Association of Serum Free Light Chain Level with Long-Term Kidney Function in Patients with Newly Diagnosed Multiple Myeloma.

INTRODUCTION: Multiple myeloma (MM) frequently involves the kidneys, resulting in acute, subacute, or chronic kidney disease (CKD). Patient- and treatment-related factors are associated with the long-term development of CKD. The aim of our study was to examine the association of serum free light chain (FLC) levels, measured at the time of diagnosis of MM, and CKD at subsequent follow-up. METHODS: Patients with newly diagnosed MM were identified using cancer registries at five hospitals. The primary outcome was low eGFR (<60 mL/min/1.73 m2) or dialysis dependence and a secondary composite outcome of low eGFR, dialysis dependence, or death at the last follow-up, up to 12 months from diagnosis. Logistic regression analyses were performed. RESULTS: A total of 149 patients met the inclusion criteria. Patients with an FLC level above the median had a higher frequency of hypertension (54% vs. 81%; p < 0.001), hyperlipidemia (37% vs. 56%; p = 0.018), low eGFR at the time of diagnosis (43% vs. 66%; p = 0.006), and a higher MM stage (p = 0.018). On multivariable analyses, after adjustment for several covariates, serum FLC level (per each 100 mg/L) was independently associated with low eGFR or dialysis dependence at follow-up (adjusted odds ratio [aOR] 1.021; 95% CI: 1.002, 1.041; p = 0.033). This association persisted for the composite outcome of low eGFR, dialysis dependence, or death (aOR 1.034; 95% CI: 1.006, 1.063; p = 0.018). DISCUSSION/CONCLUSION: Higher serum FLC level measured at the time of MM diagnosis is independently associated with CKD at up to 12 months of follow-up.

Combined serum free light chain predicts prognosis in acute kidney injury following cardiovascular surgery.

OBJECTIVES: Increased polyclonal free light chains (FLCs) are found in inflammatory conditions. Inflammation is recognized in the progression of acute kidney injury (AKI). This study was aimed to determine whether polyclonal combined FLC (cFLC) was associated with prognosis of AKI patients. METHODS: This prospective cohort included 145 adults with hospital-acquired AKI following cardiovascular surgery between 2014 and 2016, according to the KDIGO creatinine criteria. The primary end point of the study was all-cause death during follow-up. RESULTS: The median of serum cFLC concentration in the cohort was 42.0 (31.9-60.3 mg/L) and levels of cFLC in patients with AKI stage 3 were higher than those in AKI stage 1 and stage 2. cFLC levels correlated significantly with renal function biomarkers, high sensitivity C-reactive protein (hsCRP), and sequential organ failure assessment (SOFA) score. Patients were organized into the following two groups: the low-cFLC group (cFLC <43.3 mg/L) and the high-cFLC group (cFLC ≥ 43.3 mg/L). A total of 17 (11.0%) patient deaths occurred within 90 d, 13 (18.8%) in the high-cFLC group. Kaplan-Meier analysis revealed that the two groups differed significantly with respect to 90-d survival (log-rank p = .012), and Cox regression analysis showed that an cFLC level ≥43.3 mg/L was significantly associated with a 5.0-fold increased risk of death (adjusted hazard ratio [HR], 5.95; 95% confidence interval [CI], 1.04- 33.91; p = .045) compared with an cFLC level <43.3 mg/L. CONCLUSIONS: Serum cFLC levels were significantly elevated and might be an independent predictor of mortality in patients with AKI following cardiovascular surgery.

Detection of Plasma Cell Disorders by Mass Spectrometry: A Comprehensive Review of 19,523 Cases.

