Dual examinations for identification of urine as being of human origin and for DNA-typing from small stains of human urine

Concurrent methods for identification of urine as being of human origin, and for DNA-typing from small stains of human urine were examined. A urine stain was extracted with phosphate-buffered saline (PBS), and the extract was filtered using a Centricon-100 device. The filtrate was subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) for identification of human urine and a DNA-typing sample was obtained by dialfiltration of the residue using a DNA purification kit. After the purified residue was treated with an AmpflSTR Profiler PCR amplification kit, the DNA-types were analyzed by capillary electrophoresis using a Genetic Analyzer. It was possible to identify a urine stain as being of human origin, and complete DNA profiles could be successfully obtained from a urine stain which had been created by 50 microL of female urine. Serial analyses of urine stains found at a crime scene provide effective information for forensic investigation. This method is recommended for stain identification and for DNA-typing from a urine stain.     

尿液及尿斑DNA分型的研究

目的研究尿液及尿斑的DNA提取及其检验。方法用Chelex100法及QIAampMiniKit提取尿液及尿斑样本中的DNA,进行PCR扩增及STR检验。结果新鲜的及存放时间在12h以内的尿液样本能得到较好的分型结果;存放2d左右的尿液样本有50%能检出基因型;存放7d及更长时间的尿液样本全部不能检出基因型;尿斑样本的分型成功率很低。结论较新鲜的尿液样本均能进行DNA分型,在法医检案中具有应用价值。     

Successful DNA typing of urine stains using a DNA purification kit following dialfiltration

To evaluate the utility of DNA polymorphism typing of urine stains in forensic investigations, the amplifiable amount of DNA was estimated in 20 urine specimens obtained from 10 male and 10 female volunteers using a DNA purification kit following dialfiltration. DNA obtained from both urine and urine stains was amplified with the AmpflSTR Profiler PCR Amplification Kit, and was analyzed by capillary electrophoresis using the Genetic Analyzer. The amount of male and female urine necessary for obtaining a complete DNA profile was 0.2 mL and 0.08 mL, respectively. When 0.2 mL of male urine were used to create urine stains, complete DNA profiles could be obtained from just some of the stains. However, when only 0.1 mL of female urine was used, complete profiles could be successfully obtained from all of the stains. DNA on bleached cotton remained amplifiable for 3-6 weeks. This method using a DNA purification kit following dialfiltration can be recommended for the genotyping of urine stains.     

Detection of drugs of abuse in simultaneously collected oral fluid, urine and blood from Norwegian drug drivers

Blood and urine samples are collected when the Norwegian police apprehend a person suspected of driving under the influence of drugs other than alcohol. Impairment is judged from the findings in blood. In our routine samples, urine is analysed if morphine is detected in blood to differentiate between ingestion of heroin, morphine or codeine and also in cases where the amount of blood is too low to perform both screening and quantification analysis. In several cases, the collection of urine might be time consuming and challenging. The aim of this study was to investigate if drugs detected in blood were found in oral fluid and if interpretation of opiate findings in oral fluid is as conclusive as in urine. Blood, urine and oral fluid samples were collected from 100 drivers suspected of drugged driving. Oral fluid and blood were screened using LC-MS/MS methods and urine by immunological methods. Positive findings in blood and urine were confirmed with chromatographic methods. The analytical method for oral fluid included 25 of the most commonly abused drugs in Norway and some metabolites. The analysis showed a good correlation between the findings in urine and oral fluid for amphetamines, cocaine/benzoylecgonine, methadone, opiates, zopiclone and benzodiazepines including the 7-amino-benzodiazepines. Cocaine and the heroin marker 6-monoacetylmorphine (6-MAM) were more frequently detected in oral fluid than in urine. Drug concentrations above the cut-off values were found in both samples of oral fluid and urine in 15 of 22 cases positive for morphine, in 18 of 20 cases positive for codeine and in 19 of 26 cases positive for 6-MAM. The use of cannabis was confirmed by detecting THC in oral fluid and THC-COOH in urine. In 34 of 46 cases the use of cannabis was confirmed both in oral fluid and urine. The use of cannabis was confirmed by a positive finding in only urine in 11 cases and in only oral fluid in one case. All the drug groups detected in blood were also found in oral fluid. Since all relevant drugs detected in blood were possible to find in oral fluid and the interpretation of the opiate findings in oral fluid was more conclusive than in urine, oral fluid might replace urine in driving under the influence cases. The fast and easy sampling is time saving and less intrusive for the drivers.     

