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Quantifying errors without random sampling.

BACKGROUND: All quantifications of mortality, morbidity, and other health measures involve numerous sources of error. The routine quantification of random sampling error makes it easy to forget that other sources of error can and should be quantified. When a quantification does not involve sampling, error is almost never quantified and results are often reported in ways that dramatically overstate their precision. DISCUSSION: We argue that the precision implicit in typical reporting is problematic and sketch methods for quantifying the various sources of error, building up from simple examples that can be solved analytically to more complex cases. There are straightforward ways to partially quantify the uncertainty surrounding a parameter that is not characterized by random sampling, such as limiting reported significant figures. We present simple methods for doing such quantifications, and for incorporating them into calculations. More complicated methods become necessary when multiple sources of uncertainty must be combined. We demonstrate that Monte Carlo simulation, using available software, can estimate the uncertainty resulting from complicated calculations with many sources of uncertainty. We apply the method to the current estimate of the annual incidence of foodborne illness in the United States. SUMMARY: Quantifying uncertainty from systematic errors is practical. Reporting this uncertainty would more honestly represent study results, help show the probability that estimated values fall within some critical range, and facilitate better targeting of further research.

Bias↗

Polymerase chain reaction compared with dot blotting for the determination of N-myc gene amplification in neuroblastoma.

The magnitude of N-myc amplification (NMA) influences the treatment strategy of localized neuroblastomas. Reliable assays are therefore needed for all types of tumor samples. The aim of this comparative study of 119 tumor samples was to determine whether a polymerase chain reaction (PCR)-based assay could replace the current dot blot assay as a routine and reliable means of determining NMA. The 2 assays exhibited comparable sensitivity and were completely concordant for samples containing at least 20% neuroblastoma cells. In their present state, both assays remain semi-quantitative since an absolute quantification of the N-myc copy number in clinical samples is limited by uncertainty about the amplification level of reference cell lines and by the estimation of the proportion of malignant cells. However, PCR offers several advantages over dot blotting, such as feasibility on minute samples, simplicity, standardization, rapidity and cost effectiveness.

Cost-Benefit Analysis↗

Mitotic counting in surgical pathology: sampling bias, heterogeneity and statistical uncertainty.

Mitotic counting in surgical pathology: sampling bias, heterogeneity and statistical uncertainty Although several articles on the methodological aspects of mitotic counting have been published, the effects of macroscopic sampling and tumour heterogeneity have not been discussed in any detail. In this review the essential elements for a standardized mitotic counting protocol are described, including microscopic calibration, specific morphological criteria, macroscopic selection, counting procedure, effect of biological variation, threshold, and the setting of an area of uncertainty ('grey area'). We propose that the use of a standard area for mitotic quantification and of a grey area in mitotic counting protocols will facilitate the application of mitotic counting in diagnostic and prognostic pathology.

Data Interpretation, Statistical↗

Analytical aspects of the CEEM soil project.

In the past, exercises aiming at an assessment of data uncertainty in environmental analysis were usually restricted to the analysis step, while sampling and pre-analytical sample treatment was largely ignored. Collaborative studies on the quantification of sampling errors require, besides a suitable and well characterized test site, the availability of a reference laboratory for the analysis of all of the samples taken in the context of the study by all participants and also test methods which do not contribute large and variable uncertainties due to long and complex analytical methodologies. Here we summarize the major analytical aspects of a European project on the identification and quantification of sampling influences on the determination of lead, cadmium, copper and zinc in soil. The participant group included the leading soil analysis laboratories in Europe; the test site at Dornach (CH) was well suited for the purpose and showed high metal gradients and differentiated land use. The analytical methods (wavelength-dispersive X-ray fluorescence spectrometry and solid-state Zeeman AAS) used in the study showed stable performance characteristics within the confidence interval of the certified reference materials used for the measurement quality control over the entire project period. Additionally, double-blind tests on split samples showed agreement of data in very narrow limits thus demonstrating the reliability of the reference database.

Databases, Factual↗

Quasi-independent monitor unit calculation for intensity modulated sequential tomotherapy.

