Biochemical profile of Bence-Jones type multiple myeloma.

Multiple myeloma (MM) is a malignant tumor occurring from plasma cells that produce an abnormal monoclonal immunoglobulin - a paraprotein. A distinctive feature of Bence-Jones myeloma is the excretion of monoclonal free light chains of immunoglobulins with 24h urine, and the absence of monoclonal intact immunoglobulins secretion. Comprehensive analysis of biochemical parameters in blood serum and 24h urine in patients with Bence-Jones multiple myeloma using electrophoretic and immunoturbidimetric methods to assess their sensitivity as biomarkers. 50 patients with a morphologically confirmed diagnosis of MM of the Bence-Jones immunochemical type were examined. 28 people without oncological diseases were examinedas a control. Detection of monoclonal secretion in blood serum and daily urine was performed by immunofixation electrophoresis on the Hydrasys 2 electrophoretic system (Sebia). The determination of free light chains of immunoglobulins (FLC) was performed by the immunoturbidimetric method (Binding Site) on an Advia 1800 analyzer (Siemens). Analysis of IgG, IgA, IgM, ß2-microglobulin and C-reactive protein was performed on Cobas 6000 analyzer (Roche). The median excretion of Bence-Jones protein in 24h urine of MM patients was 0.49 g/24h (0.06-2.45 g/24h). In the blood serum, in 86% of cases, the presence of paraproteinemia, represented by κ and λ type light chains of immunogloublins was detected. At the same time, the frequency of detection of monoclonal secretion in blood serum in Bence-Jones type λ myeloma was 95.7%, which was statistically significantly higher than the frequency of detection of monoclonal secretion of type κ - 77.8%. In patients with identified paraproteinemia, Bence-Jones protein excretion in daily urine (median 0.82 g/day) was statistically significantly higher than in patients without a monoclonal component detected in blood serum (median 0.04 g/24h). The levels of FLC in blood serum obtained by immunoturbidimetry in Bence-Jones myeloma of the corresponding type were higher than the reference levels in 100% of cases. The median level of κ-FLC reached 4358 mg/l, λ-FLC - 2225 mg/l, which was statistically significantly higher than the control levels. The median concentrations of IgG, IgA and IgM in patients with Bence-Jones myeloma were statistically significantly lower than in the control group, while the medians of ß2-microglobulin and C-reactive protein were significantly higher than in the control. Our investigation showed high diagnostic efficiency of electrophoretic and immunoturbidimetric analysis of monoclonal secretion in patients with Bence-Jones MM, while FLC analysis demonstrated maximum sensitivity. Bence-Jones MM revealed biochemical signs of secondary immunodeficiency and general inflammatory syndrome.

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

Circulating free light chains (FLCs), considered biomarkers of B cell activity, are frequently elevated in patients affected by systemic inflammatory autoimmune diseases. As the systemic sclerosis (SSc) clinical course can be variable, this study is aimed at evaluating FLCs levels in 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 κ and λ FLC in serum significantly higher than HC (p = 0.0001) alongside the mean values of free κ/λ ratio and κ + λ sum (p = 0.0001). SSc patients showed increased free κ in urine with a κ/λ higher than HC (p = 0.0001). SSc patients with increased κ + λ in serum showed that erythro-sedimentation rate (p = 0.034), C-reactive protein (p = 0.003), DAI (p = 0.024) and DSS (p = 0.015) were higher if compared to SSc patients with normal levels of FLC. A positive linear correlation was found between serum levels of free κ and DAI (r = 0.29, p = 0.014). In addition, SSc patients with increased free κ in urine had higher DAI (p = 0.048) than SSc patients with normal κ levels. Our results strengthen the role of serum FLC as useful biomarker in clinical practice to early diagnosis and monitor disease activity, showing for the first time that also urine FLC levels correlated with disease activity in SSc patients.

