Evaluation of a new free light chain ELISA assay: bringing coherence with electrophoretic methods

2018 ◽  
Vol 56 (2) ◽  
pp. 312-322 ◽  
Author(s):  
Joannes F.M. Jacobs ◽  
Corrie M. de Kat Angelino ◽  
Huberdina M.L.M. Brouwers ◽  
Sandra A. Croockewit ◽  
Irma Joosten ◽  
...  
Keyword(s):  
Light Chain ◽  
Method Comparison ◽  
Protein Electrophoresis ◽  
Free Light Chain ◽  
Reference Ranges ◽  
Clinical Value ◽  
Serum Free Light Chain ◽  
Prognostic Stratification ◽  
Consistency Method ◽  
Assay Interference

Abstract Background: Serum free light chain (sFLC) measurements are increasingly important in the context of screening for monoclonal gammopathies, prognostic stratification, and monitoring of therapy responses. At the same time, analytical limitations have been reported with the currently available nephelometric and turbidimetric sFLC assays. We have evaluated a new quantitative sFLC ELISA for its suitability in routine clinical use. Methods: Reference ranges of the Sebia FLC assay were calculated from 208 controls. Assay interference, reproducibility, lot-to-lot variability, and linearity were assessed. Method comparison to the Freelite assay (Binding Site) was conducted by retrospective analysis of 501 patient sera. Results: Reference ranges of the Sebia κ/λFLC-ratio were 0.37–1.44. We observed good sensitivity (1.5 mg/L) and linearity in both polyclonal and monoclonal sFLC samples and never experienced antigen excess. Sebia FLC reproducibility varied between 6.7% and 8.1% with good lot-to-lot consistency. Method comparison with Freelite showed the following correlations: κFLC R=0.94, λFLC R=0.92 and κ/λFLC-ratio R=0.96. The clinical concordance of the κ/λFLC-ratio of both methods was 94%. Significant quantitative differences were observed between both methods, mainly in sera with high FLC concentrations. The Sebia monoclonal FLC concentrations were coherent with those obtained by serum protein electrophoresis (SPE). Freelite monoclonal FLC concentrations were consistently higher, with a mean 12-fold overestimation compared to SPE. Conclusions: The Sebia FLC assay provides a novel platform for sensitive and accurate sFLC measurements. The Sebia FLC showed good clinical concordance with Freelite. Further studies are warranted to confirm the clinical value of this assay.

N Latex FLC – new monoclonal high-performance assays for the determination of free light chain kappa and lambda

2011 ◽  
Vol 49 (8) ◽  
Author(s):  
Henk te Velthuis ◽  
Ingrid Knop ◽  
Peter Stam ◽  
Monic van den Broek ◽  
Hannie Klaasse Bos ◽  
...  
Keyword(s):  
Light Chain ◽  
Method Comparison ◽  
High Serum ◽  
Free Light Chain ◽  
Plasma Cell Dyscrasia ◽  
High Dose ◽  
Reference Ranges ◽  
Clinical Value ◽  
Serum Samples

AbstractHigh serum concentrations of monoclonal free light chain (FLC) kappa or lambda are markers of plasma cell dyscrasia.We developed new, latex-enhanced, specific nephelometric assays based on monoclonal antibodies for the determination of FLC kappa and lambda in serum, EDTA plasma and Li-heparin plasma for use on the Siemens BN™ systems.Reference ranges were determined from 369 samples: FLC kappa 6.7–22.4 mg/L, FLC lambda 8.3–27.0 mg/L and kappa/lambda ratio 0.31–1.56. Protection from falsely low results due to antigen excess is obtained with a built-in pre-reaction in the assay protocols. Lot-to-lot consistency between three different lots of reagent, calibrators and supplementary reagent lots showed normalized differences <7.5%. The reproducibility of serum samples varied between 4% and 7%. The method comparison with Freelite™ assays showed normalized differences of 19.7%, 32.7% and 21.7%, respectively, for FLC kappa, lambda and ratio, correlations of 0.94, 0.77 and 0.73, and concordance rates of 99.2%, 94.2% and 95%.N Latex FLC demonstrates high precision, good lot-to-lot consistency and freedom from a high-dose hook effect. The method comparison between Freelite™ and the N Latex FLC assays showed good clinical concordance. Further studies need to reveal the clinical value of the new FLC assays.

