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False negative GC/MS assay for carboxy THC due to ibuprofen interference.

A case of a false negative THC metabolite confirmation by GC/MS is presented. A urine specimen testing positive by the EMIT d.a.u. Cannabinoid 100-ng assay was subjected to confirmation by a GC/MS procedure designed to detect the methyl derivative of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH). No methylated THC-COOH was found. Analysis of the specimen by the TOXI-LAB Cannabinoids TLC procedure yielded a positive confirmation. Subsequent work-up of the specimen revealed a high concentration of ibuprofen, which is shown to interfere with the methylation of THC-COOH. Reanalysis of the specimen by GC/MS with an increased amount of methylating reagent yielded a positive result.

Dronabinol↗

Do breath tests really underestimate blood alcohol concentration?

It has been reported that in the fully postabsorptive state, breath test results underestimate actual blood alcohol concentration (BAC) in 86% of the population. Reanalysis of the data on which this conclusion was based indicates 77% of these subjects were actually underestimated, and 23% were overestimated. Further refinements indicate 68% had their actual BAC underestimated, 16% were acceptably close to the actual BAC, and 16% were overestimated. Perhaps more importantly, comparison of this data with other results indicates fully postabsorptive status may not occur until more than three hours after drinking. Consequently, breath test results may tend to overestimate actual BAC for significant amounts of time after the peak BAC has been reached.

Breath Tests↗

Two fatalities from ingestion of acetonitrile: limited specificity of analysis by headspace gas chromatography.

A married couple was found dead in bed at their home, and the police investigation indicated that they might have ingested methanol and/or ethanol the previous evening. Postmortem examinations were performed and samples of femoral vein blood, urine, and stomach contents were submitted for toxicological analysis. Headspace gas chromatography (HSGC) using two different column packings (Carbopak B and Carbopak C) was used for qualitative and quantitative analysis of organic volatile substances. The apparent concentrations of ethanol in blood and urine were 0.20 and 0.27 g% w/v, respectively. On reanalysis of body fluids by another HSGC method and gas chromatography/mass spectrometry (GC/MS), the presence of ethanol was not confirmed. Instead, acetonitrile was identified at concentrations of 0.8 g/L in blood, 1.0 g/L in urine, and 1.3 g/L in stomach contents. Inorganic cyanide, the toxic metabolite of acetonitrile, was identified in blood from both victims at concentrations of 2.4 micrograms/g (female) and 4.5 micrograms/g (male). Despite the use of two different column packing materials for HSGC, acetonitrile was mistakenly identified as ethanol.

Acetonitriles↗

Evaluation of phencyclidine by EMIT d.a.u. utilizing the ETS analyzer and a 25-ng/mL cutoff.

This study evaluates the usefulness of the EMIT d.a.u. phencyclidine assay using the Syva ETS instrument to reliably detect phencyclidine in urine specimens at 25 ng/mL and above. More than 1600 urine specimens were screened over a one-month period. Fifty three (53) specimens screened positive (25 of these were previously tested positive by EMIT and GC/MS at or above the 25-ng/ml level and resubmitted as unknowns). Of the 53 specimens, 52 were confirmed positive by GC/MS. Reanalysis by EMIT of the one sample that was confirmed negative by GC/MS yielded a negative result. The absorbance difference between a negative sample and a sample containing 25 ng/mL of phencyclidine averaged 53 absorbance units with a standard deviation of 7.7 units. The absorbance difference between a negative sample and a sample containing 75 ng/mL of phencyclidine averaged 105 absorbance units with a standard deviation of 6.8 units.

Gas Chromatography-Mass Spectrometry↗

A 5-year stability study of common illicit drugs in blood.

The present study was designed to determine the stability of common illicit drugs in stored blood at various time intervals for a period of up to 5 years. The drugs of interest were cocaine and benzoylecgonine, methamphetamine and amphetamine, nonconjugated morphine and codeine, and phencyclidine (PCP). All specimens were from live individuals and were collected in gray-top Vacutainer tubes containing sodium fluoride and potassium oxalate; the tubes were stored at ambient temperature. The results of the study showed that cocaine and benzoylecgonine have poor stability and require quantitative confirmation within a reasonable time period for reliable interpretation. Methamphetamine and PCP were both fairly stable and had a high probability of confirmation upon reanalysis. The stability of nonconjugated morphine showed wide variation throughout the study. Initially, the morphine concentration decreased, then increased at the 3-year interval, and finally decreased at the 4- and 5-year intervals. The significance of the analytical findings are discussed in this report.

