Lambda monoclonal free light chain abnormalities detected by a serum immunofixation electrophoresis assay are underrepresented by quantitative serum free light chain results
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Abstract Protein and immunofixation (IFIX) electrophoresis are used to diagnose and monitor monoclonal gammopathies. While IFIX detects clonal production of intact immunoglobulins and free light chains (FLC), the latter can also be quantified using a serum free light chain (SFLC) assay, in which polyclonal antisera detects epitopes specific for free kappa (KFLC) or lambda light chains (LFLC). An abnormal KFLC: LFLC ratio (KLR) serves as a surrogate for clonality. While the SFLC assay is highly sensitive, normal LFLC (<2.63mg/dL) and KLR results (>0.26 & <1.65) were found in samples with distinct lambda monoclonal free light chains visualized by IFIX (X-LMFLC). To investigate this discordance, contemporaneous SFLC or KLR values were evaluated for their ability to accurately classify monoclonal FLCs identified by IFIX. We performed a retrospective analysis of serum and urine IFIX (Sebia Hydrasys) and SFLC (Freelite®, Binding Site) results from our institution between July 2010 through December 2020, using R 4.0.2 and Tidyverse packages. From among 9,594 encounters in which a single monoclonal component was initially identified by IFIX, 157 X-LMFLC and 131 X-KMFLC samples were analyzed. Elevated LFLC with normal KFLC was identified in 105/157 X-LMFLC samples (67%), while both LFLC and KFLC were elevated in 42/157 samples (27%). Concordance between X-KMFLC and KFLC was markedly higher, where 122/131 samples (93%) displayed elevated kappa FLC (>1.94mg/dL) with normal LFLC, and only 7/131 X-KMFLC samples (5%) possessed both elevated KFLC and LFLC. The use of KLR to identify pathogenic monoclonal free light chains improved lambda concordance to 85%; however, 19/157 (12%) of X-LMFLC samples still exhibited normal KLR. High concordance of 98% was again observed for X-KMFLC with abnormal KLR. When samples were segregated according to normal or impaired renal function (eGFR > or ≤60mL/min/1.73m², respectively), this disparate identification of X-LMFLC and X-KMFLC by the SFLC assay persisted, suggesting that renal dysfunction (as measured by eGFR) does not underlie this phenomenon. Lastly, we corroborated the above findings in a larger sample population by examining patients with urine Bence Jones FLC identified by IFIX who had free or intact monoclonal components in serum (N=724), grouped by lambda or kappa light chain involvement. The cause(s) of the discrepant performance by the Freelite® SFLC assay, relative to the Sebia Hydrasys IFIX assay, for identifying lambda FLC components is currently unclear. Possible contributory factors include assay reference range cutoffs, other patient disease parameters, and differences in assay-specific polyclonal antisera. Future analyses of these factors will help to further characterize SFLC assay performance and elucidate how interpretation of composite serum FLC test results can be improved to better guide patient management.Keywords:
Immunofixation
Polyclonal antibodies
Concordance
Kappa
Serum protein electrophoresis
Objective To explore the clinical value of the immunofixation electrophoresis(IFE) for the diagnosis and prognosis of multiple myeloma.Methods Serum of 67 cases of multiple myeloma patients was detected by immunofixation electrophoresis,serum protein electrophoresis,and quantitative detection of immunoglobulin.5 patients of the 67 cases were implemented with autologous stem cell transplant,then the serum of them were tracked and detected.Results The M protein was detected in 62 of the 67 patients by serum protein electrophoresis.Serum immunofixation electrophoresis types of 67 patients were respectively IgG type in 38 cases(56.7%),including 20 cases of κ light chain and 18 cases of λ light chain;IgA type in 22 cases(32.8%),including 12 cases of κ light chain,and 10 cases of λ light chain;IgM type 1 case(1.49%);Free light chain type in 6 cases(8.96%),including 3 cases of κ light chain and 3 cases of λ light chain.Conclusion IFE shows high sensitivity and specificity in the determination and classification of multiple myeloma.Additionally,IFE also plays an irreplaceable role in the diagnosis,judgment and prognosis of multiple myeloma.
