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A partial bloody fingerprint has both a bloody part and a non-bloody part, each of which should be developed for visualization. Traditional techniques employ developmental cascades, whereby a sequence of development techniques is used to develop both parts of the fingerprint. As a one-step alternative, a columnar thin film (CTF) was deposited on each of several partial bloody prints from one finger of one donor on several nonporous substrates. A split-print methodology was adopted to compare the CTF technique to several traditional development techniques, using both objective computer-based grading and subjective grading by a lay examiner. CTF development was found to be superior to development with the traditional techniques employed in this study for partial bloody fingerprints on brass, anodized aluminum, hard plastics, and clear sandwich bags. Unlike the traditional techniques used, the CTF technique was able to develop the entire fingerprint on many of these substrates.

A form of physical vapor deposition, called the conformal-evaporated-film-by-rotation (CEFR) method, was optimized for the conformal deposition of columnar thin films (CTFs) on sebaceous fingermarks. Relying on the surface topology of the fingermark, the CTF development technique is different from traditional development techniques. After the optimization of the development conditions, the CTF development technique was found to be superior to traditional development methods on several nonporous substrates: the smooth side of Scotch (R) Multitask, Gorilla (R), and Scotch (R) Duct tapes; clear and black soft plastics; stained and sealed walnut and cherry woods; partial bloody fingermarks on stainless steel; and discharged cartridge casings. It was equally as good as other development techniques on other substrates, but worse on a few. The optimization study is expected to assist in designing a mobile CEFR apparatus capable of on-scene development of fingermarks.

The grading of fingerprint quality by fingerprint examiners as currently practised is a subjective process. Therefore, an objective system was devised to remove the subjectivity. The devised grading system is quantitative and uses three separate, easily available, software packages to ultimately identify the portions of a fingerprint that correspond to low-, medium-, and high-quality definitive minutiae as defined on the Universal Latent Workstation of the US Federal Bureau of Investigation.

Despite advances in DNA, fingermarks remain one the best forms of evidence available. While fingermarks are routinely analyzed in terms of their patterns, it may be possible to obtain additional information in terms of their chemical composition. If successful, a chemical analysis of the constituents of a fingermark may give scientists additional information that may help in the identification of a person. The results presented herein describe the initial investigation into the analytical determination of some of these compounds, specifically the fatty acids. This study was specifically aimed at identifying possible fatty acids, which could aid in profiling or perhaps uniquely identifying an individual. Preliminary data obtained in this study suggests that this may in fact be possible, though additional research is certainly necessary. Utilizing gas chromatography-mass spectrometry, significant differences in the ratios of several fatty acid methyl esters were found when comparing individuals of varying race and gender. In addition, large intervariability and intravariability was discovered for some compounds, suggesting the possibility of being able to individualize based on chemical profile. Follow-up investigations will continue to determine whether this continues to be the case as greater numbers of individuals are sampled and more extensive control and information on the subjects is obtained.

Various vacuum techniques are employed to develop fingermarks on evidentiary items. In this work, a vacuum was used to deposit columnar thin films (CTFs) on untreated, cyanoacrylate-fumed or dusted fingermarks on a limited selection of nonporous surfaces (microscope glass slides and evidence tape). CTF deposition was not attempted on fingermarks deposited on porous surfaces. The fingermarks were placed in a vacuum chamber with the fingermark side facing an evaporating source boat containing either chalcogenide glass or MgF(2). Thermal evaporation of chalcogenide glass or MgF(2) under a 1 μTorr vacuum for 30 min formed dense CTFs on fingermark ridges, capturing the topographical features. The results show that it is possible to capture fingermark topology using CTFs on selected untreated, vacuumed cyanoacrylate-fumed or black powder-dusted nonporous surfaces. Additionally, the results suggested this might be a mechanism to help elucidate the sequence of deposition.

