Antigen excess pitfall for free light chains measurements solved by ELISA assay
Authors: Jean‐David Pekar, Susanna Schraen, Guillaume Grzych, Salomon Manier, Brigitte Onraed
Journal: American Journal of Hematology (2019)

To the Editor: The role of serum free light chains (SFLC) assessment is increasingly important in the management of patients with multiple myeloma (MM), as well as other monoclonal gammopathies. For instance, about 15% of MM secretes light chain only and must be followed using SFLC assays. In other cases, a free light chain escape is observed at relapse. Moreover, the new IMWG criteria also include the ratio of SFLC to define symptomatic MM and to evaluate the response (stringent complete remission). Nonetheless, FLC measurements remain challenging is some cases especially in the context of antigen excess.1 In this case report, we emphasize that this issue can be addressed by using a novel specific SLFC assay. Antigens at high concentrations can interfere with the antigen-antibody reaction and lead to false low SFLC results in patients with MM. Underestimation of SFLC could lead to inappropriate disease management. Some studies have tried to quantify this phenomenon and reported a frequency between 0.12% and 5.4%.1, 2 The immunoturbidimetry Freelite assay (The Binding Site, Birmingham, UK) includes a control step for antigen excess in order to prevent SLFC underestimation. The assay can detect atypical reaction kinetic by measuring the rate of the reaction at three different time windows in order to identify samples that have a relatively high initial rate of reaction. Suspects' samples are re-assayed at the higher re-dilution.2 However, this automated detection of antigen excess remains uncompleted and prone to error. In addition to systematic dilutions difficult to perform routinely on all samples, few solutions exist to solve this pitfall but not prevent it.3 Recently, a new sandwich ELISA assay was developed by Sebia (Lisses, France) can avoid this problem. Indeed, the higher sensitivity of the test allows to use larger dilutions than Freelite (100 times) so that the risk of antigen excess is significantly reduced. Here, we report three cases of antigen excess pitfall with Freelite assay, resolved by Sebia FLC. All data were retrieved from a human biological database authorized by the French Ministry of Research (No.DC-2008-642). Therefore, no written informed consent was collected from this population. The first patient (P1) is a 55-year-old male with asthenia and renal failure. Serum protein electrophoresis (SPEP) and immunofixation (Sebia Hydrasys 2scan) revealed a monoclonal peak in the beta1 globulins area and showed an excess of lambda light chains in addition to a monoclonal IgA lambda. The second patient (P2) is a 74-year-old male with similar symptoms. SPEP revealed two monoclonal peaks in gamma globulins area. The immunofixation test revealed two bands with the anti-lambda antiserum without similar bands with anti-gamma, anti-mu, and anti-alpha antisera. Samples were sent to our laboratory in order to rule out monoclonal IgD or IgE gammopathies. The third patient (P3) is a 62-year-old male with a 10-year history of IgA MM. He was treated with autologous stem-cell transplantation and VTD (bortezomib, thalidomide, and dexamethasone). Second-line treatment consisted of 66-cycles carfilzomib-lenalidomide-dexamethasone. Third-line was pomalidomide-dexamethasone, fourth-line by daratumumab-bortezomib-dexamethasone, and fifth line with doxorubicin-ixazomib-dexamethasone. Finally, he was treated with chimeric antigen receptor T (CAR-T) cells in September 2018. The SPEP and immunofixation revealed a monoclonal peak in the gamma globulins area and showed a monoclonal IgA kappa. A free light chain assay was performed according to the two methods: immunoturbidimetry Freelite assay on the SPAplus analyzer at the recommended initial dilution of 1/10 and ELISA assay Sebia FLC on the das AP22 ELITE at the recommended initial dilution of 1/1000. Lambda free light chains concentrations with Freelite assay were moderately increased with 131 mg/L for P1 and 159 mg/L for P2 at initial dilution (Table 1). Kappa free light chains concentration with Freelite assay was lower than 4.1 mg/L for P3. The serum involved/uninvolved free light chain ratios (iFLC/uFLC) were lower than 100. The analyzer SPA Plus did not report any alarm for antigen excess. Given the results of serum immunofixation, additional manual dilutions were performed at 1/1000 and 1/10 000, providing very high concentrations of involved free light chains: 29 238 mg/L for P1, 57 922 mg/L for P2, and 2630 mg/L for P3. At the last dilution, iFLC/uFLC ratios were higher than 100. Comparatively, Sebia FLC assay gave values above 90 mg/L for all samples at initial dilution. The final results of involved free light chains after automatic dilutions at 1/100 000 were 1791 mg/L for P1, 1138 mg/L for P2, and 3339 mg/L for P3. Our report illustrates the possible underestimation of Freelite SFLC in case of antigen excess. In these three cases, the new Sebia FLC assay overcame this difficulty without any antigen detection system like Freelite by using an initial dilution 100 times greater combined with multiples wash steps. We noticed significant differences between the final results of the two methods. This is consistent with recent studies reported that Freelite assay provide higher values than Sebia FLC, and the bias increased proportionally with SFLC levels.4 However, differences observed between the two assays in our cases could not completely relate to the negative bias in high values described for the Sebia method. Measurements obtained with Freelite reagent are obviously overestimated and quantitatively inconsistent with the electrophoretic profiles and could be explained by FLC polymerization. Indeed, FLC molecules are usually monomers or dimers5 but higher polymeric forms frequently occur. The polymers act as multiantigenic targets in immunoprecipitation assays, which accelerate aggregates formation and leads to overestimation of antigen measurement.6 These three cases also highlight the importance of a careful analysis of SFLC results, in conjunction with other tests such as SPEP, urine electrophoresis, immunotyping, bone marrow biopsy immunohistochemistry, and clinical history; especially since the phenomenon can even occur for commonly measured SFLC concentrations for patient 3. In conclusion, our report shows that the new sandwich ELISA Sebia FLC is a good alternative to SFLC assessment and can avoid the problem of antigen excess. The results also emphasize the variability of SFLC measurements depending on SFLC assays and related to polymerization. With the development of new tests for SFLC measurement, a standardization of these assays will therefore be necessary. Nothing to report. Pekar: designed and performed the research study, main writer. Grzych and Manier: advised and corrected the manuscript. Schraen and Onraed: designed the research study and corrected the manuscript.