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Results for “Automated phenotyping”

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PubMed1993

A simple method for genetic typing of transferrins in nonhuman primates.

The serum protein transferrin (Tf) is a valuable marker for genetic studies of primates, because it is usually polymorphic, exhibiting as many as 13 allelic forms with high heterozygosity. The standard procedure to detect the different phenotypes requires vertical electrophoresis on polyacrylamide gels for 18 h at 4 degrees C. We have simplified the procedure by using the automated programmable PhastSystem for electrophoresis, which uses precast miniature gels and takes less than 2 h. Also, we have developed a pool of sera that displays the 6 most common alleles in rhesus monkeys. This pool allows for a more accurate assignment of phenotypes. Using this simplified procedure, we have typed over 3000 monkeys of various species and have confirmed the reliability of this technique for parentage determination.

Alleles↗

PubMed1997

Genetic typing of human platelet antigen 1 (HPA-1) by oligonucleotide ligation assay in a specific and reliable semi-automated system.

Genotyping of platelet alloantigens with the possibility of using any type of cellular material as a source of DNA has become a preferred procedure, particularly in thrombocytopenic patients when platelet counts are too low for phenotyping. Recently human platelet antigen 1 (HPA-1) has been identified as an inherited risk factor for coronary thrombosis. The different detection methods currently used have disadvantages for large-scale DNA diagnosis, including the need for electrophoresis (allele-specific restriction enzyme analysis, amplification with sequence-specific primers) or the potential risk of reduced specificity (allele-specific oligonucleotide hybridization). In this report we describe the adaptation of an automated oligonucleotide ligation assay to genotype HPA-1 in polymerase chain reaction (PCR)-amplified DNA samples. HPA-1a and HPA-1b phenotypes corresponded to the results of the different genotyping assays. The genotypes determined with the ELISA-based PCR-oligonucleotide ligation assay were in 100% concordance with the results obtained by conventional allele-specific restriction enzyme site analysis and PCR amplification with sequence-specific primers. The automated oligonucleotide ligation assay provides a rapid, reliable, nonisotopic method to genotype human platelet antigens that can rapidly be applied to large population screening.

Antigens, Human Platelet↗

PubMed2005

[Automation of the immunohematology laboratory].

Automation finds a particularly justified application in the field of immunohematology in which any human failure can have dramatic consequences for the patients. The purposes of automation, indissociable from computerization, were reminded by the French regulatory: to decrease the risks of human error linked to each step of the tests performed; to guarantee a reliable traceability of all the elements having contributed to the test process; to manage all the alarms of dysfunction of the system. The main devices available today permit automation of a part of the pre-analytical phase and of all the steps of the analytical phase by the realization in routine of ABO-RH1 grouping, phenotyping, of irregular antibody screening and of compatibility testing. They use the reaction of agglutination either in micro-plates or in filtration. One distinguishes two types of materials: full automates managing all the steps from the sample positioning on the carrier down to the final result without any handling of the samples (Autovue, Galileo, ID gel station, Qwalys, Tango, Techno) and the semi-automates, which require intervention of an operator for the phases of centrifugation, stirring and incubation (Mitis 2, Hemos, Rosys, Swing) the reading being automated for all. All include the possibility of a connection to the central data processing system . If some devices allow the choice of reagents, others can only work with the reagents supplied by the manufacturer. In all the cases, optimization of the automated systems implies a specific training of the staff and the strict compliance with the standard operating procedures by each individual.

Automation↗

PubMed2005

Otago Diagnostic Laboratories' (ODL) Method for the detection of beta-lactamases in Enterobacteriaceae.

AIM: The rapid evolvement of beta-lactamases in Enterobacteriaceae is an important concern and the clinical microbiology laboratory is required to detect them, where possible, using a rapid, reliable, simple and low cost methodology. MATERIALS AND METHODS: A disc diffusion method using NCCLS breakpoints, Jarlier's principle and cefoxitin test for AmpC was carried out. It incorporated seven antimicrobial discs in one agar plate: cefotaxime, aztreonam, amoxicillin-clavulanate, ceftazidime, cefpodoxime, cefepime and cefoxitin. NCCLS disc confirmation test for extended-spectrum beta-lactamase (ESBL) was carried out simultaneously. RESULTS: AmpC, ESBL, CTX-M, and K1 were detected using these tests. The prevalence of ESBL was <1% in the hospital. CONCLUSION: The method is recommended for the phenotypic detection of beta-lactamases in Enterobacteriaceae or for confirmation after the results are obtained by conventional automated systems.

