121st SEMI-ANNUAL SEMINAR (Spring 2013)
CALIFORNIA ASSOCIATION OF CRIMINALISTS
May 20-24, 2013
Pasadena, California

ARTISTS' PAINTS IN FOCUS
Alan Phenix, Getty Conservation Institute, Los Angeles

Many of the methods used in forensic science for examination and analysis of objects and samples also find use in the cultural heritage sector for the study of painted works of art, to support understanding of artists' techniques and materials, and to investigate deterioration or alteration phenomena that have a bearing on conservation and/or restoration of the paintings. While a variety of noninvasive methods of examination and chemical analysis (including infrared, ultraviolet and X-ray imaging; X-ray fluorescence) are routinely used in the technical study of works of art, analysis of tiny samples of paint typically remains the main approach used in such investigations. Paint binder identification is usually achieved by combinations of instrumental analytical methods, usually FTIR microscopy, GC-MS, py-GC-MS, plus other techniques such as ELISA (enzyme-linked immunosorbent assay).

However, samples prepared, examined and analyzed as cross-sections are often central to technical studies of paintings, since both layer structure and pigment composition can be visualized by optical and electron microscopy. Alan will illustrate the application of microscopical examination of paint cross-sections in the technical study of works of art using examples from a variety of different projects undertaken at the Getty over the past few years. This survey will cover over three thousand years of paint: from the wall paintings in the tomb of Egyptian pharaoh Tutankhamen, to European medieval and Renaissance devotional works and altarpieces, through to the American abstract painters of the mid-late 20th century, like Jackson Pollock and Clyfford Still. Issues such as the constraints on sampling and on the interpretation of analytical findings will be considered in passing in relation to particular objects under study.


THE ANALYSIS OF METAMERIC BLUE FIBERS AND THEIR FORENSIC SIGNIFICANCE
Dr. Paul Martin, Craic Technologies

Metamerism is a phenomenon where two or more colored items with different chemistries appear to be the same color. However, those differences should result in different UV-visible spectra and the literature on color science states that metameric samples will have spectra that intersect at three or more loci. Metameric samples of blue textile fibers, which were created using different coloring agents or different relative concentrations of the coloring agents, were studied to demonstrate that they could be differentiated by obtaining their spectra using UV-visible microspectrophotometry. However, while some of the metameric samples tested did intersect at three or more loci, others did not intersect at all. In the spectra that did intersect, no correlation was found between either the dye chemistries or the relative component concentrations.


THE ANALYSIS OF FUEL MARKERS BY SURFACE ENHANCED RAMAN SPECTROSCOPY (SERS)
Mark Baron, Kelly Whittingham, Nick Meakin,Tim Wilkinson, University of Lincoln, UK

Markers are added to subsidised fuels by governments to detect illegal activity such as deliberate misuse and smuggling to other countries. Fuel marking is an international business and there are a number of markers used globally. Historically, these have been solvent dyes that are fuel soluble and detectable by simple spectrophotometric techniques. One analytical challenge is the need to disperse the marker into a highly non-polar fuel but then extract into a more polar phase for analysis. Molecule design has provided molecules with high solvent solubility that can undergo chemical change for easier extraction into an aqueous phase for analysis. In this presentation strategies for marker phase transfer will be discussed with the aim of providing sensitive measurement in an aqueous phase. The detection technique is Surface Enhanced Raman Spectroscopy and we have demonstrated that parts per billion detection levels are possible. Analytical challenges when applied to real fuel samples will also be discussed.


ORANGE COUNTY’S FIRST FAMILIAL DNA HIT
Mary Hong, Orange County Crime Laboratory

On July 10, 1978 the partially clothed body of Linda Saunders was found behind a restaurant in Santa Ana, California. She had a blunt force trauma injury to her head, was shot once in the chest, and had been sexually assaulted. Her male companion was shot once in the head but survived. He gave conflicting statements regarding the description of the assailant(s). A DNA profile was developed from the semen recovered from Saunders body and was submitted to CODIS in 1999. The case remained unsolved and was submitted to the CA DOJ DNA Laboratory in 2010 for familial searching. A familial search result identifying a suspect was obtained in July, 2012 and, following an investigation and confirmation through DNA testing, the case was closed by the Santa Ana Police Department in December, 2012. The forensic technology utilized over the 34 years since the crime occurred and the investigative resources expended by DOJ and the local police agency will be discussed.


NIST RESEARCH, GUIDELINES, AND TOOLS THAT SUPPORT FORENSICS SCIENTISTS
John Paul Jones, NIST

The National Institute of Standards and Technology (NIST) has developed a large number of reference documents, tools and physical standards that support the forensic science community. The Forensic Science Program (FSP) at the Law Enforcement Standards Office (OLES) within NIST conducts and coordinates research and provides technical services to address the needs of the forensic science community. The FSP focuses on creating new material standards; initiating metrology research; evaluating technologies; and establishing expert working groups to facilitate knowledge exchange and identify best practices. These activities have been used to support forensic science disciplines such as: arson; digital and multimedia forensics; DNA; fingerprints; firearms and toolmarks; odontology; controlled and dangerous substances; toxicology and trace analysis.

Topics that will be covered during this lecture include:


THE BLACK DAHLIA: AN HISTORICAL PERSPECTIVE
Joan Renner

It was after 10 a.m. on January 15, 1947 when a local housewife and her three-year-old toddler discovered the naked, bisected body of a young woman in a vacant lot near 39th and Norton in the Los Angeles neighborhood of Leimert Park. No paper identification or clothing was found near the body, and the Coroner tagged the victim Jane Doe #1. The police lifted the dead woman’s fingerprints and submitted them to the FBI. Within two days, she was identified as twenty-two year old Elizabeth Short from Medford, Massachusetts. By the end of the month, she had become known as The Black Dahlia.

Elizabeth Short's murder was not the first unsolved homicide of a woman in Los Angeles during the 1940s; in fact, there were more than a dozen. At the end of the decade, the 1949 Los Angeles Grand Jury issued a report in which they determined that: "Criminals are using varied techniques in writing a record of crime that includes murders, mysterious disappearances of persons and loathsome sex crimes." The report concluded with "...there is something radically wrong with the present system for apprehending the guilty, and the alarming number of unsolved murders and other major crimes reflects ineffectiveness in law enforcement agencies and the courts that should not be tolerated."

What was happening in Los Angeles during the 1940s that would account for the number of violent crimes against women? Was law enforcement lax in its investigation of major crimes? Who were the victims and what, if anything, did they have in common?


THE HURRICANE MEETS CSI: HOW FORENSIC SCIENCE CAN CONVICT – AND EXONERATE – THE INNOCENT
Alexander Simpson, Legal Director, California Innocence Project

Forensic science has had a profound impact on the criminal justice system since its inception, and as methods for analyzing crime scenes and forensic evidence grow more sophisticated, their use in courtrooms means that judges and juries increasingly rely upon expert testimony to render verdicts in criminal cases. The reliance upon expert testimony has led many legal analysts to acknowledge the "CSI Effect" – that tendency of juries to demand forensic evidence before they convict, even when the case does not involve forensic analysis, and elevating the standard of proof prosecutors must meet. But the "CSI Effect" also means jurors place more faith in the abilities of forensic sciences to solve crimes, and rely upon experts more heavily than even the experts actually intended.

This presentation will discuss the use of forensic science in a number of wrongful conviction cases in California and across the country. Forensic science has advanced dramatically in recent years, but its possibilities (and limitations) are often misunderstood and incorrectly applied by those working in the criminal justice system, including prosecutors, defense attorneys, and judges. The presentation will dissect a number of cases of wrongful conviction, paying close attention to how forensic science has been used or misused in these cases. Forensic evidence has led to the exoneration of hundreds of innocent individuals in the last three decades. It has also led to an unknown number of convictions of innocent individuals in that same time. This presentation will explore the way forensic evidence is handled in criminal cases, and how criminalists can ensure their analyses are correctly understood and applied by attorneys, juries, and judges.