OBJECTIVES: To verify the analytical performance of a new mass spectrometry-based method, termed MASS-FIX, when screening for plasma cell disorders in a routine clinical laboratory. PATIENTS AND METHODS: Results from 19,523 unique patients tested for an M-protein between July 24, 2018, and March 6, 2020, by a combination serum protein electrophoresis (SPEP) and MASS-FIX were examined for consistency with pretest implementation performance. MASS-FIX's ability to verify abnormal results from SPEP and free light chain measurements was then compared with that of immunofixation electrophoresis (IFE) using a separate cohort of 52,586 patients tested by SPEP/IFE during the same period. RESULTS: Overall, 62.4% of our cohort was negative for an M-protein. Importantly, 7.3% of all specimens had an M spike on SPEP (0.1 to 8.5 g/dL) and MASS-FIX detected an M-protein in all these samples. Of all samples, 30.3% had M-proteins that were detected by MASS-FIX but the SPEP finding was too small for quantification. Of the positive samples, 5.7% contained a therapeutic monoclonal antibody. Of the positive samples, 4.1% had an N-glycosylated light chain (biomarker of high-risk plasma cell disorders). MASS-FIX confirmed a higher percentage of SPEP abnormalities than IFE. MASS-FIX was slightly more sensitive than IFE when confirming an M-protein in samples with an abnormal free light chain ratio. MASS-FIX had a very low sample repeat rate (1.5%). MASS-FIX was highly automatable resulting in a higher number of samples/technologist/day than IFE (∼30% more). CONCLUSION: Overall, MASS-FIX was successful in maintaining validation characteristics. MASS-FIX was more sensitive in confirming SPEP abnormalities when compared with IFE. Ability to detect therapeutic monoclonal antibodies and glycosylated light chains was distinctly advantageous.

The Increasing Role of Kappa Free Light Chains in the Diagnosis of Multiple Sclerosis.

Free light chains (FLC) are a promising biomarker to detect intrathecal inflammation in patients with inflammatory central nervous system (CNS) diseases, including multiple sclerosis (MS). The diagnostic use of this biomarker, in particular the kappa isoform of FLC ("KFLC"), has been investigated for more than 40 years. Based on an extensive literature review, we found that an agreement on the correct method for evaluating KFLC concentrations has not yet been reached. KFLC indices with varying cut-off values and blood-CSF-barrier (QAlbumin) related non-linear formulas for KFLC interpretation have been investigated in several studies. All approaches revealed high diagnostic sensitivity and specificity compared with the oligoclonal bands, which are considered the gold standard for the detection of intrathecally synthesized immunoglobulins. Measurement of KFLC is fully automated, rater-independent, and has been shown to be stable against most pre-analytic influencing factors. In conclusion, the determination of KFLC represents a promising diagnostic approach to show intrathecal inflammation in neuroinflammatory diseases. Multicenter studies are needed to show the diagnostic sensitivity and specificity of KFLC in MS by using the latest McDonald criteria and appropriate, as well as standardized, cut-off values for KFLC concentrations, preferably considering non-linear formulas such as Reiber's diagram.

Silver Nanoparticles as a Tool for the Study of Spontaneous Aggregation of Immunoglobulin Monoclonal Free Light Chains.

Some misfolded proteins, e.g., immunoglobulin monoclonal free light chains (FLC), tend to form fibrils. Protein deposits in tissue may lead to amyloidosis and dysfunction of different organs. There is currently no technique allowing for the identification of FLC that are prone to aggregate. The development of such a method would enable the early selection of patients at high risk of developing amyloidosis. The aim of this study was to investigate whether silver nanoparticles (AgNPs) could be a useful tool to study the process of aggregation of FLC and their susceptibility to form the protein deposits. Mixtures of AgNPs and urine samples from patients with multiple myeloma were prepared. To evaluate the aggregation process of nanoparticles coated with proteins, UV-visible spectroscopy, transmission electron microscopy, and the original laser light scattering method were used. It has been shown that some clones of FLC spontaneously triggered aggregation of the nanoparticles, while in the presence of others, the nanoparticle solution became hyperstable. This is probably due to the structure of the chains themselves, unique protein-AgNPs interactions and perhaps correlates with the tendency of some FLC clones to form deposits. Nanoparticle technology has proven to be helpful in identifying clones of immunoglobulin FLC that tend to aggregate.

Cell Population Data and Serum Polyclonal Immunoglobulin Free Light Chains in the Assessment of COVID-19 Severity.