Anti-Bacterial Agents↗

PubMed1987

[Evaluation of the ATB system and the 32 GN tests for the identification and determination of the antibiotic sensitivity of Acinetobacter calcoaceticus var. anitratum. Comparison with the reference dilution method in a gel medium].

The authors determined the susceptibility to 24 antibiotics of 95 strains of Acinetobacter calcoaceticus variety anitratum with the automated API ATB System in comparison with the agar dilution method for MIC measurement. The strains have been identified with API 32 GN (automated system) and API NE. There was a good agreement between the two sets of identification results. In susceptibility tests the discrepancies ranged from 0.86% to 3% with a mean value of 1.8%. The overall agreement was 95% between the two methods. This study allowed the authors to define susceptibility phenotypes to the three major antibiotic classes: beta-lactams, aminoglycosides and quinolones. The use of 32 GN tests and ATB in the automated system seems very useful, especially in epidemiologic studies in order to analyse numerous data in a short time.

Acinetobacter↗

PubMed2005

Screens using RNAi and cDNA expression as surrogates for genetics in mammalian tissue culture cells.

We have developed methods for the automation of transfection-grade DNA preparation, high-throughput retroviral preparation, and highly parallel phenotypic screens to establish approaches that will allow investigators to examine in an unbiased manner the roles of proteins in mammalian cells. These methods have been used to raise or lower the levels of individual kinases in individual micro-well cultures either by cDNA or short hairpin RNA expression and will allow investigators to treat mammalian cells in culture in manners that are analogous to genetic screens in yeast. Our proof-of-principle experiments have been performed in human cells using repositories that represent over 75% of the protein, nucleotide, carbohydrate, lipid, and amino acid kinases in the human genome. These initial experiments have demonstrated the feasibility of two general types of screens. We have performed phenotypic screens to identify proteins with specific roles in a chosen function and genetic interaction screens to establish epistatic relations between different proteins. The results suggest that any phenotype that can be scored by a robust assay in tissue culture is amenable to these types of screens and that interactions between mammalian proteins can be established. These results point to the near-term goal of establishing comprehensive, unbiased screens that will allow queries on the roles of all human proteins.

Carcinoma, Non-Small-Cell Lung↗

PubMedDate not supplied

Regional heterogeneity of mtDNA heteroplasmy in parkinsonian brain.

There is increasing evidence for a role of defects of mitochondrial DNA in the etiology of neurodegenerative disorders such as Parkinson's and Alzheimer's disease as well as in normal aging. In several studies a biochemical defect of complex I of the respiratory chain (NADH dehydrogenase, EC 1.6.5.3) has been found in the substantia nigra of Parkinsonian brains. Thus, mutations of mitochondrial genes encoding subunits of complex I could contribute to the pathogenesis of Parkinson's disease. A heteroplasmic G5460A mutation affecting the ND2 subunit of NADH dehydrogenase was detected in several brains of patients with idiopathic Parkinson's disease. Since this mutation is heteroplasmic we were interested in the distribution of mutated and wild-type mitochondrial DNA in different brain areas. Relative levels of mutated DNA were quantified in a large number of anatomical regions using DNA extracted from formalin-fixed and paraffin-embedded brain tissue. DNA was amplified by the polymerase chain reaction and digested employing the restriction enzyme Hphl. The proportion of mutated DNA was determined by laser densitometry. In addition, genotype-phenotype analyses were performed on sections of the substantia nigra with the aid of an automated image analysis system. Ratios of mutant to wild-type DNA varied between 44% and 98%. However, there was no systematic relationship between mutated DNA ratios and ontogenetically related brain areas suggesting that the observed regional heterogeneity of mitochondrial DNA heteroplasmy is most likely due to random segregation during development. Therefore, tissue-specific differences in the sensitivity to pathogenic effects of the ND2(5460) mutation or the influence of additional susceptibility genes may be envisioned.

Brain↗

PubMed1994

Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome): six unique arylsulfatase B gene alleles causing variable disease phenotypes.

