This study was aimed at determining the effect of seven blood enhancement reagents on the subsequent Profiler Plus™ fluorescent STR DNA analysis of fresh or aged bloody fingerprints deposited on various porous and nonporous surfaces. Amido Black, Crowle's Double Stain, 1,8-diazafluoren-9-one (DFO), Hungarian Red, leucomalachite green, luminol and ninhydrin were tested on linoleum, glass, metal, wood (pine, painted white), clothing (85% polyester/15% cotton, 65% polyester/35% cotton, and blue denim) and paper (Scott ® 2-ply and Xerox-grade). Preliminary experiments were designed to determine the optimal blood dilutions to use to ensure a DNA typing result following chemical enhancement. A 1:200 blood dilution deposited on linoleum and enhanced with Crowle's Double Stain generated enough DNA for one to two rounds of Profiler Plus™ PCR amplification. A comparative study of the DNA yields before and after treatment indicated that the quantity of DNA recovered from bloody fingerprints following enhancement was reduced by a factor of 2 to 12. Such a reduction in the DNA yields could potentially compromise DNA typing analysis in the case of small stains. The blood enhancement chemicals selected were also evaluated for their capability to reveal bloodmarks on the various porous and nonporous surfaces chosen in this study. Luminol, Amido Black and Crowle's Double Stain showed the highest sensitivity of all seven chemicals tested and revealed highly diluted (1:200) bloody fingerprints. Both luminol and Amido Black produced excellent results on both porous and nonporous surfaces, but Crowle's Double Stain failed to produce any results on porous substrates. Hungarian Red, DFO, leucomalachite green and ninhydrin showed lower sensitivities. Enhancement of bloodmarks using any of the chemicals selected, and short-term exposure to these same chemicals (i.e., less than 54 days), had no adverse effects on the PCR amplification of the nine STR systems surveyed (D3S1358, HumvWA, HumFGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) or of the gender determination marker Amelogenin. The intensity of the fluorescent signals was very similar and the allele size measurements remained constant and identical to those of untreated bloody fingerprints. No additional background fluorescence was noted. Continuous exposure (for 54 days) to two of the seven enhancement chemicals se-354

An automated process has been developed for the analysis of forensic casework samples using TECAN Genesis RSP 150/8 or Freedom EVO liquid handling workstations equipped exclusively with nondisposable tips. Robot tip cleaning routines have been incorporated strategically within the DNA extraction process as well as at the end of each session. Alternative options were examined for cleaning the tips and different strategies were employed to verify cross-contamination. A 2% sodium hypochlorite wash (1/5th dilution of the 10.8% commercial bleach stock) proved to be the best overall approach for preventing cross-contamination of samples processed using our automated protocol. The bleach wash steps do not adversely impact the short tandem repeat (STR) profiles developed from DNA extracted robotically and allow for major cost savings through the implementation of fixed tips. We have demonstrated that robotic workstations equipped with fixed pipette tips can be used with confidence with properly designed tip washing routines to process casework samples using an adapted magnetic bead extraction protocol.

A bioinformatic tool was developed to assist with the victim identification initiative that followed the Swissair Flight 111 disaster. Making use of short tandem repeat (STR) DNA typing data generated with AmpFlSTR R Profiler Plus TM (PP) and AmpFlSTR R COfiler TM (CO) kits, the software systematically compared each available STR genotype with every other genotype. The matching algorithm was based on the search for: (i) direct matches to genotypes derived from personal effects; and (ii) potential kinship associations between victims and next-of-kin, as measured by allele sharing at individual loci. The software greatly assisted parentage analysis by enabling kinship evaluation in situations where complete parentage trios were unavailable and, in some situations, with distantly related relatives. Exclusion of fortuitous kinship associations (FKA) was made possible through the recovery at the disaster site of at least one remains for every sought-after victim, and was incorporated into the kinship software. The data from the 13 combined STR loci produced 6 and 23 times fewer FKAs when compared with PP alone and AmpFlSTR R Profiler TM (PR) alone, respectively. Identification leads or confirmations of identification were obtained for 218 victims for which DNA reference samples (personal effects and kin) had been submitted. Confirmation of an inferred kinship association was sought through frequency and likelihood calculations, as well as corroborative data from other identification modalities. The use of a simple, yet powerful, automated genotype comparison approach and the use of megaplexes with high power of discrimination (PD) values extended considerably the identification capabilities in the case of the Swissair disaster. The DNA typing identification modality proved to be a valuable component of the large arsenal of identification tools deployed in the aftermath of this disaster.

To assist the interpretation of STR DNA typing results from forensic casework samples containing mixtures, the range of heterozygous allele peak height and peak area ratios (HR) and stutter percentages (stutter %) for the loci comprised in the AmpF STR R Profiler Plus TM (PP) kit were assessed on 468 database and 275 casework single source samples. Stutter % medians were similar for database and casework samples, ranging from 2% to 7%. The upper limit of the stutter value range was 16%, calculated as median +3 SD, although lower locus-specific values could be used. HR medians were 93 ± 6.5% for database samples, 88 ± 12% for casework samples. For casework samples, the maximum signal imbalance noted was 52%, calculated as median −3 SD. No significant difference was observed between peak height and peak area calculated values. This study shows the importance of selecting the proper reference database for the establishment of HR threshold values.

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