Bioinformatics and human identification in mass fatality incidents: the world trade center disaster.

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.

Columnar-thin-film-assisted visualization of depleted sebaceous fingermarks on nonporous metals and hard plastics.

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.

Comparison of the columnar-thin-film and vacuum-metal-deposition techniques to develop sebaceous fingermarks on nonporous substrates.

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(™) tape. Neither technique worked well for fingermarks on black garbage bags.

Evaluation of an automated liquid hybridization method for DNA quantitation.

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.

Characterization of a novel dimorphism in the 5' flanking region of the short tandem repeat (STR) locus, c-fes/fps (FES).

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.

Optimized development of sebaceous fingermarks on nonporous substrates with conformal columnar thin films.

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(®) Multitask, Gorilla(®) , and Scotch(®) 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 evaluation of fatty acid ratios in latent fingermarks by gas chromatography/mass spectrometry (GC/MS) analysis.

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.

An objective fingerprint quality-grading system.

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.

Solid-state acquisition of fingermark topology using dense columnar thin films.

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.

Measurement of substance P and met-enkephalin in the serum of violent death 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.

Optimization of a simple, automatable extraction method to recover sufficient DNA from low copy number DNA samples for generation of short tandem repeat profiles.

AIM: To develop an automated, high throughput extraction protocol in order to produce database eligible profiles from fingerprints and other low copy number (LCN) DNA sources. METHODS: Extraction of either purified control DNA or buccal cells, for example, with commercial kits was compared to extraction with a simple digestion buffer and a subsequent concentration and purification. Results were evaluated based on the amount of DNA recovered and the completeness of the DNA profiles produced. RESULTS: Simple procedures with fewer steps were superior to commercial kits, such as DNA IQ (Promega, Madison, WI, USA) and QiaAmp (Qiagen, Valencia, CA, USA), and other protocols with many manipulations. The optimized protocol included a thirty-minute incubation with 0.01% SDS and proteinase K at 56 degrees C, followed by an incubation at 100 degrees C for 10 minutes. Concentration of the extract and removal of the SDS was accomplished with a Microcon 100 (Millipore, Bedford, MA, USA), which can be assembled into a 96 well plate, the Microcon-96 Retentate Assembly Plate (Millipore) for automation. The addition of 1 ng Poly A RNA to the Microcon significantly improved DNA recovery. CONCLUSION: A one-step sample digestion followed by sample concentration/purification minimized sample loss and maximized amplification input. Moreover, this methodology can be easily adapted for automation. Implementation of this protocol, due to the numerous potential sources of LCN DNA samples, will enhance the recovery of biological evidence from crime scenes and may be a source of database profiles.

Epidemiology. DNA identifications after the 9/11 World Trade Center attack.

The attack on the World Trade Center on 9/11/2001 challenged current approaches to forensic DNA typing methods. The large number of victims and the extreme thermal and physical conditions of the site necessitated special approaches to the DNA-based identification. Because of these and many additional challenges, new procedures were created or modified from routine forensic protocols. This effort facilitated the identification of 1594 of the 2749 victims. In this Policy Forum, the authors, who were were members of the World Trade Center Kinship and Data Analysis Panel, review the lessons of the attack response from the perspective of DNA forensic identification and suggest policies and procedures for future mass disasters or large-scale terrorist attacks.

World Trade Center human identification project: experiences with individual body identification cases.

AIM: To present individual body identification efforts, as part of the World Trade Center (WTC) mass disaster identification project. METHODS: 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. RESULTS: 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