Mucopolysaccharidosis type VI, or Maroteaux-Lamy syndrome, is a lysosomal storage disorder caused by a deficiency of the enzyme arylsulfatase B (ASB), also known as N-acetylgalactosamine-4-sulfatase. Multiple clinical phenotypes of this autosomal recessively inherited disease have been described. Recent isolation and characterization of the human ASB gene facilitated the analysis of molecular defects underlying the different phenotypes. Conditions for PCR amplification of the entire open reading frame from genomic DNA and for subsequent direct automated DNA sequencing of the resulting DNA fragments were established. Besides two polymorphisms described elsewhere that cause methionine-for-valine substitutions in the arylsulfatase B gene, six new mutations in six patients were detected: four point mutations resulting in amino acid substitutions, a 1-bp deletion, and a 1-bp insertion. The point mutations were two G-to-A and two T-to-C transitions. The G-to-A transitions cause an arginine-for-glycine substitution at residue 144 in a homoallelic patient with a severe disease phenotype and a tyrosine-for-cysteine substitution at residue 521 in a potentially heteroallelic patient with the severe form of the disease. The T-to-C transitions cause an arginine-for-cysteine substitution at amino acid residue 192 in a homoallelic patient with mild symptoms and a proline-for-leucine substitution at amino acid 321 in a homoallelic patient with the intermediate form. The insertion between nucleotides T1284 and G1285 resulted in a loss of the 100 C-terminal amino acids of the wild-type protein and in the deletion of nucleotide C1577 in a 39-amino-acid C-terminal extension of the ASB polypeptide. Both mutations were detected in homoallelic patients with the severe form of the disease. Expression of mutant cDNAs encoding the four amino acid substitutions and the deletion resulted in severe reduction of both ASB protein levels and arylsulfatase enzyme activity in comparison with a wild-type control. The six mutations described in the present study were unique among 25 unrelated mucopolysaccharidosis VI patients, suggesting a broad molecular heterogeneity of the Maroteaux-Lamy syndrome.

Alleles↗

PubMed1993

Gamma delta + T cells from patients with psoriatic and rheumatoid arthritis respond to streptococcal antigen.

OBJECTIVE: To investigate cellular immune responses to streptococcal antigens in patients with psoriatic arthritis (PsA). To specifically examine responses of the gamma delta + T cell subset. METHODS: Proliferation of PsA synovial fluid lymphocytes (SFL) and peripheral blood lymphocytes (PBL) cultured with streptococcal antigen was measured using a 3H thymidine (3HTdr) uptake assay system. gamma delta + T cells from PsA PBL and SFL were phenotyped by flow cytometry. Following culture with streptococcal antigen, gamma delta + enriched SFL were sorted by automated flow cytometry and 3HTdr uptake measured. RESULTS: Patients with PsA and the control group did not differ significantly in their PBL responses to 2 strains of streptococci, one of which was isolated from a patient with guttate psoriasis (Strep 1) and the other from a patient with rheumatic fever (Strep 2). There was also no difference in their responses to a cell wall preparation derived from the former strain. SFL from 8 of 9 patients with PsA responded to both streptococcal strains as did SFL from 3 patients with rheumatoid arthritis (RA). gamma delta + SFL from 7 patients with PsA 3 patients with RA responded only to the psoriasis associated strain. CONCLUSIONS: PsA PBL and SFL responded to stimulation by streptococcal antigen but this reactivity was not disease specific. We have demonstrated that gamma delta + T cells from PsA SF proliferated when cultured with a psoriasis associated strain of streptococcus (Strep 1). However, RA gamma delta + SFL responded similarly suggesting that gamma delta + T cell reactivity to streptococcal antigen may be a feature of inflammatory arthritis.

Adult↗

PubMed2004

An imaging system for standardized quantitative analysis of C. elegans behavior.

BACKGROUND: The nematode Caenorhabditis elegans is widely used for the genetic analysis of neuronal cell biology, development, and behavior. Because traditional methods for evaluating behavioral phenotypes are qualitative and imprecise, there is a need for tools that allow quantitation and standardization of C. elegans behavioral assays. RESULTS: Here we describe a tracking and imaging system for the automated analysis of C. elegans morphology and behavior. Using this system, it is possible to record the behavior of individual nematodes over long time periods and quantify 144 specific phenotypic parameters. CONCLUSIONS: These tools for phenotypic analysis will provide reliable, comprehensive scoring of a wide range of behavioral abnormalities, and will make it possible to standardize assays such that behavioral data from different labs can readily be compared. In addition, this system will facilitate high-throughput collection of phenotypic data that can ultimately be used to generate a comprehensive database of C. elegans phenotypic information. AVAILABILITY: The hardware configuration and software for the system are available from wschafer@ucsd.edu.

Animals↗

PubMed1983

Influence of hemoglobin phenotype on the mean erythrocyte volume.

