113th SEMI-ANNUAL SEMINAR (Spring 2009)
May 11-15, 2009
Lake Arrowhead, California

Bill Matty, State of California - DOJ-CCI

Several different fabrics of differing colors and patterns were photographed under varying light conditions in order to determine optimum photographic conditions to enhance gunpowder pattern visualization. Depending on the type of dye used, the use of infrared photography can enhance the ability to see the gunpowder pattern (or other stain) to a remarkable degree. Infrared photography is a very useful tool for the detection and recording of gunpowder patterns on cloth which has a dark color or a multi-colored pattern.

Lucien Haag, Forensic Science Services

The idea of sound as form of physical evidence may seem questionable to many yet sound is an integral and daily aspect of the physical world. Witnesses may claim to have heard what they characterize as one or more gunshots. Gunshots or similar high amplitude impulse sounds may, on occasion, be inadvertently recorded. This presentation provides some insight into the nature of such sounds, the limited instrumentation available to accurately measure them and the behavior of these sounds as they propagate over distance.

Jack Wallace, Ventura County Crime Laboratory

Current ISO standards require that laboratories report the uncertainty of measurement in a manner useful to their clients, and the recent NAS report on Strengthening Forensic Science makes a similar recommendation. However, there has been little discussion of this topic in the forensic literature, and consequently many forensic scientists are concerned regarding how these requirements might be satisfied. Fortunately, procedures for estimating uncertainties have long been of interest to the larger testing community, and this paper summarizes several such methods that can be helpful to forensic laboratories. These approaches are based on (1) within-laboratory precision control samples, with and without estimates of extramural sources or error; (2) proficiency test results; (3) readability limits; (4) propagation of error considerations; (5) external standard reference materials; (6) standardized methods with known uncertainties; (7) expert judgment; (8) comparison to similar methods and analytes; (9) operational limits; (10) ruggedness testing. For each of these approaches we identify the principal underlying assumptions and limitations, and provide limited examples from the forensic arena. The goal of this presentation is to enable forensic scientists to recognize those approaches that apply to their discipline, and to avoid those that do not.

Helene Jensen, Drug Enforcement Administration

Oxycodone is a powerful synthetic opiate that is legally used in the long term treatment of pain. Oxycodone is classified as a Schedule II drug by the United States Code of Federal Regulations and the California Health and Safety Code 11055 due to its highly addictive properties and its high abuse potential. In recent years the 80 mg controlled release tablets, have become a popular drug of abuse. In particular, many abusers crush the tablets and ingest, inhale or inject the dissolved powder, or more recently, have started to smoke these oxycodone tablets. The smokeability of the tablets was tested by utilizing Purdue manufactured 80 mg controlled release, oxycodone HCl tablets (OC80). The tablets are commonly smoked by cutting them into quarters and heating a quarter tablet at a time on a piece of aluminum foil. As the tablet starts to melt, it slides down the foil leaving a "skid mark" and the smoke that is produced is inhaled through a straw by the user. This process was repeated in the lab wherein the emitted smoke was collected into methanol and the presence of oxycodone was confirmed by gas chromatography coupled with a mass spectrometer (GC-MS). Further testing of the "skid marks" on the foil by GC-MS also indicated the presence of oxycodone residue. In addition, a vacuum pump, utilizing a Buchner funnel with a moist piece of filter paper was used to collect the smoke as a quarter of an OC80 tablet was heated on foil. The smoke on the filter paper was then quantitated by gas chromatography with a flame ionization detector (GC-FID) and found to contain approximately 35-40% of the available oxycodone HCl. Thus, based on the experiment, a quarter of an OC80 tablet is equivalent to the user getting approximately 7-8 mg of oxycodone HCl for one "hit."

