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Results for “multiplex imaging”

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PubMed1997

[Proposal on new formulas for renal depth in the technetium-99m-mercaptoacetyltriglycine (MAG3) scintigraphy].

UNLABELLED: Recently, camera-based techniques to measure effective renal plasma flow (ERPF) have become more popular than single plasma sample techniques because camera-based measurements avoid the necessity of delayed plasma samples and in vitro techniques. The measurements of ERPF are used to estimate the clearance of technetium-99m-mercaptoacetyltriglycine (MAG3). However, camera-based techniques are dependent on an accurate estimate of renal depth to correct for soft-tissue attenuation. Then, new formulas for renal depth correction of technetium-99m-MAG3 clearance in place of Tønnesen's, M. Ito's, K. Itoh's, and Taylor's methods were tried to establish in this paper. PATIENTS AND METHODS: Eleven hundred and seventy patients without any renal disease were objected. The data from measurement of renal depth using X-ray CT in supine position were analyzed statistically. RESULTS: The depths of right kidney (Dr) and left kidney (Dl) were 7.33 +/- 1.27 and 7.07 +/- 1.27 cm. The correlation coefficients between Dr and height (H) body weight (W), body surface area (BSA: W0.425 x H0.725 x 0.007184 m2), age, and abdominal thickness (Ta) were 0.275, 0.709, 0.615, 0.087, and 0.743. The correlation coefficients between Dl and H, W, BSA, age, and Ta were 0.269, 0.732, 0.629, 0.029, and 0.812. Ta had best correlation with both Dr and Dl. The calculation formulas for Dr and Dl using Ta were as follows: Dr = 0.32 x Ta + 0.87 cm, and Dl = 0.36 x Ta - 0.08 cm. On the other hand, the multiplex calculation formulas of Dr or Dl with H, W, and Ta were as follows: Dr = 0.18Ta + 8.54 x (W/H) + 0.75 (r = 0.768), and Dl = 0.26Ta + 5.90 x (W/H)-0.16 (r = 0.823). CONCLUSION: The new regression equations provide superior estimates of renal depth compared to conventional equations. Application of these new formulas into camera-based protocols to determine renal clearances may lead to more accurate measurements of ERPF using technetium-99m-MAG3 scintigraphy.

Adolescent↗

PubMed2004

Development of a functionalized xenon biosensor.

NMR-based biosensors that utilize laser-polarized xenon offer potential advantages beyond current sensing technologies. These advantages include the capacity to simultaneously detect multiple analytes, the applicability to in vivo spectroscopy and imaging, and the possibility of "remote" amplified detection. Here, we present a detailed NMR characterization of the binding of a biotin-derivatized caged-xenon sensor to avidin. Binding of "functionalized" xenon to avidin leads to a change in the chemical shift of the encapsulated xenon in addition to a broadening of the resonance, both of which serve as NMR markers of ligand-target interaction. A control experiment in which the biotin-binding site of avidin was blocked with native biotin showed no such spectral changes, confirming that only specific binding, rather than nonspecific contact, between avidin and functionalized xenon leads to the effects on the xenon NMR spectrum. The exchange rate of xenon (between solution and cage) and the xenon spin-lattice relaxation rate were not changed significantly upon binding. We describe two methods for enhancing the signal from functionalized xenon by exploiting the laser-polarized xenon magnetization reservoir. We also show that the xenon chemical shifts are distinct for xenon encapsulated in different diastereomeric cage molecules. This demonstrates the potential for tuning the encapsulated xenon chemical shift, which is a key requirement for being able to multiplex the biosensor.

Avidin↗

PubMed1998

Application of fluorescence resonance energy transfer in the clinical laboratory: routine and research.

