79th SEMI-ANNUAL SEMINAR (Spring 1992)
CALIFORNIA ASSOCIATION OF CRIMINALISTS
May 7-9,1992
Bass Lake, California

THE PH PEN - A MEANS OF COMPARING PAPER PRODUCTS
R. D. Blackledge, N.I.S. Regional Forensic Laboratory, San Diego, CA; Mark N. Gernandt, N.I.S. Regional Forensic Laboratory, San Diego, CA

For the production of fine white paper many companies are switching from a process that uses titanium dioxide and produces a paper having a residual acidity (acid paper), to a process that uses calcium carbonate and produces a paper having a residual alkalinity (alkaline paper). Because of this change, printers need to use a pH pen to test the alkaline/acid content of paper stock to determine if it is compatible with a given formulation of printing ink. Questioned document examiners may find the pH pen useful as an additional test when attempting to determine if two paper products could have originated from a common source. The authors attempted to discriminate among sixty-eight different fine white paper products using a combination of physical characteristics, fluorescence under UV light, and the pH pen. From a total of 1278 possible pairs (Questioned and Known), physical characteristics alone could discriminate all but 55 pairs. All but 20 pairs were discriminated by physical characteristics plus fluorescence, and adding the pH pen reduced the number to just six groups of pairs.


THE NEW 3M DISPOSABLE IR CARD FOR QUALITATIVE MID-INFRARED ANALYSIS
James GAGNON, 3M DPD New Products Group; Neale POVEY, 3M MISD Lab

The new 3M Disposable IR card is described for use in mid-infrared spectra photometric analysis of liquids, semi solids, and soluble solids. The sample is applied to the unique sample application area and any volatiles are rapidly evaporated. Infrared spectra obtained contain minimal spectral interferences, thereby facilitating spectral manipulations such as ratios, base line corrections, library searching, etc. Infrared spectra obtained can be searched against standard condensed phase spectral libraries of samples prepared by using conventional sample accessories. Examples of commercially available products analyzed using the sample support system will be presented. Various techniques successfully employed in conjunction with the new sample holder will also be shown.


THE 3M FTIR SAMPLE WINDOW: TRIAL RESULTS
R. D. Blackledge, N.I.S. Regional Forensic Laboratory, San Diego, CA

Prototypes of a new FTIR sampling window developed by Jame's E. Gagnon of 3M Company were made available to the author for testing in the fall of 1991. Properties of the sampling window were first compared with a similar, previously-reported IR sampling method involving stretched PTFE ("Teflon") tape. The sample windows were then tested with a variety of samples including several from current cases. Results which compared well with conventional IR sampling methods were obtained for samples which could either be dissolved or finely dispersed in a volatile solvent, deposited on the sample window, and then remained un-changed upon solvent evaporation. Less successful results were obtained with samples which were dissolved in a volatile solvent, deposited on a window and crystallized upon solvent evaporation. Although the sample windows are not likely to supplant KBr discs for routine drug cases, they have advantages for many trace evidence applications. An especially important advantage in forensic science is that they are not fragile, and once prepared may be labeled and retained for subsequent evaluation.


QUANTITATIVE VARIATION IN MOLECULAR WEIGHT OF ENVIRONMENTALLY ABUSED SAMPLES SUBJECTED TO RFLP ANALYSIS
Keith Inman, Department of Justice, DNA Laboratory

Samples subjected to controlled environmental abuse conditions (laboratory-induced) and uncontrolled environmental abuse conditions (samples from "non-probative" cases) were examined for the amount of variation produced in the calculated molecular weights of DNA digested with HAE III and probed for various VNTR's (D2S44, D10S28, D17S79, D1S7, D4S139). Controlled abuse experiments included stressing known samples with controlled amounts of heat, humidity, UV, and sunlight as well as different substrates, for limited periods of time. Uncontrolled abuse samples consisted of vaginal, oral, and rectal samples from rape kits compared to reference blood samples from the victim. Experience with the process resulted in modification of the protocol for casework to incorporate double Pro-K digestion, multiple organic extraction, Centricon dialysis and concentration, double restriction enzyme digestion, and electrophoresis on 20 cm agarose gels without ethidium bromide. The variation in calculated molecular weight size for the controlled-abuse samples, and between the evidence and reference blood samples was compared to the variation seen among control samples. This constitutes a portion of the information used in the development of the "match" criteria for the laboratory.


