<i>Implementation of a Mass Spectroscopic Method (Mass-Fix) to Replace Traditional Electrophoresis Methods for Patients with Monoclonal Gammopathies</i>

Background The detection, isotyping and quantitation of over-expressed immunoglobulin(s) (Igs) (M-protein) is key for diagnosing and monitoring patients with monoclonal gammopathies. Traditionally, our lab has used a combination of agarose gel protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE) for these purposes. In 2018, a mass spectrometric method (Mass-Fix) was implemented to replace IFE for M-protein isotyping. This work documents the validation of a quantitative version (Q-Mass-Fix) to replace both SPEP and IFE. Methods 24,645 samples submitted for M-protein testing between 7/2018 and 9/2023 with quantitative IgG, IgA and IgM measurements, Mass-Fix analysis and SPEP results were utilized for this study. A separate cohort of 100,000 M-protein negative samples were utilized to mathematically model the spectral shapes IgG, IgA and IgM polyclonal background. To detect M-proteins, software was developed that fits the polyclonal Ig portion of the spectra and M-proteins are detected as peaks above the polyclonal background. M-proteins isotypes are determined by matching similar peaks in spectra in 5 sperate immuno enrichments (IgG, IgA, IgM, kappa and lambda). M-protein quantitation was obtained by multiplying total M-protein isotype quantity by the fraction area of the M-protein in the spectra. M-protein dilutional studies were used to examine the linearity and level of detection of Q-Mass-Fix. Results SPEP and Q-Mass-Fix M-protein quantitation had the best agreement between the range of 10 to 40 g/L. Below 10 g/L, Q-Mass Fix had on average, lower M-protein values. Above 40 g/dL, Q-Mass-Fix values were on average, higher than SPEP. The automated isotyping assignment was 98.8 percent consistent with the isotype assigned by visual inspection. Dilutional studies revealed that Q-Mass-Fix quantitation was linear over a range of 70 to 0.04 g/L (slope 1.0, r2 0.99) with an analytical limit of quantitation of 0.1 g/L and a limit of detection of 0.04 g/L. Conclusion Q-Mass-Fix represents a significant improvement in comparison to our current SPEP method. Q-Mass-Fix is more capable of removing the interference of polyclonal Igs below 10 g/L and is not subjective to the dye saturation effect at high M-protein levels. The increased analytical measuring range and improved limit of detection of detection could be beneficial in tracking patient's response. Q-Mass Fix is more automated than SPEP and the workflow in our laboratory.