Comparative analysis of three different serum-free light chain assays in the diagnosis of multiple myeloma

Abstract

Background

The lack of analysis methods and standardization are the core problems of serum free light chain (sFLC) detection in Multiple myeloma (MM). This study validated a new KHB sFLC assay through comparative analysis with conventional assays.

Materials and methods

Serum samples from 97 hospitalized MM patients were continuously collected. KHB, Freelite and N Latex assays were used to detect sFLC. The Bland-Altman and Passing-Bablok regressions were used for methodological comparison and bias evaluation. Spearman’s test and Cohen’s kappa coefficients were used to evaluate the correlation and clinical concordance.

Results

The sFLC results for KHB, Freelite, and N Latex showed a significant correlation. Passing-Bablok regression analysis revealed strong concordance between the KHB and N Latex for κFLC, and between KHB and Freelite assays for λFLC and FLC-ratio (κ/λ). When using N Latex and Freelite assays for sFLC determination, selecting iFLC/niFLC ≥ 20 or iFLC/niFLC ≥ 100 could lead to different clinical treatment decisions for approximately 9%∼12% of patients. When using KHB and Freelite assays for sFLC determination, selecting iFLC/niFLC ≥ 20 or iFLC/niFLC ≥ 100 could lead to different clinical treatment decisions for approximately 5%∼7% of patients.

Conclusion

KHB, as a sFLC detection method based on polyclonal antibodies and immunoturbidimetric principles, has a good correlation between its detection results and freelite and N Latex. The absolute difference in sFLC results among the three assays increased with increasing sFLC concentration, and selecting the same cutoff value for iFLC/niFLC may lead to inconsistent clinical treatment decisions in some patients.

Introduction

Multiple myeloma (MM) is a malignant proliferative disease originating from B lymphocytes capable of continuously producing monoclonal immunoglobulin proteins (M-proteins). MM is the second most common hematological malignancy, with an annual increase of more than 170,000 cases. Owing to its incurable nature, MM has a high mortality rate, with approximately 110,000 people dying from this disease worldwide each year [1–3]. Early diagnosis, accurate risk stratification, and adoption of active and effective therapy could significantly prolong the median survival time (MST) and improve the prognosis of patients [4]. However, common clinical symptoms of MM, such as anemia, bone pain, hypercalcemia, renal dysfunction, and recurrent infections, are often unrelated to hematological diseases, which could easily lead to missed diagnosis and delayed treatment of MM patients [5,6].

Immunoglobulin secreted by B cells and plasma cells is mainly composed of heavy and light chains, which are connected by interchain disulfide bonds. Under normal physiological conditions, the synthesis rate of the light chain is slightly faster than that of the heavy chain, which results in a small portion of the light chain being unable to bind with the heavy chain and exists in a free form in the blood, but is quickly metabolized and excreted through the kidney. Malignant proliferation of B lymphocytes in MM patients, with free light chain (FLC) levels increasing to over 40 times that of normal individuals, greatly exceeding the compensatory capacity of the kidney, leads to sustained high concentrations of FLC in serum (serum free light chain (sFLC)) and urine [7]. The guidelines of the National Comprehensive Cancer Network (NCCN) and the International Myeloma Working Group (IMWG) [8], as well as the latest revised ‘Guidelines for the Diagnosis and Management of MM in China’ in 2024 [9], recommend using FLC and the FLC ratio as biomarkers for diagnosis, risk stratification, and efficacy assessment of MM.