Fluorescence resonance energy transfer (FRET) phenomenon has been applied to a variety of scientific challenges in the past. The potential utility of this biophysical tool will be revisited in the 21st century. The rapid digital signal processing in conjunction with personal computers and the wide use of multicolor laser technology in clinical flow cytometry opened an opportunity for multiplexed assay systems. The concept is very simple. Color-coded microspheres are used as solid-phase matrix for the detection of fluorescent labeled molecules. It is the homogeneous assay methodology in which solid-phase particles behave similarly to the dynamics of a liquid environment. This approach offers a rapid cost-effective technology that harnesses a wide variety of fluorochromes and lasers. With this microsphere technology, the potential applications for clinical flow cytometry in the future are enormous. This new approach of well-established clinically proven methods sets the stage to briefly review the theoretical and practical aspects of FRET technology. The review shows various applications of FRET in research and clinical laboratories. Combination of FRET with monoclonal antibodies resulted in a boom of structural analysis of proteins in solutions and also in biological membranes. Cell surface mapping of cluster of differentiation molecules on immunocompetent cells has gained more and more interest in the last decade. Several examples for biological applications are discussed in detail. FRET can also be used to improve the spectral characteristics of fluorescent dyes and dye combinations, such as the tandem dyes in flow and image cytometry and the FRET primers in DNA sequencing and polymerase chain reactions. The advantages and disadvantages of donor-acceptor dye combinations are evaluated. In addition, the sensitivity of FRET provides the basis for establishing fast, robust, and accurate enzyme assays and immunoassays. Benefits and limitations of FRET-based assays are thoroughly scrutinized. At the end of the paper we review the future of FRET methodology.

Diagnostic Tests, Routine↗

PubMed2002

Particles for multiplexed analysis in solution: detection and identification of striped metallic particles using optical microscopy.

In this report, we present data demonstrating that cylindrical metallic particles, with various submicrometer striping patterns, may be readily distinguished in an optical microscope. Accurate particle identification is discussed relative to synthesis reproducibility and the limitations of optical microscopes. Results from a library of these particles, of which over 100 different striping patterns have been produced, are presented. For these particles, made with Au and Ag stripes, more than 70 patterns may be identified with greater than 90% accuracy. The ability to chemically modify the surface of these particles, making them useful for bioanalytical measurements, is also demonstrated. Finally, we discuss improvements in our manufacturing and identification processes that will lead to both larger numbers of striping patterns and improved identification accuracy.

Diagnostic Imaging↗

PubMed2006

Effects of storage time and exogenous protease inhibitors on plasma protein levels.

Plasma biomarker analysis requires intact unbiased starting material. We analyzed the effects on plasma protein profiles of protease inhibitor cocktails and preprocessing storage. Plasma from 12 healthy subjects collected with and without protease inhibitors was prepared immediately and after 2 hours of room temperature storage. The samples were analyzed by a multiplexed enzyme-linked immunosorbent assay that captured 99 chemokines and cytokines. Unsupervised hierarchical analysis clustered the samples into 4 groups; one composed predominantly of samples processed immediately, another of samples processed after 2 hours, and the remaining two were a mix of immediate and 2-hour samples. The mixed and immediate group protein profiles were similar. However, among the immediate and 2-hour samples, the levels of 37 factors differed significantly: all were greater after 2 hours. The dramatic changes in protein levels during storage were independent of protease inhibitors and are likely due to cytokine production and/or release by leukocytes and platelets.

Blood Preservation↗

PubMed1992

Discordance of the T-cell receptor alpha-chain gene in familial multiple sclerosis.

We studied restriction fragment length polymorphisms of the T-cell receptor alpha-chain (TCR alpha) gene in DNA obtained from 99 individuals of 14 multiplex families with multiple sclerosis (MS). Thirty-four family members had definite MS and two had probable MS. Six normal family members had abnormal cranial MRIs. Linkage analysis utilized constructed haplotypes of EcoRV, Sst I, and Taq I polymorphisms. With penetrance values from 0.1 to 0.7, and scoring the normal individuals with abnormal MRIs as either unknown or affected, LOD scores were between -3.16 and -7.95 for the autosomal dominant model. For the autosomal recessive model with a penetrance range from 0.1 to 1, the LOD scores ranged from -6.77 to -23.08. These findings do not support a direct role of TCR alpha in the inheritance of MS.

