B-316 Evaluation of an FDA-cleared Chromogranin A Assay on an Automated Analyzer

Abstract

Abstract Background Chromogranin A (CgA) is a hydrophilic and acidic protein, present in the chromaffin granules of neuroendocrine cells. It is the preferred tumor marker for the monitoring of neuroendocrine tumors (NETS), which are often found in the lungs, appendix, pancreas, and gastrointestinal tract. Previous CgA assays were performed by manual ELISA testing, and were not FDA-approved, leading to a longer, more rigorous validation process and longer run times. The introduction of an automated, FDA-cleared CgA assay allows the laboratory to process patient samples more quickly and reliably, shortening a multiple hour process to minutes. The objective of this study was to evaluate the performance of an automated CgA assay recently cleared by the FDA. Methods We validated the the B·R·A·H·M·S™ Chromogranin A II (CGAII) assay on the automated B·R·A·H·M·S KRYPTOR Compact PLUS (Thermo Fisher Scientific Inc.). The CGAII assay is an immunofluorescent assay, which uses Time-Resolved Amplified Cryptate Emission (TRACE) to quantitate CGA in human serum. Assay performance characteristics were evaluated including measuring range (MR) and maximum dilution, precision, method comparison, stability, reference interval, stability, and carryover. Results The MR for CGAII was established with triplicate analysis of serum at 8 levels, ranging from 25.8 - 2,700 ng/mL. Linearity was demonstrated with a slope of 0.907, intercept of 1.948 and observed error of 2.54 ng/mL. Recovery at each point ranged from 83.3-110.3%. Carryover (<5.0 ng/mL) was not observed at a concentration approximately 2 times the upper limit of the MR. A 1:25 or 1:500 automatic dilution was determined to be acceptable (% bias <16%). Intraday precision coefficient of variation (CV) ranged from 4.1-1.7% (SDs from 2.32-6.64 ng/mL) at concentrations of 56.7 and 393.0 ng/mL. The interday CVs ranged from 6.0-7.1% (SDs from 3.43 - 28.84 ng/mL) at concentrations of 57.5 and 403.8 ng/mL. Twenty samples with concentrations ranging from 56.4-2,907 ng/mL were compared to a reference laboratory utilizing a similar methodology. The correlation coefficient was 0.9934, with a slope of 1.014, and intercept of -32.95. An additional twenty samples with concentrations ranging from 67.0-2,446 ng/mL were compared to a CgA ELISA kit (CGA-ELISA-NG, CISBIO) to determine the bias between the two methodologies. The correlation coefficient was 0.9902, with a slope of 0.845, and intercept of -15.31, and a bias of -19.9%. The serum samples were stable for 3 days when stored at 2-8°C. Recovered CgA concentrations fell outside of the 95% confidence interval after day 3 at 2-8°C. The manufacturer provided reference interval of <187 ng/mL was verified by testing 20 healthy adult samples, all of which fell within this range. Conclusions This assay has been validated and shown to be an accurate and precise method for monitoring CgA. Further, the method was validated on an automated analyzer to allow expeditious turnaround time in a core laboratory.