Measurement of free light chains in Urine.
We read with interest the report by Bradwell et al. (1) describing a sensitive immunoassay for free light chains (FLCs) in serum and urine. We agree that sensitive and accurate immunoassays for FLCs can be useful in clinical laboratories for studying the renal handling of polyclonal FLCs in various conditions. We do not agree, however, that this type of assay can be used to detect Bence Jones protein (BJP) in serum or urine.
The identity of BJP was established in 1962 as FLCs synthesized by a single clone of B cells (2). The product of a single clone of B cells is a unique protein with its own structural peculiarity of the variable region of the molecule. For a protein immunoassay to be accurate, the immunoreactivity of the antigen in the sample must be identical to the immunoreactivity of the antigen in the calibrator. In other words, a criterion for a valid immunoassay is that the dose response of the calibrator should parallel that of the serum or urine specimen. This is impossible to achieve in the measurement of a monoclonal protein that exhibits structural differences from the polyclonal light chains in the calibrator.
The imbalance of antigenic determinants between polyclonal and monoclonal light chains makes methods susceptible to the risk of antigen excess. Problems of antigen excess occur, with even robust immunoassays for total light chains yielding falsely low light-chain concentrations and thus missing the diagnosis of potentially serious conditions such as light chain myeloma or light chain amyloidosis. The authors (1) report the lack of antigen excess up to 40 g/L for is and 60 g/L for [gamma] FLCs, but because they used polyclonal light chains as the reference material, this does not necessarily apply to a single monoclonal protein.
The molecular mass of BJP is quite variable; in urine it can appear as monomers (22 kDa), dimers (44 kDa), or low-molecular mass fragments and can show a high degree of polymerization (3); the state of aggregation/fragmentation of FLCs in urine is highly variable and unpredictable, depending on many factors (e.g., light chain concentration and pH). This can influence the immune reaction and may invalidate the calibration, making the quantification of urinary monoclonal light chains unreliable.
Bradwell et al. (1) acknowledge the limitations of applying a quantitative method to determine the quality (clonality) of a protein. They list several limitations (e.g., normal is/. ratios in early disease) and conclude that "In all of these situations, clonality must be confirmed by electrophoresis". Unfortunately, in most cases, these situations cannot be predicted.
As a minor point, we would like to comment on the sensitivity of immunofixation for BJP. Bradwell et al. (1) report values >100 mg/L, but detection limits of ~10 mg/L for both light chains have been published (4, 5).
The drawbacks of immunoassays in evaluating monoclonal proteins in serum or urine are so essential that none of the currently available guidelines on laboratory evaluation of monoclonal gammopathies (6-8) recommends such an analytical approach to detect or quantify the monoclonal protein.
(1.) Bradwell AR, Carr-Smith HD, Graham PM, Tang LX, Showell PJ, Drayson MT, Drew R. Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine. Clin Chem 2001;47:673-80.
(2.) Edelman GM, Gaily JA. The nature of Bence Jones protein: chemical similarities to polypeptide chains of myeloma globulins and normal gamma globulins. J Exp Med 1962;116:202-27.
(3.) Solomon A. Bence Jones proteins and light chains of immunoglobulins. N Engl J Med 1976; 294:17-23.
(4.) Martin SM, Kohn J. The sensitivity of gold staining in the detection of Bence Jones proteinuria. Ann Clin Biochem 1987;24(Suppl 1):120-1.
(5.) Lott JA, Stephan VA, Pritchard KA. Evaluation of the Coomassie Brilliant Blue G-250 method for urinary protein. Clin Chem 1983;29:1946-50.
(6.) Merlini GP, Aguzzi F, Whicher J. Monoclonal gammopathies. J Int Fed Clin Chem 1997;9: 171-6.
(7.) Keren DF, Alexanian R, Goeken JA, Gorevic PD, Kyle RA, Tomar RH. Guidelines for clinical and laboratory evaluation on patients with monoclonal gammopathies. Arch Pathol Lab Med 1999; 123:106-7.
(8.) Graziani MS, Merlini GP, Petrini C. Linee guida per la ricerca della proteina di Bence Jones. Gruppo di Studio Proteine--Societa Italiana di Biochimica Clinica. Biochim Clin 2001;25:23-32.
Giampaolo Merlini 
 Laboratorio di Analisi Chimico
Cliniche ed Ematologiche
Ospedale Civile Maggiore
Piazza Stefani 1
37126 Verona, Italy
 Laboratori di Ricerca e Biotecnologie
IRCCS Policlinico San Matteo
Dipartimento di Biochimica
Universita di Pavia
* Author for correspondence. Fax 39045-8072155 and 39-045-8072157; e-mail firstname.lastname@example.org. Drs. Bradwell, Drayson, and Mead respond:
To the Editor:
We cannot concur with many of the points raised in the letter by Graziani and Merlini. We have rigorously validated the assay, as shown in our article (1), and subsequently evaluated it in several clinical situations (2-6). Although perfect quantification may be elusive, the assay has considerable clinical value for detecting monoclonal free light chain diseases. Our disagreements with their letter are as follows:
They state that our immunoassays cannot detect Bence Jones proteins in serum or urine. This is clearly incorrect. Bence Jones proteins contain epitopes on their common region that are present on all immunoglobulin light chains and are the antigen targets in all light chain immunoassays whether they measure total or free light chains. Of more relevance is whether individual clones of light chain molecules can be accurately quantified because abnormal molecular shapes or the copresence of polyclonal free light chains will distort the results.
