Comparison of 2 Anti-PLA2R Immunoassays for the Diagnosis of Primary Membranous Nephropathy

Comparison of 2 Anti-PLA2R Immunoassays for the Diagnosis of Primary Membranous Nephropathy Abstract Background Anti–phospholipase A2 receptor (anti-PLA2R) is a promising biomarker for diagnosis, activity evaluation, therapy monitoring, and prognostic estimation of primary membranous neuropathy (pMN). Difference in methodology may be one cause of the discrepancy in anti-PLA2R–positive percentages in reported studies. In this study, we evaluated the determination consistency of anti-PLA2R using indirect immunofluorescence assay (IIF) and enzyme-linked immunosorbent assay (ELISA). Methods A total of 113 patients with pMN, 34 with secondary membranous neuropathy (sMN), and 53 healthy control individuals were enrolled. We tested their circulating anti-PLA2R, along with other biochemical parameters, using IIF and ELISA. Results The sensitivity of anti-PLA2R for pMN was 70.8% by IIF and 67.3% by ELISA, with no statistically significant difference. The overall qualitative agreement of anti-PLA2R was 91.15% (95% confidence interval [CI], 85.91%–96.39%), and the correlation coefficient was 0.79 for IIF versus ELISA. The overall correlation coefficient of anti-PLA2R titers by IIF and concentration by ELISA was 0.85. Through ROC curve analysis, anti-PLA2R as measured by IIF and ELISA showed larger areas under the curve (AUCs) than other biochemical parameters. Conclusion ELISA shows similar performance to IIF, with the advantages of quantitative results and suitability for high throughput. anti-PLA2R, membranous nephropathy, indirect immunofluorescence assay, enzyme-linked immunosorbent assay, agreement, diagnosis Membranous nephropathy (MN) is a common cause of nephritic syndrome in adults.1 Primary membranous nephropathy (pMN), as an organ-specific autoimmune disease, accounts for approximately 80% of all MN cases, whereas secondary MN (sMN) explains the other 20%.2 The differential diagnosis of pMN and sMN is largely based on exclusion of secondary causes such as malignant neoplasms, systemic lupus erythematosus (SLE), bacterial infection, and drug intoxication.3,4 Patients with pMN need to undergo immunosuppressive therapy, whereas sMN therapy is mainly aimed at the treatment of underlying disease. The etiology of pMN was obscure until the landmark study by Beck et al5in 2009, the results of which demonstrated that M-type phospholipase A2 receptor (PLA2R), a transmembrane glycoprotein expressed on podocytes, was the main target antigen of the autoantibody in pMN. Lately, thrombospondin type-1 domain-containing 7A (THSD7A) was identified as the second target antigen, with a prevalence of 2% to 3% among patients with MN.6,7 Circulating anti-PLA2R is presently considered to be a promising protential serological diagnostic biomarker of pMN.7,8 The results of 2 studies9,10 indicate that 52% to 82% of patients with pMN had circulating anti-PLA2R in accordance with geographic regions. Moreover, anti-PLA2R is now expected to be a biomarker of pMN activity, a cue for immunosuppressive therapy monitoring, and a tool for predicting disease outcomes.11–13 Western blot (WB), enzyme-linked immunosorbent assay (ELISA), and indirect immunofluorescence assay (IIF) are the 3 main methods of detecting anti-PLA2R; the difference in methodology may be a cause for the discrepancy in anti-PLA2R–positive percentages in reported studies. WB is labor-intensive and unsuitable for the evaluation of large numbers of specimens. To date, ELISA and IIF are widely used assays for detecting anti-PLA2R in clinical laboratories. The purpose of the current study was to evaluate the determination consistency of anti-PLA2R by using commercially available ELISA and IIF kits. Materials and Methods Patients and Specimens This study was carried out at The Second Hospital of Hebei Medical University in Shijiazhuang, China from April 2016 through June 2017. A total of 147 patients with MN, including 113 patients with pMN and 34 with sMN, were consecutively enrolled. PMN was diagnosed in patients with biopsy-proven MN but without laboratory or clinical signs of known underlying diseases or suspected drug exposure. No immunosuppressive therapy for patients with pMN was allowed before inclusion. Patients were diagnosed with sMN according to renal biopsy findings of MN and secondary causes. Among the 34 patients with sMN, the underlying diseases included SLE (n = 7), autoimmune thyroid disease (n = 10), rheumatoid arthritis (n = 1), hepatitis B virus infection (n = 9), hepatitis C virus infection (n = 1), gastric cancer (n = 3), multiple myeloma (n = 1) and intoxication with nonsteroidal anti-inflammatory drugs (n = 2). Also, 53 serum specimens from healthy volunteers were included as healthy control specimens. Serum was isolated within 1 hour after venous blood collection. From each blood specimen, 500 μL of serum was separated and stored at −70°C until the measurement of anti-PLA2R. Biochemistry parameters, including blood urea nitrogen (BUN), serum creatinine, total protein (TP), albumin, cholesterol, triglycerides, and 24-hour urine protein, were determined within 2 hours after specimen collection. The study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University (approval no. 2016248), and written informed consent was obtained from all patients. Determination of Biochemistry Parameters Serum analysis for BUN, creatinine, TP, albumin, cholesterol, triglycerides, and 24-hour urine protein was performed via the Cobas 6000 automatic biochemistry analyzer (F. Hoffman-La Roche Ltd), using corresponding reagents produced by the manufacturer. Reference ranges of various parameters were applied according to the protocols of the manufacturer. Anti-PLA2R Determination via IIF We performed circulating anti-PLA2R testing using a recombinant cell-based IIF test (EUROIMMUN AG) according to the manufacturer-provided protocol. The test used a 5-mm biochip mosaic with a composite substrate containing formalin-fixed human embryonic kidney 293 (HEK293) cells, which were transfected with full-length complementary DNA, encoding PLA2R and nontransfected HEK 293 cells as a negative control. Fluorescein isothiocyanate (FITC)–labeled goat antihuman immunoglobulin (Ig)G conjugate was used as the revealing reagent. We diluted serum specimens 1:10 in specimen buffer (0.05% Tween 20, 1% casein in PBS) and then applied 30 μL of diluted serum to the reaction fields, followed by incubation at room temperature for 30 minutes. After rinsing slides with specimen buffer and immersing them in specimen buffer for 5 minutes or more, 25 μL of revealing reagent was added, and the resulting product was incubated for 30 minutes. Immunostain was evaluated using a fluorescence microscope (EUROStar III plus, EUROIMMUN AG), with excitation of 460 to 490 nm. Specific fluorescence in the cytoplasm of transfected cells but not in negative control cells at a dilution of 1:10 or higher was considered to indicate a positive result. Further dilution of 1:100, 1:1000, or 1:10000 was performed for specimen with positive results to determine the final titer of anti-PLA2R. Anti-PLA2R Determination via ELISA We used a commercial quantitative ELISA assay (EUROIMMUN AG) for the IgG-specific isotype of anti-PLA2R, according to manufacturer instructions. In brief, serum specimens were diluted 1:100 in PBS-Tween. Then, 100-μL diluted specimens; calibrators with anti-PLA2R concentration of 2, 100, 500, and 1500 RU per mL; and controls with negative and positive results were added to microplate wells and incubated for 30 minutes at room temperature, followed by 3 washing steps. Anti–human-IgG horseradish peroxidase (HRP) conjugate diluted 1:1000 in specimen buffer was transferred to every microplate well for another 30 minutes of incubation, followed by 3 washing steps and the addition of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate for 15 minutes incubation subsequently. Optical density at 450 nm was read using a microplate