Comparison of serum free light chain and urine electrophoresis for the detection of the light chain component of monoclonal immunoglobulins in light chain and intact immunoglobulin multiple myeloma.

Response criteria for multiple myeloma are based upon changes in monoclonal protein levels quantified using serum and/or urine protein electrophoresis. The latter lacks sensitivity at low monoclonal protein levels and since 2001, the serum free light chain test has been available and its clinical utility proven, yet guidelines have not recommended it as a replacement for urine assessment. Herein we evaluated responses using serum free light chain measurements and serum and urine electrophoresis after 2 and 4 cycles of therapy and after stem cell transplantation in 25 light chain and 157 intact immunoglobulin myeloma patients enrolled in the IFM 2007-02 MM trial. All 25 light chain patients had measurable disease by serum free light chain and urine methods at presentation. By contrast 98 out of 157 intact immunoglobulin patients had measurable disease by serum free light chain compared to 55 out of 157 by urine electrophoresis. In all patients there was substantial agreement between predicate (serum/urine protein electrophoresis) and test (serum protein electrophoresis and serum free light chain) methods for response assessment (Weighted Kappa=0.83). Urine immunofixation became negative in 47% light chain and 43% intact immunoglobulin patients after 2 cycles of therapy. At this time the serum free light chain ratio normalised in only 11% and 27% patients, respectively. In summary we found good agreement between methods for response assessment, but the serum free light chain test provided greater sensitivity than urine electrophoresis for monitoring. To our knowledge this is the first report comparing both methods for response assignment based on the International Myeloma Working Group guidelines. (Clinical Trials Register.eu identifier: 2007-005204-40).

Monitoring free light chains in serum using mass spectrometry.

BACKGROUND: Serum 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Analytical issues of serum free light chain assays and the relative performance of polyclonal and monoclonal based reagents.

Serum free light chain (FLC) assays have been incorporated into routine clinical practice and their use is recommended in international guidelines for the management of monoclonal gammopathies. Given that FLCs are not simple analytes, laboratories should be aware of potential analytical issues when using FLC assays, including antigen excess, lot-to-lot variation and non-linearity. Whilst manufacturers of monoclonal antibody-based assays claim that they overcome such issues, the evidence available to date does not support this. Here we review and compare the technical performance of both polyclonal and monoclonal antibody-based assays. The evidence suggests that the Freelite assay, based on polyclonal antisera, gives a broader recognition of monoclonal FLCs than the N Latex assay, based on monoclonal antisera, and despite being cited as a technical concern, we show that lot-to-lot variation of the Freelite assay is good. Both non-linearity and antigen excess are characteristic of FLC analysis and laboratories should be aware of these phenomena regardless of the assay system they use. Comparisons of the absolute values of sFLCs determined using monoclonal and polyclonal antibody-based assays show poor quantitative agreement and, because current guidelines have been established using the polyclonal antibody-based Freelite assay, it should not be assumed that assays utilizing monoclonal antibodies will give compliance with these guidelines.

Measurement of free light chains with assays based on monoclonal antibodies.

Recently, serum free light chain (FLC) assays incorporating anti-kappa (κ) and anti-lambda (λ) FLC monoclonal antibodies have become available: N Latex FLC assay (Siemens) and Seralite® (Abingdon Health). The purpose of this review is to provide an overview of these two new monoclonal antibody-based methods. In doing so, the review will outline the performance characteristics of each method, including a summary of: assay principles, antibody specificity, analytical performance and assay performance in disease. Additionally, the review will describe the potential user benefits of adopting these new generation FLC assays, which are designed to overcome the established limitations of existing polyclonal antibody based FLC assays.

Strengths and weaknesses of methods for identifying monoclonal free light chains of Ig: examples from two cases with renal disease.