The influence of the hemoglobin (Hgb) phenotype on mean erythrocyte volume was re-examined in the light of recent evidence indicating a poor correlation between manual and more precise automated methods of measurement. The present studies identified a 17.6% frequency of microcytosis in a population of nonanemic Black males, a value higher than previously appreciated. In addition, it was shown that the frequency of microcytosis was also a function of the Hgb phenotype. Thus, 9.4% of Hgb AA, 18.6% of AS and 26.5% of AC subjects had microcytic erythrocytes. The data presented are consistent with the hypothesis that both the alpha-gene complement and the beta-chain phenotype contribute to the erythrocytic mean corpuscular volume and that the frequency of microcytosis in a patient population is a function of both of these variables.

Adult↗

PubMed1998

Characterization of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Greece.

There is a geographic distribution of Mycobacterium tuberculosis strains with various rpoB gene mutations that account for rifampin resistance. We studied 17 rifampin-resistant clinical isolates from patients in Greece to identify rpoB mutations. The aim of our study was the evaluation of a commercially available line probe assay kit (INNO-LiPA Rif. TB) to detect rpoB mutations and rifampin resistance. The results obtained with the commercially available assay were compared to those obtained by automated DNA sequence analysis of amplified PCR products. Randomly amplified polymorphic DNA (RAPD) analyses of the isolates were also performed. The overall concordance of the line probe assay with phenotypic rifampin susceptibility test was 94%. Three distinct rpoB mutations in codons Ser531, His526, and Asp516 were correctly identified with the kit, but mutations in external regions and insertions were detected only by automated DNA sequence analysis. The changes in codons Ser531 and His526 accounted for the majority of rifampin resistance, as previously described for isolates from other geographic areas. The results obtained by RAPD analyses of the isolates suggested that clonally related M. tuberculosis strains can have subclones bearing distinct mutant rpoB alleles. We conclude that this line probe assay kit, which is fast and with which tests are easy to perform, can be used for the rapid detection of rifampin resistance in M. tuberculosis before the availability of results by conventional methods and for epidemiological studies but that negative results obtained by this method do not rule out rifampin resistance.

Amino Acid Sequence↗

PubMedDate not supplied

Quantitative studies on the Rh-antigen D. Effect of the C gene.

Using an automated antiglobulin consumption test and Scatchard's analysis, the authors measured the number of D antigen epitopes on each of 46 samples of washed red cells (RBC) that represented six different Rh phenotypes. A clear D dosage effect was observed only when C was missing from the Rh genome, which confirmed the previously recognized suppressive effect of C. Thus, RBC of the Rh phenotype R2R2 had almost twice as many D epitopes (mean, 30,300/cell) as RBC of the Rh phenotypes R2r and R0r (means, 17,200 and 16,500 respectively). The data indicated that C in trans and cis suppressed similar amounts of D epitopes.

Automation↗

PubMed2003

Validation of PAH genotype-based predictions of metabolic phenylalanine hydroxylase deficiency phenotype: investigation of PKU/MHP patients from Lithuania.

BACKGROUND: Phenylalanine hydroxylase (PheOH) deficiency is inherited as an autosomal recessive trait. The associated hyperphenylalaninemia phenotype is highly variable, primarily due to great allelic heterogeneity in the PAH locus. The goal of our study was to assess the relationship between individual PAH locus mutations and biochemical and metabolic phenotypes in phenylketonuria (PKU) and mild hyperphenylalaninemia (MHP) patients. MATERIAL/METHODS: In this study, a total of 184 independent PAH chromosomes (92 unrelated patients with PKU and MHP residing in Lithuania) were investigated. All 13 exons of the PAH gene of all PKU probands tested were scanned for DNA sequence alterations by denaturing gradient gel electrophoresis (DGGE); mutations were identified by direct fluorescent automated sequencing or by restriction enzyme digestion analysis of the relevant exons. PAH genotype-based prediction of metabolic PhOH deficiency phenotype in PKU/MHP patients form Lithuania was estimated by the assigned value (AV) and functional hemizygosity methods. RESULTS: Our data provide evidence that a simple genotype-phenotype correlation does exist in most patients with PheOH deficiency: we observed a perfect match between the expected and observed phenotypes in 96% of the cases investigated. CONCLUSIONS: The results obtained confirm that methods of functional hemizygosity and AV sum are applicable for the estimation of the genotype-phenotype correlation in the investigated group of PKU/MHP patients.

Female↗

PubMed1996

Rapid molecular diagnosis of mutations associated with generalized thyroid hormone resistance by PCR-coupled automated direct sequencing of genomic DNA: detection of two novel mutations.

