Delayed ethanol analysis of breath specimens: long-term field experience with commercial silica gel tubes and breathalyzer collection

Delayed ethanol analysis was performed on breath specimens collected with commercial silica gel tubes using multiple Breathalyzer instruments. Eleven hundred and nine results were obtained from an ethanol testing program over a five-year period. Only 2.5% of the specimens had apparent collection errors. For the valid specimens, the most frequent result was 0.11 g/210 L and the mean result was 0.14 g/210 L. For 642 specimens, delayed results were compared with direct results. Direct results were greater than delayed results for 55%, less than for 27%, and equal to for 18% of the pairs. When fixed tolerance limits of +/- 0.03 were used, 81% of the direct results were confirmed. The confirmation percentage was best in the critical range of direct results, 0.05 to 0.15 g/210 L. The collection tubes showed no substantial variability in retaining ethanol during storage and releasing ethanol for analysis.     

采血管中添加剂对血样中乙醇含量的影响

目的：探讨不同种类采血管对血样中乙醇含量检测结果的影响。方法分别用7种一次性真空采血管[无抗凝剂管、促凝剂管、分离胶-促凝剂管、枸橼酸钠（1∶4）管、枸橼酸钠（1∶9）管、柠檬酸钠（9∶1）管、EDTA-K2管]采集10名志愿者饮酒后2 h血液,用顶空气相色谱法检测血样中乙醇含量。结果相同血样用不同的采血管,其乙醇含量检测结果不同,依次为分离胶-促凝剂管〉促凝剂管〉无抗凝剂管〉EDTA-K2管〉枸橼酸钠（1∶9）管〉枸橼酸钠（1∶4）管,柠檬酸钠（9∶1）管与枸橼酸钠（1∶9）管检测结果基本一致。结论采集涉嫌酒后驾驶的驾驶员血样,应选用一次性真空采血管,首选EDTA-K2管。     

The tendency of individuals to transfer DNA to handled items

This research investigates factors influencing the transfer of DNA to handled objects and the process known as 'shedding'. Volunteers were recruited to hold sterile plastic tubes using experiments originally designed by Lowe et al. [A. Lowe, C. Murray, J. Whitaker, G. Tully, P. Gill, The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces, Forensic Sci. Int. 129 (2002) 25-34]. Transferred cellular material was collected from the tubes and STR profiles generated using the AmpFlSTR SGM Plus multiplex with 28 and 34 PCR cycles. Volunteers were asked to hold the tubes with each hand, and to participate in a series of handwashing experiments. The DNA profiling results obtained from the transferred skin cells were compared. An attempt was made to characterize the volunteers as 'good' or 'bad' shedders and to establish which, if any, of the experimental variables were associated with 'good' shedding. Our results suggest that many factors significantly influence shedding, including which hand an individual touches an item with and the time that has elapsed since they last washed their hands. We have found that it may be more complicated than previously reported to categorise a person as being either a 'good' or a 'bad' shedder and that if truly 'good' shedders exist they may be significantly rarer than some have estimated. In the current research no 'good' shedders were observed in a group of 60 volunteers. Given these results, it seems that rather than being applied to individual forensic cases, knowledge of shedding characteristics will be most useful in providing general background data for the interpretation of trace DNA evidence.     

