The effect of paraprotein polymerisation on quantitation by capillary zone electrophoresis and Hevylite®

Abstract

Objectives Up to 3% of patients with monoclonal gammopathies have multiple serum paraproteins. This article investigates whether multiple isotype-matched paraproteins, as seen on capillary zone electrophoresis, are truly biclonal. Methods Serum samples containing multiple isotype-matched paraproteins were treated with the reducing agent dithiothreitol, and capillary zone electrophoresis was performed pre- and post-treatment. Band resolution and effect of resolution on quantitation of paraprotein burden were assessed. The Hevylite® turbidimetric assay was also evaluated for ability to quantify such paraproteins. Results Among patients with biclonal isotype-matched paraproteins, 23/24 (96%) IgA paraproteins resolved into a single band following treatment with dithiothreitol compared with only 1/12 (8%) IgG paraproteins. Daratumumab therapy accounted for the second band in 5/9 non-resolving IgGκ paraproteins. Where initially quantified as a single IgA ‘complex’ (multiple bands in close proximity), the single postdithiothreitol band averaged 2.8 g/L less ( P<0.001), likely due to inclusion of lower amounts of underlying serum proteins (y = 0.97x–2.03, R2=0.993). Quantitating IgA biclonal isotype matched ( n = 58) using the Hevylite® assay gave higher results ( P = 0.002) than capillary zone electrophoresis (y = 1.48x–7.13, R2=0.959). In contrast, single IgA paraprotein results ( n = 48) did not differ between the two methods ( P = 0.466; y = 1.24x–2.74, R2=0.898), suggesting that polymerisation enhances Hevylite® quantitation. Conclusions These results suggest that disulphide-mediated polymerisation of IgA paraproteins is more common than true biclonal gammopathy and support dithiothreitol treatment of samples with isotype-matched IgA bands before quantifying by capillary zone electrophoresis. The Hevylite® assay should be utilized with caution where polymerisation is likely. Where IgGκ biclonal isotype-matched paraproteins appear on capillary zone electrophoresis, daratumumab therapy should be considered.