Identifying therapeutic monoclonal antibodies using target protein collision electrophoresis reflex assay to separate the wheat from the chaff.

Monoclonal gammopathies are characterized by the presence of monoclonal immunoglobulins, also known as M-proteins. Therapeutic monoclonal antibodies (t-mAbs) can interfere in laboratory assays used to monitor the state of disease, such as serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE). To establish a correct interpretation of IFE, Target protein-Collision Immunofixation Electrophoresis Reflex Assay (T-CIERA) was developed to identify t-mAbs in IFE. Here we demonstrate that T-CIERA is applicable to a wide variety of t-mAbs for which the target protein is commercially available. Moreover, the shift observed was characteristic for each t-mAb, and T-CIERA enabled the identification of multiple t-mAbs sharing a common target protein. Additionally, the lower limit of detection (LLOD) was determined objectively, and T-CIERA demonstrated an adequate LLOD for all tested t-mAbs. Furthermore, T-CIERA was also successfully applied to serum samples obtained from patients receiving daratumumab, isatuximab, elotuzumab, and durvalumab treatment. In conclusion, T-CIERA is a suitable reflex assay for identifying a wide variety of t-mAbs, including those for which no commercial assay is available to deal with their interference. Moreover, CD38-CIERA could serve as an alternative or complementary test to the commercially available Hydrashift assay kits. T-CIERA would enable laboratories without mass spectrometry equipment and expertise in this area to distinguish between drug and disease to improve clinical response monitoring and diagnosis of monoclonal gammopathies.

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.

Expert-Level Immunofixation Electrophoresis Image Recognition based on Explainable and Generalizable Deep Learning.

BACKGROUND: Immunofixation electrophoresis (IFE) is important for diagnosis of plasma cell disorders (PCDs). Manual analysis of IFE images is time-consuming and potentially subjective. An artificial intelligence (AI) system for automatic and accurate IFE image recognition is desirable. METHODS: In total, 12 703 expert-annotated IFE images (9182 from a new IFE imaging system and 3521 from an old one) were used to develop and test an AI system that was an ensemble of 3 deep neural networks. The model takes an IFE image as input and predicts the presence of 8 basic patterns (IgA-, IgA-, IgG-, IgG-, IgM-, IgM-, light chain and ) and their combinations. Score-based class activation maps (Score-CAMs) were used for visual explanation of the models prediction. RESULTS: The AI model achieved an average accuracy, sensitivity, and specificity of 99.82, 93.17, and 99.93, respectively, for detection of the 8 basic patterns, which outperformed 4 junior experts with 1 years experience and was comparable to a senior expert with 5 years experience. The Score-CAMs gave a reasonable visual explanation of the prediction by highlighting the target aligned regions in the bands and indicating potentially unreliable predictions. When trained with only the new system images, the models performance was still higher than junior experts on both the new and old IFE systems, with average accuracy of 99.91 and 99.81, respectively. CONCLUSIONS: Our AI system achieved human-level performance in automatic recognition of IFE images, with high explainability and generalizability. It has the potential to improve the efficiency and reliability of diagnosis of PCDs.

[Detection of paraprotein in plasma cell tumors].

Paraprotein is a laboratory biomarker of plasma cell tumors and other lymphoproliferative diseases. Its determination is necessary for diagnosing, monitoring and assessment of therapy effectiveness. The lecture presents the main methods of qualitative and quantative analysis of monoclonal proteins: gel electrophoresis, capillary electrophoresis, immunofixation and nephelometry features, possibilities and limitations are reviewed. The main sources of errors and artifacts during these studies are considered. Also the difficulties in the diagnosis and interpretation of the results of serum and urine tests are highlighted.

Mitigating the interference of Daratumumab with immunofixation electrophoresis. A single-center experience using Hydrashift 2/4 kit.