Method comparison of four clinically available assays for serum free light chain analysis

2019 ◽  
Vol 58 (1) ◽  
pp. 85-94
Author(s):  
Chérina K.A. Fleming ◽  
Tim Swarttouw ◽  
Corrie M. de Kat Angelino ◽  
Joannes F.M. Jacobs ◽  
Henk Russcher
Keyword(s):  
Light Chain ◽  
Method Comparison ◽  
Free Light Chain ◽  
M Protein ◽  
Clinical Use ◽  
Serum Free ◽  
Monoclonal Gammopathies ◽  
Serum Free Light Chain ◽  
Chain Analysis ◽  
Prognostic Stratification

Abstract Background Serum free light chain (sFLC) measurements are increasingly important in the context of screening for monoclonal gammopathies, prognostic stratification and monitoring of therapy responses. In this study we have performed a method comparison of four sFLC assays that are currently available for routine clinical use. Methods In a retrospective study, sFLC analyses were performed on a cohort that included 139 patients with various monoclonal gammopathies and 54 control sera without an M-protein. Method comparisons of the following four FLC assays were performed: Freelite (Binding Site), N-Latex FLC (Siemens), Seralite (Abingdon Health) and Sebia FLC (Sebia). Results Bland-Altman agreement analysis showed biases varying between −0.1 and 16.2 mg/L for κFLC, −6.0 and 6.8 mg/L for λFLC and −0.04 and 0.38 for the ratio of the involved to uninvolved FLC. Strong agreements were observed for FLC-concentrations below 100 mg/L. The clinical concordance of the κ/λFLC-ratio of the four methods varied between 86% and 92%. Significant quantitative differences were observed between the different methods, mainly in sera with high FLC concentrations. Most assays consistently overestimated FLC concentrations compared to SPE. Conclusions Good overall clinical concordances were observed between the four sFLC assays that were compared in this study. Although good agreements were observed between the FLC assays, significant absolute differences in FLC concentrations in individual patients can be seen, particularly at higher FLC concentrations. Because of inequivalent absolute sFLC values between the methods in individual patients, none of the four sFLC assays can be used interchangeably.

scholarly journals In-house age-specific reference ranges for free light chains measured on the SPAPlus® analyser

2020 ◽  
Vol 57 (2) ◽  
pp. 138-143
Author(s):  
Lauren Campbell ◽  
Dawn Simpson ◽  
Adrian Shields ◽  
Berne Ferry ◽  
Karthik Ramasamy ◽  
...  
Keyword(s):  
Glomerular Filtration Rate ◽  
Light Chain ◽  
Filtration Rate ◽  
Estimated Glomerular Filtration Rate ◽  
Free Light Chain ◽  
Light Chains ◽  
Reference Ranges ◽  
Free Light Chains ◽  
Serum Free ◽  
Serum Free Light Chain

Background The measurement of monoclonal free light chains is being increasingly utilized since the introduction of serum-based assays. It is important for laboratories to determine their own reference ranges in order to reflect the local population. The aim of this study was to determine if age-adjusted reference ranges for serum free light chains would have implications for demand management of further laboratory investigations including immunofixation. Methods After certain exclusions, 4293 samples from individuals seen in primary care across Oxfordshire between 2014 and 2016 were identified for analysis of patient characteristics, serum free light chain results and estimated glomerular filtration rate. Results We found age to be an independent variable when considering serum free light chain concentrations, ratio and estimated glomerular filtration rate. The reference ranges derived from our data differ markedly from the original Binding Site ranges. When the age-specific ranges are retrospectively applied to our population, there is a 38% decrease in follow-up testing with no loss of specificity. Conclusion We feel confident implementing new age-specific serum free light chain reference ranges in our laboratory. We have developed a simple algorithm for evaluating serum free light chains based on age and estimated glomerular filtration rate. We encourage laboratories to establish their own local reference ranges using large cohorts and their chosen serum free light chain assay platform.

