Sample non-linearity can be defined as a sample that when measured at different dilutions gives a substantially different result. Two examples are shown in Table 7.3. Sample A is an example of a non-linear sample, while sample B shows a linear response (i.e. further dilution [1/100] gives a value close to that obtained at the initial dilution [1/10]).

Non-linear sampleDilution Result
Sample A1/10 50
1/100 100
Linear sampleDilution Result
Sample B1/10 50
1/100 60

Table 7.3. Examples of non-linear and linear sample behaviour.

Sample non-linearity should not be confused with assay linearity; if an assay is non-linear all samples will give substantially different results when measured at different dilutions. Assessment of Freelite assay linearity forms an important part of immunoassay development and validation (Section 5.6).

As with all immunoassays, FLC assays are potentially prone to sample non-linearity. Pretorius et al. [175] concluded that non-linearity of FLC assays was a property of the individual sample, and not a method-specific phenomenon. Sample specific non-linearity may occur in some samples when measured with Freelite FLC assays due to: 1) non-specific interference (matrix effects); or 2) the inherent structural diversity of monoclonal FLCs (Chapter 3).

7.3.1. Managing non-linearity

Binding Site recommend following the sample dilution protocol as shown in each product insert and reporting the first plausible result only. The use of non-standard sample dilutions, or skipping dilutions should be avoided. Examples of non-linear samples are shown in Table 7.4 and 7.5.

Dilution Result
Sample A1/100 95.3
1/2000 202
Dilution Result
Sample B1/100 145
1/2000 467

Table 7.4. Examples of non-linear κ Freelite results using a Siemens BNII analyser. The first plausible result should be reported (values in bold).

Sample A1/10>165

Table 7.5. Example of non-linear λ Freelite results using a Binding Site SPAPLUS analyser. The first plausible result should be reported (value in bold).

It is important to note that Freelite results should not be interpreted in isolation and other laboratory findings and clinical symptoms should be considered when evaluating the status of the patient. In addition, the results from two consecutive measurements should be assessed when interpreting disease response [21].