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

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 (CZE), are truly biclonal. Methods Sera containing multiple isotype-matched paraproteins were treated with the reducing agent dithiothreitol (DTT) and CZE performed pre- and post-treatment. Band resolution and effect of resolution on quantitation of paraprotein burden was assessed. The Hevylite® turbidimetric assay was also evaluated for ability to quantify such paraproteins. Results Among patients with biclonal isotype-matched (BIM) paraproteins, 23/24 (96%) IgA paraproteins resolved into a single band following treatment with DTT 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 post-DTT 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 BIM (n=58) using the Hevylite® assay gave higher results (P=0.002) than CZE (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 DTT treatment of samples with isotype-matched IgA bands before quantifying by CZE. The Hevylite® assay should be utilised with caution where polymerisation is likely. Where IgGκ BIM paraproteins appear on CZE, Daratumumab therapy should be considered.