B-102 The Impact of Protein / Protein Interactions on Serum Free Light Chain Assay Calibration

Abstract

Abstract Background Since 2001, Serum Free Light Chain measurements have become established as routine clinical laboratory tests. Katzmann (2002) published a reference interval based on 282 normal samples establishing a ratio reference range 0.26-1.65. Whilst kappa and lambda measurements are of little diagnostic value, kappa/lambda ratio gives a strong indication of monoclonal gammopathy. In recent years, it has been suggested that the ratio may have changed. Here we present investigations identifying protein/protein interactions that may account for the discrepancies between QC release and real-world data; identifying a remediation step which will allow verification of the published ratio. Methods The manufacturers QC release data was reviewed for kappa, lambda and kappa/lambda ratio for all lots from 2018-2023, and compared to published data. To mimic field observation, an accelerated stability study was performed on standard calibrators and protein/protein interactions identified using western blot. Subsequently, calibrators were depleted of alpha-1 antitrypsin (A1AT) and other serum proteins using specific affinity chromatography; accelerated stability was repeated. A calibrator of pure polyclonal free light chains (FLC) in human serum albumin was produced in the presence/absence of A1AT and additional interfering serum proteins. Results At release, the average kappa reference range was 7.2-29.5 mg/L (62 kits, 9 different US blood donor sera collections and 3852 data points) and 7.7-24 mg/L for lambda (78 kits, 9 different US blood donor sera and 4664 data points), with a kappa/lambda ratio of 0.45-1.69. There was minimal change over 5yrs to these measurements. Analysis of FLC calibrators during an accelerated stability identified interactions between kappa FLC and ubiquitous serum proteins, including A1AT. The standard calibrator activity declined by 14.9±0.1% (mean±SEM) over a 4-month accelerated period @22oC, equivalent to 16 months real time. Calibrators depleted of A1AT and specific additional serum proteins showed significantly less decline in activity 1.4±1.4%, p<0.001. When blood donor sera were analysed using the standard calibrators following 4 months at 22oC, the results showed an increase in the reported kappa concentrations compared to those generated from frozen calibrators (19.0[15.4-22.5] to 21.3[17.0-26.0] mg/L, 12.5±3.8%, p<0.001). This observation was mirrored in kappa/lambda ratio (1.3[1.1-1.5] to 1.4[1.2-1.7] mg/L, 12.5±3.8%, p<0.001) and when clinical patient samples were assessed (30.1[14.0-47.1] to 34.3[15.5-56.9] mg/L, 13.8±1.4%, p<0.001). The depleted calibrator did not report an increase in sample results. FLC+HSA calibrators, when spiked with either physiological concentrations of A1AT or other serum proteins, showed a 16.7% and 15.3% decrease in activity respectively, compared to FLC+HSA calibrators without additional proteins. Conclusions At release, US blood donor sera have different absolute kappa mg/L compared to the published reference interval, but these changes do not invalidate the published kappa/lambda ratio, in contrast to recent reports and have not changed since 2018. An unpredictable kappa/protein interaction was observed, particularly with A1AT in a time dependent fashion, which correlated to a reduction in calibrator activity. Removal of A1AT and other specific serum proteins remediated the issue, and the kappa/lambda ratio was verified irrespective of the age of the calibrator.