Despite structural variations, light chains rarely have their constant regions completely distorted, as is apparent by the satisfactory use of antisera for detecting free light chains in immunofixation techniques. Inaccurate quantification will also occur if the light chains are polymerized. This is a frequent occurrence, as we found (2), and is well documented (7, 8), but those polymers are detected by the immunoassay (1).
As already indicated, another factor leading to inaccurate measurements is the presence of polyclonal light chains. Their occurrence will inevitably lead to an overestimation of Bence Jones proteins. However, the concentrations typically are quite low in patients with Bence Jones myeloma, particularly when normal bone marrow function is suppressed. Indeed, in many patients the concentrations of the nonclonal light chains are below normal, thereby enhancing the clinical utility of the is [kappa]/[lambda]. ratio (2).
Another source of inaccuracy, as the authors point out, may be antigen excess. We carefully considered this during assay development and found that the assay is usually stable at high antigen concentrations. This is because the assay uses latex to enhance sensitivity, and this produces greater stability in the immune complexes. In contrast, assays for total light chains are not latex enhanced and are susceptible, as the authors point out, to antigen-excess errors. We used polyclonal free light chains to initially test for antigen excess and showed that the assay was robust. Since then we have assessed parallelism of the calibration curves against 10 different Bence Jones proteins and have not identified significant differences either in antigen detection limits or parallelism. In addition, we measured free light chain concentrations in the sera of 102 patients (Fig. 1) (3), and have since extended the measurements to 224 patients presenting with Bence Jones myeloma. All patients had increased concentrations of free light chains and abnormal is/. ratios. We observed a wide range of free light chain concentrations, and some samples exhibited antigen excess, requiring high dilutions to achieve the correct results, but all were identified as abnormal. In the absence of a reference method for serum free light chains, the absolute accuracy of the results will remain uncertain, but the method clearly has clinical accuracy and utility (2-6).
[FIGURE 1 OMITTED]
The clinical application of serum free light chain measurements was particularly apparent for detecting and monitoring patients with nonsecretory myeloma and primary amyloidosis. The results obtained by the free light chain assay were abnormal for the sera of 19 of 28 patients with nonsecretory myeloma (2), which by definition were normal by routine electrophoretic and immunofixation methods. In light chain amyloidosis, the test was more sensitive at the time of diagnosis and clinically more useful during patient follow-up than existing electrophoretic techniques (5).
To us, it appears that serum free light chain assays will have wide application. Serum protein electrophoresis is the usual screening test for whole immunoglobulin monoclonal gammopathies and should remain so. Testing for Bence Jones myeloma and other light chain diseases is by serum immunofixation or urine tests, none of which is ideal. An alternative strategy is to assess these diseases with serum (or urine) free light chain immunoassays that are sensitive, automated, and quantitative (8). Immunofixation might be reserved for typing the heavy chains of the monoclonal proteins or as a confirmatory procedure.
(1.) Bradwell AR, Carr-Smith HD, Mead GP, Tang LX, Showell PJ, Drayson MT, Drew R. Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine. Clin Chem 2001;47:673-80.
(2.) Drayson MD, Tang LX, Drew R, Mead GP, Carr-Smith HD, Bradwell AR. Serum free light-chain measurements for identifying and monitoring patients with nonsecretory multiple myeloma. Blood 2001;97:2900-2.
(3.) Carr-Smith HD, Smith L, Showell P, Mead GP, Drayson MT, Bradwell AR. Development of serum free light chain immunoassays for the detection and monitoring of patients with Bence Jones myeloma [Abstract]. Clin Chem 2001;47: A33.
(4.) Abraham RS, Katzmann JA, Clark RJ, Lymp JF, Dispenzieri A, Lust JA, Bradwell AR. Light chain myeloma: correlation of serum nephelometric analysis for the quantitation of immunoglobulin free light chain with urine Bence Jones protein [Abstract]. Clin Chem 2001;47:A33.
(5.) Abraham RS, Katzmann JA, Clark RJ, Dispenzieri A, Lust JA, Bradwell AR. Detection of serum immunoglobulin free light chains in primary amyloidosis and light chain deposition disease by nephelometry [Abstract]. Clin Chem 2001;47:A32.
(6.) Clark RJ, Katzmann JA, Abraham RS, Lymp JF, Kyle RA, Bradwell AR. Detection of monoclonal free light chains by nephelometry: normal ranges and relative sensitivity [Abstract]. Clin Chem 2001;47:A27.
(7.) Soiling K, Soiling J, Lanng Nielsen J. Polymeric Bence Jones proteins in myeloma patients with renal insufficiency Acta Med Scand 1984;216: 495-502.
(8.) Diemert MC, Musset L, Gaillard O, Escolano S, Baumelou A, Rousselet F, Galli J. Electrophoretic study of the physico-chemical characteristics of Bence-Jones proteinuria and its association with kidney damage. J Clin Pathol 1994;47:1090-7.
Mark T. Drayson 
Graham P. Mead 
 Department of Immunology
Division of Infection and Immunity
University of Birmingham
Birmingham B15 2TT,
 The Binding Site Ltd.
PO Box 4073
Birmingham B29 6AT,
* Author for correspondence. Fax 44121-415-5163; e-mail a.r.Bradwell@bham.ac.uk.
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|Author:||Graziani, Maria Stella; Merlini, Giampaolo|
|Article Type:||Letter to the editor|
|Date:||Nov 1, 2001|
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