From the Department of Pathology, Division of Clinical Pathology, Augusta University, Medical College of Georgia, Augusta.

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1 AJCP /ORIGINAL ARTICLE Serum Free Light Chain Assay and j/k Ratio Performance in Patients Without Monoclonal Gammopathies High False-Positive Rate Gurmukh Singh, MD, PhD, MBA From the Department of Pathology, Division of Clinical Pathology, Augusta University, Medical College of Georgia, Augusta. Key Words: Serum protein electrophoresis; Serum protein immunofixation electrophoresis; Serum free light chain assay; Urine protein electrophoresis; Urine protein immunofixation electrophoresis; j/k; ratio; Monoclonal gammopathy; Serum free light chains; Protein electrophoresis; Multiple myeloma Am J Clin Pathol August 2016;146: DOI: /AJCP/AQW099 ABSTRACT Objectives: Serum free light chain assay is a recommended screening test for monoclonal gammopathies. Anecdotal observations indicated a high rate of false-positive abnormal j/k ratios. This study was undertaken to ascertain the magnitude of the false-positive rate and factors contributing to the error rate. Methods: Results of serum protein electrophoresis, serum free light chains, and related tests, usually done for investigation of suspected monoclonal gammopathy, were reviewed retrospectively for 270 patients and 297 observations. Results: Using the conventional j/k ratio, 36.4% of the ratios were abnormal, in the absence of monoclonal gammopathy. When the renal j/k ratio was used, the rate of abnormal j/k ratios was 30.1%. In patients with a c-globulin concentration of 1.6 g/dl or more, the usual j/k ratio was abnormal in 54.8% of the patients. Urine protein electrophoresis was used in 53 (19.6%) instances, whereas bone marrow examination was done in 65 (24.1%) cases. Conclusions: Usual j/k ratio was abnormal in 36.4% of the observations in patients without evidence of monoclonal gammopathy, and an abnormal j/k ratio should not be used as the sole indicator for diagnosis of neoplastic proliferation of the lympho-plasmacytic system. Hypergammaglobulinemia is associated with a higher rate of false-positive abnormal j/k ratios. Examination of urine for monoclonal immunoglobulins may be underused, and recommendations by some to use serum free light chain assay in place of, rather than as an adjunct to, urine electrophoresis are not warranted. Upon completion of this activity you will be able to: discuss the clinical usefulness of serum free light chain assay and j/k ratio. outline the limitations of the serum free light chain assay in investigation of monoclonal gammopathies. apply urine protein electrophoresis and urine protein immunofixation electrophoresis in investigation of monoclonal gammopathies. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit TM per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Exam is located at Serum protein electrophoresis (SPEP) and serum protein immunofixation electrophoresis (SIFE) are commonly used as screening tests in the diagnosis of monoclonal gammopathies, and the findings of SIFE are currently the gold standard for identification of monoclonal immunoglobulins, both intact immunoglobulins as well as free light chains. 1 Additional investigations recommended by the International Myeloma Workshop Consensus Panel 3 are serum free light chain quantification (SFLC), urine protein electrophoresis (UPEP), and urine immunofixation electrophoresis (UIFE). 1,2 Bone marrow examination is often carried out to study the morphologic characters, immunoglobulin markers, and cytogenetic and genetic/dna markers. 3 The common disorders associated with monoclonal immunoglobulin production are monoclonal gammopathy of undetermined significance, smoldering plasma cell myeloma, CME/SAM American Society for Clinical Pathology, All rights reserved. For permissions, please journals.permissions@oup.com 207 Am J Clin Pathol 2016;146: DOI: /ajcp/aqw099