Amphetamine↗

Stability of phencyclidine in stored blood samples.

A retrospective study was conducted to determine the stability of phencyclidine (PCP) in whole blood samples stored for 1-3 years at both 4 degrees C and -20 degrees C. The PCP concentrations of these samples were initially determined in 1993, 1994, or 1995, then stored until 1996, when the PCP concentrations were determined again. All quantitations of PCP were by means of gas chromatography-mass spectrometry using the internal standard method. PCP was extracted from the blood by solid-phase extraction followed by liquid-liquid back extraction. The method of quantitation for reanalysis was identical to that used in the initial PCP analyses. The results of this study reveal a tendency for the concentrations of PCP to decrease gradually over a period of 3 years. Samples stored for 1 year did not show a significant change in PCP concentration, whereas samples stored for 2 years showed a 9.6% average decrease, and samples stored for 3 years showed a 17.9% average decrease. Following storage, PCP was present in all samples in which it had been originally detected. The results of this study indicate that PCP, at the concentrations in this study, remains sufficiently stable to be detected within 3 years of storage at 4 degrees C and -20 degrees C.

Blood Preservation↗

Identification and analysis of the major metabolites of cocaine in meconium.

A gas chromatographic-mass spectrometric (GC-MS) method was used for the simultaneous analysis of cocaine and its metabolites (norcocaine, benzoylecgonine, norbenzoylecgonine, ecgonine methylester, cocaethylene, and the o-, m, and p-hydroxy derivatives of cocaine and benzoylecgonine) in meconium specimens collected from newborns prenatally exposed to the drug in an effort to determine which of these metabolites are more relevant in the confirmation of immunoassay-positive specimens. Significant metabolites included benzoylecgonine and its m- and p-hydroxy derivatives. Reanalysis of immunoassay-positive meconium specimens that previously failed to confirm by GC-MS for benzoylecgonine revealed that the GC-MS confirmation rate could be substantially enhanced by inclusion of m- and p-hydroxybenzoylecgonine in the analysis along with benzoylecgonine. Several specimens were found to be positive for hydroxylated derivative(s) in the absence of benzoylecgonine itself.

Cocaine↗

Carbon monoxide stability in stored postmortem blood samples.

Carbon monoxide (CO) poisoning remains a common cause of both suicidal and accidental deaths in the United States. As a consequence, determination of the percent carboxyhemoglobin (%COHb) level in postmortem blood is a common analysis performed in toxicology laboratories. The blood specimens analyzed are generally preserved with either EDTA or sodium fluoride. Potentially problematic scenarios that may arise in conjunction with CO analysis are a first analysis or a reanalysis requested months or years after the initial toxicology testing is completed; both raise the issue of the stability of carboxyhemoglobin in stored postmortem blood specimens. A study was conducted at the Bexar County Medical Examiner's Office to evaluate the stability of CO in blood samples collected in red-, gray-, and purple-top tubes by comparing results obtained at the time of the autopsy and after two years of storage at 3 degrees C using either an IL 282 or 682 CO-Oximeter. The results from this study suggest that carboxyhemoglobin is stable in blood specimens collected in vacutainer tubes, with or without preservative, and stored refrigerated for up to two years.

Carbon Monoxide↗

LC-MS-(TOF) analysis method for benzodiazepines in urine samples from alleged drug-facilitated sexual assault victims.

In recent years there has been an increase in the number of reports in the U.S. of the use of drugs to commit sexual assault. In 1994, a nationwide urine testing program was developed to assess the incidence of the use of drugs to facilitate sexual assault and provide information for use in the investigation of these crimes. Urine samples were collected from victims of suspected drug-induced sexual assault by law enforcement agencies, emergency rooms, and rape crisis centers. The most implicated drug class was benzodiazepines, either alone or in combination with alcohol. In this report, a procedure was developed for the screening of 22 benzodiazepines in human urine by liquid chromatography-time of flight-mass spectrometry [LC-MS-(TOF)]. The limit of quantitation for all benzodiazepines ranged from 2 to 10 ng/mL, and the limit of detection was 0.5 to 3.0 ng/mL. These results suggest that the method sensitivity is suitable to screen for all 22 benzodiazepines in human urine at low levels. The method was used to analyze samples previously reported to have screened positive for benzodiazepines by immunoassay at 50 ng/mL cut off but failed to confirm by a gas chromatography-MS method. The results of reanalysis of these samples using this LC-MS method are reported.

