Performance evaluation of the Elecsys PIVKA-II and Elecsys AFP assays for hepatocellular carcinoma diagnosis.

Background and Aims: Prothrombin induced by vitamin K absence-II (PIVKA-II) is a serum biomarker linked to hepatocellular carcinoma (HCC), showing superiority to alpha-fetoprotein (AFP) for early disease detection. We aimed to assess the clinical and analytical performance of the Elecsys® PIVKA-II immunoassay in diagnosing HCC and evaluate PIVKA-II's technical performance. Methods: Serum samples from adult cases (i.e. patients with a first-time HCC diagnosis; n = 168) and disease controls (i.e. patients without HCC with an at-risk condition; n = 208) were assessed. An AFP cut-off of 20 ng/mL was used to differentiate between HCC cases and disease controls. Clinical performance of the Elecsys PIVKA-II assay was compared with that of comparator assays (Lumipulse G PIVKA-II, µTASWako DCP, ARCHITECT PIVKA-II) using receiver operating characteristic curve analysis to determine the area under the curve (AUC) values. Results: The Elecsys PIVKA-II assay compared favorably with comparator assays. Using a 28.4 ng/mL cut-off, the Elecsys PIVKA-II assay detected HCC with 86.9% sensitivity and 83.7% specificity. Clinical performance of the Elecsys PIVKA-II assay (AUC: 90.8%) was equivalent to that of comparator assays (AUC: 88.3-89.6%). Relatively high PIVKA-II concentrations were observed for cholangiocarcinoma and pancreatic cancer with the Elecsys assay in specificity panel analyses, indicating that high PIVKA-II concentrations should not be used alone in the absence of other clinical data. Conclusions: The Elecsys PIVKA-II assay showed good analytical performance under routine laboratory conditions, comparing favorably with comparator assays. These findings support the suitability of the Elecsys PIVKA-II assay as an aid in HCC diagnosis.

Multicenter evaluation of a new progastrin-releasing peptide (ProGRP) immunoassay across Europe and China.

BACKGROUND: We performed a multicenter evaluation of the Elecsys® progastrin-releasing peptide (ProGRP) immunoassay in Europe and China. METHODS: The assay was evaluated at three European and two Chinese sites by imprecision, stability, method comparison and differentiation potential in lung cancer. RESULTS: Intermediate imprecision across five analyte concentrations ranged from 2.2% to 6.0% coefficient of variation. Good stability for plasma and serum samples was shown for various storage conditions. There was excellent correlation between the Elecsys® and ARCHITECT assays in plasma (slope 1.02, intercept -2.72pg/mL). The Elecsys® assay also showed good correlation between serum and plasma samples (slope 0.93, intercept 2.35pg/mL; correlation coefficient 0.97). ProGRP differentiated small-cell and non-small-cell lung cancer (NSCLC; area under the curve 0.90, 95% CI 0.87-0.93; 78.3% sensitivity, 95% specificity; at 84pg/mL), with no relevant effects of ethnicity, age, gender or smoking. Median ProGRP concentrations were low in benign diseases (38pg/mL), other malignancies (40pg/mL) or NSCLC (39pg/mL), except chronic kidney disease above stage 3 (>100pg/mL). CONCLUSIONS: Increased stability of the Elecsys® ProGRP assay in serum and plasma offers clear benefits over existing assays. This first evaluation of a ProGRP assay in China demonstrated comparable differentiation potential among different ethnicities.

High expression of 5-lipoxygenase in normal and malignant mantle zone B lymphocytes.