Freelite (The Binding Site (TBS)) [10] and N Latex FLC (Siemens Healthineers) [11] are currently commonly used FLC assay methods, but they have the disadvantages of not being interchangeable between different assays and the assay equipment being incompatible with foreign reagents [12]. At present, only a few large-scale hospital laboratories in China are equipped with specific protein analyzers for FLC assay. Due to high equipment procurement costs and limited target populations, many low-scale hospitals and primary healthcare institutions have not conducted FLC assay, which has also caused delay in diagnosis and treatment of potential MM patients [4]. The reagent kit based on immunological transmission turbidimetry for FLC assay is constantly emerging, which could be adapted to multiple automatic biochemical analyzer at the same time, which is very beneficial for the promotion and use of FLC assay in primary hospitals [13]. This study referred to the EP9-A3 [14] published by the Clinical and Laboratory Standards Institute (CLSI) to compare three FLC assays kits and observe the comparability of their results, in order to understand the correlation and bias between different assays. Some newly diagnosed MM patients have an excess of kappa FLC (κFLC) or lambda FLC (λFLC) antigens in their serum, so it is necessary to continuously increase the dilution factor to eliminate the effects caused by prozone phenomenon. This study also compared the average dilution times, average detection time, and reagent consumption required for sFLC detection in three assays. This study will also provide valuable information and assistance for the application of appropriate sFLC assays in various scales of hospital laboratories.

Subjects, materials and methods

sFLC assays

Freelite reagents (κFLC and λFLC) based on Turbidimetry were purchased from TBS (Birmingham, UK), which was conducted using an Optilite system (TBS, Optilite Type 864). N Latex reagents (κFLC and λFLC) based on Nephelometry were purchased from Siemens (Marburg, Germany) and analyzed on a BNII analyzer (Siemens, BN II). κFLC and λFLC reagents based on Turbidimetry were purchased from Shanghai Kehua Bio-Engineering Co., Ltd. (Shanghai, China) and were analyzed using an automatic biochemical analysis system (KHB, ARCHITECT c16000). Detailed performance parameters of the three assay reagents are listed in Table 1.

Table 1.

Performance parameters of three FLC assay reagents.

Property parameter	Freelite	N Latex	KHB
Detection method	Scattering Turbidimetry	Scattering Turbidimetry	Transmission Turbidimetry
Antibodies	Goat Anti-Human Polyclonal Antibody	Mouse Anti-Human Monoclonal Antibody	Rabbit Anti-Human Polyclonal Antibody
Calibrator	Polyclonal FLC	Monoclonal FLC	Polyclonal FLC
Lot No.	κ: 544492–1, λ: 547886–1	κ: 473197 A, λ: 473204	κ: 20240522, λ: 20240512
Sample volume	κ: 20 μL, λ: 20 μL	κ: 83 μL, λ: 62 μL	κ: 6 μL, λ: 5 μL
Regent volume	κ: 150 μL, λ: 166 μL	κ: 60 μL, λ: 70 μL	κ: 160 μL, λ: 160 μL
Reference values	κ: 3.30–19.40 mg/L λ: 5.71–26.30 mg/L κ/λ: 0.26–1.65	κ: 6.7–22.4 mg/L λ: 8.3–27.0 mg/L κ/λ: 0.31–1.56	κ: 7.7–21.5 mg/L λ: 11.4–28.2 mg/L κ/λ: 0.29–1.65
Linearity range	κ: 2.9–127 mg/L λ: 5.2–139 mg/L	κ: 3.5–111.5 mg/L λ: 1.93–61.6 mg/L	κ: 6–200 mg/L λ: 10–160 mg/L
Default dilution	κ: 1:10 λ: 1:8	κ: 1:100 λ: 1:20	κ: 1:21 λ: 1:26
Extended dilution	κ: 1:100, 1:1000, 1:5000 λ: 1:80, 1:800, 1:8000	κ: 1:400, 1:2000, 1:8000, 1:40000, 1:160000 λ: 1:100, 1:400, 1:2000,1:8000,1:40000,1:160000	κ: 1:200, 1:1000, 1:2000 λ: 1:200, 1:2000, 1:3000
Detection duration	κ: 15 min λ: 15 min	κ: 12 min λ: 13 min	κ: 10 min λ: 10 min