Ethanol stability in unpreserved refrigerated antemortem blood

Ethanol stability in preserved antemortem blood has been widely studied since it is a common practice in cases involving suspected impaired driving to collect antemortem blood in evacuated blood tubes containing sodium fluoride. In some situations, antemortem blood is submitted to a forensic laboratory for ethanol analysis in evacuated blood tubes that contain only an anticoagulant. There has been limited research on ethanol stability in antemortem blood stored without a preservative. On two occasions, antemortem blood was collected from five ethanol-free individuals into 6-ml Vacutainer® tubes containing only 10.8 mg potassium EDTA. The blood tubes were spiked with ethanol to approximately either 0.08 or 0.15 g/dl. Dual-FID headspace gas chromatography was used to analyze 58 blood tubes, 29 from each session, for ethanol 1 day after sample collection and again after 1 year of refrigerated storage (~4°C). Statistically significant decreases in ethanol were detected at the 0.05 level of significance. Mean decreases in ethanol after 1 year of storage for the 0.08 and 0.15 g/dl samples were 0.013 and 0.010 g/dl, respectively. The mean ethanol decrease across all tubes was 0.012 g/dl. The range of decreases for the 58 blood tubes was 0.003–0.018 g/dl. The mean ethanol decreases measured in this unpreserved antemortem blood are comparable in magnitude to those previously observed in antemortem blood containing sodium fluoride after 1 year of refrigerated storage. Ethanol did not increase in the antemortem blood samples despite the absence of sodium fluoride.     

The application of ultraviolet irradiation to exogenous sources of DNA in plasticware and water for the amplification of low copy number DNA

Using high sensitivity forensic STR polymerase chain reaction (PCR) typing procedures, we have found low concentrations of DNA contamination in plasticware and water assumed to be sterile, which is not detected by standard DNA procedures. One technique commonly used to eliminate the presence of DNA is ultraviolet (UV) irradiation; we optimized such a protocol used in the treatment of water, tubes, plates, and tips for low copy number DNA (LCN) amplification. UV light from a Stratalinker((R)) 2400 was administered to 0.2, 1.5 mL tubes, and PCR plates contaminated with up to 500 pg of DNA. They were subsequently quantified with an ALU-based real-time PCR method using the Rotorgene 3000. Overall, there was a decrease in concentration of DNA recovered as the duration of treatment increased. Nonetheless, following 45 min of irradiating a PCR plate with 500 pg of DNA, nearly 6 pg were still detected. However, when the plate was raised within an inch of the UV source, less than 0.2 pg of DNA was detected. Additionally, lining the area around the samples with aluminum foil further reduced the amount of time necessary for irradiation, as only 30 min eliminated the presence DNA in the raised PCR plate. Similar experiments were conducted using tubes filled with a solution of DNA and water in equivalent concentrations for 50, 15, and 1.5 mL tubes with comparative results. It is plausible that the aluminum foil increased the amount of reflection in the area thereby enhancing penetration of UV rays through the walls of the plasticware. This protocol was tested for the possibility of inhibitors produced from irradiation of plastic tubes. As our protocols require less irradiation time than previous studies, PCR sensitivity was not affected. Moreover, the lifespan of the UV lamps was extended. Our findings demonstrate that this method is useful as an additional precautionary measure to prevent amplification of extraneous DNA from plasticware and water without compromising the sensitivity of LCN DNA amplifications.     

豚鼠过敏性休克类胰蛋白酶活力测定

目的探索过敏性休克法医学客观诊断标准。方法建立豚鼠异种血清过敏性休克模型,采用专性底物对模型鼠的血清,肺,气管类胰蛋白酶活力进行测定。结果过敏性休克豚鼠血清,肺,气管类胰蛋白酶活力均增加,且三者增加的程度是平行的。结论过敏性休克时,血清,肺,气管类胰蛋白酶活力增加可作为法医检案客观诊断标准。     

The killer outfit and timing: Impact of the fabric and time in body fluid identification and DNA profiling

The present work aimed to study the detection, through lateral flow immunochromatographic (LFI) tests, of saliva samples over time in three different types of fabrics, as well as, the possibility of DNA isolation and characterization from the sample tubes and the cassettes. Fifty microliters of saliva (three samples/time) were deposited in denim, cotton, and polyester. Saliva was identified by SERATEC® Amylase Test and the Crime Scene version SALIVA CS, being able to detect it up to six months of deposition, although with different band intensities. Polyester showed stronger bands than cotton, probably due to its synthetic nature, and denim, as an inked fabric, showed less band intensities. Statistical analyses confirmed significant differences among fabrics, but not over time in the same type of fabric. Total DNA from the sample tubes was successfully recovered, in contrast, from the cassettes, only polyester retrieved amplifiable DNA. These findings indicated that it is possible to recover and identify saliva up to six months after deposition, also obtaining DNA. Future research will be able to expand these results, analyzing the stability of other body fluids, and the sensitivity of lateral flow immunochromatographic tests to detect them.     