BACKGROUND: Human B lymphocytes can produce leukotriene B4 but the biological function of the 5-lipoxygenase (5-LO) pathway in B cells is unclear. In order to better understand and define the role of 5-LO in B cells, we investigated the expression of 5-LO mRNA and protein in subsets of B cells from human tonsils and different types of B cell lymphoma. RESULTS: Based on RT-PCR and western blot/immunohistochemical staining, with a polyclonal antibody raised against 5-LO, high expression of 5-LO was found in mantle zone B cells from tonsils. By contrast, only a weak expression of 5-LO was detected in germinal centre cells and no expression in plasma cells from tonsils. This pattern of 5-LO expression was preserved in malignant lymphoma with high expression in mantle B cell lymphoma (MCL) and weak or no expression in follicular lymphoma. Primary leukemized MCL, so called B-prolymphocytic leukaemia cells, and MCL cell lines also expressed 5-LO and readily produced LTB4 after activation. CONCLUSION: The present report demonstrates the expression of 5-LO mainly in normal and malignant mantle zone B cells while the expression is low or absent in germinal centre B cells and plasma cells, indicating a role of the 5-LO pathway in B cells before the cells finally differentiate to plasma cells.

In vivo near-infrared fluorescence imaging of carcinoembryonic antigen-expressing tumor cells in mice.

PURPOSE: To prospectively depict carcinoembryonic antigen (CEA)-expressing tumors in mice with a high-affinity probe consisting of a near-infrared (NIR) fluorochrome and the clinically used anti-CEA antibody fragment arcitumomab. MATERIALS AND METHODS: This study was approved by the regional animal committee. By coupling a NIR fluorescent (NIRF) cyanine dye (DY-676) to a specific antibody fragment directed against CEA (arcitumomab) and a nonspecific IgG Fab fragment, a bio-optical high-affinity fluorescent probe (anti-CEA-DY-676) and a low-affinity fluorescent probe (FabIgG-DY-676) were designed. The dye-to-protein ratios were determined, and both probes were tested for NIRF imaging in vitro on CEA-expressing LS-174T human colonic adenocarcinoma cells and CEA-nonexpressing A-375 human melanoma cells by using a bio-optical NIR small-animal imager. In vivo data of xenografted LS-174T and A-375 tumors in mice (n = 10) were recorded and statistically analyzed (Student t test). RESULTS: The dye-to-protein ratios were determined as 3.0-3.5 for both probes. In vitro experiments revealed the specific binding of the anti-CEA-DY-676 probe on CEA-expressing cells as compared with CEA-nonexpressing cells; the FabIgG-DY-676 probe showed a markedly lower binding affinity to cells. In vivo LS-174T tumors xenografted in all mice could be significantly distinguished from A-375 tumors with application of the anti-CEA-DY-676 but not with that of the FabIgG-DY-676 at different times (2-24 hours, P < .005) after intravenous injection of the probes. Semiquantitative analysis revealed maximal fluorescence signals of anti-CEA-DY-676 to CEA-expressing tumors about 8 hours after injection. CONCLUSION: Findings of this study indicate the potential use of the high-affinity probe anti-CEA-DY-676 for specific NIRF imaging in in vivo tumor diagnosis.

Fluorescent bacterial magnetic nanoparticles as bimodal contrast agents.