photometer (Multiskan FC, ThermoFisher Scientific Inc). We applied a cutoff value of 20 RU per mL for diagnosis according to manufacturer protocol. Statistical Analysis Data are presented as mean (SD) for normally distributed values or median with interquartile range (IQR) for non–normally distributed values; qualitative variables were described as numbers and percentages. We performed statistical comparisons among 3 groups using 1-way analysis of variance (ANOVA) and Scheffe posthoc tests for values with normal distributions or Kruskal-Wallis H tests for data with non-normal distributions. We used χ2 testing to compare differences between qualitative variables. We used Spearman correlation analyses to assess the correlations between portions and Cohen kappa values to analyze the agreement between the 2 studied immunoassays. To assess the diagnostic performance of biomarkers, we conducted receiver operating characteristic (ROC) curve analysis and compared the areas under the curves (AUCs). All tests were 2-sided; P values less than .05 were considered statistically significant. We performed statistical analyses using SPSS statistical software, version 22.0 (IBM). Results Baseline Clinical Characteristics and Laboratory Findings The baseline clinical characteristics of and laboratory findings for the study and control populations are shown in Table 1. Overall, pMN was predominant in men (56.6%) and sMN was predominant in women (52.9%). Healthy control individuals comprised 34 men and 19 women, with mean (SD) age of 53.5 (16.5) years. There was no significant difference in age and sex distributions (x2 = 0.483, P = .29). TP and albumin levels in patients with sMN were found to be higher than those levels in the pMN group and lower than in controls. Patients with sMN had lower cholesterol and 24-hour urine protein levels than patients with pMN and higher levels than in controls. Patients with pMN and sMN had higher triglyceride levels and lower creatinine levels than controls (Table 1). Table 1. Characteristics in Patients With Membranous Neuropathy and Control Individualsa Characteristic  Patient Group  Controls (n = 53)  P Value  pMN (n = 113)  sMN (n = 34)  pMN vs sMN  pMN vs Controls  sMN vs Controls  Male sex, no. (%)  64 (56.6%)  16 (47.1%)  34 (64.1%)  NA  NA  NA  Age (y), mean (SD)  52.3 (9.4)  49.4 (13.9)  53.5 (16.5)  .49  .87  .42  BUN (mmol/L), Median (IQR)  4.70 (3.90–5.99)  5.79 (4.28–7.40)  4.50 (4.10–5.20)  .06  .44  .06  Creatinine (μmol/L), Median (IQR)  58.3 (47.0–73.5)  60.9 (42.3–91.8)  72.0 (63.0–81.5)  .20  <.001  .03  Total protein (g/L), mean (SD)  48.7 (10.1)  54.1 (11.9)  73.4 (4.11)  <.05  <.001  <.001  Albumin (g/L), mean (SD)  25.8 (6.97)  30.3 (9.53)  46.7 (3.46)  .04  <.001  <.001  Cholesterol (mmol/L), mean (SD)  7.16 (2.19)  5.46 (1.82)  4.54 (0.98)  <.001  <.001  .03  Triglyceride (mmol/L), Median (IQR)  2.26 (1.70–2.91)  1.61 (0.96–2.50)  1.34 (1.03–1.85)  .002  <.001  .79  24-hour urine protein (g/24 h), Median (IQR)  7.26 (4.24–11.2)  3.07 (1.08–7.41)  0.08 (0.05–0.11)  .06  <.001  <.001  PLA2R-Ab (RU/mL), Median (IQR)  78.2 (4.24–222.1)  1.85 (0.36–12.78)  9.02 (5.67–14.1)  <.001  <.001  .02  Characteristic  Patient Group  Controls (n = 53)  P Value  pMN (n = 113)  sMN (n = 34)  pMN vs sMN  pMN vs Controls  sMN vs Controls  Male sex, no. (%)  64 (56.6%)  16 (47.1%)  34 (64.1%)  NA  NA  NA  Age (y), mean (SD)  52.3 (9.4)  49.4 (13.9)  53.5 (16.5)  .49  .87  .42  BUN (mmol/L), Median (IQR)  4.70 (3.90–5.99)  5.79 (4.28–7.40)  4.50 (4.10–5.20)  .06  .44  .06  Creatinine (μmol/L), Median (IQR)  58.3 (47.0–73.5)  60.9 (42.3–91.8)  72.0 (63.0–81.5)  .20  <.001  .03  Total protein (g/L), mean (SD)  48.7 (10.1)  54.1 (11.9)  73.4 (4.11)  <.05  <.001  <.001  Albumin (g/L), mean (SD)  25.8 (6.97)  30.3 (9.53)  46.7 (3.46)  .04  <.001  <.001  Cholesterol (mmol/L), mean (SD)  7.16 (2.19)  5.46 (1.82)  4.54 (0.98)  <.001  <.001  .03  Triglyceride (mmol/L), Median (IQR)  2.26 (1.70–2.91)  1.61 (0.96–2.50)  1.34 (1.03–1.85)  .002  <.001  .79  24-hour urine protein (g/24 h), Median (IQR)  7.26 (4.24–11.2)  3.07 (1.08–7.41)  0.08 (0.05–0.11)  .06  <.001  <.001  PLA2R-Ab (RU/mL), Median (IQR)  78.2 (4.24–222.1)  1.85 (0.36–12.78)  9.02 (5.67–14.1)  <.001  <.001  .02  pMN, primary membranous nephropathy; sMN, secondary membranous nephropathy; NA, nonapplicable; BUN, blood urea nitrogen; IQR, interquartile range; PLA2R-Ab, phospholipase A2 receptor antibodies. aVariables expressed as mean (SD) are compared using the Scheffe posthoc test; variables expressed as median (IQR) are compared using Kruskal-Wallis H tests. Categorical variables are expressed as no. (%) and compared using Pearson χ2 testing. View Large Table 1. Characteristics in Patients With Membranous Neuropathy and Control Individualsa Characteristic  Patient Group  Controls (n = 53)  P Value  pMN (n = 113)  sMN (n = 34)  pMN vs sMN  pMN vs Controls  sMN vs Controls  Male sex, no. (%)  64 (56.6%)  16 (47.1%)  34 (64.1%)  NA  NA  NA  Age (y), mean (SD)  52.3 (9.4)  49.4 (13.9)  53.5 (16.5)  .49  .87  .42  BUN (mmol/L), Median (IQR)  4.70 (3.90–5.99)  5.79 (4.28–7.40)  4.50 (4.10–5.20)  .06  .44  .06  Creatinine (μmol/L), Median (IQR)  58.3 (47.0–73.5)  60.9 (42.3–91.8)  72.0 (63.0–81.5)  .20  <.001  .03  Total protein (g/L), mean (SD)  48.7 (10.1)  54.1 (11.9)  73.4 (4.11)  <.05  <.001  <.001  Albumin (g/L), mean (SD)  25.8 (6.97)  30.3 (9.53)  46.7 (3.46)  .04  <.001  <.001  Cholesterol (mmol/L), mean (SD)  7.16 (2.19)  5.46 (1.82)  4.54 (0.98)  <.001  <.001  .03  Triglyceride (mmol/L), Median (IQR)  2.26 (1.70–2.91)  1.61 (0.96–2.50)  1.34 (1.03–1.85)  .002  <.001  .79  24-hour urine protein (g/24 h), Median (IQR)  7.26 (4.24–11.2)  3.07 (1.08–7.41)  0.08 (0.05–0.11)  .06  <.001  <.001  PLA2R-Ab (RU/mL), Median (IQR)  78.2 (4.24–222.1)  1.85 (0.36–12.78)  9.02 (5.67–14.1)  <.001  <.001  .02  Characteristic  Patient Group  Controls (n = 53)  P Value  pMN (n = 113)  sMN (n = 34)  pMN vs sMN  pMN vs Controls  sMN vs Controls  Male sex, no. (%)  64 (56.6%)  16 (47.1%)  34 (64.1%)  NA  NA  NA  Age (y), mean (SD)  52.3 (9.4)  49.4 (13.9)  53.5 (16.5)  .49  .87  .42  BUN (mmol/L), Median (IQR)  4.70 (3.90–5.99)  5.79 (4.28–7.40)  4.50 (4.10–5.20)  .06  .44  .06  Creatinine (μmol/L), Median (IQR)  58.3 (47.0–73.5)  60.9 (42.3–91.8)  72.0 (63.0–81.5)  .20  <.001  .03  Total protein (g/L), mean (SD)  48.7 (10.1)  54.1 (11.9)  73.4 (4.11)  <.05  <.001  <.001  Albumin (g/L), mean (SD)  25.8 (6.97)  30.3 (9.53)  46.7 (3.46)  .04  <.001  <.001  Cholesterol (mmol/L), mean (SD)  7.16 (2.19)  5.46 (1.82)  4.54 (0.98)  <.001  <.001  .03  Triglyceride (mmol/L), Median (IQR)  2.26 (1.70–2.91)  1.61 (0.96–2.50)  1.34 (1.03–1.85)  .002  <.001  .79  24-hour urine protein (g/24 h), Median (IQR)  7.26 (4.24–11.2)  3.07 (1.08–7.41)  0.08 (0.05–0.11)  .06  <.001  <.001  PLA2R-Ab (RU/mL), Median (IQR)  78.2 (4.24–222.1)  1.85 (0.36–12.78)  9.02 (5.67–14.1)  <.001  <.001  .02  pMN, primary membranous nephropathy; sMN, secondary membranous nephropathy; NA, nonapplicable; BUN, blood urea nitrogen; IQR, interquartile range; PLA2R-Ab, phospholipase A2 receptor antibodies. aVariables expressed as mean (SD) are compared using the Scheffe posthoc test; variables expressed as median (IQR) are compared using Kruskal-Wallis H tests. Categorical variables are expressed as no. (%) and compared using Pearson χ2 testing. View Large Clinical Performance of Specimens that Tested Positive for Anti-PLA2R For anti-PLA2R–positive specimens tested via IIF assay, specific cytoplasmic fluorescence could be observed in HEK293 cells expressing PLA2R protein (Image 1A), and no fluorescence was observed in nontransfected cells (Image 1B). Both biochip sections showed no fluorescence in cytoplasm for serum that had tested negative for anti-PLA2R. Image 1 View largeDownload slide Results of indirect immunofluorescence (IIF) assay to determine expression of phospholipase A2 receptor (PLA2R) protein. A, Visible fluorescence in human embryonic kidney 293 (HEK293) cells indicate presence of