BACKGROUND: Multiple myeloma (MM) accounts for approximately 10% of hematological malignancies. The monoclonal immunoglobulin G kappa (IgG-κ) daratumumab can bind to CD38 on MM cells and be detected in serum immunofixation (IF), causing pitfalls in M-protein quantification. OBJECTIVES: To determine the efficacy of mitigating the interference of IgG MM treated with daratumumab. METHODS: Levels of Ig, free light chains (FLC) kappa (κ) and lambda (λ), serum protein electrophoresis (SPE)/IF, and Hydrashift 2/4 assays were assessed following manufacturer's instructions in three patients. RESULTS: Patient 1 was a 70-year-old male diagnosed with IgG-λ MM. The IF distinguished two monoclonal bands (IgG-κ and IgG-λ). With the Hydrashift assay, the daratumumab-anti-daratumumab immune complex shifted the IgG-κ to the α zone, suggesting that the monoclonal IgG-κ band corresponded to daratumumab. Patient 2 was a 63-year-old male with IgG-κ MM who was receiving daratumumab once every other week. SPE/IF assay revealed a faint monoclonal IgG-κ band in the  zone. A stronger monoclonal band was observed after administration. The IgG-κ band disappeared on the Hydrashift assay, while the daratumumab-anti-daratumumab complex appeared as a broad smear in the α-region. Patient 3, a 63-year-old male diagnosed with IgG-λMM, was receiving daratumumab once every other month. The IF assay showed two distinct bands (IgG-κ and IgG-λ) post-daratumumab administration. The shift to the α zone of the IgG-κ bands on the Hydrashift assay confirmed that the additional band observed post-infusion was due to the daratumumab. CONCLUSIONS: The Hydrashift assay can help distinguish daratumumab from endogenous M-spike.

Monoclonal Immunoglobulin Associated with Monoclonal Free Light Chains Invisible on Capillary Electrophoresis.

BACKGROUND: We report the case of a monoclonal immunoglobulin of IgM Lambda isotype associated with monoclonal lambda-type free light chains not detected by capillary electrophoresis but identified by immunofixation. METHODS: Capillary electrophoresis showed hypoproteinemia and an inflammatory syndrome. The IF realized on Hydrasys 2 Scan Focusing Sebia® reveals an IgM monoclonal band and two monoclonal bands in the total lambda. A second IF is performed using anti IgM, anti IgD, anti IgE and anti-total and -free lambda light chains as antisera. It reveals the presence of a monoclonal protein isotype IgM Lambda with free light chains. In view of these discordant results, an immunosubtraction was performed on the same sample showing no abnormality. RESULTS: Our patient has a monoclonal IgM Lambda with lambda monoclonal free light chains all masked on capillary electrophoresis and therefore not detected. CONCLUSIONS: Capillary electrophoresis techniques are incrementally becoming the techniques of choice in medical laboratories as a replacement for gel electrophoresis, due to their automation and better sensitivity. However, in some cases, a monoclonal immunoglobulin may not be detected by capillary technique and may cause an inaccurate interpretation.

Huge discrepancy between serum immunoglobulin concentration and proteinemia due to heavy chain disease.

Immunoassays are widely used in clinical laboratories because of their ease of use and low cost. These tests are based on antigen-antibody binding. However, clinicians and laboratory personnel may be confronted with immunoassay interference leading to difficulties in medical care. Here, we report a huge analytical discrepancy with IgG concentration higher than proteinemia in a 75-year-old man. Serum electrophoresis and immunofixation diagnosed γ-heavy chain disease. After investigation by different methods, the assay discrepancy was still present. We hypothesize that the interference is related to the truncated immunoglobulin secreted by the lymphoproliferative disorder.

Detection and Characterization of Paraproteinemia in Canine Chronic B-cell Lymphocytic Leukemia Using Routine and Free Light Chain Immunofixation.

