Detection of various performance enhancing substances in specimens collected from race horses in Illinois: a five-year experience.

In order to protect the integrity of horse racing in Illinois, a complex testing of urine and blood specimens collected post-race from winning and special designation horses is continuously conducted. The initial screening by immunoassays was followed by the confirmation on presumptive positive samples. Instrumental screening was also conducted. Perimortem and postmortem specimens and special exhibits (syringes, needles, etc.) were also analyzed. The administration of alkalinizing agents was detected by measuring the total plasma carbon dioxide concentration. The laboratory analyzed specimens collected post race from winning horses and special designation horses at eight race tracks in the State of Illinois over the five-year time period (2004-2009). The total number of specimens collected was 91,808, comprising 45,210 urine specimens and 46,598 blood specimens. The total number of violations was 413 (0.45% of the total number of specimens analyzed); 207 were blood specimens (0.44% of the total blood specimens analyzed), and 206 were urine specimens (0.45% of the total urine specimens analyzed). A total of 220 violations were reported for harness horses, and 193 were reported for Thoroughbred horses. The number of reported violations of the total tested specimens in Illinois was small, but a wide variety of performance-enhancing drugs was shown.

Detection and quantification of low levels of benzoylecgonine in equine urine.

Cocaine (COC) is a highly addictive plant alkaloid expressing strong psychostimulatory effect. It has no medical use in equine veterinary practice. The contamination of the environment with cocaine such as its presence on the US paper currency has been reported few times. There are anecdotal reports of low benzoylecgonine (BE) concentrations (usually much less than 100 ng/mL) being found in urine of race horses. In order to protect horsemen against harsh penalties associated with the presence of trace amounts of BE in horse urine as a result of environmental contamination, in February 2005 the Illinois Racing Board issued new medication rules that established the threshold level of 150 ng/mL for BE in equine urine. The penalties associated with this rule provide for increasing fines ($250, $500, $1000) with successive positive reports against a trainer for levels of BE below 150 ng/mL. A total of 19,315 urine samples were collected over the 2-year period of time from winning horses (both harness and thoroughbred) at race tracks in Illinois for routine drug screening (ELISA). The presence of BE was confirmed by GC/MS in 28 urine samples (0.14%). The concentration range for BE in harness horses (21 detections) was < 5-91 ng/mL, and for thoroughbred (seven detections) was 7-52 ng/mL. To date, the laboratory has not reported concentrations of BE that exceed the established threshold concentration of 150 ng/mL.

An estimate of the proportion of drug-facilitation of sexual assault in four U.S. localities.

In recent years, drugs including flunitrazepam, gamma-hydroxybutyrate, ketamine, and ethanol, have become popularly associated with drug-facilitated sexual assault. Other drugs are also candidates as factors in "drug facilitated sexual assault" (DFSA). The true extent of DFSA is not known, and is difficult to estimate. We recruited sexual assault complainants at four clinics in different parts of the U.S. to anonymously provide urine and hair specimens, and to answer questions about suspected drugging, drug use, and the sexual assault incident. Urine and hair specimens were tested for 45 drugs, including ethanol, and those pharmacologically capable of inducing sedation, amnesia, or impairment of judgment. Analytical test results were used to estimate the proportion of subjects, and the proportion of all complainants to the clinic in the same time period, who were victims of DFSA. Overall, cases of 43% of 144 subjects, and 7% of 859 complainants, were characterized as DFSA. Subjects underreported their use of drugs. The role of toxicological results and history in characterizing DFSA cases is discussed.

Development of an analytical approach to the specimens collected from victims of sexual assault.

The objective of this study was to develop a screening process for the analysis of sexual assault samples. Recently, the Society of Forensic Toxicologists created a committee to address the issue of drug-facilitated sexual assault (DFSA) in the toxicology field. This committee prepared a list of drugs that could be, or have been, used in DFSAs. The list comprises about 50 compounds, including illicit, prescription, and over-the-counter drugs. Using this list, our laboratory wanted an easy, fast, and sensitive method to analyze a urine sample for all 50 of these drugs. We screened and confirmed for 20 compounds, including cocaine, amphetamines, benzodiazepines, barbiturates, opiates, methadone, alcohol, and PCP. A gas chromatographic-mass spectrometric screening method that was able to detect the remaining 30 compounds following 1 extraction and using only 2 mL of urine was developed. The process is inexpensive and uses equipment available in most forensic toxicology laboratories. This method is recommended for any laboratory that commonly receives specimens collected from sexual assault victims and is interested in a more thorough analysis.