Sample collection

A total of 97 MM patients who were hospitalized in the Hematology Department of Henan Provincial People’s Hospital from August 1, 2024, to January 31, 2025, were selected as the study subjects, including 53 males and 44 females, with an average age of (56.5 ± 12.6) years. The inclusion criteria were as follows: (1) patients who met the diagnostic criteria for MM in the ‘Guidelines for the Diagnosis and Management of MM in China’ in 2024 [9]; (2) MM patients with complete clinical data; the exclusion criteria were as follows: (1) MM patients with concomitant malignant tumors; (2) MM patients with organic lesions of important organs such as the heart, liver, and kidney; and (3) MM patients with other blood system diseases. Venous blood (3 ml of venous blood from MM patients was collected on an empty stomach and placed in a coagulation-promoting tube. After centrifugation for 5 min (3500 r/min, r = 8 cm), the serum was separated and transferred to a 1.2 mL EP tube, which was frozen at −80 °C in an ultra-low temperature freezer until analysis.

sFLC methods comparison and consistency assessment

According to CLSI EP9-A3 [14], the Spearman test was used to analyze the correlation between the different assays. The Bland–Altman and Passing–Bablok regressions were used for methodological comparison and bias evaluation. Three detection methods were compared pairwise, and a total of three pairs of analysis combinations were obtained. According to the reference range provided by the Freelite, N Latex, and KHB reagents, the FLC ratio (κ/λ) values of 97 patients with MM were divided into abnormal and normal values.

According to the cutoff value of the FLC ratio of involved and non-involved FLC (iFLC/niFLC ratio) recommended by the IMWG [15], the iFLC/niFLC ratio values of 97 MM patients measured by different reagents were divided into two categories: <the cutoff value and ≥ the cutoff value. Cohen’s Kappa (κ) consistency test [16] was used to evaluate qualitative concordance of the sFLC assays: moderate consistency as 0.81≤ κ coefficient <1, good consistency when 0.61≤ κ coefficient <0.8, and very good consistency as ≥0.81.

Comparison of sFLC assays and serum protein electrophoresis

The measurable iFLC peak (M protein peak) values on serum protein electrophoresis (SPE, Hydrasys, Sebia) were converted into quantitative results and compared with the iFLC concentration values measured using three different sFLC assays.

Comparison of turnaround time (TAT) and assay cost

During detection, the sFLC results of 50 patients required at least one assay to eliminate the prozone phenomenon by increasing the dilution factor. Different dilution times and detection times of different reagents will affect the issuance of inspection reports (TAT), and an increase in dilution times will also increase reagent consumption, leading to an increase in inspection costs. The average dilution time, average detection time, and average reagent consumption of the three sFLC assays were compared.

Statistical analysis

Statistical analysis and plotting were performed using Med Cale 18.2, and GraphPad Prism 8.0. Non-normally distributed data are represented by M (P25, P75). Mann–Whitney U test and Kruskal Wallis H test were used for comparison between two groups and multiple groups. Normally distributed data are represented by χ ®± S: Independent sample t test and one-way ANOVA are used for comparison between two groups and multiple groups. The Cohen’s kappa (κ) consistency test was used to evaluate the qualitative concordance of the sFLC assays. p < 0.05.

Results

Clinical data analysis of 97 MM patients

Among the 97 MM patients, 71 were newly diagnosed, 7 relapsed and 19 were in complete remission. According to the M protein type, there are 47 cases of IgG type, 18 cases of IgA type, 14 cases of IgM type, 1 case of IgD type, 14 cases of light chain type, 1 case of double-clonal type, and 2 cases of oligo-secretory type (Table 2). The results of the Kolmogorov–Smirnov (K–S) test indicated that the results of κFLC, λFLC, and FLC ratio (κ/λ) by the Freelite, N Latex FLC, and KHB assays showed a skewed distribution (Z = 0.403, Z = 0.424, Z = 0.467, all p < 0.01). The above results showed no statistically significant differences (Table 3).

Table 2.

Clinical characteristic of 97 patients with MM.

Clinical characteristic (n = 97)	No. (%)
Gender
Male	53 (54.6)
Female	44 (45.4)
Age (years)
≥60	59 (60.8)
<60	38 (39.2)
Type
IgA	18 (18.5)
IgG	47 (48.5)
IgM	14 (14.4)
IgD	1 (1.0)
Light chain	14 (14.4)
Biclonal	1 (1.0)
Oligo-secretory	2 (2.2)
Setting
Newly diagnosed	71 (73.2)
Refractory/relapsed	7 (7.2)
Complete remission	19 (19.6)

Table 3.

