Diagnostic performance of serum free light chain measurement in patients suspected of a monoclonal B-cell disorder

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1 research paper Diagnostic performance of serum free light chain measurement in patients suspected of a monoclonal B-cell disorder Pieter Vermeersch, 1 * Lieve Van Hoovels, 1 * Michel Delforge, 2 Godelieve Mariën 1 and Xavier Bossuyt 1 1 Department of Laboratory Medicine, Immunology, and 2 Department of Haematology, University Hospitals Leuven, Catholic University of Leuven, Leuven, Belgium Received 9 May 2008; accepted for publication 9 July 2008 Correspondence: Xavier Bossuyt, MD, PhD, Laboratory Medicine, Immunology, University Hospitals Leuven, Herestraat 49, B-3000 Leuven, Belgium. xavier.bossuyt@uz.kuleuven.be *Both authors contributed equally. Summary The present study aimed to determine the diagnostic performance of different testing strategies to diagnose malignant B-cell disorder or monoclonal gammopathy of unknown significance (MGUS). Sensitivity and specificity were determined in 833 consecutive patients investigated for a monoclonal gammopathy. Serum protein electrophoresis (PE), serum j/k free light chain (FLC) ratio, and serum and urine immunofixation electrophoresis (IFE) were performed in all patients. Twenty-eight patients were diagnosed with a malignant plasma cell disorder, 25 with B-cell non- Hodgkin lymphoma and 156 with MGUS. Serum PE (with follow-up IFE) plus FLC had a sensitivity of 82Æ3% and a specificity of 96Æ8% and missed one plasmacytoma and 23 patients with MGUS. Serum IFE plus urine IFE had a sensitivity of 92Æ3% and a specificity of 100% and missed two MGUS patients. Serum IFE plus FLC had a sensitivity of 93Æ8% and a specificity of 96Æ8% and missed one MGUS patient. Serum PE plus FLC had a significantly lower sensitivity than serum IFE plus FLC or serum IFE plus urine IFE for the diagnosis of MGUS. The sensitivity of serum IFE plus FLC was comparable to the sensitivity of serum IFE plus urine IFE. The specificity of serum IFE plus FLC, however, was lower than the specificity of serum IFE plus urine IFE. Keywords: haemotological malignancies, multiple myeloma, immunohaemotology, B cells. The International Myeloma Working Group algorithm requires assessment of both serum and urine in patients suspected of a malignant B-cell disorder, such as multiple myeloma (MM), light chain amyloidosis (AL) or Waldenström macroglobulinaemia (The International Myeloma Working Group, 2003). While malignant plasma cell disorders are the most frequent malignant cause of a monoclonal gammopathy, B-cell non-hodgkin lymphoma (B-NHL) can also be associated with a monoclonal gammopathy. Serum and urine immunofixation electrophoresis (IFE) are considered the gold standard (The International Myeloma Working Group, 2003; Smith et al, 2006), but may be negative in patients with nonsecretory MM, light chain MM, AL amyloidosis, and light chain deposition disease. Performing additional measurements of serum free light chains (FLC) can increase the diagnostic sensitivity for these disorders as they often do not have sufficiently high concentrations of circulating serum light chains to be detected by serum or urine IFE (Katzmann, 2006). As serum j FLC and k FLC tend to increase with population age, the ratio of serum j FLC to k FLC (j/k ratio) is used for diagnostic purposes (Katzmann et al, 2002). Analysis of sera from patients diagnosed with MM and AL amyloidosis found that urine IFE provided no additional diagnostic information compared to serum IF and serum FLC (Bradwell et al, 2003; Katzmann et al, 2005). It has therefore been suggested that serum protein electrophoresis (PE) plus serum FLC measurement could be an attractive approach for the diagnosis of plasma cell disorders (Pratt, 2008). Although several studies have evaluated this approach (Bakshi et al, 2005; Katzmann et al, 2005; Hill et al, 2006; Beetham et al, 2007), there are no studies that have compared the performance of screening strategies using serum PE plus serum FLC or serum IFE plus serum FLC to serum IFE plus urine IFE for the diagnosis of B-cell disorders in consecutive patients suspected of a B-cell disorder (Vermeersch et al, 2008). The study by Katzmann et al (2005) is the only study which First published online 22 August 2008 doi: /j x Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 143,