Victim identification initiatives undertaken in the wake of Mass Fatality Incidents (MFIs) where high-body fragmentation has been sustained are often dependent on DNA typing technologies to complete their mandate. The success of these endeavors is linked to the choice of DNA typing methods and the bioinformatic tools required to make the necessary associations. Several bioinformatic tools were developed to assist with the identification of the victims of the World Trade Center attacks, one of the most complex incidents to date. This report describes one of these tools, the Mass Disaster Kinship Analysis Program (MDKAP), a pair-wise comparison software designed to handle large numbers of complete or partial Short Tandem Repeats (STR) genotypes, and infer identity of, or biological relationships between tested samples. The software performs all functions required to take full advantage of the information content of processed genotypic data sets from large-scale MFIs, including the collapse of victims data sets, remains re-association, virtual genotype generation through gap-filling, parentage trio searching, and a consistency check of reported/inferred biological relationships within families. Although very few WTC victims were genetically related, the software can detect parentage trios from within a victim's genotype data set through a nontriangulated approach that screens all possible parentage trios. All software-inferred relationships from WTC data were confirmed by independent statistical analysis. With a 13 STR loci complement, a fortuitous parentage trio (FPT) involving nonrelated individuals was detected. Additional STR loci would be required to reduce the risk of an FPT going undetected in large-scale MFIs involving related individuals among the victims. Kinship analysis has proven successful in this incident but its continued success in larger scale MFIs is contingent on the use of a sufficient number of STR loci to reduce the risk of undetected FPTs, the use of mtDNA and Y-STRs to confirm parentage and of bioinformatics that can support large-scale comparative genotyping schemes capable of detecting parentage trios from within a group of related victims.

Very often the allocation of putative damages for wrongful death and the determination of aggravating factors in the sentencing of an individual convicted of homicide by a jury is based on a subjective determination of the amount of pain suffered by the victim. This study was designed to determine whether the quantitative determination of peptides involved in nociception and inflammation offer the potential to provide an objective basis for an assessment of pain prior to death. Two peptides. substance P and met-enkephalin, were quantitated using radioimmunoassay (RIA) in the serum of 131 autopsy subjects. Cases were selected that presented decedents who underwent a violent death resulting in extensive trauma to tissue. Only decedents with no known prior clinical manifestation and no indication of prior drug use were selected. Of 131 cases selected, 59 died from blunt trauma deaths, 47 from gunshot deaths, and 25 from stabbing deaths. Cases were selected without regard to whether the death was accidental, or by homicide or suicide. Values from cases having similar incident-death time intervals were pooled and then compared. Results show that an observable pattern exists between the concentrations of substance P and met-enkephalin and the incident-death time interval. Data showed that the concentrations of substance P and met-enkephalin vary with the incident-death time interval. The amount of serum substance P initially increases with increasing incident-death time interval but begins to decrease at longer incident-death time intervals. In contrast, the serum concentration of met-enkephalin continues to show increased concentration as the incident-death time intervals become greater. The Kruskal-Wallis test was performed to determine the level of significance of the variation in both peptide concentrations within four consecutive time intervals. Variation in substance P concentration was statistically significant in all comparisons performed with 0.01 being the lowest level of significance of any four consecutive groups tested. Conversely, intervals encompassing incident-death time intervals of 1-2 hours to 5-10 days did not demonstrate significant variation in met-enkephalin concentration. However, groups with smaller and larger time intervals than the nonsignificant groups did show statistical variation. Although owing to a number of variables, a direct correlation between peptide concentrations and the level of pain may not be possible, the results of the study indicate that a presumption of antemortem pain may be possible with future study.

The AluQuant™ (Promega Corporation) liquid hybridization DNA quantitation method was evaluated on an automated robotic platform (Biomek® 2000, Beckman Coulter, Fullerton, CA) for use in forensic PCR-STR systems. DNA from bloodstains and buccal swabs was extracted by three different methods: Chelex, Qiagen™ and DNA IQ (Promega). Samples were quantitated using both the Quantiblot and the AluQuant™ systems.Concordance between methodswas determined by comparing the average AluQuant™ DNA concentrations for samples having matching (binned) Quantiblot values. Studies testing the “accuracy” (STR analysis), precision, sensitivity, and specifies specificity of the AluQuant™ method were also conducted. The effect of inhibitors (carpet, denim, and suede) was evaluated. The results indicate that the AluQuant™ quantitation system equals the Quantiblot system in “accuracy”, sensitivity, precision, and primate-specificity. While extracts from denim and suede affected (inhibited) both systems minimally, the carpet extracts produced a sharp increase in DNA quantitation values in the AluQuant™ but not the Quantiblot system. The speed and user-friendliness of the AluQuant™ system on a robotic platform offer specific advantages to the forensic community.