The difference analysis of sFLC results between Freelite, N Latex and KHB sFLC assays.

FLC assay	κFLC (mg/L)	λFLC (mg/L)	FLC ratio (κ/λ)
Freelite	30.07 (12.43, 91.11)	22.58 (8.90, 86.89)	0.98 (0.37, 7.15)
N Latex	30.20 (13.65, 93.00)	26.60 (13.15, 71.65)	0.90 (0.53, 4.32)
KHB	31.08 (14.76, 93.90)	26.75 (9.54, 72.51)	1.11 (0.47, 4.94)
K/P value	0.372/0.946	3.493/0.322	0.700/0.873

Methodological comparison

The Bland–Altman plot was used to analyze the differences in the results between two pairs of the three reagent kits. The Median Absolute Deviation (MAD) of κFLC were −0.25 (Freelite vs N Latex), −0.76 (Freelite vs KHB), and −0.63 mg/L (N Latex vs KHB), respectively. Relatively small absolute deviations were observed among the three assays for κFLC concentrations below 100 mg/L. As the concentration of κFCL significantly increased, the absolute deviation between the three assays gradually increased, and the results for Freelite were significantly higher than those for N Latex and KHB. When the κFLC exceeds 10000 mg/L, the absolute deviation between the three assays exceeded the 95% confidence interval (CI) (Figure 1A). The MAD of λFLC were −3.49 (Freelite vs. N Latex), −0.59 (Freelite vs. KHB), and 2.09 mg/L (N Latex vs. KHB), respectively. Similar to the κFLC results, relatively small absolute deviations among the three assays were observed for λFLC-concentrations below 100 mg/L. As the concentration of λFCL increased significantly, the absolute deviation between the three assays gradually increased, and the range of increase was significantly higher than that of κFLC. The results showed that Freelite was the highest, N Latex was the lowest, and KHB was between Freelite and N Latex. When the average detection concentration was 10,000 mg/L, the difference in the results between the two assays was 10,000 mg/L (Figure 1B). The FLC ratio (κ/λ) is an independent prognostic factor for patients with MM [17]. When the ratio was < 10, the absolute deviation between the three detection methods was small. Relatively small absolute deviations among the three assays were observed for the FLC ratio (κ/λ) below 10. As the FLC ratio (κ/λ) significantly increased, the absolute deviation between the three assays gradually increased (Figure 1C).

Figure 1.

Bland–Altman plot was used to analyze the absolute difference of κFLC (A), λFLC (B) and FLC ratio (κ/λ) (C) among Freelite, N Latex and KHB sFLC assays. In Figure 1A and 1B, the dotted lines represent the 2.5th and 97.5th percentile, in Figure 1C, the dotted lines represent the median.

The results of Passing–Bablok regression analysis showed that the slope and intercept of κFLC in N Latex and KHB assays were 1.10 (95% CI: 0.93–1.02) and −0.05 (95% CI: −0.86–1.03). The slope and intercept of λFLC and FLC ratio (κ/λ) in Freelite and KHB assays were 0.93 (95% CI: 0.88–1.01) and 0.14 (95% CI: −0.56–0.88), 1.11 (95% CI: 0.99–1.23) and −0.04 (95% CI: −0.16–0.01), respectively. The 95% CI of the slope mentioned above contained 1, and the 95% CI of the intercept contained 0, indicating the comparability of results between assays (Figure 2; Table 4). In addition, Spearman correlation analysis showed that the R values of κFLC, λFLC, and FLC ratio (κ/λ) among the three assays were highest at 0.98 (λFLC, Freelite vs KHB, p < 0.001) and lowest at 0.93 (λFLC, Freelite vs N Latex, p < 0.001). The sFLC results of one assay correlated with those of the other two assays (Figure 2).

Figure 2.

The consistency of κFLC (A), λFLC (B) and FLC ratio (κ/λ) (C) among Freelite, N Latex and KHB sFLC assays by Passing–Bablok regression.

Table 4.

Passing–Bablok regression for κFLC, λFLC and FLC ratio (κ/λ) among Freelite, N Latex and KHB sFLC assays.

