Abstract
Background
The automated immunochemical serum free light chains (FLC) assays, Freelite (a polyclonal antiserum) and N Latex FLC (a mixture of monoclonal antibodies), are not interchangeable, as they may provide different results on a same sample. This study was aimed to establish if the calibrators contain FLC oligomers, and if different reactivity against monomers and dimers contributes to the discrepancy.
Methods
Gel filtration chromatography fractions of the calibrators were subjected to a Western blot (WB) and analyzed by each reagent. The procedure was repeated after pretreating the N Latex FLC calibrator with the reducing agent dithiothreitol (DTT).
Results
Both calibrators contain FLC dimers and monomers. Both reagents detect (with different sensitivity) FLC kappa monomers and dimers; instead, Freelite detects only FLC lambda dimers, while N Latex FLC detects only FLC monomers. After DTT treatment, only the N Latex lambda still detects FLC with reduced protein thiols, while the reactivity of all other reagents is abolished.
Conclusions
Due to their different reactivity against FLC monomers and oligomers, the Freelite and N Latex FLC are calibrated against different components of their own calibrators, making the two reagents not equivalent. The redox status of FLC determines the immunoreactivity not only of FLC dimers, but also of the monomers.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: Institutional funding of University of Pisa.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
References
1. Rajkumar SV, Dimopoulos MA, Palumbo A, Blade J, Merlini G, Mateos MV, et al. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma. Lancet Oncol 2014;15:e538–48.10.1016/S1470-2045(14)70442-5Search in Google Scholar PubMed
2. Graziani MS, Merlini G. Serum free light chain analysis in the diagnosis and management of multiple myeloma and related conditions. Expert Rev Mol Diagn 2014;14:55–66.10.1586/14737159.2014.864557Search in Google Scholar PubMed
3. Jenner E. Serum free light chains in clinical laboratory diagnostics. Clin Chim Acta 2014;427:15–20.10.1016/j.cca.2013.08.018Search in Google Scholar PubMed
4. Mollee P, Tate J, Pretorius CJ. Evaluation of the N Latex free light chain assay in the diagnosis and monitoring of AL amyloidosis. Clin Chem Lab Med 2013;51:2303–10.10.1515/cclm-2013-0361Search in Google Scholar PubMed
5. Kim HS, Kim HS, Shin KS, Song W, Kim HJ, Kim HS, et al. Clinical comparisons of two free light chain assays to immunofixation electrophoresis for detecting monoclonal gammopathy. Biomed Res Int 2014;647238:1–7.10.1155/2014/647238Search in Google Scholar PubMed PubMed Central
6. Carr-Smith HD, Jenner EL, Evans JA, Harding SJ. Analytical issues of serum free light chain assays and the relative performance of polyclonal and monoclonal based reagents. Clin Chem Lab Med 2016;54:997–1003.10.1515/cclm-2015-1068Search in Google Scholar PubMed
7. Caponi L, Franzini M, Koni E, Masotti S, Petrini M, Paolicchi A. Discrepancy between FLC assays: only a problem of quantification? Clin Chem Lab Med 2016;54:1111–3.10.1515/cclm-2015-1262Search in Google Scholar PubMed
8. Bossuyt X, Delforge M, Reynders M, Dillaerts D, Sprangers B, Fostier K, et al. Diagnostic thresholds for free light chains in multiple myeloma depend on the assay used. Leukemia 2017. doi: 10.1038/leu.2017.335. [Epub ahead of print].10.1038/leu.2017.335Search in Google Scholar PubMed
9. Di Noto G, Cimpoies E, Dossi A, Paolini L, Radeghieri A, Caimi L, et al. Polyclonal versus monoclonal immunoglobulin-free light chains quantification. Ann Clin Biochem 2015;52:327–36.10.1177/0004563214553808Search in Google Scholar PubMed
10. Kaplan B, Ramirez-Alvarado M, Sikkink L, Golderman S, Dispenzieri A, Livneh A, et al. Free light chains in plasma of patients with light chain amyloidosis and non-amyloid light chain deposition disease. High proportion and heterogeneity of disulfide-linked monoclonal free light chains as pathogenic features of amyloid disease. Br J Haematol 2009;144:705–15.10.1111/j.1365-2141.2008.07522.xSearch in Google Scholar PubMed
11. Kaplan B, Golderman S, Aizenbud B, Esev K, Kukuy O, Leiba M, et al. Immunoglobulin-free light chain monomer-dimer patterns help to distinguish malignant from premalignant monoclonal gammopathies: a pilot study. Am J Hematol 2014;89:882–8.10.1002/ajh.23773Search in Google Scholar PubMed
12. te Velthuis H, Knop I, Stam P, van den Broek M, Bos HK, Hol S, et al. N Latex FLC – new monoclonal high-performance assays for the determination of free light chain kappa and lambda. Clin Chem Lab Med 2011;49:1323–32.10.1515/CCLM.2011.624Search in Google Scholar PubMed
13. Baden EM, Owen BA, Peterson FC, Volkman BF, Ramirez-Alvarado M, Thompson JR. Altered dimer interface decreases stability in an amyloidogenic protein. J Biol Chem 2008;283:15853–60.10.1074/jbc.M705347200Search in Google Scholar PubMed PubMed Central
14. Palladini G, Jaccard A, Milani P, Lavergne D, Foli A, Bender S, et al. Circulating free light chain measurement in the diagnosis, prognostic assessment and evaluation of response of AL amyloidosis: comparison of Freelite and N latex FLC assays. Clin Chem Lab Med 2017;55:1734–43.10.1515/cclm-2016-1024Search in Google Scholar PubMed
15. Pretorius CJ, Klingberg S, Tate J, Wilgen U, Ungerer JP. Evaluation of the N Latex FLC free light chain assay on the Siemens BN analyser: precision, agreement, linearity and variation between reagent lots. Ann Clin Biochem 2012;49:450–5.10.1258/acb.2012.011264Search in Google Scholar PubMed
16. Hoedemakers RM, Pruijt JF, Hol S, Teunissen E, Martens H, Stam P, et al. Clinical comparison of new monoclonal antibody-based nephelometric assays for free light chain kappa and lambda to polyclonal antibody-based assays and immunofixation electrophoresis. Clin Chem Lab Med 2011;50:489–95.10.1515/cclm.2011.793Search in Google Scholar PubMed
17. Lock RJ, Saleem R, Roberts EG, Wallage MJ, Pesce TJ, Rowbottom A, et al. A multicentre study comparing two methods for serum free light chain analysis. Ann Clin Biochem 2013;50:255–61.10.1177/0004563212473447Search in Google Scholar PubMed
18. Messiaen AS, De Sloovere MM, Claus PE, Vercammen M, Van Hoovels L, Heylen O, et al. Performance evaluation of serum free light chain analysis: nephelometry vs turbidimetry, monoclonal vs polyclonal reagents. Am J Clin Pathol 2017;147:611–22.10.1093/ajcp/aqx037Search in Google Scholar PubMed
19. Palumbo A, Rajkumar SV, San Miguel JF, Larocca A, Niesvizky R, Morgan G, et al. International Myeloma Working Group consensus statement for the management, treatment, and supportive care of patients with myeloma not eligible for standard autologous stem-cell transplantation. J Clin Oncol 2014;32:587–600.10.1200/JCO.2013.48.7934Search in Google Scholar PubMed PubMed Central
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