78th SEMI-ANNUAL SEMINAR (Fall 1991)
CALIFORNIA ASSOCIATION OF CRIMINALISTS
October 17-19, 1991
A PCR-BASED SYSTEM FOR DETERMINING THE QUALITY OF FORENSIC DNA SAMPLES
Rebecca Reynolds, Cetus Corporation, 1400 53rd Street, Emeryville, CA 94608
The quality of a DNA sample determines, in large part, the success of amplification of both sequence and length polymorphisms by the PCR. Samples containing inhibitors of Taq polymerase or significantly degraded DNA are amplified less efficiently than samples containing highly purified, high molecular weight DNA. In addition, amplification of AMP-FLPs using degraded DNA can lead to ambiguous or erroneous types. Clearly, to obtain the most discriminating information from a sample, it is important to assess the quality of the DNA prior to analysis. A system for determining the quality or "amplifiability" of a sample will be discussed in addition to a DNA sample analysis strategy.
DEVELOPING RACIAL PROFILES FROM BLOODSTAINS AS A TOOL FOR INVESTIGATIVE LEADS
David C. Stockwell, San Bernardino Sheriff's Forensic Science Laboratory, 200 S. Lena Rd., San Bernardino, CA 92415-0056
One concern of law enforcement investigators is describing the physical attributes of suspects. Glance at a police report face page sometime and the cardinal descriptions are readily apparent: race, gender, and age. Forensic serologists also deal in determining physical attributes of suspects, however our descriptions, based on genetic markers, are generally of no use in describing the visible characteristics which the investigators use. In the past my laboratory has used such genetic markers as Peptidase A, Carbonic Anhydrase II, Glucose-6-phosphate Dehydrogenase, Hemoglobin, Haptoglobtn, and Transferrin in an attempt to determine the racial origin of a bloodstain. Each of these markers pose the same problems: 1) they are racially discriminating only in the black population, and 2) the frequency of the racially significant allele in each system is quite low, providing few "hits" even when the bloodstain did originate from a black person.
A different approach to this problem is to seek out genetic markers which show a skewed phenotypic frequency between populations while maintaining a high probability of discrimination among all races. By combining such markers as Group Specific Component (Gc) subtype, Gamma marker (Gm), Kappa marker (Km), and HLA DQa the individually skewed phenotypic frequency may provide a combined profile which has a higher frequency of occurrence in one race as compared to the other races. This paper will also introduce a method for evaluating genetic markers which will be useful in this type of testing. Finally, I will discuss how this technique has played a part in several investigations.
RE-EVALUATION OF A RABBIT ANTI-GOAT/ALKALINE PHOSPHATASE (RAG-ALP) CONJUGATE METHOD FOR DETECTING GROUP SPECIFIC COMPONENT GO AFTER ISOELECTRIC FOCUSING
David C. Stockwell, San Bernardino Sheriff's Forensic Science Laboratory, 200 S. Lena Rd., San Bernardino, CA 92475-0056
The detection of Gc proteins using the RAG-ALP method of Pfiug has been reviewed at a previous seminar [White, 1988]. At that time he noted a selective lessening of the Gc 2 band intensity in relation to the 1 F and IS bands. In his blind trial, 1 of 51 samples was incorrectly typed, presumably due to this artifact. White concluded that the RAG-ALP method should only be used if the conventional Gc type is known. The increase in sensitivity of the RAG-ALP method over conventional Coomasie Blue or even silver staining warrants a second look at this method. This study was designed as a comparison test between Gc subtyping using the RAG-ALP detection method and conventional Gc results utilizing Coomasie Blue and silver staining. Gc subtyping was performed using the isoelectric focusing method of Kuo, followed by the RAG-ALP detection method of Pflug. Conventional Gc results were obtained using Group III methodology [agarose gel electrophoresis, Tris glycine at pH 8.4]. Concordant results were obtained between the 221 blood samples tested. Of these, 79 were conventionally typed as Gc 2-1. No selective lessening of the 2 band intensity was observed in these samples. Population frequencies for whites (N=209), blacks (N=108), and Hispanics (N=108) were also determined.
