Introduction

1. Evaluation of latex-enhanced nephelometric reagents for measuring free immunoglobulin light-chains on the Radim Delta P.J. Showell1, E.A. Lynch1, H.D. Carr-Smith1, A.R. Bradwell2 1The Binding Site Ltd., PO Box 11712, Birmingham, B29 6AT, UK, 2Division of Immunity and Infection, The Medical School, University of Birmingham, Birmingham, B15 2TT, UK (Presented at AACC 2004, Los Angeles, USA. 2004 (Clin Chem 2004, 50, No.6 Supplement C-40, pA81) • Introduction • Assays specific for serum immunoglobulin free light chains (FLC) that are compatible with commonly used laboratory nephelometric and turbidimetric analysers have recently become available. Studies with these assays have shown that serum FLC measurement is useful for the diagnosis and monitoring of patients with primary amyloidosis, non-secretory myeloma and light chain myeloma. Here we describe development of serum FLC assays for use on the Radim Delta, a medium-sized, fully automated bench-top nephelometer. • The assay was tested for possible interference from co-existing substances by adding haemoglobin (3g/L), bilirubin (200mg/L) and triglyceride (Intralipid; 0.5%) to serum samples and comparing to an equivalent sample blank. Comparison was made with The Binding Site FLC assays for the Dade Behring BN™II by measuring serum samples from normal subjects, patients with systemic lupus erythematosus and multiple myeloma on both systems and carrying out regression analysis. • The assays were found to be linear over their measuring ranges for both kappa and lambda assays (Figure 1). Kappa Free Linearity Lambda Free Linearity 180 180 y = 1.0032x - 1.1933 y = 0.9981x - 0.2097 160 160 2 R = 0.9992 2 R = 0.9999 140 140 120 120 Observed results (mg/L) Observed results (mg/L) 100 100 80 80 60 60 40 40 20 • Results • Assay imprecision expressed as % coefficient of variation was from 2.0 – 7.7% for within-run and 5.02 – 9.0% for between-run studies (Table 2). 20 0 0 0 50 100 150 200 0 50 100 150 200 Method The instrument was programmed to construct a calibration curve from dilutions of a single calibration fluid. The standard curves were validated by assay of control fluids. Samples were initially measured at the standard programmed sample dilution and if out of range the instrument automatically re-measured the sample at appropriate alternative dilutions. All dilutions were made with the instrument’s on-board pipetting system, which was able to make dilutions between neat and 1/2000. The main assay characteristics are summarized in the table below. Calculated results (mg/L) Calculated results (mg/L) • Figure 1: The assay linearity of The Binding Site FLC assays for use on the Radim Delta. • Good agreement was observed when these assays were compared with The Binding Site FLC assays for the Dade-Behring BN™II (Figure 2). Kappa Free Comparison Lambda Free Comparison 1400 3000 y = 0.9487x + 2.8621 1200 y = 0.918x + 22.885 r = 0.986 2500 r = 0.99 • Table 2: Intra- and inter-assay precision at three antigen concentrations for The Binding Site FLC assays on the Radim Delta. Kappa Free Radim Delta (mg/L) 1000 2000 Lambda Free Radim Delta (mg/L) 800 1500 • Interference was within ±4% when haemoglobin, bilirubin or chyle were added to serum samples of known free light chain concentrations (Table 3). 600 1000 400 200 500 0 0 0 500 1000 1500 0 1000 2000 3000 Kappa Free BNII (mg/L) Lambda Free BNII (mg/L) Table 1: The assay parameters of The Binding Site FLC assays for use on the Radim Delta. • Figure 2: Comparison of results for The Binding Site FLC assays on the Radim Delta with existing Binding Site FLC assays for use on the Dade-Behring BN™II. Intra- and inter-assay precision were assessed at three antigen levels for both assays. The assay linearity was calculated by assaying serially diluted serum samples over an antigen concentration range of 0.5 – 168mg/L for Kappa free and 6.6 – 170mg/L for Lambda free, and comparing with expected results. • Table 3: The interference test results for The Binding Site FLC assays for use on the Radim Delta. The percentage differences between samples diluted with saline and with the potentially interfering substances are shown. Conclusion The assays for the Radim Delta provide a rapid and precise method of measuring FLC in serum and show good agreement with existing assays. • Trademarks: BN™II is a registered trademark of Dade Behring GmbH, Marburg, GmbH