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Hematology |
and 
Free Light Chain Assays in Clinical Practice
Division of Clinical Biochemistry and Immunology, Department of Laboratory Medicine and Pathology, and Division of Hematology, Department of Internal Medicine, Mayo Clinic College of Medicine, Rochester, MN.
aAddress correspondence to this author at: Department of Laboratory Medicine and Pathology, Hilton 210, Mayo Clinic, Rochester, MN 55905. Fax 507-266-4088; e-mail katzmann{at}mayo.edu.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: All FLC clinical test results generated in 2003 were abstracted from the Laboratory Information System. Diagnoses were obtained from the Dysproteinemia database and the patient medical history.
Results: In 2003, we received samples for FLC assays from 1020 Mayo Clinic patients. The majority of these patients (88%) had bone marrow-derived monoclonal plasma cell disorders (PCDs). The 121 patients who did not have monoclonal gammopathy all had FLC 
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ratios within the range of values obtained for a reference population in our laboratory. Among the patients with monoclonal gammopathies were patients with multiple myeloma (330), AL (269), monoclonal gammopathy of undetermined significance (114), smoldering multiple myeloma (72), plasmacytoma (22), NSMM (20), macroglobulinemia (9), LCDD (7), and a variety of other PCDs. Among the 110 AL patients who had not been previously treated and who had a FLC assay performed within 120 days of diagnosis, the FLC / ratio was positive in 91% compared with 69% for serum immunofixation electrophoresis (IFE) and 83% for urine IFE. The combination of serum IFE and serum FLC assay detected an abnormal result in 99% (109 of 110) of patients with AL.
Conclusion: The performance of the FLC assay in this analysis of clinical laboratory data is consistent with results from published retrospective validation studies.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and light chains that circulate as light chain monomers or dimers and are not bound to immunoglobulin heavy chain. Quantification of the and FLCs and calculation of the FLC / ratio have been reported to be sensitive and specific for detection of excess monoclonal FLCs. We have recommended a diagnostic range for the FLC / ratio that included 100% of a 282-sample reference population to maximize the diagnostic specificity and minimize false-positive results (1). Retrospective studies using stored serum from populations of patients with nonsecretory multiple myeloma (NSMM) (2), primary systemic amyloidosis (AL) (3)(4), light chain deposition disease (LCDD) (1), and light chain multiple myeloma (LCMM) (5) have documented the sensitivity of these assays and established their use as a complement to immunofixation electrophoresis (IFE). In addition to its diagnostic use in the FLC diseases, the assay is used for monitoring disease course in AL, LCDD, NSMM, and LCMM, in which there may be a band detected on IFE that cannot be quantified by protein electrophoresis. Our clinical laboratory implemented FLC testing in late 2002. In 2003, we performed FLC assays on 1020 samples from Mayo Clinic patients. These patients were seen predominantly by clinicians in the Division of Hematology. To assess the performance of the FLC assay in our routine clinical laboratory practice, we reviewed the diagnoses and FLC results for these 1020 patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and FLCs from January 1, 2003, to December 31, 2003. The list of 1020 patients was merged with data from the Dysproteinemia database, which contained each patient’s diagnosis, date of diagnosis, and serum and urine IFE results. Individual patient histories were reviewed for any patients not contained in the database. If a patient had more than one sample tested, only the initial 2003 sample was included in this study. The samples were obtained at the patient’s initial presentation, during disease monitoring, or depending on the diagnosis, post treatment. The treatment status of the AL and NSMM patients was determined from the patient history. All queries to the Laboratory Information System, Dysproteinemia database, or patient histories followed a protocol approved by the Mayo Institutional Review Board.
The serum FLC assay was performed on the same day as the venipuncture and was reported to the patient’s medical record. The FLC assay (FREELITETM; The Binding Site Ltd.) (6) was performed on a Dade Behring BNII automated nephelometer. This assay consists of two separate measurements: one to quantify FLCs and the other to quantify FLCs. In addition to reporting the and FLCs, the assay report also contains the FLC / ratio. Patients with ratios >1.65 have excess FLCs and are presumed to be producing clonal FLCs. Patients with ratios <0.26 have excess FLCs and are presumed to be producing clonal FLCs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Among the PCD patients, the most common diagnoses were multiple myeloma (37%), AL (30%), monoclonal gammopathy of undetermined significance (MGUS; 13%), and smoldering multiple myeloma (SMM; 8%). There were 121 patients with FLC results who did not have a monoclonal gammopathy. These patients included 52 non-AL amyloidosis patients who were diagnosed with localized amyloid (n = 23), hereditary amyloid (n = 16), senile amyloid (n = 6), secondary amyloid (n = 3), or amyloid of unknown type (n = 4). The 69 remaining patients with nonmonoclonal gammopathy had peripheral neuropathy, anemia, proteinuria, lymphoproliferative disease, possible AL, and several other miscellaneous conditions.
