Utilizing Mass Spectrometry to Detect and Isotype Monoclonal Proteins in Urine: Comparison to Electrophoretic Methods.

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.

Characterizing M-protein light chain glycosylation via mass spectrometry.

OBJECTIVES: Monoclonal gammopathy of undetermined significance (MGUS) patients with M-proteins containing n-glycosylated light chains (GLC) have an increased risk for progression to symptomatic plasma cell disorders (PCD). Large-scale research involving the determination of glycan specific moieties is understudied due to the lack of clinically viable methods. This report documents a proof-of-concept glycan characterization method for patients with M-protein GLCs. DESIGN AND METHODS: Twenty-three previously characterized MGUS patients with glycosylated light chains identified by MASS-FIX were used for this study. Glycosylated light chains were enriched from patient serum using light chain (LC) specific Sepharose nanobody beads (NB), followed by glycan digestion via PNGase F. Glycan moieties were derivatized on-target using Girard's reagent T for MALDI-TOF analysis and confirmed with top-down GLC LC-ESI-Q-TOF-MS analysis. RESULTS: Intact GLC LC-ESI-Q-TOF-MS and cleaved glycan MALDI-TOF MS analysis had 100% agreement for the top three intensity glycans between spectra and 88 percent agreement for all reported glycan moieties. GLC moieties among patients were similar with fucosylation being the only notable difference. Additionally, doubly glycosylated light chains were observed in two patients. CONCLUSIONS: The MALDI-TOF method provides the tools to characterize and evaluate GLCs in a clinical setting as it is adaptable to our clinical MASS-Fix assay, relatively cheap, and accurate in glycan moiety assignments as confirmed by top-down GLC LC-ESI-Q-TOF-MS.

Mu heavy chain disease with MYD88 L265P mutation: an unusual manifestation of lymphoplasmacytic lymphoma.

BACKGROUND: Mu heavy chain disease is a rare lymphoid neoplasm characterized by vacuolated bone marrow plasma cells and secretion of defective mu immunoglobulin heavy chains. The biological basis of mu heavy chain disease is poorly understood. CASE PRESENTATION: We report a case of mu heavy chain disease with MYD88 L265P mutation and deletion of 6q, genetic aberrations that are both strongly associated with lymphoplasmacytic lymphoma/Waldenström macroglobulinemia. Identification of the truncated mu immunoglobulin was facilitated by mass spectrometric analysis of the patient's serum. CONCLUSIONS: Mu heavy chain disease has been described as similar to chronic lymphocytic leukemia; however, the frequency of lymphocytosis in mu heavy chain disease has not been previously reported. We reviewed all previously published mu heavy chain disease reports and found that lymphocytosis is uncommon in the entity. This finding, along with the emerging genetic feature of recurrent MYD88 mutation in mu heavy chain disease, argues that at least a significant subset of cases are more similar to lymphoplasmacytic lymphoma than to chronic lymphocytic leukemia.

Mass-Fix better predicts for PFS and OS than standard methods among multiple myeloma patients participating on the STAMINA trial (BMT CTN 0702 /07LT).

Measuring response among patients with multiple myeloma is essential for the care of patients. Deeper responses are associated with better progression free survival (PFS) and overall survival (OS). To test the hypothesis that Mass-Fix, a mass spectrometry-based means to detect monoclonal proteins, is superior to existing methodologies to predict for survival outcomes, samples from the STAMINA trial (NCT01109004), a trial comparing three transplant approaches, were employed. Samples from 575 patients from as many as three time points (post-induction [post-I; pre-maintenance [pre-M]; 1 year post enrollment [1YR]) were tested when available. Four response parameters were assessed: Mass-Fix, serum immunofixation, complete response, and measurable residual disease (MRD) by next generation flow cytometry. Of the four response measures, only MRD and Mass-Fix predicted for PFS and OS at multiple testing points on multivariate analyses. Although MRD drove Mass-Fix from the model for PFS at post-I and pre-M, 1YR Mass-Fix was independent of 1YR MRD. For OS, the only prognostic pre-I measure was Mass-Fix, and the only 1YR measures that were prognostic on multivariate analysis were 1YR MRD and 1YR Mass-Fix. SIFE and CR were not. Mass-Fix is a powerful means to track response.

Detection of Plasma Cell Disorders by Mass Spectrometry: A Comprehensive Review of 19,523 Cases.