Distinguishing between severe and nonsevere COVID-19 to ensure adequate healthcare quality and efficiency is a challenge for the healthcare system. The aim of this study was to assess the usefulness of CBC parameters together with analysis of FLC serum concentration in risk stratification of COVID-19. MATERIALS AND METHODS: CBC was analyzed in 735 COVID ICU, COVID non-ICU, and non-COVID ICU cases. FLC concentration was analyzed in 133 of them. RESULTS: COVID ICU had neutrophils and lymphocytes with the greatest size, granularity, and nucleic acid content. Significant differences in concentrations of κ and λ FLCs were shown between COVID ICU and COVID non-ICU. However, no difference was found in the κ/λ ratio between these groups, and the ratio stayed within the reference value, which indicates the presence of polyclonal FLCs. FLC κ measurement has significant power to distinguish between severe COVID-19 and nonsevere COVID-19 (AUC = 0.7669), with a sensitivity of 86.67% and specificity of 93.33%. The κ coefficients' odds ratio of 3.0401 was estimated. CONCLUSION: It can be concluded that the results obtained from the measure of free light immunoglobulin concentration in serum are useful in distinguishing between severe and nonsevere COVID-19.

Hantavirus infection-induced B cell activation elevates free light chains levels in circulation.

In humans, orthohantaviruses can cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). An earlier study reported that acute Andes virus HPS caused a massive and transient elevation in the number of circulating plasmablasts with specificity towards both viral and host antigens suggestive of polyclonal B cell activation. Immunoglobulins (Igs), produced by different B cell populations, comprise heavy and light chains; however, a certain amount of free light chains (FLCs) is constantly present in serum. Upregulation of FLCs, especially clonal species, associates with renal pathogenesis by fibril or deposit formations affecting the glomeruli, induction of epithelial cell disorders, or cast formation in the tubular network. We report that acute orthohantavirus infection increases the level of Ig FLCs in serum of both HFRS and HPS patients, and that the increase correlates with the severity of acute kidney injury in HFRS. The fact that the kappa to lambda FLC ratio in the sera of HFRS and HPS patients remained within the normal range suggests polyclonal B cell activation rather than proliferation of a single B cell clone. HFRS patients demonstrated increased urinary excretion of FLCs, and we found plasma cell infiltration in archival patient kidney biopsies that we speculate to contribute to the observed FLC excreta. Analysis of hospitalized HFRS patients' peripheral blood mononuclear cells showed elevated plasmablast levels, a fraction of which stained positive for Puumala virus antigen. Furthermore, B cells isolated from healthy donors were susceptible to Puumala virus in vitro, and the virus infection induced increased production of Igs and FLCs. The findings propose that hantaviruses directly activate B cells, and that the ensuing intense production of polyclonal Igs and FLCs may contribute to acute hantavirus infection-associated pathological findings.

Comparison of Freelite and N-Latex serum free light chain assays: a critical review.

INTRODUCTION: The measurement of serum free light chain (FLC) represents a fundamental aspect on the assessment of patients with monoclonal gammopathies (MG). Different analytical methods for FLC have become available with the possibility to obtain different value with a substantial impact on the assessment of patients with MG. This study aimed to evaluate FLC results obtained with two different assays and how the difference value obtained can impact in the patient's assessment. MATERIALS AND METHODS: Ninety-three patient serum samples that underwent analysis for FLC with two different methods, Serum Freelite (The Binding Site, Birmingham, UK) and N-Latex FLC (Siemens, Marburg, Germany), were included in this retrospective study. Statistical analysis was performed to evaluate correlation, difference, and the grade of concordance between the results obtained with the two methods. RESULTS: Significant statistical differences between the results obtained from the two methods were found (P < 0.05). A good correlation was found (0.99 for κ FLC, 0.95 for λ FLC, and 0.94 for the κ/λ ratio, respectively). We found a weighted kappa value of 0.65 for κ/λ ratio, 0.65 for λ FLC and 0.90 for κ FLC. A positive bias found with the Bland-Altman plot mirrors overestimation of κ FLC and κ/λ ratio with Freelite compared to N-Latex, whilst a negative bias underscores underestimation of λ FLC by Freelite compared to N-Latex. CONCLUSION: Although in general the concordance between Freelite and N-Latex appears satisfactory, several discrepancies could be evidenced and consequently the two assays are not interchangeable.

Immunoparesis defined by heavy/light chain pair suppression in smoldering multiple myeloma shows initial isotype specificity and involves other isotypes in advanced disease.