Todd Davis, Drug Enforcement Administration

X-ray techniques have been used in forensic sciences for several decades. Powder data has been used for the identification of unknown materials or mixture of phases since the 1930's. X-ray Diffraction (XRD) techniques can be used as a nondestructive method of analysis and is especially suited for the determination of evidentiary materials that must not be destroyed. Previous forensic approaches for XRD included the analysis of paint, glass, fiber, metals, and soils. Now, with the development of ultra-fast X-ray detectors, it has allowed the technique to become a useful tool for the efficient screening of drug analysis. Identification of powder data is achieved by comparison of experimental data with standard data in crystallographic databases. The ICDD currently has one of the largest PDF libraries containing over 199,000 substances. However, current adulterants and illicit preparations were not available in PDF which prompted the DEA to create a working library that would fill in the necessary gaps. Most powder diffraction applications include phase identification, quantification, and determination of crystallite and particle size. Qualitative phase identification of polycrystalline organic, inorganic, and metallic substances has been studied with numerous controlled and non-controlled substances. New techniques have allowed the identification of the components present in pharmaceutical tablets directly through the blister packaging. These techniques have also been explored for the identification of illicit pharmaceutical preparations received from clandestine laboratories in addition to ecstasy whole tablet analysis. An evaluation of samples encountered at the DEA ranging from milligram residues to kilogram seizures has been studied. Relatively simple modifications of the diffractometer will increase its sensitivity to small amounts of sample to improve the lower limits of quantitation. X-ray diffraction in combination with the Rietveld method will also yield a reliable and accurate method to quantify the relative phase abundances in drug samples.

Moderator: Peter Barnett, Panel: Carolyn Gannett, John Murdock, Jim White, Peter Deforest, Jasmine Jefferson, Jeff Thompson

The National Academy of Sciences' recent report, Strengthening Forensic Science in the United States: A Path Forward, calls for the development of a national code of ethics for forensic scientists. Recommendation 9 is to establish "a national code of ethics for all forensic science disciplines. . . . Such a code of ethics could be enforced through a certification process." (Another recommendation in the report calls for mandatory certification of all forensic scientists.) An initial draft of a code of ethics, developed by a CAC committee, will be presented to the CAC membership for review and comment. Comments offered by criminalists during the panel presentation, and solicited from all CAC members over the next several months, will be used to develop a final draft of the document which will be presented for approval of the membership at the Fall seminar. Exactly how, when, or if the report's recommendation will be implemented is not known at the present time. But as the process evolves it is hoped that a document developed by working criminalists represented by members of the California Association of Criminalists will play a substantial role in the development of the national ethics code called for by the National Academy's report.

Natasha Poe, Coroner Forensic Science Center DNA Laboratory-St. Tammany Parish

In 2004, St. Tammany Louisiana parish residents identified the need for forensic DNA services and voted for a property tax to support a state-of-the art forensic science center in the coroner's office. The tax specified that the center provide DNA profiling, toxicology and pathology for the residents of the parish for twenty years. After delays due to hurricane Katrina, the tax collection finally began in October 2006 and funds became available for laboratory implementation. In order to provide law enforcement agencies and the district attorney's office of St. Tammany Parish with results as soon as possible, the coroner implemented the DNA laboratory in an expedited fashion. The coroner hired seasoned forensic analysts and additionally contracted with outside subject matter experts to assist the DNA analysts in validating the procedures and equipment. This novel implementation approach allowed the laboratory to begin receiving and testing evidence in August of 2007, less than one year after the tax was implemented and four months after laboratory building renovations began. The DNA laboratory received ASCLD/LAB accreditation in June 2008 and is currently waiting for their CODIS hardware to be installed. The rapid implementation of the St Tammany Parish Coroner Forensic Science Center DNA laboratory comes at a time when many DNA laboratories in the United States are struggling to reduce DNA backlogs, while concurrently dealing with increased numbers of case submissions due to the increased importance of DNA evidence in solving crimes. The novel approach taken by the coroner to rapidly implement and expand laboratory operations will be discussed.

Gregory Laskowski, Kern County District Attorney

In July of 2003, five people were found murdered in their home in Bakersfield, California. The husband of the deceased was developed as a suspect, but claimed he had an ironclad alibi; he was over 2,000 miles away in Columbus, Ohio at the time of the murders. This presentation will discuss the crime scene, the physical evidence collected and analyzed including novel geographical forensic entomological evidence, and some of the expert testimony given at the sensational murder trial.