2 Singh /SERUM FREE LIGHT CHAIN ASSAY AND j/k RATIO PERFORMANCE and multiple myeloma/symptomatic plasma cell myeloma. Monoclonal immunoglobulins are also present in other disorders, such as Waldenström macroglobulinemia, chronic lymphocytic leukemia, lymphoma, amyloidosis, autoimmune disorders, light chain deposition disease, heavy chain deposition disease, light and heavy chain deposition disease, and POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes). 1-8 The quantity of monoclonal immunoglobulin (M-protein) in plasma cell myeloma serves as a tumor marker, and quantity of the monoclonal protein reflects tumor mass. 6,9 The excessive production of one type of light chain, as part of monoclonal gammopathy, often distorts the ratio of free j and k light chains in serum. In normal circumstances, j light chains are produced in greater abundance than k light chains. The usual ratio of j/k light chains, in healthy individuals, in serum is between 0.26 and In patients with renal failure, there is greater retention of serum free light chains. Due to differences in the polymerization propensity of the two chains, with k chains being more prone to polymerization, the usually accepted normal ratio of j/k chains has been revised. The renal ratio of 0.37 to 3.17 is recommended for determination of an abnormal j/k ratio in samples from patients with elevated serum creatinine. The ratio of serum free j and k chains is also distorted in circumstances associated with a polyclonal increase in immunoglobulins as part of the response to infections, autoimmune disorders, chronic liver disease, and so on The two tests, SPEP and SIFE, are effective in recognizing more than 90% of the specimens with a monoclonal immunoglobulin. 21 These methods identify virtually all specimens with intact monoclonal immunoglobulins and about half of the disorders with monoclonal light chain production. Monoclonal gammopathies with intact immunoglobulins account for around 80% of the lesions. In the remaining 20% or so of the cases, some of the light chain monoclonal proteins and nonsecretory lesions may escape detection by SPEP/SIFE alone. Adding UPEP and UIFE is likely to improve the diagnostic yield by recognizing light chain disorders. However, UPEP and UIFE are not performed routinely and are underused tests, and data have been presented to counter the suggestion that SFLCA could replace urine electrophoretic studies. 23 The International Myeloma Workshop Consensus Panel 3 has recommended performing the SFLC assay to facilitate the recognition of nonsecretory and light chain disorders. 1 The US Agency for Healthcare Research and Quality, under its Effective Health Care Program, noted, among others, the following gaps: 1. Lack of studies of a non-diseased population as a comparison group (in order to accurately assess outcomes) (e.g., false positives, and true negatives). Studies of only patients with disease reflect the extreme end of the spectrum of disease severity and overestimate the proportion of patients with positive results. (The preceding statement was read to mean that serum free light chain quantification should be carried out in patients without monoclonal gammopathies to assess the false-positive rate in a control group of patients and not just healthy participants.) 2. Of the topics nominated for future research, the following was ranked at number 7 of 16 total recommendations: Context of diagnostic testing: What is the diagnostic performance of the SFLC assay either alone or in combination with traditional tests, compared with traditional tests, in a high-risk population with regard to: a. Conditions such as antigen excess and polyclonal gammopathy (which can result in false negative and false positives, respectively) b. Diminished kidney function and the need for different reference ranges in kidney failure c. Technical variations in commercial assays, assay platforms, or in house (hospital) vs. central lab testing d. Resolving discordant SFLC and traditional testing results in different PCDs (Plasma cell dyscrasias) e. Diagnostic reclassification based on SFLC testing. 24 In the routine processing of SPEP/SIFE tests, it was noted that patients with polyclonal gammopathy and elevated serum creatinine often had abnormal j/k ratios. Therefore, a retrospective analysis of the results of SFLC assays, j/k ratios, findings on SPEP/SIFE and UPEP/UIFE, bone marrow examination, immunoglobulin quantification, and clinical data was carried out to assess the performance of SFLC assay in patients who were determined not to have monoclonal immunoglobulin, neoplastic lymphocyte, or plasma cell disorders. Materials and Methods The study was conducted at a tertiary care, medical school affiliated, 500-bed medical center with a bone marrow transplant program, located in the Southeastern United States. The protocol was approved by the institutional review board. Serum and urine protein electrophoreses and immunofixation electrophoreses were carried out using a Helena SPIFE 3000 instrument. Serum free light chain assays were performed on an Advia 1800 analyzer, with reagent kits from The Binding Site (Birmingham, UK). The laboratory performing the analyses is a Clinical Laboratory Improvement Amendments certified laboratory in a medical school affiliated hospital. 208 Am J Clin Pathol 2016;146: American Society for Clinical Pathology 208 DOI: /ajcp/aqw099