Use of the "SMITEST" PSA card to identify the presence of prostate-specific antigen in semen and male urine

To determine whether the prostate-specific antigen (PSA) could be identified in semen using the "SMITEST" PSA immunochromatographic membrane test card, we examined semen and other body fluids, including urine. Although PSA activity was detected in semen with high sensitivity using the "SMITEST" PSA card, it was also detected in adult male urine. However, the lower detectable limit in the urine was 1000-fold lower than that in semen. The concentration of PSA in adult male urine was found to be 800 ng/ml using the card. PSA activity usually can be detected in urine of individuals over 14 years old and it has been detected in urine from children as young as 11 years old. Therefore, the appearance of PSA in urine may occur anytime between the age of 12 and 14 years. To determine the stability of PSA activity in urine, dried samples of urine on filter paper were kept at room temperature for up to 3 years. Although the immunoreactive line showing PSA activity became weak after storage, it was still detectable, but faint, after 3 years. In addition, PSA activity was not detected in male serum or saliva and in the urine from human females, male cats or male dogs using the PSA card. We conclude that the PSA card is useful for identification of PSA in both semen and adult male urine.     

血液、尿液中氯胺酮及其代谢物去甲氯胺酮的HPLC分析

目的 建立血液、尿液中氯胺酮及其代谢物去甲氯胺酮的高效液相色谱(HPLC)分析方法.方法 以非那西丁为内标,检材加入10%的氢氧化钠溶液调节pH值为14,用甲苯提取,离心后取有机层,水浴下吹干,乙腈定容后进HPLC仪分析.结果 检测血液中氯胺酮和去甲氯胺酮的线性范围均是0.05～10μg/mL(r2＞0.999 3),检测尿液中氯胺酮和去甲氯胺酮的线性范围均是0.01～50 μg/mL(r2＞0.999 5).氯胺酮和去甲氯胺酮在血液和尿液中的检测限分别是0.006 μg/mL和0.003 μg/mL.血液和尿液中氯胺酮和去甲氯胺酮的回收率不低于82.4%.检测血液和尿液中氯胺酮和去甲氯胺酮的日内精密度和日间精密度均小于10.0%.将所建的方法应用于给大鼠氯胺酮后的血液和尿液中的氯胺酮和去甲氯胺酮的测定,得到了氯胺酮和去甲氯胺酮在大鼠的药时曲线和尿排药速率曲线. 结论本方法简便、快捷,适用于血液、尿液中氯胺酮及其代谢物去甲氯胺酮的分析.     

Development of a radial gel diffusion technique for the identification of urea in urine stains

A radial gel diffusion method utilizing urease and bromthymol blue has been developed for urine stain identification. Urea, present in urine in relatively high concentrations, can be detected from urine stain extracts. This technique provides both qualitative and quantitative results, and is sensitive enough to detect 0.078 micrograms/microliter of urea.     

Detection of haptoglobin from concentrated urine samples by enzyme immunoassay

The detection of haptoglobin (Hp) from serum and bloodstains is utilized extensively in forensic science laboratories in order to include or exclude possible donors. There is an increasing need to make the same discriminations utilizing genetic markers from urine samples. This paper describes the use of enzyme immunoassay and Western blotting (electrophoretic) techniques to determine Hp phenotypes from concentrated urine samples. Serum and urine specimens were collected from volunteer donors. The serum sample from each donor was typed for Hp. The urine specimens were concentrated 3000-fold from the starting volume of 15 mL to a final volume of 5 microL and applied to the gradient polyacrylamide gels. This procedure allows the separation of Hp samples into the three common phenotypes as well as the other rare variants found in humans. The Western blotting electrophoretic technique was used to achieve the transfer of Hp bands from the gels to the nitrocellulose membranes. Enzyme immunoassay with goat anti-Hp antiserum and rabbit anti-goat immunoglobulin alkaline phosphatase conjugate were used to identify the Hp bands from the concentrated samples. Specimens stored for six months at -22 degrees C were also concentrated and typed successfully. Recent implementation of drug-screening policies has resulted in an increase in the submission of substituted urine specimens. The above procedure can be used to detect an additional genetic marker from urine samples and thus facilitate the identity of the donor.     

DNA typing: an accessory evidence in doping control

A clear positive case for anabolic steroids doping was confounded by alleged urine tampering during doping control procedures. Review of the chain of custody showed no flaws, but nevertheless the athlete was adamant that the urine sample should be analyzed for DNA in order to support her contention that she was not the donor of the sample. The results obtained showed that the urine sample that scored positive for steroids contained nuclear DNA that could not be matched to the DNA obtained from the athlete's blood. On the other hand, the same urine sample contained mitochondrial DNA whose nucleotide sequences spanning the hyper variable regions HV1 and HV2 proved to be identical to those determined in mitochondrial DNA amplified from the athlete's blood. The occurrence of an extraneous genotype is compatible with exogenous nuclear DNA admixture to the athlete's urine. Alternatively, taking in consideration the mitochondrial DNA, we could not exclude that a sibling or a maternal relative of the athlete could have acted as a donor of the urine utilized for doping control and DNA analysis. Both situations point to possible tampering of the urine by the athlete. Adjudication at CAS maintained previous national and international federation decision that there was no proof of a chain of custody flaw to justify the athlete's allegation of urine substitution after collection.     

