Development and analytical performance evaluation of an automated chemiluminescent immunoassay for pro-gastrin releasing peptide (ProGRP)

Background: Pro-gastrin releasing peptide ( ProGRP) concentrations in blood play an important role in the diagnosis and treatment of patients with small cell lung cancer (SCLC). The automated quantitative ARCHITECT (R) ProGRP assay was developed to aid in the differential diagnosis and in the management of SCLC. The purpose of this study was to evaluate the analytical performance of this chemiluminescent microparticle immunoassay at multiple sites. Methods: ARCHITECT ProGRP measures ProGRP using a two-step sandwich using monoclonal anti-ProGRP antibodies coated on paramagnetic microparticles and labeled with acridinium. Analytical performance of the assay was evaluated at four sites: Abbott Japan, Denka Seiken, the Johns Hopkins University, and the University of Munich. Results: Total precision (%CV) for nine analyte concentrations was between 2.2 and 5.7. The analytical sensitivity of the assay was between 0.20 pg/mL and 0.88 pg/mL. The functional sensitivity at 20% CV was between 0.66 pg/mL and 1.73 pg/mL. The assay was linear up to 50,000 pg/mL using a 1:10 autodilution protocol. The calibration curve was stable for 30 days. Comparison with the Fujirebio microtiter plate enzyme-linked immunosorbent assay (EIA) ProGRP assay gave a slope of 0.93 and a correlation coefficient (r) of 0.99. Conclusions: These results demonstrate that the ARCHITECT ProGRP assay has excellent sensitivity, precision, and correlation to a reference method. This assay provides a convenient automated method for ProGRP measurement in serum and plasma in hospitals and clinical laboratories. Clin Chem Lab Med 2009;47:1557-63

Monitoring of Serum DNA Methylation as an Early Independent Marker of Response and Survival in Metastatic Breast Cancer: TBCRC 005 Prospective Biomarker Study

Epigenetic alterations measured in blood may help guide breast cancer treatment. The multisite prospective study TBCRC 005 was conducted to examine the ability of a novel panel of cell-free DNA methylation markers to predict survival outcomes in metastatic breast cancer (MBC) using a new quantitative multiplex assay (cMethDNA)

Multicentre performance evaluation of the E170 Module for MODULAR ANALYTICS

The E170 module was evaluated at 13 sites in an international multicentre study. The objective of the study was to assess the analytical performance of 49 analytes, and to collect feedback on the system's reliability and practicability. The typical, within-run coefficients of variation (CVs) for most of the quantitative assays ranged between 1 and 2% while a range of 2-4% was achieved with the infectious disease methods. Total precision CVs were found to be within the manufacturer's expected performance ranges, demonstrating good concordance of the system's measuring channels and a high reproducibility during the 2-4-week trial period. The functional sensitivity of 11 selected assays met the clinical requirements (e.g., thyreotroponin (TSH) 0.008 mU/l, troponin T 0.02 µg/l, total prostate-specific antigen (PSA) 0.03 µg/l). The E170 showed no drift during an 8-hour period and no relevant reagent carryover. Accuracy was confirmed by ring trial experiments and method comparisons vs. Elecsys® 2010. The reliability and practicability of the system's hardware and software met with, or even exceeded, the evaluator's requirements. Workflow studies showed that E170 can cover the combined workload of various routine analysers in a variety of laboratory environment. Throughput and sample processing time requirements were achieved while personnel ‘hands-on-time' could be reduce

Validation of Serum Biomarkers That Complement CA19-9 in Detecting Early Pancreatic Cancer Using Electrochemiluminescent-Based Multiplex Immunoassays