OBJECTIVES: The purpose of this study was to assess whether fluorochrome-coupled bacterial magnetic nanoparticles can be used as bimodal contrast agent for both magnetic resonance imaging (MRI) and near-infrared fluorescence optical (NIRF) imaging of cultured macrophages. MATERIALS AND METHODS: Bacterial magnetic nanoparticles (magnetosomes, particle diameter: 42 nm) were harvested from Magnetospirillum gryphiswaldense and characterized by using MRI. After covalent coupling to the fluorescent dye DY-676 (lambdaabs./lambdaem.= 676 nm/701 nm, Dyomics, Jena, Germany), the fluorescent magnetosomes were analyzed by fluorescence-activated cell sorting. Subsequently, murine macrophages J774 were incubated with the bimodal contrast agent (3 hours) and examined by a whole-body near infrared small animal imaging system as well as by using a 1.5 T clinical MR system. Moreover, labeled cells were characterized using confocal laser scanning microscopy (CLSM) and ultrathin section transmission electron microscopy. RESULTS: Characterization of the nanoparticles by MRI revealed R1 and R2 relaxivities of 3.2 mMs and 526 mMs, respectively. Fluorochrome-coupled magnetosomes exhibited increased fluorescence intensities at wavelengths >670 nm. Macrophages that were incubated with the contrast agent showed a significant fluorescence emission in the near infrared range as imaged with a whole body NIR imaging system, FACS analysis and CLSM. Moreover, CLSM data showed the greatest fluorescence intensities within intracellular compartments and colocalized with the magnetosomes. With MRI, both T1 and T2 relaxation times were substantially shortened at concentrations greater than 600 cells/microL. DISCUSSION AND CONCLUSION: Macrophages could be labeled with fluorescent magnetosomes, and they were successfully imaged using both a 1.5 T MR scanner as well as with NIRF optical methods. The use of this bimodal contrast agent for diagnostic purposes may benefit from the excellent spatial resolution of the MRI and the high sensitivity of the fluorescence imaging.

MR imaging of Her-2/neu protein using magnetic nanoparticles.

The aim of this study was to assess whether Her-2/neu expressing tumour cells can be detected in vitro as well as in animal tumour models with magnetic resonance imaging at 1.5 T. Magnetic nanoparticles (with relaxivities R 1, R 2 of 3.7 ± 0.4 (mM s)(-1), 277 ± 32 (mM s)(-1) at 21 °C, respectively) coupled to anti-Her-2/neu antibodies or gamma globulin IgG (high or non-affinity probe, respectively) were used. After incubation of Her-2/neu expressing cells (SKBR3) with high or non-affinity probes (20 min), values of R 1 = 0.34 ± 0.02 (mM s)(-1) and R 2 = 63.02 ± 30 (mM s)(-1) were obtained. Electron microscopy and atomic absorption spectrometry examinations verified the presence of relatively high iron levels in cells incubated with the high affinity probe compared to controls. For in vivo MRI, high or non-affinity probes (≈1.7 mg Fe/animal) were injected into the tail vein of mice (n = 16) bearing SKBR3 tumours. A distinct decrease in the normalized MR signal ratio between tumour and reference area (approximately -17 ± 2%) after application of the high affinity probe was observed. In conclusion, in vivo detection of Her-2/neu expressing tumours is feasible in a clinical MR scanner by using immunoconjugated magnetic nanoparticles.

Diagnosis of peritonitis using near-infrared optical imaging of in vivo labeled monocytes-macrophages.

Peritonitis is an inflammatory process characterized by massive monocytes-macrophages infiltration. Since early diagnosis is important for a successful therapeutic outcome, the feasibility for a selective labeling and imaging of macrophages for highly sensitive optical imaging was assessed. After in vitro incubation of mouse macrophages J774A.1 with the far-red/near-infrared fluorochrome DY-676, distinct fluorescence intensities (1026+/-142 a.u.) were detected as compared to controls (552+/-54 a.u.) using a whole-body small animal near-infrared fluorescence (NIRF) imaging system. Macrophage labeling was confirmed by confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting, (FACS). The fluorochrome was also found to be predominantly distributed within compartments in the cytoplasm. Additionally, peritonitis was induced in mice by intraperitoneal injection of zymosanA. After intravenous injection of fluorochrome (55 nmol/kg) and using whole-body fluorescence imaging, higher fluorescence intensities (869+/-151 a.u.) were detected in the peritoneal area of diseased mice as compared to controls (188+/-41 a.u.). Furthermore, cells isolated from peritoneal lavage revealed the presence of labeled monocytes-macrophages. The results indicate that in vivo diagnosis of peritonitis by near-infrared optical imaging of labeled monocytes-macrophages is feasible. Possibly, early stages of other inflammatory diseases could also be detected by the proposed diagnostic method in the long term.