Free Light Chains of Immunoglobulins in Normal Serum and Urine Determined by Radioimmunoassay

1975 ◽  
Vol 35 (5) ◽  
pp. 407-412 ◽  
Author(s):  
K. Söulling
Keyword(s):  
Normal Serum ◽  
Light Chains ◽  
Free Light Chains ◽  
Serum And Urine

scholarly journals Quantitative Immunoprecipitation Free Light Chain Mass Spectrometry (QIP-FLC-MS) Simplifies Monoclonal Protein Assessment and Provides Added Clinical Value in Systemic AL Amyloidosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4375-4375 ◽  
Author(s):  
Faye Amelia Sharpley ◽  
Hannah Victoria Giles ◽  
Richa Manwani ◽  
Shameem Mahmood ◽  
Sajitha Sachchithanantham ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Light Chain ◽  
Residual Disease ◽  
Renal Involvement ◽  
Light Chains ◽  
Al Amyloidosis ◽  
Free Light Chains ◽  
Accurate Identification ◽  
Monoclonal Protein ◽  
Serum And Urine

Introduction Early diagnosis, effective therapy and precise monitoring are central for improving clinical outcomes in systemic light chain (AL) amyloidosis. Diagnosis and disease response assessment is primarily based on the presence of monoclonal immunoglobulins and free light chains (FLC). The ideal goal of therapy associated with best outcomes is a complete responses (CR), defined by the absence of serological clonal markers. In both instances, detection of the monoclonal component (M-component) is based on serum FLC assessment together with traditional serum and urine electrophoretic approaches, which present inherent limitations and lack sensitivity particularly in AL where the levels are typically low. Novel mass spectrometry methods provide sensitive, accurate identification of the M-component and may prove instrumental in the timely management of patients with low-level amyloidogenic light chain production. Here we assess the performance of quantitative immunoprecipitation FLC mass spectrometry (QIP-FLC-MS) at diagnosis and during monitoring of AL amyloidosis patients treated with bortezomib-based regimens. Methods We included 46 serial patients with systemic AL amyloidosis diagnosed and treated at the UK National Amyloidosis Centre (UK-NAC). All patients had detailed baseline assessments of organ function and serum FLC measurements. Baseline, +6- and +12-month serum samples were retrospectively analysed by QIP-FLC-MS. Briefly, magnetic microparticles were covalently coated with modified polyclonal sheep antibodies monospecific for free kappa light chains (anti-free κ) and free lambda light chains (anti-free λ). The microparticles were incubated with patient sera, washed and treated with acetic acid (5% v/v) containing TCEP (20 mM) in order to elute FLC in monomeric form. Mass spectra were acquired on a MALDI-TOF-MS system (Bruker, GmbH). Results were compared to serum FLC measurements (Freelite®, The Binding Site Group Ltd), as well as electrophoretic assessment of serum and urine proteins (SPE, sIFE, UPE and uIFE). Results Cardiac (37(80%) patients) and renal (31(67%) patients) involvement were most common; 25(54%) patients presented with both. Other organs involved included liver (n=12), soft tissue (n=4), gastrointestinal tract (n=3) and peripheral nervous system (n=2). Baseline Freelite, SPE, sIFE and uIFE measurements identified a monoclonal protein in 42(91%), 22(48%), 34(74%) and 21(46%) patients, respectively. A panel consisting of Freelite + sIFE identified the M-component in 100% of the samples. QIP-FLC-MS alone also identified an M-component in 100% of the samples and was 100% concordant with Freelite for typing the monoclonal FLC (8 kappa, 34 lambda). In 4 patients, QIP-FLC-MS identified an additional M-protein that was not detected by the other techniques. In addition, 4/8(50%) kappa and 4/38(11%) lambda patients showed a glycosylation pattern of monoclonal FLCs at baseline by mass spectrometry. Interestingly, the frequency of renal involvement was significantly lower for patients with non-glycosylated forms (25% vs 76%, p=0.01), while no similar relationship was found for any other organs. During the 1-year follow-up period, 17 patients achieved a CR; QIP-FLC-MS identified serum residual disease in 13(76%) of these patients. Conclusion In our series, QIP-FLC-MS was concordant with current serum methods for identifying the amyloidogenic light chain type and provided, against all other individual tests, improved sensitivity for the detection of the monoclonal protein at diagnosis and during monitoring. The ability to measure the unique molecular mass of each monoclonal protein offers clone-specific tracking over time. Glycosylation of free light chains is over-represented in AL patients which may allow earlier diagnosis and better risk-assessment of organ involvement. Persistence of QIP-FLC-MS positive M component in patients otherwise in CR may allow targeted therapy. Overall, QIP-FLC-MS demonstrates potential to be exploited as a single serum test for precise serial assessment of monoclonal proteins in patients with AL amyloidosis. Disclosures Wechalekar: GSK: Honoraria; Janssen-Cilag: Honoraria; Amgen: Research Funding; Takeda: Honoraria; Celgene: Honoraria.