A series of validation experiments was performed for a Y chromosome specific STR multiplex system following the suggestions made by the Technical Working Group DNA Analysis Methods (TWGDAM). The multiplex PCR products were detected on Perkin-Elmer 373 and 377 automated sequencers using two labeling colors. No problems regarding the stability, robustness and sensitivity of the Y STR multiplex were observed. Mixture studies revealed a cut off rate similar to autosomal STRs for mixtures of male DNAs and no interference of any female admixture. The comparison of the Y STR results to the autosomal typing results for 56 nonprobative semen stains and swabs, showed a slightly higher success rate in detecting the semen donor’s alleles for the Y STR multiplex. Two examples are shown to illustrate the usefulness of Y STR typing for DNA mixtures. In one case the Y STR results confirmed an isolated exclusion; in the other case, the interpretation of a mixture was clarified since the Y STR results proved the presence of DNA from at least two semen donors. Y STR typing is a valuable addition to the forensic DNA testing panel.

In order to produce database-eligible DNA profiles from touched objects, each testing procedure including sample recovery, extraction, amplification and separation was evaluated and optimized. The developed methodologies were tested on control samples as well on fingerprints deposited on a variety of substrates such as credit cards, keys, and pens. All samples were amplified in triplicate to confirm the presence of each allele and to detect drop-ins. Overall the modifications implemented produced reproducible results for DNA titrated to 20 pg. For DNA dilutions, 25 pg routinely resulted in full profiles, and 12.5 pg determined 76.9% of the database loci tested. Similarly, for the touched objects, 75.8% of the 20-pg to 100-pg samples yielded database-eligible profiles; the remaining samples either were mixtures or contained an insufficient number of allelic calls. Here, the three-amplification approach was crucial and produced more complete profiles with confidence in the allelic assignments. DNA amounts below 20 pg did show partial profiles with correct allelic determinations that could have been compared in a specific case but were often too incomplete for database entry.

Sudden fatal pulmonary thromboembolism (PE) is very common in Caucasians and results in over ∼ 120,000 deaths per year. There are both acquired and inherited risk factors for PE. The three most common mutations are Factor V Leiden G1691A, Prothrombin G20210A, and MTHFR C677T. We have developed an in-house molecular testing methodology using polymerase chain reaction (PCR) and automated DNA sequencing technologies. The method was validated on postmortem tissue samples, such as heart, spleen, and liver. Tissues were stored in RNAlater solution for up to 2 years. The method has also been validated on blood specimens, which were dried on staincards and stored at room temperature for up to 2 years. We obtained results for all tested specimens, including those displaying varying degrees of decomposition. The analytic sensitivity, specificity, and reproducibility show that the method is highly sensitive, and very specific for all three mutations.

Both the columnar-thin-film (CTF) and the vacuum-metal-deposition (VMD) techniques for visualizing sebaceous fingermarks require the deposition of a material thereon in a vacuum chamber. Despite that similarity, there are many differences between the two techniques. The film deposited with the CTF technique has a columnar morphology, but the film deposited with the VMD technique comprises discrete islands. A split-print methodology on a variety of fingermarked substrates was used to determine that the CTF technique is superior for developing fingermarks on clear sandwich bags and partial bloody fingermarks on stainless steel. Both techniques are similar in their ability to develop fingermarks on glass but the CTF technique yields higher contrast. The VMD technique is superior for developing fingermarks on white grocery bags and the smooth side of Gloss Finish Scotch Multitask TM tape. Neither technique worked well for fingermarks on black garbage bags.