PLA2R protein. B, Absence of visible fluorescence indicates lack of PLA2R protein in nontransfected cells. Image 1 View largeDownload slide Results of indirect immunofluorescence (IIF) assay to determine expression of phospholipase A2 receptor (PLA2R) protein. A, Visible fluorescence in human embryonic kidney 293 (HEK293) cells indicate presence of PLA2R protein. B, Absence of visible fluorescence indicates lack of PLA2R protein in nontransfected cells. Among 113 specimens from patients with pMN, 80 tested positive for anti-PLA2R via IIF; of these, 28, 40, 9, and 3 specimens tested positive at the dilution levels of 1:10, 1:100, 1:1000, and 1:10000, respectively. For ELISA assays, we discovered that 76 patients tested positive for anti-PLA2R using the cutoff value established by the assay manufacturer. The diagnosis sensitivity of anti-PLA2R for pMN was 70.8% (80/113) via IIF and 67.3% (76/113) via ELISA. In the sMN group, 33 specimens were demonstrated to be anti-PLA2R negative via ELISA and IIF, except 1 specimen, which tested positive at the dilution of 1:10 by IIF vs 59.80 RU per mL via ELISA. All 53 control specimens showed negative anti-PLA2R results via IIF and ELISA. Using χ2 testing, no significant difference of anti-PLA2R between IIF and ELISA was revealed (Table 2). Table 2. Qualitative Agreements of IIF and ELISA Results in Patients With MNa Variable  IIF  Total  Percentage Agreement (95% CI)  P Value b  Positive  Negative  ELISA  .23  Positive  73  3  76  90.12% (81.38%–98.56%)  Negative  8  63  71  95.45% (90.42%–100.48%)  Total  81  66  147  92.52% (76.38%–88.66%)  Variable  IIF  Total  Percentage Agreement (95% CI)  P Value b  Positive  Negative  ELISA  .23  Positive  73  3  76  90.12% (81.38%–98.56%)  Negative  8  63  71  95.45% (90.42%–100.48%)  Total  81  66  147  92.52% (76.38%–88.66%)  IIF, indirect immunofluorescence assay; ELISA, enzyme-linked immunosorbent assay; MN, membranous neuropathy. aKappa = 0.850 (95% CI, .77–.93). bDetermined via NcNemar χ2 testing. View Large Table 2. Qualitative Agreements of IIF and ELISA Results in Patients With MNa Variable  IIF  Total  Percentage Agreement (95% CI)  P Value b  Positive  Negative  ELISA  .23  Positive  73  3  76  90.12% (81.38%–98.56%)  Negative  8  63  71  95.45% (90.42%–100.48%)  Total  81  66  147  92.52% (76.38%–88.66%)  Variable  IIF  Total  Percentage Agreement (95% CI)  P Value b  Positive  Negative  ELISA  .23  Positive  73  3  76  90.12% (81.38%–98.56%)  Negative  8  63  71  95.45% (90.42%–100.48%)  Total  81  66  147  92.52% (76.38%–88.66%)  IIF, indirect immunofluorescence assay; ELISA, enzyme-linked immunosorbent assay; MN, membranous neuropathy. aKappa = 0.850 (95% CI, .77–.93). bDetermined via NcNemar χ2 testing. View Large Agreements Between IIF and ELISA Results For serum anti-PLA2R titers determined by IIF and concentration measured by ELISA in patients with pMN and sMN, the overall qualitative agreement of anti-PLA2R was 92.52% (95% confidence interval [CI], 76.38% to 88.66%), and the difference between the 2 methods was statistically insignificant (P> .05). The overall correlation coefficient was 0.85 for IIF vs ELISA, which showed good correlation between the 2 assays (P <.001) (Figure 1). Nevertheless, 3 specimens that tested negative via IIF yielded positive results via ELISA, with a mean concentration of 80.47 (10.51) RU per mL. Four specimens with titer of 1:10 and 3 specimens with titer of 1:100 tested negative via ELISA. Figure 1 View largeDownload slide Serum anti–phospholipase A2 receptor (anti-PLA2R) titers, as determined via indirect immunofluorescence assay (IIF) and enzyme-linked immunosorbent assay (ELISA). Figure 1 View largeDownload slide Serum anti–phospholipase A2 receptor (anti-PLA2R) titers, as determined via indirect immunofluorescence assay (IIF) and enzyme-linked immunosorbent assay (ELISA). Evaluating Diagnostic Performance With ROC Curves To identify the value of anti-PLA2R tested via ELISA and IIF as markers to differentiate pMN from sMN, we carried out ROC curve analysis. In comparisons between patients with pMN and those with sMN, anti-PLA2R values determined via ELISA (AUC = 0.90 [95% CI, 0.85–0.95]; P <.001) and IIF (AUC = 0.85 [95% CI, 0.79–0.91]; P <.001) were both statistically significant in the differential diagnosis of pMN and sMN. The difference between AUCs of anti-PLA2R determined via ELISA and IIF was statistically insignificant (P = .45). Compared with TP (AUC = 0.65 [95% CI, 0.53–0.76], P = .01), albumin (AUC = 0.65 [95% CI, 0.52–0.77]; P = .01), cholesterol (AUC = 0.73[95% CI, 0.63–0.84]; P <.001), triglycerides (AUC = 0.68 [95% CI, 0.57–0.79]; P = .001) and 24-hour urine protein (AUC = 0.69 [95% CI, 0.57–0.81]; P = .001), anti-PLA2R determined via IIF and ELISA showed larger AUC values, which indicate better performance, to differentiate pMN from sMN (Figure 2). Figure 2 View largeDownload slide Results of receiver operating characteristic (ROC) curve analysis of various analyte values, including phospholipase A2 receptor (PLA2R), to differentiate primary membranous neuropathy (pMN) from secondary membranous neuropathy (sMN). TP indicates total protein; ELISA, enzyme-linked immunosorbent assay; and IIF, indirect immunofluorescence assay. Figure 2 View largeDownload slide Results of receiver operating characteristic (ROC) curve analysis of various analyte values, including phospholipase A2 receptor (PLA2R), to differentiate primary membranous neuropathy (pMN) from secondary membranous neuropathy (sMN). TP indicates total protein; ELISA, enzyme-linked immunosorbent assay; and IIF, indirect immunofluorescence assay. Discussion Since the study of anti-PLA2R by Beck et al5 in 2009, an increasing amount of study results have verified that anti-PLA2R plays a pivotal role in the pathogenesis of pMN, although the exact mechanism is still under investigation. A widely accepted viewpoint is that in patients with pMN, subepithelial deposition occurs in situ by the binding of circulating anti-PLA2R on podocytes, leading to activation of complement and a cascade of events subsequent to nephritic syndrome.3 Moreover, it is now assumed that conformational change of PLA2R induced by molecular mimicry of exogenous antigens or by unknown causes could participate in the etiology of pMN.1 Consistent with the findings of previous studies,11–13 when laboratory values from patients with pMN and those with sMN were compared in the present study, the anti-PLA2R levels were significantly higher in patients with pMN. Although the exact mechanisms and the role of PLA2R in pathogenesis of pMN are currently still unknown,7 our data strengthen the role of PLA2R as a target antigen in pMN. Through ROC analysis, we discovered that the AUC of anti-PLA2R determined via ELISA or IIF was higher than that of TP, albumin, cholesterol, triglycerides, or 24-hour urine protein. Therefore, these data suggested that circulating anti-PLA2R can serve as an alternative diagnostic biomarker for pMN. However, the reported prevalence of anti-PLA2R in patients with pMN varied from 52% to 82%. The heterogeneity among studies probably implied that some potential factors might impact the diagnostic accuracy of anti-PLA2R assays. Initially, researchers believed that the interval between the time of diagnosis and blood collection might induce this inconsistency. In the study by Svobodava et al,10 the sensitivity of anti-PLA2R was reported to be 64% when blood was taken at the time of biopsy, compared with a positive rate of 22% several months after kidney biopsy due to spontaneous or drug-induced remission in some patients. Detecting anti-PLA2R at the time of initial diagnosis is recommended, to avoid possible confounding of disease progression and therapy.2 Also, the effect of immunosuppressive therapy should be taken into account. Moreover, differences in test methodology could contribute to observed discordance in results. The detection efficiency of the 3 dominant methods for anti-PLA2R testing, namely, WB, IIF, and ELISA, remain