Impact of Novel M-Component Based Biomarkers On to Progression Free Survival After Treatment in Intact Immunoglobulin Multiple Myeloma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2927-2927
Author(s):  
Efstathios Koulieris ◽  
Dimitrios Maltezas ◽  
Nikolaou Eytychia ◽  
Vassiliki Bartzis ◽  
Tatianna Tzenou ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Binding Site ◽  
Progression Free Survival ◽  
Normal Serum ◽  
Light Chains ◽  
Free Light Chains ◽  
Serum Free ◽  
Depth Of Response ◽  
Serum Free Light Chains

Abstract Abstract 2927 Background and Aims: Multiple Myeloma (MM) is characterized by bone marrow (BM) plasma cell infiltration and the presence of serum/urine monoclonal immunoglobulin (Ig). The depth of response has been associated with longer PFS in MM causing subsequent prolonged survival. Recently novel M-based biomarker immunoassays have been developed (Freelite™, Hevylite™) and their significance in MM diagnosis and prognosis has been demonstrated.1,2 Furthermore serum Free Light Chains (sFLC) are used for better assessment of treatment response, thus patients are considered to achieve stringed Complete Response (sCR) by having CR criteria plus normal serum Free Light Chains Ratio (sFLCR) and absent clonal cells on BM.3 The significance of Hevylite™ on response has not been assessed so far. Patients in nCR or better do not automatically restore their ratio of intact monoclonal Ig/intact polyclonal Ig of the same class (Hevylite™ or HLCR). We therefore investigated the importance of sFLCR and HLCR normalisation at plateau on PFS, in a series of patients with intact Ig MM. Patients and Methods: 50 intact immunoglobulin MM patients were studied from diagnosis to last follow up. Immunofixation was IgG (26 -kappa and 12 –lamdba) and IgA (6 –kappa and 6 -lambda). All patients were symptomatic at diagnosis. Sera samples (n=312) were analyzed for sFLC-kappa and sFLC-lambda with Freelite™ and sFLCR were calculated, and for IgGkappa, IgGlambda IgAkappa, IgAlambda with Hevylite™ and ratios IgGkappa/IgGlambda, IgGlambda/IgGkappa, IgAkappa,/IgAlambda and IgAlambda/IgAkappa (HLCRs) were calculated. sFLCRs and HLCRs values above the 95%-ile of normal individuals were considered abnormal. Statistical analysis was performed using SPSS ver 15.0. File data were reviewed. Results: At diagnosis sFLCR was abnormal in 86% of patients while HLCR was abnormal in all. All treatment lines were initiated according to standard criteria and median lines of therapy were 2 (range 1–11). Median follow up was 33 months (7–145). During patients' cumulative follow-up, 145 lines of therapy were studied and the subsequent responses were estimated. Thirty eight percent of responses were sCR, CR and nCR, 20% PR, 18% MR and 24% refractory and progressive disease. HLCR normalized in 44% of patients with sCR, CR and nCR. The depth of response correlated to PFS and patients in sCR, CR and nCR had longer PFS than the others (p<0.001). Serum FLCR and HLCR normal values at response were both strong parameters of increased PFS after treatment at any line (p=0.035 and p=0.046 respectively). Conclusion: Serum HLCR normalization at plateau reflects prolonged responses in intact Ig MM. Disclosures: Harding: Binding Site: Employment. Bradwell:The Binding Site: shareholder Other.