2 Performance of Serum FLC for Diagnosis of B-Cell Disorders compared the diagnostic performance of serum PE plus serum FLC j/k ratio to serum IFE plus urine IFE. This study, however, does not allow a good assessment of the false-positive rate since 88% of the patients in this study had a malignant B-cell disorder or a monoclonal gammopathy of unknown significance (MGUS), indicating a selection bias. The falsepositive rate in routine clinical practice needs to be assessed to determine whether serum FLC could replace urinalysis for Bence Jones proteins in clinical practice (Pratt, 2008). The present study determined the performance of different strategies to diagnose malignant B-cell disorders and MGUS. The evaluation was carried out on 833 different consecutive patients in whom clinicians suspected a B-cell disorder. Patients with known B-cell disorders were excluded. In all patients, serum PE and serum and urine IFE were performed as part of the routine laboratory investigation for monoclonal gammopathies. In addition to serum IFE and urine IFE, serum FLC was performed in all patients. The fact that (i) patients in follow-up were excluded and (ii) all blood samples were accompanied by a urine sample are major strengths of this study and allowed us to unambiguously compare the sensitivity and specificity of different testing strategies. The final clinical diagnosis was considered the gold standard. Materials and methods Patients Serum FLC j/k ratio was determined in 833 consecutive patients investigated for a monoclonal gammopathy (a specific laboratory request) between 1 January 2004 and 28 February The patients attended the outpatient clinic or were hospitalized at the University Hospitals Leuven. At our institution, the routine laboratory investigation in patients suspected of a monoclonal gammopathy includes serum PE, serum IFE and urine IFE. All laboratory requests are made electronically and the specific laboratory request for suspicion of a monoclonal gammopathy requires a serum and urine sample. Patients that were previously diagnosed with a malignant plasma cell disorder, a B-NHL or MGUS were excluded. Thirteen patients with an isolated monoclonal light chain on urine IFE that were not diagnosed with a B-cell disorder were excluded as the clinical significance of these findings is currently unknown. The study was approved by the Institutional Ethics Committee of the University Hospitals Leuven. Analytical methods Serum capillary zone electrophoresis (CZE) was performed using the Paragon CZE 2000 (Beckman Coulter, Fullerton, CA, USA) and total protein content was determined using Modular P (Roche Diagnostics, Basel, Switzerland). Based on the electropherogram, sera were categorized as M-peak present, possible M-peak, disturbed morphology of the beta and/or gamma region, acute or chronic reactive process without a disturbed morphology or no abnormality detected. When the gamma-fraction on CZE was equivalent to <6Æ4 or >14Æ4 g/l and no abnormality was detected on CZE, the serum was categorized as hypogammaglobulinaemia or hypergammaglobulinaemia respectively. Serum and urine IFE were performed in all patients using the semi-automated Hydrasys Ò electrophoresis apparatus (Sebia, Evry, France). Urine was concentrated 25 times unless urinary total protein was 1 g/l or antigen excess was suspected. Identification of monoclonal bands was performed by direct visual inspection of the gels by two immunologists with more than 8 years of experience. Sera were subsequently frozen and stored at )20 C. Sera were blinded and serum FLC measurements were performed using the Freelite assay in December 2006 by The Binding Site Ltd. (Birmingham, UK) on a Dade-Behring BNII nephelometer (Deerfield, IL, USA). For serum FLC, the reference values established by Katzmann et al were used (Katzmann et al, 2002). Sera with an abnormal FLC j/k ratio (<0Æ26 or >1Æ65) were considered positive. Analysis and statistical methods Medical