|Allele frequencies are:|
|Whites: ||1 F = .163 ||1 S = .577 ||2 = .256 ||Other = .009 |
|Blacks: ||1 F = .639 ||1 S = .190 ||2 = .148 ||Other = .037 |
|Hispanics: ||1 F = .267 ||1 S = .523 ||2 = .208 || |
A SYSTEMATIC STUDY OF THE EFFECT OF VARIOUS ENVIRONMENTAL ABUSES ON RFLP AND PCR ANALYSIS OF FORENSIC SAMPLES
Norah Rudin, Kenneth Konzak, Lance Gima, Lisa Brewer, Martin Buoncristiani, Michele Home, Keith Inman, Maosheng Ma, Mary Pierson, Gary Sims, Jan Bashinski;
California Department of Justice DNA Laboratory, c/o Lawrence Berkeley Laboratory, Mail Stop 934-47A, Berkeley, CA 94720
A series of experiments have been designed in order to investigate the effect of various environmental abuses on DNA. Specifically, we address the question of how much variation in RFLP band size estimates or ability to obtain a PCR DQα type, if any, might occur before non-detectabillty. Samples were exposed to environmental conditions commonly encountered in casework, including varying. temperatures, varying humidities, UV and sunlight exposures, different chemicals and different substrates. Unpreserved blood and semen samples from the same donor were spotted onto cotton cloth or a particular substrate and incubated under specific conditions. Duplicate samples were removed at particular time points and frozen until the DNA was Isolated. Five VNTR loci, in addition to a monomorph, were analyzed in order to evaluate a large range of band sizes. DNA was Isolated from the samples using a procedure that allows aliquots to be taken from both RFLP and PCR analysis. Stains were incubated at -70°C, -20°C, 4°C, 20°C, 37°C and 65°C. Results indicate that degradation of DNA from bloodstains occurs at 65°C after 1 month. This result is most clearly observed on the yield gels, where the degraded DNA can be visualized. On the autoradiograms this translates into a loss of signal. This study continues through a one year incubation time. Sunlight hours were defined as exposure to direct sun. Duplicate controls were wrapped in foil so that the same conditions would be experienced except for the light and all samples were brought indoors and stored at room temperature during the night or cloudy days. Although DNA from bloodstains began to show significant degradation by 48 hours as analyzed on a yield gel, DNA from the longest exposure of 96 hours was still easily typable by RFLP analysis. Exposure to UV light of 254 nm resulted in much reduced yield at 10,000-50,000 joules/m2 but no observable degradation. Previous experiments (Buoncristiani and Sensabaugh) have suggested that DNA is cross-linked to protein under these circumstances, and becomes trapped at the interface of a phenol extraction, which Is part of the DNA isolation procedure. When samples are amplified by PCR and typed using the Cetus HLA DQα Amplitype procedure, reduced yields of product are observed for the more severe exposures of UV, but through 96 hours of direct sunlight and 50,000 joules/m2 of 254 nm UV light, the samples remain easily typable. Stains were incubated at relative humidities of approximately 0%, 37%. 66% and 98% as defined at 20°C. By 1 month, there is clear evidence of discoloration at 66% humidity, and diffusion and mold growth at 98% humidity. Some of the bloodstains that diffused, probably as a result of actual drops of water coalescing at 98% humidity, showed evidence of degradation. DNA from samples held at 66% humidity up to at least one week type cleanly. Even in the degraded DNA from 98% humidity, the main bands were readily visible. This study continues through a 6 month incubation time. The most striking result obtained thus far from samples incubated on different substrates is that the amount of DNA recovered from semen is widely disparate, with fibrous and/or absorbent substrates in particular giving extremely low yields. A "bookkeeping" experiment was performed, and showed that in these cases, most of the sperm remained on the fabric, and therefore never made it into the lysis buffer. This phenomenon is not nearly as dramatic for the bloodstains. These results have led us to revise our differential extraction protocol to include a Proteinase-K digestion of the sample on the actual substrate before attempting to recover the sperm. In addition, two substrates, asphalt and fir wood, showed degradation in DNA from duplicate samples from semen, but not in the equivalent bloodstains. Bloodstain DNA from blue denim, asphalt, and a wool army blanket showed almost complete resistance to digestion by HaeIII. We are in the process of completing these long term experiments and quantifying the results.