Adult↗

PubMed2001

Ischemic peripheral neuropathy.

Ischemic neuropathy from sources other than diabetes is less common, but can be encountered in clinical practice. Diagnosis can be challenging, and many patients may be referred to the electrodiagnostic laboratory. Overlapping mononeuritis multiplex is a common presentation, but distal symmetric polyneuropathy and monomelic neuropathy patterns can be seen. Depending on the disease associated with ischemic neuropathy, a mononeuropathy or a sensory-motor, axonal-demyelinating peripheral neuropathy may be seen as well. The treatment of ischemic neuropathy varies depending on the associated disease. Prognosis can be poor in the case of amyloidosis and the primary vasculitides. The literature is limited to cross-sectional case series and rare longitudinal studies likely related to the incidence of the diseases. Further study is needed to fully define the extent of the neurologic consequences of peripheral ischemia and its significance clinically.

Acute Disease↗

PubMed1999

Two-dimensional gene scanning: exploring human genetic variability.

Current methods for mutation detection are not optimized for the generation of highly accurate data on multiple genes of hundreds of individuals in population-based studies. Two-dimensional gene scanning (TDGS) is a high-resolution system for detecting mutational variants in multiple genes in parallel. TDGS is based on a combination of extensive multiplex polymerase chain reaction (PCR) and two-dimensional (2-D) DNA electrophoresis. The latter involves a size separation step followed by denaturing gradient gel electrophoresis (DGGE). TDGS tests for a number of large human disease genes have been designed, using a computer program to optimally position PCR primers around the relevant target sequences (e.g., exons) and evaluated using panels of samples with previously detected mutations. The results indicate a high sensitivity and specificity, equal to nucleotide sequencing, which is generally considered as the gold standard. Here, we describe the different components of the TDGS process and its potential application as a high-throughput system for the systematic identification of human gene variants.

Automation↗

PubMed2005

Schizophrenia and related disorders in children and adolescents.

This paper reviews the concept and recent studies on childhood and adolescent psychoses with special reference to schizophrenia. After a short historical introduction, the definition, classification, and epidemiology of child- and adolescent-onset psychoses are described, pointing out that some early-onset psychotic states seem to be related to schizophrenia (such as infantile catatonia) and others not (such as desintegrative disorder). The frequency of childhood schizophrenia is less than 1 in 10,000 children, but there is a remarkable increase in frequency between 13 and 18 years of age. Currently, schizophrenia is diagnosed according to ICD-10 and DSM-IV criteria. The differential diagnosis includes autism, desintegrative disorder, multiplex complex developmental disorder (MCDD) respectively multiple developmental impairment (MDI), affective psychoses, Asperger syndrome, drug-induced psychosis and psychotic states caused by organic disorders. With regard to etiology, there is strong evidence for the importance of genetic factors and for neurointegrative deficits preceding the onset of the disorder. Treatment is based upon a multimodal approach including antipsychotic medication (mainly by atypical neuroleptics), psychotherapeutic measures, family-oriented measures, and specific measures of rehabilitation applied in about 30% of the patients after completion of inpatient treatment. The long-term course of childhood- and adolescent-onset schizophrenia is worse than in adulthood schizophrenia, and the patients with manifestation of the disorder below the age of 14 have a very poor prognosis.

Adolescent↗

PubMed2003

Frequency-domain multiplexing system for in vivo diffuse light measurements of rapid cerebral hemodynamics.

A novel frequency-domain multiplexing system has been developed for in vivo measurements of rapid cerebral hemodynamics. The instrument operates in the frequency-domain with three optical wavelengths, six source positions, and two detectors. Frequency-division multiplexing was used to modulate three wavelengths (690, 786, and 830 nm) at slightly different frequencies around 70 MHz. The three laser output beams were combined and switched into different source positions by use of fast optical switches (switch time <10 ms). Three narrowband, in-phase and in-quadrature demodulators decode the modulated signals. Our full-frame-acquisition rate is 2.5 Hz, with flexibility for acquisition rates greater than 50 Hz with smaller detection areas. We evaluate the performance of the instrument with tissue phantoms, and then employ the system to measure in vivo cerebral blood oxygenation during forepaw stimulation of a rat's brain.

