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

BACKGROUND: The increase in lung cancer screening is intensifying the need for a noninvasive test to characterize the many indeterminate pulmonary nodules (IPN) discovered. Correctly identifying non-cancerous nodules is needed to reduce overdiagnosis and overtreatment. Alternatively, early identification of malignant nodules may represent a potentially curable form of lung cancer. OBJECTIVE: To develop and validate a plasma-based multiplexed protein assay for classifying IPN by discriminating between those with a lung cancer diagnosis established pathologically and those found to be clinically and radiographically stable for at least one year. METHODS: Using a novel technology, we developed assays for plasma proteins associated with lung cancer into a panel for characterizing the risk that an IPN found on chest imaging is malignant. The assay panel was evaluated with a cohort of 277 samples, all from current smokers with an IPN 4-30 mm. Subjects were divided into training and test sets to identify a Support Vector Machine (SVM) model for risk classification containing those proteins and clinical factors that added discriminatory information to the Veteran's Affairs (VA) Clinical Factors Model. The algorithm was then evaluated in an independent validation cohort. RESULTS: Among the 97 validation study subjects, 68 were grouped as having intermediate risk by the VA model of which the SVM model correctly identified 44 (65%) of these intermediate-risk samples as low (n=16) or high risk (n=28). The SVM model negative predictive value (NPV) was 94% and its sensitivity was 94%. CONCLUSION: The performance of the novel plasma protein biomarker assay supports its use as a noninvasive risk assessment aid for characterizing IPN. The high NPV of the SVM model suggests its application as a rule-out test to increase the confidence of providers to avoid aggressive interventions for their patients for whom the VA model result is an inconclusive, intermediate risk.

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

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.

Performance characteristics of the Beckman Coulter total ßhCG (5th IS) assay.

BACKGROUND: Beckman Coulter recently released the first commercially available hCG reagent calibrated against the 5th International Standard (IS) for hCG (total ßhCG (5th IS)). We performed a comprehensive analytical validation of this reagent. METHODS: Precision experiments were completed using 3 concentrations of commercial quality control material. Linearity, sample stability, and analytical sensitivity were evaluated using pools of human serum. Reportable range was assessed by comparing manual dilutions to those performed by the instrument. Male and female reference intervals were established using residual serum specimens submitted for routine testing. Inter-lot variability of hCG assay reagent was assessed by analyzing serum specimens with detectable hCG using 2 different reagent lots. Inter-assay variability was established using 203 serum specimens analyzed for hCG on 6 different reagent platforms. RESULTS: Inter-day precision showed a CV of <6.0% for all concentrations of QC material. LOB, LOD, and LOQ were determined to be 0.3, 0.4, and 0.6 U/l, respectively. The Access (5th IS) reagent has an average positive bias of approximately 20% when compared to most platforms and the previous generation Beckman hCG assay. Results were consistent between lots. Female reference intervals varied by age: <1.0 U/l (<41 y), <6.0 U/l (41-50 y), and <8.0 U/l (>50 y). Male reference intervals were <2.0 U/l. CONCLUSIONS: The analytical performance of the total ßhCG (5th IS) was established. Care should be taken to re-baseline patients needing serial monitoring and/or notify physicians when transitioning to the total ßhCG (5th IS) reagent.

Analytical sensitivity of four commonly used hCG point of care devices.

BACKGROUND: Point of care (POC) hCG assays are often used to rule-out pregnancy and therefore diagnostic sensitivity, especially at low concentrations of hCG, is important. There are very few studies in the literature that seek to verify the claimed analytical sensitivity of hCG POC devices. METHODS: The analytical sensitivity of four commonly used hCG POC devices (Alere hCG Combo Cassette, ICON 20 hCG, OSOM hCG Combo Test, and Sure-Vue Serum/Urine hCG-STAT) was challenged using urine samples (n=50) selected based on quantitative hCG concentrations. The majority of these specimens (n=40) had an hCG concentration between 20 and 200 U/L. Each specimen/device combination was reviewed by three individuals. Statistical calculations were performed using Stata 12. RESULTS: The analytical sensitivity of the OSOM was significantly lower inferior than that of the other POC devices. There was no significant difference in the sensitivity of the Alere, ICON 20 and Sure-Vue devices. There was no significant difference in the individual interpretation of the hCG POC results. CONCLUSIONS: All hCG POC devices evaluated in this study were susceptible to false negative results at low concentrations of urine hCG. Laboratorians and clinicians should be aware that there are limitations when using urine hCG POC devices to rule out early pregnancy.

Age, sex, and racial influences on the Beckman Coulter AccuTnI+3 99th percentile.

BACKGROUND: Beckman Coulter recently released a new cardiac troponin I (cTnI) assay, AccuTnI+3, for the Access 2 and DxI platforms. We validated the stated 99th percentile (20 ng/l) using a large population of healthy adults representative of the Northern California population. METHODS: Within a large sample of healthy adult members receiving care at Kaiser Permanente, cTnI was quantified in residual specimens using the AccuTnI+3 assay. Patients were selected based on pre-defined criteria extracted from a comprehensive electronic medical record. All specimens with a cTnI concentration >30ng/l were repeated; specimens that had a reproducible result >30 ng/l were subject to heterophile blocking procedure. 99th percentiles were calculated based on age, sex, race and body mass index categories. RESULTS: Among 1764 tested subjects, the 99th percentile for all samples was 25 ng/l. Sex differences were observed; the male and female 99th percentiles were 31 and 21 ng/l, respectively (p=0.001). Age (range evaluated 18-89y, median 47y) also had a significant influence on the value (p=0.003), but there were no significant differences by race. False positive results were detected in 0.9% of specimens (0.6% "fliers" and 0.3% heterophile antibodies), corresponding to 52% of all results >30 ng/l. CONCLUSIONS: Among a large, representative cohort of healthy adults, we found a 99th percentile value consistent with prior studies based on highly selected small patient samples. Sex and age-specific upper reference limits for cTnI should be considered. In this cohort, about half the findings above the 99th percentile were false positives. Avoiding reporting erroneous results requires implementation of quality indicators.

Limitations in qualitative point of care hCG tests for detecting early pregnancy.

BACKGROUND: Detection of early pregnancy in the healthcare setting is important for proper patient management. Qualitative point-of-care (POC) human chorionic gonadotropin (hCG) testing is often used. There is a gap in the literature for evaluating patient samples with low (<300 IU/l) hCG concentration using POC devices. METHODS: Specimens (urine, n=289; serum, n=269) with hCG concentrations between 2 and >5000 IU/l were evaluated by hCG POC testing. Approximately half of these had an hCG concentration<300 IU/l. All specimens were evaluated using the OSOM and QuickVue+ POC hCG devices. Pregnancy status was evaluated by patient chart review. RESULTS: The OSOM and QuickVue+ devices were able to detect hCG in 53% (OSOM) and 78% (QuickVue+) of urine samples with an hCG concentration range between 20 and 300 IU/l. Serum samples with an hCG concentration range between 10 and 300 IU/l gave positive results in 79 and 91% of samples tested using the OSOM and QuickVue+, respectively. False negative results could not be attributed to the high-dose hook effect, the hCG variant effect, or the interpreters' evaluation. The mean gestational age for specimens corresponding to the false negative results was 4 weeks. CONCLUSIONS: The sensitivity of 2 commonly used hCG POC devices is insufficient for detecting very early pregnancy.