Method comparison of three serum free light chain assays on the Roche Cobas 6000 c501 chemistry analyzer

Free light chain assays

Serum free light chain measurements were performed on the Cobas 6000 c501 using Freelite (reagent lot number 712007 for FLC kappa and 711571 for FLC lambda, The Binding Site, Birmingham, UK), Diazyme (reagent lot number 491684 for FLC kappa and 479603 for FLC lambda, Diazyme Laboratories, Poway, USA) and the KLoneus assay (reagent lot number 511SK-54 for FLC kappa and 511SL-54 for FLC lambda, Trimero Diagnostics, Barcelona, Spain) according to the manufacturer’s guidelines. Characteristics of the different sFLC assays are presented in Table 1.

Samples

In this study, 145 patients are evaluated, which were used in a previous method comparison of different free light chain assays [10]. This includes 119 patients with monoclonal gammopathies, including multiple myeloma (MM, n=62 of which five newly diagnosed patients), kappa light chain multiple myeloma (LCMM, n=19), lambda LCMM (n=8), smoldering multiple myeloma (sMM, n=7), AL amyloidosis (n=13 of which two newly diagnosed patients), Waldenström macroglobulinemia (n=5) and MGUS (n=5). The diagnosis was performed according to the criteria of the International Myeloma Working Group [1], including performing immunofixation of serum and/or urine. The patients were not treated with monoclonal antibodies during collection of the samples. In total, 26 control patients are included from patients without M-protein (n=11) and patients with chronic kidney disease without M-protein (n=15). The samples were collected in 8.5 mL SST vacutainers (Becton Dickinson, UK) and separated within 1 h upon arrival at the laboratory. Samples were stored at −20 °C until analysis and thawed prior to analysis. An additional 25 reference sera were derived from healthy controls for the validation of the reference intervals of the three assays. The healthy adult controls were volunteers from our laboratory, who had no active disease and were not on medication.

The study was performed in according to the Helsinki guidelines and was approved by the institutional Medical Ethics Review Board (Erasmus MC 2017-415).

sFLC method comparison

Method comparison between the three different FLC assays on the Cobas 6000 c501 were performed according to the CLSI EP9 guidelines. The entire dynamic range of FLC concentrations was covered by the 145 selected serum samples. The reference ranges from the package inserts were used (see Table 1). The adjusted FLC-ratio was used in the case of patients with renal impairment defined as eGFR <60 mL/min/1.73 m² using creatinine concentration. The reference interval for creatinine is 55–90 μmol/L for females and 65–115 μmol/L for males (Roche Diagnostics, Almere, The Netherlands). The FLC-ratio (κ/λ) is calculated and divided into three groups: below (low), above (high) or within the reference range (normal). In patients with renal impairment, adjusted reference ranges (if applicable) were applied to categorize the FLC-ratio.

Validation of reference intervals

Validation of the reference intervals mentioned in the kit-inserts of all three methods was performed based on CLSI C28-A3 guidelines [11]. Reference samples from 25 healthy volunteers were measured with the three methods. The percentage of results that fell within the reference limit of each test was determined. When ≥90% of these samples fell within the reference interval the reference intervals were considered as verified.

Statistics

Bland-Altman evaluation, Passing-Bablok regression and Kendall’s Tau correlation coefficient analysis were performed with the results for κFLC and λFLC obtained in three FLC assays. Nonparametric methods were used since the results are not normally distributed. The results of the categorized FLC-ratio (κ/λ) were used to perform concordance analysis. The Cohen kappa coefficient (κ) is used to interpret the concordance analysis. Values between 0.81 and 1.00 are defined as almost perfect agreement, between 0.61 and 0.80 as high agreement, between 0.41 and 0.60 as moderate agreement, between 0.21 and 0.40 as fair agreement [12]. A κ-value less than 0.20 is defined as none to slight agreement. Statistical analysis was performed using Analyse-IT for Microsoft Excel (v5.40, Analyse-IT Software Ltd., Leeds, UK).

Method comparisons

Three different FLC assays were used to measure 119 sera from patients with monoclonal gammopathies and 26 sera from control patients without M-protein. In Table 2, the agreement of κFLC, λFLC and FLC-ratio are presented by Bland-Altman and Passing-Bablok for all combinations. Graphical representation of the Bland-Altman and Passing-Bablok analysis of κFLC and λFLC are represented in Supplementary Figure 1 and Supplementary Figure 2. Results of the FLC-ratio are represented in Figure 1.

Figure 1:

FLC-ratio of the 145 samples between the assays. Freelite vs. Diazyme (A), Freelite vs. KLoneus (B) and Diazyme vs. KLoneus (C). The blue dotted lines are the reference limits of the corresponding assay, the red dotted lines are the adjusted reference limit for patients with renal impairment. Values from patients with renal impairment are represented as red dots.