Benzodiazepines↗

Green turtle (Chelonia mydas) foraging and nesting aggregations in the Caribbean and Atlantic: impact of currents and behavior on dispersal.

Although significant amounts of research have been dedicated to increasing the knowledge of the life history of green turtles (Chelonia mydas), large gaps exist in our understanding of juvenile migratory behavior. These gaps can be filled by genetic studies of foraging ground aggregations. Using mitochondrial DNA markers and Bayesian analyses, samples (n = 106) from a foraging aggregation in North Carolina indicated that animals from the east coast of the United States (54%) and Mexico (27%) dominate the composition with the remainder coming from other Caribbean and Atlantic nesting aggregations. These findings prompted a reanalysis of 4 regional foraging aggregations using Bayesian mixed stock analysis, analysis of molecular variance, and diversity measures. Significant regional population structure between northern and southern foraging aggregations in the Caribbean was detected (phiST = 0.27, P = 0.000) in addition to significant nesting aggregation structure (phiST = 0.87, P = 0.000). Haplotype diversity levels were highest at foraging aggregations located within the confluence of major current systems. These findings indicate that both currents and behavior have strong influences on the composition of foraging aggregations. In addition, our results provide evidence of juvenile homing to regional foraging grounds and highlight the difficulties of separating historical and current effects on recruitment patterns at foraging locations.

Animal Population Groups↗

The combined effects of dietary protein and fat on 7,12-dimethylbenz(a)anthracene-induced breast cancer in rats.

A 3 X 3 factorial experiment was conducted to examine how protein content (8, 16, 32% of kilocalories from casein) and fat content (12, 24, 48% of kilocalories from corn oil) interact to influence 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast carcinogenesis in rats. Forty weanling Sprague-Dawley rats were assigned to each of 9 diets fed ad libitum. After 4 weeks each rat received DMBA (20 mg/kg) via gastric intubation. No substantial statistical interactions of protein and fat were observed on tumor incidence. Increasing dietary corn oil increased the percentage of rats with palpable tumors. Rats fed diets containing 12, 24 and 48% of kilocalories from corn oil showed 35, 49 and 70% tumor prevalence at necropsy, and the total number of tumors per fat level was 65, 81 and 182, respectively. Each doubling of dietary fat concentration approximately doubled the odds of a rat developing a tumor. Multiple tumors were more common with the highest corn oil intake. The odds of finding a second tumor in rats with one tumor increased by a factor of 7.5 when fat kilocalories were increased from 24 to 48% compared to a decrease of one-third when fat kilocalories were increased from 12 to 24%. Dietary corn oil significantly increased the prevalence of adenocarcinomas and adenomas but not fibroadenomas. Dietary protein did not significantly affect tumor prevalence. However, tumors palpated in rats fed 16% of kilocalories as protein regressed more frequently than in rats fed low or high protein diets. Multiple logistic-regression results indicate that, in addition to the response to dietary corn oil, tumorigenesis was increased in rats with greater ad libitum food consumption. This conclusion is supported by reanalysis that used direct rate adjustment and average partial association tests.

9,10-Dimethyl-1,2-benzanthracene↗

Statistical analysis of nitrogen balance data with reference to the lysine requirement in adults.

Statistical analysis of nitrogen balance data is an important approach to the estimation of human nutrient requirement. The usual procedure is to regress nitrogen (N) balance on intake and to define the requirement as intake that would produce zero balance. In the actual application of this methodology, there are a number of options, and in the present study we explore the sensitivity of the regression approach to those options. To put this problem into a realistic context, we examine the current controversy over the lysine requirements of healthy adults. From early N balance studies, investigators concluded that the mean requirement was generally less than 10 mg. kg(-1). d(-1), whereas based on recent (13)C-tracer and metabolic studies, we propose a tentative mean requirement of approximately 30 mg. kg(-1). d(-1). Jones et al. (1956) conducted careful N balance studies from which they derived an estimate of lysine requirement of less than about 8 mg. kg(-1). d(-1). We reanalyzed these data with different choices of modes of analysis, mathematical models, and different assumptions concerning the magnitude of miscellaneous N losses. We find that for these data the choice of a specific mathematical model has only a small effect on resultant estimates of requirement, while estimated requirements are very sensitive to amount of unmeasured losses that are assumed and how the model is applied (whether the aggregate data are fitted in one pass to a single model, or the data for each individual subject are fitted to that individual's unique model). Moreover, our reanalysis suggests that the population requirement for lysine is in the range of 17 to 36 mg. kg(-1). d(-1) and strongly supports a lysine requirement value of about 30 mg. kg(-1). d(-1). In general, our results indicate that whenever possible, N balance data should be analyzed using a square root model fitted to individual data and that the median of the individual requirements so derived be used as the estimate of population requirement. Moreover, clearly any statistical analyses of N balance data should include a sensitivity analysis to determine the influence of underlying assumptions. Finally, the finding that these estimates are highly dependent on the assumed amount of N miscellaneous losses recommends that further studies on these losses and of the factors that influence them are essential.

