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A new in vivo interdental sampling method comparing a daily flossing regime versus a manual brush control.

OBJECTIVE: The impact of flossing the interdental space is typically assessed by visual methods of questionable reliability, such as clinician observations and evaluations for scoring of plaque on visual tooth surfaces. The objective of this study was to develop and validate a method to measure interproximal quantities of dental plaque, thereby permitting quantitative evaluations of between-teeth cleaning and biofilm removal. METHODOLOGY: Laboratory studies were performed correlating the quantity of bacteria to the amount of measured protein in a sample using a standardized protein assay (Bio-Rad Protein Assay) and samples containing single bacterial species and saliva/bacterial pellets. Analysis of the plaque was standardized against a bovine serum albumin (BSA) control. Pilot studies helped develop a technique for hygienist-applied interproximal flossing, reducing contamination from saliva and other possible sources of indeterminate error. Repeated sampling experiments were carried out to optimize the technique needed to remove the majority of quantifiable plaque, the best choice of flossing material, and establish the technique for collection. A pilot clinical trial assessed the benefits of daily flossing in reducing measurable quantities of interproximal plaque using the developed method. Thirty-nine subjects had six interdental sites (distributed across all four quadrants, pre-molar and molar boundaries) sampled on either side, over a three-week treatment period, in two balanced and equally sized treatment groups, with twice-daily manual brushing with or without daily flossing. RESULTS: Laboratory studies confirmed the accuracy of the protein detection kit in assaying bacterial plaque loads from single species or complex biofilms. In pilot developmental screens, single flossing sweeps with appropriate dental floss were sufficient to remove the majority of quantifiable biomass from interproximal sites. The clinical study demonstrated that after three weeks, interdental plaque in floss users was significantly reduced versus baseline scores. Non-floss users showed no significant reduction. In direct comparison, less interdental plaque was recovered from subjects who had followed a daily flossing regime compared to subjects who had used a manual toothbrush alone (p = 0.0866). CONCLUSION: The sampling method quantified the cleaning effects of daily flossing on interproximal plaque levels. Daily flossing significantly reduced the amount of plaque found between the teeth compared to a manual brushing regimen alone. This new method should be useful in future studies on the evaluation of mechanical or chemical means of interproximal plaque control.

Adult↗

Rapid genotyping of human platelet antigen 1 (HPA-1) with fluorophore-labelled hybridization probes on the LightCycler.

Genotyping of human platelet alloantigens (HPA) has become an important procedure in the diagnosis and prevention of disorders such as neonatal alloimmune thrombocytopenic purpura, post-transfusion purpura, and refractoriness to platelet transfusion therapy. We present a single-tube method for HPA-1 genotyping that combines rapid-cycle PCR with allele-specific fluorescent probe melting profiles for product genotyping. A fragment covering the polymorphic site is amplified in the presence of two fluorescently-labelled hybridization probes. During the annealing step of the thermal cycling, both probes bind to their complementary sequences in the amplicon resulting in resonance energy transfer, thus providing real-time fluorescence monitoring of PCR. Continuous aquisition of fluorescence data during a melting curve analysis at the completion of PCR revealed that loss of fluorescence occurred in an allele-specific manner as the detection probe, which was fully complementary to the HPA-1b allele, melted off the template. By determining the temperature at which maximum melting of the hybrids occurred, the two alleles were readily distinguishable. Using this method, genotyping of 32 samples was completed within 30 min without the need for any post-PCR sample manipulation, thereby eliminating the risks of end-product contamination and sample tracking errors. The genotypes determined with the LightCyclerTM were identical when compared with a conventional PCR and restriction fragment length polymorphism technique. The genotyping of HPA-1 on the LightCycler is a rapid and reliable method that is suitable for typing both small and large numbers of samples.

Antigens, Human Platelet↗

Effect of infusion of insulin into portal vein on hepatic extraction of insulin in anesthetized dogs.