3 AJCP /ORIGINAL ARTICLE Data were collected on all patients who had SPEP/SIFE and SFLC assays from 2012 to September Medical records were reviewed to ascertain the diagnoses and patients with documented or probable monoclonal gammopathy, including patients with amyloidosis, and lymphomas/leukemias were excluded. A total of 270 patients met these criteria and had 297 instances in which the SFLC assay was done. The following additional information was collected through chart review: age, race, sex, serum concentrations of j and k light chains and j/k ratio, concentration of b- andcglobulins as determined from SPEP analysis, and serum creatinine levels. Performance of UPEP/UIFE tests, serum immunoglobulin quantification, and bone marrow examinations was noted, if performed. Clinical information was reviewed to assess main diagnoses/findings and the putative reason for SPEP/SIFE and SFLC assay requests. Clinical notes and data from SPEP/SIFE, UPEP/UIFE, and bone marrow examinations were used to exclude monoclonal gammopathies. Patients were further stratified into seven groups based on the main diagnosis and/or reason for SPEP and SFLC assays. The j/k ratio was considered normal if within 0.26 to 1.65 for patients with a serum creatinine value of 1.3 mg/dl or less. The renal j/k ratioof0.37to3.17wasappliedtopatientswith a serum creatinine of more than 1.3 mg/dl. The renal ratio was ignored in some analyses, as indicated in the Results section. Descriptive findings about the proportion of patients with an abnormal j/k ratio for usual and renal ratios were calculated, and k and j dominant ratios were identified separately. Patients were stratified into quartiles based on their c-globulin concentration, and prevalence of abnormal j/k ratios was compared among the quartiles using the v 2 test. The observation set was also divided into those with a c-globulin concentration of 1.6 g/dl or more and those with c-globulin levels of less than 1.6 g/dl. The usual and renal j/k ratios between the two groups were compared by the v 2 test. Results The salient clinical findings in the patient population who had SPEP/SIFE and SFLC assays are listed in Table 1 The corresponding findings for dominant j and k light chains and percent abnormal usual j/k ratios for the various groups are also presented in Table 1. In patients without neoplastic disorders of the lymphoid and plasma cell system, one of the most common categories of patients was one with multiple disorders common in the adult US population seeking health care at a tertiary care facility (eg, obesity, diabetes, hypertension, chronic obstructive airway disease, congestive heart failure, gastroesophageal reflux disease, hyperlipidemia, hypothyroidism, and renal impairment). Patients with neurologic disorders comprised the largest group tested. All patients with unexplained neuropathy and many patients with other disorders, such as motor neuron disease and multiple sclerosis, had SPEP/SIFE performed at initial contact with the patient. Patients referred to the hematology/oncology service for investigation of cytopenias were the next largest group, after the miscellaneous group. Elevated c-globulins or abnormal albumin/globulin ratio often prompted rheumatology to order these tests. The category labeled myeloma symptoms usually consisted of patients with hypercalcemia and/or lytic bone lesions seen in radiologic examinations done for other reasons. Infections accounting for the trigger for SPEP/SIFE and SFLC were generally hepatitis C and human immunodeficiency virus (HIV), with septicemia adding a smaller number. Hepatitis C and HIV infections usually prompted the study due to elevated c-globulins. Patients from the gastroenterology and liver service, other than patients