290.2 STORAGE AND PRESERVATION EXPERIMENT: PART A&B
Mary Pierson & Michele Horne, Department of Justice, DNA Laboratory

The purpose of this experiment was to examine what effects, if any, storage of blood samples has on DNA analysis. The samples were collected in either ACD or EDTA tubes and stored refrigerated in liquid form or frozen as dried stains. The study was completed in two parts. Part A studied bloodstains stored over a period of time and Part B compared the various storage conditions for a single individual's blood drawn at a specific time point.
Part A: Thirty-eight (38) bloodstains were selected from the 290.2 Program and divided into donor sets. A donor set is defined as a collection of three (3) or four (4 ) blood samples drawn over a period of time from one individual. The period of stain storage dated from August 1986 through March 1991. The blood samples from a donor set were spaced from 5 to 36 months apart. Obtaining stains from donor sets to represent each year of storage was not possible due to the randomness of the blood drawings. The stains were analyzed in duplicate for DMA profiling by RFLP and HLA DQalhpa PCR methods. The results demonstrate that aged 290.2 Stains may be typed up to approximately five (5) years.
Part B: Twelve (12) storage condition sets were selected from the 290.2 Program for analysis. Each set represents an individual and is defined as a liquid blood sample stored in EDTA, a liquid blood sample stored in ACD, and a stain made from the EDTA whole blood. The period of storage for the samples analyzed ranged from January 1990 through May 1991. The samples were analyzed in duplicate for DNA profiling using both RFLP and PCR methods. The results demonstrate that dried bloodstains are the best method of long term preservation for DNA analysis. The results also indicate that EDTA is a better preservative than ACD for liquid whole blood stored for long periods of time. Liquid blood samples stored longer than 1 year in ACD tubes yield no RFLP DNA typing results; however PCR DQ alpha results were obtained.


CALIFORNIA DOJ DNA DATABASE PROGRAM PROGRESS REPORT
Lance Gima, Department of Justice, DNA laboratory

In 1983 SB 809 was enacted requiring convicted sex offenders to provide a blood and saliva sample prior to their release from prison. For several years conventional serology was conducted on these examples and the data became an important part of investigations across the state. In 1990 SB 1408 was passed by the legislature. This bill established the California DNA Database Program which became an extension of the program established in 1983. Although SB 1408 was passed in 1990, the database program was never funded. The current status of this program will be discussed along with the steps the California Department of Justice DNA Laboratory is taking to make this program as useful as possible in light of the budget problems. Issues such as sample verification, racial origin information, analysis prioritization and connection to the national database program will be discussed.


ENHANCEMENT OF FAINT AND DILUTE BLOODSTAINS WITH FLUORESCENCE REAGENTS
Louis A. Maucieri, California Criminalistics Institute, Sacramento; Jamie W. Monk, University of Strathclyde, Glasgow Scotland

This paper describes experiments with bloodstain detection reagents that fluoresce. The intended application was for field use on faint, obliterated, or otherwise latent bloodstains. We sprayed various test reagents on stains dried upon several surfaces (made by serial dilution). Many of these tests produced reactions resulting from the heme-peroxide catalyzed oxidation of the reagent. Resulting complexes fluoresced with the irradiation from the handheld ultraviolet (UV) lamp. The dye fluorescin exhibited good sensitivity and ease of application to visualize faint or dilute bloodstains.


THE DEATH OF SHIRLEY DANIELS: A RECONSTRUCTIVE BEHAVIORAL EXAMINATION
Michael Prodan, Special Agent, California Department of Justice; Gary V. Cortner, Senior Criminalist, California Department of Justice