Hepatic extraction of insulin was examined in anesthetized dogs before and after constant infusion of insulin (20 and 50 mU/min) with use of samples from the portal vein, mesenteric vein, left common hepatic vein, and the femoral artery. In 19 dogs, measurement of portal vein insulin concentration indicated an overall recovery of 110% of the insulin infused. The range varied from 9 to 303%, indicating the potential for serious error in sampling the portal vein. Equilibrium arterial insulin concentrations were achieved 20 min after starting the infusion. Prior to insulin infusion, hepatic extraction of insulin averaged 4.56 plus or minus 0.43 mUmin, representing an extraction coefficient of 0.42 of the insulin presented to the liver. The proportion of insulin extracted by the liver did not change significantly during insulin infusion despite a 10-fold increase in portal vein insulin concentrations. During the infusion of insulin, a significant proportion of the extraheptic clearance of insulin occurred in the mesenteric circulation. Infusion of insulin was associated with a significant increase in insulin extraction by tissues other than the liver and splanchnic beds. Initially, hepatic glucose output average 36 plus or minus 3 mg/min; by 20 min after insulin infusion, it was 16 plus or minus 5 mg/min. Despite continuation of insulin infusion, hepatic glucose output returned to control values even though arterial glucose concentration continued to fall. Hepatic glucose output increased with termination of insulin infusion.

Animals↗

To err is human nature. Can transfusion errors due to human factors ever be eliminated?

BACKGROUND: Fatal hemolytic transfusion reaction due to ABO incompatibility occurs mainly as a result of clerical errors. Blood sample drawn from the wrong patient and labeled as another patient's specimen will not be detected by the blood bank unless there is a previous ABO grouping result. METHODS: In Hong Kong, we had designed a transfusion wristband system--portable barcode scanner system to detect such clerical errors. The system was well accepted by the house staff and had prevented two BO mismatched transfusion. Other current system of patient's identification may have similar results, but the wristband system has the advantages of being simple, inexpensive and easy to implement. The Hong Kong Government is planning to replace the personal identity card for all citizens with an electronic smart card by 2003. If the new card contains the person's detailed red cell phenotypes in digital code, then the phenotypes of all blood donors and admitted patients will be readily available. It is feasible to issue phenotype-matched blood to patients without any need of pre-transfusion testing, therefore eliminating mismatched transfusions for most patients. RESULTS: Our pilot study of 474 patients showed that the system was safe and up to 98% of admitted patients could be transfused without delays. CONCLUSIONS: Patients with rare phenotypes, visitors or illegal immigrants may still need pre-transfusion antibody screen, but if most patients can be issued blood units without testings, the potential savings in health care amount to US$14 million/year.

ABO Blood-Group System↗

Evaluation of 38 years of radiological environmental data for the nuclear research facility in South Africa.

Environmental monitoring has been conducted at the South African National Nuclear Research Facility (Necsa site) for the past 38 years. Included in this monitoring programme was the assessment of water, fish and sediment samples. The objective of this project was to review the data of these assessments to establish if the Necsa activities had any impact on the environment. An assessment of the management of discharge limits was included in the review. Fluctuations in the data reviewed can partly be ascribed to errors in sampling techniques and analysis methods, but mostly to external factors. Two main external factors identified during the review were: dilution effects based on the flowrate in the Crocodile River and the percentage of full capacity of the Hartbeespoort Dam, and the atmospheric fallout from nuclear weapons testing. In this project, the impact of these factors were investigated with the help of correlation coefficient calculations and graphs. It was concluded that the flowrate of the Crocodile River and percentage full capacity of the Hartbeespoort Dam did have an impact on the beta activity measured in water and fish samples, and the (nat)U activities measured in water samples. The measured fallout from nuclear weapons testing in the southern hemisphere also had an impact on the beta activity in water. The assessment of the environmental monitoring data also showed that accidental releases were measurable in the environment. The added routine impact to a member of the public downstream from Necsa was on average an annual dose of 0.54 microSv more than that to a person living upstream from Necsa, which is considered insignificant in international radiation protection norms. The conclusion can be made that the monitoring programme is successful in satisfying its main objective, which is to determine the effects of the discharges on the environment and the immediate population.