records of all patients (i) who were positive on serum or urine IFE, (ii) who had an abnormal j/k ratio, (iii) who underwent a bone marrow aspiration, or (iv) who had immunophenotyping on bone marrow or peripheral blood were checked to determine whether they had a malignant B-cell disorder or MGUS. To calculate sensitivity and specificity of a testing strategy in which serum IFE was performed on samples with an abnormal serum PE (SPE ± IFE), we assumed that follow-up serum IFE was performed in sera that were categorized as M peak present, possible M peak, disturbed morphology, or hypogammaglobulinaemia. The sensitivity of the different strategies for the diagnosis of patients with a malignant plasma cell disorder, B-NHL or MGUS was compared using Fischer exact test. Results Patients diagnosed with a malignant B-cell disorder or MGUS Of the 833 patients suspected of a monoclonal gammopathy, 28 patients were diagnosed with a malignant plasma cell disorder (including 18 patients with an intact MM, two with a light chain MM, and three with AL amyloidosis), 156 with MGUS (including three patients suffering from IgM-related polyneuropathy) and 25 with B-NHL. Sensitivity and specificity of serum FLC j/k ratio The overall sensitivity of serum FLC j/k ratio for the diagnosis of malignant monoclonal B-cell disorders or MGUS Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 143,

3 P. Vermeersch et al Table I. Comparison of different strategies for the diagnosis of malignant monoclonal B-cell disorders and MGUS. Strategy Sensitivity (%) Specificity (%) Missed monoclonal B-cell disorders and MGUS FLC j/k ratio 36Æ8 96Æ8 3 MM, 1 PC, 112 MGUS, 16 B-NHL SPE 80Æ4 77Æ9 1 MM, 1 AL-A, 1 PC, 25 MGUS, 13 B-NHL SPE ± IFE 78Æ MM, 1 AL-A, 1 PC, 26 MGUS, 16 B-NHL SPE ± IFE + UIFE 81Æ MGUS, 14 B-NHL SPE ± IFE + FLC j/k ratio 82Æ3 96Æ8 1 PC, 23 MGUS, 13 B-NHL SIFE 91Æ B-NH L, 2MGUS SIFE + FLC j/k ratio 93Æ8 96Æ8 12 B-NHL, 1 MGUS SIFE + UIFE 92Æ B-NHL 2 MGUS AL-A, AL amyloidosis; PC, plasmacytoma; MM, multiple myeloma; SIFE, serum immunofixation electrophoresis; UIFE, urine immunofixation electrophoresis; SPE ± IFE, serum IFE on positive serum PE (SPE) samples. was 36Æ8% (Table I). Serum FLC j/k ratio was abnormal (i) in 24 of the 28 patients (86%) diagnosed with a malignant plasma cell disorder (including 18 patients with an intact MM, two with a light chain MM, and three with AL amyloidosis), (ii) in nine of the 25 patients with a B-NHL (36%), and (iii) in 44 of the 156 patients (28%) diagnosed with MGUS (Table II). Twenty-three patients had an abnormal j/k ratio but no monoclonal band on serum or urine IFE. Of these 23 patients, three were diagnosed with a B-NHL (Table III, patients 1 3). The results and clinical data of the other 20 patients that were considered false-positive are summarized in Table III (Patients 4 23). The specificity of serum FLC j/k ratio in our study was 96Æ8%. Six of the 20 (30%) false-positive patients had a polyclonal increase of immunoglobulins compared to 99 of the 604 (16%) true-negative patients. Nine of the 20 (45%) falsepositive patients had a glomerular filtration rate (GFR) of <60 ml/min/1æ73 m 2 compared to 243 of the 604 (40%) truenegative patients. Five (20%) false-positive patients had a GFR of <30 ml/min/1æ73 m 2 compared to 97 (16%) true-negative patients. The percentage of false-positive results in patients with a GFR of GFR of 60, <60 and <30 ml/min/1æ73 m 2 was 2Æ9%, 3Æ6% and 4Æ9% respectively. Serum FLC j/k ratio in patients with a malignant plasma cell disorder or MGUS Serum FLC j/k ratio was abnormal in 77 of the 209 patients (36Æ8%) with a malignant plasma cell disorder, B-NHL or MGUS (Tables I and II). Of these 77 patients, 74 had a monoclonal band on serum and/or urine IFE. The change in serum FLC j/k ratio was concordant with serum and/or urine IFE in 73 of the 74 patients. One patient who was diagnosed with a IgGk MGUS (reconfirmed 6 months later) had an increased serum FLC j/k ratio with individual concentrations of j and k within the normal reference range. The sensitivity of serum FLC j/k ratio in patients with a monoclonal gammopathy on serum and/or urine IFE was 38Æ3%. Three patients diagnosed