controversial. Contrary to the findings of Hoxha et al,14 which claimed that the discrepancy was not associated with different sensitivities of test methods, Behnert et al15 demonstrated higher diagnosis sensitivity in IIF than in ELISA. In this study, we report that the sensitivity of anti-PLA2R was 70.8% (80/113) by IIF and 67.3% (76/113) by ELISA, to differentiate pMN from sMN, but that the difference was statistically insignificant. By ROC curve analysis, no significant difference was demonstrated between anti-PLA2R via ELISA and via IIF in the differential diagnosis of pMN and sMN. Although we observed a statistically similar performance of anti-PLA2R via ELISA and via IIF, we note that, in the present study, 3 specimens that tested negative via IIF yielded a positive result via ELISA, and 4 specimens that tested positive via IIF tested negative via ELISA. These inconsistent results probably could be explained by the differences in the antigen-binding matrices utilized in each platform. In ELISA, purified recombinant PLA2R antigens are absorbed passively to the plastic matrix, and the reactivity of antibodies is dependent mostly on the number of sufficiently exposed epitopes for binding.16 In IIF, overexpression of recombinant PLA2R in transfected HEK239 cells is applied, and anti-PLA2R is probably prone to combine its native cellular domain, which had more native expression of epitopes. Further, specimens with unclear anti-PLA2R titer readings are more prone to yield inconsistent results due to the differences in sensitivity and specificity of anti-PLA2R testing. When the results of anti-PLA2R testing are inconsistent with pathological or clinical indications, clinicians should consider whether this discrepancy is caused by differences of methodologies. Comparing with ELISA, IIF is a semiquantitative test with less operation steps and shorter incubation time. We can obtain IIF results through the observation of fluorescence under microscope and standard curve is not needed. Determination of anti-PLA2R via IIF is widely used by small scale medical laboratories due to its simplicity. Although it has good agreement with ELISA, IIF is limited due to its weak adaptability to high-throughput clinical laboratories. The superior characteristic of quantitative ELISA is that it provides more-accurate monitoring of changes in anti-PLA2R concentration, which is helpful for immunosuppressive therapy monitoring and monitoring of disease progression.14,17 Currently, ELISA assay to determine anti-PLA2R levels is widely used in clinical practice, due to its advantages. In summary, our data demonstrate that anti-PLA2R levels were significantly higher in pMN compared with sMN; therefore, anti-PLA2R levels may serve as an alternative diagnostic biomarker for pMN. Although similar diagnostic performance of anti-PLA2R testing via ELISA and IIF was observed statistically, we remind readers that a small portion of specimens showed inconsistent results via 2 immunoassays regarding the differences in antigen-binding matrices utilized in the platforms. Compared with IIF, anti-PLA2R determination via ELISA represents the more promising method, with the advantages of offering quantitative results and suitability for high-throughput clinical laboratories. Abbreviations MN membranous nephropathy pMN primary membranous nephropathy sMN secondary MN SLE systemic lupus erythematosus PLA2R phospholipase A2 receptor THSD7A thrombospondin type-1 domain-containing 7A WB Western blot ELISA enzyme-linked immunosorbent assay IIF indirect immunofluorescence assay BUN blood urea nitrogen TP total protein HEK293 human embryonic kidney 293 FITC fluorescein isothiocyanate Ig immunoglobulin HRP horseradish peroxidase TMB 3,3′,5,5′-tetramethylbenzidine IQR interquartile range ANOVA analysis of variance ROC receiver operating characteristic AUCs areas under the curve CI confidence interval NA nonapplicable References 1. Ronco P, Debiec H. Pathogenesis of membranous nephropathy: recent advances and future challenges. Nat Rev Nephrol . 2012; 8( 4): 203– 213. Google Scholar CrossRef Search ADS PubMed  2. Du Y, Li J, He F, et al.   The diagnosis accuracy of PLA2R-AB in the diagnosis of idiopathic membranous nephropathy: a meta-analysis. PLoS One . 2014; 9( 8): e104936. Google Scholar CrossRef Search ADS PubMed  3. Ronco P, Debiec H. Antigen identification in membranous nephropathy moves toward targeted monitoring and new therapy. J Am Soc Nephrol . 2010; 21( 4): 564– 569. Google Scholar CrossRef Search ADS PubMed  4. Hoxha E, Kneißler U, Stege G, et al.   Enhanced expression of the M-type phospholipase A2 receptor in glomeruli correlates with serum receptor antibodies in primary membranous nephropathy. Kidney Int . 2012; 82( 7): 797– 804. Google Scholar CrossRef Search ADS PubMed  5. Beck LHJr, Bonegio RG, Lambeau G, et al.   M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med . 2009; 361( 1): 11– 21. Google Scholar CrossRef Search ADS PubMed  6. Tomas NM, Beck LHJr, Meyer-Schwesinger C, et al.   Thrombospondin type-1 domain-containing 7A in idiopathic membranous nephropathy. N Engl J Med . 2014; 371( 24): 2277– 2287. Google Scholar CrossRef Search ADS PubMed  7. Hoxha E, von Haxthausen F, Wiech T, Stahl RAK. Membranous nephropathy—one morphologic pattern with different diseases. Pflugers Arch . 2017; 469( 7-8): 989– 996. Google Scholar CrossRef Search ADS PubMed  8. Xun C, Shuai L, Wang W, Jiang Y. Comparison of biomarkers between PLA2RAb+ and PLA2RAb− in patients with idiopathic membranous nephropathy. Int Urol Nephrol . 2015; 47( 5): 831– 835. Google Scholar CrossRef Search ADS PubMed  9. Hofstra JM, Wetzels JF. Anti-PLA₂R antibodies in membranous nephropathy: ready for routine clinical practice? Neth J Med . 2012; 70( 3): 109– 113. Google Scholar PubMed  10. Svobodova B, Honsova E, Ronco P, Tesar V, Debiec H. Kidney biopsy is a sensitive tool for retrospective diagnosis of PLA2R-related membranous nephropathy. Nephrol Dial Transplant . 2013; 28( 7): 1839– 1844. Google Scholar CrossRef Search ADS PubMed  11. Schlumberger W, Hornig N, Lange S, et al.   Differential diagnosis of membranous nephropathy with autoantibodies to phospholipase A2 receptor 1. Autoimmun Rev . 2014; 13( 2): 108– 113. Google Scholar CrossRef Search ADS PubMed  12. Radice A, Trezzi B, Maggiore U, et al.   Clinical usefulness of autoantibodies to M-type phospholipase A2 receptor (PLA2R) for monitoring disease activity in idiopathic membranous nephropathy (IMN). Autoimmun Rev . 2016; 15( 2): 146– 154. Google Scholar CrossRef Search ADS PubMed  13. Jullien P, Seitz Polski B, Maillard N, et al.   Anti-phospholipase A2 receptor antibody levels at diagnosis predicts spontaneous remission of idiopathic membranous nephropathy. Clin Kidney J . 2017; 10( 2): 209– 214. Google Scholar CrossRef Search ADS PubMed  14. Hoxha E, Harendza S, Zahner G, et al.   An immunofluorescence test for phospholipase-A₂-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis. Nephrol Dial Transplant . 2011; 26( 8): 2526– 2532. Google Scholar CrossRef Search ADS PubMed  15. Behnert A, Schiffer M, Müller-Deile J, Beck LHJr, Mahler M, Fritzler MJ. Antiphospholipase A₂ receptor autoantibodies: a comparison of three different immunoassays for the diagnosis of idiopathic membranous nephropathy. J Immunol Res . 2014; 2014: 143274. Google Scholar CrossRef Search ADS PubMed  16. Mahler M, Gascon C, Patel S, et al.   Rpp25 is a major target of autoantibodies to the Th/To complex as measured by a novel chemiluminescent assay. Arthritis Res Ther . 2013; 15( 2): R50. Google Scholar CrossRef Search ADS PubMed  17. Hofstra JM, Beck LHJr, Beck DM, Wetzels JF, Salant DJ. Anti-phospholipase A₂ receptor antibodies correlate with clinical status in idiopathic membranous nephropathy. Clin J Am Soc Nephrol . 2011; 6( 6): 1286– 1291. Google Scholar CrossRef Search ADS PubMed  © American Society for Clinical Pathology, 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Laboratory Medicine Oxford University Press

