Kidney filtration markers in human saliva: Accuracy and reproducibility of novel salivary cystatin c measurements

Background: Invasive phlebotomy followed by laborious blood specimen processing is the only reliable approach to assess routinely measured kidney filtration markers including cystatin C (CysC) Non-invasive testing of these markers is urgently needed particularly during the COVID-19 pandemic to enhance social-distancing Methods: We developed novel Enhanced Enzyme and Immunoassay-based Lateral Flow (ELF) assays to measure concentrations of CysC in human saliva Highly selective binding reagents were screened for optimum specificity, followed by applying sample treatment steps to mitigate sample to sample variability using healthy donor saliva samples spiked with known levels of the filtration markers Standard calibration curves (SCCs) for each marker was developed with nonlinear 4-parameter logistic curve fitting to triplicate measurements at each spiked concentration level Accuracy/fit of the SCC was assessed using the coefficient of determination (R2) Intra-assay repeatability was assessed using coefficient of variation (CV) and studies of inter-assay repeatability over time (over ∼8 days) examined reproducibility of whole experimental protocol Results: SCC fitted to relative optical intensities (ratio of test to control lines vs spiked CysC (0-14 ng/ml) was excellent (R2=0 991) and provided accurate estimates of spiked/ true CysC levels (R2=0 994) Assessment of intra-assay variation showed that repeatability is very good with CV <10% throughout the dynamic range of measurements Assessment of inter-assay variation (measurements over 8 days) showed that reproducibility is acceptable with CV <13% throughout most of the dynamic range Preliminary assessment of long-term reproducibility (stability) out to 258 days indicated similar performance [figure] Conclusions: We demonstrated feasibility of CysC measurements in human saliva samples with acceptable ELF assay characteristics including accuracy, repeatability, reproducibility, and long-term stability Validation studies are ongoing to optimize the saliva testing framework for kidney function markers