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Results for “Automated phenotyping”

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At least 55 records · Page 3Linked to original sources

PubMed2000

Distribution of haptoglobin phenotypes in a Korean population, using the semi-automated PhastSystem.

We have established a new phenotyping method for haptoglobin, based on sodium dodecyl sulphate-polyacrylamide gel electrophoresis using the PhastSystem (Pharmacia Biotech, Uppsala, Sweden), followed by immunoblotting for detection. We measured haptoglobin concentrations and determined the haptoglobin phenotypes of 316 healthy Koreans using this method: 31 (9.8%) were of Hp 1-1 type, 140 (44.3%) of Hp 2-1 type and 145 (45.9%) of Hp 2-2 type. The haptoglobin allele frequencies were calculated to be 0.32 for Hp1 and 0.68 for Hp2. We were able to visualize up to 12 bands from the human Hp 2-2 polymeric series, with molecular weights in the range 171.9 x 10(3) to 802.2 x 10(3). The reference range of serum haptoglobin concentrations obtained by the IFCC (International Federation of Clinical Chemistry) standard method was 0.27-2.14 g/L. The serum haptoglobin concentration in Koreans was similar to that of Caucasians, but the Hp1 allele frequency was lower in Koreans. Our method could be used in clinical laboratories as a simple and practical method of haptoglobin phenotyping. In addition, the Hp 2-2 polymeric series could be used as high molecular weight standards.

Alleles↗

PubMed2006

Use of calibrated automated thrombinography +/- thrombomodulin to recognise the prothrombotic phenotype.

There is currently no validated method to detect a prothrombotic phenotype. The question remains, can tissue factor (TF) induced thrombin generation (TG), as measured with the calibrated automated thrombinography (CAT) technique, according to Hemker et al., recognise a prothrombotic state either as such, or when the activated protein C (APC)-system is boosted with thrombomodulin (TM)? We determined the normal range of CAT-TG +/- TM in a group of 71 healthy blood donors, in 11 healthy women using oral contraceptives (OC), and in 89 patients with a history of venous thromboembolism (VTE), divided into a group of 50 in which a prothrombotic risk factor could be found (VTEprf+) and 39 others (VTEprf-). The endogenous thrombin potential (ETP) in the OC, VTEprf+ and VTEprf- group was significantly higher than for the controls. In the presence of TM, the differences were significantly higher than in its absence. The VTEprf+ group had a higher ETP, +/- TM than the VTEprf-group. In conclusion, TG, measured with the CAT technique in the presence of TM is capable of detecting the prothrombotic phenotype with a high sensitivity of 0.93 (95% confidence limits 0.82-0.99).

Adult↗

PubMed1978

Evaluation of an automated immunoassay procedure for apo-LDL for phenotyping type II hyperlipoproteinemia.

Apo-low density lipoproteins were determined by an automated immunoassay procedure on serum samples from 88 normolipidemic individuals and 84 hyperlipoproteinemic subjects, to establish whether this method was useful in the routine detection of type II hyperlipoproteinemia. The results obtained were compared with the cholesterol levels of the same specimens. In subjects with type II hyperlipoproteinemia, the apo low density lipoprotein levels, as well as the ratio of low density lipoprotein cholesterol/apo-low density lipoprotein were higher, as expected, than in normals or in subjects with other types of hyperlipoproteinemia. However, there was considerable overlap in individual values of both these parameters, between patients with type II hyperlipoproteinemia and normals or subjects with other types of hyperlipoproteinemia, suggesting that apo low density lipoprotein levels alone were not sufficiently discriminatory for the laboratory determination of type II hyperlipoproteinemia.

Adult↗

PubMed2025

Tribus: semi-automated discovery of cell identities and phenotypes from multiplexed imaging and proteomic data.

