To the Editor: Laboratory diagnostic methods of bullous pemphigoid (BP) include direct immunofluorescence (DIF) microscopy, BP180 NC16A enzyme-linked immunosorbent assays (ELISA), and indirect immunofluorescence using salt-split skin (ssIIF) in our daily clinical practice. Immunofluorescence (IF) studies are important parts of the laboratory workup for BP. DIF is regarded as the gold standard for BP diagnosis; however, it is not specific and can also be found in other acquired autoimmune subepidermal blistering dermatosis. ssIIF was found to have a specificity of 100% for BP diagnosis and in some guidelines, it is recommended to perform ssIIF in every patient with clinically suspected BP. To date, it is still not defined whether ssIIF can be used as a routine diagnosis method of BP. The BP180 NC16A ELISA is a practical and reliable diagnostic test for BP; however, the false-positive results of BP180 NCA6A ELISA were recently reported in a wide range of dermatoses, which might cause its uncertainty in the diagnosis of BP. Thus, we conducted a single center retrospective study to compare diagnostic values of these tests for BP from Hospital for Skin Diseases, Chinese Academy of Medical Sciences between January 2014 and January 2019. It was approved by the local medical ethical committee (No. 2017-KY-022) and consent was obtained from patients prior to serum collection.
In this study, we analyzed the data of 569 patients who were tested by ssIIF, DIF, BP180 NC16A ELISA and histopathology at the initial disease stage prior to systemic therapy, and were followed for at least 1 year, and BP diagnosis was made on the basis of the following criteria: (1) tense bullae on body without scaring, and Nikolsky sign is negative; (2) histopathological finding shows subepidermal blister formation; (3) DIF shows linear deposits of IgG and/or C3 along the basement membrane zone (BMZ); (4) ssIIF shows linear IgG and/or C3 reaction with the epidermal side of BMZ in the salt-split skin; (5) index value of BP180 ELISA is more than 9 U/mL. Diagnosis of BP requires at least two columns, (4) and (5), or requires three columns, (1), (2), and (3) if it meets one column of (4) and (5). Based on these diagnosis criteria, 164 of 569 suspected BP patients were diagnosed with BP, and the remaining 405 patients were used as non-BP controls. Study flowchart and characteristics of patients are described in Supplementary Figure 1, https://links.lww.com/CM9/B92 and Supplementary Table 1, https://links.lww.com/CM9/B92.
Sensitivity and specificity were calculated for assessing diagnostic values of DIF, ssIIF and BP180 NC16A ELISA, and Kappa test was used to evaluate the consistency between these tests. Measurement data were shown as mean ± standard deviation, and were compared by Mann-Whitney U test or paired t-test. Numeration data with rate or composition ratio were analyzed by McNemar test, χ2 test or Fisher's exact test. Statistical analyses were done using SPSS statistics 22 (IBM Corp, Armonk, NY, USA). All tests were two-sided, and P < 0.05 was considered as statistically significant.
For diagnosis of BP, the sensitivity of DIF, ssIIF, and BP180 ELISA were 78.05%, 97.56%, 93.90%; and their specificity were 93.58%, 99.75%, and 95.55%, respectively [Supplementary Table 2, https://links.lww.com/CM9/B92]. In previous studies, the specificity of DIF and ssIIF was 98% to 99%, 97% to 100%, and the sensitivity was 88% to 91%, 73% to 97%, respectively.[2,3] The specificity of DIF was similar to the findings of the studies mentioned above, and further proved that DIF was not specific enough for BP diagnosis. Some researchers found routine serration pattern analysis can help to improve the specificity of DIF in BP. The sensitivity of DIF in our data is lower than the studies mentioned above, and the reason may be due to availability of serologic test data, or ethnic differences, which was supported by another domestic article, and their positive rate of DIF is 82%. Some researchers found IgG4 testing may be helpful in definitively diagnosing BP in patients with negative or equivocal linear IgG deposition on DIF. In this study, the high specificity of ssIIF was consistent with the reports mentioned above, and the sensitivity was similar to the recent study from 13 international centers, but higher than the previous report. The relative higher sensitivity may be caused by transporting and tackling skin tissue timely which can avoid protein denaturation and degradation. Given all the data of ssIIF on BP diagnosis, we considered ssIIF as a valuable method for BP diagnosis. In the present study, we found that BP180 ELISA results were negative in 6.1% BP patients, which might be caused by lower titers of anti-BP180 NC16A antibodies or existence of autoantibodies recognizing BP180 on non-NC16A domain and/or BP230 in BP sera. The results indicated that it needed to be combined with IF to avoid a misdiagnosis.
Next, the sensitivity and specificity of combined tests were analyzed. The sensitivity of ssIIF/BP180 ELISA (91.66%) is statistically significantly higher than those of ssIIF/DIF (75.61%), DIF/BP180 ELISA (71.95%), and the specificities of three combined tests are all 100% [Supplementary Table 2, https://links.lww.com/CM9/B92]. These results indicated that combined tests could obtain perfect specificity, although the sensitivity is decreased a little bit, and combination of ssIIF/BP180 ELISA has the highest sensitivity in BP diagnosis. In addition, Kappa coefficients between ssIIF/BP180 ELISA, ssIIF/DIF, and DIF/BP180 ELISA were 0.86, 0.706, 0.622, respectively (all P < 0.001). And BP180 ELISA was positively correlated with ssIIF in BP patients [Supplementary Table 3, https://links.lww.com/CM9/B92].
The limitation of this study is the absence of diagnostic criteria as a reference standard for the diagnosis of BP. Patients were diagnosis as BP by at least both positive ssIIF and positive BP180 NC16A ELISA, or a combination of clinical bulla, subepidermal bulla on routine histopathology and positive DIF with either positive ssIIF or positive BP180 NC16A ELISA which may bias the sensitivity and specificity of these three diagnostic methods.
In summary, ssIIF could be used as a routine laboratory diagnosis method for BP, and the combination of ssIIF and BP180 ELISA is better for diagnosis of BP.
This work was supported by grants from Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (Nos. CIFMS-2017-I2M-1-017, CIFMS-2021-I2M-1-059), and Scientific Research Project of Jiangsu Provincial Health Commission (No. ZD2021035).
Conflicts of interest
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