with a B-NHL had an abnormal j/k ratio but no monoclonal band on serum or urine IFE. The Table II. Results of different strategies using serum FLC, serum PE, serum IFE and/or urine IFE. Number of positive patients n j/k ratio SPE* SPE ± IFE* SIFE UIFE Malignant plasma cell disorders Intact MM (1) 17 (1) Light chain MMà Plasmacytoma Osteosclerotic myeloma Plasma cell leukaemia WM Primary amyloidosis All B non-hodgkin lymphoma (B-NHL) B-CLL (1) 3 (1) 3 2 B-NHL (other) (2) 6 (1) 7 7 All (3) 9 (2) 10 9 Monoclonal gammopathy of unknown significance (MGUS) All (3) 130 (3) Other patients All (47) WM, Waldenstrom macroglobulinaemia; B-CLL, B-cell chronic lymphocytic leukaemia; SIFE, serum immunofixation electrophoresis; UIFE, urine immunofixation electrophoresis; SPE ± IFE, serum IFE on positive serum PE (SPE) samples. *Values within parentheses indicate the number of patients in which hypogammaglobulinaemia on CZE was the only abnormality. Monoclonal component in patients with intact MM on SIFE: 9 IgGj, 3 IgGk, 4 IgAj, 1 IgAk, 1 IgDk. àmonoclonal component in patients with light chain MM on SIFE: 1 j, 1k. Monoclonal component in MGUS patients with SIFE: 59 IgGj, 40 IgGk, 1 IgG(k?), 29 IgMj, 8 IgMk, 2 IgM(?), 4 IgAj, 9 IgAk, 2 IgMj+k, 1 IgMj+IgGk, and 1 j (also in urine). Two patients with IgMk were negative with normal SIFE but positive when serum was kept at 37 C. change in serum FLC j/k ratio in these three patients was concordant with the clonality of the malignant B-cell disorder (Table III, patients 1 3). 498 Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 143,

4 Performance of Serum FLC for Diagnosis of B-Cell Disorders Table III. Results of 23 patients with abnormal j/k ratios and negative serum and urine IFE. No Age (years) Sex j (mg/l) k (mg/l) j/k ratio GFR Diagnosis/other details 1 71 M 13Æ0 5Æ7 fl 2Æ30 60 B-CLL (BM: s-j) 2 56 F 3Æ57 21Æ3 0Æ17 fl 51 Mantle cell lymphoma (stage IVb) (BM: s-k) 3 46 F 10Æ0 5Æ0 fl 2Æ02 30 Follicular lymphoma (stage IIa) (lymph node: k) 4 55 M 57Æ9 31Æ8 1Æ82 55 Hodgkin disease 5 47 M 16Æ6 9Æ1 1Æ82 60 Acute myeloblastic leukaemia, minimally differentiated (AML-M0) 6 71 M 9Æ0 5Æ3 fl 1Æ71 60 Normochromic, normocytic anaemia of unknown origin 7 78 M 1Æ8 fl 22Æ1 0Æ08 fl 60 Large cell carcinoma of the lung 8 66 F 56Æ0 31Æ0 1Æ81 25 Chronic kidney disease of unknown origin 9 80 F 30Æ1 17Æ0 1Æ77 38 Chronic kidney disease due to ischaemic renovascular disease M 87Æ7 43Æ4 2Æ02 9 Acute respiratory distress syndrome and acute tubular necrosis M 76Æ9 42Æ5 1Æ81 4 Acute kidney failure due to oligomeganephronia M 64Æ4 38Æ9 1Æ66 11 Acute kidney failure due to diabetic nephropathy, DM2, recent weight loss F 69Æ9 40Æ4 1Æ73 14 Diabetic nephropathy, DM F 29Æ Æ04 fl 43 Diabetic nephropathy, DM M 8Æ7 4Æ7 fl 1Æ86 60 Non-malignant lymphadenopathy F 8Æ9 4Æ6 fl 1Æ96 60 Bone pain M 12Æ2 5Æ7 2Æ16 60 Referral for investigation of mild fl decrease in GFR M 7Æ4 81Æ0 0Æ09 fl 60 DM2, obesitas, arterial hypertension, lower limb ulcers of unknown origin M 83Æ9 33Æ0 2Æ54 55 Polyclonal hypergammaglobulinaemia F Æ1 1Æ69 60 Chronic fatigue, rheumatoid arthritis M 10Æ6 5Æ0 fl 2Æ10 60 Demyelinating polyneuropathy F 3Æ3 19Æ4 0Æ17 fl 60 Urticarial vasculitis F 9Æ0 5Æ1 fl 1Æ75 60 Urticarial skin lesions B-CLL, B-cell chronic lymphocytic leukaemia; BM, bone marrow; DM2, diabetes mellitus type 2; GFR, glomerular filtration rate. Serum PE with serum IFE on samples with an abnormal serum PE A testing strategy in which serum IFE was performed on samples with an abnormal serum PE (SPE ± IFE) had a sensitivity of 78Æ5% and a specificity of 100% (Tables I and II). This strategy (serum PE with follow-up IFE) missed three patients with a malignant plasma cell disorder (one patient with MM, one patient with a AL amyloidosis and one patient with a plasmacytoma) and 26 patients with MGUS. Specificity of serum PE alone was 77Æ9%. Serum PE (with follow-up IFE) plus urine IFE A testing strategy that performed urine IFE in all patients in addition to screening with serum PE and confirmed abnormal PE samples with IFE had a sensitivity of 81Æ8% and a specificity of 100%. This strategy missed no patients with a malignant plasma cell disorder, 24 