Freelite sFLC assays available on other instruments, such as the Siemens BNII, do not include automatic antigen excess checks. On these instruments, samples should be tested for antigen excess following advice given in the product insert. Section 7.4.2 has further information on distinguishing antigen excess from sample non-linearity.
7.4.1. Incidence of antigen excess
Several studies have evaluated the incidence of antigen excess in large numbers of consecutive patients. Murata et al.  studied 7,538 serum samples over a 4-month period using 1:100 and 1:400 sample dilutions on a Siemens BNII. There were nine samples with κ antigen excess but no samples with λ antigen excess giving an incidence of 1/840 (0.12%). Importantly, all the antigen excess samples had elevated FLC concentrations or abnormal κ/λ ratios at the initial dilution of 1:100 so they would not have been classified as normal. Bosmann et al.  studied the incidence of antigen excess in 91 patients. Samples from two patients (2.2%) exhibited antigen excess: one, a patient with λ FLC-monoclonal gammopathy of undetermined significance (Chapter 13) and the other, a κ FLC patient with a known IgAκ monoclonal gammopathy. The authors concluded that the interpretation of FLC measurements is facilitated in many cases, when combined with electrophoresis results and clinical information.
Vercammen et al.  studied the incidence of antigen excess in 865 patient samples using 1:100 and 1:2000 sample dilutions on a Siemens BNII. Antigen excess was defined as a greater than 4-fold difference between the results obtained at the two dilutions. This approach improves the consistency of reporting FLC values and is discussed further in Section 7.4.2 below. A total of 5.4% (44/811) and 1.2% (9/773) of κ and λ samples exhibited antigen excess respectively, which was much higher than that reported by others . A follow on study of 3,645 samples by the same group identified sample carryover by BNII cuvettes as a cause of false antigen excess . This phenomenon was reduced by batch analysis and the introduction of a cleaning and rinsing protocol, and the incidence of true antigen excess was recalculated as 0.25% and 0.03% for κ and λ sFLCs, respectively. Vercammen et al.  also reported that true antigen excess was not observed in samples with normal κ and λ sFLC concentrations.
7.4.2. Distinguishing between non-linearity and antigen excess
To check for antigen excess, Binding Site recommend performing an antigen excess check dilution in addition to the initial sample dilution, as described in the relevant product insert (Table 7.6). Such a protocol minimises reagent usage and ensures consistency.
for κ and λ Freelite assays
|Antigen excess check dilution
for κ and λ Freelite assays
|Binding Site SPAPLUS||1/10||1/1000*|
Table 7.6. Antigen excess check dilutions on Binding Site SPAPLUS and Siemens instruments. * This is the overall dilution: 1/10 initial dilution + 1/100 manual dilution
Distinguishing between non-linearity and antigen excess is important because with non-linearity the lower result should be reported, whereas with antigen excess the higher result should be reported. The Binding Site recommends that when the result obtained at the antigen excess check dilution is more than 4-fold greater than the result obtained at the initial sample dilution, this sample should be considered to be in antigen excess. In this case the result obtained at the higher dilution should be reported. On the other hand, if the result from the antigen excess check dilution is less than 4-fold greater than that from the initial dilution, the sample should be considered non-linear and the value at the initial dilution should be reported. Examples of the use of this guidance to distinguish between antigen excess and non-linearity are shown in Table 7.7.
|Dilution results ratio*||2.1|
|Dilution results ratio*||3.2|
|Dilution results ratio*||55|
Table 7.7. Examples of κ FLC non-linearity and antigen excess on a Siemens BNII. *Results from antigen excess check dilution (1/2000) divided by those from the initial dilution (1/100). The results in bold should be reported.