California Association of Criminalists

THE ANALYSIS OF DRIED BLOOD SAMPLES BY TANDEM MASS SPECTROMETRY
Gary Davis, CA-DOJ Toxicology Lab, Sacramento, CA

In April 1989, Ramon Salcido, a Sonoma County winery worker murdered several people, including his wife and two of his young daughters. One piece of evidence collected by criminalists from our Santa Rosa regional laboratory was a shotgun shell box which was stained with the suspect's dried blood. 55 milligrams of this dried blood were submitted to our laboratory to determine if cocaine and/or its metabolites were present in the sample. A technique was developed for this analysis which utilized tandem mass Spectrometry (MS/MS). The sample, plus dried blood standards, was reconstituted with saline and extracted at a basic pH with ethyl acetate. After extraction, the samples were derivatized with pentafluoropropionic anhydride. The samples were then analyzed for cocaine and ecgonine methyl ester (EME), an enzymatic metabolite of cocaine, utilizing positive chemical ionization and multiple reaction monitoring. The first quadrupole was set to transmit the molecular ions of cocaine and EME-PFP, 304 and 346 amu respectively. After an argon induced dissociation in a reaction region, the second quadrupole was set to transmit only the daughter ion of both compounds, 182 amu. Cocaine and ecgonine methyl ester were detected in the dried blood sample at approximate concentrations of 100 and 40 nanograms per gram respectively. The work done in this case led to the development of a method for the analysis of blood samples for cocaine and its metabolites in our routine driving under the influence cases.

ETHYL CHLORIDE: POSSIBLE MISIDENTIFICARION AS ETHANOL IN A BLOOD SAMPLE ANALYZED BY HEADSPACE GAS CHROMATOGRAPHY
Pennie Lafferty, Orange Co. Sheriff, Santa Ana, CA

Using head space gas chromatography, ethyl chloride in a blood sample was quantitated as ethanol on three out of six occasions. The component tolerance and component % tolerance parameters on the gas chromatograph were set at 0.020 and 0.500 respectively to establish the acceptable window for the relative retention time of ethanol. Because ethyl chloride and ethanol had similar retention times and relative retention times, the sample fell within the acceptable ethanol range three times. To prevent ethyl chloride from being identified as ethanol, the component tolerance and component % tolerance were changed to 0.010 and 0.400 respectively. Using the new system parameters, mixtures of ethyl chloride and ethanol were identified and quantitated as ethanol only. The peak areas of the two components were additive and gave falsely elevated ethanol results; however, the mixtures had different retention times and relative retention times than that of ethanol or ethyl chloride alone. Using our current methodology, there was no apparent way to separate the compounds in mixture.

NEWLY DISCOVERED EVIDENCE IN THE DEATHS OF SENATOR HUEY LONG AND DR. CARL WEISS
Lucien C. Haag, Forensic Science Services, Carefree, AZ

On the night of September 8, 1935 a fatal confrontation took place in a marbled hallway of the Louisiana State Capitol building between Dr. Carl Austin Weiss and Senator Huey P. Long. Former Governor Long is believed to have sustained a single perforating abdominal gunshot wound from Dr. Weiss' Model 1910 FN .32 automatic pistol. Dr. Weiss was shot numerous times by Senator Long's bodyguards. Neither victim was autopsied, and shortly after this doubly fatal incident the files, photographs and physical evidence disappeared. Recently (1991), much of this material was found in the possession of Mabel Guerre Binnings, the daughter of General Louis Guerre-the Superintendent of the Bureau of Criminal Investigation in 1935. The items of physical evidence included Dr. Weiss' pistol with six rounds of vintage .32 automatic ammunition in the magazine and a single, fired vintage .32 automatic bullet. In October of 1991 the body of Dr. Weiss was exhumed, autopsied for the first time, and a number of bullets and bullet fragments were recovered which possessed surviving class characteristics and important trace evidence. This presentation will chronicle the recent discoveries in this long unsolved Southern murder mystery and illustrate the enduring value of physical evidence.

