In addition to linearity, spike recovery can be evaluated by adding the same recombinant protein into the sample and comparing that value to a reference determined by spiking the sample diluents. All analytes in the R&D Systems kit demonstrated mean recovery between 70%–130%. As shown in Table 1B, recovery performance varied widely in kits from the other vendors (Table 1B). This lack of accuracy in sample values may increase the discrepancy in results between studies. This is especially true if the studies used kits from a variety of vendors.
Diluents used in multiplex assays must be optimized not only for accuracy, but also to minimize interferences. Heterophilic antibodies have long been a challenge in single two-site immunoassays. These antibodies can cause interference and produce error in the analytical results. This interference can be amplified when using a multiplex assay, especially in studies that evaluate samples from patients with a variety of disease states, including inflammatory and autoimmune diseases.
Such patients may have high levels of rheumatoid factor or heterophilic antibodies, particularly if they have been treated using monoclonal antibody therapy. Samples from these patients may result in false positive values unless interference is minimized.