John Jermain, Bureau of Alcohol, Tobacco, Firearms and Explosives (ATF)

Due to the powerful psychoactive chemical ingredients found in Salvia divinorum, Federal and state legislatures, including California, have been considering prohibition of the sale of the herb and making it a Schedule I Controlled Substance. Because of this legislation and the current legal status of the plant throughout the remainder of the country, the San Bernardino County Sheriff's Department's Scientific Investigations Division has taken steps to insure proper testing procedures are available to analyze Salvia divinorum and its active chemical ingredient salvinorin A. The studies described herein were performed due to the interest in developing procedures to identify the presence of salvinorin A. The two techniques that were utilized for this study were TLC and GC/MS. Raw dried leaves of Salvia divinorum, commercial Salvia divinorum extracts ("5x", "10x", and "20x"), Cannabis sativa L., and thirteen other species of Salvia were examined by TLC in order to determine if salvinorin A could be distinguished from the chemical components in the other plant species. While GC/MS is the preferred method used by surveyed crime laboratories to identify salvinorin A, there are a wide variety of extraction techniques utilized. Several of these extraction procedures were studied in order to determine which technique is best suited in analyzing salvinorin A.

Melissa Kramer, Los Angeles Police Department

California Vehicle Code (V.C.) 23152(a) states, "It is unlawful for any person who is under the influence of any alcoholic beverage or drug, or under the combined influence of any alcoholic beverage and drug, to drive a vehicle." At the Los Angeles Police Department Scientific Investigation Division, blood samples from individuals arrested in violation of a variety of laws, including V.C. 23152, and measuring at or below 0.08% BAC are subjected to a routine ELISA blood screen using Immunalysis kits for PCP, cocaine, opiates, amphetamine, methamphetamines, THC, barbiturates, and benzodiazepines. We have chosen to conduct a study to expand the window to include 0.15% BAC and below in order to ascertain what drugs, if any, are found in addition to higher alcohol levels. The data collected included 431 cases over a 7-month period from October 2007 to April 2008. This information comprised a majority of all samples analyzed in the Blood Alcohol Section of the Toxicology Unit that met the above criteria. Of the 431 samples, 40% of cases with an alcohol level of 0.09- 0.15% BAC screened positive for at least one of the eight drugs included in the screen. The most prevalent drugs detected at these levels were THC, cocaine, and benzodiazepines with percentages of 62%, 24.5%, and 17.5%, respectively. Information regarding drug-positive cases can be useful not only in a court of law as an addition to driving under the influence of alcohol, but also as an explanation of symptoms inconsistent with ethanol impairment at the time of arrest. From these results, the number drivers under the influence of drugs is significantly underestimated in the city of Los Angeles due to the policy of drug testing only those samples that lie at or below the statutory alcohol limit. These data are important as law-makers consider the necessity of expanding legislation with respect to the drug impaired driver. The data presented her may also be an underestimation of drug impairment because of the limitations of the ELISA panel. Toxicology laboratories commonly restrict the cases that undergo drug analysis because of limited resources; however, in this era of increased prescription drug abuse this topic deserves greater attention.