Immunofixation
Serum protein electrophoresis
Myeloma protein
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Sera from patients with multiple myeloma usually display a single monoclonal immunoglobulin band on serum protein immunofixation electrophoresis. Multiple bands may be seen if the myeloma is bi- or triclonal or if the monoclonal immunoglobulin has rheumatoid factor activity. We describe a patient with light chain-predominant IgA lambda myeloma; the patient's serum displayed 2 spatially distinct bands reacting for alpha heavy and lambda light chains. The methods used to establish monoclonality are addressed.
Immunofixation
Serum protein electrophoresis
Myeloma protein
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ABSTRACT Recently, turbidimetric immunoassays for detecting and quantifying κ and λ free light chains (FLC) have become available and are promoted as being more sensitive than immunofixation electrophoresis (IFE) in detecting FLC monoclonal proteins. In this study, we assessed the ability of these turbidimetric assays to detect serum monoclonal proteins involving both free and heavy-chain-bound κ and λ light chains compared to standard immunofixation electrophoresis. Sera demonstrating a restricted band of protein migration (other than a definite M spike) by serum protein electrophoresis (SPE), which may represent early monoclonal proteins, were also examined. When compared to IFE, percent agreement, sensitivity, and specificity for the κ-FLC and λ-FLC were 94.6, 72.9, and 99.5% and 98.5, 91.4, and 99.7%, respectively, in detecting monoclonal proteins involving free and heavy-chain-bound light chains. The majority of sera (73.7%) demonstrating a restricted band of protein migration on SPE demonstrated abnormal IFE patterns suggestive of multiple myeloma or monoclonal gammopathy of unknown significance, but gave normal κ/λ FLC ratios using the turbidimetric immunoassays. In conclusion, the κ and λ FLC assays are significantly less sensitive (72.9 to 91.4%) than IFE, but specific in detecting serum monoclonal proteins. Moreover, the κ/λ ratio has little value in routine screening since the majority of sera with abnormal IFE patterns had normal κ/λ FLC ratios.
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Kappa
Serum protein electrophoresis
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Background: Quantitative evaluation of serum free light chains is recommended for the work up of monoclonal gammopathies. Immunoglobulin light chains are generally produced in excess of heavy chains. In patients with monoclonal gammopathy, κ/λ ratio is abnormal less frequently with lambda chain lesions. This study was undertaken to ascertain if the levels of overproduction of the two light chain types and their detection rates are different in patients with neoplastic monoclonal gammopathies. Methods: Results of serum protein electrophoresis (SPEP), serum protein immunofixation electrophoresis (SIFE), urine protein electrophoresis (UPEP), urine protein immunofixation electrophoresis (UIFE), and serum free light chain assay (SFLCA) in patients with monoclonal gammopathies were examined retrospectively. Results: The kappa/iambda ratios were appropriately abnormal more often in kappa chain lesions. Ratios of kappa/iambda were normal in about 25% of patients with lambda chain lesions in whom free homogenous lambda light chains were detectable in urine. An illustrative case suggests underproduction of free lambda light chains, in some instances. Conclusions: The lower prevalence of lambda dominant kappa/iambda ratio in lesions with lambda light chains is estimated to be due to relative under-detection of lambda dominant kappa/iambda ratio in about 25% of the patients and because lambda chains are not produced in as much excess of heavy chains as are kappa chains, in about 5% of the patients. The results question the medical necessity and clinical usefulness of the serum free light chain assay. UPEP/UIFE is under-utilized. J Clin Med Res. 2018;10(7):562-569 doi: https://doi.org/10.14740/jocmr3383w
Immunofixation
Kappa
Serum protein electrophoresis
Bence Jones protein
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Immunofixation
Serum protein electrophoresis
Kappa
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Although the presence of oligoclonal IgG with abnormal kappa/lambda light-chain ratio in multiple sclerosis (MS) has been known for many years, this finding has not been put to diagnostic use in most routine clinical laboratories. In a retrospective study we report differences in the oligoclonal banding patterns between multiple sclerosis and non-MS patients. We had sufficient cerebrospinal fluid (CSF) on 36 from 71 patients with oligoclonal bands for immunofixation for kappa and lambda light chains, and for free kappa and free lambda. Thirteen out of 14 patients with clinically confirmed MS had predominantly IgG (kappa) banding. In contrast, in seven out of eight patients with diagnoses other than MS the IgG was linked to both kappa and lambda light chains in approximately equal proportions. Nine out of 14 patients with probable/possible/suspected MS showed predominantly IgG (kappa) banding; five others in this group had both IgG (kappa) and IgG (lambda) and free lambda light chains. The finding of IgG (kappa) bands in CSF samples with oligoclonal bands supports a diagnosis of MS.