FORENSIC SCIENCE AND NATIONAL POLICY
Anna-Marie Mazza, Ph.D., Director of Science,Technology, and the Law at the National Academies, American Association for the Advancement of Science

This past February, the US Department of Justice and National Institute of Standards and Technology announced the establishment of the National Commission on Forensic Science as part of a new federal initiative to strengthen and enhance the practice of forensic science. The Commission comes four years after the Congressionally-requested National Academy of Sciences report, Strengthening Forensic Science in the United States: A Path Forward, which found “that the forensic science system... has serious problems that can only be addressed by a national commitment to overall the current structure that supports the ... community...” Since release of the report, the needs of the forensic science community have received a great deal of attention from Congress, the White House, and the forensic science community.


THE BROCCOLI FIELD MURDER
Samantha Skotarczyk, DOJ Santa Barbara Laboratory

Pedro Gonzalez’s body was found in an open broccoli field in Nipomo, CA on February 15th, 2010. Autopsy findings revealed a through-and-through gunshot wound to the head; however, no bullets or casings were ever located and physical evidence was minimal. The investigation led detectives to suspect a top-ranking Guadalupe gang member who was responsible for collecting drug taxes from local drug dealers. After screening items from the suspect, the crime scenes, and the alleged transport vehicle, only one sub-mm bloodstain had the potential to link the suspect to the crime. This case presentation will describe the crime, the evidence, laboratory results, and courtroom testimony in a highprofile case.


VALIDATION TESTS AND ERROR RATE CALCULATIONS FOR THE CONGRUENT MATCHING CELLS (CMC) METHOD USING CARTRIDGE CASES FIRED WITH CONSECUTIVELY MANUFACTURED PISTOL SLIDES
John Song, NIST

NIST has developed the NIST Ballistics Identification System (NBIS) based on 3D topography measurements on correlation cells. The Congruent Matching Cells (CMC) method is used for ballistics identifications using three identification parameters of the paired correlation cells including the cross correlation function maximum CCFmax, registration angle, and x-y registration position. The numerical identification criterion is suggested as C ≥ 6, by which the correlated topography pair is considered as a Match. An error rate calculation procedure based on the CMC method is also proposed for the error rate report of ballistics identifications.

In order to test the CMC method and to verify the proposed numerical identification criterion, 40 cartridge cases fired from handguns with 10 consecutively manufactured pistol slides are measured by a 3D confocal microscope and correlated by the CMC method. The breech face topographies are divided into cell arrays (7 X 7 or 6 X 6) for correlation. There are a total of 780 correlations including 63 known-matchings (KM) and 717 known-non-matchings (KNM).

The initial test results show that the numbers of congruent matching cell pairs (CMCs) for all 63 matching topography pairs are distributed in a range of 9 to 29; while the numbers of CMCs for all 717 nonmatching topography pairs are distributed in a range of 0 to 3. More tests conducted at different correlation conditions also show significant separations between the KM and KNM distributions without any false positive or false negative identification. That represents the highest identification accuracy for the same set of cartridge cases that have been tested at NIST thus far. Based on the CMC method, the collective error rates for the 63 KM (false negative error rate E2) and 717 KNM (false positive error rate E1) are calculated. The individual error rates for the KM correlations with the minimum CMC = 9, and for the KNM correlations with the maximum CMC = 3 are also calculated.

Initial tests support both the proposed CMC method and the numerical identification criterion C ≥ 6 for ballistics identifications. The proposed CMC method suggests that an empirical Identification Criterion of at least 6 matching cell pairs be used for separating the matching and non-matching topographies. With this criterion there are no false positive or false negative identifications using the NIST proposed CMC method for ballistics identification for this set of cartridge cases. The identification accuracy can be further improved by optimization of the cell size and the thresholds of the identification parameters. The proposed numerical identification criterion (C ≥ 6) for the CMC method is based on the identification criterion for the Consecutively Matching Striae (CMS) method developed by Biasotti and Murdock, which has been widely accepted by firearm examination community for bullet signature identifications. The CMC method using the same numerical identification criterion C ≥ 6 has expanded the CMS method from 2D bullet signature correlations to 3D casing signature correlations.


INITIAL CORRELATION TESTS AND ANALYSIS FOR CARTRIDGE CASE INTENSITY IMAGES USING THE CONGRUENT MATCHING CELLS (CMC) METHOD
Robert Thompson, NIST

The NIST Ballistics Identification System (NBIS) is developed based on 3D topography measurements on correlation cells. The NBIS aims to provide objective, high-accuracy and high-speed ballistics identifications. The National Ballistics Evidence Search Engine (NBESE) is proposed by NIST for ballistics evidence searches using the Congruent Matching Cells (CMC) method with system interoperability and error rate report. Feasibility of applying correlation cells for 3D topography correlation is successfully demonstrated by validation tests using 40 cartridge cases fired with 10 consecutively manufactured slides. The results show a significant separation between the known-matching (KM) and known-non-matching (KNM) distribution. However, most existing ballistic identification systems are based on intensity image comparisons; and a huge number of intensity images are stored in the National ballistic database. It is necessary to conduct a validation test that demonstrates the feasibility of the CMC method for the identifications of optical intensity images.

A Leica comparison microscope is used to capture the breech face intensity images of the same set of 40 cartridge cases fired from handguns with 10 consecutively manufactured pistol slides. To confirm the equipment accuracy, repeatability and reproducibility tests are performed. In the correlation tests using CMC method, a total of 780 correlations including 63 KM and 717 KNM were implemented. The effects of different lighting conditions for the image correlations were also tested and analyzed. In the initial tests using the single correlation parameter of the whole optical image: the cross correlation function maximum CCFmax, the KM and KNM distributions cannot be separated. However, by using the CMC method with three identification parameters (the correlation function maximum CCFmax, the registration angle and registration position in x, y), the KM and KNM distributions show clear separations without any overlap. There is no false positive or false negative identification in all 780 correlations.

The results show that the CMC method works well for correlation of both the 3D topographies and optical intensity images. Significant improvement can be achieved by applying CMC method with three identification parameters on intensity image data compared with the correlation of whole image using a single correlation parameter CCFmax. The CMC method also shows a good robustness to lighting condition variance. Optimization of the cell numbers and the thresholds of the correlation parameters can further improve the identification accuracy. Certain commercial equipment, instruments, or materials are identified in this paper to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.


USING DIGITAL IMAGING TOOLS TO ENHANCE RESTORED SERIAL NUMBERS
Greg Laskowski

Many valuable objects have serial numbers, so that should they become lost or stolen, then are later recovered, the manufacturer or owner can be identified. These numbers can be stamped, etched, inscribed, or engraved. The processes used to restore removed or obliterated serial numbers are mainly by mechanical action followed by some form of chemical, electrochemical, magnetic, and by heat. In some cases, the restored serial numbers are clearly visible, while in many instances there are only partial numbers that can be visualized or the restored numbers are barely visible or legible. The introduction of digital imaging technology can assist the examiner in enhancing the visibility of restored serial numbers through a series of software tools available through such standalone photographic programs as Adobe PhotoshopTM or an integrated program offered by Mideo Systems in their CASEWORKSeisTM. This paper will discuss the use of the imaging tools available in CASEWORKSeisTM to enhance and record restored serial numbers observed in casework.