BACKGROUND: Hyperglobulinemia is reported in 26% of canine chronic B-cell lymphocytic leukemia (B-CLL) cases. However, few cases have been characterized by protein electrophoresis and immunofixation (IF), and the incidence of a monoclonal protein (M-protein) is unknown using these techniques. OBJECTIVE: To characterize and determine the proportion of canine B-CLL cases with an M-protein using plasma protein electrophoresis (PPE), routine and free light chain (fLC) IF, and to assess if productive B-CLL cases express MUM1/IRF4 by cell tube block (CTB). METHODS: PPE, routine (targeting IgG, IgA, IgM, IgG4, and light chain) and fLC IF were performed using 48 dog B-CLL plasma samples from patients diagnosed via peripheral blood flow cytometry. CTB was performed on a separate cohort of 15 patients. RESULTS: Hyperproteinemia (>7.5 g/dL) was present in 17/48 cases (35%). An M-protein was detected in 32/48 cases (67%). Of these, 19/32 cases (59%) had only complete (monoclonal heavy and light chain) M-proteins detected, 10/32 cases (31%) had both complete and fLC M-proteins detected, and 3/32 cases (9%) had only an fLC M-protein detected. IgM was the most common clonal immunoglobulin isotype detected (23 cases). CD21+ cell counts were higher in cases with detectable M-protein. Plasma fLC IF suggested ß-γ region interference, likely caused by clotting proteins. All B-CLL cases consistently expressed PAX5 and did not express MUM1/IRF4. CONCLUSIONS: Most B-CLL cases had an M-protein and were not hyperproteinemic. Most cases with paraproteins had a complete IgM monoclonal gammopathy; a subset had documented fLCs. The prognostic significance of heavy and fLC presence should be evaluated.

A rare case of the IgE prozone phenomenon in IgE multiple myeloma.

IgE multiple myeloma is a rare subtype of myeloma, accounting for <0.1% of all myeloma cases. Difficulties in diagnosing and monitoring this rare myeloma type are recognized, including the need of heightened awareness for initial diagnosis, performing a reflex immunofixation test using an anti-IgE antisera, and recognizing the possibility of the prozone phenomenon. Here, we report a rare case of IgE multiple myeloma with the prozone phenomenon. This case was characterized by a paradoxical increase in IgE levels with a progressive increase in the dilution factor. Moreover, serial monitoring of IgE levels correlated with the trend in the serum free light chain ratio, especially when the monoclonal protein was no longer detectable. This case highlights the need for laboratory professionals to be vigilant about the occurrence of the prozone phenomenon in IgE multiple myeloma.

Pilot Verification of a Novel Approach to Remove Electrophoretic Interference of the Therapeutic Monoclonal Antibody Daratumumab.

INTRODUCTION: The advent of therapeutic monoclonal antibodies (tmAbs) in treatment of multiple myeloma poses unique challenges for the clinical laboratory. These tmAbs may appear as a detectable monoclonal protein by electrophoretic methods resulting in misinterpretation or inability to measure therapeutic responses in some patients, and there are currently limited techniques for identifying interference. In this study we performed a preliminary assessment of the SPIFE anti-daratumumab (SPIFE anti-Dara) reagent to determine whether it would be a feasible aid in resolving the interference of tmAbs with serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE). METHODS: We performed a pilot study with 20 serum samples and clinical correlates. All samples had a characteristic daratumumab electrophoretic pattern (cathodal IgG/κ). A pre-electrophoretic sample treatment was performed with SPIFE anti-Dara. The reagent is a derivatized anti-Dara that forms multiple antibody/daratumumab complexes. SPE and IFE technical procedures were performed on Helena SPIFE 3000 according to the manufacturer instructions. RESULTS: Of the 20 patients, 14 patients were identified to be on daratumumab therapy. In 14/14 of cases, the daratumumab interference was successfully removed both from SPE and IFE assays. Disease associated M-protein was still visible after pretreatment, and quantification of M-protein may be possible with the use of SPIFE anti-Dara procedure. DISCUSSION: SPIFE anti-Dara is a promising method to remove the interference of therapeutic monoclonal antibody daratumumab with SPE and IFE results in clinical laboratories and warrants further assessment.