with hepatitis C, usually had chronic liver disease, often due to alcohol abuse, resulting in elevated serum globulins. Unlike in patients with monoclonal gammopathies, the patients without monoclonal gammopathy generally did not Table 1 Patient Distribution by Pathology/Diagnoses and Incidence of Abnormal j and k Light Chain Ratios a Diagnostic Group No. (%) of Patients SFLC Assays, No. Dominant k, No. Dominant j, No. % Abnormal j/k Neurology 72 (26.8) Hematology/oncology 46 (17.1) Rheumatology 33 (12.3) Myeloma symptoms 23 (8.6) Infection 21 (7.8) Gastrointestinal/liver 14 (4.8) Other/miscellaneous 61 (22.7) Total SFLC, serum free light chain quantification. a The diagnostic group was assigned based on the dominant disease process or the service requesting the laboratory test. Dominant k included the observations with a j/k ratio of - less than 0.26, and dominant j included those with a j/k ratio of more than The neurology patients included those with peripheral neuropathy, motor neuron disease, multiple sclerosis, and seizures. Hematology/oncology patients were usually referred for investigation of cytopenias. Myeloma symptoms included anemia, bone lesions, and hypercalcemia. Elevated creatinine alone was not a usual reason for serum protein electrophoresis/serum protein immunofixation electrophoresis examination. The other/miscellaneous group had the usual cohort of patients, in tertiary care, with multiple diagnoses (eg, obesity, hypertension, congestive heart failure, chronic obstructive airway disease, hypothyroidism, renal failure). The usual j/k ratio of 0.26 to 1.65 was used to assess dominant j and k light chains and abnormal j/k ratio. American Society for Clinical Pathology Am J Clin Pathol 2016;146: DOI: /ajcp/aqw099

4 Singh /SERUM FREE LIGHT CHAIN ASSAY AND j/k RATIO PERFORMANCE have repeat studies of the SPEP/SIFE and/or SFLC assay. Of the 270 unique patients, 18 had repeat studies done. One patient had seven observations, another had five, two had three observations each, and 14 had two observations each. The raw data about the various observations are presented in tables. The largest numbers of missing observations were for SIFE. SIFE was often done at the discretion of the sign-out pathologist, and as alluded to in earlier publications, the specimens that did not warrant SIFE examination were not tested by immunofixation electrophoresis. 8,25 In two instances, SPEP data were missing, but SIFE had been done, as sometimes only SIFE is requested by the ordering physician. Creatinine data were missing in five instances, usually in patients who had limited contact with the medical center Table 2. The missing findings are responsible for the minor variations in the results among various tables. For example, the missing creatinine data (n ¼ 5) in observations listed in Table 2 and exclusion of these patients in the data in Table 2 Missing Data and Relative Utilization of Urine and Bone Marrow Examinations a Characteristic No. Patients 270 Observations 297 Missing data Creatinine 5 SPEP 2 SIFE 52 UPEP and UIFE Attempted 49 Done 53 Bone marrow Done 65 SIFE, serum protein immunofixation electrophoresis; SPEP, serum protein electrophoresis; UIFE, urine immunofixation electrophoresis; UPEP, urine protein electrophoresis. a The missing data account for the minor variations in the number of patients and observations among the different tables. Urine specimens were submitted for electrophoretic analysis in 102 instances, but the specimen was suitable for analysis in 53 of these cases; thus, UPEP/UIFE was done in only 19.6% of the patients. Bone marrow examination was done in 24.1% of the patients. Table 3 j/k Ratios Stratified by Normal and Elevated Serum Creatinine Levels a Characteristic Creatinine 1.3 mg/dl (n ¼ 211) k Dominant, No. 5 3 j Dominant, No Abnormal j/k ratio, % Creatinine >1.3 mg/dl (n ¼ 72) a Total abnormal j/k ratio ¼ 30.1%. Renal j/k ratio (reference range, ) was used for patients with serum creatinine of more than 1.3 mg/dl. The total j/k abnormal ratio represents a conservative interpretation of the data. The total number of observations (292) reflects five missing creatinine values. Even at a conservative estimate of 30%, the false-positive j/k ratio requires that an abnormal j/k ratio be followed up by more definitive investigation to ascertain the presence of a monoclonal gammopathy. Table 