BACKGROUND: Serum free light chain (FLC) analysis with ratio and urine immunofixation electrophoresis (IFE) are both available for routine use in helping to detect plasma cell dyscrasia and related diseases. CASES: Case reports showing one serum positive for serum FLC but that showed a hook effect and overestimated the amount of monoclonal FLC while urine IFE was negative for Bence Jones protein, and a second serum that showed elevated FLC κ and λ but a normal κ/λ ratio, while urine IFE was positive for Bence Jones protein. CONCLUSIONS: These two techniques complement one another. Neither of the techniques is truly quantitative, and both exhibit methodological defects.

Combined light chain immunofixation to detect monoclonal gammopathy: a comparison to standard electrophoresis in serum and urine.

BACKGROUND: The purpose of this study was to evaluate a combined κ and λ light chain immunofixation (CLIF) as a screening tool to detect monoclonal immunoglobulins in serum and urine. A secondary aim was to investigate the impact on workflow and reagent utilisation of a systematic implementation of CLIF in addition to routine protein electrophoresis (PE) on all samples. METHODS: Light chain antisera (κ and λ) were mixed in a 1:1 ratio and loaded in the same sequence as the PE to create a superimposable image. RESULTS: The CLIF procedure agreed significantly better with standard immunofixation procedures in the serum and urine. In 33 (22%) new patients and in 114 (15%) follow-up patients CLIF detected a band missed by PE in serum. In 34 (4.5%) of previously categorised cases the monoclonal band was below the detection limit of CLIF in serum, but still detectable by conventional immunofixation electrophoresis. In one case (0.7%) a band in a urine specimen was missed by CLIF compared to 70 (49%) missed by PE. After the systematic introduction of CLIF turn-around-times (TATs) and utilisation of laboratory consumables decreased significantly (p<0.001). CONCLUSIONS: A systematic implementation of CLIF led to the detection of monoclonal bands missed by PE with an improvement in TATs and a decrease in cost.

Light chain crystalline kidney disease: diagnostic urine microscopy as the "liquid kidney biopsy".

Multiple myeloma (MM) is a plasma cell disorder, which often causes parenchymal kidney disease. Light chain (LC) cast nephropathy represents the most common renal lesion. In some instances, LC crystals precipitate within renal tubular lumens and deposit within proximal tubular cell cytoplasms. Importantly, urine microscopy in such patients can provide insight into the underlying LC-related lesion. Here we present two patients with MM complicated by acute kidney injury (AKI) where LC crystalline casts were observed on urinary sediment analysis. Kidney biopsy revealed acute tubular injury with LC crystal casts within both tubular lumens and renal tubular epithelial cell cytoplasms. These findings suggest that the urinary sediment may be a non-invasive way to diagnose LC crystalline-induced AKI in patients with MM.

Urinary gamma-glutamyl transferase-to-creatinine ratio as an indicator of tubular function in Bence Jones proteinuria.

Increased urinary gamma-glutamyl transferase (GGT) activity suggests early renal tubular damage. The aim of this study was to evaluate the urinary GGT activity as a marker of renal injury in different types of Bence Jones Proteinuria (BJP). One hundred and three individuals with BJP were included in the study. Urinary GGT activity, urinary GGT-to-creatinine ratio and urinary protein-to-creatinine ratio were studied. Urine samples were tested by immunofixation agarose gel electrophoresis. Total urinary excretion of kappa and lambda light chains were measured by nephelometric method. There were no significant differences in demographic characteristics of the patients in Lambda BJP, Kappa BJP and Control groups. GGT-to-creatinine ratio of the Lambda BJP group was significantly higher than Kappa BJP group and controls (p = 0.018 and 0.002, respectively). There was no correlation between the quantitative kappa and lambda BJP and urinary GGT-to-creatinine ratio. Our data have demonstrated that urinary GGT-to-creatinine ratio could be a tubular damage marker of lambda light chain proteinuria.

Mono/polyclonal free light chains as challenging biomarkers for immunological abnormalities.