DIVERSITY IN CRACK PRODUCTION FOR USE IN STING OPERATIONS (POSTER SESSION)
Lynn J. Willers, Los Angeles County Sheriffs Department, Scientific Services Bureau, 202 W. Beverly Blvd., Los Angeles, CA 90057

Due to the high number of drug related crimes in the Los Angeles area, various operations are frequently used to affect multiple arrests in the "war on drugs." The "reverse sting" is one such operation that utilizes the services of the crime laboratory. Forensic chemists are often requested to generate cocaine base from the hydrochloride salt, for use in narcotics investigations. As officers have indicated the need for a "crack" product with reduced cocaine content, cocaine base adulteration experiments were conducted to obtain products which would satisfy these criteria. Several adulterants were utilized in weight ratios of 2:1, 1:1 and 1:1.5 (cocaine base to adulterant, respectively). The products were compared against street samples of cocaine base in respect to their color, texture and homogeneity. Most products compared well, although a few demonstrated slight color differences. To determine the extent of product uniformity, five random portions from each were quantitated. Most of the quantitation results agreed with the expected values showing the adulterated products to be fairly homogenous. The products obtained demonstrate the ability to control the cocaine content as well as the appearance of the adulterated product. As the characteristics of crack cocaine can vary between geographical locations, the ability to provide products conforming to the request of the investigator, is dearly an advantage.

CALVIN GODDARD, ET AL AND THE CRITERIA FOR IDENTIFICATION AN HISTORICAL PERSPECTIVE
Paul M Dougherty, Ventura Sheriffs Crime Lab, 800 S. Victoria Ave., Ventura, , A 93009

Calvin Goddard is considered the father of modern Firearms Identification. This paper will review some of the steps that he undertook in order to develop his criteria for identification. From that point to the present day there will be an outline of the controversy over the criteria for identification and why much of it is due to a lack of understanding of the basic process by which an identification is made by a qualified firearms examiner.

FRANK'S GREATEST HITS
Frank Cassidy, CA-DOJ Santa Barbara, 820 Francis Botello Rd., Goleta, CA 93017

Over the past 20 years, I have generated many unique ideas in several areas of criminalistics. These have helped me-and hopefully others-in saving time and lessening frustration while performing casework. Many of these suggestions have been published in early editions of D.O.J.'s TIELINE-before TIELINE was circulated to other laboratories outside of D.O.J. Thus, the information may not have been promulgated to laboratories outside of D.O.J. Also, some of the recently hired criminalists may not have had the chance to peruse issues of TIELINE where some of the articles were published. It is hoped that these ideas will help criminalists on the bench and also promote improved ideas and incubate seeds of originality to produce new improvements in the field of criminalistics. The hints and tips to be presented will cover the areas of blood alcohol, serology, trace evidence, firearms/toolmarks and some miscellaneous areas.

GC-MS ANALYSIS OF LSD
Raymond A. Schep and Hoa Dug Nguyen, Diagnostic Products Corporation, Los Angeles, CA 90045

There is need for a reliable GC-MS method to determine LSD in urine using bench-top instruments. Immunoassay results and emergency room visits indicate that LSD is freely available for abuse. Efficient removal of interfering substances during extraction is required to detect LSD at the low dosage (100µg) allowed by its potency. An RIA positive sample is spiked with 20 ng/mL internal standard and buffered to a pH of 10. It is extracted with a 7:3

MATCH CRITERIA DETERMINATION FOR RFLP-DNA ANALYSIS USING FIVE MOLECULAR WEIGHT ZONES
Donald T. Jones, Daniel J. Gregonis and Donna J. Minnillo, Riverside/San Bernardino Regional Forensic DNA Laboratory, 200 South Lena Road, San Bernardino, CA, 92415-0056