PCR-STR分型技术在尿样DNA分型中的应用

目的　对尿样的DNA分型进行研究。　方法　 10份尿样随机收集于无关个体 ,同时采集血液样本做DNA分型对照 ,用PCR -STR分型技术对尿样DNA进行FIBRA和D18S5 35基因座分型。　结果　 10份尿样 10ml、1ml及 0 .2ml体积均获得准确的分型结果 ,且与同一个体的血样DNA分型结果完全相同 ;室温储存 4天及 4℃保存 4周的尿样均分型成功。　结论PCR -STR分型技术对尿样DNA分型是一种有效的方法 ,在尿样的个人识别中具有极高的实用价值。     

Blood alcohol concentration determined from urine samples as a practical equivalent or alternative to blood and breath alcohol tests

The value of urine tests for determining an equivalent blood alcohol concentration in driving under the influence (DUI) enforcement cases is reviewed from a historical, theoretical, and practical perspective. The limits of precision and accuracy that can be ascribed to urine alcohol results are demonstrated through an evaluation of actual case results wherein both a first void and a subsequent urine sample were analyzed and converted to an equivalent blood alcohol concentration (BAC) using a urine to blood conversion factor of 1.3:1.     

A 6-year experience with urine drug testing by family service agencies in Nova Scotia, Canada

The objective of this study is to describe a urine drug-testing program implemented for parents with a history of substance abuse by family service agencies in the province of Nova Scotia, Canada. Nurse collectors went to the parents' home to obtain urine specimens under direct observation and then delivered the specimens to the toxicology laboratory or arranged shipment by courier under chain of custody. Each urine specimen was screened for cannabinoids, cocaine metabolite, opiates, amphetamines and benzodiazepines, ethyl alcohol and creatinine. All positive screening tests were confirmed by another method such as gas chromatography-mass spectrometry (GC-MS). In 15,979 urine specimens collected from 1994 to 1999, the percent positive rate for one (or more) drugs/metabolites ranged from 45.6% (1994-1996) to 30.0% (1998, 1999). A total of 575 specimens (3.7%) were dilute (urine creatinine <25mg/dl). Positive rates in 15,404 non-dilute specimens from 1994 to 1999 were as follows: cannabinoids - 11.7%, benzodiazepines - 11.3%, cocaine metabolite - 3.7%, and ethyl alcohol - 2.6%. Most clients provided less than 20 urine specimens for testing but some individuals submitted urine specimens more than 100 times in a 12-15-month period. Urine drug screening in parents with a history of substance abuse provided an objective and reliable indication of recent drug use in this population.     

The analysis of the methylenedioxy derivatives of amphetamine]

Methods for analysis of narcotics belonging to amphetamine methylene dioxy derivatives (MDD) are reviewed. The characteristics of these agents, their metabolism, and methods used for their detection and identification (TLC, GC, HPLC, GC/MS) are described. Methods for their extraction from biological objects (human urine and hair) are described. Efficacy of MDMA and MDEA from the urine by different extractants is assessed. The data demonstrate different potentialities for detection and identification of amphetamine MDD, including those in biological specimens (human urine and hairs), by numerous chromatographic methods.     

尿液中几种碱性麻醉药物的检验

本文介绍了一种用 Sep-Pak C_(18)小柱,液-固萃取法提取尿液中几种碱性麻醉药物,以大口径毛细管气相色谱-氢火焰离子化检测器检验的方法。操作过程简单、迅速,各药物回收率高。     

Quantitative determination of strychnine in blood and urine by gas chromatography with mass-selective detector

A method for the quantitative determination of strychnine in biological fluids by gas chromatography--mass spectrometry is proposed. The preparation of samples for the analysis included extraction of strychnine from blood and urine with the use of AccuBond(II) EVIDEX cartridges for solid-phase extraction and SPEC MP3 disks respectively. The efficiency of extraction was estimated at 0.05 mg/l for blood and 0.02 mg/l for urine. The detection limit was 0.10 mg/l in blood and 0.05 mg/l in urine.     

