Comparison of two serum free light chain assays for the diagnosis of primary plasma cell malignant proliferative disease

Abstract Background and aims The serum free light chain assay (sFLC) is well established for aiding in the diagnosis, prognosis, and monitoring of plasma cell proliferative disorders. There are currently two commercially available sFLC immunoassays, Freelite, based on polyclonal antibody technology, and N Latex FLC, based on monoclonal antibodies. This study aimed to compare the analytical and clinical performance of these two assays in a Chinese population. Methods This study included 74 consecutive patients who were newly diagnosed with monoclonal gammopathies (MGs) including multiple myeloma (MM), AL amyloidosis, and light chain deposition disease (LCDD) between January 2014 and May 2015 at the First Affiliated Hospital of Zhejiang University. Alongside serum and urine electrophoresis analysis, the serum samples were retrospectively tested with both sFLC assays according to the manufacturers' instructions. Results The two sFLC assays showed a moderate correlation for κFLC (Passing‐Bablok slope = 0.645, coefficient of determination (R 2) = 0.83, and Spearman coefficient = 0.904). However, for λFLC, a poor correlation was found (Passing‐Bablok slope = 0.690, R 2 = 0.39, and Spearman coefficient = 0.852). The concordance rate of κFLC, λFLC, and κ/λ FLC ratio were 83.78%, 75.68%, and 86.49%, respectively. The clinical sensitivity of the κ/λ ratios were 83.8% for the Freelite assay and 75.7% for the N Latex FLC assay. Conclusion Although the concordance and the clinical sensitivity of the two assays appeared comparable, a number of discrepancies were observed. There is a low correlation between the two assays in clinical practice, suggesting that the assays are not equivalent and, thus, current IMWG guidelines, based on Freelite, cannot be cross‐applied to N Latex FLC.


| INTRODUCTION
Monoclonal plasma proliferative disorders include monoclonal gammopathy of undetermined significance (MGUS), solitary plasmacytoma, multiple myeloma (MM), and AL amyloidosis (AL). 1 In the past, tests for measuring the circulating monoclonal immunoglobulins, such as serum electrophoresis and immunofixation, have been used alongside urine electrophoresis for the identification of such disorders. [1][2][3] However, these traditional methods are not sensitive enough to identify nonsecretory MM, many AL patients, and other light chain disorders. 1,[3][4][5] In 2001, a new assay based on the use of polyclonal antisera for the detection of serum free light chains (sFLCs) was developed (Freelite; The Binding Site Group Ltd, UK). 6 The Freelite assay can accurately detect and quantify both kappa (κ) and lambda (λ) free light chains (FLC) through polyclonal antibodies recognizing a variety of FLC epitopes. The ratio of κ/λ FLC is a sensitive marker of monoclonality, which is key to the clinical utility of the assay. Because of the greater analytical sensitivity of the Freelite assay for identifying monoclonal sFLC, the International Myeloma Working Group (IMWG) have recommended that sFLC testing is included as part of the screening algorithm for MM and related disorders, alongside serum protein electrophoresis (SPE) and serum immunofixation electrophoresis (IFE). 1,7 The IMWG recently updated the MM diagnostic criteria to include biomarkers of malignancy (also known as the SLiM criteria), which include an involved/uninvolved Freelite serum FLC ratio greater than or equal to 100 (involved FLC should more than 100 mg/L). 7

| Immunofixation electrophoresis
Serum and urine IFE analyses were performed using the Helena SPIFE 3000 system (Helena, USA), according to the manufacturer's instructions. All results were evaluated by two independent readers.

| Statistics
The Clinical and Laboratory Standards Institute (CLSI)(EP09-A2-IR) guide to Method Comparison and Bias Estimation using Patient Samples (EP-09-A2-IR) states that an R 2 ≥ 0.95 is required for establishing that two assays are equivalent. 12 We, therefore,

| Patient characteristics
Samples were from 74 patients, who were recently diagnosed with an MG using commonly accepted criteria. 1,13 The median age was 63 years (interquartile range 56.5 to 68). The male to female ratio

| Correlation between the two FLC assays
The recommended reference ranges for the Freelite assay and the N Latex FLC assay were used for the analysis. The concentrations of the κ and λ sFLCs as measured by the two assays are plotted in

| Concordance between the two FLC assays
The data was analyzed according to the manufacturer's reference ranges and categorized into low (below the reference range), normal (within the reference range), and high (above the reference range), to assess qualitative concordance.
The concordance rate of κ FLC, λ FLC, and κ/λ FLC ratio were 83.78%, 75.68%, and 86.49%, respectively (Figure 4). Concordance for κ FLC was good, with Cohen kappa of 0.75, but it was only moderate for λ FLC, with a Cohen kappa of 0.644. For κ/λ FLC ratio, the Cohen kappa was 0.59, based on whether the results were simply normal (within the manufacturer's reference range for the κ/λ FLC ratio) or abnormal (outside the reference ranges) and, thus, highlighting only moderate concordance between the two assays.
For 10 patients, the results for the κ/λ FLC ratio were discordant (13.5% of patients) ( Table 1). Eight had an abnormal Freelite κ/λ FLC ratio and a normal N Latex FLC ratio, whereas two were normal according to Freelite and abnormal according to N Latex FLC. Two of the patients with discordant results had negative electrophoresis, and in both cases, the monoclonal FLCs were identified by the Freelite assay, but the N Latex FLC ratio was within the normal range. The Freelite results were in agreement with the clinical diagnosis of NSMM and LCDD for these patients.

| Clinical sensitivity of the κ/λ ratio of FLC assays
On the basis of the clinical diagnosis, the sensitivity of the κ/λ FLC ratio of the two methods to detect the presence of monoclonal FLCs was calculated. For the Freelite assay, sensitivity was 83.8% (62/74), and for N Latex FLC assay, it was 75.7% (57/74), indicating the Freelite assay has a higher clinical sensitivity. One patient with renal     The Freelite assay has shown greater sensitivity than the N latex FLC assay in the detection of monoclonal FLCs in serum. 9 We observed that in samples with extremely high levels of FLC, the difference between the results reported by the two assays was more pro- So, in considering measurable disease, Freelite appears to have more sensitivity. The data presented here indicates that because of the differences between the involved FLC concentrations measured by the two assays, guidelines either need to consider different cutoffs for the different FLC assays or a single cutoff for both assays that includes as many patients with measurable disease as possible.
Some limitations in the present study should be noted. First, our sample size was relatively small, although our conclusions are in accordance with other studies. Secondly, this is a retrospective study, and the two FLC assays were performed on frozen serum and run on different platforms. These factors may introduce bias into the study, but we believe they should not significantly affect the comparison of the two FLC assays.
Our clinical study demonstrated that although the two assays perform similarly in the detection of FLC, a number of discrepancies were observed. There is a low correlation between the two assays in clinical practice, suggesting the assays are not equivalent. Therefore, the current international guidelines based on the Freelite assays cannot be cross-applied to the N Latex FLC assay. More data are required to establish FLC criteria for N Latex FLC assay.

FUNDING
This work is supported by the Natural Science Foundation of Zhejiang Province (LY18H080001). The funder played no role in study design; collection, analysis, and interpretation of data; writing of the report; or the decision to submit the report for publication.