Comparison of 2 Anti-PLA2R Immunoassays for the Diagnosis of Primary Membranous Nephropathy

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Abstract

Abstract Background Anti–phospholipase A2 receptor (anti-PLA2R) is a promising biomarker for diagnosis, activity evaluation, therapy monitoring, and prognostic estimation of primary membranous neuropathy (pMN). Difference in methodology may be one cause of the discrepancy in anti-PLA2R–positive percentages in reported studies. In this study, we evaluated the determination consistency of anti-PLA2R using indirect immunofluorescence assay (IIF) and enzyme-linked immunosorbent assay (ELISA). Methods A total of 113 patients with pMN, 34 with secondary membranous neuropathy (sMN), and 53 healthy control individuals were enrolled. We tested their circulating anti-PLA2R, along with other biochemical parameters, using IIF and ELISA. Results The sensitivity of anti-PLA2R for pMN was 70.8% by IIF and 67.3% by ELISA, with no statistically significant difference. The overall qualitative agreement of anti-PLA2R was 91.15% (95% confidence interval [CI], 85.91%–96.39%), and the correlation coefficient was 0.79 for IIF versus ELISA. The overall correlation coefficient of anti-PLA2R titers by IIF and concentration by ELISA was 0.85. Through ROC curve analysis, anti-PLA2R as measured by IIF and ELISA showed larger areas under the curve (AUCs) than other biochemical parameters. Conclusion ELISA shows similar performance to IIF, with the advantages of quantitative results and suitability for high throughput. anti-PLA2R, membranous nephropathy, indirect immunofluorescence assay, enzyme-linked immunosorbent assay, agreement, diagnosis Membranous nephropathy (MN) is a common cause of nephritic syndrome in adults.1 Primary membranous nephropathy (pMN), as an organ-specific autoimmune disease, accounts for approximately 80% of all MN cases, whereas secondary MN (sMN) explains the other 20%.2 The differential diagnosis of pMN and sMN is largely based on exclusion of secondary causes such as malignant neoplasms, systemic lupus erythematosus (SLE), bacterial infection, and drug intoxication.3,4 Patients with pMN need to undergo immunosuppressive therapy, whereas sMN therapy is mainly aimed at the treatment of underlying disease. The etiology of pMN was obscure until the landmark study by Beck et al5in 2009, the results of which demonstrated that M-type phospholipase A2 receptor (PLA2R), a transmembrane glycoprotein expressed on podocytes, was the main target antigen of the autoantibody in pMN. Lately, thrombospondin type-1 domain-containing 7A (THSD7A) was identified as the second target antigen, with a prevalence of 2% to 3% among patients with MN.6,7 Circulating anti-PLA2R is presently considered to be a promising protential serological diagnostic biomarker of pMN.7,8 The results of 2 studies9,10 indicate that 52% to 82% of patients with pMN had circulating anti-PLA2R in accordance with geographic regions. Moreover, anti-PLA2R is now expected to be a biomarker of pMN activity, a cue for immunosuppressive therapy monitoring, and a tool for predicting disease outcomes.11–13 Western blot (WB), enzyme-linked immunosorbent assay (ELISA), and indirect immunofluorescence assay (IIF) are the 3 main methods of detecting anti-PLA2R; the difference in methodology may be a cause for the discrepancy in anti-PLA2R–positive percentages in reported studies. WB is labor-intensive and unsuitable for the evaluation of large numbers of specimens. To date, ELISA and IIF are widely used assays for detecting anti-PLA2R in clinical laboratories. The purpose of the current study was to evaluate the determination consistency of anti-PLA2R by using commercially available ELISA and IIF kits. Materials and Methods Patients and Specimens This study was carried out at The Second Hospital of Hebei Medical University in Shijiazhuang, China from April 2016 through June 2017. A total of 147 patients with MN, including 113 patients with pMN and 34 with sMN, were consecutively enrolled. PMN was diagnosed in patients with biopsy-proven MN but without laboratory or clinical signs of known underlying diseases or suspected drug exposure. No immunosuppressive therapy for patients with pMN was allowed before inclusion. Patients were diagnosed with sMN according to renal biopsy findings of MN and secondary causes. Among the 34 patients with sMN, the underlying diseases included SLE (n = 7), autoimmune thyroid disease (n = 10), rheumatoid arthritis (n = 1), hepatitis B virus infection (n = 9), hepatitis C virus infection (n = 1), gastric cancer (n = 3), multiple myeloma (n = 1) and intoxication with nonsteroidal anti-inflammatory drugs (n = 2). Also, 53 serum specimens from healthy volunteers were included as healthy control specimens. Serum was isolated within 1 hour after venous blood collection. From each blood specimen, 500 μL of serum was separated and stored at −70°C until the measurement of anti-PLA2R. Biochemistry parameters, including blood urea nitrogen (BUN), serum creatinine, total protein (TP), albumin, cholesterol, triglycerides, and 24-hour urine protein, were determined within 2 hours after specimen collection. The study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University (approval no. 2016248), and written informed consent was obtained from all patients. Determination of Biochemistry Parameters Serum analysis for BUN, creatinine, TP, albumin, cholesterol, triglycerides, and 24-hour urine protein was performed via the Cobas 6000 automatic biochemistry analyzer (F. Hoffman-La Roche Ltd), using corresponding reagents produced by the manufacturer. Reference ranges of various parameters were applied according to the protocols of the manufacturer. Anti-PLA2R Determination via IIF We performed circulating anti-PLA2R testing using a recombinant cell-based IIF test (EUROIMMUN AG) according to the manufacturer-provided protocol. The test used a 5-mm biochip mosaic with a composite substrate containing formalin-fixed human embryonic kidney 293 (HEK293) cells, which were transfected with full-length complementary DNA, encoding PLA2R and nontransfected HEK 293 cells as a negative control. Fluorescein isothiocyanate (FITC)–labeled goat antihuman immunoglobulin (Ig)G conjugate was used as the revealing reagent. We diluted serum specimens 1:10 in specimen buffer (0.05% Tween 20, 1% casein in PBS) and then applied 30 μL of diluted serum to the reaction fields, followed by incubation at room temperature for 30 minutes. After rinsing slides with specimen buffer and immersing them in specimen buffer for 5 minutes or more, 25 μL of revealing reagent was added, and the resulting product was incubated for 30 minutes. Immunostain was evaluated using a fluorescence microscope (EUROStar III plus, EUROIMMUN AG), with excitation of 460 to 490 nm. Specific fluorescence in the cytoplasm of transfected cells but not in negative control cells at a dilution of 1:10 or higher was considered to indicate a positive result. Further dilution of 1:100, 1:1000, or 1:10000 was performed for specimen with positive results to determine the final titer of anti-PLA2R. Anti-PLA2R Determination via ELISA We used a commercial quantitative ELISA assay (EUROIMMUN AG) for the IgG-specific isotype of anti-PLA2R, according to manufacturer instructions. In brief, serum specimens were diluted 1:100 in PBS-Tween. Then, 100-μL diluted specimens; calibrators with anti-PLA2R concentration of 2, 100, 500, and 1500 RU per mL; and controls with negative and positive results were added to microplate wells and incubated for 30 minutes at room temperature, followed by 3 washing steps. Anti–human-IgG horseradish peroxidase (HRP) conjugate diluted 1:1000 in specimen buffer was transferred to every microplate well for another 30 minutes of incubation, followed by 3 washing steps and the addition of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate for 15 minutes incubation subsequently. Optical density at 450 nm was read using a microplate photometer (Multiskan FC, ThermoFisher Scientific