Free light chain (FLC) kappa (k) and lambda (λ) consist of low molecular weight proteins produced in excess during immunoglobulin synthesis and secreted into the circulation. In patients with normal renal function, over 99% of FLCs are filtered and reabsorbed. Thus, the presence of FLCs in the serum is directly related to plasma cell activity and the balance between production and renal clearance. FLCs are bioactive molecules that may exist as monoclonal (m) and polyclonal (p) FLCs. These have been detected in several body fluids and may be key indicators of ongoing damage and/or illness. International guidelines now recommend mFLC for screening, diagnosis and monitoring multiple myeloma and other plasma cell dyscrasias. In current clinical practice, FLCs in urine indicate cast nephropathy and other renal injury, whereas their presence in cerebrospinal fluid is important for identifying central nervous system inflammatory diseases such as multiple sclerosis. Increased pFLCs have also been detected in various conditions characterized by B cell activation, i.e., chronic inflammation, autoimmune disease and HCV infection. Monitoring the coronavirus (COVID-19) pandemic by analysis of salivary FLCs presents a significant opportunity in clinical immunology worthy of scientific pursuit.

Influence of immunoglobulin light chain dimers on the results of the quantitative nephelometric assay.

BACKGROUND: Some polymeric forms of immunoglobulin free light chains (FLCs) have been identified, such as dimer, trimer and tetramer, and the quantitative nephelometric assay of kappa and lambda light chains is very important to the diagnosis and management of multiple myeloma (MM), primary systemic amyloidosis (AL), etc. But, whether polymeric molecules could affect the results of the nephelometric assay, is rarely reported. METHODS: 12 urinary samples from patients with light-chain MM were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and every sample was run with reduced and nonreduced sample buffer in one gel. Urinary kappa and lambda light chains were measured on the BNII nephelometer. We then added 3 microL beta-mercaptoethanol (2-ME) to the samples to transform the dimers into monomeric forms and measured them again. RESULTS: 10 samples showed obvious dimers by SDS-PAGE, of which 2 samples were kappa light chain dimers. Six samples had decreased quantitative results contrasted with the former values. The two samples of kappa light chain dimers decreased more obviously. CONCLUSIONS: The light chain dimer combined with more antibodies than the monomer and a larger antigen-antibody complex was formed. The larger complex could magnify the reaction between antigen and antibody of the nephelometric assay and therefore false elevated results could measured.

Urinary κ and λ immunoglobulin light chains in normoalbuminuric type 2 diabetes mellitus patients.

BACKGROUND AND AIM: Kidney disease is one of the major chronic microvascular complications of diabetes. Tubular involvement may precede glomerular involvement and the appearance of microalbuminuria. The aim of the study was to evaluate quantitatively immunoglobulin light chains (IgLCs), kappa and lambda excretion in the urine of patients with type 2 diabetes mellitus with normoalbuminuria and with microalbuminuria compared to control group. RESULTS: Urinary IgLCs levels of the control group were significantly lower than diabetic patients with normoalbuminuria and diabetic patients with albuminuria. IgLCs were significantly associated with the duration of disease and negatively with estimated glomerular filtration rate. CONCLUSION: Type 2 diabetic patients can have significantly raised concentrations of urinary IgLCs before microalbuminuria or renal disease occurs. Further investigations are recommended to assess LC evaluation in the early management of diabetic renal disease.

Chronic lymphocytic leukemia with associated lambda-light-chain and IgG lambda paraproteins simulating a biclonal gammopathy.