Smoldering multiple myeloma (SMM) is an asymptomatic and biologically heterogeneous plasma cell disorder, with a highly variable clinical course. Immunoparesis, defined by total immunoglobulin measurements, has been shown to be an independent risk factor for progression to symptomatic disease. The heavy/light chain (HLC) assay allows precise measurement of the polyclonal immunoglobulin of the same isotype, enabling the evaluation of isotype-matched immunoparesis (IMI). In this study, we prospectively characterized immunoparesis, as determined by HLC measurements, in 53 SMM patients. Severe IMI was present in 51% of patients, while severe IP of uninvolved isotypes (HLC IP) was present in 39%. Most of the patients with severe HLC IP presented with severe IMI, but not the other way around. Isotype specificity of immune suppression was suggested by lower relative values of isotype-matched HLC pairs, both for IgG and IgA SMM. Severe IMI was associated with other risk factors for progression while patients with severe IMI and severe HLC IP showed an even higher risk profile. Both severe IMI and severe IgM HLC IP showed a significantly shorter time to progression. Finally, gene expression analysis demonstrated differences in the bone marrow microenvironment between patients with IMI and IMI plus HLC IP, with an increased expression of genes associated with cytolytic cells. In conclusion, our data supports isotype specificity of early immunoglobulin suppression mechanisms. While suppression of both involved and uninvolved isotypes is associated with risk of progression, the later appears to develop with more advanced disease and could be mediated by different mechanisms.

Evaluation of immunoglobulins subclasses and free-light chains in non-obese patients with polycystic ovary syndrome and correlations with hormonal and metabolic parameters: preliminary data.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism and hyperinsulinemia that contribute to create a state of chronic low-grade inflammation. We performed an observational case-control study to investigate inflammatory and immunological parameters, such as IgG subclasses and free light chains (FLCs) and hemolytic complement activity (CH50) in non-obese PCOS, evaluating their relations with metabolic and hormonal parameters. PATIENTS AND METHODS: 36 subjects were studied: 16 PCOS patients (mean±SEM 27.13±1.82 age; BMI 24.1±0.9 kg/m2); 20 controls (aged 26.05±0.73; BMI 20.8 ± 0.4 kg/m2). The blood sample was collected for metabolic and hormonal parameters, IgG subclasses, k and λ FLCs, CH50. Hormones were measured by immunochemiluminometric assays; metabolic parameters by enzymatic assays; subclasses of IgG, FLCs, and CH50 were evaluated by the turbidimetric method. RESULTS: PCOS patients showed vs. controls lower IgG1, IgG2, IgG3 (mean±SEM 3.76±0.29 g/l, 2.63±0.20, 0.62±0.06, 0.34±0.08 vs. 6.49±0.35, 4.28±0.25, 0.84±0.07, 0.33±0.04, respectively) and higher levels of FLCs (k 12.22±0.71 vs. 6.03±0.30, λ 10.10±0.79 vs. 8.04±0.48 g/l) and CH50 (48.64±2.65 vs. 36.51±1.38 U/ml); we found correlation between IgG2 and free-testosterone (r=0.72, p=0.005) and CH50 and vitamin D (r=0.54, p=0.04); an inverse correlation was found between IgG1 and, respectively, ACTH (r=-0.57, p=0.02) and cortisol (r=0.78, p=0.001) in PCOS. CONCLUSIONS: In the complex scenario of low-grade inflammation in non-obese PCOS, we showed lower levels of main subclasses of IgG and higher CH50 levels, suggesting the involvement of other mechanisms other than the "classical" pathway of complement activation; FLCs could be attractive to monitor inflammation degree, disease activity and influence on hormonal status.

Serum free light chain is associated with histological activity and cirrhosis in patients with chronic hepatitis B.

