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8.5. Clinical performance of N Latex FLC and Freelite assays

Chapter 8

8.5.1. Normal reference intervals

Published reference intervals for Freelite and N Latex FLC assays are similar (Table 8.5) [199][19]. Freelite assay standardisation and normal ranges are further discussed in Chapters 5 and 6, respectively.

κ sFLC (mg/L) λ sFLC (mg/L) κ/λ sFLC ratio
Freelite95% reference interval 3.3 - 19.45.7 - 26.30.3 - 1.2
Diagnostic range 0.26 - 1.65
N Latex FLC95% reference interval 6.7 - 22.48.3 - 27.00.50 - 1.27
Diagnostic range 0.31 - 1.56

Table 8.5. A comparison of reference intervals for Freelite and N Latex FLC assays. Figures in bold indicate the limits for result interpretation. The diagnostic range includes 100% of the reference population.

8.5.2. Renal reference interval

Patients with renal impairment but no evidence of monoclonal gammopathy may have κ/λ sFLC ratios (measured by Freelite) which are slightly above the normal range [96][161][162]. The reason is that as renal clearance of sFLCs declines, the sFLC concentrations increase to more closely reflect the production rates, which are higher for κ than λ FLCs. Consequently, a modified renal reference range for the Freelite κ/λ sFLC ratio (0.37 – 3.10) has been proposed [165]. Use of the renal reference range when screening for monoclonal gammopathy in patients with renal impairment may increase specificity and is further discussed in Section 6.3.

Jacobs et al. [206] studied the effect of renal impairment on N Latex FLC concentrations in 284 patients with chronic kidney disease (CKD, stage 1-5). κ and λ FLC concentrations increased through each CKD stage. However, whilst κ N Latex FLC assays gave similar results to Freelite, the increase in λ FLC concentrations in the CKD5/dialysis groups was significantly greater for the N Latex FLC assays (Figure 8.5, Figure 8.6A, Figure 8.6B). As a result, none of the patients with CKD had a κ/λ sFLC ratio exceeding the N Latex FLC reference interval and, in fact, the ratio was significantly lower in the CKD5/dialysis group compared to healthy controls (p<0.0001; Figure 8.6C).

Berlanga et al. [207] re-analysed the data reported by Jacobs et al., and plotted the percentage increase in sFLC levels relative to controls for both assays (Figure 8.7). Freelite measured a progressive elevation of κ over λ FLC with worsening renal function, which resulted in some patients having an abnormal κ/λ sFLC ratio. By contrast, N Latex FLC assays indicated an approximately equal increase of both FLCs throughout CKD1-4, but a sharp increase in λ FLC levels in the fifth group, that comprised both CKD5 and dialysis patients. One potential explanation for this anomaly was provided by Kennard et al. [922] who compared pre- and post-hemodialysis sFLC concentrations in 105 patients with end-stage kidney disease and no known lymphoplasmacytic disorder. Pre-dialysis λ sFLC concentrations were much higher as measured by the N Latex FLC assays than Freelite, but post-haemodialysis, a larger reduction in λ sFLCs was indicated by the N Latex FLC assays (Table 8.6). This resulted in similar post-dialysis λ sFLC concentrations as measured by both assays. One possibility proposed by the authors is that a positively interfering substance may have been present in pre-dialysis samples that selectively affected the λ N Latex FLC assay (causing an over-read). If this interfering substance was of low molecular weight and was removed by haemodialysis, this would explain the greater fall in λ FLC concentrations indicated by the N Latex FLC assays. Further investigations are now required to try and identify the putative interfering substance and test this hypothesis.

N Latex FLC Freelite
κ
(mg/L)
λ
(mg/L)
κ/λ (IQR) κ
(mg/L)
λ
(mg/L)
κ/λ (IQR)
Pre-haemodialysis
130
250 0.56 (0.47-0.63) 160 110 1.44 (1.14-1.74)
Post-haemodialysis 44 91 45 65

Table 8.6. Comparison of Freelite and N Latex sFLC concentrations pre- and post-haemodialysis. Median (interquartile range, IQR) values are reported [922].