	κFLC	95% CI	λFLC	95% CI	FLC ratio (κ/λ)	95% CI
	Slope B	Intercept A	Slope B	Intercept A	Slope B	Intercept A
Freelite vs N Latex	1.06–1.18	−4.22 to –1.08	−11.37to –4.16	1.06–1.45	−0.66 to –0.17	1.59–2.13
Freelite vs KHB	1.05–1.22	−5.22 to –1.31	−0.56–0.88	0.88–1.01	−0.16–0.01	0.99–1.23
N Latex vs KHB	0.93–1.02	−0.86–1.03	4.42–8.00	0.61–0.83	0.03–0.15	0.60–0.74

Accuracy of sFLC assay

The absolute sFLC differences between the various assays were mainly visible at the high end of the concentration range. Up to 20-fold differences in sFLC concentrations between sFLC assays were regularly observed in this study. Among the 97 samples, 11 samples had a κFLC concentration > 1000 mg/L measured in at least one of the sFLC assays (highest 13300 mg/L measured with Freelite) and 13 samples had a λFLC concentration above 1000 mg/L (highest 18006.4 mg/L measured with KHB). The above 24 sample results were obtained by continuously increasing the dilution gradient in different sFLC assays, and no hook effect was found (Figure 3A).

Figure 3.

The concentration of sFLC detected by three sFLC assay were compared with the quantitative results of M protein peak in SPE. (A) Differential expression of κFLC or λFLC concentration among three methods (above 1000 mg/L was observed in at least one method) in 24 case of MM patients. (B) Quantitative results of M peak in SPE were obtained in 6 cases of MM patients, no bands were present in their IFE results that co-migrated with intact M proteins or other proteins in the beta region. The red arrow points to the M-protein peak.

In six out of 24 samples, significant absolute differences in the detection results of sFLC among different assays were observed. A monoclonal FLC band on immunofixation electrophoresis (IFE) was observed because it was either a faint band or a band that co-migrated with an intact M-protein or other proteins in the β-region. The measurable M protein peak values on serum protein electrophoresis were converted into quantitative results and compared with the iFLC concentration values measured using three different sFLC assays. The results showed that among the six samples, one sFLC result by KHB was close to its SPE value, and one sFLC result of N Latex was close to its SPE value. The detection results of Freelite were the highest among the four methods, whereas the SPE detection results were the lowest among the four methods. The average absolute difference in the FLC concentration between these two methods was 8295.5 mg/L (Figure 3B).

Consistency evaluation of FLC ratio (κ/λ) and the ratio of the involved to the non-involved FLC (iFLC/niFLC)

The results of Cohen’s κ consistency test (Table 5) showed that Freelite revealed a very good concordance for the abnormal κ/λ ratio with KHB (kappa coefficient 0.89), N Latex showed good concordance with Freelite (kappa coefficient 0.79), and N Latex showed good concordance with KHB (kappa coefficient 0.77). The IMWG recommends an iFLC/niFLC ratio ≥100 as a diagnostic criterion for the determination of active myeloma, and an iFLC/niFLC ratio ≥20 as diagnostic criteria for the determination of high-risk smoldering MM (SMM).

Table 5.

The concordance of FLC ratio (κ/λ) and iFLC/niFLC among Freelite, N Latex and KHB sFLC assays.

FLC κ/λ ratio	Freelite		FLC κ/λ ratio	Freelite		FLC κ/λ ratio	N Latex
N Latex	Normal	Abnormal	KHB	Normal	Abnormal	KHB	Normal	Abnormal
Normal	39	7	Normal	38	1	Normal	37	2
Abnormal	3	48	Abnormal	4	54	Abnormal	9	49
Cohen’s Kappa coefficient: 0.79			Cohen’s Kappa coefficient: 0.89			Cohen’s Kappa coefficient: 0.77
iFLC/niFLC	Freelite		iFLC/niFLC	Freelite		iFLC/niFLC	N Latex
N Latex	<100	≥100	KHB	<100	≥100	KHB	<100	≥100
<100	76	12	<100	74	5	<100	79	0
≥100	0	9	≥100	2	16	≥100	9	9
Cohen’s Kappa coefficient:0.54			Cohen’s Kappa coefficient:0.78			Cohen’s Kappa coefficient:0.62
iFLC/niFLC	Freelite		iFLC/niFLC	Freelite		iFLC/niFLC	N Latex
N Latex	<20	≥20	KHB	<20	≥20	KHB	<20	≥20
<20	64	7	<20	64	3	<20	65	2
≥20	2	24	≥20	2	28	≥20	6	24
Cohen’s Kappa coefficient:0.78			Cohen’s Kappa coefficient:0.88			Cohen’s Kappa coefficient:0.80