Comparison of Free Light Chain Assay with Protein Electrophoresis for Screening and Monitoring PTLD

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5883-5883 ◽  
Author(s):  
Deborah Kuhn ◽  
Ping Wang ◽  
Irene Shu ◽  
Jie Xuan ◽  
Zheng Cao ◽  
...  
Keyword(s):  
Liver Transplant ◽  
B Cell ◽  
Light Chain ◽  
Protein Electrophoresis ◽  
Free Light Chain ◽  
Healthy Population ◽  
Solid Organ ◽  
Reference Ranges ◽  
Time Points ◽  
Post Transplant

Abstract Background: Post-transplant lymphoproliferative disorder (PTLD) is primarily diagnosed histologically using tissue biopsy. Free light chain (FLC) assay and serum protein electrophoresis (SPE) have both been studied as tools to screen and monitor PTLD. However, limited data are available to compare these two assays in a well characterized patient population. It is also not clear what reference ranges should be adopted for the FLC assay in a post-transplant population. Method: Blood samples from 169 patients receiving a variety of solid organ transplants were analyzed for FLCs and screened for gammopathies by SPE/IFE. Results: Compared with non-PTLD patients, PTLD patients had higher mean, median and upper 95 percentile range of both κ and λ FLCs (p ranging from 0.0002 to 0.024). The mean, median and 95 percentile range of κ:λ ratio were similar between the two groups. PTLD patients were more likely to have polyclonal or monoclonal FLC elevations (p = 0.04). They also showed a higher frequency of gammopathy abnormalities (p = 0.0052). Nonetheless, neither FLC assay nor SPE demonstrated a clear association with the timing of PTLD diagnosis. FLC concentrations in non-PTLD recipients were higher than those in the general healthy population (95 percentile range: κ, 0.60-8.33 mg/dL vs. 0.33-1.94 mg/dL; λ, 0.77-7.08 mg/dL vs. 0.571-2.63 mg/dL) but the κ:λ ratio was similar to that of the healthy group (0.26-1.65). Conclusions: Our results suggested that elevated FLC concentrations and gammopathy abnormalities were both associated with PTLD. Therefore, FLC assay and SPE should be used conjunctively for screening PTLD among solid organ transplant recipients. For this application, the data showed that a higher upper limit of κ and λ FLC levels and normal κ:λ ratio should be used as diagnostic reference ranges. Additionally, neither method was clearly associated with the timing of PTLD diagnosis, indicating that they may be unsuitable for monitoring PTLD in the post-transplant population. Table 1. Longitudinal measurements of serum/plasma free light chains and SPE/IFE in eight PTLD cases. Type of transplant and type of PTLD Samples Days from PTLD diagnosisa κ FLC, mg/dL λ FLC, mg/dL κ /λ SPE/IFE Liver transplant, B-cell PTLD 1.1 1.2 – 616 2.33b 1.82 4.96 6.01 0.47 0.303 no band –c Liver transplant, B-cell PTLD 2.1 2.2 2.3 2.4 2.5 -145* -126 84 141 428 0.338 0.79 0.335 0.476 2.53 0.62 1.02 1.06 1.15 1.98 0.545 0.775 0.316 0.414 1.28 no band no band 1 IgG ©µ, 1 IgG λ 1 IgG ©µ, 1 IgG λ no band Liver transplant, polymorphic hyperplasia 3.1 3.2 -77 208 4.19 7.18 5.9 3.38 0.71 2.12 2 IgG ©µ, 2 λ FLC– Liver transplant, B-cell PTLD 4.1 4.2 4.3 61 272 537 4.64 3.25 7.1 11.1 6.65 10.96 0.418 0.489 0.648 no band no band no band Liver transplant, B-cell PTLD 5.1 5.2 5.3 5.4 5.5 9 12 393 429 476 305.5 957 0.721 1.01 1.24 74.25 192 1.67 1.72 2.56 4.11 4.98 0.432 0.587 0.484 1 IgG ©µ, 1 λ FLC 2 IgM ©µ no band no band no band Liver transplant, B-cell PTLD 6.1 6.2 6.3 6.4 62 153 174 188 3.16 5.58 3.97 2.14 2.92 3.91 3.81 3.37 1.08 1.43 1.04 0.635 – 1 IgG ©µ 1 IgG ©µ 1 IgG ©µ Kidney transplant, PTLD 7.1 7.2 15 30 1.4 1.68 1.58 2.06 0.89 0.82 no band no band Lung transplant, Non-Hodgkin lymphoma 8.1 -60 4.28 4.58 0.94 no band a Positive values indicate time points before PTLD diagnosis, while negative values indicate time points after PTLD diagnosis. b Numbers in bold format indicates values above ULN. c SPE/IFE results not available due to insufficient sample volume. Disclosures Kuhn: The Binding Site, Inc: Employment.