Generalized thyroid hormone resistance (GTHR) is a syndrome characterized by tissue nonresponsiveness to thyroid hormones and by variable clinical phenotype manifestations. This syndrome has also been implicated as a predisposing factor in some cases of attention deficit-hyperactivity disorder (ADHD). GTHR results from single mutations in the gene encoding the thyroid hormone receptor. These mutations are clustered in two major sites surrounding the ligand-binding domain. Mutations in 10 previously described patients as well as in five new THR cases have been identified using PCR amplification of genomic DNA coupled with automated direct sequencing with commercially available "universal" fluorescent dye-labeled primers. This strategy allows for the accurate and automated base-calling of normal and mutated nucleotides at the same position in a heterozygote. The rapid molecular diagnostic protocol, from whole blood to DNA sequence data, takes approximately 15 hr, allowing for rapid, efficient, and unambiguous direct detection of the mutant alleles.

Adolescent↗

PubMed2004

Multidimensional drug profiling by automated microscopy.

We present a method for high-throughput cytological profiling by microscopy. Our system provides quantitative multidimensional measures of individual cell states over wide ranges of perturbations. We profile dose-dependent phenotypic effects of drugs in human cell culture with a titration-invariant similarity score (TISS). This method successfully categorized blinded drugs and suggested targets for drugs of uncertain mechanism. Multivariate single-cell analysis is a starting point for identifying relationships among drug effects at a systems level and a step toward phenotypic profiling at the single-cell level. Our methods will be useful for discovering the mechanism and predicting the toxicity of new drugs.

Automation↗

PubMed2004

Computer-assisted phenotype characterization for genetic research in psychiatry.

Psychiatric disorders differ from other complex phenotypes in their lack of objectively assessable biological markers that contribute to the establishment of a research diagnosis for genetic studies. To nevertheless allow for the delineation of genetically meaningful diagnostic entities for psychiatric genetic research, comprehensive phenotype characterization procedures are required. It is widely agreed that these should include the standardized assessment of life-time clinical symptomatology, sociodemographic, and environmental factors. Data should be based on several sources, i.e. diagnostic interviews with probands and their relatives as well as a thorough review of medical records, and final assignment of diagnosis should follow robust algorithms (i.e. best-estimate procedures, consensus diagnosis). Here, we outline a practical implementation of such a phenotype characterization strategy, including patient recruitment, study enrolment procedures, comprehensive diagnostic assessment, and data management. We argue that successful psychiatric phenotype characterization requires flexible tools. For this purpose, we have developed a computer-assisted phenotype characterization inventory, built around the backbone of a relational database. It allows for the straightforward assessment of symptoms, automated error checks and diagnostic assignment, easily manageable data storage and handling, and flexible data transfer between various research centers even across language barriers, while at the same time keeping up with the highest standards for the protection of sensitive patient data.

Algorithms↗

PubMed1999

Combined molecular and clinical approaches for the identification of families with familial adenomatous polyposis coli.

OBJECTIVE: Using an interdisciplinary clinical and molecular approach, the authors identified APC germline mutations in families with familial adenomatous polyposis (FAP). Correlation of mutation site with disease manifestation and the impact of molecular data on clinical proceedings were examined. SUMMARY BACKGROUND DATA: Germline mutations in the APC gene predispose to FAP. Established and proposed genotype-phenotype correlations as well as the influence of mutation site on surgical procedures have been reported. The predictive value of APC mutation analysis for disease manifestation and therapeutic decision making needs to be investigated further. METHODS: One hundred twenty-three kindreds of the local FAP registry were included in this study. CHRPE phenotype was defined as at least one large characteristic lesion or a total of four lesions in both eyes. APC mutations were identified by protein truncation test and automated DNA sequencing from patient lymphocyte DNA and RNA. RESULTS: APC germline mutations were identified in 85/123 families with FAP. They were located between codons 213 and 1581 of the APC gene and displayed distinct genotype-phenotype correlations. CHRPE status facilitated mutation analysis by discriminating regions of interest within the APC coding region. Severe manifestations of desmoids were restricted to mutations between codons 1444 through 1581. Whereas 91% (75/82) of at-risk persons were excluded as mutation carriers, APC germline mutations were detected before clinical examination in 9% (7/82) of at-risk persons. One patient agreed to endoscopy only after mutation detection. CONCLUSIONS: This study supports the feasibility of combined molecular and clinical screening of families with FAP and may provide a guideline for routine presymptomatic molecular diagnostics in a clinical laboratory.

Adenomatous Polyposis Coli↗