Among 97 MM samples, the detection rates of iFLC/niFLC ≥20 by Freelite N Latex, and KHB were 32.0% (31/97), 26.8% (26/97), and 30.9% (30/97), respectively. Freelite revealed very good concordance with KHB (kappa coefficient 0.88), N Latex showed good concordance with Freelite (kappa coefficient 0.78), and N Latex showed good concordance with KHB (kappa coefficient 0.80). The detection rates of iFLC/niFLC ≥100 by Freelite N Latex, and KHB were 21.7% (21/97), 9.3% (9/97), and 18.6% (18/97). Freelite revealed good concordance with KHB (kappa coefficient 0.78), N Latex showed moderate concordance with Freelite (kappa coefficient 0.54), and N Latex showed good concordance with KHB (kappa coefficient 0.62).

Comparison of TAT time and detection cost for sFLC

For the sFLC assay, the KHB assay had the lowest average dilution time, the shortest average detection time, and significantly lower TAT time compared to the other two detection systems. There was less reagent to complete single sample detection in KHB than in Freelite, but higher than in N Latex. For κFLC detection, N Latex had the highest average dilution time, while Freelite had the longest average detection time. For κFLC detection, N Latex had the highest average dilution time, whereas Freelite had the longest average detection time. For λFLC detection, Freelite had the highest average dilution and detection times, whereas TAT time was the slowest (Figure 4).

Figure 4.

Comparison of TAT time and reagent consumption for FLC assay in 97 MM samples with different FLC assays. (A) The average dilution times of κFLC detection in 97MM samples by three assays. (B) The average detection time of κFLC detection in 97MM samples by three assays. (C) The volume of reagent required for κFLC analysis of an individual sample by three assays. (D) The average dilution times of λFLC detection in 97MM samples by three assays. (E) The average detection time of λFLC detection in 97MM samples by three assays. (F) The volume of reagent required for λFLC analysis of an individual sample by three assays.

Discussion

MM is a common hematologic malignancy that tends to occur in middle-aged and elderly individuals. The incidence in men was slightly higher than that in women. In recent years, with the aggravation of the aging process in China, the incidence of MM has increased [1]. Owing to the complex and various clinical characteristics and the lack of characteristic clinical characteristics in the early stages, misdiagnosis is prone to occur. Commonly used laboratory detection methods include SPE [18], IFE [19], and FLC [20]. The sensitivity of SPE analysis is between 500 and 2000 mg/L, which is mainly used for the initial screening of MM patients, but it cannot detect low-level monoclonal proteins. The sensitivity of IFE is much higher than that of SPE, and it can detect other monoclonal proteins that SPE cannot. It is mainly used to confirm cloning and typing identification, but its diagnostic role in low secretory plasma cell diseases, such as light chain MM (LCMM), light-chain amyloidosis (AL-amyloidosis), and light-chain deposition disease (LCDD), is limited [21]. The emergence of sFLC detection kits has compensated for the shortcomings of electrophoresis detection technology and has played an important role in the diagnosis [8,9], efficacy evaluation [22] and prognosis [23] of MM and monoclonal immunoglobulin diseases.