OBJECTIVES: To verify the analytical performance of a new mass spectrometry-based method, termed MASS-FIX, when screening for plasma cell disorders in a routine clinical laboratory. PATIENTS AND METHODS: Results from 19,523 unique patients tested for an M-protein between July 24, 2018, and March 6, 2020, by a combination serum protein electrophoresis (SPEP) and MASS-FIX were examined for consistency with pretest implementation performance. MASS-FIX's ability to verify abnormal results from SPEP and free light chain measurements was then compared with that of immunofixation electrophoresis (IFE) using a separate cohort of 52,586 patients tested by SPEP/IFE during the same period. RESULTS: Overall, 62.4% of our cohort was negative for an M-protein. Importantly, 7.3% of all specimens had an M spike on SPEP (0.1 to 8.5 g/dL) and MASS-FIX detected an M-protein in all these samples. Of all samples, 30.3% had M-proteins that were detected by MASS-FIX but the SPEP finding was too small for quantification. Of the positive samples, 5.7% contained a therapeutic monoclonal antibody. Of the positive samples, 4.1% had an N-glycosylated light chain (biomarker of high-risk plasma cell disorders). MASS-FIX confirmed a higher percentage of SPEP abnormalities than IFE. MASS-FIX was slightly more sensitive than IFE when confirming an M-protein in samples with an abnormal free light chain ratio. MASS-FIX had a very low sample repeat rate (1.5%). MASS-FIX was highly automatable resulting in a higher number of samples/technologist/day than IFE (∼30% more). CONCLUSION: Overall, MASS-FIX was successful in maintaining validation characteristics. MASS-FIX was more sensitive in confirming SPEP abnormalities when compared with IFE. Ability to detect therapeutic monoclonal antibodies and glycosylated light chains was distinctly advantageous.

MALDI-TOF mass spectrometry can distinguish immunofixation bands of the same isotype as monoclonal or biclonal proteins.

BACKGROUND: Plasma cell disorders (PCDs) are typically characterized by excessive production of a single immunoglobulin, defined as a monoclonal protein (M-protein). Some patients have more than one identifiable M-protein, termed biclonal. Traditional immunofixation electrophoresis (IFE) cannot distinguish if two bands of the same isotype represent biclonal proteins or M-proteins with some other feature. A novel assay using immunoenrichment coupled to matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (Mass-Fix) was applied to determine whether two bands of the same isotype represented (1) monomers and dimers of a single M-protein, (2) an M-protein plus a therapeutic monoclonal antibody (t-mAb), (3) an M-protein with light chain glycosylation, or (4) two distinct biclonal M-proteins. METHODS: Patient samples with two bands of the same isotype identified by IFE were enriched using nanobodies against IgG, IgA, IgM, or κ and λ light chains then analyzed by Mass-Fix. Light chain masses were used to differentiate IgGκ M-proteins from t-mAbs. Mass differences between peaks were calculated to identify N-glycosylation or matrix adducts. High-resolution mass spectrometry was used as a comparator method in a subset of samples. RESULTS: Eighty-one residual samples were collected. For IgA, 93% (n = 25) were identified as monoclonal. For IgG, 67% (n = 24) were monoclonal, and 33% (n = 12) were truly biclonal. Among the monoclonal IgGs, the second band represented a glycosylated form for 21% (n = 5), while 33% (n = 8) had masses consistent with a t-mAb. 44% (n = 8) of IgM samples were biclonal, and 56% (n = 10) were monoclonal, of which one was glycosylated. CONCLUSIONS: We demonstrate the utility of mass spectrometry in the characterization of multiple IFE bands of the same isotype. Improved reporting accuracy of M-proteins is useful for monitoring of patients with PCDs.

Clearing drug interferences in myeloma treatment using mass spectrometry.

OBJECTIVE: To explore the possibility of using a combination of a rapid MALDI-TOF-MS method (Mass-Fix) in conjunction with higher resolution LC-ESI-QTOF-MS (miRAMM) measurements to discriminate the IgG kappa M-protein from daratumumab, elotuzumab and isatuximab in myeloma patients. DESIGN & METHODS: 86 patients with an IgG kappa M-protein were spiked with therapeutic levels of the drugs and examined by Mass-Fix and miRAMM to establish the percent of cases that could be resolved by each method. The method was then applied to 21 samples from patients receiving one of the drugs. RESULTS: Mass-Fix was capable of resolving the t-mAb from M-protein for 87 percent of the spiked samples. For the cases unresolved by Mass-Fix, miRAMM was capable of resolving the remaining drug interferences. The 21 IgG kappa myeloma patients that were receiving the drugs were all resolved by Mass-Fix. CONCLUSION: This proposed algorithm allows use of a clinical available assay (Mass-Fix) while maximizing the number of cases that can accurately resolve the t-mAb from the M-protein.

MASS-FIX for the detection of monoclonal proteins and light chain N-glycosylation in routine clinical practice: a cross-sectional study of 6315 patients.

Immunoenrichment-based matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), termed MASS-FIX, offers several advantages over immunofixation for the detection and isotyping of serum monoclonal protein, including superior sensitivity and specificity, the ability to differentiate therapeutic monoclonal antibodies, and the rapid identification of light chain (LC) N-glycosylation. We identified 6315 patients with MASS-FIX performed at our institution since 2018. Of these, 4118 patients (65%) with a wide array of plasma cell disorders (PCD), including rare monoclonal gammopathies of clinical significance, had a positive MASS-FIX. Two-hundred twenty-one (5%) of the MASS-FIX positive patients had evidence of LC N-glycosylation, which was more commonly identified in IgM heavy chain isotype, kappa LC isotype, and in diagnoses of immunoglobulin light chain (AL) amyloidosis and cold agglutinin disease (CAD) compared to other PCD. This cross-sectional study describes the largest cohort of patients to undergo MASS-FIX in routine clinical practice. Our findings demonstrate the widespread utility of this assay, and confirm that LC N-glycosylation should prompt suspicion for AL amyloidosis and CAD in the appropriate clinical context.

