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.