DNA Analysis and Document Examination: The Impact of Each Technique on Respective Analyses.

Threatening letters, counterfeit documents, and anonymous notes can commonly be encountered in criminal situations. Such handwritten documents may encourage DNA to transfer from the writer's hands and lower arms when these areas come into contact with the document. As any DNA transferred is likely to be at a low level, sensitive low copy number (LCN) DNA analysis can be employed for testing document exhibits. In this study, we determine locations on the document that are most commonly touched during writing and handling and compare DNA recovery from these sites. We describe the impact of DNA sampling on subsequent document examination techniques including the ESDA(®) and likewise the effect of the ESDA(®) and two other document examination techniques on subsequent DNA analysis. The findings from this study suggest that DNA results can be obtained through targeted sampling of document evidence, but that care is required when ordering these examination strategies.

STR profiling of epithelial cells identified by X/Y-FISH labelling and laser microdissection using standard and elevated PCR conditions.

During the investigation of allegations of sexual assault, samples are frequently encountered that contain DNA from a female and a male donor. These may represent contributions of DNA from the complainant and potentially, the offender. Many semen stained samples successfully undergo DNA analysis and interpretation using a differential extraction method that separates sperm from the epithelial cells present in the stain. However, for those mixed cell samples that contain only epithelial cells, separation of any male cells from female cells is problematic. This paper describes the application of fluorescent in situ hybridisation (FISH) for the gender identification of epithelial cells and subsequent recovery of target cells using laser microdissection (LMD). The profiling results obtained from samples of known cell numbers using the Identifiler™ multiplex at standard 28-cycle PCR conditions and, when cell numbers are low, the SGM Plus™ multiplex at elevated 34-cycle PCR conditions (also known as Low Copy Number DNA analysis (LCN)) are described.

The use of photographs to record variation in bruising response in humans.

There is considerable value in developing tools capable of accurately and reliably determining when bruises were inflicted in humans. Previous work has focused on the visual changes observed in a bruise as the injury develops and heals. However, due to variables such as how and where on the body the bruise was inflicted, differing tissue compositions at the injured skin site between individuals and inter- and intra-observer variation; a technique sufficiently robust for use in a clinical or medicolegal setting has not yet been identified. In this study we present a series of photographs taken under controlled conditions illustrating standardised bruises induced on participants using a weight dropping mechanism. We show that variation in the appearance of bruises over time across individuals is large and, although photography may be a suitable technique for the recording of injuries, it is not sufficiently reliable for determining the age of a bruise.

Efficacy of several candidate protein biomarkers in the differentiation of vaginal from buccal epithelial cells.

Currently, there is no accurate method to differentiate vaginal epithelial cells from buccal epithelial cells in biological samples typically encountered in forensic casework. This study tested the expression of a selection of candidate proteins in buccal and vaginal epithelial cells. We investigated six candidate biomarkers, such as loricrin, vimentin, stratifin, cytokeratin 4, cytokeratin 13, small proline-rich protein 2, and involucrin, using Western blot analysis on whole protein extracts and immunohistochemistry (IHC) on intact cells in an attempt to identify cell-specific markers that would differentiate these cells by microscopy. Involucrin, loricrin, and stratifin showed differential expression during Western blot analysis and were carried through to IHC. Although proteins unique to vaginal epithelial cells and buccal epithelial cells were not identified from among the proteins tested, the increased expression levels of two proteins, loricrin and stratifin in vaginal cells, when compared to buccal cells, do provide encouraging results in the search for epithelial cell-specific markers.

Development of a one-tube extraction and amplification method for DNA analysis of sperm and epithelial cells recovered from forensic samples by laser microdissection.