Inc). We applied a cutoff value of 20 RU per mL for diagnosis according to manufacturer protocol. Statistical Analysis Data are presented as mean (SD) for normally distributed values or median with interquartile range (IQR) for non–normally distributed values; qualitative variables were described as numbers and percentages. We performed statistical comparisons among 3 groups using 1-way analysis of variance (ANOVA) and Scheffe posthoc tests for values with normal distributions or Kruskal-Wallis H tests for data with non-normal distributions. We used χ2 testing to compare differences between qualitative variables. We used Spearman correlation analyses to assess the correlations between portions and Cohen kappa values to analyze the agreement between the 2 studied immunoassays. To assess the diagnostic performance of biomarkers, we conducted receiver operating characteristic (ROC) curve analysis and compared the areas under the curves (AUCs). All tests were 2-sided; P values less than .05 were considered statistically significant. We performed statistical analyses using SPSS statistical software, version 22.0 (IBM). Results Baseline Clinical Characteristics and Laboratory Findings The baseline clinical characteristics of and laboratory findings for the study and control populations are shown in Table 1. Overall, pMN was predominant in men (56.6%) and sMN was predominant in women (52.9%). Healthy control individuals comprised 34 men and 19 women, with mean (SD) age of 53.5 (16.5) years. There was no significant difference in age and sex distributions (x2 = 0.483, P = .29). TP and albumin levels in patients with sMN were found to be higher than those levels in the pMN group and lower than in controls. Patients with sMN had lower cholesterol and 24-hour urine protein levels than patients with pMN and higher levels than in controls. Patients with pMN and sMN had higher triglyceride levels and lower creatinine levels than controls (Table 1). Table 1. Characteristics in Patients With Membranous Neuropathy and Control Individualsa Characteristic  Patient Group  Controls (n = 53)  P Value  pMN (n = 113)  sMN (n = 34)  pMN vs sMN  pMN vs Controls  sMN vs Controls  Male sex, no. (%)  64 (56.6%)  16 (47.1%)  34 (64.1%)  NA  NA  NA  Age (y), mean (SD)  52.3 (9.4)  49.4 (13.9)  53.5 (16.5)  .49  .87  .42  BUN (mmol/L), Median (IQR)  4.70 (3.90–5.99)  5.79 (4.28–7.40)  4.50 (4.10–5.20)  .06  .44  .06  Creatinine (μmol/L), Median (IQR)  58.3 (47.0–73.5)  60.9 (42.3–91.8)  72.0 (63.0–81.5)  .20  <.001  .03  Total protein (g/L), mean (SD)  48.7 (10.1)  54.1 (11.9)  73.4 (4.11)  <.05  <.001  <.001  Albumin (g/L), mean (SD)  25.8 (6.97)  30.3 (9.53)  46.7 (3.46)  .04  <.001  <.001  Cholesterol (mmol/L), mean (SD)  7.16 (2.19)  5.46 (1.82)  4.54 (0.98)  <.001  <.001  .03  Triglyceride (mmol/L), Median (IQR)  2.26 (1.70–2.91)  1.61 (0.96–2.50)  1.34 (1.03–1.85)  .002  <.001  .79  24-hour urine protein (g/24 h), Median (IQR)  7.26 (4.24–11.2)  3.07 (1.08–7.41)  0.08 (0.05–0.11)  .06  <.001  <.001  PLA2R-Ab (RU/mL), Median (IQR)  78.2 (4.24–222.1)  1.85 (0.36–12.78)  9.02 (5.67–14.1)  <.001  <.001  .02  Characteristic  Patient Group  Controls (n = 53)  P Value  pMN (n = 113)  sMN (n = 34)  pMN vs sMN  pMN vs Controls  sMN vs Controls  Male sex, no. (%)  64 (56.6%)  16 (47.1%)  34 (64.1%)  NA  NA  NA  Age (y), mean (SD)  52.3 (9.4)  49.4 (13.9)  53.5 (16.5)  .49  .87  .42  BUN (mmol/L), Median (IQR)  4.70 (3.90–5.99)  5.79 (4.28–7.40)  4.50 (4.10–5.20)  .06  .44  .06  Creatinine (μmol/L), Median (IQR)  58.3 (47.0–73.5)  60.9 (42.3–91.8)  72.0 (63.0–81.5)  .20  <.001  .03  Total protein (g/L), mean (SD)  48.7 (10.1)  54.1 (11.9)  73.4 (4.11)  <.05  <.001  <.001  Albumin (g/L), mean (SD)  25.8 (6.97)  30.3 (9.53)  46.7 (3.46)  .04  <.001  <.001  Cholesterol (mmol/L), mean (SD)  7.16 (2.19)  5.46 (1.82)  4.54 (0.98)  <.001  <.001  .03  Triglyceride (mmol/L), Median (IQR)  2.26 (1.70–2.91)  1.61 (0.96–2.50)  1.34 (1.03–1.85)  .002  <.001  .79  24-hour urine protein (g/24 h), Median (IQR)  7.26 (4.24–11.2)  3.07 (1.08–7.41)  0.08 (0.05–0.11)  .06  <.001  <.001  PLA2R-Ab (RU/mL), Median (IQR)  78.2 (4.24–222.1)  1.85 (0.36–12.78)  9.02 (5.67–14.1)  <.001  <.001  .02  pMN, primary membranous nephropathy; sMN, secondary membranous nephropathy; NA, nonapplicable; BUN, blood urea nitrogen; IQR, interquartile range; PLA2R-Ab, phospholipase A2 receptor antibodies. aVariables expressed as mean (SD) are compared using the Scheffe posthoc test; variables expressed as median (IQR) are compared using Kruskal-Wallis H tests. Categorical variables are expressed as no. (%) and compared using Pearson χ2 testing. View Large Table 1. Characteristics in Patients With Membranous Neuropathy and Control Individualsa Characteristic  Patient Group  Controls (n = 53)  P Value  pMN (n = 113)  sMN (n = 34)  pMN vs sMN  pMN vs Controls  sMN vs Controls  Male sex, no. (%)  64 (56.6%)  16 (47.1%)  34 (64.1%)  NA  NA  NA  Age (y), mean (SD)  52.3 (9.4)  49.4 (13.9)  53.5 (16.5)  .49  .87  .42  BUN (mmol/L), Median (IQR)  4.70 (3.90–5.99)  5.79 (4.28–7.40)  4.50 (4.10–5.20)  .06  .44  .06  Creatinine (μmol/L), Median (IQR)  58.3 (47.0–73.5)  60.9 (42.3–91.8)  72.0 (63.0–81.5)  .20  <.001  .03  Total protein (g/L), mean (SD)  48.7 (10.1)  54.1 (11.9)  73.4 (4.11)  <.05  <.001  <.001  Albumin (g/L), mean (SD)  25.8 (6.97)  30.3 (9.53)  46.7 (3.46)  .04  <.001  <.001  Cholesterol (mmol/L), mean (SD)  7.16 (2.19)  5.46 (1.82)  4.54 (0.98)  <.001  <.001  .03  Triglyceride (mmol/L), Median (IQR)  2.26 (1.70–2.91)  1.61 (0.96–2.50)  1.34 (1.03–1.85)  .002  <.001  .79  24-hour urine protein (g/24 h), Median (IQR)  7.26 (4.24–11.2)  3.07 (1.08–7.41)  0.08 (0.05–0.11)  .06  <.001  <.001  PLA2R-Ab (RU/mL), Median (IQR)  78.2 (4.24–222.1)  1.85 (0.36–12.78)  9.02 (5.67–14.1)  <.001  <.001  .02  Characteristic  Patient Group  Controls (n = 53)  P Value  pMN (n = 113)  sMN (n = 34)  pMN vs sMN  pMN vs Controls  sMN vs Controls  Male sex, no. (%)  64 (56.6%)  16 (47.1%)  34 (64.1%)  NA  NA  NA  Age (y), mean (SD)  52.3 (9.4)  49.4 (13.9)  53.5 (16.5)  .49  .87  .42  BUN (mmol/L), Median (IQR)  4.70 (3.90–5.99)  5.79 (4.28–7.40)  4.50 (4.10–5.20)  .06  .44  .06  Creatinine (μmol/L), Median (IQR)  58.3 (47.0–73.5)  60.9 (42.3–91.8)  72.0 (63.0–81.5)  .20  <.001  .03  Total protein (g/L), mean (SD)  48.7 (10.1)  54.1 (11.9)  73.4 (4.11)  <.05  <.001  <.001  Albumin (g/L), mean (SD)  25.8 (6.97)  30.3 (9.53)  46.7 (3.46)  .04  <.001  <.001  Cholesterol (mmol/L), mean (SD)  7.16 (2.19)  5.46 (1.82)  4.54 (0.98)  <.001  <.001  .03  Triglyceride (mmol/L), Median (IQR)  2.26 (1.70–2.91)  1.61 (0.96–2.50)  1.34 (1.03–1.85)  .002  <.001  .79  24-hour urine protein (g/24 h), Median (IQR)  7.26 (4.24–11.2)  3.07 (1.08–7.41)  0.08 (0.05–0.11)  .06  <.001  <.001  PLA2R-Ab (RU/mL), Median (IQR)  78.2 (4.24–222.1)  1.85 (0.36–12.78)  9.02 (5.67–14.1)  <.001  <.001  .02  pMN, primary membranous nephropathy; sMN, secondary membranous nephropathy; NA, nonapplicable; BUN, blood urea nitrogen; IQR, interquartile range; PLA2R-Ab, phospholipase A2 receptor antibodies. aVariables expressed as mean (SD) are compared using the Scheffe posthoc test; variables expressed as median (IQR) are compared using Kruskal-Wallis H tests. Categorical variables are expressed as no. (%) and compared using Pearson χ2 testing. View Large Clinical Performance of Specimens that Tested Positive for Anti-PLA2R For anti-PLA2R–positive specimens tested via IIF assay, specific cytoplasmic fluorescence could be observed in HEK293 cells expressing PLA2R protein (Image 1A), and no fluorescence was observed in nontransfected cells (Image 1B). Both biochip sections showed no fluorescence in cytoplasm for serum that had tested negative for anti-PLA2R. Image 1 View largeDownload slide Results of indirect immunofluorescence (IIF) assay to determine expression of phospholipase A2 receptor (PLA2R) protein. A, Visible fluorescence in human embryonic kidney 293 (HEK293) cells indicate presence of PLA2R protein. B, Absence of visible fluorescence