Physicochemical Properties of IgD-type M Protein by Electrophoretic Analysis.

BACKGROUND: We examined the physicochemical properties of IgD-type M protein from 10 patients with IgD-type M protein and those with other types of M protein. METHODS: Identification of the L-chain type by routine methods (Immunoelectrophoresis: IEP, Immunofixation electrophoresis; IFE), detection of IgD-IgG complexes and the changes in the mobility by treatment with acid. RESULTS: Identification of the L-chain type by both IEP and IFE was impossible. Only one patient revealed the possibility of IgD-IgG complex bands in 6 of the 10 samples. Changes in mobility by treatment with acid were observed in 8 of the 10 samples of IgD-type M protein. CONCLUSIONS: These abnormalities are caused by the primary structure of IgD, which may be related to the well-known weak reactivity with L-chain antibody.

Impact of M-protein detection on the response evaluations of patients undergoing treatment with the IgG-κ monoclonal antibodies daratumumab or isatuximab, and discrepancies between immunofixation electrophoresis (IFE) systems and reagents.

BACKGROUND: Immunofixation electrophoresis (IFE) is the standard method for confirming the presence of a monoclonal protein (M-protein) at multiple myeloma (MM) diagnosis. IFE is also essential at assessment of complete response (CR) and stringent CR during treatment. As the CR assessment is influenced by daratumumab and isatuximab, HYDRASHIFT assays were developed. METHODS: Samples from patients under treatment that included daratumumab or isatuximab were tested and monitored by IFE on the HYDRASYS system using HYDRASHIFT assays (HYDRASYS/HYDRASHIFT) and by IFE on the Epalyzer2 system (Epalyzer). RESULTS: The IFE using HYDRASYS/HYDRASHIFT avoided a false positive caused by drug-related IgG-κ and contributed to accurate assessment of CR. Furthermore, HYDRASYS/HYDRASHIFT detected small M-proteins at early relapse and detected free light chains (FLCs) in patients with renal impairment exhibiting high serum FLCs despite being often missed on Epalyzer. CONCLUSION: Sensitivity and specificity of M-protein detection vary greatly depending on the IFE system and reagents used.

Serum monoclonal immunoglobulin light-chain detection differs between immunofixation electrophoresis methods in patients with AL amyloidosis.

Serum immunofixation electrophoresis (IFE) is often performed for screening monoclonal proteins (M proteins) in immunoglobulin light-chain amyloidosis (AL amyloidosis). However, the performance of serum IFE for detecting M protein in AL amyloidosis patients is often insufficient. In this study, we examined the detection rate of serum M protein in newly diagnosed AL amyloidosis patients and analyzed differences in M protein detection between IFE methods. Among 60 patients newly diagnosed with AL amyloidosis, 22 had undetectable serum M protein by IFE with the Epalyzer2 system. Samples with undetectable M protein had significantly lower involved serum-free light-chain (iFLC) and a smaller difference between involved and uninvolved serum-free light-chain (dFLC) values than samples with IFE-detectable monoclonal light chains. When samples that tested negative for M protein by the Epalyzer2 system were retested by IFE with the HYDRASYS 2 system, 50% had IFE-detectable monoclonal light chains. The IFE system and reagents used may affect serum monoclonal immunoglobulin light-chain detection in AL amyloidosis patients, especially those with low iFLC or low dFLC samples. More attention should be paid to the performance of IFE systems, since it may affect the diagnostic and therapeutic evaluation of AL amyloidosis patients.

Isatuximab-Specific Immunofixation Electrophoresis Assay to Remove Interference in Serum M-Protein Measurement in Patients with Multiple Myeloma.

BACKGROUND: Isatuximab, an IgG-kappa (IgGκ) anti-cluster of differentiation 38 (CD38) monoclonal antibody approved for use in patients with relapsed or refractory multiple myeloma (MM), can potentially interfere with the visualization of endogenous monoclonal protein (M-protein) on standard immunofixation electrophoresis (IFE) and lead to inaccurate classification of a patient's response to therapy. The Hydrashift 2/4 isatuximab IFE assay (Hydrashift isatuximab assay) removes isatuximab interference from IFE. Using samples from patients enrolled in clinical trials of isatuximab-based therapy for MM, we demonstrate how the Hydrashift isatuximab assay improves the ability to detect residual M-protein and offer recommendations for when the assay is most useful. METHODS: Samples from 141 patients with a variety of known M-protein isotypes were selected and analyzed by standard IFE and the Hydrashift isatuximab assay. A positive control containing isatuximab was run on every standard IFE and Hydrashift gel. RESULTS: The Hydrashift isatuximab assay reliably shifted the migration of isatuximab in patient samples. Standard IFE was adequate for determining 104 patients' M-protein status, and the Hydrashift isatuximab assay confirmed these results. In samples from 37 patients with a history of IgGκ MM and a single IgGκ band visible on standard IFE near the isatuximab migration site, the Hydrashift isatuximab assay was able to separate isatuximab from endogenous M-protein, identifying residual M-protein in 17 samples and preventing false-positive interpretations of standard IFE in 20 samples. CONCLUSIONS: The Hydrashift isatuximab assay is most useful in patients with known IgGκ MM when a single IgGκ band appears near the isatuximab migration site on standard IFE during isatuximab-based therapy. CLINICALTRIALS.GOV REGISTRATION NUMBERS: NCT03275285 and NCT03319667.

