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At least 955 records · Page 53Linked to original sources
PubMed1996

Microtitre-plate enzyme-linked ligand-sorbent assay of riboflavin (vitamin B2) in human plasma and urine.

Enzyme-linked ligand-sorbent assay (ELLSA) of riboflavin was performed in standard, multi-well microtitre plates. 3-Carboxymethylriboflavin was carbodiimide-coupled to bovine serum albumin and the conjugate was adsorbed on the well surface. Riboflavin-binding protein from egg-white was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester. The assay was based on competition of riboflavin analyte with the immobilized flavin for the biotinylated binder. Secondary adsorption of the biotinylated riboflavin-binding protein was measured by using avidin-bearing horseradish peroxidase label. The optimized method had a detection limit of 0.8 pmol of riboflavin and was expected to work within a riboflavin concentration range of 2 X 10(-8)(-4) X 10(-6) mol l-1. Preliminary trials suggested that ELLSA was suitable for determining riboflavin in human urine and the sum of riboflavin and flavin nucleotides in human plasma. The analytical performance of ELLSA for those materials was characterized by good consistency of the results with those obtained by conventional, fluorimetric methods, a mean recovery of riboflavin supplement of over 90% and a within-plate relative standard deviation below 20%. Some unique samples of both urine and plasma were assayed with between-plate relative standard deviations higher than 30%, implicating further modification of this version of ELLSA. The method intended for routine control determinations of vitamin B2 status in human subjects and is addressed to laboratories that routinely perform automated, microplate-based enzyme-linked assays.

Chromatography, High Pressure Liquid↗
PubMed2003

Computer screen as a programmable light source for visible absorption characterization of (bio)chemical assays.

Visible absorption features suitable for color recognition and micro-plate reading of a standard bioassay are performed by the combination of a computer screen used as a programmable light source and a web camera as detector. The method provides in this way a highly available platform for 'home tests' or 'self-tests', where the requirement is to monitor well defined assays and the use of economical instrumentation is advantageous.

Automation↗
PubMed2004

Quantitative assays of chemotaxis and chemokinesis for human neural cells.

Cell migration is vital for many physiological processes, and its modulation is likely to be of therapeutic benefit. In this study we have developed fluorescence image-based chemokinesis and chemotaxis assays on the Cellomics (Pittsburgh, PA) ArrayScan platform, which would be suitable for industrial drug discovery in a variety of fields. Studying the migratory characteristics of neural stem cells is of interest for understanding the therapeutic potential of these cells, in terms of both cellular transplantation and the activation of endogenous populations of stem cells. Growth conditions were identified whereby human neural precursors could be maintained as neurospheres and plated out into microtitre plates for high-content assays. Chemokinesis was assessed using fluorescent bead-coated 96-well microtitre plates, whilst chemotaxis was assessed using BD Biosciences (Oxford, UK) Fluoroblok 24-well plates. Assays for both chemokinesis and chemotaxis were developed that were quantified automatically using the ArrayScan and appropriate algorithms. Using the two complementary techniques, foetal bovine serum was observed to have chemokinetic effects on the cells, whilst platelet-derived growth factor isoform AB was chemotactic. The two assays described here are suitable for screening for novel modulators of cell migration, or for performing more detailed mechanistic follow-up studies. These assays enable us to perform cell motility studies with minimal laboratory handling, in an automated manner, thereby allowing quantitative studies of cell behaviour to be incorporated in a routine drug discovery screening cascade.

Cell Count↗
PubMed1997

The clinical application of a new specific functional assay to detect the factor V(Leiden) mutation associated with activated protein C resistance.

Resistance to activated protein C (APC) is the most common defect found in patients who have venous thromboembolism. The molecular basis of APC resistance is a single-point mutation (arginine506-glutamine) in the gene that encodes for coagulation factor V. This mutation results in a factor V molecule (factor V(Leiden)) that is less effectively downregulated by APC than is normal factor V. The gold standard for the detection of this defect is DNA analysis. Several functional tests, which are based on activated partial thromboplastin time clotting assays, are also commercially available for the detection of APC resistance. These tests, however, have not been satisfactory. Compared with the results of DNA analysis, the results of these tests are frequently discordant. Further, in some patients (eg, those who have lupus anticoagulant or have been receiving heparin), these tests cannot be performed at all. DNA analysis is therefore required in most patients to distinguish congenital APC resistance (factor V(Leiden)) from other causes of abnormal response in functional APC-resistance tests. The purpose of this study was to investigate the clinical use of a new chromogenic APC-resistance assay that is based on direct measurement of the effect of APC on factor Va cofactor activity in highly diluted, thrombin-activated plasma specimens. All individuals who provided plasma samples for the study underwent DNA analysis to detect the presence of the factor V mutation. In all patient subgroups, including patients who had lupus anticoagulant and those who were receiving unfractionated heparin or coumarin derivatives, the chromogenic test showed excellent discrimination between normal individuals and those who were heterozygous or homozygous for the factor V(Leiden) mutation. No discordant results with DNA analysis were found in 150 cases. The new test easily can be incorporated in any laboratory that has an automated coagulation apparatus with an option for chromogenic measurements. All reagents are commercially available at low cost, and the test is easy to perform and is not time-consuming. This new, sensitive, and specific test allows large-scale screening for the factor V(Leiden) mutation without the need for DNA analysis.