Adult↗

Fecal occult blood screening in the Minnesota study: sensitivity of the screening test.

BACKGROUND: In the Minnesota Colon Cancer Control Study, which used guaiac slides to annually screen stool samples for blood, mortality from colorectal cancer was reduced by 33.4%. The reported sensitivity of this test for colorectal cancer was about 90%. However, results from another study estimated the sensitivity to be 25%-33%; other investigators have reported intermediate values. Given these contradictions, we examined screening sensitivity for colorectal cancer in the Minnesota study by several direct and indirect methods. METHODS: In this reanalysis of data from the Minnesota study, we distinguished between sensitivity for colorectal cancer of the screening test (composed of six slides) and of the screening program (a series of such tests). We estimated screen sensitivity by adjusting the crude estimate from the final tests in each screening phase for colorectal cancer incidence in 5 years of follow-up, by modeling guaiac slide results at each screen as a function of the presence of occult blood, and by incorporating sensitive detection into a modification of a mathematical model developed by Lang and Ransohoff. Program sensitivity was estimated from the fraction of screen-detected cancers among all cancers diagnosed in screened individuals. RESULTS: The crude estimate of program sensitivity was 89.4%, whereas the modified Lang-Ransohoff model estimates screen sensitivities at 94.1%-96.2%, consistent with the estimates from the other methods. Indirect measures, such as the association between the number of positive slides among the six slides in each set and the positive predictivity for colorectal cancer, are consistent with these estimates. CONCLUSIONS: The Minnesota study reduced mortality from colorectal cancer through use of a screening test with average screen and program sensitivities of about 90%.

Colorectal Neoplasms↗

Stay wet or else: three ways in which plants can adjust hydraulically to their environment.

The literature on whole-plant acclimation to drought is reviewed and it is proposed that leaf-level homeostasis in water status is attained during ontogeny largely thanks to whole-plant changes in physical resistance to liquid water flow caused by morphological and anatomical adjustments. It is shown that, in response to water deficits, plant resistance changes at different levels (tissue, organ, individual), levels that are correlated with the time scale of the response. It was found that such adjustments apparently tend to increase resistance to flow in the short term and to reduce it in the long term. A critical view of those findings is provided based on the principle that drought-induced changes cannot be analysed separately from the allometric changes that take place through ontogeny, as for example proposed by the widely cited hydraulic limitation hypothesis. A graphic synthetic model is presented according to which developmental responses to water deficits operate largely through reductions in whole-plant water transport capacity, combined with more or less strong reductions in leaf area (different "hydraulic allometries"), depending on the intrinsic tolerance of leaf tissues to partial desiccation. The model is used to show that, as the result of such adjustments, the water transport capacity per unit leaf area can decrease, remain constant, or increase, and it is argued that the expected leaf-level response would be different in each case, respectively involving a decreased, constant, or increased potential for transpiration. The article ends with a plea to collect the evidence needed to evaluate the occurrence of these three different response types across taxa and their association with different environments, including the reanalysis of existing data.

Acclimatization↗

Reconstructing the evolutionary history of China: a caveat about inferences drawn from ancient DNA.

The decipherment of the meager information provided by short fragments of ancient mitochondrial DNA (mtDNA) is notoriously difficult but is regarded as a most promising way toward reconstructing the past from the genetic perspective. By haplogroup-specific hypervariable segment (HVS) motif search and matching or near-matching with available modern data sets, most of the ancient mtDNAs can be tentatively assigned to haplogroups, which are often subcontinent specific. Further typing for mtDNA haplogroup-diagnostic coding region polymorphisms, however, is indispensable for establishing the geographic/genetic affinities of ancient samples with less ambiguity. In the present study, we sequenced a fragment (approximately 982 bp) of the mtDNA control region in 76 Han individuals from Taian, Shandong, China, and we combined these data with previously reported samples from Zibo and Qingdao, Shandong. The reanalysis of two previously published ancient mtDNA population data sets from Linzi (same province) then indicates that the ancient populations had features in common with the modern populations from south China rather than any specific affinity to the European mtDNA pool. Our results highlight that ancient mtDNA data obtained under different sampling schemes and subject to potential contamination can easily create the impression of drastic spatiotemporal changes in the genetic structure of a regional population during the past few thousand years if inappropriate methods of data analysis are employed.

