Serum Free Light Chain (FLC) Measurement Can Aid Capillary Zone Electrophoresis in Detecting Subtle FLC-Producing M Proteins

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1 Immunopathology / DETECTING SUBTLE FLC-PRODUCING M PROTEINS Serum Free Light Chain (FLC) Measurement Can Aid Capillary Zone Electrophoresis in Detecting Subtle FLC-Producing M Proteins Nasir A, Bakshi, MD, 1 Ronald Gulbranson, MT(ASCP), 2 Daniel Garstka, MT(ASCP), 3 Arthur R. Bradwell, MD, 4 and David F. Keren, MD 2 Key Words: Serum free light chain; Monoclonal gammopathy; M protein; Multiple myeloma; Chronic lymphocytic leukemia; Lymphocytosis; Serum protein electrophoresis; Capillary zone electrophoresis; Immunofixation DOI: /XE3UDARKW1B9EMWM Abstract We hypothesized that using a free light chain (FLC) assay as an adjunct to capillary zone electrophoresis (CZE) could improve detection of lymphoplasmacytic processes. We prospectively studied 1,003 consecutive serum samples submitted for routine protein electrophoresis and/or immunofixation electrophoresis by CZE and FLC. Samples from patients previously characterized as having M proteins were excluded. Protein electrophoresis was read by a pathologist unaware of the FLC results. Sixteen cases revealed an abnormal free κ/λ ratio in which CZE did not demonstrate an M protein. Nine cases of B-lymphocyte or plasma cell proliferative processes were detected by an abnormal free κ/λ ratio in which CZE did not demonstrate an M protein. Cases with low free κ/λ ratios included 1 chronic lymphocytic leukemia (CLL), 1 IgM λ with aplastic anemia, and 1 λ light chain myeloma. Cases with high free κ/λ ratios included 2 CLL, 1 lymphocytosis (possibly early CLL), 1 κ light chain myeloma, 1 atypical lymphoma with neuropathy, and 1 nonsecretory myeloma. Addition of the free κ/λ ratio to CZE increases the yield of lymphocyte and plasma cell proliferative processes detected by 56%. Capillary zone electrophoresis (CZE) is a sensitive electrophoresis technique for the detection of monoclonal M proteins in serum or urine samples. As with gel-based electrophoresis, when compared with immunofixation electrophoresis (IFE), CZE fails to detect M proteins in 5% of samples. 1,2 Most often, these false-negative results occur when small M proteins are obscured by β region proteins or, in the case of light chain myeloma, when the small free light chains (FLCs) pass readily into the urine, leaving too little in the serum to create an observable M protein deflection. A recently developed nephelometric method for the detection of FLCs in serum and urine samples is a sensitive automated immunoassay that could supplement CZE findings. It can detect the M proteins present in some samples that produce false-negative results in CZE. This method permits detection of free immunoglobulin light chains only (not bound to heavy chain). Recent studies have shown that the measurement of FLCs can be useful for the diagnosis and monitoring of patients with light chain myeloma, AL amyloidosis, and nonsecretory myeloma. 3-5 Moreover, it has been reported that the quantification of FLCs in serum by nephelometry correlates with changes in urinary FLC excretion. 4-6 Katzmann et al 7 established reference ranges for κ and λ serum FLCs and demonstrated that the serum FLC assay recognizes monoclonal light chains in some patients in whom IFE results are negative. The objective of the present study was to determine whether the serum FLC assay could serve as an adjunct to routine CZE in the detection of subtle cases of monoclonal lymphoplasmacytic processes. 214 Am J Clin Pathol 2005;124: DOI: /XE3UDARKW1B9EMWM

