Analytical validation of a novel multi-analyte plasma test for lung nodule characterization

Abstract

Background: In the National Lung Screening Trial, 96.4% of nodules had benign etiology. To avoid unnecessary actions and exposure to harm, individuals with benign disease must be identified. We describe herein the analytical validation of a multi-analyte immunoassay for characterizing the risk that a lung nodule found on CT is malignant. Those at lower risk may be considered for serial surveillance to avoid unnecessary and potentially harmful procedures. While those nodules characterized at higher risk may be appropriate for more aggressive actions. Objective: To validate the analytical performance of multiplexed plasma protein assays used in a novel test for lung nodule characterization. Methods: A multiplexed immunoassay panel for the measurement of plasma proteins in current smokers who present with a lung nodule on CT scan was evaluated in a clinical testing laboratory. Assay analytical sensitivity, reproducibility, precision, and recovery of Epidermal Growth Factor Receptor (EGFR), Prosurfactant protein B (ProSB), and Tissue Inhibitor of Metalloproteinases 1 (TIMP1) from human EDTA plasma samples were evaluated across multiple runs, lots, and technicians. Interfering substances and sample pre-analytical storage conditions were evaluated for their effect on analyte recovery. The lung nodule risk score reproducibility was assessed across multiple lots. Results: The assay sensitivities were 0.10 ng/mL EGFR, 0.02 ng/mL ProSB, and 0.29 ng/mL TIMP1 with over three orders of magnitude in the assay dynamic ranges. The assays and analytes are robust to pre-analytical sample handling and the plasma can be stored for up to 4 days at 4°C either when freshy collected or thawed after long-term storage at −80°C. Total imprecision after 20 days of testing remained under 9% for all three assays. Risk score variability remained within a ± 10% risk score range. Conclusions: The three protein assays comprising the multi-analyte plasma test for lung nodule characterization performed quite acceptably in a clinical laboratory.

Accurate diagnosis of pulmonary nodules using a noninvasive DNA methylation test

“…This suggests that PulmoSeek alone could reduce more than 40% of unnecessary invasive procedures on benign nodules with less than a 0.3% false-negative rate. The superior NPV and sensitivity of PulmoSeek compare favorably to other published rule-out models to date, for which the NPV range is between 85% and 98% (depending on the prevalence) and sensitivity is between 85% and 97% (24)(25)(26)(27). Those models are usually used in combination with clinical parameters, such as age, smoking status, nodule size/location, and classic cancer biomarkers (e.g., CEA).…”

Risk assessment for indeterminate pulmonary nodules using a novel, plasma-protein based biomarker assay

“…The three protein assays comprising the lung nodule characterization test show acceptable analytical performance demonstrating the necessary sensitivity, precision and reproducibility for use in a commercial clinical laboratory to calculate the probability of malignancy for indeterminate lung nodules found on CT scans [ 15 ].…”

“…The processing included thawing the samples and then preparing 100 μL aliquots that were refrozen to allow all testing and retesting to occur with the same number of 2 freeze-thaw cycles. Studies with freshly collected, never-frozen samples demonstrated analyte stability for up to 4 days at 2–8°C before freezing at-80°C, and up to 4 freeze-thaw cycles with no significant change in the measured analyte values [ 15 ].…”

Nanoparticle‐Based Bioaffinity Assays: From the Research Laboratory to the Market

“…[45,78] For example, MagArrays are based on measuring the change of magnetoresistance when superparamagnetic beads come close to magnetoresistive multilayer thin film (sensor surface). [79,80] Mag Arrays have mainly been used to study one specific interaction between an immobilized molecule and an NP-labeled molecule.…”