Asian People↗

Patterns of protein evolution in Tetrahymena thermophila: implications for estimates of effective population size.

High levels of synonymous substitutions among alleles of the surface antigen SerH led to the hypothesis that Tetrahymena thermophila has a tremendously large effective population size, one that is greater than estimated for many prokaryotes (Lynch, M., and J. S. Conery. 2003. Science 302:1401-1404.). Here we show that SerH is unusual as there are substantially lower levels of synonymous variation at five additional loci (four nuclear and one mitochondrial) characterized from T. thermophila populations. Hence, the effective population size of T. thermophila, a model single-celled eukaryote, is lower and more consistent with estimates from other microbial eukaryotes. Moreover, reanalysis of SerH polymorphism data indicates that this protein evolves through a combination of vertical transmission of alleles and concerted evolution of repeat units within alleles. SerH may be under balancing selection due to a mechanism analogous to the maintenance of antigenic variation in vertebrate immune systems. Finally, the dual nature of ciliate genomes and particularly the amitotic divisions of processed macronuclear genomes may make it difficult to estimate accurately effective population size from synonymous polymorphisms. This is because selection and drift operate on processed chromosomes in macronuclei, where assortment of alleles, disruption of linkage groups, and recombination can alter the genetic landscape relative to more canonical eukaryotic genomes.

Alleles↗

Statistical issues arising from mouse micronucleus assay experiments of Ashby and co-workers.

Data from a series of mouse micronucleus assays have been reanalysed to illustrate various statistical issues raised by Ashby and co-workers during the development of the assay. Most of the statistical points discussed in these earlier papers can be explained by the stochastic nature of the data. Reanalysis shows that the type of data collected in mammalian micronucleus assays is amenable to analysis by standard biometric methods. It is concluded that statistical analysis has an important role in the exploration and interpretation of data from the micronucleus assay.

Analysis of Variance↗

Disturbed latex immunoassays for C-reactive protein and ferritin in a renal transplant patient due to polyclonal IgM hypergammaglobulinaemia.

BACKGROUND: C-reactive protein (CRP) and ferritin serum levels represent routine laboratory parameters in the monitoring of renal failure patients. Analysis of CRP, ferritin and other serum proteins can be performed using latex-enhanced or non-latex-enhanced immunoassays. We report on a renal transplant patient with polyclonal IgM hypergammaglobulinaemia having markedly elevated serum CRP and ferritin levels (as detected by latex-enhanced immunoassays) in the absence of clinical signs of an infectious or malignant disorder. METHODS: CRP and ferritin serum levels were determined with various immunoassays with and without latex enhancement. To characterize the causative agent for the elevated CRP and ferritin values, the patient's and a control serum were fractionated by gel filtration on a Sephacryl S-300 column. Serum fractions were subjected to further analysis for reactivity in CRP and ferritin assays. In addition, patient's serum samples were investigated for reactivity with various other latex-based immunoassays (rheumatoid factor, antistreptolysin O, antistreptococcal DNase B). RESULTS: Using latex-enhanced CRP and ferritin immunoassays, markedly elevated serum levels were obtained (CRP 726 mg/l determined by turbidimetry, 398 mg/l determined by nephelometry; ferritin, 20,000 micrograms/l determined by turbidimetry). In contrast, assays without latex enhancement revealed levels within the normal range for both serum proteins (CRP < 5 mg/l, ferritin 52 micrograms/l). The analysis of the patient's serum by gel filtration revealed an interference of the patient's IgM with latex particles used in the CRP and ferritin assays. CONCLUSION: Our study demonstrates that even polyclonal IgM hypergammaglobulinaemia can disturb a large array of latex-enhanced immunoassays used for routine diagnostic procedures. This is of particular interest for the management of allograft recipients in whom monoclonal and polyclonal gammaglobulinaemia are frequently observed. We therefore recommend reanalysis of the respective plasma proteins by latex-free assays in patients with hypergammaglobinaemia showing no clinical signs of an acute infectious disease or malignant disorder.

C-Reactive Protein↗