The number of linac monitor units (MU) from intensity modulated sequential tomotherapy (IMST) is substantially larger than the MU delivered in conventional radiation therapy, and the relation between MU and dose is obscure due to complicated variation of the beam intensities. The purpose of this work was to develop a practical method of verifying the MU and dose from IMST so that the MU of each arced beam could be double-checked for accuracy. MU calculations for 41 arced beams from 14 IMST patients were performed using the variables of vane open fraction time, field size, target depth, output factor, TMR, and derived intensity distribution. Discrepancy between planned and checked MU was quantified as 100 (MU(cal)-MU(plan))/MU(plan) percent. All 41 discrepancies were clustered between -5% to +4%, illustrated in a Gaussian-shaped histogram centered at -1.0+/-3.5% standard deviation indicating the present MU calculations are in agreement with the planned expectations. To confirm the correctness of the present calculated MUs of the IMST plans, eight of the calculated IMST plans are performed dose verifications using their hybrid plans, which are created by transporting patient's IMST plan beams onto a spherical polystyrene Phantom for dose distribution within the Phantom. The dose was measured with a 0.07 cc ionization chamber inserted in the spherical Phantom during the hybrid plan irradiation. Average discrepancy between planned and measured doses was found to be 0.6+/-3.4% with single standard deviation uncertainty. The spread of the discrepancies of present calculated MUs relative to their planned ones are attributed to uncertainties of effective field size, effective planned dose corresponding to each arc, and inaccuracy of quantification of scattered dose from adjacent arced beams. Overall, the present calculation of MUs is consistent with what derived from treatment plans. Since the MUs are verified by actual dose measurements, therefore the present MU calculation technique is considered adequate for double-checking planned IMST MUs.

Humans↗

Reference methods for the measurement of free thyroid hormones in blood: evaluation of potential reference methods for free thyroxine.

In this paper, a review of the literature on the methods for the detection of FT(4) is provided. Furthermore, an overview of the most important uncertainties and interferences of the potential reference methods for determination of free thyroxine (free triiodothyronine) are also presented. Especially, the separation step-dialysis, or ultrafiltration-implies important technical and theoretical uncertainties and interferences as to the methods.Furthermore, a short review of methods based on isotope dilution/mass spectrometry (ID/MS) to quantify thyroxine is provided. There has so far been no attempt to quantify the free fraction of thyroxine by ID/MS, probably due to lack of sensitivity, but if quantification of the free fraction of thyroxine can be done with ID/MS, then this step seems the least compromised step only including minor uncertainties in comparison to the separation step.

Calibration↗

Isotope dilution determination of polycyclic aromatic hydrocarbons in olive pomace oil by gas chromatography-mass spectrometry.

A gas chromatographic (GC) method with mass spectrometry detection (MS) for the determination of eight polycyclic aromatic hydrocarbons (PAHs) in olive pomace oil has been developed. The oil was diluted with n-pentane and extracted by liquid-liquid partition with dimethyl sulphoxide (DMSO). After water addition and back-extraction with cyclohexane, a thin-layer chromatography on silica gel was performed as a further purification step. The PAHs spot was scraped off from the plate and the final extract was concentrated and analysed by GC-MS in full scan mode. The eight PAHs under investigation were determined in the presence of the corresponding labelled compounds added as internal standards to the sample at the beginning of the analytical process. The identified PAHs were then quantified by the isotope dilution methodology assuring the compensation of the concentration of each analyte for any variation in the sample preparation. The method precision was satisfactory with relative standard deviation (R.S.D.) values in the range 3.6-12.7% for all PAHs. The average recovery rates ranged from 69.0 to 97.5%. Accuracy was also calculated for benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene and benzo[ghi]perylene by analysing a certified reference material (CRM 458, coconut oil) with adequate results. All response curves exhibited a linear fit from 0.1 to 10 microg ml(-1) and the determination coefficients R2 were better than 0.9942. The limits of detection (0.1-0.4 microg kg(-1)) were acceptable when compared with the maximum permitted limit of 2 microg kg(-1) for each of the eight considered PAHs and 5 microg kg(-1) for the sum of the eight PAHs established by the Italian legislation. Measurement uncertainty was finally calculated identifying and quantifying the uncertainty components of the analytical process. The relative expanded uncertainties (Uc), expressed as percent values were in the range 8.5-11.4% thus appropriate for residues quantification in the range of concentrations considered in the present study.

Gas Chromatography-Mass Spectrometry↗

Future research needs associated with the assessment of potential human health risks from exposure to toxic ambient air pollutants.

This paper presents key conclusions and future research needs from a Workshop on the Risk Assessment of Urban Air, Emissions, Exposure, Risk Identification, and Quantification, which was held in Stockholm during June 1992 by 41 participants from 13 countries. Research is recommended in the areas of identification and quantification of toxics in source emissions and ambient air, atmospheric transport and chemistry, exposure level assessment, the development of improved in vitro bioassays, biomarker development, the development of more accurate epidemiological methodologies, and risk quantification techniques. Studies are described that will be necessary to assess and reduce the level of uncertainties associated with each step of the risk assessment process. International collaborative research efforts between industry and government organizations are recommended as the most effective way to carry out this research.