Dan Gregonis, Los Angeles County Sheriff's Department

The Centricon 100 centrifugal filtration device has been used in forensic science to help purify and concentrate DNA for over 20 years. With the discontinuation of these devices new methods/apparatuses must be verified to meet the needs of the concentration and clean-up steps following phenol/ chloroform/isoamyl alcohol (PCI) "organic" extraction. Pall Life Sciences corporation produces a product called "Microceps ™" and Sartorius Stedim biotech produces a product called Vivacon 2 (in 30, 50 and 100K molecular weight cutoffs) that are very similar in design and function to the Centricon® 100 which were manufactured by Millipore. Furthermore, Millipore is now manufacturing a product called "Amicon® Ultra-4" as a replacement for their Centricon series and also has the Microcon 30 and Microcon 100 filtration devices for use in a micro centrifuge. Robotic post digest cleanup is also available through the use of the Qiagen Biorobot EZ1 using the Investigator Kit. This study is designed to define analysis parameters for the Microceps™, Vivacon 2 (30k, 50k and 100k), Ultra-4, Microcon 30 and Microcon 100 as a potential replacement for the Centricon® 100s and to determine if any of these devices are as good as or superior to the Centricon® 100 for recovery of DNA. The Qiagen EZ1 using the Investigator kit was also compared. The centrifuges available for this study included the IEC Centra MP 4R and IEC CL31R at Los Angeles Sheriff's Department, the IEC Centra CL2 at Los Angeles Police Department and the Beckman Allegra 6R, Beckman TJ-6 and the Thermo Scientific Megafuge 11R at the San Bernardino County Sheriff's Department. All of these centrifuges had fixed angle rotors. The study itself consisted of several smaller studies. The first was to determine the optimal RCF and times required to obtain a usable volume after concentration. The second study involved the recovery of DNA from extracted samples. The third study estimated the efficacy of recovering different sized DNA fragments and the final part of the verification measured the various devices in cleaning up known inhibitors. The Vivacon 100 by Sartorius is a suitable replacement for the Centricon 100. This device performs at an equal or better level in the recovery of DNA after a Phenol- Chloroform extraction and takes less time (three 15 minute spins compared with three 20 minute spins). For challenged samples (either low level or degraded samples) the Microcon 30s are recommended since they are superior to either the Centricon 100s or the Vivacon 100's in the recovery of total DNA and the recovery of smaller fragments of DNA. The Vivacon 30 and 50, the Microcon 100 and the EZ1 using the Investigator kit are also suitable replacements for the Centricon 100's although the EZ1 is not recommended for very low level and/or potentially degraded samples. Neither the Microcep 30 nor the Amicon Ultra-4 performed as well as the Centricon 100. Advantages and disadvantages of each device will be discussed.

Rochelle Hranac, Arizona Department of Public Safety (Part 1);
Lon Anderson, Drug Enforcement Administration (Part 2)

In January 2009, the khat plant (Schedule II) and its active components, cathinone (Schedule II) and cathine (Schedule IV), were added to California Health and Safety Code as controlled Substances. Cathine (d Norpseudo-ephedrine) is an isomer of a non- controlled substance, l-norpseudoephedrine, thereby requiring the forensic analyst to perform testing to identify the isomer present. This presentation presents both sample preparation and instrumental techniques to identify cathine as well as the isomer. Sample preparation using acid/base extraction and/or dry-solvent extraction techniques are discussed. Four instrumental techniques to determine the isomer are presented: Gas chromatography with derivation, chiral gas chromatography, liquid chromatography with circular dichroism detection, and capillary electrophoresis.

Meiling Cabral, Los Angeles Police Department

The ability to recover a genetic profile from the backs of self-adhesive stamps holds significant implications to the field of forensic science. This knowledge is pertinent in cases in which stamp evidence is commonly encountered; examples include extortion, threats, and kidnapping, where identifying the individual source of DNA may prove pivotal in criminal investigations. In 1994, the U.S Postal Service discontinued the sale of water-activated stamps and replaced them with a pressure sensitive self-adhesive stamp. The self-adhesive stamp provides an alternative evidentiary source of DNA - DNA from fingerprint residues or "touch" DNA. Thus, there is a need to develop a method to successfully obtain a DNA profile from this alternate source of evidence. To determine the feasibility of recovering "touch" DNA from self-adhesive stamps, ten research subjects were instructed to affix self-adhesive stamps to a total of twenty envelopes. Prior to extraction, the image-side of the self-adhesive stamps was exposed to UV light for 10 minutes to decontaminate the external surface of the stamp. The stamps were extracted with phenol: chloroform: isoamyl alcohol and the extracts were purified and concentrated using Centricon® 100 microconcentrators. The extracted products were amplified and a haplotype was obtained using the LINEAR ARRAY™ Mitochondrial DNA HVI/HVII Region-Sequence Typing Kit. Three hypotheses were tested to examine the factors that may influence the recovery of mtDNA profiles. The recovery success for each research subject was calculated to determine whether the recovery of mtDNA profiles varies among subjects. Chi square analysis was performed to test the null hypothesis that there is no difference in recovery between self-adhesive stamps affixed to envelopes in the morning as opposed to the afternoon/ evening. Chi square analysis was also used to evaluate the null hypothesis that there is no difference in recovery between freezer-stored and mailed samples. When considering full and partial profiles, the overall recovery success was found to be 54%. The results were inconclusive in determining whether recovery of mtDNA profiles varied among subjects. The ability to successfully recover mtDNA profiles from self-adhesive stamps was found to be independent of the time of day the stamp was affixed to the envelope. Recovery success was also found to be independent of the stamp's exposure conditions, whether freezer stored or transported via the U.S Postal Service. The results of this study demonstrate that it is feasible to recover mtDNA from the attached side of self adhesive stamps.