BACKGROUND: The antiviral immune response is the main cause of hepatocyte damage and inflammatory necrosis. The serum free light chain, reflecting the immune function of B-cells, is strongly associated with inflammation and disease activity. We aimed to investigate the association of serum free light chain with the progression of chronic hepatitis B. METHODS: A total of 208 eligible chronic hepatitis B patients who had undergone a liver biopsy were studied. Serum free light chains of all patients were measured by turbidimetry using an immunoassay. Liver histology was assessed according to the METAVIR scoring system (which grades the stage of fibrosis on a five-point scale, F0 = no fibrosis to F4 = cirrhosis, and histological activity on a four-point scale, A0 = no activity to A3 = severe activity). The association of serum free light chains with histological activity and fibrosis progression was evaluated. RESULTS: The concentration of serum free light chains in CHB patients increased gradually with histological activity and fibrosis progression. The intensity of histological activity was significantly correlated with the serum free kappa chain (r = 0.658, P < 0.001) and the serum free lambda chain (0.675, P < 0.001). The stages of fibrosis were correlated with the serum free kappa chain (r = 0.683, P < 0.001) and serum free lambda chain (0.664, P < 0.001). After adjusting for age, sex and other synergic factors, the serum free kappa chain remained a potential risk factor, but the serum free lambda chain was no longer associated with liver cirrhosis. Similar to FIB-4 and RPR, the serum free kappa chain exhibited excellent performance in the prediction of liver cirrhosis. The AUCs of serum free Kappa chain, FIB-4 and RPR were 0.873, 0.880 and 0.895, respectively, which were significantly higher than those of the AAR and APRI (0.718 and 0.746). CONCLUSION: Our work revealed that serum free light chains were associated with histological activity and cirrhosis in chronic hepatitis B, which could play a crucial role in the immunopathogenesis of HBV-associated cirrhosis. In addition, free kappa light chain could be a useful predictor of liver cirrhosis.

Application of Capillary Electrophoresis in Monoclonal Gammopathies and the Cutoff Value of Monoclonal Protein in Differential Diagnosis of Multiple Myeloma and Other Monoclonal Gammopathies.

OBJECTIVE: Monoclonal protein (MP) exists in various diseases, and capillary electrophoresis (CE) has been widely used to detect MP. However, there is not much research on the application value of MP in the differential diagnosis of monoclonal gammopathies. This study aimed to explore MP's cutoff value for the differential diagnosis of multiple myeloma (MM) and other monoclonal gammopathies (MGs). METHODS: A retrospective analysis of 8167 cases was conducted. Serum MP was detected by CE, and the patients' clinical information was collected from the clinical database of our hospital. RESULTS: 985 cases had MP with high peaks, and 91.1% were diagnosed with malignant diseases. The MP showed small peaks in 471 cases, and only 24.4% were diagnosed with malignant diseases. Among the MPs, the IgG-κ type was the most common type, followed by the IgG-λ, IgA-κ, IgA-λ, free λ light chain, IgM-κ, free κ light chain, double clone, and IgM-λ types. Differences in the MP of the IgG, IgA, IgM, and FLC types between the MM group and MGUS group were statistically different (P<0.01). The MP of the IgG, IgA, and FLC types showed clear specificity and sensitivity in discriminating MM from other monoclonal gammopathies in ROC curve analysis. Serum IgM had statistical significance in the differential diagnosis between WM and other MGs (P<0.01). However, there was no statistical significance in the differential diagnosis between MM and other MGs (P=0.140). The cutoff values of the MP of the IgG, IgA, and FLC types were >18.67g/L, >13.86g/L, and >10.15g/L, respectively, for the differential diagnosis of MM and other MGs. The cutoff value of the MP of IgM for the WM diagnosis was >37.75 g/L. CONCLUSION: CE has good clinical application value in the diagnosis of monoclonal gammopathies, and MP can be used in the differential diagnosis of MM and other monoclonal gammopathies.

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.

Monoclonal free light chain detection and quantification: Performances and limits of available laboratory assays.

The detection and quantification of immunoglobulin free light chains in serum and urine is recommended for the diagnosis and monitoring of monoclonal gammopathies according to the guidelines of the International Myeloma Working Group (IMWG). Several tests are currently available in the clinical laboratory to detect and quantify free light chains but although quality, efficiency, and effectiveness have been improved, the results are still variable and poorly harmonized and standardized. The present review article wants to analyze these aspects, with a keen eye on techniques, such as mass spectrometry, that could replace in the practical clinical laboratory the current methods including Bence-Jones protein assay and free light chain immunoassays.