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Abstract Objective Measurement of monoclonal immunoglobulins is a reliable estimate of the plasma cell tumor mass. About 15% of plasma cell myelomas secrete light chains only. The concentration of serum free light chains is insufficient evidence of the monoclonal light chain burden. A sensitive quantitative estimate of serum free monoclonal light chains could be useful for monitoring patients with light chain myeloma. We describe such an assay that does not require mass-spectrometry equipment or expertise. Methods Serum specimens from patients with known light chain myelomas and controls were subjected to ultrafiltration through a membrane with pore size of 50 kDa. The filtrate was concentrated and tested by immunofixation electrophoresis. The relative area under the monoclonal peak, compared to that of the total involved light chain composition, was estimated by densitometric scanning of immunofixation gels. The proportion of the area occupied by the monoclonal peak in representative densitometric scans was used to arrive at the total serum concentration of the monoclonal serum free light chains. Results Using an ultracentrifugation and concentration process, monoclonal serum free light chains were detectable, along with polyclonal light chains, in all 10 patients with active light chain myelomas. Monoclonal light chains were identified in serum specimens that did not reveal monoclonal light chains by conventional immunofixation electrophoresis. The limit of detection by this method was 1.0 mg/L of monoclonal serum free light chains. Conclusion The method described here is simple enough to be implemented in academic medical center clinical laboratories and does not require special reagents, equipment, or expertise. Even though urine examination is the preferred method for the diagnosis of light chain plasma cell myelomas, measurement of the concentration of serum free light chains provides a convenient, albeit inadequate, way to monitor the course of disease. The method described here allows effective electrophoretic differentiation of monoclonal serum free light chain from polyclonal serum free light chains and provides a quantitation of the monoclonal serum free light chains in monitoring light chain monoclonal gammopathies.
Immunofixation
Ultrafiltration (renal)
Polyclonal antibodies
Immunoradiometric assay
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Serum free light chain assay is used in the diagnosis and monitoring of monoclonal gammopathic manifestations. For the kappa (κ)/lambda (λ) ratio, there is a 36% false-positive rate in patients without monoclonal gammopathic manifestations and a 30% false-negative rate in patients with monoclonal gammopathic manifestations. This study was undertaken to address the higher false-negative rate in λ chain-associated monoclonal lesions.Results of serum protein electrophoresis, serum immunofixation electrophoresis, and serum free light chain assays were reviewed retrospectively. The results for serum free light chains in cases of intact immunoglobulin monoclonal gammopathic manifestations only were analyzed.Concentrations of involved serum free light chains were significantly higher in κ chain-associated lesions than in λ chain-associated lesions. The concentration of uninvolved light chains was significantly higher in λ chain-associated lesions.κ light chains are present in significantly greater abundance than are λ chains in their respective monoclonal lesions. Moreover, κ and λ light-chain levels are not comparable for similar quantitative levels of monoclonal immunoglobulins. The findings warrant a reconsideration of the role of serum free light chain concentrations and involved to uninvolved serum free light chain ratio in designation of myeloma-defining conditions and other diagnostic criteria based on serum free light chain assay.