2 Immunopathology / ORIGINAL ARTICLE Materials and Methods We prospectively studied 1,003 consecutive samples of serum submitted for routine serum protein electrophoresis and/or IFE by CZE and FLC measurement. Samples from patients previously characterized as having M proteins were excluded. The study was conducted in accordance with a protocol approved by the institutional review board of the participating hospital. Capillary Zone Electrophoresis CZE was performed in all 1,003 cases using the automated Sebia Capillarys System (Sebia, Norcross, GA). Manufacturer guidelines were followed in performing the analysis as previously described. 8 CZE interpretation was carried out in all cases by an experienced pathologist (D.F.K.) familiar with the technique. The pathologist interpreting the CZE results had no knowledge of the FLC data. Immunofixation Electrophoresis IFE was performed with the Sebia Capillarys system using agarose gels. The precipitated protein was visualized with acid violet stain. Initially, a screening IFE using pentavalent antisera (anti-igg, anti-igm, anti-iga, anti-κ, and anti-λ) or Penta Screen (Sebia) was performed in all cases with suspicious -appearing bands on CZE. A complete IFE assay using specific antisera to determine the exact type of immunoglobulin heavy or light chain was done subsequently in cases that showed monoclonal bands on Penta Screen. IFE was done directly when a band suspected as M protein was seen on CZE to identify the heavy and light chain type. Urine IFE was performed in appropriate cases when urine samples were made available for evaluation. FLC Measurements Serum samples from all 1,003 patients were assessed for FLCs using a BN II nephelometer (Dade Behring, Deerfield, IL). The FLC reagent sets (FREELITE) were provided by The Binding Site, Birmingham, England. The assays were done using latex-enhanced immunoassays. The principle and protocol of this technique have been described by Bradwell et al. 9 The diagnostic interval for κ/λ FLC ratios used in the study was 0.26 to The 95% reference interval for κ FLC was 3.3 to 19.4 mg/l and that for λ FLC, 5.7 to 26.3 mg/l. These ranges were established by Katzmann et al. 7 Follow-up Relevant follow-up clinical and laboratory data were retrieved for all cases with an abnormal κ/λ ratio whether or not they also were identified in routine CZE. Results Of the 1,003 cases, 55 (5.48%) had an abnormal κ/λ ratio with increased concentrations of κ or λ FLCs and/or M protein by CZE and IFE. By comparing individual serum FLC results with the reference ranges, we identified 33 serum samples with abnormal FLC κ/λ ratios. We identified M proteins by CZE in 22 cases that had normal κ/λ ratios. In 13 cases, both κ and λ FLC concentrations were increased with κ/λ ratios within normal limits. In 3 cases, there was suppression of one or both FLCs with a normal κ/λ ratio, whereas 6 cases had normal levels of κ and λ FLCs. Of 33 cases with abnormal κ/λ ratios, 10 had a low κ/λ ratio and 23 a high κ/λ ratio. We detected 16 cases with an abnormal κ/λ ratio in which the CZE did not demonstrate an M protein. These included 5 (50%) of 10 samples with low κ/λ ratios and 11 (48%) of 23 samples with high κ/λ ratios Table 1. For all cases with a κ/λ ratio outside the normal range that also demonstrated an M protein in CZE, the monoclonal typing (κ or λ) by FLC data was in agreement with serum IFE results. Table 2 shows the electrophoretic findings and FLC data for 23 cases with high κ/λ ratios. Table 3 shows data for 10 cases with low κ/λ ratios. Follow-up information from the referring physicians and additional confirmatory studies revealed that 9 (56%) of 16 cases with abnormal κ/λ ratios but no abnormality by CZE were B-lymphocyte or plasma cell proliferative processes. These included the following: (1) cases with low κ/λ ratios: chronic lymphocytic leukemia (CLL), 1; IgM λ with aplastic anemia, 1; and λ light chain myeloma, 1; (2) cases with high κ/λ ratios: CLL, 2; lymphocytosis (possibly