Free light chain EQA: Analytical variability with clinical consequences - Data from 25 Italian EQA exercises (2021-2025).

Serum free light chains (FLC) κ and λ, and the κ/λ ratio, are key biomarkers for the diagnosis, risk stratification and monitoring of monoclonal gammopathies. However, substantial between-method variability still limits harmonised interpretation. We analysed five years of external quality assessment (EQA) data from an Italian FLC scheme. Twenty-five EQA rounds (2021-2025) yielded 2218 results: 746 for κ FLC, 746 for λ FLC and 726 for the κ/λ ratio. Data were processed according to ISO 13528 using robust statistics and method-specific peer groups. Inter-laboratory precision within homogeneous peer groups was generally acceptable, with typical coefficients of variation around 10-20% for κ and λ FLC. By contrast, marked between-method differences were observed. Compared with Siemens N Latex, Binding Site Freelite assays yielded approximately two-fold higher mean κ FLC concentrations (relative mean bias ≈ +94%) and lower mean λ FLC concentrations (relative mean bias ≈ -82%), leading to substantial discrepancies in κ/λ ratio interpretation. Stratified analysis across low, intermediate and high concentration bands showed that the positive κ bias of Freelite persisted throughout the analytical range, whereas the global negative λ bias was largely driven by a subset of high-concentration samples with very high N Latex values. Bland-Altman analysis confirmed wide limits of agreement for both analytes. These findings indicate that current FLC assays are not interchangeable and that κ/λ ratios should not be interpreted using universal reference intervals. Method-specific cut-offs, method-consistent follow-up and method-stratified EQA schemes are required pending international standardization of FLC measurement.