Studies on the stability and detection of cocaine, benzoylecgonine, and 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid in whole blood using Abuscreen radioimmunoassay

A study was undertaken to assess the stability and the radioimmunoassay (RIA) detection of cocaine, benzoylecgonine (BZE), and 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in whole blood while stored in 4 different kinds of blood collection tubes for up to 30 days at refrigeration and room temperatures. At various intervals, the tubes were sampled and analyzed using Abuscreen RIA. Also, semi-quantitative data derived from RIA analysis of forensic blood specimens were compared with quantitative data acquired using gas chromatography (GC) or GC/mass spectrometry (GC/MS) on the same specimens. RIA and chromatographic studies revealed that BZE and THC-COOH were stable in blood under all conditions studied. Cocaine, however, was found not to be stable in blood, especially when stored at room temperatures. Despite cocaine's instability in blood, RIA was able to detect the presence of cocaine and its breakdown products in blood under all conditions studied.     

A light stabilizer Tinuvin 770-induced toxic injury of adult rat cardiac myocytes

Tinuvin 770/bis(2,2,6,6-tetramethyl-4-piperidinyl)sebacate is a worldwide used light stabilizer for plastic materials like polyolefins. Tinuvin 770 is a biologically active component of polypropylene tubes. Glossmann and his study group managed to extract this compound by aqueous or organic solvents from laboratory plastic tubes, and propose that Tinuvin 770 is a potent blocker of L-type Ca(2+)-channel through the phenylalkylamine and benzothiazepine-selective drug binding domains of the alpha(1) subunit of the receptor [Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 9523].We examined the direct morphological effect of Tinuvin 770 in give 25nmol, 0, 30, 60, 120 minute exposure time in isolated cardiomyocytes from adult rats. Incubation of myocytes with Tinuvin resulted in a progressive decline of rod-shaped and viable cells. It was accompanied by an increase in number of hypercontracted myocytes with microbleb formation compared to control and depletion of ATP level.In summary, our results demonstrate that plasma membrane damage and hypercontraction are manifestations of Tinuvin-induced injury of isolated cardiomyocytes.     

An improved density gradient system for forensic science soil studies.

The construction and use of the apparatus required for an improved density gradient system is described. The system is a combination of existing procedures, resultin in a system more suited for forensic science soil studies. The advantages consist of rapid, simultaneous filling of several tubes with identical density gradients and of the capability to remove individual fractions from the tubes for further study. Disadvantages are that an extra quantity of liquids is required and only a preset number of tubes can be filled at one time.     

Complications following insertion and replacement of percutaneous endoscopic gastrostomy (PEG) tubes

Percutaneous endoscopic gastrostomy (PEG) tube insertion was introduced in 1980 as an alternative to nasogastric tubes and surgically placed gastrostomy tubes. The procedure is indicated in those patients who have an inability to sustain adequate nutrition in the presence of a functioning gastrointestinal tract. We report four deaths that arose within a ten-week period in 1998.     

HPLC analysis of ballpoint pen inks stored at different light conditions

A method for comparison of ink entries on documents stored in different light conditions is presented. Various blue inks were exposed to light, both daylight and artificial light from fluorescent tubes. Inks were then extracted from the document and analyzed by HPLC (high performance liquid chromatography). Significant changes in composition were noted on exposure to light. These changes were followed by using ternary diagrams constructed for dyes generally present in blue-colored inks--Crystal Violet, Methyl Violet, and Tetramethyl Para Rosaniline. Also, the amount of the various compounds formed by decomposition of these dyes on exposure to light was measured and employed for comparison of inks. An example of the use of the proposed method in casework is given.     