THE ROLE OF DNA IN PROSECUTING NONSTRANGER SEXUAL ASSAULT: PERSPECTIVES FROM RESEARCH, THE FORENSIC SCIENCE LABORATORY, LAW ENFORCEMENT, AND THE DA’S OFFICE
PANELISTS:
Carol Burke, Sex Crimes Head Deputy, Los Angeles County District Attorney’s Office
Dean Gialamas, Director, Los Angeles County Sheriff’s Department, Scientific Services Bureau
Michel Moore, Assistant Chief, Los Angeles Police Department
Joseph Peterson, Ph.D., Professor, California State University, Los Angeles
Daniel J. Scott, Detective Sergeant, Los Angeles County Sheriff’s Department, Special Victims Bureau Katharine Tellis, Ph.D., Assistant Professor, California State University Los Angeles

PURPOSE AND OBJECTIVES The purpose of this panel is to facilitate a dialogue between criminal justice researchers and practitioners to consider the evidentiary role of DNA in prosecuting nonstranger sexual assault from various perspectives. In the post-backlog era and often in response to pressure from victim advocates, many law enforcement agencies test every sexual assault evidence kit as a measure of commitment to public safety for all victims. However, the effectiveness of this approach must be examined amidst the context of increasingly limited state and local resources, along with the actual value gained from such expenditures in terms of increasing the successful prosecution of nonstranger sexual assault. Panelists will discuss the issues associated with testing all nonstranger sexual assault evidence kits, such as practical considerations regarding kit-testing priority based on caseload and suspect/victim relationship, and whether the salience of DNA in these cases is obviated by the consent defense.


MODERN TELEVISION PROCEDURALS AS SCIENCE FICTION
Dr. Patrick Sharp, California State University, Los Angeles

No Abstract Submitted.


DETECTING AND PREVENTING CRIME IN HOSPITALS
Beatrice Crofts Yorker, Dean of the College of Health and Human Services, California State University, Los Angeles

Crimes committed in the context of healthcare have been documented for centuries; however the advent of high tech and intensive care environments has created an alarming increase in both occurrence and the identification of criminal acts in hospitals. There have been over 90 healthcare providers prosecuted for serial murder of patients in their care since the 1970s. These cases are extremely complicated to prosecute as evidence ranges from post-mortem toxicology to statistical analysis to case review and eyewitness testimony. In addition to murder by healthcare providers, child abuse also occurs in hospital settings in the form of Munchausen Syndrome by Proxy and child sexual abuse.

The presenter has studied the prevalence of crime in hospitals since 1986, and she will provide data on prosecutions in 26 different countries around the world. She has testified in cases such as a mother who injected her child's intravenous line with fecal material to cause sepsis and apnea caused by suffocation. Other examples of criminal acts include a nurses' aide who was prosecuted for distributing child pornography after he took photos of developmentally delayed children with his cell phone in a pediatric hospital and the recent case of a physician in Brazil being investigated for hastening the deaths of up to 300 patients in order to “free up hospital beds”. While these cases are shocking, there are ways to collect evidence such as covert or overt video surveillance, pharmacological tracers, and death review that can assist with prosecution and deter crime.


UNUSUAL WAYS TO COMMIT SUICIDE
Margaret A Kaleuati, County of Los Angeles Department of Coroner

This presentation reviews multiple unusual cases of suicide including a gunshot wound to the top of the head with a downward and slightly back to front trajectory and the application of a chainsaw to the neck. It will also discuss the findings of each investigation including the initial death scene examination, autopsy results and subsequent analysis of trauma in the biological tissues. Only two suicides by chainsaw have occurred in Los Angeles County; suicide utilizing firearms is much more frequent. The presentation will review the mechanisms of suicide utilizing guns and chainsaws and compare them to other cases reported in the literature. Additionally, it will review the Los Angeles County Coroner’s experience with suicidal gunshot wounds over the last 10 years to highlight how unusual a gunshot wound to the top of the head is for a suicide.


ETHICAL DILEMMAS CREATED BY EMPLOYER OBJECTIVES AND PRESSURES
Gregory B. Matheson

The concept of criminalistics laboratories being totally independent of law enforcement and prosecution has been discussed for years. However, the publishing of the National Academy of Sciences report "Strengthening Forensic Science in the United States: A Path Forward" has prompted national level discussion of the concept. Recommendation #4 of the report states - "To improve the scientific basis of forensic science examinations and to maximize independence from or autonomy within the law enforcement community, Congress should authorize and appropriate incentive funds to the National Institute of Forensic Science (NIFS) for allocation to state and local jurisdictions for the purpose of removing all public forensic laboratories and facilities from the administrative control of law enforcement agencies or prosecutors’ offices." Many reasons are given for the need for independence but of primary importance is the belief that moving crime labs out of law enforcement will reduce or eliminate biases and pressures created by the close interrelationship between a scientific laboratory and law enforcement.

It is unlikely the majority of crime laboratories are going to be removed from law enforcement agencies in the foreseeable future so it is important to investigate and discuss the issues associated with the current organizational location of crime laboratories. It is my belief most criminalists will experience some type of pressure to amend their notes or report to better reflect and support the theory of how a criminal event occurred which is promoted by their employer. To test this belief, I requested CAC criminalists to provide me with their experiences of pressure to change their analytical conclusions from laboratory supervision and/or management, detectives, agency management, prosecuting attorneys or defense attorneys. In addition to many examples of pressure, I also received many comments as to how they handled the situation. Using these examples, along with my personal experiences and published information on the subject, this presentation will explore the types of real and/ or perceived pressures analysts, supervisors and managers may face when working within a law enforcement agency. We will also discuss ways to mitigate potentially negative influence of the work environment on scientific work, and how having a strong understanding of ethics and participating in strengthening an ethical culture in the work place can minimize the issues and concerns.


FIXING CRIMINALISTICS
Gregory B. Matheson and Peter R. De Forest

"Criminalistics, properly practiced, is a challenging and a profoundly intellectual endeavor. Unfortunately, it is not always viewed this way by those who hold the title Criminalist. Compounding the problem further is the fact that some criminalists are constrained from practicing criminalistics the way it should be practiced."

The foregoing quote is taken from the abstract for a presentation one of us made at the Fall 2006 CAC Meeting in Temecula. Well prior to the current session we would like you to read the full abstract and other reading materials that were supplied to you electronically by Kathy Roberts in advance of the meeting. The readings detail impediments to criminalistics realizing its full potential. This presentation will focus on what can be done to remove the impediments and is intended to be primarily a discussion session facilitated by your two presenters. Each of us will make a short introductory presentation before initiating what we hope will be a lively, informed, and constructive discussion.

Please come prepared to discuss the issues and put forward recommendations. Funding has been a perennial problem. Criminalistics has been playing "catch-up" for decades as the demands have increased. In our discussions, let us focus on fundamental changes. Some of these may impact the economics of Criminalistics positively or negatively. With the CAC's long history of leadership, hopefully, this discussion will contribute to positive changes in the field for the future.


2D/3D TOPOGRAPHY COMPARISONS OF TOOLMARKS GENERATED BY CONSECUTIVELY MANUFACTURED CHISELS AND PUNCHES
Robert Thompson, John Villanova, NIST

This study seeks to evaluate whether a mathematically objective metric, the maximum value (CCFmax) of the Cross Correlation Function, can be used to identify a tool that generated a striated or impression toolmark. The study addresses two types of tools: chisels for striated toolmarks and punches for impression toolmarks. Ten consecutively manufactured chisels and punches were obtained from Western Forge (a supplier of CraftsmanTM Tools). We separated the study into striated toolmarks generated by the chisels and impressed toolmarks generated by the punches. For each of the ten 1⁄2” (12.7 mm) chisels, a set of two known toolmarks were created on a polished copper plate though a controlled dragging motion produced by a motorized jig. After the identities of the chisels were randomized and hidden, we created an additional set of 20 unknown toolmarks (two marks per chisel). For each toolmark, we used a 2D stylus probe instrument to measure the surface topography along a line orthogonal to the striations. A total of 1600 profile comparisons were performed in a 40 x 40 matrix. For each comparison, the two profiles were automatically registered to obtain the maximum value (CCFmax) of the Cross Correlation Function. The CCFmax values for the known match and known non-match comparisons were used to establish test statistics for the reliable identification of the unknown toolmarks based on CCFmax values. For each of the ten punches, two known toolmarks were created on a polished copper plate through a controlled drop of the punch, which was mounted on a linear rail. After the identities of the punches were randomized and hidden, a set of 20 unknown toolmarks (two marks per punch) were created. A disk scanning confocal microscope was used to measure the 3D topography of the toolmarks. The data were then trimmed to remove any uninformative areas. Before the comparisons, each measurement was automatically pre-processed. A total of 1600 surface comparisons were performed in a 40 x 40 matrix. For each comparison, the pre-processed data sets were automatically registered inposition and orientation to obtain the maximum value (ACCFmax) of the Areal Cross Correlation Function. The known match and known non-match ACCFmax values were used to establish test statistics for the reliable identification of the unknown toolmarks based on ACCFmax values.