8.5.3. Comparison studies

A number of studies and EQA schemes have compared the absolute values returned by the Freelite and N Latex FLC assays (Section 39.3) [199][922][201][175][203][209][210][211][212][921][926]. All have reached similar conclusions: the two assays do not compare well and are not interchangeable. For example, a well-designed study by Lock et al. [201] compared quantitative results of Freelite and N Latex FLC assay for 327 serum samples submitted for analysis to four routine diagnostic laboratories in the UK; a total of 79% were from patients with known monoclonal gammopathies. Comparing the results produced by the two assays, standard linear regression gave r2 values of 0.86 and 0.71 for κ and λ sFLCs, respectively. This level of agreement is well below the requirements of the Clinical Laboratory Standards Institute (which requires an r2 ≥0.95 to establish that two assays are equivalent) [213]. Bland-Altman plots identified 17/327 (5.2%) samples that had the most discrepant results (Figure 8.8). This included 14 patients whose sera contained clonal FLC that were “poorly detected” by the N Latex FLC but not the Freelite assays [201]. Of most concern was the fact that 2 of the 14 patients had LCMM (prior to treatment) but their N Latex FLC assay results indicated normal sFLC ratios while their Freelite assay results were clearly abnormal (Table 8.8). These 2 patients and other examples of missed diagnoses are discussed further in Section 8.5.6 below. Whilst some comparison studies have been limited to small numbers of patients with monoclonal gammopathy [208] or a lack of clinical information [200] all have reported a similar poor concordance of Freelite and N Latex FLC assay results. This lack of agreement can be clinically important and is discussed further in Section 8.5 below.

8.5.4. Compliance with guidelines

Freelite assays are the only FLC assays recommended by name in International Myeloma Working Group guidelines, and an involved/uninvolved Freelite sFLC ratio ≥100 is designated as a biomarker of malignancy (Chapter 25) [167][42]. Guidelines and response criteria that are based on Freelite data should not be applied on the basis of N Latex FLC results because of the poor concordance between the absolute values [117][215][25]. A summary of clinical definitions that are based upon quantitative Freelite results are shown in Table 8.7.

Guideline Definition sFLC concentration
AL amyloidosis [117][114][116]Measurable disease dFLC >50 mg/L
VGPR dFLC <40 mg/L
MM [115][21][905]Measurable disease 100 mg/L
Progressive disease 25% increase in dFLC from lowest confirmed response (provided absolute increase >100 mg/L)

Table 8.7. A summary of international guidelines based on quantitative Freelite results. VGPR: very good partial response; dFLC: the difference between involved and uninvolved sFLC concentrations.

Popat et al. [215] compared the assessment of response indicated by N Latex and Freelite FLC assays for 42 MM patients who had been treated after relapse. The response criteria used were those defined by Rajkumar et al. [21]. A total of 17/42 patients had measurable disease by both assays and 11 by neither; one patient was evaluable by N Latex FLC only but 13 had measurable disease by Freelite only. For the 17 patients with measurable disease by both assays, there was poor agreement in the assigned response to treatment (Figure 8.9, weighted Kappa 0.75).

Dejoie et al. [923] compared the ability of N Latex and Freelite FLC assays to identify disease progression in LCMM patients according to IMWG guidelines. At clinical progression, Freelite confirmed progressive disease in 15/19 patients, compared to 11/19 by N Latex FLC assays. This study further supports the recommendation that IMWG guidelines based on Freelite data cannot be applied to the monoclonal assays.

8.5.5. Diagnostic performance in LCMM

In a total of six studies published to date, which incorporated approximately 80 LCMM patients, the Siemens N Latex FLC assays failed to detect abnormalities in six λ LCMM and one κ LCMM patients (Table 8.8) [175][201][209][923][140][219]. By comparison, the same six studies reported that all of these LCMM patients were correctly identified by the Freelite assays. The best described cohorts of confirmed cases of LCMM were included in the studies by Schneider and Hoedemakers [209][219] in which 3/26 of confirmed LCMM cases were not detected by the N Latex FLC assays. In contrast, an abnormal Freelite κ/λ sFLC ratio has been detected in all of 692 LCMM patients at diagnosis (Chapter 15). The recommendation that sFLC analysis plus serum electrophoresis constitutes a suitable primary screening protocol for monoclonal gammopathies [136][167] should, arguably, only be applied to FLC analysis with Freelite assays and not N Latex FLC or any other FLC assays, which are not validated in this context.

