Analytical performance of thrombospondin-1 and cathepsin D immunoassays part of a novel CE-IVD marked test as an aid in the diagnosis of prostate cancer

Abstract

The Prostate Specific Antigen (PSA) test suffers from low specificity for the diagnosis of Prostate Cancer (PCa). We originally discovered two cancer-related proteins thrombospondin-1 (THBS1) and cathepsin D (CTSD) using a mass-spectrometry-based proteomics approach. The two serum proteins were shown to improve the diagnosis of high-grade PCa. Thus, we developed quantitative ELISAs for the determination of their concentration in human serum. Here we report their analytical performance in terms of limit of detection, specificity, precision, linearity and interferences, which were determined based on CLSI guidelines. Further, we investigated the influence of pre-analytical factors on concentration measurements. For this, blood from 4-6 donors was collected in different tubes and stored at room temperature for different times prior to centrifugation at different centrifugal forces and temperatures. Stability of THBS1 and CTSD under different storage temperatures was also evaluated. Our results show that the assays are specific, linear and sensitive enough to allow measurement of clinical samples. Precision in terms of repeatability and total withinlaboratory coefficient of variation (CV) are 5.5% and 8.1% for THBS1 and 4.3% and 7.2% for CTSD, respectively. Relative laboratory-to-laboratory differences were -6.3% for THBS1 and -3% for CTSD. Both THBS1 and CTSD were stable in serum samples, with 80-120% recoveries of concentrations across donors, sample preparation and storage. In conclusion, the ELISAs as part of the novel commercial in vitro diagnostic test Proclarix are suitable for the use in clinical practice. THBS1 and CTSD can be accurately measured for their intended use independent of the lot and laboratory when conditions consistent with routine practice for PSA sampling and storage are used.

PROPOSe: A Real-life Prospective Study of Proclarix, a Novel Blood-based Test to Support Challenging Biopsy Decision-making in Prostate Cancer

“…The technical success rate for Proclarix was high, with a result available for 98% of tested samples (424 of 436 samples, 12 samples not measured due to technical limitations). This is consistent with the test robustness confirmed earlier by varying sampling tubes, storage temperature, and processing time [13]. Values of tPSA and fPSA were remeasured centrally in order to assure data completeness and reproducibility.…”

“…Values of tPSA and fPSA were remeasured centrally in order to assure data completeness and reproducibility. However, the Proclarix algorithm has been validated to incorporate PSA results obtained with testing kits from different manufacturers [13].…”

“…Proclarix kit (Proteomedix) as described before [13]. Serum tPSA and fPSA were reanalysed for all samples using the Roche Cobas immunoassay system (Roche Diagnostics, Rotkreuz, Switzerland).…”

“…It should also be noted that the Proclarix algorithm is based on demographic data (i.e. age) and normalised PSA information [13] obtained from validated laboratory platforms, which are not sensitive to intersite interpretation. Multiparametric MRI was included only for a part of the cohort once the EAU guidelines recommended its use not only in men with a previous negative biopsy, but also for all men considered for biopsy, and our study may have investigated an obsolete scenario.…”

“…The analytical and clinical performances of Proclarix have been established [13,14], and the test is CE marked. In order to reliably rule out clinically significant PCa (csPCa) defined as GG !2, the risk score's cut-off was set at 10%, which results in both high sensitivity and a high negative predictive value (NPV; 90% and 95%, respectively).…”