Prior to the use of Restriction Fragment Length Polymorphism (RFLP) technology on DNA from case samples, it is necessary to study the concordance between reference blood samples and forensic samples. A match criteria study will determine the acceptable examinations and statistical interpretation. Vaginal swabs from 72 sexual assault kits were examined as well as non-probative bloodstains from crime scenes. A differential extraction provided the vaginal epithelial cell DNA which was compared to the corresponding reference blood. The crime scene bloodstains were compared to reference samples from autopsies. All samples were processed according to our protocol and probed with a cocktail of four probes; YNH24, TBQ7, MS-1, and pH30. Corresponding bands between reference samples and stains were sized. The data were analyzed in groups relating to five zones of molecular weight sizes: less than 1250 bp, 1250 to 2499 bp, 2500 to 4715 bp, 4716 to 9416 bp, and greater than 9416 bp. For each zone the average relative difference between reference and forensic sizing was determined. A window of three standard deviations about the average relative difference provided the range for considering a match between two bands. Different criteria were determined depending on the stain type and whether the reference sample was analyzed on the same gel as the stain. The high molecular weight zone (greater than 9416 bp) had the highest range of variability, about a 13° /o window. The other molecular weight zones had windows ranging from 3 to 7 percent.

DNA ANALYSIS IN THE RIVERSIDE/SAN BERNARDINO REGIONAL FORENSIC DNA LABORATORY
Donald T. Jones, Daniel J. Gregonis and Donna J. Minnillo, Riverside/San Bernardino Regional Forensic DNA Laboratory, 200 South Lena Road, San Bernardino, CA 92415-0056

The Riverside/San Bernardino Regional Forensic DNA Laboratory opened for casework acceptance in April 1992 using Restriction Fragment Length Polymorphism technology. Preparation prior to this date involved several studies which included sample extraction protocols, match criteria determination, and population frequencies studies. Our extraction protocol follows that of the FBI with several modifications. These include the use of Centricon microconcentrators instead of ethanol precipitation, analytical gels run with circulation buffer but without ethidium bromide, Southern blots done on platforms, a hybridization oven, and membrane stripping without formamide. The match criteria were developed using the same approach as the FBI, examining epithelial fractions of vaginal swabs and comparing them to the female's standard blood. The membranes were probed with a cocktail of the four probes we use: YNH24, TBQ7, MS-1, and pH30. Additionally, several secondary standard blood samples from crime scenes were compared to the reference blood samples. Various zones of the gel were individually examined for differences between the stains and standards. Blood samples were collected from both counties and grouped into four categories: Caucasian, Hispanic, Black, and Other. At least 200 samples in each category were analyzed using sequential hybridization with the four probes. The data were examined by a population genetics expert who performed several statistical tests.

COMPARISON OF CENTRICON MICROCONCENTRATORS WITH ETHANOL PRECIPITATION FOR POST RESTRICTION CLEANUP AND CONCENTRATION
Daniel J. Gregonis, Donna J. Minnillo, and Donald T. Jones, Riverside/San Bernardino Regional Forensic DNA Laboratory, 200 South Lena Road, San Bernardino, CA 92415-0056

Because forensic samples are many times limited in quantity, care should be taken when selecting routine techniques in order to optimize recovery of the substance to be analyzed. This study compares the use of a standard (FBI protocol) ethanol precipitation versus a Centricon 30 microconcentration technique to purify and concentrate DNA following restriction with HaeIII. K562 human cell line DNA was serially diluted prior to restriction for 5 hours at 37°C with Hae III in 100 µl total volume. After restriction, 50 µl of the sample was transferred to a new Centricon 30 microconcentrator tube containing 2.0ml of TE-4 for Centricon dialysis and 50ul was transferred to a new 1.5ml Eppendorf tube for ethanol precipitation. Following the concentration of samples, a test gel was run to assure that the K562 restricted properly.

After the test gel, the samples were loaded onto an analytical gel for electrophoresis. Southern blotting onto a charged nylon membrane, sequential hybridization with four DNA probes, and autoradiography. Results on the autoradiographs show significantly stronger signal from the Centricon 30 concentrated samples when compared with the ethanol precipitated samples. Sizing data on the various samples shows that there is no significant difference in electrophoretic mobility between two concentration techniques. Because there is an increased recovery of the restricted DNA and no significant differences in the sizing of bands, it is highly recommended that Centricon dialysis be used in place of ethanol precipitation for post restriction cleanup of DNA, toluene:methylene chloride mixture. A Bond Elut Certify cartridge is treated as follows: 3 mL of 1:1 MeOH:pH 6 buffer, the LSD toluene extract, 1 mL HOAc, 8 ml MeOH, and 2 mL of fresh 2% NH₄OH in EtOAc, from which the LSD is recovered by evaporation. 30 µL of BSTFA is added to the LSD and heated at 70°C for 30 min. 3 µL of BSTFA is injected with scanning of ions 395,293, and 253. EMV is increased by 300. The inlet temp is 270°C, and the starting temperature of the 10 M HP-1 column is 175°C, programmed 25°C/min to 300°C. Critical to success is the use of a new column, conditioned by injection of 1ug of internal standard in BSTFA. The detection limit is 100ng/mL. Of 11 RIA positive samples obtained from a metropolitan crime lab, all were confirmed positive, some samples testing as high as 22,000ng/mL. The method has been used for 6 months.