early CLL), 1; κ light chain myeloma, 1; atypical lymphoma with neuropathy, 1; and nonsecretory myeloma, 1. These findings are given in Table 4. One case (case 17, Table 2) had a high abnormal FLC κ/λ ratio of CZE did not detect any M protein, and IFE results were normal. Clinically, the patient was asymptomatic, and the total and differential peripheral WBC count was within normal limits. A follow-up FLC measurement at 12 months revealed a persistently increased FLC κ/λ ratio of Table 1 Cases With Abnormal κ/λ Free Light Chain Ratio * M Protein Detected by Capillary Zone Electrophoresis κ/λ ratio Yes No Increased (n = 23) 12 (52) 11 (48) Decreased (n = 10) 5 (50) 5 (50) * Data are given as number (percentage). Cases were interpreted as follows: normal, 4; polyclonal with chronic inflammation, 2; hypogammaglobulinemia, 2; acute inflammation and mild protein loss pattern, 2; with oligoclonal bands, 1. Cases were interpreted as follows: normal, 3; hypoalbuminemia, 1; hypoalbuminemia with β-γ fusion, 1. Am J Clin Pathol 2005;124: DOI: /XE3UDARKW1B9EMWM 215

3 Bakshi et al / DETECTING SUBTLE FLC-PRODUCING M PROTEINS Table 2 FLC and Electrophoretic Data for 23 Cases With a High κ/λ Ratio CZE and FLC Case No./Sex/Age(y) κ * λ * κ/λ Ratio * CZE Discrepancy IFE (g/dl) 1/F/ Polyclonal consistent with chronic Yes Normal inflammation 2/M/ Monoclonal gammopathy No IgM κ (1.2) 3/F/ Polyclonal consistent with active Yes Broad IgG κ (tiny) chronic inflammation 4/M/ Normal pattern Yes Normal 5/M/ Normal pattern Yes Normal 6/F/56 2, Mild protein loss pattern/reactive Yes Free κ in serum and urine samples 7/F/ Monoclonal gammopathy No IgG κ (3.7) 8/F/ Monoclonal gammopathy No IgA κ (5.2) 9/F/ Monoclonal gammopathy No IgG κ (1.5) 10/M/ Monoclonal gammopathy No IgG κ (3.9) 11/M/ Mild protein loss pattern Yes ND 12/M/ Normal pattern Yes Normal 13/F/ Monoclonal gammopathy No IgG κ (1.8) 14/M/ Monoclonal gammopathy No IgG κ (2.7) 15/M/ Normal pattern Yes Normal 16/F/ Hypogammaglobulinemia Yes Normal 17 /F/ Hypogammaglobulinemia Yes Normal 18/M/ Monoclonal gammopathy No IgG κ (2.3) 19/M/ Monoclonal gammopathy No IgG κ (4.8) 20/M/ Monoclonal gammopathy No IgA κ (0.7) 21/M/ Monoclonal gammopathy No IgG κ (0.4) 22/M/ Oligoclonal bands Yes Normal 23/F/74 12, , Monoclonal gammopathy No κ 2 (0.1 each) CZE, capillary zone electrophoresis; FLC, free light chains; IFE, immunofixation electrophoresis; ND, not done. * The 95% reference interval for κ FLC was mg/l and for λ FLC, mg/l. These ranges were established by Katzmann et al. 7 The diagnostic range for the κ/λ FLC ratio was Follow-up FLC measurements at 12 mo revealed the following: κ, 13.8; λ, 0.89; and κ/λ ratio, This case was interpreted to represent monoclonal gammopathy of undetermined significance (MGUS). Discussion FLCs that are not bound covalently to heavy chains represent a quantifiable unique biomarker of neoplastic B cell related disorders. Accurate and automated quantification of FLCs in serum and urine by a nephelometric immunoassay has been developed and is available commercially. Table 3 FLC and Electrophoretic Data for 10 Cases With a Low κ/λ Ratio Measurement of FLCs in serum by nephelometric immunoassays has been shown to be useful for the diagnosis and monitoring of light chain (Bence Jones) myeloma, nonsecretory myeloma, and AL amyloidosis. Bradwell et al 9 showed that all 224 patients with light chain myeloma could be identified based on abnormal serum concentrations of FLCs at diagnosis, without a requirement for urine testing. Owing to the kidneys high capacity of protein catabolism, the serum FLC measurements actually were more sensitive than the urine IFE. 9 Lachmann et al 10 reported that serum FLC CZE and FLC Case No./Sex/Age (y) κ * λ * κ/λ Ratio * CZE Discrepancy IFE (g/dl) 1/F/ Hypoalbuminemia Yes Normal 