A fingermark on a nonporous substrate can be developed by depositing a columnar thin film (CTF) on it, but the CTF technique's sensitivity for low-quality fingermarks is unknown. The optimized CTF and traditional development of several depletion series of sebaceous-loaded fingermarks were compared using a split-print methodology as well as subjective and objective grading schemes, in a limited laboratory trial. CTF development was superior to development with selected traditional techniques on brass, anodized aluminum, black acrylonitrile butadiene styrene (ABS), and white nylon. On white ABS and black nylon, the CTF technique performed poorly but still as well as the best-performing traditional development technique. The CTF technique was more sensitive on brass and anodized aluminum than, and as sensitive on the four hard plastics and stainless steel as, the best-performing traditional technique. Thus, the CTF technique is useful to develop friction-ridge detail from limited fingermark residue on some smooth substrates.

Validation experiments were performed to evaluate the HLA-DQ alpha DNA typing system for forensic casework. Temperature profiles for two Perkin Elmer TC-1 Thermal Cyclers were measured, and the efficiency of the instruments was tested by amplifying a control sample (DQ alpha 1.2,4) susceptible to allelic drop-out. DNA extraction using chelex was compared to non-organic extraction, and the amplification and hybridization procedures were evaluated at extremes of time and temperature. With the protocol in place, samples exposed to stresses commonly encountered in forensic casework were typed to determine the flexibility of the system. Mixed samples of two different bloods and blood mixed with saliva were typed to determine the threshold at which mixtures could be resolved using the reverse dot blot method. Different storage conditions were evaluated using a set of control bloodstrains, and a set of 12 postmortem blood samples was typed repeatedly over the course of 5 months to determine the effects of natural degradation on the DQ alpha results. Finally, casework stains on a variety of substrates were typed. These experiments demonstrate the flexibility of the HLA-DQ alpha system. Based upon these results, a comprehensive quality assurance program was developed to ensure the integrity of typing results for casework.

Various vacuum techniques are employed to develop fingermarks on evidentiary items. In this work, a vacuum was used to deposit columnar thin films (CTFs) on untreated, cyanoacrylate-fumed or dusted fingermarks on a limited selection of nonporous surfaces (microscope glass slides and evidence tape). CTF deposition was not attempted on fingermarks deposited on porous surfaces. The fingermarks were placed in a vacuum chamber with the fingermark side facing an evaporating source boat containing either chalcogenide glass or MgF(2). Thermal evaporation of chalcogenide glass or MgF(2) under a 1 μTorr vacuum for 30 min formed dense CTFs on fingermark ridges, capturing the topographical features. The results show that it is possible to capture fingermark topology using CTFs on selected untreated, vacuumed cyanoacrylate-fumed or black powder-dusted nonporous surfaces. Additionally, the results suggested this might be a mechanism to help elucidate the sequence of deposition.

A radial diffusion assay in an agarose/starch gel utilizing crude kidney bean extract and a commercially prepared alpha-amylase inhibitor isolated from wheat seeds was developed and assessed to determine its ability to differentiate alpha-amylase from various sources. Kidney bean extract was found to have a greater inhibitory effect on AMY2, while the wheat lectin inhibitor was found to have a greater inhibitory effect on AMY1. Neither inhibitor was found to have any effect on commercially prepared bacterial alpha-amylase extract in both liquid preparations and dried stains. Mixtures of varying concentrations of pancreatic and salivary extracts also gave interpretable results. Additionally, dried stains prepared from human body fluids having high levels of AMY2 were differentiated from dried stains prepared from human body fluids containing high levels of AMY1.