Animals↗

Analytical performance of a direct assay for LDL-cholesterol: a comparative assessment versus Friedewald's formula.

OBJECTIVE: Because low density lipoprotein-cholesterol (LDL-C) is a modifiable risk factor for coronary artery disease (CAD), its routine measurement is recommended in the evaluation and management of hypercholesterolemia. Concentrations of LDL-C are commonly monitored by means of the Friedewald formula (FF), which provides a relative estimation of LDL-C concentration when the triglyceride (TGs) concentration is <200 mg/dl and there are no abnormal lipids. Because of the limitations of the Friedewald calculation, direct methods for an accurate quantification of LDL-C are needed. METHODS: We critically examined an immunoseparation method for direct assay of LDL-C in a comparison with FF. 1) We measured intraassay and interassay precision using quality-control sera and patient serum pools. Accuracy was evaluated from total error analyses. Sample stability was examined over 2 months. 2) The LDL-C levels obtained with direct assay were compared with those calculated by the FF in 47 randomly chosen patient samples. The samples were classified as group 1 (patients with TGs 60-308 mg/dl, n=25) and group 2 (patients with TGs 320-695 mg/dl, n=22). RESULTS: The direct immunoseparation assay displayed an excellent precision (total coefficient of variance (CV)<2.5%, intraassay CV<1.5% and interassay CV<1.5%). Mean total error was 4.34%. The direct assay met the current National Cholesterol Education Program (NCEP) requirements for LDL-C testing for precision and accuracy. The results of direct method (x) and the FF (y) were highly correlated (r=0.9908, y=1.030 x -0.289, n=25) in group 1, but the results of two methods disagreed (r=0.716, y=0.956 x -24.869, n=22) in group 2 (patients with TGs 320-695 mg/dl). CONCLUSION: The direct immunoseparation assay meets the currently established analytical performance goals and may be useful for the diagnosis and management of hyperlipidemic patients.

Biomarkers↗

Source contributions to the mutagenicity of urban particulate air pollution.

Using organic compounds as tracers, a chemical mass balance model was employed to investigate the relationship between the mutagenicity of the urban organic aerosol sources and the mutagenicity of the atmospheric samples. The fine particle organic mass concentration present in the 1993 annual average Los Angeles-area composite sample was apportioned among eight emission source types. The largest source contributions to fine particulate organic compound mass concentration were identified as smoke from meat cooking, diesel-powered vehicle exhaust, wood smoke, and paved road dust. However, the largest source contributions to the mutagenicity of the atmospheric sample were natural gas combustion and diesel-powered vehicles. In both the human cell and bacterial assay systems, the combined mutagenicity of the composite of primary source effluents predicted to be present in the atmosphere was statistically indistinguishable from the mutagenicity of the actual atmospheric sample composite. Known primary emissions sources appear to be capable of emitting mutagenic organic matter to the urban atmosphere in amounts sufficient to account for the observed mutagenicity of the ambient samples. The error bounds on this analysis, however, are wide enough to admit to the possible importance of additional mutagenic organics that are formed by atmospheric reaction (e.g., 2-nitrofluoranthene has been identified as an important human cell mutagen in the atmospheric composite studied here, accounting for approximately 1% of the total sample mutagenic potency).

Air Pollutants↗

Tradeoff between no-call reduction in genotyping error rate and loss of sample size for genetic case/control association studies.