2/F/ Polyclonal with β-γ bridging Yes IgM λ (tiny) 3/F/ Monoclonal gammopathy No IgD λ (0.2) 4/M/ Monoclonal gammopathy No IgG λ (<1) 5/M/ Monoclonal gammopathy No IgG λ (tiny) 6/M/ Normal pattern Yes λ light chain (urine) 7/M/ , Monoclonal gammopathy No IgG λ (2.1) 8/F/ Normal pattern Yes IgA λ (too small to measure) 9/F/ Normal pattern Yes Normal 10/F/ Monoclonal gammopathy No IgG λ (0.4) CZE, capillary zone electrophoresis; FLC, free light chains; IFE, immunofixation electrophoresis. * The 95% reference interval for κ FLC was mg/l and for λ FLC, mg/l. These ranges were established by Katzmann et al. 7 The diagnostic range for the κ/λ FLC ratio was Am J Clin Pathol 2005;124: DOI: /XE3UDARKW1B9EMWM

4 Immunopathology / ORIGINAL ARTICLE Table 4 Clinical and Laboratory Data for Nine Cases With an Abnormal κ/λ Ratio Determined to Have a Monoclonal Lymphoplasmacytic Disorder Case No./Sex/ Relevant Confirmatory Age (y) Clinical Features CZE κ/λ Ratio * Studies Diagnosis Low κ/λ ratio 1 (1)/F/58 Replacement therapy for Decreased albumin level 0.24 Flow cytometric B-cell CLL/SLL hypogammaglobulinemia 2 (6)/M/88 Workup for weight loss Normal pattern 0.03 Serum and urine IFE λ light chain myeloma 3 (2)/F/58 Aplastic anemia Decreased albumin level 0.24 Serum and urine IFE Aplastic anemia with β-γ bridging High κ/λ ratio 4 (15)/M/56 Lymphocytosis Normal pattern 3.04 Flow cytometric B-cell CLL/SLL 5 (16)/F/74 Lymphocytosis Hypogammaglobulinemia 4.94 Flow cytometric B-cell CLL/SLL 6 (4)/M/74 Lymphocytosis; smudge Normal pattern 1.90 Peripheral blood smear Possible early B-cell cells on blood smear flow cytometry CLL/SLL 7 (11)/M/59 Lymphadenopathy, Decreased albumin level; 1.69 Lymph node and bone B-cell NHL, possible MZL neuropathy acute infection pattern marrow biopsies or LPL 8 (6)/F/56 NA Hypogammaglobulinemia Serum and urine IFE κ light chain myeloma 9 (22)/M/57 NA Decreased albumin level; Bone marrow biopsy Nonsecretory myeloma oligoclonal pattern CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; CZE, capillary zone electrophoresis; FLC, free light chains; IFE, immunofixation electrophoresis; LPL, lymphoplasmacytic lymphoma; MZL, marginal zone lymphoma; NA, not available; NHL, non-hodgkin lymphoma. * The diagnostic range for the κ/λ FLC ratio was Parenthetical numbers correspond to case numbers in Table 3. Parenthetical numbers correspond to case numbers in Table 2. assays were more sensitive than electrophoresis or IFE of serum and urine samples in patients with AL amyloidosis. By quantifying FLCs in the serum, Drayson et al 5 demonstrated abnormal κ/λ FLC ratios in 19 of 28 patients with nonsecretory myeloma. Whereas the usefulness of serum FLC assays has been established in these disorders, not much attention has been directed to the potential use of this method as a screening test for monoclonal lymphoplasmacytic diseases or its role when combined with routine electrophoresis in enhancing the detection rate of M protein. We studied the usefulness of serum FLC measurements for screening in conjunction with routinely performed serum CZE and IFE in our laboratory by prospectively analyzing serum samples from 1,003 cases concurrently for FLCs by nephelometry and by CZE. The pathologist interpreting the CZE was blinded to the FLC data. Previously established cutoff ranges for the κ/λ ratio were used. The latter have so far proven to be good discriminators between monoclonal and polyclonal FLCs. 7 We identified 16 cases with abnormal κ/λ ratios in which CZE failed to detect any M protein. As shown in Table 1, these cases had been called normal pattern, hypogammaglobulinemia, or one of the reactive patterns by the interpreting pathologist. Clinical follow-up revealed that 9 (56%) of 16 cases with abnormal FLC ratios showed evidence of monoclonal B- cell processes or