Based on the CCFmax metric, and a statistical analysis of the known match and known non-match scores, all of the unknown striated chisel toolmarks and impressed punch toolmarks were correctly identified back to the tool that created them.

In a blind study of ten consecutively manufactured chisels and punches, the maximum value (CCFmax) of the Cross Correlation Function was successfully applied to link the measured surface topography of a toolmark to the tool that created it. These results provide an objective mathematical validation of the science for both striated and impressed toolmark comparisons.


THE ROLE OF THE MEDICAL EXAMINER–CORONER, INCLUDING SOLVING 'UNSOLVED HOMICIDES' - THE LOS ANGELES CORONER EXPERIENCE
Dr. Lakshmanan Sathyavagiswaran MD, FRCP(C)FCAP,FACP, Chief Medical Examiner-Coroner/Interim Director, Department of Coroner, Clinical Professor USC Keck School and UCLA Geffen School of Medicine Los Angeles County, California, USA

Instructional Goals of Presentation:

  1. Coroner law mandate, including local practices for accepting Coroner cases in Los Angeles County.
  2. Team approach in medico-legal death investigation.
  3. Learn the benefits of autopsies in evaluating natural and unnatural deaths.
  4. Attendees will understand the importance of consistently taking DNA specimens during medico legal death investigation and autopsy.
  5. Attendees will understand the importance of following procedures as they relate to evidence collection/submission and maintenance of chain of custody.

Coroner law mandate including local practices for accepting Coroner cases in Los Angeles County, interesting cases emphasizing teamwork, scene investigation, appropriate consultation in medico legal death investigation will be discussed. The investigation of various long-unsolved murders in Los Angeles County using modern forensic science techniques, especially recent advances in DNA, will be discussed. Case examples will be used to discuss the challenges involved, including verification of identification, retrieval of records and photos, authentication of physical and sexual assault evidence, chain of custody documentation, need for following procedures, and maintaining an archive of old procedure manuals including testing techniques/standards. The need to withhold information from family members in the interest of not jeopardizing an investigation is paramount.

Challenges in handling high profile cases including Trial techniques used by the Chief Medical Examiner-Coroner emphasizing interagency cooperation/scene visits to the scene of death so that environmental factors like location of plants, size of area possible confrontation, and location and position of bodies when found all could be evaluated will be discussed. This allowed for proper correlation in opining injury patterns observed on the decedents.

Challenges of intense media scrutiny due to high profile nature of the case will be discussed i.e. all requests were coordinated through the office of the Chief Medical Examiner-Coroner. This will include evaluation of evidence by experts, release of autopsy reports, photographs, all department manuals, and specimens to both prosecution and defense. There was also intense competition among media, with each of them competing to get the “scoop of the day” for the prime time news.


DO YOU USE THE DUQUENOIS TEST? WHICH ONE?
Hiram Evans

The vast majority of forensic laboratories utilize the Duquenois test as part of their analytical method for the identification of marijuana. There are, however, at least five “Duquenois” tests, each incorporating slightly different extraction techniques and/or mechanical applications (e.g., spot plates, test tubes). Laxity in nomenclature allows challengers to call into question the specificity of the technique and muddles communication among forensic scientists. Each of the five variations of the “Duquenois Test,” Duquenois-Negm, Duquenois-Mustafa, Dueuenois-Mustapha, Duquenois-Levine, and de Fauber Maunder Modification of Duquenois-Levine will be presented along with literature references to each variation.


DESIGNER DRUGS 201
Nathan Lind, LA County Sheriff's Department, Controlled Substances Section

Research chemicals, AKA synthetics, have been sweeping across the globe in recent years. Originally born for legitimate research ideals, these drugs have become the norm for individuals looking to achieve the perfect legal high. In many states and countries, these drugs have little to no restrictions yet show frightening consequences at different levels of abuse. The large percentage of abusers appear to be younger individuals looking to extend long nights of partying, dancing and socializing without the repercussions of being in possession of a controlled substance. These synthetic drugs are first developed and tested in the European social scene as club drugs used to sustain long nights of raves and parties. After continued use, these drugs find their way to our shores, streets, bars, clubs and schools. They can be easily purchased from internet venders, head shops or other nontraditional means of purchase, i.e. street chemists.

Identification of these designer drugs is often a difficult and daunting task. Much like technology of today, these drugs can change and reconfigure under the whim of any street chemist. Their goal is always to stay one step ahead of law enforcement’s attempts to clamp down and regulate these synthetic substances. Given the ever changing appearance of these drugs, identification can become a difficult process to obtain reference samples needed to positively identify these dangerous substances. Synthetic drugs are designed to mimic naturally occurring or other known narcotic families. Synthetic cannabinoids (spice, K2) have been found to give effects similar to delta-9 tetrahydrocannabinol by interacting with the same body receptors that delta-9 THC bind with. The substituted cathinones (bath salts) have been found to be a cross between methamphetamine and/or cocaine use. However, these synthetic drugs have a more dangerous and sinister effects give the many observable adverse reactions that users face which include excited delirium and in some cases death. Much of these can be attributed to not just the designer drug abused but also from the fact that there is no quality control associated with the production and distribution of these chemicals.


MICROCRYSTALLINE TESTS - UPDATING A CLASSIC TECHNIQUE
Leonie Elie, Mathieu Elie, Dr. Ruth Croxton, Dr. Mark Baron, University of Lincoln, UK

Microcrystalline tests are rapid, non-instrumental tests using the formation of specific crystals on a micro scale for identification of compounds when mixed with a chosen reagent. They have been used for decades aiding analysis of seized samples of suspected drugs of abuse. Due to the nature of the test, a definite yes or no answer is always provided when a test is carried out. In principle, an unknown powder sample is mixed with water and a small volume of this solution is applied to a microscope glass slide. A reagent is then added and microcrystal formation is observed using optical microscopy. Results are compared to microcrystals obtained with standards, which are carried out alongside. With the proliferation of novel psychoactive substances as legal high products on the internet and the subsequent attempts to control these, quick and conclusive tests for forensic drug analysis are required. The authors have recently published microcrystalline tests for three relatively novel substances, mephedrone, MDAI, and benzyplpiperazine; and research to find new tests for emerging substances is constantly on going. It was proven that microcrystalline identification is possible on legal high samples purchased over the internet, which are often mixtures of active ingredients and cutting agents such as caffeine and benzocaine. Furthermore, a novel method of performing microcrystalline tests with reagents integrated into capillary tubes will be presented. Results show that using capillary tubes instead of glass slides neither affects size and crystal shape nor habit and detectability but improves portability and minimizes cross-contamination. Additionally, a simple but effective method of preservation of microcrystals has been developed to aid long-term storage. Modern imaging technology was used to improve the quality of micrographs taken throughout the observations. Multiple images of different focal planes of samples were taken with a digital SLR camera mounted to a microscope. Images were combined using software-based stacking techniques, which greatly enhanced the depth of field allowing better interpretation of the micrographs obtained. The presented material provides an update on microcrystalline research and presents new developments on the technique that should assist Forensic Service providers in improving routine procedures of microcrystalline testing as well as aid with long-term storage and preparation of evidence.


