The positive rate and level of anti-PLA2R in our study were significantly higher in IMN patients than in non-IMN patients, which indicated that it was a specific biomarker in the differential diagnosis of IMN. Analyzing the baseline characteristics of the participants enrolled in our study, no significant differences were observed in the Scr, U-RBC, hypertension rate and eGFR measurements among the three groups. The high titer IMN group had a higher urinary protein, and a lower albumin than those in the low and middle titer groups. It was similar to the study by Kim et al. which found that albumin was lower in the high titer group and that no significant difference existed in serum creatinine. In both Chinese studies, the high titer group also had higher levels of 24-hour urinary protein, but there was no difference in albumin [15, 16]. And Li et al. found that the positive group had a higher baseline urine protein level, a lower albumin level, but a lower eGFR than the negative group . For these differences, the effect of ethnic background cannot be ignored. Different stages and statuses of disease when subjects were enrolled also account for the discrepancies.
Anti–phospholipase A2 receptor was a biomarker for diagnosis, activity evaluation, therapy monitoring, and prognostic estimation of IMN. The optimal cutoff value of anti-PLA2Rvalue by ELISA was variable in different studies from different countries. The EUROIMMUN manufacturer suggests reporting anti-PLA2R values higher than 20RU/ml as positive. In our study, using cut-off of 20 RU/ml the specificity can be high to 98.8%, but the sensitivity was just 67.5%. Behnert reported a sensitivity of 82.2%, with a specificity of 89.7% respectively using cut-off value of 20 RU/ml in American and German cohorts of patients. In a Chinese cohort, Dou et al. reported that at different cut-off value of 20 RU/ml, specificity was 97.3%, while sensitivity was 60.2%. But there were also studies from China[1, 14] and Japan when using the cutoff value of 20 RU/ml the sensitivity was just between 44.1% and 50.9%, what’s more the sensitivity in a study from Australia was just 25%.
Anti-PLA2R values higher than 14RU/ml were considered as borderline to diagnosis IMN. Behnert et al.  reported a sensitivity of 86.1%, with a specificity of 84.5% using cut-offs of 14 in American and German cohorts of patients. Dou et al.  reported that at cut-off value of 14RU/ml, specificity was 97.3%, while sensitivity was 65.3%, suggesting that 14 RU/ml should be applied to obtain higher sensitivity with no change in the specificity values in China.
In order to get a higher diagnosis sensitivity, researchers have been exploring whether a lower cutoff value can be available. We found that 2.5RU/mL was the optimal cutoff with the highest Youden index (0.740), the sensitivity was 85.7%, the specificity was 88.3%, the area under the ROC curve was 0.916 (95% CI 0.890−0.942, p=0.000). In the study by Timmermans et al. that sensitivity could be improved to 72% without affecting the specificity by reducing the cut-off value to 2 RU/ml. Furthermore, Liu et al.  found that the optimal cut-off value was 2.6 RU/ml with a sensitivity of 78.9% and a specificity of 91.7% in 57 Chinese IMN patients and two studies indicated that the optimal cut-off value was 2.7 RU/ml with a sensitivity 83.3%-88.1% and a specificity 95.1%-96%[16, 19].
The optimal cutoff value of 7.45 RU/mL was preferred in a China study which sensitivity was 83.3% and specificity was 95.1%. In our study, when the cutoff value was 6.3, the sensitivity was 77.8%, the specificity was 96.0%, and the Youden index was 0.737. Take altogether, 2.5 RU/ml and 6.3 RU/ml were both optimal cutoff values, 2.5 RU/ml can get higher sensitivity and 6.3 can get higher specificity. In our mind, we thought that maybe 6.3 RU/ml seemed to be much better to be the optimal cutoff value which can get a higher specificity and a not much lower sensitivity. The cut-off value of 6.3 RU/ml was recommended for the use of anti-PLA2R for the diagnosis of IMN in Chinese patients based on the ELISA.
Can we use double criterions for blood PLA2R? Anti-PLA2R>20RU/ml is used as the specific index to diagnose PLA2R related IMN without renal biopsy pathology, and the error probability is less than 2%; Anti-PlA2R<2RU/ml is used as the sensitivity index to deny PLA2R related IMN. If the clinical characteristics of patients with anti-PLA2R 2-20RU/ml are consistent with IMN, it may still be PLA2R related IMN. Whether renal biopsy is needed should be depend on the patient's clinical characteristics.
In our study, the remission rate and immunosuppressive therapy in the high titer group were higher than those in the low and middle titer groups, but there was no significant difference. Multivariable Cox regression analysis showed that immunosuppressive therapy was the only factor that associated with higher remission rate of IMN patients. In other words, if the severe IMN patients receive treatment in time, they also can get a high remission rate .So we should pay more attention at the immunosuppressive therapy in order to get better prognosis. However, it was quite different from these studies. For example, a reduction in the anti-PLA2R value correlated with better prognosis in IMN patients [6, 21-23], while persistence of anti-PLA2R value was associated with clinical resistance[22, 23]. Lower anti-PLA2R value at baseline and after 6 months were associated with IMN remission. What caused these different results in different studies?
First, in our study anti-PLA2R was correlated with 24-hour urinary protein, albumin, eGFR, TC and LDL. However, correlations between anti-PLA2R and albumin was negative, and others were positive. Furthermore, there were significant differences in 24-hour urinary protein, albumin, and eGFR, TC and LDL among the low titer subgroup and high titer subgroup. Other studies were different with our results. For example, Li et al found that 24-hour urinary protein positively correlated with anti-PLA2R, while albumin, and serum creatinine did not correlate. Another study showed that serum anti-PLA2R antibody levels were correlated with serum albumin, serum creatinine, eGFR, and proteinuria . Two Japanese studies indicated that anti-PLA2R negatively correlated with albumin, but its correlations with serum creatinine and 24-hour urinary protein were also different in these two studies[18, 25]. Li YQ revealed that the correlations of anti-PLA2R antibody value with urine protein and cholesterol were positive, but the correlations of anti-PLA2R antibody value with albumin and eGFR were negative. Secondly, the IMN patients were from different races in different countries; Third, small sample size and patient selection bias may also be important reasons for the difference in results.
This study suffers from several limitations. Firstly, it is a single-center retrospective study. Secondly, because patients receiving immunosuppressive therapy were excluded, anti-PLAR level of them at onset could not be analyzed and selection bias could not be neglected in this retrospective study. Third, the findings may reflect practice patterns in an East Asian population. A large-sample multi-center prospective study is needed to further validate these findings.
In conclusion, this study showed that the best cutoff value of anti-PLA2R antibodies for the widely used ELISA method to detect anti-PLA2R antibodies in IMN patients is probably much lower (around 2-7RU/ml) than that indicated by the manufacturer, thereby relevantly increasing assay sensitivity, the best cut-off value could be positioned at 6.3 RU/ml in our study. If the IMN patients were treated actively, they maybe can get better prognosis.