Antigen excess causes immunoassays to underestimate high concentrations of protein (Section 7.5). Freelite antigen excess checks can be performed on all platforms, and are carried out in one of two ways: 1) automatic prozone parameters, e.g. Binding Site SPAPLUS® and Optilite®; or 2) a sample dilution protocol detailed in the product insert, e.g. Siemens BN™II. Freelite antigen excess is further discussed in Section 7.5.

The Siemens N Latex FLC assays include antigen excess detection parameters based on a pre-reaction protocol. For the pre-reaction, a small volume of sample is initially added, followed by the rest of the sample for the primary reaction. If the signal at the end of the pre-reaction is above a certain threshold value, the instrument automatically performs a higher dilution [1046]. te Velthuis et al. [199] described the initial validation of N Latex FLC antigen excess parameters. In that study, over 2,000 serum samples were screened to identify the two with the highest concentration of κ or λ sFLCs: 23,000 and 57,000 mg/L, respectively. Serial dilutions of these two high samples failed to demonstrate antigen excess. However, this protocol does not provide a good assessment of antigen excess susceptibility because 1) only two samples were tested; and 2) they were chosen in a manner that was most likely to select samples not subject to antigen excess.

Jacobs et al. [203] compared the results from Freelite and N Latex FLC assays for 46 samples containing polyclonal or monoclonal sFLCs and using multiple sample dilutions. For monoclonal κ FLCs measured by Freelite or N Latex FLC, the discrepancies of concentrations between different dilutions were sometimes large. In addition, λ sFLC concentrations were underestimated at higher concentrations by both methods. However, this study failed to give a clear definition of non-linearity or antigen excess and the incidence of neither phenomenon was reported.

Two further studies compared the incidence of antigen excess and gross non-linearity for Freelite and Siemens N Latex FLC assays by testing samples at higher sample dilutions on the BNII [204][205]. Harding et al. [205] reported that κ sFLC antigen excess was observed in 3/91 (3%) samples by Freelite and 4/91 (4%) by N Latex FLC; all cases of Freelite antigen excess were correctly identified using the manufacturer’s dilution protocol. λ sFLC antigen excess was not observed with Freelite assays, in keeping with previous reports that suggest that these λ assays are less prone to antigen excess than κ assays (Section 7.5.3). In contrast, 3/91 (3%) of samples showed λ sFLC antigen excess with the N Latex FLC assay. Burden et al. [204] reported that a proportion of samples measured with κ or λ N Latex FLC assays gave results that were 5-9 times greater at the higher sample dilution compared with the result at the initial dilution (Table 8.4); this is indicative of antigen excess despite the fact that the assays should have automatic protection from antigen excess issues.

N Latex FLC Freelite
Dilution κ FLC (mg/L) Dilution κ FLC (mg/L)
1:100 71:100 >171
1:400 NR 1:400 >684
1:2000 2891:2000 617
N Latex FLC Freelite
Dilution λ FLC (mg/L) Dilution λ FLC (mg/L)
1:20 41:100 >155
1:100 61 1:400 >620
1:400 1581:2000 1020

Table 8.4. sFLC concentrations measured by N Latex FLC and Freelite assays at several sample dilutions [204]. NR: not reported.

Therefore, it should be noted that: 1) both Freelite and N Latex FLC assays show antigen excess in a small number of samples; 2) antigen excess parameters utilised in the N-Latex assays do not prevent all samples from exhibiting antigen excess; and 3) discrepant results between the two assays could be due to antigen excess. This third point is discussed further in Section 8.6.3.

Evidence of antigen excess for the N Latex FLC k assay was reported by Bossuyt et al. [1046]. The study included a total of 730 samples (n=525 MGUS and n=205 MM), which were measured at two dilutions on the BN ProSpec: the standard dilution and a 20-fold higher dilution. Antigen excess was missed for six samples, all of which contained monoclonal k sFLCs, as well as a monoclonal IgAk intact immunoglobulin.

The antigen excess capacity of the Sebia FLC assays has not been rigorously tested. The combined number of samples included in the two studies published to date totals only 30 [1178][175]. Therefore, further studies are required before conclusions can be drawn about the antigen excess protection in these ELISA-based assays.