Impact of Normalized Free Light Chain Ratio for Assessment of Monoclonal Protein in Patients with Light Chain Only and Oligosecretory Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2074-2074
Author(s):  
Kentaro Narita ◽  
Yoshiaki Usui ◽  
Yoshiaki Abe ◽  
Masami Takeuchi ◽  
Kosei Matsue
Keyword(s):  
Light Chain ◽  
Urine Analysis ◽  
Residual Disease ◽  
Response Assessment ◽  
Protein Electrophoresis ◽  
Free Light Chain ◽  
Reference Ranges ◽  
Immunofixation Electrophoresis ◽  
The Impact ◽  
Monoclonal Component

Abstract Background: Monitoring of serum free light chain (sFLC) ratio after treatment in multiple myeloma (MM) patients is valuable for assessing monoclonal component of free light chain (FLC). However, the recent International Myeloma Working Group guidelines did not recommend replacing 24-hour urine analysis with FLC analysis in diagnosis or response assessment of MM, and previous studies indicated discordance between urine analysis and sFLC levels in light chain-only MM (LCMM). This is clinically relevant because sFLC normalization was considered a surrogate for improved outcome in both LCMM and intact immunoglobulin MM (IIMM). The clinical impact of FLC ratio normalization on detection of monoclonal component may differ between LCMM or oligosecretory myeloma (OSMM) and IIMM. This study explored the utility of sFLC ratio as a surrogate for residual clonal monoclonal component compared with 24-hour urine immunofixation electrophoresis (uIFx) after treatment. We evaluated the impact of normalization of sFLC ratio in patients with LCMM/OSMM that obtained very good partial response (VGPR), complete response (CR), and immunophenotypic CR (iCR; sIFx/uIF negative plus ≤ 10-4 clonal PCs) determined by multicolor flow cytometry (MFC). Methods: We included 176 patients (51 with LCMM and OSMM, 125 with IIMM) treated between April 2006 and January 2016 at Kameda Medical Center, Japan. Immunoglobulin levels in serum and urine samples were examined by serum protein electrophoresis (SPEP), serum immunofixation electrophoresis (sIFx), urine protein electrophoresis (UPEP), uIFx, and sFLC for response assessment. Minimal residual disease (MRD) assessments after treatment were performed by 6-color MFC and the results were compared to other tests of monoclonal components, including SPEP, UPEP, sIFx, uIFx, and FLC. Agreement between sFLC normalization and MRD by MFC was assessed using kappa statistic. Disease response was evaluated using IMWG criteria. sFLC was measured by Fleelite® assay (The Binding Site Group Ltd.). Reference ranges for sFLC have been previously published. Statistical analyses were performed with EZR, which is a graphical user interface for R ver. 3.2.1. Ethical considerations: This