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 (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.

Monoclonal protein polymerisation is the cause of IgM multiple isotype-matched monoclonal proteins

“…The recently published study by Valentine and Dawney 1 contained interesting insights about multiple isotype-matched monoclonal proteins (MIMMP) and the effect of treatment with a reducing agent. Their findings suggested that, in most cases, reducing agent treatment caused IgA but not IgG MIMMP to resolve into a single band.…”