Validation of the novel GLAS algorithm as an aid in the detection of liver fibrosis and cirrhosis based on GP73, LG2m, age, and sex.

BACKGROUND: Diagnosis of liver disease at earlier stages can improve outcomes and reduce the risk of progression to malignancy. Liver biopsy is the gold standard for diagnosis of liver disease, but is invasive and sample acquisition errors are common. Serum biomarkers for liver function and fibrosis, combined with patient factors, may allow for noninvasive detection of liver disease. In this pilot study, we tested and validated the performance of an algorithm that combines GP73 and LG2m serum biomarkers with age and sex (GLAS) to differentiate between patients with liver disease and healthy individuals in two independent cohorts. METHODS: To develop the algorithm, prototype immunoassays were used to measure GP73 and LG2m in residual serum samples collected between 2003 and 2016 from patients with staged fibrosis and cirrhosis of viral or non-viral etiology (n = 260) and healthy subjects (n = 133). The performance of five predictive models using combinations of age, sex, GP73, and/or LG2m from the development cohort were tested. Residual samples from a separate cohort with liver disease (fibrosis, cirrhosis, or chronic liver disease; n = 395) and healthy subjects (n = 106) were used to validate the best performing model. RESULTS: GP73 and LG2m concentrations were higher in patients with liver disease than healthy controls and higher in those with cirrhosis than fibrosis in both the development and validation cohorts. The best performing model included both GP73 and LG2m plus age and sex (GLAS algorithm), which had an AUC of 0.92 (95% CI: 0.90-0.95), a sensitivity of 88.8%, and a specificity of 75.9%. In the validation cohort, the GLAS algorithm had an estimated an AUC of 0.93 (95% CI: 0.90-0.95), a sensitivity of 91.1%, and a specificity of 80.2%. In both cohorts, the GLAS algorithm had high predictive probability for distinguishing between patients with liver disease versus healthy controls. CONCLUSIONS: GP73 and LG2m serum biomarkers, when combined with age and sex (GLAS algorithm), showed high sensitivity and specificity for detection of liver disease in two independent cohorts. The GLAS algorithm will need to be validated and refined in larger cohorts and tested in longitudinal studies for differentiating between stable versus advancing liver disease over time.

Serum PreS1 and HBsAg ratio reflects liver fibrosis and predicts the development of hepatocellular carcinoma in chronic hepatitis B patients.

While the preS1 region of the large hepatitis B surface protein plays an essential role in hepatitis B virus (HBV) infection, the effect of preS1 on liver fibrosis and hepatocarcinogenesis in chronic hepatitis B (CHB) patients is not well known. In this study, we measured serum preS1 levels by chemiluminescent immunoassay technology in 690 CHB patients and evaluated the correlation between serum preS1 levels and HBV, liver function markers and liver inflammation, fibrosis assessed by histological findings. Predictive factors for hepatocellular carcinoma (HCC) development in patients who had no previous history of HCC at the time of preS1 level measurement were also analysed. Median hepatitis B surface antigen (HBsAg) and preS1 levels were 3.08 log IU/mL and 98 ng/mL, respectively. PreS1 values were significantly correlated with serum HBsAg (p <0.001), hepatitis B core-related antigen (HBcrAg) (p <0.001) and HBV DNA levels (p <0.01). PreS1 values were also significantly correlated with serum alanine aminotransferase levels (p <0.001) and were significantly higher in patients who had higher grading of liver inflammatory activity (p <0.05). HBsAg level was correlated, but preS1/HBsAg ratio reflected liver fibrosis staging more directly than HBsAg alone. Multivariate analysis identified age ≥53 years (hazard ratio [HR], 18.360 for <53 years; p = 0.021) and preS1/HBsAg ratio ≥0.12 (HR, 6.205 for <0.12; p = 0.040) as significant and independent factors for HCC development in CHB patients. The preS1/HBsAg ratio directly reflects liver fibrosis, and the ratio might be a predictive marker for HCC development in CHB patients.

Epitope characterization of an anti-PIVKA-II antibody and evaluation of a fully automated chemiluminescent immunoassay for PIVKA-II.

OBJECTIVES: Protein induced by vitamin K absence or antagonist-II (PIVKA-II) has been used as a tumor marker to aid in the diagnosis of hepatocellular carcinoma (HCC). We developed an anti-PIVKA-II monoclonal antibody, 3C10, and a fully automated quantitative immunoassay for PIVKA-II on the ARCHITECT® i-systems. The aim of this study was to characterize the epitope of 3C10 and to evaluate the reactivity to PIVKA-II of this assay. METHODS: The epitope characterization was examined by using prothrombin γ-carboxyglutamic acid residues (Gla) domain polypeptides which are amino acid residues 17-27 that include four Gla residues at positions 19, 20, 25 and 26. The correlation with Picolumi PIVKA-II MONO (Eidia, Tokyo, Japan) and tube type equivalency was evaluated by using the developed fully automated quantitative immunoassay. RESULTS: Peptides having glutamic acid residues (Glu) at Gla domains strongly reacted to 3C10 but lost reactivity when the Glu at positions 19 or 20 was changed to Gla. The results were equivalent with an existing in vitro diagnostics product for PIVKA-II using the MU-3 antibody. A correlation study with the Picolumi PIVKA-II MONO gave a correlation coefficient of 0.99 and a regression slope of 0.92. No difference between a plain serum tube and a rapid serum tube including thrombin (RST) was observed on ARCHITECT PIVKA-II. CONCLUSIONS: The results demonstrate that this anti-PIVKA-II antibody detects equivalent epitopes with MU-3 and has equivalent reactivity to PIVKA-II as MU-3. Moreover, the ARCHITECT PIVKA-II assay has good correlation with the existing PIVKA-II product, and is applicable for use with RST.