MOTIVATION: Multiplexed imaging and single-cell analysis are increasingly applied to investigate the tissue spatial ecosystems in cancer and other complex diseases. Accurate single-cell phenotyping based on marker combinations is a critical but challenging task due to (i) low reproducibility across experiments with manual thresholding, and, (ii) labor-intensive ground-truth expert annotation required for learning-based methods. RESULTS: We developed Tribus, an interactive knowledge-based classifier for multiplexed images and proteomic datasets that avoids hard-set thresholds and manual labeling. We demonstrated that Tribus recovers fine-grained cell types, matching the gold standard annotations by human experts. Additionally, Tribus can target ambiguous populations and discover phenotypically distinct cell subtypes. Through benchmarking against three similar methods in four public datasets with ground truth labels, we show that Tribus outperforms other methods in accuracy and computational efficiency, reducing runtime by an order of magnitude. Finally, we demonstrate the performance of Tribus in rapid and precise cell phenotyping with two large in-house whole-slide imaging datasets. AVAILABILITY AND IMPLEMENTATION: Tribus is available at https://github.com/farkkilab/tribus as an open-source Python package.

Proteomics↗

PubMed2002

Development of a rapid and sensitive high-performance liquid chromatographic method to determine CYP2D6 phenotype in human liver microsomes.

Dextromethorphan is a probe substrate to determine CYP2D6 phenotype. The conversion of dextromethorphan to dextrorphan by CYP2D6 accounts for approximately 60% of total metabolism. Most analytical methods utilize complicated labor- and time-intensive sample processing methods with several liquid-liquid extraction (LLE) steps. Our goal was to develop a non-LLE based rapid and sensitive HPLC method, to measure dextromethorphan metabolism in human liver microsomes. A solid-phase filtration based reverse-phase HPLC method with fluorescence detection was developed and validated. Human liver (n = 6) microsomal incubations were carried out with dextromethorphan, under optimum conditions. The analytes were separated by one-step centrifugal filtration with Nanosep separation units. The filtrate was injected ( 50 microL) into a Waters Alliance 2690 HPLC system. Metabolic incubations were also conducted to determine levels using LLE for comparisons. The Nanosep separation step reduced the extraction time from 3h to 40 min. The limit of quantitation was 23.8 nM (9.7 ng/mL), recovery was approximately 98%, the mean precision values were <10% RSD for the controls (80, 320 and 640 nM) and mean percentage error was <5%. Michaelis-Menten parameters were determined to distinguish CYP2D6 phenotypes. A rapid and sensitive HPLC method is reported, which may be suitable for automation and allows phenotyping of human liver microsomes.

Antitussive Agents↗

PubMed2003

Phenotypic resistance testing.

The development of automated assay technology for rapid genotypic and phenotypic characterization of human immunodeficiency virus (HIV) in plasma samples now makes it feasible to use these assays in the management of antiretroviral therapy. In particular, phenotypic assays measure the drug susceptibility of the virus by determining the concentration of drug that inhibits viral replication in tissue culture. Recent experiences suggest that interpreting phenotypes may be more complicated than originally believed. Thus, rather than considering drugs as active or inactive on the basis of cut-offs, it would be more reasonable to consider their relative activity. To complicate matters further, a relatively high incidence of discordance between phenotypic and genotypic results when both tests were performed on plasma samples of antiretroviral treated patients has been reported. These findings could have considerable clinical significance, because the choice of drug regimen could differ depending on which test is used. The result of phenotypic testing may be more important when considering discordant results, because this provides a more quantitative assessment of resistance. However, clinical validation will be necessary to delineate which approach is better.

Anti-HIV Agents↗

PubMed2001

Phenotype microarrays for high-throughput phenotypic testing and assay of gene function.

The bacterium Escherichia coli is used as a model cellular system to test and validate a new technology called Phenotype MicroArrays (PMs). PM technology is a high-throughput technology for simultaneous testing of a large number of cellular phenotypes. It consists of preconfigured well arrays in which each well tests a different cellular phenotype and an automated instrument that continuously monitors and records the response of the cells in all wells of the arrays. For example, nearly 700 phenotypes of E. coli can be assayed by merely pipetting a cell suspension into seven microplate arrays. PMs can be used to directly assay the effects of genetic changes on cells, especially gene knock-outs. Here, we provide data on phenotypic analysis of six strains and show that we can detect expected phenotypes as well as, in some cases, unexpected phenotypes.

Escherichia coli↗

PubMed1979

A semi-automated Bratton & Marshall micromethod for determining acetylator phenotype of rabbits using the Abbott Bichromatic Analyzer-100.

