Calibration Differences and the Prozone Phenomenon in IgG4‐Related Disease: Comment on the Article by Khosroshahi et al
Authors: Antony R. Parker, Hugh D. Carr‐Smith
Journal: Arthritis & Rheumatology (2014)

The recent report by Khosroshahi and colleagues (1) discusses the limitations of The Binding Site BNII Human IgG4 Immunoassay for operation on a BNII analyzer (Siemens). Khosroshahi et al reported the phenomenon of antigen excess in 26% of patients (10 of 38 samples) with IgG4-related disease (IgG4-RD). Here we address the technical aspects raised and provide guidance for the optimal use of The Binding Site IgG4 Immunoassays. Deficiency in IgG4 is the most common subclass deficiency, with levels varying during childhood (ages 0–18 years; 0–1,961 mg/liter) and stabilizing to 52–1,250 mg/liter in adults (2). Measurement of IgG4 concentrations is recommended in guidelines for the diagnosis of primary antibody deficiency (3). Hyper-IgG4 syndrome or IgG4-RD is a recently described type of disease that is characterized by dense organ infiltration of IgG4-positive plasma cells and elevated serum IgG4 (4-8). The measurement of elevated serum IgG4 has been included in guidelines for the diagnosis of IgG4-RD (7, 8). Khosroshahi et al erroneously report that both The Binding Site and Siemens IgG4 immunoassays are calibrated to the same International Federation of Clinical Chemistry standard. Calibration of these IgG4 assays can be traced to different reference materials; The Binding Site IgG4 Immunoassays were calibrated to CRM470, now superseded by ERM-DA470k due to depletion of CRM470 stocks (2, 9). Siemens IgG4 assays were calibrated to WHO67/97 (10), which was later replaced by the commercial calibration material Sanquin M1590. The impact of the different calibration material has recently been reported (11-13). A comparison of IgG4 values in samples from patients illustrated significant differences between the manufacturers' assays (Deming regression slopes 0.50–0.63 [13] and 0.57–0.66 [12]). As measured by the Siemens assay, the concentration of IgG4 in ERM-DA470k was 607 mg/liter, while an IgG4 concentration of 374 mg/liter was measured with The Binding Site assay (13). Therefore, the difference in calibration between assays accounts for the 2-fold variability between IgG4 measurements observed by Khosroshahi and colleagues. The authors are correct that The Binding Site BNII Human IgG4 Immunoassay (LK009.TB; measuring range 38–613 mg/liter) used in their study does not have protection against antigen excess for samples with elevated IgG4 concentrations. The LK009.TB assay was designed for the detection of IgG4-deficient individuals, and so it has antigen excess capacity up to 2,400 mg/liter. If there is suspicion of antigen excess caused by elevated polyclonal IgG4 due to IgG4-RD or monoclonal IgG4, it is recommended in these cases that the result be verified by reassay of the patient sample at a dilution of 1:2,000. In addition, new parameters have been developed on the BNII analyzer that provide protection against antigen excess up to 49,000 mg/liter (Table 1). The principle of the new parameter involves using a 2-step reaction. Table 2 shows 5 selected patient sera with elevated IgG4 levels. Using the standard parameters, 5 of 5 were in antigen excess; however, all samples were correctly reported using the new parameters. In summary, calibration differences between the 2 assays need to be considered when evaluating patient sera. In addition, a simple alteration to the parameters of The Binding Site IgG4 assay, which protects against elevated IgG4 concentrations while still providing the sensitivity for detection of patients with primary immunodeficiency, is now available.