Animals↗

PubMed2001

Small marker chromosome identification in metaphase and interphase using centromeric multiplex fish (CM-FISH).

Multicolor karyotyping procedures, such as multiplex fluorescence in situ hybridization (M-FISH), spectral karyotyping, or color-changing karyotyping, can be used to detect chromosomal rearrangements and marker chromosomes in prenatal diagnosis, peripheral blood cultures, leukemia, and solid tumors, especially in cases where G-banding is not sufficient. A regular M-FISH analysis requires relatively large amounts of labeled DNA (microgram quantities), is not informative in interphase nuclei, hybridization can take up to 2 to 3 days, and unlabeled human chromosome-painting probes are not available commercially. Unique probes (plasmids, PAC), specific for centromeric or subtelomeric chromosomal regions, can replace the painting probes in M-FISH to address specific issues, such as the identification of marker chromosomes and aneuploidies. A set of plasmid probes carrying repetitive sequences specific for the alpha-satellite region of all human chromosomes were combined in a metaphase assay and an interphase assay, allowing identification of aneuploidies in one hybridization step, on a single cytogenetic slide. The fluorophore-dUTP and the labeled antibodies required to label and detect the DNA probes can be prepared in any laboratory. All DNA probes can be easily isolated and labeled using common molecular cytogenetic procedures. Because of the repetitive nature of the probes, hybridization time is short, usually less than 1 hour, and the analysis can be performed with nonspecialized image-processing software.

Aneuploidy↗

PubMed2005

Cost comparison of genetic and clinical screening in families with hereditary hemorrhagic telangiectasia.

Endoglin (ENG) and ALK-1 mutations cause hereditary hemorrhagic telangiecstasia (HHT), an autosomal dominant disorder leading to vascular dysplasia in the form of mucocutaneous telangiectasia and visceral arteriovenous malformations (AVMs). We proposed to compare two alternative strategies for management of HHT: screening HHT families with molecular diagnostic tests followed by targeted clinical screening versus conventional clinical screening. A decision analytic model was constructed to compare screening strategies for a hypothetical HHT family. The family consists of 1 index case and 13 relatives. The clinical screening protocol in use at the Canadian HHT Center in Toronto was assumed to be the standard of care. Unit costs for clinical screening (in Canadian dollars) were obtained from the 2003 Ontario Health Insurance Schedule of Benefits. Genetic screening costs were estimated for quantitative multiplex PCR and sequencing of Endoglin (ENG) and ALK-1 genes, as performed at HHT Solutions, Toronto. The genetic screening strategy resulted in a net cost of $4,060 per individual versus $5,975 for the clinical screening strategy. The genetic screening strategy would save $1,915 per family member or $26,810 saved per family. Sensitivity analyses revealed that the genetic screening strategy was cost saving over all plausible ranges of input variables for all hypothetical families tested. We concluded that a genetic screening strategy with targeted clinical screening is more economically attractive than conventional clinical screening and results in a reduction in the number of clinical tests for family members who do not have HHT.

Adolescent↗

PubMed2000

Micromachined ultrasound transducers with improved coupling factors from a CMOS compatible process

