A novel and robust approach to defining population-based reference ranges for a serum free light chain assay in a clinical laboratory
Authors: Treger Rebecca , Mathias Patrick , Hutchinson Kathleen , Bryan Andrew , Chaudhary Anu , Fink Susan , Wener Mark , Morishima Chihiro 
Published: November 2022

Abstract
Serum protein electrophoresis and immunofixation are used to detect the presence of monoclonal heavy and light immunoglobulin chains for the diagnosis of plasma cell neoplasms (PCN). In conjunction, the Freelite® assay has been used to detect excess free kappa and lambda light chains produced by PCN, where the kappa:lambda free light chain ratio (KLR) serves as a surrogate marker of clonality and disease. The manufacturer-defined KLR reference range of 0.26-1.65 is used in internationally recognized criteria for PCN diagnosis and monitoring, and KLR results affect clinical decision making, disease categorization, and response assessment. Our laboratory has verified the manufacturer-defined Freelite® KLR reference range three times using healthy control sera since assay implementation in 2006. Despite this, high percentages of abnormal KLR have consistently been observed among immunofixation-defined monoclonal-negative specimens. We therefore retrospectively interrogated 48,012 serum free light chain clinical results reported at our institution between 2010-2020 by extracting results from our laboratory information system and analyzing population distributions using the R programming language. For all specimens, the distribution of KLR values was shifted towards the towards the upper limit of the manufacturer-defined reference range, indicating that it poorly describes both our monoclonal-negative and monoclonal-positive patient populations. Furthermore, &gt;80% of monoclonal-negative specimens exhibited KLR above the median of the manufacturer-defined reference range (0.26-1.65), while 24% (1,226/5,057) unexpectedly exhibited KLR &gt;1.65. We found that this phenomenon has been present consistently since 2010 and is not affected by patient sex or age, impaired renal function, lot-to-lot variation, or assay drift. We therefore sought to establish an institution-appropriate KLR reference range by defining a reference population from within our existing dataset. We leveraged International Classification of Diseases codes, estimated glomerular function data, and serum/urine immunofixation results to exclude specimens from individuals with PCN- or renal-related diagnoses, impaired renal function, and prior, current, or subsequent monoclonal components. The resultant 1,536 monoclonal-negative reference specimens were then classified according to the manufacturer-defined KLR reference range, where 13% remained abnormally elevated, consistent with the published false positive rate in healthy volunteers. Lastly, we determined a new KLR reference range of 0.66-2.21 using the central 95% of KLR values from these reference specimens. When applied to our dataset, this institution-specific KLR reference range improved the accurate classification of specimens containing monoclonal free light chains by immunofixation, while reducing the false-positive rate in monoclonal-negative specimens. Together, our findings support the use of historical KLR and immunofixation data from routine clinical testing to derive institution-appropriate KLR reference ranges, which can then be used for continuous quality improvement. Implementation of this more-accurate KLR reference range should improve subsequent test utilization by non-specialist providers, reduce concern for/over-diagnosis of kappa-involved disease, and improve the detection and monitoring of lambda-involved disease.