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Biomedical subjects

R O Allen

Publications and source records attributed to R O Allen.

13 recordsLinked to original sources
PubMed2001

Forensic determination of ricin and the alkaloid marker ricinine from castor bean extracts.

Liquid chromatography/mass spectrometry (LC/ MS) and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS methods were developed for the presumptive identification of ricin toxin and the alkaloid marker ricinine from crude plant materials. Ricin is an extremely potent poison, which is of forensic interest due to its appearance in terrorism literature and its potential for use as a homicide agent. Difficulties arise in attempting to analyze ricin because it is a large heterogeneous protein with glycosylation. The general protein identification scheme developed uses LC/MS or MALDI-TOF for size classification followed by the use of the same instrumentation for the analysis of the tryptic digest. Fragments of the digest can be searched in an online database for tentative identification of the unknown protein and then followed by comparison to authentic reference materials. LC fractionation or molecular weight cutoff filtration was used for preparation of the intact toxin before analysis. Extracts from two types of castor beans were prepared using a terrorist handbook procedure and determined to contain 1% ricin. Additionally, a forensic sample suspected to contain ricin was analyzed using the presented identification scheme (data not shown). The identification of the alkaloid ricinine by GC/MS and LC/MS was shown to be a complementary technique for the determination of castor bean extracts.

Alkaloids↗
PubMed1998

Analysis of two multiplexed short tandem repeat systems using capillary electrophoresis with multiwavelength fluorescence detection.

A series of experiments was performed to analyze the utility of capillary electrophoresis (CE) with multiwavelength detection capabilities for multiplex typing of short tandem repeat loci. Characteristics of the sieving polymer, hydroxyethylcellulose, which affect resolution of single strand (ss) DNA fragments were examined. Additionally, the effects of denaturant in the polymer system, separation voltage, and analysis temperature were studied to ascertain their effects on DNA separations and capillary lifetime. The use of elevated run temperature (60 degrees C) was found to improve sizing precision, to increase the lifetime of capillaries (100 runs or more per capillary), and to provide runtimes of under 20 min. Finally, 100 individual human DNA samples were typed successfully using CE. The average resolution obtained was 1.4 bases for a 200 base fragment with a standard deviation of sizing of 0.2 bases, allowing all alleles examined to be distinguished clearly.

DNA, Single-Stranded↗
PubMed1996

DNA typing of a polymerase chain reaction amplified D1S80/amelogenin multiplex using capillary electrophoresis and a mixed entangled polymer matrix.

In this study, a technique was developed to separate by capillary electrophoresis (CE) the widely varying DNA fragment sizes produced by a multiplex polymerase chain reaction (PCR) amplification of the loci D1S80 and amelogenin. Experiments were performed to analyze different buffer systems and obtain optimal resolution for the separation. A matrix composed of two different molecular weights of the same polymer was constructed to separate the DNA fragments with baseline resolution, and a cubic spline fit was used to estimate the size of DNA fragments over 350 base pairs. Over 100 samples were examined to demonstrate the rapid, robust and precise characteristics of this CE system. An average relative standard deviation of 0.3% was obtained for the sizing of the D1S80 alleles in these samples. DNA from mixed body fluid samples, samples subjected to environmental insult, and D1S80 sequence variants were also typed successfully. These results demonstrate that CE is a viable method for analysis of D1S80 and amelogenin forensic DNA samples.

Alleles↗
PubMed1995

Application of dual internal standards for precise sizing of polymerase chain reaction products using capillary electrophoresis.

Capillary electrophoresis (CE) is an analytical technique which provides rapid, high resolution analysis of amplified DNA fragments produced by the polymerase chain reaction (PCR). In this study, two internal standards are used as size markers to bracket und precisely size PCR products. The technique is applied to typing PCR products from the short tandem repeat locus HUMTH01. HUMTH01 consists of five to seven major alleles in the size range of 179-203 bp, with each allele four bp apart. Using this genetic marker, a population containing 97 individuals was examined with both polyacrylamide gel electrophoresis and CE. Identical genotypes were obtained with both techniques demonstrating the reliability of CE in DNA typing applications. The DNA analysis took place in sets of 10 with a calibration of the CE being performed between each set of samples. For the 97 samples examined, the pooled standard deviation was 0.3 bp. The observed genotype frequencies determined from the sample set did not deviate significantly from Hardy-Weinberg expectations. From these CE results, we conclude that HUMTH01 PCR products can be accurately and precisely sized by capillary electrophoresis using the method described.

Base Sequence↗
PubMed1994

Quantitation of polymerase chain reaction products by capillary electrophoresis using laser fluorescence.

In samples where the amount of DNA is limited, the polymerase chain reaction (PCR) can amplify specific regions of the DNA. A quantitative analysis of the PCR product would be desirable to ensure sufficient DNA is available for analysis. In this study, we examine the use of capillary electrophoresis (CE) with laser fluorescence detection for quantitation of PCR products. A coated open tubular capillary was used with a non-gel sieving buffer and a fluorescent intercalating dye to obtain results within 20 minutes. Using an internal standard, peak migration time was below 0.1% relative standard deviation (R.S.D.) with a peak area precision of 3% R.S.D. In comparison to quantitation by hybridization, (i.e., slot blot) and spectrophotometric analysis, capillary electrophoresis shows distinct advantages due to its ability to separate unincorporated primers and PCR byproducts from the targeted PCR product. The results demonstrate that CE can be used to monitor the quality and quantity of the PCR product.

