| 1. |
- Fredriksson, Simon, et al.
(författare)
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Multiplexed proximity ligation assays to profile putative plasma biomarkers relevant to pancreatic and ovarian cancer
- 2008
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 54:3, s. 582-589
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Sensitive methods are needed for biomarker discovery and validation. We tested one promising technology, multiplex proximity ligation assay (PLA), in a pilot study profiling plasma biomarkers in pancreatic and ovarian cancer. METHODS: We used 4 panels of 6- and 7-plex PLAs to detect biomarkers, with each assay consuming 1 mu L plasma and using either matched monoclonal antibody pairs or single batches of polyclonal antibody. Protein analytes were converted to unique DNA arnplicons by proximity ligation and subsequently detected by quantitative PCR. We profiled 18 pancreatic cancer cases and 19 controls and 19 ovarian cancer cases and 20 controls for the following proteins: a disintegrin and metalloprotease 8, CA-125, CA 19-9, carboxypeptidase A1, carcinoembryonic antigen, connective tissue growth factor, epidermal growth factor receptor, epithelial cell adhesion molecule, Her2, galectin-1, insulin-like growth factor 2, interleukin-1 alpha, interleukin-7, mesothelin, macrophage migration inhibitory factor, osteopontin, secretory leukocyte peptidase inhibitor, tumor necrosis factor a, vascular endothelial growth factor, and chitinase 3-like 1. Probes for CA-125 were present in 3 of the multiplex panels. We measured plasma concentrations of the CA-125-mesothelin complex by use of a triple-specific PLA with 2 ligation events among 3 probes. RESULTS: The assays displayed consistent measurements of CA-125 independent of which other markers were simultaneously detected and showed good correlation with Luminex data. In comparison to literature reports, we achieved expected results for other putative markers. CONCLUSION: Multiplex PLA using either matched monoclonal antibodies or single batches of polyclonal. antibody should prove useful for identifying and validating sets of putative disease biomarkers and finding. multimarker panels.
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| 2. |
- Geistanger, Andrea, et al.
(författare)
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Statistical methods for monitoring the relationship between the IFCC reference measurement procedure for hemoglobin A(1c) and the designated comparison methods in the United States, Japan, and Sweden
- 2008
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 54:8, s. 1379-1385
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The American Diabetes Association (ADA)/European Association for the Study of Diabetes (EASD)/International Diabetes Federation GDFAFCC Consensus Statement on the worldwide standardization of HbA(1c) states that"...[HbA(1c)] results are to be reported world-wide in IFCC units...and derived NGSP units...,using the IFCC-NGSP master equation." METHODS: We describe statistical methods to evaluate and monitor the relationships as expressed in master equations (MEs) between the IFCC Reference Measurement procedure (IFCC-RM) and designated comparison methods (DCMs) [US National Glycohemoglobin Standardization Program (NGSP), Japanese Diabetes Society/Japanese Society for Clinical Chemistry (JDS/JSCC), and Mono-S in Sweden]. We applied these statistics, including uncertainty calculations, to 12 studies in which networks of reference laboratories participated, operating the IFCC-RM and DCMs. RESULTS: For NGSP and Mono-S, slope, intercept, and derived percentage HbA(1c) at the therapeutic target show compliance with the respective MEs in all 12 studies. For JDS/JSCC, a slight deviation is seen in slope and derived percentage HbA(1c) in 2 of the 12 studies. Using the MEs, the uncertainty in an assigned value increases from 0.42 mmol/mol HbA(1c) (IFCC-RM) to 0.47 (NGSP), 0.49 (JDS/JSCC), and 0.51 (Mono-S). CONCLUSIONS: We describe sound statistical methods for the investigation of relations between networks of reference laboratories. Application of these statistical methods to the relationship between the JFCC-RM and DCMs in the US, Japan, and Sweden shows that they are suitable for the purpose, and the results support the applicability of the ADA/EASD/lDF/lFCC Consensus Statement on HbA1c measurement. (C) 2008 American Association for Clinical Chemistry.