Study Total no. of
LCMM patients
Missed LCMM N Latex FLC Freelite
κ FLC
mg/L
λ FLC
mg/L
κ/λ ratio κ FLC
mg/L
λ FLC
mg/L
κ/λ ratio
Lock 2013 [201]n.s
(189 MM)
λ LCMM 19.5 59.7 0.33
Normal
4.66 6070 <0.01
Abnormal
λ LCMM 12.8 40.2 0.32
Normal
7.9 422 0.02
Abnormal
Hoedemakers 2011 [209]3 κ LCMM
6 λ LCMM
λ LCMM n.s n.s n.s
Normal
n.s n.s n.s
Abnormal
Schneider 2013 [219]17 LCMM λ LCMM n.s n.s 0.8
Normal
n.s n.s 0.02
Abnormal
κ LCMM n.s n.s 0.8
Normal
n.s n.s 1.89
Abnormal
Cavalcanti 2013 [140]

1 λ LCMM λ LCMM 16.5 23.0 0.71
Normal
9.14 818.8 0.01
Abnormal
Pretorius 2012 [175]n.s λ LCMM 29 84 0.35
Normal
18 464 0.04
Abnormal

Table 8.8. Summary of missed cases of LCMM by N Latex FLC assays. Patients were categorised as misclassified if the N Latex FLC assays reported a normal κ/λ ratio (n.s. = not stated).

8.5.6. Rationale for the diagnoses of LCMM missed by N Latex FLC assays

The clinical studies discussed above have provided a number of examples where patients known to have LCMM have had normal N Latex FLC results but abnormal results with Freelite assays. There are two reasons why FLC assays may fail to identify the presence of monoclonal sFLCs: 1) antigen excess (Section 8.4.4), or 2) complete non-reaction, in which the monoclonal antibodies fail to recognise a particular FLC clone.

Pretorius et al. [175] performed a comparison of Freelite and N Latex FLC results for 116 samples sent to the laboratory for routine FLC analysis (after exclusion of samples with Freelite <50 mg/L), and investigated any samples that were highly discordant. For 6/116 (5.2%) samples, N Latex FLC assays gave markedly higher κ results (Figure 8.10). When these six samples were further diluted and retested using Freelite assays, the results significantly increased, consistent with antigen excess in the Freelite assays. In contrast, 4/116 samples (3.4%) had markedly higher λ Freelite results (Figure 8.10B). Further dilution did not increase the N Latex FLC λ results for these samples. These four patients included one with confirmed λ LCMM for whom the κ/λ sFLC ratio was normal by N Latex FLC assays, but abnormal by Freelite (Table 8.8).

It is noteworthy that the majority of diagnoses that were missed by the N Latex FLC assays were of λ type. Whilst the κ constant domain is typically encoded by a single C gene segment, the λ constant domain is encoded by one of a number of C gene segments (Section 3.3). It is probable that the monoclonal antibody-based assays fail to detect all polymorphic forms of FLCs, particularly λ FLCs that are more genetically diverse.

A further patient with λ LCMM that was not diagnosed by the N Latex FLC assays is presented as a clinical case study below. In this example, serum immunofixation confirmed the presence of monoclonal FLCs that were detected by the Freelite sFLC assays, but not the monoclonal antibody-based N Latex FLC assays.

Clinical case history

A patient with λ LCMM identified by Freelite but not N Latex FLC assays [140].

A 47-year-old woman was admitted to the Istituto Nazionale Tumori, Naples, with bone pain. An X-ray of her pelvis revealed osteolytic lesions, and a serum protein electrophoresis (SPE) was ordered but this revealed no obvious monoclonal protein. Subsequently, sFLC analysis was performed using both Freelite and N Latex FLC assays. Whilst the Freelite assay identified monoclonal λ sFLCs, the N Latex FLC assay results were normal (Table 8.9). High-resolution agarose electrophoresis of serum and urine samples identified a monoclonal protein band Figure 8.11A), which was typed by immunofixation electrophoresis (IFE) as monoclonal λ FLCs (in the absence of intact immunoglobulins including IgD/IgE; Figures 8.11B and C). A bone-marrow biopsy confirmed the diagnosis of λ LCMM. The patient was admitted to the Hematology-Oncology Unit, and one month after the start of therapy, serum and urine IFE became negative and the Freelite sFLC assay values returned to normal.