STR复合扩增技术在亲子鉴定中的应用

采用12个荧光标记STR位点复合扩增,进行双亲亲子鉴定和单亲亲子鉴定,取得满意效果.     

Multiple homicides as a result of chloroform poisoning: case report and experimental study.

Two cases (involving five murder victims) of multiple homicide by inhalational chloroform intoxication are reported. In the discussion of the findings the valence of toxicological analyses is underlined with regard to the possibility of forcible external suffocation due to occlusion of the respiratory orifices by means of a chloroform-soaked soft covering. In addition storage experiments were performed at +4, +20 and -20 degrees C with cadaver blood mixed with chloroform. The optimal solution for avoiding volatile losses was stored in glass tubes with ground glass stoppers. In cases of unclear death in which involvement of volatile substances is suspected it is, therefore, advisable to preserve an additional blood sample at -20 degrees C in glass tubes that are only opened for the analysis of volatile substances.     

Stability of Morphine,Codeine, and 6‐Acetylmorphine in Blood at Different Sampling and Storage Conditions

The stability of drugs in biological specimens is a major concern during the evaluation of the toxicological results. The stability of morphine, codeine, and 6‐acetyl‐morphine in blood was studied after different sampling conditions: (i) in glass, polypropylene or polystyrene tubes, (ii) with addition of dipotassium ethylene diamine tetraacetic acid (K₂EDTA) or sodium oxalate (Na₂C₂O₄), and (iii) with or without the addition of sodium fluoride (NaF). Spiked blood samples were stored at two different temperatures (4 and ?20^°C), analyzed after different storage times and after three freeze–thaw cycles. Opiate concentrations were decreased in all conditions, but the most unstable was 6‐acetyl‐morphine. The addition of NaF as preservative improved the stability of opiates at all conditions studied, whereas the type of anticoagulant did not affect the stability of opiates. It was concluded that blood samples should be stored at ?20^°C in glass tubes containing oxalate and NaF for maximum stability.     

Results from the NIST 2004 DNA Quantitation Study

For optimal DNA short tandem repeat (STR) typing results, the DNA concentration ([DNA]) of the sample must be accurately determined prior to the polymerase chain reaction (PCR) amplification step in the typing process. In early 2004, the National Institute of Standards and Technology (NIST) conducted an interlaboratory study to help assess the accuracy of DNA quantitation in forensic DNA laboratories. This study was designed with four primary purposes: (1) to examine concentration effects and to probe performance at the lower DNA concentration levels that are frequently seen in forensic casework; (2) to examine consistency with various methodologies across multiple laboratories; (3) to examine single versus multiple source samples; and (4) to study DNA stability over time and through shipping in two types of storage tubes. Eight DNA samples of [DNA] from 0.05 ng/microL to 1.5 ng/microL were distributed. A total of 287 independent data sets were returned from 80 participants. Results were reported for 19 different DNA quantitation methodologies. Approximately 65% of the data were obtained using traditional slot blot hybridization methods; 21% were obtained using newly available quantitative real-time PCR (Q-PCR) techniques. Information from this interlaboratory study is guiding development of a future NIST Standard Reference Material for Human DNA Quantitation, SRM 2372.     

Evaluation of breath-alcohol instruments. IV. Roadside tests with Alcolmeter pocket model