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| 3. |
- Gustafsdottir, Sigrun M, et al.
(författare)
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Use of Proximity Ligation to Screen for Inhibitors of Interactions between Vascular Endothelial Growth Factor A and Its Receptors
- 2008
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 54:7, s. 1218-1225
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Improved methods are required to screen drug candidates for their influences on protein interactions. There is also a compelling need for miniaturization of screening assays, with attendant reductions in reagent consumption and assay costs. METHODS: We used sensitive, miniaturized proximity ligation assays (PLAs) to monitor binding of vascular endothelial growth factor A (VEGF-A) to 2 of its receptors, VEGFR-1 and VEGFR-2. We measured the effects of proteins and low molecular weight compounds capable of disrupting these interactions and compared the results with those obtained by immunoblot analysis. We analyzed 6 different inhibitors: a DNA aptamer, a mixed DNA/RNA aptamer, a monoclonal VEGF-A neutralizing antibody, a monoclonal antibody directed against VEGFR-2, a recombinant competitive protein, and a low molecular weight synthetic molecule. RESULTS: The PLAs were successful for monitoring the formation and inhibition of VEGF-A-receptor complexes, and the results correlated well with those obtained by measuring receptor phosphorylation. The total PLA time is just 3 hours, with minimal manual work and reagent additions. The method allows evaluation of the apparent affinity [half-maximal inhibitory concentration (IC(50))] from a dose-response curve. CONCLUSIONS: The PLA may offer significant advantages over conventional methods for screening the interactions of ligands with their receptors. The assay may prove useful for parallel analyses of large numbers of samples in the screening of inhibitor libraries for promising agents. The technique provides dose-response curves, allowing IC(50) values to be calculated.
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| 4. |
- Hartmann, Michael, et al.
(författare)
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Expanding assay dynamics : A combined competitive and direct assay system for the quantification of proteins in multiplexed Immunoassays
- 2008
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 54:6, s. 956-963
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The concurrent detection and quantification of analytes that vary widely in concentration present a principal problem in multiplexed assay systems. Combining competitive and sandwich immunoassays permits coverage of a wide concentration range, and both highly abundant molecules and analytes present in low concentration can be quantified within the same assay. METHODS: The use of different fluorescence readout channels allows the parallel use of a competitive system and a sandwich configuration. The 2 generated assay signals are combined and used to calculate the amount of analyte. The measurement range can be adjusted by varying the competitor concentration, and an extension of the assay system's dynamic range is possible. RESULTS: We implemented the method in a planar protein microarray-based autoimmune assay to detect autoantibodies against 13 autoantigens and to measure the concentration of a highly abundant protein, total human IgG, in one assay. Our results for autoantibody detection and IgG quantification agreed with results obtained with commercially available assays. The use of 2 readout channels in the protein microarray-based system reduced spot-to-spot variation and intraassay variation. CONCLUSIONS: By combining a direct immunoassay with a competitive system, analytes present in widely varying concentrations can be quantified within a single multiplex assay. Introducing a second readout channel for analyte quantification is an effective tool for spot-to-spot normalization and helps to lower intraassay variation.
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| 5. |
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| 6. |
- Lakso, Hans-Ake, et al.