Lisa Anderson, California State University, Los Angeles, School of Criminal Justice and Criminalistics

Mitochondrial DNA (mtDNA) has played an increasing role in the analysis of biological evidence. Its application in identifying decomposed remains and victims of mass disasters has been extensively documented and investigated. MtDNA can also be used in general forensic casework when nuclear DNA is not available or may be too degraded to interpret. While it is not possible to individualize evidence to the exclusion of all other individuals, mtDNA profiles may provide identifying criteria relating to the family origin of the individual who deposited the evidence. However, since mtDNA is inherited from an individual's mother, relatives generally cannot be distinguished among family pedigrees in the maternal linage. Studies have demonstrated that mtDNA is susceptible to genetic mutation. In particular, a high degree of polymorphism is observed within hypervariable region I (HVI) and II (HVII) of the control region. The mutations may be somatic (mitotic) or may originate in the female germ-line (meiotic). Generally, only a single mtDNA type is detected within an individual, a condition referred to as homoplasmy. However, when a mutation arises, but is only reflected in some copies of an individual's mtDNA, the result is a combination of normal (wild) and mutant types. This mixture of haplotypes on mtDNA sequences is referred to as heteroplasmy. The presence of heteroplasmic mutations can present advantages and disadvantages in a forensic context. The present study examines mtDNA polymorphisms in human head hair, saliva and bloodstains with respect to their potential for forensic application. MtDNA was isolated and polymorphisms were detected by applying sequence-specific oligonucleotide probe analysis. The particular focus was to characterize heteroplasmic mutations as germ- line or somatic in origin. This distinction is important in a forensic context because if transmission of a mutation occurs along the germ-line, this would support the collection of samples from maternal relatives or any tissue source associated with the decedent. However, if transmission is somatic in origin, intra- and inter tissue variation may exist and reference samples must correspond to the tissue analyzed in the case sample. Failure to do so would potentially result in a false exclusion. Bloodstains, head hairs, and buccal swabs representing the U.S. Caucasian population group were typed using the LINEAR ARRAY™ mtDNA HVI/HVII Region-Sequence Typing Kit. For the purposes of this study a sample was scored as heteroplasmic if two probe signals were visible within a single probe region (either with equal or uneven intensity). The results of this study demonstrate heteroplasmic expression in hair, tissue, blood and saliva within transmission of a particular mutation.

Katherine Roberts, California State University, Los Angeles

The successful resolution of crime scene investigations often depends on the ability to identify and individualize biological evidence. Fundamental to this analysis is the stabilization of biological evidence, because testing generally does not proceed immediately after collection. Crime scene samples and liquid extract derivatives are routinely stored frozen in forensic laboratories, to be thawed at the time of analysis. However, numerous lines of evidence indicate that the process of freezing and thawing has detrimental effects on biological samples. Many crime scene samples are sub-optimal for analysis-the further degradation of the samples by current storage systems only exasperates the difficulty of identifying and individualizing biological evidence. Biomatrica, Inc. has developed a proprietary platform technology for the dry storage of biological materials at ambient temperatures. The key component of this technology is SampleMatrix™, which was derived from studies on extremophile organisms. These organisms can survive extreme environmental conditions. SampleMatrix™ consists of protective agents developed from combining small molecule chemistry with advanced polymer chemistry. This product has multiple components: 1) the dissolvable polymer in a stabilization buffer adjusted for the different biological samples; 2) a stabilizing solution containing small synthetic molecules. We have conducted research on the effectiveness of SampleMatrix™ to stabilize biological evidence as compared to conventional storage methods. The study tests the hypothesis that the storage of biological samples, swabs and liquid DNA extracts, in the SampleMatrix™ polymer at room temperature reduces the exogenous degradation of DNA by minimizing the adverse effects of hydrolysis and freeze-thawing. Blood, semen, and saliva stains of different concentrations were initially deposited on five different substrates. Each stain was sampled by swabbing with water or Sample Matrix™ as the wetting agent and then exposed to room temperature v. freezer storage for longitudinal study. In addition, we have conducted parallel studies whereby blood, semen, and saliva were directly applied to swabs and subsequently stored for longitudinal study. This presentation will discuss the results of the experimental conditions that were evaluated by Real-Time and STR analysis following standard forensic protocols.