For medical high frequency acoustic imaging purposes the reduction in size of a single transducer element for one-dimensional and even more for two-dimensional arrays is more and more limited by fabrication and cabling technology. In the fields of industrial distance measurement and simple object recognition low cost phased arrays are lacking. Both problems can be solved with micromachined ultrasound transducers (MUTs). A single transducer is made of a large number of microscopic elements. Because of the array structure of these transducers, groups of elements can be built up and used as a phased array. By integrating parts of the sensor electronics on chip, the cabling effort for arrays can be reduced markedly. In contrast to standard ultrasonic technology, which is based on massive thickness resonators, vibrating membranes are the radiating elements of the MUTs. New micromachining technologies have emerged, allowing a highly reproducible fabrication of electrostatically driven membranes with gap heights below 500 nm. A microelectronic BiCMOS process was extended for surface micromechanics (T. Scheiter et al., Proceedings 11th European Conference on Solid-State Transducers, Warsaw, Vol. 3, 1997, pp. 1595-1598). Additional process steps were included for the realization of the membranes which form sealed cavities with the underlying substrate. Membrane and substrate are the opposite electrodes of a capacitive transducer. The transducers can be integrated monolithically on one chip together with the driving, preamplifying and multiplexing circuitry, thus reducing parasitic capacities and noise level significantly. Owing to their low mass the transducers are very well matched to fluid loads, resulting in a very high bandwidth of 50-100% (C. Eccardt et al., Proceedings Ultrasonics Symposium, San Antonio, Vol. 2, 1996, pp. 959-962; P.C. Eccardt et al., Proceedings of the 1997 Ultrasonics Symposium, Toronto, Vol. 2, 1997, pp. 1609-1618). In the following it is shown how the BiCMOS process has been modified to meet the demands for ultrasound generation and reception. Bias and driving voltages have been reduced down to the 10 V range. The electromechanical coupling is now almost comparable with that for piezoelectric transducers. The measurements exhibit sound pressures and bandwidths that are at least comparable with those of conventional piezoelectric transducer arrays.

Journal Article↗

PubMed2007

A glycoproteome database of normal human liver tissue.

PURPOSE: To extensively investigate the glycoproteins of normal human liver tissue, constructing the glycoprotein profile and database of the normal human liver tissue. METHODS: The total proteins were extracted from the normal human liver tissue and then subjected to two-dimensional electrophoresis (2-DE). Finally, 2-DE gels were stained according to the methods of multiplexed proteomics (MP) technology. Glycoprotein spots were excised from 2-DE gel and then characterized by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). RESULTS: The PDQuest software detected 1,011 glycoprotein spots and 1,923 total protein spots in the 2-DE gels of sample from the normal human liver tissue. Furthermore, 116 species of glycoproteins were successfully identified via peptide mass profiling using MALDI-TOF-MS/MS and annotated to our databases. In addition, we also applied bioinformatics softwares to predict N- or O-glycosylation sites of identified glycoproteins. CONCLUSION: This study demonstrates the feasibility of a novel technological platform to contruct glycoprotein databases. These results lay the foundation for future physiological and pathological studies of the human liver.

Databases, Protein↗

PubMed2002

Anatomical, physiological, molecular and circuit properties of nest basket cells in the developing somatosensory cortex.

Anatomical, electrophysiological and molecular diversity of basket cell-like interneurons in layers II-IV of rat somatosensory cortex were studied using patch-clamp electrodes filled with biocytin. This multiparametric study shows that neocortical basket cells (BCs) are composed of three distinct subclasses: classical large (LBC) and small (SBC) basket cells and a third subclass, the nest basket cell (NBC). Anatomically, NBCs were distinct from LBCs and SBCs in that they formed simpler dendritic arbors and an axonal plexus of inter-mediate density, composed of a few long, smooth axonal branches. Electrophysiologically, NBCs exhibited diverse discharge responses to depolarizing current injections including accommodation, non-accommodation and stuttering. Single-cell multiplex RT-PCR revealed distinct mRNA expression patterns for the calcium binding proteins parvalbumin (PV), calbindin (CB) and calretinin (CR), and the neuropeptides somatostatin (SOM), vasoactive intestinal peptide (VIP), cholecystokinin (CCK) and neuropeptide Y (NPY) for each BC-subclass. SBCs lacked NPY expression but invariably expressed VIP, whereas neither VIP, CR nor SOM expression was detected in LBCs, and VIP and CR expression was absent in NBCs. Electro-physiologically distinct types of NBCs formed GABAergic synapses with specific dynamics onto pyramidal cells (PCs) and received either strongly facilitating or depressing synaptic inputs from PCs. Finally, NBCs were found to be the most common basket cell in layers II/III, while LBCs were the most common in layer IV. These data provide multiparametric distinguishing features of three major subclasses of basket cells and indicate that NBCs are powerful interneurons that provide most of the (peri-)somatic inhibition in the supragranular layers.