A micromethod for the determination of acetylator phenotype using the method of Bratton & Marshall (1939) J. Biol. Chem. 128, 537--550) was adapted to the Abbott Bichromatic Analyzer (ABA-100). Advantages of this procedure include: (a) the requirement for small blood samples (20 microliter) making it a suitable pediatric method and eliminating the need for venipunctures; (b) elimination of tedious manual reading of absorbances of samples and mathematical manipulation of data by the automated capabilities of the ABA-100; (c) an increase in the accuracy of the assay by reading samples at two wavelengths thereby correcting for differences in background.

Acetylation↗

PubMed2006

Automated local bright feature image analysis of nuclear protein distribution identifies changes in tissue phenotype.

The organization of nuclear proteins is linked to cell and tissue phenotypes. When cells arrest proliferation, undergo apoptosis, or differentiate, distribution of nuclear proteins changes. Conversely, forced alteration of the distribution of nuclear proteins modifies cell phenotype. Immunostaining and fluorescence microscopy have been critical for such findings. However, there is increasing need for quantitative analysis of nuclear protein distribution to decipher epigenetic relationships between nuclear structure and cell phenotype and to unravel the mechanisms linking nuclear structure and function. We have developed imaging methods to quantify the distribution of fluorescently stained nuclear protein NuMA in different mammary phenotypes obtained using 3D cell culture. Automated image segmentation of DAPI-stained nuclei was generated to isolate thousands of nuclei from 3D confocal images. Prominent features of fluorescently stained NuMA were detected by using a previously undescribed local bright feature analysis technique, and their normalized spatial density was calculated as a function of the distance from the nuclear perimeter to its center. The results revealed marked changes in the distribution of the density of NuMA bright features when nonneoplastic cells underwent phenotypically normal acinar morphogenesis. Conversely, we did not detect any reorganization of NuMA during formation of tumor nodules by malignant cells. Importantly, the analysis also discriminated proliferating nonneoplastic from proliferating malignant cells, suggesting that these imaging methods are capable of identifying alterations linked not only to the proliferation status but also to the malignant character of cells. We believe that this quantitative analysis will have additional applications for classifying normal and pathological tissues.

Antigens, Nuclear↗

PubMed2003

PowerTrim: An automated decision support algorithm for preprocessing family-based genetic data.

Statistical genetics software packages for linkage analysis have their own unique constraints on the size and shape of the pedigrees they can process. As a result, researchers are often forced to exclude from analysis some individuals in a given family. Existing procedures for reducing pedigree size to fit computational constraints use arbitrary rules and are not interactive. However, judicious evaluation of which subject(s) to remove to minimize loss of information involves consideration of many factors, including informativeness owing to position in pedigree, availability of genotypic information, and quality of phenotypic information. Thus, automation of this task would be of significant benefit. We designed an interactive algorithm (PowerTrim) that provides the user access to detailed information with which to make informed decisions. In addition, PowerTrim checks for transcriptional and data-entry errors, which can be very time-consuming to localize manually.

Algorithms↗

PubMed2003

Mapping dominant-negative mutations of anthrax protective antigen by scanning mutagenesis.

The protective antigen (PA) moiety of anthrax toxin transports edema factor and lethal factor to the cytosol of mammalian cells by a mechanism that depends on its ability to oligomerize and form pores in the endosomal membrane. Previously, some mutated forms of PA, designated dominant negative (DN), were found to coassemble with wild-type PA and generate defective heptameric pore-precursors (prepores). Prepores containing DN-PA are impaired in pore formation and in translocating edema factor and lethal factor across the endosomal membrane. To create a more comprehensive map of sites within PA where a single amino acid replacement can give a DN phenotype, we used automated systems to generate a Cys-replacement mutation for each of the 568 residues of PA63, the active 63-kDa proteolytic fragment of PA. Thirty-three mutations that reduced PA's ability to mediate toxicity at least 100-fold were identified in all four domains of PA63. A majority (22) were in domain 2, the pore-forming domain. Seven of the domain-2 mutations, located in or adjacent to the 2beta6 strand, the 2beta7 strand, and the 2beta10-2beta11 loop, gave the DN phenotype. This study demonstrates the feasibility of high-throughput scanning mutagenesis of a moderate sized protein. The results show that DN mutations cluster in a single domain and implicate 2beta6 and 2beta7 strands and the 2beta10-2beta11 loop in the conformational rearrangement of the prepore to the pore. They also add to the repertoire of mutations available for structure-function studies and for designing new antitoxic agents for treatment of anthrax.