MICROCRYSTALLINE TEST-RAMAN SPECTROSCOPY:A NOVEL HYPHENATING APPROACH FOR THE DETECTION OF DRUGS OF ABUSE
Leonie Elie, Mathieu Elie, Dr. Gareth Cave, Dr. Ruth Croxton, and Dr. Mark Baron. University of Lincoln, UK

Microcrystalline tests (MT) are a powerful non-instrumental technique for the analysis of bulk samples of drugs of abuse. Despite being largely used as screening tests to gain preliminary results, they hold higher discriminating power than other screening techniques ultimately providing identification of active ingredients. The formation of microcrystals is a separation of molecules from the sample matrix. This was proven by analysing microcrystals by Raman micro-spectroscopy and comparing results to those obtained from standards. The crystal lattice cell unit was determined by X-ray diffraction confirming the presence of only drug and reagent and therefore making the microcrystals highly specific. Model mixtures of two commonly found cutting agents with novel psychoactive substances (NPS) were analysed using microcrystalline test – Raman spectroscopy (MT-RS) and spectra obtained from mixture crystals were similar to those of pure standards proving the separation of molecules by forming microcrystals. Additionally, microcrystalline tests are very powerful when used for the separation and identification of isomers. The differentiation between optical isomeric forms of amphetamines and methamphetamines is well published, and it indicates that results can be transferred to other isomeric forms. NPS are cleverly designed molecules, which often have multiple forms of region- and/or stereoisomers. Legislation often specifies the region- and/or stereo-isomeric forms so there is a need for conclusive identification of the isomeric form present. Microcrystalline tests offer a solution with the development of different crystal habits depending on the molecular structure. In combination with a consecutive analysis by Raman spectroscopy, an identification and confirmation of a psychoactive substance can be achieved. Microcrystals formed between drugs of abuse and their corresponding reagents are the result of addition reactions where drug and reagent form a complex. The bond between drug and reagent molecules can be broken easily by adding solvent subsequently dissolving the crystals and releasing the drug molecules back into solution. Solvents in which the analyte has optimum solubility are the most appropriate to choose. MTs were coupled with GC and LC-MS creating simple offline methods to screen and separate, identify, confirm and quantify drug samples and legal high products. Therefore, microcrystalline tests are reversible and non-destructive in the sense that the analyte molecule can still be detected by other techniques even though the sample may be contaminated with reagents. The recycling of samples could be of use where seized samples have been insufficient to carry out confirmation tests.


THE PAT TILLMAN CASE
Michael Kelley

Corporal Patrick Daniel "Pat" Tillman was an American Football Player who left his professional career to enlist in the United States Army in June 2002 in the aftermath of the September 11 hijacking of US commercial airliners and subsequent attacks. Pat Tillman joined the United States Army Rangers and served several combat tours in Iraq and Afghanistan before he was killed by friendly fire in the mountains of Afghanistan on April 22, 2004. In order for Pat Tillman to be awarded the Silver Star medal, the Army at first reported that Tillman had been killed by enemy fire, and Lieutenant General Stanley A. McChrystal approved the award of a Silver Star. As a result of the incorrect reporting of Pat Tillman’s death, the Army launched a couple of investigations, the earliest of which fell short of true fact finding investigations. The Department of Defense ordered a final on-site investigation by Army Criminal Investigation Command (CID) special agents and forensic investigators in order to fully document the events surrounding Ranger Tillman's death and to assess the actual cause of death.


POSTER SESSION

OPTIMIZATION OF A PROBE CAPTURE/NGS ASSAY FOR SEQUENCING THE WHOLE MITOCHONDRIAL GENOME OF LIMITED AND DEGRADED SAMPLES
Daniela Cuenca1,2, Valerie McClain1,2, George Sensabaugh1,3, D.Crim., Cassandra Calloway2, PhD

1. University of California Davis, Davis, California
2. Children’s Hospital Oakland Research Institute, Oakland, California
3. University of California Berkeley, Berkeley, California

Samples containing degraded and limited amounts of DNA are often encountered in forensic casework. The application of mini STR and bi-allelic SNPs have increased the success in analyzing these samples. However, these methods target nuclear DNA, and although they offer high discrimination power, degraded samples often cannot be effectively analyzed. Sequence analysis of the hypervariable regions of mitochondrial DNA (mtDNA) can be beneficial in these cases because the mtDNA copy number per cell is much higher than nuclear DNA, but it has limited discrimination power. We have developed a probe capture/next generation sequencing (NGS) approach that allows sequence analysis of the entire mitochondrial genome in degraded and limited samples, increasing the discrimination power of mtDNA analysis. This assay has the potential for improving the analysis of degraded DNA because the probe capture step circumvents the inefficiency of PCR on highly degraded DNA. We have further optimized the assay focusing on DNA fragmentation to adapt it for the use of limited and degraded samples, and the results are presented here.

The Probe Capture/NGS assay is divided into three main steps: library preparation, probe capture, and 454 NGS sequencing. A critical step for the quality and success of the entire assay is the preparation of the sample library. The purpose of the library preparation step is to produce unbiased DNA fragments of a specific size. Nebulization and enzymatic fragmentation are two methods that have been successfully used in a research setting. However, they are dependent on the quality and quantity of the initial DNA input and are not appropriate for limited or degraded DNA. Therefore, a different fragmentation method needed to be explored.

We explored the Covaris ultrasonicator for DNA library preparation of limited and degraded samples, due to its Adaptive Focusing Acoustic (AFA) technology for mechanical DNA shearing. This technology is concentration independent due to its cavitation bubble mechanism and it was this aspect of the technology that led us to test if it was also quality independent. Naturally and artificially degraded DNA was used to test the Covaris mechanical shearing technology on limited and degraded samples. This DNA shearing method proved to be not only concentration independent but also yielded fragments of uniform size independent of the degree of degradation of the starting sample. The incorporation of mechanical shearing to the probe capture/NGS assay has been tested on limited, partially degraded, and mixture samples and the results will be presented. Successful incorporation of mechanical shearing to our assay enhances its capability to analyze samples found in a forensic setting.

Key Words: Next Generation Sequencing, Probe Capture, Fragmentation, mtDNA, Limited and Degraded DNA.


ENVIRONMENTAL EFFECTS ON THE STABILITY OF SEMINAL FLUID IN LUBRICATED CONDOMS
Erik J. Haw1 and Katherine A. Roberts1 School of Criminal Justice and Criminalistics, California State University, Los Angeles

Recovery of physical evidence is particularly important in incidents of rape because there is substantial reliance on witness or victim testimony in these cases. This study evaluated the detection of acid phosphatase and prostate specific antigen, in addition to assessing cellular morphology, and DNA recovery with samples placed in environments that simulate crime scene scenarios. Seminal fluid was placed into Trojan ENZ® Lubricated condoms and exposed on a soil substrate while measuring the temperature, humidity, and daylight hourly. Each biomarker began to exhibit false positives at: 95 days (acid phosphatase), 63 days (prostate specific antigen), 127 days (cellular morphology), and 30 days (DNA recovery). As a general trend, increasing temperature, humidity, and daylight exposure negatively affected the detection of each biomarker. However, exposure of samples to increasing humidity over short durations improved AP and PSA detection. Lubricated condoms containing seminal fluid that have been exposed to the environment for longer durations may require enhanced methods of recovery.