BACKGROUND: Monoclonal components (MCs) are frequently detected in the sera of patients with B-cell malignancies, by techniques that are getting more and more sensitive. Only few chronic lymphocytic leukemia (CLL) patients with multiple serum paraproteins are reported in the literature. METHODS: In this case report we present a 71-year-old woman with CLL and serum MCs. Immunofixation was performed on agarose film using anti-sera monospecific for the heavy and light chains of human immunoglobulins (anti-gamma, -alpha, -mu, -delta, -epsilon, -kappa, -lambda). Serum free light chains (FLCs) were quantified nephelometrically. Immunofluorescence analysis was performed using fluorochrome-conjugated goat antibodies specific for human mu, gamma or alpha immunoglobulin heavy chains and K or lamda light chains. RESULTS: Immunofixation revealed two different MCs (IgGlambda + lambda light-chains) in the serum and only one MC (lambda light chains) in concentrated urine. Serum lamda FLCs were 206 mg/L. The bone marrow aspiration and biopsy revealed a 38 % interstitial and nodular infiltration of mature small lymphocytes expressing IgG lambda surface immunoglobulins CD 19, CD20, CD5, and CD23, with negative BCL-1, t(11, 14) and cyclin D1. The plasma cells were less than 1%. Final diagnosis was CLL (Rai stage I) with IgG lamda plus lamda serum paraproteins. Three years later, the patient died because of myocardial infarction after a follow-up period with no need for CLL therapy. CONCLUSIONS: Our hypothesis is that the double MC may be the result of an unbalanced immunoglobulin chain synthesis by the leukemic B-cell clone, resulting in IgGlamda and excess of lambda FLCs.

κ and λ urine free light chains: a new method for quantification.

BACKGROUND: Immunofixation electrophoresis of urinary proteins, coupled with densitometric analysis, is the gold standard method for determining urinary monoclonal free light chains (FLCs), i.e. Bence Jones protein. Recently, immunochemical methods have been developed for Bence Jones protein quantification, but no such method has been widely adopted. This study evaluated a new antibody-based immunoturbidimetry method for urinary FLC quantification, using immunofixation electrophoresis as reference. METHODS: κ and λ FLCs were measured in urine specimens from 95 (training cohort) and 103 (testing cohort) patients by both immunofixation electrophoresis and immunoturbidimetry. RESULTS: There was almost perfect concordance in the training cohort between the new immunoturbidimetry assay and immunofixation electrophoresis and substantial agreement, with Cohen kappa of 0.85 and 0.75, for κ and λ FLC determination, respectively. Results were confirmed in the testing cohort, where Cohen kappa was 0.86 for κ and 0.94 for λ FLCs. The κ FLC assay had 88% sensitivity and 98%-100% specificity; the λ FLC assay had 94% and 96% sensitivity and 91% and 99% specificity in the training and testing cohorts, respectively. CONCLUSIONS: The new immunochemical method has a satisfactory performance and almost perfect agreement with immunofixation electrophoresis and gives the advantage of FLC quantification.

Quantitation of serum free light chains does not compensate for serum immunofixation only when screening for monoclonal gammopathies.

BACKGROUND: Detection of plasma cell dyscrasias (PCD) requires screening of serum and urine for monoclonal proteins. Several studies have demonstrated increased sensitivity and specificity when measurement of serum free light chain (SFLC) is part of the screening protocol. In addition, omission of immunofixation (IFE) in the standard work-up that includes SFLC assay has been proposed. This study attempts to define the role of the SFLC assay in a screening strategy limited to serum only. It compares outcomes to a serum-only screening strategy that omits serum IFE. METHODS: Serum from 691 patients was analysed for the presence of monoclonal protein using standard serum IFE, serum protein electrophoresis (SPE) and measurement of SFLC. Data were analysed retrospectively. RESULTS: Specificity and sensitivity of abnormal SFLC-ratios for the detection of monoclonal protein using IFE were 96% and 41%, respectively. Eighteen patients with negative monospecific and Bence Jones IFE, but abnormal SFLC-ratios were identified. In most cases, this could be attributed to kidney and inflammatory disease or haematological disorders. In four cases, this resulted in further diagnostic investigation and light chain disease was later detected in two cases. Light chain disease was confirmed in one case but not confirmed in the other patient. In 14 patients, Bence Jones IFE was negative, although the concentrations of SFLC suggested the presence of monoclonal Bence Jones protein at concentrations detectable by IFE. Thus, either the anti-serum failed at detection, there was polymerisation of the free light chains or the SFLC assay overestimated protein concentrations. Simulating a work-up that included IFE only if abnormalities were detected by SPE or the SFLC assay would have resulted in 26% fewer IFEs being performed, but three patients with monoclonal proteins by IFE would have been missed. CONCLUSIONS: Abnormal SFLC concentrations are neither sensitive nor specific for the detection of monoclonal proteins by IFE. Not all PCD are accompanied by excessive production of SFLC, and several other conditions, such as renal disease are associated with increased SFLC concentrations. An abnormal SFLC-ratio is a specific marker for PCD, and occurs primarily in patients with haematological disease. If renal and inflammatory diseases are excluded, this should prompt further diagnostic investigation. Screening of serum without performing an IFE as a standard procedure leads to a reduction of sensitivity when compared to screening of serum that includes IFE.