Freelite (TBS, UK) [24] and N Latex (Siemens, Germany) [25] are the two most commonly used commercial FLC detection kits for monoclonal immunoglobulin diseases. The former uses polyclonal antibodies (targeting hidden epitopes of light chains) to detect the free and partially bound forms of κ and λ light chains with a wider recognition range [26]. The latter uses monoclonal antibodies (targeting exposed epitopes of light chains) to specifically bind to FLC, avoiding cross-reactivity with intact immunoglobulin-binding light chains, and has better specificity and precision [27]. As of now IMWG, NCCN, and the Chinese National Guidelines for Myeloma recommend the use of the Freelite FLC Detection Kit but do not explicitly exclude other reagents. Clinicians need to select reagents based on local laboratory conditions, but it should be noted that the results cannot be directly exchanged. In recent years, with the rapid development of in vitro diagnostic technologies, FLC detection reagents based on turbidimetry have gradually entered the market. Owing to their advantages of good compatibility with the equipment, low detection cost, and fast detection speed, they compete with imported reagents [28].

This study collected serum samples from 97 patients with MM (including newly diagnosed, treatment, complete remission, and recurrence), detected sFLC using Freelite, N Latex, and KHB assays, and conducted methodological comparisons based on the CLSI EP9-A3. R values of κFLC, λFLC, and FLC ratio (κ/λ) among the three assays were highest at 0.98 (λFLC, Freelite vs KHB) and lowest at 0.93 (λFLC, Freelite vs N Latex). Relatively small absolute deviations among the three assays were observed for κFLC and λFLC concentrations below 100 mg/L and FLC ratio (κ/λ) below 10. As the concentration of sFCL or FLC ratio (κ/λ) significantly increased, the absolute deviation between the three assays gradually increased up to 20 times, which is consistent with the results of Fleming et al. [29]. In addition, Schieferdecker et al. [30] found that the concentration of sFLC detected by Freelite was significantly higher than that of N Latex, especially in MM patients with κ light chain, which may be related to differences in antibody design and calibration methods of the reagents. Both KHB and Freelite are assays based on polyclonal antibodies and turbidimetry; however, for patients with abnormally high sFLC values, the results of Freelite in this study were significantly higher than those of KHB, which may be related to different antigen design sites. Given that there is currently no internationally standardized reference method for the sFLC assay, it is difficult to evaluate which assay produces more accurate detection results. However, by comparing the quantitative results of the M peak in SPE, we found that the iFLC results of the three free FLC assays were significantly higher than those of SPE, with Freelite having the highest detection result. To further evaluate the consistency of sFLC among the three assays, Passing–Bablok regression was performed. The 95% CI of the slope and intercept of κFLC in the N Latex and KHB assays were 1 and 0, respectively. The 95% CI of the slope and intercept of the λFLC and FLC ratio (κ/λ) in the Freelite and KHB assays were 1 and 0, respectively. The above results showed the comparability of κFLC results between the N Latex and KHB assays and the comparability of λFLC and FLC ratio (κ/λ) results between the Freelite and KHB assays. In addition, after converting the FLC ratio (κ/λ) to binary classification results based on the reference range provided by the kit, the abnormal FLC ratio (κ/λ) showed good consistency among the three assays, especially for KHB and Freelite, with a Kappa coefficient as high as 0.89.