Characteristics of isatuximab-derived interference in serum protein electrophoresis and immunofixation, and an absence of sustained in vivo interference due to belantamab mafodotin and denosumab.

OBJECTIVES: Some therapeutic monoclonal antibodies, like daratumumab and elotuzumab, produce interfering monoclonal bands on serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE). Whether other common therapeutic antibodies also produce interference has not been systematically evaluated. DESIGN AND METHODS: SPEP/IFE from patients receiving isatuximab (48 patients), belantamab mafodotin (BM; 41), and denosumab (41) were retrospectively reviewed for therapeutic antibody interference. Cases exhibiting isatuximab interference were quantified and the maximum duration of isatuximab effect was evaluated. To characterize band position, neat human serum was spiked with BM or denosumab at supratherapeutic concentrations. Band migration patterns were compared on SPEP and IFE, with band position expressed relative to other constant protein fractions. RESULTS: Isatuximab-induced IFE interference was common (81.3 % of evaluated patients) with a maximum observed duration of 8 weeks. 10.4 % of isatuximab patients had IgG kappa monoclonal gammopathies that co-migrated with the drug; this subset could benefit from HYDRASHIFT 2/4 isatuximab testing. 8.3 % of IFE cases were negative for an isatuximab band but showed large, endogenous M-spikes migrating elsewhere. All patients in this group expired within 1 year of this finding. We hypothesize that an inability to detect isatuximab in this setting corresponds to a large residual myeloma burden that reduces isatuximab serum concentration. This observation may serve as a negative prognostic factor. Spiking studies demonstrated that BM and denosumab produce interference in vitro, but sustained interference was not observed in >40 treated patients. CONCLUSIONS: Therapeutic antibody interference in patients receiving isatuximab is common, and can persist for at least 8 weeks after administration. >10 % of patients receiving isatuximab may benefit from HYDRASHIFT testing post-therapy. In contrast, BM and denosumab fail to produce sustained interference in treated patients.

Urine immunofixation electrophoresis and serum free light chain analyses benefit diagnosis of multiple myeloma in orthopedic patients with normal serum total proteins, creatinine, calcium, and hemoglobin.

BACKGROUND: A substantial number of patients with multiple myeloma (MM) who have bone destruction are initially admitted into the orthopedic service at the hospital. However, routine laboratory testing usually fails to identify these patients, thus delaying optimal therapy. Therefore, there is a clear medical need for early diagnosis of MM in these patients. METHODS: Between 2019 and 2021, 42 patients receiving treatment for orthopedic conditions had normal hemoglobin (Hb), total protein (TP), albumin (ALB), creatinine (CREA), and blood calcium (Ca) levels before their surgical procedure(s) but were subsequently pathologically confirmed to have MM, based on their presenting orthopedic symptoms. During the same period, 52 patients with orthopedic conditions were pathologically excluded from the diagnosis of MM and were recruited into our control group. Serum free light chain (sFLC) testing was performed in 94 consecutive patients in the orthopedic service using Siemens N Latex FLC kits. The levels of Hb, TP, ALB, CREA, and Ca were also measured. All 42 patients with MM were divided into group A (n = 25: κ proliferation) and group B (n = 17: λ proliferation) by the pathology department. RESULTS: There were no significant differences in levels of Hb, TP, ALB, CREA, and Ca between group A and group B and the control group. However, the sFLC κ/λ ratio of group A and B was also significantly different from that of the control group (P < .001). The results of serum immunofixation electrophoresis (IFE) testing demonstrated negative results in 14 cases (58.3%) in group A and 4 cases (25.0%) in group B. CONCLUSIONS: Some patients with orthopedic conditions who do not have typical MM laboratory results, such as those with abnormal Hb, TP, ALB, CREA, and Ca levels before their operation(s), actually have MM. MM should be highly suspected in patients with unexplained bone lesions and with an abnormal sFLC κ/λ ratio. Further tissue or bone marrow biopsy is needed in these patients even if serum and urine IFE results are negative and light chain ratio is normal.

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.

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.

[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.

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.

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.

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.