On June 22, 1977, the body of Shirley Daniels was found by an irrigation district laborer alongside a road in rural Kings County with the murder weapon across her lap. The murder weapon was a .22 caliber rifle that she had purchased herself the day before her death. During the initial investigation, the Kings Co. Sheriff's Department's detectives felt that this was a suicide until they learned that she was 1 1/2 months pregnant. At that point their feelings changed and the case was not closed. While attending Corcoran Union High School, the victim met Daniel Richards, a married teacher with children, who taught and supervised the agricultural courses at the high school. Richards also supervised the Future Farmers of America's "field trips." It was reported the victim's feelings about Richards grew from a "crush" to infatuation to love. Naturally, the only suspect in the case was Richards. He was questioned by detectives but never arrested. Eleven years after her death had been described as a possible murder, the Kings Co. Sheriff's Office decided to declare the case a suicide. But the debate about what happened to Daniels continued. Finally, in an attempt to resolve the case, Sheriff Tom Dark announced that he would ask the California Dept. of Justice to conduct an independent investigation. Special Agent Supervisor Michael Prodan and Senior Criminalist Gary Cortner examined photographs, physical evidence and reports from the earlier investigations. Their findings showed that it was physically possible for Daniels to shoot herself between the eyes with the rifle and moreover a short story, diary and letter written by Daniels support the conclusion that she killed herself.


FORENSIC ANTHROPOLOGY AND ITS APPLICATIONS: ILLUSTRATIONS FROM THE WARREN CHASE CASE
Roger Marks La Jeunesse, PhD., Professor of Anthropology, California State University, Fresno

This presentation will illustrate anthropological problems encountered in a homicide case, including the morphological analysis of hand and footprint data. Although the focus of the discussion will be the Chase case, supplementary examples will be drawn upon from other cases that the author has worked on to illustrate the applications of anthropological knowledge in the resolution of homicide investigations.


DESIGNING AND BUILDING A PCR-BASED DNA LABORATORY
Theresa Spear, Santa Clara County Laboratory

Properly designing a laboratory requires a thorough understanding of the work that will be performed. The design must facilitate the proper execution of the analytical steps and maximize efficiency by taking into account work flow. This paper will describe the process the Santa Clara County Crime Laboratory went through to design a PCR-based DNA laboratory, remodel existing space, select and purchase equipment, install laboratory benches and equipment, and implement DNA extraction, amplification and typing procedures.


COMPARATIVE STABILITY OF THE ACIDIC (+) AND BASIC (-) ALLELES OF PHOSPHOGLUCOMUTASE IN HETEROZYGOUS SEMEN SAMPLES
J.M. White & M. M. Hong, Orange County Sheriff-Coroner

Ten semen samples which were heterozygous PGM types and contained one acidic (+) and one basic (-) allele were selected from semen samples collected from patients at a fertility clinic. The samples were aliquoted and stored either liquid or dried on cotton cloth at 37 degrees C, room temperature, refrigerated and in a -20 degree C freezer. Samples were removed from storage periodically and relative activity of the two PGM isozymes were judged following agarose gel electrophorisis at pH 5.5. Samples removed from storage and not immediately analyzed were stored at -70 degrees C until analyzed. In some cases the samples remained typable until the storage collection for that sample was used up, while others eventually lost all activity. In broad terms, the 37 degree liquids were typeable for several days, the stains for several weeks; the room temperature liquids for a week and the stains for 2 to 3 months; refrigerated liquids for 2 months and the stains past 5 months. Testing of the freezer samples is ongoing. At present, after 6 months, the liquid samples are retaining typeable activity. In most samples the activity of the two isozymes deteriorated at a relatively constant rate. In some samples, differential enyzme activity loss was noted. Most frequently the acidic (+) activity exceeded the basic (-) activity, however one semen in the study had the (-) activity exceed the (+) in some samples. In no cases examined to date was there total loss of the weaker isozyme, but several samples required prolonged staining time to detect this band. This study provides further evidence for the cautious interpretation of weak enzyme banding patterns.


IDENTIFICATION OF OXAZOLIDINES AS A NEW CLASS OF IMPURITIES FOUND IN METHAMPHETAMINE
Mark F. Kalchik, California Department of Justice, Fresno Regional Lab

Over the past year a new class of compounds have been found in ephedrine and methamphetamine samples. These compounds belong to a class of compounds called oxazolidines. Oxazolidines are reaction products of ephedrine and usually an aldehyde or a ketone. The basic structure is 3,4-dimethyl-5-phenyl-1,3-oxazolidine. Variation is at the 2 position and is dependent on the starting aldehyde or ketone. They may appear as only trace components to significant compounds.