The bias between the Freelite and Diazyme assays is 5.1% and −29.2% for kappa and lambda, respectively, according to the Bland-Altman evaluation. The agreement of these two methods is quite high, especially for kappa. The slopes of the Passing-Bablok regression for Freelite vs. Diazyme were 1.05 (Kendall’s τ=0.888, p<0.001) and 0.96 (Kendall’s τ=0.869, p<0.001) for kappa and lambda, respectively. The bias between the Freelite and Diazyme assays is 30.9% for the FLC-ratio. The Passing-Bablok regression slope is 1.47 (Kendall’s τ=0.761, p<0.001). Figure 1A shows higher elevated FLC-ratios when using the Diazyme assay. For the Freelite vs. KLoneus assays, there is a bias of −56.8% for kappa and 54.9% for lambda which results in less agreement between these two assays. The slope was 0.51 (Kendall’s τ=0.847, p<0.001) for kappa and 1.33 (Kendall’s τ=0.605, p<0.001) for lambda when comparing Freelite with the KLoneus assay. The bias for the FLC-ratio is −99.3% for these two assays. The slope of the Passing-Bablok regression is 0.221 (Kendall’s τ=0.703, p<0.001). Comparing the Diazyme with the KLoneus assay, there is a bias of −62.7% for kappa and 80.5% for lambda. The slope for this comparison was 0.54 (Kendall’s τ=0.900, p<0.001) for kappa and 1.39 (Kendall’s τ=0.666, p<0.001) for lambda. For the FLC-ratio, the bias is −123.9% with a Passing-Bablok regression slope of 0.156 (Kendall’s τ=0.755, p<0.001). The analysis is also performed on the results of the multiple myeloma patients only, but no large differences were detected compared to the all patients with monoclonal gammopathies (Supplementary Table 1).

When inspecting the results of the Passing-Bablok analysis of the comparison between the Freelite assay and the Diazyme assay, there are some samples with discrepancy. These samples have high concentrations of FLC kappa in the Freelite assay, but low concentrations of FLC kappa in the Diazyme assay. Therefore, we diluted three samples manually and higher concentrations of FLC kappa were measured with the Diazyme assay, indicating a high-dose hook effect (Table 3).

Concordance analysis FLC-ratio

For the concordance analysis, the FLC-ratios (κ/λ) are classified according to the kit-insert into three groups: below (low), above (high) or within the reference range (normal) (Figure 2).

Figure 2:

Concordance analysis of the FLC-ratio between the three FLC assays. (A) For all patients (n=145) and (B) for patients without renal impairment (n=74). Grey boxes indicate agreement between the assays. The concordance is represented in percentages, and the Cohen kappa coefficient is also displayed.

The concordances for the Freelite and Diazyme assay is 63% and a Cohen’s coefficient of 0.43, corresponding to a moderate agreement (Figure 2A). Most of the mismatches were observed in samples that were classified as normal with the Freelite assay, but high with the Diazyme assay. There was good agreement between the Freelite and the KLoneus assay (Cohen’s κ=0.61) with a concordance of 75%. Most of the discrepant samples are below the reference interval of the FLC-ratio of the KLoneus assay. When comparing the results of the FLC-ratio of the Diazyme method with the KLoneus assay, there is none to slight agreement (Cohen’s κ=0.20) with a concordance of 41%. Most of the samples are classified as normal or high FLC-ratio with the Diazyme assay, but with lower FLC-ratios with the KLoneus assay. When concordance analysis is performed in patients without renal impairment (eGFR >60 mL/min/1.73 m²) only, there are some minor changes in concordance and agreement between the methods (Figure 2B). The results of the FLC-ratio of the Diazyme method are still higher compared to the Freelite assay, and the results of the FLC-ratio are lower with the KLoneus assay in patients without renal impairment. The concordance analysis is also performed on multiple myeloma patients only, there are minor changes in the concordances (Supplementary Table 1).

Validation of reference intervals

Both kappa and lambda concentration were measured with the three methods in sera from 25 healthy volunteers. Table 4 shows the number and percentage of the samples which fell within the reference intervals. Validation was considered successful when ≥90% of the reference samples fell within the reference interval. The reference interval for both the Freelite method and the KLoneus method can be verified (Supplementary Table 2). In contrast with that, all healthy volunteers have an elevated FLC-ratio when tested with the Diazyme assay. The reason for this increased FLC-ratio is that the Diazyme assay consistently measured FLC kappa values in the upper-range of normal (and 24% of samples above). Additionally, the FLC lambda values were consistently in the lower range of normal (and 12% of samples below).