patients with MGUS and 14 patients with a B-NHL (Table I). Serum PE (with follow-up IFE) plus serum FLC j/k ratio Performing serum FLC j/k ratio on all samples in addition to screening with serum PE and confirming abnormal PE samples with IFE had a sensitivity of 82Æ3% and a specificity of 96Æ8%. This strategy missed one patient with a plasmacytoma and 23 patients with MGUS, but no patients with MM (Table I). Serum FLC j/k ratio identified one patient with MM, one patient with AL amyloidosis and two patients with MGUS with a M-protein on serum IFE who were negative with serum PE. Serum IFE plus urine IFE Serum IFE plus urine IFE, which is traditionally considered the most sensitive technique for the diagnosis of monoclonal gammopathies, had an overall sensitivity of 92Æ3% and a specificity of 100%. This strategy missed two MGUS patients with a monoclonal cryoglobulin who were negative on routine serum IFE, but no patients with a malignant plasma cell disorder (Table I). Serum IFE plus serum FLC j/k ratio A strategy performing serum IFE plus serum FLC j/k ratio in all patients had a sensitivity of 93Æ8% and a specificity of 96Æ8%. It missed one MGUS patient with a monoclonal cryoglobulin and was positive in 13 of the 25 patients with a B-NHL (Table I). The sensitivity of serum IFE plus FLC j/k ratio was comparable to the sensitivity of serum IFE plus urine IFE, but the specificity was somewhat lower. Serum FLC for the diagnosis of B-NHL Our patient population comprised 25 patients with B-NHL. Serum IFE was negative in 15 of these patients (Table IV). It is Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 143,

5 P. Vermeersch et al Table IV. Results of 25 patients newly diagnosed with a B non-hodgkin lymphoma (including Ann Arbor stage at diagnosis). No Age (years) Sex SPE SIFE j (mg/l) k (mg/l) j/k ratio GFR UIFE Diagnosis/other details 1 74 F MP IgG-j 52Æ7 11Æ3 4Æ66 60 j B-CLL (BM: s-j) 2 66 F HypoG IgG-j 12Æ9 4Æ6 fl 2Æ B-CLL (BM: s-j) 3 66 M pmp IgM-k 12Æ8 42Æ5 0Æ30 52 k B-CLL (BM: cy-k) 4 71 M Æ0 5Æ7 fl 2Æ B-CLL (BM: s-j) (Table III, patient 1) 5 58 M pmp 0 18Æ1 18Æ2 0Æ B-CLL (BM: s-k) 6 58 F 0 0 9Æ4 10Æ0 0Æ B-CLL (BM: s-j) 7 58 M Æ9 14Æ3 0Æ B-CLL (BM: s-j) 8 64 M Æ9 32Æ4 0Æ B-CLL (BM: s-j) 9 64 F MP IgM-j 68Æ0 39Æ4 1Æ73 46 j B-NHL: marginal zone lymphoma? (stage IVa) F pmp IgM-k 28Æ Æ29 fl 51 k Mantle cell lymphoma (stage IVb) (BM: s-k) F 0 0 3Æ57 21Æ3 0Æ17 fl 51 0 Mantle cell lymphoma (stage IVb) (BM: s-k) (Table III, Patient 2) F Æ4 41Æ8 0Æ30 60 k Mantle cell lymphoma (stage IVb) (BM: s-k) M Æ7 22Æ7 1Æ Mantle cell lymphoma (stage IVb) M 0 IgM-j 14Æ2 8Æ8 1Æ61 60 j Mantle cell lymphoma (stage IVa) (BM: s-j) F GM 0 14Æ3 19Æ4 0Æ MALT-lymphoma (stage IVa) F Æ5 38Æ0 1Æ MALT-lymphoma (stage Ia) M Æ3 15Æ2 0Æ Follicular lymphoma (stage IIIa) F HypoG 0 10Æ0 5Æ0 fl 2Æ Follicular lymphoma (stage IIa) (lymph node: k) (Table III, patient 3) F Æ9 15Æ8 1Æ Follicular lymphoma (stage IIa) F MP IgG-j Æ3 11Æ71 60 j Diffuse large B-cell lymphoma (stage IVb) M pmp IgG-j 24Æ3 20Æ7 1Æ Diffuse large B-cell lymphoma (stage IIIsb) F 0 0 8Æ9 11Æ2 0Æ Diffuse large B-cell lymphoma (stage IIea) M MP IgM-j 34Æ4 11Æ7 2Æ94 46 j Diffuse large B-cell lymphoma (stage IIea) F HypoG IgM-j 3Æ6 4Æ5 fl 0Æ Diffuse large B-cell lymphoma (stage Ib) M RP 0 34Æ4 62Æ8 0Æ Post-transplant lymphoproliferative disorder (stage IVa) Results of immunophenotyping are given within parentheses. +, abnormal (cfr. methods); 0, no abnormality detected; s-j, surface IgKappa; cy-k, cytoplasmatic IgLambda; DD, differential diagnosis; SPE, serum protein electrophoresis; SIFE, serum immunofixation electrophoresis; UIFE, urine immunofixation electrophoresis; B-CLL, B-cell chronic lymphocytic leukaemia; MALT-lymphoma, mucosa-associated lymphoid tissue lymphoma. important to note that, in contrast to malignant plasma cell disorders, which are almost always associated with a monoclonal gammopathy, B-NHL often do not secrete a monoclonal immunoglobulin. Serum IFE plus urine IFE detected a monoclonal protein in 11 of the 25 patients with B-NHL, while serum IFE plus serum FLC j/k ratio was positive in 13 patients. Serum FLC j/k ratio was, however, negative in one patient with B-NHL who was