Fingerprint visualization obtained from physical evidence taken from crime scenes for subsequent comparison typically requires the use of physical and chemical techniques. One physical technique to visualize or develop sebaceous fingerprints on various surfaces employs the deposition of metals such as gold and zinc thereon. We have developed a different vacuum technology: the conformal-evaporated-film-by-rotation technique to deposit dense columnar thin films (CTFs) on latent fingerprints on different types of surfaces. Sample fingerprints, acting as nonplanar substrates, deposited on different surfaces were placed in a vacuum chamber with the fingerprint side facing a boat containing an evaporant material such as chalcogenide glass. Thermal evaporation of the solid material led to the formation of a dense CTF on the fingerprint, thereby capturing the topographical texture with high resolution. Our results show that it is possible to acquire the topology of latent fingerprints on non-porous surfaces. Additionally, deposition of CTFs on overlapping fingerprints suggested ours may be a technique for elucidating the sequence of deposition of the fingerprints at the scene.

This chapter proceeds in two sections. The first section provides a historical overview of the process used to identify victims’ remains from the WTC disaster. This was an evolving process that adjusted over time, as new techniques were developed to address novel challenges. The second section provides a series of case examples illustrating some mistakes encountered with the identification of WTC victims and how the problems were resolved. As discussed in the following sections, the science of DNA identification was pushed forward by the efforts and expertise of those working on this project. The overriding purpose of this chapter is to provide future practitioners and policymakers a means of learning from the adaptations made during the early months of the WTC identification project as they plan and implement their own programs to prepare for future events.

The AluQuant (Promega Corporation) liquid hybridization DNA quantitation method was evaluated on an automated robotic platform (Biomek 2000, Beckman Coulter, Fullerton, CA) for use in forensic PCR-STR systems. DNA from bloodstains and buccal swabs was extracted by three different methods: Chelex, Qiagen and DNA IQ (Promega). Samples were quantitated using both the Quantiblot and the AluQuant systems. Concordance between methods was determined by comparing the average AluQuant DNA concentrations for samples having matching (binned) Quantiblot values. Studies testing the "accuracy" (STR analysis), precision, sensitivity, and specifies specificity of the AluQuant method were also conducted. The effect of inhibitors (carpet, denim, and suede) was evaluated. The results indicate that the AluQuant quantitation system equals the Quantiblot system in "accuracy", sensitivity, precision, and primate-specificity. While extracts from denim and suede affected (inhibited) both systems minimally, the carpet extracts produced a sharp increase in DNA quantitation values in the AluQuant but not the Quantiblot system. The speed and user-friendliness of the AluQuant system on a robotic platform offer specific advantages to the forensic community.

To present individual body identification efforts, as part of the World Trade Center (WTC) mass disaster identification project.More than 500 samples were tested by using polymerase chain reaction (PCR) amplification and short tandem repeat (STR) typing. The extent to which the remains were fragmented and affected by taphonomic factors complicated the identification project. Anthropologists reviewed 19,000 samples, and detected inconsistencies in 69, which were further split into 239 new cases and re-sampled by DNA specialists.The severity and nature of the disaster required an interdisciplinary effort. DNA profiling of 500 samples was successful in 75% of the cases. All discrepancies, which occurred between bone and tissue samples taken from the same body part, were resolved by re-sampling and re-testing of preferably bone tissue. Anthropologists detected inconsistencies in 69 cases, which were then split into 239 new cases. Out of 125 "split" cases, 65 were excluded from their original case. Of these 65 cases, 37 did not match any profiles in M-FISys, probably because profiles were incomplete or no exemplar for the victim was available. Out of the 60 remains not excluded from their original case, 30 were partial profiles and did not reach the statistical requirement to match their original case, because the population frequency of the DNA profile had to be/=1 in 10(9) for men and/=1 in 10(8) for women.Due to transfer of soft tissue and other commingling of remains, DNA testing alone would have led to problems if only soft tissue would have been tested. This was one of the reasons that forensic anthropologists were needed to evaluate the consistency between all linked body parts. Especially in disasters with a high potential for commingling, the described anthropological review process should be part of the investigation.