indicates lack of PLA2R protein in nontransfected cells. Image 1 View largeDownload slide Results of indirect immunofluorescence (IIF) assay to determine expression of phospholipase A2 receptor (PLA2R) protein. A, Visible fluorescence in human embryonic kidney 293 (HEK293) cells indicate presence of PLA2R protein. B, Absence of visible fluorescence indicates lack of PLA2R protein in nontransfected cells. Among 113 specimens from patients with pMN, 80 tested positive for anti-PLA2R via IIF; of these, 28, 40, 9, and 3 specimens tested positive at the dilution levels of 1:10, 1:100, 1:1000, and 1:10000, respectively. For ELISA assays, we discovered that 76 patients tested positive for anti-PLA2R using the cutoff value established by the assay manufacturer. The diagnosis sensitivity of anti-PLA2R for pMN was 70.8% (80/113) via IIF and 67.3% (76/113) via ELISA. In the sMN group, 33 specimens were demonstrated to be anti-PLA2R negative via ELISA and IIF, except 1 specimen, which tested positive at the dilution of 1:10 by IIF vs 59.80 RU per mL via ELISA. All 53 control specimens showed negative anti-PLA2R results via IIF and ELISA. Using χ2 testing, no significant difference of anti-PLA2R between IIF and ELISA was revealed (Table 2). Table 2. Qualitative Agreements of IIF and ELISA Results in Patients With MNa Variable  IIF  Total  Percentage Agreement (95% CI)  P Value b  Positive  Negative  ELISA  .23  Positive  73  3  76  90.12% (81.38%–98.56%)  Negative  8  63  71  95.45% (90.42%–100.48%)  Total  81  66  147  92.52% (76.38%–88.66%)  Variable  IIF  Total  Percentage Agreement (95% CI)  P Value b  Positive  Negative  ELISA  .23  Positive  73  3  76  90.12% (81.38%–98.56%)  Negative  8  63  71  95.45% (90.42%–100.48%)  Total  81  66  147  92.52% (76.38%–88.66%)  IIF, indirect immunofluorescence assay; ELISA, enzyme-linked immunosorbent assay; MN, membranous neuropathy. aKappa = 0.850 (95% CI, .77–.93). bDetermined via NcNemar χ2 testing. View Large Table 2. Qualitative Agreements of IIF and ELISA Results in Patients With MNa Variable  IIF  Total  Percentage Agreement (95% CI)  P Value b  Positive  Negative  ELISA  .23  Positive  73  3  76  90.12% (81.38%–98.56%)  Negative  8  63  71  95.45% (90.42%–100.48%)  Total  81  66  147  92.52% (76.38%–88.66%)  Variable  IIF  Total  Percentage Agreement (95% CI)  P Value b  Positive  Negative  ELISA  .23  Positive  73  3  76  90.12% (81.38%–98.56%)  Negative  8  63  71  95.45% (90.42%–100.48%)  Total  81  66  147  92.52% (76.38%–88.66%)  IIF, indirect immunofluorescence assay; ELISA, enzyme-linked immunosorbent assay; MN, membranous neuropathy. aKappa = 0.850 (95% CI, .77–.93). bDetermined via NcNemar χ2 testing. View Large Agreements Between IIF and ELISA Results For serum anti-PLA2R titers determined by IIF and concentration measured by ELISA in patients with pMN and sMN, the overall qualitative agreement of anti-PLA2R was 92.52% (95% confidence interval [CI], 76.38% to 88.66%), and the difference between the 2 methods was statistically insignificant (P> .05). The overall correlation coefficient was 0.85 for IIF vs ELISA, which showed good correlation between the 2 assays (P <.001) (Figure 1). Nevertheless, 3 specimens that tested negative via IIF yielded positive results via ELISA, with a mean concentration of 80.47 (10.51) RU per mL. Four specimens with titer of 1:10 and 3 specimens with titer of 1:100 tested negative via ELISA. Figure 1 View largeDownload slide Serum anti–phospholipase A2 receptor (anti-PLA2R) titers, as determined via indirect immunofluorescence assay (IIF) and enzyme-linked immunosorbent assay (ELISA). Figure 1 View largeDownload slide Serum anti–phospholipase A2 receptor (anti-PLA2R) titers, as determined via indirect immunofluorescence assay (IIF) and enzyme-linked immunosorbent assay (ELISA). Evaluating Diagnostic Performance With ROC Curves To identify the value of anti-PLA2R tested via ELISA and IIF as markers to differentiate pMN from sMN, we carried out ROC curve analysis. In comparisons between patients with pMN and those with sMN, anti-PLA2R values determined via ELISA (AUC = 0.90 [95% CI, 0.85–0.95]; P <.001) and IIF (AUC = 0.85 [95% CI, 0.79–0.91]; P <.001) were both statistically significant in the differential diagnosis of pMN and sMN. The difference between AUCs of anti-PLA2R determined via ELISA and IIF was statistically insignificant (P = .45). Compared with TP (AUC = 0.65 [95% CI, 0.53–0.76], P = .01), albumin (AUC = 0.65 [95% CI, 0.52–0.77]; P = .01), cholesterol (AUC = 0.73[95% CI, 0.63–0.84]; P <.001), triglycerides (AUC = 0.68 [95% CI, 0.57–0.79]; P = .001) and 24-hour urine protein (AUC = 0.69 [95% CI, 0.57–0.81]; P = .001), anti-PLA2R determined via IIF and ELISA showed larger AUC values, which indicate better performance, to differentiate pMN from sMN (Figure 2). Figure 2 View largeDownload slide Results of receiver operating characteristic (ROC) curve analysis of various analyte values, including phospholipase A2 receptor (PLA2R), to differentiate primary membranous neuropathy (pMN) from secondary membranous neuropathy (sMN). TP indicates total protein; ELISA, enzyme-linked immunosorbent assay; and IIF, indirect immunofluorescence assay. Figure 2 View largeDownload slide Results of receiver operating characteristic (ROC) curve analysis of various analyte values, including phospholipase A2 receptor (PLA2R), to differentiate primary membranous neuropathy (pMN) from secondary membranous neuropathy (sMN). TP indicates total protein; ELISA, enzyme-linked immunosorbent assay; and IIF, indirect immunofluorescence assay. Discussion Since the study of anti-PLA2R by Beck et al5 in 2009, an increasing amount of study results have verified that anti-PLA2R plays a pivotal role in the pathogenesis of pMN, although the exact mechanism is still under investigation. A widely accepted viewpoint is that in patients with pMN, subepithelial deposition occurs in situ by the binding of circulating anti-PLA2R on podocytes, leading to activation of complement and a cascade of events subsequent to nephritic syndrome.3 Moreover, it is now assumed that conformational change of PLA2R induced by molecular mimicry of exogenous antigens or by unknown causes could participate in the etiology of pMN.1 Consistent with the findings of previous studies,11–13 when laboratory values from patients with pMN and those with sMN were compared in the present study, the anti-PLA2R levels were significantly higher in patients with pMN. Although the exact mechanisms and the role of PLA2R in pathogenesis of pMN are currently still unknown,7 our data strengthen the role of PLA2R as a target antigen in pMN. Through ROC analysis, we discovered that the AUC of anti-PLA2R determined via ELISA or IIF was higher than that of TP, albumin, cholesterol, triglycerides, or 24-hour urine protein. Therefore, these data suggested that circulating anti-PLA2R can serve as an alternative diagnostic biomarker for pMN. However, the reported prevalence of anti-PLA2R in patients with pMN varied from 52% to 82%. The heterogeneity among studies probably implied that some potential factors might impact the diagnostic accuracy of anti-PLA2R assays. Initially, researchers believed that the interval between the time of diagnosis and blood collection might induce this inconsistency. In the study by Svobodava et al,10 the sensitivity of anti-PLA2R was reported to be 64% when blood was taken at the time of biopsy, compared with a positive rate of 22% several months after kidney biopsy due to spontaneous or drug-induced remission in some patients. Detecting anti-PLA2R at the time of initial diagnosis is recommended, to avoid possible confounding of disease progression and therapy.2 Also, the effect of immunosuppressive therapy should be taken into account. Moreover, differences in test methodology could contribute to observed discordance in results. The detection efficiency of the 3 dominant methods for anti-PLA2R testing, namely, WB, IIF, and ELISA, remain controversial. Contrary to the findings of Hoxha et al,14 