Blood Coagulation Disorders↗
PubMed1998

Evaluation and interference study of hemoglobin A1c measured by turbidimetric inhibition immunoassay.

The technical performance of the turbidimetric immunoinhibition (TI) assay for hemoglobin (Hb) A1c (Tina-quant Hb A1c, Boehringer Mannheim, Indianapolis, Ind) was evaluated by using the BM/Hitachi 911 analyzer. Intra-assay imprecision was less than 2.7%, and interassay imprecision was less than 2.8% as measured by coefficient of variation. In 93 subjects with diabetes who did not have hemoglobin variants, results of the TI assay for Hb A1c correlated strongly with those obtained by using a high-performance liquid chromatography analyzer (Diamat, BioRad Laboratories, Hercules, Calif). Among 241 subjects who had or did not have hemoglobin variants, the TI assay for Hb A1c correlated strongly with results of affinity chromatography for total glycated hemoglobin (Glyc-Affin GHb, IsoLab, Akron, Ohio). We also studied the effect of various percentages of hemoglobin S, C, E, and F on the accuracy of the TI Hb A1c assay. Only high hemoglobin F percentages caused interference. More than 14 times as many samples can be analyzed per hour by using the TI Hb A1c assay than can be analyzed by using the HPLC assay. For high-volume reference laboratories, using the fully automated TI Hb A1c assay to monitor glycemic control in patients with diabetes may be preferable to using the conventional ion-exchange high-performance liquid chromatography Hb A1c assay because the TI assay measures Hb A1c more accurately in patients with diabetes who have hemoglobin variants, and it requires less time.

Autoanalysis↗
PubMedDate not supplied

Interference of herbal drinks with urinalysis for drugs of abuse.

Recently there have been claims among drug users that some herbal drinks interfere with urinalysis for drugs of abuse and yield false positive results. Proof of such claims has yet to be shown. Screening for drugs of abuse is usually carried out using fluorescence polarization immunoassay (FPIA) or thin-layer chromatography (TLC). Fifty herbal samples which are considered among the most purchased herbs in the consumer market were used to investigate such claims. The drug groups that were tested for included amphetamines, opiates, barbiturates, cocaine metabolite, methadone, and their analogs. The herbs were analyzed at different concentrations (0.1, 1, 3, and 5 g/100 mL of distilled water) using TLC and FPIA to determine if any interfere with urinalysis for drugs of abuse and yield false positive results. For the FPIA test, the sample infusions were analyzed directly using the automated ADX analyzer (Abbott Laboratory). For TLC, infusions of the herbs were added to a solid-phase extraction column (pH 9.25), then extracted with a methylene chloride-isopropanol solvent system. At this pH, neutral, basic, and acidic drugs of abuse are extractable. The developed chromatographic plates were sprayed sequentially with several reagents. None of the herbs in the concentration ranges screened showed any interference with TLC or FPIA, indicating the invalidity of such claims.

Beverages↗
PubMed2004

Quality of depression care in a population-based sample of patients with diabetes and major depression.

OBJECTIVES: Major depression occurs in approximately 11% to 15% of patients with diabetes and is associated with poor glycemic control and adverse medical outcomes. This study examined the rates and predictors of recognition of depression among primary care patients with diabetes and comorbid major depression and the quality of depression care provided during a 12-month period. METHODS: This study used automated utilization, pharmacy, and laboratory data from a health maintenance organization to describe the rate of recognition of depression and quality of care provided for patients with major depression and diabetes in the 12-month period before diagnosis. Major depression was diagnosed based on the Patient Health Questionnaire (PHQ-9) that was included in a mail survey sent to 9063 patients on the Group Health diabetes registry from 9 primary care clinics. RESULTS: Approximately 51% of patients with major depression and diabetes were recognized as depressed by the health care system. Women were more likely to be recognized (odds ratio [OR] 1.58, 95% confidence interval [CI 1.26-1.97]), as were those with dysthymia (OR 3.44, 95% CI 2.08-5.72), panic attacks (OR 1.55, 95% CI 1.19-2.19), patients with more than 7 primary care visits (OR 1.42, 95% CI 1.06-1.91) and patients reporting poor health (OR 1.62, 95% CI 1.04-2.53). Of the 51% of patients with major depression who were recognized, 43% received 1 or more antidepressant prescriptions but only 6.7% received 4 or more psychotherapy sessions during a 12-month period. DISCUSSION: There were large gaps in both recognition and quality of depression care provided to patients with major depression and diabetes within a health maintenance organization system.

