Abstract:
Hairs transferred through contact are a common type of trace evidence observed in many cases, specifically hairs in the telogen growth phase which are held the most loosely within the hair follicle and are therefore the most likely to be shed. Telogen hairs, however, may produce very little evidential value in a case, due to the low success rate of nuclear DNA (nuDNA) profiling. If hairs from relevant individuals overlap in the range of discriminating characteristics they may not be able to differentiated, particularly when using low power microscopy. Gender identification of the donor of the hair might be a useful tool, providing information as to whether or not high power comparison microscopy is appropriate in cases where the relevant individuals are of opposite gender. This is useful for hairs which have failed to produce or are not expected to produce results from the gender determining nuDNA site Amelogenin using standard DNA profiling analysis. Fluorescent in situ Hybridisation is a cytological technique used to localise fragments of DNA specified by the probe that is used. Gender identification of hair samples was achieved with the use of a chromosome enumeration probe (CEP). This probe is specific for the AT rich α satellite DNA sequence on the X chromosome giving an orange fluorescent signal and the satellite III DNA sequence, located in the Yq12 region of the Y chromosome giving a green fluorescent signal. This method is currently also being tested for use in forensic casework for the isolation of male and female epithelial cells in cellular mixtures. The aim of this project was to develop a robust and reliable screening method for the determination of the gender of the donor telogen hair roots without any visible adhering cellular material using low microscopic examination. This would enable scientists to assess the benefits of further microscopic comparisons or specialised DNA profiling tests such as LCN, Low Copy Number DNA analysis. Telogen hair roots were prepared using one of three methods, to investigate the appropriateness of each method in relation to success of downstream FISH X/Y signal numbers. These methods included the use of a microtome to produce thin cross sections of hair root for processing, a whole mount intact hair method utilising a permeabilisisation reagent on the hair root, and lastly a novel suspension approach using a dissociating reagent. The pre-processing wash steps currently employed at Environmental Science Research Ltd (ESR) were assessed to determine whether foreign DNA could lead to erroneous gender determination. The utilisation of the microtome for the production of telogen hair root cross sections appeared to be an inappropriate preparation method for FISH processing. Despite the positive DAPI staining observed in these samples, no FISH signals were seen. The results from the whole mount approach indicated that permeabilising the hair root did not increase the number of DAPI stained nuclei in the root and no FISH signals were observed. Lastly the suspension approach appeared promising as a preparation method for FISH processing. TrypLE™ express was a suitable dissociating reagent generating adequate numbers of cellular clumps from the telogen hair roots. Cellular clumps displayed sufficient DAPI staining and an adequate number of FISH X/Y signals for the determination of the gender of the hair donors. The pre-processing wash steps used at ESR appeared to be adequate for the removal of foreign DNA and the subsequent correct identification of the gender of the hair donor was made. The results from this pilot study indicate that this method could prove reliable and robust enough for forensic applications following successful outcomes from future validation work on a larger number of replicate hairs.