M-protein quantification is routinely performed by demarcating serum protein electrophoresis (SPE) regions. However, quantification of β-migrating M-protein is hindered by overlapping nonimmunoglobulin protein. Immunosubtraction (ISUB) on capillary electrophoresis is a method currently used qualitatively to subtract out (and therefore highlight) immunoglobulin isotypes in serum, thus reducing the masking effect of normal serum proteins. This study expands on traditional ISUB by developing a quantitative immunosubtraction (qIS) methodology.
qIS is achieved by estimating the monoclonal class-specific immunoglobulin contribution to the SPE region containing the M-protein. We conducted a recovery study by use of serial dilutions from 3 patients with β-region M-proteins (n = 22), performing SPE and ISUB on each dilution. We visualized the difference between the ISUB electrophoresis trace and the involved ISUB isotype-subtracted trace to distinguish M-protein and background polyclonal immunoglobulins, which was demarcated independently by 3 pathologists. The M-protein contribution to the β-region was calculated and applied to the β-region protein concentration producing the quantitative M-protein concentration, while minimizing contamination by nonimmunoglobulin or polyclonal immunoglobulin proteins.
Using a quality target of 25% error, we determined that our analytical measurable range spanned the maximum concentration tested (0.81 g/dL) to 0.05 g/dL. Passing–Bablok regression between qIS and the expected M-protein produced a slope of 1.04 (95% CI, 0.94–1.09), r = 0.99. Total CV was 4.8% and intraclass correlation between pathologists was 0.998.
qIS promises quantification of β-migrating M-proteins at concentrations an order of magnitude lower than traditional SPE methodology, allowing earlier detection of increasing or decreasing M-protein.