scholarly journals Serum and urine free light chains measurements in patients with systemic sclerosis: novel biomarkers for disease activity

2021 ◽  
Author(s):  
Antonietta Gigante ◽  
Chiara Pellicano ◽  
Giorgia Leodori ◽  
Cecilia Napodano ◽  
Lorenzo Vantaggio ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
Disease Activity ◽  
Light Chains ◽  
Free Light Chains ◽  
Novel Biomarkers ◽  
Serum And Urine

scholarly journals Assessment of immunoglobulins free light chains in serum and urine of patients with system sclerosis: novel biomarkers for activity disease

2021 ◽  
Author(s):  
Antonietta Gigante ◽  
Chiara Pellicano ◽  
Giorgia Leodori ◽  
Cecilia Napodano ◽  
Lorenzo Vantaggio ◽  
...  
Keyword(s):  
Disease Activity ◽  
Activity Index ◽  
Cell Activity ◽  
Disease Activity Index ◽  
Serum Levels ◽  
Light Chains ◽  
Free Light Chains ◽  
Laboratory Findings ◽  
Positive Linear Correlation ◽  
Serum And Urine

Free light chains (FLC), considered a biomarker of B cell activity, are frequently elevated in different systemic inflammatory-autoimmune diseases. As systemic sclerosis (SSc) clinical course may be variable, this study aimed to assess FLCs levels in serum and urine of affected individuals, as biomarkers of disease activity. We assessed FLC levels in serum and urine of 72 SSc patients and 30 healthy controls (HC). Results were analyzed in comparison with overall clinical and laboratory findings, disease activity index (DAI) and disease severity scale (DSS). SSc patients displayed increased levels of k and λ FLC in serum, significantly higher than HC (p=0.0001) alongside with the mean levels of free k/λ ratio and of the k+λ sum (p=0.0001). In addition, SSc patients had significant higher levels in the urine of free k and of k/λ ratio than HC (p=0.0001). SSc patients with increased k+λ sum in serum showed a statistically higher levels of erythro-sedimentation rate (p=0.034), C-reactive protein (p=0.003), higher DAI (p=0.024) and DSS (p=0.015) than SSc patients with normal levels of FLC. A positive linear correlation was found between serum levels of free k and DAI (r=0.29, p=0.014). SSc patients with increased free k in urine had statistically higher DAI (p=0.048) than SSc patients with normal level of free k. The significant increased levels of FLC correlated with disease activity in SSc patients. Our results strengthen the role of FLC as useful biomarkers in clinical practice to early diagnosis and monitor disease activity with an improvement of SSc patients’ management.

Serum Free Light Chain Levels in Asymptomatic Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4880-4880 ◽  
Author(s):  
Bradley M. Augustson ◽  
Steven D. Reid ◽  
Graham P. Mead ◽  
Mark T. Drayson ◽  
J. Anthony Child ◽  
...  
Keyword(s):  
Median Time ◽  
Light Chain ◽  
Normal Serum ◽  
Free Light Chain ◽  
Light Chains ◽  
Time To Progression ◽  
Free Light Chains ◽  
Serum Free ◽  
Serum Free Light Chain ◽  
Serum Free Light Chains