THE FORENSIC USE OF POLARIZED LIGHT PHOTOGRAPHY
Torey D. Johnson, California Criminalistics Institute, 4949 Broadway, Room A104, Sacramento, CA 95820

Forensic scientists photograph difficult subjects under a variety of working conditions ranging from a well equipped photo studio to a night crime scene. Ring flash attachments may be used for even, shadow free illumination of otherwise difficult to light subjects. Polarizing filters are commonly used to control reflection. A combination of the two techniques, the use of a ring flash with a polarizing filter, yields an extremely useful technique for the forensic scientist/photographer. The ring flash technique is especially useful when documenting three dimensional, hi relief features where normal closeup flash photography produces deep shadows. In combination with the polarizing filters, it readily allows photography of flat, reflective materials such as printed material and art work that might otherwise be plagued with "bounced" reflections.

STABILITY OF CHELEX EXTRACTIONS STORED AT VARIOUS TEMPERATURES
Erin Riley, LAPD-SID, 555 Ramirez St., Space 270, Los Angeles, CA 90012

DNA isolated from a sample, whether known or questioned, may sometimes need to be stored for an indefinite period of time before amplification and/or detection is performed. This study addressed the question of the integrity of the Chelex extracted DNA, which was stored at three different temperatures (Room Temp., 4°C, and -80°C), and stored for various time intervals prior to amplification and detection by HLA DQalpha and D1S80. Each set of extracts was amplified and detected by the two methods above, several times during an eight week period. Each extracted sample gave the same results throughout the entire study. In addition, the same results were achieved at all three storage temperatures used.

AN EXAMINATION OF BIN BOUNDARY EFFECTS IN AN RFLP DATABASE BY COMPARISON OF ALLELE FREQUENCIES USING FIXED VERSUS FLOATING BINS
Rob Keister and E. Buse, Orange County Sheriff-Coroner Department, 320 N. Flower St., Santa Ana, CA 92705

Because the bin boundaries for determining RFLP allele frequencies in a fixed bin system are arbitrary with respect to the distribution of the database alleles, there may be chance clustering of alleles near fixed bin boundaries. Selecting the larger frequency of two adjacent fixed bins may underestimate the allele frequency compared to counting the number of database alleles that satisfy a numerical match criteria (a floating bin). This is likely the unstated reason behind the recommendation by the recent National Research Council (NRC) report to always combine adjacent fixed bin allele frequencies when an allele for comparison falls near a bin boundary. This study compares allele frequencies determined by both methods at each rebin boundary using four loci for each of seven racial/ethnic RFLP databases compiled by our laboratory. Out of a total of 477 rebin boundaries examined, there were 32 cases (6.7%) where the floating bin value exceeded the larger of the two adjacent fixed bin values (+/- 3.4% floating bin width). The largest variation occurred where the floating bin gave a value 1.34 times larger than the greater of the two adjacent fixed bins allele frequencies. The mean ratio of floating bin to fixed bin was 0.59. Given the low frequency and small magnitude of this phenomenon, implementing the NRC recommendation in this regard does not seem justified. A more reasoned approach would be to examine alleles in each particular case and only combine adjacent fixed bins when appropriate, or alternatively, use a floating bin system to determine allele frequencies in all cases.

