Antigen excess occurs when an antigen is present in such high levels that it interferes with antigen-antibody crosslinking, resulting in the formation of smaller immune complexes (Figure 7.4). This causes immunoassays to underestimate high concentrations of protein. sFLC concentrations can range from <1 mg/L to >10,000 mg/L. This is a greater range than almost any other serum protein test. Consequently, a small proportion of samples may be underestimated because of antigen excess. Monoclonal sFLCs produced by different patients can exhibit considerably different points of equivalence (the concentrations above which the assay is in antigen excess). It is therefore not possible to predict the concentration at which Freelite antigen excess may occur – it could be 300, 3000 or 30,000 mg/L for different patients. It is important to note that for the majority of patients, their samples will never demonstrate Freelite antigen excess.

Assessment of antigen excess forms an important part of immunoassay development and validation (Section 5.6). Freelite sFLC assays available on some instruments include prozone parameters (Chapter 37). For example, the Binding Site SPAPLUS instrument monitors the initial reaction kinetics of each sample at three separate time intervals (Figure 7.5) and compares the results with reaction limits set by the manufacturer through testing of an extensive myeloma library. Samples detected as being in antigen excess are automatically flagged by the instrument and retested at a higher sample dilution. A very small proportion of samples in antigen excess have normal reaction kinetics so will not prompt the flag. Such undetected antigen excess is a rare event but cannot be excluded. Therefore, it is recommended that the following statement accompanies all FLC results “Undetected antigen excess is a rare event but cannot be excluded. If these free light chain results do not agree with other clinical or laboratory findings, or if the sample is from a patient that has previously demonstrated antigen excess, the result must be checked by retesting at a higher dilution”.

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. [177] 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. [178] 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. [179] 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 [177][178]. 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 [180]. 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. [180] 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.

Instrument Initial dilution
for κ and λ Freelite assays
Antigen excess check dilution
for κ and λ Freelite assays
Binding Site SPAPLUS1/10 1/1000*
Siemens BNII/ProSpec1/100 1/2000

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 Result
Sample 11/100 95.3
1/2000 202
Dilution results ratio* 2.1
Sample 21/100145
Dilution results ratio*3.2
Sample 31/10078.1
Dilution results ratio*55
Antigen excess

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.