positive with urine IFE but negative with serum IFE. This patient had a monoclonal k band on urine IFE and was surface k positive with immunophenotyping (Table IV, patient 12). Serum FLC in patients with a urinary monoclonal protein There were 106 patients in whom a monoclonal protein was found in the urine. Serum FLC j/k ratio was abnormal in 62 (58Æ5%) of these patients and serum IFE was abnormal in 105 (99Æ1%) of these 106 patients. One patient, who was diagnosed with a B-NHL, had a urinary monoclonal protein, but was negative with serum IFE and serum FLC j/k ratio (Table IV, patient 12). Discussion The present study evaluated the sensitivity and specificity of different strategies to detect monoclonal proteins. Serum CZE, serum IFE, serum FLC and urine IFE were performed in 833 consecutive patients suspected of a monoclonal gammopathy. Of the 833 patients, 28 patients were diagnosed with a malignant plasma cell disorder, 156 with MGUS, and 25 with B-NHL. Serum FLC j/k ratio was abnormal in 77 of the 209 patients (36Æ8%) with a malignant plasma cell disorder, B-NHL or MGUS (Tables I and II). The sensitivity of serum FLC j/k ratio in patients with a monoclonal gammopathy on serum or urine IFE was 38Æ3%, which was comparable to the sensitivity of 40Æ0% reported in the study by Hill et al (2006). The main reason for the low overall sensitivity of serum FLC j/k ratio was the low sensitivity of serum FLC j/k ratio for MGUS. Serum FLC j/k ratio was abnormal in only 44 of the 156 patients (28%) diagnosed with MGUS in the present study (Table II), which was comparable to the 33% reported by Rajkumar et al (2005) in a study of 1148 patients diagnosed 500 Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 143,

6 Performance of Serum FLC for Diagnosis of B-Cell Disorders with MGUS. Rajkumar et al (2005) found that an abnormal serum FLC j/k ratio at the time of diagnosis of MGUS was an independent prognostic marker for progression to multiple myeloma. The specificity of serum FLC j/k ratio in the present study was 96Æ8%, which is similar to the 95Æ8% specificity reported by Hill et al (2006). The lower positive predictive value in the study by Hill et al (2006) (51%) compared to our study (81%) was the result of the lower prevalence of patients diagnosed with a B-cell monoclonal disorder (8%) in their study compared to our study (25%). Hill et al (2006) included all patients who had a serum PE request (irrespective of the clinical indication) and did not perform serum and urine IFE in all patients. The present study included only patients with a suspected monoclonal B-cell disorder. All patients had serum and urine IFE performed as part of the routine laboratory investigation of a monoclonal gammopathy. Decreased renal function and a polyclonal increase of immunoglobulins are associated with increased serum free j and free k concentrations. According to Katzmann et al (2002), serum FLC j/k ratio is not affected by a decreased renal function or a polyclonal increase in polyclonal immunoglobulins. However, Hill et al (2006) found that 14% of the patients with a polyclonal increase in immunoglobulins had a falsely elevated serum FLC j/k ratio and almost all patients who were false-positive had a GFR below the mean for age and sex, suggesting that renal insufficiency and polyclonal increase of immunoglobulins can cause false-positive results for serum FLC j/k ratio. In the present study, 6% of the diseased control patients had a polyclonal increase of immunoglobulins and an abnormal serum FLC j/k ratio. Three per cent of the diseased control patients without a polyclonal increase of immunoglobulins had an abnormal serum FLC j/k ratio, suggesting that a polyclonal increase of immunoglobulins might be associated with an increased occurrence of false-positive results. It should, however, be noted that most false-positives for FLC j/k ratio (70%) did not have a polyclonal increase in immunoglobulins. We did not observe an important increase in false-positive results in patients with reduced renal function and most falsepositive patients (75%) had a GFR of 30 ml/min/1æ73 m 2. Serum PE with confirmation of abnormal PE samples with serum