study was approved by the local ethics committee and conducted in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines. Results: All of 51 LCMM/OSMM patients (100%) and 95 of the 125 IIMM patients (72%) had measurable and abnormal involved sFLC (≥ 100 mg/L) and positive uIFx at presentation. VGPR, CR, and iCR were obtained in 31 (61%), 25 (49%), and 14 (27%) patients with LCMM/OSMM, respectively, and normalization of sFLC ratio at VGPR, CR and iCR was seen in 1/31 (3%), 13/25 (48%), and 8/14 (57%) of these patients, respectively. Among the LCMM/OSMM patients with iCR, 4 patients obtained deeper iCR (≤ 10-5 clonal PCs) and all of them had normal sFLC ratio, while sFLC ratio remained abnormal in the rest of 10 iCR patients that did not achieve deeper iCR. In IIMM patients, VGPR, CR, and iCR were obtained in 78 (61%), 52 (42%), and 20 (16%) patients, respectively. In contrast to the LCMM/OSMM patients, normalization of the sFLC ratio at VGPR, CR, and iCR was seen in 52/78 (67%), 39/52 (75%), and 17/20 (85%) of IIMM patients, respectively. Thirteen of the 14 IIMM patients (93%) that obtained deeper iCR had normal sFLC ratio. Among the patients with IIMM, percentage of patients with normalized sFLC ratio did not differ between the response groups (p=0.11), while it was significantly different in LCMM/OSMM patients (p<0.001) (Figure 1). These observations indicated that the normalization of sFLC ratio is significantly associated with deeper response in LCMM/OSMM patients, but not in IIMM patients. Conclusions: Our observations indicated that sFLC test has greater sensitivity than urine immunofixation for detection of the monoclonal component of sFLC, especially in patients with LCMM/OSMM. In addition, we also showed that normalization of sFLC ratio is correlated with the depth of response assessed by MFC in patients with LCMM/OSMM, but not in IIMM patients. These findings suggest that FLC ratio provides greater sensitivity for residual disease monitoring than uPEP or uIFx in patients with LCMM and OSMM, and therefore could be considered as an alternative to urine analysis for monitoring of LCMM/OSMM patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Comparing the Performance of Serum Free Light Chain Measurements with Urine Electrophoresis and Immunofixation for Monitoring and Assessing Response to Therapy in Patients with Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3347-3347 ◽  
Author(s):  
Thomas Dejoie ◽  
Michel Attal ◽  
Philippe Moreau ◽  
Jean-Luc Harousseau ◽  
Herve Avet-Loiseau
Keyword(s):  
Light Chain ◽  
Conflicts Of Interest ◽  
Free Light Chain ◽  
Response To Therapy ◽  
Stage I ◽  
Reference Ranges ◽  
Serum Free ◽  
Post Transplant ◽  
Serum Free Light Chain ◽  
Measurable Disease