Pitfalls in Assessing Measurable Residual Disease in Plasma Cell Myeloma: a Comparison between Common Laboratory Tests.

BACKGROUND: Measurable residual disease (MRD) in plasma cell myeloma is one of the most important determinants for patients' outcome. Several laboratory tests exist to assess for the presence of MRD with variable accuracy. The aim of this study is to examine the sensitivity of immunofixation electrophoresis (IFE), serum free light chain (FLC), bone marrow immunohistochemistry (IHC), and multicolor flow cytometry (FC) and to address potential caveats of each test. METHODS: Forty patients of plasma cell myeloma who were diagnosed with a positive MRD were retrospectively included in this study. The results of IFE and serum FLC at the time of bone marrow biopsy were collected. RESULTS: In all cases, malignant plasma cells constituted less than 5% of bone marrow cells. MRD was detected by FC in 38 cases (95%) and by IHC in 28 cases (70%). In 2 cases, residual malignant plasma cells appeared in the subcortical area which is difficult to aspirate, and thus they were detected by IHC but not by FC. Among the entire cohort, 38 patients (95%) had positive IFE at the time of bone marrow biopsy, while serum FLC abnormality was detected in 19 patients (48%) only. CONCLUSIONS: Both FC and IFE exhibited high sensitivity in detecting MRD in plasma cell myeloma with comparable results. IFE remains less invasive and less expensive than FC. Despite the lower sensitivity of bone marrow IHC staining, its diagnostic role is essential and can be superior to FC in a subset of cases, for which its routine examination is recommended. Serum FLC test provided the least sensitivity among all tests.

[Application of Hydrashift 2/4 daratumumab assay in eliminating interference of daratumumab on serum immunofixation electrophoresis].

Objective: To investigate the interference of daratumumab on immunofixation electrophoresis after treating plasma cell diseases and methods to eliminate the interference. Methods: Serum samples of eight patients with plasma cell diseases treated with daratumumab in Peking University People's Hospital from April 2020 to March 2021 were collected for standard immunofixation electrophoresis and Hydrashift 2/4 daratumumab assay. Results: After treatment, 81.3% (13/16) of the samples showed drug-induced monoclonal antibodies (IgG-κ) . The samples without drug-induced monoclonal bands were related to individual differences, administration intervals, and immunoglobulin levels. Among the samples with IgG-κ monoclonal bands, 76.9% (10/13) could be directly identified as endogenous or exogenous monoclonal bands by immunofixation electrophoresis, and the others (3/13) could be identified by Hydrashift 2/4 daratumumab assay. Conclusion: Hydrashift 2/4 daratumumab assay can remove the band of daratumumab on the immunofixation electrophoresis and help with efficacy evaluation.

In vitro evaluation of potential interference of lokivetmab with protein electrophoresis and immunofixation.

OBJECTIVE: In humans, misdiagnoses of monoclonal gammopathy after use of therapeutic monoclonal antibodies has been documented. This triggers concerns for similar misdiagnoses in animals treated with monoclonal antibodies. The aim of this study was to evaluate if lokivetmab interferes with serum protein electrophoresis and immunofixation electrophoresis in dogs. MATERIAL AND METHODS: Residual sera from 25 client-owned, healthy blood donor dogs from 2 veterinary hospitals in Germany were used. The residual sera were analysed with serum protein electrophoresis and immunofixation electrophoresis before and after being spiked with lokivetmab at a concentration of 10 µg/ml (corresponding to the mean peak serum concentration after a subcutaneous injection of 2 mg/kg lokivetmab). RESULTS: No monoclonal gammopathy was observed on serum protein electrophoresis and all proteins had a normal distribution pattern without any pathologic bands on immunofixation electrophoresis. The absolute γ-globulin values of spiked samples, however, were significantly higher than in the native sera although they remained within the reference interval. No other globulin fractions were significantly different. CONCLUSION AND CLINICAL RELEVANCE: This study suggests that lokivetmab at a dose of 2 mg/kg is not detected as a monoclonal peak on serum protein electrophoresis or immunofixation electrophoresis, and thus is unlikely to lead to a misdiagnosis of other diseases that are characterised by monoclonal gammopathies.