Freelite
N Latex FLC
Results
Reference
interval
Results
Reference
interval
κ sFLC
(mg/L)
9.1 3.3 - 19.4 16.4 6.7 - 22.4
λ sFLC
(mg/L)
818.8 5.7 - 26.3 23.0 8.3 - 27.0
κ/λ sFLC
ratio
0.01 0.26 - 1.65 0.71 0.31 - 1.56

Table 8.9. Freelite and N Latex FLC patient results and reference intervals.

8.5.7. Diagnostic performance in cast nephropathy

The International Kidney and Monoclonal Gammopathy Research Group (IKMGRG) recommend the use of SPE and sFLC analysis to screen for monoclonal disease in patients presenting with acute kidney injury (Chapter 27) [22]. The IKMGRG suggest that if the concentration of monoclonal FLCs is ≥500 mg/L in patients with acute kidney injury (AKI), a diagnosis of tubular interstitial pathology is likely, and the most common renal lesion in such cases is cast nephropathy [213]. For such patients, further haematological work-up and prompt treatment, to reduce FLC production, is essential.

In the only study performed to date using the Siemens N Latex FLC assays in the context of AKI, the authors concluded that the IKMGRG recommendations could not be carried out satisfactorily [220]. In this study, five of the 28 patients (18%) with AKI secondary to MM were misclassified by the N Latex FLC assays. For one patient, the N Latex FLC assay reported a λ FLC concentration of 1 mg/L, whereas a value of 1810 mg/L was reported by the Freelite assay. Once again, this suggests that the pathogenic monoclonal λ FLC clone was not recognised by the monoclonal antibody-based assay.

8.5.8. Diagnostic performance in AL amyloidosis

The largest study that has compared the performance of N Latex FLC and Freelite assays in AL amyloidosis was published by Palladini et al. [221]. This included 426 patients with newly-diagnosed AL amyloidosis from two specialist centres (Pavia, n=353; and Limoges, n=73). A poor agreement of quantitative results between the two methods was observed but the diagnostic sensitivity of the Freelite (82%) and N-latex (84%) assays was similar, and both improved to 98% in combination with serum and urine immunofixation. Similar results were reported by Mollee et al. [211].

Mahmood et al. [924] confirmed that absolute values reported by Freelite and N Latex FLC assays do not always compare well in AL amyloidosis. In this study, the agreement between κ sFLC results at presentation (n=90) was better than that of λ (κ sFLC R2 = 0.91; λ sFLC R2 = 0.52), as discussed above (Section 8.5.3.). κ/λ sFLC ratios were also discordant between the two assays: 10/90 patients were abnormal by Freelite but normal by N Latex, and 11/90 abnormal by N Latex but normal by Freelite. Similar results were reported by Mollee et al. [211], but the reason for these differences is not understood.

8.5.9. Monitoring AL amyloidosis

Freelite sFLC assays are well established for monitoring haematological response in AL amyloidosis, and have been incorporated into a number of national and international guidelines (Section 28.6). By contrast, FLC assays from other manufacturers, including N Latex FLC assays, have not been formally validated. A study by Mahmood et al. [924] was the first to compare haematological responses assigned (using consensus criteria) with Freelite or N Latex FLC assays in 90 newly diagnosed AL amyloidosis patients. Although there was broad agreement in responses at 2, 4, and 6 months, the N Latex assay response was often earlier: 18 patients reached a PR or VGPR earlier by N Latex FLC than by Freelite. The authors highlight that patients classed as early responders by the N Latex FLC assay may get under-treated, and hence carry a risk of shorter time to disease progression.

Palladini et al [925] reported that during follow-up, the reduction in dFLC concentration measured using either method was of prognostic significance in AL amyloidosis, although the optimal cut off values was different (>50% dFLC decrease by Freelite; >33% dFLC decrease by N Latex FLC). In conclusion, Freelite and N Latex assays cannot be used interchangeably when monitoring haematological response in AL amyloidosis, and new response criteria would need to be derived and validated for the N Latex FLC assays if they are to be used in routine clinical practice.


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