(författare)
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Quantification of methylmalonic acid in human plasma with hydrophilic interaction liquid chromatography separation and mass spectrometric detection
- 2008
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Ingår i: Clinical Chemistry. - Washington, DC : American Association for Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 54:12, s. 2028-2035
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Tidskriftsartikel (refereegranskat)abstract
- Background: Measurement of methylmalonic acid (MMA) in serum or plasma is useful for diagnosing cobalamin deficiency. We developed a method for quantifying MMA in plasma based on hydrophilic interaction liquid chromatography (HILIC) and single-stage negative electrospray ionization (ESI) mass spectrometry. Methods: We deproteinized plasma samples (200 microL) with 800 microL acidified acetonitrile containing 0.17 micromol/L deuterated MMA (D(3)-MMA) internal standard, centrifuged the samples, and injected 4 microL of the supernatant into the LC-MS instrument. Separation was achieved within 3 min on a Merck SeQuant ZIC-HILIC column with a mobile phase consisting of 4 volumes acetonitrile plus 1 volume 100 mmol/L ammonium acetate buffer, pH 4.5, at a flow rate of 400 microL/min. Subsequent column washing and reconditioning contributed to a total run time of 10 min. MMA and D(3)-MMA were quantified by single-ion monitoring (m/z 117.2 and 120.2, respectively) in negative ESI mode at a drying-gas flow rate of 10 L/min, 300 degrees C, and a capillary voltage of 3.0 kV. Results: The estimated limits of MMA quantification and detection were 0.09 micromol/L and 0.03 micromol/L, respectively, in plasma. The assay was linear to 200 micromol/L. Interassay and intraassay CVs were < or = 5% at all tested concentrations. Recoveries were 90%-93%. Conclusions: This robust assay allows analysis of MMA in human plasma without derivatization. Sample preparation is simple and suitable for automation.
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| 7. |
- Lee, Jin-Moo, et al.
(författare)
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The brain injury biomarker VLP-1 is increased in the cerebrospinal fluid of Alzheimer disease patients.
- 2008
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Ingår i: Clinical chemistry. - : Oxford University Press (OUP). - 1530-8561 .- 0009-9147. ; 54:10, s. 1617-23
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Definitive diagnosis of Alzheimer disease (AD) can be made only by histopathological examination of brain tissue, prompting the search for premortem disease biomarkers. We sought to determine if the novel brain injury biomarker, visinin-like protein 1 (VLP-1), is altered in the CSF of AD patients compared with controls, and to compare its values to the other well-studied CSF biomarkers 42-amino acid amyloid-beta peptide (Abeta(1-42)), total Tau (tTau), and hyperphosphorylated Tau (pTau). METHODS: Using ELISA, we measured concentrations of Abeta(1-42), tTau, pTau, and VLP-1 in CSF samples from 33 AD patients and 24 controls. We compared the diagnostic performance of these biomarkers using ROC curves. RESULTS: CSF VLP-1 concentrations were significantly higher in AD patients [median (interquartile range) 365 (166) ng/L] compared with controls [244 (142.5) ng/L]. Although the diagnostic performance of VLP-1 alone was comparable to that of Abeta, tTau, or pTau alone, the combination of the 4 biomarkers demonstrated better performance than each individually. VLP-1 concentrations were higher in AD subjects with APOE epsilon4/epsilon4 genotype [599 (240) ng/L] compared with epsilon3/epsilon4 [376 (127) ng/L] and epsilon3/epsilon3 [280 (115.5) ng/L] genotypes. Furthermore, VLP-1 values demonstrated a high degree of correlation with pTau (r = 0.809) and tTau (r = 0.635) but not Abeta(1-42) (r = -0.233). VLP-1 was the only biomarker that correlated with MMSE score (r = -0.384, P = 0.030). CONCLUSIONS: These results suggest that neuronal injury markers such as VLP-1 may have utility as biomarkers for AD.
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| 8. |
- Lempiäinen, Anna, et al.
(författare)
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Free beta-subunit of human chorionic gonadotropin in serum is a diagnostically sensitive marker of seminomatous testicular cancer
- 2008
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 54:11, s. 1840-3
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: We studied whether measurement of the free beta subunit of human chorionic gonadotropin (hCGbeta) in serum offers additional diagnostic information compared to determination of intact hCG alone in testicular cancer. METHODS: We determined hCG and hCGbeta with ultrasensitive assays in 94 serum samples obtained preoperatively, 22 samples obtained during relapse, and 3687 samples obtained during routine follow-up of 351 patients with testicular tumors. RESULTS: In preoperative samples, isolated increases of hCGbeta were seen in 40% of the samples from seminoma patients (n = 42) and in 8% of those from patients with nonseminomatous testicular cancer (NSGCT) (n = 51). Both markers were increased in 12% of the seminoma and 71% of the NSGCT patients and were within reference intervals in 43% of the seminoma and 20% of the NSGCT patients. Specific determination of hCGbeta increased the frequency of marker-positive seminomas from 17% to 57% and of marker-positive relapses from 32% to 59% (n = 22). Theoretically, about 40% of marker-positive seminomas and relapses would have been missed with an assay measuring hCG and hCGbeta together. Preoperative hCG and hCGbeta concentrations correlated with stage, tumor histology, and disease-related mortality. Additionally, hCGbeta correlated with tumor size. CONCLUSIONS: hCGbeta is a diagnostically sensitive marker for testicular cancer. In patients with seminomatous testicular cancer, hCGbeta is superior to hCG, and in some NSGCT patients it provides additional information.