Key Words: Semen, biomarker stability, environmental conditions, lubricated condoms


THE ANALYSIS OF MARKERS IN FUELS
Kelly Whittingham1, Mark Baron1, Nick Meakin,1 and Tim Wilkinson1
1. University of Lincoln, United Kingdom

Fuel used for heating, cooking, or in self-producing sectors such as arming, is often subsidised by governments. These subsidised fuels are used illegally either in the parent country or after smuggling into other countries. This is major organised criminal activity and is responsible for significant amounts of lost revenue for governments. Some countries, such as the UK, mark subsidised fuel by the addition of a coloured dye that makes the cheaper fuel distinguishable from ‘unsubsidised’ fuel; illegal activity can be detected through the presence of the marker. However, this has led to the criminal act of marker removal using chemical reagents or adsorbents that leaves the subsidised fuel unmarked. These laundering procedures often leave traces of chemicals in the fuel and hazardous by-products are frequently discarded into the environment. Dyes that remain in fuel after removal attempts would render laundering as ineffective and increase detection of the misuse of subsidised fuel. Various markers are being investigated and characterised for this purpose. Markers are subjected to a range of laundering techniques and their behaviour is observed. Raman spectroscopy can be used detect markers in fuel and is now being commercially applied in the field. A portable system using a silver colloid for surface enhanced Raman scattering (SERS) has been developed for at-the-scene analysis. Markers that are selected are required to be stable in fuel and yet must phase transfer from the hydrophobic fuel into an aqueous silver colloid for analysis, requiring contradicting properties.


INVESTIGATIONS ON MIRNA TISSUES MARKERS TO CORRELATE BLOODSTAINS WITH WOUNDS
Cheryl Andersen1, Katherine Scriven1, Amberly Klein1, Stella (Mo) Choi1, and Donald Johnson1

1. School of Criminal Justice and Criminalistics, California State University, Los Angeles

The body of a homicide victim is sometimes removed from the primary scene by the perpetrator to be disposed of elsewhere. The location of the murder then becomes an important fact to establish. Murders often result in significant bloodshed, which can allow investigators to establish the location of the murder based on bloodstain pattern interpretation and knowledge of the victim’s wounds. However, the circumstances of other homicide cases are such that little blood is shed by the victim, or discovered by the investigators, due to the nature of the victim’s injuries or the alteration of the scene by the perpetrator. Additionally, the suspected murder scene is often a place where the victim is known to have a history of physical activities; the suspected murder scene may be, for example, the victim’s residence. The authors have encountered situations in casework where blood from the victim was found at the victim’s residence or frequented locations, but it was in the form of a non -specific bloodstain pattern (a smear for example) and in small quantities. The cause of the bloodshed was not indicated by the blood evidence. Accordingly, the finding of nondescript bloodstains from the victim at the victim’s residence, or place of visit, raises questions as to the relationship of the bloodstains to the crime. The bloodstains may have been the result of the homicide or the result of some prior accidental injury sustained by the victim. The significance of the blood evidence, and therefore the location of the murder, was uncertain in these cases. Furthermore, the authors have encountered situations where blood from the victim was found on an item in the suspect’s possession, but the victim and suspect had a history of physical contact, and they likely shared the item in question. Again, the victim’s blood was present on the item as an uninformative pattern, and consequently, the relevance of the bloodstained item to the case was in question. The blood may have been related to prior activities of the victim or the blood may have been related to the homicide; the size, shape, and distribution of the bloodstains on the item were consistent with both possibilities.

In this proof-of-concept study, we examined a molecular approach to correlate bloodstains with injuries using the rat as a model. Specifically, investigations were conducted on the rat brain marker, rno-miR-124-3p, with the QIAGEN miScript System and real-time PCR analysis. Rno-miR-124-3p was detected in brain homogenates diluted 100,000 times; in 3 week old, room temperature stored, simulated brain-blood stains; and in bloodstains from head gunshot wounds collected with swabs and subsequently frozen for 9-18 months; however, rno-miR-124-3p was not detected in whole blood. Proof-of -principle was demonstrated by the ability to distinguish bloodstains from a gunshot wound to the head versus bloodstains from a gunshot wound to the chest, by the testing of otherwise identical bloodstains from the two patterns.


EVALUATION OF THE OXYVU IMAGING SYSTEM AS A METHOD TO DETERMINE TIME OF DEATH AND THE AGE OF BLOODSTAINS
Angela Bittrolff1, Vanessa Esparza1, and Donald Johnson1

1. School of Criminal Justice and Criminalistics, California State University, Los Angeles

This research investigated the use of the HyperMed OxyVu Hyperspectral Imaging System as a method to determine time of death and the time since deposition of bloodstains. The OxyVu Imaging System combines spectroscopy with remote sensing technologies to measure dermal oxyhemoglobin (oxy) and deoxyhemoglobin (deoxy) levels. The instrument is used in clinical medicine to non-invasively assess the viability of the superficial dermis. Current methods used to determine time of death (TOD) in medicolegal investigations are overall inaccurate and unreliable, and many methods required invasive procedures and laboratory instrumentation. The limitations in TOD estimates have prevented the successful resolution of criminal cases. In the TOD study, nine amputation specimens were examined from diabetic patients at the Olive View-UCLA Medical Center. The time of amputation for each specimen was known and represented the time of death. The best specimen in size and condition included the first and second digits of a right foot. The larger size of the specimen permitted imaging over several ~ 1 cm diameter sites, and the calculated oxy/(oxy + deoxy) values for the sites were then averaged for each time point. The results showed a linear relationship (R2 = 0.9374) between the oxy/(oxy + deoxy) values and the time elapsed after amputation over a 58 hour period. The OxyVu Imaging System was additionally investigated as a method to determine the age of bloodstains. In this study, venous blood was collected in clot tubes from four patients at known times. Each blood sample was used to create bloodstains of known volume and diameter on glass slides. Stains ~17 mm in diameter made from ~15 μl of blood were determined to be optimal for the study. Bloodstains were subjected to three different conditions: room temperature, 37°C, and 4°C environments. The stains were imaged every 15 minutes for the first 1.5 hours, then every 30 minutes for the next 1.5 hours, then hourly for up to seven hours and at least once a day for up to 7 days post-deposition. The oxy/(oxy + deoxy) values for the bloodstains kept at room temperature increased logarithmically for the initial ~6.5 hours, then decreased in a polynomial pattern. Moreover, the effect was found to be temperature dependent. The OxyVu System is a non-destructive and easy to use technology, and a handheld instrument is now available. The results of both preliminary studies are encouraging, but further research is needed to fully evaluate the application of the OxyVu technology to time-of-death and time-since-deposition determinations.


X-RAY PHOTOELECTRON SPECTROSCOPY (XPS) CHARACTERIZATION OF FIBER SURFACE MODIFICATIONS FOR FORENSIC SCIENCE APPLICATIONS
Robert Blackledge1 Katherine A. Roberts2, and Chistopher Deeks3

1. Retired, Naval Criminal Investigative Service Regional Forensic Laboratory, San Diego
2. School of Criminal Justice & Criminalistics, California State University, Los Angeles
3. Thermo Fisher Scientific, United Kingdom.

Single fibers epitomize the desirable characteristics of trace evidence in as much as they are readily transferred and retained but are invariably difficult to detect by the unaided eye. This poses a challenge to efforts by the assailant to remove any extraneous fibers from a particular substrate. Further, individual fibers may exhibit distinct properties that can be highly probative. However, the current analytical techniques employed by forensic laboratories to analyze fibers do not exploit certain characteristics associated with recent advances in the textile industry. This point is best-illustrated using cotton as an example. Cotton fibers continue to be a common source for clothing materials; these fibers are renewable, biodegradable and readily available. However, from a forensic perspective, the ubiquity of un-dyed cotton fibers also diminishes their probative value.

In recent years, the textile industry has made tremendous advances in developing new products intended to impart specific performance properties. For example, modifying the surface properties of fibers using nanotechnology can provide high durability to fabrics without impacting breathability and texture. Current nanotechnology permits the deposition of extremely thin films on a fiber surface; consequently, an analytical method capable of characterizing the outer 2-10 nm of the sample surface is necessary in order to detect minor qualitative and/or quantitative differences in the chemical composition of the film. The “proof-of principle” research presented here evaluated X-ray photoelectron spectroscopy (XPS) as an analytical method to discriminate fibers cotton obtained from different manufacturers. The basis for this discrimination is the chemical characterization of surface modifications deposited on the fibers.

