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.

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.

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.

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.

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.

Clinical Presentation of Tubulointerstitial Nephritis Caused by Amyloid Light-chain Amyloidosis in a Patient with Sjögren's Syndrome.

We report a 70-year-old woman with Sjögren's syndrome who had severe renal dysfunction with mild proteinuria and elevated urinary low-molecular-weight proteins. Based on these clinical presentations, interstitial nephritis due to Sjögren's syndrome was strongly suspected. Unexpectedly, renal pathology revealed amyloid light-chain (AL) lambda-type depositions predominantly in the vasculatures with severe tubulointerstitial damage. Concentrated urine immunofixation was positive for Bence Jones lambda-type monoclonal proteins. Given the involvement in other organs, systemic AL amyloidosis was diagnosed. The patient underwent chemotherapy, but hemodialysis was ultimately instituted. It should be remembered that renal amyloidosis occurs as a clinical presentation of interstitial nephritis.

A novel approach for the chromatographic purification and peptide mass fingerprinting of urinary free light chains.

We describe a chromatographic approach for the purification of urinary free light chains (FLCs) viz., lambda free light chains (λ-FLCs) and kappa free light chains (κ-FLCs). Isolated urinary FLCs were analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular masses of λ-FLC and κ-FLC are 22,933.397 and 23,544.336Da respectively. Moreover, dimer forms of each FLC were also detected in mass spectrum which corresponds to 45,737.747 and 47,348.028Da respectively for λ-FLCs and κ-FLCs. Peptide mass fingerprint analysis of the purified λ-FLCs and κ-FLCs has yielded peptides that partially match with known light chain sequences viz., gi|218783338 and gi|48475432 respectively. The tryptic digestion profile of isolated FLCs infers the exclusive nature of them and they may be additive molecules in the dictionary of urinary proteins. This is the first report of characterization and validation of FLCs from large volume samples by peptide sequencing. This simple and cost-effective approach to purification of FLCs, together with the easy availability of urine samples make the large-scale production of FLCs possible, allowing exploration of various bioclinical as well as biodiagnostic applications.

The significance and predictive value of free light chains in the urine of patients with chronic inflammatory rheumatic disease.

In patients with rheumatic diseases, reliable markers for determining disease activity are scarce. One potential parameter is the level of immunoglobulin free light chains (FLCs), which is known to be elevated in the blood of patients with certain rheumatic diseases. Few studies have quantified FLCs in urine, a convenient source of test sample, in patients with different rheumatic diseases. We carried out a retrospective analysis of patients with rheumatic disease attending the University hospital of Goettingen, Germany. Subjects were included if they had urine levels of both κ and λ FLCs available and did not have myeloma. Data regarding systemic inflammation and kidney function were recorded, and FLC levels were correlated with inflammatory markers. Of the 382 patients with rheumatic disease, 40.1 % had chronic polyarthritis, 21.2 % connective tissue disease, 18.6 % spondyloarthritis and 15.7 % vasculitis. Elevated levels of κ FLCs were found for 84 % of patients and elevated λ for 52.7 %. For the patients with rheumatoid arthritis, FLCs correlated with C-reactive protein (κ, r = 0.368, p < 0.001; λ, r = 0.398, p < 0.001) and erythrocyte sedimentation rate (κ, r = 0.692, p < 0.001; λ, r = 0.612, p < 0.001). Patients being treated with rituximab displayed FLC levels similar to those of the reference group. There were clear elevations in both κ and λ FLCs in patients with rheumatic disease, but not in κ/λ ratio. The correlation between FLCs and inflammatory markers in patients with rheumatoid arthritis demonstrates their potential for predicting disease activity.

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.

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.

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.

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.

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).

[A Case of Primary Gastric Amyloidosis with Fulminant Heart Failure].

A 53-year-old woman was admitted with epigastric discomfort and weakness. Laboratory examination at admission showed mild anemia and proteinuria. Esophagogastroduodenoscopy revealed marked mucosal atrophy, diffuse nodularity and granular appearance with mucosal friability. Biopsy was performed on the antrum and body of the stomach. On the next day, the patient began to complain of severe dyspnea, and hypoxia was present on pulse oximetry. Therefore, emergency echocardiography was conducted and it showed restrictive cardiomyopathy along with thrombus in the left atrium. With time, heart failure was aggravated despite intensive management. The result of gastric biopsy revealed amyloid deposits which stained positively with Congo red. On immunohistochemistry study, kappa and lambda chain were present. In addition, kappa chain was significantly elevated in urine and serum on electrophoresis. Although the patient was finally diagnosed as having primary gastric amyloidosis with restrictive cardiomyopathy, her general condition rapidly deteriorated and died at 12th hospital day. When obscure gastric lesion is encountered, performing gastric biopsy is strongly recommended since it be primary gastric amyloidosis. Herein, we present an unusual case of primary gastric amyloidosis.

The serological diagnostic challenges of multiple myeloma.

Serological screening tests for multiple myeloma are commonly requested by physicians in both primary and secondary care to investigate patients presenting with anaemia or renal impairment of unknown cause. This article reviews the interpretation of these tests.

Differences identified between serum and urine immunofixation electrophoresis.

BACKGROUND: Different patterns between serum immunofixation electrophoresis (SIFE) and urine immunofixation electrophoresis (UIFE) happen occasionally and laboratorians should understand the mechanisms behind the differences and additional tests required for complicated cases. METHODS: We investigated a complicated multiple myeloma case that showed inconsistent patterns of SIFE and UIFE. To differentiate monoclonal proteins (M-proteins), the urine sample was treated with dithiothreitol to open up the IgA molecule and urine free light chain assay was used for free light chain analysis. RESULTS: The patient's SIFE indicated 2 IgA λ and 1 IgM κ M-proteins, while UIFE revealed monoclonal free λ light chains and a suspicious monoclonal IgA κ protein. Subsequent treatment of the urine sample with dithiothreitol and urine free light chain assay demonstrated that the suspicious monoclonal IgA κ protein was actually a monoclonal IgA λ and a free κ light chain that had similar electrophoretic mobility. CONCLUSIONS: The differences identified between SIFE and UIFE in this case are due to the limitation of immunofixation electrophoresis on different specimen types and intra-molecular disulfide bonds formation in IgA. The laboratorians must be cognizant of the strengths and limitations of the procedure being performed and the ancillary testing techniques available to solve the problem.