Single nucleotide polymorphisms (SNP) may be genotyped for use in case-control designs to test for association between a SNP marker and a disease using a 2 x 3 chi-squared test of independence. Genotyping is often based on underlying continuous measurements, which are classified into genotypes. A "no-call" procedure is sometimes used in which borderline observations are not classified. This procedure has the simultaneous effect of reducing the genotype error rate and the expected number of genotypes observed. Both quantities affect the power of the statistic. We develop methods for calculating the genotype error rate, the expected number of genotypes observed, and the expected power of the resulting test as a function of the no-call procedure. We examine the statistical properties of the chi-squared test using a no-call procedure when the underlying continuous measure of genotype classification is a three-component mixture of univariate normal distributions under a range of parameter specifications. The genotype error rate decreases as the no-call region is increased. The expected number of observations genotyped also decreases. Our key finding is that the expected power of the chi-squared test is not sensitive to the no-call procedure. That is, the benefits of reduced genotype error rate are almost exactly balanced by the losses due to reduced genotype observations. For an underlying univariate normal mixture of genotype classification to be analyzed with a 2 x 3 chi-squared test, there is little, if any, increase in power using a no-call procedure.

Case-Control Studies↗

Estimation of growth hormone secretory patterns in children with use of a numerical deconvolution technique: experimental design with use of computer simulation.

Growth hormone (GH) secretion rates were estimated from 24-hour plasma GH concentration profiles using a model-based numerical deconvolution method. The basic kinetic parameters describing GH distribution and elimination were obtained from studies using single intravenous injections in GH-deficient children. Computer simulation techniques were used to produce 24-hour GH secretion profiles, which could serve as reference curves for analysis of the impact of certain practical aspects of the estimation of GH secretion, such as the method of blood sampling (discrete or integrated), sampling frequency and analytical errors. The results show that sampling intervals of 20 min are acceptable even in the presence of analytical errors, and that with this sampling interval, discrete blood sampling does not seem to be preferable to continuous withdrawal of blood.

Blood Specimen Collection↗

Diagnostic errors after inferior petrosal sinus sampling.

Although inferior petrosal sinus sampling (IPSS) is useful in the evaluation of Cushing's syndrome, false negative cases have been described, and many patients presumed to have ectopic tumors based upon negative IPSS remain without a final diagnosis. These patients are often managed as if they have as yet undiscovered ectopic tumors. To test this assumption, we conducted a retrospective review of our results to determine the ultimate diagnoses after IPSS. Between 1986 and 2002, 179 patients underwent 185 IPSS procedures as part of their evaluation for Cushing's syndrome. Confirmed diagnoses were available for 149 patients (83%): 139 patients had pituitary adenomas (94%), eight had bronchial carcinoids (5%), and two had adrenal tumors (1%). Threshold criteria for a pituitary source were defined as an inferior petrosal sinus to peripheral (IPS:P) basal ratio of 2:1 or greater without CRH or an IPS:P ratio of 3:1 or greater after CRH stimulation. There were nine patients in whom the IPS:P ratio failed to meet threshold criteria after successful sampling, but were nonetheless found to have pituitary tumors after transsphenoidal exploration (false negatives). Eight of these had received CRH and had a significant rise (>35%) in peripheral ACTH levels after CRH treatment, even though the IPS:P ratio did not reach the threshold. There were two patients in whom the IPS:P ratio reached threshold criteria, and ectopic tumors were demonstrated as the source of ACTH overproduction (false positives). The sensitivity after CRH stimulation was 90% (95% confidence interval, 80.8-95.5%) with a specificity of 67% (95% confidence interval, 11.4-94.5%). The positive and negative predictive values were 99 and 20%, respectively. Our data show that patients with an IPS:P ratio suggestive of a nonpituitary source of ACTH overproduction may still have Cushing's disease. Analyzing the CRH-stimulated peripheral ACTH levels in addition to the standard IPS:P ratios may provide improved diagnostic accuracy. Transsphenoidal exploration should be considered in all cases of unsuccessful sampling and in those cases for which no ectopic source can be identified after further body imaging, even if the IPSS is negative, and especially if peripheral ACTH levels rise significantly with CRH stimulation.

Adolescent↗

A new human genetic resource: a DNA bank established as part of the Avon longitudinal study of pregnancy and childhood (ALSPAC).

