Analyte-containing dilution extends the functional range of clinical immunoassays for volume-limited specimens

Accurate quantification of hormone concentrations using clinical immunoassays is often limited by minimum specimen volume requirements, particularly with microvolume collection devices such as microtainers and capillary microsampling systems. When volume is insufficient to meet assay requirements, dilution is necessary; however, conventional dilution with analyte-free diluent proportionally reduces analyte concentration and may result in measurements falling below the assay's functional sensitivity, preventing reliable quantification. We evaluated an analyte-containing diluent strategy designed to preserve measurable signal and enable accurate hormone quantification from minimal serum volumes. Pooled human serum specimens were serially diluted and analyzed for estradiol (E2), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) using the Beckman Coulter DxI 600 platform. Reverse-calculated concentrations from analyte-free and analyte-containing dilutions were compared with undiluted measurements using Passing-Bablok regression and recovery analysis. Analyte-containing dilution kept the measured concentration above the limit of quantitation at dilution factors up to 1:128, exceeding the dilution range over which conventional analyte-free dilution remained quantifiable. A hybrid correction model restored agreement with undiluted measurements, yielding recovery rates of 95.5% for hCG, 116.5% for E2, and 105.3% for LH across evaluable dilution ranges. This approach extends the functional analytical range of immunoassays and enables accurate hormone quantification from substantially reduced specimen volumes. Analyte-containing dilution provides a practical method compatible with existing clinical laboratory workflows and may enhance diagnostic testing reliability in microvolume and minimally invasive sampling applications.