Monitoring the M-protein of multiple myeloma patients treated with a combination of monoclonal antibodies: the laboratory solution to eliminate interference.

OBJECTIVES: The therapeutic monoclonal antibody (t-mAb) daratumumab, used to treat multiple myeloma (MM) patients, interferes with routine, electrophoretic based M-protein diagnostics. Electrophoretic response assessment becomes increasingly difficult when multiple t-mAbs are combined for use in a single patient. This is the first study to address the analytical challenges of M-protein monitoring when multiple t-mAbs are combined. METHODS: In this proof-of-principle study we evaluate two different methods to monitor M-protein responses in three MM patients, who receive both daratumumab and nivolumab. The double hydrashift assay aims to resolve t-mAb interference on immunofixation. The MS-MRD (mass spectrometry minimal residual disease) assay measures clonotypic peptides to quantitate both M-protein and t-mAb concentrations. RESULTS: After exposure to daratumumab and nivolumab, both t-mAbs become visible on immunofixation electrophoresis (IFE) as two IgG-kappa bands that migrate close to each other at the cathodal end of the γ-region. In case the M-protein co-migrates with these t-mAbs, the observed interference was completely abolished with the double IFE hydrashift assay. In all three patients the MS-MRD assay was also able to distinguish the M-protein from the t-mAbs. Additional advantage of the MS-MRD assay is that this multiplex assay is more sensitive and allows quantitative M-protein-, daratumumab- and nivolumab-monitoring. CONCLUSIONS: Daratumumab and nivolumab interfere with electrophoretic M-protein diagnostics. However, the M-protein can be distinguished from both t-mAbs by use of a double hydrashift assay. The MS-MRD assay provides an alternative method that allows sensitive and simultaneous quantitative monitoring of both the M-protein and t-mAbs.

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.

Sensitivity of Capillary Electrophoresis to Detect Monoclonal Immunoglobulins: a Report of Two Cases and a Systematic Review.

BACKGROUND: The detection of monoclonal immunoglobulins (MIg) is a key element in the diagnosis of monoclonal gammopathies. METHODS: Here we report two cases of high concentration serum IgM-kappa MIgs (6.4 g/L and 6.8 g/L) not detected with capillary electrophoresis (CE). A systematic literature search was conducted to assess the sensitivity of CE to detect MIgs. RESULTS: The CE sensitivity to detect MIgs did not exceed 0.9 to 0.95 compared to immunofixation as standard. On the one hand, MIgs with blood concentrations below 1 g/L may be hidden by other serum proteins. On the other hand, some MIgs of high concentrations are not detected due to their insolubility in the electrophoresis buffer. CONCLUSIONS: Performing a second SPE with agarose gel electrophoresis method or modifying buffer properties may reveal some MIgs not detected by a first SPE alone.

Serum Protein Electrophoresis and Immunofixation Electrophoresis Detection in Multiple Myeloma.

The objective of this study was to investigate the diagnostic significance of serum protein electrophoresis and immunofixation electrophoresis detection in diagnosis of multiple myeloma (MM). One hundred and five patients were investigated. The detection rate of M protein by immunofixation electrophoresis detection was better (105 cases, 100%) than that of serum protein electrophoresis (101 cases, 96.19%, p<0.001). The M band was not detected by serum protein electrophoresis in four cases (3.81%), among which one case (0.95%) was identified as IgA type and 3 cases (2.86%) as light chain type after immunoglobulin analysis. Immunofixation electrophoresis detection technique can be used for screening M protein in patients with atypical MM; and immunofixation electrophoresis detection technique can increase the diagnosis accuracy in patients with atypical MM. Key Words: Multiple myeloma (MM), Serum protein electrophoresis, Immunofixation electrophoresis, Monoclonal immunoglobulin.