Animals↗

PubMed2002

Fully automated three-dimensional tracking of cancer cells in collagen gels: determination of motility phenotypes at the cellular level.

We developed a fully automated three-dimensional cell tracking system that quantified the effect of extracellular matrix components on the infiltration and migration of tumor cells. The three-dimensional trajectories of two highly invasive cell lines, the human HT-1080 fibrosarcoma and the human MDA-MB-231 adenocarcinoma, were determined for long-term infiltration in plain or Matrigel-containing collagen type I gels. We modeled the trajectories with a novel formulation of the continuous Markov chain model that can distinguish between the tendencies for infiltration or lateral motion. Parameters such as the speed of subpopulations, the persistence of motion in certain directions, the turning frequency of the cells, the ultimate direction of motion, and the cell distribution with the infiltration depth were obtained to quantify the migration and infiltration at the cellular level. Distinct migratory and infiltration phenotypes were identified for the two cell types that were significantly dependent on gel composition. The HT-1080 cell line expressed a high motility phenotype on the plain collagen gel surface. The Matrigel-containing gel significantly enhanced the infiltration and the turning frequency of the HT-1080 cells. This study shows that tumor cell infiltration and migration are dynamic processes that depend significantly on the cell type and the microenvironment.

Adenocarcinoma↗

PubMed2006

Small molecule-based reversible reprogramming of cellular lifespan.

Most somatic cells encounter an inevitable destiny, senescence. Little progress has been made in identifying small molecules that extend the finite lifespan of normal human cells. Here we show that the intrinsic 'senescence clock' can be reset in a reversible manner by selective modulation of the ataxia telangiectasia-mutated (ATM) protein and ATM- and Rad3-related (ATR) protein with a small molecule, CGK733. This compound was identified by a high-throughput phenotypic screen with automated imaging. Employing a magnetic nanoprobe technology, magnetism-based interaction capture (MAGIC), we identified ATM as the molecular target of CGK733 from a genome-wide screen. CGK733 inhibits ATM and ATR kinase activities and blocks their checkpoint signaling pathways with great selectivity. Consistently, siRNA-mediated knockdown of ATM and ATR induced the proliferation of senescent cells, although with lesser efficiency than CGK733. These results might reflect the specific targeting of the kinase activities of ATM and ATR by CGK733 without affecting any other domains required for cell proliferation.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2004

Automatic identification of subcellular phenotypes on human cell arrays.

Light microscopic analysis of cell morphology provides a high-content readout of cell function and protein localization. Cell arrays and microwell transfection assays on cultured cells have made cell phenotype analysis accessible to high-throughput experiments. Both the localization of each protein in the proteome and the effect of RNAi knock-down of individual genes on cell morphology can be assayed by manual inspection of microscopic images. However, the use of morphological readouts for functional genomics requires fast and automatic identification of complex cellular phenotypes. Here, we present a fully automated platform for high-throughput cell phenotype screening combining human live cell arrays, screening microscopy, and machine-learning-based classification methods. Efficiency of this platform is demonstrated by classification of eleven subcellular patterns marked by GFP-tagged proteins. Our classification method can be adapted to virtually any microscopic assay based on cell morphology, opening a wide range of applications including large-scale RNAi screening in human cells.

Artifacts↗

PubMed1989

Applications of automated simultaneous double fluorescence (SDF). II. HLA class I phenotyping using immunomagnetically separated T lymphocytes.

HLA class I phenotyping was performed using T-lymphocyte populations isolated by immunomagnetic beads (IMBs) coated with monoclonal antibodies with specificity for CD2, CD4 or CD8. The results were compared to those obtained using density gradient-separated lymphocytes (PBL). The typing trays were read by the automated simultaneous double-fluorescence (SDF) technique previously established in our laboratory using an Astroscan 2100 system. The aims of the present study were to establish whether the advantages of IMB lymphocyte separation and automated plate reading by SDF were complementary and whether the results obtained by IMB-SDF and PBL-SDF were concordant. Similarity coefficients for paired results obtained by IMB-SDF and PBL-SDF varied between 0.825 using anti-CD8-coated IMBs and 0.914 using anti-CD4-coated IMBs with a consistent excess of stronger results observed with the PBL-SDF technique. The variations observed did not result in incorrect phenotype assignment but would significantly influence a cross-matching test. These results illustrate the feasibility of using IMB-separated lymphocytes for HLA phenotyping by SDF.

