Immunoglobulin clonality analysis. Resolution of ambiguities in immunofixation electrophoresis results by high-resolution, two-dimensional electrophoretic analysis of paraprotein bands eluted from agarose gels.

This article describes evaluation of the clonality of origin of immunoglobulin (Ig) chains by strategies that include qualitative analysis of paraproteins with high-resolution, two-dimensional electrophoresis (2DE) and silver staining. This approach is helpful in evaluating specimens in which standard immunoelectrophoresis or immunofixation electrophoresis (IFE) techniques do not provide definitive Ig typing results. One method the authors developed involves a "band elution" procedure, in which proteins present in high-resolution agarose gel electrophoresis bands are cut from the agarose gel, eluted with a denaturing buffer, and subjected to 2DE. The microheterogeneity patterns of the Ig light chains, heavy chains, or both are evaluated for their relationship regarding mass, charge, and, by inference, number of genes of origin. When necessary, determination of charge relationships may be aided by urea-mediated carbamylation of lysine residues, which introduces single-charge shifts to the individual protein subunits. Overall, these adjunctive techniques are particularly useful in cases with multiple bands of identical immunologic types (eg, several IgG1 bands) on IFE before and after sulfhydryl reduction (with dithiothreitol or 2-mercaptoethanol). The authors present procedural details and examples of the 2DE band elution patterns of serum and urine samples from four patients with B-cell dyscrasias, including the first reported case of POEMS syndrome with biclonal gammopathy, to the best of their knowledge.