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.

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.
UNASSIGNED: NCT03275285 and NCT03319667.

OBJECTIVE: 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.
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.

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.

Sarcoidosis is a systemic disorder characterized by noncaseating granuloma formation, which can affect any organ in the body; however, skeletal involvement is relatively uncommon. This case report presents a rare case of sarcoidosis primarily affecting the skeletal system in a 39-year-old man with a history of neurosarcoidosis. The patient presented with symptoms of nausea, vomiting, fatigue, weight loss, and lower back and pelvic pain, which were initially suspicious for malignancy. Computed tomography scans revealed lytic bone lesions and lymphadenopathy. However, a biopsy of a left inguinal lymph node confirmed necrotizing granulomatous lymphadenitis, which was consistent with necrotizing sarcoid granulomatosis - a rare variant of sarcoidosis. The patient was treated with systemic corticosteroids, which led to clinical improvement. The prognosis of sarcoidosis is generally good, with spontaneous remission occurring in up to two-thirds of patients; however, some patients may develop chronic and/or progressive disease. In particular, patients with a history of neurosarcoidosis may be at an increased risk for chronic or recurrent disease. This case highlights the importance of considering sarcoidosis in the differential diagnosis of patients presenting with nonspecific symptoms and lymphadenopathy, even in the absence of pulmonary involvement.

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.

Myasthenia gravis (MG) is a disease with impaired transmission at the neuromuscular junction, characterised by weakness and fatigability of skeletal muscles. In acquired autoimmune MG, antibodies against acetylcholine receptor (AChRAb) or muscle-specific tyrosine kinase (MuSKAb) are present. There is not much data about immunoglobulin G (IgG) galactosylation in MG, and none based on interactions with lectins. This study aims to examine IgG galactosylation in two types of myasthenia, using affinity immunoelectrophoresis with lectin concanavalin A (Con A). Affinity of Con A-IgG interaction, expressed as retardation coefficient (R), indicated the presence of degalactosylated IgG. The average R values were significantly different between three examined groups, being the lowest in controls (healthy subjects), higher in acetylcholine receptor (AChR) MG, and the highest in muscle-specific tyrosine kinase (MuSK) MG (ANOVA, p < .05). This indicated decreased galactosylation of IgG in both types of MG compared to controls, more pronounced in MuSK MG. IgG galactosylation was also investigated in relation to the disease severity score, determined according to the Myasthenia Gravis Foundation of America (MGFA) criteria, at the time of diagnosis, nadir of the disease and last check-out visit. The average R values for mild disease (stages I-IIIa) were significantly lower than for severe disease (stages IIIb-V), both at the time of diagnosis (p < .05), and at the nadir of the disease (p < .05). Thus, IgG galactosylation was associated with the presence of specific autoantibodies in MG, as well as with disease severity for both types of MG, and may be a predictive marker of MG outcome.

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.
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.
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.
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.

Cardiac amyloidosis is a life-threatening disease that occurs when amyloid proteins, most commonly immunoglobulin light chain or transthyretin, mutate or become unstable, misfold, deposit as amyloid fibrils, and accumulate in the myocardium. Early diagnosis of cardiac amyloidosis is hindered by insufficient awareness, specifically regarding clinical red flags and diagnostic pathways. Cardiac amyloidosis diagnosis comprises two important phases, clinical suspicion (phase one) followed by definitive diagnosis (phase two). Each phase is associated with specific clinical techniques. For example, clinical features, electrocardiography, echocardiography, and cardiac magnetic resonance imaging serve to raise suspicion of cardiac amyloidosis and facilitate early diagnosis, whereas laboratory tests (i.e., blood or urine electrophoresis with immunofixation), biopsy, scintigraphy-based nuclear imaging, and genetic testing provide a definitive diagnosis of cardiac amyloidosis. In Egypt, both the lack of cardiac amyloidosis awareness amongst healthcare providers and the unavailability of clinical expertise for the use of diagnostic techniques must be overcome to improve the prognosis of cardiac amyloidosis in the region. Previously published diagnostic algorithms for cardiac amyloidosis have amalgamated techniques that can raise clinical suspicions of cardiac amyloidosis with those that definitively diagnose cardiac amyloidosis. Though such algorithms have been successful in developed countries, diagnostic tools like echocardiography, scintigraphy, and cardiac magnetic resonance imaging are not ubiquitously available across Egyptian facilities. This review presents the current state of knowledge regarding cardiac amyloidosis in Egypt and outlines a new diagnostic algorithm which leverages regional nuclear imaging expertise. Importantly, the proposed diagnostic algorithm guides accurate amyloid-typing to mitigate misdiagnosis and erroneous treatment selection and improve the cardiac amyloidosis diagnostic accuracy in Egypt.
Diagnostic algorithms are useful tools for guiding clinical diagnosis by summarizing diagnostic approaches and defining the patient pathway. The diagnostic algorithms for cardiac amyloidosis amalgamate techniques that raise suspicion of the disease with those that can definitively diagnose the disease. These algorithms, for the early detection and diagnosis of cardiac amyloidosis, are designed in accordance with developed healthcare systems that have the resources and infrastructure for diagnostic equipment and clinical expertise. There are limited financial resources across healthcare facilities in Egypt for diagnostic equipment like echocardiograms (ECHO), scintigraphy, and cardiac magnetic resonance imaging (cMRI), and the required clinical training for the diagnosis of cardiac amyloidosis. This reduces the possibility of early diagnosis of the disease and subsequent early intervention. Evidently, there is a significant unmet clinical need to develop an algorithm for the diagnosis of cardiac amyloidosis in accordance with the Egyptian healthcare system. This review article details the current awareness regarding the diagnosis of cardiac amyloidosis and the associated challenges in Egypt. Accordingly, a diagnostic algorithm that leverages nuclear imaging expertise to guide accurate amyloid-typing in order to mitigate misdiagnosis and erroneous treatment, and also improve the diagnostic accuracy of cardiac amyloidosis, has been proposed.

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