Air Pollutants↗

Quantifying local lung perfusion and ventilation using correlated SPECT and CT data.

UNLABELLED: A clinically applicable method for quantifying lung perfusion and ventilation on a subregional (local) scale from SPECT scans in order to estimate local lung function in patients with pre-existing pulmonary disease and to monitor local treatment effects was developed and evaluated. METHODS: SPECT 99mTc perfusion and 81mKr ventilation images were corrected for photon attenuation and scatter effect with a postreconstruction correction method incorporating a variable-effective linear-attenuation coefficient calculated from spatially-correlated CT data. A new algorithm was developed to quantify local ventilation from the SPECT data, which, in contrast with other algorithms, makes no assumptions on ventilation homogeneity over the lung. The quantification procedure was applied to clinical data from patients with a normal lung function and from patients suffering from radiation-induced pulmonary dysfunction. RESULTS: The calculated attenuation correction factors on the observed number of counts in the lung range from 2.0 to 3.0 and 2.3 to 3.5 for 81mKr and 99mTc, respectively, showing a systematic increase from the diaphragm to the lung apex. As a result of this correction, the values of local perfusion and ventilation differ 10%-15% from values calculated without attenuation correction. The calculated values of the local ventilation are 10%-50% lower than those found by quantification algorithms which assume homogeneous ventilation. CONCLUSIONS: The methods presented here are robust with respect to uncertainties in the input parameters and yield realistic values for perfusion and ventilation distribution in the lung with an intrinsic accuracy (largely determined by count statistics) of about 10%.

Algorithms↗

Rapid gas chromatographic method for the determination of famoxadone, trifloxystrobin and fenhexamid residues in tomato, grape and wine samples.

Trifloxystrobin, fenhexamid and famoxadone belong to the generation of fungicides acting against a broad spectrum of fungi and widely used in Integrated Pest Management strategies in different agricultural crops but mainly in viticulture. In the present work, a gas chromatographic (GC) method for their determination was developed and validated on tomato, grape and wine matrices. The method was based on a simple one step liquid-liquid microextraction with cyclohexane/dichloromethane (9+1, v/v) and determination of fungicides by gas chromatography with nitrogen phosphorous (NP-) and electron capture (EC-) detection, and ion trap mass spectrometry (ITMS) for confirmation. The method was validated by recovery experiments, assessment of matrix effect and calculation of the associated uncertainty. Recoveries for GC-NPD and GC-ECD were found in the range of 81-102% with RSD <12%, while matrix-matched calibration solutions were imposed for quantification. LOQs ranged from 0.005 to 0.05 mg/kg and 0.01 to 0.10 mg/kg for the GC-ECD and GC-NPD, respectively, depending on the sensitivity of each compound with trifloxystrobin being the most sensitive. The expanded uncertainty, calculated for a sample concentration of 0.10 mg/kg, ranged from 4.8 to 13% for the GC-ECD and from 5.4 to 29% for the GC-NPD. The concentration levels for famoxadone residues found in tomato and grape samples from field experiments were clearly below the EU established MRL values, thus causing no problems in terms of food safety.

Acetates↗

Measurement uncertainty from physical sample preparation: estimation including systematic error.

A methodology is proposed, which employs duplicated primary sampling and subsequent duplicated physical preparation coupled with duplicated chemical analyses. Sample preparation duplicates should be prepared under conditions that represent normal variability in routine laboratory practice. The proposed methodology requires duplicated chemical analysis on a minimum of two of the sample preparation duplicates. Data produced from the hierarchical design is treated with robust analysis of variance (ANOVA) to generate uncertainty estimates, as standard uncertainties ('u' expressed as standard deviation), for primary sampling (ssamp), physical sample preparation (sprep) and chemical analysis (sanal). The ANOVA results allow the contribution of the sample preparation process to the overall uncertainty to be assessed. This methodology has been applied for the first time to a case study of pesticide residues in retail strawberry samples. Duplicated sample preparation was performed under ambient conditions on two consecutive days. Multi-residue analysis (quantification by GC-MS) was undertaken for a range of incurred pesticide residues including those suspected of being susceptible to loss during sample preparation procedures. Sampling and analytical uncertainties dominated at low analyte concentrations. The sample preparation process contributed up to 20% to the total variability and had a relative uncertainty (Uprep%) of up to 66% (for bupirimate at 95% confidence). Estimates of systematic errors during physical sample preparation were also made using spike recovery experiments. Four options for the estimation of measurement uncertainty are discussed, which both include and exclude systematic error arising from sample preparation and chemical analysis. A holistic approach to the combination and subsequent expression of uncertainty is advised.