ISOENZYMES OF DENTAL PULP (POSTER SESSION)
Kathyrn Marks, Patricia Lough and David Stockwell, San Bernardino S.O., S.I.D., 200 S. Lena Rd., San Bernardino, CA 92415-0056

The isoenzymes of dental pulp were compared to those of reference blood standards using a nonequilibrium isoelectric focusing electrophoresis multisystem. The 20 samples were analyzed for the isoenzymes Esterase D (EsD), Red Cell Add Phosphatase (AcP1), Phosphoglucomutase (PGM1), Adenylate Kinase (AK), and Adenosine Deaminase (ADA). These 5 enzymes were identified in both the blood standards and the dental pulp samples. A great increase in PGM activity was seen in the dental pulp, requiring a reduction in sample concentration. It was determined that IEF is a suitable method for identifying the genetic markers present in dental pulp.

THE BAR-STO BARREL
Starr Sachs, LAPD-SID/Firearms, 3401 San Fernando Road, Los Angeles, CA 90065

Bar-Sto Precision Machine is a small (eight employees) family-owned company located in Twenty-nine Palms, CA, which produces custom manufactured and fitted barrels for revolvers and semi-automatic pistols of various calibers. None of the products are made by automated methods, but only by hand. Their Products have been received in casework by examiners in the LAPD firearms unit. A video presentation demonstrating the manufacture and precision machining of stainless-steel pistol barrels is presented. The .45 auto is featured in this demo, but other calibers including a .38 revolver are manufactured.

CRIME LABORATORIES IN THE PEOPLE'S REPUBLIC OF CHINA
Diana K. Holsinger, LAPD-SID/Firearms, 3401 San Fernando Rd., Los Angeles, CA 90065

A delegation of 18 members of the American Academy of Forensic Sciences, led by Dr. Marina Stajic of the New York City Medical Examiner's Office visited the People's Republic of China for two weeks in June of 1992. The purpose of the tour was to compare training, procedures and research in the State Laboratories of Beijing, Shanghai, and Guangzhou. Also accompanying them were Judy Erickson, Washington State Patrol Crime Lab, and others including a professor of pathology from France. The overall impression was one of a laboratory system employing state-of-the-art equipment and research techniques. It was interesting to note that the bulk of crime in China was against property and traffic-related, rather than against persons.

APPLICATION OF THE MILENIA COCAINE METABOLITE KINETIC ENZYME IMMUNOASSAY TO SCREENING WHOLE BLOOD SAMPLES (POSTER SESSION)
Renee Artman, Debra Mittelbrun and Norman Wade, Ventura Co. Sheriff, 800 S. Victoria Ave., Ventura, CA 93009

The objective of this study was to adapt a commercially available kinetic enzyme immunoassay (EIA) to screening whole blood (DUID and post mortem specimens) for cocaine, benzoylecgonine and cocaine metabolites. Calibrators were made by spiking blood bank blood with benzoylecgonine. Sample volume was increased to 50µl. The blank response (mOD/min) for blood bank blood was found to be not significantly different than that for negative post mortem blood or negative ante mortem blood collected on NaF/Oxalate from laboratory volunteers. The blank mean was 206.4 mOD/min, SD=7.4 mOD/min, CV=3.6%.. The limit of detection determined as the mean blank response minus 3 SD was 10 ng/ml. From the blood calibration curve a cutoff concentration for positives of 50 ng/ml was selected. The precision at the cutoff was: mean=159.6 mOD/min, SD=4.2, CV=2.6%. The difference between the negative blood reference and the cutoff reference was 46 mOD/min or greater than ten SDs. In another run, interindividual variation for negative specimens collected from 15 individuals was: mean=336 mOD/min, SD=44. There was no overlap at 3 SD with the cutoff response. Forty-six DUID samples were analyzed by RIA (cutoff 100 ng/ml) and by the modified EIA (cutoff 50 ng/ml). The two methods agreed in 14 positive samples and 26 negative samples. Two samples were positive by RIA but negative by the EIA. Four samples were positive by EIA but negative by RIA. Cross-reactivity of the kit for drugs spiked into blood bank blood was benzoylecgonine 100%, Cocaine 153%, ethylcocaine 60%, ecgonine methyl ester not detected at 100,000 ng/ml (ND), benzphetamine 0.11%, procaine 0.02%, lidocaine ND, amitriptyline ND.