WHEN IS A DRUG STANDARD A STANDARD
Mark F. Kalchik, California Department of Justice, Fresno Regional Lab

All laboratories use standards which are purchased from chemical supply companies. We trust them to provide properly labeled compounds. However before using any standards care should be taken to confirm the identity of the compound before using. Care must be taken to either find known physical data or to be able to predict properties that can be measured. The properties to be checked should be appropriate to the use of the standard. On rare occasions an incorrectly labeled standards will be supplied. These have to be found and eliminated. Examples will be reviewed.


CONSIDERATIONS IN THE SELECTION AND PREPARATION OF A DRUG ANALYSIS PROFICIENCY TEST SAMPLE
John P. Bowden, California Criminalistics Institute, Sacramento

The California Department of Justice has cooperated with Collaborative Testing Service (CTS) by preparing a Drug Analysis Proficiency Sample for each of the past four years. The author formulated these proficiency samples. The most recent sample, number 91-5, was distributed to 238 participating laboratories. This sample consisted of several pieces of printed "blotter paper" which were impregnated with either Lysergic Acid Diethylamide (LSD) or Lysergol. The selection process, preliminary testing, and logistics of preparation and packaging of more than 250 duplicate-test samples will be discussed. The results received from the 157 laboratories responding to the test sample will be summarized.


BOOTSTRAP CONFIDENCE INTERVALS FOR FBI FIXED BIN GENOTYPE PROBABILITY POINT ESTIMATES WITH MODELING OF BOTH SAMPLING AND MEASUREMENT VARIANCE
J. M. Hartmann, B. T. Houlihan, R. S. Keister; Orange County Sheriff-Coroner

A computer program has been developed, which enables modeling of three sources of variance in estimating genotype probabilities using FBI-type fixed bin allele frequency estimates. These three sources of variance are; sampling, and both database sample and case sample band size measurements. Individual band size may be perturbed by a precision based independent normally distributed random factor. Genotype probabilities are calculated assuming independence within and across loci. The computer program was applied to a set of 1000 four loci genotypes that were generated by resampling an Hispanic database of 250 individuals using each of the following four conditions: perturbation of the database alleles, perturbation of the four loci genotype being compared to the database, perturbation of both the database and the sample, and perturbation of neither the database nor the sample. The sizes of the generated confidence intervals were compared to the mean point estimate. Allowing for sampling variance alone, the four locus genotype probability 95% confidence intervals relative to the point estimates are approximately one order of magnitude out of nine, which is of no practical significance for a mean genotype probability of 10-8. For two loci, the size of the confidence interval increases to one order of magnitude out of 6.7, illustrating the benefit of increasing the number of loci employed. Database measurement variance has no effect on confidence intervals. Measurement variance of the case sample being compared to the database has a minor effect on the confidence intervals, increasing the mean relative size of the confidence interval by about 10%.


CRITICAL ISSUES IN APPLICATION OF THE EMPIRICAL SCIENTIFIC METHOD IN DNA PROFILING
Talib ul Haq, California State University, Sacramento

There has been a legacy of neglect in applying the fundamental canons and critical concepts of the empirical scientific method in the area of measurement in the forensic field and their usage in establishing the sought-after actuality of uniqueness (individuality) of entities. This paper will address some of the critical issues involved in the application of the empirical scientific method in forensic problem solving endeavor. This discussion will be exemplified by a critical examination of the work being done in DNA-profiling endeavor in the criminal investigation arena. A number of violations of the empirical scientific method protocol which render this work scientifically unacceptable will be discussed. For example, not attempting to falsify basic theoretical concepts, not carrying out independent verification of observed phenomena, accepting unverified theoretical concepts as empirical facts, making scientifically unwarranted conclusions, etc.


PHENOTYPING OF GC USING A DOUBLE ANTIBODY TECHNIQUE
Brenda Markham, Fresno County Sheriff's Office