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| 9. |
- Peter Goetze, Jens, et al.
(författare)
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Impact of Epitope Specificity and Precursor Maturation in Pro-B-Type Natriuretic Peptide Measurement
- 2008
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 54:11, s. 1780-1787
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Tidskriftsartikel (refereegranskat)abstract
- Background: Cardiac-derived natriuretic peptides are sensitive plasma markers of cardiac dysfunction. Recent reports have disclosed a more complex molecular heterogeneity of B-type natriuretic peptide precursor (proBNP)-derived peptides than previously Suggested. In this study, we examined the impact of epitope specificity and precursor maturation oil plasma measurement of proBNP-derived peptides. Methods: We compared 2 assays, N-terminal proBNP and proBNP 1-76, in a randomly collected set of human plasma specimens (n = 370). Additionally, we evaluated the clinical performance of 4 assays with different epitope specificities in a cohort of elderly patients presenting with symptoms associated with heart failure (n = 415). Results: Comparison of N-terminal proBNP with proBNP 1-76 measurement in plasma revealed a high correlation on regression analysis (r(2) = 0.91, p < 0.0001). Nevertheless, the proBNP 1-76 assay measured lower concentrations in the high range than the N-terminal proBNP assay. Correlations between assay measurements in a clinical setting were comparable for all the assays (r(2) approximately 0.57-0.83), and ROC analyses revealed area-under-the-curve values ranging between 0.77 and 0.81 for identifying reduced left ventricular ejection fraction. In parallel, all assays displayed comparable abilities in predicting long-term mortality. Conclusions: Our results reveal marked assay differences in analytical assay comparison, contrasting the overall comparable clinical performance in cardiovascular diagnostics or prognosis in the elderly.
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| 10. |
- Russom, Aman, et al.
(författare)
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Microfluidic leukocyte isolation for gene expression analysis in critically ill hospitalized patients
- 2008
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 54:5, s. 891-900
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Microarray technology is becoming a powerful tool for diagnostic, therapeutic, and prognostic applications. There is at present no consensus regarding the optimal technique to isolate nucleic acids from blood leukocyte populations for subsequent expression analyses. Current collection and processing techniques pose significant challenges in the clinical setting. Here, we report the clinical validation of a novel microfluidic leukocyte nucleic acid isolation technique for gene expression analysis from critically ill, hospitalized patients that can be readily used on small volumes of blood. METHODS: We processed whole blood from hospitalized patients after burn injury and severe blunt trauma according to the microfluidic and standard macroscale leukocyte isolation protocol. Side-by-side comparison of RNA quantity, quality, and genome-wide expression patterns was used to clinically validate the microfluidic technique. RESULTS: When the microfluidic protocol was used for processing, sufficient amounts of total RNA were obtained for genome-wide expression analysis from 0.5 mL whole blood. We found that the leukocyte expression patterns from samples processed using the 2 protocols were concordant, and there was less variability introduced as a result of harvesting method than there existed between individuals. CONCLUSIONS: The novel microfluidic approach achieves leukocyte isolation in <25 min, and the quality of nucleic acids and genome expression analysis is equivalent to or surpasses that obtained from macroscale approaches. Microfluidics can significantly improve the isolation of blood leukocytes for genomic analyses in the clinical setting. (c) 2008 American Association for Clinical Chemistry.
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