DNA TYPING USING THE AMPLITYPE POLYMARKER SYSTEM
N. Fildes, B. S., R. Saiki, B.S., R. Reynolds, Ph.D.; Cetus Corporation, 1400 53rd Street, Emeryville, CA 94608
The AmpliType PolyMarker Amplification and Typing System was developed for use in the genetic analysis of forensic biological evidence. The polymerase chain reaction (PCR) was used to coamplify six genetic loci, including DQα. The additional markers - LDLr, gypA, Ggamma, D7S8 and Gc - are detected using sequence-specific immobilized probes on a single strip in the same manner as DQα. LDLr, gypA and D7S8 are biallellc markers, while Ggamma and Gc are triallelic. To date we have typed 300 individuals from Black, Caucasian and Hispanic populations for all 6 loci and the combined Pd value ranges from 0.9995-0.9997. We have demonstrated concordance between conventional serological gypA (M/N) and electrophoretic Gc (1F/1S/2) types and those obtained using the PolyMarker system. The sensitivity of the system has been established and is equivalent to that of the AmpliType DQα kit. We have examined amplification of the markers individually and as part of the coamplified system and found them to be essentially equivalent. In addition, we have begun validation studies following the TWGDAM-CAC guidelines.
STATUS OF CERTIFICATION AND THE AMERICAN BOARD OF CRIMINALISTICS
Greg Matheson, Los Angeles Police Department, Scientific Investigations Division, 555 Ramirez Street, Space 270, Los Angeles, California, 90012
The current status of certification and the American Board of Criminalistics (ABC) will be presented. Certification by the ABC is approaching the point where a formal program must be adopted. A draft of the current ABC Certification Program will be provided to all attendees. Included in this draft is; a description of the ABC and the certification concept, the general knowledge areas, proficiency testing guidelines, the recertification process, Rules of Professional Conduct, and a survey form. The purpose of this presentation is to keep the profession informed of the progress of the ABC, and to offer a process for direct input by interested parties.
DETERIORATED FORMS OF OVOID PIGMENT BODIES IN HUMAN HEAD HAIR
James G. Bailey, Los Angeles Sheriff's Dept, Criminalistics Laboratory, 2020 W. Beverly Blvd., Los Angeles, CA 90057
Ovoid pigment bodies are microscopical structures often used in the forensic comparison of human hair, but little reported in the scientific literature. The classical description is of an ovoid-shaped mass of melanin pigment ranging in size from 3 to 30 Am. It was recently observed that human hairs which once had classic ovoid bodies, after five years had deteriorated into a collection of smaller, lighter fragments of pigment. These fragments had been observed earlier in casework samples, but had not been recognized as ovoid body fragments. Photomicrographs will be presented showing some of these deteriorated structures.
THE IRUS SCANNING IR MICROPROBE (SIRM)
Dahlia H. Riley, Dr. John Reffner, 480 Lancashire Pl, Pasadena, CA 91103
A system will be described which consists of a research grade optical microscope with an FT-IR modulator integrated within it. Optical Pathlength and number of reflecting surfaces are minimized thus maximizing sensitivity reproducibility and quantitative accuracy. The IRUS SIRM is optimized and prealligned for FTIR microscopy in four analyses modes: Near Normal Reflectance, Transmission, Grazing angle reflectance and Attenuated total reflectance. This speeds up the analysis time of different types of samples. Extensive automation of data acquisition and spectral mapping of specimen, make it possible to measure at multiple points on samples and controls, obtaining data which is more statistically significant. Analysis is faster, more reproducible and done with minimal operator intervention in unattended mode. The IRUS is operated from a 486 PC based workstation and an on board computer which can be unlinked during lengthy experiments retaining full speed and access to all data reduction functions while a mapping experiment is in progress. The software is mouse driven, intuitive and easy to use. It also provides flexible data presentation modules including customizable functions to allow better correlation of spectral data with physical evidence. The system signifi-cantly enhances the performance and throughput of a forensic laboratory.