Group Specific Component (Gc), also known as Vitamin D Binding Protein, is found in various body fluids; including serum, urine, vaginal secretions and semen. Utilization of isoelectric focusing as a method for separating Gc into its phenotypes results in high discriminating power of 0.74. Because of this, Gc is useful in forensics. Phenotyping of Gc in secretions from vaginal fluids and semen is virtually impossible using a single antibody detection system. A double antibody technique, which uses an enzyme labeled second antibody, satisfies the need for detecting semen and minute bloodstains. (Gc is analogous to PGM in that it is found in the seminal plasma and can therefore be detected in vasectomized individuals}. The method employed utilizes isoelectric focusing as a means of separating Gc into its phenotypes. Following separation, the Gc proteins are passively transferred onto a Nitrocellulose (NC) membrane for detection purposes. Nonspecific binding sites on the NC are then blocked overnight using PBS (Phosphate Buffered Saline) solution. After blocking, the NC is removed from the blocking solution and put in a PBS buffer containing the first of two antibodies (anti-Gc IgG antiserum). This antibody binds with the Gc Proteins on the membrane, providing a "link" for the second antibody. After washing off excess antibody, the membrane is placed in PBS containing the second antibody, which is labeled with alkaline phoshatase. This enzyme-labeled antibody attaches to the first antibody. To visualize the Gc proteins, the NC membrane is submerged in a substrate buffer for the enzyme to act on. Consequently, the Gc alleles appear as blue bands on a white NC membrane. This method can detect blood stains up to at least a 1:300 dilution. Because of its sensitivity, it is possible to detect both male and female phenotypes from post-coital swabs. Whether the sample is a semen stain from a sexual assault case or a minute blood stain from a homicide, the method looks promising for casework.


SLOW MOTION BLOOD DROP STUDIES
Jerry Chisum, California Criminalistics Institute, Sacramento; Ed Shipp, Washoe County Sheriff's Office, Reno

Blood drops on various surfaces at various heights and angles, from different objects. The studies that criminalists do to prepare them for bloodstain interpretation show surprising phenomena when a high speed video camera recorded the event. The camera is Kodak's, capable of 30 to 6000 fps. Most of the experiments were recorded at 1000 fps. which is enough to see bullets in flight.


RESOLUTION OF EXCESSIVE HOMOZYGOSITY AT THE LOCUS D2S44 IN A VNTR DATABASE
L D. Thompson, E.M. Steinberger, J. M. Hartmann; Orange County Sheriff-Coroner

A southern California database of Red Cross blood donors was obtained and analyzed for RFLP distribution using the restriction enzyme HaeIII and a D2S44 probe (YNH24), which recognizes both the consensus repeat unit and a flanking region. Twenty-three African-American single-banded patterns (sbp) were reanalyzed to detect closely spaced pairs (coalesced) and bands too small to be detected originally (nulls). When HaeIII and HindI restricted DNA was separated in 1.5% agarose, seven of the sbp were found to be due to a previously unobserved 'null' band. Two of the sbp had been the result of coalescence, as shown through prolonged electrophoresis in 2% agarose. All samples were restricted with Pstl, which revealed a Pstl restriction site polymorphism among null-allelic samples as well as four of the remaining unresolved sbp. The existence of a 'Pstl heterozygote' in a 'HaeIII homozygote' shows that even if the Haelll-cut VNTR alleles are the same length, the two VNTR alleles are not necessarily of common descent. In such a situation, it is important to distinguish between independence testing for genotype frequency determinations and detection ofsubpopulations.


A COMPARISON OF ETHNIC AND RACIAL DATABASES AND THE IMPACT OF POPULATION SUBSTRUCTURING ON MULTILOCUS GENOTYPE PROBABILITY ESTIMATES
J. M. Hartmann, B. T. Houlihan, E. L Buse; Orange County Sheriff-Coroner

HaeIII VNTR were used to characterize four Asian ethnic and three other racial databases. Comparison of band size distributions with the Smirnov two-sample test and of FBI fixed-bin allele frequencies with a log-likelihood test were made using a Monte Carlo technique to account for measurement as well as sampling variance. The ethnic groups displayed greater homogeneity than was observed among the racial groups. Measurement variance had only a very slight effect on test results. Using 1990 U. S. Census figures for Southern California, the same ethnic databases were used to obtain Asian FBI fixed-bin average allele frequency sets, which ignore ethnic substructure. Four-locus genotype frequencies were determined using these sets as well as with the original ethnic allele frequencies and population-weighted (stratified) genotype frequencies derived from them. The same type of analyses were performed at the total population level with Asian, Black. Hispanic, and White databases. The relative difference of the four-locus genotype probability estimates determined by the three methods rarely exceeded one order of magnitude out of ten, which is of no practical significance. Although some of the ethnic and most of the racial databases differed significantly in a statistical sense, such differences have only a minimal impact on the greater majority of multilocus probability estimates; and are of negligible significance in a decision making sense.