Animals↗

PubMed2006

Holoprosencephaly and preaxial polydactyly associated with a 1.24 Mb duplication encompassing FBXW11 at 5q35.1.

Holoprosencephaly (HPE) is the most common developmental defect affecting the forebrain and midface in humans. The aetiology of HPE is highly heterogeneous and includes both environmental and genetic factors. Here we report on a boy with mild mental retardation, lobar HPE, epilepsy, mild pyramidal syndrome of the legs, ventricular septal defect, vesicoureteral reflux, preaxial polydactyly, and facial dysmorphisms. Genome-wide tiling path resolution array based comparative genomic hybridisation (array CGH) revealed a de novo copy-number gain at 5q35.1 of 1.24 Mb. Additional multiplex ligation-dependent probe amplification screening of a cohort of 31 patients with HPE for copy-number changes at the 5q35.1 locus did not reveal any additional genomic anomalies. This report defines a novel 1.24 Mb critical interval for HPE and preaxial polydactyly at 5q35.1. The duplicated region encompasses seven genes: RANBP17, TLX3, NPM1, FGF18, FBXW11, STK10, and DC-UbP. Since FBXW11 is relatively highly expressed in fetal brain and is directly involved in proteolytic processing of GLI3, we propose FBXW11 as the most likely candidate gene for the HPE and prexial polydactyly phenotype. Additional research is needed to further establish the role of genes from the 5q35.1 region in brain and limb development and to determine the prevalence of copy number gain in the 5q35.1 region among HPE patients.

Adolescent↗

PubMed2002

A prototype microfluidic platform for miniaturization and automation of serial dilution and dose-response assays.

A novel microfluidic device was designed and developed to miniaturize, multiplex, and automate serial dilution and three-reagent dose-response assays using submicroliter quantities of reagents. This prototype microfluidic device can be used to measure enzyme kinetics and to test a chemical lead's response to a target by fluorescent readout using common plate readers and detection systems. The prototype microfluidic system yielded serial dilution and dose-response assay data comparable to results obtained from manual titrations and reagent additions performed using a microwell plate. Enzyme kinetics were highly reproducible using these devices, although Michaelis-Menten kinetics results differed from those obtained in the microwell plate. In all cases reported here, assays performed on the microfluidic format required lower volumes of reagents compared with the microwell plate. In addition to savings in reagent consumption, the microfluidic devices and bench-top instruments offer additional advantages over conventional solutions, including a small footprint and compatibility with commercially available fluorescence detectors. Future directions for the prototype technology are discussed.

Automation↗

PubMed2006

Emerging use of nanoparticles in diagnosis and treatment of breast cancer.

The biological application of nanoparticles is a rapidly developing area of nanotechnology that raises new possibilities in the diagnosis and treatment of human cancers. In cancer diagnostics, fluorescent nanoparticles can be used for multiplex simultaneous profiling of tumour biomarkers and for detection of multiple genes and matrix RNA with fluorescent in-situ hybridisation. In breast cancer, three crucial biomarkers can be detected and accurately quantified in single tumour sections by use of nanoparticles conjugated to antibodies. In the near future, the use of conjugated nanoparticles will allow at least ten cancer-related proteins to be detected on tiny tumour sections, providing a new method of analysing the proteome of an individual tumour. Supermagnetic nanoparticles have exciting possibilities as contrast agents for cancer detection in vivo, and for monitoring the response to treatment. Several chemotherapy agents are available as nanoparticle formulations, and have at least equivalent efficacy and fewer toxic effects compared with conventional formulations. Ultimately, the use of nanoparticles will allow simultaneous tumour targeting and drug delivery in a unique manner. In this review, we give an overview of the use of clinically applicable nanoparticles in oncology, with particular focus on the diagnosis and treatment of breast cancer.

Animals↗