We describe a unique human DNA resource forming part of the Avon Longitudinal Study of Pregnancy and Childhood (ALSPAC), a longitudinal cohort study involving 14 000 children and their families living in a geographically defined area of England. The DNA bank will underpin the search for associations between genetic polymorphisms and common health outcomes. The opportunities to collect blood samples suitable for DNA extraction are necessarily limited, and the samples themselves have often been treated in different ways and have varied storage histories. With the need to maximise yields, the choice of DNA extraction method is critical to the success of the bank and we have investigated the suitability of various commercial and in-house methods of DNA extraction. Various steps have been taken to minimise errors in sample address and identification, including the use of a pipetting robot for dilution and transfer of samples between 96-well arrays to provide aliquots suitable for PCR. The robot has been programmed to cope with concentrated viscous DNA solutions.

Animals↗

Measurement of insulin sensitivity by the minimal model method using a simplified intravenous glucose tolerance test: validity and reproducibility.

This study aimed at testing whether 12 rather than 26 plasma glucose and insulin determinations can be used to calculate the indices of insulin sensitivity and of glucose effectiveness using Bergman's minimal model during a simple intravenous glucose tolerance test performed without tolbutamide injection. Two intravenous glucose tolerance tests (separated by 1 week) were performed in 7 lean normal subjects and a single test was performed in 9 severely obese non-diabetic subjects. Intra-subject reproducibility of insulin sensitivity was not significantly different when 26 or 12 time-points were analyzed (CV = 16.8 +/- 3.4 versus 18.9 +/- 3.8% respectively). Compared with the insulin sensitivity of the lean subjects, that of obese subjects was significantly (P < 0.001) and similarly reduced when using 12 (2.14 +/- 0.34 versus 7.97 +/- 1.29 10(-4)min-1/mU.1-1) rather than 26 determinations (2.13 +/- 0.42 versus 6.95 +/- 1.12 10(-4) min-1/mU.1-1) respectively. Glucose effectiveness was less reproducible than insulin sensitivity and was slightly diminished by the reduction of blood samples (relative error: -9.7 +/- 4.4%; P < 0.05). Finally, glucose effectiveness tended to be slightly lower in the morbidly obese subjects than in the lean controls with both modes of calculation. In conclusion, in non-diabetic subjects, the insulin sensitivity index can be accurately measured during a simple intravenous glucose tolerance test, without tolbutamide injection and with only 12 blood samples. The possibility of performing a simplified test should contribute to increase the use of the minimal model method for estimating insulin sensitivity in clinical practice.

Adult↗

A calculation and number processing battery for clinical application in illiterates and semi-literates.

Ten simple tasks assessing counting, number processing, elementary calculation and quantity estimation were proposed to 122 normal Brazilian adults aged between 18 and 58 years with 0, 1, 2, 3 or 4 years of education. Tasks such as counting the number of elements in small sets were almost perfectly mastered by these illiterate or semi-literate normal subjects; however in other tasks (e.g. those assessing knowledge of the correspondence between numbers and banknotes) a sizeable proportion of the sample showed errors. The pattern of errors was analysed to identify difficulty factors. A strong gender effect with better performance in men than women was observed, which was even greater than the expected effect of educational level. Results in normals allowed to propose cut-off scores for neuropsychological assessment in brain-damaged patients with very low levels of education, which were tested in a small sample of illiterate or semi-literate patients with cerebrovascular accident. It is argued that the relatively neglected area of neuropsychological assessment in illiterates is of great practical and theoretical interest.

Adolescent↗

Plant DNA flow cytometry and estimation of nuclear genome size.

