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Chapter 7

Summary:

  • FLCs are stable in serum stored at 2 - 8 °C for up to 21 days.
  • Freelite® assays available on some instruments include prozone parameters for antigen excess detection.
  • FLC polymerisation leads to over-estimation of antigen concentrations determined by immunoassay.
  • Freelite non-linearity may occur in some samples due to non-specific interference (matrix effects) or the inherent variability of monoclonal FLCs.

Freelite serum free light chain (sFLC) assays are polyclonal antisera-based immunoassays, and can be performed on a number of automated laboratory instruments. κ and λ sFLCs are measured separately, then results can be expressed as a κ/λ sFLC ratio, or when there is monoclonal production of a FLC, as the difference (dFLC) between the involved (iFLC) and uninvolved (uFLC) concentrations. This chapter discusses both the practical aspects of implementation of Freelite assays (including choice of instrument, sample types and biological variation) and interpretation of results.

7.1.1. Choice of instrument

Freelite immunoassays are available for the majority of nephelometric and turbidimetric laboratory instruments (Chapter 37). Between-platform agreement of sFLC results is good (Section 5.8). Factors that may influence a laboratory’s choice of instrument include features of the sFLC assays on a particular platform (assay time, prozone parameters etc.), as well as those related to general laboratory organisation (workload, complete testing menu offered, existing platforms already present etc.). It is recommended that all laboratories performing sFLC assays participate in external quality assurance (EQA) schemes. These are further discussed in Chapter 39.

7.1.2. Reporting units

It is important to ensure that sFLC concentrations are reported in consistent units. In the UK the preferred reporting units are mg/L. Within the USA, results may be either in mg/L or mg/dL.

7.1.3. Choice of sample

All Freelite sFLC assays are validated for the quantification of κ or λ FLCs in serum. In addition, Freelite assays are also available for the measurement of FLCs in urine (Section 4.5.3) or cerebrospinal fluid (Chapter 36).

In general, samples that are haemolysed, lipaemic, or with highly elevated bilirubin should be avoided. The maximum concentration of interfering substance that can be reliably assessed is stated in the product insert. An example of interference testing of κ and λ sFLC assays is shown for the Binding Site SPAPLUS® in Table 7.1.
Interfering substance
Concentration Deviation from target value
κ sFLC λ sFLCs
Haemoglobin 3 g/L 2.1% -1.6%
Intralipid 0.3% -9.1% -3.0%
Bilirubin 300 mg/L -5.0% -2.4%

Table 7.1. Freelite assay interference for κ and λ Freelite assays on the Binding Site SPAPLUS. Interference was tested using a control serum containing ≤10 mg/L sFLCs, tested at the minimum sample dilution (1/1).

7.1.4. Sample and reagent stability

An in-house study was conducted to assess the stability of FLCs in unpreserved serum samples (n=30) stored at 2 to 8°C for up to 7 weeks. For each sample, κ and λ sFLCs were measured at regular intervals. After 3 weeks there was a significant drop in the κ sFLC concentrations (Figure 7.1). Therefore, it is recommended that samples are stored for a maximum of 21 days at 2 to 8 °C prior to analysis.

A second study assessed the stability of FLCs in unpreserved serum samples stored at -20°C. At each time point, an aliquot was defrosted and κ and λ sFLCs were measured. There was no deterioration in κ or λ sFLC concentrations over 7 months (Figure 7.2). Therefore, for long term storage of serum samples prior to sFLC analysis, it is recommended that samples are stored frozen at ≤-20°C.

Stability of the Freelite reagent is also an important issue. “Open-vial stability” refers to the shelf-life of the antisera after their first use. This is a minimum of 40 days for the SPAPLUS, but for other instruments the open vial stability may vary due to differences in instrument storage conditions (Chapter 37).

7.1.5. Changing batch of reagent or instrument

Great effort is made during Freelite assay manufacture to maintain batch-to-batch consistency (Section 5.5). Moving from one batch to the next should not present the laboratory with any issues. However, it is recommended that identical internal quality control samples are measured with both the new batch and the existing batch to confirm consistency, before moving entirely over to the new batch. Correlation of Freelite sFLC measurements obtained using different platforms is good (Section 5.8). However, before a laboratory changes platform, it is recommended that they compare the results obtained using the new and existing platforms as part of their validation protocol.

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