Validation of Leica LMD 6000 Microscope for the Separation of Sperm and Epithelial Cells.




Subramanyam Reddy, Lakshmi


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This project conducted at the Southwest Louisiana Criminalistics Laboratory in Lake Charles, Louisiana involved validating the Leica LMD 6000 Microscope for the separation of the sperm and epithelial cells. Separation of the perpetrator’s sperm DNA from the victim’s epithelial cell DNA is a crucial step in sexual assault cases. Traditionally, preferential extraction methods have been used to separate the sperm cells from the epithelial cells of the victim. Although this technique has been successful in separating the epithelial cells from the sperm cells, there is periodically carryover between the two cell fractions, making the interpretation of results challenging. The Laser Microdissection technique, which was introduced in the mid-nineties, can improve and expedite the separation of the sperm and the epithelial cells, thereby enhancing the capabilities of analyzing difficult mixture samples. The validation involved assessing the reproducibility of the profiles obtained, sensitivity of the system and stochastic effects, separation of male sperm cells and female epithelial cells in a slide of mixed samples with a contamination assessment. Based on the results obtained, each of the samples with a sperm count of 250 and above generated a complete profile. The validation study suggests that 250 sperm would be sufficient to obtain a full genetic profile. A major issue of concern is that though the quantification step indicated the presence of DNA in the Laser microdissected samples, only one of the duplicates of the samples generated full profiles. Further studies with more replicates and conducting experiments using direct amplification or increased PCR cycles can be suggested to generate full profiles. Further work needs to be done to develop more consistency in the process, before applying the Laser Microdissection technique to real cases.