Analytical developments in toxicological investigation of drug-facilitated sexual assault.

This paper gives a general overview of the drug-facilitated sexual assault phenomenon. Sexual assault perpetrated on both women and men, while incapacitated by so-called date-rape drugs, recently became the focus of many investigations conducted by law enforcement agencies in the US throughout the 1990s; an alarming increase in reports of this crime as well as in the number of scientific publications on drug-facilitated sexual assault has been observed. The list of drugs reportedly associated with sexual assault is long and among others includes flunitrazepam with other benzodiazepines such as diazepam, temazepam, clonazepam, oxazepam, as well as gamma-hydroxybutyrate (GHB), ketamine, and scopolamine. We discuss the most recent analytical developments in the toxicological investigation of drug-facilitated rape designed to reveal drug presence and that may help successfully prosecute perpetrators.

The feasibility of external blind DNA proficiency testing. I. Background and findings.

We describe the origins, purposes, and findings of a national study to determine whether a large-scale program of blind proficiency testing in U.S. DNA laboratories is feasible and/or practical. Proficiency testing in clinical laboratories is reviewed, particularly as mandated by the Clinical Laboratory Improvement Acts and its role in the regulation of those laboratories. Proficiency testing in forensic urine drug testing labs is also briefly reviewed. Studies involving comparisons between open and blind proficiency testing are discussed. The clinical laboratory proficiency testing and regulation landscape provides the background for the DNA Act of 1994, and the congressional mandate to investigate blind proficiency testing in forensic DNA laboratories. Four models of blind proficiency testing are defined and discussed, along with the advantages and disadvantages of each and estimates of the costs of a large-scale program. The purposes of proficiency testing in a quality-assurance context are likewise discussed and related to the models and the arguments generally proffered for and against blind vs. open proficiency testing.

The feasibility of external blind DNA proficiency testing. II. Experience with actual blind tests.

The background and goals of a national study to determine the feasibility of blind proficiency testing in U.S. forensic DNA laboratories are discussed. Part of the project involved designing and executing a series of fifteen blind proficiency tests. Execution included biological specimen donor recruitment and case evidence manufacturing. Simulated cases were submitted to DNA laboratories by law enforcement agencies and in some cases by other forensic-science laboratories. Replicate-manufactured evidence was submitted to reference laboratories to simulate the workings of a larger-scale program. Ten tests were straightforward, and essentially tested analytical ability. Five tests involved selecting on the basis of case facts appropriate bloodstains for typing from a bloodstain pattern. We describe in detail our experience in designing and conducting these blind proficiency test trials, and relate those experiences to the overall issue of blind proficiency testing as a quality-assurance tool in forensic DNA laboratories. In this feasibility test series, one blind test was detected by a laboratory, a second one was shown to the lab by law enforcement, and a third was never completed because of lapses in communication. Turnaround times were relatively fast in the independent/commercial labs and relatively slow in the larger public laboratories. Two cross-state case-to-case CODIS "hits" were "planted" among the first series of ten blind tests. One pair was detected. One member of the second pair went to a lab that was not CODIS-ready.

World population data for the HLA-DQA1, PM and D1S80 loci with least and most common profile frequencies for combinations of loci estimated following NRC II guidelines.

All published and unpublished gene frequency data for the PCR-based loci HLA-DQA1, LDLR, GYPA, HBGG, D7S8, GC, and D1S80 that could be located are presented in summary tables. These gene frequencies provide the data necessary for estimating probabilities of chance match according to NRC II guidelines for any DNA profile that includes any combination of these loci for any of the populations. To illustrate the range of polymorphism for combined locus profiles, least and most common profile frequencies were estimated following NRC II guidelines for: the PM loci for all populations for which PM data were available; and for combinations of HLA-DQA1/PM, HLA-DQA1/D1S80, PM/D1S80, and HLA-DQA1/ PM/D1S80 for populations for which data were available for the relevant combinations. The profile frequencies were calculated at theta values of zero and 0.01. Minimum allele frequencies (MAF) were calculated, and are shown, for each data set for which the MAF was greater than the lowest observed allele frequency. Least common profile frequencies were calculated using MAF in those cases to illustrate a conservative estimate. The effect of using MAF versus lowest observed allele frequency in estimating least common profile frequencies is briefly illustrated as well. We finally show that aggregate U.S. gene frequency data for the classical MN and GC polymorphisms for both Caucasian and African-American populations is fully in accord with the DNA-based gene frequency data obtained from PM reverse dot-blot strips for GYPA and GC, respectively.