3 account for the differences in the abnormal j/k ratio figures and the number of observations. Exclusion of two additional patients who did not have c-globulin quantification due to lack of SPEP examination reduced the total number of valid observations to 290, as presented in Table It is noteworthy that UPEP/UIFE were used infrequently. In 37.8% of the instances, a request for UPEP/UIFE was received, and in nearly half of the instances, 49 of 102, the specimens were deemed unsuitable for UPEP due to inadequate volume and/or low protein concentration, and only 19.6% of the samples had a valid UPEP/UIFE examination. As a result of this review, the laboratory has changed its policy of not performing UPEP/UIFE if the specimen had a protein concentration of less than 15.0 mg/dl. All specimens will henceforth be processed for UPEP/UIFE irrespective of the protein concentration by concentrating the urine sample as needed. Bone marrow examination was done in 24.1% (65 of 270) of patients. It is worth noting that even in the patients without evidence of neoplastic disorders, bone marrow examination was done in 13% of the patients. It is not implied that bone marrow examination is done too frequently since these patients often had cytopenias and/or lytic bone lesions that warranted a bone marrow examination; nevertheless, the low frequency of a noninvasive urine examination is worth emphasizing. The incidence of abnormal j/k ratios, using the usual yardstick of a normal j/k ratio of 0.26 to 1.65, was 36.4% (Table 1). If a more conservative yardstick is used for Table 4 Effect of Normal and Elevated Polyclonal c-globulin Levels on j/k Ratios in Patients With Normal and Elevated Creatinine Levels a Abnormal j/k Ratio, No. c-globulin <1.6 g/dl c-globulin 1.6 g/dl (n 5 197) b (n 5 93) c Characteristic Dominant k Dominant j Dominant k Dominant j Usual j/k ratio d Renal j/k ratio e a A c-globulin concentration greater than 1.6 g/dl was considered elevated (ie, hypergammaglobulinemia). 27 The abnormal j/k ratio is significantly more prevalent in samples with a c-globulin of 1.6 g/dl or more when using the usual j/k reference range ( ) as well as the renal j/k ratio ( ) for values of serum creatinine more than 1.3 mg/dl. For the usual j/k, the reference range of 0.26 to 1.65 was used for all creatinine values in each of the c-globulin groups (ie, <1.6 g/dl and 1.6 g/dl). For the renal ratio, the values in each of the two c-globulin groups were stratified into two groups each, one with a serum creatinine of 1.3 mg/dl or less and the other with a serum creatinine of more than 1.3 g/dl. The reference range of 0.23 to 1.65, as applied to the observations with a serum creatinine of 1.3 mg/dl or less and a renal reference range of 0.37 to 3.17, was applied to the observation with a serum creatinine of more than 1.3 mg/dl. The number (290) is smaller than the number (295) in Table 5 due to five missing creatinine values. b Percent abnormal j/k ratio: usual, 30.5%; renal, 23.9%. c Percent abnormal j/k ratio: usual, 54.8%; renal, 36.6%. d Usual j/k ratio: comparison of less than 1.6 g/dl and 1.6 g/dl or more for c-globulins, v 2 test: P ¼ e Renal j/k ratio: comparison of less than 1.6 g/dl and 1.6 g/dl or more for c-globulins, v 2 test: P ¼ Am J Clin Pathol 2016;146: American Society for Clinical Pathology 210 DOI: /ajcp/aqw099

5 AJCP /ORIGINAL ARTICLE patients with elevated creatinine, the incidence of an abnormal j/k ratio was still remarkably high at 30.1%. The renal j/k ratio value of 0.37 to 3.17 was applied to samples with a creatinine value of more than 1.3 mg/dl, as shown in Table 3 and Table 5. The incidence of abnormal j/k ratios appeared to increase with increasing c-globulin concentration. This increase in abnormal j/k ratios was almost entirely due to j light chain dominance of the j/k ratio. When observations were stratified into quartiles, based on the concentration of c-globulins in the sample, the differences among the quartiles were statistically significant. However, most of the difference seems to reside in the quartile with a c-globulin concentration of more than 1.78 g/dl. There was a significantly higher rate of abnormal j/k ratios among patients with elevated (>1.6 g/dl) c-globulins but with normal serum creatinine (54.8%) compared with those with an elevated creatinine (36.6%). When c-globulin levels