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Serum protein electrophoresis
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Serum free light chain (SFLC) assay has been promoted for diagnosing, determining the prognosis, and monitoring of monoclonal gammopathies. Currently, serum protein electrophoresis (SPEP) and serum protein immunofixation electrophoresis (SIFE) are commonly used as screening tests, and the findings of SIFE are the gold standard for identification of monoclonal immunoglobulins according to the International Myeloma Workshop Consensus Panel 3. Urine protein electrophoresis (UPEP) and urine protein immunofixation electrophoresis (UIFE) are also recommended. Immunoglobulin light chains are generally produced in excess of heavy chains. In patients with monoclonal gammopathy, κ/λ ratio is abnormal less frequently with lambda chain lesions. This study was undertaken to ascertain if the high false-negative rate for SFLC results for lambda chain lesions was due to underdetection of lambda light chains or underproduction of lambda light chains or both. Data from 482 patients comprising 2,448 observations were reviewed retrospectively from January 2010 through September 2017 in a 480-bed tertiary medical center. Of these, 249 observations corresponding to 175 patients with neoplastic monoclonal gammopathies (NMGs), including monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, or multiple myeloma, were examined. SPEP, SIFE, UPEP, and UIFE were carried using a Helena (Beaumont, TX) SPIFE 3000. SFLC assay was done by using reagent kits from The Binding Site, (Birmingham, UK) on an Optilite instrument. Comparison of results of different groups was done by the chi-square test. UIFE results were divided into three categories: UIFE0, when no monoclonal immunoglobulin (Ig) or free light chains were detected; UIFE1, when monoclonal free light chains were detected with or without intact monoclonal Ig; and UIFE2, when only intact monoclonal Ig was detected. κ/λ ratios were abnormal more often in kappa chain lesions with χ2 (10.4–12.9) and P value (0.0003–0.0006). κ/λ ratios were normal in about 25% of UIFE1 category of patients (10 out of 31) with lambda chain lesions in whom free homogeneous lambda light chains were detectable in urine. None of the patients with kappa chain lesions and UIFE positive for monoclonal kappa light chains showed a normal κ/λ ratio. An illustrative case is identified in which a patient with biclonal gammopathy with a dominant IgG λ lesion suggests underproduction of free lambda light chains, in some instances. The 30% false-negative κ/λ ratio in patients with lambda chain monoclonal gammopathy is due to underdetection of lambda light chains, by the Binding Site assay, in about 25% of the patients. In the remaining 5% of patients, the false-negative κ/λ ratio is estimated to be due to underproduction of excess free lambda light chains. The results question the medical necessity and clinical usefulness of the SFLC assay as well as arguing for the greater use of UPEP/UIFE.
Immunofixation
Serum protein electrophoresis
Bence Jones protein
Myeloma protein
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Serum immunoglobulin free light chain assay has proved to be an invaluable biological tool for diagnosis and monitoring of monoclonal gammopathies including multiple myeloma, primary amyloidosis, solitary plasmocytoma or monoclonal gammopathy of undetermined significance. Free light chain quantification, although essential, cannot be achieved by serum protein electrophoresis either because there is no monoclonal peak or because the peak is hidden in beta or alpha-globulin fraction. As for serum protein immunofixation, this major test allows the typing of the paraprotein but does not provide any quantitative evaluation. Hence, the development of free light chain assays constitutes a significant improvement in the management of these patients. In this context, we compared the results of serum free light chains quantification and of calculation of the ratio kappa/lambda, indicator of monoclonality, in forty samples performed on BN ProSpec(®) analyzer, with the two methods available on the European market. This comparative analysis provided evidence of a good correlation of results between the two methods. However, we noticed clinically significant differences in four samples. In addition, this evaluation highlighted the fact that all free light chain results must be biologically validated on the light of different criteria such as serum protein electrophoresis, serum protein immunofixation, presence of proteinuria, presence of renal failure, and additional clinical data, in order to ascertain the best interpretation for clinical use.
Serum protein electrophoresis
Immunofixation
Bence Jones protein
AL amyloidosis
Myeloma protein
Cryoglobulins
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