B-349 Improving identification of monoclonal IgM associated light chains by MASS-FIX

Abstract

Background Quantitative M-protein Study, Serum (QMPSS), a laboratory developed test, is used to identify and quantify monoclonal immunoglobulins by MALDI-TOF MS MASS-FIX. Monoclonal IgM proteins with glycosylated light chains (LC) are rare (5% of total M-proteins identified). In a given month approximately 3 cases of the 71 reported IgM monoclonal proteins with glycosylated light chains present with indeterminate kappa or lambda LC. The structure of the intact IgM monoclonal protein may restrict access to the Ig-LC constant region resulting in inefficient isolation. The laboratory uses immunofixation electrophoresis (IFE, Sebia) to confirm the Ig-LC isotype in this scenario. This study evaluates a reduction step prior to LC isolation to enhance the detection of the IgM isotype. Methods For QMPSS analysis, serum is added to five immunoaffinity matrices that target IgG, IgA, IgM, Ig-K, and Ig-L (CaptureSelect). After a binding phase, the serum/bead mixtures are washed to remove unbound protein. Finally, Igs are eluted, and the disulfide bonds are reduced, resulting in purified Ig-components. This step is followed by MALDI-TOF MS (Bruker) analysis. M-protein isotypes are determined by pairing each monoclonal protein’s unique LC mass detected in both heavy chain and LC Ig isolates. Twenty de-identified, residual waste serum samples with IgM-M-proteins without clear LC isotype were collected over several months. The proof-of-concept method modification studied to decrease reliance on IFE included use of 2.5 mcL of 0.5M dithiothreitol (DTT) added to kappa and lambda affinity matrix beads prior to sample addition followed by the QMPSS pre-analytical processing. Results For the 20 IgM glycosylated M-proteins, M-spike concentrations ranged from 0.026 to 0.506 g/dL. Using QMPSS and IFE lab routine protocol, the IgM samples were reported as glyco MK (n=17), biclonal MK+ML (n=1), and 2 samples were reported as negative. 16 of the 20 samples used in the study had IgM kappa monoclonal proteins that were identifiable on IFE. 13 of these IgM kappa monoclonal proteins were also identifiable by QMPSS-DTT. Additionally, 1 IgM kappa monoclonal protein was identifiable on QMPSS-DTT but was reported as equivocal on IFE. Of the 6 samples that the DTT method could not resolve, IFE was positive for 3. As shown by the 3 samples in which the light chain was identified only by IFE, the need for IFE as a confirmatory assay is not fully eliminated. The 3 false negative samples had M-quant values of 0.036g/dL, 0.043g/dL, and 0.117g/dL. More extensive testing would be required to determine the cause and prevalence of the observed discrepancy between IFE and QMPSS-DTT. Conclusion The use of the alternative QMPSS-DTT method shows potential to improve the capabilities of the QMPSS assay to identifying IgM monoclonal proteins with glycosylated light chains and would effectively remove the need for IFE in 70% of cases, improving test turnaround time. Further experimentation and optimization could decrease the number of discrepancies.