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Technical Briefs |
1 Laboratory Medicine, Immunology, University Hospitals Leuven, Herestraat 49, B-3000 Leuven, Belgium;
2
Department of Immunology, University of Birmingham, The Medical School, Birmingham B15 2TJ, United Kingdom
aaddress correspondence to this author at: Laboratoriumgeneeskunde, Immunologie, Universitaire Ziekenhuizen Leuven, Katholieke Universiteit Leuven, Herestraat 49, 3000 Leuven, Belgium; fax 32-16-347-931, e-mail Xavier.bossuyt{at}uz.kuleuven.ac.be
Capillary zone electrophoresis (CZE) is increasingly used in clinical laboratories for serum protein separation. The technique is a sensitive method for the detection of monoclonal (M) proteins. However, when compared with immunofixation, CZE fails to detect M proteins in 5% of samples (1)(2). These so-called "false-negative" results encompass low-concentration and "hidden" M proteins (e.g., in the transferrin peak).
The goal of this study was to evaluate whether an assay for the detection of free light chains (FLCs) that has recently become available (Freelite®; The Binding Site) can detect the M proteins present in samples that produce false-negative results in CZE. The Freelite assay is a sensitive automated immunoassay for 
and 
FLCs in serum (3) and has been useful for the diagnosis and monitoring of patients with nonsecretory myeloma (4). Moreover, it has been reported that the quantification of FLCs in serum by nephelometry correlates with changes in urinary FLC excretion (5).
Frozen sera from 54 patients that had previously been shown to contain M proteins by immunofixation (Sebia), but not by CZE (Paragon 2000TM CZE, software Ver. 1.5; Beckman-Coulter), were assayed by nephelometry (Immage; Beckman-Coulter) for FLCs (Freelite). All assays were performed according to the manufacturers’ instructions. The M proteins were of various types and included examples of both intact immunoglobulins and FLCs. The samples were from patients with (a) (chronic) immune stimulation [n = 7; Sjögren syndrome, polyarteritis, pneumonia, Staphylococcus aureus sepsis, chronic hepatitis B infection, post-renal transplantation (n = 2)], (b) monoclonal gammopathy of unknown significance (MGUS; n = 8), (c) primary (AL) amyloidosis (n = 1), and (d) B-cell-derived malignant disease (n = 34). The last group included patients with multiple myeloma (n = 20), smoldering myeloma (n = 1), plasma cell leukemia (n = 1), plasmacytoma (n = 3), and lymphoproliferative disease, including Waldenström macroglobulinemia (n = 2), non-Hodgkin lymphoma (n = 6), and chronic lymphocytic leukemia (n = 1). Eleven of the 34 patients with B-cell-derived malignant disease had received a transplant. For four patients, medical records could not be consulted.
Sera from 20 controls were analyzed as well. The free /free ratios for these samples were within the reference interval as specified by the manufacturer (0.359–1.01).
The results of the free and free quantification are shown in Fig. 1
. Sera from patients with free light chain M proteins (n = 9) all showed an increased free / ratio (between 9.5 and 793) and increased absolute values for the relevant free chain. Sera from patients with free light chain M proteins (n = 12) all showed a low free / ratio (between 0.001 and 0.036) and increased concentrations of the relevant free chain. In 16 of these samples in which monoclonal FLCs were present, quantification of total and was performed by nephelometry using Beckman-Coulter reagents on the Immage instrument. Quantification of the FLCs revealed an abnormal free /free ratio in all samples, whereas quantification of the total light chains revealed an abnormal / ratio in only 5 of the 16 samples (data not shown).
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Of 12 sera from patients with an intact immunoglobulin M protein of type (1 IgG, 6 IgA, and 5 IgM), only 4 had an increased free / ratio (between 1.5 and 5.2). Nine of 21 sera from patients with an intact immunoglobulin M protein of type (6 IgG, 8 IgA, 6 IgM, and 1 IgD) had decreased free / ratios (between 0.037 and 0.2).
The distribution of diagnoses with an indication of the number of abnormal FLC ratios is shown in Table 1
. Of seven patients with chronic stimulation of the immune system, the free /free ratio was abnormal in four (57%). In one of these four cases, the ratio was only modestly increased (1.09). In patients with MGUS and B-cell-derived malignant disease, the free / ratio was abnormal in 75% and 71%, respectively. Concentrations of both free and free were increased in five of the seven patients (71%) with chronic immune stimulation. By contrast, concentrations of both free and free were increased in only 7 of the 43 patients (16%) with MGUS, B-cell-derived malignancy, or primary amyloidosis.
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In conclusion, FLC nephelometric measurement in serum seems to be a convenient assay for detection of FLC M proteins in serum. This technique is more reliable than CZE and nephelometric measurement of total and . For intact immunoglobulin M proteins, the free / ratio was within reference values in 60% of the cases in which CZE failed to detect an M protein. Future studies should address the question whether the presence of increased FLCs together with a low concentration intact immunoglobulin M protein has a prognostic clinical value.
References
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