Evaluation of indirect transfer mechanisms of semen under varying test conditions.

There is limited data available on the subject of indirect transfer of non-visible body fluids, particularly semen, and often forensic science practitioner experience alone must be used to guide expectations. It can be difficult to assess the likelihood of proposed transfer scenarios without knowledge of how different variables can affect a transfer. The following work carried out by the Association of Forensic Service Providers UK and Ireland Body Fluid Forum explores how the features of transferred semen change with differences in the primary and secondary surface (porous and non- porous), different contact types (passive, pressure and pressure+) and with wet and dry primary stains. It was concluded that the primary surface type and whether the stain was wet or dry when contact occurred had the most significant effect on the transfer of semen, with wet transfers and transfers from the tested non-porous surface producing significantly more, and larger, visible stains under white light, stains with stronger fluorescence as viewed using Crime-lite® ML2, stains with stronger and faster acid phosphatase reactions and greater numbers of spermatozoa viewed using high power microscopy, compared to dry transfers and transfers from the tested porous surface. Pressure with movement transfers resulted in significantly more visible stains under white light and greater numbers of spermatozoa viewed using high power microscopy compared to passive transfers, however this only occurred when transfers were from a porous primary surface. The secondary surface type was not found to have a significant effect on the numbers of spermatozoa viewed using high power microscopy.

Enzyme activity, DNA degradation and drying times of semen, saliva and vaginal material.

The stability of enzyme activity and the amount of detectable DNA within liquid samples of semen, saliva and vaginal material were tested across a number of days. The concentration of DNA within neat semen and saliva samples fell significantly after one week of refrigeration. No apparent change in acid phosphatase or amylase enzyme activity was observed in neat semen and saliva samples over 96 or 72 h respectively. Changes in the enzyme activity of most of the dilute semen and saliva samples, as well as the neat vaginal material sample, were noted after 24 h. The drying times and sizes of stains produced from various volumes of neat semen, saliva and vaginal material were tested on porous and non-porous surfaces at room temperature. Larger volumes of body fluid took longer to dry and generally resulted in larger stains. Body fluids on a non-porous surface took longer to dry than on the porous surface tested.

Y-STR analysis of digital and/or penile penetration cases with no detected spermatozoa.

This forensic casework trial involved Yfiler(®) testing samples from 47 digital and/or penile penetration cases where the medical examination had occurred within 48h of the alleged incident and no spermatozoa had been detected following Sperm Elution(©). 30% of these cases yielded at least one Y-STR profile comprising three or more alleles per profile and 21% yielded at least one Y-STR profile of ten or more alleles per profile. This trial further investigated the persistence of male DNA in different case types, the location of samples submitted for testing and whether samples from different locations benefit from being combined prior to testing. The data supports the use of Y-STR profiling to provide scientific evidence to investigate whether the alleged sexual activity had occurred as well as to obtain probative evidence in spermatozoa negative penetration cases.

Comparison of methods for visualizing blood on dark surfaces.

Difficulties can arise when screening dark casework items for blood, a poor contrast between blood and the background can mean stains are not always evident. Typical indirect searching methods can be time consuming and may result in potentially important bloodstains being missed. Luminol, fluorescein, hydrogen peroxide, ultraviolet light and infrared photography were tested in an effort to find a rapid and efficient blood search tool for direct application to dark surfaces. Methods were compared in their sensitivity, specificity, ability to work on various surface types and their effect on DNA extraction and typing. Along with experimental results, the ease of use, costs and the health and safety considerations were also compared. Hydrogen peroxide was determined to be the most effective method. However, where blood was likely to be dilute, luminol was proposed due its greater sensitivity.

Sperm elution: an improved two phase recovery method for sexual assault samples.

This report describes the validation of a two phase cell recovery technique for the elution of two common cell types, epithelia and spermatozoa, from frequently examined items submitted as part of sexual assault casework. Furthermore, separation of cell types prior to microscopic examination of cell pellets improves the scientist's confidence in observing and scoring spermatozoa that may be present. During the validation, Orchid Cellmark's Sperm Elution© method consistently recovered a greater number of spermatozoa from simulated sexual assault items and swabs taken following consensual sexual intercourse compared to a water extraction technique. On average the Sperm Elution method recovered over twice the number of spermatozoa compared to the water method. The ability to separate the cell types present allows a rapid microscope slide search for spermatozoa and faster DNA extraction protocol in comparison to Cellmark's previous preferential method.

Tissue preparation using Arabidopsis.

The ability to track changes in the levels of many metabolites in plants has great utility in a number of biological contexts. A metabolomics experiment usually requires the comparison of different varieties in either a functional genomics context or in response to perturbation by an external treatment. Such treatments can result in subtle changes in the final chemical signature of the plant tissue, and therefore, any unwanted variance produced in the generation of that tissue must be minimised. Procedures for plant growth, harvesting, preparation of extracts, and the subsequent collection of data have been optimised to minimise experimental variation within the dataset. This chapter describes in detail how to generate reproducible Arabidopsis tissue suitable for a typical plant metabolomics experiment. Issues concerned with tissue sampling, harvesting, and storage are also discussed.

Assessment of 1H NMR spectroscopy and multivariate analysis as a technique for metabolite fingerprinting of Arabidopsis thaliana.

An approach to metabolite fingerprinting of crude plant extracts that utilizes 1H nuclear magnetic resonance (NMR) spectroscopy and multivariate statistics has been tested. Using ecotypes of Arabidopsis thaliana as experimental material, a method has been developed for the rapid analysis of unfractionated polar plant extracts, enabling the creation of reproducible metabolite fingerprints. These fingerprints could be readily stored and compared by a variety of chemometric methods. Comparison by principal component analysis using SIMCA-P allowed the generation of residual NMR spectra of the compounds that contributed significantly to the differences between samples. From these plots, conclusions were drawn with respect to the identity and relative levels of metabolites differing between samples.