Fluorescence in situ Hybridisation labelling and Laser Microdissection of male and female cells from Forensic Samples

Abstract

Forensic case samples can often comprise mixtures of body fluids and/or skin cells that have been deposited on surfaces. These typically contain cells from both the complainant and the assailant. Therefore, biological samples recovered as forensic evidence may include gender cell mixtures and many also comprise different cell sources. DNA analysis of case samples is dependent on the efficiency by which the biological material can be recovered from a sample, such as a cotton swab. The method used to remove cells from cotton swabs is therefore a vital step to the success of DNA analysis. A method that maximises the intact epithelial cells recovered will assist in the successful DNA analysis of samples where intact cells are required, such as fluorescent in situ hybridisation (FISH) and laser microdissection (LMD). The addition of 2% sodium dodecyl sulphate (SDS) to buffers was investigated to determine if it improved intact cell recovery from cotton swabs. It was found that SDS led to a decrease in the percentage of intact cells recovered when compared to the buffers alone. DNA profiles of the perpetrator can be obtained from most sexual assault samples by the separation of sperm from epithelial cells. However current methods of separation do not take into account non sperm cells present and can result in mixed DNA profiles, which can be challenging to interpret. It is therefore desirable to explore alternative methods that enable the separation of cell samples prior to analysis. The main aim of this research was to combine FISH and LMD techniques for potential use on casework samples at ESR. Investigation into an X/Y FISH method, using the CEP® X SpectrumOrange / Y SpectrumGreen DNA probe kit, showed that male and female epithelial cells could be differentiated based on coloured fluorescent signals. Laser microdissection, using the Leica LMD6000 at ESR, allowed isolation of XX and XY FISH labelled cells for further DNA analysis. Full DNA profiles were generated from epithelial cells that were X/Y FISH labelled on PET membrane slides using standard Identifiler. The X/Y FISH method correctly identified the gender of buccal cells and some skin cells. All profiling results obtained from the X/Y FISH labelled cell samples recovered by LMD corresponded to the donor of the cells. X/Y FISH combined with targeted LMD cell selection and subsequent DNA profiling will expand ESR's cell identification and DNA profiling capability, while removing the challenges encountered with mixed profile interpretation.