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy; its early detection is critical for improving prognosis. Electrochemiluminescent-based multiplex immunoassays were developed with high analytical performance. All proteins were analyzed in sera of patients diagnosed with PDAC (n = 138), benign pancreatic conditions (111), and healthy controls (70). The clinical performance of these markers was evaluated individually or in combination for their complementarity to CA19-9 in detecting early PDAC. Logistic regression modeling including sex and age as cofactors identified a two-marker panel of CA19-9 and CA-125 that significantly improved the performance of CA19-9 alone in discriminating PDAC (AUC: 0.857 vs. 0.766), as well as early stage PDAC (0.805 vs. 0.702) from intraductal papillary mucinous neoplasm (IPMN). At a fixed specificity of 80%, the panel significantly improved sensitivities (78% vs. 41% or 72% vs. 59%). A two-marker panel of HE4 and CEA significantly outperformed CA19-9 in separating IPMN from chronic pancreatitis (0.841 vs. 0.501). The biomarker panels evaluated by assays demonstrated potential complementarity to CA19-9 in detecting early PDAC, warranting additional clinical validation to determine their role in the early detection of pancreatic cancer

A multiplex immunoassay of serum biomarkers for the detection of uveal melanoma

Abstract Background Approximately 50% of uveal melanoma (UM) patients develop metastases preferentially in the liver leading to death within 15 months. Currently, there is no effective treatment for metastatic UM, in part because the tumor burden is typically high when liver metastases are detected through abnormal liver function tests (LFTs) or imaging studies. The use of LFTs results followed by diagnostic tests has high specificity and predictive values but low sensitivity, and better tests are needed for early diagnosis of the primary tumor as well as its metastatic spread. To evaluate serum biomarkers for the early detection of UM, multiplex immunoassays were developed. Methods Magnetic bead-based multiplex immunoassays were developed for the selected serum biomarkers using a Bio-Plex 200 system. The dynamic ranges, lower limits of detection and quantification, cross-reactivity, and intra- and inter-assay precision were assessed. All proteins were analyzed in sera of 48 patients diagnosed with UM (14 metastatic, 9 disease–free (DF) ≥ 5 years, 25 unknown) and 36 healthy controls. The performance of the biomarkers was evaluated individually and in combination for their ability to detect UM. Results A 7-plex immunoassay of OPN, MIA, CEACAM-1, MIC-1, SPON1, POSTN and HSP27 was developed with negligible cross-reactivity, recovery of 84–105%, and intra-assay and inter-assay precision of 2.3–7.5% or 2.8–20.8%, respectively. Logistic regression identified a two-marker panel of HSP27 and OPN that significantly improved the individual biomarker performance in discriminating UM from healthy controls. The improved discrimination of a two-marker panel of MIA and MIC-1 was also observed between metastatic UM and DF, however not statistically significant due to the small sample size. Conclusions The multiplex immunoassay provides sufficient analytical performance to evaluate serum biomarkers that complement each other in detection of UM, and warrants further validation with a larger number of patient samples

Non-linearity within the primary measurement range of a lipase assay as the cause of a gap in the interpatient lipase results distribution.

OBJECTIVES: Interpatient distribution data for lipase (Roche Cobas® assay) showed an unexpected data gap, where no results were reported. This gap occurred beginning at a point just above the assay\u27s primary measurement range (i.e., above the cutoff (300U/L) for automated repeat-on-dilution). Calculation or other errors within the automated dilution process were ruled out. Linearity of assay results was investigated. DESIGN AND METHODS: Linearity of experimental sample dilution series data was assessed by correlation coefficient, intercept, and constancy of slope. RESULTS: Dilution experiment data demonstrated a discontinuity of results between 300 and 400U/L consistent with the observed gap in patient data. Although data within the presumed linear range of the assay had a high linear correlation coefficient (r2\u3e0.99), a non-zero intercept and progressively variable slope were inconsistent with linearity. Although the assay was assessed as linear by the College of American Pathology linearity survey, survey data also demonstrated non-linearity for this assay when analyzed for slopes and intercept. CONCLUSIONS: Non-linearity in the presumed linear range of an assay can produce gaps in patient data above a repeat-on-dilution cutoff. As in this instance, CAP linearity surveys may not identify certain forms of non-linearity