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In all 121 patients with nonmonoclonal gammopathy, the FLC / ratio was within the values obtained for our reference population (Table 2
). Among the PCD patient groups, the FLC results were comparable to published retrospective data for patients with NSMM, AL, LCDD, and MGUS. The 5 untreated NSMM patients all had an abnormal FLC / ratio. The 6 NSMM patients with normal FLC / ratios had all received a stem cell transplant, and 5 of the 6 had achieved a complete bone marrow response. Among the 269 AL patients, 110 had not yet received treatment, and the FLC assay had been performed within 120 days of diagnosis. The FLC / ratio was abnormal in 100 (91%) of these patients. Among the LCDD patients, all 7 patients had abnormal FLC / ratios. Among the 114 patients with MGUS, 44% had an abnormal FLC ratio, a percentage almost identical to that in a retrospective cohort study of MGUS patients that was balanced regarding whether the patients had progressed to malignant disease (7). Among the 72 untreated SMM patients, 88% had abnormal FLC ratios. The performance of the FLC assay in patients with SMM has not been reported previously.
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The assay results of the serum FLC / ratio, serum IFE, and urine IFE for the 110 untreated AL patients are shown in Table 3
. As we have seen in retrospective studies, the FLC assay is more sensitive (91%) than the serum or urine IFE assay (69% and 83%, respectively). In addition, the 3 assays are complementary for the detection of monoclonal FLCs in AL patients. If serum and urine IFE assays are evaluated, 95% of AL patient had an abnormal result in at least 1 of the 2 assays. If serum IFE and FLC assays are evaluated, 109 of the 110 AL patients (99%) had an abnormal result in at least 1 of the 2 tests, and use of the urine IFE did not add any information.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ ratio as the interval that included all of the reference population to ensure high specificity (1). The absence of abnormal results in the 121 patients with no monoclonal gammopathy in this study, however, was unexpected. We assume that 2 factors may have contributed to the absence of false-positive results. The first is that most of the requests were from the Division of Hematology and not from general medical practice. As the assay becomes more widely requested, we expect to see false-positive FLC results. The second, and perhaps more important, factor is that in this study some of the clinical diagnoses may have been influenced by the FLC results. (It should be remembered, however, that in AL the diagnosis requires histopathologically confirmed AL amyloid.) With these 2 caveats in mind, we are reassured regarding the diagnostic use of the FLC assay as a tool that is complementary to other laboratory tests. The current gold standard for detection of a monoclonal FLC is IFE. IFE assays are qualitative, and although their sensitivities may vary among laboratories and among antiserum lots, nonlaboratorians tend to think of them as black or white with no ambiguity in results. The use of a quantitative assay with defined normal cutoffs relies on low assay variability and long-term reagent stability that will yield consistent results as new reagents are prepared. The FLC assay is currently produced by a single manufacturer, and there is no defined international standard. Although we are reassured by the performance of the FLC assay during this 1-year study period, a verifiable standard needs to be developed.
In addition to the absence of false-positive results, this study has also validated use of the FLC / ratio as a diagnostic tool in the light chain diseases. The identification of abnormal results in various disease groups closely matches the published retrospective data. The diagnostic results in AL are similar to those in 2 published studies (3)(4). In this study and in our previous retrospective study (4), the sensitivity of the FLC assay in AL was lower than the sensitivity of 98% reported by Lachmann et al. (3). That study used a 95% reference interval for the FLC / ratio (0.3–1.2) vs our use of a range that encompassed 100% of the reference population (0.26–1.65). None of the 110 AL patients, however, had borderline FLC / ratios that would have been categorized differently by the 2 criteria. Any differences in the diagnostic sensitivity therefore cannot be explained by the different criteria used to define normal FLC / ratios and may reflect differences in the patient populations. The slightly higher sensitivity in the group of 5 untreated NSMM patients compared with the sensitivity reported by Drayson et al. (2) is most likely an artifact attributable to the small number of patients in this study. In addition, the FLC and IFE data validate our retrospective studies that indicated that the FLC assays are more sensitive than individual serum or urine IFE assays for detection of FLCs in AL and LCDD (1).
Multiple myeloma patients were the largest group of patients in which FLC assays were ordered. We are not aware of any data that suggest that the FLC assay is useful for the diagnosis or monitoring of most myeloma patients. Some observations suggest that measurement of serum FLC may be better than urine protein electrophoresis for monitoring monoclonal light chains in LCMM patients, but no published studies have validated this approach. We assume that during the first year the FLC assay was introduced into routine practice, our hematologists were ordering the assay in myeloma patients simply to get a better understanding of the assay performance.
The third and fourth largest patients groups were MGUS and SMM. We have recently published evidence that results of the FLC assay provide a prognostic indicator for progression of MGUS to malignant disease (7). Those data, however, were not available until late in 2003. The use of FLC assays as part of risk stratification of patients with MGUS may become part of the clinical management of these patients. In addition, the collection of FLC data on the 72 patients with SMM will allow us to evaluate the use of FLC assays as a prognostic marker for progression in SMM.