尿中氯喹定性定量分析方法的研究

建立了人尿中氯喹的定性定量分析方法,2ml尿样用2ml×2环己烷：乙酸乙酯（8：2）提取净化后,60℃水浴室气吹干,残留物定容溶解后,气相色谱分析,氯喹的保留时间为9．44min。方法最低检测限为200ng／ml,回收率为87．0％,RSD＝7．9％（n＝5）,在0～50μg／ml浓度范围内,有良好的线性关系：A＝1778．9＋13686C,r＝0．999。方法同时可用于血中氯喹的分析。附一例应用报告,测得尿中氯喹的含量为0．745mg／ml,血中氯喹的含量为3．68μg／ml。尿液中同时检出氯喹的N－去单已基代谢物。定性结果经质谱法验证。     

A simple method of DNA extraction and STR typing from urine samples using a commercially available DNA/RNA extraction kit

We devised a simple DNA extraction procedure suitable for STR typing of urine sample. Use of a commercially available DNA/RNA extraction kit equipped with a silica-gel-based membrane made it possible to omit the recovery of urinary nucleated cells by sedimentation before the extraction. Successful genotyping of the TH01, HumTPO and multiplex STRs was achieved using aliquots of urine as small as 100 microL. Furthermore, application of this DNA extraction procedure to frozen urine samples provided STR allele results comparable to results obtained from fresh samples. Therefore, this extraction procedure is considered to be effective for STR typing of urine samples in both the frozen and aqueous state. Furthermore, addition of sodium azide to fresh urine samples prolonged their storage duration even at room temperature.     

Rapid colorimetric screening test for gamma-hydroxybutyric acid (liquid X) in human urine

A rapid colorimetric test for the detection of gamma-hydroxybutyric acid (GHB) is described. The ferric hydroxamate test for ester detection has been adapted to detect GHB in human urine samples from a healthy female and a healthy male subject. The assay can be performed within 5 min and with a GHB detection limit of 0.5 mg/ml when 0.3 ml of human urine is used and a GHB detection limit of 0.1 mg/ml when 1 ml of human urine is used. The colored complex indicating the presence of GHB is purple according to the assay conditions. Test results are free from the interference by alcohol, phenolic compounds and other biological chemicals under the assay conditions. In addition, the colorimetric test is free from the potential false-positive test result that could result from physiological concentrations of GHB.     

Detection of human chorionic gonadotropin (HCG) in fabric urine stains older than five years

Human chorionic gonadotropin (HCG) is used in clinical medicine as a particularly important indicator to determine pregnancy. In this study, it was necessary to determine whether the urine spots on car seat fabric from a murder 5 years previously were from a pregnant woman. The HCG in the dried urine spot on a car seat was detected using an immunochromatography kit. It was found that the HCG in urine can be detected for much longer periods of time than the previously reported period of approximately 6 months.     

Comparisons of protocols for enhanced DNA profile quality from FTA®-deposited urine samples

Disputes over the identity of a urine sample donor have been reported, and urine authentication by genetic profiling has helped resolved the cases. However, since genotyping of urine is not always required, many drug-testing laboratories may face sample storage issues. Several studies have investigated the use of FTA® cards as a convenient tool for keeping specimen at room temperature for extended periods of time. However, generating complete STR profile from some FTA®-deposited urine samples remains challenging due to low levels of genetic material content, necessitating amendments to the laboratory’s standard protocols. This work therefore aims to evaluate the effects of two DNA template preparation methods, both employing FTA® cards as the storage medium, on the success rates of STR profiling from urine. Specimen from a female volunteer, representing a particularly low-yield sample, was employed. Aliquots of 1 and 2 mL were used as the starting material to evaluate DNA template preparation using the FTA® manufacturer’s protocol for disc purification against elution of DNA from the FTA® using Prepfiler™ Forensic DNA Extraction Kit. AmpFℓSTR™ Identifiler™ Plus PCR Amplification Kit was used to amplify the STR markers, and the PCR products were analysed using Applied Biosystems™ 3500xL Genetic Analyzer. The DNA profile qualities were examined in terms of number of loci detected and peak height balance. Comparisons with the profiles obtained from DNA isolated using QIAamp® DNA Micro Kit from 1 and 2 mL of the same batch of urine were also made. The optimised protocol was then tested on urine samples from three male volunteers. The results showed that the purification of FTA® punches according to the manufacturer’s protocol enabled full DNA profiles to be obtained from both 1 and 2 mL of urine from all samples tested, including male samples. In contrast, no DNA profile could be generated from the DNA eluted with the Prepfiler™ kit. When compared with the more conventional solid-phase DNA extraction method, the profiles generated from the FTA® punches exhibited similar reproducibility and quality to those from the template isolated by the QIAamp® Kit. This work further demonstrated the feasibility of FTA® cards as a tool for specimen storage and DNA template preparation from small volumes of urine for authentication by STR profiling. Full STR profiles could be generated from sample from both sexes without modification of the PCR conditions or injection time.