A PCR-based strategy for ABO genotype determination.

The ABO blood group system has been widely used in forensic serology. Several techniques have been developed which detect ABH antigens. To overcome the problems associated with conventional methods such as bacterial contamination, extreme environmental conditions, antigen activity, non-secretor issues, and non-specific absorption, several new strategies have been employed to detect ABO genotypes by PCR. We have developed improved amplimers for the glycosyl transferase locus on chromosome 9 and examined the suitability of PCR-based ABO genotyping for forensic identification. We show that the ABO system is primate specific and that DNA extracted from various tissues commonly encountered in criminal cases can be quickly and reliably typed by ABO-PCR. The results indicate that ABO genotyping by PCR and restriction enzyme digestion of the amplified product is a useful procedure for forensic analysis that can provide additional discriminating power compared to conventional immunological methods.

Heat-soaked PCR: an efficient method for DNA amplification with applications to forensic analysis.

Heat-soaked PCR (HS-PCR) is a method for enhancing amplification performed by heating the DNA sample at 94 degrees C in 90 microliters 1.1 x buffer for 30 min. A 10-microliters bolus of concentrated (10x) deoxynucleotides, Taq DNA polymerase and primers prepared without buffer is then added just prior to thermal cycling. We have investigated the application of this method in a variety of forensically important DNA samples and compared it with regular PCR (R-PCR). DNA samples extracted from bone, postmortem tissues, bloodstains and hair contained low concentrations of human DNA or were contaminated with either non- human DNA or hemoglobin degradation products. Optimal conditions for HS-PCR were determined for the 3' ApoB VNTR locus and applied to a centromeric repeat element and to a single-copy locus. HS-PCR consistently and reproducibly enhanced product yield and specificity over R-PCR at all three loci in the entire set of DNA samples. HS-PCR was also effective in overcoming the inhibitory effect of hemoglobin at concentrations that fully impeded R-PCR.

A polymerase chain reaction (PCR) method for sex and species determination with novel controls for deoxyribonucleic acid (DNA) template length.

Human X and Y chromosome alpha-satellite sequences lying within higher order repeats were amplified by the polymerase chain reaction (PCR) in genomic deoxyribonucleic acid (DNA) isolated from blood, bone, and several other tissues and specimens of potential forensic science interest. X and Y sequences could be coamplified under some of the PCR conditions employed. Monomorphic sequences in the 3'-apolipoprotein B gene (designated "H") and in an alpha-satellite higher order repeat on Chromosome 17 (p17H8, D17Z1) were likewise amplified in the specimens. X and Y sequence amplification can provide information about the sex of origin. Amplification of the X, H, and D17Z1 sequences was found to be primate-specific among the common animals tested and can thus provide species of origin information about a specimen. The authors suggest that amplification of X and D17Z1 or H sequences might provide "relaxed" and "stringent" controls for appropriate PCR amplification tests on forensic science specimens. Testing was carried out using PCR protocols that employed Thermophilus aquaticus (Taq) and Thermus flavis (Replinase) thermostable DNA polymerases.

Genetic markers in human bone: II. Studies on ABO (and IGH) grouping.

A combination absorption-elution, two-dimensional absorption-inhibition procedure was used to determine the ABH antigen composition of a series of human bone specimens of known ABO type that had been aged up to nine months under dry and humid conditions at ambient temperature, 37 degrees C, and 56 degrees C; at ambient temperature in dry and wet soil; and buried in soil outdoors. Grouping data for the separate elution and inhibition testing, as well as for the combination procedure, are given. The combination method was found to be a highly reliable procedure for bone tissue ABH typing. Some data on microbial contaminants of human bone specimens aging in soil, and their effects on ABH typing results, are presented. No direct correlation between the properties of microbial contaminants and specific changes in the ABH antigenic composition of aging bone tissue specimens could be ascertained. Data on IGH antigen determination and on the quantitation of immunoglobulin G (IgG) in human bone tissue extracts indicated that immunoglobulin levels were typically too low to expect routinely successful Gm antigen testing results. However, these factors can sometimes be determined in fresh bone tissue extracts, particularly if the extracts are concentrated.