Based on a large amount of reliable experimental data from previous clinical studies on MM [31–33], IMWG recommends abnormal sFLC ratio (<0.26 or > 1.65) and increased levels of iFLC (in patients with a ratio > 1.65, there is an increase in κFLC; in patients with a ratio < 0.26, there is an increase in λFLC) as necessary diagnostic indicators for light-chain immunoglobulin monoclonal gammopathy of undetermined significance (LC-MGUS). IMWG recommends an iFLC/niFLC ratio ≥100 as a diagnostic criterion for the determination of active myeloma, and an iFLC/niFLC ratio ≥20 as a diagnostic criterion for the determination of high-risk SMM [31–33]. In recent years, FLC assays, such as N Latex and Seralite^™, have been increasingly utilized in clinical practice [34]. These methods demonstrate excellent analytical performance and strong correlations in identifying abnormal monoclonal sFLC [35]. However, when the sFLC concentration exceeds a certain threshold (often above 100 mg/L), significant absolute discrepancies between different assay results can emerge, leading to inconsistencies in the iFLC/niFLC. Consequently, the continued application of IMWG-recommended cutoff values may result in delayed treatment or diagnosis for MM patients monitored using alternative FLC methods, ultimately impacting patient prognosis. Therefore, conducting independent clinical validation studies for each specific FLC assay is of paramount importance. For example, Henriot et al. [36] reported that in the diagnosis and efficacy observation of SMM patients, N Latex could exert the same effect as Freelite, but the cutoff value of iFLC/niFLC needs to be reduced. The research by Henriot B [36] showed that when the cutoff value of iFLC/niFLC is 35, the N Latex method can have the same effect as Freelite. The research by Caillon H [37] showed that when the cutoff value of iFLC/niFLC is 16, Elisa method developed by Sybia can have the same effect as Freelite. Among the 97 MM samples in this study, 31 of 97, 26 of 97, and 30 of 97 samples showed an iFLC/niFLC ratio ≥20 in the Freelite, N Latex, and KHB assays. 21 of 97, nine of 97, and 18 of 97 samples showed an iFLC/niFLC ratio ≥100 in the Freelite, N Latex, and KHB assays, respectively. Freelite revealed good concordance with KHB, and N Latex showed moderate concordance with Freelite, which was also consistent with the study by Kubicki et al. [30]. It should be noted that although there is good consistency between Freelite and KHB, 5% of patients may still have treatment indications affected by different detection methods, ultimately affecting their prognosis. KHB is a new method for detecting FLC, and there is currently no research reporting on its unique cutoff value, which is also our next research direction. Previous studies [38] have focused more on bias and consistency when comparing different FLC detection methods, while paying less attention to TAT time and reagent consumption. Different FLC analyses eliminated the prozone phenomenon by setting multiple dilution gradients. Increasing the number of dilutions can prolong the TAT time, consume more reagents, and increase detection costs. This study found that for the same number of patients with MM undergoing the sFLC assay, the KHB assay had the lowest average dilution times and the shortest average detection time. However, there was less reagent to complete single-sample detection in KHB than in Freelite, but higher than in N Latex. Considering the detection method and principle of KHB, its reagent cost is much lower than that of imported reagents; therefore, KHB is still a detection method that balances efficiency and economy.

Conclusions

In summary, the detection results of sFLC showed a good correlation between Freelite, N Latex, and KHB. The detection results of κFLC are comparable between N Latex and KHB, and the detection results of λFLC and the FLC ratio (κ/λ) are comparable between Freelite and KHB. According to the FLC ratio (κ/λ) reference range and the iFLC/niFLC cutoff value recommended by the IMWG, there is good consistency between reelite and KHB. KHB also has certain advantages in terms of the TAT time and reagent cost. Based on the above results, we believe that Freelite and KHB assays can be used interchangeably for the FLC ratio (κ/λ) and iFLC/niFLC will not affect the diagnosis of patients with MM and the correct classification and response evaluation of treatment outcomes during follow-up. However, there are still some shortcomings to this study. The number of samples included in this study is insufficient, and the number of samples needs to be continuously increased to ensure the authenticity and reliability of the above conclusions.

Glossary

Abbreviations

MM

Multiple myeloma

sFLC

serum free light chain

MST

median survival time

FLC

free light chain

NCCN

National Comprehensive Cancer Network

IMWG

International Myeloma Working Group

TBS

the binding site

CLSI

Clinical and Laboratory Standards Institute

κFLC

kappa FLC

λFLC

lambda FLC

SPE

serum protein electrophoresis

TAT

turnaround time

MAD

Median Absolute Deviation

CI

confidence interval

IFE

immunofixation electrophoresis

SMM

smoldering multiple myeloma

LCMM

light chain multiple myeloma

LCDD

light-chain deposition disease

Funding Statement

No funding was received for this research.

Ethics statement

Written informed consent was obtained from patients or guardians. The protocol of the current study was approved by the Ethics Committee of Henan Provincial People’s Hospital (Approved number: 2021-193). The studies were conducted in accordance with the Declaration of Helsinki.

Disclosure statement

The authors declare no conflict of interest.

Data availability statement

The data generated in this study are available upon request from the corresponding author.