Automation and validation of a MALDI-TOF MS (Mass-Fix) replacement of immunofixation electrophoresis in the clinical lab.

Objectives: A matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) method (Mass-Fix) as a replacement for gel-based immunofixation (IFE) has been recently described. To utilize Mass-Fix clinically, a validated automated method was required. Our aim was to automate the pre-analytical processing, improve positive specimen identification and ergonomics, reduce paper data storage and increase resource utilization without increasing turnaround time. Methods: Serum samples were batched and loaded onto a liquid handler along with reagents and a barcoded sample plate. The pre-analytical steps included: (1) Plating immunopurification beads. (2) Adding 10 µl of serum. (3) Bead washing. (4) Eluting the immunoglobulins (Igs), and reducing to separate the heavy and light Ig chains. The resulting plate was transferred to a second low-volume liquid handler for MALDI plate spotting. MALDI-TOF mass spectra were collected. Integrated in-house developed software was utilized for sample tracking, driving data acquisition, data analysis, history tracking, and result reporting. A total of 1,029 residual serum samples were run using the automated system and results were compared to prior electrophoretic results. Results: The automated Mass-Fix method was capable of meeting the validation requirements of concordance with IFE, limit of detection (LOD), sample stability and reproducibility with a low repeat rate. Automation and integrated software allowed a single user to process 320 samples in an 8 h shift. Software display facilitated identification of monoclonal proteins. Additionally, the process maintains positive specimen identification, reduces manual pipetting, allows for paper free tracking, and does not significantly impact turnaround time (TAT). Conclusions: Mass-Fix is ready for implementation in a high-throughput clinical laboratory.

Blood mass spectrometry detects residual disease better than standard techniques in light-chain amyloidosis.

In patients with immunoglobulin light-chain (AL) amyloidosis, depth of hematologic response correlates with both organ response and overall survival. Our group has demonstrated that screening with a matrix-assisted laser desorption/ionization-time-of-flight (TOF) mass spectrometry (MS) is a quick, sensitive, and accurate means to diagnose and monitor the serum of patients with plasma cell disorders. Microflow liquid chromatography coupled with electrospray ionization and quadrupole TOF MS adds further sensitivity. We identified 33 patients with AL amyloidosis who achieved amyloid complete hematologic response, who also had negative bone marrow by six-color flow cytometry, and who had paired serum samples to test by MS. These samples were subjected to blood MS. Four patients (12%) were found to have residual disease by these techniques. The presence of residual disease by MS was associated with a poorer time to progression (at 50 months 75% versus 13%, p = 0.003). MS of the blood out-performed serum and urine immunofixation, the serum immunoglobulin free light chain, and six-color flow cytometry of the bone marrow in detecting residual disease. Additional studies that include urine MS and next-generation techniques to detect clonal plasma cells in the bone marrow will further elucidate the full potential of this technique.

Direct Detection of Monoclonal Free Light Chains in Serum by Use of Immunoenrichment-Coupled MALDI-TOF Mass Spectrometry.

BACKGROUND: Free light chain (FLC) quantification is the most analytically sensitive blood-based method commercially available to diagnose and monitor patients with plasma cell disorders (PCDs). However, instead of directly detecting monoclonal FLCs (mFLCs), FLC assays indirectly assess clonality based on quantifying κ and λ FLCs and determination of the к/λ FLC ratio. Often an abnormal FLC ratio is the only indication of a PCD, and confirmation by a direct method increases diagnostic confidence. The aim of this study was to develop an analytically sensitive method for direct detection of mFLCs. METHODS: Patient sera (n = 167) previously assessed by nephelometric FLC quantification and immunofixation electrophoresis (IFE) were affinity enriched for IgG, IgA, and total and free κ and λ light chains and subjected to MALDI-TOF MS. Relative analytical sensitivity of these methods was determined using serially diluted sera containing mFLCs. RESULTS: In sera with abnormal FLC ratios (n = 127), 43% of monoclonal proteins were confirmed by IFE, 57% by MALDI-TOF MS without FLC enrichment, and 87% with FLC enrichment MALDI-TOF MS. In sera with normal FLC ratios (n = 40), the FLC MALDI-TOF MS method identified 1 patient with an mFLC. Serial dilution and analysis of mFLC containing sera by IFE, nephelometry, and FLC MALDI-TOF MS demonstrated that FLC MALDI-TOF MS analysis had the highest analytical sensitivity. CONCLUSIONS: FLC immunoenrichment coupled to MALDI-TOF MS enables direct detection of mFLCs and significantly increases the confirmation of abnormal serum FLC ratios over IFE and MALDI-TOF MS without FLC enrichment, thereby providing added confidence for diagnosing FLC PCDs.