Abstract The introduction of the serum free light chain (sFLC) changed the diagnostic paradigm for patients with B cell disorders. IMWG guideline recommends the assay as a replacement for 24h urine at diagnosis, however with the exception of oligosecretory disease the assay is not recommended as a tool to monitor patients. One rationale for this recommendation is that to date, studies have compared the concentrations of FLC as measured by the two tests rather than determine which test provides the more reliable clinical assessment. Here we compare the sensitivities of FLC and 24h urine and comment on the reliability of each to monitor patients. Sequential sera from 25 LCMM (14 FLCκ, 11 FLCλ; stage I: 10, II: 10, III: 5) and 157 IIMM patients (79 IgGκ, 34 IgGλ, 26 IgAκ, 18 IgAλ; Stage I: 46, II: 75, III: 35, 1 missing) enrolled onto the IFM 2007-02 MM trial were analysed. Serum FLCκ and FLCλ levels were measured by Freelite® in samples collected at presentation, after cycles 2 and 4 of therapy and post ASCT. Results were compared to previously published sFLC reference ranges (sFLCκ 3.3-19.4 mg/L, sFLCλ 5.7-26.3 mg/L, sFLCκ/λ ratio 0.26-1.65), SPEP, UPEP sIFE and uIFE. IMWG guidelines were used to define measurable disease and to assess response the therapy. Quadratic Weighted Kappa (WK) analysis was performed to assess agreement in responses assigned by sFLC and urine tests. All 25 LCMM patients had abnormal sFLC ratios (14 FLCκ, 11 FLCλ) and measurable disease at presentation (iFLC 3620 (689-22000) mg/L). Similarly, all patients were positive by uIFE and had measurable disease by UPE (1940 (490-42000) mg/24h). However, in keeping with previous reports quantitative correlation between the two assays was poor (r=0.27). Responses assigned by sFLC and UPEP were concordant in 11/25 (44%) patients, although in 4/11 (40%) timing of the response was different (UPEP 89 (58-118) days; sFLC 226 (216-227) days). In the remaining 14/25 patients the responses assigned using the two tests differed. In 7/14 patients UPEP and uIFE became negative whilst the FLC ratio remained abnormal; in 1/7 patient sIFE confirmed the presence of the M protein. In 3 patients FLC identified relapse, while UPEP was negative or indicated response. In a further 2 patients FLC identified no response whilst UPEP initially identified a response and subsequent relapse. Overall, a moderate concordance was identified between the responses assigned by sFLC and urine tests (WK (95% CI): 0.59 (0.36-0.82)). At presentation, 154/157 (98%) IIMM patients had abnormal FLC ratios (FLCκ ratio 57 (2-33191); FLCλ ratio 0.009 (0.00003-0.25)), whereas only 85/157 (54%) patients were positive by uIFE and 67/157 (43%) by UPEP. 98/157 (62%) had measurable disease using sFLC (κFLC 491 (101-15600) mg/L; λFLC 441 (101-14100) mg/L), and 55/157 (35%) had measurable disease by UPEP (1000 (210-9200) mg/24h). 53/157 (34%) patients had measurable disease by both methods. The correlation between sFLC and UPEP measurements was poor (r=0.36) as was the correlation between intact immunoglobulin measurements by SPEP and sFLC (r=-0.06) or UPEP (r=-0.26). In 53 IIMM patients with measurable disease by both FLC and UPEP, sFLC ratios normalised in 14/53 patients (in 8/14 sIFE remained positive) while uIFE became negative in 33/53 patients (20/33 remained sIFE positive). WK showed better agreement for response assignment between intact immunoglobulin and sFLC measurements (WK (95% CI): 0.63 (0.48-0.79); substantial agreement) than with urine tests (0.49 (0.27-0.72); moderate agreement). Additionally, there was an association between depth of response by sFLC pre- and post-transplant: patients achieving >VGPR before transplant were more likely to achieve >VGPR post-transplant compared with patients who achieved <VGPR prior to transplant (96.2% vs. 63.2%, respectively; p=0.001). Finally 5/157 IIMM patients were oligosecretory and had measurable levels of disease by both UPEP and sFLC, but not by SPEP. In all 5 patients UPEP became negative by cycle 2; however, an abnormal sFLC ratio and positive sIFE indicated persistent disease. sFLC was a more sensitive tool and showed a greater degree of concordance with IFE and SPEP than UPEP in LCMM and IIMM patients respectively during patient monitoring. Furthermore, >90% reduction in sFLC prior to transplant was associated with post-transplant response in IIMM patients. Larger studies with patient outcome are required to validate our findings. Disclosures No relevant conflicts of interest to declare.

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