The results demonstrate that differences in elemental concentrations distinguish individual cotton fibers with a surface modified treatment from untreated fibers. Further, the XPS data distinguishes between two fibers with the same chemical surface modification that were prepared using different deposition techniques (wet v. plasma processing). If XPS technology is as beneficial as our preliminary data indicate, we propose that this analytical approach may herald applications for other sources of trace evidence.

Key Words: X-ray photoelectron spectroscopy, textile fibers, surface analysis, surface modifications, nanotechnology


INVESTIGATIONS ON THE UV-VIS ABSORPTION SPECTRUM OF HEMOGLOBIN IN POSTMORTEM RAT BLOOD SAMPLES AS A METHOD TO DETERMINE TIME OF DEATH
Helen Wolcott1 and Donald Johnson1

1. School of Criminal Justice and Criminalistics, California State University, Los Angeles

The purpose of this research was to evaluate the hypsochromic shift of the hemoglobin Soret band as a method to determine time of death. Four experiments were conducted to examine the Soret band shift over time, the degree of inter-subject variation, the precision of the technique, and the behavior of liquid blood in situ and as dried stains. Using three different erythrocyte lysing methods, blood from nine euthanized rats and samples drawn from a living human subject, were tested at different times with an Implen NanoPhotometerTM P 300 to obtain an absorption spectrum. The three lysing reagents were 0.2 M Tris-HCl [pH = 8.0], 1% SDS Detergent, and Nanopure water. The Tris-HCl buffer samples were first either frozen or dried and then extracted to assist in the lysing of the erythrocytes. The first three experiments utilized six of the euthanized rats, while the fourth experiment utilized the last three euthanized rats and the human samples. For the first three experiments, cardiac blood was collected from multiple rats from the time of death to up to 84 hours after death. There was no significant change in the λmax of the Soret band at the measured postmortem intervals. Also, the results from the six rats were consistent with each other and showed there was little to no intersubject variation. Furthermore, the method was determined to be precise between the duplicated samples. The fourth experiment compared liquid blood samples obtained directly from the rats versus rat and human bloodstains. The results from liquid blood samples were not significantly different from those of the previous experiments. Conversely, the rat and human bloodstain samples showed a small decrease in the λmax of the Soret band over time. Furthermore, Nanopure water was found to be the best lysing reagent in that it produced clean spectra and demonstrated the best examples of the hypsochromic shift. In conclusion, this research showed that the hypsochromic shift of the Soret band does not occur at a rate to be useful as a time of death indicator; however, the research does support previous findings that this phenomenon could be used to estimate the age of bloodstains.


Mitochondrial DNA Analysis of Complex Mixtures and Heteroplasmy Using 454 Next Generation Sequencing
Hanna Kim1, B.A. and Cassandra D Calloway, Ph.D1.

1. Children’s Hospital & Research Center Oakland

Mixed DNA samples are often encountered in forensic cases and pose great interpretational challenges particularly with mtDNA analysis. The standard approach is to analyze the hypervariable regions (HVI/HVII) of the mitochondrial genome using Sanger sequencing. However, Sanger sequencing has limited sensitivity in minor component detection (~10-20%) of mixture samples and difficulty separating components in a mixture. Discerning sequence haplotyes in a mixture using this approach is challenging because peak heights don’t always reflect the true component ratio. Given the limitations associated with the current standard method, there is an immense need for the development of an improved system for mixture analysis in the field.

To address this need, we have developed a “front-end” duplex PCR assay targeting the non-coding HVI and HVII regions of the mitochondrial genome for use with the 454 next generation sequencing (NGS) technology. The assay uses eight sets of duplex multiplex identifier (MID) tagged fusion primers optimized for forensic applications and tested for low input sensitivity. These primers can be used to generate 64 different combinations of forward and reverse tagged primer sets. Use of fusion primers consisting of the MID, 454 library key, and target specific sequence enable us to prepare ready-to-sequence libraries with one PCR step while the combinatory approach of the assay enables us to sequence 64 different samples in a single run. The “clonal sequencing” (amplification of a single DNA fragment) aspect of the NGS technologies enables separation and characterization of individual components of a mixture which allows for haplotype identification of each composequence. These technologies offer a greater sensitivity for low level mixtures as well as a quantitative estimate of the relative proportions of DNA from different contributors of a mixture due to their ability to massively parallel sequence each DNA molecule with high read depths.

Mixture detection sensitivity of the assay was determined by testing artificially mixed DNA samples with various composition ratios, mixtures composed of 3-5 individuals from four different population groups, and heteroplasmic samples. Preliminary results show that the HVI/HVII 454 NGS assay has increased sensitivity for detecting low level mixtures (~1%) with a read depth of 500-1000. These results show that multiple contributors to a complex mixture can be detected and mtDNA sequence haplotypes can be distinguished. Additionally, the detection of low level heteroplasmy well below the limits of current methods further shows the high sensitivity of the assay. In conclusion, these results show that the HVI/HVII 454 NGS assay has higher sensitivity and resolution compared to the standard method and can be applied to the analysis of complex mixtures encountered in forensic cases.

Key Words: mtDNA, mixtures, Next Generation Sequencing


COLLECTION OF SEXUAL ASSAULT EVIDENCE FROM A TOILET
Alex White, Samantha Saitman, and Ruth E Ballard, Ph.D.; California State University, Sacramento; Forensic Biology Program

After sexual assaults in California, victims are given a Sexual Assault Forensic Examination (SAFE). During this exam, a forensic nurse or physician takes a history of the patient, including information about post-assault hygienic activities. A typical time between an assault and a SAFE is 6-12 hours, and most victims report that they have not showered or changed their clothes during the interim. However, most report that they have urinated. The activity of urinating, wiping, and flushing a toilet is a concern because semen or saliva from the perpetrator could be inadvertently discarded. In addition, there is no research indicating whether it is possible to retrieve biological evidence from a toilet and crime scene investigators do not routinely attempt to do so. Our project tested whether it is possible to collect sexual assault evidence from a toilet. We stained toilet paper with small amounts of semen, allowed it to soak in a clean toilet bowl for 60 seconds and then flushed the toilet. We then waited either 6 or 12 hours and collected the toilet contents (water) and took swabs of the inside surface of the bowl. We were able to get DNA from the semen donor at both time intervals, presumably because some of the sperm in the semen stain were agitated back up into the water as the toilet refilled after flushing. These results are surprising and show that toilets are a possible source of DNA evidence in sexual assault cases. Indeed, in instances where the victim has urinated and wiped, a toilet may be the only source of DNA evidence to solve the case.


IDENTIFICATION OF SPERM IN SEMEN-VOMIT MIXTURES
Jonathan Charron, Raymond Tan, and Ruth E Ballard Ph.D.; California State University, Sacramento; Forensic Biology Program

After forced oral copulation, victims often vomit. Currently, forensic analysts use a nuclear fast red and picroindigocarmine staining procedure (commonly called the "Christmas Tree Stain") to screen for sperm in semen-vomit mixtures. Recently, a new sperm detection method was developed that is more sensitive than Christmas Tree staining. This method, called Sperm Hy-Liter, uses a monoclonal, fluorescently-labeled antibody that recognizes human sperm heads. The efficacy of this system has been well-studied in sperm/epithelial cell mixtures (which are frequently found on vaginal swabs from rape victims). However, the system was recently shown to fail in the presence of fecal material. To further this research, our study examined how well the system detects sperm in the presence of vomit. We collected sperm from a donor and incubated it with human vomit for various lengths of time. We then performed sperm counts using either light (Christmas Tree stain) or immunofluorescent (Sperm HY-Liter) microscopy to compare the two systems. We found that vomit interferes with the Sperm Hy-Liter system. We are currently attempting to rescue the system by raising the pH of the semen-vomit mixtures prior to exposing it them to the antibody. Our theory is that the acidic nature of gastric fluids interferes with the attachment of the antibody to the sperm head and that activity can be restored by raising the pH to physiological levels (7-8).


