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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 7.5). 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).

7.5.1. Instruments with automated antigen excess checking

Freelite sFLC assays available on some instruments include prozone parameters (Section 37.2.3). For example, the Binding Site SPAPLUS instrument monitors the initial reaction kinetics of each sample at three separate time intervals (Figure 7.6.) and compares the results with defined reaction limits established by testing of an extensive library of myeloma and normal samples (Section 37.3.3). Samples detected as being in antigen excess are automatically flagged by the instrument and retested at a higher sample dilution. The Binding Site Optilite has three different methods of antigen excess detection, including; control addition, sample addition and review of the reaction kinetics in a similar method to the SPAPLUS. Whilst the incidence of Freelite antigen excess on the SPAPLUS and Optilite is very low (see Section 7.5.3), some samples may generate such unusual kinetics that the reaction does not prompt a flag, and may result in undetected antigen excess. 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”.

7.5.2. Instruments without automated antigen excess checking

Freelite sFLC assays are also available on some other instruments, such as the Siemens BNII, which do not include automatic antigen excess checks. On these instruments, samples should be tested for antigen excess following advice given in the product insert. This involves retesting the sample at a higher dilution (known as the antigen excess check dilution). These protocols minimise reagent usage and ensure consistency of sample measurement. Whilst these protocols will ensure that the vast majority of samples in antigen excess are detected, Rassner et al. [1158] reported a rare sample (~1/10,500 samples) with monoclonal κ sFLCs that was still in antigen excess when tested at a higher dilution. However, a number of other findings were present that were suggestive of MM disease (λ sFLCs were undetectable, the κ/λ sFLC ratio was highly abnormal, κ urinary Bence Jones protein was detected, and renal failure and osteolytic lesions were present). The authors conclude that this case highlights that “clinical decisions should not be based on a single assay…”.

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, the sample should be considered to be in antigen excess and 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
(mg/L)
Sample 11/100 95.3
1/2000 202
Dilution results ratio* 2.1
Non-linear
DilutionResult
(mg/L)
Sample 21/100145
1/2000467
Dilution results ratio*3.2
Non-linear
DilutionResult
(mg/L)
Sample 31/10078.1
1/20004310
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 were reported.

7.5.3. 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 7.5.2. 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.

Investigations have also been carried out into the incidence of antigen excess on Binding Site analysers with automated antigen excess checks. Bossuyt et al. [1046] evaluated automatic antigen excess detection by the Freelite Optilite assays. A total of 730 samples (n=525 MGUS and n=205 MM) were measured at two dilutions: the standard dilution and a 10-fold higher dilution. Initially, 3 κ and 5 λ samples were in antigen excess, but as a result of this study, the antigen excess detection protocol was optimised, and an improvement in antigen excess detection was subsequently reported [1046]. These improved parameters have been applied to all subsequent kit lots. The authors also stated that a number of samples were in antigen excess by the N Latex FLC assays, which is further discussed in Section 8.4.4. Hitchcock et al. [1086] studied the incidence of antigen excess flags generated against 1452 serum samples analysed on the SPAPLUS. Flags were generated against 1.1% (16/1451) samples, and the authors concluded that the incidence of antigen excess was low. Coley et al. [1091] reviewed the incidence of antigen excess flags on the Optilite for over 10,000 sample results. The proportion of samples that demonstrated antigen excess flags on the κ and λ Freelite assays were 11% and 6%, respectively, and these were correctly identified in at least 98% of cases. A separate study by Walker et al. [1092] investigated the Optilite performance across over 18,000 κ and λ measurements and reported a very similar incidence of antigen excess flags, and antigen excess was confirmed in 95 and 99% of κ and λ cases, respectively. These authors observed only one instance of undetected antigen excess during the 1 year study period. These results confirm that the antigen excess detection for Freelite Optilite assays is robust and that undetected antigen excess is a rare event, but laboratories should remain vigilant if other laboratory tests results or clinical findings are inconsistent with Freelite results.

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References