Turbidemetric and nephelometric homogeneous immunoassays are popular test formats involving the addition of patient sample to a solution of analyte-specific reagent antibodies. If present, the target analyte (aka the antigen) will crosslink the antibodies to form light scattering immune complexes. Quantitation is achieved by assuming a linear relationship between the analyte concentration and the signal produced within a short time interval.
In reality, the endpoint response curve is bell shaped1 and only approximated by a line within a limited range of analyte concentrations called the analytical measuring range (AMR). Unfortunately, for many clinically relevant analytes, the AMR is not wide enough to cover the span of biological variation. These shortcomings typically manifest as underestimation of the analyte concentration in samples with high analyte concentration, also known as antigen excess (the hook effect). Errors caused by antigen excess can be so extreme that a sample with grossly elevated analyte concentration can be mistaken for normal.
In order to prevent clinical confusion and potential patient harm, clinical laboratories have implemented various strategies to detect antigen excess based upon the endpoint or kinetic characteristics of the measurement. Samples that are flagged for antigen excess are then diluted to reduce the antigen concentration to within the AMR and the measurement is repeated. These methods can be very effective, but they come at high costs and do not detect all cases of antigen excess. Sample dilutions consume precious laboratory resources including technician time, instrument time, and reagents, and they introduce additional opportunities for laboratory errors.
We endeavored to develop a data-driven approach to dealing with antigen excess without sample dilution. Taking advantage of the fact that chemistry analyzers monitor the reaction at regular time intervals, we retrieved the kinetic records from our routine clinical measurements. We were able to model the assay behavior using a simple chemical reaction scheme and then we used our model to explore the relationship between antigen concentration and reaction kinetics. Similar to prior reports, we found that the curvature of the reaction differentiated cases of antigen excess from normal samples, even when the endpoint signal failed to do so. We developed a novel derived kinetic parameter, the Area Under the CUrvature (AUCU), and demonstrated in silico that it 1) provides a more robust measure of assay curvature than prior methods and 2) demonstrates a log-linear relationship with antigen concentrations in a range beyond the AMR of conventional endpoint methods, providing an opportunity to directly quantify analyte concentration in the zone of antigen excess without sample dilution. Finally, we validated the predicted log-linear relationship in vitro using consecutive clinical records for two different immunoassays.
Additional studies are needed before this kinetic calibration method is applied clinically. These studies will address the safety and accuracy of the method as well as establishing a threshold AUCU value above which antigen excess is deemed likely and the kinetic calibration curve is used in place of the traditional endpoint calibration curve. Finally, regulatory approval by FDA will likely be required for on label utilization of this novel calibration method.
In reality, our method is not a paradigm shift. We are currently fitting a nonlinear (endpoint) response curve with a line in an approximately linear regime. Our method simply provides another nonlinear (kinetic) response curve that is approximately linear in a different regime. The real paradigm shift, that our study is just one example of, is that clinical laboratorians are being more proactive about how to leverage the data owned by the clinical laboratory to improve the quality and efficiency of clinical laboratory testing.
References
- Heidelberger M, Kendall FE. A quantitative study of the 394 precipitin reaction Between type III Pneumococcus polysaccharide and purified homologous antibody. J Exp Med1929;50:809-823.
- Kinetic Approach Extends the Analytical Measurement Range and Corrects Antigen Excess in Homogeneous Turbidimetric Immunoassays. J Appl Lab Med. 2019 Sep;4(2):214-223. doi: 10.1373/jalm.2019.029256.