plasma cell related clonal abnormalities (Table 4). Of the 9 cases, 3 were B-cell CLL/SLL (cases 1, 4, and 5), whereas 1 case demonstrated smudge cells on the peripheral blood smear and the case was designated as atypical lymphocytosis or early CLL at the time of last follow-up (case 6). CZE in case 7 showed increased α 1 - and α 2 -globulin levels and decreased albumin and transferrin levels, which we regard as consistent with acute inflammation. No IFE was recommended in this case. Additional clinical information from the referring physician revealed that the patient had lymphadenopathy and features of neuropathy at initial examination. At the time of last follow-up, the patient was diagnosed with B-cell non-hodgkin lymphoma, possibly lymphoplasmacytic lymphoma or marginal zone B-cell lymphoma. The serum κ FLC level was elevated and the λ FLC level, mildly suppressed, giving an abnormal κ/λ ratio of The presence of monoclonal immunoglobulin FLCs in patients with different types of B-cell proliferative disorders other than multiple myeloma indicates a much broader use of the FLC assay than previously thought. Recently, Mead et al 11 used FLC assays to follow-up and monitor all multiple myeloma cases with intact immunoglobulin proteins and to demonstrate that the changes in serum FLC concentrations provided a rapid indication of the response to treatment. Two cases of λ and κ light chain myeloma also were identified by FLC assay (cases 2 and 8, respectively). In case 8 with κ light chain disease, CZE revealed hypogammaglobulinemia resulting in reflex IFE testing, which confirmed it being a case of κ light chain myeloma. However, in case 2, the CZE was regarded as having a normal pattern, so no urine IFE was ordered. In this case, the λ FLC levels were about 20 Am J Clin Pathol 2005;124: DOI: /XE3UDARKW1B9EMWM 217

5 Bakshi et al / DETECTING SUBTLE FLC-PRODUCING M PROTEINS times higher than normal. Consequently, we assume that there would be some cases of light chain myeloma that are missed by routine electrophoresis techniques, and, in these cases, FLC measurements might be extremely valuable in facilitating early detection. A case of nonsecretory myeloma also was detected by FLC assay (case 9). In this case, CZE showed only oligoclonal bands, and IFE results were normal. FLC quantitation showed predominance of κ light chains with a κ/λ ratio of 10.00, confirming the clonal nature. These findings also demonstrate the usefulness of following the recommendation to perform serum and urine electrophoresis when a monoclonal gammopathy is suspected. 12 In a retrospective study that evaluated an FLC assay in samples that produced false-negative results in CZE, Marien et al 13 showed that the former is more reliable than CZE and nephelometric measurement of total κ and λ. It has been shown that the FLC ratio can be abnormal in even nonneoplastic B-cell proliferative disorders (chronic immune stimulation) such as systemic lupus erythematosus, polyangiitis, Sjögren syndrome, and hepatitis B. However, the κ/λ ratios in such cases were altered only modestly. 13,14 In both of our cases of light chain myeloma (1 κ and 1 λ), the FLC ratios were grossly abnormal whether detected initially by CZE or not. These findings, however, by themselves do not imply that FLC assays will detect all cases of clonal lymphoplasmacytic processes, including light chain myelomas. Indeed, in the present study, we detected 22 cases with M proteins by CZE that had normal κ/λ ratios. Furthermore, theoretically polymerization or truncation of some light chains with consequent loss of antigenic sites could affect the sensitivity of FLC assays. Therefore, we consider FLC quantitation to be an adjunctive procedure to CZE. It is interesting that IFE was unable to detect a clonal M protein in any cases except 