CAN DNA BE TRANSFERRED BETWEEN SWABS IN A SWAB DRYER?
Cindy Palomar, Sing Chau, and Ruth Ballard Ph.D.; California State University, Sacramento; Forensic Biology Program

During Sexual Assault Forensic Examinations (SAFEs), swabs are collected for DNA. Such swabs, especially those taken from body cavities, contain copious amounts of bacteria, and must be dried thoroughly to prevent bacterial degradation of the human cells (and DNA). In California, this is achieved by drying them in a swab drying box upright, next to one another but not touching, in the presence of a gentle airflow provided by a fan. Current guidelines forbid drying swabs from the victim and suspect at the same time but allow swabs from different areas of the victim to be dried together. However, cross-contamination between swabs taken from the same person could mislead an investigation, and there is no research showing whether cross-contamination is possible/probable. Our study examined whether swabs stained with semen or saliva can cross-contaminate sterile swabs while drying in a swab dryer. Six different spatial set-ups were tested and cross-contamination was assessed after drying by DNA analysis. We found no evidence of cross-contamination during our experiments, suggesting that DNA transfer between swabs in a swab dryer is unlikely and that current practices are probably sufficient to protect the integrity of swabs collected during SAFEs.


FORENSIC BIOLOGY EDUCATION AT SACRAMENTO STATE: A SCAFFOLD MODEL
Kelly Beeson, Chelsea Oga, Sara Tribble, and Ruth Ballard Ph.D. California State University, Sacramento; Forensic Biology Program

No Abstract Submitted.


A REEXAMINATION OF THE UTILITY OF SWAB DRYERS IN THE PRESERVATION OF SEXUAL ASSAULT EVIDENCE
Tak Hou Fong, Edward Panacek MD MPH, William Green MD, and Ruth E Ballard PhD University of California, Davis

The California standard for preserving swabs collected during a sexual assault examination is drying them for 60 minutes in a “swab dryer” (room temperature in an enclosed box in the presence of a fan). The swabs are then packaged in paper or cardboard for transport to a crime lab for serological and/or DNA testing. Drying swab in a swab dryer s is time-consuming for the forensic examiners as they must remain in the room to maintain chain-of-custody. However, there is no published research demonstrating that drying sexual assault swabs in a swab dryer is necessary or whether the current drying conditions (time and temperature) are optimal for preserving evidence. This study compared the quantity and quality of human DNA generated from mock sexual assault swabs saturated with either saliva or semen and air-dried overnight (no swab dryer) or dried in a swab dryer. Three different drying times (15, 30, and 60 minutes) and three different temperatures (ambient, 37 deg C, and 56 deg C) were examined for the swab dryer. The study also examined how different drying conditions affected swab dehydration as measured by water loss during the drying process. Our results indicate that swabs dried under standard conditions (60 minutes, swab dryer) retain a mean of 40% of their water and are not maximally dried until after they have been packaged and allowed to air dry for several hours. By contrast, swabs dried at higher temperatures (37 deg and 56 deg) are completely dry in less than 60 minutes. In addition, swabs that are air-dried only (no swab dryer) yield the same amount of DNA as swabs that are dried in a swab dryer (at different temperatures and for different times) and then allowed to air-dry. DNA quality was likewise unaffected by drying method; all samples yielded full STR profiles with no sign of degradation. Because the swabs were thoroughly saturated with semen or saliva prior to drying, our study mimicked the wettest swabs likely to be gathered from sexual assault victims. Therefore, our results suggest that drying swabs in a swab-dryer may not be necessary under most casework conditions


FAST GC-MS OF ‘LEGAL HIGHS’
Mathieu Elie, Leonie Elie, and Dr. Mark Baron
University of Lincoln, School of Life Sciences, Brayford Pool, Lincoln, UK

In recent years the availability of so called legal highs over the internet has hugely increased. Numerous online legal high retailers market a broad variety of products which are advertised as research chemicals, bath salts or plant food although clearly intended for human consumption as recreational drug replacements. No guidelines exist as to what is sold and in what purity. Consumers are lead to believe that purchased goods are entirely legal. The continuous appearance of novel psychoactive substances (NPS) in legal high products presents a challenge for the routine analytical laboratory.

Methods using fast gas chromatography mass spectrometry (fast GC-MS) are now replacing conventional capillary column GC methods. Here we present a rapid screening method for NPS analysis using fast GC-MS. Twenty-three analytes, including 5-iodo-2-aminoindane (5-IAI), 1-(thiophen-2-yl)-2-methylaminopropane (MPA), 1-benzylpiperazine (BZP), 4-methylmethcathinone (mephedrone), 5,6-methylenedioxy-2-aminoindane (MDAI) and methoxetamine (MXE) were separated within 4 min. The method was used to analyse 35 Internet and head shop purchased legal high products with the successful identification of their active ingredients.

Legal high products do not always contain their stated ingredients. Of the group of products purchased as 5-IAI not one contained 5-IAI with several containing mixtures of substances either already controlled in the UK or under consideration by the Advisory Council on Misuse of Drugs (ACMD).

The low bleed and high inertness of the chromatography column used ensured clean high quality mass spectrometry data which when combined with the short run time resulted in an efficient tool for NPS screening, even when standards were unavailable. Electron impact and chemical ionization mass spectra used in combination for the identification of unknown NPS are presented.


INVESTIGATION INTO THE SUITABILITY OF CAPILLARY RUBES FOR MICROCRYSTALLINE TESTING
Leonie Elie, Mathieu Elie, Dr. Ruth Croxton, and Dr. Mark Baron; University of Lincoln, School of Life Sciences, Brayford Pool, Lincoln, UK

Microcrystalline testing is a rapid technique for identifying drugs of abuse in forensic casework. Microcrystalline tests are conventionally carried out on simple glass microscope slides. A small volume of dissolved sample or a few grains of solid sample are applied onto the slide and mixed with an equal volume of a specifically chosen reagent of suitable concentration. After seconds to minutes, microcrystals grow within the applied droplet and can be observed under a microscope. Positive identification of a substance is made by comparing habit, shape and colour to reference standards tested alongside the samples with the same reagent. Pictures and videos of the microcrystals are recorded and can be used as permanent records to be presented in Court.

Despite the apparent simplicity of the test set-up which theoretically makes the technique favourable for rapid on-site testing, limited portability has always been a hindrance and seized samples are typically field tested with non-specific presumptive colour tests before being sent off to laboratories for further analysis if found suspicious. The specificity and discrimination capacity of microcrystalline tests could reveal interesting results in the field and could facilitate crime scene sampling for law enforcement forces.

Three different microcrystalline tests were selected to demonstrate that results obtained can be applied to a wider range of microcrystalline test types and drugs using similar reagents. Firstly, each of the tests uses a different reagent to form microcrystals with mephedrone, cocaine and phencyclidine (PCP). Secondly, each test presents a different crystal growth speed and very different crystal shape and habit: mephedrone-reagent crystals form slowly as colourless blades often joined in paddlewheels or rosettes while cocaine-reagent crystals rapidly grow as skeletonised feathers after mixing drug and reagent, and PCP-reagent crystals develop slightly slower as purple razor blades and crossed squares. Finally, the choice of drug was influenced by the recent Advisory Council on the Misuse of Drugs report stating that with the constant flow of novel psychoactive substances (NPS), so called “legal highs”, all three drugs chosen remained of interest either as direct precursors or structurally related substances.

The study examined the suitability of flat capillary tubes for microcrystalline tests, described the handling of the tubes and the effects of the confined space on crystal growth and limit of detection. It also explored the possibility of seeding the tubes with reagents prior to testing and investigated the preservation of microcrystals. In the end the technique was tested on a range of six online purchased legal high products identifying active ingredients using microcrystalline tests in flat capillary tubes and comparing results to those obtained with gas chromatography mass spectrometry (GC-MS).