THE HISTORY OF THE COMPARISON MICROSCOPE; PART III COMPARISON RESULTS AND PHOTOGRAPHS IN THE COURTS
Duayne J. Dillon, P.O. Box 488, Courthouse Station, Martinez, CA 94553
The "Waite Group", despite the publicity received for their adaptation of the comparison microscope to firearms identification, were not the first to apply this technique to a major criminal case. The first court introduction of photographs taken through this instrument, also resulted from the efforts of another researcher. Edward O. Heinrich, a generalist practicing in Berkeley, California, had distinction of initially introducing the comparison microscope to the American criminal justice system. Heinrich overcame fundamental deficiencies associated with basic firearms knowledge, individualization concepts, and equipment hardware, to introduce both the use of comparison microscope for bullet identification and photographs of bullet matches a 1926 California murder trial. Subsequently, he was responsible for the dissemination of his techniques to the pioneers in the field of firearms identification.
CONCEPTS OF ELECTRONIC INFORMATION MANAGEMENT: FURTHER IMPACT ON THE MICROSCOPIST
Stephen A. Shaffer, MicroDataware, 2894 Tribune Avenue, Hayward, CA, 94542
We have all watched or participated in the steady invasion of computers and computer-assisted processes into our analytical laboratories. This process, going on intensely over the last decade, has brought electronic catalogs to infrared spectroscopy, automated data manipulation to gas chromatography, and automated spectral analysis to energy dispersive x-ray spectrometry, among many other developments. The optical microscopist has, until recently, largely been left out of this process, but that is beginning to change. New developments will place vast amounts of highly useful information at the microscopist's fingertips, organized in a manner which makes it quickly accessible and which associates related information. This paper will survey some of the current developments of electronic information management for the microscopist and attempt to prepare the audience for some of the exciting things coming in the near future.
SOLID-PHASE EXTRACTION FOR ACID AND NEUTRAL DRUGS UTILIZING ZYMARK’S BENCHMATE
Manuel J. Munoz, Los Angeles County Department of the Coroner, 1104 N. Mission Road, Los Angeles CA 90033
Solid-Phase extraction is a very efficient, sensitive and rapid technique for drug extraction. At the Los Angeles County Coroner's office, the huge case load forced us to acquire and implement a robotic system, with large sample size capabilities, reproducibility, efficiency, and most important, cost effectiveness. The Benchmate is a relatively new robotic system manufactured by Zymark. During the past three years, at the Los Angeles Department of the Coroner, good results have been produced utilizing solid-phase extractions. The capability of implementing the existing procedure to robotic system was our goal. The Benchmate has analyzed over two hundred coroner's cases; averaging 40 samples per run. Each run employs 4 standards (50, 25, 5 mcg/ml barbiturate mix, and a 3.3 mcg/ml neutral mix). Over the last three months, the Benchmate has achieved high reliability and sensitivity with minimal analyst hands on time. The detection limit by utilizing the GC-MS for acid and neutral drug analyses is 0.6 mcg/ml in the total Ion mode. The majority of the extractions were performed overnight.
ANALYSIS OF INORGANIC PAINT CONSTITUENTS BY SCANNING ELECTRON MICROSCOPY-ENERGY DISPERSIVE X-RAY SPECTROMETRY
Richard S. Brown, Thomas J. Hopen and Tim B. Vander Wood; Millette, Vander Wood & Associates, Inc., 5500 Oakbrook Parkway, Suite 200, Norcross, GA 30093
Paint fillers and extenders are sometimes overlooked when paint fragments and traces are examined in the crime laboratory. Previous work done in this area (Light and Electron Microscopy of Paint Pyrolysis Residue) described how inorganic paint constituents can be characterized. Due to the extreme temperatures produced, a less harsh preparation is desirable. A video tape demonstrating how paint crossections, dispersions and low temperature ashing preparations appear in the Philips 525M scanning electron microscope will be presented.
A STATEWIDE EXPERT TESTIMONY TRAINING COURSE: COURT ROOM PRESENTATION OF EVIDENCE
Louis A. Maucieri, California Criminalistics Institute, 4949 Broadway, Room A 104
Sacramento, California 95820
This paper reports on progress toward the objective of delivering a positive and effective course on expert court testimony. Fashioned after training given to staff of the Arizona Department of Public Safety Labs, a California version was crafted and piloted in May 1991 for students from various agencies. Teaching methods include classroom instruction by communication specialists, group exercises, student presentations, and constructive team evaluations of video recorded testimony. This course enhances the presentation skills for the forensic and law enforcement witness. It differs from the traditional moot court. Witness effectiveness is increased by enhancing positive skills and revealing/modifying subconscious behaviors that impede this goal. This course is certified under POST Plan IV. It is intended for forensic or law enforcement specialists called to give expert testimony.