Abstract Introduction: Patients with asymptomatic myeloma fulfil two of the diagnostic criteria for myeloma having more than 10% bone marrow plasma cells and an M protein of greater than 30g/l, but they are asymptomatic with no evidence of end organ or tissue damage. The median time to disease progression is 12–32 months. These patients do not require treatment but do require monitoring for progression to symptomatic myeloma. Predicting progression of asymptomatic myeloma would be of clinical benefit to optimise monitoring and initiate treatment prior to substantial end organ damage. However monoclonal spike, plasma cell labelling index, bone marrow plasmacytosis, immunoparesis and the presence of Bence Jones protein have limited value in predicting progression. Abnormal levels of serum free light chains are present in 95% of all multiple myeloma patients and have clinical benefit in diagnosis and monitoring of disease. In monoclonal gammopathy of undetermined significance (MGUS) 60% of patients have abnormal serum free light chain ratios and are an independent risk factor for progression to myeloma. The aim of this study was to examine the serum of asymptomatic patients for serum free light chains at diagnosis and to determine if they are predictive of disease progression. Methods: Archived presentation sera were studied from forty three asymptomatic myeloma patients who had been registered into United Kingdom Medical Research Council trials (1980 – 2002). Archived presentation sera were assayed for serum free light chains using the serum free light chain assay on an Olympus AU400 analyzer. Times to progression for those with abnormal versus normal serum free light chain ratios were compared. Times to progression were examined by Kaplan-Meier survival curves and log-rank sum statistical analysis. Results: Abnormal serum free light ratios were present in 36/43 (84%) of asymptomatic myeloma patients at the time of diagnosis and the remaining 7 patients had normal ratios. The median follow-up time for all 43 patients was 2807 days. Six patients with a normal kappa/lambda ratio had a median time to progression of 1323 days. In contrast, 26 patients with abnormal serum free kappa/lambda ratios had a median time to progression of 713 days. Ten patients who had an abnormal kappa/lambda ratio had not progressed at the time of follow-up. Although the median time to progression of patients with normal serum free light chain ratios was greater than those with abnormal ratios, this did not reach statistical significance (p<0.13). Conclusions: In summary, 84% of asymptomatic myeloma patients have an abnormal kappa/lambda ratio at diagnosis, in comparison with 95% of multiple myeloma and 60 % of (MGUS) patients. Furthermore, our data suggest that those with normal serum free light chain ratio may progress more slowly than those with abnormal ratios. Due to the small number of patients in this study, this did not reach statistical significance. In the spectrum of malignancy from MGUS to asymptomatic and symptomatic myeloma serum free light chain levels have an increasing frequency of abnormality and are associated with increased risk of progression to symptomatic myeloma.

Immunochemical quantification of free immunoglobulin light chains from an analytical perspective

2006 ◽  
Vol 44 (5) ◽  
Author(s):  
Takanari Nakano ◽  
Shuichi Miyazaki ◽  
Hidenori Takahashi ◽  
Akira Matsumori ◽  
Taro Maruyama ◽  
...  
Keyword(s):  
Reference Intervals ◽  
Normal Serum ◽  
Free Form ◽  
Light Chains ◽  
Immunoglobulin Light Chains ◽  
Heavy Chains ◽  
Excess Production ◽  
Analytical Perspective ◽  
Recent Developments ◽  
Serum And Urine

AbstractImmunoglobulin light chains are components of antibodies, but some exist in a free form in serum and urine as a result of their excess production over heavy chains. Free light chain (FLC) levels are of the order of milligram per liter in normal serum and urine, but marked increases have been observed in various disease conditions. It has now been established that the measurement of FLC levels contributes to diagnosis and clinical management in monoclonal gammopathies. Recent developments in FLC assays have been adapted to several automated platforms and they have now become available in laboratories. There have, however, been some concerns regarding the analytical aspects. The current assay specificity appears to be insufficient to prevent the influence of intact light chains of several orders of magnitude greater than FLCs in serum. Moreover, the heterogeneous nature of light chains makes accurate quantification unreliable. FLC assays have never been standardized because of the lack of an international reference calibrator. In this review, we summarize the reports on FLC measurements and examine the specificity of anti-FLC antibodies and the reliability of FLC assays. We also discuss difficulties in the standardization and setting of normal reference intervals for FLC assays.