were normal (<1.6g/dL), 30.5% of those with normal serum creatinine levels had increased j/k ratios compared with 23.9% of those with abnormal creatinine values. The differences in the rates of abnormal j/k ratios in low and high c-globulin groups were statistically significant at P <.05 for the usual as well as renal ratios (Table 4). Discussion The assay for serum free light chains has been a valuable addition to the laboratory diagnosis of monoclonal immunoglobulin disorders. The altered ratio of j and k light chains reflects overproduction of one type of light chain and suggests that there may be a monoclonal overproduction of that light chain or an immunoglobulin with that particular light chain It is generally recognized that adequate screening for monoclonal immunoglobulins should include SPEP/SIFE and UPEP/UIFE. However, urine examination often does not take place due to multiple, generally logistical, issues. SFLC assays can be carried out on the same sample as that used for SPEP/SIFE, and a separate sample, namely urine, is not required. The recommendation of the International Myeloma Workshop Consensus Panel 3 includes routine testing for Table 5 Frequency of Abnormal j/k Ratios Among Different Quartiles for c-globulin Concentrations a SFLC in the screening for monoclonal gammopathy. It could be argued that UPEP/UIFE is a better test than the SFLC assay since the former discloses the actual presence of a monoclonal immunoglobulin or monoclonal light chain, not just alteration in the ratio of the concentration of two chains without direct evidence for the presence of a monoclonal light chain. 27 Even the International Myeloma Workshop Consensus Panel 3 states that serum-free light chain estimation does not obviate the need for 24-hour urine studies. 1 Urine studies are particularly valuable in patients with amyloidosis However, as has been pointed out and noted in the data in this communication, urine examination is often not carried out and has been noted to be more expensive than the SFLC assay. 28 Some of this underutilization may be due to restrictive practices in the laboratories. For example, this laboratory did not conduct UPEP/UIFE if the total protein concentration was below 15.0 mg/dl. The change in this practice will be monitored to determine if testing all urine samples, irrespective of the protein concentrations, will be an improvement. Immunoglobulin light chains are produced in excess of heavy chains, and all individuals have free light chains in circulation as well as in urine. However, these proteins are not monoclonal. The j lights are produced in excess of k chains in that the former account for about 60% of the total light chains. This ratio is much higher in some animals (eg, in mice, the usual ratio of j to k light chain production is 95:5). 29,30 SIFE provides a rough estimate of the light chain content of the various immunoglobulins, and usually j chain staining is stronger than that for k light chains. It is understood that the staining intensity depends on other factors as well, in addition to the light chain content of immunoglobulin molecules being stained after SIFE. In a polyclonal increase in immunoglobulins that usually accompanies inflammatory responses, especially chronic inflammation and chronic liver disease, there is usually a greater production of immunoglobulins with j light chains. This excessive production of j chain immunoglobulins results in increased amounts of serum free j light chains, and this excess may be sufficient to result in an abnormal SFLC Characteristic Quartile 1 Quartile 2 Quartile 3 Quartile 4 Quartiles 1-3 b c-globulin range, g/dl k Dominant abnormal, No j Dominant abnormal, No Observations, No a Observation by c-globulin concentration (n ¼ 295), using the usual j/k ratio (reference range, ). Using the usual j/k ratio, there is statistically significant increase in the rate of the false-positive j/k ratio with increasing serum c-globulin concentration. There appears to be sharp demarcation in the increase in the false-positive j/k ratio in patients with a serum c-globulin concentration of 1.78 g/dl or more. The normal c-globulin reference range is 0.6 to 1.7 g/dl. Comparison among quartiles by v 2 test: P ¼ b Comparison of quartiles 1 to 3 vs quartile 4 by v 2 test: P ¼ American Society for Clinical Pathology Am J Clin Pathol 2016;146: DOI: /ajcp/aqw099