AUTOMATED GUN SHOT RESIDUE ANALYSIS—YET ANOTHER SYSTEM
Richard S. Brown, Thomas J. Hopen and Tim B. Vender Wood; Millette, Vander Wood & Associates, Inc., 5500 Oakbrook Parkway, Suite 200, Norcross, GA 30093
Dust samples were collected from a local indoor shooting range. All types of particle morphologies and elemental combinations were found. The Philips 525M scanning electron microscope coupled with an ECON IV light element detector and an EDAX PV9900 energy dispersive x-ray spectrometer (SEM-EDS) utilizing HAX software was evaluated for automated gun shot residue analysis. The analysis time, software capabilities and ease of operation were all found to be excellent. GSR analysis from the particle analyst's point of view is ideally suited for automated SEM-EDS due to its high atomic number and its existence as discrete particles. The speed at which area search routines are carried out, as in all automated systems, is dependent on the minimum particle size defined.
SOME THOUGHTS, OBSERVATIONS AND RECOMMENDATION ON GIVING TECHNICAL PAPERS AT SEMINARS
Lucien C. Haag, Forensic Science Services, 4034 W. Luke Avenue, Phoenix, AZ 85019
This slide presentation with accompanying handout will cover the conceptual and practical aspects of developing, assembling and presenting technical papers at seminars.
Once the basic script has been lined out, carefully chosen and properly prepared slides should be made to support the narrative. After sufficient private rehearsals the slides become orderly visual cues for the delivery. Specific techniques for the preparation of quality slides will be illustrated. Common mistakes and procedures for overcoming them will complete this slide presentation.
THE CHARACTERIZATION OF "COLOR RELEASE" LIPSTICKS
R.D. Blackledge(1), M. Fink(2), M. Grieve(3), & B. Garland(4)
(1) Naval Investigative Service Laboratory, PO Box 220, San Diego, CA, 92136
(2) San Diego Sheriff's Laboratory, 3520 Kurtz Street, San Diego, CA, 92110
(3) USACII-Europe, APO, New York, 09757-5272
(4) B. Garland, Nicolet Instruments, 215 Fourier Way, Fremont, CA, 94539
Lipstick traces may be an important form of associative evidence found at crime scenes (especially those involving assaults on women), or found on the clothing of the suspect.
Avon Products has recently introduced a new line of lipsticks called Color Release. These lipsticks have properties that should increase the likelihood that identifiable traces will be left on contact surfaces. Color Release lipsticks have a formulation that includes thousands of microspheres in which color and emollients are encapsulated. Simple acts such as talking and drinking will crush the micro-spheres, thereby renewing the lipstick throughout the day. Color Release lipsticks were examined by several analytical methods including TLC/HPTLC, microspectrophotometry, SEM/EDX, and FTIR. TLC/HPTLC, microspectrophotometry, SEM/EDX, and FTIR provide a good basis for lipstick comparisons and the presence of microspheres appear to make the Avon Color Release lipsticks unique.
POSSIBLE ERRORS IN USING VOIDED URINE FOR PREDICTION OF THE PHASE OF ALCOHOL METABOLISM
Ronald L. Moore, Orange County Sheriff-Coroner's Department, Forensic Science Services, 550 N. Flower St., Santa Ana, CA, 72703
Using an example from a recent alcohol metabolism study conducted by the Orange County Sheriff-Coroner's Forensic Science Services, an argument is presented against using the difference between void and second sample urine specimens to predict the phase or direction of alcohol metabolism, i.e., absorption or elimination phase. In the example, a pair of urine samples seems to show an increasing BAC, thus the absorptive phase, while a BAC curve based on breath tests show both void and second sample to have been collected in the elimination phase. It is concluded using the example for support that since the void concentration depends on behavior (frequency of urination before arrest) that this is too unpredictable on which to base forensic opinions.