Calibration↗

A rapid method to quantify nitrifiers in activated sludge.

Quantification of bacteria using Fluorescence In Situ Hybridization (FISH), confocal laser scanning microscopy (CLSM) and image analysis is very time consuming and requires the availability of an expensive microscope. Therefore, a rapid method to quantify nitrifying bacteria in activated sludge using FISH and epifluorescence microscopy was developed. The quantification of the biovolume is based on manual counting of the aggregates formed by nitrifying bacteria and determination of their size. The overall uncertainty of the method was evaluated as a function of the number of analyzed microscopic fields. It was found that 10-15 microscopic fields for ammonia-oxidizing bacteria and 6-8 microscopic fields for nitrite-oxidizing bacteria per sample were optimal regarding effort and accuracy. Accordingly, the time needed for one sample was only 5-15 min, compared to about 1h for the quantification with CLSM and image analysis. As a consequence, this method also allows for the measurement of extended time series with a reasonable effort. The comparison of the determined biovolume and the measured activity showed an explicit correlation.

Automation↗

Investigating brain metabolism at high fields using localized 13C NMR spectroscopy without 1H decoupling.

Most in vivo 13C NMR spectroscopy studies in the brain have been performed using 1H decoupling during acquisition. Decoupling imposes significant constraints on the experimental setup (particularly for human studies at high magnetic field) in order to stay within safety limits for power deposition. We show here that incorporation of the 13C label from 13C-labeled glucose into brain amino acids can be monitored accurately using localized 13C NMR spectroscopy without the application of 1H decoupling. Using LCModel quantification with prior knowledge of one-bond and multiple-bond J(CH) coupling constants, the uncertainty on metabolites concentrations was only 35% to 91% higher (depending on the carbon resonance of interest) in undecoupled spectra compared to decoupled spectra in the rat brain at 9.4 Tesla. Although less sensitive, 13C NMR without decoupling dramatically reduces experimental constraints on coil setup and pulse sequence design required to keep power deposition within safety guidelines. This opens the prospect of safely measuring 13C NMR spectra in humans at varied brain locations (not only the occipital lobe) and at very high magnetic fields above 4 Tesla.

Animals↗

Quantitative nuclear magnetic resonance (QNMR) spectroscopy for assessing the purity of technical grade agrochemicals: 2,4-dichlorophenoxyacetic acid (2,4-D) and sodium 2,2-dichloropropionate (Dalapon sodium).

Comparison of quantitative NMR spectroscopy (QNMR) with chromatographic methods such as gas chromatography (GC) or high-pressure liquid chromatography (HPLC) for the determination of the purity of and impurities in technical grade agrochemicals, 2,4-dichlorophenoxyacetic acid (2,4-D), 1, and Dalapon sodium (sodium 2,2-dichloropropionate), 10, has revealed that QNMR is more precise and accurate than the chromatographic methods. Quantitative impurity profiling of technical grade 1 is rapid and accurate using 600 MHz (1)H NMR. Extra dispersion at the relatively high frequency allowed full assignment of the NMR spectrum of 1 and its related organic impurities in technical samples. The percentage purity of 1 was measured by the difference QNMR method, which involves summing the amounts of impurities and subtracting from 100%. Results are superior in consistency to those obtained by chromatographic methods. The percentage purity of Dalapon sodium, 10, in technical grade batches is readily obtained by (1)H QNMR, using either the difference method or the internal standard method, using dimethyl sulfone (DMSO2) internally as a reference material, that is chemically unrelated to the analyte. The latter method also allows the simultaneous identification and quantification of impurities, many of which are either not accessible to or detectable by the chromatographic methods. Uncertainty budgets for the QNMR method are presented and demonstrate that the major contributors to uncertainty lie in the weighing of the chemicals and in purity of the standard reference material prior to the QNMR experiment.

2,4-Dichlorophenoxyacetic Acid↗

A methodology for specifying PET VOI's using multimodality techniques.

Volume-of-interest (VOI) extraction for radionuclide and anatomical measurements requires correct identification and delineation of the anatomical feature being studied. We have developed a toolset for specifying three-dimensional (3-D) VOI's on a multislice positron emission tomography (PET) dataset. The software is particularly suited for specifying cerebral cortex VOI's which represent a particular gyrus or deep brain structure. A registered 3-D magnetic resonance image (MRI) dataset is used to provide high-resolution anatomical information, both as oblique two-dimensional (2-D) sections and as volume renderings of a segmented cortical surface. VOI's are specified indirectly in two dimensions by drawing a stack of 2-D regions on the MRI data. The regions are tiled together to form closed triangular mesh surface models, which are subsequently transformed into the observation space of the PET scanner. Quantification by this method allows calculation of radionuclide activity in the VOI's, as well as their statistical uncertainties and correlations. The methodology for this type of analysis and validation results are presented.