We conclude that the FLC assay is a reliable clinical laboratory test and is performing as predicted by the retrospective validation studies. In current practice, it is reasonable to perform serum and urine protein electrophoresis and IFE in patients suspected of having multiple myeloma or AL and in patients with unexplained renal disease, cardiac failure, bone fractures, osteolytic lesions, or immune deficiency. If these assays are negative and clinical suspicion remains high, the FLC assay may be performed as an additional diagnostic test. In addition to these diagnostic uses, the FLC assay provides prognostic information in patients with newly diagnosed MGUS and can be performed at diagnosis to identify patients at low risk for transformation to malignant disease. Lastly, the FLC assay is useful for monitoring disease activity in patients with NSMM, LCMM, AL, and LCDD in whom there is no measurable M-spike on serum or urine protein electrophoresis.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and free immunoglobulin light chains: relative sensitivity for detection of monoclonal light chains. Clin Chem 2002;48:1437-1444.The following articles in journals at HighWire Press have cited this article:
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  R. B Fulton and S. L Fernando Serum free light chain assay reduces the need for serum and urine immunofixation electrophoresis in the evaluation of monoclonal gammopathy Ann Clin Biochem, September 1, 2009; 46(5): 407 - 412. [Abstract] [Full Text] [PDF]
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 J. A. Katzmann, R. A. Kyle, J. Benson, D. R. Larson, M. R. Snyder, J. A. Lust, S. V. Rajkumar, and A. Dispenzieri Screening Panels for Detection of Monoclonal Gammopathies Clin. Chem., August 1, 2009; 55(8): 1517 - 1522. [Abstract] [Full Text] [PDF]
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 S. Singhal, E. Vickrey, J. Krishnamurthy, V. Singh, S. Allen, and J. Mehta The relationship between the serum free light chain assay and serum immunofixation electrophoresis, and the definition of concordant and discordant free light chain ratios Blood, July 2, 2009; 114(1): 38 - 39. [Abstract] [Full Text] [PDF]
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G. Palladini, P. Russo, T. Bosoni, L. Verga, G. Sarais, F. Lavatelli, M. Nuvolone, L. Obici, S. Casarini, S. Donadei, et al. Identification of Amyloidogenic Light Chains Requires the Combination of Serum-Free Light Chain Assay with Immunofixation of Serum and Urine Clin. Chem., March 1, 2009; 55(3): 499 - 504. [Abstract] [Full Text] [PDF]
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A. P. Piehler, N. Gulbrandsen, P. Kierulf, and P. Urdal Quantitation of Serum Free Light Chains in Combination with Protein Electrophoresis and Clinical Information for Diagnosing Multiple Myeloma in a General Hospital Population Clin. Chem., November 1, 2008; 54(11): 1823 - 1830. [Abstract] [Full Text] [PDF]
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A. Dispenzieri, L. Zhang, J. A. Katzmann, M. Snyder, E. Blood, R. DeGoey, K. Henderson, R. A. Kyle, M. M. Oken, A. R. Bradwell, et al. Appraisal of immunoglobulin free light chain as a marker of response Blood, May 15, 2008; 111(10): 4908 - 4915. [Abstract] [Full Text] [PDF]
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M. A. Gertz, M. Q. Lacy, A. Dispenzieri, S. R. Hayman, S. K. Kumar, N. Leung, and D. A. Gastineau Effect of hematologic response on outcome of patients undergoing transplantation for primary amyloidosis: importance of achieving a complete response Haematologica, October 1, 2007; 92(10): 1415 - 1418. [Abstract] [Full Text] [PDF]
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S. Singhal, R. Stein, E. Vickrey, and J. Mehta The serum-free light chain assay cannot replace 24-hour urine protein estimation in patients with plasma cell dyscrasias Blood, April 15, 2007; 109(8): 3611 - 3612. [Full Text] [PDF]
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E. S.K. Ma and E. T.K. Lee A Case of IgM Paraproteinemia in Which Serum Free Light Chain Values Were Within Reference Intervals Clin. Chem., February 1, 2007; 53(2): 362 - 363. [Full Text] [PDF]
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 J. A. Katzmann, A. Dispenzieri, R. A. Kyle, M. R. Snyder, M. F. Plevak, D. R. Larson, R. S. Abraham, J. A. Lust, L. J. Melton III, and S. V. Rajkumar Elimination of the Need for Urine Studies in the Screening Algorithm for Monoclonal Gammopathies by Using Serum Immunofixation and Free Light Chain Assays Mayo Clin. Proc., December 1, 2006; 81(12): 1575 - 1578. [Abstract] [Full Text] [PDF]
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V. Sanchorawala Light-Chain (AL) Amyloidosis: Diagnosis and Treatment Clin. J. Am. Soc. Nephrol., November 1, 2006; 1(6): 1331 - 1341. [Abstract] [Full Text] [PDF]
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