The FES short tandem repeat (STR) locus contains seven to 14 repeats of the tetranucleotide sequence ATTT. A novel 10 base pair dimorphism in the 5' flanking region of the FES locus was characterized in four broad populations: African-American, Hispanic, Caucasian, and Asian. The absence of the 10 base pair sequence, or (-) allele, was closely linked to FES STR alleles with 10 or fewer repeats. The presence of the 10 base pair sequence, or (+) allele, was closely linked to FES STR alleles with 12 or more repeats. The (-) and (+) alleles occurred equally often in FES STR allele 11. The nucleotide sequence (5'-GGCTGTTTTG-3') of the (+) allele, located 179 base pairs upstream of the FES STR, was determined to be consistent within and among the four populations. Statistical and sequence analysis confirmed the linkage between the two polymorphic sites. The results indicate that the exclusion rate of the FES locus is increased, above that for the STR alone, when both polymorphic characteristics are considered.

The analysis of genetic markers for the purpose of individualization of semen specimens is extremely important in cases of sexual abuse and assault. The serological analysis of sexual assault evidence can sometimes be complicated because stains are often composed of a mixture of spermatozoa, vaginal epithelial cells and white and red blood cells. A filtration method has been developed to cleanly separate spermatozoa from epithelial cells based upon differences in size and shape. Nylon mesh filters of the appropriate pore size can be used to separate the smaller oval shaped spermatozoal cells from the larger and flatter epithelial cells. The former pass freely through the membrane while the latter are retained on the filter. In this study, cell separation was demonstrated by (a) microscopic observation of stained cells, (b) amplified fragment length polymorphism analysis of DNA obtained from separated cells. The results of these analyses indicate that: (1) Approximately 70% of spermatozoa in the mixed cell sample will penetrate the 10 microns pore size filter, (2) Only about 1-2% of intact epithelial cells will do so, and (3) A small number of nuclei from spontaneously lysed epithelial cells will cross the filter. Experimental results using mixtures of spermatozoa and vaginal epithelial cells prepared in different ratios support the conclusion that the filtration process is an efficient and reliable method to separate spermatozoa from epithelial cells in casework specimens for subsequent DNA analysis.

Fingerprint visualization obtained from physical evidence taken from crime scenes for subsequent comparison typically requires the use of physical and chemical techniques. One physical technique to visualize or develop sebaceous fingerprints on various surfaces employs the deposition of metals such as gold and zinc thereon. We have developed a different vacuum technology: the conformal-evaporated-film-by-rotation technique to deposit dense columnar thin films (CTFs) on latent fingerprints on different types of surfaces. Sample fingerprints, acting as nonplanar substrates, deposited on different surfaces were placed in a vacuum chamber with the fingerprint side facing a boat containing an evaporant material such as chalcogenide glass. Thermal evaporation of the solid material led to the formation of a dense CTF on the fingerprint, thereby capturing the topographical texture with high resolution. Our results show that it is possible to acquire the topology of latent fingerprints on nonporous surfaces. Additionally, deposition of CTFs on overlapping fingerprints suggested ours may be a technique for elucidating the sequence of deposition of the fingerprints at the scene.

A Dot Blot format using allele specific oligonucleotides is commonly used to identify base pair substitution polymorphisms following the PCR amplification of target genomic DNA. A common application of this technique in forensic science has been to identify polymorphisms associated with the DQa region of the major histocompatibility complex (HLA DQa).

DNA evidence holds an important position in criminal investigations and proceedings. The polymerase chain reaction (PCR) is often utilized to amplify polymorphic regions of DNA which are subsequently typed to produce distinct genotypes. The sensitivity of PCR-based techniques provides a major advantage over other DNA or conventional serological typing systems. Samples containing quantities of DNA in the picogram range are often typed. However, the unprecedented sensitivity of PCR is often cited as a criticism. One concern is that the interpretation of PCR typing can be affected by DNA contaminants from foreign sources. In this report, the level of DNA contamination in New York City Medical Examiner facilities and its potential affects on HLA-DQA1 typing were assessed. Two related studies conducted over a five week period measured and typed HLA-DQA1 from accumulated DNA on autopsy room and Forensic DNA Laboratory structures. The potential for DNA contamination from airborne sources was also evaluated in the autopsy suites. This study demonstrated the presence of small amounts of DNA on structural surfaces, but little evidence of airborne DNA contamination.