which claimed that the discrepancy was not associated with different sensitivities of test methods, Behnert et al15 demonstrated higher diagnosis sensitivity in IIF than in ELISA. In this study, we report that the sensitivity of anti-PLA2R was 70.8% (80/113) by IIF and 67.3% (76/113) by ELISA, to differentiate pMN from sMN, but that the difference was statistically insignificant. By ROC curve analysis, no significant difference was demonstrated between anti-PLA2R via ELISA and via IIF in the differential diagnosis of pMN and sMN. Although we observed a statistically similar performance of anti-PLA2R via ELISA and via IIF, we note that, in the present study, 3 specimens that tested negative via IIF yielded a positive result via ELISA, and 4 specimens that tested positive via IIF tested negative via ELISA. These inconsistent results probably could be explained by the differences in the antigen-binding matrices utilized in each platform. In ELISA, purified recombinant PLA2R antigens are absorbed passively to the plastic matrix, and the reactivity of antibodies is dependent mostly on the number of sufficiently exposed epitopes for binding.16 In IIF, overexpression of recombinant PLA2R in transfected HEK239 cells is applied, and anti-PLA2R is probably prone to combine its native cellular domain, which had more native expression of epitopes. Further, specimens with unclear anti-PLA2R titer readings are more prone to yield inconsistent results due to the differences in sensitivity and specificity of anti-PLA2R testing. When the results of anti-PLA2R testing are inconsistent with pathological or clinical indications, clinicians should consider whether this discrepancy is caused by differences of methodologies. Comparing with ELISA, IIF is a semiquantitative test with less operation steps and shorter incubation time. We can obtain IIF results through the observation of fluorescence under microscope and standard curve is not needed. Determination of anti-PLA2R via IIF is widely used by small scale medical laboratories due to its simplicity. Although it has good agreement with ELISA, IIF is limited due to its weak adaptability to high-throughput clinical laboratories. The superior characteristic of quantitative ELISA is that it provides more-accurate monitoring of changes in anti-PLA2R concentration, which is helpful for immunosuppressive therapy monitoring and monitoring of disease progression.14,17 Currently, ELISA assay to determine anti-PLA2R levels is widely used in clinical practice, due to its advantages. In summary, our data demonstrate that anti-PLA2R levels were significantly higher in pMN compared with sMN; therefore, anti-PLA2R levels may serve as an alternative diagnostic biomarker for pMN. Although similar diagnostic performance of anti-PLA2R testing via ELISA and IIF was observed statistically, we remind readers that a small portion of specimens showed inconsistent results via 2 immunoassays regarding the differences in antigen-binding matrices utilized in the platforms. Compared with IIF, anti-PLA2R determination via ELISA represents the more promising method, with the advantages of offering quantitative results and suitability for high-throughput clinical laboratories. Abbreviations MN membranous nephropathy pMN primary membranous nephropathy sMN secondary MN SLE systemic lupus erythematosus PLA2R phospholipase A2 receptor THSD7A thrombospondin type-1 domain-containing 7A WB Western blot ELISA enzyme-linked immunosorbent assay IIF indirect immunofluorescence assay BUN blood urea nitrogen TP total protein HEK293 human embryonic kidney 293 FITC fluorescein isothiocyanate Ig immunoglobulin HRP horseradish peroxidase TMB 3,3′,5,5′-tetramethylbenzidine IQR interquartile range ANOVA analysis of variance ROC receiver operating characteristic AUCs areas under the curve CI confidence interval NA nonapplicable References 1. Ronco P, Debiec H. Pathogenesis of membranous nephropathy: recent advances and future challenges. Nat Rev Nephrol . 2012; 8( 4): 203– 213. Google Scholar CrossRef Search ADS PubMed  2. Du Y, Li J, He F, et al.   The diagnosis accuracy of PLA2R-AB in the diagnosis of idiopathic membranous nephropathy: a meta-analysis. PLoS One . 2014; 9( 8): e104936. Google Scholar CrossRef Search ADS PubMed  3. Ronco P, Debiec H. Antigen identification in membranous nephropathy moves toward targeted monitoring and new therapy. J Am Soc Nephrol . 2010; 21( 4): 564– 569. Google Scholar CrossRef Search ADS PubMed  4. Hoxha E, Kneißler U, Stege G, et al.   Enhanced expression of the M-type phospholipase A2 receptor in glomeruli correlates with serum receptor antibodies in primary membranous nephropathy. Kidney Int . 2012; 82( 7): 797– 804. Google Scholar CrossRef Search ADS PubMed  5. Beck LHJr, Bonegio RG, Lambeau G, et al.   M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med . 2009; 361( 1): 11– 21. Google Scholar CrossRef Search ADS PubMed  6. Tomas NM, Beck LHJr, Meyer-Schwesinger C, et al.   Thrombospondin type-1 domain-containing 7A in idiopathic membranous nephropathy. N Engl J Med . 2014; 371( 24): 2277– 2287. Google Scholar CrossRef Search ADS PubMed  7. Hoxha E, von Haxthausen F, Wiech T, Stahl RAK. Membranous nephropathy—one morphologic pattern with different diseases. Pflugers Arch . 2017; 469( 7-8): 989– 996. Google Scholar CrossRef Search ADS PubMed  8. Xun C, Shuai L, Wang W, Jiang Y. Comparison of biomarkers between PLA2RAb+ and PLA2RAb− in patients with idiopathic membranous nephropathy. Int Urol Nephrol . 2015; 47( 5): 831– 835. Google Scholar CrossRef Search ADS PubMed  9. Hofstra JM, Wetzels JF. Anti-PLA₂R antibodies in membranous nephropathy: ready for routine clinical practice? Neth J Med . 2012; 70( 3): 109– 113. Google Scholar PubMed  10. Svobodova B, Honsova E, Ronco P, Tesar V, Debiec H. Kidney biopsy is a sensitive tool for retrospective diagnosis of PLA2R-related membranous nephropathy. Nephrol Dial Transplant . 2013; 28( 7): 1839– 1844. Google Scholar CrossRef Search ADS PubMed  11. Schlumberger W, Hornig N, Lange S, et al.   Differential diagnosis of membranous nephropathy with autoantibodies to phospholipase A2 receptor 1. Autoimmun Rev . 2014; 13( 2): 108– 113. Google Scholar CrossRef Search ADS PubMed  12. Radice A, Trezzi B, Maggiore U, et al.   Clinical usefulness of autoantibodies to M-type phospholipase A2 receptor (PLA2R) for monitoring disease activity in idiopathic membranous nephropathy (IMN). Autoimmun Rev . 2016; 15( 2): 146– 154. Google Scholar CrossRef Search ADS PubMed  13. Jullien P, Seitz Polski B, Maillard N, et al.   Anti-phospholipase A2 receptor antibody levels at diagnosis predicts spontaneous remission of idiopathic membranous nephropathy. Clin Kidney J . 2017; 10( 2): 209– 214. Google Scholar CrossRef Search ADS PubMed  14. Hoxha E, Harendza S, Zahner G, et al.   An immunofluorescence test for phospholipase-A₂-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis. Nephrol Dial Transplant . 2011; 26( 8): 2526– 2532. Google Scholar CrossRef Search ADS PubMed  15. Behnert A, Schiffer M, Müller-Deile J, Beck LHJr, Mahler M, Fritzler MJ. Antiphospholipase A₂ receptor autoantibodies: a comparison of three different immunoassays for the diagnosis of idiopathic membranous nephropathy. J Immunol Res . 2014; 2014: 143274. Google Scholar CrossRef Search ADS PubMed  16. Mahler M, Gascon C, Patel S, et al.   Rpp25 is a major target of autoantibodies to the Th/To complex as measured by a novel chemiluminescent assay. Arthritis Res Ther . 2013; 15( 2): R50. Google Scholar CrossRef Search ADS PubMed  17. Hofstra JM, Beck LHJr, Beck DM, Wetzels JF, Salant DJ. Anti-phospholipase A₂ receptor antibodies correlate with clinical status in idiopathic membranous nephropathy. Clin J Am Soc Nephrol . 2011; 6( 6): 1286– 1291. Google Scholar CrossRef Search ADS PubMed  © American Society for Clinical Pathology, 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Laboratory MedicineOxford University Press

Published: Apr 23, 2018

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