Laser microdissection can be used in forensic casework to isolate specific cell types from mixtures of biological samples. Extraction of DNA from selected cells is still required prior to STR amplification. Because of the relatively pristine nature of the recovered cells, laser microdissection is more sensitive than more traditional methods of DNA analysis, theoretically resulting in DNA profiles from less cellular material. A one-tube extraction and amplification method minimises loss of DNA through liquid transfers and reduces the potential for contamination events occurring. In this paper, the development of a one-tube method for the effective extraction of DNA from laser microdissected sperm and epithelial cells is described. The performance of the in-house method was compared to that of a commercial DNA extraction kit for extraction of DNA from sperm and the downstream compatibility with STR amplification was determined for both sperm and epithelial samples. Full Identifiler™ profiles after 28 amplification cycles were obtained from as few as 15 epithelial cells and 30 sperm.

Effects of histological staining on the analysis of human DNA from archived slides.

Archived slides of cell smears treated with histological stains for sperm detection are often the only source of DNA available when cold cases are reopened. There have been conflicting reports as to the negative effects of particular histological stains on DNA recovery and quality from human cells, making stain selection an important consideration for forensic laboratories. This study investigates the effect of several staining systems on DNA recovery from histological slide samples stored from 0 to 10 weeks. DNA profiles obtained after analysis of these samples with AmpFlSTR(®) Identifiler™ and increased cycle AmpFlSTR(®) SGM Plus™ short tandem repeat (STR) profiling systems and the effects that these stains have on DNA quantity and quality over time are described. Results indicate that Christmas Tree and Hematoxylin and Eosin stains do not have significantly different effects on DNA quality after 10-week storage of slides. This research will assist scientists to select staining systems that have minimal deleterious effects on the DNA recovered.

Validation and development of interpretation guidelines for low copy number (LCN) DNA profiling in New Zealand using the AmpFlSTR SGM Plus multiplex.

The characteristics of STR profiles produced from approximately 1 ng starting template using the AMPFlSTR SGM Plus multiplex and 28 PCR cycles, are well documented. However, the analysis of samples perceived as low in starting template (less than 100 pg), and referred to as low template DNA (LTDNA), can require a test of higher sensitivity in order to achieve successful results. One way of increasing this sensitivity is to increase the number of PCR amplification cycles from 28 to 34. This type of analysis has become known as low copy number, or LCN, DNA profiling. Amplification of LTDNA under LCN conditions can result in increased incidents of profile characteristics such as allelic 'drop-in' and allelic 'drop-out'. Adopting a testing regime which includes duplicate analysis, and maintaining a laboratory environment of stringent and monitored cleanliness, enables the scientist to identify and control these phenomena for a reliable interpretation of the DNA profiling results. A recent court ruling has questioned the reliability of LCN analysis and commented on the paucity of publications surrounding the validation of the technique. We present data for the LCN validation undertaken in our laboratory, and describe the guidelines and working practices we have developed for the analysis and interpretation of profiles generated after LCN profiling. This study augments the published record relating to LCN validation and should act as a useful guide for other laboratories who are considering implementing LCN profiling.

Evaluation of the BioSign PSA membrane test for the identification of semen stains in forensic casework.

AIM: To evaluate BioSign prostate specific antigen (PSA), a membrane test device used as a clinical aid in the diagnosis of prostate cancer, to determine whether it can be used in forensic laboratories for identifying semen stains. METHODS: Biological fluids were obtained under ethical approval from anonymous consenting donors. BioSign PSA was evaluated in terms of its specificity, sensitivity and cost to replace an ELISA (enzyme linked immunosorbent assay) method of PSA detection. RESULTS: Semen stain extracts and semen diluted 10(5) tested positive with BioSign PSA. Animal semen, other human body fluids and commonly encountered household products tested negative. Anomalous results were observed with semen-free condoms containing nonoxynol-9. The cause of these false positive results is not known. CONCLUSIONS: These results and the ease of use of the BioSign PSA kit indicate that it is a valuable addition to forensic laboratories and can adequately replace the ELISA method of PSA detection. BioSign PSA was not suitable for testing condoms for semen.