Antibodies, Monoclonal↗

PubMed1997

Screening with monoclonal anti-Fy3 to provide blood for phenotype-matched transfusions for patients with sickle cell disease.

BACKGROUND: In the United States, there is a shortage of blood group phenotype-matched red cells (RBCs) for patients with sickle cell disease (SCD). A protocol designed to supply phenotype-matched RBCs for these patients by combining the recruitment of African American blood donors and automated testing of RBCs for these patients for the presumptive Fy(a-b-) phenotype using monoclonal anti-Fy3 was evaluated. STUDY DESIGN AND METHODS: African American donors were recruited, to increase the likelihood of phenotype matches in the donor population. Samples of RBCs were tested for the presumptive Fy(a-b-) phenotype by using monoclonal anti-Fy3 and an automated blood typing analyzer. RBCs confirmed to be Fy(a-b-) were retyped for selected Rh, MNS, Kell, Duffy, and Kidd blood system antigens. The extended phenotypes were matched with those of 41 SCD patients requiring transfusions. RESULTS: Of 8323 blood donations during the study, approximately 40 percent (3329) were made by African Americans. Approximately 22 percent (737) of African Americans were identified as Fy(a-b-) by this protocol and 12 percent (410) were phenotype matches for the 41 SCD patients. CONCLUSION: Combining the recruitment of African American blood donors and automated phenotyping using monoclonal anti-Fy3 offers a practical, relatively low-cost strategy for supplying phenotype-matched RBCs for SCD patients. This protocol increases the options for addressing the shortage of phenotype-matched RBCs for SCD patients.

Anemia, Sickle Cell↗

PubMed1984

Disease activity markers in multiple sclerosis. Another look at suppressor cells defined by monoclonal antibodies OKT4, OKT5, and OKT8.

Here we report our experience in profiling peripheral blood T-cell subsets with the monoclonal antibodies OKT4, OKT5 , and OKT8. Lymphocyte surface phenotype was measured by automated cytofluorometry. In a population survey, we were unable to detect differences between patients with multiple sclerosis (MS) and control subjects when we compared ratios of lymphocytes of helper cell phenotype (OKT4) to those with suppressor cell phenotype ( OKT5 and OKT8). No differences could be established between patients with stable disease, chronic progressive disease, or those with active disease. In a study of 10 patients followed through an exacerbation, we were also unable to define perturbations in these lymphocyte ratios that correlated with disease activity. Detailed analysis of the fluorescence histogram, which examines the entire spectrum of cell surface fluorescence intensity in a population of lymphocytes, was also not useful in predicting disease activity in these patients. The discrepancies between these data and other reports in the literature are discussed. We propose that these reagents are inadequate indices of disease activity, and that until other monoclonal reagents are developed and studied, the suppressor cell compartment is best assessed by assays of function.

Antibodies, Monoclonal↗

PubMed2026

Genetic basis of escape-related locomotor performance in a wild-introgressed sheep population.

Rapid running and jumping are core components of escape responses in prey animals and provide measurable traits for studying locomotor performance in large mammals. The genetic basis of these escape-related locomotor traits remains poorly understood in large mammals, partly because repeated, standardized phenotyping under field conditions is challenging. Here, we leveraged a sheep population carrying argali-introgressed genetic components to map genetic variations associated with running speed and jumping height. Through controlled field experiments, automated high-resolution phenotyping, whole-genome analysis, and gene-edited mouse models, we identified two loci associated with escape-related locomotor traits: one in ABCC4 (Chr10:71,849,347; p = 5.03 × 10-7) linked to maximum running speed and another in GRID2 (Chr6:32,120,477; p = 1.30 × 10-9) associated with jumping height. Functional assays in knockout mice reveal that disruption of Grid2 reduces jumping ability, whereas Abcc4 knockout and knockdown increase running speed through enhanced heart contractility under stress. These results elucidated the genetic bases of wild-derived variations in affecting locomotor performance.

Animals↗