BACKGROUND: DNA flow cytometry describes the use of flow cytometry for estimation of DNA quantity in cell nuclei. The method involves preparation of aqueous suspensions of intact nuclei whose DNA is stained using a DNA fluorochrome. The nuclei are classified according to their relative fluorescence intensity or DNA content. Because the sample preparation and analysis is convenient and rapid, DNA flow cytometry has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathway, and estimation of absolute DNA amount or genome size. While the former applications are relatively straightforward, estimation of absolute DNA amount requires special attention to possible errors in sample preparation and analysis. SCOPE: The article reviews current procedures for estimation of absolute DNA amounts in plants using flow cytometry, with special emphasis on preparation of nuclei suspensions, stoichiometric DNA staining and the use of DNA reference standards. In addition, methodological pitfalls encountered in estimation of intraspecific variation in genome size are discussed as well as problems linked to the use of DNA flow cytometry for fieldwork. CONCLUSIONS: Reliable estimation of absolute DNA amounts in plants using flow cytometry is not a trivial task. Although several well-proven protocols are available and some factors controlling the precision and reproducibility have been identified, several problems persist: (1) the need for fresh tissues complicates the transfer of samples from field to the laboratory and/or their storage; (2) the role of cytosolic compounds interfering with quantitative DNA staining is not well understood; and (3) the use of a set of internationally agreed DNA reference standards still remains an unrealized goal.

Cell Nucleus↗

Refractive errors in twin studies.

It is estimated that 1.6 billion people worldwide have myopia, a refractive error, and this number is expected to increase to approximately 2.5 billion by the year 2020. It is now well established that both the environment and genetics play a role in the development of myopia. However, the exact contribution of each of these components to myopia development has yet to be completely determined. Twin studies (classical twin model) are commonly used to determine the weighting of genetic and environmental components in disease. Over the last century, twin studies have investigated the heritability of refractive errors in different sample populations and have collectively supported a genetic basis to refractive errors. However, different sample populations and methods of data collection have produced a wide range of heritability estimates ranging from .5 to .9. This article will review those twin studies that have investigated refractive error, particularly myopia, as well as biometric measures linked to refractive error, to compare heritability estimates and methodology designs.

Diseases in Twins↗

Prediction of elasticity constants in small biomaterial samples such as bone. A comparison between classical optimization techniques and identification with artificial neural networks.

Measuring the elasticity constants of biological materials often sets important constraints, such as the limited size or the irregular geometry of the samples. In this paper, the identification approach as applied to the specific problem of accurately retrieving the material properties of small bone samples from a measured displacement field is discussed. The identification procedure can be formulated as an optimization problem with the goal of minimizing the difference between computed and measured displacements by searching for an appropriate set of material parameters using dedicated algorithms. Alternatively, the backcalculation of the material properties from displacement maps can be implemented using artificial neural networks. In a practical situation, however, measurement errors strongly affect the identification results, calling for robust optimization approaches in order accurately to retrieve the material properties from error-polluted sample deformation maps. Using a simple model problem, the performances of both classical and neural network driven optimization are compared. When performed before the collection of experimental data, this evaluation can be very helpful in pinpointing potential problems with the envisaged experiments such as the need for a sufficient signal-to-noise ratio, particularly important when working with small tissue samples such as specimens cut from rodent bones or single bone trabeculae.

Algorithms↗

Estimation of attributable number of deaths and standard errors from simple and complex sampled cohorts.

Estimates of the attributable number of deaths (AD) from all causes can be obtained by first estimating population attributable risk (AR) adjusted for confounding covariates, and then multiplying the AR by the number of deaths determined from vital mortality statistics that occurred in the population for a specific time period. Proportional hazard regression estimates of adjusted relative hazards obtained from mortality follow-up data from a cohort is combined with a joint distribution of risk factor and confounders to compute an adjusted AR. Two estimators of adjusted AR are examined. These estimators differ according to which reference population is used to obtain the joint distribution of risk factor and confounders. Two types of reference populations were considered: (i) the population represented by the baseline cohort and (ii) a population that is external to the cohort. Methods used in survey sampling are applied to obtain estimates of the variance of the AD estimator. These variances can be applied to data that range from simple random samples to multistage stratified cluster samples, which are used in national household surveys. The variance estimation of AD is illustrated in an analysis of excess deaths due to having a non-ideal body mass index using the second National Health and Examination Survey (NHANES) Mortality Study and the 1999-2002 NHANES. These methods can also be used to estimate the attributable number of cause-specific deaths and their standard errors when the time period for the accrual of deaths is short.

Cohort Studies↗