6 Singh /SERUM FREE LIGHT CHAIN ASSAY AND j/k RATIO PERFORMANCE j/k ratio even with normal renal function and more excretion of j chains due to their usual monomeric configuration, in contrast to the dimeric configuration of k chains. The often cited claim that renal failure and a polyclonal increase in c- globulins due to inflammation increase the concentrations of both j and k light chains, without altering their ratio, needs to be reexamined in light of the finding of abnormal j/k ratios, particularly in patients with a polyclonal increase in c-globuglobulins. 21,22,28,31,32 A polyclonal increase in c-globulins has been shown to interfere with the expected j/k ratio in patients with monoclonal gammopathy and produce false-positive and false-negative j/k ratios. Primary antibody deficiency has also been shown to produce abnormal j/k ratios. 16,32-35 Impairment of renal function often results in increased serum levels of free immunoglobulin light chains. The glomerular filtration is dependent on protein size, and variable polymerization of the light chains differentially affects the retention of free light chains. The k light chains tend to dimerize more than the j light chains and may be retained preferentially. Usual normal range for the j/k ratio of 0.26 to 1.65 has been modified to address the changes imposed by renal impairment, and a renal j/k ratio of 0.37 to 3.17 has been proposed for patients with azotemia The SFLC assay results may be further affected by the polymerization/aggregation of serum free light chains by impairing the antibody recognition of the sites specifically targeted by the antibodies used in the assay. The visual recognition of a monoclonal intact immunoglobulin or monoclonal light chain in SPEP/SIFE and UPEP/ UIFE is generally an incontrovertible evidence for the presence of a monoclonal immunoglobulin. The altered j/k ratio does not provide the same information and merely suggests an alteration in the relative serum content of the two light chains. This altered ratio may be due to excessive monoclonal production of one of the light chains, excessive production of one of the light chains as part of an acute or more generally chronic immune response, impairment of renal function, or a combination of all of these. Serum free light chain assay does not detect all instances of monoclonal gammopathy of either intact immunoglobulins or even light chain monoclonal gammopathy. 33,35 Thus, the presence of an abnormal SFLC ratio does not equate to the presence of a monoclonal gammopathy, and observation of a normal ratio does not exclude the presence of a monoclonal gammopathy. It is not the intent of the foregoing statement to imply that the SFLC assay does not have clinical utility or merit. The statement is meant to emphasize that an abnormal SFLC ratio requires further study to establish the presence of a monoclonal gammopathy. The starting point of that additional investigation ought to be examination of urine by UPEP/UIFE. Urine examination ought to precede bone marrow examination. It is also understood that in many clinical circumstances, bone marrow examination may be warranted due to other reasons (eg, cytopenias and lytic bone lesions). The observation, as noted in this study, of an incidence of an abnormal j/k ratio of 30.1%, using the conservative criteria, without any evidence of monoclonal proliferation of plasma cells, represents a high rate of a spurious signal in a screening assay. It appears that not all patients with an abnormal SFLC ratio warrant further investigation, especially in the presence of a polyclonal immune response. Patients with monoclonal gammopathy generally have reduction in the normal or background immunoglobulins, further complicating the interpretation of the j/k ratio Thus, patients with hypogammaglobulinemia and an abnormal SFLC ratio should have further investigation that should start with UPEP/UIFE. In patients with a normal immunoglobulin concentration and lacking other indications for plasma cell proliferative disorder, yearly follow-up may be sufficient. False-positive and falsenegative results with SFLC assay have been reported earlier, including a false-positive rate of 34.1%. 16,27,32-35 SFLC assay results have been shown to have other uses (eg, as an indicator for worse outcome in patients with multiple disorders), not just those associated with monoclonal gammopathy. 