This paper reports results from a field trial with a breath-alcohol screening device--Alcolmeter pocket model. Breath tests were made with drivers apprehended during routine controls (road-blocks), for traffic violations and those involved in traffic accidents. Of 908 roadside breath tests made with chemical reagent tubes, 343 showed zero alcohol (no colour change) and these results were confirmed by Alcolmeter. Alcohol was detected in 191 tests but the level was judged as being below the legal limit of 0.50 mg/ml. The Alcolmeter results, however, ranged from 0 to 1.22 mg/ml (mean 0.21 mg/ml) and 15 individuals (7.8%) were above the legal limit. There were 373 positive chemical tube breath screening tests whereas in 5 cases (1.3%) Alcolmeter indicated a blood-alcohol level below 0.50 mg/ml. Duplicate determinations with the Alcolmeter device were highly correlated r = 0.93 +/- 0.02 (+/- S.E.), P less than 0.001. The standard deviation of a single breath-alcohol analysis under field conditions was +/- 0.10 mg/ml which corresponds to a coefficient of variation of 10%. The time interval between positive roadside breath test and blood-sampling ranged from 5 to 220 min (median 62 min). The results were therefore adjusted by 0.15 mg/ml per hour to compensate for ethanol metabolised between the time of sampling blood and breath. The corrected blood and breath values were well correlated r = 0.84 +/- 0.03, P less than 0.001 but the predictive power of the regression relationship was poor. The regression equation was y = 0.27 +/- 0.65x and the standard error estimate was +/- 0.21 mg/ml at the mean concentration of ethanol of 1.0 mg/ml.     

Tenax GR吸附解析技术在火场汽油残留物提取检验中的应用研究

目的针对火灾现场常规提取检验方法操作复杂、容易导致汽油残留物有效成分丢失等问题,研究建立采用Tenax GR吸附解析技术简便、高效提取检验汽油残留物的方法。方法将Tenax GR吸附管放入相关检材中,密封后在60℃烘箱中加热1h,经热脱附仪解析后使用气质联用仪分析。结果Tenax GR吸附解析技术可以检验汽油残留物中特征组分,主要包括甲苯、二甲基苯、三甲基苯、四甲基苯、萘、甲基萘及茚满类化合物。该技术的检测限为0.25pL/mL,同时放入6根吸附管做稳定性实验,其相对标准偏差(RSD)为2.43%。该技术进行7、15、30、60、90d等不同阶段的保存实验,其相对偏差分别为6.3%、14.4%、8.7%、18.3%、11.6%。结论该技术操作方便、灵敏度高、稳定性好,适用于火灾现场中汽油残留物的提取检验。     

5个STR基因座的遗传多态性及其法医学应用

目的获得D11S4951、D11S4957、GATA193H05、D2S2951、D6S2421基因座的群体遗传学数据,并分析其在法医学中的应用价值。方法随机抽取成都地区汉族群体无血缘关系个体的静脉血,EDTA抗凝,用Chelex-100法提取DNA,应用PCR技术,扩增上述5个短串联重复序列基因座,聚丙烯酰胺凝胶垂直板电泳分型。结果5个基因座在中国成都汉族人群中分别发现了7、10、8、6、8个等位基因,5个基因座的基因型分布符合Hardy-Weinberg平衡(P>0.05)。各基因座的杂合度分别为0.743、0.772、0.833、0.650和0.800;非父排除概率分别为0.497、0.549、0.662、0.356和0.599;个人识别几率分别为0.863、0.912、0.947、0.829和0.931。结论5个基因座在中国汉族群体中具有法医学应用价值。     

AutoMate Express™ forensic DNA extraction system for the extraction of genomic DNA from biological samples

Abstract: The AutoMate Express? Forensic DNA Extraction System was developed for automatic isolation of DNA from a variety of forensic biological samples. The performance of the system was investigated using a wide range of biological samples. Depending on the sample type, either PrepFiler? lysis buffer or PrepFiler BTA? lysis buffer was used to lyse the samples. After lysis and removal of the substrate using LySep? column, the lysate in the sample tubes were loaded onto AutoMate Express? instrument and DNA was extracted using one of the two instrument extraction protocols. Our study showed that DNA was recovered from as little as 0.025 μL of blood. DNA extracted from casework‐type samples was free of detectable PCR inhibitors and the short tandem repeat profiles were complete, conclusive, and devoid of any PCR artifacts. The system also showed consistent performance from day‐to‐day operation.