Defining the impact of individual sample variability on routine immunoassay of serum free light chains (sFLC) in multiple myeloma

Abstract

SummarySerum free light chain (sFLC) measurement has gained widespread acceptance and is incorporated into various diagnostic and response criteria. Nonlinearity and antigen excess are the main causes of 'variability' in the measurement of sFLC using immunoassay, but the impact of these on measurement has been unclear. We performed a retrospective evaluation using a dilutional strategy to detect these phenomena. A total of 464 samples in 2009 and 373 samples in 2010 were analysed for sFLC. Non-linearity was detected in both high and apparently normal sFLC. Major non-linearity of more than twofold is common in high kappa (20·2%) and lambda (14·1%). It is less common in samples with apparently normal levels -kappa (6·4%) and lambda (9·5%). 9·4% of kappa and 15·5% of lambda showed antigen excess at screening dilutions. 34·4% of the samples had either non-linearity or antigen excess. We conclude that significant measurement variability is common in the measurement of sFLC. There is currently no reliable technique to detect non-linearity phenomena unless a serial dilution strategy is applied to every analysis. We recommend that laboratories routinely reporting sFLC results for clinical services need appropriate strategies for addressing these issues. Clinicians should be aware of these limitations in interpretation of sFLC assay for individual patients. Future guidelines should adopt action thresholds which are grounded firmly in test performance parameters.

Overestimation of free light chain antigen excess rate

“…These numbers surpassed the estimates of two earlier reports [9,10] but were later confirmed in a retrospective analysis (also on BNII) showing antigen excess in 5% to 8% of the samples for κ FLC and in 1.5% to 4.5% of the samples for λ FLC [11]. In the latter study, nonlinearity was detected in 5% to 6.9% of the samples for κ FLC and in 2% to 4.2% of the samples for λ FLC [11]. A multicentre study found 5% antigen excess for both κ and λ FLC [12].…”

“…Non-linearity, defined as a more than 2-fold but less than 4-fold difference between the results obtained at the 2000-fold and 100-fold dilution, was found in 6% of the samples for κ FLC and in 0.5% of the samples for λ FLC [8]. These numbers surpassed the estimates of two earlier reports [9,10] but were later confirmed in a retrospective analysis (also on BNII) showing antigen excess in 5% to 8% of the samples for κ FLC and in 1.5% to 4.5% of the samples for λ FLC [11]. In the latter study, nonlinearity was detected in 5% to 6.9% of the samples for κ FLC and in 2% to 4.2% of the samples for λ FLC [11].…”

Serum-free light-chain assay: clinical utility and limitations

“…80 A second analysis showed that when the same high concentration sample was distributed on two separate occasions to users of the scheme, the majority of the laboratories incorrectly reported the sample on one of those occasions with 10% of users reporting the result incorrectly on both occasions. 81 The other recognised issue contributing to imprecision with the sFLC assay has been non-linearity on sample dilutions. 77,83,84 This may be a combination of the immunoreactivity of the monoclonal FLC itself as well as lot-to-lot variations in the reagents and performance characteristics of the analysers.…”

“…Previous work analysing the variation generated across centres measuring the same sample have produced large coefficient of variance (CV) % values. 80,81 Both of these datasets however focused on samples that displayed antigen excess properties rather than more linear samples. Due to the relatively recent release of the N-latex assay, there is significantly more data and more participants of the EQA scheme using the Freelite TM assay.…”

Time to redefine Myeloma

“…Patients with elevated sFLC are more likely to present with renal damage and, in the Mayo series, 27% of patients presented with acute renal failure, underlying the fact that this biomarker is also a marker of renal risk (Larsen et al , ). There is inter‐test variability with the sFLC assay and confirmatory testing is required (Murng et al , ). The IMWG definition incorporates both a sFLC ratio of >100 and, in addition, the minimum involved sFLC must be >100 mg/l (Rajkumar et al , ) and this cut‐off increases the specificity and predictive power.…”