Separation of sperm and epithelial cells based on fluorescence-activated cell sorting

The detection and separation of spermatozoa is crucial in the forensic investigation of alleged sexual assault cases. Differential lysis and microscopy-based techniques are conventional methods for the isolation of spermatozoa, though are time-consuming and frequently fail with samples containing an unfavourable sperm/epithelial cell ratio. Successful separation by means of fluorescence-activated cell sorting (FACS) has previously been reported, yet little efforts have been dedicated towards the further improvement or routine implementation of this technique. With this ongoing research, a methodology is being developed to sort sperm from epithelial cells by combining the Sperm Hy-Liter™ staining kit and FACS. Sorted sperm cells are then subjected to a direct lysis and low volume PCR (LV-PCR) protocol. Preliminary results demonstrate the successful separation of both cell types at a sperm/epithelial cell ratio of up to 1:500. The direct lysis and LV-PCR protocol allows to produce full haploid profiles from single sperm cells and mostly full diploid profiles from 10 spermatozoa. These data suggest that the proposed methods are potentially viable for forensic casework, yet additional testing is required for validation purposes.