29,37-42 Although it is not clear if the excess light chains reflect the underlying disorder or are a contributory factor in the worse outcome (eg, by impairing renal function), an abnormal SFLC ratio has also been cited as a marker for higher risk for progression of smoldering myeloma to plasma cell myeloma. 43 Due to the shorter half-life of light chains compared with intact immunoglobulins, it has been proposed that monitoring the SFLC may provide an earlier indication of response, or lack thereof, to treatment. 44,45 However, other investigators did not find any advantage to using the SFLC assay over monitoring the concentration of intact monoclonal protein. 46,47 Quantification of Hevylite chains (The Binding Site) has been proposed for quantification of immunoglobulin A monoclonal immunoglobulins. The latter often migrate in the b region and overlap bands for transferrin or complement. Most laboratories deal with this by measuring the concentration of the combined peak and noting the change in the concentration of the peak as an indicator of the progress of the disease. 48 As in the case of SFLC, the Hevylite chain does not measure the concentration of the monoclonal protein, just a specific heavy and light chain combination. The clinical usefulness of the assay remains to be determined. Whether excess polyclonal serum free light chains induce renal tubular damage, like monoclonal light chains, has not been established. Monoclonal light chains may be more toxic due to their propensity to crystalize. It would be difficult to distinguish renal damage from excess polyclonal light chains from that resulting from the underlying pathology associated with polyclonal gammopathy. 49, Am J Clin Pathol 2016;146: American Society for Clinical Pathology 212 DOI: /ajcp/aqw099

7 AJCP /ORIGINAL ARTICLE This study shares the general weaknesses of retrospective observational studies, which may not be confirmed in a controlled randomized trial. In a prospective trial, collection of urine specimens and immunofixation electrophoresis would help in excluding monoclonal light chain disorders that may have been missed by SPEP/SIFE. It would be unreasonable to perform bone marrow examination solely based on an abnormal j/k ratio, since more than one study has shown a sizable false-positive rate for the j/k ratios. 15,32 34 Around 13% of the patients in this study did have bone marrow examination for other reasons, usually cytopenias and lytic bone lesions. Most of the patients had only one SPEP/SIFE and SFLC study and only a short-term, unstructured follow-up based on clinical needs. Longer term follow-up would have been desirable to rule out the development of monoclonal plasma cell disorders in patients with abnormal j/k ratios. Abnormalities in serum free light chains have been noted in the preclinical phase of monoclonal lymphoid disorders and autoimmune disorders. 51,52 The retrospective observational nature of the study is also its strength in that all patients who had the SFLC assay done were included, unlike the contrived circumstance of a controlled trial with its inclusion and exclusion criteria, which are seldom, if ever, replicated in routine practice. Review of the 2,409 episodes of testing in 468 patients with monoclonal gammopathy revealed a high false-negative rate for SFLC assay, especially for lesions with k light chains. Because (1) an abnormal j/k ratio is not diagnostic of monoclonal gammopathy, (2) a normal j/k ratio does not exclude monoclonal gammopathy, (3) there is a high falsepositive rate for abnormal j/k ratio in samples without monoclonal gammopathy, and (4) a high rate of false-negative results for j/k ratio is noted in samples from patients with monoclonal gammopathy, the usefulness of SFLC assay, in routine clinical testing, is questionable. Corresponding author: Gurmukh Singh, MD, PhD, MBA, Dept of Pathology, Augusta University, Medical College of Georgia, Division of Clinical Pathology, th St, BI 2008A, Augusta, GA 30912; gurmukhsinghmdphd@yahoo.com. Acknowledgments: The technical assistance of Teresa Thomas, John Anderson, Valerie Payne, and Joyce Oliver is gratefully acknowledged. Natasha Savage, MD, kindly reviewed and edited the manuscript. References 1. Dimopoulos M, Kyle R, Fermand JP, et al. 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