Lotte Downey, Promega Corporation

Short tandem repeat (STR) analysis remains the primary method for human identification. Forensic Typing, criminal databasing and relationship testing laboratories in the US and many other regions of the world use a standard set of 13 STR markers selected by the US Federal Bureau of Investigation for the Combined DNA Indexing System (CODIS). The PowerPlex® 16 HS System co-amplifies these 13 loci (D18S51, D21S11, TH01, D3S1358, FGA, TPOX, D8S1179, vWA, CSF1PO, D16S539, D7S820, D13S317, and D5S818) plus the low-stutter Penta E and Penta D markers and the gender-determining Amelogenin locus. One primer for each of these loci is labeled with fluorsecein, carboxy-tetramethylrhodamine (TMR) or 6-carboxy-4', 5'-dichloro-2',7'-dimethoxy-fluorescein (JOE). Amplicon size is determined by comparison with the Internal Lane Standard 600 (ILS) labeled with carboxy-X-rhodamine (CXR). This four-color chemistry can be analyzed on the ABI Prism® 310, 3100 and 3100-Avant genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers using existing dye matrix standards. The PowerPlex® 16 HS System provides a hot-start Taq DNA polymerase in a modified master mix to provide increased ease-of-use and performance over previous PowerPlex® systems. This assay has increased tolerance to common forensic sample inhibitors known to reduce genotyping success rates. The presentation will share results from sensitivity and inhibitor studies along with developmental validation results.

Bob Blackledge, Forensic Chemist Consultant

Gas chromatography combustion stable isotope ratio mass spectrometry, GC-C-IRMS, is the analytical method used as a confirmatory test for the presence of exogenous anabolic steroids in urine. This analytical technique should especially be of interest to CAC members because it has many potential applications to questions addressed in forensic science. This presentation will explain the scientific basis for GC-C- IRMS and use the data from the Floyd Landis sports doping case to illustrate possible pitfalls.

Mehul B. Anjaria, Kellie A. Fenesan, Chantel M. Giamanco, Human Identification Technologies, Inc.

In November 2007 Human Identification Technologies, Inc. (HIT) was forced to abandon use of CENTRICON® Centrifugal Filter Devices for organic DNA extraction clean up due to their unavailability in the marketplace. The MICROCON® Centrifugal Filter Device was assessed and found to be a suitable replacement, and in fact produced slightly better DNA yields than did the Centricon. Initially, the Microcon appeared to perform well with casework samples. However, over time an increase in apparent PCR inhibition was observed. This was generally overcome by amplifying a reduced amount of DNA extract. The practice of exposing Microcons to ultraviolet light prior to use in casework was investigated as a possible aggravating factor, but experiments showed that the ultraviolet treatment did not have a detrimental effect. As the inhibition reached unacceptable levels, HIT began validating the VIVACON 2 as a replacement for the Microcon. The Vivacon performed well during the validation for both extraction and the concentration of extracts, and therefore HIT has abandoned the Microcon device in favor of the Vivacon. The troubleshooting approach used to deal with the inhibition encountered will be described and data from the Vivacon validation will be presented. Results of a literature search on ultraviolet treatment of consumables prior to PCR will also be described.