IFE (SPE ± IFE) had a sensitivity of only 78Æ5% and missed three patients with a malignant plasma cell disorder (one patient with MM, one patient with AL amyloidosis, and one patient with a plasmacytoma) and 26 patients with MGUS. Performing serum j/k ratio in addition to screening with serum PE and confirming abnormal PE samples with serum IFE had a sensitivity of 82Æ3% and a specificity of 96Æ8%. Serum FLC j/k ratio identified four patients with a M-protein on serum IFE who were negative with serum PE (one patient with MM, one patient with AL amyloidosis and two patients with MGUS), confirming previous reports that the serum FLC j/k ratio helps to identify patients with a M-protein that were negative for serum PE (Marien et al, 2002; Hill et al, 2006). The positive predictive value of an abnormal FLC j/k ratio in a patient with no abnormalities on serum PE was only 29%. It is therefore recommended to perform additional serum and urine IFE in patients that only have an abnormal FLC ratio. This approach will help to positively identify some patients with B-cell disorders including light chain MM and AL amyloidosis. The sensitivity of serum IFE plus serum FLC j/k ratio was comparable to the sensitivity of serum IFE plus urine IFE (93Æ8% vs. 92Æ3%), which is traditionally considered the gold standard. Both strategies did not miss patients with a malignant plasma cell disorder. The specificity of serum IFE plus serum FLC j/k ratio was, however, lower. Our study is the first to determine the false-positive rate of serum FLC j/k ratio in consecutive patients in a routine laboratory setting that were tested with serum PE, serum IFE, and urine IFE. When used in routine clinical practice, the reduced specificity of serum FLC j/k ratio compared to serum and urine IFE needs to be taken into account. An important argument for replacing urine IFE with serum FLC j/k ratio is that compliance for urine samples is not high (Pratt, 2008). In the study by Hill et al (2006), paired urine samples were available for only 40% of the consecutive patients tested with serum PE. Beetham et al (2007) obtained urine samples in 52% of the patients tested with serum PE. In our hospital, we have a specific laboratory request for suspicion of a monoclonal gammopathy which requires a serum and urine sample. Requests are made electronically and the requestors understand the need to provide a serum and urine sample. The sensitivity of 58Æ5% for serum FLC j/k ratio in patients with a urinary monoclonal gammopathy in our study was significantly lower than the 85Æ7% reported by Katzmann et al (2006) in patients with a urinary monoclonal protein who also had a FLC measurement, serum PE and serum IFE requested within 30 d of diagnosis. The selection criteria used by Katzmann et al (2006) caused a bias towards more severe pathology. Only 16% of the 428 patients with a urinary monoclonal protein had MGUS compared to 67% in our study. Since most patients diagnosed with MGUS do not have an abnormal serum FLC j/k ratio at the time of diagnosis (Rajkumar et al, 2005), this selection bias most likely explains the observed differences. Our study is the first to evaluate the value of serum FLC j/k ratio for the diagnosis of B-NHL in a screening setting. Our results indicate that a significant number of patients diagnosed with different subtypes of B-NHL have an abnormal serum FLC j/k ratio at the time of diagnosis. It remains to be determined in a larger group of patients whether serum FLC j/k ratio provides additional diagnostic information and whether an abnormal serum FLC j/k ratio is an independent prognostic factor for certain subtypes of B-NHL. Taken together, serum PE (with follow-up IFE) combined with serum FLC j/k ratio had a sensitivity of only 82Æ3% for the diagnosis of malignant plasma cell disorders, B-NHL or MGUS. It missed 23 patients with MGUS and one patient with Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 143,