Development and evaluation of analytical performance of a fully automated chemiluminescent immunoassay for protein induced by vitamin K absence or antagonist II.

OBJECTIVES: Protein induced by vitamin K absence or antagonist II (PIVKA-II), an abnormal form of prothrombin, has been used as an aid in the diagnosis of hepatocellular cancer (HCC) as a tumor marker. We developed a fully automated quantitative immunoassay for PIVKA-II on the ARCHITECT® i systems. The aim of this study was to evaluate the analytical performance of this assay. DESIGN AND METHOD: Assay imprecision, sensitivity, dilution linearity, high dose hook effect, sample type equivalency, assay interferences of potential interfering materials and correlation with Picolumi PIVKA-II (Eidia, Tokyo, Japan) were evaluated. RESULTS: The percentage coefficient of variation (%CV) of total imprecision ranged from 2.8% to 5.4% with 10 levels of samples. The limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) were less than 0.63 mAU/mL, 1.62 mAU/mL, and 8.25 mAU/mL, respectively. Linearity up to 30,000 mAU/mL, no high dose hook effect, no difference among sample types and no interference of common drugs and endogenous substances were observed. Correlation study with the Picolumi PIVKA-II gave a correlation coefficient of 0.93 and a regression slope of 1.07. CONCLUSIONS: The results demonstrate that the fully automated prototype ARCHITECT PIVKA-II assay is an accurate, highly sensitive and precise assay for the measurement of PIVKA-II levels in human sera and plasmas.

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 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.

A new sensitive and automated chemiluminescent microparticle immunoassay for quantitative determination of hepatitis C virus core antigen.

A new sensitive and automated chemiluminescent assay was developed for the quantitative determination of hepatitis C virus (HCV) core antigen (Ag) in human sera or plasma: the Abbott ARCHITECT HCV Ag test. The assay sensitivity was determined by testing 10 commercial HCV seroconversion panels. Without exception, a positive result for HCV core Ag was observed before anti-HCV detection, resulting in an average reduction in the period between exposure and detection of 35.8 days. Both HCV core Ag and HCV RNA were detected in the panels at the same time, indicating equivalent sensitivity and detectability. A total of 197 HCV specimens comprising genotypes 1a, 1b, 2a, 2b, 3a, 3k, 4a, 5a and 6a were evaluated. Among these, 196 (99.5%), 191 (97%) and 193 (98%) were reactive using the HCV Ag, the immunoradiometric HCV Ag and the Amplicor HCV Monitor 2 assays, respectively. A comparison with the Amplicor HCV Monitor 2 showed a correlation coefficient (r) of 0.74. The specificity of the assay was established at 99.8% by testing 5403 specimens from US volunteer blood donors, hospitalized patients and individuals with medical conditions unrelated to HCV infection, in addition to specimens containing potentially interfering substances.

Cyclic AMP/protein kinase a signal attenuates Ca(2+)-induced fibroblast growth factor-1 synthesis in rat cortical neurons.

Fibroblast growth factor (FGF)-1 is increased in particular brain regions after birth, suggesting an involvement of some regulatory neuronal circuits. To address the neuronal activity responsible for FGF-1 synthesis, effects of various neurotransmitter receptor activation on cellular FGF-1 content were examined using cultured rat cortical neurons. Histamine, glutamate, carbachol, serotonin or gamma-aminobutyric acid (GABA) caused an increase of FGF-1 content. Because this effect was mimicked by (1) N-methyl-D-aspartate, a glutamatergic agonist; (2) Ca(2+) ionophore; (3) depolarization with high concentration of KCl, but was abolished in Ca(2+)-free medium, Ca(2+) influx was thought to trigger FGF-1 synthesis. Such Ca(2+)-mediated enhancement of FGF-1 synthesis, however, did not occur in the presence of norepinephrine (NE), but was restored by KT-5720, an inhibitor of protein kinase A (PKA), suggesting an interplay between Ca(2+)-activated and cAMP/PKA signals for neuronal FGF-1 synthesis. This mechanism was proved to function in vivo by stimulation of FGF-1 expression in neurons of the cerebral cortex after intracerebral administration of propranolol, an antagonist of adrenergic beta receptors. This demonstrates that FGF-1 synthesis is essentially upregulated by Ca(2+) influx through excitatory neuronal activities, but such an effect is abolished by neurotransmission that evokes cAMP/PKA signals. FGF-1 produced is thought to act on establishment and maintenance of particular neuronal circuits in the brain, which may be one of the ways neurotransmitters regulate brain function.