Clinical Comparison of a New Serum Free Light Chain (FLC) Assay Based on the Seralite Monoclonal Anti-FLC Antibodies with the Freelite and N-Latex FLC Assays in the Diagnosis of AL Amyloidosis

Aims: Accurate quantitation of FLC is essential for the diagnosis and monitoring of patients with AL amyloidosis. Current assays, however, have their limitations. Recently a new assay based on monoclonal antibodies (Seralite, Abingdon Health Ltd, UK) has been developed which appears to have some analytical advantages over existing assays and has been clinically validated in patients with myeloma (BJH 2017;178:220). We compared the antibodies used in this novel assay for FLC quantitation to the established monoclonal (N-Latex) and polyclonal (Freelite) antibody-based assays in AL amyloidosis.

Methods: 61 diagnostic samples from patients with AL amyloidosis were analysed. Serum FLC concentration was measured using Seralite monoclonal anti-FLC antibodies on an in-house ELISA platform in the Clinical Immunology Service Birmingham, and Freelite (The Binding Site, UK) and N-Latex (Siemens, Germany) immunoassays on a BNII nephelometer in the Pathology Queensland laboratory, Brisbane. Serum and urine immunofixation electrophoresis (IFE) were performed on Hydrasys gel systems (Sebia, France).

Results: For AL of kappa type (n=18), the median involved FLC (iFLC) was non-significantly lower by the Seralite assay than by the N-Latex or Freelite assay (220 vs 289 vs 667mg/L, p=0.64 and 0.27 for Seralite vs N-Latex and Freelite, respectively). In AL of lambda type (n=43) the values were significantly different (37 vs 144 vs 156mg/L, p=0.005 and 0.006 for Seralite vs N-Latex and Freelite, respectively). Measurable disease is currently defined as a difference between the involved and uninvolved FLC (dFLC) of > 50mg/L. By these criteria, 54% of AL would be measurable by the Seralite FLC assay compared to 82% by the N-Latex assay and 89% by the Freelite assay indicating that different cut-offs are likely to be required for different FLC assays.

In terms of the diagnostic sensitivity, the κ:λ FLC ratio was normal in 30%, 21% and 15% of patients by the Seralite, N- Latex and Freelite assays, respectively. All patients with AL amyloidosis of kappa type had an abnormal κ:λ ratio by all 3 assays. This was not the case for the 43 patients with lambda AL amyloidosis where the Seralite determined κ:λ FLC ratio was normal in 18 (42%) of cases compared with 30% and 21% for the N-Latex and Freelite assays, respectively. Providing greater detail of these 18 cases with a normal κ:λ ratio by the Seralite reagents: in 5 cases the κ:λ ratio was normal by all 3 assays; in 9 cases the κ:λ ratio was abnormal by both Freelite and N Latex assays; 1 case had a normal κ:λ ratio by Freelite and abnormal by N Latex; and 3 cases had an abnormal κ:λ ratio by Freelite and normal by N latex assays. For many of these cases the Seralite reagent appeared not to be reacting well with a lambda light chain clearly identifiable by one of the alternate assays. The combination of serum and urine IFE with either FLC assay, however, allowed identification of the amyloidogenic clone in 95%, 98% and 98% producing comparable overall diagnostic sensitivity for the methods. An attempt by the Birmingham laboratory to modify the assay to improve lambda FLC reactivity failed to improve the results.

Conclusions: There are significant differences between iFLC as measured by this in-house ELISA assay using Seralite monoclonal antibodies as compared to the existing N Latex and Freelite assays. In particular, the Seralite assay appears to react poorly with amyloidogenic lambda light chains and the current in-house ELISA assay is not suitable for diagnosis or monitoring of AL amyloidosis. It may be that a combination of anti-FLC monoclonal antibodies is required to improve detection of lambda clones by the assay. This data also highlights that different cut-offs and response criteria will be required depending on the assay used in patients with AL amyloidosis which should be a focus of further research.