This study used three experiments to assess the feasibility of applying laser microdissection (LMD) technology to precisely separate sexual assault cell mixtures by visual inspection coupled with laser dissection.
This study shows that through short-tandem-repeat (STR) genotyping, the LMD of mixed cell population achieves pure separation of sperm with no DNA contamination from external buccal epithelial cells. Hematoxylin/eosin (H&E) staining can be effectively used for sperm identification in conjunction with LMD separation for STR genotyping. Used in combination with the Lyse-N-Go extraction procedure, the LMD method is a simple, low-manipulation method for the analysis of sperm cells. This has the potential to facilitate analysis of low numbers of cells. An additional benefit of using the LMD method, whether it is for mixture separation or single source cell recovery, is that laborious intermediary DNA quantification analysis may be eliminated. Instead, cells can easily be counted during LMD collection and extrapolated into an estimate of DNA quantity added to the PCR reaction. This work will continue with the testing of mixtures with high concentrations of epithelial cells and its application to recover a small number of sperm cells for LCN analysis. Further studies are needed to establish whether testing of genuine casework specimens responds to LMD separation in a manner similar to the mock mixed-cell specimens of this study. The description of materials and methods addresses sample collection, samples/LMD slide preparation, H&E staining, LMD, DNA isolation, PCR conditions, electrophoresis conditions, data analysis, LMD histology comparison, and LMD mixture study. 2 tables, 9 figures, and 28 references