Monitoring free light chains in serum using mass spectrometry

2016 ◽  
Vol 54 (6) ◽  
Author(s):  
David R. Barnidge ◽  
Angela Dispenzieri ◽  
Giampaolo Merlini ◽  
Jerry A. Katzmann ◽  
David L. Murray
Keyword(s):  
Mass Spectrometry ◽  
Light Chain ◽  
Disulfide Bonds ◽  
Plasma Cells ◽  
Mass Measurement ◽  
Normal Serum ◽  
High Mass ◽  
Light Chains ◽  
Plasma Cell Dyscrasia ◽  
Free Light Chains

AbstractSerum immunoglobulin free light chains (FLC) are secreted into circulation by plasma cells as a by-product of immunoglobulin production. In a healthy individual the population of FLC is polyclonal as no single cell is secreting more FLC than the total immunoglobulin secreting cell population. In a person with a plasma cell dyscrasia, such as multiple myeloma (MM) or light chain amyloidosis (AL), a clonal population of plasma cells secretes a monoclonal light chain at a concentration above the normal polyclonal background.We recently showed that monoclonal immunoglobulin rapid accurate mass measurement (miRAMM) can be used to identify and quantify a monoclonal light chain (LC) in serum and urine above the polyclonal background. This was accomplished by reducing immunoglobulin disulfide bonds releasing the LC to be analyzed by microLC-ESI-Q-TOF mass spectrometry. Here we demonstrate that the methodology can also be applied to the detection and quantification of FLC by analyzing a non-reduced sample.Proof of concept experiments were performed using purified FLC spiked into normal serum to assess linearity and precision. In addition, a cohort of 27 patients with AL was analyzed and miRAMM was able to detect a monoclonal FLC in 23 of the 27 patients that had abnormal FLC values by immunonephelometry.The high resolution and high mass measurement accuracy provided by the mass spectrometry based methodology eliminates the need for κ/λ ratios as the method can quantitatively monitor the abundance of the κ and λ polyclonal background at the same time it measures the monoclonal FLC.

Detection of Bence-Jones Protein in Serum by Immunoblotting

1996 ◽  
Vol 33 (2) ◽  
pp. 132-138 ◽  
Author(s):  
E T Backer ◽  
A Brand
Keyword(s):  
Alkaline Phosphatase ◽  
Present Method ◽  
Light Chains ◽  
Agarose Gel ◽  
Bence Jones Protein ◽  
Free Light Chains ◽  
Polyvinylidene Difluoride ◽  
Background Staining ◽  
Urine Testing ◽  
Serum And Urine

To detect Bence-Jones protein (BJP) in serum we precipitated intact immunoglobulins (Ig) using polyethylene glycol (PEG) and subjected the BJP in solution to electrophoresis in agarose gel, followed by transfer to a polyvinylidene difluoride membrane, and immunoenzymatic staining (successively using rabbit anti-human light/heavy chain of Ig, biotinylated swine anti-rabbit Ig, and alkaline phosphatase-conjugated streptavidin). Treatment with PEG effectively reduced background staining of polyclonal Ig in the immunoblots, although intact monoclonal Ig was not always completely removed. To compare the present method with immunoelectrophoresis (IEP), we selected samples from patients demonstrating BJP by IEP in both serum and urine ( n = 40), serum only ( n = 18), and urine only ( n = 32); 21 of these patients had BJP alone and 69 had BJP in addition to intact monoclonal Ig. Efficiency of detection of BJP in serum was increased by the present method: Serum BJP was detected in 70 patients by the present method versus 58 by IEP. The present method demonstrated single BJP bands in the samples from 16 patients (κ, n = 7; λ, n = 9) and multiple BJP bands (range: 2–9) in the samples from 54 patients (κ, n = 31; λ, n = 23). This method could be useful for detecting BJP in serum from patients suspected of having light chain gammopathy (without the need for urine testing) and may complement urine testing in patients excreting polyclonal free light chains of Ig.

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