3d and C4d on paraffin blocks can be used to confirm the diagnosis of bullous pemphigoid without the need for fresh frozen tissue and direct immunofluorescence, but the negative results of immunohistochemistry should be confirmed by immunofluorescence.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
Comments (0)