Adult↗
PubMedDate not supplied

Influence of patient attachment style on self-care and outcomes in diabetes.

OBJECTIVE: Difficulties collaborating with providers and important others may adversely influence self-management in patients with diabetes. We predicted that dismissing attachment style, characterized by high interpersonal self-reliance and low trust of others, would be associated with poorer self-management in patients with diabetes. METHODS: A population-based mail survey was sent to all patients with diabetes from nine primary care clinics of a health maintenance organization. We collected data on attachment style, self-care behaviors, the patient provider relationship and depression status and accessed automated diagnostic, pharmacy, and laboratory data to measure diabetes treatment intensity, medical comorbidity, glycosylated hemoglobin levels, and diabetes complications. We used logistic regression to determine whether dismissing attachment style was associated with poorer diabetes self-care behaviors, adherence to medication, smoking status, and higher glycosylated hemoglobin. RESULTS: In 4095 primary care patients with diabetes, prevalence rates for secure, dismissing, preoccupied, and fearful attachment styles were 44.2%, 35.8%, 7.9%, and 12.1%, respectively. When compared with secure attachment style, dismissing attachment style was associated with significantly lower levels of exercise (p =.005), foot care (p <.05), diet (p =.001), and adherence to oral hypoglycemic medications (p <.05), and with higher rates of smoking (p <.05), and these associations were mediated through the patient-provider relationship. Preoccupied attachment style, characterized by overreliance on others, was associated with a significantly lower risk of having glycosylated hemoglobin levels >8%, compared with secure attachment style. CONCLUSION: Attachment style is significantly associated with diabetes self-management and outcomes.

Adult↗
PubMed2006

Assessment of MagNA pure LC extraction system for detection of human papillomavirus (HPV) DNA in PreservCyt samples by the Roche AMPLICOR and LINEAR ARRAY HPV tests.

Roche Molecular Systems recently released two PCR-based assays, AMPLICOR and LINEAR ARRAY (LA), for the detection and genotyping, respectively, of human papillomaviruses (HPVs). The manual specimen processing method recommended for use with both assays, AmpliLute, can be time-consuming and labor-intensive and is open to potential specimen cross-contamination. We evaluated the Roche MagNA Pure LC (MP) as an alternative for specimen processing prior to use with either assay. DNA was extracted from cervical brushings, collected in PreservCyt media, by AmpliLute and MP using DNA-I and Total Nucleic Acid (TNA) kits, from 150 patients with histologically confirmed cervical abnormalities. DNA was amplified and detected by AMPLICOR and the LA HPV test. Concordances of 96.5% (139 of 144) (kappa=0.93) and 95.1% (135 of 142) (kappa=0.90) were generated by AMPLICOR when we compared DNA extracts from AmpliLute to MP DNA-I and TNA, respectively. The HPV genotype profiles were identical in 78.7 and 74.7% of samples between AmpliLute and DNA-I or TNA, respectively. To improve LA concordance, all 150 specimens were extracted by MP DNA-I protocol after the centrifugation of 1-ml PreservCyt samples. This modified approach improved HPV genotype concordance levels between AmpliLute and MP DNA-I to 88.0% (P=0.043) without affecting AMPLICOR sensitivity. Laboratories that have an automated MP extraction system would find this procedure more feasible and easier to handle than the recommended manual extraction method and could substitute such extractions for AMPLICOR and LA HPV tests once internally validated.

Cervix Uteri↗
PubMed1981

Rapid and direct staphylocoagulase assay that uses a chromogenic substrate for identification of Staphylococcus aureus.

A rapid and direct assay of staphylocoagulase that uses chromogenic substrate Chromozym TH was developed for the routine identification of Staphylococcus aureus. False-positive and false-negative reactions were eliminated by measuring the activity of staphylocoagulase-prothrombin complex directly. The method was compared with three clotting assays. The results show that the new assay is specific, quantitative, and easily automated. If offers clinical laboratories a more reliable assay of staphylocoagulase and is a valuable aid for the rapid identification of S. aureus.

Blood Coagulation↗