Murray, David L

Abstract Introduction Multiple myeloma (MM) is characterized by the abnormal proliferation of plasma cells, resulting in the overproduction of distinctive monoclonal proteins (M-protein). Suspected MM necessitates screening for M-protein through a combination of serum protein electrophoresis, serum immunofixation (SIFE), and serum free light chain (SFLC) determination. An M-protein appears as a relatively restricted band on agarose gel, where migration in ɑ-2 is rare. Methods A 55-year-old man with pulmonary tuberculosis presented with severe lower back pain. On examination, he appeared chronically ill, with conjunctival pallor. X-rays revealed vertebral compression fractures. The full blood count confirmed anemia; however, serum calcium and creatinine levels did not meet myeloma-defining event criteria. Results The serum protein electrophoresis revealed hypogammaglobulinemia, with the SIFE demonstrating unusual unrestricted κ staining in the ɑ-2 region. A markedly elevated κ SFLC and κ:λ ratio were found. Bone marrow examination demonstrated approximately 90% plasmacytosis. Urine immunofixation revealed a small, restricted κ band disproportionate to the κ SFLC. Notably, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified only polyclonal κ SFLC. Discussion Given the absence of a discernible M-protein on SIFE, a small κ restriction on urine immunofixation, and a polyclonal increase in κ SFLCs, the patient’s condition is being managed as an oligosecretory MM.

Abstract Background Immunoglobulin (Ig) measurements in the clinical laboratory have been traditionally performed by nephelometry, turbidimetry, electrophoresis, and ELISA assays. Mass spectrometry (MS) measurements have the potential to provide deeper insights on the nature of these markers. Content Different approaches—top-down, middle-down, or bottom-up—have been described for measuring specific Igs for endogenous monoclonal immunoglobulins (M-proteins) and exogenous therapeutic monoclonal antibody therapies (t-mAbs). Challenges arise in distinguishing the Ig of interest from the polyclonal Ig background. MS is emerging as a practical method to provide quantitative analysis and information about structural and clonal features that are not easily determined by current clinical laboratory methods. This review discusses clinically implemented examples, including isotyping and quantification of M-proteins and quantitation of t-mAbs within the polyclonal Ig background, as examples of how MS can enhance our detection and characterization of Igs. Summary This review of current clinically available MS proteomic tests for Igs highlights both analytical and nonanalytical challenges for implementation. Given the new insight into Igs from these methods, it is hoped that vendors, laboratorians, healthcare providers, and payment systems can work to overcome these challenges and advance the care of patients.

Abstract Background New immunoglobulin free light chain (FLC) assays are available. Despite analytical differences, it seems possible to use free light chain ratios (FLCr) generated by different assays and apply similar cut-points for the diagnosis of multiple myeloma. It is still unknown if we can use different assays for risk stratification of patients with monoclonal gammopathy of undetermined significance (MGUS). Methods Patients diagnosed with MGUS (N = 923) had FLC tested using a nephelometric FreeLite (Binding Site) assay on BNII instruments (Siemens) and a Sebia FLC assay (Sebia) on a DS2 ELISA analyzer (Dynex). Patients were followed up for progression to any plasma cell dyscrasia (PCD) for several decades. The Mayo MGUS risk stratification model for progression was assessed with both assays (M-spike >1.5 g/dL; non-IgG isotype and abnormal FLCr), using package insert reference intervals (RI) and a new metric called principal component 2 (PC2). Results There were 94 events of progression to PCD in the cohort during a median of 38 years of follow-up. Freelite and Sebia FLC showed similar hazard ratios in the risk models for elevated FLCr. An alternative clinical decision point lower than the package insert RI was evaluated for the Sebia assay, which improved risk stratification for patients with a low FLCr. The PC2 metric showed similar performance to the FLCr in models, without superior benefit. Conclusions The Sebia ELISA-based FLC assay can be employed in an MGUS risk stratification model with similar performance to the original 2005 risk stratification model using the FreeLite assay.

Abstract Background Matrix assisted laser desorption ionization time of flight mass spectrometry coupled to immune enrichment (MASS-FIX) as an alternative to serum immunofixation electrophoresis has demonstrated increased sensitivity in monoclonal protein (MP) detection with improved laboratory workflow. This study explored similar replacement of urine immunofixation electrophoresis (u-IFE) with urine MASS-FIX (u-MASS-FIX) by method comparison. Methods Residual urine (n = 1008) from Mayo Clinic patients with a known plasma cell disease were assayed neat by u-MASS-FIX analysis. Each sample was paired with the following: u-IFE, urine total protein, urine protein electrophoresis, serum κ/λ free light chain (LC) ratio (rFLC), and serum MASS-FIX (s-MASS-FIX). Analytical sensitivities were measured in pooled urine spiked with daratumumab. Results u-IFE and u-MASS-FIX had 91% agreement in determining the presence/absence of MPs (Cohen kappa = 0.8200). In discrepant cases, serum rFLC statistically aligned more closely with positive u-MASS-FIX cases than u-IFE. Patients positive by both s-MASS-FIX and u-MASS-FIX had matching MP masses (±20 daltons) in 94% of cases. The u-MASS-FIX spectra further identified κ/λ LC fragments and glycosylated LCs not appreciated on u-IFE. The unconcentrated u-MASS-FIX limit of detection of 0.156 mg/mL was determined equivalent to 100× concentrated u-IFE. Conclusion u-MASS-FIX is a reliable alternative to u-IFE with the added benefits of LC glycosylation detection and MP mass tracking between serum and urine. Furthermore, u-MASS-FIX is performed using neat urine. Eliminating the need to concentrate urine for u-IFE has potential to increase productivity by decreasing labor minutes per test.

Abstract Background Multiple laboratory tests are employed for detection of monoclonal proteins in patients and include serum protein electrophoresis (SPEP), immunofixation electrophoresis, free light chain (FLC) immunoassay, and mass spectrometry (Mass-Fix). Recently, reports on a drift in FLC quantitation results have been brought to light. Methods We studied a cohort of 16 887 patients whose sera were tested for a monoclonal protein by a FLC assay, serum protein electrophoresis, and Mass-Fix. This is a retrospective study designed to assess the impact of a drift on the performance of FLC ratio (rFLC) in groups of patients with and without detectable plasma cell disorders (PCDs). Results The results demonstrated that 63% of patients with monoclonal protein equal or higher than 2 g/L (by SPEP) had an abnormal rFLC (reference range 0.26–1.65). Conversely, 16% of patients with undetectable monoclonal protein by other methods (i.e., SPEP and Mass-Fix) who also had no record of treated PCD had an abnormal rFLC. In these cases, there was an imbalance in the number of kappa high rFLCs to lambda low rFLCs of 201 to 1. Conclusions The results of this study suggest decreased specificity of rFLC for a monoclonal kappa FLC in the 1.65 to 3.0 range.