1 case, which had a markedly elevated κ/λ ratio of (case 8, Table 4). In the other cases, the κ/λ ratio, although abnormal, was only marginally outside normal range. This finding might indicate that a greater amount of FLC is required before the M protein can be detected by IFE than by FLC assay. One case in our series (case 17, Table 2) showed no M protein by CZE, whereas the IFE result was normal. This case, however, had a persistently high κ/λ ratio, even at the 12-month follow-up. Cases in which IFE showed no evidence of an M protein could represent a previously unrecognized entity of FLC MGUS. Recently, Rajkumar et al 15 indicated that the presence of an abnormal κ/λ ratio was associated with a significantly higher risk of MGUS progression. Our study emphasizes the usefulness of FLC assays for detecting the purely monoclonal cases, in which CZE was essentially nondiagnostic. From the 1 University of Michigan, Ann Arbor; 2 Warde Medical Laboratory, Ann Arbor; 3 The Binding Site, Birmingham, England; and 4 Department of Immunology and Infection, the Medical School, University of Birmingham, Birmingham, England. Address reprint requests to Dr Keren: Warde Medical Laboratory, 5025 Venture Dr, Ann Arbor, MI References 1. Katzmann JA, Clark R, Sanders E, et al. Prospective study of serum protein capillary electrophoresis immunotyping of monoclonal proteins by immunosubtraction. Am J Clin Pathol. 1998;110: Bossuyt X, Mariën G. False negative results in detection of monoclonal proteins by capillary zone electrophoresis: a prospective study. Clin Chem. 2001;47: Bradwell AR, Carr-Smith HD, Mead GP, et al. Highly sensitive automated immunoassay for immunoglobulin free light chains in serum and urine. Clin Chem. 2001;47: Abraham RS, Katzmann JA, Clark RJ, et al. Quantitative analysis of serum free light chains; a new marker for the diagnostic evaluation of primary systemic amyloidosis. Am J Clin Pathol. 2003;119: Drayson M, Tang LX, Drew R, et al. Serum free light-chain measurement for identifying and monitoring patients with nonsecretory multiple myeloma. Blood. 2001;97: Abraham RS, Clark RJ, Bryant SC, et al. Correlation of serum immunoglobulin free light chain quantification with urinary Bence Jones protein in light chain myeloma. Clin Chem. 2002;48: Katzmann JA, Clark RJ, Abraham RS, et al. Serum reference intervals and diagnostic ranges for free kappa and free lambda immunoglobulin light chains: relative sensitivity for detection of monoclonal light chains. Clin Chem. 2002;48: Bossuyt X, Lissoir B, Mariën G, et al. Automated serum protein electrophoresis by Capillarys. Clin Chem Lab Med. 2003;41: Bradwell AR, Carr-Smith HD, Mead GP, et al. Serum test for assessment of patients with Bence Jones myeloma. Lancet. 2003;361: Lachmann HJ, Gallimore R, Gillmore JD, et al. Outcome in systemic AL amyloidosis in relation to changes in concentration of circulating free immunoglobulin light chains following chemotherapy. Br J Haematol. 2003;122: Mead GP, Carr-Smith HD, Drayson MT, et al. Serum free light chains for monitoring multiple myeloma. Br J Haematol. 2004;126: Keren DF, Alexanian R, Goeken JA, et al. Guidelines for the clinical and laboratory evaluation of patients with monoclonal gammopathies. Arch Pathol Lab Med. 1999;123: Mariën G, Oris E, Bradwell AR, et al. Detection of monoclonal proteins in sera by capillary zone electrophoresis and free light chain measurements. Clin Chem. 2002;48: Tate JR, Gill D, Cobcroft R, et al. Practical considerations for the measurement of free light chains in serum. Clin Chem. 2003;49: Rajkumar SV, Kyle RA, Therneau TM, et al. Presence of monoclonal free light chains in the serum predicts risk of progression in monoclonal gammopathy of undetermined significance. Br J Haematol. 2004;127: Am J Clin Pathol 2005;124: DOI: /XE3UDARKW1B9EMWM