Chapter 7


  • Freelite® assays include prozone parameters for instruments with automated antigen excess detection.
  • Monoclonal FLCs may form polymers, which accelerate the formation of aggregates in Freelite immunoassays and produce an over-estimation of antigen concentrations.
  • Sample non-linearity may occur in some samples due to non-specific interference (matrix effects), the inherent variability of monoclonal FLCs, sample polymerisation or other unknown factors.

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 sFLC, 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 a range of nephelometric and turbidimetric laboratory instruments, including the Binding Site SPAPLUS® and Optilite® 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 38.

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 plasma, urine (Section 4.5.3) or cerebrospinal fluid (CSF, 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 and for the Binding Site Optilite in Table 7.2.
Interfering substance
Concentration Deviation from target value
κ sFLC λ sFLC
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).

Interfering substance
ConcentrationMaximum deviation from target value
κ sFLCλ sFLC
Haemoglobin1.25 g/L7.9%5.7%
Intralipid5 g/L-7.5%-7.3%
Bilirubin200 mg/L-3.2%2.5%
Triglyceride10 g/L6.5%-2.9%

Table 7.2. Freelite assay interference for κ and λ Freelite assays on the Binding Site Optilite. Interference was tested across a range of serum samples, including normal, abnormal and samples close to medical decision points.

7.1.4. Sample and reagent stability

A study by Coley et al. [1085] demonstrated that κ sFLCs stored at 22°C are only stable for up to 4 days, but their stability is prolonged when stored at lower temperatures. An in-house study was conducted to assess the stability of sFLCs 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). These findings were confirmed by Coley et al. [1085]. Therefore, samples should be stored for a maximum of 21 days at 2 to 8°C prior to analysis.

A second study assessed the stability of sFLCs 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.

Gurtner et al. [1025] studied the stability of κ FLCs in CSF. At ambient temperature, κ CSF FLCs were stable for 1 day (5% mean difference from baseline), refrigerated 2 – 8°C for up to 4 days (3%) and frozen at -20°C for up to 28 days (-5%). There was no deterioration in κ FLC concentrations after two free/thaw cycles (3%).

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. Once opened, Freelite reagents may be used for up to three months, providing that when not in use, the reagent vials are stored at 2 – 8°C, securely capped to prevent evaporation (Chapter 37).

7.1.5. Changing reagent lot or instrument

Great effort is made during Freelite assay manufacture to maintain lot-to-lot consistency (Section 5.5). Moving from one lot to the next should not present the laboratory with any issues. Correlation of Freelite sFLC measurements obtained using different platforms is good (Section 5.8) [965]. 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.