Chapter 5

Assay accuracy is defined as the degree of closeness of achieved results relative to their actual (true) values. Unfortunately, international standards do not exist for FLC measurements, so there are no reference points from which to assess the accuracy of results. In order to ensure accuracy in Freelite immunoassays, a suitable basis for standardisation and calibration was required. It was considered that polyclonal FLCs should be used in order to minimise any potential problems that might arise from the use of unique monoclonal proteins.

The production of assay calibrators was achieved in the following manner: 1) production and accurate quantification of pure polyclonal, primary κ and λ FLC standards; 2) production of secondary internal reference standards; and 3) production of calibration materials for use in the FLC kits (calibrated to the internal reference) (Figure 5.6).

Each primary standard was found to be greater than 99% pure by silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), whilst the alternate FLC was not detected by haemagglutination-inhibition and dot blot assays. The amino acid content of each primary standard was then determined in order to produce an accurate estimation of the protein content. Secondary standards were initially prepared from pools of different monoclonal κ and λ proteins. These were not considered ideal for use as working calibrators, so additional polyclonal standards were prepared from a pool of sera that contained elevated polyclonal sFLCs. These standards are known as κ or λ Internal Reference standards. The primary standards were used to assign κ and λ FLC concentrations to the Internal Reference standards by nephelometry. Each stage of the value transfer was completed at three dilutions and repeated three times. The final FLC values for the Internal Reference standards were 46.0 mg/L and 71.4 mg/L for κ and λ sFLCs, respectively. These values were used for all subsequent laboratory and clinical studies and used to assign values to kit calibrators and controls.

The measuring ranges of Freelite nephelometric/turbidimetric assays are dependent upon two factors: the slope of the respective calibration curve and the portion selected for the assay. The latter should be chosen to allow the maximum number of normal and abnormal clinical samples to be measured at the initial sample dilution. Typical analytical ranges (at the standard dilution) on the Binding Site SPAPLUS® for κ and λ sFLCs are 4.0 - 180 mg/L and 4.5 - 165 mg/L, respectively (Figure 5.7). Samples containing higher concentrations require further dilution. Calibration curves are validated by the measurement of high and low control samples. It is also recommended that all laboratories take part in external quality assurance schemes, to allow comparison of performance and results among different test sites (Chapter 39).