7 P. Vermeersch et al plasmacytoma. Serum IFE plus urine IFE had a sensitivity of 92Æ3%. It missed no patients with a malignant plasma cell disorder, but missed two patients with MGUS with a monoclonal cryoglobulin. Serum IFE combined with serum FLC had a sensitivity of 93Æ8% for the diagnosis of malignant B-cell disorder or MGUS. It missed one patient with MGUS with a monoclonal cryoglobulin and no patients with a malignant plasma cell disorder. The sensitivity of serum PE plus serum FLC j/k ratio was significantly lower than the sensitivity of serum IFE plus FLC j/k ratio or serum plus urine IFE (P <0Æ01 each). Serum FLC j/k ratio identified three patients diagnosed with B-NHL and one patient diagnosed with MGUS that were negative with serum and urine IFE. Serum FLC j/k ratio, however, had a slightly lower specificity than IFE. We conclude that serum PE plus FLC j/k ratio has a significantly lower sensitivity than serum IFE plus FLC j/k ratio or serum IFE plus urine IFE for the diagnosis of MGUS. The sensitivity of serum IFE plus FLC j/k ratio is comparable to the sensitivity of serum IFE plus urine IFE. When used in routine clinical practice, the reduced specificity of FLC j/k ratio compared to serum and urine IFE needs to be taken into account. Acknowledgement We thank The Binding Site Ltd. for performing the determinations of FLC. Grants/funding support Not applicable. References Bakshi, N.A., Gulbranson, R., Garstka, D., Bradwell, A.R. & Keren, D.F. (2005) Serum free light chain (FLC) measurement can aid capillary zone electrophoresis in detecting subtle FLC-producing M proteins. American Journal of Clinical Pathology, 124, Beetham, R., Wassell, J., Wallage, M.J., Whiteway, A.J. & James, J.A. (2007) Can serum free light chains replace urine electrophoresis in the detection of monoclonal gammopathies? Annals of Clinical Biochemistry, 44, Bradwell, A.R., Carr-Smith, H.D., Mead, G.P., Harvey, T.C. & Drayson, M.T. (2003) Serum test for assessment of patients with Bence Jones myeloma. Lancet, 361, Hill, P.G., Forsyth, J.M., Rai, B. & Mayne, S. (2006) Serum free light chains: an alternative to the urine Bence Jones proteins screening test for monoclonal gammopathies. Clinical Chemistry, 52, Katzmann, J.A. (2006) Serum free light chain specificity and sensitivity: a reality check. Clinical Chemistry, 52, Katzmann, J.A., Clark, R.J., Abraham, R.S., Bryant, S., Lymp, J.F., Bradwell, A.R. & Kyle, R.A. (2002) Serum reference intervals and diagnostic ranges for free kappa and free lambda immunoglobulin light chains: relative sensitivity for detection of monoclonal light chains. Clinical Chemistry, 48, Katzmann, J.A., Abraham, R.S., Dispenzieri, A., Lust, J.A. & Kyle, R.A. (2005) Diagnostic performance of quantitative kappa and lambda free light chain assays in clinical practice. Clinical Chemistry, 51, Katzmann, J.A., Dispenzieri, A., Kyle, R.A., Snyder, M.R., Plevak, M.F., Larson, D.R., Abraham, R.S., Lust, J.A., Melton, III, L.J. & Rajkumar, S.V. (2006) Elimination of the need for urine studies in the screening algorithm for monoclonal gammopathies by using serum immunofixation and free light chain assays. Mayo Clinic Proceedings, 81, Marien, G., Oris, E., Bradwell, A.R., Blanckaert, N. & Bossuyt, X. (2002) Detection of monoclonal proteins in sera by capillary zone electrophoresis and free light chain measurements. Clinical Chemistry, 48, Pratt, G. (2008) The evolving use of serum free light chain assays in haematology. British Journal of Haematology, 141, Rajkumar, S.V., Kyle, R.A., Therneau, T.M., Melton, Jr, L., Bradwell, A.R., Clark, R.J., Larson, D.R., Plevak, M.F., Dispenzieri, A. & Katzmann, J.A. (2005) Serum free light chain ratio is an independent risk factor for progression in monoclonal gammopathy of undetermined significance. Blood, 106, Smith, A., Wisloff, F. & Samson, D. (2006) Guidelines on the diagnosis and management of multiple myeloma British Journal Haematology, 132, The International Myeloma Working Group (2003) Criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International Myeloma Working Group. British Journal of Haematology, 121, Vermeersch, P., Marien, G. & Bossuyt, X. (2008) More studies are needed to assess the performance of serum free light chain measurement for the diagnosis of B-cell disorders in routine clinical practice. British Journal of Haematology, (in press). 502 Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 143,