A-138 Implications of native protein conformation in the IgM quantification: total Ig vs HLC methods comparison

Abstract

Abstract Background For IgM monoclonal gammopathies such as Waldenström Macroglobulinemia, the diagnosis, risk stratification and monitoring response to therapy is based on the detection and quantification of the secreted IgM monoclonal protein. Accurate IgM measurement is challenging due to its complex nature and the occurrence in higher polymeric structures (i.e., pentamers or hexamers), as well as heterogeneity and inherent subjectivity of the selected common methodologies. The recently developed Heavy/Light chain assay (Binding Site, part of Thermofisher Scientific) allows the separate determination of the IgM-Kappa and IgM-Lambda molecules, offering an alternative method to the serum electrophoresis (SPE) with reported higher sensitivity (Ref. Boyle E. et al, Clin Cancer Research 2016). In this study we explore the effect of the molecular structure on 3 different analytical techniques for accurate IgM measurements Methods 214 serum samples from patients (age median 63yo) under follow-up at our hospital were tested for: IgG, IgA, and IgM levels (immunoturbidimetry, either on a Beckman Coulter AU5800 analyser; Nyon, Switzerland; or a Cobas c 702 system, Roche Diagnostics); Hevylite assay (on an Optilite, Binding Site); and SPE and Immunofixation (Capillarys 3 Octa and Hydrasys 2, Sebia Inc.). The effect of the molecular conformation was evaluated through sample depolymerization with N-Acetylcysteine (NAC) according to the manufacturer’s instruction (Sebia Inc.) and IgM quantification by: tIgM (immunoturbidimetric total Ig); red-Ig (idem after NAC reduction); and, HLC-Ig (i. e. sum of the IgM-K+IgM-L heavy/light chain pair measured by Hevylite (Binding Site)). Results Total-Ig, red-Ig, and HLC-Ig returned similar results for IgG and IgA immunoglobulins (Kruskal-Wallis test, p-value > 0.05), and Passing Bablok regression analysis showed good agreement between the three methods, except for higher IgA levels. In the later, both HLC-IgA levels red-IgA values were higher than total-IgA. Regarding the IgM, PB regression revealed a significant proportional bias, with obtained values approx. 2,5x higher by red-IgM and approx. 1,4x by HLC-IgM methods than tIgM (red-IgM=-5.5+2.42*tIgM, Spearman’s r=0.995; HLC-IgM=-0,77+1.34*tIgM, Spearman’s r=0.978; red-IgM=-6.29+1.82*HLC-IgM, Spearman’s r=0.98). So, both red-IgM and HLC-IgM results are higher than total IgM. Of note, 1 sample presented antigen excess, resulting in unusually low IgM levels by tIgM but high red-IgM and high HLC-IgM levels coherent with the patient’s clinical condition. 75 samples had monoclonal protein measurable by SPE, showing good correlation with the three methods, however with greater proportional errors between measurements with other techniques Conclusions IgM molecular conformational status may affect the epitopes exposed and, therefore, available for the different assays’ recognition. Higher IgM complexes depolymerization into monomeric conformation results in higher IgM levels, which agree better with the Hevylite than with the total IgM measured previous NAC reduction, reflecting the impact of the native protein conformation in the IgM precise detection. Although also affected by the conformational status, Hevylite IgM results were closer to the reduced IgM levels than the obtained by the total IgM assay, therefore offering a sensitive assay that does not need a reduction treatment for a more accurate IgM quantification, optimizing the laboratory workflow