Urinary Alpha-1-Acid Glycoprotein Is a Sensitive Marker of Glomerular Protein Leakage at Altitude.

Talks, Ben J., Susie B. Bradwell, John Delamere, Will Rayner, Alex Clarke, Chris T. Lewis, Owen D. Thomas, and Arthur R. Bradwell. Urinary alpha-1-acid glycoprotein is a sensitive marker of glomerular protein leakage at altitude. High Alt Med Biol. 19:295-298, 2018.-Proteinuria is an established feature of ascent to altitude and may be caused by a loss of negative charges on glomerular capillary walls (GCWs). To test this hypothesis, we measured two similar sized but oppositely charged proteins in urine: negatively charged alpha-1-acid glycoprotein (α1-AGP, 41-43 kDa) and positively charged dimeric lambda free light chains (λ-FLCs, 50 kDa). Twenty-four-hour urinary leakage was compared with albumin, a 66 kDa negatively charged protein. We studied 23 individuals (ages 23-78 years, male = 17) at baseline (140 m) and daily during an expedition to 5035 m. The results showed a significant increase in median urinary leakage of α1-AGP (p < 0.0001; 6.85-fold) and albumin (p = 0.0006; 1.65-fold) with ascent to altitude, but no significant increase in leakage of λ-FLCs (p = 0.39; 1.14-fold). α1-AGP correlated with the daily ascent profile (p = 0.0026) and partial pressure of oxygen (p = 0.01), whereas albumin showed no correlation (p = 0.19). Urinary α1-AGP was a more sensitive marker of altitude proteinuria than urinary albumin and λ-FLCs, and supported the possibility of loss of GCW negative charges at altitude.

Intravenous infusion of pituitary adenylate cyclase-activating polypeptide (PACAP) in a patient with multiple myeloma and myeloma kidney: a case study.

We have recently shown significant renoprotective effects with the administration of pituitary adenylate cyclase-activating polypeptide (PACAP) in models of myeloma kidney. PACAP markedly inhibited the production of proinflammatory cytokines stimulated by immunoglobulin light chains in human renal proximal tubule epithelial cells and in the kidneys of rats infused with myeloma light chains. PACAP was also shown to suppress the proliferation of human kappa and lambda light chain-secreting multiple myeloma-derived cells. In this case study, an 81-year-old male patient with active multiple myeloma and myeloma kidney was infused intravenously with synthetic human PACAP38 at a rate of 4 pmol/kg/min for 120 min. The continuous infusion increased the level of PACAP38 in blood, with a plateau at about 0.2 nM during the infusion. The level of PACAP in the blood rapidly declined after the cessation of administration with a half-life of about 5-10 min. The continuous infusion did not significantly alter the basal glucose level, blood gases or blood pressure. There was a large reduction in free lambda light chains in urine after the start of the treatment with PACAP. These studies show that PACAP can be safely used in humans and suggest that it could be used as a novel therapeutic agent for the treatment of multiple myeloma and myeloma kidney.