DNA, Viral↗
PubMed1994

Rapid analysis of the short tandem repeat HUMTH01 by capillary electrophoresis.

Using capillary electrophoresis, we demonstrate separation and analysis of the short tandem repeat HUMTH01 in under 10 min with 3 bp resolution. Separation of the PCR products, which range in size from 179 to 203 bp, is achieved using hydroxyethyl cellulose as the separation medium and a novel single-step voltage gradient. Internal standards on either side of the alleles are used to size the PCR products with an average standard deviation of 0.5 bp. DNA typing patterns obtained with this system are compared to samples separated by polyacrylamide slab gel electrophoresis.

Cellulose↗
PubMedDate not supplied

A mass spectrometric method for quantitation of intact insulin in blood samples.

An analytical method was developed for the quantitation of intact insulin in blood samples. Solid-phase extraction (SPE) was used to purify and concentrate the protein after the plasma is separated. Analysis is performed by electrospray liquid chromatography-mass spectrometry (LC-MS) using a trifluoroacetic acid mobile phase. The limit of quantitation of the SPE LC-MS method has been determined to be 1.0 ng/mL for endogenous levels of insulin. Base levels of human insulin in plasma have been quantitated, and values ranging from 1.0 to 1.4 ng/mL were observed. In a single analysis, the method can determine human, porcine, and bovine insulin. Reproducibility was tested for both blood samples and aqueous standards and produced relative standard deviations of approximately 10% and lower. Calibration curves were constructed corresponding to plasma levels of 0.4 to 80 ng/mL and found to be linear with R2 values greater than 0.99. Stability studies of human and porcine insulin were performed over a period of 21 days for whole human blood samples stored at both room temperature and 4 degrees C. Hemolyzed blood samples were also analyzed using the developed method and were found to produce quantitatable levels of insulin. The advantage of the application of SPE and LC-MS for the quantitation of insulin is the high specificity compared to other techniques such as radioimmunoassay (RIA). In addition, the developed LC-MS method is not subject to interferences that cause problems with RIA, such as hemolysis. The method is efficient and rapid and produces results more specific than those obtained with RIA.

Animals↗
PubMedDate not supplied

EtO personnel monitoring devices: the state of the art.

Results from a single monitor worn by an employee become a part of his record of previous EtO exposure and a measure of the conditions in the central sterile supply area. It is obviously important that these results be accurate. Random errors in the results of analytical measurements are allowed for in the NIOSH criteria for overall accuracy of a technique. The sensitivity and accuracy of devices used to monitor TWA personnel exposure to EtO are only as good as the collection efficiency of the technique and the analytical instrumentation it utilizes. The active sampling charcoal absorption tube, impinger, and Tedlar bag methods are common air quality monitoring tools within the occupational health field. In the hands of experienced industrial hygienists and analytical chemists, these devices may be used with accurate EtO collection and analytical results. Passive sampling diffusion monitoring devices obtain the same results through simplicity of use and ease of providing analysis. However, the user must be careful to follow the manufacturer's instructions exactly if the results are to be accurate and meaningful. With these facts in mind, and in light of recent studies showing continued improvements in the technique's precision, it is likely that most hospitals will turn to the diffusion monitor methods as their primary means of documenting compliance with the new OSHA monitoring standard.

Ethylene Oxide↗
PubMedDate not supplied

An evaluation of the accuracy and reliability of ethylene oxide diffusion badge monitors.

Ethylene oxide (EO) gas is widely used in hospitals to sterilize certain moisture- and heat-sensitive materials. Based on scientific studies indicating its potential as a human carcinogen and mutagen, and the possible genotoxic, reproductive, neurologic, and sensitization hazards associated with EO exposure, the Occupational Safety and Health Administration has recently lowered the permissible exposure limit (PEL) from 50 ppm to 1 ppm as an 8-hour time-weighted average (TWA). This standard also established an "action level" of 0.5 ppm for an 8-hour TWA, below which employers are exempted from such requirements as periodic employee exposure monitoring or medical surveillance. These much lower concentrations of EO in air now place greater demands upon the analytical techniques used to monitor exposure of hospital personnel to EO. In this study, the capabilities of five EO diffusion monitors were examined in the TWA concentration range of 0.25-3.7 ppm. Both accuracy and precision were tested by exposing these devices simultaneously to measured concentrations of EO in a stainless steel exposure chamber. Temperature and humidity conditions were controlled, as was the flow rate of the gases across the sampling areas of the diffusion monitors. All of the monitors tested were sensitive enough to measure EO at the new PEL level, but in this laboratory investigation only one type of monitoring badge was able to meet the National Institute for Occupational Safety and Health criteria of +/- 25% overall system accuracy at the 95% confidence level in the exposure range of 0.5-2.0 X the OSHA PEL.

Chromatography, Gas↗