Brain↗

A 2-year dose-response study of lesion sequences during hepatocellular carcinogenesis in the male B6C3F(1) mouse given the drinking water chemical dichloroacetic acid.

Dichloroacetic acid (DCA) is carcinogenic to the B6C3F(1) mouse and the F344 rat. Given the carcinogenic potential of DCA in rodent liver and the known concentrations of this compound in drinking water, reliable biologically based models to reduce the uncertainty of risk assessment for human exposure to DCA are needed. Development of such models requires identification and quantification of premalignant hepatic lesions, identification of the doses at which these lesions occur, and determination of the likelihood that these lesions will progress to cancer. In this study we determined the dose response of histopathologic changes occurring in the livers of mice exposed to DCA (0.05-3.5 g/L) for 26-100 weeks. Lesions were classified as foci of cellular alteration smaller than one liver lobule (altered hepatic foci; AHF), foci of cellular alteration larger than one liver lobule (large foci of cellular alteration; LFCA), adenomas (ADs), or carcinomas (CAs). Histopathologic analysis of 598 premalignant lesions revealed that (a)) each lesion class had a predominant phenotype; (b)) AHF, LFCA, and AD demonstrated neoplastic progression with time; and (c)) independent of DCA dose and length of exposure effects, some toxic/adaptive changes in non-involved liver were related to this neoplastic progression. A lesion sequence for carcinogenesis in male B6C3F(1) mouse liver has been proposed that will enable development of a biologically based mathematical model for DCA. Because all classes of premalignant lesions and CAs were found at both lower and higher doses, these data are consistent with the conclusion that nongenotoxic mechanisms, such as negative selection, are relevant to DCA carcinogenesis at lower doses where DCA genotoxicity has not been observed.

Adenoma, Acidophil↗

Uncertainty and the value of diagnostic information, with application to axillary lymph node dissection in breast cancer.

In clinical decision making, it is common to ask whether, and how much, a diagnostic procedure is contributing to subsequent treatment decisions. Statistically, quantification of the value of the information provided by a diagnostic procedure can be carried out using decision trees with multiple decision points, representing both the diagnostic test and the subsequent treatments that may depend on the test's results. This article investigates probabilistic sensitivity analysis approaches for exploring and communicating parameter uncertainty in such decision trees. Complexities arise because uncertainty about a model's inputs determines uncertainty about optimal decisions at all decision nodes of a tree. We present the expected utility solution strategy for multistage decision problems in the presence of uncertainty on input parameters, propose a set of graphical displays and summarization tools for probabilistic sensitivity analysis in multistage decision trees, and provide an application to axillary lymph node dissection in breast cancer.

Axilla↗

Spatial contaminant heterogeneity: quantification with scale of measurement at contrasting sites.

Material within the terrestrial environment is rarely homogeneously distributed, either spatially or temporally. One consequence of heterogeneity is that uncertainty is usually generated in measurements that are taken with the aim of characterising the environment. For example, a measurement of analyte concentration within soil taken from one sampling location on contaminated land can vary substantially when compared against another sample taken at effectively the same nominal location. The measurement uncertainty arising from the heterogeneity can substantially limit the reliability of the interpretations made upon environmental investigations. The sampling uncertainty usually outweighs the analytical uncertainty from the laboratory, often by a factor of 20 or more. One approach to reducing the uncertainty is to design a more suitable sampling strategy. This might be achieved by predicting the degree of heterogeneity prior to the investigation, but this is often difficult to achieve accurately. Another approach, which was investigated here, is to actually characterise the heterogeneity prior to the main investigation using rapid and inexpensive technology, such as in situ measurement techniques. In situ portable X-ray fluorescence (PXRF) and X-ray microprobe (XMP) techniques were employed to test the feasibility of this approach. Two contrasting contaminated land sites were chosen to characterise the two-dimensional spatial heterogeneity of heavy metal contamination in topsoil at a range of scales (50 m to 0.001 m). The spatial heterogeneity of contaminants, expressed as relative standard deviations, was found to differ between the two sites by a factor of two, largely due to the mode of deposition of pollution. The study also indicated that the heterogeneity did not change systematically with the scale of measurement between sampling locations at either site.

England↗